Realgar Transforming Solution (RTS)-induced lysosomal pH perturbation activates a non-canonical Ca²⁺-associated...

Dysregulation of intracellular Ca²⁺ signaling is a critical determinant of cell fate; however the contribution of non-canonical Ca²⁺ reservoirs to cancer-selective apoptosis remains incompletely understood. In this study, realgar transforming solution (RTS), a microbially processed arsenical, was employed as a biologically informative perturbation to investigate the potential link between lysosomal pH dysregulation and a Ca²⁺-associated mitochondrial apoptotic program in triple-negative breast cancer (TNBC) cells. RTS displayed selective inhibitory activity compared with inorganic arsenic trioxide (ATO) and paclitaxel, leading to reduced viability of TNBC cells (MDA-MB-231, BT-549, and MDA-MB-468) while showing minimal impact on non-malignant MCF-10 A cells. RTS-induced cell death was linked to a Ca²⁺-mediated mitochondrial program-marked by cytochrome c release and caspase-9 activation-while showing limited correlation with reactive oxygen species (ROS) accumulation or p53 signaling. Mechanistically, RTS triggered sustained cytosolic and mitochondrial Ca²⁺ overload derived primarily from lysosomal mobilization rather than extracellular influx or endoplasmic reticulum depletion. Time-course profiling observed lysosomal acidic intensification as an early event, preceding TRPML1-mediated Ca²⁺ efflux and subsequent lysosomal membrane permeabilization (LMP). Consistently, pharmacological neutralization of the acidic shift (BafA1) or TRPML1 inhibition (ML-SI1) significantly attenuated the cytosolic Ca²⁺ elevation observed at the measured intervals. Collectively, these in vitro findings highlight a potential"lysosome-mitochondria" signaling axis in which early pH perturbation may represent a vulnerability in TNBC. While the multicomponent nature of RTS requires further characterization, this study provides preliminary insights into targeting organelle-specific Ca²⁺ hubs as a possible complementary strategy for refractory solid tumors.