Mechanism Of Natural Product Ainsliadimer A Against Prostate Cancer By Inhibiting HSP90

Objective: To study the anti-tumor effect of Ainsliadimer A from natural product Ainsliaea,and reveal the target and mechanism of Ainsliadimer A-induced apoptosis in prostate cancer DU145 cells.Methods:(1)The anti-tumor activities of Ainsliadimer A on the different cancer cells were detected by CCK-8 assay,and the effect of DU145 cell colony forming ability was detected by colony formation experiment.(2)The effects of Ainsliadimer A on apoptosis,cycle and mitochondrial membrane potential of DU145 cells were detected by flow cytometry.(3)The expressions of cleaved-caspase 9 and cleaved-PARP proteins in DU145 cells were investigated by western blot.(4)Possible targets of Ainsliadimer A were predicted by CMAP database and Molecular Docking technique.(5)Western blot assay was used to verify the effect of Ainsliadimer A on the target-associated protein pathway.(6)DARTS and CETSA techniques were used to verify Ainsliadimer A binding to the target protein.Results:(1)The results of CCK-8 assay showed that Ainsliadimer A could inhibit the proliferation of hepatoma cells,melanoma cells,breast cancer cells,colon cancer cells,lung cancer cells and prostate cancer cells.Among them,Ainsliadimer A had the best effect on prostate cancer DU145 cells.The results of colony formation experiment revealed that Ainsliadimer A could inhibit the colony forming ability of DU145 cells in a concentration dependent manner.(2)The results of flow cytometry proveded that the natural product Ainsliadimer A could significantly promote the apoptosis of prostate cancer DU145 cells,block cell cycle arrest at G2 and S phases,and change the mitochondrial membrane potential.(3)The results of Western blot showed that Ainsliadimer A could up-regulate the expression of apoptotic proteins cleaved-PARP and cleaved-caspase 9 in concentration-dependence.(4)The prediction results of CMAP database and Molecular Docking experiments indicated that the target of Ainsliadimer A might be HSP90 protein.(5)Western blot assay confirmed that Ainsliadimer A could significantly down-regulate the expression of HSP90 client proteins IKK?,p-STAT3 and p-AKT and up-regulate HSP90 marker protein HSP70.(6)The results of DARTS and CETSA showed that Ainsliadimer A enhanced the stability of HSP90 protein,indicating that Ainsliadimer A had the ability to bind to HSP90 protein.Conclusion: The results of this study showed that Ainsliadimer A had a good anti-tumor effect and could inhibit the proliferation of many kinds of tumor cells.Among them,Ainsliadimer A had the best effect on prostate cancer DU145 cells.In addition,Ainsliadimer A significantly promoted the apoptosis of DU145 cells in a concentrationdependent manner,blocked the cell cycle arrest at G2 and S phases,and affected the mitochondrial membrane potential.Finally,we verified that Ainsliadimer A could bind to HSP90 protein and inhibit the activity of HSP90 by CMAP,Molecular Docking,Western blot,DARTS and CETSA experiments on prostate cancer.