Biomedical subjects

Stephen Safe

Publications and source records attributed to Stephen Safe.

At least 19 recordsLinked to original sources

PubMed2006

17beta-estradiol (E2) induces cdc25A gene expression in breast cancer cells by genomic and non-genomic pathways.

Cdc25A is a potent tyrosine phosphatase that catalyzes specific dephosphorylation of cyclin/cyclin-dependent kinase (cdk) complexes to regulate G1 to S-phase cell cycle progression. Cdc25A mRNA levels are induced by 17beta-estradiol (E2) in ZR-75 breast cancer cells, and deletion analysis of the cdc25A promoter identified the -151 to -12 region as the minimal E2-responsive sequence. Subsequent mutation/deletion analysis showed that at least three different cis-elements were involved in activation of cdc25A by E2, namely, GC-rich Sp1 binding sites, CCAAT motifs that bind NF-Y, and E2F sites that bind DP/E2F1 proteins. Studies with inhibitors and dominant negative expression plasmids show that E2 activates cdc25A expression through activation of genomic ERalpha/Sp1 and E2F1 and cAMP-dependent activation of NF-YA. Thus, both genomic and non-genomic pathways of estrogen action are involved in induction of cdc25A in breast cancer cells.

Base Composition↗

PubMed2006

Molecular mechanism of inhibitory aryl hydrocarbon receptor-estrogen receptor/Sp1 cross talk in breast cancer cells.

The trifunctional carbamoylphosphate synthetase/aspartate transcarbamyltransferase/dihydroorotase (CAD) gene is hormone responsive in MCF-7 and ZR-75 breast cancer cells, and this response is inhibited by the aryl hydrocarbon receptor (AhR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Estrogen-dependent induction of CAD mRNA and reporter gene activity in cells transfected with constructs (pCAD) containing hormone-responsive GC-rich CAD promoter inserts involves estrogen receptor alpha (ERalpha)/Sp1 interactions with these proximal GC-rich motifs. TCDD also inhibits hormone-induced transactivation in MCF-7 and ZR-75 cells transfected with pCAD constructs. The mechanism of inhibitory AhR-ERalpha/Sp1 cross talk was further investigated by chromatin immunoprecipitation (ChIP), and the results show that ERalpha/Sp1 and the AhR are constitutively bound to the CAD gene promoter and only minor changes are observed after treatment with 17beta-estradiol, TCDD, or their combination. However, examination of interactions of these transcription factors by fluorescence resonance energy transfer shows that E2 enhances ERalpha-Sp1 interactions, whereas cotreatment with TCDD significantly decreases interaction of these proteins. These results suggest that inhibitory AhR-ERalpha/Sp1 cross talk is due, in part, to enhanced association of AhR and ERalpha (also determined by fluorescence resonance energy transfer), which coordinately dissociates ER and Sp1 and decreases ERalpha/Sp1-mediated transactivation, whereas remaining associated with the CAD promoter. This represents a novel interaction between two ligand activated receptors where one receptor inhibits activation of the second receptor.

Animals↗

PubMed2006

Structure-dependent Ah receptor agonist activities of chlorinated biphenylenes.

Polychlorinated biphenylenes (PCBP) have been identified as combustion by-products that bind the aryl hydrocarbon receptor (AhR) and exhibit 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-like activity. This study investigates the Ah-responsiveness of 2,3,6,7-tetrachlorobiphenylene (2,3,6,7-CBP), 2,3,6-CBP, 2,3-CBP and 2-CBP in breast cancer cells. MCF-7 or ZR-75 cells were treated with different concentrations (1-100 nM) of the compounds alone to determine their activity as inducers of CYP1A1 protein expression or luciferase activity in cells transfected with a construct (pDRE(3)) containing three tandem dioxin responsive elements (DREs) linked to a luciferase reporter gene. In both assays, the order of potency was 2,3,6,7-CBP>2,3,6-CBP>2,3-CBP approximately 2-CBP, and 2,3,6,7-CBP and TCDD were equipotent. Similar results were also observed in an antiestrogenic assay in MCF-7 cells, confirming the high AhR agonist activity of 2,3,6,7-CBP in breast cancer cells.

Blotting, Western↗

PubMed2006

Regulation of vascular endothelial growth factor receptor-2 expression in pancreatic cancer cells by Sp proteins.

Vascular endothelial growth factor receptor-2 (VEGFR2/KDR) is an important mediator of angiogenesis, and VEGFR2 mRNA is expressed in several pancreatic cancer cell lines. Deletion analysis of the VEGFR2 promoter in Panc-1, AsPC-1, and MiaPaCa-2 pancreatic cancer cells shows that the proximal region of the promoter is primarily responsible for VEGFR2 expression, and two GC-rich sites at -58 and -44 are critical elements in all three cell lines. Panc-1, AsPC-1, and MiaPaCa-2 cells also express Sp1, Sp3, and Sp4 proteins which bind to the GC-rich region of the VEGFR2 promoter in electrophoretic mobility shift and chromatin immunoprecipitation assays. RNA interference with small inhibitory RNAs for Sp1, Sp3, and Sp4 decreases VEGFR2 mRNA and reporter gene activity in transfection assays, confirming that VEGFR2 expression in pancreatic cancer cells is regulated by Sp proteins. These results suggest that VEGFR2 cannot only be targeted by receptor tyrosine kinase inhibitors but also by drugs that downregulate Sp proteins or block Sp-dependent transactivation.

Cell Line, Tumor↗

PubMed2006

Vascular endothelial growth factor receptor-2 expression is induced by 17beta-estradiol in ZR-75 breast cancer cells by estrogen receptor alpha/Sp proteins.

Vascular endothelial growth factor receptor-2 kinase insert domain receptor (VEGFR2/KDR) is critical for angiogenesis, and VEGFR2 mRNA and protein are expressed in ZR-75 breast cancer cells and induced by 17beta-estradiol (E2). Deletion analysis of the VEGFR2 promoter indicates that the proximal GC-rich region is required for both basal and hormone-induced transactivation, and mutation of one or both of the GC-rich motifs at -58 and -44 results in loss of transactivation. Electrophoretic mobility shift and chromatin immunoprecipitation assays show that Sp1, Sp3, and Sp4 proteins bind the GC-rich region of the VEGFR2 promoter. Results of the chromatin immunoprecipitation assay also demonstrate that ERalpha is constitutively bound to the VEGFR2 promoter and that these interactions are not enhanced after treatment with E2, whereas ERalpha binding to the region of the pS2 promoter containing an estrogen-responsive element is enhanced by E2. RNA interference studies show that hormone-induced activation of the VEGFR2 promoter constructs requires Sp3 and Sp4 but not Sp1, demonstrating that hormonal activation of VEGFR2 involves a nonclassical mechanism in which ERalpha/Sp3 and ERalpha/Sp4 complexes activate GC-rich sites where Sp proteins but not ERalpha bind DNA. These results show for the first time that Sp3 and Sp4 cooperatively interact with ERalpha to activate VEGFR2 and are in contrast to previous results showing that several hormone-responsive genes are activated by ERalpha/Sp1 in breast cancer cell lines.

Base Sequence↗

PubMed2006

Effects of benzo-a-pyrene on oxytocin-induced Ca2+ oscillations in myometrial cells.

Benzo-a-pyrene (BaP) is a polycyclic aromatic hydrocarbon that exists as a major environmental pollutant. The effect of this carcinogen/mutagen upon myometrial Ca(2+) signaling in a human myometrial cell line (PHM1) was examined. Exposure of cells to BaP did not alter basal Ca(2+) levels or the inositol(1,4,5) trisphosphate-releasable Ca(2+) pool. However, BaP significantly decreased the initial oxytocin-induced Ca(2+) transient and the frequency of oxytocin-induced Ca(2+)oscillations as well as delayed their onset. To determine the specific effects of BaP, pharmacologic agents that target intracellular Ca(2+) homeostasis mechanisms were used. Genistein (a non-specific tyrosine kinase inhibitor) and AG1478 (an epidermal growth factor receptor blocker) markedly reduced the oxytocin-induced Ca(2+) oscillations in control, but had no effect in BaP treated cells. Addition of epidermal growth factor or serum before or after oxytocin restored the Ca(2+) oscillations in BaP treated cells to a level similar to control cells, while the K(+) channel blocker tetraethylammonium chloride, partially restored the Ca(2+) response. These data suggest that the tyrosine kinase pathway, which is part of the G-protein coupled receptor pathway response to oxytocin in PHM1 cells, is a target of BaP action and that EGF or serum can restore the oxytocin-induced Ca(2+) oscillations.

Adult↗

PubMed2006

3-Methylcholanthrene and other aryl hydrocarbon receptor agonists directly activate estrogen receptor alpha.

3-Methylcholanthrene (3MC) is an aryl hydrocarbon receptor (AhR) agonist, and it has been reported that 3MC induces estrogenic activity through AhR-estrogen receptor alpha (ER alpha) interactions. In this study, we used 3MC and 3,3',4,4',5-pentachlorobiphenyl (PCB) as prototypical AhR ligands, and both compounds activated estrogen-responsive reporter genes/gene products (cathepsin D) in MCF-7 breast cancer cells. The estrogenic responses induced by these AhR ligands were inhibited by the antiestrogen ICI 182780 and by the transfection of a small inhibitory RNA for ER alpha but were not affected by the small inhibitory RNA for AhR. These results suggest that 3MC and PCB directly activate ER alpha, and this was confirmed in a competitive ER alpha binding assay and in a fluorescence resonance energy transfer experiment in which PCB and 3MC induced CFP-ER alpha/YFP-ER alpha interactions. In a chromatin immunoprecipitation assay, PCB and 3MC enhanced ER alpha (but not AhR) association with the estrogen-responsive region of the pS2 gene promoter. Moreover, in AhR knockout mice, 3MC increased uterine weights and induced expression of cyclin D1 mRNA levels. These results show that PCB and 3MC directly activate ER alpha-dependent transactivation and extend the number of ligands that activate both AhR and ER alpha.

Animals↗

PubMed2006

Activation of kinase pathways in MCF-7 cells by 17beta-estradiol and structurally diverse estrogenic compounds.

17beta-Estradiol (E2) activates non-genomic pathways in MCF-7 cells, and this study investigates the effects of structurally-diverse estrogenic compounds on activation of mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase (PI3-K), protein kinase C (PKC), PKA, and calcium calmodulin-dependent kinase IV (CaMKIV). Activation of kinases was determined by specific substrate phosphorylation and transactivation assays that were diagnostic for individual kinases. The compounds investigated in this study include E2, diethylstilbestrol (DES), the phytoestrogen resveratrol, and the following synthetic xenoestrogens, bisphenol-A (BPA), nonylphenol, octylphenol, endosulfan, kepone, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), and 2',3',4',5'-tetrachloro-4-biphenylol (HO-PCB-Cl(4)). With the exception of resveratrol, all the compounds activated PI3-K and MAPK. Activation of PKC by the xenoestrogens was structure-dependent since resveratrol, kepone and HO-PCB-Cl(4) were inactive and only minimal activation of PKA was observed. CaMKIV was activated only by E2 and DES, and HO-PCB-Cl(4) was a potent inhibitor of CaMKIV-dependent activity. These results demonstrate that activation of estrogen receptor-alpha-mediated non-genomic pathways by estrogenic compounds in MCF-7 cells is structure-dependent and can result in activation or inhibition of kinase activities.

Benzhydryl Compounds↗

PubMed2006

Inhibition of bladder tumor growth by 1,1-bis(3'-indolyl)-1-(p-substitutedphenyl)methanes: a new class of peroxisome proliferator-activated receptor gamma agonists.

1,1-Bis(3'-indolyl)-1-(p-substitutedphenyl)methanes containing p-trifluoromethyl (DIM-C-pPhCF3), p-t-butyl (DIM-C-pPhtBu), and phenyl (DIM-C-pPhC6H5) substituents have been identified as a new class of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists that exhibit antitumorigenic activity. The PPARgamma-active C-DIMs have not previously been studied against bladder cancer. We investigated the effects of the PPARgamma-active C-DIMs on bladder cancer cells in vitro and bladder tumors in vivo. In this study, the PPARgamma-active compounds inhibited the proliferation of KU7 and 253J-BV bladder cancer cells, and the corresponding IC50 values were 5 to 10 and 1 to 5 micromol/L, respectively. In the less responsive KU7 cells, the PPARgamma agonists induced caveolin-1 and p21 expression but no changes in cyclin D1 or p27; in 253J-BV cells, the PPARgamma agonists did not affect caveolin-1, cyclin D1, or p27 expression but induced p21 protein. In KU7 cells, induction of caveolin-1 by each of the PPARgamma agonists was significantly down-regulated after cotreatment with the PPARgamma antagonist GW9662. DIM-C-pPhCF3 (60 mg/kg thrice a week for 4 weeks) inhibited the growth of implanted KU7 orthotopic and s.c. tumors by 32% and 60%, respectively, and produced a corresponding decrease in proliferation index. Treatment of KU7 cells with DIM-C-pPhCF3 also elevated caveolin-1 expression by 25% to 30%, suggesting a role for this protein in mediating the antitumorigenic activity of DIM-C-pPhCF3 in bladder cancer.

Animals↗

PubMed2006

1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes inhibit colon cancer cell and tumor growth through PPARgamma-dependent and PPARgamma-independent pathways.

1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes containing p-trifluoromethyl, t-butyl, and phenyl [1,1-bis(3'-indolyl)-1-(p-phenyl)methane (DIM-C-pPhC(6)H(5))] substituents induce peroxisome proliferator-activated receptor gamma (PPARgamma)-mediated transactivation in SW480 colon cancer cells. These PPARgamma-active compounds also inhibit cell proliferation and modulate some cell cycle proteins. At concentrations from 2.5 to 7.5 micromol/L, the PPARgamma agonists induce caveolin-1 and phosphorylation of Akt and cotreatment with the PPARgamma antagonist GW9662 inhibited the induction response. In contrast, higher concentrations (10 micromol/L) of 1,1-bis(3'-indolyl)-1-(p-substituted phenyl)methanes containing 1,1-bis(3'-indolyl)-1-(p-trifluoromethyl)methane and DIM-C-pPhC(6)H(5) induce apoptosis, which is PPARgamma independent. This was accompanied by loss of caveolin-1 induction but induction of proapoptotic nonsteroidal anti-inflammatory drug activated gene-1. In athymic nude mice bearing SW480 cell xenografts, DIM-C-pPhC(6)H(5) inhibits tumor growth at doses of 20 and 40 mg/kg/d and immunohistochemical staining of the tumors showed induction of apoptosis and nonsteroidal anti-inflammatory drug activated gene-1 expression. Thus, the indole-derived PPARgamma-active compounds induce both receptor-dependent and receptor-independent responses in SW480 cells, which are separable over a narrow range of concentrations. This dual mechanism of action enhances their antiproliferative and anticancer activities.

Animals↗

PubMed2005

3,3'-diindolylmethane (DIM) and its derivatives induce apoptosis in pancreatic cancer cells through endoplasmic reticulum stress-dependent upregulation of DR5.

3,3'-Diindolylmethane (DIM), ring-substituted DIMs and 1,1-bis(3'-indolyl)-1-(p-substitutedphenyl)methanes (C-DIMs) inhibit growth of Panc-1 and Panc-28 pancreatic cancer cells. Although DIMs (diarylmethanes) and selected C-DIMs (triarylmethanes), such as the p-t-butyl derivative (DIM-C-pPhtBu), activate the aryl hydrocarbon receptor and peroxisome proliferator-activated receptor gamma, respectively, this study shows that both DIM and DIM-C-pPhtBu induce common receptor-independent pathways. Both DIM and DIM-C-pPhtBu increased endoplasmic reticulum (ER) staining and ER calcium release in Panc-1 cells, and this was accompanied by increased expression of glucose related protein 78 and C/EBP homologous transcription factor (CHOP/GADD153) proteins. Similar results were observed after treatment with thapsigargin (Tg), a prototypical inducer of ER stress. The subsequent downstream effects of DIM/DIM-C-pPhtBu- and Tg-induced ER stress included CHOP-dependent induction of death receptor DR5 and subsequent cleavage of caspase 8, caspase 3, Bid and PARP. Activation of both receptor-dependent and receptor-independent (ER stress) pathways by DIM and DIM-C-pPhtBu in pancreatic cancer cells enhances the efficacy and potential clinical importance of these compounds for cancer chemotherapeutic applications.

Anticarcinogenic Agents↗

PubMed2005

Conjugated linoleic acid stimulates an anti-tumorigenic protein NAG-1 in an isomer specific manner.

Conjugated linoleic acids (CLAs), naturally occurring fatty acids in ruminant food products, have anti-tumorigenic and pro-apoptotic properties in animal as well as in vitro models of cancer. However, the cellular mechanism has not been fully understood. NAG-1 (non-steroidal anti-inflammatory drug-activated gene-1) is induced by several dietary compounds and belongs to a TGF-beta superfamily gene associated with pro-apoptotic and anti-tumorigenic activities. The present study was performed to elucidate the molecular mechanism by which CLA stimulates anti-tumorigenic activity in human colorectal cancer (CRC) cells. The trans-10, cis-12-CLA (t10,c12-CLA) repressed cell proliferation and induced apoptosis, whereas linoleic acid or c9,t11-CLA showed no effect on cell proliferation and apoptosis. We also found that t10,c12-CLA induced the expression of a pro-apoptotic gene, NAG-1, in human CRC cells. Inhibition of NAG-1 expression by small interference RNA (siRNA) results in repression of t10,c12-CLA-induced apoptosis. Microarray analysis using t10,c12-CLA-treated HCT-116 cells revealed that activating transcription factor 3 (ATF3) was induced and its expression was confirmed by western analysis. The t10,c12-CLA treatment followed by the overexpression of ATF3 increased NAG-1 promoter activity in HCT-116 cells. We further provide the evidence that t10,c12-CLA inhibited the phosphorylation of AKT and the blockage of GSK-3 by siRNA abolished t10,c12-CLA-induced ATF3 and NAG-1 expression. The current study demonstrates that t10,c12-CLA stimulates ATF3/NAG-1 expression and subsequently induces apoptosis in an isomer specific manner. These effects may be through inhibition of AKT/GSK-3beta pathway in human CRC cells.

Activating Transcription Factor 3↗

PubMed2005

Regulation of KiSS-1 metastasis suppressor gene expression in breast cancer cells by direct interaction of transcription factors activator protein-2alpha and specificity protein-1.

KiSS-1 has been shown to function as a tumor metastasis suppressor gene and reduce the number of metastases in different cancers. The expression of KiSS-1 or KiSS1, like other tumor suppressor, is commonly reduced or completely ablated in a variety of cancers via an unknown mechanism. Here we show that the loss of KiSS-1 expression in highly metastatic breast cancer cell lines correlates directly with the expression levels of two transcription factors, activator protein-2alpha (AP-2alpha) and specificity protein 1 (Sp1), which synergistically activate the transcriptional regulation of KiSS-1 in breast cancer cells. Although the KiSS-1 promoter contains multiple AP-2alpha binding elements, AP-2alpha-mediated regulation occurs indirectly through Sp1 sites, as determined by deletion and mutation analysis. Overexpression of AP-2alpha into highly metastatic breast cell lines did not alter KiSS-1 promoter-driven luciferase gene activity. However, co-transfection of AP-2alpha wild-type or the dominant negative form of AP-2 lacking its C-terminal DNA-binding domain, AP-2B, together with Sp1, increased KiSS-1 promoter activity dramatically, suggesting that AP-2alpha regulation of KiSS-1 transcription does not require direct binding to the KiSS-1 promoter. Furthermore, we demonstrated that AP-2alpha directly interacted with Sp1 to form transcription complexes at two tandem Sp1-binding sites of the promoter to activate KiSS-1 transcription. Together, our results indicate that AP-2alpha and Sp1 are strong transcriptional regulators of KiSS-1 and that loss or decreased expression of AP-2alpha in breast cancer may account for the loss of tumor metastasis suppressor KiSS-1 expression and thus increased cancer metastasis.

Breast Neoplasms↗

PubMed2005

Sp transcription factor family and its role in cancer.

Specificity protein 1 (Sp1) and other Sp and Krüppel-like factor (KLF) proteins are members of a family of transcription factors which bind GC/GT-rich promoter elements through three C(2)H(2)-type zinc fingers that are present at their C-terminal domains. Sp1-Sp4 proteins regulate expression of multiple genes in normal tissues and tumours. There is growing evidence that some Sp proteins play a critical role in the growth and metastasis of many tumour types by regulating expression of cell cycle genes and vascular endothelial growth factor. Sp/KLF proteins are also potential targets for cancer chemotherapy.

Cell Survival↗

PubMed2005

1,1-Bis(3'-indolyl)-1-(p-substitutedphenyl)methanes are peroxisome proliferator-activated receptor gamma agonists but decrease HCT-116 colon cancer cell survival through receptor-independent activation of early growth response-1 and nonsteroidal anti-inflammatory drug-activated gene-1.

1,1-Bis-(3'-indolyl)-1-(p-substitutedphenyl)methanes containing p-trifluoromethyl (DIM-C-pPhCF3), p-t-butyl (DIM-C-pPhtBu), and phenyl (DIM-C-pPhC6H5) substituents decrease survival of HCT-116 colon cancer cells and activate peroxisome proliferator-activated receptor (PPAR) gamma in this and other cancer cell lines. These PPARgamma-active compounds had minimal effects on expression of cell cycle proteins and did not induce caveolin-1 in HCT-116 cells. However, these compounds induced nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) and apoptosis in HCT-116 cells, and in time-course studies, the PPARgamma agonists maximally induced early growth response-1 (Egr-1) protein within 2 h, whereas a longer time course was observed for induction of NAG-1 protein. These data, coupled with deletion and mutation analysis of both the Egr-1 and NAG-1 gene promoters, indicate that activation of NAG-1 by these compounds was dependent on prior induction of Egr-1, and induction of these responses was PPARgamma-independent. Results of kinase inhibitor studies also demonstrated that activation of Egr-1/NAG-1 by methylene-substituted diindolylmethanes (C-DIMs) was phosphatidylinositol 3-kinase-dependent, and this represents a novel receptor-independent pathway for C-DIM-induced growth inhibition and apoptosis in colon cancer cells.

Apoptosis↗

PubMed2005

2-Cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) directly targets mitochondrial glutathione to induce apoptosis in pancreatic cancer.

Surgical resection is the only curative strategy for pancreatic cancer (PC). Unfortunately, >80% of pancreatic cancer patients bear inoperable, locally advanced, chemoresistant tumors demonstrating the urgent need for development of novel therapeutic approaches to treat this disease. Here we report that the synthetic triterpenoid 2-cyano-3,12 dioxooleana-1,9 dien-28-imidazolide (CDDO-Im) antagonizes PC cell growth by inducing apoptosis at submicromolar concentrations. Notably, we demonstrate for the first time that the cytotoxicity of CDDO-Im is accompanied by the rapid and selective depletion of mitochondrial glutathione that results in accumulation of reactive oxygen species, oxidation of the cellular glutathione pool, loss of mitochondrial membrane potential, and phosphatidylserine externalization. The parent compound CDDO as well as the methyl ester of CDDO also depleted mitochondrial glutathione, demonstrating that this effect is mediated by the triterpenoid nucleus of these agents. Co-treatment with sulfhydryl nucleophiles completely prevented apoptosis and loss of viability induced by CDDO-Im, whereas alkylation of intracellular thiols by diethylmaleate or co-treatment with dithiothreitol decreased the accumulation of a biotinylated derivative of CDDO, TP-301, in PC cells, suggesting that intracellular reduced thiols are functional targets of the electrophilic triterpenoid nucleus of CDDO and its derivatives. In conclusion, our report is the first to identify mitochondrial glutathione as a target of CDDO and its derivatives and demonstrates that depletion of this antioxidant in the mitochondria is an effective strategy to induce cell death in PC cells. These results suggest that CDDO and its derivatives may offer a clinical benefit for the treatment of PC.

Antineoplastic Agents↗

PubMed2005

Inhibition of breast cancer cell growth and induction of cell death by 1,1-bis(3'-indolyl)methane (DIM) and 5,5'-dibromoDIM.

1,1-Bis(3'-indolyl)methane (DIM) and the 5,5'-dibromo ring substituted DIM (5,5'-diBrDIM) inhibited growth of MCF-7 and MDA-MB-231 breast cancer cells, and IC50 values were 10-20 and 1-5 microM, respectively, in both cell lines. DIM and 5,5'-diBrDIM did not induce p21 or p27 protein levels or alter expression of Sp1 or Sp3 proteins in either cell line. In contrast, 10 microM 5,5'-diBrDIM downregulated cyclin D1 protein in MCF-7 and MDA-MB-231 cells 12 and 24 h after treatment. DIM (20 microM) also decreased cyclin D1 in MCF-7 (24 h) and MDA-MB-231 (12 h), and the DIM/5,5'-diBrDIM-induced degradation of cyclin D1 was blocked by the proteasome inhibitor MG132. Both DIM and 5,5'-diBrDIM induced apoptosis in MCF-7 cells and this was accompanied by decreased Bcl-2, release of mitochondrial cytochrome c, and decreased mitochondrial membrane potential as determined by the red/green fluorescence of JC-1. DIM and 5,5'-diBrDIM induced extensive necrosis in MDA-MB-231 cells; however, this was accompanied by decreased mitochondrial membrane potential primarily in cells treated with 5,5'-diBrDIM but not DIM. Thus, DIM and 5,5'-diBrDIM induce cell death in MCF-7 and MDA-MB-231 cells by overlapping and different pathways, and the ring-substituted DIM represents a novel class of uncharged mitochondrial poisons that inhibit breast cancer cell and tumor growth.

Antineoplastic Agents↗

PubMed2005

Cyclooxygenase-2 inhibitors decrease vascular endothelial growth factor expression in colon cancer cells by enhanced degradation of Sp1 and Sp4 proteins.

Cyclooxygenase 2 (COX-2) inhibitors, such as celecoxib (Cel), nimesulfide (NM), and NS-398 [NS; N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulfonamide] and other nonsteroidal anti-inflammatory drugs inhibit colon cancer growth and angiogenesis; however, the mechanism of this response is not well defined. Treatment of SW-480 colon cancer cells with Cel, NS, or NM decreased vascular endothelial growth factor (VEGF) mRNA and immunoreactive protein expression. This was also accompanied by decreased transactivation in cells transfected with constructs containing VEGF gene promoter inserts. Deletion analysis of the VEGF promoter indicated that decreased VEGF expression by COX-2 inhibitors was associated with the proximal -131 to -47 GC-rich region of the VEGF promoter that binds Sp proteins. Treatment of SW-480 cells with Cel, NM, and NS also decreased Sp1 and Sp4 protein expression but not that of Sp2 or Sp3. Similar results were observed in RKO, HT-29, and DLD colon cancer cells demonstrating comparable responses in COX-2-expressing and -nonexpressing colon cancer cell lines. COX-2 inhibitors do not affect Sp1 or Sp4 mRNA levels in SW-480 cells; however, decreased expression of both proteins was accompanied by increased protein ubiquitination and inhibited by the proteasome inhibitor gliotoxin. These results suggest that the antiangiogenic activity of COX-2 inhibitors in colon cancer cells is linked to activation of proteasome-dependent degradation of Sp1 and Sp4 proteins.

Cell Line, Tumor↗