Selenium disrupts estrogen receptor (alpha) signaling and potentiates tamoxifen antagonism in endometrial cancer cells and tamoxifen-resistant breast cancer cells

Tamoxifen, a selective estrogen receptor (ER) modulator, is the most widely prescribed hormonal therapy treatment for breast cancer. Despite the benefits of tamoxifen therapy, almost all tamoxifen-responsive breast cancer patients develop resistance to therapy. In addition, tamoxifen displays estrogen-like effects in the endometrium increasing the incidence of endometrial cancer. New therapeutic strategies are needed to circumvent tamoxifen resistance in breast cancer as well as tamoxifen toxicity in endometrium. Organic selenium compounds are highly effective chemopreventive agents with well-documented benefits in reducing total cancer incidence and mortality rates for a number of cancers. The present study shows that the organic selenium compound methylseleninic acid (MSA, 2.5 micromol/L) can potentiate growth inhibition of 4-hydroxytamoxifen (10(-7) mol/L) in tamoxifen-sensitive MCF-7 and T47D breast cancer cell lines. Remarkably, in tamoxifen-resistant MCF-7-LCC2 and MCF7-H2Delta16 breast cancer cell lines and endometrial-derived HEC1A and Ishikawa cells, coincubation of 4-hydroxytamoxifen with MSA resulted in a marked growth inhibition that was substantially greater than MSA alone. Growth inhibition by MSA and MSA + 4-hydroxytamoxifen in all cell lines was preceded by a specific decrease in ER(alpha) mRNA and protein without an effect on ER(beta) levels. Estradiol and 4-hydroxytamoxifen induction of endogenous ER-dependent gene expression (pS2 and c-myc) as well as ER-dependent reporter gene expression (ERE(2)e1b-luciferase) was also attenuated by MSA in all cell lines before effect on growth inhibition. Taken together, these data strongly suggest that specific decrease in ER(alpha) levels by MSA is required for both MSA potentiation of the growth inhibitory effects of 4-hydroxytamoxifen and resensitization of tamoxifen-resistant cell lines.     

Inhibition of the phosphatidylinositol 3-kinase/Akt pathway sensitizes MDA-MB468 human breast cancer cells to cerulenin-induced apoptosis

Fatty acid synthase is overexpressed in cancer especially in tumors with a poor prognosis. The specific fatty acid synthase inhibitor cerulenin can induce apoptosis in cancer cells. Likewise, phosphatidylinositol 3-kinase (PI3K)/Akt kinase activities are elevated in primary tumors and cancer cell lines. Here, we tested whether inhibition of PI3K/Akt pathway would sensitize cancer cells to cerulenin-induced apoptosis. We show that LY294002, an inhibitor of PI3K, sensitized MDA-MB468 breast cancer cells to cerulenin-induced apoptosis. In MDA-MB468 cells, cerulenin- and LY294002-mediated apoptosis was associated with caspase-3 activation and the release of cytochrome c from mitochondria to cytosol. In addition, we observed additional species of Bak in mitochondria, suggesting a possible Bak activation. Treatment of cells with cerulenin and LY294002 down-regulated the protein levels of X chromosome-linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis 1 (cIAP-1), and Akt, whereas the levels of mitogen-activated protein/extracellular signal-regulated kinase kinase and other antiapoptotic Bcl-2 family proteins (Bcl-2 and Bcl-xl) did not change. Interestingly, the nonspecific caspase inhibitor, z-VAD-FMK, inhibited the down-regulation of Akt, XIAP, and cIAP-1 in cerulenin- and LY294002-treated cells. In conclusion, these studies show that inhibition of PI3K can sensitize cerulenin-induced apoptosis in MBA-MB468 breast cancer cells via activation of caspases, down-regulation of antiapoptotic proteins, such as XIAP, cIAP-1 and Akt, and possibly, activation of Bak in mitochondria.     

Epigallocatechin-3-gallate induces apoptosis in estrogen receptor-negative human breast carcinoma cells via modulation in protein expression of p53 and Bax and caspase-3 activation

Epigallocatechin-3-gallate (EGCG) has been shown to have anticarcinogenic effects in in vitro and in vivo models, and this effect is mediated at least in part by its ability to induce apoptosis in cancer cells without affecting normal cells. It has been recognized that estrogen receptor (ER)-dependent breast cancers generally have a better prognosis and are often responsive to antiestrogen therapy; however, ER-independent breast cancers are more aggressive and unresponsive to antiestrogens. Using the MDA-MB-468 human breast cancer cell line as an in vitro model of ER-negative breast cancers, we found that treatment of EGCG resulted in dose-dependent (5-80 microg/mL) and time-dependent (24-72 hours) inhibition of cellular proliferation (15-100%) and cell viability (3-78%) in MDA-MB-468 cells. Decrease in cell viability was associated with the induction of apoptosis (18-66%) which was analyzed by DNA ladder assay, fluorescence staining, and flow cytometry. Induction of apoptosis by EGCG could be corroborated to the increased expression of tumor suppressor protein p53 and its phosphorylation at Ser 15 residue. EGCG decreased the expression of antiapoptotic protein Bcl-2 but increased proapoptotic protein Bax in these cells. The increased ratio of Bax/Bcl-2 proteins after EGCG treatment may have resulted in increased release of cytochrome c from mitochondria into cytosols, increased expression of Apaf-1, and activation of caspase-3 and poly(ADP-ribose) polymerase, which may lead to apoptosis in MDA-MB-468 cells. Together, the results of this study provide evidence that EGCG possesses anticarcinogenic effect against ER-negative breast cancer cells and thus provide the molecular basis for the future development of EGCG as a novel and pharmacologically safe chemopreventive agent for breast cancer prevention.     

Tamoxifen-resistant human breast cancer cell growth: inhibition by thioridazine, pimozide and the calmodulin antagonist, W-13.

Estrogen receptor (ER)-negative human breast cancer cell lines (MDA-MB-231 and MDA-MB-435) and ER-positive derivatives of the MCF-7 cell line selected for growth in the presence of antiestrogens (LY2 and RR) were used as in vitro models of tamoxifen-resistant human breast cancer in this study. The sensitivity of the tamoxifen-sensitive (MCF-7) and tamoxifen-resistant human breast cancer cell growth to two noncytotoxic neuroleptic drugs, pimozide and thioridazine, and the anticalmodulin agent, W-13, were compared. Inhibition of cell growth was measured as a decrease in cell number following a 72-h incubation with drug. Growth of the ER-negative cell lines MDA-MB-231 and MDA-MB-435 was inhibited by all three drugs. The average Ki values in these two lines were 6.3 and 3.8 microM for pimozide and 4.1 and 15 microM for thioridazine, respectively. Both ER-negative cell lines were more sensitive than MCF-7 cells to growth inhibition by W-13. MCF-7 cells selected for antiestrogen resistance were sensitive to growth inhibition by W-13 and thioridazine (LY2, average Ki = 10.4 microM; RR, average Ki = 5.2 microM). LY2 and RR cells were resistant to pimozide except when treated with estradiol (Ki = 4.6 and 7.9 microM, respectively). Pimozide, thioridazine and W-13 all exerted different effects on the distribution of human breast cancer cells within the cell cycle, suggesting that each drug may utilize a distinct pathway for inhibition of cell growth. We conclude that all three drugs are potential noncytotoxic alternatives to tamoxifen for the treatment of tamoxifen-resistant human breast cancer.     

Synthesis and in vitro cytotoxicity evaluation of 4-aminoquinoline derivatives.

A series of 4-aminoquinoline derivatives were synthesized by the reaction of 4-chloro-7-substituted-quinolines with the corresponding mono/dialkyl amines. The structures of the synthesized compounds were confirmed by NMR and FAB-MS spectral and elemental analyses. Subsequently, the compounds were examined for their cytotoxic effects on two different human breast tumor cell lines: MCF7 and MDA-MB468. Although all compounds examined were quite effective on both cell lines, the compound N'-(7-chloro-quinolin-4-yl)-N,N-dimethyl-ethane-1,2-diamine emerged as the most active compound of the series. It was particularly potent against MDA-MB 468 cells when compared to chloroquine and amodiaquine. The compound butyl-(7-fluoro-quinolin-4-yl)-amine showed more potent effects on MCF-7 cells when compared to chloroquine. Therefore, 4-aminoquinoline can serve as the prototype molecule for further development of a new class of anticancer agents.     

Methylseleninic acid synergizes with tamoxifen to induce caspase-mediated apoptosis in breast cancer cells

Tamoxifen has efficacy as a breast cancer therapy and chemoprevention agent. However, toxicity and resistance to tamoxifen limit its clinical application. There is an urgent need to develop compounds that may be combined with tamoxifen to improve efficacy and overcome toxicity and resistance. We showed previously that the organoselenium compound methylseleninic acid (MSA) increased the growth-inhibitory effect of tamoxifen and reversed tamoxifen resistance in breast cancer cells. In this study, we examined the mechanism for induction of apoptosis by MSA combined with tamoxifen in tamoxifen-sensitive and tamoxifen-resistant breast cancer cells. 4-hydroxytamoxifen (TAM; 10(-7) mol/L) alone resulted in cell cycle arrest but no apoptosis, whereas MSA alone (10 mumol/L) induced apoptosis in tamoxifen-sensitive cells. Combination of MSA with TAM resulted in a synergistic apoptosis in both tamoxifen-sensitive and tamoxifen-resistant breast cancer cells compared with either agent alone. MSA and MSA combined with TAM induced apoptosis through the intrinsic, mitochondrial apoptotic pathway. MSA induced a sequential activation of caspase-9 and then caspase-8. These results indicate that the growth inhibition synergy and reversal of tamoxifen resistance by combination of selenium with tamoxifen occurs via a tamoxifen-induced cell cycle arrest, allowing more cells to enter the intrinsic apoptotic pathway elicited by selenium. [Mol Cancer Ther 2008;7(9):3056-63].     

Sulforaphane induces cell type-specific apoptosis in human breast cancer cell lines

Sulforaphane, an isothiocyanate found in cruciferous vegetables, has been shown to induce phase 2 detoxication enzymes and inhibit the growth of chemically induced mammary tumors in rats, although the exact mechanisms of action of sulforaphane are not understood. In this study, we evaluated the effects of sulforaphane on cell growth and death in several human breast cancer cell lines and examined the hypothesis that sulforaphane acts as a histone deacetylase (HDAC) inhibitor in these cell lines. Sulforaphane treatment inhibited cell growth, induced a G(2)-M cell cycle block, increased expression of cyclin B1, and induced oligonucleosomal DNA fragmentation in the four human breast cancer cell lines examined, MDA-MB-231, MDA-MB-468, MCF-7, and T47D cells. Activation of apoptosis by sulforaphane in MDA-MB-231 cells seemed to be initiated through induction of Fas ligand, which resulted in activation of caspase-8, caspase-3, and poly(ADP-ribose) polymerase, whereas apoptosis in the other breast cancer cell lines was initiated by decreased Bcl-2 expression, release of cytochrome c into the cytosol, activation of caspase-9 and caspase-3, but not caspase-8, and poly(ADP-ribose) polymerase cleavage. Sulforaphane inhibited HDAC activity and decreased the expression of estrogen receptor-alpha, epidermal growth factor receptor, and human epidermal growth factor receptor-2 in each cell line, although no change in the acetylation of H3 or H4 was seen. These data suggest that sulforaphane inhibits cell growth, activates apoptosis, inhibits HDAC activity, and decreases the expression of key proteins involved in breast cancer proliferation in human breast cancer cells. These results support testing sulforaphane in vivo and warrant future studies examining the clinical potential of sulforaphane in human breast cancer.     

Brachyury promotes tamoxifen resistance in breast cancer by targeting SIRT1

Tamoxifen is effective for treating estrogen receptor-alpha (ERα)-positive breast cancers. However, few molecular mediators of tamoxifen resistance have been elucidated. In the present study, we determine the underlying roles of Brachyury in tamoxifen resistance. Loss- and gain-of-function assay are utilized to confirm the oncogenic roles of Brachyury in breast cancer. Compared with the normal MCF10A cells, Brachyury is commonly overexpressed in breast cancer cell lines. Knockdown of Brachyury inhibits tamoxifen resistance, whereas overexpression of Brachyury enhances tamoxifen resistance as demonstrated increased cell viability and reduced cell apoptosis. Mechanistically, we demonstrate for the first time that Brachyury mediates tamoxifen resistance by regulating Sirtuin-1 (SIRT1). Collectively, our data, as a proof of principle, indicate that Brachyury is a candidate marker for predicting the clinical efficacy of tamoxifen and targeting SIRT1 could overcome resistance to tamoxifen in breast cancer cells.     

Benzyl isothiocyanate-induced apoptosis in human breast cancer cells is initiated by reactive oxygen species and regulated by Bax and Bak

Epidemiologic studies have revealed an inverse correlation between dietary intake of cruciferous vegetables and the risk of breast cancer. We now show that cruciferous vegetable constituent benzyl isothiocyanate (BITC) effectively suppresses growth of cultured human breast cancer cells (MDA-MB-231 and MCF-7) by causing G(2)-M phase cell cycle arrest and apoptosis induction. On the other hand, a normal mammary epithelial cell line (MCF-10A) is significantly more resistant to growth arrest and apoptosis by BITC compared with breast cancer cells. The BITC-mediated cell cycle arrest was associated with a decrease in levels of proteins involved in regulation of G(2)-M transition, including cyclin B1, cyclin-dependent kinase 1, and cell division cycle 25C. The BITC-induced apoptosis correlated with induction of proapoptotic proteins Bax (MCF-7) and Bak (MDA-MB-231 and MCF-7) and down-regulation of antiapoptotic proteins Bcl-2 and Bcl-xL (MDA-MB-231). The SV40-immortalized mouse embryonic fibroblasts derived from Bax and Bak double knockout mice were significantly more resistant to BITC-induced DNA fragmentation compared with wild-type mouse embryonic fibroblasts. The BITC treatment caused rapid disruption of the mitochondrial membrane potential, leading to cytosolic release of apoptogenic molecules, which was accompanied by formation of autophagosome-like structures as revealed by transmission electron microscopy. The BITC-mediated apoptosis was associated with generation of reactive oxygen species and cleavage of caspase-9, caspase-8, and caspase-3. Apoptosis induction by BITC was significantly attenuated in the presence of a combined superoxide dismutase and catalase mimetic EUK134 as well as caspase inhibitors. In conclusion, the present study reveals a complex signaling leading to growth arrest and apoptosis induction by BITC.