Study On The Mechanism Of Liver Cancer Cell Death Induced By Berberine Through Nrf2/GSK3β Pathway

Background Primary liver cancer is the sixth most common cancer worldwide and the third leading cause of cancer death,with an estimated 906,000 new cases and 830,000 deaths worldwide in2020.Primary liver cancer is currently the fourth most common malignant tumor and the second most lethal cause of cancer in China,putting a heavy burden on the family and the country.The treatment of primary liver cancer can be local hepatectomy,local chemoembolization,liver transplantation,as well as immunotherapy,targeted therapy and transformation therapy which are emerging in recent years.However,due to the hidden onset of hepatocellular carcinoma,most patients are diagnosed in the middle and late stage,so many treatment methods are limited.In addition to improving the early diagnosis rate,so that more patients can be found early,early treatment,exploring more diverse treatment is also the current urgent task of many scientific research and clinical workers.The curative effect of traditional Chinese medicine on cancer has been paid more and more attention by researchers in recent years.Berberine,a traditional Chinese medicine,has been proved to have good anti-cancer activity against a variety of malignant tumors,such as pancreatic cancer,cervical cancer,colorectal cancer,breast cancer and ovarian cancer.Studies have reported that berberine can regulate the life cycle of liver cancer cells by promoting apoptosis and autophagy of liver cancer cells,inhibiting the proliferation of liver cancer cells,regulating cell cycle,inhibiting tumor cell migration,inducing mitochondrial dysfunction and other ways to play an anticancer role.In our preliminary study,berberine was found to inhibit the activity of liver cancer cells while the reactive oxygen species(ROS)levels of liver cancer cells were elevated.There are still few studies on whether Nrf2/GSK3βand Nrf2/Keap1 signaling pathways related to ROS level changes are involved in the inhibitory process of berberine on liver cancer cells.Under the conditions of low glucose and low oxygen which are more suitable for tumor microenvironment,or even under the extreme glucose-free environment,there are no reports on the studies on the inhibitory activity of berberine on liver cancer and the induction of liver cancer cell death.Aims To study the effects of berberine on the cell activity and proliferation ability of liver cancer cell in normal medium and glucose-free medium,explore the changes of ROS related proteins in hepatocellular carcinoma after Berberine incubation,and reveal whether berberine induces liver cancer cell death through Nrf2/GSK3β and/or Nrf2/Keap1 signaling pathway and its mechanism.Methods1.The effects of berberine on the cell activity and proliferation of human normal liver cell lines MIHA and hepatoma cell lines Hep3B and HepG2 were studied by CCK8 assay and clonal formation assay.2.SYTOX Green nucleic acid dye kit was used to detect cell death of hepatoma cell lines Hep3B and HepG2 after incubation with berberine.3.The ROS levels of Hep3B and HepG2 were detected by flow cytometry after berberine incubation.4.Western blot method was used to detect the changes of Nrf2,HO-1,c-Myc,Keap1,GSK3β,P-GSK3β(Ser9),P-GSK3β(Tyr216)proteins after berberine incubation.5.The proteasome inhibitor MG132 and protein synthesis inhibitor Cycloheximide(CHX)were co-cultured with berberine to study its effects on Nrf2 and related proteins of hepatoma cell line Hep3B.6.Real-time quantitative fluorescence PCR(qRT-PCR)was used to investigate the changes of Nrf2 and HO-1 mRNA levels in hepatoma cell line Hep3B cells after the intervention of berberine.7.Transcriptional sequencing technology was applied to study the changes in Nrf2,HO-1and Keap1 gene expression levels of hepatoma cell line Hep3B cells after incubation with berberine.8.Hep3B cells were injected subcutaneously into BALB/c nude mice to establish tumor formation model.In the tumorigenic process,berberine or equal amount of normal saline was injected into the abdominal cavity of the experimental group and control group respectively to observe the effect of berberine on tumor growth in nude mice.9.Subcutaneous tumor tissues of nude mice were removed,paraffin-embedded sections were performed hematoxylin-eosin(HE)staining and immunohistochemical staining,and the effects of berberine on Nrf2 and related proteins were studied in nude mice.Results1.CCK8 experiment indicated that the activities of hepatoma cell lines Hep3B and HepG2 gradually decreased after berberine incubation.However,berberine had no significant inhibitory effect on the cellular activity of human normal liver cells MIHA.Berberine can significantly inhibit the clonal formation of hepatocellular carcinoma cells in the glucose-free environment,and with the increase of berberine concentration,the inhibitory effect on the clonal formation was strengthened.However,berberine did not significantly inhibit the cloning of human normal liver cells MIHA.2.SYTOX nucleic acid staining suggested that berberine incubation could promote the death of Hep3B and HepG2 cells,and the death of cells increased with the increase of berberine concentration.3.Flow cytometry indicated that the ROS levels of hepatoma cell lines Hep3B and HepG2 increased after berberine incubation,and the ROS levels gradually increased with the increase of berberine concentration.4.Western blot experiments indicated that after berberine treatment,Nrf2,HO-1 and cMyc protein levels of hepatoma cell lines Hep3B and HepG2 decreased,while Keap1 protein had no significant change.Further studies showed that protein GSK3β remained unchanged,while the inactivated state protein of P-GSK3β(Ser9)decreased and activated state protein of P-GSK3β(Tyr216)increased.5.The co-incubation of MG132 with berberine in Hep3B hepatoma cell line could significantly save the Nrf2 protein reduction of Hep3B cells induced by berberine;the Nrf2 protein downregulation of Hep3B cells induced by berberine could be further enhanced by CHX co-incubation with berberine.6.The qRT-PCR results showed that mRNA level of Nrf2 increased and mRNA level of HO-1 decreased after treatment with berberine in Hep3B cell line.7.Transcriptional sequencing results showed that after berberine incubation,Nrf2 gene expression in Hep3B cells increased,HO-1 gene expression decreased,and Keap1 gene expression level did not change significantly.8.The tumor formation experiment of nude mice showed that berberine could significantly inhibit the growth of tumor cells during the tumor formation process of nude mice by intraperitoneal injection of berberine compared with the injection of the same amount of normal saline.9.HE staining showed that the subcutaneous tumor necrosis increased after injection of berberine;immunohistochemical staining showed that Nrf2 and c-Myc were decreased and Keap1 remained unchanged after intraperitoneal injection of berberine in nude mice.Conclusion1.Berberine can inhibit the activity and proliferation of liver cancer cells and promote the death of liver cancer cells;but it had no obvious effect on human normal liver cells.2.Berberine can significantly inhibit the growth of tumor cells in nude mice,resulting in increased necrosis of tumor cells.3.Berberine may decrease the deactivated P-GSK3β(Ser9)and increase the activated PGSK3β(Tyr216)through the non-Keap1-dependent Nrf2/GSK3β pathway,resulting in the degradation of Nrf2 protein through ubiquitin-proteasome system and the decrease of HO-1 protein.Thus,the antioxidant stress ability of tumor cells is reduced,and the invasion ability of tumor cells is weakened by the decreased level of c-Myc protein,which ultimately leads to the death of liver cancer cells.