Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0006142 (breast cancer)
 160,383 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Urokinase-type plasminogen activator (uPA) binds to the uPA receptor (uPAR) and activates the Ras-extracellular signal-regulated kinase (ERK) signaling pathway in many different cell types. In this study, we demonstrated that endogenously produced uPA functions as a major determinant of the basal level of activated ERK in MDA-MB-231 breast cancer cells. When these cells were cultured in the presence of antibodies that block the binding of uPA to uPAR, the level of phosphorylated ERK decreased substantially. Furthermore, conditioned medium from MDA-MB-231 cells activated ERK in MCF-7 cells and this response was blocked by uPA-specific antibody. The mitogen-activated protein kinase kinase inhibitor, PD098059, decreased expression of uPA and uPAR in MDA-MB-231 cells. Thus, uPA and the uPAR-ERK signaling pathway form a positive feedback loop in these cells. When this feedback loop was disrupted with uPA- or uPAR-specific antibody, uPA mRNA-specific antisense oligodeoxynucleotides or PD098059, cell growth was inhibited and apoptosis was promoted, as determined by the increase in cytoplasmic nucleosomes and caspase-3 activity. Treating the cells simultaneously with PD098059 and uPA- or uPAR-specific antibody did not further promote apoptosis, compared with either reagent added separately, supporting the hypothesis that uPAR and ERK are components of the same cell growth/survival-regulatory pathway. The ability of uPA to signal through uPAR, maintain an elevated basal level of activated ERK and inhibit apoptosis represents a novel mechanism whereby the uPA-uPAR system may affect breast cancer progression in vivo.
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PMID:Endogenously produced urokinase-type plasminogen activator is a major determinant of the basal level of activated ERK/MAP kinase and prevents apoptosis in MDA-MB-231 breast cancer cells. 1159 26

Apoptosis is meticulously controlled in living organisms. Its dysregulation has been shown to play a key role in a number of human diseases, including neoplastic, cardiovascular, and degenerative disorders. Bcl-2 family member proteins and inhibitors of apoptosis proteins are two major negative regulators of apoptosis. We report here the characterization of novel antiapoptotic protein, fortilin, which we identified through yeast two-hybrid library screening. Sequence analysis of fortilin revealed it to be a 172-amino acid polypeptide highly conserved from mammals to plants. Fortilin is structurally unrelated to either Bcl-2 family member proteins or inhibitors of apoptosis proteins. Northern blot analysis showed the fortilin message to be ubiquitous in normal tissue but especially abundant in the liver, kidney, and small intestine. Western blot analysis using anti-fortilin antibody showed more extensive expression in cancerous cell lines (H1299, MCF-7, and A549) than in cell lines derived from normal tissue (HEK293). Immunocytochemistry using HeLa cells transiently expressing FLAG-tagged fortilin and immunohistochemistry using human breast ductal carcinoma tissue and anti-fortilin antibody both showed that fortilin is predominantly localized in the nucleus. Functionally, the transient overexpression of fortilin in HeLa cells prevented them, in a dose-dependent fashion, from undergoing etoposide-induced apoptosis. Consistently, U2OS cells stably expressing fortilin protected the cells from cell death induced by etoposide over various concentrations and durations of exposure. In addition, fortilin overexpression inhibited caspase-3-like activity as assessed by the cleavage of fluorogenic substrate benzyloxycarbonyl-DEVD-7-amido-4-(trifluoromethyl)coumarin. Furthermore, the antisense depletion of fortilin from breast cancer cell line MCF-7 was associated with massive cell death. These data suggest that fortilin represents a novel antiapoptotic protein involved in cell survival and apoptosis regulation.
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PMID:Characterization of fortilin, a novel antiapoptotic protein. 1159 39

MCF-7 human breast cancer cells are widely utilized to study apoptotic processes. Recent studies demonstrated that these cells lack procaspase-3. In the present study, caspase activation and activity were examined in this cell line after treatment with the microtubule poison paclitaxel. When cells were harvested 72 h after the start of a 24-h treatment with 100 nm paclitaxel, 37 +/- 5% of the cells were nonadherent and displayed apoptotic morphological changes. Although mitochondrial cytochrome c release and caspase-9 cleavage were detectable by immunoblotting, assays of cytosol and nuclei prepared from the apoptotic cells failed to demonstrate the presence of activity that cleaved the synthetic caspase substrates LEHD-7-amino-4-trifluoromethylcoumarin (LEHD-AFC), DEVD-AFC, and VEID-AFC. Likewise, the paclitaxel-treated MCF-7 cells failed to cleave a variety of caspase substrates, including lamin A, beta-catenin, gelsolin, protein kinase Cdelta, topoisomerase I, and procaspases-6, -8, and -10. Transfection of MCF-7 cells with wild type procaspase-3 partially restored cleavage of these polypeptides but did not result in detectable activities that could cleave the synthetic caspase substrates. Immunoblotting revealed that caspase-9, and -3, which were proteolytically cleaved in paclitaxel-treated MCF-7/caspase-3 cells, were sequestered in a salt-resistant sedimentable fraction rather than released to the cytosol. Immunofluorescence indicated large cytoplasmic aggregates containing cleaved caspase-3 in these apoptotic cells. These observations suggest that sequestration of caspases can occur in some model systems, causing tetrapeptide-based activity assays to underestimate the amount of caspase activation that has occurred in situ.
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PMID:Lack of correlation between caspase activation and caspase activity assays in paclitaxel-treated MCF-7 breast cancer cells. 1167 38

We have shown previously that (NOHA) an intermediate in the nitric oxide (NO) synthetic pathway and an inhibitor of arginase significantly reduced intracellular polyamines, activated caspase-3 and induced apoptosis in the human breast cancer cell line MDA-MB-468. These actions of NOHA were abolished in the presence of exogenous L-ornithine suggesting that a reduction in the intracellular polyamine content might be responsible for the activation of caspase-3 and apoptotic actions of NOHA. In order to further explore this possibility, we used SAM-486A and alpha-difluoromethylornithine (DFMO), which are inhibitors of S-adenosylmethionine decarboxylase (SAMDC), and ornithine decarboxylase (ODC), respectively, either alone or in combination to reduce the intracellular polyamine levels. We then assessed whether a reduction in polyamine levels by these two compounds to a similar degree to that produced by NOHA activated caspase-3 which occurs prior to the onset of apoptosis. We observed that both SAM-486A and DFMO, either alone or in combination, inhibited cell proliferation, induced p21 and arrested cells in the G(0)-G(1) phase of the cell cycle but failed to activate caspase-3 as assessed by enzymatic assay of caspase-3, western blot analysis of the proteolytic cleavage of caspase-3 protein as well as TUNEL assay. Furthermore, pre-incubation of the cells with SAM-486A and DFMO for 4 days, either alone or in combination significantly inhibited the activation of caspase-3 and apoptosis by NOHA when compared with that observed with cells treated with NOHA alone. Our results, therefore, indicate that the activation of caspase-3 and apoptosis observed with NOHA cannot be solely explained by a reduction in intracellular polyamine levels and that other mechanisms need to be also considered.
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PMID:Activation of caspase-3 activity and apoptosis in MDA-MB-468 cells by N(omega)-hydroxy-L-arginine, an inhibitor of arginase, is not solely dependent on reduction in intracellular polyamines. 1169 50

Heregulins are a group of growth factors that play diverse and critical roles in the signaling network of the human epidermal growth factor receptor (HER or EGFR) superfamily. Our earlier studies have shown that recombinant heregulinbeta1 (HRG) induces apoptosis in SKBr3 breast cancer cells that overexpress HER2. Here we report molecular mechanisms of HRG-induced apoptosis. HRG treatment of SKBr3 cells for 72 h decreased the level of Bcl-2 protein. HRG treatment led to degradation of poly (ADP-ribose) polymerase (PARP) and activated both caspase-9 and caspase-7. No significant activation of caspase-3, -6, or -8 was detected. Expression of exogenous caspase-7 by adenovirus-caspase-7 (Ad-casp-7) in SKBr3 cells resulted in apoptosis, which mimicked the effect of HRG treatment. Expression of exogenous caspase-7 had no impact on Bcl-2 expression, but promoted PARP degradation. Two highly selective inhibitors of protein kinase C (PKC), GF109203X (GF) and Ro318425 (Ro), significantly enhanced HRG-induced apoptosis as determined by flow cytometric analysis and DNA fragmentation assay. Accordingly, the PKC inhibitor GF further decreased the level of Bcl-2 protein and further degraded PARP in HRG-treated cells. Assay of PKC activity indicated that HRG activated PKC in SKBr3 cells, predominantly affecting the PKCalpha isoform. To confirm which PKC isoform(s) mediated potentiation of HRG-induced apoptosis, the profile of PKC isoforms was measured in SKBr3 cells. Five PKC isoforms, PKCalpha, PKCiota, PKCzeta, PKClambda, and PKCdelta as well as their receptors (RACK1) were expressed in this cell line. Treatment with PKC inhibitors GF and Ro decreased protein levels of both PKCalpha and PKCdelta at 24 h. PKCalpha levels were still depressed at 72 h. GF and Ro had little effect on the expression of other PKC isoforms. An inhibitor of classical PKC isoforms (Go6976) enhanced HRG-induced apoptosis, whereas the PKCdelta selective inhibitor rottlerin did not. As PKCalpha was the only classical isoform expressed in SKBr3 cells, the effect of Go6976 on HRG-induced apoptosis largely related to inhibition of PKCalpha. Constitutive expression of wild-type PKCalpha attenuated the apoptosis produced by HRG and GF. Consequently, HRG-induced apoptosis in SKBr3 cells appeared to involve down-regulation of Bcl-2 protein, activation of caspase-9 and caspase-7, and degradation of PARP. Inhibition of PKC function enhanced HRG-induced apoptosis, leading to synergistic down-regulation of Bcl-2 expression. Impairment of the PKCalpha isoform alone was sufficient to potentiate HRG-induced apoptosis.
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PMID:Heregulin-induced apoptosis is mediated by down-regulation of Bcl-2 and activation of caspase-7 and is potentiated by impairment of protein kinase C alpha activity. 1178 40

We previously demonstrated that the forced expression of pro-caspase-3 can revert acquired chemoresistance in MT1-Adr breast cancer cells which show a defective activation of the mitochondrial pathway of apoptosis. We now asked whether the manipulation of mitochondrial apoptosis signaling can revert different types of drug resistance, i.e. the resistance due to impaired mitochondrial activation in the MT1-Adr cells and the resistance in MT3-Adr cells which is caused by increased expression of the Mdr-1/p-glycoprotein ABC transporter. Here we show that Bcl-2 overexpression is the underlying cause for the resistant phenotype in the MT1-Adr cells. Overexpression of the apoptosis-promoting Bax homologue Bak or the BH3 only protein Nbk/Bik reverts, as expected, acquired drug resistance in the MT1-Adr cells as recently demonstrated for pro-caspase-3. Moreover, we show that both apoptosis-promoters, Nbk/Bik and Bak, antagonize acquired chemoresistance for epirubicin-mediated apoptosis in MT3-Adr breast cancer cells. Neither drug uptake nor drug efflux were influenced by Bak or Nbk/Bik. Thus, our data show that manipulation of the downstream apoptosis signaling cascade by Bak and Nbk/Bik can overcome not only drug resistance due to mitochondrial apoptosis deficiency (in the MT1-Adr cells) but also classical, i.e. efflux-mediated, resistance for drug-induced cell death in the MT3-Adr cell line. Nbk/Bik and Bak could therefore be target genes to increase chemosensitivity and overcome different types of drug resistance.
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PMID:The apoptosis promoting Bcl-2 homologues Bak and Nbk/Bik overcome drug resistance in Mdr-1-negative and Mdr-1-overexpressing breast cancer cell lines. 1180 66

The spermine analogue N(1),N(11)-diethylnorspermine (DENSPM) efficiently depletes the cellular pools of putrescine, spermidine and spermine by down-regulating the activity of the polyamine biosynthetic enzymes and up-regulating the activity of the catabolic enzyme spermidine/ spermine N(1)-acetyltransferase (SSAT). In the breast cancer cell line L56Br-C1, treatment with 10 microm DENSPM induced SSAT activity 60 and 240-fold at 24 and 48 h after seeding, respectively, which resulted in polyamine depletion. Cell proliferation appeared to be totally inhibited and within 48 h of treatment, there was an extensive apoptotic response. Fifty percent of the cells were found in the sub-G(1) region, as determined by flow cytometry, and the presence of apoptotic nuclei was morphologically assessed by fluorescence microscopy. Caspase-3 and caspase-9 activities were significantly elevated 24 h after seeding. At 48 h after seeding, caspase-3 and caspase-9 activities were further elevated and at this time point a significant activation of caspase-8 was also found. The DENSPM-induced cell death was dependent on the activation of the caspases as it was inhibited by the general caspase inhibitor Z-Val-Ala-Asp fluoromethyl ketone. The results are discussed in the light of the L56Br-C1 cells containing mutated BRCA1 and p53, two genes involved in DNA repair.
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PMID:Rapid caspase-dependent cell death in cultured human breast cancer cells induced by the polyamine analogue N(1),N(11)-diethylnorspermine. 1184 6

The human breast cancer cell line MCF-7 is deficient in procaspase-3 and in caspase-3-dependent steps in apoptosis due to deletion of the CASP-3 gene. We previously found that the cells transfected with empty vector (MCF-7v cells) were considerably less sensitive to photodynamic treatment in vitro with the phthalocyanine photosensitizer Pc 4 than were the cells stably transfected with human procaspase-3 cDNA (MCF-7c3 cells); however, overall cell killing, as determined by a clonogenic assay, was not affected by the presence of procaspase-3. The present study was undertaken to determine whether photodynamic therapy (PDT) in vivo was dependent on the ability of the cells to carry out the late steps in apoptosis that are catalyzed by this caspase. Xenografts of MCF-7 cells and the isogenic-derived MCF-7v and MCF-7c3 cells were generated in female athymic nude mice implanted with an estrogen pellet. MCF-7c3 xenografts, but not those of the other two lines, continued to express procaspase-3, as revealed by Western blots of proteins from the cells and the xenografts. When the xenografts reached 50-120 mm(3), some were treated with PDT (1mg/kg Pc 4 i.v. followed 48 h later by 150 J/cm(2) light at 672 nm and 150 mW/cm(2)), while others served as controls (no treatment, light alone, or Pc 4 alone). All Pc 4-PDT-treated tumors and none of the controls exhibited either complete or strong partial responses, and complete responses were durable for the entire observation period of 16 days. The responses were not dependent upon the presence of procaspase-3 in the xenografts. The results indicate that the rapid response of Pc 4-PDT-treated tumors in vivo is not due to their ability to carry out the major caspase-3-mediated late steps in apoptosis.
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PMID:Photodynamic therapy of human breast cancer xenografts lacking caspase-3. 1188 Jan 81

Caspase-3 deficiency can limit the efficiency of pro-apoptotic anticancer treatments. Irofulven (hydroxymethylacyl-fulvene, HMAF. MGI 114, NSC 683863) is an antitumor drug, currently in a Phase III and multiple Phase II trials, which can differentiate between tumor and normal cells in apoptosis induction. This study investigated whether apoptosis induced by irofulven requires caspase-3. Irofulven action was compared in breast cancer cells differing in caspase-3 status: deficient MCF-7 cells and proficient MDA-MB-231 cells and in normal human mammary epithelial cells, HMEC. Irofulven induces significant, concentration and time-dependent apoptotic DNA fragmentation in breast cancer cell lines, regardless of caspase-3 status. After 12, 24 and 48 h incubation at 1 microM irofulven (approximately 3 x GI50), fragmented DNA comprised 3.7, 14.1 and 34.6% and 8.4, 12.6 and 20.3% of total DNA in MCF-7 and MDA-MB-231 cells, respectively. Cell viability (trypan blue exclusion) remained largely unaffected during the first 24 h but decreased markedly after 48 h, indicating secondary necrosis. Net losses in cell numbers were apparent at 48 h. Normal HMEC cells were refractory to 1 microM drug with only approximately 3-9% fragmented DNA after 12-48 h, although apoptosis was observed at drug levels >3 microM. The broad-spectrum caspase inhibitor Z-VAD-fmk inhibited irofulven-induced apoptosis of all cell lines at 20 microM with nearly complete abrogation of apoptosis at 100 microM. Irofulven treatment resulted in marginal caspase-3 processing in MDA-MB-231 and HMEC cells. These results indicate that whereas the caspase cascade mediates irofulven- induced apoptosis, caspase-3 is dispensable (supported by NIH CA70091 and CA78706).
Breast Cancer Res Treat 2002 Jan
PMID:Irofulven induces apoptosis in breast cancer cells regardless of caspase-3 status. 1188 39

Exposure of mammalian cells to agents that induce apoptosis results in a rapid and substantial inhibition of protein synthesis. In MCF-7 breast cancer cells, tumor necrosis factor alpha (TNFalpha) and TNF-related apoptosis-inducing ligand inhibit overall translation by a mechanism that requires caspase (but not necessarily caspase-3) activity. This inhibition is associated with the increased phosphorylation of eukaryotic initiation factor (eIF2) alpha, increased association of eIF4E with the inhibitory eIF4E-binding protein (4E-BP1), and specific cleavages of eIF4B and eIF2alpha. All of these changes require caspase activity. The cleavage of eIF4GI, which specifically needs caspase-3 activity, is dispensable for the inhibition of translation in MCF-7 cells. Similar experiments with embryonic fibroblasts from control mice and animals defective for expression of the double-stranded RNA-regulated protein kinase (PKR) reveal requirements for both caspase activity and PKR for inhibition of protein synthesis in response to TNFalpha. In contrast, treatment of cells with the DNA-damaging agent etoposide inhibits protein synthesis equally well in the presence of a pan-specific caspase inhibitor and in the presence or absence of PKR. Surprisingly, the ability of etoposide to cause increased association of eIF4E with 4E-BP1 does require PKR activity. However, our data suggest that neither increased phosphorylation of eIF2alpha nor increased [eIF4E.4E-BP1] complex formation is essential for the inhibition of overall translation by the DNA-damaging agent.
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PMID:Inhibition of protein synthesis in apoptosis: differential requirements by the tumor necrosis factor alpha family and a DNA-damaging agent for caspases and the double-stranded RNA-dependent protein kinase. 1195 83


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