Lysine p-nitroanilide impairs cellular energetics and potentiates statin-induced cytotoxicity in RD rhabdomyosarcoma cells

Abstract

Statins are clinically effective drugs for treating dyslipidemia and have been proposed as promising antineoplastic and adjuvant agents in cancer therapy for years due to their impact on dysregulated cell growth processes, including cell signaling, energetics, and membrane synthesis. Despite being potent inhibitors of mevalonate synthesis and its downstream products, their limited clinical success highlights the need to further explore their mechanistic effects. Leveraging the observed sensitivity of muscle cells to atorvastatin in clinical settings and utilizing untargeted metabolomic analysis of atorvastatin-treated RD rhabdomyosarcoma cells, we identified reduced levels of aminoadipic acid, an intermediate in lysine catabolism. We investigated whether metabolic sensitization of RD cells to lysine-related metabolites (lysine, aminoadipic acid, pipecolic acid, glutamic acid, α-ketoglutarate, and lysine-p-nitroanilide) prior to atorvastatin treatment enhances its cytotoxic effects. Metabolic sensitization or reprogramming involves cellular processes wherein cells adapt their metabolism to environmental changes, reflecting alterations in enzymatic activity, transport, and stress response thresholds. These adaptations enable cells to cope with specific environmental pressures but may impair their ability to respond to other stressors or stimuli. To evaluate the impact of metabolic supplementation, we analyzed cellular stress response markers via western blot. The results revealed that lysine-p-nitroanilide increased BiP, the master regulator of the unfolded protein response, and augmented the phosphorylation at threonine 172 of AMPK, an indicator of altered cellular energetics. Further analysis demonstrated that combining lysine-p-nitroanilide with atorvastatin disrupted mitochondrial homeostasis and reduced glycolysis, both desirable outcomes in antineoplastic treatments. Lysine-p-nitroanilide acts as an in vitro inhibitor of α-aminoadipic semialdehyde synthase, enzyme essential for lysine metabolism via the saccharopine pathway. However, we demonstrated that it is catabolically cleaved to p-nitroanilide, with this molecule driving the cytotoxic activity observed in our experiments. Although lysine metabolism was not fully suppressed by lysine-p-nitroanilide, these findings provide valuable insights for developing novel therapies for rhabdomyosarcoma.