In this study, we aimed to examine the anti-cancer effects of WA in ERα-positive MCF-7 breast cancer cells and explore the role of ERα and pathways associated with it in mediating these effects. We found that WA has potent inhibitory effects on the protein expression of ERα as well as RET tyrosine kinase that interacts with ERα pathway. WA also strongly up-regulated the protein levels of p53 and its down-stream target p21. Furthermore, WA at 2.5 or 5 μM markedly decreased HSF1 and survivin protein levels. These molecular changes correlate well with WA-induced dose- and time-dependent alterations in cell viability and apoptosis, suggesting that these are key factors involved in the anti-cancer effects of WA.
Most of breast cancer cases are hormone-dependent and higher levels estrogen are linked to increased risk of breast cancer . ERα is a transcription factor that plays a key role in mediating estrogen signaling. Recent research revealed that ERα also plays a critical role in breast cancer progression via estrogen-independent mechanisms . Knockdown of ERα expression using siRNA for ERα has been found to inhibit cell proliferation and induce apoptosis in MCF-7 breast cancer cells . As such, ERα has become a molecular target in the development of therapeutics for breast cancer. In this study, WA treatment caused drastic decrease in ERα protein levels, which most likely contributed to growth inhibition and apoptosis.
RET is a receptor tyrosine kinase that mediates the proliferative and pro-survival effects of GDNF family growth factors in breast cancer. It is over-expressed in breast cancer, with higher levels in ERα-positive tumors as compared to ERα-negative tumors. Activation of RET stimulates MCF-7 breast cancer cell proliferation, survival and scattering [5, 15]. RET pathway functionally interacts with ERα pathway . In the present study, we found that WA down-regulates RET protein levels in parallel with ERα depletion. Down-regulation of both ERα and RET pathways may thus provide more effective anti-tumor activity.
The tumor suppressor p53 exerts its anti-proliferative action by inducing reversible or irreversible cell cycle arrest or apoptosis . P53 can block cell cycle progression and/or induce apoptosis by transactivation of specific target genes, including p21Waf-1/Cip1 (p21), Gadd45, cyclin G and Bax [25, 26]. Loss of p53 activity inhibits apoptosis and accelerates the appearance of tumors in transgenic mice . The p53 gene is mutated in approximately 20% of breast cancers. Thus, although the majority of breast cancers have wild-type p53, its anti-cancer function remains suppressed. Additionally, p21 is a critical cell cycle inhibitor in many cancer cells including MCF-7 cells . It has been found to be essential for G2/M cell cycle arrest upon DNA damage . Although its role in apoptosis is controversial in general, it has been reported to induce apoptosis in MCF-7 cells [19, 30]. In the present study, we found that WA increased the protein levels of p53 and p21, suggesting that they are involved in the growth inhibition and pro-apoptotic effects of WA. The pattern of p21 correlated well with that of p53, indicating that p53 may play a role in p21 up-regulation by WA. An up-regulation of phospho-p38 MAKP was observed at 1 h and 3 h post WA treatment, preceding the up-regulation of p53. Since phopho-p38 MAPK has been demonstrated to up-regulate p53 expression , and WA is able to induce apoptosis by activating p38 in leukemic cells , it appears that the p38 pathway may be an up-stream pathway involved in the anti-cancer effects of WA in MCF-7 cells.
Accumulating evidence indicates that there is a cross-talk between pathways mediated by ERα and p53. ERα and p53 exert opposing effects on breast cancer cell proliferation . ERα binds directly to p53, leading to down-regulation of transcriptional activation or depression by p53. For example, wild-type p53 can be functionally inhibited by ERα leading to up-regulation of survivin and suppression of apoptosis . RNA interference-mediated knockdown of ERα insulted in reduced survivin expression and enhanced apoptosis in MCF-7 breast cancer cells . The depletion of ERα in combination with the up-regulation of p53 following WA treatment in MCF-7 cells may render the cells more sensitive to apoptosis. The fact that WA can also induce apoptosis, although not as effective, in MDA-MB-231 breast cancer cell line , and unpublished data from our lab] which is ERα negative and p53-mutant, suggests that WA targets multiple pathways in its anti-cancer function, and neither ERα nor wild-type p53 is indispensable for the anti-cancer effect of WA. However, it is reasonable to postulate that in ERα-positive and p53-wild-type breast cancer cells, depletion of ERα and up-regulation of p53 substantially contribute to the anti-cancer effects of WA.
HSF1, the transcription factor for heat shock proteins (HSP), has recently been shown as a facilitator of transformation in breast cancer [33, 21]. HSF1 inactivation inhibits the progression of a wide spectrum of cancers [34, 35]. As the central regulator of HSP expression, HSF1 is also a target in designing anti-breast cancer therapies. HSF1 is another up-stream regulator of survivin. Down-regulation of HSF1 leads to decreased survivin expression in breast cancer cells . In the present study, 2.5 or 5 μM of WA markedly decreased HSF1 and survivin expression at 24 h, suggesting that HSF1 may play a role in WA-induced apoptosis in part by regulating survivin expression. At lower concentrations or early time points, WA induced HSF1 phosphorylation, which is likely due to a transient defensive response of cells to the stress caused by WA.
In this study, WA induction of apoptosis in MCF-7 cells was confirmed by Annexin V assay as well as PARP cleavage and decreased expression of the pro-caspase3 and pro-caspase7. The amount of pro-caspase7 is much more abundant as compared to pro-caspase3 in MCF-7 cells, and this is in agreement with earlier reports . At 1 μM, WA induced mainly early apoptosis. At higher concentrations (2.5 and 5 μM), the number of early apoptotic cells decreased whereas more late apoptotic cells were detected. It appears that whether it is early apoptosis or late apoptosis primarily depends on the concentration of WA, as mainly late apoptosis was detected with 2.5 μM WA at 12 h when apoptosis became detectable. WA causes minimal necrosis in MCF-7 cells. The small number of cells gated to the necrosis fraction after treatment with WA is most likely artifact caused by trypsinization, because when we tried Annexin V experiments at 36 h after 2.5 μM WA treatment without trypsinization (cells were all detached by 36 h), nearly 100% of cells were gated to the last apoptosis fraction (data not shown).
A recently published paper by Hahm et al.  also reported down-regulation of ERα and up-regulation of p53. However, they declared that WA-mediated decline in ERα protein level is caused by transcriptional repression, instead of proteasomal degradation. Our data showed that WA decreased ERα at post-translational level by inducing aggregation and proteasome-dependent degradation of ERα protein. The reason why they failed to see any effect of MG132 on WA-induced decline in ERα protein level is most likely that they only examined ERα protein in Triton-soluble fraction but not in Triton-insoluble fraction.