| Gastric cancer is one of the most common cancers in our country.Chemotherapy play an important role in the therapy of gastric cancer especially advanced gastric cancer.However,gastric carcinoma chemotherapy has the natural drug tolerance.To improve the effect and reduce the toxicity,lots of research have been studied, including combined chemotherapy suitably,and progress have been made continually.Platinum is cycle-non-specific antitumoral drug,and DNA is its effect target spot. The platinum is hit by the chlorine dissociation in low chlorine environment.Platinum induced cell apoptosis through the combination of biological macromolecules such as DNA chain.Since the discovery of that cisplatin can inhibit the activity of the growth of tumor cells in 1967,the platinum-based drugs' research and their clinical applications in cancer therapy are developing rapidly,and now they have became indispensable clinical first-line chemotherapies.At present,the commonly used platinum-based drugs are cisplatin(DDP),carboplatin(CBP) and oxaliplatin(OXA). The clinical dose of DDP is subject to certain restrictions because of its serious ear-and-nephrotoxicity,so that a new type of platinum anti-cancer drug(OXA) is developed to affect the efficacy and low toxicity.1,2-diamino cyclohexane(DACH) of OXA is used to replace NH4 amino of DDP.The mechanism of oxaliplatin is the same with cisplatin,but its anti-tumor activity is higher than cisplatin,and it is also useful to cisplatin-resistant tumor cells.The major dose-limiting toxicity of oxaliplatin is peripheral nerve toxicity,without oto-renal toxicity.Superposition of their side effects,it is worth further research to explore the joint application of oxaliplatin and cisplatin to play a synergistic clinical effect program.It has been demonstrated by several clinical trials that the combination of carboplatin and oxaliplatin is effective against some of malignant tumors,including gastric and esophageal cancer,lung cancer,ovarian cancer,et al.Today there are some study on the combination of oxaliplatin and cisplatin against esophageal and gastroesophageal cancer,ovarian cancer,germ cell tumors,et al.Howerver,study on the combination of oxaliplatin and cisplatin in gastric cancer is rarely published. Oxaliplatin,cisplatin and 5-fluorouracil are critical component in most chemotherapy regimens for gastric cancer,PF is the basis of first-line treatment of gastric cancer,but their toxicity limit their high dose clinical use.To design combined therapeutic regimens with better antineoplastic activity and/or less toxicity,a trial were performed to observed the antineoplasic activity of the combined chemotherapy with oxaliplatin and cisplatin to evaluated the value of the regimens containing OXA/DDP.We expect that this research could provide the theoretical basis for clinical therapy of gastric carcinoma.Methods:1.Human gastric tumor cell line was used in this study and cultured with DMEM and 10%calf serum and antibiotics.2.Drugs included oxaliplatin(OXA),cisplatin(DDP) and 5-fluorouracil(5-FU).3.MTT assay was used to examine the suppressive rate of cell growth dealt with different concentration of OXA alone and in combination,and dose-response curves for proliferation inhibition were drew according to MTT data at 48h.4.The development of cell cycle induced by OXA alone and in combination was examined by flow cytometry at 12,24,48h.5.Bcl-2,Bax,Caspase-3 protein expression was detected by immunochemistry assay.6.The data of variance-samples was performed by one-way analysis,and paired-samples with T test,using SPSS 13.0.Statistics were expressed as(?)±S. In all statistical analyses,P≤0.05 was considered statistically significant.Results:1.MTT assay showed that various concentration of OXA single could effectively inhibit the growth of human gastric carcinoma cell line,compared all of the management groups with control groups,the difference is significant(P＜0.05). The results suggested that treatment of human gastric carcinoma cell line with various concentrations of OXA alone resulted in a concentration-dependent increase in the suppressive rate of growth of cell line.2.The project of OXA/DDP could also effectively inhibit the growth of human gastric carcinoma cell line in vitro,compared with all of the management groups with control groups(P＜0.05).The results suggested that treatment of human gastric carcinoma cell line with various concentrations of OXA/DDP resulted in a concentration-dependent increase in the suppressive rate of growth of cell line. More obvious cellular proliferation inhibition induced by dealing with OXA/DDP treatment compared with OXA alone.The value of IC50 of OXA in OXA/DDP regime was lower than OXA alone.3.The project of OXA/DDP/5-FU could also effectively inhibit the growth of human gastric carcinoma cell line in vitro,compared with all of the management groups with control groups(P＜0.05).The results suggested that treatment of human gastric carcinoma cell line with various concentrations of OXA/DDP/5-FU resulted in a concentration-dependent increase in the suppressive rate of growth of cell line.More obvious cellular proliferation inhibition induced by dealing with OXA/DDP/5-FU treatment compared with either OXA alone or OXA/DDP combination.The value of IC50 of OXA in OXA/DDP/5-FU regime was lower than that of OXA alone and OXA/DDP combination.4.Dose-response curves for proliferation inhibition show that various concentration of OXA alone,OXA/DDP and OXA/DDP/5-FU inhibit the growth of human gastric carcinoma cell line at 48h.The results suggested that treatment of human gastric carcinoma cell line with various concentrations of OXA alone,OXA/DDP and OXA/DDP/5-FU resulted in a dose-dependent inhibition of cell proliferation. The sequence of OXA single and combined chemotherapy in intensity of effect is OXA/DDP/5-FU＞OXA/DDP＞OXA.5.Cell cycle distribution of human gastric carcinoma cell infected with OXA alone, OXA/DDP and OXA/DDP/5-FU were assayed by flow cytometry:After exposed to OXA,the distribution of S phase gradually increased,and the percent of S phase was(21.40±0.46)%,(46.30±1.68)%,(64.93±0.68)%at 12,24,48 hour, respectively.Combined administration of OXA and DDP remarkably arrested in S, and cells in G2/M phase significantly decreased,and the percent of S phase was (31.67±0.72)%,(49.73±0.76)%,(81.80±0.96)%at 12,24,48 hour,respectively. Combined administration of OXA,DDP and 5-FU remarkably arrested in S at 12, 24 hour,and the percent of S phase was(44.87±1.59)%,(58.60±0.85)% respectively.The percent of S phase was remarkably decreased(37.73±0.71)%at 48 hour.6.By immunochemistry,cultures of the human gastric carcinoma cell line were dealed with OXA alone,OXA/DDP and OXA/DDP/5-FU 48h later,the Bcl-2, Bax,Caspase-3 protein was expressed in BGC-823 cell line as shown by immunochemistry staining.6.1 Bcl-2 expression.After exposed to the drugs,Bcl-2 protein expression ratio of OXA group was(52.46±2.07)%lower than that of control group(73.73±2.24) %(P＜0.01).OXA/DDP management group was(37.44±2.42)%lower than that of control group and OXA alone(P＜0.01).OXA/DDP/5-FU management group was(21.91±2.38)%lower than that of control group,OXA alone management group and OXA/DDP management group(P＜0.01).6.2 Bax expression.After exposed to the drugs,Bax protein expression ratio of OXA group was(40.21±0.93)%higher than that of control group(19.91±0.70)% (P＜0.01).OXA/DDP management group was(56.36±0.67)%higher than that of control group and OXA group(P＜0.01).OXA/DDP/5-FU management group was (75.88±0.58)%higher than that of control group,OXA group and OXA/DDP management group(P＜0.01).6.3 Caspase-3 expression:After exposed to the drugs,Caspase-3 protein expression ratio of OXA group was(22.20±1.18)%higher than that of control group(9.45±1.63)%(P＜0.01).OXA/DDP management group was(40.91±1.58)%higher than that of control group and OXA alone management group(P＜0.01). OXA/DDP/5-FU management group was(65.68±1.57)%higher than that of control group,OXA alone management group and OXA/DDP management group(P＜0.01).Conclusion1.The growth of BGC-823 cell can be inhibited by OXA,OXA/DDP and OXA/DDP/5-Fu all,the suppressive rate of growth is dose-dependent.The value of IC50 of OXA was lower in combined regime than single chemical drug.2.Cell cycle was arrested in S phase exposed to OXA and OXA/DDP.When exposed to OXA/DDP/5-Fu,cell cycle was strongly arrested in S phase at 12h, 24h.At 48h the percent of S phase was remarkably decreased.3.Immunochemistry showed that OXA alone could down-regulated the express rate of Bcl-2 protein,and up-regulated the express rate of Bax and Caspase-3 protein after exposed to OXA 48h.It also well be shown that the regulation was strengthened when the cell exposed to OXA/DDP and OXA/DDP/5-FU 48h via the mitochondrial apoptosis pathway... |
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