The Effects And Molecular Mechanism Research On Ganoderic Acid A Derivatives Against Cervical Cancer

Aim:In this study,we synthesized 22 kinds of ganoderic acid A derivatives through structural modification,explored their anti-cervical cancer effects by using in vitro and in vivo models,and clarifies its possible mechanism,so as to provide scientific basis for the discovery of more effective anti-cervical cancer drugs with lower side effects.Methods:1.Through reduction modification,amidation modification and esterification modification,ganoderic acid A derivatives were synthesized.The anti-tumor activity was screened by CCK-8 method.The effect of GaAD19,a bromoaniline ester derivative with the strongest anti-tumor activity,on Hela cell colony formation was determined by cell cloning assay.Apoptosis was detected by AnnexinV/PI staining,Western blot,TUNEL and immunohistochemistry.2.The effect of GaAD19 on the cell cycle distribution of Hela cells was detected by flow cytometry,and the effect of GaAD19 on the mRNA level of Hela cell cycle regulator was detected by q-PCR.3.Target prediction analysis and molecular docking were used to predict the signal pathway of Hela cells acted by GaAD19,and Western blot was used to detect the effects of GaAD 19 on the expression of signal pathway proteins and upstream genes MAP2K4 and MAP2K7 of Hela cells.q-PCR was used to detect the effects of GaAD19 on the expression of JNK pathway membrane protein receptors in Hela cells.JNK agonist Anisomycin and inhibitor SP600125 were added to study the regulating effects of GaAD19 on proliferation,apoptosis and cell cycle of Hela cells.4.The in vivo antitumor activity of GaAD19 was studied through constructing U14 transplanted tumor model in nude mouse.The expression of tumor tissue protein was detected by Western blot.Liver,kidney and spleen were examined by HE staining to clarify the mechanism of anti-tumor effect of GaAD19 in vivo.Results:1.Synthesize 22 ganoderic acid A derivativesThrough reduction modification,amidation modification and esterification modification,22 ganoderic acid A derivatives were synthesized.2.GaAD19 induced apoptosis of cervical cancer Hela cellsThe results of CCK-8 showed that GaAD19 had obvious inhibitory activity on Hela cells,but had weak inhibitory effect on normal cell lines.Cell cloning assay showed that GaAD19 had significant inhibitory effect on the clonal formation of Hela cells.In terms of anti-apoptosis,GaAD19 could up-regulate the expression of proapoptotic proteins,Cleaved Caspase 9,Cleaved PARP-1 and Bax,and down-regulate the expression of anti-apoptotic protein Bcl-2.The results of flow cytometry,TUNEL and immunohistochemistry showed that GaAD 19 induced Hela cell apoptosis in a dose-dependent manner.3.GaAD19 induced cell cycle arrest of cervical cancer Hela cellsFlow cytometry showed that GaAD19 blocked the cell cycle transition from G1 phase to S phase.Meanwhile,q-PCR results showed that GaAD19 could significantly down-regulate the mRNA levels of Hela cell cycle regulatory proteins CDK1,CDK2,CDK4,CDK6,CDK7,CDK8,ccne1,PCLAF and Ube2c.4.GaAD19 inhibited the JNK pathway in cervical cancer Hela cellsThrough target prediction analysis,it was found that MAPK8 is a hub gene of GaAD19 acting on Hela cells.The lowest binding energy obtained by molecular docking of GaAD19 with JNK pathway is-12.31 kcal/mol.Western blot results showed that the GaAD19 dose-dependent inhibited the activation of JNK pathway and its upstream genes,but had no significant differences in the expression of ERK,p38,AMPKα1-S485,AMPKα1-S496,AMPKβ1-S108 and Akt pathway proteins.q-PCR results showed that GaAD19 mainly acted on JNK pathway growth factors,cell stress and G12/13-coupled receptors membrane protein receptors,down-regulating mRNA levels of Crk,CrkL,Shc,GRB2,Sos,Ras,Rac,cdc42,Ga12 and Gβ.The addition of JNK agonist Anisomycin and inhibitor SP600125 could weaken and enhance the effects of GaAD19 on Hela cell proliferation,apoptosis and cell cycle,respectively.5.GaAD19 inhibited the growth of U14 xenografts in nude mouseThe results of in vivo experiments showed that GaAD19 had a good inhibitory effect on U14 xenografts in nude mouse,and had no obvious toxic effect on body weight,liver,kidney and spleen of nude mouse.In addition,GaAD19 significantly reduced the expression of p-JNK protein in tumor tissues,which was consistent with the results of in vitro experiments.Conclusions:GaAD19 can significantly inhibit the proliferation of Hela cells,promote apoptosis of Hela cells,and induce the arrest of G1/S phase of Hela cells.In addition,GaAD19 significantly inhibited the growth of U14 xenografts in nude mouse.The mechanism is related to the inhibition of JNK signal pathway.