Study On TRAIL On Expression Of Multidrug Resista Genes In SGC7901/VCR And TRAIL Plus Cisplatin Killing SGC7901/VCR

BackgroundRemains high incidence of gastric cancer, the diagnosis and treatment of gastric cancer is still one of the challenges faced in the cancer diagnosis and treatment. Diagnosis and treatment of early gastric cancer,10-year survival rate of nearly90%, but the domestic early detection of gastric cancer is still low. Already in the middle and late diagnosis of the majority of patients with gastric cancer and had to undergo chemotherapy which for many years a important method of treatment of gastric cancer. But a frustrating problem in cancer chemotherapy patients is on chemotherapy drug resistance problem, also known as multi-drug resistant (multidrug resistance, MDR). Therefore, the phenomenon of drug resistant gastric cancer as the research direction, exploring its internal mechanism and looking for intervention against drug-resistant tumor cells was of great value, and lay the foundation for the screening of drugs against resistant or reversal of drug resistance.The previous series of studies have shown that resistance genes such as LRP. GST-π high expression was positively correlated with COX-2in human gastric cancer cells. The equally resistant gastric cancer cell lines in vitro SGC7901/VCR also was observed a similar phenomenon. With COX-2inhibitors celecoxib down this expression, confirmed that COX-2activity relationship of high expression of the resistance genes, at the same time show that the intervention of foreign material from the resistant gene is effective. Tumor necrosis factor-related apoptosis-inducing ligand on normal cells almost no effect, but can induce apoptosis in some types of tumor cells, while also direct killing effect on tumor cells. Recently found that some of the phenomena that TRAIL has increased the effectiveness of chemotherapy drugs kill tumor cells,that the tumor cells become more sensitive to chemotherapeutic drugs. TRAIL mechanism to increase the effectiveness of chemotherapy drugs killing tumor cells lie? Is the classic role of the pro-apoptotic effect on tumor cell, and the synergistic effect of the antineoplastic agents or by inhibiting the expression of the resistance genes of the tumor cells thereby reversing the drug resistance of the tumor cells? By the results of our previous studies, we assume that the tumor cells by inhibiting tumor cell resistance gene leave to become sensitive to chemotherapeutic drugs. If this assumption is true, it will provide good prospects solve troubled by stomach cancer chemotherapy drug problem.This study is divided into two parts:Part Ⅰ:TRAIL resistant gastric cancer cell lines SGC7901/VCR common culture, to explore the effect of TRAIL on the expression of MDR1, LRP and GST-π in drug-resistant gastric cancer Cell SGC7901/VCR; Part Ⅱ:Explore sub-toxic doses of tumor necrosis factor-related apoptosis ligand (TRAIL) in combination with low-dose chemotherapy drug cisplatin (DDP) on gastric cancer cell lines SGC-7901/VCR activity, apoptosis rate and multidrug resistance gene1(MDR1) expression.Part Ⅰ:The effect of TRAIL on the expression of multidrug resistant genes MDR1, LRP and GST-π in drug-resistant gastric cancer Cell SGC7901/VCRObjective:Research TRAIL on gastric cancer cell line SGC7901/VCR multidrug resistance gene MDR1, LRP and GST-π expression of, and to explore the possible mechanism of action. The research applications of TRAIL to provide experimental evidence to resolve the multidrug resistance of gastric cancer cells.Method:Resistant gastric cancer cell line SGC7901/VCR with different concentrations of TRAIL cultured48h, no dosing cell line as a control, RT-PCR was used to detect the groups resistant gastric cancer cell line resistance gene MDR1, LRP, GST-πimRNA expression.ELISA was used to detect the three types of resistance protein P-gp, LRP, GST-π content of resistant gastric cancer cell lines, t-test and one-way ANOVA statistical methods for analysis. The purpose is to understand the TRAIL on resistant gastric cancer cell line expression of multidrug resistance gene.Results:1. RT-PCR results:The control group three resistant gastric cancer cell line resistant gene MDR1, LRP, GST-π were positive and mRNA values were0.88±0.01,0.91±0.04and1.01±0.13. With TRAIL action, gastric resistant cell line of MDR1, LRP, GST-π mRNA expression was inhibited, and with the increase of concentration, inhibited stronger, but did not show a linear relationship. The TRAIL group MDR1mRNA value in four concentrations of the experimental group than in the control group was significantly lower (0.78±0.02;0.69±0.05;0.55±0.01and0.53±0.01,respectively, all four groups are p<0.01). With increasing concentrations of TRAIL, MDR1mRNA be suppressed amplitude increasing trend, showed a significant difference (P<0.01) among the respective groups. But when the concentration was increased to400μg/L and200μg/L, inhibition of MDR1mRNA amplitude contrast, there was no significant difference (p>0.05). Similarly, the TRAIL-group LRPmRNA value in the experimental group of four concentrations significantly lower than those of the control group, the values were0.85±0.05;0.76±0.05;0.59±0.04and0.58±0.02, all four groups p<0.01. With TRAIL concentration of50μg/L,100μg/L.200μg/L increments. LRPmRNA decreases in each concentration groups showed a significant differences(P<0.01). But when the concentration was increased to400μg/L, the inhibition of LRPmRNA compared with200μg/L, no significant difference (p>0.05). Performance similar to the first two: The TRAIL-group GST-πmRNA value in the experimental group of four concentrations significantly lower than those of the control group (respectively0.89±0.04,0.77±0.08,0.65±0.06and0.61±0.03, p <0.01). With the TRAIL concentration of50ug/L,100μg/L,200ug/L increments, GST-π mRNA was inhibited more significantl and showed a significant difference among various concentrations (P <0.01). But when the concentration was increased to400μg/L and200μg/L, contrast, there was no significant difference inhibition of GST-πmRNA amplitude (p>0.05).2. ELISA results:P-gp, LRP, GST-π protein expression in the control group were49.04±2.07ug/L,119.35±1.04ug/L and58.62±1.38ug/L. Compared with the control group, the concentration of50ug/L of TRAIL group of P-gp, LRP and GST-π content were40.42±1.89ug/L,112.51±19ug/L and57.56±1.19ug/L (all p <0.01). Concentration of100ug/L of TRAIL group of P-gp, LRP and GST-71content were36.49±0.94ug/L,106.69±1.51ug/L,52.30±0.80ug/L and also significantly lower than the control group (p <0.01). At the same time the concentration of100ug/L TRAIL group compared with50ug/L TRAIL group was also significantly decreased (p <0.01). For200ug/L TRAIL group, P-gp, LRP and GST-π values were25.15±0.69ug/L,83.54±2.15ug/L and31.41±1.65ug/L. They were significantly lowered than the control group (p <0.01). P-gp, LRP and GST-π values in200ug/L TRAIL group was significantly lower than in100ug/L TRAIL group also (p <0.01). P-gp, LRP and GST-π levels in400ug/L TRAIL group were24.45±1.41ug/L.82.63±1.35ug/L.30.80±1.34ug/L respectively and they were significantly lowered than in the control group,50ug/L TRAIL group and100ug/L TRAIL (p <0.01;). However, there was no significant difference of P-gp, LRP and GST-π’s values between400ug/L TRAIL group and200ug/L TRAIL group(p>0.05).Conclusion: 1. The gastric cancer resistance gene MDR1, LRP, GST-π in gastric cancer resistant cell line SGC7901/VCR expression.2. Four concentrations of TRAIL intervention, SGC7901/VCR resistance gene MDR1,LRP and GST-π expression decreased and their decrease amplitudes were related to the TRAIL concentrations.3. In addition to promoting apoptosis of tumor cells, TRAIL may also reduce MDR1, LRP, GST-πexpressions of tumors resistant cell line SGC7901/VCR and enhanced the efficacy of chemotherapy drugs. Part II:Effect of TRAIL in combination with DDP on the expression of MDR1gene in gastric cancer cellsObjective:To study the effect of tumor necrosis factor related apoptosis inducing ligand (TRAIL) combined with chemotherapeutic drug cisplatin(DDP) on activity, apoptosis rate, and expression of multidrug resistance gene MDR1in the gastric cancer cell line SGC-7901/VCR.Methods:SGC-7901/VCR cells were cultured with DDP and TRAIL in different concentrations. The apoptosis rate was separately measured by a flow cytometer in DDP (sub-toxic dose) alone, TRAIL ((200μg/L) alone and in the combination of the two. Expression level of MDR1mRNA and P-gp protein were detected by RT-PCR and ELISA analysis, respectively.Results:The apoptosis rate in combination group was significantly higher than that in the other groups (P<0.05). According to the results of RT-PCR and ELISA, the expression of MDR1mRNA and P-gp protein of combination group were statistically significant different compared with other groups (P<0.05).Conclusion:1. MDR1high expression in resistant gastric cancer cell line SGC7901/VCR MDR1resistance gene indicated that MDR1was resistance gene in gastric cancer With relevant secondary multidrug resistance.2. TRAIL can enhance the effect of DDP in anticancer therapy. Cisplatin and TRAIL synergistic down MDRlgene expression.3. The down-regulate of multidrug resistance gene MDR1may play a potential role in overcoming the chemotherapeutic resistance of gastric cancer cells.