Investigation Of The Role And Mechanisms Of PTPMT1 Inhibitor Alexidine In Sensitizing Hepatocellular Carcinoma Cells To Ferroptosis

Background and Objective:Hepatocellular carcinoma（HCC）stands as the predominant primary liver cancer,exhibiting a rising incidence trend.This invasive malignancy is associated with poor prognosis,particularly in advanced stages,where effective treatment options are lacking.Addressing this challenge requires the exploration of novel therapeutic approaches and targets.Ferroptosis is a different form of cell death from apoptosis,necrosis,and pyroptosis,and studies have found that many existing anticancer drugs in clinical practice can also exert their anti-cancer effects by inducing ferroptosis.However,chemotherapy resistance remains a significant clinical hurdle,underscoring the need to unravel ferroptosis regulatory mechanisms and establish innovative sensitization models,particularly in the realm of cancer biology.Mitochondria,recognized for their pivotal role in ferroptosis,spotlight Protein Tyrosine Phosphatase Mitochondrial 1（PTPMT1）,a member of the protein tyrosine phosphatase superfamily residing in the inner mitochondrial membrane.Targeting PTPMT1 presents an opportunity to forge a unique regulatory framework for ferroptosis.Alexidine dihydrochloride（AD）,the small molecule inhibitor targeting PTPMT1,shows promise in cancer treatment.However,the precise regulatory functions and key mechanisms of AD in ferroptosis necessitate thorough investigation.Thus,this study aims to delve into the role and core mechanisms of the PTPMT1 inhibitor AD in inducing ferroptosis in liver cancer,providing fresh potential targets and theoretical support for hepatocellular carcinoma treatment.Methods:（1）Evaluate liver cancer cell sensitivity to cysteine-deficient ferroptosis induced by the PTPMT1 inhibitor AD,utilizing techniques like cell viability analysis and lipid peroxidation detection.（2）Explore the effects and key regulatory mechanisms of AD on the stability and half-life of PTPMT1 protein from the perspectives of PTPMT1 synthesis and degradation.（3）Investigate the impact of AD on classical ferroptosis-related targets, uncovering potential mechanisms of AD in sensitizing cysteine-deficient ferroptosis.（4）Analyze the influence of AD on the expression of functional genes in liver cancer cells through next-generation transcriptome sequencing,presenting volcano plots,GO enrichment analysis,and heatmaps to elucidate the biological effects of AD on liver cancer cells.（5）Examine the effects of AD on mitochondrial morphology,biogenesis, autophagy,fission,and fusion,probing its impact on mitochondrial tricarboxylic acid（TCA）cycle metabolism.（6）Construct a hepatocellular carcinoma-bearing mouse model to systematically evaluate the effects of AD and Erastin combination therapy on tumor growth and TCA cycle metabolism in mice.Results:（1）In wild-type liver cancer cells,the PTPMT1 inhibitor AD increases sensitivity to Erastin-mediated ferroptosis.Conversely,in PTPMT1 knockout cells,AD fails to exhibit a similar sensitization effect,highlighting the dependence of AD’s ferroptosis-sensitizing action on the PTPMT1 signaling pathway.（2）AD does not affect the transcriptional level of PTPMT1 protein but rather promotes its degradation,significantly reducing the half-life of PTPMT1 protein,leading to a substantial decrease in intracellular PTPMT1 protein content.（3）AD does not impact the expression levels of classical ferroptosis regulatory factors（GPX4,SLC7A11,DHODH,FSP1）.Under conditions of unaffected cell autophagy,AD reduces the protein level of ferritin light chain（FTL）while increasing the content of unstable iron pools within cells.（4）RNASeq results demonstrate that AD treatment significantly affects the transcriptional levels of mitochondrial-related genes.GO enrichment analysis indicates that related genes predominantly cluster into pathways related to mitochondrial m RNA stability,ATP metabolism,mitochondrial membrane stability,and mitochondrial autophagy.（5）AD treatment induces reduced connectivity of the mitochondrial network in liver cancer cells,resulting in mitochondrial fragmentation in the form of large vacuoles and donut-shaped structures.AD treatment increases the content of PINK1 in cells,promoting the recruitment of MFN1 to mitochondria.Additionally,AD activates the enzymatic activity of succinate dehydrogenase（SDH）,facilitating the conversion of succinate to fumarate,and increasing intracellular fumarate content,thereby enhancing cell sensitivity to Erastin-induced ferroptosis.（6）The combined application of AD and Erastin significantly inhibits tumor growth in mice,with results aligning with cellular-level research.In the in vivo environment,AD also promotes the conversion of succinate to fumarate,thereby enhancing the sensitivity of liver cancer cells to ferroptosis.Conclusions:This study establishes that the PTPMT1 inhibitor AD increases HCC sensitivity to cysteine-deficient ferroptosis by regulating mitochondrial TCA cycle and unstable iron pools.AD decreases FTL protein levels,promotes the conversion of ferritin-bound Fe³⁺to free Fe²⁺.Simultaneously,AD induces the formation of swollen and donut-shaped mitochondria,enhances SDH enzymatic activity,promoting the metabolic process of succinate to fumarate in the mitochondrial TCA cycle.The upregulation of fumarate content enhances HCC cell sensitivity to ferroptosis induced by cysteine deficiency.In summary,our work unveils the close association of the PTPMT1 inhibitor AD with cysteine-deficient ferroptosis,providing new insights for HCC chemotherapy strategies and offering theoretical references and technical support for treating other drug-resistant tumors.