| Objective: China is a country with a high prevalence of gastric cancer. The gastric carcinoma in our country is with higher morbidity and mortality, lower positive rate in early. It seriously influences life and health for citizen. At present, the gastric cancer treatment is mainly by operation. But chemotherapy is the most important comprehensive treatment of post operation for advanced gastric cancer. However, the effect of chemotherapy on gastric cancer is limited, the toxic, and side effects bring serious consequences. Traditional Chinese medicine combination with chemotherapy drugs can improve the life quality, prolong the survival time, reinforce immune function and relief the myelosuppression it is superior to chemotherapy alone. Curcumol, is also known as turmeric ring the alcohol, hydrogenated compounds with hemiketal, and is isomerized by curdione which is a part of the traditional Chinese medicine biologics curcuma zedoary oil, which is studied under the action of heating or lewis acid catalysis. Several studies found that curcumol has obviously therapeutic effect for cervical cancer, ovarian cancer, stomach cancer, esophageal cancer, liver cancer and other cancers, and it also has obviously effect for antibacterial, antivirus, anti-liver fibrosis, promoting blood circulation to remove blood stasis, etc. So it is a good application value medicine for disease. But most studies for curcumol in gastric cancer are still focused on the in-vitro cell culture experiments, the study for the in-vitro antitumor effects relatively rare, and its mechanism is not yet very clear.The aim of our trial is to study the effect of curcumol combined with oxaliplatin on the apoptosis of human gastric cancer cell line BGC-823 xenograft transplanted in nude mice on the growth and the impact of Caspase-3 and Bcl-2 expression, and to investigate its molecular mechanisms.Methods:1 The models of the human gastric cancer cell line BGC-823 xenograft transplanted in nude mice were established.Chose two male nude mice, four to five weeks old.Breeding them under the environment of SPF. Inoculated the human gastric cancer BGC-823 cells which were in the logarithmic phase in nude mice right axillary subcutaneous to establish human gastric cancer xenograft transplanted in nude mice.Every two days measuring the length of the subcutaneous tumor diameter when the subcutaneous tumor appeared. When the tumor diameter reached average of 10 mm, then removed and cut into the size of about 1mm3. Then inoculated it into the twenty male nude mice right axillary subcutaneously, four to five weeks old. Similarly, every two days measuring the length of the subcutaneous tumor diameter when the subcutaneous tumor appeared. After the tumor diameter reached average of 10 mm, it indicated that the models of the human gastric cancer cell line BGC823 xenograft transplanted in nude mice were established.2 Set up experimental group and drug intervention. Twenty nude mice models were randomly divided into control group(n=5),curcumol group(100mg/kg;n=5),Oxaliplatin group(30mg/kg;n=5) and curcumol combined with Oxaliplatin group( curcumol 100mg/kg, Oxaliplatin 30mg/kg;n=5).The drug was deliveryed for four times and once every four days. And at the same time the control group took sterile distilled water.Every four days, the body weight and tumor volume of the tumor-bearing nude mice were observed closely. The seventeenth day the tumor-bearing nude mice were executed by cervical dislocation and the whole tumor was cut off. Stored them respectively,according to the requirements of subsequent experiments.3 Statistics and analysis. Calculated the tumor volume and then draw the curve according to the tumor volume.Weighed the tumor weight and calculated anti-tumor rate. Determined by MTT colorimetric method to explore the growth of tumor cells in nude mice in vivo. By using immunohistochemistry and fluorescence quantitative RT-PCR to detect the cysteinyl aspartate specific proteinase-3(Caspase-3), the B-cell lymphoma-2(Bcl-2) protein expression and m RNA and calculat the difference between groups. The data were presented with mean and standard deviation, analyzed with One-way ANOVA and chi-square test by Statistic software of SPSS 21.0 edition, P<0.05 was considered statistically significant.Results:1 The growth curve and the change of the xenograft transplanted tumor in tumor-bearing nude mice.There was no statistically significant difference of tumor volume before each group nude mice were deliveryed(P>0.05).From the first deliverying, the tumor volume was measured every four days. In the seventeenth day, the average tumor volume among curcumol group, Oxaliplatin group and curcumol combined with Oxaliplatin group were smaller than control group, it has a statistically significant difference(P<0.05). The average tumor volume of curcumol group was larger than Oxaliplatin group,it has a statistically significant difference(P<0.05). The average tumor volume of curcumol group and Oxaliplatin group were larger than curcumol combined with Oxaliplatin group,it has a statistically significant difference(P<0.05).2 The tumor weight and tumor inhibition rate of the tumor-bearing nude mice.In the seventeenth day the tumor-bearing nude mice were executed by cervical Dislocation, and the whole tumor was cut off. Weighed the tumor weight and calculated anti-tumor rate. The tumor weight of curcumol group,Oxaliplatin group and curcumol combined with Oxaliplatin group were lighter than control group, it has a statistically significant difference(P<0.05).The tumor weight of curcumol group compared to Oxaliplatin group has not a statistically significant difference(P>0.05). The tumor weight of curcumol group and Oxaliplatin group were heavier than curcumol combined with Oxaliplatin group, it has a statistically significant difference(P<0.05).3 The effect of curcumol combined with Oxaliplatin on the growth of the nude mice transplantation tumor cells.Determined by MTT colorimetric method, it is explored the growth of tumor cells treated with medicine in nude mice in vivo. The OD values of curcumol group was lower than control group in 24 h, 48 h, 72 h respectively,it has a statistically significant difference(P<0.05).The cell survival rate of curcumol group, Oxaliplatin group and curcumol combined with Oxaliplatin group were lower than control group in 24 h,48h and 72 h respectively, it has a statistically significant difference( P < 0.05). The effect of growth inhibition of cell in Combination group was more obviously than curcumol group, and it has a statistically significant difference in 24h(P<0.05). But the effect of growth inhibition of cell in Combination group compared to Oxaliplatin group has not a statistically significant difference in 24h(P>0.05). The effect of growth inhibition of cell in Combination group were more obviously than monotherapy group, and it has a statistically significant difference in 48 h and 72h(P<0.05).4 The Bcl-2 and Caspass-3 m RNA expression of tumor in nude mice were detected by fluorescent real-time PCR method.The Caspass-3 m RNA expression in curcumol group,Oxaliplatin group and curcumol combined with Oxaliplatin group were higher than control group, it has a statistically significant difference(P<0.01).The Bcl-2 m RNA expression in curcumol group,Oxaliplatin group and curcumol combined with Oxaliplatin group were lower than control group, it has a statistically significant difference(P<0.01).5 The Bcl-2 and Caspass-3 protein expression of tumor in nude mice were detected by immunohistochemistry method.The immunohistochemical study revealed that the positive expression of Bcl-2 is cytoplasm and cell membrane was dyed to a yellowish-brown, and the positive expression of Caspase-3 is cytoplasm was dyed to a yellowish- brown.The results showed that the strong positive rates of Bcl-2 in control group,curcumol group,Oxaliplatin group and curcumol combined with Oxaliplatin group were 86.67%, 23.33%, 25.00% and 13.33% respectively.The strong positive rates of Caspase-3 in group,curcumol group,Oxaliplatin group and curcumol combined with Oxaliplatin group were 26.67%, 66.67%, 86.67% and 93.33%respectively.The Bcl-2 protein expression in the curcumol group,Oxaliplatin group and curcumol combined with Oxaliplatin group was lower than control groups(P<0.05). The Caspase-3 protein expression of the curcumol group,Oxaliplatin group and curcumol combined with Oxaliplatin group was higher than control groups(P<0.05).Conclusion:1 The curcumol,Oxaliplatin and curcumol combined with Oxaliplatin can inhibit the growth of the human gastric cancer cell line BGC-803 xenogratft transplanted in nude mice. The average tumor volume and the tumor weight of curcumol group and Oxaliplatin group were larger than curcumol combined with Oxaliplatin group. The curcumol can inhance the growth inhibition of gastric cancer induced by oxaliplatin.2 The curcumol combined with oxaliplatin can enhance the inhibitory effect of oxaliplatin into gastric cancer cell line BGC-823 xenogratft transplanted in nude mice.3 The mechanism of the curcumol combined with oxaliplatin induced apoptosis may be related to up-regulation of caspase-3 and down-regulation of Bcl-2. |
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