Abstract
Breast cancer is a significant cause of death worldwide. Recent research has focused on identifying natural compounds for developing effective cancer treatments. Resiniferatoxin, a transient receptor potential vanilloid 1 (TRPV1) agonist, is a common diterpene in Euphorbia bicolor Engelm. & A. Gray (Euphorbiaceae), 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 xylene 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 xylene extract resulted in apoptosis associated with reactive oxygen species (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 N-acetyl-l-cysteine (NAC), a ROS inhibitor. Both cell lines underwent apoptosis via multiple mitochondrial- and endoplasmic reticulum stress-mediated pathways. This was supported by the activation of caspases 3, 8, and 9; increased expression of FAS, XBP1s, and CHOP; upregulation of BAX; and downregulation of BCL-2. 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 xylene extract could become promising ingredients for developing breast cancer therapeutics.