Preliminary Study On The Mechanism Of Shikonin Inducing Pyroptosis In Gastric Cancer Cells

Gastric cancer is the fourth most common cancer and the third leading death caused by cancer in the world,approximately 950,000 new cases of gastric cancer are diagnosed each year.Chinese gastric cancer cases account for up to 40% of the world,which become a public health problem that cannot be ignored.Gastric cancer is difficult to cure due to postoperative recurrence and metastasis.The current treatments for gastric cancer mainly include surgical resection,radiotherapy,chemotherapy and immunotherapy,but the shortcomings are also certain and obvious.Therefore,it is urgent to invent new drugs with better pharmacological properties for cancer treatment.At present,many types of anti-cancer drugs are discovered and extracted from natural plants,and applied to the clinical treatment of cancer through structural modification and verification.In this article,we anlayze shikonin has anti-tumor effects,which is an active substance extracted from the roots of comfrey.Previous studies have shown that the anti-tumor effect of shikonin involves multiple mechanisms such as apoptosis,necrosis,pyroptosis and anti-tumor angiogenesis.We takes gastric cancer cells as the research object,using bioinformatics analysis methods,molecular biology experimental methods such as cell culture,Western Blot,Immunofluorescence staining and so on.To conduct a preliminary study on the mechanism of shikonin-induced pyroptosis of gastric cancer cells.The study results as follows:1、Exploring the effect of the survival rate and death types of SGC-7901 and BGC-823 cells after treating with shikonin.We found that high concentrations of shikonin(20μM and 40 μM)had a significant inhibitory effect on the survival of SGC-7901 and BGC-823 cells,and the cell survival rate showed a downward trend as the concentration of shikonin increased.Through Hoechst/PI staining,lactate dehydrogenase(LDH)release experiment and flow cytometry,we verify that the type of cell death induced by shikonin is pyroptosis.2、Western Blot of GSDME protein which is highly expressed in SGC-7901 and BGC-823 cells and as a pyroptosis fuction protein.We found that shikonin can induce cleavage of GSDME protein in SGC-7901 and BGC-823 cells.To knock down the expression of GSMDE protein by RNA interference technology reduced the proportion of pyrolyzed cells.After that,over-expression of GSDME gene in AGS cells,,the type of cell death changes from the apoptosis induced by shikonin to the pyroptosis that depends on the shearing of GSDME protein.To knock down of Caspase-3 and Bax proteins by RNA interference technology also inhibited pyroptosis,indicating that BaxCaspase-3-GSDME is involved in shikonin-induced pyroptosis.3、The expression changes of LC3 were detected by Western Blot and the fusion protein of m RFP-GFP-LC3 dual fluorescence system was used to examine the shikonin-induced autophagy.We indicate that shikonin can promote the conversion of LC3-I to LC3-II and the formation of autolysosome has been observed.Through RNA interference technology to knock down the expression of autophagy-related proteins ATG5 and Beclin-1 to understand the relationship between pyroptosis and autophagy.We verify that inhibiting autophagy can increase shikonin-induced pyroptosis.4、Using reactive oxygen species(ROS)inhibitor NAC to research the relationship between ROS on pyroptosis and autophagy,we show that shikonin-induced pyroptosis and autophagy depend on the generation of ROS.Therefore,NAC pre-treatment can significantly inhibit gastric cancer cell from pyroptosis and autophagy.5、Using mass spectrometry and bioinformatics analysis to explore the effect of shikonin on the expression of proteins in gastric cancer cells and the signal pathways constituted by these proteins.We found that these proteins are mainly involved in metabolic pathways,Huntington’s disease and endometrial cancer pathways.In addition,endometrial cancer includes CTNNA1,CTNNA2 and CTNNB1.All the proteins are closely related to cancers,such as bladder cancer,ovarian cancer and colorectal cancer.The results can be used as prognostic biomarkers for patients.