Bisphenol A suppresses colon epithelial cell responses via G0/G1-phase arrest, MAPK and PI3K/AKT pathway modulation, and MMP-2/9 Inhibition by upregulating p21WAF1

Abstract

Bisphenol A (BPA) is a non-steroidal endocrine-disrupting chemical compound with applications in the production of epoxy resins and polycarbonates. Accumulating evidence suggests that BPA damages various organs and tissues, including those of the reproductive, immune, and neuroendocrine systems. However, the mechanisms by which BPA affects the intestinal tract have not been fully elucidated. We explored the adverse effects of BPA on human colonic epithelial cells in vitro by performing comprehensive viability, proliferation, invasion, and migration assays on HCT 116 and HCT-8 cells. BPA suppressed the proliferation of both cell types by regulating cell cycle progression and modulating the mitogen-activated protein kinase and phosphatidylinositol 3-kinase/protein kinase B pathways. Furthermore, BPA treatment significantly reduced the induction of matrix metalloproteinases-2 and -9 by inhibiting the binding activity of specificity protein-1, nuclear factor kappa B, and activator protein-1, thereby interfering with cell migration and invasion. This BPA-induced regulation of colonic epithelial cell proliferation, migration, invasion, and MAPK/AKT pathway was reversed by silencing p21WAF1 with siRNA. Collectively, our data indicate that BPA inhibits the proliferation and mobility of human colonic epithelial cells via p21WAF1 induction. This study provides valuable information into the precise mechanisms underlying the adverse effects of BPA on colonic epithelial cells.

Keywords: Bisphenol A; Cell cycle; Human colonic epithelial cells; Migration; Proliferation; Signaling pathway.

Fig. 1

BPA suppresses colon cell proliferation. HCT 116 (0, 60, 90, and 120 µM) and HCT-8 (0, 70, 140, and 210 µM) cells were cultured with various concentrations of BPA for 24 h. (A) Cell viability was evaluated using an MTT assay. (B) Cells were counted using a hemocytometer. The counts are shown in the bar graphs. (C) Morphological changes in both cells were monitored at 200× magnification. Values are displayed as mean ± standard deviation from three experimental data. *p < 0.05 compared with the control.

Fig. 2

BPA induces arrest of cell cycle at the G₀/G₁ phase in both colon cell lines. Designated concentrations of BPA were incubated with HCT 116 and HCT-8 cells for 24 h. Flow cytometric analysis was conducted to identify the cell cycle proportion in HCT 116 (A) and HCT-8 (B) cells treated with BPA. The distributions of cell cycle DNA contents are shown in the bar graphs. Values are represented as mean ± standard deviation from triplicate data. *p < 0.05 compared with the control.

Fig. 3

BPA modulates cell cycle regulatory proteins in colon cells. (A, B) The protein levels of CDK2, CDK4, cyclin D1, cyclin E, p27KIP1 (p27), p21WAF1 (p21), and p53 in BPA-treated colonic cells were determined by immunoblotting. Protein levels were quantified by β-actin. (C, D) To evaluate the binding affinity between CDK and CKI proteins, immunoprecipitation was performed. Bar graphs represent the ratios of relative protein levels. Results are depicted as mean ± standard deviation from triplicate data. *p < 0.05 compared with control. Original blots/gels are presented in Supplementary Fig. 4–6.

Fig. 4

BPA regulates PI3K/AKT and MAPK phosphorylation in colon cells. A The phosphorylation of AKT, ERK1/2, JNK, and p38 MAPK (p38) was measured in colon cells exposed to designated concentrations of BPA for 12 h. B Both cell lines were pretreated with the indicated concentrations of inhibitors, such as LY2780301 (AKT inhibitor), SP600125 (JNK inhibitor), U0126 (ERK inhibitor), and SB203580 (p38 inhibitor), for 40 min followed by incubation in the absence or presence of BPA for 12 h. The phosphorylated (p-AKT, p-ERK1/2, p-JNK, and p-p38) and total forms (AKT, ERK1/2, JNK, and p38) of signaling molecules were analyzed by immunoblotting. The relative protein expression is plotted on the bar graphs. All values are shown as mean ± standard deviation from experiments of three data. *p < 0.05 compared with control, #p < 0.05 compared with BPA treatment alone. Original blots/gels are presented in Supplementary Fig. 7–10.

Fig. 5

BPA affects migration and invasion capacity in both colon cell lines. Both cells were exposed to various concentrations of BPA. After 24 h, densities of metastatic potential in BPA-treated cells were determined by optical microscopy. The ratios of relative migratory (A) and invasive (B) ability are shown in the bar graphs. All values are shown as mean ± standard deviation from three repeated data. *p < 0.05 compared with control.

Fig. 6

BPA suppresses MMP-2 and − 9 expression via inhibition of AP-1, Sp-1, and NF-κB binding in colon cells. (A) The activity and protein levels of MMP-2 and − 9 in HCT 116 and HCT-8 cells were assessed under gelatin zymography and immunoblotting. As the loading control, β-actin was employed in the immunoblot experiment. (B) The DNA-binding affinities of Sp-1, AP-1, and NF-κB in colon cells were determined by EMSA. All values from three triplicate experiment are expressed as mean ± standard deviation. *p < 0.05 compared with control. Original blots/gels are presented in Supplementary Fig. 11.

Fig. 7

p21WAF1 silencing reverses BPA-induced interference of proliferation and metastatic potential in colon epithelial cells. MTT (A) and cell counting (B) assays were performed in BPA-treated colon epithelial cells after p21WAF1 siRNA (si-p21) or scramble siRNA transfection. Wound-healing migration (C) and invasion assays (D) in BPA-treated colon epithelial cells following si-p21 or scramble siRNA transfection. All values from triplicate results are indicated as mean ± standard deviation. *p < 0.05 compared with control, #p < 0.05 compared with BPA treatment alone.

Fig. 8

p27KIP1 silencing with siRNA had no effect on the BPA-induced colon epithelial cell responses. Colon cells were transfected with either scramble siRNA or p27KIP1 siRNA (si-p27), and then treated with BPA for 24 h. Transfected cells were analyzed by MTT (A) and cell counting assays (B). Wound-healing migration (C) and invasion (D) assays were performed to estimate the metastatic ability using transfected cells. All values from triplicates data are designated as mean ± standard deviation. *p < 0.05 compared with control, #P < 0.05 compared with BPA treatment.