Background High production of hydrogen sulfide (H₂S), an endogenous gasotransmitter, has been suggested to promote chemoresistance in non-muscle-invasive bladder cancer (NMIBC), resulting in high recurrence rate and limited long-term success of current therapies. The present study investigated the therapeutic potential of targeting H₂S production through pharmacological inhibition of the H₂S-producing enzyme cystathionine γ-lyase (CSE) using DL-propargylglycine (PAG) in both in vitro and in vivo models of NMIBC. Methods In vitro, human bladder cancer cells (5637 & T24) were treated with 20, 60, and 100 mM PAG and proliferation was measured using a CCK-8 assay over 72 h. Migration was also measured using a scratch wound-healing assay in cells treated with 100 mM PAG. 5637 cells were treated with 1 μM gemcitabine (GEM) with or without 100 mM PAG. After 24 and 48 h, cell viability, H2S and ROS levels, and qPCR of H₂S-producing enzymes were measured. In vivo, male C57BL/6 mice received 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) for 12 weeks to induce bladder tumours. Tumor burden was measured by MRI before and after 6 weeks of weekly intravesical saline, PAG, GEM, Bacillus Calmette–Guérin (BCG), or combination therapy. Results In vitro, treatment of human bladder cancer cells with PAG significantly attenuated cell proliferation and migration. Exposure to GEM increased CBS and CSE expression at 24 and 48 h (p < 0.05). Also, GEM treatment significantly increased intracellular H₂S levels while co-treatment with PAG markedly suppressed this response (p < 0.05), leading to enhanced GEM cytotoxicity, mediated by increased ROS production. In vivo, GEM + PAG was associated with reduced tumour growth compared with either treatment alone, while the addition of BCG exhibited the lowest overall tumour growth. Conclusions PAG attenuated human bladder cancer cell proliferation and migration, enhanced the cytotoxic effects of GEM, and was associated with reduced tumour growth when combined with GEM and BCG. Our findings warrant further investigation of targeting H₂S-producing enzymes and intracellular H2S production as a novel pharmacological approach to enhance the cytotoxic effects of GEM in the treatment of NMIBC.
Bedaquiline approval paved the way for the development of drug candidates against energy metabolism pathways of TB-causing organisms, and oxidative phosphorylation was proven to be a relevant target domain. While bedaquiline acts on the F1F0 ATP synthase, Q203 targets the cytochrome bc1-aa3 terminal oxidase, which mediates electron transport to O2 in Mycobacterium tuberculosis. However, due to functional redundancies between cytochrome bc1-aa3 and a second terminal oxidase, the cytochrome-bd, the sterilization effect of Q203 is impaired. Here, we describe the identification of BD-2, a novel small molecule inhibitor of the cytochrome-bd in a cell-based screening campaign. Alone, BD-2 does not exhibit any significant activity. When paired with Q203, however, BD-2 strongly inhibits Mycobacterium tuberculosis respiratory chain function and ATP synthesis. Moreover, BD-2 combined with Q203 was shown to possess potent bactericidal effect on both replicating and nonreplicating Mycobacterium tuberculosis, including those living under nutrient starved and hypoxic conditions, representing the persistent forms found in granulomas of patients. Mechanistic analysis included molecular docking studies using the known structure of Mycobacterium tuberculosis cytochrome-bd, in vitro activity testing on purified enzyme, all of which confirmed that BD-2 is a direct cytochrome-bd inhibitor. Furthermore, the BD-2/Q203 combination was effective against intracellular bacteria in an in vitro macrophage infection model based on THP-1 cells, with over 90% reduction in bacterial load achieved at clinically relevant concentrations. This study supports the hypothesis of combining a cytochrome-bd inhibitor and Q203 in tuberculosis therapy as a rational, mechanistically driven approach that might result in more efficient treatment and overcome antibiotic tolerance.
As a lactate-derived post-translational modification, lactylation plays a vital role in connecting cellular glycolysis with the regulation of cuproptosis, a unique form of cell death triggered by copper overload. The article explores the growing relationship between lactylation and cuproptosis, highlighting the potential for lactate metabolism to influence cellular responses to copper toxicity. We further discuss the potential roles of lactylation writers, erasers and readers in modulating key cuproptosis-related proteins, such as FDX1, DLAT and CTR1, through the specific actions of molecular players including METTL16, NAT10, and NUDT21 across transcriptional regulation, alternative polyadenylation (APA), and m6A modification pathways. Further analysis of the lactate paradox involves exploring the roles lactate plays in various tumor microenvironments and disease contexts. Clinically, this proposed metabolic-epigenetic interplay could be targeted with lactate metabolism inhibitors or copper ionophores to potentially provide synergistic therapeutic strategies for cancer and metabolic diseases. This metabolic–epigenetic interplay could shed light on disease progression and open up new possibilities for precision medical approaches.
Background Oxidative stress is a central mechanism of chronic toxicity, disrupting metabolic, inflammatory, apoptotic, mitochondrial, and epigenetic processes. Potassium bromate (KBrO₃) induces oxidative stress, but its systemic effects following chronic exposure and modulation by metformin remain insufficiently characterized. This study evaluated chronic KBrO₃ toxicity in zebrafish, the effects of concurrent metformin exposure, and molecular responses in F1 offspring following parental exposure. Methods Adult zebrafish were exposed to KBrO₃ for 28 days with or without metformin co-treatment. Histopathological analyses of liver, ovary, intestine, and testis were performed. IL-1β, insulin, and vitellogenin were analyzed by ELISA, wheras oxidative stress, metabolic, inflammatory, apoptotic, and epigenetic markers by qPCR. Global DNA methylation, mitochondrial DNA copy number, and relative telomere length were evaluated in F0 and F1 cohorts. Data were analyzed using conventional statistics, principal component analysis, and Spearman correlation. Results KBrO₃ exposure was associated with alterations in antioxidant defence, metabolism, inflammation, apoptosis, mitochondrial and epigenetic endpoints, and tissue pathology. Metformin co-treatment attenuated multiple KBrO₃-associated molecular and histopathological alterations. In F1 offspring, selected gene-expression and DNA methylation alterations were observed, whereas mtDNA copy number and relative telomere length did not differ significantly among groups, indicating endpoint-specific intergenerational responses. Multivariate analyses identified coordinated biomarker patterns associated with treatment. Conclusion Chronic KBrO₃ exposure produced coordinated molecular and tissue alterations, while metformin co-treatment attenuated several associated responses. Selected F1 molecular and epigenetic alterations indicate intergenerational responses following parental exposure, although F2/F3 generations were not assessed. These findings support further investigation of metformin as a pharmacological modulator of oxidative stress-associated toxicity.
Given the expanding clinical use of orexin-modulating drugs, this study investigated whether orexin agonism or antagonism influences inflammatory responses in vitro and in vivo. PBMCs stimulated with LPS or TNF were treated with orexin agonists or antagonists. In rats, pharmacokinetic/pharmacodynamic studies assessed OXA 17–33 pharmacokinetics and effects on adrenocorticotropin (ACTH) and corticosterone. In collagen-induced arthritis (CIA), rats received OXA 17–33, lemborexant, methylprednisolone, placebo, or no treatment. Disease progression was evaluated using clinical scores, paw width, mechanical allodynia, serum cytokines, and synovial gene and protein expression. OXA 17–33 reduced TNF production in LPS-stimulated PBMCs up to 33.4 ± 12.3% of control (P < 0.001) at a 5 µM concentration. Its pharmacokinetics resembled those of orexin A, with a half-life of 0.48 ± 0.07 h, volume of distribution of 1.84 ± 1.38 L/kg, and clearance of 103.74 ± 45.18 mL/min/kg. Following intravenous administration, OXA 17–33 increased ACTH and corticosterone (57.6 ± 15.0 pg/mL and 256.6 ± 85.9 ng/mL vs 23.9 ± 10.0 pg/mL and 79.4 ± 39.5 ng/mL; both P < 0.05). Subcutaneous dosing significantly increased corticosterone, with a nonsignificant ACTH increase. In CIA, OXA 17–33 produced modest improvements versus placebo, including lower arthritis scores (8.6 ± 0.5 vs 12.0 ± 3.4; P = 0.08), reduced synovial STAT3 expression (P < 0.05), and decreased ERK1/2 and pERK1/2 expression. OXA 17–33 exerts modest anti-inflammatory effects in PBMCs, shows orexin-like pharmacokinetics, and activates the HPA axis. It provided modest prophylactic benefit in CIA, less pronounced than corticosteroid treatment. Lemborexant showed no detrimental effect of OX1R/2 R blockade.
