Inactivation of PI3K/Akt pathway and upregulation of PTEN gene are involved in daucosterol, isolated from Salvia sahendica, induced apoptosis in human breast adenocarcinoma cells

Abstract

Ethnopharmacological relevanceSalvia sahendica has been traditionally used for antibacterial, anti-fungal, and anti-cancer purposes and treatment of dyspepsia in many parts of Iran. Aim of the studyOur aim was to investigate the mechanism by which daucosterol a β-sitosterol glycoside isolated from S. sahendica induced apoptosis against human breast adenocarcinoma MCF-7 cells. MethodsMCF-7 cells were treated with 5, 10, 20 and 40μM of daucosterol for 24h, and proliferation, cytotoxicity, and apoptosis in human breast adenocarcinoma MCF-7 cells were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) leakage assay, acridine orange (AO)/ethidium bromide (EB) double staining and flow cytometry. Phosphoinositide 3-kinase (PI3K), p-Akt, poly (ADP-ribose) polymerase (PARP), Bax, Bcl2, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and cytochrome c were analyzed by western blotting and mRNA expression of PTEN was detected by RT-PCR. ResultsThe results showed that daucosterol suppresses the proliferation and induces the cell cytotoxicity of MCF-7 cells in a dose-dependent manner. Cells treated with different concentrations of daucosterol also showed changes typical of apoptosis: morphological changes and DNA damage. Further analysis revealed a modulation of Bax, Bcl2, and PARP as well as losing mitochondrial membrane potential, and cytochrome c (Cyt c) release in breast cancer cells induced by daucosterol. Our results also showed that cell death in MCF-7 breast tumor cells was achieved through the induction of apoptosis via upregulation of the PTEN gene and inhibition of the PI3K/Akt pathway. To further explore the proapoptotic mechanism of daucosterol, we investigated the role of the reactive oxygen species (ROS) in the apoptosis induced by daucosterol in MCF-7 cells. The generation of ROS was about 6 to 8-fold as compared to control cells after treatment with daucosterol (20 and 40μM) for 24h. Glutathione (GSH) depletion was consistent with ROS generation after treatment with daucosterol. Reversal of apoptosis in antioxidant (NAC and catalase) pretreated cells indicated the involvement of ROS in daucosterol-induced apoptosis. ConclusionTaken together, our results suggest that daucosterol significantly inhibits the growth and induces the apoptosis of human breast cancer cells in vitro.