The Effects Of Allicin Combinated With Oxaliplatin On The Growth Of Hct-8 Cell And Its Mechanisms

BackgroundThe incidence rate of colorectal cancer has a tendency of increasing recent years with the change of both life style and diet structure as well as the aging of population. The tumor tissue of more than 30% of colorectal cancer patients has metastasized when the disease is diagnosed. More than 50% of colorectal cancer will recur in five years after eradicative operation. Chemotherapy plays an important role in the comprehensive treatment of the recurrent or metastatic colorectal cancer. Oxaliplatin is the preferred chemotherapy medicine to cure colorectal cancer. But it will cause serious side reaction which is related with cumulative dose if we use Oxaliplatin for a long time. So it is very urgent to look for a kind of medicine that has little toxicity and side reaction and can add the sensitivity of tumor cells to the chemotherapy medicine. Allicin,which is a kind of sulfur-containing compound extracted from garlic bulbs , is famous known as the natural antibiotic drug with broad antibacterial spectrum .In addition to anti-inflammatory effects, recent reserches have showed that the allicin also has the effect of anti-cancer. Although there are reports about allicin combinated with paclitaxel, vincristine or cisplatin in cervical cancer, prostate cancer, esophageal cancer and gastric cancer cells, the reserch of the function of allicin combinated with Oxaliplatin on colorectal cancer is rare.ObjectiveThe objective of this study was to investigate the effect of allicin alone and combined with Oxaliplatin on the proliferation,apoptosis and cell cycle of human colorectal cancer cell line HCT-8.Materials and Methods1:The effects of proliferation inhibition of different concentrations of allicin and Oxaliplatin on the cell line of HCT-8 were evaluated by MTT assay when the cells were treated for 24,48,72 hours. And then calculated the half inhibitory concentration of 24h of allicin and Oxaliplatin on the HCT-8 cell.2 MTT assay was used to detect the effects of proliferation inhibition of allicin combined with Oxaliplatin on the HCT-8 cells 24h after the cells were treated with allicin and Oxaliplatin respectively and both of them. In this part of experiment, the cells were divided into 5 groups:the allicin group,the Oxaliplatin group,the combination group, the control group and the blank group.3. Inverted microscope was used to observe the morphological changes of cells before and 24, 48,72 h after the cells were treated with allicin and Oxaliplatin respectively and both of them.4.The rate of apoptosis and the change of the cell cycle of HCT-8 cells treated with allicin and Oxaliplatin respectively and both of them were detected by flow cytometry. HCT-8 cells were cultured with serum-free medium for 24h in order to reach the statement of synchronization of cell growth, followed by conventional culture. The cells were divided into following 4 groups: the allicin group, the Oxaliplatin group, the combination group and control group when the cells coverd 70% paries of the culture flask. Expriried culture 24h after treatment. Collected each group of cells .Some of the cells were used to detectapoptosis,while the other part of cells were fixed by cold ethanol to detect cell cycle.5 The protein expression of caspase-3 of HCT-8 cells after treated by allicin and Oxaliplatinwas studied by immunocytochemistry staining. If there are brown granules in cytoplasm and nucleus ,it is caspase-3 positive cell.Results1 .Allicin and Oxaliplatin displayed respectively the effection of proliferation inhibition on the HCT-8 cells, and the effection of proliferation inhibition increased as the increse of drug concentration and the extension of time . 2. Allicin and Oxaliplatin displayed a synergistic effect of proliferation inhibition on HCT-8 cells. The inhibition rate of proliferation of the combination group was higher than the sum of the inhibition rate of allicin group and Oxaliplatin group.3. Seen from the inverted microscope, cells expanded and showed anomalous polygon in the control group. As time went on,cell number increased gradually. In the allicin group and the Oxaliplatin group, with the extension of treatment time, cell number was less and less , and volume of cell was smaller. Vacuole could be seen in cytoplasm and suspension cells could be seen in the culture medium. In combination group , these changes were more obvious.. And suspension cells in the culture medium increased markedly.4.The detection results of flow cytometry showed that the percentage of cells of G₂/M phase increased,while that of G₀/G₁ phase decreased and that of S phase showed no significant change for both the allicin group and the combination group. As for the Oxaliplatin group,the distribution of cell cycle showed no significant difference from that of the control group.5. Compared with the control group, the apoptosis rate of the allicin group,the Oxaliplatin group and the combination group was higher. When compared with the allicin group and Oxaliplatin group respectively ,the apoptosis percentage of the combination group increased dramaticaly.6. Compared with the control group, the rate of the caspase-3 positive cells of the allicin group,the Oxaliplatin group and the combination group was higher. The apoptosis rate of the combination group increased when compared with the allicin group and Oxaliplatin group respectively.Conclusion1. Allicin could inhibit the proliferation of the HCT-8 cell lines.2. Allicin and Oxaliplatin displayed a synergistic effect of proliferation inhibition on HCT-8 cells. Garlicin could promote the apoptosis of the HCT-8 cell line.3. The apoptosis rate of the cells treated with both allicin and Oxaliplatin was significantly increased.4. Allicin blocked the cell cycle at the G₂/M phase and the percentage of the G₀/G₁ phase cells decreased while the percentage of the S period showed no significant change. 5. The application of allicin combinated with Oxaliplatin could reduce the dose of Oxaliplatin.