We are reporting that both estradiol and the endocrine disruptor BPA at very low concentrations confer resistance against cisplatin in breast cancer cells. Both compounds antagonize cisplatin cytotoxicity by increasing cell proliferation and viability and decreasing apoptosis. As judged by the use of ERα and ERβ specific antagonists, ERα-negative MDA-MB-468 cells, and ERβ-knockdown T47D cells, BPA and E2 do not appear to mediate their effects through the classical ERs. Whereas BPA and E2 do not affect the entry of cisplatin into the nucleus, they increase the expression of Bcl-2, suggesting that their action is exerted downstream of cisplatin binding to DNA. The fact that a Bcl-2 inhibitor completely blocked the protective effect of BPA while only partially antagonizing that of E2 suggests a somewhat different mechanism by which each compound confer chemoresistance.
Although the role of hormones in chemoresistance has not received considerable attention, several reports have shown that E2 opposes anti-cancer drugs in breast cancer cells. Based on viability and apoptotic endpoints, E2 at 1–100 nM protected cells from toxicity by taxol, a microtubule altering drug that is widely used in breast cancer treatment [13
]. Additionally, Teixeira et al
reported that MCF-7 breast cancer cells were less sensitive to doxorubicin when the medium was supplemented with estrogen [14
]. We are the first to report that the survival effect of E2 extends to cisplatin, and that such protection is observed with as little as 0.01 nM E2. In spite of the growing health concerns attributed to endocrine disruptors, little has been done to address their potential role in chemoresistance. We recently reported that BPA opposed the actions of several anti-cancer drugs with a focus on doxorubicin [27
]. We now extended these studies to cisplatin by finding that BPA is just as effective as E2 at antagonizing the drug. Importantly, BPA is effective at environmentally relevant concentrations of 0.01–10 nM.
Estradiol is well known as a mitogen for normal breast epithelial cells as well as breast cancer cells [30
], while BPA activity as a mitogen has been controversial. Although BPA increased MCF-7 cell proliferation, its relative mitogenic activity was 60,000 times lower than that of E2 [31
]. A confounding issue is the lack of linear dose-dependence of BPA, which often shows a ‘U’ or an inverted ‘U’ shaped curves [32
]. Thus, extrapolation from an action, or a lack of action, of BPA at high doses to its presumed bioactivity at low doses is unwarranted. Indeed, Sameuelson et al.
reported maximal MCF-7 cell proliferation in response to 1 µM BPA, an effect which was not observed at higher or lower doses [33
]. For these reasons, our studies utilized only low doses of BPA. The finding that BPA and E2 opposed cisplatin cytotoxicity by increasing cell viability could be due to increased cell proliferation, decreased apoptosis, or both. As judged by both BrdU incorporation and flow cytometry, E2 alone increases cell proliferation, while BPA alone increases survival. This suggests that in antagonizing cisplatin, E2 may act primarily as a mitogen while BPA may act more as an anti-apoptotic factor.
Our next goal was to determine which receptor(s) mediate the protective effects of E2 and BPA. ERα and ERβ, often referred to as classic ERs, are well characterized in terms of their ability to transduce the actions of E2. The relative binding affinity of BPA to either receptor is 1,000 to 10,000 times lower than that of E2, suggesting that BPA would have to be at the µM range to activate these receptors [24
]. Our studies clearly show that BPA and E2 exert their effects when ERα or ERβ were blocked by specific inhibitors. This was supported by the use of MDA-MB-468 cells, which express ERβ but not ERα [27
], and ERβ-depleted T47D cells. Our results do not agree with Sui et al
reporting that estradiol significantly reduced taxol cytotoxicity in breast cancer cells overexpressing ERα but has no effect on the ER-negative parental cells. Their study showed that ICI sensitized MCF-7 and T47D cells to taxol [29
There are several non-classical ERs through which BPA and E2 can signal, including yet unidentified membrane-associated estrogen binding proteins, GPR30 and members of the ERR family. For example, E2 rapidly stimulates ERK1/2 phosphorylation in MCF-7 cells via GPR30 [34
]. BPA binds to GPR30 with an IC50
of 630 nM, as compared to 17.8 nM for E2 [25
]. BPA binds strongly to ERRγ with a Kd of 5.5 nM, while E2 presumably does not bind to this receptor [26
]. ERRγ is expressed in 75% of breast tumors as compared to normal mammary epithelial cells [35
]. We have previously shown by Real-Time PCR that both T47D and MDA-MB-468 cells express several of the non-classical receptors [27
]. We postulate that non-classical ERs, or as yet an unidentified receptor, mediate the protective effects of these compounds. This issue is currently under investigation.
One of the mechanisms underlying resistance to chemotherapeutic agents is by conjugation of electrophilic drugs such as cisplatin to glutathione, thereby facilitating their extrusion from the cell and reducing their availability to enter the nucleus [36
]. The use of inductively coupled mass spectroscopy which can measure platinum reveals that the amount of cisplatin bound to DNA was not affected by either E2 or BPA, suggesting that their protective actions occur downstream of DNA damage rather than by regulating detoxification enzymes or membrane transporters.
Teixeira et al
reported that an E2-induced increase in Bcl-2 plays a role in antagonizing doxorubicin in MCF-7 cells [14
]. This is supported by another study underscoring the importance of increased Bcl-2 to Bax ratio for protecting MCF-7 cells against taxol-mediated cytotoxicity [13
]. Our data demonstrate that BPA and E2 alone or in combination with cisplatin dramatically increased Bcl-2 expression without altering Bcl-xL or Bax. The effects of the Bcl-2 inhibitor, which completely prevented BPA actions while only partially affecting E2, further supports the notion that the two compounds may activate somewhat different mechanisms, i.e., proliferation vs anti-apoptosis, in opposing cisplatin cytotoxicity.
In conclusion, we have demonstrated that BPA and E2 confer resistance against cisplatin, likely through their ability to increase Bcl-2 expression, thereby preventing drug-induced apoptosis. Since their actions appear to be independent of ERα and ERβ, patients with ER-negative tumors may show high resistance to cisplatin because of their endogenous estrogens. These studies also highlight an harmful effect of the endocrine disruptor BPA. Blockade of BPA and E2 actions should be beneficial for breast cancer patients by increasing the sensitivity of their tumors to anti-cancer drugs and allowing for the introduction of cisplatin into treatment regimens.