Induction of cell cycle arrest and apoptosis in human breast cancer cells by quercetin

Quercetin, a widely distributed bioflavonoid, has been shown to induce growth inhibition in certain cancer cell types. In the present study we have pursued the mechanism of growth inhibition in MCF-7 human breast cancer cells. Quercetin treatment resulted in the accumulation of cells specifically at G2/M phase of the cell cycle. Mitotic index measured by MPM2 staining clearly showed that cells were transiently accumulated in M phase, 24 h after treatment. The transient M phase accumulation was accompanied by a transient increase in the levels of cyclin B1 and Cdc2 kinase activity. However, 24 h or longer treatment caused a marked accumulation of cells in G2 instead of M phase. Levels of cyclin B1 and cyclin B1-associated Cdc2 kinase activity were also decreased. We also found that quercetin markedly increased Cdk-inhibitor p21CIP1/WAF1 protein level after treatment for 48 h or longer, and the induction of p21CIP1/WAF1 increased its association with Cdc2-cyclin B1 complex, however, up-regulation of p53 by quercetin was not observed. Quercetin also induced significant apoptosis in MCF-7 cells in addition to cell cycle arrest, and the induction of apoptosis was markedly blocked by antisense p21CIP1/WAF1 expression. The present data, therefore, demonstrate that a flavonoid quercetin induces growth inhibition in the human breast carcinoma cell line MCF-7 through at least two different mechanisms; by inhibiting cell cycle progression through transient M phase accumulation and subsequent G2 arrest, and by inducing apoptosis.     

Cucurbitacin D induces growth inhibition, cell cycle arrest, and apoptosis in human endometrial and ovarian cancer cells

Cucurbitacin D, a newly isolated triterpenoid cucurbitacin, has been found to possess anticancer effects. The purpose of this study was to elucidate the effects of cucurbitacin D on human endometrial and ovarian cancer cells. Human endometrial and ovarian cancer cells were treated with various concentrations of cucurbitacin D, and its effects on cell growth, the cell cycle, apoptosis, and their related measurements were investigated in vitro. All endometrial and ovarian cancer cell lines were sensitive to the growth-inhibitory effect of cucurbitacin D. Cell cycle analysis indicated that their exposure to cucurbitacin D increased the proportion in the sub-G0/G1 phases and G2/M phases of the cell cycle. Induction of apoptosis was confirmed by annexin V staining of externalized phosphatidylserine and loss of the transmembrane potential of mitochondria. This induction occurred in concert with altered expression of genes related to cell growth, malignant phenotype, and apoptosis. Our results suggest that cucurbitacin D might be a new therapeutic option for the treatment of endometrial and ovarian cancers.     

Monensin-mediated growth inhibition of SNU-C1 colon cancer cells via cell cycle arrest and apoptosis

Previously, we showed that monensin, Na+ ionophore, potently inhibited the growth of acute myelogenous leukemia and lymphoma cells. Here, we demonstrate that monensin inhibited the proliferation of solid tumor cells with IC50 of about 2.5 micro M. Monensin induced a G1 or a G2-M phase arrest in these cells. When we examined the effects of this drug on SNU-C1 cells, monensin decreased the levels of CDK2, CDK4, CDK6, cyclin D1 and cyclin A proteins. While p27 was increased by monensin, p21 was not. In addition, monensin markedly enhanced the binding of p27 with CDK2, CDK4 and CDK6. Furthermore, the activities of CDK2-, CDK4- and CDK6-associated kinase were reduced in association with hypophosphorylation of Rb protein. Monensin also induced apoptosis in solid tumor cells. Apoptotic process of SNU-C1 cells was associated with the changes of Bax, caspase-3 and mitochondria transmembrane potential (deltapsim). Taken together, these results demonstrated for the first time that monensin inhibited the growth of solid tumor cells, especially SNU-C1 cells, via cell cycle arrest and apoptosis.     

Reversine induces cell cycle arrest,polyploidy, and apoptosis in human breast cancer cells

Background

Reversine, a small synthetic purine analogue, has been reported to be effective in tumor suppression. In the present study, we demonstrated an antitumor activity of reversine that could suppress cellular proliferation and induce cell cycle arrest and apoptosis in human breast cancer cell lines.

Methods

To evaluate whether reversine could suppress cell growth of MCF-7 and MDA-MB-231 cells and induce cell death, the cell viability, cell cycle, and apoptosis were determined in this study.

Results

Reversine treatment in human breast cancer cells reduced cell viability in a dose-dependent manner. Cell cycle accumulation at the G₂/M phase in reversine-treated cells was also determined. Moreover, polyploidy was also found in reversine-treated cells. Apoptosis in reversine-treated cells was exhibited with PARP cleavage and caspase-3 and caspase-8 activation, but not caspase-9 activation, indicating that caspase-dependent apoptosis mediated by an extrinsic pathway took place in reversine-treated cells. Furthermore, reversine attenuated cell death in cells pretreated with a pan-caspase inhibitor before reversine treatment.

Conclusions

In the present study, we demonstrated that reversine contributes to growth inhibition in human breast cancer cells through cell cycle arrest, polyploidy, and/or apoptosis induction. The apoptosis mediated by reversine was induced by the mitochondria-independent pathway. Therefore, the potential role of reversine as a novel therapeutic agent for the treatment of breast cancer is worthy of further investigation.     

Cranberry phytochemical extracts induce cell cycle arrest and apoptosis in human MCF-7 breast cancer cells

Breast cancer is the most commonly diagnosed cancer in women in the US and is one of the leading causes of death due to cancer. Epidemiological studies have consistently suggested the inverse association between cancer risk and intake of fruits and vegetables. These health benefits have been linked to the additive and synergistic combination of phytochemicals in fruits and vegetables. Cranberries have been shown to possess anti-carcinogenic activities such as inhibition of growth of several cancer cell lines, and inhibition of ornithine decarboxylase (ODC) activity in vitro. However, the molecular mechanisms of the anti-cancer properties of cranberry phytochemical extracts have not been completely understood. Our data showed that cranberry phytochemical extracts significantly inhibited human breast cancer MCF-7 cell proliferation at doses of 5 to 30mg/mL (P<0.05). Apoptotic induction in MCF-7 cells was observed in a dose-dependent manner after exposure to cranberry phytochemical extracts for 4h. Cranberry phytochemical extracts at a dose of 50mg/mL resulted in a 25% higher ratio of apoptotic cells to total cells as compared to the control groups (P<0.05). Cranberry phytochemical extracts at doses from 10 to 50mg/mL significantly arrested MCF-7 cells at G0/G1 phase (P<0.05). A constant increasing pattern of the G1/S index was observed in the cranberry extract treatment group while the G1/S ratio of the control group decreased concomitantly between 10 and 24h treatment. After 24-h exposure to cranberry extracts, the G1/S index of MCF-7 cells was approximately 6 times higher than that of the control group (P<0.05). These results suggest that cranberry phytochemical extracts possess the ability to suppress the proliferation of human breast cancer MCF-7 cells and this suppression is at least partly attributed to both the initiation of apoptosis and the G1 phase arrest.     

The mechanism of ellipticine-induced apoptosis and cell cycle arrest in human breast MCF-7 cancer cells

Ellipticine, a cytotoxic plant alkaloid, is known to inhibit topoisomerase II. Here, we first report the molecular mechanism of ellipticine's apoptotic action in human breast MCF-7 cancer cells. Treatment of cells with ellipticine resulted in inhibition of growth, and G2/M phase arrest of the cell cycle. This effect was associated with a marked increase in the protein expression of p53 and, p21/WAF1 and KIP1/p27, but not of WAF1/p21. Ellipticine treatment increased the expression of Fas/APO-1 and its ligands, mFas ligand and sFas ligand, and subsequent activation of caspase-8. The mitochondrial apoptotic pathway amplified the Fas/Fas ligand death receptor pathway by Bid interaction. This effect was found to result in a significant increase in activation of caspase-9. Taken together, we have concluded that the molecular mechanisms during ellipticine-mediated growth inhibition and induction of apoptosis in MCF-7 cells were due to (1) cell cycle arrest and induction of apoptosis, (2) induction of p53 and KIP1/p27 expression, (3) triggering of Fas/Fas ligand pathway, (4) disruption of mitochondrial function, and (5) the apoptotic signaling was amplified by cross-talk between Fas death receptor and mitochondrial apoptotic pathway.     

Acacetin-induced cell cycle arrest and apoptosis in human non-small cell lung cancer A549 cells

In this study, we examined acacetin (5,7-dihydroxy-4'-methoxyflavone), a flavonoid compound, for its effect on proliferation in human non-small cell lung cancer A549 cells. The results first reported that acacetin not only inhibited A549 cell proliferation but also induced apoptosis and blocked cell cycle progression in the G1 phase. ELISA assay demonstrated that acacetin significantly increased the expression of p53 and p21/WAF1 protein, which caused cell cycle arrest. An enhancement in Fas and its two forms of ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), might be responsible for the apoptotic effect induced by acacetin. Taken together, p53 and Fas/FasL apoptotic system may participate in the antiproliferative activity of acacetin in A549 cells.     

Monensin-mediated growth inhibition in NCI-H929 myeloma cells via cell cycle arrest and apoptosis

Previously, we showed that monensin, Na+ ionophore, potently inhibited the growth of acute myelogenous leukemia and lymphoma cells. Here, we investigated the antiproliferative effect of monensin on human myeloma cell lines. Monensin significantly inhibited the proliferation of myeloma cell lines examined with IC50 of about 1 micro M. Cell cycle analysis indicated that monensin induced a G1 and/or a G2-M phase arrest in these cell lines. To address the mechanism of the antiproliferative effect of monensin, we examined the effect of this drug on cell cycle-related proteins in NCI-H929 cells. Monensin decreased the levels of CDK2, CDK6, cdc2, cyclin A, cyclin B1, cyclin D1 and cyclin E proteins but did not alter CDK4 protein. While p21 was increased by monensin, p27 was not. In addition, monensin markedly enhanced the binding of p21 with CDK6 and cdc2. Furthermore, the activities of CDK2- and CDK6-associated kinases were reduced in association with hypophosphorylation of Rb protein. The activity of cdc2-associated kinase was decreased, which was accompanied by reduction of cdc25C phosphatase. Also, monensin induced apoptosis in myeloma cells, as evidenced by annexin V binding assay and flow cytometric detection of sub-G1 DNA content. This apoptotic process was associated with down-regulation of Bcl-2, loss of mitochondria transmembrane potential (Deltapsim) and an increase of caspase-3 activity. In addition, monensin caused the up-regulation of ERK and p38 kinase activities. Taken together, these results have demonstrated for the first time that monensin potently inhibited the proliferation of human myeloma cell lines, especially NCI-H929 cells, via cell cycle arrest in association with p21 and apoptosis.     

Monensin-mediated growth inhibition in acute myelogenous leukemia cells via cell cycle arrest and apoptosis

Monensin, an Na(+) ionophore, regulates many cellular functions including apoptosis. However, there has been no report about the antitumoral effect of monensin on acute myelogenous leukemia (AML). Here, we investigated the antiproliferative effect of monensin on AML cells in vitro and in vivo. Monensin efficiently inhibited the proliferation of all of 10 AML cell lines, with IC(50) of about 0.5 microM. DNA flow cytometric analysis indicated that monensin induced a G(1) and/or a G(2)-M phase arrest in these cell lines. To address the mechanism of the antiproliferative effect of monensin, we examined the effect of monensin on cell cycle-related proteins in HL-60 cells. The levels of CDK6, cyclin D1 and cyclin A were decreased. In addition, monensin not only increased the p27 level but also enhanced its binding with CDK2. Furthermore, the activities of CDK2- and CDK6-associated kinases reduced by monensin were associated with hypophosphorylation of Rb protein. Monensin also induced apoptosis in AML cells including HL-60 cells. The apoptotic process of HL-60 cells was associated with changes in Bax, caspase-3, caspase-8 and mitochondria transmembrane potential (Deltapsi(m)). In particular, monensin (i.p. at a dose of 8 mg/kg thrice weekly) significantly reduced the tumor size of BALB/c mice that were inoculated s.c. with its derived cell line, WEHI-3BD cells (69% growth inhibition relative to control group; p < 0.05). Tumors from monensin-treated mice exhibited increased apoptosis, and these tumor were immunohistochemically more stained with Bax, Fas and p53 antibodies than control tumors. In conclusion, this is the first report that monensin potently inhibits the proliferation of AML cells.     

Isoobtusilactone A induces cell cycle arrest and apoptosis through reactive oxygen species/apoptosis signal-regulating kinase 1 signaling pathway in human breast cancer cells

This study is the first to investigate the anticancer effect of isoobtusilactone A (IOA) in two human breast cancer cell lines, MCF-7 and MDA-MB-231. IOA exhibited effective cell growth inhibition by inducing cancer cells to undergo G(2)-M phase arrest and apoptosis. Further investigation revealed that IOA's inhibition of cell growth was also evident in a nude mice model. Cell cycle blockade was associated with increased levels of p21 and reduced amounts of cyclin B1, cyclin A, cdc2, and cdc25C. IOA also enhanced the levels of inactivated phosphorylated cdc2 and cdc25C. IOA triggered the mitochondrial apoptotic pathway, as indicated by a change in Bax/Bcl-2 ratios, resulting in mitochondrial membrane potential loss, cytochrome c release, and caspase-9 activation. We also found that the generation of reactive oxygen species (ROS) is a critical mediator in IOA-induced cell growth inhibition. Enhancement of ROS by IOA activated apoptosis signal-regulating kinase 1 (ASK1) resulted in the increased activation of c-Jun NH(2)-terminal kinase and p38. Antioxidants EUK8 and N-acetyl cystenine significantly decreased apoptosis by inhibiting the ASK1 dephosphorylation at Ser(967) and subsequently increased the interaction of ASK1 with thioredoxin or 14-3-3 proteins. Moreover, blocking ASK1 by small interfering RNA inhibition completely suppressed IOA-induced apoptosis. Taken together, these results imply a critical role for ROS and ASK1 in IOA's anticancer activity.     

A steroid-binding protein mediates estrogen-dependent inhibition of growth of MCF-7 breast cancer cells

We have described two different proteins that mediate MCF-7 cell growth inhibition (1). One is estrogen-dependent and is a heterodimer consisting of a heavy (54 kDa) and light (29 kDa) chain. This protein is distinct from cortisol-binding globulin (CBG), sex hormone-binding protein (SBP or SHBG) and albumin (HSA). It may be fetal steroid binding protein (FSBP). The other is not estrogen dependent, but may be related to the estrogen-dependent protein. Since these are large proteins they must be acting at the level of the plasma membrane, not traditional intracellular estrogen receptors, and inhibit MCF-7 cell growth.     

Cryptophycin-induced hyperphosphorylation of Bcl-2, cell cycle arrest and growth inhibition in human H460 NSCLC cells

Bcl-2 has been described as a factor that can protect from apoptosis. The protective effect of Bcl-2 may be lost if the protein is phosphorylated. Bcl-2 phosphorylation can be induced by agents that affect microtubule depolymerization or prevent microtubule assembly. In 13 human tumor cell lines there was a high degree of heterogeneity in Bcl-2 protein expression. Human H460 non-small-cell lung carcinoma (NSCLC) cells expressed high levels of Bcl-2 and were selected for study. Western blot analysis for Bcl-2 phosphorylation was carried out after 4 h and 24 h of exposure to cryptophycin 52, cryptophycin 55, paclitaxel or vinblastine. Cryptophycin 52 and cryptophycin 55 were very potent inducers of Bcl-2 phosphorylation. After 4 h of exposure, Bcl-2 phosphorylation was evident with 0.05 nM cryptophycin 52, 0.25 nM cryptophycin 55, 5 nM vinblastine and 50 nM paclitaxel. The hyperphosphorylated form of Bcl-2 was evident after 24 h exposure of H460 cells to 0.25 nM cryptophycin 52 or cryptophycin 55 and 50 nM vinblastine or paclitaxel. The effects of the compounds on the cell cycle paralleled those on Bcl-2 phosphorylation. In H460 cells 90% cell killing was obtained with 0.13 nM cryptophycin 52, 0.2 nM cryptophycin 55, 20 nM paclitaxel and > 100 nM vinblastine after 24 h of exposure as determined by colony formation. In Bcl-2-negative Calu-6 NSCLC cells, 90% cell killing was obtained with 0.03 nM cryptophycin 52, 0.1 nM cryptophycin 55, 11 nM paclitaxel and 0.5 nM vinblastine using the same experimental design. Thus, cryptophycins are potent inducers of Bcl-2 phosphorylation. The cryptophycins were more potent cytotoxic agents in Bcl-2-negative Calu-6 cells than in Bcl-2-positive H460 cells indicating that pathways triggered by Bcl-2 phosphorylation are involved in cryptophycin-induced lethality.     

RETRACTED ARTICLE: Erucylphosphocholine induces growth inhibition,cell cycle arrest,and apoptosis in human choriocarcinoma cells

A membrane-targeted, lipophilic ether lipid of synthetic phospholipid analog, erucylphosphocholine (ErPC), induces apoptosis in some lines of human tumor cells. We investigated the effect of ErPC in the choriocarcinoma cell line, BeWo. BeWo cells were treated with various concentrations of ErPC, and changes in cell growth, the cell cycle, apoptosis, and related parameters were examined. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that BeWo cells were sensitive to the growth inhibitory effect of ErPC. Cell cycle analysis indicated that exposure to ErPC decreased the proportion of cells in the S phase and increased the proportion in the G0/G1 phases of the cell cycle. Induction of apoptosis was confirmed by Annexin V staining of externalized phosphatidylserine and by the loss of mitochondrial transmembrane potential. This induction occurred in conjunction with the altered expression of genes related to cell growth, malignant phenotype, and apoptosis. These results suggest that ErPC may serve as a therapeutic agent for the treatment of choriocarcinoma.     

Deguelin inhibits the growth of colon cancer cells through the induction of apoptosis and cell cycle arrest

As previously demonstrated, deguelin [(7aS, BaS)-13, 13a-dihydro-9,10-dimethoxy-3,3-dimethyl-3H-bis[1]benzo-pyrano[3,4-b:6′,5′-e]pyran-7(7aH)-one mediates anti-proliferative properties in a variety of cell types. In the present study, deguelin was found to suppress the growth of HT-29 colon cancer cells with an IC₅₀ of 4.32 × 10⁻⁸ M. The cells were arrested in the G1-S-phase of the cycle. Investigations of G1/S regulatory proteins by Western blot analyses showed an upregulation of p27, and decreased expression levels of cyclin E and CDK4. Furthermore, by 24 h, exposure to deguelin resulted in an increase in the hypophosphorylated form of Rb. Since hypophosphorylated pRb binds to and inactivates E2F1, additional studies were performed and downregulation of E2F1 was observed after 24 h of treatment with deguelin. These results are consistent with the observation that deguelin arrested cells in the G1-S- phase. In addition, based on ethidium bromide/acridine orange staining, detection of digoxigenin-labelled genomic 3′-OH DNA ends, and DNA laddering, it was found that deguelin exerts its growth inhibitory effects via the induction of apoptosis. Based on these data, the potential of deguelin to serve as a cancer chemotherapeutic agent for colon cancer may be suggested.     

Artesunate promotes G2/M cell cycle arrest in MCF7 breast cancer cells through ATM activation

Background

Recent studies have revealed that artesunate (ART) has clear anti-tumor activity, suggesting that it could be a good candidate chemotherapeutic agent. In this study, we researched the inhibitory effect of ART on MCF7 cells and explored the possible mechanisms.

Methods

MTT assay was used to detect the effect of ART on the proliferation of MCF7 cells. Crystal violet staining was used to observe morphological and quantitative changes. Flow cytometry was used to detect the cell cycle of the drug-acting MCF7 cells. In addition, western blotting was used to detect the drug influence on expression of the ATM, phospho-ATM(S1981), H2AX, γH2AX(S139), CHK2 and phospho-CHK2(T68), cdc25C, and phospho-cdc25C(S216).

Results

In the experimental groups, the proliferation of MCF7 cells was inhibited in a dose-dependent manner and the original cell morphology was lost. The number of G2/M phase cells in the experimental groups increased significantly, and the expression of DNA damage response-associated proteins was significantly increased, such as phospho-ATM(S1981), γH2AX(S139), phospho-CHK2(T68), and phospho-cdc25C(S216).

Conclusions

ART can inhibit cell proliferation and promote G2/M arrest in MCF7 cells through ATM activation and the ensuing “ATM-Chk2-Cdc25C” pathway, thus implicating ART as a novel candidate for breast cancer chemotherapy.

     

Epigallocatechin-3 gallate induces growth inhibition and apoptosis in human breast cancer cells through survivin suppression

Recent investigations have demonstrated that polyphenolic catechins inhibit cancer cell proliferation and tumor growth. However, how the major active component of tea catechins, epigallocatechin-3 gallate (EGCG), mediates anticancerous effects has not been extensively examined. We have investigated the cell growth inhibitory effects of EGCG on cell growth of the human breast cancer cell line MCF-7, and the mechanism of its action with emphasis on the regulation of tumor cell survival. A significant EGCG dose-dependent growth inhibition was observed coordinated with EGCG-induced apoptosis. Analysis of survivin expression after addition of EGCG showed that both survivin mRNA and protein were decreased. The survivin-promoter luciferase activity in EGCG-treated cells was significantly inhibited by 91+/-2.0% (P<0.001), compared with the control. Interestingly, EGCG strongly inhibited the basal activation of phospho-AKT and AKT kinase activity as early as 30 min after treatment. Furthermore, inhibition of AKT kinase activity by EGCG preceded the suppression of survivin (1 h post treatment), followed by increased caspase-9 activity (6 h post treatment). A dominant negative AKT or the phosphatidylinositol 3-kinase inhibitor, LY294002, also strongly inhibited survivin promoter activity, providing further evidence to support the hypothesis that the inhibitory effect of EGCG on survivin is mediated via the AKT pathway. Therefore, EGCG is a potent proapoptotic agent in MCF-7 breast cancer cells that targets survivin expression via suppression of the AKT pathway.     

Calcium/calmodulin-dependent kinase inhibitor induces growth inhibition, cell cycle arrest, and apoptosis in human choriocarcinoma cells

KN-93, a membrane-permeant calcium/calmodulin- dependent kinase-selective inhibitor, induces apoptosis in some lines of human tumor cells. We investigated the effect of KN-93 in the choriocarcinoma cell line, BeWo. BeWo cells were treated with various concentrations of KN-93, and changes in cell growth, the cell cycle, apoptosis, and related parameters were examined. A WST-1 assay showed that BeWo cells were sensitive to the growth inhibitory effect of KN-93. Cell cycle analysis indicated that exposure to KN-93 decreased the proportion of cells in the S phase and increased the proportion in the G0/G1 phases of the cell cycle. Induction of apoptosis was confirmed by Annexin V staining of externalized phosphatidylserine, by the loss of mitochondrial transmembrane potential, and by antibodies directed against histones from fragmented DNA. This induction occurred in conjunction with the altered expression of genes related to cell growth, malignant phenotype, and apoptosis. These results suggest that KN-93 may serve as a therapeutic agent for the treatment of choriocarcinoma.     

Ganoderma lucidum extract induces cell cycle arrest and apoptosis in MCF-7 human breast cancer cell

Although the pharmacology and clinical application of water extracts of Ganoderma lucidum have been extensively documented, little is known regarding its alcohol extract. In the present study, the anti-tumor effect of an alcohol extract of Ganoderma lucidum was investigated using MCF-7 cells. We found that the alcohol extract of Ganoderma lucidum inhibited cell proliferation in a dose- and time-dependent manner, which might be mediated through up-regulation of p21/Waf1 and down-regulation of cyclin D1. Furthermore, this compound can directly induce apoptosis in MCF-7 cells, which might be mediated through up-regulation of a pro-apoptotic Bax protein and not by the immune system. Our findings suggest that there are multiple mechanisms underlying the anti-tumor effects of Ganoderma lucidum.