The Inhibitive Effect Of Collaborative Hyperthermia And Cisplatin On Gastric Cancer Cell Line MGC-803 And To Investigative Its Mechanism

Objective: Cisplatin is a highly effective broad-spectrum anti-cancer drug and its efficacy is dose-related, but large doses can cause irreversible renal damage and severe gastrointestinal reactions, which limiting the clinical application and efficacy of the further improved. Hyperthermia in addition to the direct anti-cancer cells,but also with the sensitizing effect of chemotherapy, a combination of both can significantly improve the anti-tumor effect. This study was to explore the mechanism of proliferation inhibition of collaborative hyperthermia and cisplatin on gastric cancer cell line MGC-803, and there may be a preliminary discussion of the mechanism, so as to provid a strong basis for hyperthermia on the clinical application.Methods: 1 MGC-803 cell line were maintained in vitro, using MTT assay to detect the inhibitive effect rate among different cisplatin concentration groups (0.625,1.25,2.5,5,10,20μg/mL) , then to determine the concentration of cisplatin in the work and use the concentration to the experiment. 2 Using MTT assay to detect the survival rate of the control group and Low,medium,high concentrations of cisplatin (1.5,7,12μg/mL) and the corresponding thermo-chemotherapy group of cells. According to Weeb coefficient, we can decide whether the combination of hyperthermia and chemotherapy is synergy. 3 Experiment were randomly divided into four groups: control group, hyperthermia group, chemotherapy group, thermo- chemotherapy group, Select the inhibition rate of 30% of the drug concentration after 24 hours on the work, then the cell cycle distribution was detected by flow cytometry in each group after treatment. 4 Extracting total RNA of each group cell, assessing the integrality and content of RNA, the level of CyclinD1mRNA,PCNAmRNA expression was examined by semi-quantitative Reverse transcription polymerase chain reaction(RT-PCR) technique in the MGC-803 cells after treatment of different factors. 5 The expression of CyclinD1,PCNA protein was semi-quantitately examined by flow cytometry in MGC-803 cells in each group after treatment.Results: 1 MTT assay results: Within a certain concentration, different concentrations of DDP acting on the MGC-803 cells after 24 hours, then dose -effect positive linear correlation. Linear correlation analysis showed positive correlation with inhibition rate and drug concentration(r = 0.8551, P = 0.0300). Linear regression analysis find the regression equation of the DDP concentration (x) and the inhibition rate (y) was y = 0.1801 + 0.0444x. Choose the inhibition rate of 30% of the drug concentration after 24 hours as a working concentration and the concentration was 2.7μg/ml. 2 MTT assay results:As the cisplatin concentration increased, the cell survival rate of chemotherapy group and thermo-chemotherapy group decreased gradually, in addition the thermo-chemotherapy group was significantly lower compared to chemotherapy, the difference was statistically significant (P<0.05). Weeb coefficient used to determine the interaction of heat combined with chemotherapy, and results of the analysis showed that the high, medium and low concentrations of DDP combined with hyperthermia on the MGC-803 cell proliferation with a significant synergistic effect. 3 The results of flow cytometry detection of gastric cancer MGC-803 cell cycle distribution: hyperthermia group: compared with the control group, G0/G1 phase cells significantly increased, and S phase cells significantly reduced, the difference was statistically significant (P<0.05). chemotherapy group: compared with the control group, S-phase fraction decreased, and G0/G1 phase,G2/M phase cells significantly increased, the difference was statistically significant(P<0.05). thermo-chemotherapy group: compared to the other three groups, S phase cells significantly reduced, G0/G1 phase cells significantly increased, the difference was statistically significant (P<0.05). 4 RT-PCR detection results:CyclinD1: When compared with the control group, hyperthermia group, chemotherapy group and thermo-chemotherapy group were significantly reduced the expression of CyclinD1mRNA in volume, the difference was statistically significant (P<0.05). When thermo-chemotherapy group compared with chemotherapy group and hyperthermia group, the expression of CyclinD1mRNA were significantly reduced, the difference was statistically significant (P<0.05). PCNA: When compared with the control group, hyperthermia group, chemotherapy group and thermo-chemotherapy group were significantly reduced the expression of PCNAmRNA in volume, the difference was statistically significant (P<0.05). When thermo- chemotherapy group compared with chemotherapy group and hyperthermia group, the expression of PCNAmRNA were significantly reduced, the difference was statistically significant (P<0.05). 5 FCM assay results: CyclinD1 protein: When compared with the control group, hyperthermia group, chemotherapy group and thermo-chemotherapy group were significantly reduced the expression of CyclinD1 protein in volume, the difference was statistically significant (P<0.05). When thermo-chemotherapy group compared with chemotherapy group and hyperthermia group, the expression of CyclinD1 protein were significantly reduced, the difference was statistically significant (P<0.05). PCNA protein: When compared with the control group, hyperthermia group, chemotherapy group and thermo- chemotherapy group were significantly reduced the expression of PCNA protein in volume, the difference was statistically significant (P<0.05). When thermo-chemotherapy group compared with chemotherapy group and hyperthermia group, the expression of PCNA protein were significantly reduced, the difference was statistically significant (P<0.05).Conclusions:1 When low, medium and high concentrations of DDP Combined with hyperthermia acting on the MGC-803 cell, the inhibition of proliferation has significant synergistic effect.2 Thermo-chemotherapy changed the distribution of the cell cycle, and reducing the proliferation of cells in S phase distribution, blocking cells in S phase of DNA synthesis, extent that affecting the normal operation of the cell cycle, so that cell growth became slowed, and even apoptosis.3 Thermo-chemotherapy can reduce the expression of PCNA and CyclinD1 at the gene level and protein level. We can speculate further cell cycle arrest caused by changes in cycle-related proteins may inhibit the proliferation of MGC-803 cells, which was one of the mechanisms.4 This experiment confirmed that the combination of hyperthermia and cisplatin in vitro synergy gastric cancer MGC-803 cells significantly inhibited the proliferation, so as to clinical application of hyperthermia and chemotherapy together providing a reference in vitro.