| BackgroundGastric cancer is one of the most common malignant tumors in the world. In China, the incidence and mortality rate of gastric cancer is second only to lung cancer. At present, the optimum treatment of the patients with early gastric cancer has been achieved with a combination of surgical operation, chemotherapy, radiotherapy and biological therapy. For patients with advanced gastric cancer, chemotherapy would be the best choice. With the increased use of chemotherapeutic drugs, multidrug resistance (MDR) has been widespread in clinical tumor therapy and is the major obstacle to successful chemotherapy and patients’survival.In the past decades, many strategies have been developed to overcome this problem, notable among them are resistance reversal agents. MDR refers to resistance of tumor cells to several mechanistically and structurally unrelated chemotherapeutic drugs simultaneously. Several molecular mechanisms have been proposed to be responsible for the MDR phenomenon, and the ATP-binding cassette (ABC) proteins ABCB1(MDR1/Pgp), ABCC1-MRP1, and ABCG2-BCRP are the best characterized mediators of MDR. In particular, ABCB1(MDR1) encoded P-glycoprotein (P-gp) plays a crucial role in drug resistance by actively extruding many types of drugs from cancer cells. Known substrates of P-gp include anticancer drugs such as paclitaxel, doxorubicin and vinblastine.Cancer stem cells (CSCs) theory holds that CSCs are a small subpopulation of cells within a tumor that can self-renewal, differentiate into multiple lineages, and drive tumor growth, while the bulk of the tumor cells are differentiated cells and incapable of self-renewal. Cancer stem cells theory also suggests that CSCs are resistant to chemotherapy and may contribute to tumor metastasis and tumor recurrence after treatment since chemotherapeutic drugs kill differentiated or differentiating cells, but CSCs remain untouched.Salinomycin is a polyether anticoccidial drug produced by a Streptomyces albus strain. Recently, salinomycin was reported to possess potent anti-CSC activity. Compared to paclitaxel, a chemotherapeutic drug, salinomycin reduced the proportion of CSCs by>100-fold in breast cancer. The mechanism by which salinomycin acts as an anti-cancer and anti-CSCs agent has not yet been elucidated, but it was suggested that salinomycin might induce terminal epithelial differentiation rather than trigger cytotoxicity. Further investigation showed that salinomycin induces massive apoptosis in human cancer cells of different origin through overcoming multiple mechanisms of apoptosis resistance. A more recent study demonstrated that salinomycin acts as a potent inhibitor of multidrug resistance P-glycoprotein (P-gp), as evidenced through drug efflux assays in MDR cancer cell lines overexpressing P-gP.ObjectiveTo investigate the effct and mechanism of salinomycin as a resistance reversal agent on multi-drug resistant gastric cancer cell line SGC-7901/VCR. Methods1. SGC-7901and SGC-7901/VCR cells were were grown in RPMI1640supplemented with10%of FBS. Their growth curve, cell cycle and multidrug resistance were measured.2. Tumorigenicity of the cells was determined in a nude mouse xenograft model.3. The mechanisms of the multidrug resistance of SGC-7901/VCR cells were analyzed through a variety of experimental methods:3.1The proportion of side population in the two cell lines was measured by flow cytometry, and the expression levels of the ABC transporter protein ABCG2and MDR1-Pgp were detected by Western blot analysis.3.2The expression levels of gastric cancer stem cell marker CD44and EpCAM in the two cell lines were analyzed by flow cytometry.3.3The self-renewal ability of cancer stem cells was evaluated with tumor-sphere forming assay. Expression levels of cancer stem cell markers Nanog, SOX,-2, OCT3/4and C-myc were detected by Western blot analysis.4. Effect of salinomycin on multidrug resistance of SGC-7901/VCR and the behind mechanisms were investigated through of the following experimental methods:4.1The half maximal inhibitory concentration (IC50) of salinomycin in SGC-7901and SGC-7901/VCR cells were determined by MTT colorimetric assay.4.2The resistance reversal index was examined by MTT assay in SGC-7901/VCR cells treated in combination with salinomycin and VCR.4.3Accumulation of ADM was detected by MDR efflux assay in SGC-7901/VCR cells treated with salinomycin.4.4Apoptosis was detected by Annexin V staining in the two cell lines treated with different concentrations of salinomycin.4.5Side-population (SP) of the cells was detected by Hoechst33342staining and flow cytometry sorting. Expression of SP related ABC transporter protein was measured by Western blot.4.6EpCAM+cells were detected by flow cytomerty.4.7Anti-CSC effect of salinomycin on SGC-7901/VCR cells was evaluated with tumor-sphere forming assay. The expression levels of stem cell markers were detected by Western blot analysis.5. Statistical analysisThe experimental data were statistically analyzed using SPSS13.0software:two samples were compared using independent sample t-test analysis; multi-samples One-way ANOVA was used to compare the differences of the cells in each group, with the LSD method for multiple comparisons.Results1. Characterization of the multidrug resistant gastric cancer cell line SGC-7901/VCR(1) Cell morphologyThe parental SGC-7901cells grow as a single-layer with polygonal morphology, and cells with spindle shape were occasionally observed. SGC-7901cells proliferate rapidly and could be passaged every3or4days. SGC-7901/VCR cells also grow as a single-layer but they exhibit a spindle shape. The proliferative rate of SGC-7901/VCR is similar to that of SGC-7901.(2) Cell proliferation rateCell proliferative rate was determined by MTT assay, and the cell proliferation curve was plotted. Both of SGC-7901and SGC-7901/VCR cell have inhibition phase, the logarithmic growth phase and cell death phase. From the fifth day, SGC-7901/ VCR and SGC-7901cell in vitro proliferation ability has significant difference (F=24.755, P=0.000). Cell cycle was detected by flow cytometry analysis. The ratios of G0/G1, S and G2/M phases in SGC-7901cells was44.700+1.400%、43.366±3.197%'11.933±2.074%, respectively, and in SGC-7901/VCR cells they were37.200±1.908%、56.133+2.747%'6.667+1.518%, respectively. The proliferation indexes (PI=S+G2/G1+S+G2) of SGC-7901and SGC-7901/VCR cells respectively were55.300±1.400%and62.800±1.908%(t=-5.489, P=0.005)(3) Tumorigenesis of SGC-7901and SGC-7901/VCR cells in nude miceDecreasing numbers (5×106,1×106,1×105and1×104) of SGC-7901or SGC-7901/VCR cells were injected into nude mice. After4weeks, every group of SGC-7901/VCR cells generated a tumor, but at least1×105of SGC-7901cells were needed to form a tumor. Further study showed that the minmum number of SGC-7901/VCR cells which could form a tumor was1000. This result indicated that SGC-7901/VCR cells have a more potent tumorigenicity compared with SGC-7901cells.(4) Multidrug resistance of SGC-7901/VCR cellsWhen treated with chemotherapeutic drugs VCR, ADM, Taxol or DDP, SGC-7901/VCR cells showed resistance and their IC50values were2.530+0.287μM,9.030±0.737μM,0.583±0.076μM and3.327±0.172μM, respectively, while SGC-7901cells were relatively sensitive to these drugs and their IC50values were0.073±0.020μM,0.340±0.118μM,0.066±0.008μM and1.347±0.315μM, respectively. The IC50values of each drug were significant differences between SGC-7901and SGC-7901/VCR cells (P﹤0.05). Compared with parental cells, the resistance index of drug-resistant cells to the four chemotherapy drugs were36.0,27.0,7.10and2.46, respectively.2. Study on multidrug resistance mechanisms of SGC-7901/VCR cells(1) Flow cytometry analysis showed that the proportion of SP cells was 0.067±0.115%in SGC-7901cells, and88.900±11.966%in SGC-7901/VCR cells, a dramatic difference between the parental and drug resistant cell lines (t=-12.858, P=0.006). Since ABC transporter, especially ABCB1(MDR1-Pgp), and ABCG2, were closely related to SP phenotype, We examined the expression levels of these two proteins. Western blot analysis showed that ABCG2was not expressed in SGC-7901nor in SGC-7901/VCR cells. In contrast, MDR1-Pgp was weakly detectable in SGC-7901cells, but highly expressed in SGC-7901/VCR cells.(2). The proportion of CD44+cells was95.400±3.400%in SGC-7901, and was95.433±2.139%in SGC-7901/VCR. Both of them had high CD44expression and the difference does not have statistical significance (t=-0.014, P=0.989). Proportions of EpCAM+cell were22.733+2.554%and93.667±4.723%in SGC-7901and SGC-7901/VCR cells, respectively. The difference is statistically significant (t=-22.883, P=0.000)(3) Tumor-sphere formation assay showed that after cultured in serum-free medium for30days, SGC-7901cells could not form tumor-sphere, but SGC-7901/VCR cells formed large tumor-spheres. The tumor-sphere numbers was0and36.000±5.568per1000SGC-7901and SGC-7901/VCR cells, respectively. The difference had statistical significance (t=-11.199, P=0.000). Cancer stem cell markers were detected by Western blot. C-myc was equally expressed in SGC-7901and SGC-7901/VCR cells. Nanog was not detectable in SGC-7901, but highly expressed in SGC-7901/VCR.3. The effect and mechanism of salinomycin in reversing multidrug resistance of SGC-7901/VCR cells(1) The dose-dependant effects of salinomycin on SGC-7901and SGC-7901/VCR cells were analyzed by MTT assay. The IC50values were1.755±0.182μM and2.480±0.174μM, respectively. The difference was significant (t=-4.978, P=0.008). Under the action of1,2and4uM salinomycin, the sensitivity of the drug-resistant cell SGC-7901/VCR to VCR was enhanced, as reflected by decreased IC50values (P<0.01) of0.710+0.060Mm,0.410±0.040μM and0.210±0.020μM, respectively. And there were significant difference, compared with un-treated group (P﹤0.05). The reversal indexes were3.420、5.930and11.57. The result of MDR efflux assay on SGC-7901/VCR cells indicated that Salinomycin could markedly increase doxorubicin accumulation in drug-resistant cells.(2) After treated with,1μM,2μM, or4uM salinomycin for48h, the percentages of apoptotic cells were increased from1.700±1.300%in untreated cells to21.433±4.496%,58.800+4.331%and79.800+3.869%, respectively, in SGC-7901, while they were increased from1.767±1.850%in untreated cells to21.033±5.804%,58.200+6.596%and81.233+3.037%in SGC-7901/VCR cells. Treatment with salinomycin induced apoptosis dramatically in both SGC-7901and SGC-7901/VCR cells (F=417.850, P=0.000). The apoptosis rates are not statistically significant between SGC-7901and SGC-7901/VCR when the two cell lines were treated with the same drug concentration (F=0.069, P=0.976)(3) Salinomycin decreased SP of the drug resistant cells. when SGC-7901/VCR cells were treated with salinomycin at increasing concertrations (OμM,1μM,2μM or4μM) for48h, the proportions of SP were decreased from93.733±6.351%,54.967±5.784%,30.567±4.92%and11.70±2.17%(F=128.332, P=0.000) respectively. Accompanying the decrease of SP proportion, the expression of MDR1-Pgp was downregulated.(4) Salinomycin suppressed tumor-sphere forming capability of the drug resistant cells. SGC-7901/VCR cells were cultured with serum-free medium and treated with salinomycin at increasing concentrations (0μM,1μM,2μM and4μM) for48h. After cultured for30days, we the ability of tumor-formation was suppressed by salinomycin in a dose-dependent manner. Salinomycin inhibited not only the numbers but also the size of tumor-spheres.(5) Salinomycin suppressed EpCAM+cells of the drug resistant cells. After treated with OμM,1μM,2μM or4μM Salinomycin for48h, the percentages of EpCAM+cells were decreased from26.667+2.616%to20.600+4.444%,66.667±1.242%and2.767+0.603%, respectively, in SGC-7901cells, while they were decreased from90.500±5.839%to57.067±6.467%,21.567±6.048%and7.900±4.158%, respectively in SGC-7901/VCR cells. Salinomycin could decrease the proportion of EpCAM+cell in both SGC-7901and SGC-7901/VCR cell, following the increasing of the concentration. There were significant differences between every group in every cell (F=177.240, P=0.000)Conclusion1. Compared with parental cell line SGC-7901, drug-resistant cell line SGC-7901/VCR had multi-drug resistance phenotype, higher proliferation rate, stronger tumorigenic ability and the increased percentage of cancer stem cells. The underlying mechanism of multidrug resistance is related to overexpression of MDR1-Pgp and increased percentage of cancer stem cells.2. Salinomycin exerts its anti-cancer and resistance reversal effects in SGC-7901/VCR cells through inhibiting cancer stem cells, reducing expression of MDR1-Pgp and inducing apoptosis. |
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