<em>Euphorbia bicolor</em> Diterpene Extract Induces Mitochondrial and Endoplasmic Reticulum Stress-Mediated Apoptotic Pathways in MDA-MB-231 and T47D Cells

Breast cancer is a significant cause of death worldwide. Recent research has focused on identifying natural compounds for developing effective cancer treatments. Resiniferatoxin, a TRPV1 agonist, is a common diterpene in Euphorbia bicolor, a plant native to the southern United States that has not been studied before. We investigated the antiproliferative activities and mechanisms of action of E. bicolor diterpene extract in estrogen receptor-positive T47D and triple-negative MDA-MB-231 cell lines. The extract significantly reduced the viability of T47D and MDA-MB-231 cells in a dose-dependent manner. In MDA-MB-231 cells, the extract induced apoptosis via intracellular calcium overload, triggered by TRPV1 activation. This effect was diminished by the TRPV1 antagonist capsazepine and the calcium chelator BAPTA-AM. Intracellular calcium influx was confirmed through Fura-2 AM staining, revealing that E. bicolor phytochemicals activated TRPV1 in MDA-MB-231 cells. Treatment of T47D cells with E. bicolor diterpene extract resulted in apoptosis associated with ROS generation (10-fold higher in T47D cells than in MDA-MB-231 cells) and mitochondrial calcium overload. These effects were significantly blocked when cells were pretreated with NAC (N-acetyl-l-cysteine), a ROS inhibitor. Both cell lines initiated apoptosis through multiple mitochondrial and endoplasmic reticulum-stress-mediated apoptotic pathways, demonstrated by the expression of activated caspase 3, caspase 9, caspase 8, FAS, XBP1s, CHOP, overexpression of BAX proteins and reduction of BCL-2 levels. In addition, PI3K, AKT, and pAKT protein expressions were also reduced in both cell lines, indicating downregulation of PI3K/Akt signaling pathway. Phytochemicals in E. bicolor diterpene extract could become promising ingredients for developing breast cancer therapeutics.