Evaluation of hydrogen sulfide inhibition as an adjunct to gemcitabine and BCG therapy in non-muscle-invasive bladder cancer.

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.