RESUMO
The prerigor salting effect is known to provide superior meat processing quality. Based on the urgent need for low salt meat products, the present study was undertaken to evaluate the prerigor salting effect when basic amino acids were introduced at 1% NaCl level. Ground chicken breast meat was salted with NaCl and basic amino acids at 30 min, 60 min, and 90 min postmortem for prerigor treatments. Compared to the 1% NaCl (w/w) treatment, the introduction of 0.06% basic amino acids (w/w) in the prerigor significantly led to an increase in myofibril fragmentation, myofibrillar protein solubility, emulsion activity, storage modulus change rate, gel water-holding capacity and hardness (P < 0.05). Furthermore, smaller and more uniformly sized droplets were produced in emulsion by basic amino acids. Individual basic amino acids had different prerigor salting effects, and it was indicated that basic amino acids could play a positive role in the prerigor salting effect when NaCl was reduced.
Assuntos
Galinhas , Cloreto de Sódio , Animais , Manipulação de Alimentos , Melhoria de Qualidade , EmulsõesRESUMO
The objective of this study was to evaluate the effects of the suspended carcass deboning technique on the meat attributes of electrically stimulated cattle ( = 10). A carcass deboning technique that removes the main bones without breakdown of the entire carcass was applied to suspended sides soon after slaughter. After 3 d of aging at 4°C, the shear force of the rectus femoris, longissimus lumborum, and supraspinatus muscles of the suspended carcass deboning technique was reduced by 27%, 29%, and 23% ( < 0.05), respectively. The carcass deboning technique increased the sarcomere length by 14%, 10%, and 16% ( < 0.05), and the myofibril fragmentation index was increased by 10%, 5%, and 13% ( < 0.05), respectively, for the same 3 muscles. There was no difference in the pH, color, or cooking loss between the treatment and the control ( > 0.05). The carcass deboning technique could be a viable approach to improve beef tenderness with a relatively short aging duration.
Assuntos
Bovinos/fisiologia , Manipulação de Alimentos/métodos , Carne Vermelha/normas , Envelhecimento , Animais , Osso e Ossos , Cor , Culinária , Concentração de Íons de Hidrogênio , Miofibrilas/ultraestrutura , Sarcômeros/ultraestrutura , Fatores de TempoRESUMO
BACKGROUND: The dynamics of focal adhesion (FA) turnover is a key determinant for the regulation of cancer cell migration. Here we investigated FA turnover in a panel of breast cancer models with distinct invasive properties and evaluated the impact of reversine on this turnover in relation to cancer cell invasion in in vitro and in vivo conditions. METHODS: Live imaging and immunofluorescence assays were used to investigate FA turnover in breast cancer cells. Biochemical studies were used to investigate the impact of reversine on FA signalling and turnover. In vivo activity was investigated using orthotopic breast cancer mouse models. RESULTS: Accelerated FA disassembly from plasma membrane protrusions was observed in invasive compared with non-invasive breast cancer cells or non-immortalised mammary epithelial cells. Reversine significantly inhibited FA disassembly leading to stable FAs, which was associated with reduced cell motility and invasion. The inhibitory effect of reversine on FA turnover accounted for a large part on its capacity to interfere with FAK function on regulating its downstream targets. In orthotopic breast cancer mouse models, reversine revealed a potent inhibitory activity on tumour progression to metastasis. CONCLUSION: These results support the utility of targeting FA turnover as a therapeutic approach for invasive breast cancer.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Carcinoma Ductal de Mama/tratamento farmacológico , Adesões Focais/efeitos dos fármacos , Morfolinas/uso terapêutico , Purinas/uso terapêutico , Animais , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Células Cultivadas , Feminino , Adesões Focais/metabolismo , Humanos , Células MCF-7 , Camundongos , Camundongos SCID , Terapia de Alvo Molecular , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Anthracyclines and taxanes have remarkable anticancer efficacy, but have poor selectivity and high toxicity. Targeted delivery of chemotherapeutics has emerged as a strategy to achieve higher drug levels at the tumor site, to spare noncancerous tissue and potentially to use lower systemic drug doses, thus preventing side effects. In this study, we targeted the HER2 receptor using the monoclonal antibody (mAb) Herceptin (Trastuzumab) chemically conjugated to Doxorubicin or Taxol. In vitro, drug-Herceptin conjugates exhibited cytotoxicity comparable to equimolar concentrations of free drugs, with the benefit that the cytotoxicity of the conjugates was selective for cells expressing the HER2 target. In vivo, treatment of tumor-bearing mice with Taxol-Herceptin conjugates had a reduction of primary tumors comparable to equivalent doses of free drugs. However, Taxol-Herceptin conjugates significantly reduced metastasis compared with equivalent doses of free drugs. Thus, the data support the concept that conjugates might target metastasis better than primary tumors. This would offer a potential therapeutic approach for management of metastatic breast cancer.
Assuntos
Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , Animais , Anticorpos Monoclonais Humanizados/imunologia , Antineoplásicos/química , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Doxorrubicina/química , Doxorrubicina/farmacologia , Feminino , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos SCID , Paclitaxel/química , Paclitaxel/farmacologia , Receptor ErbB-2/imunologia , Trastuzumab , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A crucial early event by which cancer cells switch from localised to invasive phenotype is initiated by the acquisition of autonomous motile properties; a process driven by dynamic assembly and disassembly of multiple focal adhesion (FA) proteins, which mediate cell-matrix attachments, extracellular matrix degradation, and serve as traction sites for cell motility. We have reported previously that cancer cell invasion induced by overexpression of members of the ErbB tyrosine kinase receptors, including ErbB2, is dependent on FA signalling through FA kinase (FAK). Here, we show that ErbB2 receptor signalling regulates FA turnover, and cell migration and invasion through the Src-FAK pathway. Inhibition of the Src-FAK signalling in ErbB2-positive cells by Herceptin or RNA interference selectively regulates FA turnover, leading to enhanced number and size of peripherally localised adhesions and inhibition of cell invasion. Inhibition of ErbB2 signalling failed to regulate FA and cell migration and invasion in cells lacking FAK or Src but gains this activity after restoration of these proteins. Taken together, our results show a regulation of FA turnover by ErbB2 signalling.
Assuntos
Neoplasias da Mama/metabolismo , Adesões Focais/metabolismo , Regulação da Expressão Gênica , Invasividade Neoplásica , Receptor ErbB-2/metabolismo , Transdução de Sinais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/genética , Humanos , Receptor ErbB-2/genéticaRESUMO
The amplified region of chromosome 19q13.1-13.2 has been associated with several cancers. The well-characterized oncogene AKT2 is located in this amplicon. Two members of the same gene family (SERTAD1 and SERTAD3) are also located within this region. We report herein the genomic structure and potential functions of SERTAD3. SERTAD3 has two transcript variants with short mRNA half-lives, and one of the variants is tightly regulated throughout G1 and S phases of the cell cycle. Overexpression of SERTAD3 induces cell transformation in vitro and tumor formation in mice, whereas inhibition of SERTAD3 by small interfering RNA (siRNA) results in a reduction in cell growth rate. Furthermore, luciferase assays based on E2F-1 binding indicate that SERTAD3 increases the activity of E2F, which is reduced by inhibition of SERTAD3 by siRNA. Together, our data support that SERTAD3 contributes to oncogenesis, at least in part, via an E2F-dependent mechanism.
Assuntos
Fatores de Transcrição E2F/biossíntese , Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Oncogênicas/biossíntese , Proteínas Oncogênicas/genética , Transativadores/biossíntese , Transativadores/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Linhagem Celular Tumoral , Cromossomos Humanos Par 19/genética , Fatores de Transcrição E2F/metabolismo , Amplificação de Genes , Humanos , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Proteínas Oncogênicas/fisiologia , Homologia de Sequência de Aminoácidos , Transativadores/fisiologiaRESUMO
Protein kinases, including tyrosine kinases, are one of the largest classes of proteins implicated in cancer development and progression. Recent discovery of selective therapies targeting tyrosine kinase receptor signaling has provided encouraging clinical results. Clinical trials with anti-EGFR, anti-ErbB2/Her2, anti-Bcr-Abl and others have demonstrated the clinical utility of tyrosine kinases as therapeutic targets and as surrogate markers to guide the selection of patients susceptible to respond to treatment. This success has been tempered in part because resistance to targeted therapies is now documented to occur in experimental models and in patients, which hampers therapeutic efficacy. Mechanisms of resistance include cell heterogeneity in target expression, mutations in target's encoding genes, and compensatory signaling mechanisms. This paper provides a brief overview on the diversity of tyrosine kinase signaling and the impact on cancer cell response to targeted tyrosine kinase inhibitors.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/fisiologia , Transdução de Sinais , Animais , Humanos , Receptor ErbB-2/antagonistas & inibidoresRESUMO
The effect of imatinib on chlorambucil (CLB) cytotoxicity in chronic lymphocytic leukemia (CLL) lymphocytes was examined in vitro. Imatinib sensitizes the WSU and I83 human CLL cell lines, 10- and two-fold, respectively, to CLB. Furthermore, in primary cultures of malignant B-lymphocytes obtained from 12 patients with CLL (seven patients were untreated and five treated with CLB), imatinib synergistically sensitized these lymphocytes from two- to 20-fold to CLB. This synergistic effect was observed at concentrations of imatinib (=10 microM), which are achievable in patients with minimal toxicity. Moreover, the combination of both drugs results in increased apoptosis in CLL cell lines. These results suggest that imatinib should be useful in improving the therapeutic index of CLB in CLL. The mechanism of action appears to involve imatinib inhibition of c-abl kinase activity with an associated decrease in CLB-induced Rad51 phosphorylation and CLB-induced Rad51 nuclear foci, suggesting that imatinib decreases Rad51-related DNA repair of CLB-induced DNA lesions. Altogether, our results suggest that imatinib is a promising adjuvant therapy to CLB treatment of CLL.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Clorambucila/farmacologia , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Piperazinas/farmacologia , Pirimidinas/farmacologia , Apoptose/efeitos dos fármacos , Benzamidas , Ciclo Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Sinergismo Farmacológico , Humanos , Mesilato de Imatinib , Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Linfocítica Crônica de Células B/patologia , Fosforilação , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-abl/metabolismo , Rad51 Recombinase , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologiaRESUMO
The enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) expressed by the parasite Trypanosoma brucei (Tb) can convert allopurinol, a purine analogue, to corresponding nucleotides with greater efficiency than its human homologue. We have developed a retroviral system that expresses the parasitic enzyme and tested its capacity to activate the prodrug allopurinol to a cytotoxic metabolite. Cytotoxicity assays demonstrated that five non-small cell lung carcinoma cell lines transduced with the construct were sensitized to the prodrug by 2.1- to 7.6-fold compared with control values. This selectivity was not observed in seven other cell lines also expressing the construct, such as breast carcinoma. Assays indicated that enhanced cytotoxicity to allopurinol correlated with induction of apoptosis in lung cancer cells. The selectivity of this suicide gene was not explained either by the TbHGPRT expression or by the allopurinol accumulation. Our study shows that this novel system may represent a therapeutic tool for gene prodrug targeting of lung cancer, considering the fact that allopurinol is well tolerated in humans.
Assuntos
Genes Letais/genética , Terapia Genética/métodos , Hipoxantina Fosforribosiltransferase/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genética , Alopurinol/metabolismo , Alopurinol/toxicidade , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Genes de Protozoários/genética , Humanos , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/uso terapêutico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Microscopia de Fluorescência , Especificidade de Órgãos , Pró-Fármacos/metabolismo , Fatores de Tempo , Transdução Genética , Células Tumorais Cultivadas , Vírus da Estomatite Vesicular Indiana/genéticaRESUMO
Prostate cancer is the most diagnosed invasive malignancy in males. Androgens and oestrogens have been implicated in the pathogenesis of prostate cancer. We report herein that the pure anti-oestrogen ICI 182,780 (ICI) reduces Ki-67 labelling index and IGF-I receptor levels in rat prostate. Increase of IGF-I mRNA and IGF-binding protein 3 (IGFBP-3) accumulation occur without any effect on prostate weight. Finasteride significantly decreases prostate weight and inhibits IGF-I gene expression. IGFBP-3 mRNA, Akt and phospho-Akt are not affected by finasteride. Co-administration of ICI plus finasteride reduces prostate weight by approximately 50% and causes acinar dilation with decreased luminal epithelial cell thickness. The acinar epithelial cells became atrophic and inactive with minimal cytoplasm. We also demonstrate a synergistic effect of ICI and finasteride on induction of IGFBP-3 accumulation and inhibition of Akt phosphorylation. Because the IGF and IGFBP-3 system plays an important role in prostate epithelial cell proliferation, apoptosis and tumour progression, the inhibitory effects of finasteride and ICI on IGF system may contribute to their anti-proliferative activity. These observations support a potential use of ICI in conjunction with finasteride in the prevention and/or treatment of prostate cancer.
Assuntos
Inibidores de 5-alfa Redutase , Estradiol/análogos & derivados , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Finasterida/farmacologia , Fator de Crescimento Insulin-Like I/metabolismo , Próstata/metabolismo , Proteínas Serina-Treonina Quinases , Animais , Northern Blotting/métodos , Divisão Celular/efeitos dos fármacos , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Fulvestranto , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Masculino , Tamanho do Órgão/efeitos dos fármacos , Fosforilação , Próstata/anatomia & histologia , Próstata/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Receptor IGF Tipo 1/metabolismo , Estatísticas não ParamétricasRESUMO
Steroid-sensitive gene-1 (SSG1) is a novel gene we cloned, found regulated by 17beta-estradiol in the rat uterus and mammary gland, and over-expressed in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors. We show here that SSG1 mRNA and protein expression are regulated by androgens in the rat ventral prostate. Increases in SSG1 mRNA levels were detected by Northern blotting after 24 h and reached a 27-fold peak 96 h following castration, relative to SSG1 mRNA expression in sham-operated rats. Dihydrotestosterone or testosterone supplementation of castrated rats prevented this rise in SSG1 mRNA. In contrast with SSG1 mRNA expression, SSG1 protein was decreased 16-fold 2 weeks following castration but was at control levels in the prostates of castrated rats receiving dihydrotestosterone or testosterone. Although SSG1 is regulated by androgens in vivo, treatment of LnCap cells with dihydrotestosterone, cyproterone acetate or flutamide did not result in the regulation of SSG1 protein levels in vitro. Immunofluorescence studies show that SSG1 is mainly expressed in prostatic smooth muscle cells. These results indicate that SSG1 is an androgen-regulated gene that is expressed in the smooth muscle component of the rat ventral prostate in vivo.
Assuntos
Androgênios/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteínas de Neoplasias/genética , Próstata/metabolismo , Sequência de Aminoácidos , Antagonistas de Androgênios/farmacologia , Androgênios/administração & dosagem , Animais , Northern Blotting , Finasterida/farmacologia , Imunofluorescência , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Dados de Sequência Molecular , Orquiectomia , Próstata/citologia , Ratos , Ratos Sprague-Dawley , Células Tumorais Cultivadas , Proteínas Supressoras de TumorRESUMO
BACKGROUND: 1alpha,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and its analogues inhibit growth of various types of cancer cells. Although the therapeutic potential of 1,25(OH)(2)D(3) is limited by its tendency to induce hypercalcemia, analogues such as EB1089 are potent inhibitors of cell growth and exhibit reduced calcemic effects. We analyzed the antiproliferative and calcemic effects of EB1089 in tissue culture and animal models of head and neck squamous cell carcinoma (SCC) to investigate its potential as a chemotherapeutic/chemopreventive agent. METHODS: The effects of 1,25(OH)(2)D(3) and EB1089 on cell growth and expression of p21(WAF1/CIP1) and p27(KIP1), which encode cyclin-dependent kinase inhibitors, and a novel target, gadd45alpha, a growth-arrest and DNA-damage gene, were monitored in cultured murine AT-84 SCC cells. The effects of these agents on AT-84 cell growth in vitro and on growth of AT-84 tumors in syngeneic C3H mice were monitored; treatment started at the time of tumor implantation (early tumor model) or after 12 days (late tumor model). Weight and serum calcium levels were also monitored in these animals. All P values were two-sided. RESULTS: Both 1,25(OH)(2)D(3) and EB1089 arrested proliferation of AT-84 cells in G(0)/G(1) phase, inhibited p21(WAF1/CIP1) expression, and induced expression of p27(KIP1) protein. 1,25(OH)(2)D(3) also enhanced the expression of gadd45alpha, apparently by a p53-independent mechanism. There was a statistically significant decrease in tumor growth for 1,25(OH)(2)D(3)-treated mice (P<.001 for early tumor model) and EB1089-treated mice (P<.001 and P =.001 for early and late tumor models, respectively). Unlike 1,25(OH)(2)D(3), EB1089 did not induce cachexia or hypercalcemia. The effects of 1,25(OH)(2)D(3) and EB1089 on expression of p21(WAF1/CIP1) and GADD45alpha were similar in tumors and in vitro. CONCLUSIONS: EB1089 completely inhibited growth of AT-84 SCC cells at nanomolar concentrations, reduced tumor growth, and did not have calcemic effects. Our results support continued investigation of EB1089 as a chemopreventive/chemotherapeutic agent for head and neck SCC.
Assuntos
Antineoplásicos/uso terapêutico , Calcitriol/análogos & derivados , Calcitriol/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Proteínas de Ciclo Celular , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Proteínas Supressoras de Tumor , Animais , Northern Blotting , Western Blotting , Carcinoma de Células Escamosas/prevenção & controle , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Ciclinas/biossíntese , Citoplasma/metabolismo , Dano ao DNA/genética , Genes p53/genética , Neoplasias de Cabeça e Pescoço/prevenção & controle , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Camundongos Endogâmicos C3H , Proteínas Associadas aos Microtúbulos/biossíntese , Transplante de Neoplasias , Testes de Precipitina , Proteínas/metabolismo , RNA/metabolismo , Fatores de Tempo , Células Tumorais Cultivadas , Proteínas GADD45RESUMO
Estrogens play a role in mammary gland function and are implicated in mammary carcinogenesis. We report the cloning of a novel gene [steroid-sensitive gene 1 (SSG1)] that is regulated by E(2) in the rat uterus and mammary gland. The full-length SSG1 complementary DNA has an open reading frame of 1158 nucleotides encoding a putative protein of 385 amino acids. A SSG1-specific antibody recognizes a 40-kDa protein localized to myoepithelial cells of normal mammary tissue and to endothelial cells of 7,12-dimethylbenz(a)antracene-induced mammary tumors. Treatment of rats with E(2) at 1.2 or 2.4 microg/kg.day for 21 days increases SSG1 protein levels in mammary tissue by 16-fold compared with controls. Removal of E(2) after a 14-day treatment decreases SSG1 protein levels 6-fold and 3-fold at 120 and 144 h, respectively. Treatment of rats with the estrogen antagonists tamoxifen or ICI 182,780 did not affect SSG1 protein levels compared with controls. SSG1 protein levels in 7,12-dimethylbenz(a)antracene-induced rat mammary tumors were 23-fold greater than SSG1 levels in resting mammary tissue, and 8-fold higher than protein levels expressed in lactating mammary glands. We propose that SSG1 plays a role in estrogen functions, and its overexpression is correlated with mammary carcinogenesis.
Assuntos
Clonagem Molecular , Estradiol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Neoplasias Mamárias Experimentais/genética , Proteínas de Neoplasias/genética , 9,10-Dimetil-1,2-benzantraceno , Sequência de Aminoácidos , Animais , Sequência de Bases , Carcinógenos , Endotélio Vascular/química , Feminino , Imunofluorescência , Glândulas Mamárias Animais/irrigação sanguínea , Glândulas Mamárias Animais/química , Glândulas Mamárias Animais/efeitos dos fármacos , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/induzido quimicamente , Dados de Sequência Molecular , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/química , Ovariectomia , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Proteínas Supressoras de TumorRESUMO
Eight minor taxanes have been identified for the first time in Taxus canadensis needles. Four of these metabolites are new taxane analogues: 7-acetyl-10-deacetyltaxol (1), 2'-acetyl-7-epi-cephalomannine (2), 10-deacetyl-10-oxo-7-epi-cephalomannine (3), and 10-acetylglycollylbaccatin VI (4).
Assuntos
Alcaloides/isolamento & purificação , Adenocarcinoma/patologia , Alcaloides/química , Alcaloides/farmacologia , Neoplasias da Mama/patologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Árvores , Células Tumorais CultivadasRESUMO
PURPOSE: Downregulation of gap junctional intercellular communication (GJIC) has been implicated in carcinogenesis. This is a result of altered expression of connexins, the proteins that mediate GJIC, including connexin 43 (Cx43). Our aim was to evaluate the effect of known inducers of Cx43 on the chemosensitivity of the human neuroblastoma cell line IMR-32 to chemotherapeutic agents. METHODS: We examined the effect of dibutyryl-cyclic AMP (db-cAMP) and all-trans-retinoic acid (tRA) on Cx43 and GJIC, glutathione (GSH) and gamma-glutamyl-cysteine-synthetase (gamma-GCS) levels, and glutathione S-transferase (GST) activity. Finally, we performed cell survival assays to measure the response of IMR-32 cells to the chemotherapeutic drugs doxorubicin, melphalan and bis-chloronitrosourea (BCNU), after treatment with db-cAMP and/or tRA. RESULTS: Exposure to db-cAMP led to the upregulation of GJIC and Cx43 expression and phosphorylation. On the other hand, exposure to tRA led to the upregulation of GJIC but Cx43 expression and phosphorylation were not greatly affected. The combination of both agents was more potent in inducing GJIC in comparison to treatment with db-cAMP or tRA alone. Treatment with db-cAMP, but not with tRA, was associated with a significant increase in the cytotoxic effects of the anticancer drugs doxorubicin, melphalan and BCNU as shown by a decrease in their IC50 values. Concomitant exposure to db-cAMP and tRA, however, had a more pronounced effect on cell sensitization to chemotherapy drugs (particularly doxorubicin) than exposure to db-cAMP or tRA alone. Under the db-cAMP and tRA treatment conditions (which upregulate GJIC and modulate drug response), GSH levels were significantly reduced while the levels of GST and gamma-GCS activities remained unchanged. CONCLUSIONS: This study suggests that GJIC plays a role in cellular drug resistance, and highlights the potential use of GJIC modulators in combination with chemotherapy. Also, this is the first study exploring the ability of both db-cAMP and tRA to enhance cell chemosensitivity.
Assuntos
Bucladesina/farmacologia , Comunicação Celular/efeitos dos fármacos , Conexina 43/biossíntese , Junções Comunicantes/efeitos dos fármacos , Glutationa/metabolismo , Neuroblastoma/tratamento farmacológico , Tretinoína/farmacologia , Glutamato-Cisteína Ligase/metabolismo , Humanos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Human hepatitis B virus is a risk factor for the development of hepatocellular carcinoma. The hepatitis B virus x protein (HBx) has been shown to inactivate the p53 tumor suppressor protein and impair DNA repair, cell cycle, and apoptosis mechanisms. Herein we report that HBx represses two components of the transcription-repair factor TFIIH, XPB (p89), and XPD (p80), both in p53-proficient and p53-deficient liver cells. This inhibition is observed while HBx maintains its transactivation function. Expression of HBx in liver cells results in down-regulation of endogenous XPB and XPD mRNAs and proteins; this inhibition is not observed with other TFIIH subunits, XPA or PCNA. In liver tissue from HBx transgenics, XPB and XPD proteins are down-regulated in comparison to matched normal liver tissue. HBx has been shown to interact with Sp1 transcription factor and affects its DNA binding activity. Sp1 is essential for the basal promoter activity of XPB in liver cells and Drosophila SL2 cells. In the Sp1-deficient SL2 cells, HBx-induced XPB and XPD inhibition is Sp1-dependent. In summary, our results provide evidence that HBx represses the expression of key TFIIH proteins at least in part through Sp1 elements; this repression may impair TFIIH function in DNA repair mechanisms.
Assuntos
DNA Helicases , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Regulação Viral da Expressão Gênica , Fígado/metabolismo , Proteínas/metabolismo , Fatores Associados à Proteína de Ligação a TATA , Transativadores/genética , Transativadores/metabolismo , Fator de Transcrição TFIID , Fatores de Transcrição TFII , Fatores de Transcrição/genética , Transcrição Gênica , Animais , Animais Geneticamente Modificados , Apoptose , Western Blotting , Linhagem Celular , Cloranfenicol O-Acetiltransferase/metabolismo , Reparo do DNA/genética , Regulação para Baixo , Drosophila , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Modelos Genéticos , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição TFIIH , Ativação Transcricional , Transfecção , Células Tumorais Cultivadas , Proteínas Virais Reguladoras e Acessórias , Proteína Grupo D do Xeroderma PigmentosoRESUMO
Androgens play an important role in prostate gland development and function, and have been implicated in prostate carcinogenesis. We report the regulation of the gap junctional intercellular communication gene connexin 43 (Cx43) by androgens in the prostate gland. In rat ventral prostate tissue, only trace levels of Cx43 mRNA were detected. Castration, however, resulted in a high increase in Cx43 mRNA and protein. Cx32 was unchanged. Castration-induced Cx43 mRNA and protein were abolished by administration of dihydrotestosterone (DHT). Following castration, prostate weights were approximately 16% of sham-treated controls. However, DHT replacement resulted in prostate weights which were not different from sham-treated controls. Under similar castration conditions, Cx43 induction coincided with pronounced apoptosis in the prostate gland cells, and DHT prevented the induction of apoptosis. Given the physiological role of gap junctions and androgens in the regulation of prostate tissue homeostasis, our observations are relevant to the understanding of androgen-dependent prostate carcinogenesis.
Assuntos
Conexina 43/genética , Regulação da Expressão Gênica , Próstata/metabolismo , Animais , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Masculino , RNA Mensageiro/genética , Ratos , Ratos Sprague-DawleyRESUMO
Replication Protein A (RPA) is required for DNA recombination, repair and replication in all eukaryotes. RPA participation in these pathways is mediated by single-stranded DNA binding and protein interactions. We herein identify a novel protein, Replication Protein Binding Trans-Activator (RBT1), in a yeast two-hybrid assay employing the second subunit of human RPA (RPA32) as bait. RBT1-RPA32 binding was confirmed by glutathione S:-transferase pull-down and co-immunoprecipitation. Fluorescence microscopy indicates that green fluorescence protein-tagged RBT1 is localized to the nucleus in vivo. RBT1 mRNA expression, determined by semi-quantitative RT-PCR, is significantly higher in cancer cell lines MCF-7, ZR-75, SaOS-2 and H661, compared to the cell lines normal non-immortalized human mammary epithelial cells and normal non-immortalized human bronchial epithelial cells. Further, yeast and mammalian one-hybrid analysis shows that RBT1 is a strong transcriptional co-activator. Interestingly, mammalian transactivation data is indicative of significant variance between cell lines; the GAL4-RBT1 fusion protein has significantly higher transcriptional activity in human cancer cells compared to human normal primary non-immortalized epithelial cells. We propose that RBT1 is a novel transcriptional co-activator that interacts with RPA, and has significantly higher activity in transformed cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Transativadores/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Clonagem Molecular , DNA , Humanos , Dados de Sequência Molecular , Ligação Proteica , Proteína de Replicação A , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Técnicas do Sistema de Duplo-HíbridoRESUMO
Persistent infection by hepatitis B virus (HBV) and exposure to chemical carcinogens correlates with the prevalence of hepatocellular carcinoma in endemic areas. The precise nature of the interaction between these factors is not known. Glutathione S-transferases (GST) are responsible for the cellular metabolism and detoxification of a variety of cytotoxic and carcinogenic compounds by catalysis of their conjugation with glutathione. Diminished GST activity could enhance cellular sensitivity to chemical carcinogens. We have investigated GST isozyme expression in hepatocellular HepG2 cells and in an HBV-transfected subline. Total GST activity and selenium-independent glutathione peroxidase activity are significantly decreased in HBV transfected cells. On immunoblotting, HBV transfected cells demonstrate a significant decrease in the level of GST Alpha class. Cytotoxicity assays reveal that the HBV transfected cells are more sensitive to a wide range of compounds known to be detoxified by GST Alpha conjugation. Although no significant difference in protein half-life between the two cell lines was found, semi-quantitative reverse transcription-polymerase chain reaction shows a reduced amount of GST Alpha mRNA in the transfected cells. Because the HBV x protein (HBx) seems to play a role in HBV transfection, we also demonstrated that expression of the HBx gene into HepG2 cells decreased the amount of GST Alpha protein. Transient transfection experiments using both rat and human GST Alpha (rGSTA5 and hGSTA1) promoters in HepG2 cells show a decreased CAT activity upon HBx expression, supporting a transcriptional regulation of both genes by HBx. This effect is independent of HBx interaction with Sp1. Treatment with oltipraz, an inducer of GST Alpha, partially overcomes the effect of HBx on both promoters. Promoter deletion studies indicate that oltipraz works through responsive elements distinct from AP1 or NF-kappaB transcription factors. Thus, HBV infection alters phase II metabolizing enzymes via different mechanisms than those modulated by treatment with oltipraz.
Assuntos
Antivirais/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glutationa Transferase/genética , Vírus da Hepatite B/fisiologia , Isoenzimas/genética , Pirazinas/farmacologia , Hepatite B/complicações , Hepatite B/enzimologia , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/genética , Humanos , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Fator de Transcrição Sp1/fisiologia , Tionas , Tiofenos , Transativadores/genética , Transativadores/fisiologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/fisiologia , Proteínas Virais Reguladoras e AcessóriasRESUMO
The interaction between the erbB tyrosine kinase receptors and their ligands plays an important role in tumor growth via the regulation of autocrine and paracrine loops. We report the effect of heregulin beta1, the ligand for erbB-3 and erbB-4 receptors, on the regulation of vascular endothelial growth factor (VEGF) expression, using a panel of breast and lung cancer cell lines with constitutive erbB-2 overexpression or engineered to stably overexpress the erbB-2 receptor. We demonstrate that heregulin beta1 induces VEGF secretion in most cancer cell lines, while no significant effect was observed in normal human mammary and bronchial primary cells. Overexpression of erbB-2 receptor results in induction of the basal level of VEGF and exposure to heregulin further enhances VEGF secretion. This is associated with increased VEGF mRNA expression. In contrast, VEGF induction is significantly decreased in a T47D cell line where erbB-2 is functionally inactivated. Conditioned media from heregulin-treated cancer cells, but not from normal cells, stimulates endothelial cell proliferation; this paracrine stimulation is inhibited by co-exposure to a specific VEGF neutralizing antibody. Furthermore, heregulin-mediated angiogenesis is observed in the in vivo CAM assay. This study reports the first evidence of VEGF regulation by heregulin in cancer cells. Oncogene (2000) 19, 3460 - 3469