| Background:Cervical cancer is a kind of happened at cervical intraepithelial malignancies, but also the world,second only to breast cancer,a serious threat to women's health and lives of malignant tumors.Its incidence in our country accounted for the top female malignancy,according to World Health Organization estimates that every year 131,000 new cases,accounting for the world's new cases of cervical cancer of 28.8%. Women of any age can develop cervical cancer.However,it frequently strikes-and sometimes kills-women when they are relatively young,in their mid-to-late 40s.In recent years,young cancer patients have a marked increase in the trend to retain reproductive function and improve quality of life is imperative.Cervical cancer occur in low socio-economic status of women,and sexual health possible,HPV infection, early marriage,smoking and so on.Developing countries the incidence of cervical cancer is 6 times that of developed countries,but also because of early cervical cancer screening is not yet universal,80%of the patients at the time of diagnosis for invasive carcinoma has developed.Early cervical cancer is currently mainly used for surgery or radiation therapy for advanced as well as the recurrence and metastasis of many cancer patients using radiation therapy combined with systemic chemotherapy treatment.Clinical findings, between different patients,the differences in the sensitivity of radiotherapy and chemotherapy,resulting in therapeutic effects are also very different.Improve the dose of radiotherapy and chemotherapy at a certain extent,although that can enhance the effectiveness,but different tolerance to different patients,and a marked increase in toxicity,in patients with severely reduced quality of life.Bcl-2 are apoptosis-related protein Bcl-2 family of important anti-apoptotic protein,cervical cancer and other tumor at high expression,the expression of such high status and the incidence of tumor development,recurrence,as well as generated by radiotherapy and chemotherapy resistance is closely related to.At the early stages of apoptosis,mitochondrial outer membrane is located on the Bcl-2 conformational change happen,leading to PTP opening,so that mitochondrial membrane permeability increases,such as the release of cytochrome C,apoptotic protein activating factor -1(Apaf-1),as well as apoptosis-inducing factor(AIF) and other bioactive protein to promote apoptosis.Cytochrome C and Apaf-1,Caspases-9 composed of apoptosis activated complex so that caspases-9 is activated,and further activation of caspase-3,eventually leading to cell apoptosis.Gossypol is a dual-naphthol yellow polyphenol hydroxyl aldehydes,there are two optical isomers of gossypol,respectively,for L-[(-)- gossypol],d-gossypol[(+)-gossypol] At present,the main agents of gossypol has three kinds:gossypol acetic acid,and formic acid gossypol,gossypol refined.Gossypol was first used as a male contraceptive clinical,recent study found that gossypol has significant anti-tumor ability to be able to a wide range of tumor cell lines such as prostate cancer, lymphoma and colon produce inhibition and tumor cell proliferation The role of apoptosis.Studies have shown that gossypol can be through a variety of ways and molecular target for inhibiting tumor cell proliferation and(or) induction of apoptosis, including through direct inhibition of mitochondrial energy metabolism,reduce the cell cycle regulatory protein Rb and cyclinD1 protein expression,inhibition of protein kinase C activity.Substantial information indicating that the role by the gossypol-induced apoptosis in the mitochondria are the variety of ways to induce apoptosis in a most important means of cell death.Mitochondrial-dependent apoptosis pathway is the expression of Bcl-2 down,causing cytochrome C release from mitochondrial,and Caspase-9 through the combination of activated Caspase-3, Caspase-7 activation,thereby regulating cell apoptosis.Further research confirmed that gossypol are anti-apoptotic protein Bcl-2/Bcl-XL a small molecule inhibitor,the specificity can Bcl-2/Bcl-XL at the BH3 binding site(Bcl-2 homology domin3), prevent its pro-apoptotic protein Bax,such as the formation of heterodimers, Bcl-2/Bcl-XL suppression of anti-apoptotic function of tumor-induced apoptosis.ObjectiveGossypol in the past at home and abroad about the role of cervical cancer has not been reported,this experiment to make use of high expression of Bcl-2 protein in cervical cancer cell line Hela as the research object,research gossypol acetate inhibition of proliferation and apoptosis and to explore its possible mechanism,at the same time comparison study of gossypol acetate joint X-ray of human cervical cancer cell lines in vitro for the further study of gossypol and its clinical application to provide an experimental basis,to lay a theoretical basis.Methods1.Using MTT colorimetric studies of gossypol acetate on the cervical cancer cell line Hela in vitro growth inhibition,at the same time comparison study of gossypol acetate joint X-ray of human cervical cancer cell lines in vitro.2.Morphological changes in the nuclear chromatin of cells undergoing apoptosis were determined both by Hoechst33342/PI staining. 3.Flow cytometry(FCM) was used to measure DNA contents in order to analyze Hela cells apoptotic rates.4.The influence of gossypol acetate to the expression of apoptosis-associated protein Bcl-2 and Bax were detected by FCM.5.Caspase-3,Caspase-8,Caspase-9 activation was analyzed by colorimetric assay.Results1.Gossypol acetate on the growth of Hela cells growth inhibition,and this effect was time and dose-dependent effect.10μmol/L of gossypol acetate over the role of 24h can significantly inhibit the growth of Hela cells,1μmol/L of gossypol acetate on cell growth were not significantly affected.2.Radiotherapy alone group and the united X-rays gossypol acetate treatment group Hela cell growth inhibition rate was(7.97±1.43,15.83±1.16,27.96±1.25)%, (42.71±2.25,61.36±2.33,77.45±3.05)%.The results show that gossypol acetate united X-rays can significantly enhance the X-rays on Hela cells in vitro,with the interactive effect between the two.3.After treatment with 20μmol/L gossypol acetate,Hoechst33342/PI staining showed that the nuclear chromatin condensed and brightly stained,but the control group presented the well diffusion,bluish normal cell nucleus.4.Flow cytometry can detect apoptotic cells in the G0/G1 cell cycle phase before the emergence of hypodiploid DNA peak,ie peak apoptosis.This experiment, 20μmol/L of gossypol acetate after 24h role,DNA histogram on a hypodiploid peak, the apoptosis rate of gossypol acetate compared with the control group,significant differences have statistical significance(t=-19.840,P=0.000).5.20μmol/L of gossypol acetate after 24h role,Bcl-2 protein expression level from the control group(78.17±3.23)%decreased from(68.97±1.94)%,significant difference has statistical significance(t=4.239,P=0.013);Bax protein expression level from the control group(8.16±0.67)%increased to(18.88±1.36)%,significant difference has statistical significance(t=-12.254,P=0.000);Bcl-2/Bax ratio compared with the control group,there is statistical significant difference Study significance(t=17.734,P=0.000)6.20μmol/L gossypol acetate has occurred after Caspase-3,Caspase-8, Caspase-9 activation 12h,24h,48h time point Hela cells,Caspase-3(2.32±0.15,5.44±0.20,4.34±0.04),Caspase-8(3.70±0.15,5.45±0.20,2.36±0.16), Caspase-9(4.36±0.33,6.40±0.19,2.84±0.08) the degree of activation gradually increased to a peak 24h,followed by a gradual decline in activity.ConclusionThis study confirmed that gossypol acetate on Hela cervical cancer cell lines with growth inhibition and induction of apoptosis,its mechanism may be related to Bcl-2 protein expression down,Bax protein expression and Caspase-3,Caspase-8, Caspase-9 activation.Gossypol acetate induced Hela cell apoptosis by the mitochondrial pathway and death receptor pathway of common finish.Gossypol acetate can enhance the joint X-rays of cervical cancer Hela cell line proliferation inhibition with synergy between the two. |
PDF Full Text Request