Molecular mechanisms underlying the potential anticancer activity of hexane fraction in HCT116 cancer cells.

Given the high mortality rate associated with tumors and the severe side effects of current treatments, scientists are exploring alternative therapies with fewer adverse effects. They are increasingly turning to natural remedies, much like our ancestors who used plant extracts to treat various ailments long before understanding the underlying mechanisms. Even though they did not know exactly why these plants treated those diseases then, we have the privilege of testing these plants and discovering the active ingredients responsible for these effects. This study aims to investigate the anticancer mechanisms of hexane fraction (Hex F) against human colorectal cancer cells and elucidate its molecular pathways of action. The methanol extract of and its fractions were evaluated for cytotoxic activity using MTT assay against HepG2, HCT116, and Hep-2 cancer cell lines, with oral epithelial normal cells (OEC) as controls. The most potent fraction (Hex F) was further analyzed using flow cytometry for cell cycle and apoptosis analysis, qRT-PCR for gene expression profiling, ELISA for protein quantification, and biochemical assays for oxidative stress and glycolytic enzyme activities. Hex F demonstrated significant cytotoxicity against HCT116 cells with an IC of 39.4 μg/mL and a selectivity index of 1.76 indicating preferential toxicity toward cancer cells. Flow cytometry analysis revealed G/M phase cell cycle arrest and significant induction of apoptosis. Gene expression analysis showed significant upregulation of pro-apoptotic genes p53, caspase-8, and caspase-9, while anti-apoptotic Bcl2 was downregulated). Protein analysis confirmed increased caspase-3 and caspase-7 activities, accompanied by enhanced anti-inflammatory response with increased IL-10 and decreased IL-4 levels. Oxidative stress markers indicated cellular damage with decreased GSH and SOD levels, while MDA increased significantly. Glycolytic enzyme activities were substantially reduced, with PK, Aldolase, and LDH activities decreased, suggesting metabolic disruption. GC-MS analysis identified β-sitosterol (17.89%), phytol (15.65%), stigmasterol (13.13%), and lupeol (12.89%) as major bioactive compounds. These findings demonstrate that Hex F exerts anticancer effects through multiple mechanisms including cell cycle arrest, apoptosis induction, oxidative stress generation, and metabolic disruption, supporting its potential as a natural anticancer therapeutic agent.