Lysosomal acidity perturbation activates a non-canonical Ca-mitochondrial apoptotic pathway.

Dysregulation of intracellular Ca signaling is a critical determinant of cell fate, however the contribution of non-canonical Ca 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 how lysosomal pH dysregulation a Ca-associated mitochondrial apoptotic program in triple-negative breast cancer (TNBC) cells. RTS exhibited superior selectivity compared with inorganic arsenic trioxide (ATO) and paclitaxel, significantly reducing the viability of TNBC cells (MDA-MB-231, BT-549, and MDA-MB-468) while sparing non-malignant MCF-10 A cells.. RTS-induced cell death was characterized by a Ca-dependent mitochondrial program-marked by cytochrome c release and caspase-9 activation-operating independently of reactive oxygen species accumulation and p53 signaling. Mechanistically, RTS triggered sustained cytosolic and mitochondrial Ca overload originating from lysosomal mobilization rather than extracellular influx or endoplasmic reticulum depletion. Time-course profiling identified lysosomal acidic intensification as an early event, preceding TRPML1-mediated Ca efflux and subsequent lysosomal membrane permeabilization (LMP). Consistently, pharmacological neutralization of the acidic shift (BafA1) or TRPML1 inhibition (ML-SI1) significantly attenuated the cytosolic Ca elevation observed at the measured intervals. Collectively, these in vitro findings establish a "lysosome-mitochondria" signaling axis in which early pH perturbation represents a potential vulnerability in TNBC. While the multicomponent nature of RTS requires further characterization, this study provides preliminary insights into targeting organelle-specific Ca hubs as a complementary strategy for refractory solid tumors.