Thunb. Hexane fraction induces MDA-MB-231 cell apoptosis via caspases, ER stress, cell cycle arrest and attenuated Akt/ERK signaling.

Thunb. (HCT) is a perennial plant used in traditional Thai medicine for many centuries. This study aimed to investigate the antiproliferative effect of the hexane fraction, which has not been explored before. HCT ethanol extract (crude extract) was sequentially fractionated to obtain a hexane (H) fraction. GC-MS was used to determine the phytochemicals. The H fraction consisted of lipids, mainly α-linolenic acid and some terpenoids. MTT assay was used to determine the cytotoxic effects of H fraction in MCF-7, MDA-MB-231, NIH3T3 and PBMCs. The mode of cell death and cell cycle analysis were determined by flow cytometry. The mechanisms of cell death were defined by mitochondrial transmembrane potential (MTP) reduction and activation of caspase-3, -8 and -9. The expression levels of the Bcl-2 family, cell cycle-related, endoplasmic reticulum (ER) stress-associated proteins; and Akt/ERK signaling molecules were investigated by immunoblotting. The H fraction was toxic to MDA-MB-231 more than MCF-7 cells but not to NIH3T3 and PBMCs. The growth of MDA-MB-231 cells was inhibited through apoptosis. MTP was disrupted whereas caspase-3, -8 and -9 were activated. The expression of pro-apoptotic Bax and Bak was upregulated, while Bid and anti-apoptotic Bcl-xL proteins were downregulated. Cyclin D1 and CDK4 levels were downregulated. The cell cycle was arrested at G1. Moreover, GRP78 and CHOP elevation indicated ER stress-mediated pathway. The expression ratio of pAkt/Akt and pERK/ERK were reduced. Taken together, the molecular mechanisms of MDA-MB-231 cell apoptosis were via intrinsic/extrinsic pathways, cell cycle arrest, ER stress and abrogation of Akt/ERK survival pathways. According to the most current research, the H fraction may be used as an adjuvant in the BC treatment; however, before the anticancer strategy can be applied to patients, it is important to determine each active compound's effects in cell lines and when compared with a combined mixture.