RESUMO
We synthesized the novel tricyclic thiolactams 2a-d, 3d-k, having a benzyl or substituted benzyl substituent on the nitrogen of indole subunit, and their preferential cytotoxicity under both nutrient-deprived medium (NDM) and Dulbecco's modified Eagle's medium (DMEM) was evaluated against a human pancreatic cancer cell line PANC-1. Among the tested compounds, the 4'-hydroxy derivative 3d showed the most potent cytotoxicity in NDM (PC50 1.68µM) although the moderate preferential cytotoxicity (PC50 1.68µM in NDM vs PC50 20µM in DMEM). The 3'-hydroxy derivative 3e exhibited the most preferential cytotoxicity (PC50 1.96µM in NDM vs less than 50% inhibition at 30µM in DMEM). The benzyl 2a and halogenated benzyl derivatives 2b,c showed no cytotoxicity in NDM. In addition, the indole (10, PC50 173.7µM), lactone (11, PC50 131.7µM), and lactam (12, PC50 44.8µM) derivatives showed week or moderate cytotoxicity in NDM. These results indicated that the hydroxy group on the benzyl substituent and tricyclic thiolactam ring were essential for the cytotoxicity in NDM against PANC-1 cell line. Moreover, 3'-hydroxy derivative 3e compound exhibited antitumor activity against the pancreatic ductal adenocarcinoma (PDAC) xenograft model in vivo.
Assuntos
Antineoplásicos/farmacologia , Lactamas/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Compostos de Sulfidrila/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Lactamas/síntese química , Lactamas/química , Camundongos , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Neoplasias Pancreáticas/patologia , Relação Estrutura-Atividade , Compostos de Sulfidrila/síntese química , Compostos de Sulfidrila/químicaRESUMO
SNF-1/5'-AMP-activated kinase (AMPK)-related kinase (SNARK) is a member of the AMPK-related kinases. Snark(+/-) mice exhibited mature-onset obesity and related metabolic disorders. Obesity is regarded as a risk factor for colorectal cancer. To investigate whether Snark deficiency is involved in tumorigenesis in the large intestine, obese Snark(+/-) mice were treated with a chemical carcinogen, azoxymethane (AOM). The incidences of both adenomas and aberrant crypt foci (ACF) were significantly higher in Snark(+/-) mice than in their wild-type counterparts 28 weeks after the completion of AOM treatment (10 mg/kg/week for 8 weeks). Furthermore, ACF formation was enhanced in Snark(+/-) mice treated with AOM for 2 weeks, suggesting that Snark deficiency contributed to the early phase of tumorigenesis. The total number of ACF was correlated with bodyweight in Snark(+/-) and Snark(+/+) mice, suggesting that obesity was a risk factor for colorectal tumorigenesis in this model. However, the correlation coefficient was higher in Snark(+/-) mice. Moreover, AOM-induced ACF formation was also enhanced in preobese Snark(+/-) mice. Together, these findings suggest that AOM-induced tumorigenesis in Snark(+/-) mice was enhanced via obesity-dependent and -independent mechanisms.
Assuntos
Neoplasias Colorretais/genética , Predisposição Genética para Doença , Obesidade/genética , Lesões Pré-Cancerosas/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Azoximetano/toxicidade , Peso Corporal , Carcinógenos/toxicidade , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/patologia , Feminino , Trato Gastrointestinal/enzimologia , Trato Gastrointestinal/patologia , Camundongos , Camundongos Mutantes , Obesidade/complicações , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/patologia , Fatores de RiscoRESUMO
SNARK, a member of the AMPK-related kinases, has been involved in the cellular stress responses but its precise mechanisms remain unclear. Subcellular localization of SNARK protein was identified. Unlike cytoplasmic localizing AMPKalpha, SNARK was predominantly localized in the nucleus. SNARK was constitutively distributed in the nucleus even when SNARK was activated by metabolic stimuli such as AICAR and glucose-deprivation. Conserved nuclear localization signal (NLS) was identified at the N-terminal portion ((68)KKAR(71)). Deletion and point mutation of this part resulted in the cytoplasmic translocation of mutant proteins. Furthermore, GFP fused with the SNARK fragment containing (68)KKAR(71) translocated to the nucleus. A microarray analysis revealed that the nuclear localizing SNARK altered transcriptome profiles and a considerable part of these alterations were canceled by the mutation of NLS, suggesting the ability of SNARK to modulate gene expression dependent on its nuclear localization.
Assuntos
Núcleo Celular/enzimologia , Regulação da Expressão Gênica , Sinais de Localização Nuclear/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Ativo do Núcleo Celular , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Mutação , Sinais de Localização Nuclear/genética , Proteínas Serina-Treonina Quinases/genéticaRESUMO
AMP-activated protein kinases (AMPKs) are a class of serine/threonine protein kinases that are activated by an increase in intracellular AMP concentration. They are a sensitive indicator of cellular energy status and have been found to promote tumor cell survival during nutrient starvation. We recently identified a novel AMPK catalytic subunit family member, ARK5, whose activation is directly regulated by Akt, which, in turn, has been reported to be a key player in tumor malignancy. In this study, we attempted to determine whether ARK5 is involved in tumor malignancy under regulation by Akt. Matrigel invasion assays demonstrated that both overexpressed and endogenous ARK5 showed strong activity dependent on Akt. In addition, ARK5 expression induced activation of matrix metalloproteinase 2 (MMP-2) and MMP-9 following new expression of membrane type 1 MMP (MT1-MMP), and the MT1-MMP expression induced by ARK5 was initiated by rapamycin-sensitive signaling. In nude mice, ARK5 expression was associated with a significant increase in tumor growth and significant suppression of necrosis in tumor tissue. Interestingly, only the ARK5-overexpressing PANC-1 cell line (P/ARK) tumor showed invasion and metastasis in nude mice, although Akt was activated in tumors derived from both P/ARK and its parental cell line. We report that a novel AMPK catalytic subunit family member, ARK5, plays a key role in tumor malignancy downstream of Akt.
Assuntos
Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular Tumoral , Ativação Enzimática , Humanos , Metaloproteinase 14 da Matriz , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinases da Matriz Associadas à Membrana , Metaloendopeptidases/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Proteínas Quinases/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Interferência de RNA , Proteínas RepressorasRESUMO
ARK5, AMP-activated protein kinase (AMPK)-related protein kinase mediating Akt signals, is closely involved in tumor progression, and its stage-associated expression was observed in colorectal cancer. In this study, we found ARK5 expression in multiple myeloma cell lines expressing c-MAF and MAFB. In addition, gene expression profiling of 351 clinical specimens revealed ARK5 expression in primary myelomas expressing c-MAF and MAFB, suggesting that ARK5 may be a transcriptional target of the Large-MAF family. Sequence analysis of the ARK5 gene promoter revealed that it contains two putative MAF-recognition element (MARE) sequences. In support of this hypothesis, ARK5 was induced when an MAFB or c-MAF expression vector was introduced into non-ARK5-expressing colon cancer cells. Furthermore, ARK5 promoter activity was dramatically decreased by mutation or deletion of MARE sequences. Chromatin immunoprecipitation assays revealed an interaction between the Large-MAF family proteins and MARE sequences in the ARK5 promoter. Moreover, in ARK5 mRNA-expressing multiple myeloma lines, but not in ARK5-negative lines, insulin-like growth factor (IGF)-1 increased invasion activity. IGF-1-induced invasion was reproduced when ARK5 was overexpressed in Burkitt's lymphoma and plasmacytoma lines. Based on results, we conclude that ARK5 is a transcriptional target of the Large-MAF family through MARE sequence and that ARK5 may in part mediate the aggressive phenotype associated with c-MAF- and MAFB-expressing myelomas.
Assuntos
Regulação Neoplásica da Expressão Gênica/fisiologia , Fatores de Transcrição Maf Maior/fisiologia , Mieloma Múltiplo/genética , Proteínas Quinases/genética , Proteínas Repressoras/genética , Transcrição Gênica/fisiologia , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Humanos , Mieloma Múltiplo/patologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
An assay using a specific peptide (SAMS peptide) as a substrate is widely used for determination of AMP-activated protein kinases (AMPK) activity. However, it is not an efficient assay for crude AMPK preparations. In this study, we modified the assay by using the SAMS peptide fused to glutathione-S-transferase (GST-SAMS) instead of the SAMS peptide on its own. Radioactivity incorporated into GST-SAMS can be recovered easily by precipitation with glutathione-agarose. The kinetic parameters of partially purified AMPK for the GST-SAMS were as follows. The Vmax was 0.26 +/- 0.012 nmol/min/mg of total proteins and Km for GST-SAMS was 110 +/- 12 microM. The parameters for ATP were 0.40 +/- 0.016 nmol/min/mg of total proteins (Vmax) and 202 +/- 21 microM (Km). The activity of AMPK in this system was stimulated about threefold by the AMPK activators, AMP or 5-amino-4-imidazolecarboxamide ribotide (ZMP), and inhibited by the AMPK inhibitors, adenine 9-beta-D-arabinofuranoside (ara-A) and iodotubercidin. These values correlate well with those for the SAMS peptide reported previously. Thus, we successfully established a convenient and rapid method to measure AMPK applicable, even for crude enzyme preparations.
Assuntos
Glutationa Transferase/metabolismo , Complexos Multienzimáticos/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Monofosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Catálise/efeitos dos fármacos , Linhagem Celular Tumoral , Escherichia coli/genética , Glutationa Transferase/genética , Humanos , Cinética , Fragmentos de Peptídeos/genética , Fosforilação/efeitos dos fármacos , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleotídeos/farmacologia , Especificidade por Substrato , Tubercidina/análogos & derivados , Tubercidina/farmacologia , Vidarabina/farmacologiaRESUMO
The aim of this study was to evaluate the cytotoxicity of cubic boron nitride (cBN), a component of surgical cutting tools. The small quantities of cBN that typically remain on implants as a result of the manufacturing process may act as abrasives, injuring tissues surrounding the implant. To determine how cBN affects cells, we treated human neuroblastoma cells (NB-1) and human articular chondrocytes (nHAC-kn) with different concentrations of cBN powder and assessed cell growth and cell survival using the methyl-thiazol-tetrazolium (MTT) assay and a fluorescence probe assay. We also assessed the effects of tungsten carbide (WC) and cobalt (Co), two common components of joint implants, on cell growth and cell survival. Both cBN and WC moderately inhibited NB-1 and nHAC-kn cell growth. However, cBN and WC did not affect cell survival, even at high concentrations (40 microg/ml). By contrast, Co affected cell survival, inducing cell death in both cell types at increasing concentrations. These results suggest that cBN may be less toxic than WC alloys containing Co.
Assuntos
Materiais Biocompatíveis/toxicidade , Compostos de Boro/toxicidade , Células 3T3 , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/efeitos dos fármacos , Cobalto/toxicidade , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Varredura , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Tamanho da Partícula , PósRESUMO
AMPK is a serine/threonine protein kinase family and we recently identified a novel member, ARK5. The activation of ARK5 is triggered by Akt, and ARK5 induces tumor cell survival during nutrient starvation. In the current study, we investigated the mechanisms of induction of cell survival by ARK5. Human hepatoma HepG2 cells undergo necrotic cell death within 24 h after the start of glucose starvation, and the cell death signaling has been found to be mediated by death-receptor-independent activation of caspase 8. When HepG2 cells were transfected with ARK5 expression vector and subjected to several cell death stimuli, ARK5 was found to suppress cell death by glucose starvation, TRAIL, and TNF-alpha, but not by ultraviolet irradiation, camptothecin, or doxorubicin. Western blotting analysis revealed that both TRAIL and glucose starvation induced Bid cleavage and FLIP degradation following caspase 8 activation in a time-dependent manner, and ARK5 overexpression clearly delayed Bid cleavage, FLIP degradation, and caspase 8 activation. On the basis of the results of this study, we report that cell survival induced by ARK5 is, at least in part, due to inhibition of caspase 8 activation.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Inibidores de Caspase , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Quinases/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Raios Ultravioleta , Proteínas Reguladoras de Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Carcinoma Hepatocelular/patologia , Proteínas de Transporte/metabolismo , Sobrevivência Celular , Ativação Enzimática , Glucose/metabolismo , Humanos , Ligantes , Neoplasias Hepáticas/patologia , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Necrose , Proteínas Quinases/genética , Proteínas Repressoras , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismoRESUMO
As tumors grow and invade beyond their homeostatic limits, the tumor cells are subjected to insufficient nutrient and oxygen supplies because of excessive demand for nutrition and oxygen, and insufficient vascularization. We therefore hypothesized that tolerance to nutrient deprivation as well as angiogenesis may be critical in some malignancies, including pancreatic cancers, which are seen to be a hypovascular tumor. In this study, we assessed the effect of AMP-activated protein kinase (AMPK), which plays a major role in protecting cells from metabolic stresses, on tumor biology under nutrient-deprived condition. Whereas hepatic cancer cells had mostly died within 48 h during glucose deprivation, most pancreatic cancer cells survived more than 48 h. The tolerance to glucose deprivation tended to correlate with the cells level of expression of AMPK alpha1 and alpha2. The introduction of AMPK antisense RNA expression vectors into pancreas cancer cell lines, PANC-1 and AsPC-1, significantly diminished their tolerance to glucose deprivation, and the stable transfection of AMPK antisense into PANC-1 cells inhibited tumor growth in nude mice. These findings indicate that AMPK expression contributes to tolerance to nutrient starvation in cancer cells. We propose AMPK as a new target for therapeutic strategies to suppress tumor growth and invasion.
Assuntos
Complexos Multienzimáticos/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Células Tumorais Cultivadas/enzimologia , Células Tumorais Cultivadas/patologia , Proteínas Quinases Ativadas por AMP , Animais , Antineoplásicos/farmacologia , Northern Blotting , Western Blotting , Hipóxia Celular , Estruturas do Núcleo Celular , Sobrevivência Celular/fisiologia , Ensaio de Unidades Formadoras de Colônias , Meios de Cultura , Ativação Enzimática , Glucose/deficiência , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , RNA Antissenso/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Colorectal cancer cells are unique in that they escape Fas-mediated cell death in the presence of Fas ligand, and we recently reported that AMP-activated protein kinase-related kinase 5 (ARK5) suppresses cell death signaling mediated by cell death receptor in Akt-dependent manner. In the current study, therefore, we examined whether ARK5 is involved in the escape from Fas-mediated cell death of colorectal cancer cells. Among 10 cell lines, ARK5 mRNA expression was observed in LoVo, SW480, and SW1116 cell lines. Interestingly, SW480 and SW1116 cell lines, but not LoVo cell line, showed expressions of both Fas ligand (FasL) and Fas mRNAs. SW620 cell line also showed FasL mRNA; however, Fas and ARK5 mRNAs were not detected. Furthermore, well-coincided expression among ARK5, FasL, and Fas mRNAs was observed in tumor tissues from patients with colorectal cancer, suggesting the suppression of FasL/Fas system-induced cell death by ARK5 in colorectal cancer cell lines. Intensive cell death, which was dependent on the FasL/Fas system was encountered when ARK5 antisense RNA (ARK5/AS) was introduced into SW480 cells. FLIP was expressed in only ARK5 mRNA-expressing cell lines, and ARK5/AS induced FLIP cleavage in a caspase-6-dependent manner. Amino-acid sequence analysis of caspase-6 revealed two putative sites of phosphorylation by ARK5 at Ser80 and Ser257. Although active caspase-6 overexpression induced cell death in SW480 and DLD-1 cell lines, SW480 cells, but not DLD-1 cells, exhibited strong resistance to procaspase-6 overexpression. Moreover, mutant caspase-6, in which the Ser257 was substituted by Ala (caspase-6/SA), induced cell death and FLIP degradation, even in SW480 cells. Active ARK5 was found to phosphorylate wild-type caspase-6 in vitro, but not caspase-6/SA, and the prevented activation of caspase-6 was promoted due to its phosphorylation by active ARK5 in vitro. On the basis of the results of this study, we propose that ARK5 negatively regulates procaspase-6 by phosphorylation at Ser257, leading to resistance to the FasL/Fas system.
Assuntos
Adenilato Quinase/metabolismo , Caspases/genética , Regulação Neoplásica da Expressão Gênica/genética , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Caspase 6 , Inibidores de Caspase , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Dados de Sequência Molecular , Fosforilação , Interferência de RNA , RNA Mensageiro/genética , Proteínas Repressoras , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/efeitos dos fármacosRESUMO
Recently, we have shown that PFKFB4 gene which encodes the testis isoenzyme of PFKFB is also expressed in the prostate and hepatoma cancer cell lines. Here we have studied expression and hypoxic regulation of the testis isoenzyme of PFKFB4 in several malignant cell lines from a female organ--the mammary gland. Our studies clearly demonstrated that PFKFB4 mRNA is also expressed in mammary gland malignant cells (MCF-7 and T47D cell lines) in normoxic conditions and that hypoxia strongly induces it expression. To better understand the mechanism of hypoxic regulation of PFKFB4 gene expression, we used dimethyloxalylglycine, a specific inhibitor of HIF-1alpha hydroxylase enzymes, which strongly increases HIF-1alpha levels and mimics the effect of hypoxia. It was observed that PFKFB4 expression in the MCF7 and T47D cell lines was highly responsive to dimethyloxalylglycine, suggesting that the hypoxia responsiveness of PFKFB4 gene in these cell lines is regulated by HIF-1 proteins. Moreover, desferrioxamine and cobalt chloride, which mimic the effect of hypoxia by chelating or substituting for iron, had a similar stimulatory effect on the expression of PFKFB mRNA. In other mammary gland malignant cell lines (BT549, MDA-MB-468, and SKBR-3) hypoxia and hypoxia mimics also induced PFKFB4 mRNA, but to variable degrees. The hypoxic induction of PFKFB4 mRNA was equivalent to the expression of PFKFB3, Glut1, and VEGF, which are known HIF-1-dependent genes. Hypoxia and dimethyloxalylglycine increased the PFKFB4 protein levels in all cell lines studied except MDA-MB-468. Through site-specific mutagenesis in the 5'-flanking region of PFKFB4 gene the hypoxia response could be limited. Thus, this study provides evidence that PFKFB4 gene is also expressed in mammary gland cancer cells and strongly responds to hypoxia via an HIF-1alpha dependent mechanism. Moreover, the PFKFB4 and PFKFB3 gene expression in mammary gland cancer cells has also a significant role in the Warburg effect which is found in all malignant cells.
Assuntos
Hipóxia Celular/fisiologia , Fosfofrutoquinase-2/genética , Aminoácidos Dicarboxílicos/farmacologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Luciferases/genética , Dados de Sequência Molecular , RNA Mensageiro/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
The PFKFB4 gene encodes isoenzyme of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB or PFK-2/FBPase-2) which originally was found in the testes. We have studied hypoxic regulation of PFKFB4 gene in prostate cancer cell line, PC-3, and several other cancer cell lines. It was shown that hypoxia significantly induced PFKFB4 mRNA levels in PC-3 as well as in HeLa, Hep3B and HepG2 cell lines. Hypoxia increased PFKFB4 protein levels also. Moreover, desferrioxamine and cobalt chloride, which are known to mimic hypoxia, also had a stimulatory effect on the expression of PFKFB4 mRNA. In order to investigate the mechanisms of hypoxic regulation of PFKFB4 gene expression, we used dimethyloxalylglycine, which has the ability to mimic effect of hypoxia by significant induction of hypoxia-inducible factor (HIF-1alpha) protein levels. Our studies showed that PFKFB4 mRNA expression in PC-3, HeLa, Hep3B and HepG2 cell lines was highly responsive to dimethyloxalylglycine, an inhibitor of HIF-1alpha hydroxylase enzymes, suggesting that the hypoxia responsiveness of this gene is regulated by HIF proteins. To better understand the hypoxic regulation of PFKFB4 gene expression, we isolated genomic DNA, which includes the promoter region of PFKFB4. Cell transfection, deletion and site-specific mutagenesis of the PFKFB4 promoter region indicates that hypoxic induction of PFKFB4 gene expression is mediated by the hypoxia-responsive element (HRE). These experiments identified a HRE 422-429 bp upstream from the translation start site. Thus, our results indicate that testis-specific form of PFKFB or PFK-2/FBPase-2 is also expressed in several cancer cell lines and that hypoxia induces transcription of PFKFB4 gene in these cell lines by HIF-1alpha dependent mechanism. HRE in 5'-promoter region of PFKFB4 gene mediates hypoxic induction of PFKFB4 gene transcription.
Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Fosfofrutoquinase-2/biossíntese , Fosfofrutoquinase-2/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Carcinoma Hepatocelular/enzimologia , Hipóxia Celular , Linhagem Celular Tumoral , Cobalto/farmacologia , Desferroxamina/farmacologia , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glicina/análogos & derivados , Glicina/farmacologia , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Isoenzimas/biossíntese , Isoenzimas/genética , Masculino , Neoplasias da Próstata/enzimologia , RNA Mensageiro/biossíntese , Transcrição GênicaRESUMO
We previously reported that loss of heterozygosity (LOH) in tuberous sclerosis 1 (TSC1)-gene-associated region on chromosome 9q34, was frequently observed in adenocarcinoma (AC) of the lung and its putative precursor lesion, atypical adenomatous hyperplasia (AAH). Some novel tumor suppressor gene for AC of the lung may be present in this region, and the TSC1 gene located on chromosome 9q34 is one candidate. The aim of this study was to determine whether the TSC1 gene itself acts as a tumor suppressor gene in the pathogenesis of AC of the lung. DNA extracted from frozen tumor tissue was used to screen 47 ACs of the lung for the presence of mutations in 21 coding exons of the TSC1 gene. When using these bulk tissue specimens, three different types of mutations were detected in five ACs. These positive samples were then submitted to laser capture microdissection to selectively collect tumor cells, DNA extraction and finally analysis of LOH and mutations of the TSC1 gene. Mutations were detected in three ACs, and LOH was detected in another AC. No mutations or LOH was detected in the other AC. Loss of heterozygosity and mutations were not detected simultaneously in the same tumor. We concluded that the TSC1 gene itself does not act as a tumor suppressor gene in the pathogenesis of AC of the lung in accordance with classical Knudsen's two-hit hypothesis. However, the frequent LOH in AC and AAH shown in our previous studies indicate the following possibility in the etiology of AC of the lung: the presence of a novel tumor suppressor gene close to the TSC1 gene, aberrant promoter methylation, or haploid insufficiency of the TSC1 gene.
Assuntos
Adenocarcinoma/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Proteínas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Metilação de DNA , Análise Mutacional de DNA , DNA de Neoplasias , Feminino , Genes Supressores de Tumor , Humanos , Perda de Heterozigosidade , Masculino , Pessoa de Meia-Idade , Proteína 1 do Complexo Esclerose Tuberosa , Proteínas Supressoras de TumorRESUMO
We investigated the involvement of the inducible nitric oxide synthase (iNOS) gene in tumor promotion and progression. In the first study, 1 week after subcutaneous injection of benzo(a)pyrene into iNOS-deficient (iNOS) and wild-type (iNOS) mice, a foreign body (plastic plate) was subcutaneously inserted into the carcinogen injection site to evoke chronic inflammation. In the second study, primarily cultured tumor cells (PCTc) with different iNOS gene status were prepared from tumors induced in the first study, and they were implanted into the subcutaneous space of iNOS and iNOS mice, making four different combinations of iNOS gene status. Although the mice that were subjected to plastic plate-induced inflammation [p-IN(+)] exhibited significantly shorter tumor latency than those with p-IN(-), iNOS gene status did not affect it in the p-IN(-) or p-IN(+) groups. The rate of microscopic invasion and expression levels of matrix metalloproteinase mRNA were, however, higher in iNOSp-IN(+) than iNOSp-IN(+) mice. In the second study, microscopic invasion was also observed in the subcutaneously implanted tumors only in the case of PCTc with iNOS gene into iNOS mice, although iNOS gene status in PCTc or host mice did not affect the tumor growth curve. These data suggest that the iNOS gene was associated with tumor progression, rather than tumorigenesis, in this experimental model. The iNOS gene in both the stromal and cancer cells played an important role in invasion. Inhibition of iNOS gene activity might be useful for local cancer control in inflammation-associated cancers.
Assuntos
Neoplasias/genética , Neoplasias/patologia , Óxido Nítrico Sintase Tipo II/genética , Animais , Transformação Celular Neoplásica/genética , Progressão da Doença , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inflamação/genética , Masculino , Metaloproteinases da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Invasividade Neoplásica , Transplante de Neoplasias , Óxido Nítrico Sintase Tipo II/fisiologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Células Estromais/enzimologia , Células Estromais/patologia , Tela Subcutânea/patologiaRESUMO
Hypoxia inducible factor 1 (HIF-1) regulates the transcription of vascular endothelial growth factor (VEGF), which plays important roles in angiogenesis. We investigated the redox effect of glutathione (GSH) on the hypoxic induction of HIF-1 in a human oral squamous cell carcinoma (HSC-2) cell line. The maximal induction of HIF-1 in HSC-2 cells was observed 30 h after hypoxia, and VEGF mRNA was expressed after 36 h under hypoxia. GSH ethyl ester (GSHee, a membrane permeable analog of GSH) and N-acetyl-L-cysteine (NAC, a membrane permeable precursor of GSH) reduced HIF-1 binding activity in a dose-dependent manner. Further, HIF-1 dependent promoter activity was similarly reduced by GSHee and NAC. However, ebselen, which increases glutathione peroxidase activity and oxidizes GSH, negated the effect of GSHee on HIF-1 dependent promoter activity. The inhibitory effect of GSHee and NAC on HIF-1 binding activity was reversed by bis (2-chlorethyl)-nitrosourea, an oxidized glutathione (GSSG) reductase inhibitor which increases the concentration of GSSG. GSSG methyl ester (GSSGme), a membrane permeable analog of GSSG, enhanced HIF-1 dependent promoter activity and exhibited a bell-shaped concentration-dependant activity curve. The increasing effect of GSSGme on HIF-1 induction was also observed under chemically-induced hypoxia obtained using cobalt chloride. These results suggest that changes in the intracellular GSSG/GSH ratio may regulate HIF-1 induction during hypoxia.
Assuntos
Glutationa/metabolismo , Glutationa/farmacologia , Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/metabolismo , Ativação Transcricional/efeitos dos fármacos , Acetilcisteína/farmacologia , Animais , Azóis/farmacologia , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Glutationa/análogos & derivados , Dissulfeto de Glutationa/farmacologia , Glutationa Redutase/antagonistas & inibidores , Glutationa Redutase/metabolismo , Humanos , Isoindóis , Compostos Organosselênicos/farmacologia , Oxirredução , RNA Mensageiro/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
ARK5 is a tumor progression-associated factor that is directly phosphorylated by AKT at serine 600 in the regulatory domain, but phosphorylation at the conserved threonine residue on the active T loop has been found to be required for its full activation. In this study, we identified serine/threonine protein kinase NDR2 as a protein kinase that phosphorylates and activates ARK5 during insulin-like growth factor (IGF)-1 signaling. Upon stimulation with IGF-1, NDR2 was found to directly phosphorylate the conserved threonine 211 on the active T loop of ARK5 and to promote cell survival and invasion of colorectal cancer cell lines through ARK5. During IGF-1 signaling, phosphorylation at three residues (threonine 75, serine 282, and threonine 442) was also found to be required for NDR2 activation. Among these three residues, phosphorylation of serine 282 seemed to be the most important for NDR2 activation (the same as for the mouse homologue) because its aspartic acid-converted mutant (NDR2/S282D) induced ARK5-mediated cell survival and invasion activities even in the absence of IGF-1. As in the mouse homologue, threonine 75 in NDR2 was required for interaction with S100B, and binding was in a calcium ion- and phospholipase C-gamma-dependent manner. We also found that PDK-1 plays an important role in NDR2 activation especially in the phosphorylation of threonine 442. Based on the results of this study, we report here that NDR2 is an upstream kinase of ARK5 that plays an essential role in tumor progression through ARK5.
Assuntos
Neoplasias Colorretais/patologia , Fator de Crescimento Insulin-Like I/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Ácido Aspártico/química , Cálcio/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Progressão da Doença , Ativação Enzimática , Humanos , Dados de Sequência Molecular , Fosfolipase C gama/metabolismo , Estrutura Terciária de ProteínaRESUMO
The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) is responsible for maintaining the cellular levels of fructose-2,6-bisphosphate which is a key regulator of glycolysis. Here we have studied the expression of PFKFB-4 isozyme in the DB-1 melanoma cells. An additional isoform of PFKFB-4 mRNA with 148 bases insert in the amino-terminal region at high constitutive levels was identified in these cells. The expression of this splice isoform as well as main isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was responsible to hypoxia and dimethyloxalylglycine, an inhibitor of HIF-1 alpha hydroxylase enzymes, suggesting that the hypoxia responsiveness of PFKFB-4 gene in these cells is regulated by HIF-1alpha protein. Hypoxic induction of PFKFB4 mRNA in the DB-1 melanoma cells correlates with the expression of PFKFB-3 and VEGF mRNA which are known as HIF-1 dependent genes. Thus, our results clearly demonstrated the existence of splice isoform of PFKFB-4 mRNA in the DB-1 melanoma cells and its overexpression under hypoxic conditions.
Assuntos
Processamento Alternativo/genética , Hipóxia Celular/genética , Regulação Enzimológica da Expressão Gênica , Fosfofrutoquinase-2/genética , Resinas Acrílicas , Bioensaio , Western Blotting , Linhagem Celular Tumoral , Frutosedifosfatos/metabolismo , Humanos , Isoenzimas , Neoplasias Pancreáticas , RNA Mensageiro/genética , Ribonucleases/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Because survival and growth of human hepatoma cells are maintained by nutrient, especially glucose, glucose starvation induces acute cell death. The cell death is markedly suppressed by hypoxia, and we have reported involvement of AMP-activated protein kinase-alpha (AMPK-alpha), Akt, and ARK5 in hypoxia-induced tolerance. In the current study we investigated the mechanism of hypoxia-induced tolerance in human hepatoma cell line HepG2. ARK5 expression was induced in HepG2 cells when they were subjected to glucose starvation, and we found that glucose starvation transiently induced Akt and AMPK-alpha phosphorylation and that hypoxia prolonged phosphorylation of both protein kinases. We also found that hypoxia-induced tolerance was partially abrogated by blocking the Akt/ARK5 system or by suppressing AMPK-alpha expression and that suppression of both completely abolished the tolerance, suggesting that AMPK-alpha activation signaling and the Akt/ARK5 system play independent essential roles in hypoxia-induced tolerance. By using chemical compounds that specifically inhibit kinase activity of type I-transforming growth factor-beta (TGF-beta) receptor, we showed an involvement of TGF-beta in hypoxia-induced tolerance. TGF-beta1 mRNA expression was induced by hypoxia in an hypoxia-inducible factor-1alpha-independent manner, and addition of recombinant TGF-beta suppressed cell death during glucose starvation even under normoxic condition. AMPK-alpha, Akt, and ARK5 were activated by TGF-beta1, and Akt and AMPK-alpha phosphorylation, which was prolonged by hypoxia, was suppressed by an inhibitor of type I TGF-beta receptor. Based on these findings, we propose that hypoxia-induced tumor cell tolerance to glucose starvation is caused by hypoxia-induced TGF-beta1 through AMPK-alpha activation and the Akt/ARK5 system.
Assuntos
Glucose/metabolismo , Hipóxia/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Proteínas Quinases Ativadas por AMP , Proteínas de Ligação a DNA/metabolismo , Glucose/deficiência , Humanos , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Complexos Multienzimáticos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1RESUMO
BACKGROUND: Sodium chloride (NaCl) is a strong promoter of gastric cancer. We hypothesized that inducible nitric oxide synthase (iNOS) induced by NaCl may be involved in its promoting effects. We investigated iNOS expression by hypertonic NaCl solutions and its pathophysiological roles in the gastric mucosa of rats and mice. METHODS: iNOS mRNA and protein expressions were examined in the rat and mouse gastric mucosa after intragastric administration of NaCl solution by northern blot, reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. The effect on luminal pH by iNOS activity was assessed using aminoguanidine, a potent iNOS inhibitor, and iNOS gene deficient (iNOS-/-) mice. RESULTS: iNOS expression was detected at concentrations higher than 1.7 M, mainly in the cells infiltrating the damaged mucosa of rats. Administration of a higher than 3.4 M NaCl solution elevated luminal pH of the rat stomach remarkably, enabling bacterial overgrowth and dramatically increasing iNOS expression (n = 5 per concentration). Pretreatment with ampicillin (p.o), an antibiotic, attenuated the iNOS expression in duplicate experiments. Co-treatment with aminoguanidine (s.q) accelerated recovery of elevated luminal pH at 8 h and 16 h or 24 h after administration of 3.4 M (n = 8) and 5.2 M NaCl solution (n = 5), respectively. iNOS expression and luminal pH elevation were also observed in mice stomachs after administration of 3.4 M NaCl solution. The elevated luminal pH of iNOS-/- mice stomachs after the administration of NaCl solution was significantly lower at 6 h (n = 7) and at 9 h (n = 11), compared to that of wild type mice (n = 9 and 10, respectively). CONCLUSIONS: Hypertonic NaCl solutions induced iNOS expression in the gastric mucosa. iNOS activity prolonged the elevation of the luminal pH, potentially leading to bacterial overgrowth, which in turn enhanced iNOS expression. This vicious cycle might be related to the promoting effect of NaCl.
Assuntos
Óxido Nítrico Sintase/metabolismo , Cloreto de Sódio/farmacologia , Estômago/efeitos dos fármacos , Estômago/enzimologia , Animais , Bactérias/crescimento & desenvolvimento , Determinação da Acidez Gástrica , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Masculino , Camundongos , Óxido Nítrico Sintase/biossíntese , RNA Mensageiro/análise , Ratos , Ratos Wistar , Soluções , Estômago/microbiologia , Fatores de TempoRESUMO
SNARK, the fourth member of the AMPK catalytic subunit family, was originally identified in a rat kidney cDNA library, and in this study we isolated its human homologue. A BLAST search analysis using rat SNARK protein yielded a single high homology clone, DKFZp434J037, isolated from human testis, and since its hypothetical protein showed 84% homology to rat SNARK protein, we assumed DKFZp434J037 to be the human SNARK cDNA. The human SNARK cDNA is 3443bp long and encodes a 628 amino acid protein having an estimated molecular weight of 69kDa, and its chromosomal localization had been assigned to 1q32.1. The same as other members of AMPK catalytic subunit family, human SNARK showed AMP-dependent GST-SAMS phosphorylation activity and enhanced HepG2 cell survival during glucose starvation. Human SNARK-overexpressing HepG2 cells (H/SNK) showed acute cell-cell detachment when exposed to glucose-free medium and the cell-cell detachment correlated well with the detection of G-actin. Deletion mutant analysis strongly suggested that the putative catalytic domain of SNARK is necessary for the cell-cell detachment, and Western blotting analysis showed that phosphorylation of FAK and PKC, which were dramatically increased by glucose starvation in HepG2 cells, was markedly suppressed by SNARK.