| Objective: To explore whether Rosiglitazone(RSG), a selective Peroxisome proliferator-activated receptor gamma (PPARγ) ligand, can reverse the resistance to Mitomycin (MMC) of human gastric cancer SGC7901/VCR cell line which has multidrug resistant phenotype, and further to explore the possible mechanism of RSG reserving multidrug resistance.Methods: There are blank control group,MMC group,RSG group, CSA+ MMC group and RSG+MMC group, with different concentrations of RSG and MMC affecting SGC7901/VCR and SGC7901 cell lines, using: (1) The influence of RSG on human gastric cancer SGC7901/VCR cell line which had multidrug resistant phenotype and its parental generation SGC7901 cell line was observed with the analysis of cell growth curve; (2) MTT assay and clone formation rate were used to determine the influence of RSG on MMC-inhibited proliferation of the two human gastric cell lines which mentioned above; (3) The influence of RSG on MMC-induced apoptosis was determined by flow cytometry and AO-EB fluorescent staining; (4) Cell cycle was determined by flow cytometry; (5) The expressions of multidrug resistance related genes MDR1,MRP and apoptotic related gene Livin were measured by RT-PCR and western-blot, and undertaken semi quantitative analysis.Results: (1) RSG at 20,40,80,160μM had inhibitory effects on the growths of human gastric cancer SGC7901/VCR cell line and its parental generation SGC7901 cell line, but 20μmol/L RSG group and blank control group had no statistical significance (P＞0.05), while 40, 80, 160μM RSG groups had statistical significance with control group(P＜0.05), and expressing a dose-dependent relationship; (2) The IC50 of MMC to human gastric cancer SGC7901/VCR cell line and its parental generation SGC7901 cell line are 9.1mg·L-1 and 0.73mg·L-1 respectively, the multiple of drug resistance is 12.4, the two groups had statistical significance(P＜0.05), illustrating SGC7901/VCR cell line had drug resistance to MMC; The IC50 of RSG to SGC7901/VCR cell line and SGC7901 cell line are75.9μM and 58.4μM respectively, the IC50 between MMC and RSG had no statistical significance (P＞0.05); illustrating SGC7901/VCR cell line had no drug resistance to RSG. (3) In RSG+MMC group, when the concentration of RSG increased from 0μM to 80μM progressively, the IC50 of MMC to SGC7901/VCR cell line decreased from 9.1mg·L-1 to 0.87mg·L-1, the reserving multiple of RSG at 20,40,80μM to SGC7901/VCR cell line are 2.84,9.6,10.5 respectively, the difference had obvious statistical significance compared with blank control,MMC and RSG group(P＜0.01). The effect of reversing SGC7901/VCR drug resistance to MMC between RSG at 40μM and CSA at 4μg/L is familiar (P＞0.05), illustrating that RSG can reverse the resistance to MMC of the human gastric cancer SGC7901/VCR cell line, expressing an obvious dose-dependent relationship. (4) Clone formation rate indicated: the difference of colon formation rate between RSG(40μM)+MMC(1mg·L-1) and MMC(1mg·L-1),RSG(40μM) had statistical significance(P＜0.01), but it had similar effects with CSA(4mg/L)+ MMC(1mg·L-1) group (P＞0.05), illustrating that RSG combined MMC can decrease the colon formation rate of SGC7901/VCR. (5) The results of apoptosis between AO-EB fluorescent staining and flow cytometry is coincident, RSG+MMC group can increase the apoptotic rate of SGC7901/VCR cell line, compared with blank control,RSG and MMC groups, the difference had obvious statistical significance (P＜0.01), compared with CSA+MMC group the difference had statistical significance(P＜0.05), illustrating that RSG combined MMC can enhance the apoptosis of MMC induced in SGC7901/VCR cell line; RSG enhanced the G1-phase percents of gastric cancer SGC7901/VCR cell line and SGC7901 cell line obviously, indicating that RSG arrested cell growth in G1 phase. (6) RT-PCR and Western-blot indicated:①The expression of PPARγ, mRNA and PPARγ, protein of SGC7901/VCR cell line in RSG and RSG+MMC groups is increased than RSG,MMC and CSA groups, the difference had statistical significance(P＜0.05), RSG can increase the expression of PPARγ, mRNA and PPARγ, protein in SGC7901/VCR cell line;②Compared blank control group to SGC7901 cell line, the expression of MDR1 and Living mRNA all enhanced obviously(P＜0.01), showing that the expression of MDR1 and Livin mRNA in SGC7901/VCR cell line is stronger than in SGC7901 cell line;③The expression of MRP had no obvious difference between SGC7901/VCR cell line and SGC7901 cell line (P＞0.05);④The expression of MDR1 mRNA, P-gp and Livin mRNA of SGC7901/VCR cell line in RSG and RSG+MMC groups decreased than blank control and MMC groups, the difference had statistical significance(P＜0.05), but compared with CSA + MMC group, the difference had no statistical significance (P＞0.05), illustrating that RSG and CSA all can inhibit the expression of MDR1 and Livin in SGC7901/VCR cell line. (8) RSG at 40μM can down-regulate the expression of MDR1 mRNA, P-gp and Livin mRNA in SGC7901/VCR cell line, compared with blank control and MMC groups, the difference had statistical significance (P＜0.01), but compared with CSA+MMC group, the difference had no statistical significance(P＞0.05).Conclusions: (1) RSG inhibits the growth of gastric cancer SGC7901/VCR cell line and SGC7901 cell line at 40μM,80μM,160μM; And has no drug resistance in SGC7901/VCR cell line. (2) RSG can reserve the drug resistance of SGC7901/VCR cell line to MMC partly. (3) RSG can induce the apoptosis of SGC7901/VCR cell line, and has G1-phase arresting effect. (4) The over expression of multidrug resistance gene MDR1 and apoptotic inhibited gene Livin is a possible reason of multidrug resistance of SGC7901/VCR. cell line. (5) RSG can down-regulate the expression of MDR1 and Livin, this maybe an important mechanism of RSG reversing multidrug resistance of SGC7901/VCR cell line partly. |
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