Here we examined whether Intracellular ER may mediate Cd ind
Here, we examined whether Intracellular ER may mediate Cd-induced ovarian cancer proliferation. To determine this relationship, we used ICI 182,780 as a general inhibitor of ERα and ERβ. The results suggest a metalloestrogenic effect of Cd in ovarian cancer cell lines. Inconsistency with our results, Stoica et al. and Byrne et al. observed that Cd functionally acted like E2 in breast cancer 2211 as a result of its ability to bind to the ligand-binding domain of ERα with high affinity (Byrne et al., 2009; Stoica et al., 2000). Brama et al. claimed that CdCl2 stimulated an ER-dependent mitogenic signaling in breast cancer cell line and the ER-antagonist ICI 182,780 blunted it (Brama et al., 2007). Ronchetti et al. reported that nM concentrations of Cd exerted a potential estrogenic effect on cell proliferation of normal and tumor lactotrophs and its effects were blocked by ICI 182,780 (Ronchetti et al., 2013). In contrast to our study, Ali et al. reported that ICI 182,780 could not inhibit the CdCl2-induced proliferation in HepG2, MCF-7and ECC-1 cell lines. They suggested that instead of ER, a membrane G protein-coupled receptor of estrogen, GPR30, mediated these Cd effects (Ali et al., 2015). Moreover, Huff et al. observed that both ER antagonist ICI 182,780 and GPER antagonist G-15 attenuated CdCl2 and E2 stimulated proliferation in lung cancer cell lines (Huff et al., 2016). Although recent studies have suggested that GPR30 possibly has a role in proliferative effects of Cd as a xenoestrogen (Gao et al., 2015; Huff et al., 2016), the activation of ERs was considered as a critical step in metal-induced carcinogenesis (Aquino et al., 2012). More attention has been given to the roles of ER in etiology, incidence, and pathology and survival rate of ovarian tumors as a prominent hormone-related cancer. (Järvinen et al., 2000). So we also evaluated the effect of various concentrations of CdCl2 on the expression of ERα and ERβ. We found that Cd similar to E2 significantly increased the expression of ERs (α and β), but the expression of ERα was higher than ERβ. These results suggested that both subtypes of ER have possible roles in the Cd-induced proliferation of ovarian cancer cells. Ronchetti et al. claimed that Cd up-regulated mRNA expression of ERα in anterior pituitary cells at nM concentration (Ronchetti et al., 2013). Huff et al. reported that 100 nM CdCl2 and E2 increased both ERα and ERβ expression in lung cancer cell lines (Huff et al., 2016). ERα and ERβ can dimerize and associate with DNA and some transcription factors (Leclercq, 2002). The findings of Dougherty et al. demonstrated that ERβ protein was expressed higher than ERα in lung tumors and cell lines (Dougherty et al., 2006). Lindberg et al. have been suggested that ERα and ERβ were co-expressed. ERα not only was responsible to inducing cell proliferation but also it could up-regulate ERβ (Lindberg et al., 2003). The high level of ERβ expression in lung tumors and ERα expression in breast tumors can provide a good rationale that ERα and ERβ may play a critical role in cellular proliferation depending on tissue type (Brandenberger et al., 1997). ERK/MAPK, one of the most important intracellular protein kinases play a critical role in cell proliferation by phosphorylation of transcription factors and stimulation of intracellular networks of signaling cascades (Waisberg et al., 2003; Geffroy et al., 2005; Roskoski, 2012). We observed that CdCl2 activated ERK1/2 in OVCAR3 and SKOV3 cells similar to estradiol effects and ICI 182,780 pretreatment reduced ERK1/2 phosphorylation. It suggests that ERK/MAPK pathway might be involved in Cd-induced proliferation and ER is required for ERK1/2 activation. In supporting our findings, Huang et al. reported that 1 μM Cd-induced a sustained activation of ERK1/2 in mouse epidermal cells (Huang et al., 2001). Jiang et al. showed that low concentrations of Cd promoted cell proliferation by increasing in p-ERK1/2 activity whereas, at high Cd concentrations, phospho-p38 activity markedly increased and induced apoptosis in human embryo lung fibroblast cells (Jiang et al., 2009). The results of Hao et al. claimed that Cd in a biphasic effect, at higher concentrations (50 and 500 μM) induces apoptosis by increasing of JNK and P38 phosphorylation but at lower concentrations (50 and 500 nM), activated ERK/MAPK pathway lead to proliferation of HEK293 cell line (Hao et al., 2009). Gao et al. also reported that ERK1/2 /MAPK pathway is essential for Cd-induced cell proliferation in uterine cell lines. They explored that CdCl2 stimulated the growth of uterine cancer cell lines at lower concentrations (0.1 μM and 10 μM) using dose-dependent p-ERK1/2 activation and inhibited the cell proliferation at concentrations ≥5 μM by p-ERK1/2 diminution (Gao et al., 2015). Brama et al. showed that ICI 182,780 in the MCF-7 cells blocked ERK1/2 activation (Brama et al., 2007). Liu et al. described that treatment of MCF-7 cells with 0.5–10 μM CdCl2 caused rapid activation of ERK1/2 similar to E2 and partially blocked by specific siRNA of ERα (Liu et al., 2008). In accordance with previous results, our study demonstrated that low Cd concentrations activated ERK1/2 and subsequent proliferation in ovarian cancer cell lines. So ERK1/2 signal pathway might take part in hormesis phenomenon by phosphorylation of ERK1/2 and activation of dependent genes. Stebbing explained that in the lower dose of metals, cells showed an overcompensation response to protect and adapt against the damage of metals but higher doses caused severe toxic effects to induce cell death (Stebbing, 2002).