The Mechanism Of Cryptotanshinone Induced Apoptosis Of Colorectal Cancer Cells Through Endoplasmic Reticulum Stress-autophagy Pathway

Objective: To study the growth inhibition and mechanism of cryptotanshinone(CPT)on colorectal cancer cells(CRC).Method: Clinical samples: Embed the clinical samples into sections,and detect the expression of apoptosis-related protein(cleaved caspase-3)and autophagy-related protein(LC3B)in tumor tissues by immunohistochemistry.In vitro cell experiment: In the cell experiment,the rectal cancer cells HCT116 and SW620 cells cultured in vitro were treated in different ways and divided into control group,administration group(1 μM CPT and 10 μM CPT),and single inhibitor group(Spautin-1,4-PBA),combined treatment group(Spautin-1+CPT,4-PBA+CPT)and positive drug group(cisplatin).MTT method,lactate dehydrogenase(LDH)release assay,and colony formation assay were used to detect the impact of drugs on cell growth and proliferation;FITC Annexin V-PI staining and JC-1 staining were used to tect cell apoptosis;Western blot assay to detect the expression level of apoptosis-related proteins(caspase-3 and cleaved caspase-3),autophagy-related protein(LC3B)and endoplasmic reticulum stress(BIP);acridine orange staining to detect autophagic vesicles of acidic organelles in cells.Animal experiment: Choose mature AB zebrafish to establish zebrafish tumor xenograft models,and divide them into control group and administration group(50 n M and 100 n M)to detect the inhibitory effect of CPT on tumor growth in vivo.Results: 1.In clinical samples,the expression levels of apoptosis(cleaved caspase-3)and autophagy(LC3B)related proteins in tumor tissues were significantly reduced compared to the intestinal mucosal epithelial tissue adjacent to the cancer.2.In in vitro cell experiments,the results of MTT,LDH release and plate cloning experiments showed that CPT can inhibit the viability of HCT116 and SW620 cells and the formation of cell colonies,but has no inhibitory effect on SW480 cells.In in vivo experiments,CPT can significantly inhibit the growth of tumors in zebrafish.3.JC-1 staining detected that the membrane potential of mitochondria in HCT116 and SW620 cells treated with CPT decreased;Western Blot results showed that CPT can up-regulate the expression of cleaved caspase-3.CPT concentration-dependently induced apoptosis of HCT116 and SW620 cells,and promoted the activation of the intracellular apoptosis-related protein caspase-3.4.The expression of autophagy-related protein(LC3B)in HCT116 and SW620 cells treated with CPT increased in a time-dependent manner.However,the results of MTT,LDH and plate cloning experiments showed that the autophagy inhibitor Spautin-1 effectively reversed the death of HCT116 cells caused by CPT,but had no effect on SW620 cells.At the same time,the addition of Spautin-1 can down-regulate the expression of apoptosis-related protein(cleaved caspase-3)triggered by CPT and reduce cell apoptosis.5.After CPT treatment of HCT116 cells,the expression of endoplasmic reticulum stressrelated proteins(BIP and p-PERK)increased significantly.The endoplasmic reticulum stress inhibitor 4-PBA can reverse the death of HCT116 cells caused by CPT and increase the number of cell colonies formed.In addition,4-PBA can effectively inhibit the conversion of LC3-I to LC3-II and the formation of autophagolysosomes in HCT116 cells of CPT treatment,and reduce the rate of apoptosis induced by CPT.Conclusion: 1.CPT inhibits the proliferation of HCT116 and SW620 colorectal cancer cells in vitro and in vivo.2.CPT mediates the apoptosis of HCT116 and SW620 cells through the autophagy pathway.3.CPT induced endoplasmic reticulum stress regulates the apoptosis of HCT116 cells in the upstream of autophagy.