Cancer Letters

Cancer Letters

Volume 378, Issue 2, 10 August 2016, Pages 104-110
Cancer Letters

Original Articles
Progesterone receptor activation is required for folic acid-induced anti-proliferation in colorectal cancer cell lines

https://doi.org/10.1016/j.canlet.2016.05.019Get rights and content

Abstract

Previously, we demonstrated that folic acid (FA) could inhibit proliferation of colorectal cancer cell lines through activating the folate receptor (FR)α/cSrc/ERK1/2/NFκB/p53 pathway and anti-COLO-205 tumor growth in vivo. Since we recently also demonstrated that female sex hormones could affect the FA's action in regulating endothelial cell proliferation and migration, the aim of this study was to investigate the effect of progesterone (P4) on the FA-induced anti-proliferation in colorectal cancer cells. Treatment with FA significantly reduced the proliferation of the P4 receptor (PR)-positive colon cancer cell lines, COLO-205, HT-29 and LoVo, but did not significantly affect the proliferation of the PR-negative colon cancer cell lines, HCT116 and DLD-1. Pre-treatment with Org 31710, a PR specific antagonist, abolished the FA-induced proliferation inhibition and activation in the signaling pathway involved in regulating proliferation inhibition in these PR positive colorectal cancer cell lines. The involvement of PR in the FA-induced activation of cSrc and up-regulations in cell cycle inhibitory proteins (p21, p27 and p53) was confirmed by knock-down of PR expression using the siRNA technique. Importantly, we show direct protein interaction between FR and PR in COLO-205. Moreover, treatment with FA induced PR activation in COLO-205. Taken together, these data suggest that FA induced proliferation inhibition in colon cancer cells through activation of PR. This finding might explain some of the controversies of FA's effects on cancer growth and provide valuable reference for clinical applications of FA in treating colorectal cancer.

Introduction

Colorectal cancer is a major cause of morbidity and mortality throughout the world. Currently, it is the third most common cancer worldwide and the fourth most common cause of death. Although it has been indicated that incidence rates for colon cancer in both sexes are similar, accumulating evidence has suggested that sex hormones are relevant to the development of colorectal cancer. Epidemiological studies in women have shown that increases in female hormones such as estrogens and progestin are associated with a lower risk for developing colorectal cancer [1], [2], [3]. In support of these results, reports from the Women's Health Initiative trial showed an approximately 40% lower risk for colorectal cancer in the estrogen plus P4 group as compared with the placebo group [4], [5]. However, the other Women's Health Initiative trial did not show a lower risk of colorectal cancer among hysterectomized women treated with estrogen alone [6], [7] or among postmenopausal women with higher circulating levels of estradiol and estrone [8], [9]. These observations seem to suggest that P4, but not estrogen, may be the key factor for reduction of colorectal cancer risk in women.

Although intensive research has led to considerable improvement in the treatment and diagnosis at an early stage of colorectal cancer, the prognosis is still not good. Therefore, continuous searching for new therapeutic strategies is required. One approach is to identify medicinal agents capable of retarding the cell cycle and/or activating the cellular apoptotic response in the cancerous cells. Folate has been demonstrated to exert an inverse relationship between the risks of some malignancies including cancer of colon, stomach, pancreas, lung, ovary, breast and leukemia [10], [11], [12]. Epidemiological and clinical studies have shown that dietary folate supplement might decrease the risk of colorectal cancer and be involved in DNA methylation of p53 [12], suggesting a possible protective effect of folate on colorectal cancer [13], [14].

Recently, we demonstrated that FA can reduce the proliferation of cultured colon cancer cell lines, such as COLO-205, HT-29 and LoVo, and inhibit the COLO-205 tumor growth in vivo [15]. Using COLO-205 as the cell model, our results showed that FA inhibited cell proliferation through the FRα/cSrc/ERK1/2/NFαB/p53-mediated up-regulations of p21 and p27. Interestingly, we found that this signaling pathway involved in the FA-induced anti-proliferation in COLO-205 is very similar to the signaling pathway involved in the P4-induced anti-proliferation in human umbilical venous endothelial cells [16] and rat aortic smooth muscle cells [17]. These observations led us to investigate whether P4 can affect FA's action in colorectal cancer development. Here, we demonstrated that PR activation is required for the FA-induced anti-proliferation in colorectal cancer cells.

Section snippets

Chemicals

FA, P4, fetal bovine serum (FBS), dithiothreitol, phenylmethylsulphonyl fluoride, glycerol, Nonident P-40, sodium dodecyl sulfate (SDS), anti-ERK antibody and anti-p27 antibody were purchased from Sigma-Aldrich (St.Louis, MO). Trypsin-EDTA, penicillin-streptomycin, kanamycin, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT), anti-phospho-serine antibody and protein A-Sepharose were purchased from Life Technologies (Darmstadt, Germany). Anti-p53, p-ERK, p21, PR, cSrc, FR and

Involvement of PR activation in the FA-induced growth inhibition in COLO-205

Since we have demonstrated an interaction between FA and female sex hormones in regulating endothelial cell proliferation and migration, we are interested to examine whether P4 can affect the FA-induced anti-proliferation action in colorectal cancer cells. Initially, we used COLO-205 for this study. As shown in Fig. 1A, daily treatment with FA (10 µM) or P4 (50 nM) alone for 4 days caused a significant reduction in the cell number. However, co-treatment with FA and P4 did not cause any further

Discussion

FA is essential for the synthesis of adenine and thymidine and has been frequently used as nutritional supplement. Recently, we uncovered a completely novel role of FA in regulating angiogenesis and demonstrated that FA inhibits vascular endothelial cell proliferation through activating the cSrc/ERK 2/NFκB/p53 signaling pathway mediated by FRγ [19] and migration through inhibiting the RhoA activity mediated by activating the FRγ/cSrc/p190RhoGAP-signaling pathway [20]. We also demonstrated that

Conflict of interest

The authors have nothing to disclose.

Acknowledgments

This work was supported by research grants from MOST 104-2320-B-038 -030 -MY3.

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