Brucine Triggers Ferroptosis In Hepatocellular Carcinoma Cells Via Activation Of Autophagy By AMPK

Background:Primary liver cancer is one of the most prevalent malignancies with the highest mortality rates.It is the 6th most common malignant tumour diseas worldwide and the 2nd most common causes of death each year.And more than 50%of all newly confirmed deaths occur in China.Hepatocellular carcinoma(HCC)is the pathological type of 85-90%of liver cancer.There are many different treatments for HCC,including surgical procedures such as hepatectomy,liver transplantation,TACE,RFA,etc.,but they can also be combined with targeted and immunotherapy and radiotherapy.In recent years,some progress has been made in the clinical treatment of HCC,but since hepatectomy and liver transplantation have strict indications and contraindications for surgery and are only suitable for a small number of patients,and most HCC patients are already in advanced stages of tumour when diagnosed,or have severe liver insufficiency and other underlying diseases,they cannot undergo hepatectomy or liver transplantation,and need systematic systemic comprehensive anti-tumour therapy in order to reduce tumour stage,improve tumour-related symptoms,improve quality of life and prolong survival,including chemotherapy,molecular immunotherapy,radiotherapy and adjuvant anti-tumour therapy with Chinese medicine.Although new anti-cancer drugs have been introduced for HCC,the overall prognosis of HCC is still unsatisfactory,so research into new anti-cancer drugs,new targets and related mechanisms is imminent.Ferroptosis is a newly discovered form of cell death regulation that is usually accompanied by excessive intracellular production of free Fe2+ and lipid peroxidation,a form of death associated with many pathological processes,including cancer,degenerative diseases,ischaemia-reperfusion injury,etc.Morphologically,cells undergoing ferroptosis have normal-sized nuclei and normal chromatin morphology,but there are alterations in mitochondrial condensation and increased mitochondrial membrane density,so they differ from apoptosis and pyroptosis.Biochemically it is characterised by an abnormal accumulation of intracellular Fe2+ and an excessive production of lipid peroxides.Since the concept of ferroptosis was introduced,ferroptosis has been closely associated with antitumour,and is now known to act as a form of death in a variety of malignancies including lung,colorectal and breast cancers,and even as an effective antitumour agent in a number of cancers that have become resistant to conventional antitumour drug treatment.Thus,ferroptosis inducers could hold considerable promise as potential targets for therapeutic agents for HCC.As an important way for cells to maintain their own homeostasis,autophagy is closely associated with various modes of cell death.It has been reported that autophagy plays a key role in the onset and progression of ferroptosis,and the concept of autophagy-dependent ferroptosis has also been proposed,which may be related to selective ferritin autophagy,intracellular reactive ROS production,and complex redox reactions and energy changes during ferroptosis.Adenylate-activated protein kinase(AMPK)acts as an energy sensor,regulating energy homeostasis and metabolic stress through a variety of endostasis mechanisms including control of autophagy and protein degradation.mTOR plays an important role in integrating metabolic,energy,hormonal and nutrient signals to facilitate biosynthetic pathways and inhibiting autophagic catabolic processes.AMPK-mTOR signalling pathway,as an upstream pathway of autophagy,participates in and initiates autophagy.AMPK negatively regulates mTOR by promoting AMPK phosphorylation,decreasing mTOR phosphorylation levels,triggering autophagic flux and thus activating autophagy.However,whether there are changes in AMPK in ferroptosis,whether it can regulate the progression of ferroptosis,and its related specific mechanisms are not yet clarified.Brucine,a natural small molecule compound,was originally extracted from Strychnos nux-vomica L seeds and is weakly alkaline in nature.Strychnos nux-vomica L is used as a traditional herbal ingredient,the active ingredient in the treatment,Brucine,is generally used for the relief of pain associated with inflammation and trauma etc.Some studies have shown that Brucine has some antitumour activity and is now known to exert considerable antitumour effects on various types of tumours including colon adenocarcinoma,breast cancer and glioma through mechanisms such as affecting tumour cell proliferation,inhibiting cancer cell invasion and migration and anti-angiogenesis.However,it remains unclear whether Brucine has antitumour effects on HCC and whether both ferroptosis and AMPK are involved in Brucine-induced cell death in HCC.Objective:The objectives of this study were to investigate whether Brucine can induce HCC cell death,whether ferroptosis is present in the mode of death it induces,and the specific mechanism and role of AMPK in its induction of ferroptosis.Methods:1.MTT and Colony formation assay:Calculate IC50 and detect the toxic effect of Brucine on HCC cells and normal hepatocytes.2.LDH release assay:to detect the killing effect of Brucine on HCC cells and the difference in the killing effect of Brucine on HCC cells after pretreatment with inhibitors etc.3.Fe2+,ROS,MDA assay:The relationship between Brucine induced ferroptosis and autophagy in HCC cells was investigated by detecting the changes in Fe2+,ROS and the degree of lipid peroxidation in HCC cells after Brucine and Brucine combination with inhibitors.4.GSH assay:The effect of Brucine on the antioxidant system of HCC cells was verified by the effect of Brucine on the GSH content in HCC cells.5.Western Blotting:To analyse the relationship and interaction between Brucine induced ferroptosis and autophagy in HCC cells by detecting the differences in iron metabolism and autophagy related protein expression in samples after Brucine and co-added inhibitors and knockdown of ATG5/AMPK.6.C11-BODIPY581/591 staining,ROS fluorescence assay:detection of Brucine and changes in the degree of induced cellular lipid peroxidation and ROS in various HCC cell lines after pretreatment with ferroptosis specific inhibitors by fluorescence microscopy.ROS fluorescence showed that Brucine could induce elevated ROS levels in HCC cells.7.siRNA silencing method:The relationship and interaction between autophagy and ferroptosis was investigated by detecting changes in the corresponding indexes of HCC cells under Brucine induction after siRNA-targeted knockdown of ATG5 and AMPK.8.Subcutaneous tumor loading assay in nude mice:The mouse HCC metastatic tumor model was established by subcutaneous tumor loading,and the antitumor effect of Brucine in the HCC in vivo model was observed by intraperitoneal injection;the related Fe2+ and MDA contents were detected by tumor tissues,and the corresponding protein expression in tumor tissues was examined by Western Blotting;the effects of Brucine on mouse heart,liver,lung,spleen and kidney were examined by HE staining;the effects of Brucine on HCC cell proliferation in vitro were investigated by Ki 67 staining.Results:1.The results of MTT assay suggest that Brucine can inhibit the proliferation ability of HCC cells.2.The results of LDH assay suggest that Brucine can induce HCC cell death,and ferroptosis specific inhibitors(DFO,Fer-1),autophagy specific inhibitors(3MA,Baf-A1)and AMPK specific inhibitors(Dor)can effectively inhibit Brucine-induced HCC cell death;siRNA knockdown of ATG5 and AMPK can also effectively inhibit Brucine-induced HCC cell death.3.The results of Fe2+assay suggested that Brucine could induce an increase in Fe2+concentration in HCC cells in a time-and concentration-dependent manner,and that ferroptosis-specific inhibitors(DFO,Fer-1),autophagy-specific inhibitors(3-MA,Baf-A1)and AMPK-specific inhibitors(Dor)could effectively inhibit Brucine-induced increase in Fe2+ concentration in HCC cells;siRNA knockdown of ATG5 and AMPK could also effectively inhibit Brucineinduced increase in Fe2+ concentration in HCC cells.4.The ROS assay suggested that Brucine could induce an increase in ROS content in HCC cells in a time-and concentration-dependent manner,and that ferroptosis-specific inhibitors(DFO,Fer-1),autophagy-specific inhibitors(3MA,Baf-A1)and AMPK-specific inhibitors(Dor)could effectively inhibit Brucine-induced increase in ROS content in HCC cells;siRNA knockdown of ATG5 and AMPK could also effectively inhibit Brucine-induced increase in ROS content in HCC cells.5.The detection of MDA suggested that Brucine could induce lipid peroxidation in HCC cells in a time-and concentration-dependent manner,and that ferroptosis-specific inhibitors(DFO,Fer-1),autophagy-specific inhibitors(3MA,Baf-A1)and AMPK-specific inhibitors(Dor)could effectively inhibit Brucine-induced lipid peroxidation in HCC cells;siRNA knockdown of ATG5 and AMPK could also effectively inhibit Brucine-induced lipid peroxidation in HCC cells.6.The results of GSH assay suggest that Brucine can reduce the level of GSH in HCC cells in a time-and concentration-dependent manner.7.C11-BODIPY581/591 staining results suggest that Brucine-induced lipid peroxidation in HCC can be inhibited by ferroptosis-specific inhibitors(DFO,Fer-1).8.The results of western blotting suggested that there were differences in the expression of autophagy-related proteins,iron metabolism-related proteins and antioxidant-related proteins in HCC cells induced by Brucine alone and after pretreatment with specific inhibitors and siRNA-targeted knockdown at fixed concentrations for a fixed period of time.9.Subcutaneous tumor-bearing assay in nude mice:It was suggested that Brucine inhibited the proliferation of HCC cells in vivo after intraperitoneal injection and was accompanied by a concentration-dependent increase in Fe2+ and MDA levels in tumor tissues.The non-significant structural damage of Brucine on mouse heart,liver,lung,spleen and kidney was examined by HE staining;Ki 67 staining was used to investigate that Brucine significantly inhibited the proliferation of HCC cells in an in vitro assay.Conclusion:1.Brucine inhibits the proliferative capacity of HCC cells and induces HCC cell death.2.Brucine induces ferroptosis in HCC cells.3.Brucine induces autophagic cell death in HCC cells.4.Brucine mediates autophagy in HCC cells through activation of AMPK and regulates ferroptosis.