Study Of The Mechanism Of Eriocalyxin B In Inhibiting Prostate Cancer Cells Prolife Via Regulating Apoptosis And Autophagy

1 BackgroundProstate cancer is the most common cancer in men over 50 years old and is the second leading cause of cancer death in the world.Prostate cancer initially depends on androgen and is sensitive to androgen deprivation.However,the tumor was insensitive to androgen deficiency within two years.As a result,prostate cancer can slowly develop into a high-risk stage of attack.The treatment of prostate patients includes androgen biosynthesis inhibitors,chemotherapy and immunotherapy.Therefore,it is urgent to identify and develop new drugs against prostate cancerAutophagy is an important homeostasis process in health and disease.Autophagy,as a circulatory system of cells,transports cell materials to lysosomes for degradation,especially damaged proteins and organelles in cells,and then uses the constituent macromolecules for other processes.The mechanism of autophagy regulation interacts closely with the mechanism of cell death and significant dynamic interaction between these processes.Therefore,it is of great significance to investigate the mechanism of apoptosis and autophagy in tumor cells,as well as to further explore the common signal pathway that affects apoptosis and autophagyNatural extracts come from plants,animals,microorganisms,which have been widely used in traditional medicine for many years.Chinese herbal medicine is one of the main types of natural extracts.Chinese herbal medicine has been used to treat diseases for thousands of years and is an important part of Chinese medicine.Chinese herbal medicine is considered to be an important source for the discovery of modern drugs,including anticancer drugs.Ericalyxin B(EriB)is a diterpene isolated from plants,which has been shown to possess antitumor properties.EriB was isolated from the leaves of the plant Isodon eriocalyx var.Laxiflora,a perennial herb of the Labiatae family,which was used as an anti-bacterial and anti-inflammatory agent.Recent studies show that EriB can suppress the growth of several cancer cell lines.However,there are few systematic studies on the mechanism of the antitumor activity of EriB in prostate cancer cells.The aim of this study was to investigate the effect of EriB on prostate cancer cells2 ObjectiveTo explore whether EriB can inhibit the growth of prostate cancer cells by regulating apoptosis and autophagy,and further confirm the interaction between apoptosis and autophagy in this process.To provide the basic experimental theoretical basis for the mechanism of EriB in the treatment of prostate cancer3 MethodsIn this study,human prostate cancer cell lines PC-3 and 22Rv1 were used,which belonged to androgen independent and androgen dependent prostate cancer cell lines respectively3.1 Different concentrations of EriB were added to the culture medium of human prostate cancer cells,and EriB,autophagy inhibitor or ROS inhibitor were added to the culture medium of prostate cancer cells.3.2 The activity of prostate cancer cells was measured by MTT method.3.3 Annexin V-FITC/PI double staining was used to treat prostate cancer cells,and flow cytometry was used to detect the distribution and cell cycle of apoptosis cells3.4 Fluorescence microscopy was used to capture autophagy related protein LC3B-Ⅱ in prostate cancer cells.3.5 The Autophagic vacuoles in prostate cancer cells were observed by electron microscopy.3.6 Western blotting was used to detect Caspase 3,PARP,LC3B-Ⅰ,LC3B-Ⅱ,Akt,p-Akt,mTOR and p-mTOR.4 Results4.1 Effect of EriB on the proliferation of human prostate cancer cellsEriB inhibits the proliferation of human prostate cancer cells in a time and dose-dependent manner.4.2 Effect of EriB on apoptosis of human prostate cancer cellsEriB induces apoptosis of human prostate cancer cells,and EriB can induce apoptosis related proteins in human prostate cancer cells.4.3 Effect of EriB on cell cycle of human prostate cancer cellsEriB has on ablity to induce cell cycle arrest in human prostate cancer cells4.4 Effect of EriB on autophagy of human prostate cancer cellsEriB induces the increase of autophagy related protein LC3B-Ⅱ in human prostate cancer cells.Through the influence of different stages of autophagy inhibition on LC3B-Ⅱ protein and the changes of autophagy bodies observed by electron microscopy,It is clear that EriB induces autophagy in human prostate cancer cells4.5 The interaction between apoptosis and autophagy induced by EriBBy adding chloroquine,an autophagy inhibitor,to the culture medium of human prostate cancer cells treated with EriB,we found that the combination of the autophagy inhibitor and EriB could further increase the number of apoptosis cells and the expression of apoptosis related proteins.4.6 The effect of EriB on the common signal pathway of apoptosis and autophagyAkt/mTOR patheway is a common signal pathway of apoptosis and autophagy.EriB reduces the levels of p-Akt and p-mTOR,indicating that it inhibits Akt/mTOR signal pathway in human prostate cancer cells.4.7 The effect of ROC inhibitor NAC on the growth of prostate cancer cells after erib treatment.NAC blocks the growth inhibition of EriB on prostate cancer cells.4.8 The effect of ROC inhibitor NAC on the apoptosis of prostate cancer cells after EriB treatment.NAC inhibits the apoptosis of prostate cancer cells induced by EriB.4.9 The effect of ROC inhibitor NAC on the expression of autophagy protein in EriB treated prostate cancer cells.NAC inhibits the increased expression of LC3B-Ⅱ in prostate cancer cells induced by EriB.4.10 The effect of ROC inhibitor NAC on Akt/mTOR signaling pathway in EriB treated prostate cancer cells.NAC blocks inhibition of Akt/mTOR expression by EriB in prostate cancer cells5 Conclusion5.1 EriB effectively inhibits the growth of human prostate cancer cells.5.2 EriB induces apoptosis in human prostate cancer cells.5.3 EriB can not affect the cell cycle of human prostate cancer cells.5.4 EriB induces autophagy in human prostate cancer cells.5.5 Inhibition of autophagy can promote the apoptosis of human prostate cancer cells induced by EriB,and the autophagy induced by EriB in human prostate cancer cells has a cytoprotective effect.5.6 EriB induces autophagy in human prostate cancer cells by inhibiting the Akt/mTOR signal pathway.5.7 Erib inhibits Akt/mTOR signaling pathway and induces apoptosis and autophagy by inducing ROS level in prostate cancer cells.