Effect And Mechanism Of Gossypol On Apoptosis In Human Nasopharyngeal Carcinoma Cells

Background:Nasopharyngeal carcinoma(NPC) is the malignant tumor with high mortality in Southern China that threatening mankind health and life.The incidence of NPC holds the first place in cephalo-trachelian tumor.In recent years,the incidence of NPC has increasing obviously.Radiotherapy is applied to the early-stage of NPC currently, and the major treatment to the intermediate,advanced and recrudescent NPC is radiotherapy combined with chemotherapy.However,resistivity of chemo-radiotherapy in NPC often appears in clinic.Though the survival rate increases with the high dose of chemo-radiotherapy to some extent,there are different tolerances existing in different patients.The significant side effects make the quality of life descending obviously and influencing the therapeutic efficacy.Gossypol is a naturally yellow polyphenolic compound extracted from the cotton plant of malvaceae family.There are two kinds of optical isomer in gossypol, (-)-gossypol and(+)-gossypol,and it also can be divided into gossypol acetate, refined gossypol and formic acid gossypol according to the preparation.It was originally demonstrated as a male antifertility agent and applied in clinic many years ago,the recent researches find that gossypol is identified anticancer efficacy,it has been approved to have antiproliferative and apoptosis-inducing effects on some kinds of tumor cells in vitro and in vivo such as prostatic carcinoma,lymphoma and colon carcinoma.Further studies discover that gossypol is a small molecule inhibitor of Bcl-2 and Bcl-X_L proteins,it can bind to the BH3(Bcl-2 homology domain 3) binding site particularly in Bcl-2/Bcl-X_L and block the heterodimerization of Bcl-2/Bcl-X_L with proapoptotic protein such as Bax,this effect may inhibit the antiapoptotic function of Bcl-2/Bcl-X_L and induce apoptosis in tumor cells.Bcl-2 is an important antiapoptotic protein in the Bcl-2 protein family of cell apoptosis related proteins,which overexpresses in many tumors such as NPC.The state of overexpression of Bcl-2 protein closely correlates with the occurrence,progress, recurrence of tumor and resistance generated by chemo-radiotherapy.Objective:The effect of gossypol on NPC cells has not been reported before both home and abroad.In this research,we investigated and compared the antiproliferative effects and apoptosis-inducing effects of gossypol acetate and(-)-gossypol in NPC cells CNE2 overexpressed Bcl-2 protein,then we tried to explore the possible molecular mechanism.The data might provide an experiment basis for the clinical application and the theories foundation.Methods:1.MTT colorimetric assay was applied to detect the antiproliferation of gossypol in NPC cells CNE2.2.Morphological changes in the nuclear chromatin of cells undergoing apoptosis were determined both by Hoechst33342/PI staining and transmission electron microscopy detection.3.Flow cytometer(FCM) was used to measure DNA contents in order to analyze CNE2 cells apoptotic rates.4.Cell cycle regulated by gossypol was determined by FCM. 5.The influence of gossypol to the expression of apoptosis-associated protein Bcl-2 and Bax were detected by FCM.6.Caspase-3 activation was analyzed by colorimetric assay.Results:1.Gossypol could inhibite the proliferation of NPC cells CNE2 in dose and time dependent manner.More than 10μmol/L gossypol acetate or(-)-gossypol inhibited this proliferation significantly,1μmol/L gossypol acetate or(-)-gossypol did not influence the proliferation of cells.On exposing CNE2 cells to gossypol acetate for 24h,48h and 72h,the IC₅₀ were(45.30±1.53)μmol/L,(26.04±0.62)μmol/L and (17.54±0.75)μmol/L respectively.The IC₅₀ were(26.29±0.73)μmol/L,(13.30±0.51)μmol/L and(10.36±0.43)μmol/L when CNE2 cells were exposed to (-)-gossypol for 24h,48h and 72h.There was statistical difference of IC₅₀ between gossypol acetate and(-)-gossypol after exposed for 24h,48h and 72h(P＜0.001).It was clear that the antiproliferative effect of(-)-gossypol was better than gossypol acetate.2.After treated with 30μmol/L gossypol acetate or(-)-gossypol, Hoechst33342/PI staining showed that the nuclear chromatin condensed and brightly stained,but the control group presented the well diffusion,bluish normal cell nucleus. Transmission electron microscopy revealed the typical apoptotic features such as shrinkage of cytoplasm,nuclear fragmentation and chromatic agglutination,but in the control group,the morphous of nucleus presented anomalism,the nucleole could be seen obviously and chromatic was maldistribution.3.Flow cytometer(FCM) detected that the typical subdiploid peak could be observed before G₀/G₁ phases.After more than 10μmol/L gossypol acetate or (-)-gossypol treated for 24h,different proportional subdiploid peak could be seen on DNA histogram,and the cell apoptotic rates had a positive correlation relationship with the concentration of gossypol.There was statistical difference of the apoptotic rates between the gossypol and control group(P＜0.05).and there was also statistical difference of the apoptotic rates between the gossypol acetate and(-)-gossypol when the concentrations were above 10μmol/L(P=0.013,P=0.002,P＜0.001).It was indicated that(-)-gossypol had more significant apoptosis- inducing effect than gossypol acetate.4.FCM indicated that after the treatment of gossypol acetate or(-)-gossypol for 24h,the proportion of the G₀/G₁ phase cells was increasing accompanied with the concentrations,but the proportions of the G₂/M and S phases cells decreased. Gossypol blocked cells at G₀/G₁ phases,this phenomenon demonstrated that gossypol could regulate the cell cycle of CNE2 cells.5.After treatment of gossypol acetate or(-)-gossypol at the concentration of 30μmol/L for 24h,the expression level of Bcl-2 protein down regulated to(73.03±1.98)%and(58.50±4.42)%,which compared with the control group respectively, there were statistical differences among the gossypol acetate,(-)-gossypol and control group(P=0.005,P＜0.001),there was also statistical difference between the gossypol acetate and(-)-gossypol(P=0.001).The expression level of Bax protein up regulated to(17.43±1.08)%and(25.10±3.72)%,there were statistical differences among the gossypol acetate,(-)-gossypol and control group(P=0.003,P＜0.001),there was also statistical difference between the gossypol acetate and (-)-gossypol(P＝0.009).The ratio of Bcl-2/Bax down regulated to(4.20±0.30)% and(2.36±0.39)%,which relative to the control group(11.52±2.77)%.There were statistical differences among the gossypol acetate,(-)-gossypol and control group(P =0.001,P＜0.001),there was no statistical difference between the gossypol acetate and(-)-gossypol(P=0.206).6.Caspase-3 activation was confirmed at the concentration of 30μmol/L treated by gossypol acetate or(-)-gossypol.When exposed to gossypol acetate or (-)-gossypol for 6h,Caspase-3 activation was irritated at 6h and increased gradually accompanied with the time.The activity reached the peak at 24h and was(5.13±0.27) or(5.44±0.41) times relative to Oh.Then the activity showed a descending tendency, and it was(1.77±0.22) or(1.61±0.20) times at 48h relative to Oh.Conclusion:In this study,we demonstrated that gossypol was able to inhibit the proliferation and induce apoptosis of NPC cells CNE2,the mechanisms might include down regulation of Bcl-2 protein,up regulation of Bax protein and Caspase-3 activation.(-)-Gossypol had more significant anti-tumor effect than gossypol acetate.