Integrin alpha 5 beta 1 suppresses apoptosis triggered by serum starvation but not phorbol ester in MCF-7 breast cancer cells that overexpress protein kinase C-alpha

MCF-7 breast cancer cells grow as adherent cells, but following overexpression of protein kinase C-alpha these cells (MCF-7-PKC-alpha cells) become anchorage-independent and exhibit increased tumorigenicity in nude mice. MCF-7-PKC-alpha cells are also sensitized to apoptosis in response to phorbol ester but not serum starvation. Flourescence-activated cell sorting revealed that several integrin subunits were down-regulated in MCF-7-PKC-alpha cells, however, the fibronectin receptor alpha 5 beta 1 was upregulated. MCF-7-PKC-alpha cells growing under non-adherent conditions underwent cell death when antibodies to alpha 5 beta 1 were added to growth media lacking serum but not when serum was present. Addition of soluble fibronectin to cells incubated without serum suppressed apoptosis triggered by anti-alpha 5 beta 1 antibodies but not by phorbol esters. MCF-7-PKC-alpha cells also were shown to express more fibronectin on their cell surface than MCF-7V cells (MCF-7 cells transfected with pSV(2)M(2)6 vector only). This study indicates that the survival of MCF-7-PKC-alpha cells under non-adherent conditions in the absence of serum results from the ligation of alpha 5 beta 1 with surface-bound fibronectin, which may account, in part, for the increased aggressiveness of these cells.     

Expression of the invasive phenotype by MCF-7 human breast cancer cells transfected to overexpress protein kinase C-alpha or the erbB2 proto-oncogene

Transfection of the estrogen dependent and poorly invasive MCF-7 cell line to overexpress erbB2 was reported to increase athymic nude mouse mammary fat pad tumor growth; similar PKC-alpha overexpression produced a rapidly growing and metastatic transfectant. We investigated the invasive capacities of the two transfectants in vitro, their secretion of the proteolytic enzymes metalloproteinase (MMP)-9 and -2 and urokinase-type plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1), together with membrane uPA receptor (uPAR) levels as determined by ELISA. Compared with the MCF-7 cells, the erbB2 transfectant was more invasive and secreted higher levels of MMP-9 and uPA; also there was a greatly enhanced PAI-1 secretion and cellular uPAR expression. The PKC-alpha transfectant cells secreted extremely high levels of uPA and some MMP-9 and MMP-2, with an intermediate increase in uPAR; however, they were so poorly adherent that it was not possible to assess invasiveness in vitro. Thus, erbB2-overexpressing MCF-7 cells possessed several features associated with the invasive phenotype. The reportedly aggressive metastatic phenotype induced by PKC-alpha overexpression, however, was notable only for its uPA hypersecretion.     

Down-regulation of PPARgamma1 suppresses cell growth and induces apoptosis in MCF-7 breast cancer cells

Background  

Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the nuclear hormone receptor superfamily and is highly expressed in many human tumors including breast cancer. PPARγ has been identified as a potential target for breast cancer therapy based on the fact that its activation by synthetic ligands affects the differentiation, proliferation, and apoptosis of cancer cells. However, the controversial nature of current studies and disappointing results from clinical trials raise questions about the contribution of PPARγ signaling in breast cancer development in the absence of stimulation by exogenous ligands. Recent reports from both in vitro and in vivo studies are inconsistent and suggest that endogenous activation of PPARγ plays a much more complex role in initiation and progression of cancer than previously thought.     

Reconstitution of caspase 3 sensitizes MCF-7 breast cancer cells to doxorubicin- and etoposide-induced apoptosis

MCF-7, a breast cancer-derived cell line, is deficient of caspase 3 and relatively insensitive to many chemotherapeutic agents. To study the association of caspase 3 deficiency and chemotherapeutic resistance, we reconstituted caspase 3 in MCF-7 cells and characterized their apoptotic response to doxorubicin and etoposide. Western blots demonstrated that caspase 3 was constitutively expressed in the reconstituted MCF-7 cells. Both morphological observation and survival assays showed that caspase 3 reconstitution significantly sensitized MCF-7 cells to both drugs. Remarkably increased activation of caspases 3, 6, and 7, cleavage of cellular death substrates, and DNA fragmentation were detected in the reconstituted MCF-7 cells after drug treatment. Together, these data demonstrated a specific role for caspase 3 in chemotherapy-induced apoptosis and in activation of caspases 6 and 7. Our results also suggest that caspase 3 deficiency may contribute to chemotherapeutic resistance in breast cancer.     

Gold nanoparticles induce apoptosis in MCF-7 human breast cancer cells

Background: Gold nanoparticles have recently been investigated with respect to biocompatibility accordingto their interactions with cells. The purpose of this study was to examine cytotoxicity and apoptosis induction bywell-characterized gold nanoparticles in human breast epithelial MCF-7 cells. Methods: Apoptosis was assessedby TUNEL, cytotoxicity by MTT assay and caspase 3, 9, p53, Bax and Bcl expression by real-time PCR assays.Results: Gold nanoparticles at up to 200 μg/mL for 24 hours exerted concentration-dependent cytotoxicity andsignificant upregulation of mRNA expression of p53, bax, caspase-3 & caspase-9, whereas expression of antiapoptoticbcl-2 was down-regulated. Conclusion: To the best of our knowledge this is the first report showingthat gold nanoparticles induce apoptosis in MCF-7cells via p53, bax/bcl-2 and caspase pathways.     

Daidzein induces MCF-7 breast cancer cell apoptosis via the mitochondrial pathway

BackgroundIn order to study the anticancer effects and cellular apoptosis pathways induced by daidzein.Materials and methodsWe used the human MCF-7 breast cancer cell line as a model and examined the apoptosis by Hoechst–propidium iodide staining fluorescence imaging and flow cytometry.ResultsOur data indicated that daidzein induces antiproliferative effects in a concentration- and time-dependent manner. We demonstrated that daidzein-induced apoptosis in MCF-7 cells was initiated by the generation of reactive oxygen species (ROS). Furthermore, we showed that this daidzein-induced ROS generation was accompanied by disruption of mitochondrial transmembrane potential, down-regulation of bcl-2, and up-regulation of bax, which led to the release of cytochrome C from the mitochondria into the cytosol, which, in turn, resulted in the activation of caspase-9 and caspase-7, and ultimately in cell death. The induction of the mitochondrial caspase-dependent pathway was confirmed by pretreatment with pan-caspase inhibitor z-VAD-fmk and antioxidant N-acetyl-L-cysteine.ConclusionAccordingly, daidzein could induce breast cancer cell apoptosis through the mitochondrial caspase-dependent cell death pathway.     

Overexpression of bax sensitizes human breast cancer MCF-7 cells to radiation-induced apoptosis

Resistance to apoptosis plays an important role in tumors that are refractory to chemotherapy and ionizing radiation (IR). bax, which forms a heterodimer with bcl-2 and accelerates apoptosis, is not, or only weakly, expressed in most human breast cancer cells, and weak bax expression is considered to be related to the resistance of breast cancer cells to apoptosis. bax expression vector was introduced to human breast cancer MCF-7 cells, which exhibit weak expression of bax, to demonstrate its role of modulating radiation-induced apoptosis. bax overexpression in MCF-7 cells by stable transfection does not affect viability by itself, but each stable transfectant was more sensitive to IR than the parental MCF-7 cells. The degree of enhancement in radiosensitivity was dependent on the expression level of bax. IR upregulated p53 and p21^WAFI about 5- to 10-fold and downregulated bcl-2 and bcl-XL by 80–90% at 6 hr in both parent and bax stably transfected MCF-7 cells to the same degree. FACS analysis and DNA electrophoresis revealed that this sensitization was due to apoptosis. We suggest that exogenous bax expression might be one of the factors determining cellular radiosensitivity in MCF-7 breast cancer cells and may have therapeutic applications for enhancing radiation sensitivity in breast cancer cells. © 1996 Wiley-Liss, Inc.     

Stimulation of the mitogen-activated protein kinase pathway antagonizes TRAIL-induced apoptosis downstream of BID cleavage in human breast cancer MCF-7 cells

We studied the role of the mitogen-activated protein kinase (MAPK) pathway in the regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in breast tumor MCF-7 cells. We found that addition of a protein kinase C (PKC) activator to MCF-7 cultures prevented TRAIL-induced apoptosis, by inhibiting a step downstream of both caspase-8 activation and BID cleavage. TRAIL-induced translocation of Bax from cytosol to mitochondria, release of cytochrome c from mitochondria and activation of caspase-9 were all inhibited by PKC activation. PKC-mediated prevention of mitochondrial apoptotic events and apoptosis was found to be dependent on the MAPK pathway. Since TRAIL is a ligand of potential use in antineoplastic clinical trials, our findings may provide relevant information in cancer therapy.     

Insulin-like growth factor-binding protein 3 induces caspase-dependent apoptosis through a death receptor-mediated pathway in MCF-7 human breast cancer cells

Insulin-like growth factor-binding protein (IGFBP)-3 has been shown to potently inhibit cell proliferation in various cell systems. However, the specific mechanisms involved in the antiproliferative action of IGFBP-3 have yet to be elucidated. In the present study, we demonstrate that IGFBP-3 induces apoptosis in an insulin-like growth factor (IGF)-independent manner through the activation of caspases involved in a death receptor-mediated pathway in MCF-7 human breast cancer cells. Induction of IGFBP-3 using an ecdysone-inducible expression system inhibited DNA synthesis in an IGF-IGF receptor axis-independent fashion and resulted in the subsequent induction of apoptosis and an increase in caspase activity. Similar results were obtained when cells were transfected with GGG-IGFBP-3, an IGFBP-3 mutant unable to bind IGFs, corroborating the IGF-independent action of IGFBP-3. Additional caspase activity studies and immunoblot analyses using specific caspase substrates and/or caspase inhibitors revealed that the growth-inhibitory effect of IGFBP-3 results mainly from its induction of apoptosis (in particular, activation of caspase-8 and -7). Analyses of caspase-9 activity and release of cytochrome c into the cytosol confirmed that the mitochondria-mediated pathway is not involved. Taken together, these results show that IGFBP-3 expression leads to the induction of apoptosis through the activation of caspases involved in a death receptor-mediated pathway and that IGFBP-3 functions as a negative regulator of breast cancer cell growth, independent of the IGF-IGF receptor axis.     

Effects of regulatory domains of specific isoforms of protein kinase C on growth control and apoptosis in MCF-7 breast cancer cells

Protein kinase C (PKC) is a multigene family consisting of at least 11 isoforms that play key roles in growth control and tumorigenesis. To understand the roles of specific isoforms of PKC in breast cancer, we generated derivatives of the human breast cancer cell line MCF-7 that stably overexpress dominant negative mutants (REG) of PKC-alpha, -epsilon, or -zeta, which encode only the regulatory domains of the respective isoforms. When stimulated to re-enter the cell cycle after serum starvation, the MCF-7/PKC-alpha-REG cell line exhibited enhanced cell-cycle progression in comparison to the control cell line. These cells also showed increased sensitivity to growth inhibition and induction of apoptosis in response to various cytotoxic stimuli, including serum starvation, tamoxifen, and gamma-radiation. Western blot analysis indicated that the MCF-7/PKC-alpha-REG cell line displayed marked decreases in the levels of the cyclin-dependent kinase inhibitor p21CIP1 and the anti-apoptotic protein bcl-2. Similar, but less striking, effects were seen in the MCF-7/PKC-epsilon-REG cell line, and the MCF-7/PKC-zeta-REG cell line showed minimal changes, when compared to the control cells. Taken together, these results suggest that the endogenous PKC-alpha in MCF-7 cells plays a critical role in regulating cell-cycle control and apoptosis, in part through upregulating the expression of p21CIP1 and bcl-2. Therefore, inhibitors of PKC-alpha may potentiate the activity of cytotoxic agents in the therapy of breast cancer.     

下调Skp2表达诱导乳腺癌MCF-7细胞凋亡

目的 探讨在乳腺癌细胞系MCF-7中下调S期激酶相关蛋白(Skp2)表达诱导细胞凋亡及其机制。方法 应用RNAi方法在体外下调乳腺癌细胞系MCF-7中Skp2的表达水平,48小时后表阿霉素处理细胞,TUNEL和Hoechst 33258染色检测凋亡,Western Blot检测细胞周期调控相关因子及凋亡相关蛋白表达情况,研究其机制。结果 下调MCF-7中Skp2表达水平后,乳腺癌细胞凋亡增加。下调Skp2使p27、p21和CyclinE蛋白表达水平升高。表阿霉素处理MCF-7细胞后,Skp2蛋白水平下调。Skp2 siRNA与表阿霉素有协同诱导凋亡的作用,p53蛋白水平升高。结论 p27、p21和CyclinE在通过下调Skp2诱导的凋亡中发挥作用。Skp2 siRNA和表阿霉素协同诱导细胞凋亡,与p53依赖的凋亡途径有关。Skp2可能是乳腺癌治疗的靶点。     

Inhibition of SGK1 enhances mAR-induced apoptosis in MCF-7 breast cancer cells

Functional membrane androgen receptors (mAR) have previously been described in MCF-7 breast cancer cells. Their stimulation by specific testosterone albumin conjugates (TAC) activate rapidly non-genomic FAK/PI3K/Rac1/Cdc42 signaling, trigger actin reorganization and inhibit cell motility. PI3K stimulates serum and glucocorticoid inducible kinase SGK1, which in turn regulates the function of mAR. In the present study we addressed the role of SGK1 in mAR-induced apoptosis. TAC-stimulated mAR activation elicited apoptosis of MCF-7 cells, an effect significantly potentiated by concomitant incubation of the cells with TAC and the specific SGK1 inhibitors EMD638683 and GSK650394. In line with this, TAC and EMD638683 activated caspase-3. These effects were insensitive to the classical androgen receptor (iAR) antagonist flutamide, pointing to iAR-independent, mAR-induced responses. mAR activation and SGK1 inhibition further considerably augmented the radiation-induced apoptosis of MCF-7 cells. Moreover, TAC- and EMD638683 triggered early actin polymerization in MCF-7 cells. Blocking actin restructuring with cytochalasin B abrogated the TAC- and EMD638683-induced pro-apoptotic responses. Further analysis of the molecular signaling revealed late de-phosphorylation of FAK and Akt. Our results demonstrate that mAR activation triggers pro-apoptotic responses in breast tumor cells, an effect significantly enhanced by SGK1 inhibition, involving actin reorganization and paralleled by down-regulation of FAK/Akt signaling.     

Tamoxifen induces selective membrane association of protein kinase C epsilon in MCF-7 human breast cancer cells

Tamoxifen, a synthetic antiestrogen, is known for its antitumoral action in vivo; however, it is well accepted that many tamoxifen effects are elicited via estrogen receptor-independent routes. Previously, we reported that tamoxifen induces PKC translocation in fibroblasts. In the present study, we investigated the influence of tamoxifen, and several triphenylethylene derivatives, on protein kinase C (PKC) in MCF-7 human breast cancer cells. As measured by Western blot analysis, tamoxifen elicited isozyme-specific membrane association of PKC-ϵ, which was time-dependent (as early as 5 min post-treatment) and dose-dependent (5.0–20 μM). Tamoxifen did not influence translocation of α, β, γ, δ or ζ PKC isoforms. Structure-activity relationship studies demonstrated chemical requirements for PKC-ϵ translocation, with tamoxifen, 3-OH-tamoxifen and clomiphene being active. Compounds without the basic amino side chain, such as triphenylethylene, or minus a phenyl group, such as N,N-dimethyl-2-[(4-phenylmethyl)phenoxy]ethanamine, were not active. In vitro cell growth assays showed a correlation between agent-induced PKC-ϵ translocation and inhibition of cell growth. Exposure of cells to clomiphene resulted in apoptosis. Since PKC-ϵ has been associated with cell differentiation and cellular growth-related processes, the antiproliferative influence of tamoxifen on MCF-7 cells may be related to the interaction with PKC-ϵ. Int. J. Cancer 77:928–932, 1998.© 1998 Wiley-Liss, Inc.     

Enzastaurin renders MCF-7 breast cancer cells sensitive to radiation through reversal of radiation-induced activation of protein kinase C

Enzastaurin (LY317615.HCI), a protein kinase C (PKC)-β inhibitor, has a radiosensitising effect on 4T1 murine breast cancer and human glioma cells; however, the exact mechanism of this action has not been evaluated. The present study investigated the effects of enzastaurin and gamma irradiation on PKC activity in MCF-7 human breast cancer cells in vitro and in vivo. Enzastaurin (5 μM) in combination with irradiation (2–8 Gy) produced a synergistic decline in MCF-7 clonogenic cell survival. Analysis of MCF-7 cells stained with Annexin V and 7-aminoactinomycin D showed a dose-dependent increase in apoptosis in response to enzastaurin (3, 5 and 7 μM) and irradiation (10 Gy) compared to irradiation alone. This pro-apoptotic effect was confirmed by increases in caspase-3 and -9 activity. In a MCF-7 xenograft model, irradiation with 25 Gy increased PKC-α activity by 2.5-fold compared to untreated controls, whereas PKC-ε and -βII activity was increased by 1.8-fold. Radiation-induced activation of all three anti-apoptotic isoforms of PKC was reversed by pre-treatment with enzastaurin (75 mg/kg, twice daily for 3 days). We conclude that enzastaurin has a radiosensitising effect on MCF-7 human xenograft tumours through the reversal of anti-apoptotic activation of PKC isoforms.     

Reconstitution of caspase-3 sensitizes MCF-7 breast cancer cells to radiation therapy

Caspase-3 plays an important role in apoptotic execution. Caspase-3 deficiency or down-regulation has been reported in breast and other kinds of cancers. Given the redundancy of caspase cascade, however, the impact of caspase-3 deficiency/down-regulation on radiation-induced apoptosis remains to be defined. In this report, the specific role of caspase-3 in radiotherapy-induced apoptosis was studied using MCF-7 control (MCF-7/pv, caspase-3 deficient) and caspase-3 reconstituted MCF-7 (MCF-7/c3) breast cancer cells. Caspase-3 reconstitution significantly enhanced radiation-induced apoptosis, with a decrease in the survival fraction, an increase in caspase activation, cleavage of cellular death substrates and mitochondrial depolarization. We also found that the activation of caspase-7 was caspase-3-dependent in radiation-induced apoptosis, which suggests a mini-cascade among the effector caspases and that caspase-3 is essential for signal amplification. In comparing the patterns of death substrates cleavage in radiation-induced apoptosis with that in doxorubicin and TNF-alpha-induced apoptosis, we found that cleavage of lamin B and beta-actin was relatively more susceptible to radiation, which is enhanced in the presence of caspase-3, suggesting cytoskeleton proteins might be preferred markers for radiation-induced apoptosis. These data indicate that caspase-3 plays a critical role in radiotherapy-induced apoptosis, and suggest that caspase-3 deficiency may contribute to the radioresistance of breast cancers.     

唑来膦酸对乳腺癌细胞系MCF-7的凋亡作用及其机制

目的:研究唑来膦酸对乳腺癌MCF-7细胞的凋亡作用和途径.方法:培养乳腺癌MCF-7细胞株,用不同浓度梯度的唑来膦酸进行处理,MTT、Annexin V-PI双染法进行生长抑制和凋亡情况的检测和分析,Western blot检测相关蛋白表达情况.结果:唑来膦酸作用于MCF-7细胞后,MTT、Annexin V-PI双染法检测发现药物对细胞呈明显的生长抑制作用,且有明显的剂量依赖性.当作用时间为48h时抑制作用最为明显.Western blot检测发现,使用唑来膦酸后,Bcl-2、Bcl-XL和Survivin 3个凋亡抑制蛋白表达水平相较于未处理组(NM)表达降低,促凋亡蛋白Bax表达明显升高.同时,Caspase-3和Caspase-9相较于未处理组,表达水平也有明显的升高,并都呈现剂量相关性.结论:唑来膦酸通过线粒体凋亡途径促进乳腺癌MCF-7细胞凋亡,且具有剂量依赖性.     

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.