Study Of Ethanol Extract From Radix Of Actinidia Chinensis On Proliferation And Apoptosis In Colorectal Cancer Cell

Objective:Actinidiae is commonly used in clinical treatment of colorectal cancer of Chinese herbal medicine, its anti-tumor effect of pharmacology and molecular studies confirmed a lack of research, but for the colorectal cancer tumor suppressor mechanism, and therapeutic targets. In order to clarify Actinidiae extracts (Ethanol Extract from Radix of Actinidia Chinensis, EERAC)on apoptosis-related genes in colon cancer cells during apoptosis Bcl-2and Bax in the impact of caspase-3and proliferation inhibition effect, whereby understand Actinidiae extract some of the mechanisms of colorectal cancer anti-cancer dose-effect relationship, to carry out this study.Methods:1. In vitro experiment:the patent alcohol extraction process to extract Actinidiae anticancer effective active ingredient (EERAC), respectively dubbed10μg/ml,40μg/ml,160μg/ml,and320μg/ml concentration, and LoVo were cultured in vitro,the different concentrations of EERAC, and the following experiment (1) using the inverted microscope to observe the different EERAC on LoVo cell morphology changes, and chromatin changes within the two kinds of fluorescence staining nuclei by AO/EB and Hoechst33258nuclear condensation, membrane shrinkage, apoptotic bodies;(2) using different concentrations of MTT assay EERAC inhibition of LoVo cells;(3) using agarose gel electrophoresis observation EERAC in various in vitro experimental groupin the formation DNAladder LoVo cells;(4) using flow cytometry of determination EERAC on LoVo cell cycle;(5) immunochemistry (IHC) determination of the related genes Bel-different concentrations EERAC on apoptosis of LoVo cells2, Bax, Caspase-3expression levels;2In vivo experiments:the establishment of colorectal cancer cells HT-29tumor-bearing mouse model, observed:(1) different doses (5mg/kg,10mg/kg,20mg/kg) EERAC weight of tumor-bearing mice implanted tumor growth inhibitory effect on the blood, liver and renal function, immune organs;(2) and LDH release method deeterminate EERAC tumor-bearing mouse NK cell activity;(3) using the HT-IHC method determination of EERAC processing29tumor-bearing mice, apoptosis related genes Bcl-2, of Bax and Caspase-3expression. Results:Vitro experiment:(1) Compared with the control group, in different concentrations of Actinidiae extract group, LoVo cell density to reduce the proliferation slows down; cells gradually become larger, rounded, cells indirect thixotropic loose, increased particles in the cytoplasm, empty bubble, off the wall, surrounded by cell debris increased, the fluorescence staining characteristics of visible apoptosis school morphological changes appear.(2) Determination by MTT method, the extract Actinidiae time of LoVo cells for72h, and maximal inhibition rate of79.48%;(3) By agarose gel electrophoresis detection of DNA fragmentation increased significantly with the experimental drug concentration increased, the DNA ladder is the strongest expression in320μg/mL group.(4) Dropping EERAC24h increased the proportion of cells in G1/G1phase of LoVo cells, the S and G2/M phase cells reduced the apoptotic rate was6.8%,13.1%,18.2%and26.3%, and the blank group, the difference was statistically significant (P <0.05).(5)After the EERAC role in LoVo cells after24Bcl-2expression was significantly weakened, Bax and Caspase-3expression levels were significantly higher, of Bcl-2/Bax ratio decreased, the effects of concentration.In vivo experiments:(1) EERAC transplanted HT-29tumors significantly inhibited tumor inhibition rate of each dose group was9.12%,20.13%,37.81%, a positive correlation with drug concentration; different doses of EERAC tumor-bearing mice the WBC, spleen index, immune parameters were increased, no significant effect on the liver and kidney function.(2) Different doses of EERAC can NK. cell activity increased.(3) the role of EERAC LoVo cells Bel-2expression was decreased, increased levels of Bax. caspase-3expression of Bcl-2/Bax ratio decreased, the role of a concentration.Conclusion:1.Actinidiae extract inhibit the proliferation of LoVo cells, the inhibition concentration (dose) and time; its mechanism of action and promote apoptosis, mainly LoVo cells, Bcl-2expression by inhibitingenhanced Bax and Caspase-3expression levels, reduction in the Bcl-2/Bax ratio, and its role with the concentration.2.EERAC on colorectal cancer cells HT-29tumor-bearing mice with inhibition of tumor growth and induction of cancer cell apoptosis, mainly through the inhibition of tumor-bearing mice, Bcl-2expression, enhanced Bax and Caspase-3expression level, the Bcl-2/Bax the ratio down, and its role with the concentration, suggesting that its mechanism of action and promote apoptosis. Actinidiae extract can enhance the immune function of tumor-bearing mice, no significant liver and kidney toxicity.3.EERAC had no significant effect on weight of body, blood, liver and kidney function, and can increase the body's immune function. Inhibitory effect of EERAC was worse than5-FU, but it can enhance WBC, spleen index and immune in HT-29nude. EERAC can inhibit tumor and help the body to improve the immunity without obvious side effects of5-Fu.4. In vivo, the inhibition of colorectal cancer, EERAC dose certain the efficacy of high dose group, and no apparent side effects. Related conversion rules, the clinical treatment of colorectal cancer can be an appropriate increase in the amount of Actinidiae in a single prescription to improve the efficacy of cancer, preliminary proposals for the ideal anti-cancer dosage is30-35g.