Docosahexaenoic acid increases the expression of oxidative stress-induced growth inhibitor 1 through the PI3K/Akt/Nrf2 signaling pathway in breast cancer cells

Highlights

• DHA up-regulates the oxidative stress-induced growth inhibitor 1 (OSGIN1) in breast cancer cells but not normal cells.

• Induction of OSGIN1 is accompanied by elevation of Bax/Bcl-2 ratio, mitochondrial p53 accumulation and cytochrome c release.

• Induction of OSGIN1 appears to be involved in DHA-induced onset of apoptosis in breast cancer cells.

Abstract

Oxidative stress-induced growth inhibitor 1 (OSGIN1), a tumor suppressor, inhibits cell proliferation and induces cell death. N-6 and n-3 PUFAs protect against breast cancer, but the molecular mechanisms of this effect are not clear. We investigated the effect of n-6 and n-3 PUFAs on OSGIN1 expression and whether OSGIN1 is involved in PUFA-induced apoptosis in breast cancer cells. We used 100 μM of n-6 PUFAs including arachidonic acid, linoleic acid, and gamma-linolenic acid and n-3 PUFAs including alpha-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid (DHA). Only DHA significantly induced OSGIN1 protein and mRNA expression. DHA triggered reactive oxygen species (ROS) generation and nuclear translocation of Nrf2. LY294002, a PI3K inhibitor, suppressed DHA-induced OSGIN1 protein expression and nuclear accumulation of Nrf2. Nrf2 knockdown attenuated DHA-induced OSGIN1 expression. N-Acetyl-l-cysteine, a ROS scavenger, abrogated the DHA-induced increases in Akt phosphorylation, Nrf2 nuclear accumulation, and OSGIN1 expression. DHA induced the Bax/Bcl-2 ratio, mitochondrial accumulation of OSGIN1 and p53, and cytochrome c release; knockdown of OSGIN1 diminished these effects. In conclusion, induction of OSGIN1 by DHA is at least partially associated with increased ROS production, which activates PI3K/Akt/Nrf2 signaling. Induction of OSGIN1 may be involved in DHA-induced apoptosis in breast cancer cells.

Introduction

Oxidative stress-induced growth inhibitor 1 (OSGIN1), also known as OKL38, is recognized as an oxidative stress response and tumor suppressor gene (Ratliff, 2005, Li et al., 2007). According to the alternative splicing of the 5′-untranslated region of OSGIN1 mRNA, OSGIN1 transcript variants encode proteins including OSGIN1 (34 kDa), OSGIN1-1a/2a (52 kDa), OSGIN1-2b (61 kDa), and OSGIN1-2c (59 kDa) (Ong et al., 2004). Loss at the OSGIN1 genomic locus or a nucleotide variation (G to A substitution at nucleotide 1494) in the OSGIN1 coding region impairs the tumor-suppressive function of OSGIN1, which is frequently found in hepatocellular carcinoma patients compared with adjacent nontumor tissue (Liu et al., 2014). Induction of OSGIN1 expression results in growth inhibition and cellular apoptosis in a variety of carcinoma cells (Huynh et al., 2001, Hu et al., 2012), whereas knockdown of OSGIN1 expression leads to cell proliferation and survival (Hu et al., 2012). Previous studies showed that OSGIN1 is significantly induced by aqueous extract of Taheebo and that induction correlates positively with the inhibition of breast cancer cell growth and initiation of apoptosis (Mukherjee et al., 2009).

Nuclear factor erythroid 2-related factor-2 (Nrf2), a basic leucine zipper transcription factor, is necessary for the induction of numerous antioxidative genes (Lu et al., 2014). In response to oxidative stress, Nrf2 translocates from the cytosol into the nucleus and forms a heterodimer with small Maf proteins, followed by binding to the antioxidant response elements (AREs) in the promoters of its downstream target genes (McNally et al., 2007, Li et al., 2008). Extracellular signal regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and the p38 mitogen-activated protein kinases (MAPKs) as well as phosphoinositide 3-kinase/Akt (PI3K/Akt) are involved in Nrf2-mediated gene transactivation in carcinoma cells (Kocanova et al., 2007) and human endothelial cells (Yang et al., 2013). A recent study revealed that Nrf2 is essential for OSGIN1 expression in human aortic endothelial cells (Yan et al., 2014). However, the role of Nrf2 and the actual working mechanism of the regulation of OSGIN1 in cancer cells are not fully elucidated.

N-6 and n-3 PUFAs not only play crucial roles in energy metabolism and endogenous hormone synthesis (Spector and Yorek, 1985) but also regulate a number of genes (Li et al., 2006, Liu et al., 2016). Moreover, n-6 PUFAs, such as arachidonic acid (AA), linoleic acid (LA), and gamma-linolenic acid (GLA), and n-3 PUFAs, such as alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), induce apoptosis in cancer cells (Zhang et al., 2015). Several apoptosis-related proteins such as BCL-2 family proteins, p53, caspases, and cytochrome c have been shown to be up-regulated by n-3 and n-6 PUFAs, which contributes to apoptosis in various cancer cells (Kwon et al., 2008, Lee et al., 2009). Although induction of OSGIN1 has been reported to induce apoptotic cell death, whether OSGIN1 plays an essential role in the cancer cell apoptosis induced by n-3 and n-6 PUFAs is still not understood.

Breast cancer is one of the most frequently diagnosed cancers worldwide, and it has maintained a high incidence and mortality rate in women, particularly African American women (DeSantis et al., 2016). The therapeutic choices and prognostic outcomes for patients with breast cancer are usually based on predictive biomarkers and diversified biological phenotypes, including luminal A, luminal B, HER2 positivity, and basal-type tumors (triple-negative breast cancer) (Prat et al., 2013). OSGIN1 is a predictive biomarker in tumorigenesis, and a negative correlation between OSGIN1 and breast cell proliferation was demonstrated in previous studies (Huynh et al., 2001, Wang et al., 2005). In the present study, we used MCF-7 and Hs578T human breast cancer cells to study the individual effects of n-3 and n-6 PUFAs on OSGIN1 expression and the possible mechanisms involved in this gene regulation. Moreover, we further studied whether OSGIN1 is involved in the regulation of apoptosis by DHA in breast cancer cells, and compared with the difference of OSGIN1-relative effects by DHA between human breast carcinoma cells and the non-tumorigenic mammary epithelial cells.