Long term induction by pterostilbene results in autophagy and cellular differentiation in MCF-7 cells via ROS dependent pathway

Abstract

This study shows the effect of pterostilbene on intracellular neutral lipid accumulation in MCF-7 breast cancer cells leading to growth arrest and autophagy. On exposing the breast cancer cells with 30 μM pterostilbene for 72 h there was almost 2-folds increase in neutral lipids and triglycerides. Also the phytochemical caused a 4-folds increase in the expression of adipogenic differentiation marker c/EBPα. Further, pterostilbene inhibited 3β-hydroxylsterol-Δ⁷-reductase, the enzyme which catalyzes the last step conversion of 7-dehydrocholesterol to cholesterol, and thereby causes the intracellular accumulation of the former sterol. These results were associated with over-expression of oxysterol binding protein homologue and liver X receptor (LXR) by ∼7-folds. Pterostilbene also caused a simultaneous increase in the expression autophagic marker proteins Beclin 1 and LC3 II (microtubule-associated protein 1 light chain 3) by approximately 6-folds, which leads to an alternative pathway of autophagy. These effects were observed in association with the loss of mitotic and metastatic potential of MCF-7 cells which was abolished in the presence of catalase (ROS scavenger) or 3MA (autophagic inhibitor). Thus the present data shows that the long term exposure to pterostilbene causes growth arrest in MCF-7 cells which may be due to differentiation of the mammary carcinoma cells into normal epithelial cell like morphology and activation of autophagy.

Introduction

Pterostilbene (trans-3,5-dimethoxy-4′-hydroxystilbene) is an analogue of resveratrol and plays a potential role in preventing breast (Alosi et al., 2010, Chakraborty et al., 2010, Pan et al., 2009, Rimando et al., 2002), prostate (Chakraborty et al., 2010) and colon cancers (Paul et al., 2009). It also bears immuno-stimulatory and anti-inflammatory effects (Pan et al., 2008) due to multiple cellular targets of action. These activities of this phytochemical are attributed to the fact that they are mainly hydroxylated stilbene molecules which bear the capacity to form hydrogen-bonds and to generate stable resonance structures in presence of oxidative electrophilic molecules in the biological system (Stivala et al., 2001).

Our previous studies have shown that pterostilbene causes cytotoxic effects in MCF-7 breast cancer cells due to its capacity to generate reactive oxygen species (ROS) (Chakraborty et al., 2010). Among these ROS, hydrogen peroxide and singlet oxygen species were subsequently produced which activated the death signaling pathways. In normal cells the ROS is generated as a natural byproduct of cellular metabolism inside the mitochondrial matrix. If the balance of ROS increases more than the scavenging capacity of the intracellular antioxidant system, the cell undergoes a state of oxidative stress with significant impairment of cellular structures (Stadtman, 1992). Oxidative stress can lead to the auto-oxidation of sterols (Murphy and Johnson, 2008, Payre et al., 2008) thereby affecting the cholesterol biosynthetic pathway mainly the postlanosterol derivatives (Medina et al., 2009). The intracellular accumulation of oxysterols directs the cell to its autophagic fate and may also induce it to differentiate (Medina et al., 2009, Payre et al., 2008). This phenomenon was reported earlier by two different groups where tamoxifen and N-pyrrolidino-(phenylmethyphenoxy)-ethanamine–HCl (PBPE), a tamoxifen metabolite, have been reported to show differentiation of the MCF-7 human breast cancer cells to more of normal epithelial cell like morphology with accumulation of large human milk globulin proteins. The differentiated breast cancer cells tend to lose their clonal growth potential (Elstner et al., 1998). In a mutual way, it was also found that in the presence of fatty acids like docosahexaenoic acid and eicosapentaenoic acid, the synthesis of lipid increases within the breast cancer cells (Chamras et al., 2002). Although the cholesterol lipid rafts are involved in cell proliferation (Simons and Toomre, 2000), the membrane lipids phosphatidylinositol-3-phosphate (PtdIns3P), generated by class III PI3K, functions in tumor suppression (Stenmark, 2010). Lipids thus have differential effect on cancer development and cell death. The PPARγ ligands (troglitazone and all-trans retinoic acid) alter the intracellular lipid machinery and inhibit cell proliferation in breast cancer cells due to dramatic decrease of the bcl-2 protein levels only in carcinoma tissues (Elstner et al., 1998). The role of lipids in the process of differentiation in breast cancer cells is further supported by the studies of O’Rourke et al. (1997) which showed that growth arrest induces the lipogenic markers, CCAAT/enhancer binding protein (C/EBP) isoforms, in mouse mammary epithelial cells. Not only the growth in MCF-7 cancer cells is inhibited by differentiation, the ROS producers like tamoxifen and its metabolite PBPE have been found to inhibit MCF-7 proliferation by inducing self-catastrophic phenomenon of autophagy (Medina et al., 2009).

In the present study an attempt was made to link the ROS mediated effect of pterostilbene to control the growth of MCF-7 breast cancer cells through differentiation and autophagy. The specific aim of this study was to determine the role of ROS in the production of oxysterol and the effects on MCF-7 cell proliferation. The data shows pterostilbene induced ROS participation in the intracellular lipid accumulation and autophagy of the MCF-7 cells.