| Background and objectives:Based on the latest statistical data,the United States is projected to experience 82,290 new occurrences of bladder cancer,resulting in 16,710 deaths in 2023.Among male patients,bladder cancer remains among the top ten cancer types associated with high mortality rates.The preferred treatment options for bladder cancer are dependent on the tumor stage and may comprise surgical interventions,chemotherapy,radiation therapy,and immunotherapy.Despite considerable advancements in the treatment of bladder tumors in recent decades,the overall prognosis for patients with bladder cancer remains poor.Therefore,it is imperative to elucidate the underlying mechanisms of bladder cancer development and identify novel therapeutic targets or medications in order to improve patient outcomes.Tumor cells often exhibit a heightened basal level of reactive oxygen species(ROS)due to genetic mutations and abnormal growth patterns.Consequently,intracellular antioxidants,such as glutathione(GSH),are crucial for maintaining their survival and proliferation.In numerous studies,elevated GSH levels have been observed in bladder cancer,breast cancer,ovarian cancer,head and neck cancer,and lung cancer,as compared to normal tissues.GSH metabolism has been implicated in the initiation,progression,metastasis,and resistance to chemo-radiation therapy of diverse malignancies.The synthesis of GSH is influenced by several factors,including the activity of the glutamate cysteine ligase(GCL)enzyme,as well as the availability of precursor molecules.The glutamate cysteine ligase modulating subunit(GCLM)is one of the subunits of GCL and it has been revealed that GCLM-mediated GSH synthesis is essential for tumor development and has the potential to curb malignant transformation.Inhibitors of GSH synthesis can also achieve similar effects.Nuclear Factor Erythroid 2-related Factor 2(NRF2)serves as a pivotal regulator of cellular antioxidant response and activates the transcription of several genes,including Glutamate cysteine ligase catalytic subunit(GCLC),GCLM,and Glutathione synthetase(GSS),which are involved in GSH synthesis and contribute to oxidative stress resistance.Notably,functional enrichment analysis highlights the involvement of GSH metabolism and the NRF2 signaling pathway in the pathogenesis of bladder cancer.In particular,GCLM,which plays a role in ferroptosis regulation,is overexpressed in bladder cancer and associated with unfavorable clinical outcomes,as it promotes bladder cancer cell proliferation and migration.Therefore,targeting the NRF2/GCLM signaling pathway to modulate GSH metabolism may represent a promising therapeutic strategy for bladder cancer.The precise function of MTX-211,a dual inhibitor of EGFR and PI3K kinases,in bladder cancer remains incompletely understood,and its interplay with the regulation of GSH metabolism via the NRF2/GCLM signaling pathway is yet to be determined.Thus,the present study endeavors to investigate the effects of MTX-211 on bladder cancer cells through both in vitro and in vivo experimentation,to establish its impact on GSH metabolism,and elucidate the underlying mechanisms of regulation.Methods:1.The effects of MTX-211 on the biological behavior of bladder cancer cells(1)The toxicity of MTX-211 on bladder cancer cells,HUVEC,and SV-HUC-1was investigated by using a CCK8 assay to measure the drug’s effect on cell viability and determine the IC50 values for each cell type.(2)Cell cloning and EdU proliferation assays were used to assess the impact of MTX-211 on bladder cancer cell proliferation at various concentrations.(3)Flow cytometry was utilized to analyze the changes in apoptosis and cell cycle distribution of bladder cancer cells treated with MTX-211,while western blot experiments were conducted to investigate changes in the expression of apoptosis and cell cycle markers in these cells.(4)The impact of MTX-211 on subcutaneous tumor growth was determined through in vivo experiments involving nude mice,and HE staining and immunohistochemistry were performed to evaluate the drug’s potential toxic side effects on major organs and its effect on tumor Ki-67 expression.(5)The research also aimed to investigate the effect of MTX-211 on HUVEC cells’ in vitro angiogenesis ability and VEGFA expression in tumor cells.The bladder cancer cells were exposed to a combination of MTX-211 and cisplatin,and the CCK8 assay was utilized to determine the cell viability in different treatment groups.The combination index(CI)was computed based on the cell inhibition rate to assess whether MTX-211 and cisplatin had a synergistic effect.2.MTX-211 inhibits bladder cancer cell proliferation by interfering with GSH metabolism.(1)To investigate the effects of MTX-211,western blot was utilized to evaluate the expression of intracellular proteins such as EGFR,pEGFR,PI3K,pPI3K,AKT,pAKT,Erk,and pErk.(2)Transcriptome sequencing was performed to analyze changes in gene expression within cells treated with MTX-211.Bioinformatics methods were then utilized to investigate the potential signaling pathways involved.(3)The effects of MTX-211 treatment on intracellular levels of GSH,GSH/GSSG ratio,and ROS were evaluated using corresponding assay kits.(4)After supplementing with GSH in vitro,changes in cell viability following MTX-211 treatment were observed using the CCK8 assay.Apoptosis and cell cycle arrest were also analyzed using flow cytometry.3.MTX-211 suppresses GSH synthesis by promoting NRF2 ubiquitination and degradation,which inhibits GCLM transcription(1)Using a combination of joint sequencing data,a gene set related to the GSH metabolic pathway,and NRF2 target genes,we identified key genes that are influenced by MTX-211,specifically GCLM,GSR,and IDH1.We then investigated the co-expression relationship between these genes and NRF2 in bladder cancer using data from the TCGA database.To further explore this relationship,we employed RT-qPCR to assess the m RNA expression levels of NRF2,GCLM,GSR,and IDH1 in cells.And utilized western blot and immunohistochemistry assays to examine the expression of NRF2 and GCLM in cells and various groups of xenograft tumors respectively.(2)The cells were subjected to treatment with MG132,a proteasome inhibitor,to determine if the downregulated expression of NRF2 protein by MTX-211 treatment recovered.The impact of MTX-211 on the degradation rate of NRF2 protein was assessed using cycloheximide(CHX),a protein synthesis inhibitor.To evaluate the effect of MTX-211 on the ubiquitination level of NRF2 protein,co-immunoprecipitation(COIP)experiments were conducted.(3)The protein expression levels of p62,Keap1,and NRF2 within cells following exposure to MTX-211 was assessed through western blot and immunofluorescence assays.COIP experiments were also conducted to investigate the influence of MTX-211 on the binding of Keap1 and NRF2 proteins.(4)To investigate whether the induction of NRF2 ubiquitination and degradation induced by MTX-211 depends on Keap1 expression,bladder cancer cell lines with Keap1 knockdown and lung cancer cell line A549 with Keap1 mutation were subjected to MTX-211 treatment.The impact of p62 or NRF2 overexpression on the expression of proteins involved in the p62/Keap1/NRF2 signaling pathway was also analyzed.(5)We evaluated the expression of AKT,pAKT,GSK3β,pGSK3β,and β-TrCP after treatment with MTX-211 through western blot and assess the expression of β-TrCP and NRF2 using immunofluorescence experiments.We also investigated whether knocking down β-TrCP expression affected the ability of MTX-211 to inhibit NRF2 protein expression.Results:(1)The effects of MTX-211 on the biological behavior of bladder cancer cells.Bladder cancer cells exhibited greater sensitivity to MTX-211 treatment compared to normal epithelial cells,and the drug displayed concentration-and time-dependent inhibition of bladder cancer cell viability.Evidence from cell clone formation and EdU experiments suggested that the drug suppressed bladder cancer cell proliferation.Flow cytometry analysis revealed that MTX-211 induced apoptosis and G0/G1 phase arrest in bladder cancer cells and western blot results showed the upregulated expression of cleaved-caspase3,cleaved-PARP,and p21 proteins.Moreover,MTX-211 inhibited the growth of subcutaneous xenograft tumors in vivo,downregulated the expression of Ki67,and demonstrated no significant toxic side effects on vital organs in nude mice.Additionally,MTX-211 suppressed the in vitro angiogenic ability of HUV-EC cells and reduced the expression of VEGFA in tumor cells.Notably,MTX-211 showed a synergistic effect when used in combination with CDDP.(2)MTX-211 inhibits bladder cancer cell proliferation by interfering with GSH metabolism.The activation of EGFR and its downstream signaling pathways were found to be inhibited by MTX-211 via western blot assay.Transcriptional sequencing analysis was performed on 5637 cells with screening criteria of q-value < 0.05 and |log2FC| >1,revealing that MTX-211 upregulated 1536 genes and downregulated 694 genes.The bioinformatics analysis suggested that MTX-211 interfered with bladder cancer cell GSH metabolism processes and affected the expression of related genes.Further experiments confirmed that MTX-211 reduced intracellular GSH levels and GSH/GSSG ratio,and increased intracellular ROS levels.Additionally,rescue experiments demonstrated that supplementing GSH partially restored the inhibitory effect of MTX-211 on cell viability,induction of apoptosis,and G0/G1 phase arrest.(3)MTX-211 suppresses GSH synthesis by promoting NRF2 ubiquitination and degradation,which inhibits GCLM transcriptionFurther analysis of the sequencing data revealed that MTX-211 repressed the transcription of downstream target genes of NRF2.After intersecting the downregulated genes(log2FC <-1),GSH metabolism genes,and NRF2 target genes,three crucial genes,namely GCLM,GSR,and IDH1,were identified.The TCGA bladder cancer database indicated a positive correlation between NRF2 expression and the aforementioned genes.Nevertheless,further experimental validation demonstrated that only GCLM’s transcriptional expression was diminished in both 5637 and EJ cells post-treatment with MTX-211.This was confirmed by western blot and tumor immunohistochemistry,which indicated a decrease in the protein expression of both NRF2 and GCLM.To further elucidate the mechanism underlying MTX-211’s promotion of ubiquitination and degradation of NRF2,The use of MG132 fully restored the drug’s inhibitory effect on NRF2 expression.Furthermore,the combination with CHX revealed that MTX-211 increased the degradation rate of NRF2.The COIP results also indicated that MTX-211 treatment elevated the ubiquitination level of NRF2 protein.Mechanistically,MTX-211 promoted the ubiquitination and degradation of NRF2 protein by inhibiting p62 expression and increasing the expression of Keap1 protein,and facilitating the binding of Keap1 with NRF2,which was partially independent of Keap1 expression.Further experimental results indicated that MTX-211 activated GSK3β by inhibiting AKT phosphorylation,leading to the involvement of β-TrCP in the ubiquitination and degradation of NRF2 protein.Conclusion:1.MTX-211 effectively inhibits the proliferation of bladder cancer cells in vitro and in vivo,with low toxicity to normal cells or tissues.2.MTX-211 exerts its anti-proliferation effects in bladder cancer cells by interfering with intracellular GSH metabolism.3.MTX-211 inhibits the NRF2/GCLM signaling pathway by promoting the ubiquitination and degradation of NRF2 protein through the co-participation of p62/Keap1 and GSK3β/β-TrCP signaling pathways,leading to reduced intracellular GSH synthesis. |
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