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PURPOSE: We previously identified small molecules that fit into a BRCA1-binding pocket within estrogen receptor-alpha (ERα), mimic the ability of BRCA1 to inhibit ERα activity ("BRCA1-mimetics"), and overcome antiestrogen resistance. One such compound, the hydrochloride salt of NSC35446 ("NSC35446.HCl"), also inhibited the growth of antiestrogen-resistant LCC9 tumor xenografts. The purpose of this study was to investigate the down-stream effects of NSC35446.HCl and its mechanism of action. METHODS: Here, we studied antiestrogen-resistant (LCC9, T47DCO, MCF-7/RR, LY2), ERα-negative (MDA-MB-231, HCC1806, MDA-MB-468), and antiestrogen-sensitive (MCF-7) cell lines. Techniques utilized include RNA-seq, qRT-PCR, cell growth analysis, cell-cycle analysis, Western blotting, luciferase reporter assays, TUNEL assays, in silico analysis of the IKKB gene, and ChIP assays. RESULTS: SC35446.HCl inhibited proliferation and induced apoptosis in antiestrogen-resistant LCC9, T47DCO, MCF-7/RR, and LY2 cells but not in ERα-negative breast cancer cell lines. IKKB (IKKß, IKBKB), an upstream activator of NF-κB, was identified as a BRCA1-mimetic-regulated gene based on an RNA-seq analysis. NSC35446.HCl inhibited IKKB, IKKA, and IKKG/NEMO mRNA and protein expression in LCC9 cells. NSC35446.HCl also inhibited NF-κB activity and expression of NF-κB target genes. In silico analysis of the IKKB promoter identified nine estrogen response element (ERE) half-sites and one ERE-like full-site. ChIP assays revealed that ERα was recruited to the ERE-like full-site and five of the nine half-sites and that ERα recruitment was inhibited by NSC35446.HCl in LCC9 and T47DCO cells. CONCLUSIONS: These studies identify functional EREs in the IKKB promoter and identify IKKB as an ERα and NSC35446.HCl-regulated gene, and they suggest that NF-κB and IKKB, which were previously linked to antiestrogen resistance, are targets for NSC35446.HCl in reversing antiestrogen resistance.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Antagonistas de Estrogênios/administração & dosagem , Receptor alfa de Estrogênio/genética , Quinase I-kappa B/genética , Apoptose/genética , Proteína BRCA1/antagonistas & inibidores , Proteína BRCA1/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/genética , Resistencia a Medicamentos Antineoplásicos/genética , Estrogênios/genética , Estrogênios/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , NF-kappa B/genética , Regiões Promotoras GenéticasRESUMO
Previous studies indicate that BRCA1 protein binds to estrogen receptor-alpha (ER) and inhibits its activity. Here, we found that BRCA1 over-expression not only inhibits ER activity in anti-estrogen-resistant LCC9 cells but also partially restores their sensitivity to Tamoxifen. To simulate the mechanism of BRCA1 inhibition of ER in the setting of Tamoxifen resistance, we created a three-dimensional model of a BRCA1-binding cavity within the ER/Tamoxifen complex; and we screened a pharmacophore database to identify small molecules that could fit into this cavity. Among the top 40 "hits", six exhibited potent ER inhibitory activity in anti-estrogen-sensitive MCF-7 cells and four of the six exhibited similar activity (IC50 ≤ 1.0 µM) in LCC9 cells. We validated the model by mutation analysis. Two representative compounds (4631-P/1 and 35466-L/1) inhibited ER-dependent cell proliferation in Tamoxifen-resistant cells (LCC9 and LCC2) and partially restored sensitivity to Tamoxifen. The compounds also disrupted the association of BRCA1 with ER. In electrophoretic mobility shift assays, the compounds caused dissociation of ER from a model estrogen response element. Finally, a modified form of compound 35446 (hydrochloride salt) inhibited growth of LCC9 tumor xenografts at non-toxic concentrations. These results identify a novel group of small molecules that can overcome Tamoxifen resistance.
Assuntos
Proteína BRCA1/antagonistas & inibidores , Benzofenonas/farmacologia , Neoplasias da Mama/tratamento farmacológico , Chalconas/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Antagonistas de Estrogênios/química , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Piperidinas/farmacologia , Tamoxifeno/farmacologia , Animais , Apoptose/efeitos dos fármacos , Benzofenonas/química , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Chalconas/química , Ensaio de Desvio de Mobilidade Eletroforética , Estrogênios/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Piperidinas/química , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
CONTEXT: Resistance to conventional antiestrogens is a major cause of treatment failure and, ultimately, death in breast cancer. OBJECTIVE: The objective of the study was to identify small-molecule estrogen receptor (ER)-α antagonists that work differently from tamoxifen and other selective estrogen receptor modulators. DESIGN: Based on in silico screening of a pharmacophore database using a computed model of the BRCA1-ER-α complex (with ER-α liganded to 17ß-estradiol), we identified a candidate group of small-molecule compounds predicted to bind to a BRCA1-binding interface separate from the ligand-binding pocket and the coactivator binding site of ER-α. Among 40 candidate compounds, six inhibited estradiol-stimulated ER-α activity by at least 50% in breast carcinoma cells, with IC50 values ranging between 3 and 50 µM. These ER-α inhibitory compounds were further studied by molecular and cell biological techniques. RESULTS: The compounds strongly inhibited ER-α activity at concentrations that yielded little or no nonspecific toxicity, but they produced only a modest inhibition of progesterone receptor activity. Importantly, the compounds blocked proliferation and inhibited ER-α activity about equally well in antiestrogen-sensitive and antiestrogen-resistant breast cancer cells. Representative compounds disrupted the interaction of BRCA1 and ER-α in the cultured cells and blocked the interaction of ER-α with the estrogen response element. However, the compounds had no effect on the total cellular ER-α levels. CONCLUSIONS: These findings suggest that we have identified a new class of ER-α antagonists that work differently from conventional antiestrogens (eg, tamoxifen and fulvestrant).
Assuntos
Antagonistas de Estrogênios/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Tumoral , Humanos , Ligação Proteica , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Ressonância de Plasmônio de Superfície , Tamoxifeno/farmacologiaRESUMO
The breast cancer susceptibility genes BRCA1 and BRCA2 are classic tumor suppressor genes that exhibit an autosomal dominant pattern of inheritance with high penetrance. BRCA carriers inherit one mutant BRCA allele and one wild-type allele; and the wild-type allele is invariably deleted or mutated within the tumor. These genes function as caretakers in the maintenance of genomic stability, in part, by participating in homology-directed DNA repair (HDR), an error- free mechanism for the repair of double-strand breaks (DSBs). PARP1 (poly (ADP-ribose) polymerase 1) is an enzyme that functions in the base excision repair (BER) pathway, where its ability to post-translationally modify histones and DNA damage response proteins is required for repair of single-strand breaks (SSBs). In 2005, it was observed that knockdown of PARP1 or treatment with a small molecule PARP inhibitor was far more toxic to cells with BRCA1 or BRCA2 mutations than BRCA1/2-competent cells. This observation is an example of "synthetic lethality", a concept whereby two gene mutations combine to cause cell death, when neither mutation alone is lethal. These results spawned the idea to use PARP inhibitors to treat BRCA1/2 mutant cancers. Here, we will review the basic science underlying the discoveries described above, the preclinical research, and the clinical trials designed to exploit the sensitivity of BRCA1/2 mutant tumor cells to PARP inhibitors. We will also describe problems associated with the use of these agents, including development and mechanisms of drug resistance; and we will provide a forward look at new agents and strategies currently under development.
Assuntos
Marcação de Genes , Genes BRCA1 , Genes BRCA2 , Genes Supressores de Tumor , Pareamento Incorreto de Bases , Reparo de Erro de Pareamento de DNA , HumanosRESUMO
Radioprotectors are compounds that protect against radiation injury when given prior to radiation exposure. Mitigators can protect against radiation injury when given after exposure but before symptoms appear. Radioprotectors and mitigators can potentially improve the outcomes of radiotherapy for cancer treatment by allowing higher doses of radiation and/or reduced damage to normal tissues. Such compounds can also potentially counteract the effects of accidental exposure to radiation or deliberate exposure (e.g., nuclear reactor meltdown, dirty bomb, or nuclear bomb explosion); hence they are called radiation countermeasures. Here, we will review the general principles of radiation injury and protection and describe selected examples of radioprotectors/mitigators ranging from small-molecules to proteins to cell-based treatments. We will emphasize agents that are in more advanced stages of development.
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DIM (3,3'-diindolylmethane), a small molecule compound, is a proposed cancer preventive agent that can be safely administered to humans in repeated doses. We report that administration of DIM in a multidose schedule protected rodents against lethal doses of total body irradiation up to 13 Gy, whether DIM dosing was initiated before or up to 24 h after radiation. Physiologic submicromolar concentrations of DIM protected cultured cells against radiation by a unique mechanism: DIM caused rapid activation of ataxia-telangiectasia mutated (ATM), a nuclear kinase that regulates responses to DNA damage (DDR) and oxidative stress. Subsequently, multiple ATM substrates were phosphorylated, suggesting that DIM induces an ATM-dependent DDR-like response, and DIM enhanced radiation-induced ATM signaling and NF-κB activation. DIM also caused activation of ATM in rodent tissues. Activation of ATM by DIM may be due, in part, to inhibition of protein phosphatase 2A, an upstream regulator of ATM. In contrast, DIM did not protect human breast cancer xenograft tumors against radiation under the conditions tested. In tumors, ATM was constitutively phosphorylated and was not further stimulated by radiation and/or DIM. Our findings suggest that DIM is a potent radioprotector and mitigator that functions by stimulating an ATM-driven DDR-like response and NF-κB survival signaling.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Ativação Enzimática/efeitos dos fármacos , Indóis/farmacologia , Lesões Experimentais por Radiação/prevenção & controle , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular , Ensaio Cometa , Feminino , Proteínas de Fluorescência Verde , Imunoprecipitação , Indóis/uso terapêutico , Estimativa de Kaplan-Meier , Luciferases , Camundongos , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 2/metabolismo , RNA Interferente Pequeno/genética , Lesões Experimentais por Radiação/tratamento farmacológico , Radiação Ionizante , Ratos , Ratos Sprague-DawleyRESUMO
Mutations of the breast and ovarian cancer susceptibility gene 1 (BRCA1) account for about 40-45% of hereditary breast cancer cases. Moreover, a significant fraction of sporadic (non-hereditary) breast and ovarian cancers exhibit reduced or absent expression of the BRCA1 protein, suggesting an additional role for BRCA1 in sporadic cancers. BRCA1 follows the classic pattern of a highly penetrant Knudsen-type tumor suppressor gene in which one allele is inactivated through a germ-line mutation and the other is mutated or deleted within the tumor. BRCA1 is a multi-functional protein but it is not fully understood which function(s) is (are) most important for tumor suppression, nor is it clear why BRCA1-mutations confer a high risk for breast and ovarian cancers and not a broad spectrum of tumor types. Here, we will review BRCA1 functions in the DNA damage response (DDR), which are likely to contribute to tumor suppression. In the process, we will highlight some of the controversies and unresolved issues in the field. We will also describe a recently identified and under-investigated role for BRCA1 in the regulation of telomeres and the implications of this role in the DDR and cancer suppression.
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Vascular endothelial growth factor (VEGF) is a main angiogenic factor which is known to be upregulated in lung cancer. In the present study, it was demonstrated that sanguinarine, an alkaloid obtained from the bloodroot plant, markedly repressed the VEGF-induced tube formation of human microvascular endothelial cells (HMVECs) and the migration of human A549 lung cancer cells. Furthermore, sanguinarine decreased VEGF secretion and expression in HMVECs and A549 lung cancer cells in a dose- and time-dependent manner. Additionally, sanguinarine inhibited the activation of serum starvation- and hypoxia-induced VEGF promoter activity. Sanguinarine also inhibited the VEGF-mediated Akt and p38 activation, as well as VE-cadherin protein phosphorylation. To the best of our knowledge, this is the first study demonstrating that VEGF inhibition appears to be an important mechanism involved in the antiangiogenic and anti-invasive activities of sanguinarine in lung cancer treatment.
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Sanguinarine, a natural benzophenanthridine alkaloid, has been shown to possess anticancer activity in vitro and in vivo. In the present study, we demonstrated that sanguinarine caused a dose-dependent inhibition of growth in HeLa and SiHa human cervical cancer cells, i.e., 2.43 µmol/l (IC50) in HeLa cells and 3.07 µmol/l in SiHa cells. Cell cycle analysis revealed that sanguinarine significantly increased the sub-G1 population, from 1.7 to 59.7% in HeLa cells and from 1.7 to 41.7% in SiHa cells. Sanguinarine caused a dose-dependent decrease in Bcl-2 and NF-κB protein expression and a significant increase in Bax protein expression. Our findings indicate that sanguinarine as an effective anticancer drug candidate inhibits the growth of cervical cancer cells through the induction of apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Benzofenantridinas/farmacologia , Isoquinolinas/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/metabolismo , Anti-Infecciosos/farmacologia , Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Células HeLa , Humanos , NF-kappa B/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Neoplasias do Colo do Útero/patologiaRESUMO
BACKGROUND: Indole-3-carbinol (I3C) is a naturally occurring phytochemical found in cruciferous vegetables. The aim of the present study is to investigate the influence of I3C on radiosensitivity in epidermal growth factor receptor (EGFR)-positive and EGFR-negative lung cancer cell lines. METHODS: Human lung adenocarcinoma NIH-H1975 cells and human lung squamous carcinoma NIH-H226 and NIH-H520 cells were routinely cultured in RPMI-1640. MTT assay and clonogenic assay were used to detect cell growth and survival, respectively. Western blot and RT-PRC assay was employed to detect EGFR protein and mRNA expression. RESULTS: 5 µmol/L of I3C significantly reduced radiosensitivity of EGFR-positive NIH-H1975 and NIH-H226 cells, but failed to affect radiosensitivity of EGFR-negative NIH-H520 cells. Furthermore, I3C caused an increased expression of total EGFR and pEGFR (Y845) protein in NIH-H1975 and NIH-H226 cell lines, but not in NIH-H520 cell line. A reduction of EGFR expression by EGFR-siRNA significantly inhibited I3C-caused radioresistance in NIH-H1975 cells. CONCLUSIONS: Our data presented here for the first time demonstrate that I3C reduces radiosensitivity of lung cancer cells by mediating EGFR expression, indicating that EGFR may be an important target for I3C-mediated radioresistance in lung cancer.
Assuntos
Anticarcinógenos/farmacologia , Receptores ErbB/metabolismo , Indóis/farmacologia , Neoplasias Pulmonares/patologia , Tolerância a Radiação/efeitos dos fármacos , Brassicaceae/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , HumanosRESUMO
DNA damage induces multiple checkpoint pathways to arrest cell cycle progression until damage is repaired. In our previous reports, when DNA damage occurred in prometaphase, cells were accumulated in 4 N-DNA G1 phase, and mitosis-specific kinases were inactivated in dependent on ATM/Chk1 after a short incubation for repair. We investigated whether or not mitotic DNA damage causes cells to skip-over late mitotic periods under prolonged incubation in a time-lapse study. 4 N-DNA-damaged cells re-replicated without cell division and accumulated in 8 N-DNA content, and the activities of apoptotic factors were increased. The inhibition of DNA replication reduced the 8 N-DNA cell population dramatically. Induction of replication without cell division was not observed upon depletion of Chk1 or ATM. Finally, mitotic DNA damage induces mitotic slippage and that cells enter G1 phase with 4 N-DNA content and then DNA replication is occurred to 8 N-DNA content before completion of mitosis in the ATM/Chk1-dependent manner, followed by caspase-dependent apoptosis during long-term repair.
Assuntos
Dano ao DNA , Replicação do DNA/genética , Mitose/genética , Neoplasias/genética , Neoplasias/patologia , Apoptose/genética , Divisão Celular , Linhagem Celular Tumoral , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Células HeLa , HumanosRESUMO
Follistatin (FST), a folliculogenesis regulating protein, is found in relatively high concentrations in female ovarian tissues. FST acts as an antagonist to Activin, which is often elevated in human ovarian carcinoma, and thus may serve as a potential target for therapeutic intervention against ovarian cancer. The breast cancer susceptibility gene 1 (BRCA1) is a known tumor suppressor gene in human breast cancer; however its role in ovarian cancer is not well understood. We performed microarray analysis on human ovarian carcinoma cell line SKOV3 that stably overexpress wild-type BRCA1 and compared with the corresponding empty vector-transfected clones. We found that stable expression of BRCA1 not only stimulates FST secretion but also simultaneously inhibits Activin expression. To determine the physiological importance of this phenomenon, we further investigated the effect of cellular BRCA1 on the FST secretion in immortalized ovarian surface epithelial (IOSE) cells derived from either normal human ovaries or ovaries of an ovarian cancer patient carrying a mutation in BRCA1 gene. Knock-down of BRCA1 in normal IOSE cells demonstrates down-regulation of FST secretion along with the simultaneous up-regulation of Activin expression. Furthermore, knock-down of FST in IOSE cell lines as well as SKOV3 cell line showed significantly reduced cell proliferation and decreased cell migration when compared with the respective controls. Thus, these findings suggest a novel function for BRCA1 as a regulator of FST expression and function in human ovarian cells.
Assuntos
Proteína BRCA1/metabolismo , Folistatina/metabolismo , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/metabolismo , Ativinas/genética , Ativinas/metabolismo , Proteína BRCA1/genética , Western Blotting , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Movimento Celular/genética , Células Cultivadas , Análise por Conglomerados , Feminino , Folistatina/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Epiteliais e Glandulares/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Our previous studies have shown that high-mobility group box 1 (HMGB1) could physically associate with the retinoblastoma (RB) protein via an LXCXE (leucine-X-cysteine-X-glutamic; X=any amino acid) motif. An identical LXCXE motif is present in the HMGB1-3 protein sequences, whereas a near-consensus LXCXD (leucine-X-cysteine-X-asparagine; X=any amino acid) motif is found in the HMGB4 protein. In this study, we have demonstrated that like HMGB1, HMGB2-3 also associated with the RB in vitro and in vivo, as evidenced by glutathione-s-transferase capture and immunoprecipitation-Western blot assays. A point mutation of the LXCXE or LXCXD motif led to disruption of RB:HMGB1-4 interactions. Enforced expression of HMGB1-3 or HMGB4 by adenoviral-vector-mediated gene transfer resulted in significant inhibition of breast cancer cell proliferation through an LXCXE- or LXCXD-dependent mechanism and an increased radiosensitivity through an LXCXE- or LXCXD-independent mechanism. These results suggest an important role of the LXCXE/D motif in RB:HMGB1-4 association and modulation of cancer cell growth, but not radiosensitivity.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteína do Retinoblastoma/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/radioterapia , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Dados de Sequência Molecular , Ligação Proteica , Tolerância a Radiação , Retinoblastoma/genética , Retinoblastoma/metabolismo , Retinoblastoma/patologia , Proteína do Retinoblastoma/genética , TransfecçãoRESUMO
The B-cell translocation gene 2, BTG2, a member of the BTG/TOB (B-cell translocation gene/transducers of ErbB2) gene family, has been implicated in cell cycle regulation, normal development, and possibly tumor suppression. Previously, it was shown that BTG2 expression is lost or down-regulated in human breast cancers. We now report that BTG2 protects human mammary epithelial cells from oxidative stress due to hydrogen peroxide and other oxidants. BTG2 protection against oxidative stress is BRCA1-independent but requires the antioxidant transcription factor NFE2L2 and is associated with up-regulation of the expression of antioxidant enzymes, including catalase and superoxide dismutases 1 and 2. BTG2 stimulation of antioxidant gene expression is also NFE2L2-dependent. We further demonstrate that BTG2 is a binding partner for NFE2L2 and increases its transcriptional activity. In addition, BTG2 is detectable at the antioxidant response element (ARE) of several NFE2L2-responsive genes. Finally, we show that the ability of BTG2 to associate with NFE2L2, to protect cells against oxidative stress, and to stimulate antioxidant gene expression requires box B, a short highly conserved amino acid motif characteristic of BTG2/TOB family proteins, but does not require boxes A or C. These findings suggest a novel role for BTG2 as a co-activator for NFE2L2 in up-regulating cellular antioxidant defenses.
Assuntos
Antioxidantes/metabolismo , Células Epiteliais/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Glândulas Mamárias Humanas/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Motivos de Aminoácidos , Linhagem Celular Tumoral , Células Epiteliais/citologia , Feminino , Humanos , Peróxido de Hidrogênio/farmacologia , Proteínas Imediatamente Precoces/genética , Glândulas Mamárias Humanas/citologia , Fator 2 Relacionado a NF-E2/genética , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Elementos de Resposta/fisiologia , Proteínas Supressoras de Tumor/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
PURPOSE: To investigate the effect of ubiquitin-like with plant homeodomain (PHD) and ring finger domains 1 (UHRF1) overexpression on radiosensitivity to X-rays in human breast cancer MDA-MB-231 cells. MATERIALS AND METHODS: Cell survival was determined by colony formation assay; cell cycle distribution was measured by flow cytometry; apoptosis was evaluated by DNA fragmentation assay and Annexin V apoptosis detection kit; protein expression was analysed by Western blot assay; chromosome aberrations (centric rings and dicentrics) were assayed by conventional chromosome analysis. RESULTS: A significant decrease of radiosensitivity to X-rays was observed in MDA-MB-231 cells transfected with a full-length of human UHRF1 cDNA (MDA-MB-231/UHRF1) compared to the control cells (MDA-MB-231/parental and MDA-MB-231/pcDNA3 [mammalian expression vector]), and the similar results were observed in MDA-MB-468 cells. In contrast, a decreased expression of UHRF1 by a specific UHRF1-small interfering RNA (siRNA) significantly enhanced cell radiosensitivity. The UHRF1-mediated radioresistance was correlated with a G2(Ra)/M arrest, a decreased induction of apoptosis, a down-regulation of the pro-apoptotic protein anti-B cell lymphoma/leukemia 2 (bcl-2) associated X protein (Bax) and a up-regulation of the DNA damage repair proteins Lupus Ku autoantigen protein p70 (Ku-70) and Lupus Ku autoantigen protein p80 (Ku-80). Furthermore, chromosomal aberrations (centric rings and dicentrics) by X-rays were less in MDA-MB-231/UHRF1 than in MDA-MB-231/parental and MDA-MB-231/pcDNA3 control cells. CONCLUSIONS: These results suggested that UHRF1 may be a new target in the radiotherapy of breast cancer via affecting apoptosis and DNA damage repair.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/radioterapia , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Resistencia a Medicamentos Antineoplásicos , Proteínas Nucleares/metabolismo , Animais , Anexina A5/farmacologia , Antígenos Nucleares/farmacologia , Apoptose , Linhagem Celular Tumoral , Fragmentação do DNA , Proteínas de Ligação a DNA/farmacologia , Citometria de Fluxo/métodos , Humanos , Autoantígeno Ku , Camundongos , Tolerância a Radiação , Ubiquitina-Proteína Ligases , Proteína X Associada a bcl-2/metabolismoRESUMO
The tumor suppressor gene, BTG2 has been down-regulated in prostate cancer and the ectopic expression of this gene has been shown to inhibit prostate cancer cell growth. Sequence analysis revealed that the BTG2 protein contains two leucine-rich motifs ((20)LxxLL(24) and (92)LxxLL(96)), which are usually found in nuclear receptor co-factors. Based on this, we postulated that there will be an association between BTG2 and AR. In this study, we discovered that BTG2 directly bound to the androgen receptor (AR) in the absence of 5α-dihydrotestosterone (DHT), and in the presence of the androgen, this interaction was increased. BTG2 bearing the mutant (20)LxxLL(24) motif bound to AR equally efficient as the wild-type BTG2, while BTG2 bearing the mutant (92)LxxLL(96) motif failed to interact with AR. Functional studies indicated that ectopic expression of BTG2 caused a significant inhibition of AR-mediated transcriptional activity and a decreased growth of prostate cancer cells. Androgen-induced promoter activation and expression of prostate-specific antigen (PSA) are significantly attenuated by BTG2. The intact (92)LxxLL(96) motif is required for these activities. These findings, for the first time, demonstrate that BTG2 complexes with AR via an LxxLL-dependent mechanism and may play a role in prostate cancer via modulating the AR signaling pathway.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Imediatamente Precoces/metabolismo , Zíper de Leucina , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , 5-alfa-Di-Hidroprogesterona/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Proteínas Imediatamente Precoces/genética , Masculino , Mutação , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/genética , Proteínas Repressoras/genética , Transcrição Gênica , Proteínas Supressoras de Tumor/genéticaRESUMO
Inactivation of the breast cancer susceptibility gene BRCA1 plays a significant role in the development of a subset of breast cancers, although the major tumor suppressor function of this gene remains unclear. Previously, we showed that BRCA1 induces antioxidant-response gene expression and protects cells against oxidative stress. We now report that BRCA1 stimulates the base excision repair pathway, a major mechanism for the repair of oxidized DNA, by stimulating the activity of key base excision repair (BER) enzymes, including 8-oxoguanine DNA glycosylase (OGG1), the DNA glycosylase NTH1, and the apurinic endonuclease redox factor 1/apurinic endonuclease 1 (REF1/APE1), in human breast carcinoma cells. The increase in BER enzyme activity appears to be due, primarily, to an increase in enzyme expression. The ability of BRCA1 to stimulate the expression of the three BER enzymes and to enhance NTH1 promoter activity was dependent upon the octamer-binding transcription factor OCT1. Finally, we found that OGG1, NTH1, and REF1/APE1 each contribute to the BRCA1 protection against oxidative stress due to hydrogen peroxide and that hydrogen peroxide stimulates the expression of BRCA1 and the three BER enzymes. These findings identify a novel mechanism through which BRCA1 may regulate the repair of oxidative DNA damage.
Assuntos
Proteína BRCA1/fisiologia , Reparo do DNA , Regulação Neoplásica da Expressão Gênica , Transcrição Gênica , Animais , Linhagem Celular Tumoral , Dano ao DNA , DNA Glicosilases/metabolismo , Fibroblastos/metabolismo , Humanos , Peróxido de Hidrogênio/química , Camundongos , Estresse Oxidativo , RNA Interferente Pequeno/metabolismo , Fatores de TranscriçãoRESUMO
Inherited mutations of the breast cancer susceptibility gene BRCA1 confer a high risk for breast cancer development. The (300)RXKK and (266)KXK motifs have been identified previously as sites for acetylation of the estrogen receptor-alpha (ER-alpha), and (302)K was also found to be a site for BRCA1-mediated mono-ubiquitination of ER-alpha in vitro. Here we show that ER-alpha proteins with single or double lysine mutations of these motifs (including K303R, a cancer-associated mutant) are resistant to inhibition by BRCA1, even though the mutant ER-alpha proteins retain the ability to bind to BRCA1. We also found that BRCA1 overexpression reduced and knockdown increased the level of acetylated wild-type ER-alpha, without changing the total ER-alpha protein level. Increased acetylation of ER-alpha due to BRCA1 small interfering RNA was dependent upon phosphatidylinositol 3-kinase/Akt signaling and on up-regulation of the coactivator p300. In addition, using an in vitro acetylation assay, we found that in vitro-translated wild-type BRCA1 but not a cancer-associated point mutant (C61G) inhibited p300-mediated acetylation of ER-alpha. Furthermore, BRCA1 overexpression increased the levels of mono-ubiquitinated ER-alpha protein, and a BRCA1 mutant that is defective for ubiquitin ligase activity but retains other BRCA1 functions (I26A) did not ubiquitinate ER-alpha or repress its activity in vivo. Finally, ER-alpha proteins with mutations of the (300)RXKK or (266)KXK motifs showed modest or no BRCA1-induced ubiquitination. We propose a model in which BRCA1 represses ER-alpha activity, in part, by regulating the relative degree of acetylation vs. ubiquitination of ER-alpha.
Assuntos
Proteína BRCA1/fisiologia , Receptor alfa de Estrogênio/metabolismo , Genes BRCA1 , Processamento de Proteína Pós-Traducional , Acetilação , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Neoplasias da Mama , Linhagem Celular Tumoral , Estradiol/farmacologia , Feminino , Técnicas de Silenciamento de Genes , Genes Reporter , Humanos , Masculino , Mutação , Inibidores de Fosfoinositídeo-3 Quinase , Neoplasias da Próstata , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/genética , RNA Interferente Pequeno , Transfecção , Ubiquitinação , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Scatter factor (SF) and its receptor c-Met are overexpressed in various tumor types, and their expression often correlates with a poor prognosis. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), is a proposed tumor-specific chemotherapy agent, but its clinical usage is limited by acquisition of TRAIL resistance by tumors. The goals of this study were to determine whether and how SF protects tumor cells against TRAIL and whether SF-induced TRAIL resistance could be reversed. We used MTT assays, trypan blue dye exclusion assays, apoptosis assays, RNA interference, luciferase reporter assays, immunoprecipitation/western blotting, and other cell biological techniques to study SF protection of cultured human tumor cells against TRAIL. SF conferred resistance to TRAIL in various human prostate carcinoma and breast carcinoma cell lines. SF inhibited TRAIL-induced caspase-3 activation, poly (ADP-ribose) polymerase cleavage, and cell death. SF protection against TRAIL required c-Akt; but unlike protection against adriamycin, it did not require Src signaling or the classical pathway of nuclear factor-kappaB activation. Protection against TRAIL was blocked by knockdown of X-linked inhibitor of apoptosis or FLICE-inhibitor protein (FLIP) (a component of the death-inducing signaling complex). We found that c-Met physically associates with several TRAIL receptors and SF regulates their protein stability. Protection against TRAIL was blocked by a novel small molecule inhibitor of c-Met (PHA665752) and by an inhibitor of cyclooxygenase 2. In conclusion, these findings elucidate potential mechanisms of TRAIL resistance in tumors that overexpress the SF/c-Met and identify possible means of reversing this resistance.