Myc protein is differentially sensitive to quinidine in tumor versus immortalized breast epithelial cell lines

Quinidine regulates growth and differentiation in human breast tumor cells, but the immortalized mammary epithelial MCF-10A cell line is insensitive to quinidine. We found that a morphologically similar differentiation response was evoked by quinidine and c-myc antisense oligonucleotides in MCF-7 cells and this prompted us to investigate the actions of quinidine on c-myc gene expression. Myc protein levels were suppressed in human breast tumor cell lines, but not in MCF-10A cells, an observation that supports the hypothesis that suppression of c-myc gene expression is involved in the preferential growth and differentiation response of breast tumor cells to quinidine. Quinidine reduced c-myc mRNA levels in MCF-7 cells. Acute induction of c-myc mRNA by estradiol, as well as the c-myc response to sub-cultivation in fresh serum and H-ras driven elevations in c-myc mRNA were depressed by 50-60% in the presence of quinidine. Quinidine decreased c-myc promoter activity in MCF-7 cells in a transient reporter gene assay and a 168 bp region of human c-myc promoter (-100 to +68 with respect to the P1 promoter) was sufficient to confer responsiveness to quinidine. Quinidine is a potential lead compound for developing pharmacological agents to regulate Myc. In addition, the study of quinidine-regulated events is a promising approach to unravel differentiation control pathways that become disrupted in breast cancer.     

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.     

Differential chemosensitivity of breast cancer cells to ganciclovir treatment following adenovirus-mediated herpes simplex virus thymidine kinase gene transfer

The development of resistance to radiation and chemotherapeutic agents that cause DNA damage is a major problem for the treatment of breast and other cancers. The p53 tumor suppressor gene plays a direct role in the signaling of cell cycle arrest and apoptosis in response to DNA damage, and p53 gene mutations have been correlated with increased resistance to DNA-damaging agents. Herpes simplex virus thymidine kinase (HSV-tk) gene transfer followed by ganciclovir (GCV) treatment is a novel tumor ablation strategy that has shown good success in a variety of experimental tumor models. However, GCV cytotoxicity is believed to be mediated by DNA damage-induced apoptosis, and the relationship between p53 gene status, p53-mediated apoptosis, and the sensitivity of human tumors to HSV-tk/GCV treatment has not been firmly established. To address this issue, we compared the therapeutic efficacy of adenovirus-mediated HSV-tk gene transfer and GCV treatment in two human breast cancer cell lines: MCF-7 cells, which express wild-type p53, and MDA-MB-468 cells, which express high levels of a mutant p53 (273 Arg-His). Treating MCF-7 cells with AdHSV-tk/GCV led to the predicted increase in endogenous p53 and p21WAF1/CIP1 protein levels, and apoptosis was observed in a significant proportion of the target cell population. However, treating MDA-MB-468 cells under the same conditions resulted in a much stronger apoptotic response in the absence of induction in p21WAF1/CIP1 protein levels. This latter result suggested that HSV-tk/GCV treatment can activate a strong p53-independent apoptotic response in tumor cells that lack functional p53. To confirm this observation, four additional human breast cancer cell lines expressing mutant p53 were examined. Although a significant degree of variability in GCV chemosensitivity was observed in these cell lines, all displayed a greater reduction in cell viability than MCF-7 or normal mammary cells treated under the same conditions. These results suggest that endogenous p53 status does not correlate with chemosensitivity to HSV-tk/GCV treatment. Furthermore, evidence for a p53-independent apoptotic response serves to extend the potential of this therapeutic strategy to tumors that express mutant p53 and that may have developed resistance to conventional genotoxic agents.     

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.     

Inhibitory Effect of Ginseng on Breast Cancer Cell Line Growth Via Up-Regulation of Cyclin Dependent Kinase Inhibitor,p21 and p53

Objective: Breast cancer is global female health problem worldwide. Most of the currently used agents for breast cancer treatment have toxic side-effects. Ginseng root, an oriental medicine, has many health benefits and may exhibit direct anti-cancer properties. This study was performed to assess the effects of ginseng on breast cancer cell lines. Materials and Methods: Cytotoxicity of ginseng extract was measured by MTT assay after exposure of MDA-MB-231, MCF-10A and MCF-7 breast cancer cells to concentrations of 0.25, 0.5, 1, 1.5, 2 and 2.5 mg/well. Expression levels of p21WAF, p16INK4A, Bcl-2, Bax and P53 genes were analyzed by quantitative real time PCR. Results: The treatment resulted in inhibition of cell proliferation in a dose-and time-dependent manner. p53, p21WAF1and p16INK4A expression levels were up-regulated in ginseng treated MDA-MB-231 and MCF-7 cancer cells compared to untreated controls and in MCF-10A cells. The expression levels of Bcl2 in the MDA-MB-231 and MCF-7 cells were down-regulated. In contrast, that of Bax was significantly up-regulated. Conclusion: The results of this study revealed that ginseng may inhibit breast cancer cell growth by activation of the apoptotic pathway.     

Apoptotic response to camptothecin and 7-hydroxystaurosporine (UCN-01) in the 8 human breast cancer cell lines of the NCI Anticancer Drug Screen: multifactorial relationships with topoisomerase I, protein kinase C, Bcl-2, p53, MDM-2 and caspase pathways.

Derivatives of camptothecins, topoisomerase I inhibitors and 7-hydroxystaurosporine (UCN-01), a protein kinase C (PKC) inhibitor and cell cycle checkpoint abrogator, are promising anticancer drugs. We characterized the apoptotic response to camptothecin and UCN-01 for the 8 human breast carcinoma cell lines (MCF-7, MCF-7/ADR, T47D, HS578T, BT549, MDA-N, MDA MB231, MDA435) from the National Cancer Institute (NCI) Anticancer Drug Screen. MCF-7 and T47D cells exhibited marked resistance to apoptosis, whereas MCF-7/ADR (NCI/ADR-RES) and HS578T cells exhibited the most pronounced apoptotic response. Apoptotic response was not correlated with growth inhibition measured by sulforhodamine B (SRB) assay, indicating that apoptosis is not the only mechanism of drug-induced cell death. Measurements of topoisomerase I levels and cleavage complexes and of PKC isoforms demonstrated that primary target inhibition was not correlated with apoptotic response. Several key apoptotic pathways were evaluated. Only MCF-7 cells had wild-type p53, indicating that p53 is not required for drug-induced apoptosis. MCF-7 cells also showed the highest MDM-2 expression (along with T47D cells, which were also resistant to apoptosis). Bcl-2, Mcl-1 and caspases 2 and 3 protein levels varied widely, whereas Bax expression was comparable among cell lines. Interestingly, Bcl-2, Mcl-1 and Bcl-X(L) cumulative expressions were inversely correlated with apoptotic response. Our results provide a comparative molecular characterization for the breast cancer cell lines of the NCI Anticancer Drug Screen and demonstrate the diversity of cellular responses to drugs (apoptosis vs. cell cycle arrest) and the importance of multifactorial analyses for modulating/predicting the apoptotic response to chemotherapy.     

Differences in protein kinase C and estrogen receptor alpha, beta expression and signaling correlate with apoptotic sensitivity of MCF-7 breast cancer cell variants

Widespread use of MCF-7 human breast cancer cells as a model system for breast cancer has lead to variations in these cells between different laboratories. Although several reports have addressed these differences in terms of proliferation and estrogenic response, differences in sensitivity to apoptosis have just begun to be described. Based on the possible differences in apoptotic sensitivity that may arise due to the existence of MCF-7 cell variants, we determined the relative sensitivity of MCF-7 cell variants from three established laboratories (designated M, L and N) to known inducers of apoptosis. Consistent with our previous studies we demonstrate that differences exist among these variants in regards to tumor necrosis factor alpha (TNF)-induced cell death and inhibition of proliferation in a dose-dependent manner. To establish if the difference in apoptotic susceptibility was specific to TNF, the three MCF-7 cell variants were tested for their response to other known inducers of apoptosis: okadaic acid, staurosporine and 4-hydroxy-tamoxifen. Viability and DNA fragmentation analysis revealed a similar pattern of resistance to apoptosis by all agents in the MCF-7 M variant. The MCF-7 L variant was resistant to okadaic acid and 4-hydroxy-tamoxifen but not staurosporine. In contrast, MCF-7 N cells were sensitive to induction of apoptosis by all agents. The role of both protein kinase C (PKC) and estrogen signaling in the regulation of cell survival prompted investigation of these pathways as a mechanism for differential sensitivity of MCF-7 cell variants to apoptosis. While both estrogen receptor alpha (ERalpha) and ERbeta were expressed in MCF-7 M and N cells, the absence of ERbeta in MCF-7 L cells correlated with decreased estrogen responsiveness of the L variant. Variations in estrogenic responsiveness and PKC isoform expression may account for the enhanced susceptibility of both the L and N variants to staurosporine.     

Silibinin Enhances Ultraviolet B-Induced Apoptosis in MCF-7 Human Breast Cancer Cells

Purpose

Chemotherapies for breast cancer generally have strong cellular cytotoxicity and severe side effects. Thus, significant emphasis has been placed on combinations of naturally occurring chemopreventive agents. Silibinin is a major bioactive flavonolignan extracted from milk thistle with chemopreventive activity in various organs including the skin, prostate, and breast. However, the mechanism underlying the inhibitory action of silibinin in breast cancer has not been completely elucidated. Therefore, we investigated the effect of silibinin in MCF-7 human breast cancer cells and determined whether silibinin enhances ultraviolet (UV) B-induced apoptosis.

Methods

The effects of silibinin on MCF-7 cell viability were determined using the MTT assay. The effect of silibinin on PARP cleavage, as the hallmark of apoptotic cell death, and p53 protein expression in MCF-7 cells was analyzed using Western blot. The effect of silibinin on UVB-induced apoptosis in MCF-7 cells was analyzed by flow cytometry.

Results

A dose- and time-dependent reduction in viability was observed in MCF-7 cells treated with silibinin. Silibinin strongly induced apoptotic cell death in MCF-7 cells, and induction of apoptosis was associated with increased p53 expression. Moreover, silibinin enhanced UVB-induced apoptosis in MCF-7 cells.

Conclusion

Silibinin induced a loss of cell viability and apoptotic cell death in MCF-7 cells. Furthermore, the combination of silibinin and UVB resulted in an additive effect on apoptosis in MCF-7 cells. These results suggest that silibinin might be an important supplemental agent for treating patients with breast cancer.     

Heregulin-triggered Her-2/neu signaling enhances nuclear accumulation of p21WAF1/CIP1 and protects breast cancer cells from cisplatin-induced genotoxic damage

Elevated levels of p21WAF1/CIP1, an important mediator of DNA repair, have been observed in various aggressive tumors as well as linked to chemoresistance. We examined whether heregulin (HRG), a member of the EGF-like growth factor family closely related to breast cancer tumorigenesis and metastasis, modulates p21WAF1/CIP1 expression and cellular localization. We used a model system that consisted of MCF-7 cells and MCF-7 cells engineered to overexpress the full-length cDNA of the human HRG gene (MCF-7/HRG). MCF-7/HRG cells demonstrate constitutive hyperactivation of Her-2/neu receptor as well as activation of down-stream PI-3'K/AKT and MAPK signaling cascades. Immunoblotting analyses showed that MCF-7/HRG cells significantly up-regulate p21WAF1/CIP1 expression relative to control MCF-7/pBABE cells, while a strong nuclear accumulation of p21WAF1/CIP1 in MCF-7/HRG cells was revealed by immunofluorescence microscopy studies. Protein degradation analyses demonstrated that the half-life of p21WAF1/CIP1 protein was increased from approximately 35 min in control MCF-7/pBABE cells to >/=3 h in MCF-7/HRG cells. Pharmacological inactivation of the PI-3'K/AKT and MAPK completely prevented HRG-induced accumulation of p21WAF1/CIP1. A structural deletion mutant of HRG (HRG-M4) lacking the N-terminus sequence and the cytoplasmic-transmembrane region of HRG was generated to investigate whether secretion of HRG and transactivation of Her-2/neu actively contributed to HRG-regulated p21WAF1/CIP1 expression and cellular localization. MCF-7 cells engineered to overexpress HRG-M4 did not demonstrate either activation of Her-2/neu, PI-3'K/AKT, or MAPK. Remarkably, HRG-M4 overexpression completely abolished the ability of HRG to promote nuclear accumulation of p21WAF1/CIP1 and concomitantly enhanced the apoptotic effects of cisplatin towards breast cancer cells. This novel interplay between HRG and p21WAF1/CIP1 strongly suggests that one mechanism of HRG-regulated breast cancer cell proliferation, survival, and/or sensitivity to genotoxic damage is to stabilize and promote a nuclear accumulation of p21WAF1/CIP1.     

Involvement of p53 in insulin-like growth factor binding protein-3 regulation in the breast cancer cell response to DNA damage

Chemotherapy drugs that induce apoptosis by causing DNA double-strand breaks, upregulate the tumor suppressor p53. This study investigated the regulation of the growth-regulatory protein insulin-like growth factor binding protein-3 (IGFBP-3), a p53 target, by DNA-damaging agents in breast cancer cells. IGFBP-3 was upregulated 1.4- to 13-fold in response to doxorubicin and etoposide in MCF-10A, Hs578T, MCF-7 and T47D cells, which express low to moderate basal levels of IGFBP-3. In contrast, IGFBP-3 was strongly downregulated by these agents in cells with high basal levels of IGFBP-3 (MDA-MB-231, MDA-MB-436 and MDA-MB-468). In MDA-MB-468 cells containing the R273H p53 mutation, reported to display gain-of-function properties, chemotherapy-induced suppression of IGFBP-3 was not reversed by the p53 reactivating drug, PRIMA-1, or by p53 silencing, suggesting that the decrease in IGFBP-3 following DNA damage is not a mutant p53 gain-of-function response. SiRNA-mediated downregulation of endogenous IGFBP-3 modestly attenuated doxorubicin-induced apoptosis in MDA-MB-468 and Hs578T cells. IGFBP-3 downregulation in some breast cancer cell lines in response to DNA-damaging chemotherapy may have clinical implications because suppression of IGFBP-3 may modulate the apoptotic response. These observations provide further evidence that endogenous IGFBP-3 plays a role in breast cancer cell responsiveness to DNA damaging therapy.     

Potent in vivo anti-breast cancer activity of IN-2001, a novel inhibitor of histone deacetylase, in MMTV/c-Neu mice

A novel synthetic inhibitor of histone deacetylase (HDAC), 3-(4-dimethylaminophenyl)-N-hydroxy-2-propenamide (IN-2001), was examined for its antitumor activity and for the underlying molecular mechanisms of any such activity. IN-2001 effectively inhibited cellular HDAC activity (IC(50), 5.42 nmol/L) in MCF-7 human breast cancer cells. Based on the Western blot analysis, this HDAC inhibitory effect of IN-2001 was confirmed by an increase in histone H4 acetylation from the IN-2001-treated breast cancer cells. IN-2001 suppressed mammary tumor growth in MMTV/c-Neu transgenic mice and also showed higher apoptotic index and lower lymphatic invasion compared with controls. In human breast cancer cells (MCF-7, T47D, MDA-MB-231, and MDA-MB-468), IN-2001 induced cell cycle arrest at G(2)-M phase through up-regulation of p21(WAF1) and p27(KIP1) and eventually caused apoptosis. IN-2001-induced apoptosis was caspase dependent and seems mediated through an increase in Bax/Bcl-2 ratio. Taken together, our data indicate that this novel HDAC inhibitor is a promising therapeutic agent against human breast cancer.     

Effect of apoptosis and response of extracellular matrix proteins after chemotherapy application on human breast cancer cell spheroids

Multicellular tumor spheroid (MTS) represents a three-dimensional structural form of tumors in laboratory conditions, and it has the characteristics of avascular micrometastases or intervascular spaces of big tumors. Recent studies indicate that extracellular matrix (ECM) proteins play a critical role in tumor metastasis, therefore normal and cancer cells require an ECM for survival, proliferation and differentiation. Doxorubicin and Docetaxel are widely used in the therapy of breast cancer, as well as in in vivo and in vitro studies. In this study, we examined the effect of apoptosis and proliferation of cells on the human breast cancer cell line, MCF-7, by using p53, bcl-2 and Ki67 gene expression, and the tendency to metastasis with extracellular matrix proteins, laminin and type IV collagen after chemotherapy in the spheroid model. The apoptotic cell death in situ was detected by TUNEL method. TUNEL-positive cells and positive immunoreactivities of laminin, type IV collagen, p53 and, bcl-2 were detected in the control group. There was no laminin and type IV collagen immunoreactivities in spheroids of drug groups. While TUNEL-positive cells and p53 immunoreactivity were detected in Docetaxel, Doxorubicin and Docetaxel/Doxorubicin groups, p53 immunoreactivity was not observed in the Docetaxel group. There was no bcl-2 immunoreactivity in either drug group. In addition, we did not detect Ki67 immunoreactivity in both control and drug treatment groups. However, the absence of Ki67 protein in MCF-7 breast multicellular tumor spheroids is possibly related to the cells in G0 or S phase. These chemotherapeutic agents may affect the presence of ECM proteins in this in vitro model of micrometastasis of spheroids. These findings suggest that the possible mechanism of cell death in Doxorubicin and Docetaxel/Doxorubicin treatment groups is related to apoptosis through the p53 pathway. However, we considered the possibility that there is another control mechanism for the Docetaxel group.     

Endogenous p21WAF1/CIP1 status predicts the response of human tumor cells to wild-type p53 and p21WAF1/CIP1 overexpression

Expression of exogenous wild-type (wt) p53 protein can suppress the growth and/or induce apoptosis in different tumor cells. The effect of exogenous p21(WAF1/CIP1) expression is more controversial: while it can induce apoptosis in some cells, it can protect against p53-mediated apoptosis in others. We used adenoviral vectors to introduce p53 and p21(WAF1/CIP1) genes into human tumor cell lines with different p53 and/or p21(WAF1/CIP1) status. The cell growth inhibition and the induction of apoptosis were measured. Overexpression of wt p53 induced more efficient growth inhibition and apoptosis in SW 620 (mutant p53) and HeLa (inactivated p53 protein) than in MCF-7 (wt p53) and CaCo-2 cell line, which was the most resistant to p53 overexpression despite the p53 mutation. Unlike HeLa and SW 620 cells, the basal p21 protein level was readily detected in CaCo-2 and MCF-7 cells. Overexpression of p21(WAF1/CIP1) gene induced somewhat less pronounced growth inhibition of all cell lines tested, but it also induced apoptosis in HeLa and SW 620 cells. These results suggest that the basal, but not the inducible, levels of p21(WAF1/CIP1) protein in tumor cells could protect from p53-mediated apoptosis. On the other hand, overexpression of p21(WAF1/CIP1) gene itself can induce apoptosis in cells with no basal p21(WAF1/CIP1) protein level. Possible mechanisms of the differential response to these genes are discussed.     

人乳腺癌细胞株WAF1／GIP1／p21基因的表达及其与细胞生物学特性的关系

OBJECTIVE: To study the WAF1/CIP1 gene DNA status, mRNA and protein expressions in human breast cancer cell line and its significance. METHODS: Using cell culture, molecular biological techniques such as Southern blot, Northern blot and immunocytochemical methods, the WAF1/CIP1 gene DNA status, mRNA and protein expression levels in MCF-7 expressing wild type p53(wtp53) and MDA-MB-231 expressing mutant p53 (mtp53) human breast cancer cell lines were detected respectively. The p53 and mdm-2 protein expression levels and cytobiological features of the 2 cell lines were compared and correlated to their WAF1/CIP1 gene expression levels. RESULTS: (1) There was no difference in WAF1/CIP1 gene DNA status in the two breast cancer cell lines. Neither of them showed gene amplificatian or deletion. However, the WAF1/CIP1 mRNA and p21WAF1/CIP1 protein expression levels of MCF-7 cells were higer than those of MDA-MB-231 cells (P < 0.05). (2) The character and cellular distribution of p53 protein in the two cell lines were clearly different. The expression level of mdm-2 proteion was significantly higher in MCE-7 than in MDA-MB-231 cells (P < 0.05). (3) Compered to the other breast cancer cell line, MCF-7 cells were better differentiated, grew more slowly and adhered more closely with each other. CONCLUSION: The WAF1/CIP1 gene expression at mRNA and protein levels is associated with p53 phenotype and some cytobiological features of human breast cancer.     

Resveratrol inhibits cell proliferation and induces apoptosis of human breast carcinoma MCF-7 cells

Resveratrol, which is found in grapes and wine, has been reported to have a variety of important pharmacological effects including anti-inflammatory, anti-platelet, and anti-carcinogenetic properties. In this study, using the human breast cancer cell line MCF-7, we have analyzed a possible mechanism by which resveratrol could interfere with cell cycle control and induce cell death. Resveratrol treatment of MCF-7 cells resulted in a dose-dependent inhibition of the cell growth and the cells accumulated at the S phase transition of the cell cycle at low concentrations, but high concentrations do not induce S phase accumulation. The anti-proliferative effects of resveratrol were associated with a marked inhibition of cyclin D and cyclin-dependent kinase (Cdk) 4 proteins, and induction of p53 and Cdk inhibitor p21WAF1/CIP. Growth suppression by resveratrol was also due to apoptosis, as seen by the appearance of a sub-G1 fraction and chromatin condensation. In addition, the apoptotic process involves activation of caspase-9, a decrease of Bcl-2 as well as Bcl-XL levels, and an increase of Bax levels.     

Effects of sulindac and its metabolites on growth and apoptosis in human mammary epithelial and breast carcinoma cell lines

Nonsteriodal anti-inflammatory drugs (NSAIDs) are among the most commonly used medications in the United States and elsewhere, mainly for the treatment of arthritis. The NSAID sulindac causes regression and prevents the recurrence of premalignant colonic polyps in patients with familial adenomatous polyposis and inhibits colon carcinogenesis in rodents. Sulindac and sulindac sulfone, a metabolite of sulindac that lacks cyclooxygenase (cox) inhibitory activity, also inhibit mammary carcinogenesis in rats. To obtain insights into the relevance of these findings to human breast cancer, we examined the mechanism of action of sulindac and its sulfide and sulfone metabolites on the normal human mammary epithelial cell line MCF-10F and the human breast cancer cell line MCF-7. Of the three compounds, the sulfide was the most potent inhibitor of cell growth, although the sulfone and sulfoxide were also active at higher concentrations. Treatment of MCF-10F and MCF-7 cells with 100 µM sulindac sulfide resulted in accumulation of cells in the G1 phase of the cell cycle and induction of apoptosis. Apoptosis occurred within 24 h as determined by the TUNEL assay and DNA laddering was observed at 72 h. The accumulation of cells in G1 was associated with decreased levels of expression of cyclin D1 but no effect was seen on the expression of CDK4 or the immediate early response gene c-jun. Treatment with sulindac sulfide caused a striking induction of the CDK inhibitor p21^WAF1 in MCF-10F cells. The MCF-7 cell line expressed a high basal level of p21^WAF1 which did not change significantly after drug treatment. The pro-apoptotic gene BAX was not induced in either MCF-10F or MCF-7 cells by sulindac sulfide. Stable overexpression of cyclin D1, which frequently occurs in breast cancers, did not protect mammary epithelial cells from inhibition by the sulfide. These studies suggest that this class of compounds warrants further study with respect to breast cancer prevention and treatment.     

Differential sensitivity of human mammary epithelial and breast carcinoma cell lines to curcumin

Curcumin has anti-inflammatory, antiproliferative, and antitumor effects. To understand the chemopreventive mechanism of curcumin against human malignancies, the cellular and molecular changes induced by this agent in human mammary epithelial (MCF-10A) and breast carcinoma (MCF- 7/TH) cell lines were investigated. The human multidrug- resistant breast cancer cell line was 3.5 fold more sensitive to curcumin than the mammary epithelial cell line. Even though both cell lines accumulated a similar amount of curcumin, a significantly higher percentage of apoptotic cells was induced in breast cancer cells compared to a very low percentage of apoptosis in mammary epithelial cells. Incubation of breast cancer cells with 20 and 40 microM curcumin for 24 h induced G2 block and sub-G0/G1 cell population, respectively. Curcumin treatment caused a reduction in the expression of Ki67, PCNA, and p53 mRNAs in breast cancer cells. The human mammary epithelial cell line showed a down-regulation of p21 mRNA and an up-regulation of Bax mRNA expression with curcumin treatment. The results suggest that apoptosis is involved in the curcumin-induced inhibition of tumor cell growth, and genes associated with cell proliferation and apoptosis may be playing a role in the chemopreventive action of curcumin.     

Concomitant Differentiation and Partial Proteasome Inhibition Trigger Apoptosis in Neuroblastoma Cells

Proteasome activity is essential during cAMP-induced terminal differentiation of a murine neuroblastoma cell line (NBP2). However, the mechanisms through which proteasome affects NBP2 differentiation have not been characterized. We hypothesized that proteasome is required to implement the differentiation-mediated effects on cell cycle, and its partial inhibition during differentiation may have adverse consequences. Here we show that partial inhibition of proteasome during cAMP-induced differentiation of NBP2 cells causes apoptosis. Whereas differentiation induced growth arrest at G₁ phase, partial proteasome inhibition during differentiation resulted in the accumulation of cells at G₂M phase. Cell cycle data correlated with the level of cyclin-dependent kinase inhibitors p21WAF and p27Kip1, and cyclin A. While the level of p21 and p27 increased, the level of cyclin A decreased upon differentiation. In contrast, cells treated with proteasome inhibitor in the presence of cAMP-inducing agents showed increased levels of p21 and cyclin A early in the course of differentiation. However, the level of p21 and p27, but not cyclin A, decreased later during concomitant differentiation and partial proteasome inhibition when cells were undergoing apoptosis. Our data suggest that differentiation-mediated growth arrest is dependent on the temporal activity of cell cycle proteins. Partial inhibition of proteasome interferes with differentiation events partly by stabilizing cell cycle proteins and this triggers apoptosis. Thus, differentiating drugs combined with partial proteasome inhibition may impart higher therapeutic efficacy than differentiating agents alone for the treatment of neuroblastoma tumors.     

JNK/SAPK mediates doxorubicin-induced differentiation and apoptosis in MCF-7 breast cancer cells

Pharmacologic induction of cancer cell differentiation has potential in the treatment of breast cancer. Doxorubicin, a widely used anthracycline antibiotic, was previously reported to induce differentiation of MCF-7 breast cancer cells. We demonstrate in this study that inhibition of MCF-7 breast cancer cell growth by low dose doxorubicin (0.01 µg/ml) was accompanied by an increase in cytokeratin 8/18 and milk fat globule membrane protein expression, biomarkers for differentiation of breast cancer, as well as an increase in JNK/SAPK phosphorylation. High dose doxorubicin (10.0 µg/ml) induced apoptosis in these cells. Overexpression of dominant-inhibitory forms of JNK1 and c-Jun blocked both the differentiation and apoptotic effects of doxorubicin. These results suggest that JNK/SAPK pathway signaling plays a prominent role in doxorubicin-induced cell cycle withdrawal, differentiation and control of apoptosis in this cell system. These findings support the possibility that JNK/SAPK pathway activation may be a means of therapeutic intervention in breast cancer.     

Apoptosis-mediated inhibition of human breast cancer cell proliferation by lemon citrus extract

Dietary phytochemicals have a variety of antitumor properties. In the present study, the antitumor activity of methanolic extract of lemon fruit (lemon extract; LE) (LE) on the MCF-7 breast cancer cell line was investigated in vitro. Apoptotic cell death was analyzed using the TUNEL assay. In addition, the apoptosis mediated by LE extract in the MCF-7 cells was associated with the increased expression of the tumor suppressor p53 and caspase-3. Additionally, the expression of a pro-apoptotic gene, bax, was increased, and the expression of an anti-apoptotic gene, bcl-2, was decreased by LE extract treatment, resulting in a shift in the Bax:Bcl-2 ratio to one that favored apoptosis. The expression of a major apoptotic gene, caspase-3, was increased by LE extract treatment. In light of the above results, we concluded that LE extract can induce the apoptosis of MCF-7 breast cancer cells via Bax-related caspase-3 activation. This study provides experimental data that are relevant to the possible future clinical use of LE to treat breast cancer.