Troglitazone inhibits growth of MCF-7 breast carcinoma cells by targeting G1 cell cycle regulators

Peroxisome proliferator activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily. Ligand activation of PPARgamma has been shown to cause growth arrest in several human tumor cell types, but the underlying molecular mechanism has not been elucidated. We report here that the PPARgamma ligand troglitazone (TRO) inhibited MCF-7 cell proliferation by blocking events critical for G1 --> S progression. Flow cytometry demonstrated that TRO at 20 microM increased the percentage of cells in G1 from 51 to 69% after 24 h. Accumulation of cells in G1 was accompanied by an attenuation of Rb protein phosphorylation associated with decreased CDK4 and CDK2 activities. Inhibition of CDK activity by TRO correlates with decreased protein levels for several G1 regulators of Rb phosphorylation (cyclin D1, and CDKs 2, 4, and 6). Overexpression of cyclin D1 partially rescued MCF-7 cells from TRO-mediated G1 arrest. Targeting of G1 regulatory proteins, particularly cyclin D1, and the resulting induction of G1 arrest by TRO may provide a novel antiproliferative therapy for human breast cancer.     

Resveratrol chemosensitizes breast cancer cells to melphalan by cell cycle arrest

Melphalan (MEL) is a chemotherapeutic agent used in breast cancer therapy; however, MEL's side effects limit its clinical applications. In the last 20 years, resveratrol (RSV), a polyphenol found in grape skins, has been proposed to reduce the risk of cancer development. The aim of this study was to investigate whether RSV would be able to enhance the antitumor effects of MEL in MCF-7 and MDA-MB-231 cells. RSV potentiated the cytotoxic effects of MEL in human breast cancer cells. This finding was related to the ability of RSV to sensitize MCF-7 cells to MEL-induced apoptosis. The sensitization by RSV involved the enhancement of p53 levels, the decrease of procaspase 8 and the activation of caspases 7 and 9. Another proposed mechanism for the chemosensitization effect of MCF-7 cells to MEL by RSV was the cell cycle arrest in the S phase. The treatment with RSV or MEL increased the levels of p-Chk2. The increase became pronounced in the combined treatments of the compounds. The expression of cyclin A was decreased by treatment with RSV and by the combination of RSV with MEL. While the levels of cyclin dependent kinase 2 (CDK2) remained unchanged by treatments, its active form (Thr(160) -phosphorylated CDK2) was decreased by treatment with RSV and by the combination of RSV with MEL. The activity of CDK7, kinase that phosphorylates CDK2 at Thr(160), was inhibited by RSV and by the combination of RSV with MEL. These results indicate that RSV could be used as an adjuvant agent during breast cancer therapy with MEL.     

Elevated expression of the estrogen receptor prevents the down-regulation of p21Waf1/Cip1 in hormone dependent breast cancer cells

Expression of an estrogen receptor alpha (ER) transgene in hormone independent breast cancer and normal breast epithelial cells arrests cell cycling when estradiol is added. Although endogenously expressed ER does not typically affect estradiol-induced cell cycling of hormone dependent breast cancer cells, we observed that elevated expression of a green fluorescent protein fused to ER (GFP-ER) hindered entry of estrogen treated MCF-7 cells into S phase of the cell cycle. In analyses of key cell-cycle regulating proteins, we observed that GFP-ER expression had no affect on the protein levels of cyclin D1, cyclin E, or p27, a cyclin dependent kinase (Cdk) inhibitor. However, at 24 h, p21 (Waf1, Cip1; a Cdk2 inhibitor) protein remained elevated in the high GFP-ER expressing cells but not in non-GFP-ER expressing cells. Elevated expression of p21 inhibited Cdk2 activity, preventing cells from entering S phase. The results show that elevated levels of ER prevented the down-regulation of p21 protein expression, which is required for hormone responsive cells to enter S phase.     

New Castanospermine Glycoside Analogues Inhibit Breast Cancer Cell Proliferation and Induce Apoptosis without Affecting Normal Cells

sp²-Iminosugar-type castanospermine analogues have been shown to exhibit anti-tumor activity. However, their effects on cell proliferation and apoptosis and the molecular mechanism at play are not fully understood. Here, we investigated the effect of two representatives, namely the pseudo-S- and C-octyl glycoside 2-oxa-3-oxocastanospermine derivatives SO-OCS and CO-OCS, on MCF-7 and MDA-MB-231 breast cancer and MCF-10A mammary normal cell lines. We found that SO-OCS and CO-OCS inhibited breast cancer cell viability in a concentration- and time-dependent manner. This effect is specific to breast cancer cells as both molecules had no impact on normal MCF-10A cell proliferation. Both drugs induced a cell cycle arrest. CO-OCS arrested cell cycle at G1 and G2/M in MCF-7 and MDA-MB-231cells respectively. In MCF-7 cells, the G1 arrest is associated with a reduction of CDK4 (cyclin-dependent kinase 4), cyclin D1 and cyclin E expression, pRb phosphorylation, and an overexpression of p21^Waf1/Cip1. In MDA-MB-231 cells, CO-OCS reduced CDK1 but not cyclin B1 expression. SO-OCS accumulated cells in G2/M in both cell lines and this blockade was accompanied by a decrease of CDK1, but not cyclin B1 expression. Furthermore, both drugs induced apoptosis as demonstrated by the increased percentage of annexin V positive cells and Bax/Bcl-2 ratio. Interestingly, in normal MCF-10A cells the two drugs failed to modify cell proliferation, cell cycle progression, cyclins, or CDKs expression. These results demonstrate that the effect of CO-OCS and SO-OCS is triggered by both cell cycle arrest and apoptosis, suggesting that these castanospermine analogues may constitute potential anti-cancer agents against breast cancer.     

Regulation of IGF-1-dependent cyclin D1 and E expression by hEag1 channels in MCF-7 cells: The critical role of hEag1 channels in G1 phase progression

Insulin-like Growth Factor-1 (IGF-1) plays a key role in breast cancer development and cell cycle regulation. It has been demonstrated that IGF-1 stimulates cyclin expression, thus regulating the G1 to S phase transition of the cell cycle. Potassium (K⁺) channels are involved in the G1 phase progression of the cell cycle induced by growth factors. However, mechanisms that allow growth factors to cooperate with K⁺ channels in order to modulate the G1 phase progression and cyclin expression remain unknown. Here, we focused on hEag1 K⁺ channels which are over-expressed in breast cancer and are involved in the G1 phase progression of breast cancer cells (MCF-7). As expected, IGF-1 increased cyclin D1 and E expression of MCF-7 cells in a cyclic manner, whereas the increase of CDK4 and 2 levels was sustained. IGF-1 stimulated p21^WAF1/Cip1 expression with a kinetic similar to that of cyclin D1, however p27^Kip1 expression was insensitive to IGF-1. Interestingly, astemizole, a blocker of hEag1 channels, but not E4031, a blocker of HERG channels, inhibited the expression of both cyclins after 6-8 h of co-stimulation with IGF-1. However, astemizole failed to modulate CDK4, CDK2, p21^WAF1/Cip1 and p27^Kip1 expression. The down-regulation of hEag1 by siRNA provoked a decrease in cyclin expression. This study is the first to demonstrate that K⁺ channels such as hEag1 are directly involved in the IGF-1-induced up-regulation of cyclin D1 and E expression in MCF-7 cells. By identifying more specifically the temporal position of the arrest site induced by the inhibition of hEag1 channels, we confirmed that hEag1 activity is predominantly upstream of the arrest site induced by serum-deprivation, prior to the up-regulation of both cyclins D1 and E.     

Indole-3-carbinol (I3C) inhibits cyclin-dependent kinase-2 function in human breast cancer cells by regulating the size distribution, associated cyclin E forms, and subcellular localization of the CDK2 protein complex

Indole-3-carbinol (I3C), a dietary compound found in cruciferous vegetables, induces a robust inhibition of CDK2 specific kinase activity as part of a G1 cell cycle arrest of human breast cancer cells. Treatment with I3C causes a significant shift in the size distribution of the CDK2 protein complex from an enzymatically active 90 kDa complex to a larger 200 kDa complex with significantly reduced kinase activity. Co-immunoprecipitations revealed an increased association of both a 50 kDa cyclin E and a 75 kDa cyclin E immunoreactive protein with the CDK2 protein complex under I3C-treated conditions, whereas the 90 kDa CDK2 protein complexes detected in proliferating control cells contain the lower molecular mass forms of cyclin E. I3C treatment caused no change in the level of CDK2 inhibitors (p21, p27) or in the inhibitory phosphorylation states of CDK2. The effects of I3C are specific for this indole and not a consequence of the cell cycle arrest because treatment of MCF-7 breast cancer cells with either the I3C dimerization product DIM or the anti-estrogen tamoxifen induced a G1 cell cycle arrest with no changes in the associated cyclin E or subcellular localization of the CDK2 protein complex. Taken together, our results have uncovered a unique effect of I3C on cell cycle control in which the inhibition of CDK2 kinase activity is accompanied by selective alterations in cyclin E composition, size distribution, and subcellular localization of the CDK2 protein complex.     

Mechanisms of Cyclin-Dependent Kinase Inactivation by Progestins

The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. In breast cancer cells the predominant effect of synthetic progestins is long-term growth inhibition and arrest in G₁ phase. Progestin-mediated growth arrest of T-47D breast cancer cells was preceded by inhibition of cyclin D1-Cdk4, cyclin D3-Cdk4, and cyclin E-Cdk2 kinase activities in vitro and reduced phosphorylation of pRB and p107. This was accompanied by decreases in the expression of cyclins D1, D3, and E, decreased abundance of cyclin D1- and cyclin D3-Cdk4 complexes, increased association of the cyclin-dependent kinase (CDK) inhibitor p27 with the remaining Cdk4 complexes, and changes in the molecular masses and compositions of cyclin E complexes. In control cells cyclin E eluted from Superdex 200 as two peaks of ~120 and ~200 kDa, with the 120-kDa peak displaying greater cyclin E-associated kinase activity. Following progestin treatment, almost all of the cyclin E was in the 200-kDa, low-activity form, which was associated with the CDK inhibitors p21 and p27; this change preceded the inhibition of cell cycle progression. These data suggest preferential formation of this higher-molecular-weight, CDK inhibitor-bound form and a reduced number of cyclin E-Cdk2 complexes as mechanisms for the decreased cyclin E-associated kinase activity following progestin treatment. Ectopic expression of cyclin D1 in progestin-inhibited cells led to the reappearance of the 120-kDa active form of cyclin E-Cdk2 preceding the resumption of cell cycle progression. Thus, decreased cyclin expression and consequent increased CDK inhibitor association are likely to mediate the decreases in CDK activity accompanying progestin-mediated growth inhibition.     

High levels of arachidonic acid and peroxisome proliferator-activated receptor-alpha in breast cancer tissues are associated with promoting cancer cell proliferation

Fatty acids are endogenous ligands of peroxisome proliferator-activated receptor-alpha (PPARα), which is linked to the regulation of fatty acid uptake, lipid metabolism and breast cancer cell growth. This study was designed to screen candidate fatty acids from breast cancer tissue and to investigate the effects of these candidate fatty acids on PPARα expression, cell growth and cell cycle progression in breast cancer cell lines. One breast cancer tissue and one reference tissue were each taken from 30 individual breasts to examine for fatty acid composition and PPARα expression. The cancer cell lines MDA-MB-231 (ER–), MCF-7 (ER++++) and BT-474 (ER++) were used to explore the mechanisms regulating cell proliferation. We found that arachidonic acid (AA) and PPARα were highly expressed in the breast cancer tissues. AA stimulated the growth of all three breast cancer cells in a time- and dose-dependent manner. The growth stimulatory effect of AA was associated with PPARα activation, and the most potent effect was found in MCF-7 cells. The stimulation of cell proliferation by AA was accompanied by the increased expression of cyclin E, a reduced population of G1 phase cells, and a faster G1/S phase transition. In contrast, AA had no effects on the levels of CDK2, CDK4, cyclin D1, p27, Bcl-2 and Bax. Our results demonstrate that high levels of AA and PPARα expression in human breast cancer tissues are associated with ER-overexpressed breast cancer cell proliferation, which is involved in activating PPARα, stimulating cyclin E expression, and promoting faster G1/S transition.     

Synergistic proliferative action of insulin-like growth factor I and 17 beta-estradiol in MCF-7S breast tumor cells.

We have analyzed the mechanism by which the combination of insulin-like growth factor I (IGF-I) and 17 beta-estradiol (E2) induces cell cycle progression in MCF-7S cells. This cell line differs from many other breast cancer-derived cell lines in that E2 (1 nM) does not induce cell cycle progression, whereas the combination of submitogenic concentrations of IGF-I (2 ng/ml) and E2 does. We find that addition of IGF-I to MCF-7S cells leads to a dose-dependent activation of the IGF type I receptor and of the MAP kinase and PI3-kinase signaling pathways. No synergy of IGF-I and E2 was detected in the activation of these signaling cascades. In terms of cell cycle-related molecules, we find that IGF-I dose-dependently raises cyclin D1 levels in serum-starved cells. Subsequent activation of cyclin E/CDK2, hyperphosphorylation of pRb, and DNA synthesis are only induced by mitogenic concentrations of IGF-I (> or =20 ng/ml). Treatment of the cells with E2 also results in the induction of cyclin D1, but in the absence of IGF-I the cells remain arrested in G1 phase. We conclude that in MCF-7S cells, the synergistic action of E2 and IGF-I derives from the ability of both hormones to induce cyclin D1 expression. The action of IGF-I is required in these cells to induce activity of the cyclin D1/CDK4 complex, which triggers progression through the cell cycle.     

Bone morphogenetic protein-2 blocks MDA MB 231 human breast cancer cell proliferation by inhibiting cyclin-dependent kinase-mediated retinoblastoma protein phosphorylation

Bone morphogenetic protein-2 (BMP-2) has been shown to act as an antiproliferative agent for a number of different cell types. We show that BMP-2 dose-dependently inhibits growth of MDA MB 231 human breast cancer cells. Epidermal growth factor (EGF) stimulates DNA synthesis and entry of these cells into the S-phase. BMP-2 inhibits EGF-induced DNA synthesis by arresting them in G1 phase of the cell cycle. BMP-2 increases the level of cyclin kinase inhibitor p21. Furthermore, we show that exposure of MDA MB 231 cells to BMP-2 stimulates association of p21 with cyclin D1 and with cyclin E resulting in the inhibition of their associated kinase activities. Finally, BMP-2 treatment is found to cause hypophosphorylation of the retinoblastoma protein (pRb), a key regulator of cell cycle progression. Our data provide a mechanism for the antiproliferative effect of BMP-2 in the breast cancer cells.     

The mitogenic signaling pathway for fibroblast growth factor-2 involves the tyrosine phosphorylation of cyclin D2 in MCF-7 human breast cancer cells

Fibroblast growth factor-2 (FGF-2) is mitogenic for the human breast cancer cell line MCF-7; here we investigate some of the signaling pathways subserving this activity. FGF-2 stimulation of MCF-7 cells resulted in a global increase of intracellular tyrosine phosphorylation of proteins, particularly FGF receptor substrate-2, the protooncogene product Src and the mitogen-activated protein kinase (MAP kinase) cascade. A major increase in the tyrosine phosphorylation of a 30-kDa protein species was also found. This protein was identified as cyclin D2 by mass spectrometry after trypsin digestion. Immunoprecipitation of cyclin D2 and immunoblotting with anti-phosphotyrosine antibodies confirmed that the tyrosine phosphorylation of cyclin D2 was indeed induced by FGF-2 stimulation. In addition, pharmacological inhibition of Src (with herbimycin A and PP2), and of the MAP kinase cascade (with PD98059), confirmed that Src activity is required for the FGF-2-induced phosphorylation of cyclin D2 whereas MAP kinase activity is not. Thus, tyrosine phosphorylation of cyclin D2 may be a key regulatory target for FGF-2 signaling.     

Cell-cycle arrest and apoptosis induction in human breast carcinoma MCF-7 cells by carboxymethylated β-glucan from the mushroom sclerotia of Pleurotus tuber-regium

The mechanism for the anti-tumor activity of a water-soluble carboxymethylated β-glucan (CMPTR), partially synthesized from an insoluble native glucan isolated from the sclerotia of Pleurotus tuber-regium, was studied using human breast carcinoma MCF-7 breast cancer cells in vitro. CMPTR-induced anti-proliferative activity dose-dependently, with an IC₅₀ of 204 μg/ml. CMPTR inhibited the cell proliferation of MCF-7 by arresting the G₁ phase of its cell cycle after 48 h of incubation as shown by flow cytometry. Such G₁ phase arrest was associated with the down-regulation of cyclin D1 and cyclin E expressions in the breast cancer cells. In addition, the CMPTR-treated MCF-7 cancer cells were associated with decreased expression of anti-apoptotic Bcl-2 protein and increased expression of Bax/Bcl-2 ratio. This study shows that CMPTR can inhibit the proliferation of MCF-7 by cell-cycle arrest and apoptosis induction. The potential development of this mushroom polysaccharide as a water-soluble anti-tumor agent requires further investigation.     

siRNA-cyclin D1对乳腺癌MCF-7细胞增殖的抑制作用

研究小干扰RNA(small interfering RNA,siRNA)对乳腺癌MCF-7细胞株cyclin D1表达的抑制及对细胞增殖的影响。化学合成针对cyclin D1基因的siRNA,转染MCF-7细胞株;分别应用荧光定量PCR和免疫印迹测定cyclin D1 mRNA和蛋白的表达,CCK-8测定细胞的增殖活性,流式细胞仪检测细胞周期,软琼脂培养检测细胞克隆形成能力。在实验中,10、50、100 nmol/L siRNA-cyclin D1分别使MCF-7细胞cyclin D1 mRNA表达降低了57．85%、63．22%和68．02%,蛋白表达降低了51．13%、62．09%、77．68%。转染siRNA-cyclin D1后,细胞增殖受到抑制,细胞周期阻滞于G1期,软琼脂克隆形成率降低。结果提示siRNA可以有效抑制MCF-7细胞株中cyclin D1的表达,使细胞周期阻滞于G1期,从而抑制细胞增殖。     

Dissociation of bone morphogenetic protein-mediated growth arrest and apoptosis of mouse B cells by HPV-16 E6/E7

We have previously found that bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-beta family, induces cell-cycle arrest in the G1 phase and apoptotic cell death of HS-72 mouse hybridoma cells. In this study, we show that BMP-2 did not alter expression of cyclin D, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4, p27KIP1, p16INK4a, or p15INK4b, but enhanced expression of p21(CIP1/WAF1). Accumulation of p21(CIP1/WAF1) resulted in increased binding of p21(CIP1/WAF1) to CDK4 and concomitantly caused a profound decrease in the in vitro retinoblastoma protein (Rb) kinase activity of CDK4. Furthermore, the ectopic expression of human papilloma virus type-16 E7, an inhibitor of p21(CIP1/WAF1) and Rb, reverted G1 arrest induced by BMP-2. Expression of E6/E7, without increasing the p53 level, blocked inhibition of Rb phosphorylation and G1 arrest, but did not attenuate cell death in BMP-treated HS-72 cells. Taken together, these results suggest that inhibition of Rb phosphorylation by p21(CIP1/WAF1) is responsible for BMP-2-mediated G1 arrest and that BMP-2-induction of apoptosis might be independent of Rb hypophosphorylation.     

Biological effects of G1 phase arrest compound,sesquicillin, in human breast cancer cell lines

Sesquicillin, isolated from fungal fermentation broth, strongly induced G1 phase arrest in human breast cancer cells. During G1 phase arrest, the expression level of cyclin D1, cyclin A, and cyclin E was decreased, and the expression of CDK (cyclin-dependent-kinase) inhibitor, protein p21(Waf1/Cip1), was increased in a time-dependent manner in a breast cancer cell MCF-7. Interestingly, the G1 phase arrest induced by sesquicillin also occurred independently of the tumor suppressor protein, p53. Sesquicillin inhibits the proliferation of MCF-7 via G1 phase arrest in association with the induction of CDK inhibitor protein, p21(Waf1/Cip1), and the reduction of G1 phase related-cyclin proteins.