Effect And Mechanism Of AD23 Synergistic With Histone Deacetylase Inhibitor In Inhibiting Small Cell Lung Cancer

Object:The purpose of this study is to Verify that AD23 is a natural analog compound of BH3 only protein,explore whether AD23 can cooperate with HDAC inhibitor(HDACi)vorinostat(SAHA)to inhibit the proliferation of small cell lung cancer,and clarify their synergistic antitumor mechanism,so as to lay a foundation for the development of new therapeutic drugs and treatment models for small cell lung cancer.Methods:1.Molecular docking using Auto Dock Vina software,molecular dynamics simulation using GROMACS software,and surface plasmon resonance experiments to detect the binding of AD23 to BCL-2family anti-apoptotic proteins(BCL-2,BCL-XL,MCL-1)ability.2.The effect of AD23 and SAHA alone and combined administration on the viability of human small cell lung cancer H82 cells was determined by MTS experiment in vitro;according to the Chou-Talalay principle of intermediate effect,the combined administration analysis software Calcu Syn 2.1 was used to calculate the synergy of AD23 and SAHA The action index was used to determine the optimal combined administration concentration.3.Hoechst 33342 staining method was used to observe the changes of apoptosis morphology of H82 cells after administration of AD23 and SAHA alone and in combination;Annexin V-FITC/PI double staining flow cytometry was used to further explore the cells after administration of AD23 and SAHA alone and in combination Apoptosis rate.H82 cells were stained with dansylcadaverine(MDC)to detect whether AD23 cooperates with SAHA to form autophagic vesicles;transmission electron microscopy was used to observe whether AD23 combined with SAHA administration synergistically induces the formation of autophagosomes and promotes autophagy in cells.4.The expression changes of autophagy and apoptosis-related proteins were detected by Western blot.5.Establish subcutaneous H82 cell xenograft model in BALB/c nude mice to evaluate the inhibitory effect of AD23 combined with SAHA administration on tumor.6.To explore the effect of AD23 combined with SAHA on histone acetylation level and BH3-only protein expression by Western blot.7.To explore the effect of AD23 combined with SAHA on the acetylation of histones and other proteins by acetylation proteomics.Results:1.The prediction results of molecular docking and molecular dynamics simulation show that AD23 can stably bind to BCL-2,BCL-XL and MCL-1 proteins.Surface plasmon resonance experiments show that AD23 can bind to BCL-2 and BCL-XL in a concentration-dependent manner.And the high affinity binding of MCL-1 protein confirms that AD23 is a BH3-only protein mimetic compound.2.Through MTS experiment,it was found that AD23 combined with SAHA treatment group significantly increased the inhibition rate of H82 cell viability compared with the drug alone treatment group.The calculation of the two-drug combination index showed that AD23 and SAHA had synergistic inhibitory effect on H82 cell viability.3.Hochest33342 staining results showed that compared with the control group and the single drug treatment group,AD23 combined with SAHA treatment group appeared more typical apoptotic cells;Annexin V-FITC/PI double staining flow cytometry results showed that AD23 cooperated with SAHA The apoptosis rate of H82 cells was significantly increased;MDC staining results showed that AD23 cooperated with SAHA to induce the formation of autophagic vesicles;transmission electron microscopy results showed that AD23 cooperated with SAHA to induce the formation of autophagosomes and autophagolysosomes to promote autophagy.4.Western blot results showed that AD23 cooperated with SAHA to increase the expression of apoptosis-related proteins C-Caspse9 and C-PARP,and activate the apoptosis pathway.AD23 cooperates with SAHA to up-regulate the ratio of autophagy-related protein LC3II/I protein,down-regulate the expression of p62 protein,and activate the autophagy pathway.5.In the in vivo experiment,AD23,SAHA and AD23 combined with SAHA administration group can significantly inhibit the growth of orthotopic transplanted tumor of BALB/c nude mice subcutaneous H82 cells,and reduce the tumor weight index.6.Further mechanism study found: 1)AD23 cooperates with SAHA to reduce the expression of BCL-2 family anti-apoptotic proteins BCL-2,BCL-XL and MCL-1,and increase the expression of pro-apoptotic protein BAX protein.2)AD23 cooperates with SAHA to inhibit the PI3K-AKT-m TOR signaling pathway,thereby inducing autophagy in small cell lung cancer.3)AD23 cooperates with SAHA to increase histone H3K9 ac acetylation and H3K27 ac acetylation,promote chromatin loosening and DNA exposure,and increase the expression of BH3-only proteins BIM and BID.5)Acetylation proteomics suggested that AD23 cooperated with SAHA to promote the acetylation of H3C1 and other proteins in H82 cells.Conclusion:This study confirmed that AD23 is a new natural product-derived BH3 mimetic compound,which can inhibit the growth of small cell lung cancer and induce apoptosis and autophagy in small cell lung cancer in vitro and in vivo;AD23 can synergize with the HDAC inhibitor SAHA to inhibit PI3K-AKT-m TOR signaling pathway further promotes autophagy and inhibits the proliferation of small cell lung cancer;AD23cooperates with SAHA to increase the acetylation levels of H3K9 ac and H3K27 ac,promote the expression of BH3-only protein,and enhance the inhibitory effect on small cell lung cancer.