Research On The Growth Inhibition Effect Of Corosolic Acid On SMMC-7721 Cells And Approach To Its Mechanism

IntroductionHuman hepatocellular carcinoma (HCC), with 500,000-1,000,000 new cases and causing approximately 1,000,000 deaths annually, is the fifith most common malignancy in the world. Since there has not yet been a particularly effective treatment, seeking for better treatment has become the major problem facing us. Traditional Chinese Medicine (TCM) with distant history in cancer treatment has its unique advantages in liver cancer prevention, treatment and prevention of recurrence and metastasis. However, the chemical compositions of many herbals and their exact mechanisms remain unclear, which has hindered their further study and being widely recognized. Currently, the separation of effective components of Chinese medicine and researches on the pharmacological effects is becoming an important way to study how Chinese herbal medicine is treating cancer.Traditional Chinese Medicine―mao ren shen‖, the root of Actinidia valvata Dunn, is commonly used in clinical cancer treatment in Jiangsu and Zhejiang provinces. With anti-tumor and anti-inflammatory effects, it was applied to treatment of liver cancer, lung cancer and myeloma. After study on the components of―mao ren shen‖, corosolic acid (CRA), one kind of triterpenoids, was found as one of its effective components. As medicine for prevention and treatment of obesity and typeⅡdiabetes, CRA has entered the PhaseⅢclinical efficacy evaluation of U.S. FDA. Researches show that CRA has cytotoxicity effect on many human tumor cell lines. It was also found to inhibit tumor cell viability and induce tumor cell apoptosis. On the base of previous related work, this subject is to further study the growth inhibitory effect of CRA on liver cancer both in vivo and in vitro, and preliminarily discussed part of its probably relevant mechanisms.Objectives1. To study the effects of CRA on the proliferation of SMMC-7721 cells in vitro, and observe the correspondent morphological variations.2. To explore the apoptosis effect of CRA on SMMC-7721 cells.3. To evaluate the mitochondrial membrane potential changes and the expression of cytochrome C both in mitochondria and cytoplasm.4. To explore the influence of CRA on the expression of apoptosis induce protein Bax and the anti-apoptosis protein Bcl-2.5. To observe the inhibitory action of CRA on nude mouse liver subcutaneous transplanted tumor in vivo.MethodsPART ONE: Experiments of CRA on SMMC-7721 in vitro1. To observe the morphological changes of SMMC-7721 cells after being infected with CRA.2. MTT was applied to detect the proliferation inhibitory effect of CRA on SMMC-7721 at different concentrations, primarily identify the effective concentration range.3. AnnexinV/PI Apoptosis Kit and FCM were applied to detect the apoptosis inducing effect of CRA on SMMC-7721.4. Mitocapture Kit was used to detect the potential changes of mitochondrial membrane in SMMC-7721 cells after being treated wit CRA.5. Western blotting was used to analyze the expression of cytochrome C both in cytoplasm and in mitochondria.6. To detect the changes of Bax/Bcl-2 ratio in SMMC-7721 cells after being treated with CRA through applying Western blotting to observe the expression of Bax and Bcl-2.PART TWO: Researches on the growth inhibitory effect of CRA on liver cancerEstablishing nude mouse liver subcutaneous transplanted tumor model,random grouping,and regular administration.Weighing mice in each group before and after the experiment,measuring cubic capacity and weighing the weight of each tumor.ResultsPART ONE: Experiments of CRA on SMMC-7721 in vitro1. Changes in cell morphology: CRA can influence cell morphology and adherent situation being observed under phase contrast microscope. At 35μM CRA could induce an obvious cell morphological change, after 6 hours some cells start to contract, after 12 hours floating cells appeared in the culture medium, after 18 hours most cells have lost the original flat, polygonal shape.2. MTT results: CRA performs a proliferation inhibitory effect on SMMC-7721, the inhibitory rate was 31.64% after being treated 48 hours with 25μg/ml CRA.3. FCM results: the apoptosis rates of SMMC-7721 after being treated with 35μM CRA for 24 hours and 36 hours are 60.5% and 70.6% separately.4. Changes in mitochondrial membrane potential: compared with the control group, the mitochondrial membrane potential obviously dissipated after being treated with CRA for 18 hours.5. Western blot analysis: cytochrome C was gradually released form mitochondrial into cytosol with treatment of CRA.6. With treatment of CRA on SMMC-7721, the expression of Bax proteins gradually increased, while Bcl-2 remain unchanged, which indicates that CRA could upgrade the Bax/Bcl-2 ratio to induce cell apoptosis.PART TWO: Researches on the growth inhibitory effect of CRA on liver cancerThe tumor inhibitory rate of the high dose group is 58.7%,which indicate at the concentration of 50mg/kg CRA could inhibit the growth of liver tumor. Conclusions1. CRA could inhibit the proliferation of SMMC-7721 cells and produce characteristic changes of apoptosis.2. CRA could cause the mitochondrial membrane potential dissipated and cytochrome C released from mitochondrial into cytosol in SMMC-7721 cells.3. CRA could upgrade the Bax/Bcl-2 ratio to induce SMMC-7721 cell apoptosis.4. CRA could inhibit the growth of nude mouse liver subcutaneous transplanted tumor at the concentration of 50mg/kg.