Exploring the Ca2+ signaling and cytotoxicity induced by the alantolactone in breast cancer cells and its potential implications in treatment using the Ca2+ chelating agent BAPTA-AM.

Abstract

Alantolactone, a bioactive sesquiterpene lactone derived from the roots of Inula helenium (elecampane), has garnered attention in biomedical and pharmacological research for its diverse therapeutic properties, including anticancer, anti-inflammatory, antimicrobial, and antioxidant activities. Despite its well-documented bioactivity, the effects of alantolactone on calcium ion (Ca²⁺) signaling and the underlying mechanisms in human breast cancer cells remain poorly understood. This study explored how alantolactone influences intracellular Ca²⁺ levels ([Ca²⁺]_i), cell viability, and the role of Ca²⁺-dependent pathways in T-47D human breast cancer cells. Specifically, it examined the relationship between Ca²⁺ signaling and cytotoxicity in cells exposed to alantolactone, with or without the Ca²⁺ chelator BAPTA-AM. The findings reveal that alantolactone (25-75 μM) increases [Ca²⁺]_i in a concentration-dependent manner, while concentrations of 25-100 μM induce cytotoxicity, an effect that can be reversed by BAPTA-AM pre-treatment. Removing extracellular Ca²⁺ significantly inhibits Ca²⁺ influx, and both SKF96365 and 2-APB, modulators of store-operated Ca²⁺ channels, block the alantolactone-induced Ca²⁺ entry. Additionally, in a Ca²⁺-free environment, thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺ pump, suppresses the alantolactone-induced rise in [Ca²⁺]_i, while alantolactone reduces the [Ca²⁺]_i increase triggered by thapsigargin. Moreover, inhibiting phospholipase C (PLC) with U73122 abolishes the alantolactone-induced [Ca²⁺]_i elevation. These results suggest that alantolactone-induced cell death in T-47D cells is Ca²⁺-dependent, involving Ca²⁺ entry via store-operated channels and Ca²⁺ release from the endoplasmic reticulum, with PLC playing a pivotal role. Importantly, the ability of BAPTA-AM to reverse alantolactone's cytotoxic effects highlights its potential therapeutic significance in breast cancer research.