| Liver cancer is one of the most common malignant tumors,and the vast majority of primary liver cancers are hepatocellular carcinoma(HCC).On average,about 250,000 people die of liver cancer every year in the world,and our country accounts for about45% of them.The main treatment methods for liver cancer include liver resection,liver transplantation,targeted therapy,systemic immunotherapy,and chemotherapy.Cinobufacin is an extract of the dried skin of Bufo bufo,a traditional Chinese medicinal material,which has anti-tumor and immune-regulating effects.arenobufagin is the active ingredient extracted from cinobufagin.Studies have shown that arenobufagin can induce apoptosis,ferroptosis,and autophagy of tumor cells.Ferroptosis is an iron-dependent form of cell death,which is caused by abnormal metabolism of intracellular lipid oxides and imbalance of intracellular redox.More and more studies have found that autophagy is closely related to ferroptosis;for example,nuclear receptor coactivator 4(nuclear receptor coactivator 4,NCOA4),RAB7 A,and other regulated autophagy-dependent ferroptosis.Nuclear factor erythroid 2-related factor 2(nuclear factor erythroid 2-related factor2,Nrf2)is a key regulator of intracellular anti-oxidation.Studies have found that the p62-Keap1-Nrf2 pathway plays a protective role in the process of ferroptosis in liver cancer cells.p62(also known as Sequestosome-1)is encoded by the SQSTM1 gene,which increases the nuclear accumulation of Nrf2 by promoting the inactivation of Kelch-like ECH-associated protein 1(Keap1)and inhibiting the degradation of Nrf2.Protein and receptor of autophagy membrane protein microtubule-associated protein1A/1B-light chain 3(Autophagy membrane protein microtubule-associated protein1A/1B light chain 3,LC3),whose expression is closely related to autophagy.Although previous studies have elaborated on autophagy and ferroptosis induced by arenobufagin,the relationship between autophagy and ferroptosis is still unclear.Therefore,in this study,HepG2 cells and BALB/c Nude mice were used as the research objects to explore the effect and preliminary mechanism of arenobufagin-induced autophagy and ferroptosis in liver cancer cells.Objective:The purpose of this project is to explore the effect of arenobufagin on the p62-Keap1-Nrf2 pathway,to preliminarily study the relationship between arenobufagin-induced autophagy and ferroptosis in liver cancer cells,and to clarify the effect of arenobufagin on liver cancer cells.Methods A BALB/c Nude mouse liver cancer xenograft model was constructed with HepG2 cells,the size of the tumor was recorded,and the changes of the proliferation and metastasis-related protein Ki-67 were detected by immunohistochemistry,and the pathological changes of the tumor tissue were observed by HE staining;CCK8 was detected Proliferation ability of HepG2 cells;morphological observation of autophagy and ferroptosis by transmission electron microscope;microplate reader detection of oxidative stress indicators GSH,MDA,T-SOD content changes in tumor tissues and cells;flow cytometry detection of HepG2 Cytoplasmic ROS and lipid ROS levels in cells;Western blot detection of COX-2,Nrf2,Lc3,p62 expression in transplanted tumors and COX-2,Nrf2,Lc3,p62,Keap-1,HO-1 expression.HepG2 cells were pre-protected with autophagy inhibitor CQ and ferroptosis inhibitor DFO(iron chelator deferoxamine)respectively,and then treated with arenobufagin to observe the changes of autophagy and ferroptosis indicators in HepG2 cells.The nuclear protein was extracted,and the effect of arenobufagin on Nrf2 nuclear translocation in HepG2 cells was detected.The Nrf2 overexpression stably transfected HepG2 cell line was constructed by lentivirus infection,and the effects of arenobufagin on the oxidative stress indicators of Nrf2 overexpression cells and the protein expressions of Nrf2,COX-2,and HO-1 were observed.Results1.Arenobufagin inhibited the growth of transplanted tumors.5-FU was used as a positive control drug.Compared with the control group,the high-dose arenobufagin and 5-FU groups could inhibit tumor growth,and the middle-dose arenobufagin could significantly inhibit tumor growth.The difference was statistically significant.Scientific significance(P < 0.05).Combined with the results of HE staining and Ki-67 immunohistochemistry,it was shown that arenobufagin can inhibit the growth of tumors in BALB/c Nude mice.2.Arenobufagin inhibits the proliferation of HepG2 cells.Taking HepG2 cells as the research object,it was detected by CCK8 that arenobufagin had a significant inhibitory effect on the cell proliferation of HepG2 cells,and the difference was statistically significant(P < 0.05).3.Arenobufagin induces ferroptosis in HepG2 cells.Western blot was used to detect the expression of ferroptosis-related proteins Nrf2 and COX-2 in BALB/c Nude mice transplanted tumors and HepG2 cells before and after treatment with arenobufagin.The expressions of Nrf2 and COX-2 were significantly decreased,and the difference was statistically significant(P < 0.05).Immunohistochemical detection of the expression of ACSL4 in transplanted tumors in mice demonstrated that arenobufagin induces ferroptosis in HepG2 cells from the level of protein expression.Immunohistochemical detection also found that arenobufagin increased the level of 4HNE in mouse transplanted tumors,indicating that arenobufagin increased the level of lipid peroxidation in mouse transplanted tumor tissues.Then,the content of ROS and lipid ROS,MDA,T-SOD,and GSH in HepG2 cells was detected,and it was found that arenobufagin increased the level of ROS and lipid ROS in a dose-dependent manner,the content of T-SOD and GSH decreased,and the content of MDA increased.The difference was statistically significant(P < 0.05).The morphological changes of xenograft tumor tissue and HepG2 cell mitochondria before and after treatment with arenobufagin were observed by transmission electron microscope.The ferroptosis inducer DFO verified the above results and found that DFO could partially reverse the changes of ferroptosis-related proteins and lipid peroxidation indicators caused by arenobufagin,and the difference was statistically significant(P <0.05).4.Arenobufagin induces autophagy-dependent ferroptosis in HepG2 cells.Western blot was used to detect the expressions of autophagy marker proteins p62 and Lc3 in transplanted tumors and HepG2 cells before and after treatment with arenobufagin.The expression of p62 decreased,while the value of Lc3Ⅱ/Lc3Ⅰincreased,and the difference was statistically significant(P < 0.05).The expression of p62 was detected by immunohistochemistry,and the results showed that arenobufagin could induce autophagy of transplanted tumor cells and HepG2 cells.At the same time,the autophagy inhibitor CQ was used to verify this result,and the results showed that CQ partially reversed the autophagy of HepG2 cells induced by arenobufagin,and the difference was statistically significant(P < 0.05).At the same time,CQ could partially reverse the changes in the expression of COX-2 and Nrf2 and the changes in lipid peroxidation indicators caused by arenobufagin,and the difference was statistically significant(P < 0.05).It shows that arenobufagin induces autophagy-dependent ferroptosis in HepG2 cells.5.Nrf2 is involved in arenobufagin-induced autophagy-dependent ferroptosis.Another result of CQ treatment showed that CQ could partially reverse the inhibition of arenobufagin on Keap1 and HO-1 in the p62-Keap1-Nrf2 pathway,and the difference was statistically significant(P < 0.05),indicating that arenobufagin regulates p62-Keap1-Nrf2 pathway;arenobufagin treated HepG2 cells with Nrf2 overexpression,the results showed that the effect of arenobufagin on the oxidative stress of HepG2 cells with Nrf2 overexpression was significantly less than that of the control group,and the difference was statistically significant(P < 0.05),indicating that arenobufagin regulates p62-Keap1-Nrf2 pathway induces ferroptosis.Conclusion:Arenobufagin promotes autophagy-dependent ferroptosis in HepG2 cells by regulating the p62-Keap1-Nrf2 pathway through induced autophagy. |
PDF Full Text Request