The proliferation of CCA cell
The proliferation of CCA cell lines can be stimulated by estrogen and other growth factors (Alvaro et al., 2006; DeMorrow, 2009; Mancino et al., 2009). It has also been reported that estrogen positively modulates cholangiocyte proliferation, which is associated with cholangiopathies found in humans (Alvaro et al., 2004). However, the effects of glyphosate on CCA development are limited. This study aims to evaluate the effects of glyphosate compared to E2 in CCA cells. The range of concentrations that were used in this study is comparable to levels that have been reported in environmental human exposures (Acquavella et al., 2004; Bai and Ogbourne, 2016; Kongtip et al., 2017). Cell proliferation, effects on the cell cycle, and mRNA and protein expressions of various signaling proteins were analyzed. 4-hydroxytamoxifen, an ER antagonist, and U0126, an MEK inhibitor, were also used to determine effects of glyphosate exposure on the involving signaling pathways.
Materials and methods
Discussion This is the first study that demonstrates the effects of glyphosate on cholangiocarcinoma cells, and provides a better understanding of the possible mechanisms related to its effects. The low concentrations of glyphosate tested in this study are relevant to the range of levels that have been reported in the environment and detected in humans (Acquavella et al., 2004; Bai and Ogbourne, 2016; Kongtip et al., 2017). Previous studies have reported that E2 induces cell proliferation in an ERα expressing CCA, HuH28 cell line (Alvaro et al., 2006). It was reported that E2 also induced the expression levels of several proteins, including ERα, VEGF, VEGFR, IGF-1, and IGF-1R, as well as downstream signaling proteins associated to cell proliferation. In this study, we found that 10−11 to 10−5 M of glyphosate and E2 induced cell proliferation in HuCCA-1 cells under estrogen withdrawal conditions and the inductive effects were inhibited by ER antagonists. This study also demonstrated the results of various doses of glyphosate that covered no effect (10−15 M) and toxicity (10−3 to 25 × 10−3 M) in HuCCA-1 cells. These results demonstrated that low concentration of glyphosate induced bile duct cancer cell growth via the estrogen receptor which is similar to the previous report in breast cancer cell line (Thongprakaisang et al., 2013). A recent report from other researchers showed that only high concentration of glyphosate can induce cell proliferation in breast cancer SMER 3 molecular (Mesnage et al., 2017). The different results of these two studies may reflect the different estrogen withdrawal durations. This study demonstrated that the estrogen withdrawal duration had an effect on analysis of exogenous estrogen effects. We showed that glyphosate and E2 did not induce cell proliferation of HuCCA-1 cells in 2-days SF condition, which was comparable to 2-days CSS condition. Interestingly, 5-days in CSS prior to glyphosate or E2 treatment resulted in cell proliferation induction in the HuCCA-1 cells (Fig. 3B). The ERα protein expression in different culture conditions supported this data because 5 days CSS culture condition displayed higher ERα protein expression than other culture conditions (Fig. 1B). The higher levels of estrogen receptor expression might increase the response to weak binding chemicals, such as glyphosate in this study. The induction of ERα mRNA and protein levels suggests that they may have a role in CCA progression; however, we did not detect a change in ERβ in the HuCCA-1 cells after treatment with E2 and glyphosate. This result is similar to that from a previous study which showed that E2 can induce ERα expression but lower response in ERβ expression (Alvaro et al., 2006). Glyphosate-induced cell proliferation effects were also supported by cell cycle analysis and confirmed that the responses occurred via the estrogen receptor because these effects were inhibited by an ER antagonist (4-hydroxytamoxifen). In addition, the preliminary unpublished data in our study showed that ICI 182,780 alone at low concentrations (10−9- 10−8 M) act as agonist to estrogen receptor in HuCCA-1 cell line, while 4-hydroxytamoxifen did not. Previously, ICI 182,780 has been reported that it induces cell proliferation and acts as ERα agonist in the other cells beside breast cancer cells (Hanazono et al., 1998; Zhao et al., 2006). Therefore, we used only 4-hydroxytamoxifen in this study. There are some studies that support the observation that induction of cell proliferation is concomitant with an increase in S-phase cell populations (Amonyingcharoen et al., 2015; Bertoli et al., 2013). Two different cyclin proteins, cyclin A and cyclin D1, have been reported as activators of the estrogen receptor (Neuman et al., 1997; Trowbridge et al., 1997). In this study, we found that E2 and glyphosate exposure leads to an increase in the protein levels of cyclin D1, and cyclin A, as well as other cyclins involved in cell cycle regulation. In our study, glyphosate induces a higher proliferative effect than E2 in HuCCA-1 cells with a significant difference at low concentration, 10−13-10−9 M. At higher concentration (10−7-10−5 M), there is no significant difference between these two chemicals. At present, we can\'t exactly explain this higher proliferative action of glyphosate. However, in a recent study of Mesnage and colleague, they reported the molecular dynamics simulation of glyphosate and estradiol binding to human estrogen receptor alpha (ER alpha) and they concluded that glyphosate is unlikely to activate ER alpha in a similar manner to the natural hormone (Mesnage et al., 2017). Furthermore, we observed the different effects of glyphosate and E2 on the ERα expression. All tested concentrations of glyphosate increased ERα expression whereas E2 only at 10−11 M increased ERα expression (Fig. 4). Estrogen or estradiol which is natural hormone has negative feedback inhibition mechanism after binding and activate estrogen receptor. Drabovich and co-workers showed that there is a network of transcription factors with a negative feedback loop (ERα-EGR3-NAB2) regulated the estrogen activation in breast cancer cells (Drabovich et al., 2016). Glyphosate which is a chemical mimic natural hormone may not be inhibited by the activation of the negative feedback as natural hormones do. However, this hypothesis need further study.