Bisphenol-A-induced inactivation of the p53 axis underlying deregulation of proliferation kinetics, and cell death in non-malignant human breast epithelial cells

Abstract

Widespread distribution of bisphenol-A (BPA) complicates epidemiological studies of possible carcinogenic effects on the breast because there are few unexposed controls. To address this challenge, we previously developed non-cancerous human high-risk donor breast epithelial cell (HRBEC) cultures, wherein BPA exposure could be controlled experimentally. BPA consistently induced activation of the mammalian target of rapamycin (mTOR) pathway--accompanied by dose-dependent evasion of apoptosis and increased proliferation--in HRBECs from multiple donors. Here, we demonstrate key molecular changes underlying BPA-induced cellular reprogramming. In 3/3 BPA-exposed HRBEC cell lines, and in T47D breast cancer cells, proapoptotic negative regulators of the cell cycle (p53, p21(WAF1) and BAX) were markedly reduced, with concomitant increases in proliferation-initiating gene products (proliferating cell nuclear antigen, cyclins, CDKs and phosphorylated pRb). However, simultaneous exposure to BPA and the polyphenol, curcumin, partially or fully reduced the spectrum of effects associated with BPA alone, including mTOR pathway proteins (AKT1, RPS6, pRPS6 and 4EBP1). BPA exposure induced an increase in the ERα (Estrogen Receptor): ERβ ratio--an effect also reversed by curcumin (analysis of variance, P < 0.02 for all test proteins). At the functional level, concurrent curcumin exposure reduced BPA-induced apoptosis evasion and rapid growth kinetics in all cell lines to varying degrees. Moreover, BPA extended the proliferation potential of 6/6 primary finite-life HRBEC cultures--another effect reduced by curcumin. Even after removal of BPA, 1/6 samples maintained continuous growth--a hallmark of cancer. We show that BPA exposure induces aberrant expression of multiple checkpoints that regulate cell survival, proliferation and apoptosis and that such changes can be effectively ameliorated.

Fig. 1.

BPA-induced pro-survival changes in proapoptotic and cell cycle proteins; effects of curcumin. (A) Representative sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blot analysis of cell lysates of HRBECs (PA115, PA024 and PA025), and T47D after 7 day exposure to 100nM BPA and/or curcumin to evaluate alterations in proteins related to apoptosis regulation and cell cycle progression. Note downregulation of p53 by BPA alone and its induction by curcumin exposure in all cell lines. In parallel to p53, these chemicals also modulate levels of the proapoptotic protein—BAX. Similarly, BPA exposure results in the suppression of the p53-regulated CDK inhibitor, p21^WAF1, and the induction and activation of CDKs and cyclins followed by pRb phosphorylation in all cell lines to varying degrees. Accumulation of proteins associated with DNA replication, MCM2 and PCNA, is evident in the DNA-bound fraction of test cell lysates. Curcumin alone had the opposite effects for p53, p21^WAF1, cyclin D3, cyclin A, pRb, CDK6 and CDK2, with mixed effects for MCM2, BAX and PCNA. Curcumin reduces BPA-mediated pro-survival changes in all cell lines. Plots derived from the densitometric scans of blots generated from two independent runs are included as Supplementary Figure 1, available at Carcinogenesis Online. (B) Annexin V-based FACS analysis of tamoxifen (4-OHT) induced apoptosis in BPA-treated cells, with or without curcumin supplementation in four test cell lines. Scatter plot of induced apoptotic cell death plotted as a percentage of untreated control. Data represent two to three replicates from two independent experiments on each cell line; each dot represents one or more replicates in the event of overlapping values. There was a minimum of four replicates per cell line for each treatment. BPA alone treatment (blue spheres) significantly reduced the apoptotic fraction. The addition of curcumin to BPA (maroon spheres) increased apoptotic cell death to varying degrees. Two-sample t-tests were used to compare percent apoptosis induction in BPA + curcumin versus BPA alone for each cell line. Estimated improvement in apoptosis induction by curcumin in the presence of BPA is as follows: PA024: 13% (t = 4.3, P = 0.004); PA025: 34% (t = 10.2, P = 0.0001); PA115: 11.5% (t = 3.6, P = 0.007); T47D: 74% (t = 29.5, P = 0.0001).

Fig. 2.

BPA and curcumin modulation of mTOR pathway proteins and ER subtypes. (A) Representative sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blot analysis of cell lysates of spontaneously immortalized non-malignant HRBEC lines (PA115, PA024 and PA025), and T47D breast cancer cells exposed to 100nM BPA and/or curcumin for 7 days to determine effect on mTOR pathway proteins. AKT1, RPS6 (total and phosphorylated) and 4EBP1 proteins increased with BPA alone. Curcumin induced an opposite effect on RPS6, pRPS6 and 4EBP1, but not AKT expression. BPA + curcumin reduced the effect of BPA alone for all four mTOR proteins. BPA alone induced ERα and reduced ERβ, thereby raising the ERα:ERβ ratio. Adding curcumin to BPA reduced induction of ERα protein and increased ERβ protein resulting in a relative decrease in the ERα:ERβ ratio compared with BPA exposure alone. Plots derived from the densitometric scans of the blots generated from two independent runs are included as Supplementary Figure 2, available at Carcinogenesis Online. (B) Summary representation of 64 two-way ANOVA tests for change in the expression of 16 gene products in four test cell lines partially illustrated in Figure 1A and part (A) of this figure. All measurements shown represent duplicate values from two independent gel runs. A minimum of eight data points was included for ANOVA of each gene product under each test conditions (HRBECs: n = 6, T47D: n = 2). The directional effect on each gene (positive or negative) was the same across all four cell lines. Top panel—effect of BPA alone. Bottom panel—effect of curcumin in the presence of BPA. The two treatment conditions display opposite effects consistent across all test cell lines. (ANOVA, P < 0.02 for all gene products.)

Fig. 3.

BPA-induced cell cycle progression and increased rate of cell proliferation; effects of curcumin. (A) Distribution of cell cycle phases in cultures pretreated with BPA with or without concurrent curcumin exposure for 7 days. Seven cell samples were evaluated—three IMM-HRBEC lines (PA024, PA025 and PA115), three primary finite-life HRBEC cultures (PA126, PA127 and PA128) and T47D breast cancer cells. Representative pie charts derived from FACSanalysis of BrdU and PI-labeled cells demonstrate altered distribution of cell cycle phases to varying degrees in the presence of BPA and its reversal by curcumin supplementation evident as an increase in the percentage of cells in G₁ and G₂/M and a reduction in percent S phase. Statistical analysis of the data by ANOVA is summarized in Supplementary Table 1, available at Carcinogenesis Online. (B) Changes in proliferation kinetics of cultures pretreated with BPA with or without concurrent curcumin exposure analyzed as the percentage of BrdU-labeled cells over a 1–24h period. Note that the proliferation rate is highest in the presence of BPA but reverts to control or lower rates with curcumin. Arrows from each plot point to the initial replication rate of exposed cells calculated by measuring the slope of the linear regression of the curves between 0 and 1h. BPA consistently increased the replication rate, whereas concomitant exposure to BPA and curcumin reset the replication rate to values similar to or lower than control. (C) Cell counts of IMM-PA025 and primary HRBEC (PA124) at the end of a 7 day period of exposure to 5nM E2 or 100nM BPA, with or without curcumin, confirm the FACS data shown in panels A and B. The effect of curcumin in reducing cell number in the presence of BPA compared with BPA alone was statistically significant for both PA025 and PA124, as estimated by ANOVA (P = 0.0001). Control cell counts represented an average of 4500±500. In (A–C), each data set represents the averaged values from two independent experiments performed in triplicate on each immortalized cell line and in duplicate for each finite lifespan primary HRBEC culture exposed to all test conditions.

Fig. 4.

Prolonged survival and abrogation of quiescence induced by BPA in finite-life primary HRBECs. Brightfield micrographs of six independent cases of finite-life primary HRBECs (PA144, PA146, PA147, PA148, PA150 and PA151) demonstrate vigorous growth stimulated by BPA within a 40 day period (darkened squares). A minimal amount of sample precludes multiple replicates of each treatment condition to be set up directly after acquisition from volunteer donors, but six of six consecutive specimens were consistent in the results shown. (A) The growth differential induced by BPA is visible as early as day 5 after initial plating (PA145 and PA146) and later as well. (B) Forty-day cultures in the presence of curcumin show growth reduction compared with controls, and those exposed to BPA + curcumin display growth attenuation in comparison with the stimulation by BPA alone (PA147, PA148, PA150 and PA151).

Fig. 5.

Long-term effects of prior BPA exposure on the cell cycle. Primary HRBECs (PA151) exposed to BPA for 40 days, followed by growth in BPA-free and E2-free medium for an additional 30 days display S-phase characteristics of early passage finite-life primary HRBEC control cultures (see Figure 3A). In contrast, parallel controls without BPA or in BPA + curcumin cease proliferation and are represented largely by cells in G₁ and subG₁, respectively. Cells exposed for the first 30 days to curcumin alone did not survive. The differences in percent BrdU-positive cells in various treatment groups were significant (curcumin-induced reduction compared with BPA alone for G₁ phase was 51%, P = 0.0007; S phase—10.7%, P = 0.009; G₂/M—13.7%, P = 0.006; subG₁—75.5%, P = 0.0001).

Fig. 6.

Schematic model of BPA-induced cellular reprogramming and its reversal. We postulate that BPA exposure perturbs multiple regulatory hubs within target cells, thereby promoting the functional manifestation of well-accepted ‘hallmarks of cancer’ and culminating in the sustained propagation of finite-life non-malignant HRBECs. Green arrow represents activation; red ‘T’ denotes inhibition. Our findings support the antagonist effects of ER and p53 on cellular programs altered by BPA (depicted by large circles), each of which is associated with clinical breast cancer outcome. For example (i) cell survival—mediated by activation of the PI3K/AKT/mTOR pathway is known to result from estrogenic stimuli among others (31). Activation of this pathway confers a poor prognosis (32), as we demonstrated previously using a BPA-response gene signature (10). (ii) Apoptosis evasion—a role for BPA in the inhibition of p53, and downstream effects on BAX expression is consistent with its estrogenic action. TP53 loss of function is a hallmark of all cancer in general. Specifically in breast cancer, p53 status identifies women at a high risk of disease-specific mortality among African-Americans (33) and other ethnicities. (iii) Cell cycle—analogous to estrogen-stimulated early G₁ changes (34), BPA exposure induces cyclin D, inactivates p21^WAF1, facilitating cyclin E–Cdk2 activation and pRb inactivation in mid-to-late G₁, allowing S phase entry to HRBECs. Overexpression of cell cycle progression genes is prognostic in breast cancer (35). (iv) DNA replication—ERα interacts with PCNA to initiate DNA replication and repair (36). Similarly, the MCM gene family required for DNA replication is frequently upregulated in various cancers, including breast tumors (37). (v) Extended propagation potential—is often a prelude to in vitro cell immortalization and is a characteristic feature of tumor cells derived from aggressive high-grade breast cancer (38). Our hypothesis predicts that BPA-induced aberrant gene expression alters the normal regulation of ER-responsive genes that influence pathways underlying tumorigenesis. As such, this model provides a molecular basis for BPA-promoted breast carcinogenesis and a mechanistically defined role for curcumin or curcumin-like agents in its prevention.