Induction of apoptotic cell death by Pharbitis nil extract in HER2-overexpressing MCF-7 cells

Abstract

We performed this study to understand the molecular basis underlying the antitumor effects of Pharbitis nil (PN, Morning glory) which has been used for herbal medicinal treatment against cancers in East Asia. We analyzed the effects of PN extract on proliferation of breast cancer cell line, MCF-7 cells and MCF-7 HER2 cells engineered to overexpress oncogenic HER2 via retroviral infection. HER2 is a cell membrane surface-bound receptor tyrosine kinase involved in the signal transduction pathways leading to cancer cell proliferation. Interestingly, PN extract preferentially inhibited proliferation of MCF-7 HER2 cells than MCF-7 vec (vector control) cells as revealed by MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) and proliferation assays. SubG1 apoptotic fractions induced by PN extract was also greater in MCF-7 HER2 cells than MCF-7 vec cells, indicating that the decrease in MCF-7 HER2 cell proliferation was caused by preferred induction of apoptotic cell death in MCF-7 HER2 cells by PN extract. In an attempt to reveal the molecular mechanism(s) responsible for the apoptotic cell death by PN extract in MCF-7 HER2 cells, a series of western blot experiment were conducted to check the effect of PN extract on HER2 and its downstream signaling molecules. Treatment with PN extract resulted in the inhibition of phosphorylation of HER2 and its downstream effectors, ERK and Akt. Strikingly, at the concentration of 5 g/ml for 12 h, PN extract could completely shut off ERK phosphorylation in MCF-7 HER2 cells, while ERK phosphorylation in MCF-7 vec cells remained effective at the same dosage. These results might indicate that the selective inhibition of cell proliferation via apoptotic cell death in MCF-7 HER2 cells is mediated by the complete loss of ERK activity by PN extract treatment. HER2-driven up-regulation of Akt activity which still remained substantially active only in MCF-7 HER2 cells even after PN extract treatment, might in turn induce inhibitory phosphorylation on Raf, an upstream kinase of MEK/ERK and this inhibition of Raf by Akt combined with inhibition of HER2/Ras/Raf/ERK by PN may be responsible for complete loss of ERK phosphorylation and induction of apoptosis in MCF-7 HER2 cells