The Research Of Apoptosis Introduction And Inhibition Proliferation Of Hep-2Cells By Genistein

Objective: Laryngeal carcinoma is the eleventh common malignancy of theHuman being，derived from malignant tumour of the laryngeal mucosaepithelial tissue. On histopathology, the vast majority is squamous epithelialcells. Traditionally, patients with laryngeal carcinoma are treated with surgery,radiotherapy alone or adjuvant treatments. Although surgical resection of thelesions is the direct treatment approach of laryngeal cancer, but thepostoperative will has the high risk of local tumor recurrence or poor qualityof life because of the pronunciation and swallowing dysfunction. Looking fora auxiliary chemotherapy agent that can make the tumor shrink to reduce theresection range, decrease rate of recurrence, increase patients life quality andsurvival rate is becoming more and more important. Apoptosis is also knownas programmed cell death, many factors and signaling pathways involved inthis process,being carefully regulated process, that is critical for preservingnormal homeostasis and tissue and organ morphogenesis. Defects in apoptoticpathways are now considered as contributing to a number of human diseases（such as malignancies e tal）.Evasion of apoptosis is a common feature ofcancer and can contribute to the development of resistance to cytotoxicchemotherapy in neoplasms. Therefore, compounds that can induce apoptosisby blocking or suppressing the proliferation of tumor cells have the greatestpotential to become antitumor agents. Genistein, or4,5,7-trihydroxyisoflavone,is a small molecule found in high natural abundance in soy products. Genisteinconsumption have many important biological effects，primarily anti-cancerproperties. In addition, genistein has many other important health benefits,such as prevention of osteoporosis，lowering the incidence of cardiovasculardiseasesand attenuation of post-menopausal problems. A large number ofexperiments confirmed genistein has inhibitory effect on a wide variety of tumor in vivo and in vitro. Mainly antitumor mechanisms:1. Effects on cellcycle regulation: Genistein induced G2/M cell cycle arrest in cancer cells isdue to the up-regulation of p21WAF1expression and down-regulation of cyclinB.2. Effects on the induction of apoptosis: we found genistein could induceapoptosis in cancer cells by using multiple assay techniques as hallmark todetect apoptosis.3. Inhibits cell migration and cell invasion: Inhibitingphosphorylation of FAK, the canonical TGF-β, and selectively inhibitingmember of MMPs are the main mechanisms that effects of genistein oncellular adhesion,migration and invasion.4.Acts as a weak estrogen mimc:Genistein’s structural is close similarity to17-β-estradiol, leading to its weakestrogenic activity. Genistein can modulate its function via directly binding tothe estrogen receptor and, then effect the proliferation both of androgen andestrogen-mediated carcinogenesis（such as: breast cancer，prostate cancer）.5.Others: Genistein can induce angiogenesis through decreasing VEGFproduction. Genistein can protect cells against reactive oxygen species (ROS)by revoing free radicals. This experiment takes the laryngeal carcinoma cellline Hep-2as research object, using inverted-microscope and fluorescencemicroscope, cytotoxicity experiment, flow-cytometry etc. methods andtechniques,to investing the role of genistein on the proliferation，cycleblockage and apoptosis of the Human Laryngeal carcinoma cells Hep-2. Forthe application of genistein in head and neck cancer provide experiment basis.Methods:1The Human Laryngeal carcinoma cells Hep-2is cultured in vitro.Genisten was diluted into different concentrations.2Using CCK-8to detect the inhibition of Hep-2cells after exposure todifferent concentrations of genistein and then calculate the inhibition rate＆IC50.3Observing the difference of cell growth and cell morphology aftertreated with genistein by inverted microscope and fluorescence microscope4Using the flow cytometry to analyze the change of cell apoptosis andcell cycle 5Using real-time fluorescent quantitative PCR evaluate the alteration ofsurvivin mRNA, bcl-2mRNA expression in Human Laryngeal carcinoma cellHep-26Statistieal analysis: SPSS statistieal Package Program13.0were toanalysis data. The two sample data was performed with t-test. The quantitativedata were tested by one way ANOVA. The MultiPle comparisons betweengroups were compared with LSD analysis of varianc.Ｐ﹤0.05were deemedstatistically significant.Result: Observed the change of cell growth and cell morphology aftertreated with genistein by inverted microscope. In the negative control group,Hep-2cells grew as adherent, cell morphology is full, fusiform or polygonal.The outline of cells is clear. There is few suspension cell. In contrast, in theexperimental group，Edge of cells is irregular, the part of the cell surface havea budding phenomenon, some dissolve broken cells and suspension cellswhich detachment from the substrate. Using CCK-8proliferation assayassessed the effect of genistein on the cell proliferation. The experimentsshowed that genistein significantly inhibited the proliferation of Hep-2cells ina dose-dependent manner. Flow cytometric analysis showed that genisteinmodulated cell cycle progression through inducing cells to accumulate at Sand G2/M-phases with concurrent decrease of cells at G1phase. Cells weretreated by different concentration of genistein (12μg/ml,24μg/ml) after24hours, the G2phase and S phase differences are statistically significantcompared with untreated cells (Ｐ﹤0.05). In addition, genistein induced cellapoptosis. Furthermore，with the increasing of drug concentration and actiontime, the rate of apoptosis increased. The extent of apoptosis was dose–timedependent. Treatment with different concentration genistein for24and48hrespectively on laryngeal cancer cell, the difference of apoptosis rate hadstatistical significance (Ｐ<0.05), Exposure to the same drug concentration for24hours and48hours, the difference of the laryngeal cancer cells apoptosisrate was statistically significant (Ｐ<0.05). RT-qPCR demonstrate that themRNA levels of Bcl-2and Survivin conspicuous decreased in the cells treatment with genistein，compared with untreated cells, the difference hadstatistical significance (Ｐ<0.05).Conclusion:1Genistein significantly inhibit the proliferation of laryngeal carcinomacell in vitro and having concentration-dependent relation.2Genistein can make the laryngeal carcinoma cell cycle blockage, lowdose drug make cell arrest in G2phase，high dose drug hold up cell in S phase.This is the one of mechanism that genistein induce cell apoptosis.3Genistein can induce laryngeal carcinoma cells Hep-2apoptosis andhave time and dose-dependence relation,12μg/ml genistein for24hours caninduce the laryngeal carcinoma cell apoptosis significantly.4Genistein decrease the expression of Bcl-2, Survivin in laryngealcarcinoma cell, and induce cells apoptosis.