β-carotene at physiologically attainable concentration induces apoptosis and down-regulates cell survival and antioxidant markers in human breast cancer (MCF-7) cells

Although β-carotene is known for its anti-carcinogenic and antioxidant properties, a few recent epidemiological and experimental evidence show that at higher concentration it acts as pro-oxidant and induces cancer. Since the global burden of breast cancer exceeds all other types of cancer, and its incidence rates is also in increasing trend, the present study attempted to evaluate the anti-cancer molecular mechanism of β-carotene (at 1 µM concentration) isolated from Spinacia oleracea in human breast cancer (MCF-7) cells. The carotenoid was purified by open column chromatography and identified by LC-MS. The anti-proliferative effect of β-carotene at different concentrations was evaluated by WST-1 assay and the changes in cell morphology were examined by microscopic observation. The induction of apoptosis by β-carotene was observed by DAPI staining and colorimetric caspase-3 assay. The expression of cell survival, apoptotic, and antioxidant marker proteins was measured by western blot analysis. Purified β-carotene inhibited the viability of MCF-7 cells in a dose-dependent manner, which was well correlated with changes in cell morphology. Increased apoptotic cells were observed in β-carotene (1 µM)-treated cells. This apoptosis induction was associated with increased caspase-3 activity. The protein expression studies showed that β-carotene at 1 µM concentration effectively decreases the expression of the anti-apoptotic protein, Bcl-2 and PARP, and survival protein, NF-kB. It also inhibited the activation of intracellular growth signaling proteins, Akt and ERK1/2. The inhibition of Akt activation by β-carotene results in decreased phosphorylation of Bad. Further, it down-regulated antioxidant enzyme, SOD-2, and its transactivation factor (Nrf-2), and endoplasmic reticulum (ER) stress marker, XBP-1, at protein levels. These findings exhibit the key role of β-carotene even at a low physiological concentration in MCF-7 cells which further explains its predominant anti-cancer activity.