Osteopontin siRNA does not confer resistance to toxic effects of parthenolide in Jurkat cells

DOI: 10.32471/exp-oncology.2312-8852.vol-42-no-3.15180

Osteopontin (OPN), also known as secreted phosphoprotein 1 is a 34kD multidomain secreted glycoprotein that functions as a soluble cytokine and an adhesive component of the extracellular matrix [1, 2]. It is a member of small integrin-binding ligand N-linked glycoproteins (siblings), a family of five integrin binding glycophosphoproteins [3]. OPN interacts mainly with various α_v (particularly α_vβ₁, α_vβ₃, α_vβ₅) integrins and with the CD44 splice variants. Due to these properties, OPN induces the activation of signal transduction pathways leading to cell proliferation, adhesion, invasion and migration. Hence, its role in cancer is important, as it is involved in angiogenesis, and metastasis [4–7]. OPN, also called early T cell activation gene 1 (Eta-1), is expressed after activation of T cells, macrophages and in multiple tissue types. OPN is cleaved by some proteases such as thrombin, cleaved product participating in various cellular functions. Jurkat cells originating from T cell acute lymphoblastic leukemia (T-ALL) have enhanced cell adhesion to thrombin-cleaved OPN compared with full-length OPN [8, 9].

Given that the human OPN gene is encoded by a single-copy gene on chromosome 4q21-q25 and has an alternative translation start site, there are two major variants of OPN, intracellular and extracellular [1]. It is also subjected to alternative splicing as well as post-translational modifications such as phosphorylation, glycosylation and proteolytic cleavage.

Parthenolide (PTL)-containing herbs are highly bioactive and have been traditionally used for treatment of inflammations. The anti-inflammatory activities of these herbs are partially due to their anti-NF-kB effects [10]. Currently PTL is used extensively in cancer treatment [11–14]. It can induce oxidative-stress-mediated apoptosis in T cells at high doses (up to 10 µm) [15].

T-ALL represents approximately 12% to 15% of all newly diagnosed acute lymphocytic leukemia (ALL) cases in pediatric patients and about 25% of adult ALL. This aggressive malignancy is associated with a significant risk of disease relapse and does not respond well to chemotherapy [16–18].

Herein, we attempted to study the role of OPN in conferring in vitro resistance to PTL in Jurkat cells and to examine the effect of knockdown of OPN gene expression via OPN-siRNA on PTL-treated Jurkat cell survival.

MATERIALS AND METHODS

Reagents. Parthenolide was purchased from Sigma-Aldrich (USA) and dissolved in dimethyl sulfoxide (DMSO) as a 50 mM stock solution, stored at –20 °C, and diluted in DMSO before use. The Annexin V-FITC/PI kit was purchased from BD Biosciences (USA). Tripure Isolation Reagent was purchased from Roche Applied Science (Germany). The cDNA synthesis kit and SYBR^® Premix Ex Taq™ were purchased from Takara Biotechnology Co. (Otsu, Japan).

Cell culture. The human leukemic Jurkat cell line was obtained from the Iranian Biological Resource Center. RPMI 1640 supplemented with 10% fetal bovine serum medium (Invitrogen, USA) was used for culturing. The medium was supplemented with 2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂.

MTT assay. Cells were cultured in triplicate at 5 · 10³/100 μl in 96-well culture plates (SPL Life sciences, Korea) with 8, 10, 12, 15, or 20 μm PTL for 48 h. Following the culture, the cells were incubated for 4 h with 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT, 5 mg dissolved in 1 ml of PBS, Sigma, USA). The plates were centrifuged (10 min at 350 g), and the purple formazan crystals of metabolized yellow tetrazolium salt by viable cells were dissolved in DMSO. The absorbance was quantified at 570 nm using the ELISA plate reader (Microplate Reader; Bio-Rad, USA). The results were expressed as a percentage of viability, with 100% representing control cells treated with 0.1% DMSO alone.

Annexin V/Propidium iodide (PI) assay. Cells were stained with Annexin V-Alexa Fluor-488/PI according to the manufacturer’s instructions and examined by flow cytometry (Partec, Germany). Discrimination of cells was performed as apoptosis (Annexin V⁺/PI^- [early apoptosis] and Annexin V⁺/PI⁺ [late apoptosis]).

Quantitative real-time polymerase chain reaction (qRT-PCR). Total RNA of the PTL-treated and untreated cells was extracted with Tripure Isolation Reagent according to the manufacturer’s instructions, quantified on a NanoDrop ND-1000 (NanoDrop Technologies, USA), and stored at −80 °C. The cDNA synthesis kit was used to synthesize cDNA). A light cycler instrument (Roche Diagnostics, Germany) and SYBR Premix Ex Taq were used for quantitative real-time RT-PCR analysis. A final volume of 20 μl containing 2 μl of a two-fold diluted cDNA, 1 μl of 10 pmol primers (0.5 μl each forward and reverse primers), 10 μl of SYBER, and 7 μl of distilled water were used. Data were normalized to HPRT expression in each sample. Relative gene expression data were analyzed using the 2^-ΔΔCt method.

Short interfering RNA (siRNA) transfection. The siRNA against OPN was applied to cell lines using lipofectamin 2000 reagent (LF2000; 10 μg/ml; Invitrogen, USA) according to the manufacturer’s instruction. Cells were lysed 24 h post-transfection and quantitative real-time PCR was performed using following sequences of OPN siRNA primers: 5´-ggaauauuacugugggaaadtdt-3´ (sense) and 5´-uuucccacaguaauauuccdtdt-3´ (antisense). The highest transfection efficiency was obtained in the experiments within 24 h post-transfection at a final concentration of 30 pmol/ml. This optimal condition was used for subsequent experiments tested with siRNA.

Statistical analysis. Using IBM SPSS Statistics 19 software, the groups of data was presented as means ± SDs and compared by one-way analysis variance (ANOVA) or t-test.

RESULTS

Jurkat cells viability. Various concentrations of PTL were used to determine its toxic potency in Jurkat cells. The IC₅₀ values at 48 h were estimated to be 16.1 μM (Fig. 1). 15 μM concentration was chosen for other assays.

OPN-siRNA does not influence significantly growth inhibition and apoptosis-inducing effects of PTL in Jurkat cells. The viability of OPN siRNA-treated cells with or without treatment with PTL was measured using MTT assay (48 h). OPN siRNA (30 pmol/ml) did not decrease cell viability much more than control. In addition, subsequent PTL treatment decreased the cell viability up to 51% that is without any difference in comparison of PTL alone (Fig. 2).

Apoptosis assay was performed as an additional testing to contribute in evaluating the OPN function in PTL-induced death. Annexin-V/PI staining indicated that PTL induced apoptosis. The average percentage of apoptosis was 70% within 48 h (Fig. 3). OPN siRNA transfection and subsequent treatment with PTL did not increase the apoptotic cells compared to the PTL alone in Jurkat cells (see Fig. 3).

OPN gene expression is not affected by PTL in Jurkat cells. The expression of OPN was evaluated­ by qRT-PCR. As illustrated in Fig. 4, OPN mRNA expression in cells treated with PTL does not differ significantly from that in PTL-treated cells transfected with OPN siRNA. Therefore, it appears that OPN could not be crucial for the survival of Jurkat cells in the setting of PTL treatment.

DISCUSSION

Regarding the role of OPN in chemotherapy resistance and leukemia treatment, our previous study demonstrated that PTL induces apoptosis in AML cell line via a reduction in OPN gene expression [19]. In another report, we suggested that silencing of OPN gene using siRNA significantly decreased CD34+/CD38- human leukemia cells [20].

The results of this study showed that the transfection of Jurkat cells with OPN siRNA does not affect the viability of cells upon PTL treatment. It should be pointed out that OPN has three isoforms based on mRNA splice variants, including OPN-a, full‐length transcribed mRNA containing all seven exons, OPN-b, with mRNA lacking exon 5, and OPN-c with mRNA lacking exon 4 [20]. Although in our own work, OPN siRNA degrades all three isoforms of the gene, this does not contribute to PTL toxicity for Jurkat cells [20].

While little evidence is available on the genetic basis of resistance of leukemic cells to chemotherapy, multidrug resistance genes and genes involving in cell-cycle progression and apoptosis, OPN is one of the candidate genes that has been recently investigated as a target in gene therapy projects and its spliced variants possibly have their unique place as cancer markers [6]. Following the related studies on OPN isoforms, it seems that two main isoforms including OPN-b and OPN-c have an important role in angiogenesis and chemoresistance [21]. Weber et al. [11] demonstrated that in T cells only OPN-a is expressed. Besides, in a study of OPN isoforms of macrophage, it was found that OPN isoforms increase macrophage survival and decrease macrophage apoptosis [2].

Previous research reported that drug resistance is an intrinsic feature of acute myeloid leukemia cells reflected in the gene expression pattern, which makes the resistance to chemotherapy predictable prior to the treatment [22]. The development of targeted therapies, including monoclonal antibodies and gene therapy and siRNA has shown great potential, particularly in the field of cancer treatment [17, 23].

On the other hand, Huang et al. [23] demonstrated that inhibition of BCL11B (B cell chronic lymphocytic leukemia (lymphoma11b) gene by siRNA selectively inhibited proliferation and effectively induced apoptosis in T-ALL cells. They revealed that the expression of OPN gene was significantly down-regulated by BCL11B gene silencing. Supplementary OPN gene silencing significantly increased mitochondrial cytochrome c release. Thus, the OPN gene may play a consistent role in anti-apoptotic effects by the BCL11B gene [23].

To sum up, if only OPN-a isoform would have existed in Jurkat cells [11], it would have been logical to hypothesize that the OPN-survival phenomenon might play a role in T-ALL cells similar to that of macrophages [2]. However, the role of this gene in T cell malignancies is unclear due to the low expression of OPN gene in Jurkat cells [23, 24]. It seems reasonable to hypothesize that OPN is not an appropriate gene for targeting in Jurkat cells. Further investigation on the role of OPN in malignant T cells is recommended.

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