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  • It should be noted that not


    It should be noted that not only glyphosate but E2 at the same concentration range (10−11-10−7 M) also showed similar effects on the expression of the WIN 64338 hydrochloride clinical regulating proteins. Furthermore, we could not see a significant difference among this concentration range of glyphosate and E2 on other tested endpoints including cell proliferation, ERK activation and S-phase cell cycle. In contrast to our previous study in breast cancer cell lines, glyphosate at the same concentration showed lower proliferative induction than E2 (Thongprakaisang et al., 2013). Since the maximum saturation of the effects of glyphosate and E2 on cell proliferation in HuCCA-1 were observed at low concentrations (10−11-10−9 M) together with the maximum proliferative response of these two compounds were relatively low (<30% of control), we hypothesize there is a rapid estrogen receptor saturation and low sensitivity of non-genomic estrogen receptor signaling to induce cholangiocarcinoma HuCCA-1 cell growth. It is also postulated that non-genomic estrogen receptor signaling occurred faster than genomic estrogen receptor signaling pathway. Therefore, the time point that we determined the effects are occurred at the near maximal responses. However, further study to prove this hypothesis is really needed. The molecular mechanisms of estrogen activity can be categorized into two main pathways: genomic and non-genomic signaling pathways. In the genomic or classical pathway, E2 binding triggers conformational changes in the receptor leading to receptor dimerization. Active ER dimer then binds directly or indirectly to genes containing estrogen response elements (ERE). Various co-activators of transcription factors may also be recruited in this step. Transcription of genes, such as ps2 and progesterone receptor; PR, is stimulated, resulting in the activation of several cell functions, such as cell proliferation and cell survival (Heldring et al., 2007; Nilsson et al., 2001; Zhang and Trudeau, 2006). Many signaling molecules are rapidly activated by the non-genomic effects of E2 such as epidermal growth factor receptor (EGFR), insulin-like growth factor I receptor (IGF-IR), Ras/Raf-1, mitogen activating protein kinase (MAPK), Akt and protein kinase C (Cheskis, 2004; Yee and Lee, 2000). In this study, we found that estrogenic activity of glyphosate and E2 on HuCCA-1 cells are mainly associated with non-genomic estrogen signaling because we did not find the alteration of gene expressions that were regulated through ERE activation such as ps2 and PR. Rather, we demonstrated that glyphosate and E2 induced the expression levels of non-genomic signaling proteins, such as pERK, VEGFR2. Previous studies showed that estradiol induced the increase of IGF-1, IGF-1R, VEGF and VEGFR protein levels, and that was inhibited by ER- or IGF-1R-specific antagonists (Alvaro et al., 2006; Mancino et al., 2009). However, we found that E2 and glyphosate induced the protein expression of ERα, and VEGFR2, but not IGF-1R. These different results may be due to different CCA cell lines that may have different responsive mechanisms to chemicals. Moreover, the inductions of those signaling proteins were diminished by an ER antagonist. We also report here that the effects of glyphosate are mediated partly through MEK signaling pathway, as observed from the inhibitory effects of the MEK1/2 inhibitor (U0126). There are limit data of ERα protein localization in cholangiocarcinoma. We first examined ERα protein expression and localization in HuCCA-1 compared to MCF-7 using immunofluorescent staining. Previously, several studies showed the localization of breast cancer cell lines or patient samples by different techniques, including cell fractionated western immunoblotting, in situ proximity ligation assay (PLA), immuno-electron microscopy, immunohistochemistry and immunofluorescent staining (Iwabuchi et al., 2017; Kocanova et al., 2010; Li et al., 2015; Welsh et al., 2012). Li and colleague showed that there are two types of ERα localization in breast cancer cells from patients by immunohistochemical staining; in the cell nucleus and/or the cell membrane (Li et al., 2015). Iwabuchi and colleague presented the dimerization and localization of ERα on MCF-7 and T-47D cells by using double-stained ERα proteins using two different anti-ERα antibodies as well as in situ Proximity Ligation Assay (PLA) (Iwabuchi et al., 2017). They showed that ERα signals were detected in the nuclei of both cells. Moreover, cytoplasmic expression of ERα was low incident in cell lines and clinical samples of breast cancer using quantitative immunofluorescent (Welsh et al., 2012). Poulard and colleague used PLA to demonstrate that the complex of non-genomic estrogenic signaling (ERα/PI3K/Src) was presented in the cytoplasm of breast cancer cell lines as well as formalin-fixed, paraffin-embedded tumors (Poulard et al., 2012). In this study, we found the different localization of ERα expression between MCF-7 and HuCCA-1 cells. Predominant nuclear expression of ERα in MCF-7 was clearly related to genomic estrogenic signaling pathway in breast cancer cells that also showed the same ERα expressing pattern. While HuCCA-1 cells which extensively expressed ERα in the cytoplasm suggests the majority of their responses are mediated through non-genomic estrogenic signaling pathway compared to less extent in genomic associated pathway.