Approximately 3–5% of colorectal cancer cases belong to the hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome. HNPCC is an autosomal dominantly inherited disease with high penetrance due to germline mutations in mismatch repair genes. The increased overall mutation rate is associated with an elevated risk of developing early onset colorectal cancer as well as extracolonic tumors, such as endometrial and ovary cancer in women, stomach, small bowel, pancreas, and others [1]. Overall survival is better in patients with HNPCC compared to patients with sporadic cancer [2]. About 70% of HNPCC cases have developed due to the mutations distributed equally through the exons in the MLH1 and MSH2 genes [3, 4], and only some mutations have a proven founder effect [4]. HNPCC as a clinical syndrome is diagnosed according to the Amsterdam criteria and Bethesda guidelines and allow the identification of high risk families [5]. Family members with a confirmed mutation or at high risk, if the mutation is unknown but diagnosis is clinically proven, should take a screening colonoscopy every 1–2 years beginning at age 20–25 [5]. Endometrial sampling and transvaginal ultrasonography in women from HNPCC families is also considered to be useful starting at age 30–35 [1, 5], as the risk of developing endometrial cancer for a woman in a HNPCC family is 40–60% [6]. Still, due to HNPCC most endometrial cancer cases are diagnosed symptomatically, not by transvaginal ultrasound or biopsy. Transvaginal ultrasound can be more helpful in the case of ovarian cancer as the risk of developing it is about 6–12% [6]. Therefore, it is important to screen patients and their relatives for mismatch repair gene mutations in order to confirm the diagnosis of HNPCC and begin prevention measures for reducing the probability of developing cancer. This allows more accurate identification of patients from HNPCC families. In previous studies, it was concluded that the use of the Amsterdam criteria for HNPCC patient diagnosis in Latvia is limited and mutation spectrum differs from other neighboring countries [7, 8]. This study continues the research of mismatch repair gene mutations in the case of HNPCC.

The aim of this study is to investigate the mutation spectrum of MLH1 and MSH2 in high risk families and to accumulate information necessary for future diagnosis and consulting high risk patients and their family members.

PATIENTS AND METHODS

Patients with colorectal cancer corresponding to the Amsterdam criteria or Bethesda guidelines were selected from 1035 consecutive colorectal cancer patients at the Pauls Stradins Clinical University Hospital or counseled at the Hereditary Cancer cabinet during 2005–2009. Approval of Riga Stradins University Medical ethics committee was obtained and all patients who participated in this study signed an informed consent form. Patients or their relatives who participated in previous studies [7, 9] were excluded.

DNA was extracted from whole blood by the QIAgen FlexiGene DNA Kit. All DNA samples were subjected to whole sequencing of MLH1 and MSH2 as described earlier [10, 11]. Mutations were confirmed by sequencing both DNA strands on an independent PCR product. Samples with no mutation detected by sequencing were subjected to the multiplex ligation-dependent probe amplification (MLPA) analysis. MLPA and sequencing reactions were performed using the SALSA MLPA P003 MLH1/MSH2 kit (MRC-Holland, the Netherlands). MLPA reactions were analyzed using the Applied Biosystems genetic analyzer ABI3130. The following databases were used for mutation analysis: INSIGHT-group database (http://www.insight-group.org/mutations/) and NCBI SNP database (www.ncbi.nlm.nih.gov/snp/).

RESULTS

Amsterdam I criteria define HNPCC families according to family colorectal cancer history and the age of onset; at least two successive generations and three patients should be involved, at least one of which is a first degree relative to the other two, one of the cancers should be diagnosed before age 50 and family adenomatous polyposis (FAP) should be excluded. Amsterdam II criteria also include cancers that are associated with the HNPCC, such as endometrial, small intestine, stomach, and others. Bethesda guidelines are used to test colorectal cancers for microsatellite instability, and it is proven, that these are more applicable for detecting patients who should undergo genetic testing [1]. In this study, the main criteria used from the Bethesda guidelines were the young age of onset (before 50) in one of the affected family members.

Ten index patients were identified from 1035 consecutive colorectal cancer patients in Latvia by family history according to the Amsterdam I/II criteria or Bethesda guidelines. Among them 1 patient fulfilled the Amsterdam I criteria (0.1%), 5 fulfilled the Amsterdam II criteria (0.57%) and 4 fulfilled the Bethesda guidelines (0.38%). Medical and family histories are summarized in the Table.

Table.  Patients and their families data

(CRC — colorectal cancer, Ut — uterine cancer, Ov — ovarian cancer, Li — liver cancer, Pro — prostate cancer, CSU — cancer site unknown, d — died)

Seven patients out of 10 were found to harbor mutations in the MLH1 or MSH2 genes including large rearrangements. Four out of 5 patients meeting the Amsterdam II criteria were harboring mutations. Three out of 4 patients meeting the Bethesda guidelines were harboring mutations.  No mutation was detected in the only patient meeting the Amsterdam I criteria.

DNA sequencing revealed MLH1 and MSH2 mutations in five index patients. Four of those mutations including two nonsense mutations (MLH1, 37G>T and 1546C>T) and two frameshift mutations (MLH1, 1340delTGinsC and MSH2, 288delGTinsA) are clinically significant, as they result in a truncated protein. One nonsense mutation in the first exon of the MLH1 gene 37G>T (E13X) was discovered in patient E430. Patient C152 carried the nonsense mutation 1546C>T (Q516X) in the MLH1 exon 16. Patient C321 had a mutation in the MSH2 exon 2 288delGTinsA which leads to a premature stop codon at the amino acid position 173. Mutation in the MLH1 exon 12 1340delTGinsC, discovered in patient J236, truncates protein, leading to premature stop at codon 490. Patient J236’s family members were available for analysis: his son was diagnosed with colorectal cancer at age 36, and daughter (40 years old at present) is not diagnosed with any cancer. Both siblings carry the 1340delTGinsC mutation in the MLH1 gene. In patient D583, the MLH1 gene mutation 1959 G>T was found in exon 17.

MLPA analysis revealed two large rearrangements. In patients A538 and D500, large rearrangements of the MLH1 gene were found using MLPA. Patient A538 had the deletion of exon 12. Patient D500 has the duplication of exon 6.

None of all the mutations that were found in this study coincided with the previously reported mutations in Latvia [7, 9].

DISCUSSION

About 1000 new colorectal cancer cases are diagnosed in Latvia every year and approximately 100 of them at the Pauls Stradins Clinical University Hospital. Less than 1% are FAP cases [12]. As concluded before, the HNPCC rate from consecutive colorectal cancer patients in Latvia is about 2% [7] and about 20 primary diagnosed HNPCC patients can be expected in Latvia per year. The HNPCC is estimated at about 0.34% within the population of Latvia [13]. In other studies, hereditary colorectal cancer is estimated at 3–5% from all the colorectal cancer cases [14, 15] and 0.41% from the total population [16]. It is possible that the number of HNPCC cases in Latvia is underestimated due to a lower reliability of patients’ family data or the lack of full information about the medical history of a family. It has been described that finding hereditary cancer families in Latvia is a common problem because of small families, as there is small number of first degree relatives and not all patients cooperate with the doctors [17]. Families with hereditary cancer syndrome are more easily detected if the family is large. Previously in Latvia a statistically significant difference was observed between the size of the family diagnosed with hereditary cancer, according to defined criteria, and families with non-diagnostic findings. The mean numbers of blood relatives within the families with hereditary cancer syndromes were 13.6 and 12.2, while the mean number of blood relatives for the families not diagnosed with hereditary cancer syndrome was 9.5 [17]. As proven by case of hereditary breast cancer families in Latvia during population screening, the results of clinical screening and mutation screening do not overlap and molecular screening reveals more mutation carriers as clinical criteria [17]. Similar results were observed in the case of HNPCC from patients corresponding with the Amsterdam criteria — mutations were found in some of the patients, and mutation screening in consecutive patients revealed patients without familial cancer history [18]. In this study, only one patient is diagnosed according to the Amsterdam I criteria and the patient did not harbor any mutation in the MLH1 and MSH2 genes. Five patients were diagnosed according to the Amsterdam II criteria, which also included cancers that were associated with the HNPCC syndrome, not only colorectal cancer. Out of those five patients, four of them had mismatch repair gene mutations. From four patients who corresponded with the Bethesda guidelines, three patients had germline mutations in mismatch repair genes. Three families did not have any mutation in the MLH1 and MSH2 genes. The syndrome of those patients could be due to the mutations in other mismatch repair genes or associated with an unknown susceptibility locus or epimutations [19–21]. Up until now, several MLH1, MSH2 and MSH6 gene mutations in the case of HNPCC have been found in Latvia [7, 9], but none of these mutations were found in our research. None of the mutations had a proven founder effect in Latvian colorectal cancer patients, although the mutation MLH1 1409+1 A>G that was found in Latvia [7] is described in Polish and Finnish populations [8, 22].

Information about the MLH1 1959G>T mutation is not consequential and there is a possibility that the exact mutation does not affect mismatch repair. The mutation is predicted to form alternative splice site, resulting in exon skipping [23], although information available in the INSIGHT-group database does not conclude pathogenesis of this mutation in all cases. However, this mutation can be considered a rare polymorphism, as there is no phenotypic consequence [18, 24]. We concluded that 6 mutations out of 7 were pathogenic, as they resulted in altered protein, thus affecting mismatch repair and resulting in the development of cancer. The mutation MLH1 37G>T (E13X) has been reported in the INSIGHT-group database.

Mutations in the MLH1 and MSH2 genes are highly heterogeneous in Latvia. Combination of direct sequencing of the MLH1 and MSH2 genes and MLPA is the most appropriate molecular method of detecting HNPCC patients and family members at risk.

ACKNOWLEDGEMENTS

This study was supported by The National Research Programme “Development of new prevention, treatment, diagnostics means and practices and biomedicine technologies for improvement of public health”.

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Since it was shown in 1998 that the capacity of an allogeneic embryo to escape rejection by the mother may somehow involve tryptophan catabolism [1], a possible mechanism of maternal immune tole­rance has been widely investigated. Quite soon it was found that indoleamine 2,3-dioxygenase (IDO), an enzyme expressed in trophoblast and catabolizing tryptophan via the kynurenine pathway, is responsible for local depletion of the essential aminoacid tryptophan and accumulation of its toxic catabolites affecting T-cells [2, 3]. Suppression of IDO in the experiments with pregnant mice resulted in the rejection of allogeneic but not syngeneic embryos [1]. In onco­logy, these findings are critical for understanding how an intrinsically immunogenic tumor avoids or may avoid the immune attack in the host [4]. As a matter of fact, IDO was found to be expressed in various tumor cells and in tumor-infiltrating macrophages [5, 6]. In such cases high tumor IDO expression can predict unfavorable prognosis [4, 6, 7]. IDO inhibitors on their own slow down tumor progression and can enhance the therapeutic efficacy of a chemotherapy drug [8, 9]. A synthetic analog of tryptophan, D-1-methyl-tryptophan, is widely used as IDO inhibitor [9]. It is administered into animals chronically at daily doses up to 800 mg/kg b.w. [9]. However, since the IDO-induced immune tolerance to tumors can be overcome by suppressing the enzyme in the tumor cells and in tumor-infiltrating macrophages only, the dose of IDO inhibitor (and treatment costs) can be significantly reduced if the inhibitor is administered locally at tumor site. On the other hand, one of the recently discovered IDO-inhibiting compounds, ethyl pyruvate, a comparatively inexpensive and low-toxic anti-inflammatory agent, has drawn our attention [10, 11]. In the present study with the use of mice with an intrinsically immunogenic non-syngeneic tumor it was shown that chronic, systemic or local at tumor site, administration of ethyl pyruvate accelerates tumor rejection as compared with control.

Mice of C3HA strain and the transplantable tumor, Hepatocarcinoma-29 (H-29), were used [12]. All experimental procedures were perfomed in accordance with the normative rules of bioethics. The H-29 tumor originates from CBA mice but is transplantable to 100% of C3HA mice. In a considerable part of the grafted mice the tumor eventually stops to progress and regresses. At the preparatory stage, 5 x 10⁵ H-29 cells were inoculated into the femur muscle of C3HA male mice. A month later the animals with progressing tumors were culled. A considerably higher dose of tumor cells (fivefold and tenfold) was then transplanted to immune animals which rejected the tumor. After that the animals were divided into two groups. The animals of one group were administered ethyl pyruvate for IDO inhibition, the other group was used as control. In the experiment with the systemic administration of ethyl pyruvate the inhibitor was dissolved in Ringer’s solution (5 mg/ml) and administered intraperitoneally twice a day for 20 days. Control mice received no injections. In the experiment with local administration, 0.1 ml of 1% ethyl pyruvate solution was administered intramuscularly at tumor site once a day during 10 days after tumor cells inoculation. Control animals in the same manner were administered by 0.1 ml of saline. At regular intervals tumors were palpated and measured with a vernier caliper. The time of tumor resorption was registered. The experiment was concluded 10 days after regression of the last tumor in the experimental group. The φ-criterion and Fischer’s arcsin transformation were used to test the validity of differences in tumor regression values (percentage of mice with regressed tumors) between the experimental and control groups.

In our previous study, under primary H-29 transplantation to non-syngeneic C3HA mice the inhibition of IDO by D-1-methyl-tryptophan resulted in enhancement rather than suppression of tumor outgrowth [13]. The effect observed may appear paradoxical at first sight. Actually, an arising tumor and primary tumor transplants develop in the so-called pathologic immune privilege conditions [14] with a complex immune response that may suppress tumor growth or enhance it. In this case, IDO inhibition may cause not only the proliferation of T-killer and T-helper cells but may also activate suppressor T-cells (Treg) contributing to the development of immune tolerance in the host, thereby enhancing the growth of the tumor. On the contrary, the clinicians have usually to deal with tumors which immune relationships with the host are explicit and which use IDO to suppress the effector phase of immune response. For this reason in the experiments on the tumor-suppressing potential of IDO inhibitors it is reasonable to use preimmunized animals. In the present study tumor recipients were tumor-grafted mice who rejected the tumors had been transplanted earlier.

At the preparatory stage for the first experiment, H-29 tumor cells were inoculated into the femur muscle of forty intact four-month old mice of C3HA strain at a dose of 5 x 10⁵ cells. After a week tumors develo­ped in all animals; subsequently tumor progression was observed in 13 animals and tumor inhibition and eventual regression – in 27 animals. The mice with tumor progression were culled. The mice rejecting the tumor transplant were re-inoculated with a significantly higher (fivefold, 1 x 10⁶) dose of tumor cells at in the same femur muscle. Immediately after tumor cell inoculation the mice were divided into 2 groups. In the control group 13 animals were kept without any exposure. In the experimental group, 14 animals were exposed to injections of ethyl pyruvate twice a day as described in METHODS. The ethyl pyruvate injections at a single dose of 40 mg/kg were performed 40 times with a total course dose of about 1.6 g/kg b.w. As shown in Fig. 1, ten days after the transplantation tumors developed in 100% of the mice of both groups and then began to regress. During the first 4 days the rate of tumor regression was the same for both groups, and then somewhat faster in the experimental group as compared with control. Twenty six days after the transplantation the tumors regressed in all animals except two of the control group. Subsequently the tumor regressed in one animal and recurred and killed the other.

A month after tumor rejection the survived mice were used for H-29 cells inoculation at a dose higher than that used in the previous transplantation (5 x 10⁶). After the transplantation the treated and untreated mice were divided equally between the experimental and control groups of the new experiment. The groups were enlarged by 4–5 mice with regressed tumors, which became available by that time, and then exposed to treatment (daily local administration of ethyl pyruvate or saline, see MATHERIAL AND METHODS).

Six days after tumor cells inoculation, tumors appeared in all animals of both groups. The average tumor volume was 0.30 ± 0.031 cm³ for control and 0.20 ± 0.045 cm³ for the experimental group. Two days later the average tumor volume was 0.23 ± 0.031 cm³ and0.14 ± 0.034 cm³, respectively (insignificant differences). The tumors eventually diminished in both groups, but they became nonpalpable (fully regressed) at an earlier time and in more individuals treated with ethyl pyruvate as compared with control animals (Fig. 2). In both experiments ethyl pyruvate produced no notable toxic effect: both under systemic and local administration the mice’s body weight decreased by less than 4% upon completion of the course.

Thus, the results indicate that chronic, systemic or local, administration of ethyl pyruvate at a non-toxic dose to preimmunized mice results in an accelerated rejection of the repeated tumor transplants. In the present study (in particular in the first experiment) we used a cumbersome regimen of ethyl pyruvate administration recommended by the authors who first suggested it as IDO inhibitor [11]. At the same time, from the literature ethyl pyruvate is known to be a food additive [10, 11], implicating it should retain at least some of its activity under per os administration. Therefore, it can not be excluded that ethyl pyruvate may inhibit IDO not only under parenteral administration. This question requires further investigation. Thus, future research is to focus on the applicability and clinical prospects of ethyl pyruvate (and/or other IDO inhibitors) for the treatment of tumors in combination with common chemotherapy drugs.

REFERENCES

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Neuroendocrine tumors (NET) are common GI tumors and their detection and resection is like finding a needle in a hay stack. Nuclear medicine is a potential imaging tool which can be used for both diagnostic and therapeutic purposes. Many imaging modalities used in the past have a low sensitivity for NET compared to ⁶⁸Ga-DOTATOC [1]. Computed tomography with contrast can detect only up to 30% of tumors with a size of 1–3 cm [2]. Imaging techniques such as magnetic resonance imaging and computed tomography are not very adequate in detection of neuroendocrine tumours [3]. Use of specific tracers such as ⁶⁸Ga-DOTATOC in a PET/CT study has been proven to have a high sensitivity and specificity to cells expressing somatostatin receptors [5]. The use of ^99mTc-Octreotide to detect neuroendocrine tumors during surgery is an effective adjunct for therapy. We here present a clinical case where these modalities help in both diagnostic and therapeutic surgery.

Case report. In June, 2008, a 54-year-old man was referred to us with the diagnosis of urothelial carcinoma of the bladder (transitional cell carcinoma). After clinical investigations and confirmation of the diagnosis, he underwent a laparoscopic resection of the urinary bladder and prostate gland together with dissection and clearance of pelvic lymph nodes. During follow-up, in January 2010, biopsy was done on a suspicious paracaval lymph node. Histology revealed it to be a lymph node metastases of NET which did not originate from the bladder. MRI, endoscopic ultrasound, esophago-gastro-duodenal endoscopy, and colonoscopy were performed, but no primary NET was found. Besides this, the tumor marker chromogramin-A was low. The patient was considered to have cancer of unknown primary. However, the resected lymph node presented an elevated number of somatostatin receptors which are typically found in NET. Hence, a PET/CT scan was performed with ⁶⁸Ga-DOTATOC which is highly sensitive to SSR2-receptors. We found an increased uptake in paravertebral lymph node and in a small area located in the duodenum. We have successfully nailed down the primary tumor — but how could we help our surgical colleagues to resect the suspicious tumor?

We decided with our surgical team to proceed with gamma radiation probe guided surgery (Fig. 1).

^99mTc-Octreotide is also sensitive to SSR2 but emits gamma-rays instead of positrons as ⁶⁸Ga. The gamma probe was effectively used to help us detecting radiation emitted by the tumor intraoperatively and provided real-time information to navigate for tumour localization [1]. After surgical procedure the resected specimen was reported to be a 1.8 cm well-differentiated neuroendocrine (Fig. 2) carcinoma of the papilla limited to the duodenal wall and two peripancreatic lymph node metastases. Four months later on a follow-up ⁶⁸Ga-DOTATOC PET/CT imaging showed no suspicious lesion (Fig. 1).

Pathologic examination of the resected specimen confirmed as a 1.8 cm tumor of whitish cut surface and firm consistency, locating at the papilla (Fig. 2).

The tumor was limited to the duodenal wall. Microscopically, the tumor consisted of solid and trabecular formations of medium sized epithelial cells, displaying only little pleomorphism (Fig. 2, a). The tumor cells displayed prominent nucleoli; the cytoplasm was pale eosinophilic and occasionally contained clear vacuoles. The mitotic activity was low (one mitotic figure in 10 high power fields) and the tumor did not show any areas of necrosis. 2 of 18 peripancreatic lymph nodes contained metastases. The resection margins of the surgical specimen were free of tumor confirming appropriate margin clearance. Immunohistochemically, the tumor cells expressed the neuroendocrine markers chromogranin A and synaptophysin (Fig. 2, b). The proliferative activity (Mib1) was below 2%. An expression of the hormones ACTH, glucagon, gastrin, insulin, somatostatin, serotonin, or pancreatic polypeptide was not detected. Only very few tumor cells displayed a weak membranous immunoreactivity for somatostatin receptor 2 (Fig. 2, c).

Based on these pathological findings, the diagnosis of a well-differentiated neuroendocrine carcinoma of the papilla was established. According to the re­commendations of the European Neuroendocrine Tumor Society [6] the tumor was classified as pT2, pN1(2/18), G1.

Neuroendocrine tumors arise from hormone producing cells in the neuroendocrine system. They may originate anywhere in the body where there is neuroendocrine tissue. However, the location in the gastrointestinal tract is more likely because there are more neuroendocrine cells than anywhere else in the body. In early stages treatment of choice is surgical resection which subsequently results in a good prognosis.

CONFLICT OF INTEREST

No authors had any conflict of interest to declare.

REFERENCES

1. Gabriel M, Decristoforo C, Kendler D, et al. 68Ga-DOTA-Tyr3-octreotide PET in neuroendocrine tumors: comparison with somatostatin receptor scintigraphy and CT. J Nucl Med 2007; 48: 508–18.
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Ionizing radiation (IR) is one of the most studied carcinogens in the development of multiple myeloma, primary myelofibrosis, polycythemia vera, non-Hodgkin’s lymphomas, myelodysplastic syndromes and some forms of acute and chronic leukemia, especially in acute myelogenous leukemia (AML) [1, 2]. Until recently, chronic lymphocytic leukemia (CLL) has not been considered as a radiation-associated leukemia. Nevertheless, current understanding of radiation-induced tumorigenesis and the etiology of lymphatic neoplasia show that IR exposure increases CLL risk [3].

After Chernobyl nuclear accident, people living in the contaminated areas of Ukraine are still exposed to low doses of IR. Analysis of the patients with various forms of the malignancies of hematopoietic and lymphoid tissues has not revealed the differences in B-CLL percentage among Chernobyl clean-up wor­kers and Ukrainian population in whole. B-CLL was shown to be a predominant form of hematopoietic malignancies in clean-up workers as well as in general population [4]. Genetic mechanisms that result in the development and progression of CLL are mainly unknown [5]. Gene expression profiling by microarray is useful to understand B-CLL origin and development [6]. The analysis of the molecular genetic features should be advantageous in elucidating the putative association of IR and B-CLL.

Earlier, we have studied gene expressions of seve­ral apoptosis related genes in different types of tumors of hematopoietic and lymphoid tissues in 189 patients including those with B-CLL living in areas of Ukraine contaminated with radionuclides in post-Chernobyl period [7]. In the present study, we have analyzed gene expression patterns in samples from 44 B-CLL Ukrainian patients in post-Chernobyl period with the aim of identifying the genes related to or involved in this pathology in order to shed light on the biology of B-CLL.

MATERIAL AND METHODS

The samples of the peripheral blood of B-CLL patients were obtained from R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobio­logy of the National Academy of Sciences of Ukraine. All the patients were referred to Reference Laboratory of Immunocytochemistry and Oncohematology Department of the Institute for verifying the diagnosis. Bone marrow and peripheral blood smears stained by May-Grunwald-Giemsa were studied morphologically. Immunocytochemical techniques (APAAP, LSAB-AP) and a broad panel of monoclonal antibodies against lineage specific, differentiation and activation antigens of leukocytes were employed for immunophenotyping pathological cells in blood and bone marrow. The main forms and cytological variants of hematological malignancies were diagnosed according to new WHO classification [8]. All the samples were immunophenotyped, and only 44 samples from CD38-negative B-CLL patients out of 127 diagnosed patients with B-CLL/B-cell lymphoma from small lymphocytes [7] were included in the study. Control group comprised peripheral blood samples from 17 healthy donors. The ethic committees of both collaborating research institutions approved the design of the study.

Total RNA isolation. Total RNA was isolated from mononuclear cells for each patient using QIAamp RNA Blood Mini Kit (QIAGEN, Valencia, CA, USA) and treated with DNase I according to the manufacturer’s instructions. The quality of the RNA was assessed by loading 300 ng of total RNA onto an RNA Labchip (Agilent Technologies, Waldbronn, Germany), followed by analysis (A2100 Bioanalyzer; Agilent Technologies). An RNA integrity value (RIN) of 7.0 was considered acceptable.

RNAs from 44 B-CLL patients and 17 healthy donors were pooled seperately. Pooling process was performed in the way that 100 ng RNA sample was used from each B-CLL patient/healthy donor. Each RNA pool was prepared as three replicates.

Microarray analysis. Microarray analysis was performed using the Whole Human Genome Oligo Microarray (Agilent Technologies), encompassing more than 44,000 human DNA probes. The full list of cDNAs is available online (www.agilent.com). Protocols for sample preparation and hybridization of the mononuclear cells were adaptations of those in the Agilent Technical Manual. In short, first strand cDNA was trans­cribed from 300 ng of total RNA using T7-Oligo(dT) Promoter Primer. Samples were transcribed in vitro and Cy-3-labelled by using a Quick-AMP labeling kit (Agilent Technologies). Following a further clean-up round (Qiagen), cRNA was fragmented into pieces ranging from 35 to 200 bases in size. Fragmented cRNA samples (1.65 mg) were hybridized onto chips by means of 17 h of incubation at 65°C with constant rotation, followed by a two-step microarray wash of 1 min in two washing buffers (Agilent Technologies). Hybridized microarrays were scanned in a Agilent Technologies Scanner (model G2505B) and numerical results were extracted with Feature Extraction version 9.5.1.1 using 014850_D_F_20060807 grid, GE1-v5_95_Feb07 protocol and GE1_QCM_Feb07 QC metric set.

The microarray data were analyzed using GeneSpring software version 9.0 (Agilent Technologies, Santa Clara, CA). The fold changes were analyzed by filtering the dataset using P-value < 0.01 and a signal-to-noise ratio >2 for use in T-test statistical analysis. Additional filtering (minimum 2-fold change) was applied to extract the most these genes, which were analyzed using Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems, Redwood City, CA). Those genes with known gene symbols (HUGO) and their corresponding expression values were uploaded into the software. Each gene symbol was mapped to its corresponding gene object in the Ingenuity Pathways Knowledge Base. Networks of these genes were algorithmically generated based on their connectivity and assigned a score. The score is a numerical value used to rank Networks according to how relevant they are to the genes in the input dataset but may not be an indication of the quality or significance of the network. The score takes into account the number of focus genes in the network and the size of the network to approximate how relevant this network is to the original list of focus genes. The network identified is then presented as a graph indicating the molecular relationships between genes/gene products. Genes are represented as nodes, and the biological relationship between two nodes is represented as an edge (line). The intensity of the node color indicated the degree of up- or down-regulation. The node shapes are disclosed in corresponding figure legends. Canonical pathway analysis identified the pathways from the IPA library of canonical pathways, which were most significant to the input data set. The significance of the association between the data set and the canonical pathway was determined based on two parameters: (1) A ratio of the number of genes from the data set that map to the pathway divided by the total number of genes that map to the canonical pathway and (2) a P value calculated using Fischer’s exact test determining the probability that the association between the genes in the data set and the canonical pathway is due to chance alone.

Quantitative real-time PCR (Q-RT-PCR). cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit (Fermentas Inc., Maryland, USA). Q-RT-PCR was performed as we described previously for determination of MYC, BAX, BCL-2 and FAS1 gene expressions [9, 10]. Standard curves were obtained using serial dilutions of the beta-globulin gene (DNA Control Kit, Roche). Gene-specific primers (Table 1) were obtained from Integrated DNA Technologies (Iowa, USA). Obtained gene expression values were normalized using a housekeeping gene of beta2 microglobulin. Gene expression ratios were compared in patient and control groups using REST (Relative Expression Software Tool).

RESULTS

Differentially expressed genes are shown in two separate tables. The 100 most up-regulated genes are shown in Table 2. The 100 most down-regulated genes are shown in Table 3. Both sets of results were obtained based on minimum 2-fold change using GeneSpring software version 9.0 (Agilent Technologies, Santa Clara, CA). In Table 4 the gene expression results of four genes (MYC, BAX, BCL-2 and FAS1) obtained by real-time PCR and microarray methods are compared. Real-time PCR results of MYC, BCL-2 and BAX are in a good agreement with microarray expression rates.

We investigated interactions using IPA software and found 1191 network eligible up-regulated genes and 3398 Functions/Pathways eligible up-regulated genes. Fig. 1 shows the most significant four gene networks of over-expressed genes in B-CLL samples. Top functions of these genes are related to hematopoiesis, lipid metabolism, small molecule biochemistry, cancer, infectious diseases, cell cycle, cardiovascular system deve­lopment and function, gene expression, embryonic development, tissue morphology, inflammatory response. Up-regulated gene networks are identified around MYC, HNF1A and HNF4A, YWHAG, NF-κB1 and SP1.

We also found 1225 network eligible down-regulated genes, and 2657 Functions/Pathways eligible down-regu­lated genes. Fig. 2 shows four gene networks of down-regulated genes in B-CLL. The main functions of the genes are related to cellular functions and maintenance, protein synthesis, dermatological diseases and conditions, cell death, gene expression, inflammatory disease, cellular growth and proliferation, post-translational modification, cancer, infectious diseases, cell morphology, and deve­lopment. Down-regulated gene networks are identified around CEBPA, YWHAG, SATB1 and RB1.

DISCUSSION

B-CLL is a heterogeneous disease and a predominant form of hematopoietic malignancies. Despite new molecular methods identifying important prognostic and diagnostic genetic markers, genetic mechanisms involved in B-CLL origin are mainly unknown. A number of novel prognostic markers such as Bcl-2, MAP-kinase, NF-κB, ZAP-70 were identified applying gene expression profiling before [11, 12].

We have analyzed gene expression patterns in samples from 44 B-CLL Ukrainian patients in post-Chernobyl period to identify genes associated with this form of lymphoproliferative malignancy. Our study has demonstrated new genetic networks and biological pathways in both up- and down-regulated gene expression levels.

Analysis using IPA software revealed 1191 network eligible up-regulated genes and 3398 Functions/Pathways eligible up-regulated genes. The individual genes are found in multiple categories of functions related to hematopoiesis, lipid metabolism, small molecule biochemistry, cancer, infectious diseases, cell cycle, development and function of cardiovascular system, gene expression, embryonic development, tissue morphology, inflammatory response.

One important gene network is identified around the up-regulated MYC and SP1 genes (Fig. 1, a). MYC, a strong proto-oncogene, plays very important roles in cellproliferation (by upregulating cyclins, downregulating p21), controlling cell growth (by upregulating ribosomal RNA and proteins), apoptosis (by downregulating BCL-2), differentiation and stem cell self-renewal. Mutations, overexpression, rearrangement and translocation of MYC have been associated with a variety of hematopoietic tumors, leukemias and lymphomas, including Burkitt lymphoma [13]. High expression level of MYC has been reported in more aggressive and apoptosis resistant forms of B-CLL and might be used as molecular marker specific of resistant B-CLL subsets [14, 15]. SP1, a zinc finger transcription factor, is involved in cell differentiation, cell growth, apoptosis, immune response, response to DNA damage, and chromatin remodeling. SP1 and MYC are involved cooperatively in telomerase activation, which is a critical step in cellular immortalization and carcinogenesis. Kyo et al. have suggested that the level of SP1 expression might be a critical determinant of telomerase activity both in cancer and normal cells [16].

Another network is identified around NF-κB1 gene (Fig. 1, b). NF-κB regulates several genes that mediate tumorigenesis and metastasis and also plays an important role in pathogenesis of B-cell neoplasms. Carcinogens, tumor promoters, inflammatory cytokines, and chemotherapeutic agents activate NF-κB and this activation can suppress apoptosis, thus promoting chemoresistance and tumorigenesis. Bharti et al. suggested that NF-κB might be an ideal target for chemoprevention and chemosensitization [17, 18]. In addition, we have found NF-κB gene centered around two up- and down-regulated networks in our previous study on prostate cancer [19].

Canonical pathway analysis revealed that G-protein coupled receptor (GPCR) signaling is an important pathway modulated by the up-regulated genes in B-CLL. It is known that GPCRs regulate proliferation, differentiation, chemotaxis and also they play an important role in inflammatory diseases and cancer [20]. GPCRs are involved in control of transcription factors such as STAT3, NF-κB and CREB by G protein subfamilies [21]. Enhanced viability of CLL cells by the STAT3 phosphorylation and interaction between hepatocyte growth factor and its receptor (c-MET), which was found up-regulated in our study, was reported before [22]. CREB (cAMP response element binding protein) had been found overexpressed in bone marrow samples from patients with acute lymphoid and myeloid leukemia and associated with a poor outcome in AML patients according to previous studies [23].

A network is also identified around HNF1A and HNF4A (Fig. 1, c). HNF1A is a transcription factor required for the expression of several liver-specific genes and the expression of this gene is controlled by HNF4A, which may play role in development of the liver, kidney and intestines.

Another significant signaling pathway is calcium signaling involved in many processes such as cell survival/apoptosis, cell cycle progression, differentiation, cross-talk between intracellular compartments (ER, mitochondria), general metabolism and telomerase activity. The calcineurin/NFAT signaling pathway is important in lymphoma/leukemogenesis [24]. Deregulation of this signaling and/or abnormal expression of its components has been reported in solid tumors of epithelial origin, lymphoma and lymphoid leukemia. Mouse models of human T-ALL/lymphoma showed the pro-oncogenic effect of active calcineurin/NFAT signaling in vivo [25]. NFAT transcription factors form four calcium signaling responsive members: NFATc1, NFATc2, NFATc3 and NFATc4. Among these members NFATc1 and NFATc2, which are found up-regulated in our study, were reported to be involved in the development, differentiation and functioning of multiple T-and B-cell subsets in previous studies. NFATc1 was found to be expressed in a majority of aggressive B-cell lymphomas. On the other hand, NFATc2 activation was shown to be responsible in B-CLL, in cooperation with STAT6, for the high expression of CD23 [24].

Metabolism of xenobiotics by cytochrome P450 pathway has been shown as highly significant in our study. The enhanced expression of several P450s like CYP1A, CYP2C and CYP3A, that are up-regulated in our study, was reported in tumor cells elsewhere [26].

Arachidonic acid and linoleic acid metabolisms are the other significant pathways modulated by the up-regulated genes in our study.

Analysis using IPA software revealed 1225 network eligible down-regulated genes, and 2657 Functions/Pathways eligible down-regulated genes. These individual genes are related to cell functions and maintenance, protein synthesis, dermatological diseases and conditions, cell death, gene expression, inflammatory disease, cell growth and proliferation, post-translational modification, cancer, infectious diseases, cell morphology, and development.

One important down-regulated network is identified around RB1 gene (Fig. 2, a). The role of RB1 in B-CLL has been reported based on cytogenetic data [27]. RB1 deletions involved in 13q14 abnormalities have been reported in B-CLL before [28].

Another down-regulated network is identified around SATB1 gene (Fig. 2, b). SATB1 is a new type of gene regulator expressing in various human cancers and thought to be related to the malignant potential. Overexpression of this gene has been reported as a predictor of poor prognosis in lung and gastric cancers [29, 30].

An important network is identified around CEBPA gene (Fig. 2, c). CEBPA is a critical transcriptional factor and regulates the balance between cell proliferation and differentiation during early hematopoietic development and myeloid differentiation [31]. CEBPA has a tumor-suppressor function in leukemogenesis and both loss of function and gain of function have leukemogenic potential. It was reported that overexpression of CEBPA could contribute to B-ALL and loss of function could contribute to AML [32]. On the other hand, down-regulated CEBPA was found in acute promyelocytic leukemia stem cells in animal models [33].

Canonical pathway analysis revealed that oxidative phosphorylation is an important pathway modulated by the down-regulated genes in B-CLL. In fact, previous studies suggested that the oxidative phosphorylation (OXPHOS) system is severely compromised in various cancers [34].

EIF2 signaling is another significant pathway. Suppression of head and neck, colorectal carcinoma and multiple myeloma tumor growth and/or survival by phosphorylation of eIF2α was reported before [35].

IPA reveals regulation of eIF4 and p70s6K signa­ling pathway. eIF4E down-regulated in our study plays an important role in tumor initiation and progression when its overexpression cooperates with oncogenes to accelerate transformation in cell lines and animal models [36]. p70s6K is a serine/threonine kinase and its target substrate is S6 ribosomal protein [37]. Inhibition of p70s6K was related to cell cycle arrest at G0/G1 phase in human cancer cells before [38].

Protein ubiquitination is another pathway found significant in our study. Ubiquitination of key signaling molecules by E3 ubiquitin ligases forms an important regulatory mechanism for NF-κB signaling. Deubiquitinases (DUBs) counteract E3 ligases and play a substantial role in down-regulation of NF-κB signaling and homeostasis [39].

Cdc42 signaling is a highly significant pathway. Cdc42 promotes or inhibits tumor progression depending on the cellular context and contributes to cancer development through its different roles in intracellular trafficking, cell cycle regulation and survival, polarity, migration and transcriptional control [40]. Cdc42 is also important in the development and progression of lymphoma. Genetic knockdown or pharmacological inhibition of Cdc42 resulting in a cell cycle arrest and apoptosis of anaplastic large cell lymphoma cells has been reported [41].

An important network is identified around both down-regulated and up-regulated YWHAG gene in our study (Fig. 1 and Fig. 2). This gene encoding for 14–3-3 protein gamma was found highly expressed in skeletal and heart muscles. It has been suggested that this protein has an important role in muscle tissue [42, 43]. 14–3-3 proteins play critical regulatory roles in signaling pathways in cell division and apoptosis [44]. Further investigations are required to establish the function of YWHAG gene in B-CLL.

Real-time PCR confirmation results of four genes (MYC, FAS1, BAX and BCL-2) show that only MYC, BAX and BCL-2 expressions are in agreement with microarray results. Up-regulation of MYC is compatible with our expectations.

Previous studies indicate that high ratio of Bcl-2 to Bax proteins confers a poor prognosis with decreased rates of complete remission and overall survival [45]. In our study, BCL-2 upregulation level is superior to that of BAX in real-time PCR results but not in microarrays being analyzed.

FAS1 expression was found up-regulated in real-time PCR but down-regulated in microarrays in our study. It has been reported that Fas expression is not very high in B-CLL [46] that coincides with our fin­dings of relatively small up-regulation by real-time PCR. It should be noted that Fas was mentioned as apoptosis regulator [47] in CLL cells exposed to IR.

NF-κB gene network was conspicuous in terms of being determined also in our previous studies of gene expression in prostate cancer. In addition, upregulated levels of G protein coupled receptor signaling pathway, which has an important role in transcription of NF-κB, need advanced examinations. In this sense, NF-κB gene which is important in both cell cycle regulation and cancer progression deserves further study.

Our study has presented the gene expression profiling in B-CLL patients of Ukrainian population as whole. We believe that the contribution of IR as the putative factor in the origin of B-CLL should be further evaluated using such molecular genetic approach.

ACKNOWLEDGEMENTS

The study was financed within the framework of the joint research project M/32–2008 “Cytomorphological, immunocytochemical and molecular biological features of leukemias in persons exposed to ionizing radiation” according to the Agreement between the Ministry of Education and Science of Ukraine and the Scientific and Technical Research Council of Turkey (TUBITAK).

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Endoglin (CD105) is a 180-kDa cell membrane glycoprotein which serves as a coreceptor for transforming growth factor (TGF)‑b1 or TGF‑b3, in the presence of the TGF‑b type II receptor (TbRII) [1]. Endoglin is highly expressed by activated endothelial cells (ECs) [2]. Hypoxia and TGF‑b are two main factors that coope­rate to induce its expression [3]. Endoglin promotes angiogenesis mainly by activation of vascular ECs proliferation [4]. Endoglin overexpressed on endothelia of vessels in several human solid malignancies [5] and its overexpression is associated with lower patient survival rates, presence of nodes metastases and distant metastatic disease [6].

Tumor vascular endothelium shows up-regulation of various receptors, including the vascular endothelial growth factor receptor 2 (VEGF R2) and angiopoietin receptor, Tie2 [7]. Two other endothelial receptors for angiogenic factors, the VEGF receptor 1 (VEGF R1) and the orphan receptor Tie 1, are also up regulated by hypoxia [7]. So far, two different mechanisms are known which lead to the formation of soluble receptors [8]. Firstly, soluble receptors can be translated from differentially spliced pre-mRNA molecules lacking the transmembrane domain (e. g., sVEGF R1) [9] and the second mechanism involves limited proteolysis in the extracellular domain of the membrane receptor leaving the ligand-binding domain intact (e. g., sTie2) [8]. The external domain can be cleaved or shed and released into the circulation [7].

Similarly to classical receptors, accessory receptor — endoglin exists in two forms: as a membrane-bound and a soluble (s‑endoglin) in the circulation. Recent findings [10] suggest that the ectodomain of endoglin is released through proteolytic cleavage by membrane-type 1 matrix metalloproteinase (MT1‑MMP) — matrix metalloproteinase‑14 (MMP‑14). Coexpression of endoglin with MMP‑14 on the cell membrane leads to the cleavage of endoglin at the glycine-leucine bond at position 586, releasing the nearly complete endoglin extracellular domain [10]. Hawinkels` et al. [10] study also shows that MMP‑14 is the most abundantly expressed MT‑MMP in ECs and that knockdown of MMP‑14 strongly reduces s‑endoglin levels in the conditioned media of these cells cultures. Local up‑regulation of endothelial MMP‑14 expression may increase s‑endoglin, decrease membrane-localized endoglin, and transform the endothelium to a quiescent state [10]. Similarly to endoglin, MMP‑14 is highly expressed not only by ECs, but also by several other cell types, i. e. by cells of lung tumor [11]. In Atkinson’s et al. [11] studies, among all MT‑MMPs (MMPs 14‑17, 24 and 25), MMP‑14, -15 and –17 displayed higher expression in tumor relative to normal lung specimens. In addition MMP‑14 mRNA expression strongly correlated to MMP‑14 proteolytic activity in tumor models. Therefore, s‑endoglin might be shed not only from endothelial cells, but also from tumor cells. To conclude, the level of s‑endoglin could be an indicator of tumor‑activated endothelium, but it depends on shedding proteases expression, mainly MMP‑14.

The soluble receptor displays biological activity by acting as a specific endogenous antagonist complexing the corresponding ligand and thus preventing the ligand-mediated signal transduction [8]; soluble receptors are capable of scavenging circulating ligands, e. g. VEGF can be bound to sVEGF R1 [9], Ang1 and Ang2 — to sTie2 [8]. The role of s‑endoglin is not yet clear, maybe it competes with TGF‑b for TbRII binding [12]. But it is known that s‑endoglin has antiangiogenic properties; it is capable of reducing spontaneous and VEGF‑induced angiogenesis. Also, s‑endoglin fused with the Fc fragment of human immunoglobulin G strongly reduces microvessel density in a mouse model of invasive ductal breast carcinoma [10]. Experiment of Le et al. [12] showed two diffe­rent oligomeric forms of recombinant s‑endoglin. The dimeric s‑endoglin enhanced TGF‑b signalling in U937 cells, in a dose‑dependent fashion. However, tetrameric s‑endoglin was not active in this system, thus, its biological relevance is not yet clear. This form of s‑endoglin might be a resting inactive form that can undergo conformational changes into dimeric or other active forms under certain activating conditions. They concluded that the recombinant s‑endoglin is capable of modulating TGF‑b signal effectively, thus, can potentially be applied for therapeutic purposes.

The quantification of soluble forms of receptors might be interesting in terms of diagnostic and/or prognostic, but also therapeutic applications. The aim of present study was to analyze the changes of s‑endoglin level in plasma of lung cancer patients and to estimate the correlation of s‑endoglin with other soluble receptors, sTie2 and sVEGF R1.

MATERIALS AND METHODS

Study population. The study included patients with stage I non-small cell lung cancer, who underwent tumor resection without any preoperative therapy. These patients were treated in the University Hospital Department of Thoracic Surgery and Tumors, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, during the years 2008–2010. Three blood samples were taken from each patient: one prior to surgery and others on postoperative days 7 and 30.

The study protocol was approved by the Ethical Committee of the Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń (Poland). All patients gave consent.

Blood sampling and processing. s‑Endoglin, sTie2, sVEGF R1 concentrations were evaluated in plasma. Two millilitres of blood were taken from elbow vein. EDTA was used as an anticoagulant. Within 30 min after the collection, the blood samples were centrifuged at 2–8°C for 15 min at 1000 x g. The plasma was stored at ‑70°C.

s‑Endoglin determination. s‑Endoglin, sTie2, sVEGF R1 concentrations were assayed by commercially available sandwich enzyme-linked immunosorbent assay kits from R&D Systems (Quantikine Human Endoglin/CD105, sTie2, sVEGF R1 Immunoassay, R&D Systems Inc., Minneapolis, USA). Kit is designed to measure human endoglin/CD105, sTie2, sVEGF R1 in cell culture supernates, serum, and plasma.

Statistical analysis was done using Wilcoxon signed-rank test and Pearson`s linear correlation. The results were considered statistically significant for p < 0.05.

RESULTS

In Table 1 the comparison of s‑endoglin concentration before surgical treatment of lung cancer patients and on 7^th day and 30^th day after tumor resection is presented. The median of s‑endoglin concentration decreased on 7^th day when compared with preoperative level (3212.0 vs. 4112.0 pg/ml; p < 0.0001) and then it increased on 30^th day to reach greater values than on 7^th day (4447.0 vs. 3212.0 pg/ml; p < 0.01), but it was comparable with pretreatment level (4447.0 vs. 4112.0 pg/ml; p = 0.478).

In this study the estimation of correlation between s‑endoglin and other soluble receptors was accomplished (Table 2). The correlation between s-endoglin and sTie2, both before surgery (r=0.44) and on postoperative day 7 (r=0.52) and on 30^th day (r=0.58) was high. However, s‑endoglin was correlated with sVEGF R1 only on postoperative day 7, this correlation was very high (r=0.75).

DISCUSSION

Endoglin is primarily expressed in proliferating vascular endothelial cells, and its expression increases during tumor angiogenesis. Such properties have made endoglin a reliable marker of various solid tumors vasculature [13–21].

Soluble endoglin in serum is also elevated in various cancers, including breast, colorectal and liver cancers, and it correlates with the presence of metastatic disease [22–25].

Serum s‑endoglin has also prognostic value. It is an indicator of prostate cancer metastasis to the pelvic lymph nodes and of biochemical recurrence after medical prostatectomy. In multivariate analysis, only endoglin and Gleason score, but not PSA or clinical stage, were predictive of lymph node metastases [26–28]. Elevated pretreatment plasma s‑endoglin level is predictive for decreased clinical benefit and a shorter overall survival in metastatic breast cancer patients treated with 2^nd-line hormone therapy [29].

Besides that, the high level of s‑endoglin decreased in patients receiving chemotherapy. Conventional chemotherapy regimens suppress endothelial cells in tumor vasculature and consequently inhibit the release of s‑endoglin from endothelial cells [23].

In our study, after surgical resection of lung cancer the level of plasma s‑endoglin decreased on 7^th day when compared with preoperative level, and next on 30^th day — increased and it was comparable with that before surgery intervention. This problem can be explained in the follo­wing way. The decrease of s‑endoglin probably is a consequence of resection of tumor mass, highly vasculated and expressing both endoglin and shedding enzyme, MMP-14. However, increase of s‑endoglin on 30^th day in comparison with postoperative day 7 might be the result of stimulation of ECs and circulating progenitor endothelial cells (EPCs) by various tumor-derived angiogenic factors (e.g., VEGF, Ang2), which levels in circulation after surgical treatment are increased [30, unpublished own data].

Myśliwiec et al. [24] received similar results: after surgical treatment of colorectal cancer s-endoglin level on postoperative day 3 decreased when compared with preoperative level, then it increased on day 10 to reach greater values than on postoperative day 3, but lower than preoperative point. They explain these changes as follows: decrease of s‑endoglin level after surgery might be at least in part due to the action of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha). TNF‑alpha has been reported to down-regulate endoglin at post-transcriptional level [31].

In our study there were interesting correlations between plasma s‑endoglin levels with other soluble form of receptors. s‑Endoglin correlated with sTie2 both before and after surgery (BEFORE: r=0.44, AFTER (day 7): r=0.52, AFTER (day 30): r=0.58) and with sVEGF R1 — only on postoperative day 7 (r=0.75). These two soluble receptors are formed in different manner: sTie2, similarly to s‑endoglin is produced by proteolytic proces­sing, however, sVEGF R1 derived predominantly from alternative splicing. This could explain above correlations of s‑endoglin: with sTie2 — constant, and with sVEGF R1 — only in one investigated point.

The increased level of serum endoglin in various cancers compared to controls, prognostic value of this angiogenic factor and changes of its level after chemotherapy or surgical treatment suggest potential application of soluble form of endoglin as tumor marker in the future.

ACKNOWLEDGEMENT

The work was supported by the subsidy for financing the statutory activity of Nicolaus Copernicus University in Toruń, number 461/2010.

References

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4. Lebrin F, Goumans M-J, Jonker L, et al. Endoglin promotes endothelial cell proliferation and TGF-b/ALK1 signal transduction. EMBO J 2004; 23: 4018–28.
5. Miller DW, Graulich W, Karges B, et al. Elevated expression of endoglin, a component of the TGF‑b-receptor complex, correlates with proliferation of tumor endothelial cells. Int J Cancer 1999; 81: 568–72.
6. Dallas NA, Samuel S, Xia L, et al. Endoglin (CD105): A marker of tumor vasculature and potential target for therapy. Clin Cancer Res 2008; 14: 1931–7.
7. Harris AL, Reusch P, Barleon B, et al. Soluble Tie2 and Flt1 extracellular domains in serum of patients with renal cancer and response to antiangiogenic therapy. Clin Cancer Res 2001; 7: 1992–7.
8. Reusch P, Barleon B, Weindel K, et al. Identification of a soluble form of the angiopoietin receptor TIE‑2 released from endothelial cells and present in human blood. Angiogenesis 2001; 4: 123–31.
9. Wu FTH, Stefanini MO, Gabhann FM, et al. A systems biology perspective on sVEGFR1: its biological function, pathogenic role and therapeutic use. J Cell Mol Med 2010; 14: 528–52.
10. Hawinkels LJAC, Kuiper P, Wiercinska E, et al. Matrix metalloproteinase‑14 (MT1‑MMP)-mediated endoglin shedding inhibits tumor angiogenesis. Cancer Res 2010; 70: 4141–50.
11. Atkinson JM, Pennington CJ, Martin SW, et al. Membrane type matrix metalloproteinases (MMPs) show differential expression in non-small cell lung cancer (NSCLC) compared to normal lung: Correlation of MMP‑14 mRNA expression and proteolytic activity. Eur J Cancer 2007; 43: 1764–71.
12. Le BV, Franke D, Svergun DI, et al. Structural and functional characterization of soluble endoglin receptor. Biochem Biophys Res Commun 2009; 383: 386–91.
13. Marioni G, Marino F, Giacomelli L, et al. Endoglin expression is associated with poor oncologic outcome in oral and oropharyngeal carcinoma. Acta Otolaryngol 2006; 126: 633–9.
14. Oxmann D, Held-Feindt J, Stark AM, et al. Endoglin expression in metastatic breast cancer cells enhances their invasive phenotype. Oncogene 2008; 27: 3567–75.
15. Taskiran C, Erdem O, Onan A, et al. The prognostic value of endoglin (CD105) expression in ovarian carcinoma. Int J Gynecol Cancer 2006; 16: 1789–93.
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Acute lymphoblastic leukemia (ALL) is a malignant neoplasm of hematopoietic stem cells, which is the most common cancer among children with a peak incidence in 4–5 years of age [1]. It accounts for 23% of childhood cancers (children younger than 15 years) [2]. It is still the most common malignant disorder in childhood and principal cause of death due to disease in the pediatric age group, especially prevalent in males [3]. Interaction of genetic and environmental factors together may enhance the risk of leukemogenesis [3, 4].

Folate metabolism plays crucial role in cellular functioning and it serves as donor of one carbon for the synthesis of purines and pyrimidines which are used for the synthesis of RNA and DNA [3, 5–7]. Normal metabolism of folate might be affected by imbalance of nutrition, cellular transport interruption and mutation in folate-related genes. Several mutations in folate-related gene sequences have been reported. However, the effect of these mutations on folate related genes still is unclear [3, 4]. Deficiency of folate or non-homogenous distribution of folate vitamins may influence the risk of cancer by misincorporation of uracil into DNA and leading to double-strand breaks and chromosomal damage, DNA hypomethylation/demethylation of some tumor suppressor genes by which it modulate the leukemogenesis [8–11].

The methylenetetrahydrofolate reductase (MTHFR) gene is located on chromosome 1p36 [12] and translated to enzyme which catalyzes the reduction of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. 5-methyltetrahydrofolate acts as a carbon donor for de novo methionine synthesis and DNA methylation [13–15]. MTHFR influences the process of DNA methylation and distribution of uridylates and thymidylates bases for DNA synthesis and repair. Therefore due to its role in folate pathways, it makes the MTHFR an important candidate for study of cancer predisposing gene [12, 15–17]. Two frequently reported and well-studied mutations in sequence of methylenetetrahydrofolate reductase gene are (MTHFR 677C→T and 1298A→C) which reduce the activity of encoded enzyme [16, 17]. The folate used for synthesis of purine and pyrimidine is accumulated in the form of 5-methyltetrahydrofolate (the most abundant form in wild-type MTHFR 677 CC subjects) [18, 19].

5-methyltetrahydrofolate-homocysteine methyltransferase (MTR) gene is located on 1q43. It is the enzyme which catalyzes the transfer of methyl base from 5-methyl THF to homocysteine [20]. It is reported to have a polymorphism in locus 2756 (A→G i.e. glycine→aspartic acid), resulting in reduced enzyme activity and it is considered as a prime cause for elevation of homocysteine and subsequently DNA hypomethylation [20, 21],

Several reports have indicated that substitutions of C→T at locus 677, A→C at locus 1298 of MTHFR gene and A→G at locus 2567 of MTR gene reduce the enzyme activity and result in hypomethylation of DNA and subsequently reduced incidence of DNA double-strand breakage [21]. Hence, the study of these SNPs can rationally assist to determine the risk of group to ALL and also can be used for study of development and progression of childhood ALL in the population study. The aim of this work is to investigate frequency of MTHFR and MTR polymorphisms and their interaction with respect to possible effect on risk of childhood ALL among North Indian population.

METHODS

Specimens. Total 125 bone marrow aspirates were collected from the patients at initial time of diagnosis and prior to any treatment during admission to Hemato-oncology ward (2007–2009), Advanced Pediatric Center, PGIMER, Chandigarh, India. Informed consent was obtained from all patients in accordance with the institutional guidelines and the declaration of PGIMER. The ethical committees of PGIMER approved the protocol.

Diagnosis was based on morphological, cytogenetic and immunophenotypic criteria of WHO classification [39] by Department of Hematology, PGIMER. The clinical characteristic of the ALL patients are shown in Table 1.

Control samples (n=100) were obtained from blood of healthy donors with respect of median and mean of age and sex of patients. The mononuclear cell fraction was separated from bone marrow aspirate and blood by Ficoll density gradient centrifugation (Ficoll Hystopaqu, Sigma Aldrich, USA) and was used for DNA isolation. All samples contained at least 40% blast cells.

DNA isolation. DNA was extracted from mononuclear cells of bone marrow and blood by standard proteinase K digestion followed by phenol-chloroform method [27]. DNA quantity and quality was determined by spectrophotometry via measuring the optical density at 260 nm and 280 nm of the prepared diluted DNA (1 OD at 260 nm unit =50 µg/mL) and 260 nm/280 nm.

Detection of MTHFR 677C→T, 1298A→C and MTR 2756 A→G SNPs. Genomic DNA containing the polymorphic sites was amplified by polymerase chain reaction (PCR) with specific primers for each gene (Table 2) using 15 ng genomic DNA, 0.5 µM of each of the primers, 100 µM deoxyribonucleotide triphosphates (dNTPs), 10 mM Tris (tris-(hydroxymethyl) aminomethane)–HCl (pH 8.3), 1.5 mM MgCl₂, 50 mM KCl, and 0.5 units of Taq polymerase (Sigma–Aldrich, USA) in a total volume of 50 µl.

The PCR conditions were as mentioned in Table 2. RFLP was performed with restriction enzymes according to the manufacturer’s instructions (MBI, Fermentas, Burlington, ON, Canada). The restriction digestion was visualized after electrophoresis on 2.5% agarose gel. The amplified PCR product of MTHFR 677C→T, digested by HinfI restriction enzyme. Those patients carrying genotype CC showed a single band of 198 bp, whereas carrier of genotype TT showed bands of 175 and 23 bp, and three bands of 198, 175 and 23bp appeared with genotype CT. After digesting with MboII for detecting MTHFR 1298A→C, the bands 56, 31, 30, 28 and 18 bp were observed for genotype AA, whereas, the expected bands for genotype CC were 84, 31, 30 and 18 bp and for heterozygous (AC), the size of expected bands were 84, 56, 31, 30, 28 and 18 bp. The amplified product of MTR 2756A→G gene followed by digestion by HaeIII, the carrier of genotype AA showed the single band at 211 bp. Those homozygous for mutation (GG) showed bands of 131 and 80bp. Heterozygous (AG) represented bands of 211, 131 and 80 bp.

Statistical analysis. Distribution of MTHFR 677C→T and MTHFR 1298A→C and MTR 2756A→G va­riants among patients and controls were tabulated for cases and controls (Table 3). With respect to assumption which indicates that MTHFR and MTR genotypes may be associated with the risk of ALL, we tested genotypes interaction in models for acute lymphoblastic leukemia and the interaction of these genes with age, sex and hematological classification, risk group. The χ² test was used to examine differences in frequencies between MTHFR 677C→T, MTHFR 1298A→C and MTR 2756A→G genotypes in cases and controls. Fisher exact test was used when cell numbers were less than 5. The relationship between MTHFR and MTR genotypes and risk of ALL was assessed by means of the odds ratio (OR) with 95% confidence interval (95%CI). It was calculated using both conditional and unconditional logistic regression (adjusting for age and sex) by use of software version 11.5 (SPSSs, Chicago, IC), and EPI software version 3.2.

RESULTS

Demographic data of cases and controls has been summarized in Table 1. The variables have also been categorized for ALL immunophenotypes. The blast lineage, NCI risk groups of ALL patients have been tabulated in Table 1. 94.4% patients were diagnosed with B-lineage ALL and 5.6% with T-lineage ALL. The distribution of ALL patients on the basis of NCI risk groups were 82.4% in standard risk group and 17.6% in high risk group.

The mean age (±SD) of all cases and controls were 6.46 (±3.34) and 6.55 (±3.37) years (p=0.245) respectively. Around 9.6% of patients were in the age-group of age ≤2 years and 34.4% were in the age-group of 2< age ≤ 5 and 56% were in age of more than 5 years, whereas it was 11, 33 and 56% respectively for healthy controls. The number of males and females among cases and controls were almost similar (77.6: 77 for males and 22.4: 23 for females, respectively). The distribution pattern of ALL patients and controls according to place of living were divided in to two groups (a) urban (b) rural. 35.2% of patients were living in urban and 64.8% were living in rural areas. The values for controls were not different from cases (38% urban and 62% rural). The distribution of ALL cases and controls among different states of North India showed the most of the patients in present study were from Punjab (44%), followed by Haryana (27.2%), Uttar Pradesh (12.8%), Himachal Pradesh (8%), Chandigarh (4.0%) and J&K (4%). Similar distribution was seen among the controls.

The prevalence of genotypes of MTHFR and MTR genes in patients and controls were depicted in Table 3. There is no significant differences between patients and controls in this study population.The frequencies of MTHFR C677T/A, MTHFR A1298C, MTR A2756G variants in childhood ALL and healthy controls did not deviate significantly from the Hardy — Weinberg equilibrium. Interestingly, all genotypes of MTHFR 677TT, MTHFR 1298CC and MTR 2756GG show slight decreasing of the risk disease, but no significant difference (p>0.05) was observed between patients and controls in all these studied genes.

The frequencies of MTHFR 677C→T, MTHFR 1298A→C and MTR 2756A→G in stratified risk group is shown in Table 4. Significant difference of distribution of genotypes between standard and high risk group was not observed.

The comparison of the frequency distribution of MTHFR 677C→T, MTHFR 1298A→C and MTR 2756A→G polymorphisms between case and control on the bases of sex showed that the distribution of MTHFR 677 and MTHFR 1298 and MTR2756 variants among the both sex were not significantly different (p>0.05). Also on the basis of age-group (the age of the children at the time of diagnosis), distribution of genotypes of MTHFR 677C→T, MTHFR 1298A→C and MTR 2756A→G were calculated. However, no significant difference was detected (Table 5).

The effect of gene-gene interaction between MTHFR polymorphisms on susceptibility of childhood ALL was investigated further; significant association between the interaction of MTHFR variants and development and progression of ALL among the children in the present study was on observed (Table 6).

As it is shown in Table 3, individuals who carried the genotype of MTHFR 677TT, did not show and difference on risk of ALL (OR=0.61; 95% CI=0.21–1.77). The adjusted OR for carriers of MTHFR 1298CC genotype was calculated. It was 0.56 (95% CI=0.18–1.68) which statistically did not show significance reduction on susceptibility against of childhood ALL. Moreover, this trace was not observed for variants of 2756A→G gene. The significance was not altered when these 3 polymorphisms were evaluated in combination (Table 7).

DISCUSSION

Polymorphism of genes which regulate the metabolism of folate is the important phenomenon which may play role in cancer susceptibility by decreasing folate status or by interference in distribution of folate in cells could influence the risk of ALL [22]. Childhood ALL is more prone to environmental exposures during fetal and infant stage [10]. Reduction in the level of 5, 10-methylene–THF (MTHFR substrate), which is essential for synthesis of thymidylate, could lead to uracil misincorporation into DNA [23] and further diminished the DNA repair system and increased the susceptibility to double-strand breakage there by leading to the chromosomal damage and translocation [24–27]. Folate availability is critical for DNA integrity, required for the transfer of methyl groups in the biosynthesis of thymidilate. In this study we tried to find out whether MTHFR variants and MTR polymorphism play a protective role in a childhood ALL.

The decrease in activity of MTHFR could affect the nucleotide synthesis by increasing availability of the 5, 10-methylene–THF which is required for DNA synthesis and cell division. Folate distribution decreases the transmethylation capacity and thereby decreasing S-adenosylmethionine / S-adenosylhomocysteine ratio and methylation of homocysteine to methionine. Diminished folate availability increases the probability of mis-incorporation of uracil in DNA strand at the time of replication and subsequently increasing the frequency of chromosomal breakage in human leukocyte [28–31].

A hematological malignancy like ALL is more vulnerable to DNA and chromosomal damage because folate deficiency in them cannot accomplish the demand of hasty DNA synthesis in uncontrolled and rapidly proliferating hematopoitic cells. DNA hypomethylation and uracil misincorporation are considered important factors in carcinogenesis [32, 11].

Several groups have reported that the polymorphisms which reduce the MTHFR activity were associated with the reduced risk of leukemia and lymphoma [10, 33, 34]. ALL patients with polymorphisms in serine hydroxymethyl transferase gene and thymidylate synthase genes are considered as low risk group [15, 35]. ALL cases with a polymorphisms in MTR 2756A→G genotype) showed reduction by 5.6-fold in adult ALL risk [35]. Not only MTR 2756A→G variants were considered as a risk allele for ALL but also in combination with the MTHFR variants [14].

In this study, attempts were made to evaluate the associations between the MTHFR 677C→T, MTHFR 1298A→C and MTR 2756A→G polymorphisms and the risk of ALL in the 125 cases of the childhood ALL in north India compared with the sex and age matched controls. It has been reported that both MTHFR polymorphisms reduce the susceptibility to adult and childhood lymphoid leukemia however not in myeloid leukemia [10, 34, 36–38].

The significant protective effect of MTHFR 677C→T variant but not MTHFR 1298A→C in the Brazilian population to decrease the risk of childhood ALL has been reported by de Franchis et al. [13]. Recently, Sood et al. [34] demonstrated the significant protective effect of MTHFR 677C→T variant but not MTHFR 1298A→C in the north Indian population to decrease the risk of childhood ALL. Wiemels et al. [33] reported the protective effect of MTHFR 677C→T polymorphism on the risk for infant leukemia in the UK population but not for MTHFR 1298A→C polymorphism; also it was demonstrated that 677C/T frequency was higher in ALL patients with MLL rearrangements, low in ALL with hyper-diploidy indicating that impact of 677C/T variant on development and progression of leukemia might vary between different subtype of ALL [37]. Matsuo et al. [21] provided the evidence which MTHFR mutant alleles are associated with lower susceptibility to ALL. Some other studies also reconfirmed that both MTHFR polymorphisms decreased the risk for childhood ALL in French–Canadian population [5]. The recent report by Reddy et al. [39] suggested a possible protective effect of MTHFR (677C→T) and MTHFR 1298A→C) for the first time they reported gender-bias protective effect of 677CT /1298AA and 677CT/1298AC toward of female ALL patients. Yeoh et al. [40] 2010 demonstrated association between MTHFR 1298A→C and increasing the risk of ALL among male patients.

However, other research groups reported no association between MTHFR polymorphisms and the risk for childhood ALL, from different ethnic groups [13, 15, 21]. Wang et al. [41] in their recent meta-analysis reported albeit MTHFR C677T was found previously to be associated with increased risks of colorectal cancer, leukemia, and gastric cancer, but on the bases of this meta–analysis, they could not find evidence for a main role of MTHFR C677T in the leukemogesis of childhood acute lymphoblastic leukemia.

In the present study, significant difference between patients and healthy controls was not found, but also our result did not show the significant reduction in the risk of ALL and increasing the tolerance induced by T and C alleles in position of 677 and 1298 of MTHFR gene, respectively, in ALL patients in north Indian population.

We tried to analyze the influence of each polymorphism of MTHFR and MTR individually and in combination and their effect on the risk for childhood ALL. We could not observe significant associations between the genetic polymorphisms of the folate metabolizing enzymes and MTR on the risk for childhood ALL in our population of study.

However, the contradictory results were observed in association of genetic polymorphisms in the folate metabolic pathway with the risk for adult ALL [33, 35, 36, 40, 42]. Also, Gemmati et al. [36] and Lincz et al. [43] observed that MTR 2756A→G polymorphism reduced the risk for adult ALL and lymphoma. On the other hand, Skibola et al. [35] and Matsuo et al. [21] reported on increased risk for different types of adult lymphoma in carriers for at least one G allele in locus 2756 of MTR gene. No association has been observed between MTR 2756A→G polymorphism and the risk for adult ALL according to [35, 42]. Our work also showed no association between MTR 2756A→G polymorphism and the risk of childhood ALL. The inconsistencies of results might be attributed to some variables in study of population like size of sampling, age and nutritional folic acid in diet. Rosenberg et al. [44] showed that the adequate folic acid consumption in a population may increase the frequency of the MTHFR 677T allele, in contrast insufficient level of folate in diet may results in decreasing the T allele frequency. This can justify the different frequency of MTHFR allele in population of this study as compared with Reddy et al. [39] may be influenced by different level of folic acid in diet of different ethnic groups in India. Thus the polymorphism of these genes did not show any effect on ALL susceptibility, so it could be interpreted that other molecular mechanisms like hypermethylation mediated gene silencing may play an important role in etiology of ALL in north Indian population. The mechanism proposed to explain these associations was the shunt of folate metabolism versus thymidine and purine synthesis, which could reduce the possibility of the incorporation of uracil into DNA and protect against carcinogenesis. It has been proposed that shunt of folate metabolism encounters the synthesis of thymidine and purine, reduces misincorpration of uracil into DNA and protect from any damage which lead to arising cancer [10, 39].

In conclusion, the present study provided the evidence that MTHFR (677 C→T), MTHFR (1298 A→C) and MTR (2576A→C) are not associated with decreased risk of childhood ALL and did not show significant effect on progression and development of childhood ALL. In the other words, we found that MTHFR 677C→T and 1298A→C may could not be considered as a crucial and probable marker for ALL development and progression and also could not be used for treatment strategies for childhood ALL, as epigene­tic mechanisms are more important in progression of childhood ALL. The larger studies, which in turn will be able to provide support to truly significant finding through much larger combined and comparative data sets, are necessary in this regard.

Acknowledgement

We are indebted to all patients and their parents who consented to participate in this study. This work was supported by University Grand Commission (UGC) India.

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The mechanisms of peripheral tolerance that control the quality and intensity of immune responses are exerted by different types of T cells with regulatory function which include specialized subsets of CD4⁺, CD8⁺, double negative (CD4^-CD8^-) CD3⁺, γδ T cells, and natural killer T (NKT) cells [1, 2] . Among these, natural T regulatory cells (Tregs) were first described in the 1970s by Gershon [3]. They were originally identified in mice [4] but then, they were also described in rats [5] and humans [6]. Tregs exert their suppressive activity in an antigen-nonspecific way called “bystander suppression” [2]. They suppress a wide variety of immune cells including naive and memory CD4⁺ and CD8⁺ T cells, B cells, monocytes, and dendritic cells [7–12]. Tregs are characte­rized by the constitutive expression of several activation markers as CTLA-4 (CD152) and Foxp3, among others [13–15]. However, Foxp3 is the most specific marker for natural Tregs playing a precise role in the development and function of natural CD4⁺CD25⁺ Tregs [13, 14]. Another subset of regulatory cells are the Type 1 regulatory cells (Tr1) that are defined by their ability to produce high levels of IL-10 and transforming growth factor-β (TGF-β)[16–17], utilizing those cytokines to suppress Th1 and Th2-mediated immune responses. Tr1 cells are inducible, antigen-specific and express normal levels of activation markers such as CD25, following TCR-mediated stimulation. Other important regulators of the immune response are NKT cells [19]. This cell population expresses both functional TCR αβ chains, and NK receptors, such as NK1.1. The cells that recognize antigens presented by the nonpolymorfic MHC class I-like molecule CD1d are true NKT cells [20]. Although type I NKT cells have NK-like cytolytic activity, they are considered regulators of immune responses because they rapidly produce large amounts of Th1 and Th2 cytokines in autoimmune disease, infectious disease, and cancer. Activated NKT cells induce cell death in tumor cells through the expression of cell-death-inducing effector molecules [21].

Both, experimental and clinical studies, revealed that cyclophosphamide (Cy), an alkylating agent commonly used in cancer chemotherapy [22], exerts an apparently paradoxical effect on host immune responses [23]. High doses of Cy usually bring about an impairment of the host defense mechanisms, along with the reduction of primary tumor mass, therefore leading to severe immunosuppression. However, the administration of low doses of Cy leads to an enhancement of the immune response, frequently causing tumor rejection [24, 25].

Our previous results in a rat B-cell lymphoma model (L-TACB) showed that a single low-dose Cy, admini­stered to rats bearing already grown lymphomas, inhibited metastasis development [26]. The antimetastatic action of Cy was mediated by immunomodulation [27]. We demonstrated that IL-10 was the main factor responsible for the induction and development of immunosuppression in L-TACB-bearing hosts [28, 29]. In fact, Cy induced a Th2/Th1 switch in the cytokine profile and increased the proliferative rate of spleen cells [30].

Considering the already demonstrated immunomodulatory effect of low-dose Cy in a B-cell lymphoma tumor model and taking into account that Treg and NKT I cells may be involved in the regulation of the antitumor immune response, we decided to evaluate if low-dose Cy treatment was able to modulate those lymphocyte subpopulations in the same tumor model. Moreover, it has been suggested that Tregs and NKT I might regulate each other. Treg cells suppress the anti-tumor effect and reduce the number of NKT I cells. At the same time, NKT I cells regulate Treg cell function [20].

MATERIALS AND METHODS

Mice. Ten to twelve weeks old female inbred IIM e/Fm rats (e rats) [31] were housed and cared at the animal facilities of the School of Medical Sciences, National University of Rosario. Animals were fed commercial chow and water ad libitum and were maintained in a 12 h light/dark cycle. All the experiments were done during the first half of the light cycle and in accordance with animal care standards of the institution, which complies with the guidelines issued by the Canadian Council on Animal Care [32].

Tumor. L-TACB is a poorly differentiated B-cell lymphoma that arose spontaneously in an inbred e rat [33] and metastasizes exclusively to regional lymph nodes when injected subcutaneously [26]. This tumor is maintained by serial subcutaneous trocar implantation of 1 mm³ tumor fragments (approximately 10⁶ cells) in syngeneic rats.

Drugs. Cyclophosphamide (Cy) (Filaxis Lab., Argentina) was dissolved in sterile distilled water to a concentration of 20 mg/ml and injected at a dose of 10 or 20 mg/kg of body weight.

Experimental model. Circulating T regulatory and NKT I cells were quantified during tumor evolution in individual experiments. Inbred e rats were inoculated with L-TACB s.c. (day 0). On day 14 animals were distributed in two groups: control animals injected i.p. with saline and animals treated with a single low dose Cy of 10 mg/kg i.p. Blood samples were obtained on days 0; 7; 14 and 21, and were processed for flow cytometry analysis with the antibodies to the appropriated markers. Different populations of CD4⁺CD25⁺ Tregs were identified: CTLA-4⁺, Foxp3⁺ and IL-10⁺ in both experimental groups. Also, we quantified NKT I cells using anti-TCR and anti-CD161 antibodies.

Tregs and NKT I cells quantification. Blood samples from the tail vein were obtained using EDTA as anticoagulant. Peripheral blood mononuclear cells were isolated from peripheral blood samples by centrifugation, using Ficoll-Paque density gradient (Ficoll-Paque PLUS, GE Healthcare, Uppsala, Sweden). The following antibodies were used for immunophenoty­ping: CD4-PE-Cy5 (BD Pharmingen, USA), CD25-FITC (Serotec, UK), CD152-RPE (Serotec, UK), Foxp3-PE (eBioscience, USA), IL-10-PE (BD Pharmingen, USA), TCR-RPE (Serotec, UK), CD161-FITC (Serotec, UK). Samples were fixed in 1% (wt/vol) paraformaldehyde and analyzed on a Coulter Epics XL (Coulter Corp. Miami, FL, USA). Cells were permeabilized using saponin (0.1% w/v in PBS). Acquired data were analyzed with WinMD1 2.8 data analysis software (Scripps Research Institute, La Jolla, Ca, USA).

Statistics. Non-Parametric ANOVA, Mann — Whitney’s U tests were utilized using GraphPad Prism^ version 3.03 (GraphPad Software, San Diego, CA). Differences between groups were considered significant when P < 0.05.

RESULTS AND DISCUSSION

First, we studied the kinetics of different subsets of Tregs during tumor growth evolution and after the treatment with low dose Cy. We observed an increase during tumor growth in the percentage of the three cell subsets analyzed (Fig. 1).

The percentage of CD4⁺CD25⁺CTLA-4⁺ cells increased during tumor growth (P < 0.01); indeed, the levels of these cells on day 21 were higher than those on day 0 [% median (range); day 0: 0.0 (0–0) vs. day 21: 0.4 (0.2–1.4); P < 0.001]. At the end of the experiment, the percentage of CD4⁺CD25⁺CTLA-4⁺cells in animals treated with Cy was lower than that of controls, but they did not differ between each other [Day 21: Control 0.4 (0.2–1.4) vs Cy: 0.3 (0.1–0.9); ns] (Fig. 1, a). The percentage of CD4⁺CD25⁺Foxp3⁺ cell subset increased throughout the experiment (P < 0.001), being also significant the difference. On day 21, the percentage of CD4⁺CD25⁺Foxp3⁺ cells in the treated group was significantly lower compared to that of the control group [Day 21: Cy, 0.2 (0.0–0.7) vs Control, 1.1 (0.3–6.3), P < 0.01] (Fig. 1, b). Moreover, CD4⁺CD25⁺IL-10⁺ cells increased their percentage du­ring the studied period (P < 0.001), and the difference between the values on days 0 and 21 was significant [Day 0: 0.7 (0.1–1.0) vs. Day 21: 6.3 (1.0–7.4); P < 0.01]. Also, the percentage of circulating CD4⁺CD25⁺IL-10⁺ cells in the treated group was significantly lower with respect to the control group on the same day [0.7 (0.2–4.3) vs. 6.3 (1.0–7.4), P < 0.05] (Fig. 1, c).

NKT I is a T cell subset with regulatory properties which can display an antitumor function. We aimed to study the variation of this cell population during tumor evolution. We observed that while from days 0 to 7 the % of NKT I cells increased significantly [12.4 (4.0–30.8) vs. 17.8 (6.2–38.2)], (P < 0.001)], by day 14 those levels had already returned to the basal ones [8.3 (1.9–22.1)]. The analysis of the effect of Cy treatment on this cell population showed that the treatment decreases its %, although not significantly, on day 21 [Day 21: Control 8.4 (3.8–19.3) vs Cy 3.7 (2.4–6.3)] (Fig. 2, a).

As NKT I and natural Tregs cells exert opposite actions on tumor development we depicted graphically their simultaneous variations along tumor evolution (Fig. 2, b). Both cell populations increased their respective levels until day 7. From that day on, while Tregs continue increasing their percentage, NKT I cells decreased, reaching levels even lowers than the basal ones.

Taking into account the lack of modulation of NKT I cells by the antimetastatic dose of Cy, we wanted to know if a different schedule or dose of Cy would be able to increase this cell population. We decided to duplicate the dose previously used in a single injection (Cy-d): when the single dose of Cy was duplicated (20 mg/kg instead of 10), a marginally significant increase in the percentage of NKT I cells with respect to the control group was observed [day 21: Control 8.4 (3.8–19.3) vs Cy-d 12.8 (3.5–20.7), P = 0.055] (Fig. 2, a). Interestingly, when comparing the effect caused on this cell population by a single Cy dose of 10 mg/kg with that of 20 mg/kg (Cy-d), the difference was significant (P < 0.01).

The idea that the immune system can recognize and destroy nascent transformed cells was originally integrated in the cancer immunosurveillance hypothesis posed by Burnet and Thomas in 1957 [34]. Almost half a century later, Schreiber and colleagues proposed that cancer immunosurveillance may function as the elimination phase of a process called cancer immunoediting. This process is responsible for eliminating tumors and sculpting the immunogenic phenotypes of tumors that eventually appear in immunocompetent hosts [35]. The importance of the antitumor immune response in determining the fate of an immunogenic tumor makes it interesting and necessary to study the modulation of different subsets of immune cells during tumor development.

CD4⁺CD25⁺ regulatory T cells NKT are two populations of T lymphocytes that can independently regulate adaptive and innate responses. Activated NKT cells seem to modulate quantitatively Treg function through IL-2-dependent mechanisms, whereas Tregs can suppress the proliferation, cytokine release and cytotoxic activity of NKT cells by cell-contact-dependent mechanisms [36].

During L-TACB tumor evolution we observed an increase in the levels of different subsets of CD4⁺CD25⁺ Tregs, namely CTLA-4⁺, Foxp3⁺ and IL-10⁺ cells. On the other hand, the significant increase in the percentage of NKT I cells demonstrated between days 0 and 7, was completely reversed by day 14, the level of this cells being even under the basal ones. Taking into consideration that natural Foxp3⁺ Tregs and NKT I cells exert opposite actions on tumor development, we were interested in learning about their simultaneous variation during tumor growth. We observed that in the first steps of tumor development both cell types, Tregs and NKT I, increased their levels, indicating the existence of a sort of a balance, at least partial, between these cells types with opposite effects. After that moment, the balance is broken because of a significant decrease of the NKT I population, while Tregs showed increased values with respect to basal ones throughout the experiment, in spite of a transitory non significant decrease on day 14, thus contributing to an immune response modulated towards immunosuppression. We are aware that several other regulatory cell types may have influence in the modulation of the immune response to the tumor. Nevertheless, Tregs and NKT I represent two important cell types involved in innate immunity related to pro- and anti- tumor responses, respectively.

Both, experimental and clinical studies revealed that cyclophosphamide, an alkylating agent commonly used in cancer chemotherapy [22], exerts an apparently paradoxical effect in host immune response. High doses of Cy (i.e. MTD, maximum tolerated dose) bring about, along with a reduction of primary tumor mass, an impairment of the host defense mechanisms, therefore leading to immunosuppression. However, the administration of low doses of Cy leads to an enhancement of the immune response, both in experimental animals and in humans, frequently causing tumor rejection[23, 25, 28, 29].

Previously, we demonstrated that a single low-dose Cy, a treatment completely devoid of toxicity, inhibited metastasis development without affecting primary tumor growth [26]. The antimetastatic action of Cy was mediated by immunomodulation since this effect could be adoptively transferred by immune cells of Cy-treated tumor-bearing rats and the same treatment did not have any effect in immunodeficient nude mice [27]. We demonstrated that IL-10 was the main factor responsible for the induction and development of immunosuppression in L-TACB-bearing hosts and that the antimetastatic immunomodulatory effect of cyclophosphamide was mediated by a reduction in IL-10 levels produced by T-lymphocytes [28]. In fact, Cy induced a Th2/Th1 switch and increased the proliferative rate of spleen cells [30].

Interestingly, the present results showed that the antimetastatic dose of Cy (10mg/kg) induced a significant decrease of the CD4⁺CD25⁺Foxp3⁺ and CD4⁺CD25⁺IL-10⁺ cells. On the other hand, this treatment was not able to change the levels of NKT I cells. Considering the lack of modulation of NKT I cells by the antimetastatic dose of Cy, we studied the level of that cell population after a single dose of 20 mg/kg Cy, and we observed an increase compared to the percentage obtained after the treatment with half a dose. Nevertheless, when compared to the control group, the difference was not significant. Therefore, the decrease of peripheral blood NKT I observed during L-TACB tumor growth could not been reverted by the different treatments utilized.

Hence, the antimetastatic effect of a single low dose of Cy would be due, at least in part, to downregulation of natural and inducible Treg cells. The importance of the innate antitumor immune response in general, and its modulation by treatments with low dose chemotherapeutics in particular, warrants further studies in this area.

Our results may have importance on development of new therapies for metastatic lymphomas considering that a single low-dose cyclophosphamide, a treatment devoid of toxicity, would act inhibiting one of the mechanisms contributing to escape from immune rejection, thus inhibiting malignant growth and progression.

Acknowledgements

We would like to thank Cibic S.A. and especially to Dr. Ricardo Giordano, for their help in flow cyto­metry studies. This work was supported by a grant from Universidad Nacional de Rosario to O.G. Scharovsky.

Conflict of interest

The authors declare that they have no conflict of interest.

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The tumor-host interaction is a complex process that puzzles experimental and clinical oncologists for decades. The problem of interaction of the tumor with the host was well summarized by Kavetsky in 1977 [1]. Even though at present time new and more accurate techniques are being used in cancer research, much work is yet to be done to wider uncover this problem. It is not surprising that a large body of work has been focused on the study of effects of a tumor on surrounding (adjacent) tissues and vice versa, a model which is attractive for several reasons: it is convenient in terms of experimental design planning, and it is informative and precise in terms of visualization of effects and dissection of mechanisms. Tumor cells by interacting with its stroma have been found to change their phenotype and biological properties [2, 3]. On the other hand, tumors have been shown to affect surrounding noncancerous cells causing DNA damage [4−6]. As for adverse effects of tumors on distant tissues, there is the only known fact that they can induce a complex DNA damage (double strand breaks (DSBs) and oxidatively induced clustered DNA lesions (OCDLs)), particularly in proliferative tissues (skin and crypts in the gastrointestinal organs) [7]. Since a proliferative tissue contains a large fraction of S-phase cells sensitive to DNA DSB formation (as evidenced by phosphorylation of histone H2AX [8, 9]), proliferation of cells in this tissue exposed to DNA damaging agents could be delayed due to DNA repair processes. Cytotoxic/genotoxic insults frequently impair the proliferating and maturational abilities of cells.

To test whether such a highly proliferative hematopoietic tissue as bone marrow is among sensitive targets for distantly located tumors, adult male rats were subcutaneously implanted with Guerin carcinoma (GC; uterine adenocarcinoma of rats) cells, and then on the 12^th and 18^th days after implantation cytotoxic and genotoxic effects were assessed in hematopoietic cells of unfractionated bone marrow according to the techniques proposed by Criswell et al. [10, 11]. In brief, the cytotoxic effect was assessed by flow cytomety using two parameters: 1) ratio of the population of total nucleated cells (TNC) to the population of total enucleated erythrocytes (TE) composed of immature polychromatic (PCE) and mature normochromatic erythrocytes (NCE); 2) ratio of the population of PCE to the population of NCE [10]. The TNC/TE ratio is used to determine overall myelosuppression, while the PCE/NCE ratio is used to specifically determine suppression of erythropoiesis. Clastogenic agents that target the process of DNA replication are known to suppress cell proliferation thus causing decreases of these ratios [10, 12, 13]. TNC/TE and PCE/NCE ratios are key components of cytotoxicity assessment, among which the PCE/NCE ratio is most frequently used with the micronucleus (MN) test. The genotoxic effect was assessed by flow cytometric counting of micronucleated PCE (MNPCE) within the population of PCE [11].

MATERIALS AND METHODS

Animal tumor model. Adult random-bred male rats (250−300 g) were obtained from the vivarium of R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine (Kyiv, Ukraine). Guerin carcinoma (GC, or T8) cells were obtained from the Bank of Cell Lines from Human and Animal Tissues of the aforementioned Institute. Up to 90% of rats can be successfully implanted with this type of cells regardless of the strain of animals used for implantation [14]. The work with animals was performed according to the rules of local Ethic Committee. Tumors were implanted by subcutaneous injection (dorsally into the left flank) of 2.2 × 10⁶ GC cells suspended in 0.5 ml of sterile physiological solution. On the 12^th and 18^th days after implantation animals were sacrificed. Tumors attached to the inner side of the skin were carefully removed with scissors and then weighed. Although this animal tumor model may be not highly syngeneic, this is not likely to be an issue, since the goal of our work was not investigation of a specific mechanism of the tumor-host interaction or anti-cancer drug delivery.

Bone marrow isolation. Femur removal and bone marrow isolation procedures were performed as proposed [10]. Bone marrow cells were thoroughly flushed from the femur with 3 ml of FBS and kept at +4−6 °C before use.

Specimen processing and fixation. Bone marrow samples were kept in a fridge no longer than 1.5 h before they were resuspended by vortexing and centrifuged at 300 × g for 5 min. In general, specimen processing and fixation procedures were performed as proposed [10]. The supernatant was discarded followed by washing cells in 5 ml of PBS. After centrifugation them at 300 × g for 5 min, the supernatant was discarded and then the pellet was resuspended in 2 ml of PBS by vortexing. Cell aggregates were dissociated by gentle syringing of the suspension through a 21-gauge needle. While vigorous vortexing, 0.2 ml of processed whole bone marrow was added to 5 ml of fixative solution: 1% glutaraldehyde (v/v) in PBS with 30 μg/ml of SDS (Merck, Germany). In this solution, erythrocytes become spherical [15]. Cells were fixed for 5 min and then centrifuged for an additional 5 min at 300 × g. The supernatant was removed followed by resuspension of cells in 0.5 ml of PBS.

Fluorescence staining. This procedure was performed in accordance with the published protocol [11]. Solution A was prepared by dissolving in 100 ml (final volume) of distilled H₂O of the following components: 0.1 ml Triton X-100 (Loba Chemie, Austria), 8 ml 1.0 N HCl, and 0.877 g NaCl. Solution B was prepared by mixing of 37 ml 0.1 M anhydrous citric acid with 63 ml 0.2 M Na₂HPO₄ (pH 6.0) and adding 0.877 g NaCl, 34 mg EDTA disodium salt (Sigma, USA) and 0.6 ml of acridine orange (AO; Sigma) stock solution (1 mg/ml). Fixed cells (0.2 ml of cell suspension) were mixed with ice-cold Solutions A and B (0.4 and 1.2 ml, respectively) in a 12 × 75 mm centrifuge tubes. While shaking, cells were stained on ice for 30 min in the dark. They were then centrifuged at 300 × g for 5 min. After the supernatant was carefully removed, 1 ml of PBS was added to resuspend the pellet. Before flow cytometry, the cell suspension was gently syringed through a 21-gauge needle to mainly analyze single cells.

Flow cytometry. Samples were analyzed on an EPICS XL flow cytometer (Beckman Coulter, USA) equipped with a 15 mW argon-ion laser (488 nm). Instrument settings were in general the same as recommended [10, 11]. The forward light scatter (related to cell size) and the side light scatter (related to intracellular granularity) signals were collected in linear mode. The fluorescence of DNA- and RNA-bound AO was measured in the green fluorescence channel (FL1) through a 525/10-nm band-pass filter with logarithmic amplification and in the far red fluorescence channel (FL4) through a 675/10-nm band-pass filter with logarithmic amplification, respectively. An acquisition rate was ≈1000 cells per second. At least 1.5 × 10⁵ events were collected for each sample. Analysis of the data was performed with the publicly available software “WinMDI” developed by Dr. J. Trotter (http://facs.scripps.edu/software.html). Cells were gated on forward scatter versus side scatter histograms to eliminate debris and aggregates from analysis, although microscopical observation showed that their numbers were very low. On the Forward scatter versus FL1-Height histogram, events that represent populations of TNC, PCE, and NCE were compartmentalized well enough to perform analysis (Fig. 1). Although Criswell et al. [10] proposed to use the FL4- versus FL1-Height histogram for quantification of TNC, PCE, and NCE, the Forward scatter versus FL1-Height histogram (presented in Fig. 1) can also be used for this purpose (both histograms gave similar apportionments of these cells). For cytotoxicity assessment, TNC/TE (where TE = PCE + NCE) and PCE/NCE ratios were used [10]. For genotoxicity assessment, MNPCE (shown by the arrow; Fig. 1) were defined within the population of PCE (shown in the region R2; Fig. 1), and then their number was calculated per 1000 PCE [11]. Since levels of micronucleated NCE usually correlate with levels of MNPCE, they were not analyzed.

Statistical analysis. The statistical significance of differences between mean values was assessed by the Student’s t-test. Values P < 0.05 were conside­red as statistically significant.

RESULTS

In the bone marrow isolated from femurs of GC-bearing rats on the 12^th and 18^th days post-implantation there was about a 2-fold drop in both TNC/TE and PCE/NCE ratios compared to corresponding controls (P < 0.05; Fig. 2). These ratios were not sufficiently changed with the time post-implantation (P > 0.05; Fig. 2). Levels of MNPCE in the bone marrow of GC-bearing rats on the 12^th and 18^th days post-implantation were slightly higher than the level of MNPCE in the bone marrow of the control group of animals (increasing trend), although these elevations were insignificant (P > 0.05; Fig. 3).

To assess whether the cytotoxic effect depends upon the tumor progression, we generated the scatter plot of tumor masses and corresponding PCE/NCE ratios (Fig. 4). This plot shows that in the majority of GC-bearing animals the PCE/NCE ratio reversely correlated with the tumor mass. However, the slope of the regression line depended upon the time passed since implantation of tumor cells. For the data collected from animals that carried tumors for up to 18 days, compared with the data collected from animals that carried tumors for up to 12 days, the slope of the regression line was markedly steeper (Fig.4). Unlike the plot of PCE/NCE ratios versus tumor masses, the plot of TNC/TE ratios versus tumor masses showed a much sparser distribution of the data points whose regression lines were strictly horizontal, which is indicative of lack of correlation between these two parameters (data not shown). On the 12^th and 18^th days post-implantation the tumor mass values were 5.2 ± 0.6 and 6.4 ± 2.0 g, respectively (data shown are the mean ± standard error of the mean).

DISCUSSION

A significant decrease in both TNC/TE and PCE/NCE ratios is indicative of serious cytotoxic insult to the bone marrow of GC-bearing rats. This insult is likely to affect a large number of cells residing in the bone marrow as evidenced by ≈2-fold decrease in the TNC/TE ratio (Fig. 2). Cytotoxic effects in tissues distant to sites of implanted tumors have not been previously reported. As for genotoxicity in the bone marrow of GC-bearing rats, this effect, perhaps, exists to some extent (a slight elevation in the number of MNPCE; Fig. 3). More studies with a larger cohort of animals are probably needed to fully uncover this issue. At first it might seem that cytotoxicity in the bone marrow of GC-bearing animals does not depend on tumor growth (e.g., there was no further decrease in TNC/TE or PCE/NCE ratios, and there was no further increase in the level of MNPCE, if the data collected from rats that carried tumors for up to 18 days are compared with the data collected from rats that carried tumors for up to 12 days; Fig. 2). Nevertheless, correlation analysis of tumor masses and corresponding PCE/NCE ratios did reveal tumor growth dependent cytotoxicity in the bone marrow, namely erythropoietic cytotoxicity (Fig. 4). Perhaps, erythropoietic cells are more susceptible to tumor-associated cytotoxic stress than cells of other lineages. Impaired erythropoiesis is known to frequently accompany cancer-related anemia [16]. Inflammatory cytokines, whose production is induced by cancer, suppress erythroid progenitor cell proliferation and erythropoietin production as well [17]. Thus, the PCE/NCE ratio appears to be a valuable parameter to monitor progression/regression of tumors that are capable of affecting erythropoiesis.

Since cytotoxicity is often accompanied by DNA lesions, we cannot exclude the possibility of DNA damage in the bone marrow of rats implanted with GC. If a complex DNA damage occurs, its repair can be delayed thus causing suppressed cell proliferation, which is one of the cytotoxicity manifestations. An insignificant genotoxic effect observed in this study (a slight elevation in the number of MNPCE; Fig. 3), perhaps, supports an assumption that DNA damage may occur in the bone marrow of rats implanted with GC. Redon et al. [7] in their recent study on mice that were subcutaneously implanted with tumors, such as B16 melanoma, M5076 sarcoma, and COLON26 carcinoma, have reported the induction of complex DNA damage (DSBs and OCDLs) in distant proliferative tissues, particularly in skin and crypts of gastrointestinal organs. Similar DNA lesions could be in our study. However, the most intriguing issue in this “tumor-induced bystander effect” is mechanism of its induction. Tumor-associated macrophages, major players of cancer-related inflammation [18], have been found in aforementioned mouse tumors, and also, increased amounts of activated macrophages have been found in gastrointestinal tissues and skin [7]. Moreover, cytokine CCL2 (also known as monocyte chemoattractant protein-1), which is linked to chronic inflammation conditions and cancer [19], has been shown as an essential mediator in tumor-induced DNA damage in distant tissues [7]. This cytokine has been reported to be secreted by tumor cells, normal tissues, and immune cells [20]. However, in the study by Redon et al. [7], CCL2 is unlikely to be produced by the tumor cells themselves, since tumor-bearing CCL2-deficient mice, compared with tumor-bearing CCL2-proficient mice, showed neither the presence of this cytokine in the serum nor elevation of DNA damage (DSBs or OCDLs) in distant tissues [7]. To induce DNA damage in a distant tissue, tumor cells have to activate resident or distant immune cells that after being activated release genotoxic substances including ROS. Tumor cells are likely to activate immune cells via direct and indirect contacts as well [7], since tumor cells are capable of secreting a variety of cytokines and other factors [21].

The fact that a tumor together with expressed CCL2 is an inevitable prerequisite to induce in distant tissues DNA lesions (whose level correlates with proliferative state of the tissue) may also take place in our study. Tumor growth dependent erythropoietic cytotoxicity in GC-bearing rats is likely to be a part of the common mechanism of tumor-induced inflammatory response.

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The role of nitric oxide in tumor biology is ambiguous and is studied insufficiently. NO-production in human and animal organisms is based on enzymatic NO-synthase transformation of L-Arg-guanidine fragment in the reaction with oxygen [1, 2]. Long duration of NO-biosynthesis (10–100 fold longer than basic level) results in genotoxicity effect, induces dose-dependant DNA-destruction etc, leading to tumor development. Multifold increase in NO-production is considered to be a consequence of inducible NO-synthase activation (iNOS), being expressed in different cell types in normal state and pathology, including macrophages, microglyal cells, keratinocytes, hepatocytes, astrocytes, endothelial cells of blood vessels, epithelial cells and a variety of human tumor cells affected by cytokines: interleukin-1 and 2, interferon-γ or their combination with tumor necrosis factor-α or -β, etc [3–7].

Direction of NO effect is defined by many factors: concentration, exposition, reaction products with key reagents (oxygen and its active forms, carbon dioxide, etc) and targets (metals, thiol containing aminoacids, proteins, etc). Clinical investigations show that NOS expression in many tumor tissues is often disturbed. That is true for tumors of central nervous system, stomach, colon and mammary gland, and melanoma, where NOS activity is found to be elevated. A direct correlation is shown between iNOS expression in tissues of these tumors and the following parameters: stage of a disease, vessel development in tumor, high frequency of metastases, what result in poor prognosis [8–11]. Animal experiments revealed that during iNOS activation, utilization of enzyme selective inhibitors resulted in tumor growth suppression [12, 13]. Exoge­nous NO is shown to inhibit endogenous NO synthesis. Mechanisms of feedback include direct NOS inactivation via NO binding by hem-containing enzyme group and inhibition of m-RNA iNOS expression [14].

At the same time NO plays a positive role in defense reactions of an organism. Thus, NO secreted by nonspecific immunity cells, macrophages and neutrophils, shows oxidizing and antimicrobial properties and is responsible for phagocytes cytostatic and cytotoxic potential with respect to tumor cells [15]. There is another significant property of NO: apoptosis initiation, inclu­ding that in transformed cells, due to violation of mitochondrial oxidative phosphorylation, ribonucleotide reductase metabolism, etc [16]. In some instances, when NO cell production is low (what promotes survival of transformed cells), it is proposed that the activity of iNOS should be restored with the use of medicinal agents or with gene therapy [7]. NO concentration in biological fluids of organism, tumor tissues, tumor microenvironment is shown to predict the activity of key proteins for carcinogenesis — such as p53, extracellular signal-regulated kinase (ERK), hypoxic inducible factor (HIF), Akt etc. [17]. Taking into account the above information a special scientific and practical interest will include investigations of NO biosynthesis levels upon the influence of NOS modulators and natural antioxidants in different models of tumor growth.

The aim of the present study was to investigate the dynamics of nitric oxide derivatives (NOD) formation in mice with transplanted tumors and to analize whether synthetic NO-synthase inhibitors, NO-donors and natural compounds could modulate NOD synthesis.

Materials and methods

322 male F₁(C₅₇BlxCBA), CBA/Lac, BDF and Balb/c mice weighting 22–31 g were used in the work. Animals were fed with briquetted feed with constant access to water. All experimental procedures were conducted following the normative rules of bioethics.Ehrlich carcinoma (EC) and Lewis lung carcinoma (LLC) tumor strains were received from Russian Oncologic Scientific Centre of RAMS (Moscow, Russia). LLC and EC cells (5×10⁶ or 10⁶ cells per mice) were inoculated subcutaneously, in right armpit and right inguinal region.

The following reagents were used: N^g-monomethyl-L-arginine (L-NMMA), N^ώ-nitro-L-arginine (L-NNA), N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide dihydrochloride (1400W), aminoguanidine (AG) (Sigma-RBI); L-carnozine (CZ) (BioChemika). Other reagents: nitroprusside (NP), acetylsalicylic acid (ASA), etc. were of Russian production. While choosing NOS modulators, chemical properties of compounds were taken into consideration [1].

Endogenous NOD formation in the organism was evaluated on two tumor growth models — LLC (60 mice) and EC (40 mice) by analysis of nitrate (NA) and nitrite (NI) excretion with urine per day and their concentration in tumor tissue.

Gathering of diurnal urine for NA and NI analysis of mice F₁(C57BlxCBA) with LLC was achieved before tumor transplantation and after it on the 2d, 9^th, 16^th, 21^st and 30^th day. NA and NI concentrations in tumor tissue were determined on the 14^th, 21^st, 28^th and 32^nd day of tumor growth.

Gathering of diurnal urine of Balb/c-mice with EC was performed before EC transplantation and after it on the 4^th, 10^th, 17^th, 24^th and 32^nd day of tumor growth. NA and NI concentrations in tumor tissue of BDF mice with EC transplanted tumor were determined on the 32^nd day of tumor growth.

Investigation of synthetic NOS inhibitors and NO donors effect on EC growth was carried out on 102 F₁(C₅₇BlxCBA) and BDF mice. 7 groups of hybrid mice (9–10 animals per group) were formed. The control group was composed from 16 mice. BDF mice were divided into 2 groups, 10 mice per group. EC was transplanted to all mice subcutaneously. The intraperitoneal introduction was used for administration of an active compound solution, 0.1 ml, 5 days per week (except special cases discussed later) to animals from the 2–8^th groups beginning from the 8^th day after EC transplantation. In total, each animal received 14 doses. There were formed the following groups: 1) control, 2) treatment with 50 mg/kg L-NMMA, 3) 100 mg/kg L-NNA, 4) 140 mg/kg AG, 5) combined treatment with 140 mg/kg AG and 10 mg/kg ASA. The 6 group of animals received 4 mg/kg of NP on the 2^nd and 3d day after EC transplantation and later subcutaneously 40 µg/kg of NP. The 7^th group received subcutaneously 80 µg/kg of NP on the 8^th day after EC transplantation. The 8^th-group of mice received 1000 mg/kg of CZ with water since the second day after transplantation and during all the experiment.

The effect of selective inhibitor iNOS-1400W on EC growth was studied on 20 BDF mice with transplanted EC (group 9, control, n=10, and group 10, n=10, treated subcutaneously with 13 mg/kg 1400W inhibitor solution from day 4 after tumor transplantation. In total, 14 1400W administrations per animal were performed.

Investigation of effect of natural biologically active compounds was analyzed in CBA/Lac (n=50), F₁(C₅₇BlxCBA) (n=50) and Balb/c (n=20) male mice using the following preparations: pycnogenol (Pg), containing >=60% of procyanidins (Biolandes, France), diquertin (Dq) with>90% of dihydroquercetin (Flavir, Russia), indol-3-carbinol (I-3-C) (Sigma), phenolic acids: gallic (GA), tannic (TA) and also Se-enriched paprika (Se-P, 1 mg Se/kg Mayak cultivar grown on sodium selenate containing 0.03% NPK-fertilizers) and algae spirulina- Arthospira platensis (Se-Sp, 1 g Se/kg; production of “Agro-Victoria” corporation, Russia). The preparations were given per os as water suspensions 2–3 weeks before EC transplantation and after it up to the end of experiment 5 times per week. The euthanasia of animals was performed under the light ester anesthetic.

Tumor growth inhibition (TGI) under the effect of tested compounds was estimated in tumor growth dynamics using mass and volume parameters in comparison with the control group according to [18].

For diural urine sampling animals were placed in exchange cages (5 per cage) for 24 h without feeding and with free water access. To exclude NI oxidation 0.3 ml of 30% sodium hydroxide solution was used. Removal of protein-carbohydrate component in urine water extract was achieved using ZnS0₄ and potassium ferricyanide.

On the 14^th, 21^st, 28^th days after LLC transplantation (or 32^nd day for EC) the euthanasia of animals under the light ester anesthetic was performed. Tumors were removed, homogenized and the samples of tissue extracts were obtained as follows: 1–4 g of homogenized tumor tissue were placed in a calibrated vessel, bidistilled water was added (tissue/water ratio= 1:10) and the resulting mixture was heated at 60° С during 15 min. To prevent NI destruction, pH of water suspension was adjusted to 7.2–7.4 using buffer solution. Then the mixure was cooled to room temperature, protein-carbohydrate component was removed by centrifugation for 15 min at 500 g. NI and NA content in all reagents, water and materials was controlled.

NI and NA concentration in urine and tumors was determined by spectrophotometric method using Griss reagent with prereduction of NA to NI by porous cadmium [19]. Optical density of colored solutions was determined on spectrophotometer SF-46.

Statistical analysis of the data (mean value±SD) was performed by standard methods using Student’s t-test.

Results and discussion

EC growth in Balb/c mice was accompanied by increase of NA and NI excretion with urine (Table 1). By the end of experiment (day 32) urine NI and NA excretion increased by 3.6 (p<0.01) and 67.1 (p<0.01) times respectively compared to these parameters of animals with the initial tumor nods (day 4). NI excretion in EC-bearing mice was in the range 3.6х10^–7—1.3х10^–6 mol/kg bw, and NA excretion — 8.5х10^–8—5.7х10^–6 mol/kg bw. Total nitro compound excretion during EC growth increased by 15.5 fold (in N0₃^- equivalents). During this period mean tumor volume increased by 67.3 times. A positive correlation between EC volume and diurnal total NI and NA excretion with urine was revealed (r=0.99).

NOD excretion by F₁(C₅₇BlxCBA) mice with transplanted LLC demonstrated certain peculiarities (Table 2). One day after LLC inoculation maximal excretion values of NI and NA were 1.9 times higher than the respective parameters of healthy animals, and were constantly higher by 22.2–70.5% versus control at all time points during LLC growth.

NI concentration in LLC tumor tissue (Table 3) was in the range of 2.8–4.6×10^–6 mol/kg, increasing monotonously during tumor development whereas NA concentration in the majority of samples was negligible. Total NA+NI content in tumor tissue did not exceed 5.3×10^–6 mol/kg and did not depend on tumor development.

Total amount of NI and NA in EC tumor tissue isolated from BDF mice on day 32 was 5.5 times higher (p<0.01) than their maximal total content in LLC tumor tissue (Table 3).

Investigation of the effect of synthetic compounds capable to modulate NOS activity has shown that intraperitoneal administration of L-NNA (100 mg/kg), AG (140 mg/kg), AG (140 mg/kg)+ASA (10 mg/kg), to hybride mice resulted in statistically significant EC growth inhibition (Table 4). The earliest and stable TGI was demonstrated for NNA (39%, p<0.01). AG caused low TGI (14–22%, p<0.05) but in combination with ASA it inhibited EC growth more efficiently (20–42%, p<0.05). NP effect depended on the dose and rout of administration: subcutaneous administration of 80 µg/kg of NP resulted in 26% EC growth inhibition (p<0.05) but intraperitoneal administration of higher dose (4 mg/kg) on days 2 and 3 after EC transplantation stimulated tumor growth up to 124% (p<0.01). In mice treated with L-NNA and AG tumor growth inhibition by 53% (p<0.01) and a decrease of NOD formation by 26% were recorded.

It should be pointed out that L-NMMA often used in investigations as iNOS inhibitor (50 mg/kg) did not affect EC growth while N0D excretion with urine was close to that of control animals. Subcutaneous administration of 1400W, selective iNOS inhibitor (13 mg/kg), did not cause statistically significant EC tumor depression though NOD excretion was decreased by 25.0%.

Investigation of natural preparation effect on EC growth has shown that prolonged per os consumption of 150 mg PG/kg and 75 mg I-3-C/kg caused statistically significant TGI by 52% and 56% respectively (p<0.01). During the whole experiment Pg effect was stable and resulted in 31% decrease of tumor mass (p<0.01). GA (100 mg/kg), TA (100 mg/kg) and Se-Sp (1 g/kg) statistically significantly increased the latent period of tumor nod formation by 1.4–1.6 times and inhibited tumor growth during two weeks after EC transplantation. At the early stages of EC development TGI was equal to 91% (GA), 78% (TA), 75% (Sp), 89% (Se-Sp) and 65% (Se-P) (p<0.05).

Total NI and NA excretion detected on days 14, 21 or 23 of EC growth in control group of animals was 6.4×10^–6 mol/kg bw (F₁ mice); 1.11×10^–5 mol/kg bw (CBA/Lac mice) and 6.29×10^–6 mol/kg bw (Balb/c mice) (Table 5). Pg, Dq, TA, Sp and Se-P administration resulted in increase of NOD excretion by 44.2% (p<0.05), 47.3% (p<0.05), 27.7%, 65.6% (p<0.01) and 114.6% (p<0.01) accordingly compared to the control group of animals. Only GA inhibited NO formation by 32% (p<0.05) on day 14 of EC growth whereas
I-3-C and Se-Sp did not affect NO biosynthesis.

Thus, the results have shown that EC growth is accompanied by increased NOD formation whereas LLC tumor growth is characterized by depression of NOD biosynthesis. It should be noted that NO endogenous synthesis determination via NOD urine excretion is an integral parameter including also the amount of NOD, being formed from nitrogen oxide, necessary for normal function of cardiovascular, immune, endocrine and nervous systems. NO synthesis by constitutive NO-synthase isoforms is considered not to be accompanied by toxic effects of NO and its derivatives as concentration levels capable to cause necessary physiological reactions are in nano-to micromolar range [20]. There are few data on NO levels in animal tumor tissues. Study of human breast cancer MCF7 cells has shown that the activity of several key proteins (HIF-1α, ERK, and p53 ) is regulated by distinct threshold concentrations of nitric oxide. At low steady-state concentrations of NO (<50 nmol), ERK phosphorylation was induced via a guanylate cyclase-dependent mechanism. HIF-1α accumulation was associated with an intermediate amount of NO (>1.0×10^–7 M), whereas p53 serine 15 phosphorylation occurred at considerably higher levels (>3.0×10^–7 M) [17].

In our experiment endogenous formation of NOD in EC mice beginning from the 9^th to the 30^th day of tumor growth was in the range 1.1×10^–7–7.0×10^–6 mol/kg bw and for LLC-mice — 5.7×10^–7–3.71×10^–6 mol/kg bw. At the same time the total NI and NA concentration in tumor tissue was 2.91×10^–5 mol/kg, while NOD concentration in LLC tumor did not exceed 5.3×10^–6 mol/kg tissue. It is obvious that mean NOD concentration in EC and LLC tumors is significantly higher than concentrations necessary for activation of proteins responsible for cells proliferation and apoptosis.

One can assume that the ability of NOS inhibitors (L-NNA and AG) to suppress tumor growth is related to their ability to depress iNOS activity [21]. Also it has been shown that NP could have oppositely directed effects and stimulate tumor growth at the relatively high doses (4 mg/kg) and suppress it at lower doses (80 µg/kg). There are the data demonstrating that in tumor microenvironment with low content of glucose and oxygen tumor cells may be more sensitive to high concentrations of NO and peroxinitrite that normal ones. That is why in several cases NO donors are considered to be effective for promotion of chemo- and radiotherapy efficiency [16]. However, we have shown that EC has been developing against the augmented NOD biosynthesis and double intra-peritoneal injection of a high NP dosage the next day after EC inoculation, which only stimulated tumor growth.

Lack of L-NMMA-dependent inhibition of NOD endogenous formation can be explained by peculiarities of its metabolism. It is known that NOS converts L-NMMA to N-hydroxy-N-methyl-L-agrinine, which inhibits the enzyme irreversibly. But in certain tissues and cells especially in hypoxic conditions, L-NMMA metabolism may be accompanied by the formation of L-arginine or may possess other peculiarities [1].

Taking into account the NOD endogenous formation in a model of EC tumor growth, its 25–53% reduction upoin the use of L-NNA, AG and 400W appears to be insufficient to attain its normal physiological level. In conditions of developing tumor acceptable iNOS blocking will not be able to produce stable defense response of an organism, but may retard tumor growth. At the same time significant depression of iNOS activity may have also negative consequences. It is known that N0 is formed in the oxidation reaction of L-arginine, which is also a precursor of endogenous synthesis of polyamines. This supposes the existence of an integrated mechanism of nitric oxide and polyamine synthesis regulation. Polyamines are known to stimulate mammalian cells proliferation and potentiate carcinogenesis. The results show that blocking of NO-synthase may cause pretumor changes in intestine due to the decreased NO release and induction of ornitine-decarboxylase — an enzyme of polyamine synthesis [22].

A study of the effect of natural compounds with pronounced antioxidant properties on tumor growth and endogenous NOD formation has revealed that the use of 4 from 9 tested compounds resulted in tumor suppression on the background of stimulation of NOD formation. One can suppose that such effect could be related not only to direct reactions with free radicals so typical for antioxidants but also to other mechanisms involved in stimulation of detoxification defense systems through antioxidant responsive elements present in the promoter region of genes inducible upon oxidative and chemical stresses [23].

In conclusion, our study has demonstrated that EC development is accompanied by increased endo^­genous NOD formation while LLC growth — by depression of NOD synthesis. Modulators of NO-synthase activity (L-NNA and AG) decreased NOD formation, inducing simultaneously moderate, but stable decrease of tumor development in EC-bearing mice. The use of NP had oppositely directed effects on EC growth dependent on the dose and the rout of administration. Most of natural antioxidants tested demonstrated antitumor activity at the early stage of EC growth stimulating endogenous NOD formation.

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Metal nanoparticles are a principally new class of compounds that possess significant biological activity and are potentially perspective for diagnostics and treatment of diseases of different ethiology, especially cancer [1–5].

Gold nanoparticles, in comparison with other me­tals, are characterized by unique physical, chemical, biological properties and functional activity [6–10]. The nanoparticle size and shape substantially define their properties [11–15]. High affinity to tumor cells, surface modification ability and special optical properties create the basis for effective usage of GNP as vectors for target antitumor drug delivery [16, 17], in cancer phototermal therapy [18–20], as contrasting agents in magnetic resonance and computer tomography [21, 22].

The aim of this work was the synthesis of gold nanoparticles with different sizes and estimation of their biological activity in vitro and in vivo.

MATERIALS AND METHODS

GNP have been synthesized by Davis’ method from the tetrachloroauric (III) acid (HAuCl₄ · 3H₂O) (≥99.9% trace metals basis, Sigma-Aldrich) [23] and have been characterized by their size using laser-correlation spectroscopy (Zetasizer-3, Malvern Instruments Ltd, UK) and transmission electron microscopy (JEM-1230, JEOL, Japan) methods.

The concentration of obtained GNP was 38.6 μg/ml by metal for each size of preparations.

For in vitro experiments U937 (human leukemic monocyte lymphoma) cell line as model of tumor cells has been used. The cell line was obtained from the Bank of Cell Lines from Human and Animal Tissues, R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of NAS of Ukraine. The cell’ viability has been estimated by trypan blue exclusion test. The quantity of alive cells was > 90 % in all experiments.

The GNP interaction with tumor cells has been visualized by confocal microscopy method (LSM 510 META, Carl Zeiss, Germany).

U937 cell total membrane fraction isolation has been carried out by the method [24]. Protein content in membrane preparations has been evaluated by the method of Lowry [25].

Na⁺,K⁺-АТР-ase activity (E.C. 3.6.1.3) of U937 cell membrane fraction has been measured by method [26] at 37 °Сin 1 ml incubation medium (50 мМ Tris-HCl, 5 мМ MgCl₂, 100 мМ NaCl, 20 мМ KCl, 3 мМ АТР (рН=7.5)). Membrane aliquot (15–20 μg of protein) was added into incubation medium, incubated for 10 min and stopped by addition of 1 ml 10% trichlo­racetic acid. The phosphorus content has been measured by Fiske-Subbarow method [27]. For estimation of GNP influence on the enzyme activity the membrane fraction protein (150–200 μg) was mixed with GNP 3 min before the incubation (at concentration range of 0.11–1.1 μg/ml by metal). 20 мМ Тris-HCl buffer has been added in control sample instead of GNP.

For in vivo experiments white inbred rats (males and females) with average weight of 180–230 g from vivarium of R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, have been used.

All experiments with laboratory animals using have been carried out in compliance with “Guide for the Care and Use of Laboratory Animals”.

The transplantation of Guerin carcinoma to the laboratory rats has been carried out subcutaneously on the back using 23% suspension of tumor tissue in physiological solution. The study of GNP influence on the tumor growth in vivo has been initiated when tumor size reached ~ 30 mm × 40 mm × 20 mm.

Experimental tumor-bearing animals have been kept at the standard regimen and housed in four groups: control group — 3 animals without treatment; and 3 experimental groups (8 animals per group) treated with single intravenous GNP injection (1 ml of GNP, 38.6 μg/ml by metal) on the basis of 20, 30 and 45 nm preparations.

The euthanasia of animals from control and experimental groups has been performed 1 h after injection (4 animals from each experimental group) and 24 h after administration.

The GNP distribution in tissues (thymus, brain, spleen, liver, kidneys, adrenal glands, lungs, heart and tumor) has been studied using the atomic-absorption spectrometry method [28] on the complex “Graphit -2” (Ukraine). The sensitivity of the method is 6 ng/ml.

The GNP DNA-damaging activity in vivo has been estimated by “Comet-assay” method (alkaline gel-electrophoresis of isolated eukaryotic cells) [29]. Cell isolation from liver, kidneys, spleen, bone marrow, intestine and tumor tissue has been performed using standard protocols.

The statistical analysis of the obtained data has been carried out using Student’s t-criterion [30]. The differences p < 0.05 were considered as significant.

RESULTS AND DISCUSSION

Water dispersions of coagulation-resistant gold nanoparticles have been synthesized for analysis of their activity in vitro and in vivo. Average sizes of synthesized GNP preparations were 10; 20; 30 and 45 nm according to the data of laser-correlation spectroscopy and transmission electron microscopy. The electron-microscopic image of synthesized 10 nm gold nanoparticles is presented on Fig. 1.

The method of synthesis used for the GNP production is based on the conversion of dissolved gold (water solution of tetrachloroauric acid) into insoluble condition with subsequent aggregation and crystallization of insoluble particles which form dispersed phase. The peculiarities of gold nanoparticle structure play an important role in conditions of their contact with different types of biological objects. The different quantity of constituent atoms, depending on the size of nanoparticles, bind to the surface: the percentage of surface atoms is higher for smaller particles. The increase of active surface area per mass, changes in interatomic distance and crystal lattice period affect the nanoparticle ability to penetrate into the cell, their biological activity as well as chemical, physical and pharmacological properties [31, 32].

High level of the GNP accumulation in U937 tumor cells has been determined by confocal microscopy by layer-by-layer scanning (Fig. 2).

The most effective GNP accumulation by U937 tumor cells has been observed for 20 and 30 nm gold nanoparticles.

In vitro biological activity of synthesized gold nanoparticles is estimated through measuring the values of Na⁺,К⁺-АТР-ase and Mg²⁺-АТР-ase activities of membrane fraction of U937 cells treated with GNP. The obtained results have demonstrated the dependence of such activity from nanoparticles size (Fig. 3, curves 1–4). GNP with average size of 10 nm in all studied concentrations (0.11–1.1μg Au/ml) inhibited the enzyme activity by 70% in comparison with control cells (curve 1), GNP with 20 nm diameter — by 20 % (curve 2). At the same time GNP with average size 30 nm and 45 nm in concentration range 0.11–1.10μg Au/ml stimulated Na⁺,K⁺-АТР-ase activity by 30–40 % (curve 3), and 20–40% (curve 4), respectively.

Thus, in GNP concentration range of 0.11–0.28 μg Au/ml (45nm) we have registered 20% elevation of the enzyme activity, while in concentration range 0.28–0.55 μg Au/ml GNP this value increased from 20% to 40% and was equal to 40% in concentration range of 0.55–1.10 μg Au/ml.

However, treatment of U937 cells with GNP (in all investigated sizes) has no significant influence on Mg²⁺-АТР-ase activity of cell membrane fraction.

In vivo study of GNP biological activity after their intravenous injection is important for estimation of GNP perspective usage in cancer diagnostics and therapy. That’s why we have estimated the patterns of GNP distribution and accumulation in organs and tissues of experimental animals.

Via atomic absorption spectrometry the GNP distribution and accumulation in organs and tissues of experimental normal animals and Guerin carcinoma bearing animals have been studied. For gold content analysis thymus, brain, spleen, liver, kidneys, adrenal glands, lungs, heart and tumor tissue of experimental animals have been examined 1 and 24 h after single GNP i.v. injection. It has been revealed that the gold is not present in studied organs of normal experimental animals either in 1 h nor 24 h after GNP injection. It may be suggested that tumor influences gold nanoparticles distribution in tumor-bearing organism resulting in their accumulation in non tumor tissue in contrast to the non-tumor-bearing host.

However, in studied organs of tumor-bearing animals the picture was different. 20 nm GNP distribution and accumulation in different organs of experimental animals are demonstrated in Table 1. In tumor bearing animals 1 h after GNP injection gold content values were as follows: in brain — 26.09±3.15 ng Au/g tissue, kidneys — 51.40±6.23 ng Au/g tissue, spleen — 60.00±5.78 ng Au/g tissue, and liver — 37.60±4.07 ng Au/g tissue.

In 24 h after GNP injection the level of gold accumulation in liver, spleen and kidneys has been substantially decreased and amounted to 12.7±1.92 ng Au/g tissue for liver and was absent in spleen and kidneys. These data demonstrate the principal role of these organs in detoxication and clearance of GNP from the organism.

The peculiarities of GNP accumulation in brain of tumor-bearing animals have shown their ability to penetrate through hematoencephalic barrier what can be used in diagnostics and target therapy.

The highest level of the gold accumulation (78.90±5.27 ng Au/g tissue), comparatively with other organs, has been registered in tumors in 1 h after GNP injection, while in 24 h it amounted to 31.7±2.69 ng Au/g tissue, what was twice higher than its residual concentration in liver.

Thus, the GNP high clearance rate in the conditions of their i.v. injection to normal animals has been demo­nstrated while as GNP predominant accumulation has been determined in tumor tissue at both time points (1 and 24 h after intravenous injection).

GNP usage in diagnostics or therapy should be validated through their biosafety marker tests in vitro and in vivo. One of most sensitive marker tests of biosafety is genotoxicity test, which reveals agent DNA-damaging influence. For this purpose we have used “Comet assay” method.

The GNP genotoxicity, depending on their size, has been analyzed earlier [33, 34] in vitro and in vivo. The 20 nm GNP genotoxic influence on kidney cells of tumor-bearing rats in 1 h after single GNP intravenous injection has resulted in 24.04% of DNA in comet tail versus 0.24% for negative control (Table 2, Fig. 4). These data evidence on potential risk of 20 nm GNP injection for normal organs.

1 h after 20 nm GNP injection the apoptosis rate of tumor cells yielded up to 80%. It is the evidence of total tumor cells death.

The 30 and 45 nm GNP injection didn’t cause DNA damage in organs and tumor tissue of experimental animals in 1 h after i.v. injection. In other words, 30 and 45 nm nanoparticles revealed biosafety in such test.

In 24 h after i.v. injection of 20, 30 and 45 nm GNP there has been registered no genotoxic influence (Table 3).

So, the high specific size-dependent biological activity in vitro and in vivo of synthesized water dispersions of GNP has been revealed. In vitro data show that synthesized gold nanoparticles possessed by expressed size-dependent modulation of membrane Na⁺,K⁺-АТР-ase activity in U937 tumor cells. In vivo results indicate that gold nanoparticles possess high affinity to tumor cells after their i.v. injection to experimental animals. These in vitro and in vivo results point on the perspectiveness of GNP use in cancer diagnostics and treatment, although it is necessary to take into account the size-dependent biosafety level of GNP concerning normal organs.

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