W146 Di ethylhexyl phthalate DEHP is one

Di-(2-ethylhexyl) phthalate (DEHP) is one of the most widely used environmental chemicals in the production of polyvinyl chloride. After W146 into human body, DEHP can be converted to the metabolite mono-ethylhexyl phthalate (MEHP), which has higher toxicity than DEHP and can influence reproductive and development system (Wang et al., 2012). Clinical studies revealed that higher concentrations of MEHP, while not urinary cotinine, arsenic, or phthalate monoesters, were associated with a larger change in tumor size in 37 cervical cancer patients (Neamtiu et al., 2016). However, there is no study concerning the effects and related mechanisms of MEHP on the growth and biological functions of cervical cancer cells. Our present study revealed that nanomolar concentrations of MEHP can trigger the proliferation of cervical cancer cells via activation of G-protein coupled estrogen receptor (GPER)/Akt signals.
Materials and methods
Results
Discussion Increasing evidences suggested that MEHP can promote the progression of various cancers including Wilms' tumor (Wang et al., 2017), testicular (Yao et al., 2012), prostate (Wang et al., 2016), and ovarian cancer (Chang et al., 2017). Consistently, our present data revealed that nanomolar MEHP can promote the proliferation, while not invasion, of cervical cancer cells, which was confirmed by the up regulation of PCNA. MEHP treatment can activate Akt and promote its nuclear localization, while targeted inhibition or knockdown of Akt can reverse MEHP induced cell proliferation. GPER, while not ERα, mediated MEHP induced phosphorylation of Akt and cell proliferation. In addition, MEHP treatment also increased mRNA and protein expression of GPER. Collectively, our present data showed that nanomolar MEHP can promote the proliferation of cervical cancer cells via GPER/Akt signals (Fig. 6 F). Many studies suggested that MEHP can modulate the progression of cancer cells via promoting proliferation or metastasis. Our present study revealed that nanomolar MEHP can promote the proliferation while not invasion of cervical cancer cells, which was further evidenced by the results that MEHP increased the expression of PCNA, while not MMP-2 or MMP-9. This is consistent with previous data that DEHP increased mitochondrial membrane potential in Hep3B cells and promote cell proliferation (Chen et al., 2013). MEHP can also promote adipocyte differentiation and induce obesity in mice (Hao et al., 2013). Yao et al. (2012) revealed that MEHP induced the expression of MMP-2 expression in testicular embryonal carcinoma NT2/D1 cells and then promoted cell invasion and migration. Wang et al. (2017) also suggested that MEHP can increase the expression of MMP-2/-9 and trigger the invasion and migration of Wilms' tumor cells. However, our study revealed that MEHP had no effect on in vitro invasion of cervical cancer cells and the expression of MMPs. It suggested that the effect of MEHP might be cell and cancer dependent and need more investigations. Our data revealed that activation of Akt mediated the promotion effects of MEHP on cell proliferation. The phosphorylation and nuclear localization of Akt were significantly increased in cells treated with MEHP, while its inhibition abolished MEHP induced cell proliferation. The activation of Akt has been well known to promote the proliferation of cancer cells including cervical cancer (Morgensztern and McLeod, 2005). Our data are also consistent with previous study that Akt kinase activity increases in the testes of postnatal day 28 wild-type mice following exposure to 500 mg/kg MEHP (Rogers et al., 2008). In RAW264.7 cells, MEHP treatment can increase the phosphorylation of PI3/Akt and then induced the release of tumor necrosis factor (TNF)-α (Bolling et al., 2012). PI3K–AKT–mTOR pathway was also involved in DEHP-induced Hep3B cell abnormal proliferation (Chen et al., 2013). Recent study also revealed that MEHP can trigger the phosphorylation and nuclear localization, and induce nuclear accumulation of p65 in G401 cells (Wang et al., 2017), however, our data showed that MEHP had no effect on phosphorylation of p65 in cervical cancer cells. This might be due to cell dependent effects of MEHP and need further studies. Finally, our data suggested that GPER, rather than ERα, mediated MEHP induced activation of Akt and cell proliferation. GPER can mediate the estrogenic effects of environmental endocrine disruptors such as bisphenol A (Ge et al., 2014). Our data provided novel mechanisms that MEHP can modulate cancer progression via ERα-independent manners.