Enhanced cytotoxicity of рhotoexcited fullerene С60 and cisplatin combination against drug-resistant leukemic cells
Summary. Aim: To evaluate the viability of leukemic cells sensitive (L1210S) and resistant (L1210R) to cisplatin, ROS production and free cytosolic Ca2+ concentration under treatment with cisplatin or its combination with photoexcited fullerene C60. Methods: Cell viability was assessed by the MTT reduction assay. Light-emitting diode lamp (2.45 J/сm2) was used for photoexcitation of intracellular accumulated fullerene C60. Free cytosolic calcium concentration ([Ca2+]i) and ROS production in cells were estimated with the use of fluorescent probes Indo-1 and 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA), respectively. Results: It is shown that viability of L1210R cells wasn’t changed under treatment with cisplatin in concentration range 0.1–10 μg/ml. 50% and 30% decrease of L1210S cells were observed after 24 h of incubation with cisplatin at concentrations 5 and 1 μg/ml, respectively. Intensification of extranuclear cytotoxic effects (ROS production and [Ca2+]iincrease) after treatment with 1 μg/ml was detected in L1210S, but not in L1210R cells. The most strongly pronounced increase of ROS production and [Ca2+]i in both L1210 cell lines was revealed in dynamics after combined treatment with cisplatin (1 μg/ml) and photoexcited fullerene C60 (10–5 M) and was followed by decreased viability of not only L1210S, but of L1210R cells as well.. Conclusion: Combined treatment with photoexcited C60 and cisplatin allowed to decrease effective concentration of cisplatin against parental L1210 cells and to increase sensibility of resistant cells to the drug.
Submitted: June 22, 2015.
*Correspondence: E-mail: firstname.lastname@example.org
Abbreviations used: CP — cisplatin; DCF-DA — 2´,7´-dichlorodihydrofluorescein diacetate; ROS — reactive oxygen species.
Despite the fact that the most commonly used cytotoxic agents — cisplatin (CP), doxorubicin and paclitaxel, are highly efficient in current cancer therapy; their therapeutic efficiency is lowered by high toxicity and easy development of drug resistance. CP displays clinical activity against variety of human malignancies and is generally recognized as the DNA-damaging drug. Nevertheless CP is shown to induce apoptosis independently of DNA damage; and the ability to activate several different rather than a single apoptotic pathway is thought to be a distinction, which makes CP highly efficient as an anticancer drug. Cytoplasmic reactive oxygen species (ROS) production, free cytosolic Ca2+ concentration increase, modification of membrane ion channels and transport proteins, endoplasmic reticulum stress are supposed to be the pathways of CP-induced cell death signaling .
Because CP has multiple cellular targets and many different routes of cell entry, resistance to this drug is very complex and requires numerous mechanisms including reduced accumulation by impaired influx or active efflux, defective endocytosis, detoxification by GSH system, inactivation of apoptotic pathways, increased DNA repair, alterations in the expression of tumor suppressor gene p53 [2–6]. To increase the therapeutic index and to minimize CP resistance combined therapies with the use of nanotechnologies are developing now.
The recent studies have showed that combination or conjunction of anticancer drugs with the representatives of carbon nanostructures (single-walled carbon nanotubes, hydroxylated fullerene C60, metallofullerenes with gadolinium [Gd@C82(OH)22]n, etc.) could be promising for targeted drug delivery, overcoming of tumor cells drug resistance and reduction of toxic effects in normal cells [7–11].
To estimate the probability of enhancing CP proapoptotic effects we have studied the effect of combined treatment with CP and photoexcited fullerene C60 on cancer cells. Fullerene C60 demonstrates unique physicochemical properties and biological activity: spherical form, nanosize (0.72 nm in diameter), allocation inside hydrophobic regions of cell membranes and ability to penetrate into cytoplasm, compatibility with biological molecules [12, 13]. Due to extended π-conjugated system of molecular orbitals fullerene C60 absorb UV/Vis light efficiently and is able to generate cytotoxic ROS with almost 100% quantum yield. Modification of fullerene core appears to cause a perturbation of its electronic structure and hence to reduce the photodynamic potential of the molecule . Using pristine fullerene C60 we havedemonstrated its accumulation inside leukemic cells and apoptosis induction after photoexcitation .
The aim of this study was to evaluate the viability of leukemic cells sensitive (L1210S) and resistant (L1210R) to CP, ROS production and free cytosolic Ca2+ concentration under CP treatment or combined treatment with CP and photoexcited fullerene C60.
MATERIALS AND METHODS
Two leukemic L1210 cell lines — sensitive (L1210S) and resistant (L1210R) to CP were used, which were kindly provided by Dr. Liudmyla Drobot (V.A. Palladin Institute of Biochemistry, the NAS of Ukraine). Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, Germany), 50 μg/ml penicillin and 100 μg/ml streptomycin at 37 °C in a 5% CO2humidified atmosphere. Cells were treated with CP (Sigma, USA) in concentrations 0.1–10 μg/ml.
Stable water colloid solution of fullerene C60 (10–4 M, purity >99.5%, nanoparticle average size 50 nm) was synthesized in Technical University of Ilmenau (Germany) as described in [16, 17]. Cells in RPMI 1640 medium were incubated for 2 h with or without fullerene C60 (10–5 M). Photoactivation of accumulated fullerene C60 was done by probes irradiation with light-emitting diode lamp (410–780 nm light, irradiance 100 mW (2.45 J/сm2), during 2 min).
Cell viability was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide] reduction assay. At indicated time points of incubation 200 μl aliquots were removed from cell suspensions into the 96-well microplates (L1210S/R cells — 1×105/well), 20 μl of MTT solution (2.5 μg/ml) was added to each well and the plates were incubated for another 2 h. The culture medium was then replaced with 100 μl of DMSO, diformazan formation was determined by measuring absorption at 570 nm with a plate reader (μQuant, BioTek, USA).
ROS production was measured using fluorescent probe 2´,7´-dichlorodihydrofluorescein diacetate (Sigma, USA), which was added to the cell incubation medium (1×106 cells/ml) in concentration 5 mM. The fluorescent intensity was measured in real time on spectrofluorimeter Shimadzu 150 RF (Japan) λ excitation — 480 nm, λ emission — 520 nm .
The concentration of free cytosolic Ca2+ was measured using fluorescent probe Indo-1 (Sigma, USA). Cells (3×107/ml) in buffer A consisting of (mM): KCl — 5, NaCl — 120, CaCl2 — 1, glucose — 10, MgCl2 — 1, NaHCO3 — 4, HEPES — 10, pH 7.4 were loaded with Indo-1AM (in concentration 1 mM) in the presence of 0.05% Pluronic F-127 (Sigma, USA) for 40 min at 25 °C. Indo-1 fluorescence in cells was recorded using spectrophotometer (Shimadzu RF-510, Japan), λ excitation — 350 nm, λ emission — 410 and 495 nm. The concentration of free cytosolic Ca2+ was calculated as described in .
Data processing and plotting were performed by IBM PC using specialized applications Excel 2010 and Origin 7.0. Statistical analysis was performed using Statistica 6.0 computer program (StatSoft Inc.). Paired Student’s t-tests were performed. Differences values p < 0.05 were considered to be significant.
To estimate the sensitivity of two L1210 cell lines to CP, the viability of cells treated with the drug in concentration range 0.1–10 μg/ml was studied by MTT test. The viability of parental L1210 cells treated with CP appeared to be decreased in a dose and duration dependent manner (Fig. 1, a). At 24 h of incubation the viability of cells treated with 1 μg/ml was decreased by 30% while of those treated with 5 μg/ml — by 50%. No L1210S cell survival was detected at 72 h after treatment with 5 or 10 μg/ml of CP.
Treatment with CP in a range 0.1–10 μg/ml was virtually nontoxic to L1210 resistant cells. A slight decrease of viability (to approximately 80%) was observed at 48 h after the treatment with 5 and 10 μg/ml CP, but at 72 h the indexes were normalized (Fig. 1, b).
Fig. 1. Cell viability kinetics of L1210S (a) and L1210R (b) cells treated with CP in different concentrations (n = 4)
To investigate the effect of combined treatment with photoexcited fullerene C60 and CP on susceptibility of L1210 cells to the drug, we used in further experiments with long-term cell incubation the CP concentration — 1 μg/ml. Cells were preincubated with C60 nanoparticles for 2 h, irradiated for photoexcitation of accumulated fullerene C60 and incubated for 72 h. In the case of combined treatment CP was added just before C60 photoexcitation.
Irradiation of cells with or without CP did not change the viability of either L1210S or L1210R cells (data not shown), while treatment with fullerene C60 was followed by slight decrease of both cell lines viability (Fig. 2, a, b).
The cytotoxic effect of CP against L1210S appeared to be comparable with the effect of photoexcited C60; both agents cause the 40% decrease of cell viability at 72 h. However, after combined treatment with CP and photoexcited C60 the cytotoxic effect was increased and CP concentration of 1 μg/ml was proved to be sufficient to reach 50% decrease at 48 h as distinct from CP treatment alone (see Fig. 2, a).
We have demonstrated that cytotoxic effect of photoexcited fullerene C60 was realized not only against parental, but against CP resistant cells as well. Moreover while treatment with 1 μg/ml CP alone did not change the viability L1210R cells the combined treatment with photoexcited C60 and CP allowed to intensify cytotoxic effect and to decrease viability of resistant cells by 68% at 48 h of incubation.
Fig. 2. Cell viability kinetics of L1210S (a) and L1210R (b) cells exposed to fullerene C60 (10–5 M; n = 4), CP (1 μg/ml; n = 4), photoexcited C60 (n = 5) and combined treatment with CP and photoexcited fullerene C60 (n = 5)
We supposed that enhancement of cytotoxicity after combined treatment with CP and photoexcited C60 could be caused by ability to activate early cytoplasmic events leading to cell death independently of DNA damage. It has been shown, that CP requires cytoplasmic generation of ROS and [Са2+]iincrease to induce apoptosis [20–22].
As we have demonstrated earlier that fullerene C60 is accumulated in leukemic Jurkat cell lines, increased ROS production and free cytosolic calcium concentration at early period (3 h) after photoexcitation with further substantial activation of caspase-3 and cell death by mitochondrial-dependent pathway at 24 h [15, 23–25]. To confirm this assumption the experiments with CP addition to both cell lines at 3 h after photoexcitation of accumulated fullerene C60 were carried out.
Sensitive and specific method for ROS production evaluation in cells is the use of fluorescent probe 2´,7´-dichlorodihydrofluorescein diacetate (DCF-DA). When DCF-DA was added to untreated cells, the increase of fluorescence after 10 min incubation was detected corresponding to the endogenous ROS level (Fig. 3). The values of free cytosolic calcium concentration in sensitive and resistance cells in control were 125 ± 6 and 130 ± 4 nM, respectively, and weren’t changed during cells incubation (taken for 100%) (Fig. 4).
Treatment of L1210S with CP alone was shown to be followed by increasing of both ROS generation and [Са2+]i at early period after CP addition while after treatment of L1210S with the drug neither ROS nor [Са2+]i levels were changed (see Fig. 3, a, 4, a). In contrast to CP photoexcited fullerene C60 induced ROS production and [Са2+]ielevation not only in L1210S, but in L1210R cells as well (see Fig. 3, 4).
Fig. 3. The kinetics of ROS production in L1210S (a) and L1210R (b) cells after exposition to CP (n = 3), photoexcited fullerene C60 (n = 4) and combined treatment with CP and photoexcited fullerene C60 (n = 4)
Fig. 4. Ca2+ concentration in L1210S (a) and L1210R (b) cells exposed to CP — 1 (n = 3), photoexcited fullerene C60 — 2 (n = 4) and combined treatment with CP and photoexcited fullerene C60 — 3 (n = 4)
But the most strongly pronounced increase of DCF and indo-1 fluorescent signals in both L1210S and L1210R cells were detected in dynamics after combined treatment with CP and photoexcited fullerene C60 indicating on substantial disturbance of intracellular redox status and calcium homeostasis (see Fig. 3, 4).
The obtained data demonstrate the synergic cytotoxic effect of CP and photoexcited fullerene C60 against L1210R leukemic cells. The combined treatment with these agents is followed by intensification of ROS dependent pathways of cell death and allows not only to decrease effective concentration of CP against parental L1210 cells, but to restore sensibility of resistant cells to the drug.
Recent studies [3, 6, 21, 22] demonstrate that activation of the signaling pathways to apoptosis which occurs in cytoplasm independently of nuclear is the alternative to DNA damaging mechanism of CP toxicity against cancer cell. Searching for the ways to enhance CP extranuclear cytotoxic effects with the use of nanotechnology opens the possibility to increase the therapeutic efficiency of the drug.
We studied the effect of CP at a range 1–10 μg/ml against parental and drug-resistant leukemic L1210 cells. Early intensification of ROS production and Ca2+ increase after treatment with 1 μg/ml was detected in L1210S cells. No decrease of L1210R cells viability and no changes in ROS or [Ca2+]ilevels at the early period of treatment with 1 μg/ml CP were detected.
These data are in agreement with the studies which reported that CP-induced extranuclear effects involve intensification of ROS generation due to CP upregulation of nicotinamide adenine dinucleotide phosphate oxidases (NOX-1 and NOX-4) and ER stress induction [20, 26]. It is also shown that CP could provoke elevation of cytosolic calcium concentration by an increased Ca2+ uptake from the extracellular space (mediated through plasma membrane calcium channel) or by Ca2+ release through IP3 receptors of endoplasmic reticulum [21, 27].
To overcome drug resistance in L1210R cells the combined treatment with photoexcited fullerene C60 and CP was used. The results indicated that combined treatment improved the cytotoxic effect of CP in L1210R cells by increase of ROS production and free cytosolic calcium concentration and decrease of cells viability. We suggested that this alteration in L1210R cells sensitivity to CP could be explained by the ability of fullerene C60 to affect and modify some mechanisms of cell drug resistance development: platinum compounds transport through the plasma membrane, antioxidative system activity (detoxification by glutathione conjugates), induction of apoptotic pathways, calcium homeostasis maintenance [3, 28–33]. Thus, fullerene derivatives are able to reactivate endocytosis in cancer cells and to promote CP accumulation . Fullerene C60 is shown to be located in the plasma membrane and intermembrane space of mitochondria with further intensification of ROS in mitochondrial electron transport chain [24, 34]. It is also shown that cancer cells have genetically remodulated system of calcium homeostasis: underfilled ER calcium pool and reduced of SOCE (store-operated calcium entry through plasma membrane) directed to prevent Ca2+-dependent way of apoptosis. We have earlier shown that photoexcited C60 induced the increase of SOCE, depletion of mitochondrial Ca2+-pool, cytochrome c release from mitochondria and activate Ca2+-dependent apoptotic pathway .
Consequently, we supposed that sensitivity enhancement of L1210R cells to CP induced by combined treatment was connected with properties of photoexcited fullerene C60 to affect on auxiliary intracellular targets beside that which were involved in CP cytotoxic effect.
This suggests that combined treatment with CP and photoexcited fullerene C60 is followed by synergistic anticancer effect of both compounds and allows to increase cytotoxicity against leukemic L1210 cells and to restore sensibility of L1210 resistant cells to CP by enhancing extranuclear proapoptotic effects of the drug.
The proposed model of combined treatment can be helpful in developing the approaches to decrease anticancer drug’s toxic dose and to optimize the methods of photodynamic therapy.
1. Chung T-W, Choi H-J, Kim S-J, et al. The ganglioside GM3 is associated with cisplatin-induced apoptosis in human colon cancer cells. PLoS One 2014; 9: doi: 10.1371/journal.pone.0092786.
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