Abstract 4611: Novel lysosomotrophic agent inhibits in vivo tumor formation and triggers calcium-dependent cell death in a variety of human cancer cell lines

Abstract

Abstract In addition to being investigated as probes for diagnostic imaging, lanthanide family of rare earth compounds were shown to be tumor-selective, however the mechanism of anti-cancer activity remains unclear. We synthesized a conjugate of the lanthanide, praseodymium (141Pr), and observed remarkable anti-tumor activity in 2 murine xenograft models (colorectal and prostate cancer), with minimal off target effects. Importantly, Pr-mercaptopyridine oxide (Pr-MPO) elicited strong anti-cancer activity (IC50∼2.5uM) against a host of human cancer cell lines including cisplatin resistant, p53-/- and/or Bax-/- variants. Intrigued by this, we investigated the mechanisms underlying the anti-cancer activity of Pr-MPO. Our earlier findings indicated weak PPARγ agonist-like activity despite any structural similarity to known PPARγ agonists, therefore, we first investigated the effect of Pr-MPO on cell metabolism. An increase in glucose uptake and a significant decrease in pyruvate levels were observed. Indeed, pyruvate delayed cell death while restoring a regulated activity similar to non-treated conditions. Despite the PPARγ agonist-like activity, the anti-cancer effect of Pr-MPO was not dependent on PPARγ signaling. Furthermore, while exposure to Pr-MPO resulted in a drastic reduction in tumor colony forming ability, cell death was caspase independent and associated with a strong autophagic phenotype. Despite the induction of mToR-dependent autophagy, confirmed by western blot analysis of LC3II, p62, and puncta formation upon GFP-LC3 transfection, neither pharmacological inhibition nor the knockdown of Atg7/5 blocked Pr-MPO-induced cell death. Interestingly, Pr-MPO induced a significant increase in intracellular calcium fluorescence, measured by time resolved live imaging, however, unlike thapsigargin, the increase in calcium was not mediated by ER calcium stores. Instead, a significant increase in calcium fluorescence (puncta) was observed within minutes, a pattern distinctly different from the diffuse cytosolic increase upon depleting ER calcium. Notably, chelation of extracellular calcium abrogated the increase in intracellular calcium as well as rescued cells from Pr-MPO-induced cell death, thus suggesting the involvement of store operated calcium entry. Further analysis indicated these puncta being associated with the lysosomes. Therefore, we turned our focus on lysosomes as the “organelle preferé” in Pr-MPO induced cell death. Indeed, Pr-MPO resulted in the induction of lysosomal membrane permeability, which was associated with an overall increase in cathepsin L activity. As the increase in lysosomal calcium fluorescence is rapidly induced by Pr-MPO, a possible target could be a membrane ATPase or the rapid increase in fluorescence could be an indicator for the subsequent release of calcium. These possibilities are the current focus of investigation. Citation Format: Dan Liu, Gregory L. Bellot, Sanjiv K. Yadav, Mark Fivaz, Charnjit Kaur, Gautam Sethi, Shazib Pervaiz. Novel lysosomotrophic agent inhibits in vivo tumor formation and triggers calcium-dependent cell death in a variety of human cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4611. doi:10.1158/1538-7445.AM2014-4611