Experimental Research On The Effects Of The Combination Treatment Of Rapamycin And Doxorubicin To The Proliferation And Migration Of Hepatoma Cell

The incidence of Primary Liver Cancer ranked fifth at the global level.China have the highest incidence in the world.Chemotherapy is one of the most important means of Hepatic Carcinoma Patients'Comprehensive Treatment.However, long-term's application of Chemotherapy Drugs have produced obvious tolerance. Hematologic toxicity and gastrointestinal complications are more pronounced. Therefore, how to improve chemotherapy sensitivity have become the heated concern. Rapamycin is 36-member ring macrolide antibiotic,which is found have low Side-effect ,and it is widely used for anti-rejection reaction after organ transplantation.Recently, it is found that Rapamycin can induce tumor cell apoptosis, meanwhile it also can inhibit tumor cell proliferation and migration.Furthermore, Combination of Rapamycin and traditional chemotherapy drugs have synergistic effect. This news has bing glad tidings to largeness patients with Hepatocellular Carcinoma.In this study, we found that Hepatoma Cell line BEL-7402 was significantly sensitized by using Rapamycin in combination with Doxorubicin. First of all, we detected the inhibition rate of Doxorubicin to Hepatoma Cell line BEL-7402 by MTT ASSAY. After treated with 0.125, 0.25, 0.5, 1 and 2mg/L Doxorubicin for 48h, the inhibition rate of Doxorubicin to Hepatoma Cell line BEL-7402 were 12.83%,29.51%,57.41%,67.38%and77.03%, and after treated with 7000, 8000, 9000, 10000, 11000and12000nmol/L Rapamycin for 48h, the inhibition rate of Rapamycin to Hepatoma Cell line BEL-7402 were 5.32%, 12.00%, 25.47%, 34.63%, 78.21%and95%. Then the effective dose of Rapamycin (10000nmol/L) and effective dose of Doxorubicin (0.25mg/L) were combined used to Hepatoma Cell line BEL-7402, we detected the absorbance change during six days. Cell growth curve of the six days showed that the combined group was suppressed more significantly than single medication group. Besides, the different concentration of rapamycin (8000, 9000, 10000nmol/L) and different Concentration of Doxorubicin (0.125, 0.25, 0.5, 1and2mg/L) are combined used to Hepatoma Cell line BEL-7402 for 48h,we detected the inhibition rate,then drew the histogram ,evaluating the interaction of the two agents by CDI (coefficient of drug interaction). This further proved the combination treatment of two agents can improve sensitivity of Doxorubicin to Hepatoma Cell line BEL-7402. CDI analysis showed that low dose combination of Rapamycin and Doxorubicin have synergistic effect (CDI<1). A significant difference between the combined group and single medication group (P<0.05).In order to clear the proliferation and migration ability changes of BEL-7402 cells, we apply RT-PCR analysis of different treatment groups cyclind1mRNA and MMP-2mRNA, the expression results showed that the expression of cyclind1mRNA and MMP-2mRNA were most obviously decreased in the combined group. That is the combination of the two agents can significantly inhibit the transcription of cyclind1gene and MMP-2gene.Cyclind1is a kind of vital protein which is related with cell cycle regulation. cyclinD1 binds CDK4 or CDK6 in G1 period,then it combines with pRb by n-terminal motif LXCXE, this makes serine residue and tyrosine residue of pRb phosphorylate, so that transcription factor E2F is released. This process begins to promote some vital gene Transcription. This finally drives cell cycle progression and cell proliferation. We applied Western Blot to analysing the expression of cyclind1 and mTOR protein of different treatment groups,expression results showed that cyclind1 and mTOR protein decreased significantly. we can presume that the reason why the combination of rapamycin and Doxorubicin decreased the expression of cyclind1 protein is because the combind group can inactivate the mTOR protein.Based on the above experimental results, we can reach the following conclusions, The combination of the two agents can improve chemotherapy sensitivity of Doxorubicin to Hepatoma Cell line BEL-7402. The combined application have synergistic effect on inhibiting the proliferation and migration of hepatoma cell.The mechanism lies in the combination application can inhibit the transcription of MMP-2 and cyclind1 gene and the expression of mTOR protein and cyclind1 protein. The results of this study verified the finding that the synergistic effect of Rapamycin and traditional chemotherapy drugs exists. The research provide a theoretical basis for the final combination application of the two agents to clinical treatment of Hepatocellular Carcinoma.