Anti-estrogen tamoxifen has been used widely to treat the patients with ER-positive breast tumors either as adjuvant therapy following surgery or as first-line treatment for advanced disease since 1971 (Cole et al., 1971
). Tamoxifen was also approved as a chemopreventive agent for high-risk population, women who have a familial history of breast cancer. Although tamoxifen is effective as an adjuvant and chemopreventive agent, there are still a great proportion of patients who develop breast cancer or relapse breast cancer after tamoxifen treatment, and the emergence of advanced breast cancer is often not preventable (Muss, 1992
). Thus, tamoxifen treatment is usually temporary and relapse of disease is eventually inevitable.
In this study, we found that tamoxifen inhibited growth of both estrogen receptor-positive and-negative breast cancer cells at micromolar concentrations, consistent with the previous reports that tamoxifen induced growth arrest and cell apoptosis in estrogen receptor-negative breast cancer cells in vitro
(Mandlekar and Kong, 2001
; Perry et al., 1995
). Thus, induction of cell apoptotic death is one of the mechanisms involved in anti-tumor activity of tamoxifen. Tamoxifen has been used in treatment of estrogen receptor-negative tumors such as hepatoma, glioma and melanoma. However, the therapeutic efficacy of tamoxifen has been obtained at doses 4- to 8- fold higher than those used for estrogen receptor-positive tumors (reviewed in Mandlekar and Kong, 2001
), indicating that tamoxifen at higher concentrations acts in a non-estrogen receptor-mediated way to induce growth arrest and apoptotic cell death in estrogen receptor-negative breast cancer cells.
In this study, we investigated the potential of icaritin, a prenylflavonoid derivative from Chinese herb Epimedium Genus
, to inhibit growth of breast cancer cells. Previously, it was reported that icaritin exhibits estrogen-like activity in estrogen receptor-positive breast cancer MCF7 cells at sub-micromolar concentrations (Wang and Lou, 2004
). At micromolar range, however, icaritin inhibited growth of prostate cancer PC3 cells (Huang et al., 2007b
). These results indicated that icaritin has both agonist and antagonist activities depending on concentrations and may function as an estrogen receptor modulator to regulate cell growth. Here, we showed that icaritin potently inhibited growth of both estrogen receptor-positive and –negative breast cancer cells through induction of sustained ERK activation mediated G2
/M arrest of the cell cycle and cell death.
Eukaryotic cell cycle progression involves sequential activation of Cdks, which are controlled by a complex of proteins, including the cyclins. A complex formed by the association of Cdk1 (also called as p34cdc2) and cyclinB1 plays a major role during entering into mitosis (Hartwell and Kastan, 1994
). Cdc25C regulates the cdc2-cyclinB1 complex and is believed to be the rate-limiting step in the G2
/M transition (Xiao et al., 2003
). Here, we found that icaritin treatment arrested breast cancer cells mainly at the G2
/M phase of the cell cycle, which was accompanied with down-regulation of the expression levels of the proteins pivotal for the G2
/M transition. Previously, Huang et al. reported that icaritin treatment induced G1
arrest in the prostate cancer PC-3 cell line (Huang et al., 2007b
). The exact mechanism underlying this difference is not clear. One possibility is that different cell lines were used. In addition, their study also employed much higher concentrations of icaritin (10–50 μM), which may also provide an explanation to the difference we observed.
In this study, we observed that icaritin induced a different pattern of cell death in MDA-MB-453 and MCF7 cells; icaritin treatment mainly increased annexin V-positive (apoptotic) cells in MDA-MB-435 cell line while annexin V-negative/PI positive (necrotic) cells in MCF7 cell line. These experiments were repeated more than three times and similar results were consistently obtained. The exact mechanism underlying this difference is currently not clear. It was reported that as an important caspase in apoptotic cell death, caspase-3 (CPP32/Yama/apopain) is critical for phosphatidylserine externerlization in the early stage of apoptosis (Mandal D et al., 2002
). Thus, it is possible that as a caspase-3-deficient cell line (Kurokawa et al., 1999
), MCF7 cells may exhibit an annexin V/PI staining pattern different from that of MDA-MB-435 after icaritin treatment. It is also possible that icaritin may mainly choose necrosis relevant pathway to induce cell death in MCF7 cells.
ERK is a member of MAPK family that plays an essential role in many cellular events such as cell growth, differentiation, survival and apoptosis through phosphorylation of specific serines/threonines of substrates (Chang and Karin, 2001
; Platanias, 2003
). Previously, it has been reported that persistent or sustained ERK activation contributes to cell death in several systems (Ramos, 2008
; Stanciu et al., 2000
). Here, we found that icaritin induced sustained ERK activation in both estrogen receptor-positive and -negative breast cancer cells, which contributes to icaritin-induced cell cycle arrest and cell death. In this study, we did not observe that icaritin induced activation of the other members of the MAPK family, p38 and JNK. Recently, It was reported that icaritin induced cell apoptosis in the hepatoma HepG2 cell line and icaritin activated JNK1 but not ERK1/2 and p38 (He et al., 2010
), suggesting that icaritin may function through different signaling pathway in different cell context.
Accumulating evidence indicated that many types of cancer, including breast cancer, originate from and are maintained by a small population of cancer stem/progenitor cells. These cancer stem/progenitor cells are resistant to most therapeutic approaches currently used. In this study, we enriched breast cancer stem/progenitor cells using the fluorescent ALDEFLUOR assay that was used to successfully isolate breast cancer stem cells from specimens of breast tumors and established breast cancer cell lines (Ginestier et al., 2007
). These ALDH-high breast cancer cells exhibit stem/progenitor cell properties and display increased metastatic potentials (Ginestier et al., 2007
). Here, we showed that ALDH-high breast cancer stem/progenitor cells were less sensitive to anti-estrogen tamoxifen compared to ALDH-low cells, suggesting that breast cancer stem/progenitor cells are more resistant to tamoxifen compared to non-stem/progenitor cells and tamoxifen may only inhibits growth of non-stem/progenitor cells. Our finding is consistent with the previous reports that cancer stem/progenitor cells are resistant to many current cancer therapies including chemo- and radiation-therapy (Fillmore and Kuperwasser, 2008
; Hambardzumyan et al., 2006
; Shafee et al., 2008
). This suggests that many cancer therapies including hormone therapy, while killing the bulk of tumor cells, may eventually fail since they do not eradicate cancer stem/progenitor cells that survive to regenerate new tumors.
In this study, we found that unlike tamoxifen, icaritin inhibited growth of ALDH-high breast cancer stem/progenitor cells and ALDH-low differentiated breast cancer cells with the preferential targeting of breast cancer stem/progenitor cells. Here, we also found that a combination of tamoxifen and icaritin functioned more effectively to inhibit growth of breast cancer cells compared to either agent alone. Our results thus suggested that therapies that are directed against both differentiated breast cancer cells and breast cancer stem/progenitor cells may provide advantages to treat this deadly disease.