Docosahexaenoic acid increases the expression of oxidative stress-induced growth inhibitor 1 through the PI3K/Akt/Nrf2 signaling pathway in breast cancer cells
Graphical abstract
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.
Section snippets
Reagents
Albumin, essentially fatty acid–free BSA, sodium bicarbonate, PI3k inhibitor (LY294002), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were from Sigma–Aldrich (St. Louis, MO); DMEM, FBS, 25% trypsin-EDTA, and penicillin–streptomycin solution were from GIBCO/BRL (Grand Island, NY); SP600125 (JNK inhibitor II), PD98059 (ERK inhibitor), and SB203580 (p38 inhibitor) were from TOCRIS (Ellisville, MO); AA, LA, GLA, ALA, EPA, and DHA were from Cayman Chemical (Ann Arbor, MI); TRIzol
Effect of n-6 and n-3 PUFAs on OSGIN1 expression in MCF-7 and Hs578T cells
As shown by the MTT assay, the cell viabilities of MCF-7 cells treated with 100 μM AA, LA, GLA, ALA, EPA, and DHA were 115.6 ± 5.1%, 110.3 ± 20.6%, 111.8 ± 12.3%, 121.3 ± 16.0%, 107.4 ± 4.7%, and 107.4 ± 4.8%, respectively, compared with the untreated controls (100%). The corresponding cell viabilities of Hs578T cells were 96.2 ± 5.9%, 102.2 ± 6.4%, 100.6 ± 8.7%, 95.5 ± 5.2%, 103.0 ± 14.1%, and 101.1 ± 8.4%, respectively. We chose to use 100 μM PUFAs because concentrations greater than 100 μM
Discussion
The n-6 and n-3 PUFAs regulate the expression of several genes that modulate cell proliferation, angiogenesis, and metastasis in tumorigenesis (Wen et al., 2013, Galindo-Hernandez et al., 2014, Lii et al., 2016). Although OSGIN1 was recognized as tumor suppressor gene, whether it played an important role in the suppression of breast cancer by n-6 and n-3 PUFAs remained unclear. In this study, we first revealed that DHA, but not other PUFAs, dramatically induced OSGIN1 expression and that this
Conclusions
In summary, the results of the present study indicate that DHA-induced OSGIN1 expression is mediated at least in part through the PI3K/Akt/Nrf2 signaling pathway. Additionally, induction of OSGIN1 expression caused mitochondrial accumulation of p53 and OSGIN1, which are partially involved in the induction of cytochrome c release and onset of apoptosis by DHA in breast cancer cells.
Funding
This study was supported by grants from the Ministry of Science and Technology MOST 103-2313-B-040-001 and MOST 104-2320-B-040-005.
Author contributions
The author's responsibilities were as follows: Li C-C (Principal Investigator) and Yao H-T designed the study; Tsai C-H, Chen H-W, Liu K-L, Chang J-W, Chen P-Y, Lin C-Y contributed to sample measurements and data collection; Tsai C-H and Shen Y-C analyzed data and performed statistical analysis; Tsai C-H and Li C-C wrote paper; Li CC had primary responsibility for final content. All authors contributed to the manuscript review and approved the final version.
Financial disclosure
The authors have nothing to disclose.
Conflict of interest
Nothing to disclose.
Acknowledgement
The MCF-10A and H184 cell line was kindly provided by Dr. J. -W. Chang (Institute of Molecular and Genomic Medicine, National Health Research Institute, Taiwan). Chemiluminescence/fluorescence imaging analysis and flow cytometry were performed in the Instrument Center of Chung Shan Medical University, which is supported by the Chung Shan Medical University.
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These authors contributed equally to this work.