The Effect and Potential Mechanism of CP and SP on Hepatoma Carcinoma SMMC-7721 Cell
|Course||Nutrition and Food Hygiene|
|Keywords||Pueraria crude extract Puerarin Cell cycle Bax Bcl-2|
Primary liver cancer (hepatocelluar carcinoma, HCC) is one of the common malignant tumors in China. Disease is the early stage of intrahepatic metastasis, and the lack of specific symptoms, the difficult early detection, combined with the rapid progress of the disease, when diagnosed, most patients have lost the chance of operation, and the vast majority of patients with liver cancer is not sensitive to radiotherapy and chemotherapy, the short-term effect low, often in a short period after the onset of death. Seek and use of anticancer drugs, clinical found many chemical anticancer drugs act on target cells, often involving normal cells, resulting in decreased immunity, side effects and the development of expensive chemicals, so look for both non-cytotoxic drugs to inhibit the growth of tumor cells and promote apoptosis, and toxicity light, become the main direction of the research. Therefore, people turned to natural medicinal plants, trying to find drug side effects and the unique role of anti-cancer drugs from natural ingredients. Pueraria is one of the Ministry of Health announced the first batch of food and medicine plants both, Kudzu is expected to become the world's sixth-largest food crop According to authority FAO experts predict. The province Dabie Mountains the Pueraria resources lush Pueraria development is only getting started. In recent years, the anticancer effects of Pueraria increasingly widespread domestic Dodd Pueraria Extract anticancer activity of a variety of tumors, poles and other research found that Pueraria extract was inhibited by a variety of tumors. At home and abroad for Pueraria crude extract (pueraria crude extract, CP) and puerarin (standard ofpure puerarin, SP) role in human hepatoma SMMC-7721 cells in vitro inhibitory effect observed comparing the two and Pueraria crude extract component groups and the pharmacodynamics between spectral efficiency is still less. Apoptosis is controlled by the gene cell autonomous death process, a large number of studies have shown that apoptosis, cycle disorders and liver cancer occurrence and development are closely related. Therefore, the issues to be adopted of SMMC-7721 cells as target cells, integrated a variety of laboratory methods to observe, to understand the role of CP and SP SMMC-7721 cell proliferation, induction of apoptosis and cell cycle regulation, resulting in protein level and gene level preliminary elucidate its mechanism of action, and to provide a theoretical basis for clinical cancer therapy. Purpose 1. To study Pueraria crude extract and its main ingredient puerarin SMMC-7721 cell viability and cell cycle, and compare the biological effects of the two. Explore Pueraria crude extract and puerarin inhibition of SMMC-7721 cell proliferation, induction of apoptosis, change the mechanism of cell cycle. The method 1.MTT Determination of different concentrations of CP and SP respectively 24, 48, and 72 hours after the role, SMMC-7721 cell growth inhibition rate. End off nucleotide transferase-mediated dUTP nick end labeling (TUNEL) labeled apoptotic cells, the observation of the comparator CP and SP caused apoptosis of SMMC-7721 cells. Flow cytometric analysis of CP and SP role in SMMC-7721 cells, the cell cycle distribution. 4 cells immunochemical method and flow cytometry to detect cell apoptosis and cell cycle related protein expression. Using semi-quantitative RT-PCR method to monitor apoptosis-related genes Bax and Bcl-2mRNA expression. Results 1.CP and SP can be different degrees of inhibition of the growth of SMMC-7721 cells, a dose - response relationship and time - effect relationship; under the same conditions, the CP antiproliferative effect on SMMC-7721 cells stronger than SP difference statistically significance (P <0.05). The 2.CP and SP role in SMMC-7721 cells, apoptosis rates were 17.04% and 5.69% in the CP group, apoptotic rate was higher and the apoptosis rate of the SP group, the difference was statistically significant (P <0.05 ). Role 3.CP and SP SMMC-7721 cells after G 0 / G 1 constitute phase cells were 75.50% and 60.50%, the S phase cell proportions were 12.95% and 20.65%, respectively, compared with the control group, the differences were statistically significant (P <0.05); cellular proliferation index and reduced the CP group cell proliferation index is less than the SP group, the difference was statistically significant (P <0.05 ). SMMC-7721 cells after 4.CP and SP role, compared with the control group, the CP group and the SP group Bax protein upregulation and downregulation of Bcl-2 protein, Bax/Bcl-2 ratio increased, the difference was statistically significant ( P <0.05), and the CP group Bax/Bcl-2 ratio is higher than the SP group, the difference was statistically significant (P <0.05), RT-PCR detection further validate the results. Role in SMMC-7721 cells after 5.CP and SP P27 protein expression enhancement, and the CP group P27 average fluorescence intensity of the protein than the SP group CyclinD 1 protein expression was decreased, and the average CP group The fluorescence intensity lower than the SP, the differences were statistically significant (P <0.05). The conclusion 1.CP and SP have to inhibit the proliferation of SMMC-7721 cells, the inhibitory effect of CP is stronger than the SP. The 2.CP and SP role in SMMC-7721 cells extend G 0 / G 1 period shortened the S period and G , 2 / M phase. 3.CP may promote apoptosis of SMMC-7721 cells by regulating the expression of Bax and Bcl-2 protein and its mRNA proportion. The 4.CP and the SP may by reducing CyclinD 1 protein expression increased P27 protein expression and cell cycle arrest at the G 1 Phase.