RESUMO
Given our previous finding that certain tumor-suppressing functions of p53 are exerted by the p53/p21 complex, rather than p53 alone, cells may have a system to regulate the p53/p21 interaction. As p53 binds to p21 via its C-terminal domain, which contains acetylable lysine residues, we investigated whether the C-terminal acetylation of p53 influences the p53/p21 interaction. Indeed, the p53/p21 interaction was reduced when various types of cells (HCT116 colon cancer, A549 lung cancer, and MCF7 breast cancer cells) were treated with MS-275, an inhibitor of SIRT1 (a p53 deacetylase), or with SIRT1-targeting small interfering RNAs. These treatments also increased the acetylation levels of the five lysine residues (K370, K372, K373, K381, K382) in the C-terminal domain of p53. The p53/p21 interaction was also reduced when these lysine residues were substituted with glutamine (an acetylation memetic), but not arginine (an unacetylable lysine analog). While the inhibitory effect of the lysine-to-glutamine substitution was evident upon the substitution of all the five lysine residues, the substitution of only two (K381, K382) or three residues (K370, K372, K373) was less effective. Consistently, the five substitutions reduced the ability of p53 to regulate cell invasion and death by liberating Bax from Bcl-w. Overall, our data suggest that the acetylation, especially the hyperacetylation, of the p53 C-terminal domain suppresses the p53/p21-complex-dependent functions of p53 by inhibiting the p53/p21 interaction. We propose that cellular components involved in the acetylation or deacetylation of the p53 C-terminus are critical regulators of the formation of p53/p21 complex.
Assuntos
Lisina , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor p53/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Lisina/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Glutamina/metabolismo , Linhagem Celular Tumoral , Regiões Promotoras Genéticas , AcetilaçãoRESUMO
The p53 tumor suppressor regulates cell functions either by acting as a transcription factor or by interacting with other proteins. Previously, we reported that the non-transcriptional actions of p53 can be facilitated by the binding of p53 to p21. Herein, we investigated whether p53/p21 interaction influences the transcriptional activity of p53. We observed that the expression of the p53 promoter-based reporter gene is dependent on p21 levels. Moreover, using a p21 variant that is unable to bind p53, we showed that p53 promoter activity requires p53/p21 interaction. To investigate the possible role of p21 in regulating the expression of endogenous p53 targets, we analyzed mRNA levels of Puma, Mdm2, and Gadd45a in untreated control and γ-ray-irradiated cells. We observed that while Puma expression is dependent on p53 regardless of γ-irradiation, p53 mediates the expression of Mdm2 and Gadd45a only in irradiated cells. Notably, p53/p21 interaction is required only for the p53-dependent expression of the tested genes and not Mdm2 and Gadd45a in non-irradiated cells. Moreover, chromatin immunoprecipitation assay revealed that p21 is required for the binding of p53 to the promoters of Puma, Mdm2, and Gadd45a. Collectively, our data support the view that the p53/p21 complex is involved in regulating p53-dependent gene expression. These findings provide a new foundation for understanding the transcriptional action of p53.
Assuntos
Proteínas Proto-Oncogênicas c-mdm2 , Proteína Supressora de Tumor p53 , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
The objective of this study was to determine whether (5S)-5-(4-benzyloxy-3,5-dimethoxy-phenyl)-5,9-dihydro-8H-furo [3',4':6,7] naphtho [2,3-d] [1,3]dioxol-6-one (JNC-1043), which is a novel chemical derivative of ß-apopicropodophyllin, acts as a novel potential anticancer reagent and radiosensitizer in colorectal cancer (CRC) cells. Firstly, we used MTT assays to assess whether JNC-1043 could inhibit the cell proliferation of HCT116 and DLD-1 cells. The IC50 values of these cell lines were calculated as 114.5 and 157 nM, respectively, at 72 h of treatment. Using doses approximating the IC50 values, we tested whether JNC-1043 had a radiosensitizing effect in the CRC cell lines. Clonogenic assays revealed that the dose-enhancement ratios (DER) of HCT116 and DLD-1 cells were 1.53 and 1.25, respectively. Cell-counting assays showed that the combination of JNC-1043 and γ-ionizing radiation (IR) enhanced cell death. Treatment with JNC-1043 or IR alone induced cell death by 50~60%, whereas the combination of JNC-1043 and IR increased this cell death by more than 20~30%. Annexin V-propidium iodide assays showed that the combination of JNC-1043 and IR increased apoptosis by more 30~40% compared to that induced by JNC-1043 or IR alone. DCFDA- and MitoSOX-based assays revealed that mitochondrial ROS production was enhanced by the combination of JNC-1043 and IR. Finally, we found that suppression of ROS by N-acetylcysteine (NAC) blocked the apoptotic cell death induced by the combination of JNC-1043 and IR. The xenograft model also indicated that the combination of JNC-1043 and IR increased apoptotic cell death in tumor mass. These results collectively suggest that JNC-1043 acts as a radiosensitizer and exerts anticancer effects against CRC cells by promoting apoptosis mediated by mitochondrial ROS.
Assuntos
Antineoplásicos , Neoplasias Colorretais , Radiossensibilizantes , Humanos , Podofilotoxina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Anexina A5 , Acetilcisteína/farmacologia , Propídio/farmacologia , Radiossensibilizantes/farmacologia , Apoptose , Antineoplásicos/farmacologia , Proliferação de Células , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Linhagem Celular TumoralRESUMO
Downregulation of the p53 tumor suppressor in cancers is frequently accompanied by the upregulation of Wip1 (a phosphatase) and Mdm2 (an E3 ubiquitin ligase). Mdm2 binds and ubiquitinates p53, promoting its degradation by the proteasome. As the p53/Mdm2 interaction is alleviated by the phosphorylation of the serine-15 (S15) residue of p53, Wip1, which can directly dephosphorylate phospho-S15, facilitates the Mdm2-mediated degradation of p53. Here, we found that p21WAF1/CIP1, previously shown to bind p53 and Mdm2, reduces the cellular levels of p53 protein by decreasing its stability. This is accompanied by a decrease in p53-S15 phosphorylation levels. In agreement, p21 promotes the p53/Wip1 interaction. Additionally, p21 interacts with Wip1, forming a trimeric complex of p53, p21, and Wip1. Studies using a p21 deletion mutant that cannot bind p53 revealed that the p53/p21 complex is more efficient than p53 alone in facilitating the binding of p53 to Wip1 and Mdm2. These findings indicate that p21 is a novel negative regulator of p53 stability and therefore, may be used as a target to restore p53 activity by preventing the action of Wip1 and Mdm2 on p53.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Neoplasias/metabolismo , Proteína Fosfatase 2C/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias/patologia , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Proteólise , Transdução de SinaisRESUMO
Here, we demonstrate that interleukin-1ß (IL-1ß) contributes to the γ-ionizing radiation (IR)-induced increase of migration/invasion in A549 lung cancer cells, and that this occurs via RIP1 upregulation. We initially observed that the protein expression and secreted concentration of IL-1ß were increased upon exposure of A549 cells to IR. We then demonstrated that IR-induced IL-1ß is located downstream of the NF-κB-RIP1 signaling pathway. Treatments with siRNA and specific pharmaceutical inhibitors of RIP1 and NF-κB suppressed the IR-induced increases in the protein expression and secreted concentration of IL-1ß. IL-1Ra, an antagonist of IL-1ß, treatment suppressed the IR-induced epithelial-mesenchymal transition (EMT) and IR-induced invasion/migration in vitro. These results suggest that IL-1ß could regulate IR-induced EMT. We also found that IR could induce the expression of IL-1ß expression in vivo and that of IL-1 receptor (R) I/II in vitro and in vivo. The IR-induced increases in the protein levels of IL-1 RI/II and IL-1ß suggest that an autocrine loop between IL-1ß and IL-1 RI/II might play important roles in IR-induced EMT and migration/invasion. Based on these collective results, we propose that IR concomitantly activates NF-κB and RIP1 to trigger the NF-κB-RIP1-IL-1ß-IL-1RI/II-EMT pathway, ultimately promoting metastasis.
Assuntos
Interleucina-1beta/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/radioterapia , NF-kappa B/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Células A549 , Animais , Movimento Celular/efeitos da radiação , Raios gama , Humanos , Interleucina-1beta/genética , Neoplasias Pulmonares/genética , Camundongos Endogâmicos BALB C , Invasividade Neoplásica/genética , Radiação Ionizante , Regulação para Cima/efeitos da radiaçãoRESUMO
ß-apopicropodophyllin (APP), a derivative of podophyllotoxin (PPT), has been identified as a potential anti-cancer drug. This study tested whether APP acts as an anti-cancer drug and can sensitize colorectal cancer (CRC) cells to radiation treatment. APP exerted an anti-cancer effect against the CRC cell lines HCT116, DLD-1, SW480, and COLO320DM, with IC50 values of 7.88 nM, 8.22 nM, 9.84 nM, and 7.757 nM, respectively, for the induction of DNA damage. Clonogenic and cell counting assays indicated that the combined treatment of APP and γ-ionizing radiation (IR) showed greater retardation of cell growth than either treatment alone, suggesting that APP sensitized CRC cells to IR. Annexin V-propidium iodide (PI) assays and immunoblot analysis showed that the combined treatment of APP and IR increased apoptosis in CRC cells compared with either APP or IR alone. Results obtained from the xenograft experiments also indicated that the combination of APP and IR enhanced apoptosis in the in vivo animal model. Apoptosis induction by the combined treatment of APP and IR resulted from reactive oxygen species (ROS). Inhibition of ROS by N-acetylcysteine (NAC) restored cell viability and decreased the induction of apoptosis by APP and IR in CRC cells. Taken together, these results indicate that a combined treatment of APP and IR might promote apoptosis by inducing ROS in CRC cells.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Podofilina/farmacologia , Radiossensibilizantes/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Abstract: Previously, we demonstrated that γ-ionizing radiation (IR) triggers the invasion/migration of A549 cells via activation of an EGFR-p38/ERK-STAT3/CREB-1-EMT pathway. Here, we have demonstrated the involvement of a novel intracellular signaling mechanism in γ-ionizing radiation (IR)-induced migration/invasion. Expression of receptor-interacting protein (RIP) 1 was initially increased upon exposure of A549, a non-small cell lung cancer (NSCLC) cell line, to IR. IR-induced RIP1 is located downstream of EGFR and involved in the expression/activity of matrix metalloproteases (MMP-2 and MMP-9) and vimentin, suggesting a role in epithelial-mesenchymal transition (EMT). Our experiments showed that IR-induced RIP1 sequentially induces Src-STAT3-EMT to promote invasion/migration. Inhibition of RIP1 kinase activity and expression blocked induction of EMT by IR and suppressed the levels and activities of MMP-2, MMP-9 and vimentin. IR-induced RIP1 activation was additionally associated with stimulation of the transcriptional factor NF-κB. Specifically, exposure to IR triggered NF-κB activation and inhibition of NF-κB suppressed IR-induced RIP1 expression, followed by a decrease in invasion/migration as well as EMT. Based on the collective results, we propose that IR concomitantly activates EGFR and NF-κB and subsequently triggers the RIP1-Src/STAT3-EMT pathway, ultimately promoting metastasis.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/patologia , Radiação Ionizante , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/radioterapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , NF-kappa B/genética , NF-kappa B/metabolismo , Invasividade Neoplásica , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Radiotherapy is one of the most common treatments for cancer, but radioresistance and injury to normal tissue are considered major obstacles to successful radiotherapy. Thus, there is an urgent need to develop radiosensitizers to improve the therapeutic outcomes of radiotherapy in cancer patients. Our previous efforts to identify novel radiosensitizers, using high-throughput screening targeting p53 and Nrf2 revealed a promising N-phenylpyrimidin-2-amine (PPA) lead compound. In the present study, 17 derivatives of this lead compound were examined, and it was found that 4-(4-fluorophenyl)-N-(4-nitrophenyl)-6-phenylpyrimidin-2-amine (PPA5), 4-((4-(4-fluorophenyl)pyrimidin-2-yl)amino)-3-methoxy-N-methyl -benzamide (PPA13), 4-((4-(4-fluorophenyl)pyrimidin-2-yl)amino)benzenesulfonamide (PPA14), 4-((4-(2-chlorophenyl)pyrimidin-2-yl)amino)benzenesulfonamide (PPA15), and 4-((4-(2-chlorophenyl)pyrimidin-2-yl)amino)-N-methylbenzamide (PPA17) inhibited cell viability by more than 50%, with a marked increase in the proportion of cells arrested at the G2/M phase of cell cycle. Among these compounds, PPA15 markedly increased the sub-G1 cell population and increased the levels of cyclin B1 and the phosphorylation levels of cyclin-dependent kinase (CDK) 1. Combined treatment with radiation and PPA14 or PPA15 significantly decreased clonogenic survival. An in vitro kinase assay revealed that PPA15 inhibited multiple CDKs involved in cell cycle regulation. Compared with drug or radiation treatment alone, combined treatment with PPA15 and radiation resulted in the suppression of A549 tumor growth in mice by 59.5% and 52.7%, respectively. Treatment with PPA15 alone directly inhibited tumor growth by 25.7%. These findings suggest that the novel pan CDK inhibitor, PPA15, may be a promising treatment to improve the effectiveness of radiotherapy for the treatment of cancer. SIGNIFICANCE STATEMENT: Several inhibitors of CDK have been successfully evaluated in combination with other chemotherapeutics in clinical trials, but negative side effects have partially restricted their clinical use. In this study, we identified a novel pan-CDK inhibitor to increase radiosensitivity, and we hope this work will encourage the development of promising small-molecule radiosensitizers.
Assuntos
Ciclo Celular/efeitos dos fármacos , Neoplasias Pulmonares/patologia , Pirimidinas/química , Pirimidinas/farmacologia , Radiossensibilizantes/química , Radiossensibilizantes/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Feminino , Humanos , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ionizing radiation (IR) has been widely used in the treatment of cancer. Radiation-induced DNA damage triggers the DNA damage response (DDR), which can confer radioresistance and early local recurrence by activating DNA repair pathways. Since karyopherin-α2 (KPNA2), playing an important role in nucleocytoplasmic transport, was significantly increased by IR in our previous study, we aimed to determine the function of KPNA2 with regard to DDR. Exposure to radiation upregulated KPNA2 expression in human colorectal cancer HT29 and HCT116 cells and breast carcinoma MDA-MB-231 cells together with the increased expression of DNA repair protein BRCA1. The knockdown of KPNA2 effectively increased apoptotic cell death via inhibition of BRCA1 nuclear import following IR. Therefore, we propose that KPNA2 is a potential target for overcoming radioresistance via interruption to DDR.
Assuntos
Proteína BRCA1/metabolismo , Morte Celular/efeitos da radiação , Sobrevivência Celular/fisiologia , alfa Carioferinas/metabolismo , Apoptose/efeitos da radiação , Proteína BRCA1/genética , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/genética , Ensaio Cometa , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Células HCT116 , Células HT29 , Humanos , Imunoprecipitação , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Radiação IonizanteRESUMO
We previously reported that podophyllotoxin acetate (PA) inhibits the growth and proliferation of non-small cell lung cancer (NSCLC) cells and also makes them more sensitive to radiation and chemotherapeutic agents. In an attempt to enhance PA activity, we synthesized 34 derivatives based on podophyllotoxin (PPT). Screening of the derivative compounds for anti-cancer activity against NSCLC led to the identification of ß-apopicropodophyllin (APP) as a strong anti-cancer agent. In addition to its role as an immunosuppressive regulator of the T-cell mediated immune response, the compound additionally showed anti-cancer activity against A549, NCI-H1299 and NCI-460 cell lines with IC50 values of 16.9, 13.1 and 17.1â¯nM, respectively. The intracellular mechanisms underlying the effects of APP were additionally examined. APP treatment caused disruption of microtubule polymerization and DNA damage, which led to cell cycle arrest, as evident from accumulation of phospho-CHK2, p21, and phospho-Cdc2. Moreover, APP stimulated the pro-apoptotic ER stress signaling pathway, indicated by elevated levels of BiP, phospho-PERK, phospho-eIF2α, CHOP and ATF4. We further observed activation of caspase-3, -8 and -9, providing evidence that both intrinsic and extrinsic apoptotic pathways were triggered. In vivo, APP inhibited tumor growth of NSCLC xenografts in nude mice by promoting apoptosis. Our results collectively support a novel role of APP as an anticancer agent that evokes apoptosis by inducing microtubule disruption, DNA damage, cell cycle arrest and ER stress.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Podofilina/farmacologia , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Estrutura Molecular , Podofilina/síntese química , Podofilina/químicaRESUMO
The function of PSMC5 (proteasome 26S subunit, ATPase 5) in tumors, particularly with respect to cancer radioresistance, is not known. Here, we identified PSMC5 as a novel radiosensitivity biomarker, demonstrating that radiosensitive H460 cells were converted to a radioresistance phenotype by PSMC5 depletion. Exposure of H460 cells to radiation induced a marked accumulation of cell death-promoting reactive oxygen species, but this effect was blocked in radiation-treated H460 PSMC5-knockdown cells through downregulation of the p53-p21 pathway. Interestingly, PSMC5 depletion in H460 cells enhanced both AKT activation and MDM2 transcription, thereby promoting the degradation of p53 and p21 proteins. Furthermore, specific inhibition of AKT with triciribine or knockdown of MDM2 with small interfering RNA largely restored p21 expression in PSMC5-knockdown H460 cells. Our data suggest that PSMC5 facilitates the damaging effects of radiation in radiation-responsive H460 cancer cells and therefore may serve as a prognostic indicator for radiotherapy and molecular targeted therapy in lung cancer patients.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/efeitos da radiação , Proteínas com Domínio LIM/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/radioterapia , Tolerância a Radiação , Fatores de Transcrição/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Linhagem Celular Tumoral , Relação Dose-Resposta à Radiação , Humanos , Neoplasias Pulmonares/patologia , Complexo de Endopeptidases do Proteassoma , Dosagem Radioterapêutica , Resultado do TratamentoRESUMO
Members of the Bcl-2 family are established regulators of cell death. However, recent studies have shown that they can also regulate cell migration, invasion, and cancer metastasis. These functions of cancer cells are promoted by pro-survival Bcl-2 proteins (Bcl-2, Bcl-XL, and Bcl-w) but are suppressed by pro-apoptotic members (Bax and Bak). We have previously shown that Bcl-w and Bcl-XL enhance the ability of respiratory complex-I to produce reactive oxygen species (ROS), stimulating the phosphoinositide 3-kinase (PI3K)-dependent invasion pathway. Here, we show that Bcl-w overexpression increases the phosphorylation of epidermal growth factor receptor (EGFR) and Src, and their interaction. Our results show that ROS production induced by Bcl-w activates Src, which then binds to and phosphorylates EGFR, leading to stimulation of the PI3K-dependent invasion pathway. Importantly, Bcl-w-induced cell invasion was prevented by treating cells with gefitinib (Iressa, ZD1839), an anticancer drug that directly inhibits EGFR. We also show that Bcl-XL can stimulate Src and EGFR phosphorylation, and that this function of Bcl-XL and Bcl-w is antagonized by Bax and Bak. Overall, this study demonstrates the involvement of Src and EGFR in the regulation of cellular invasiveness by Bcl-2 proteins, suggesting that chemotherapeutics targeting EGFR may be useful in preventing the progression of cancers that have altered Bcl-2 protein functions.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Receptores ErbB/metabolismo , Invasividade Neoplásica , Quinases da Família src/metabolismo , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Progressão da Doença , Gefitinibe , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Quinazolinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Proteína bcl-X/metabolismoRESUMO
Here, we report a new intracellular signaling pathway involved in γ-ionizing radiation (IR)-induced migration/invasion and show that podophyllotoxin acetate (PA) inhibits the IR-induced invasion and migration of A549 cells (a non-small cell lung cancer (NSCLC) cell line). Our results revealed that IR increased the invasion/migration of A549 cells, and this effect was decreased by 10 nM PA treatment. PA also inhibited the expressions/activities of matrix metalloprotase (MMP) -2, MMP-9, and vimentin, suggesting that PA could block the IR-induced epithelial-mesenchymal transition (EMT). The IR-induced increases in invasion/migration were associated with the activation of EGFR-AKT, and PA inhibited this effect. P38 and p44/42 ERK were also involved in IR-induced invasion/migration, and combined treatments with PA plus inhibitors of each MAPK synergistically blocked this invasion/migration. In terms of transcription factors (TFs), IR-induced increases in cyclic AMP response element-binding protein-1 (CREB-1) and signal transducer and activator of transcription 3 (STAT3) increased invasion/migration and EMT. PA also inhibited these transcription factors and then blocked IR-induced invasion/migration. Collectively, these results indicate that IR induces cancer cell invasion/migration by activating the EGFR-p38/ERK-CREB-1/STAT3-EMT pathway and that PA blocks this pathway to inhibit IR-induced invasion/migration.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Transição Epitelial-Mesenquimal/efeitos da radiação , Raios gama , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Neoplasias Pulmonares/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Podofilotoxina/análogos & derivados , Transdução de Sinais/efeitos da radiação , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/efeitos da radiação , Ensaios de Seleção de Medicamentos Antitumorais , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Neoplasias Pulmonares/enzimologia , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/fisiologia , Podofilotoxina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/fisiologiaRESUMO
How the p53 transcription factor/tumor suppressor inhibits cell invasion is poorly understood. We demonstrate that this function of p53 requires its direct interaction with p21(WAF1), a transcriptional target of p53, and that both p21 and p53 bind to Slug, which promotes cell invasion. Functional studies reveal that p21 and p53 cooperate to facilitate Mdm2-dependent Slug degradation and that this p53 function is mimicked by p53(R273H), a mutant lacking trans-activating activity. These actions of p21 and p53 are induced by γ-irradiation of cells and also operate in vivo. This is the first study to elucidate a mechanism involving p53 and p21 cooperation.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Neoplasias Pulmonares/genética , Invasividade Neoplásica/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Raios gama , Humanos , Neoplasias Pulmonares/patologia , Ligação Proteica/efeitos da radiação , Proteólise/efeitos da radiação , Proteínas Proto-Oncogênicas c-mdm2/genética , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Ativação Transcricional/efeitos da radiação , Proteína Supressora de Tumor p53/genéticaRESUMO
We previously identified hepatoma-derived growth factor-related protein-3 (HRP-3) as a radioresistant biomarker in p53 wild-type A549 cells and found that p53-dependent induction of the PUMA pathway was a critical event in regulating the radioresistant phenotype. Here, we found that HRP-3 knockdown regulates the radioresistance of p53-null H1299 cells through a distinctly different molecular mechanism. HRP-3 depletion was sufficient to cause apoptosis of H1299 cells by generating substantial levels of reactive oxygen species (ROS) through inhibition of the Nrf2/HO-1 antioxidant pathway. Subsequent, ROS-dependent and p53-independent NF-κB activation stimulated expression of c-Myc and Noxa proteins, thereby inducing the apoptotic machinery. Our results thus extend the range of targets for the development of new drugs to treat both p53 wild-type or p53-null radioresistant lung cancer cells.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias Pulmonares/patologia , NF-kappa B/metabolismo , Proteínas Nucleares/genética , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular Tumoral , Proteínas do Citoesqueleto , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Pulmonares/radioterapia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Although low-dose radiation (LDR) regulates a wide range of biological processes, limited information is available on the effects of LDR on the chondrocyte phenotype. Here, we found that LDR, at doses of 0.5-2 centiGray (cGy), inhibited interleukin (IL)-1ß-induced chondrocyte destruction without causing side effects, such as cell death and senescence. IL-1ß treatment induced an increase in the expression of α-, ß-, and γ-catenin proteins in chondrocytes via Akt signaling, thereby promoting dedifferentiation through catenin-dependent suppression of Sox-9 transcription factor expression and induction of inflammation through activation of the NF-κB pathway. Notably, LDR blocked cartilage disorders by inhibiting IL-1ß-induced catenin signaling and subsequent catenin-dependent suppression of the Sox-9 pathway and activation of the NF-κB pathway, without directly altering catenin expression. LDR also inhibited chondrocyte destruction through the catenin pathway induced by epidermal growth factor, phorbol 12-myristate 13-acetate, and retinoic acid. Collectively, these results identify the molecular mechanisms by which LDR suppresses pathophysiological processes and establish LDR as a potentially valuable therapeutic tool for patients with cytokine- or soluble factors-mediated cartilage disorders.
Assuntos
Cateninas/metabolismo , Desdiferenciação Celular/efeitos da radiação , Inflamação/metabolismo , Cartilagem Articular/efeitos da radiação , Cateninas/genética , Desdiferenciação Celular/efeitos dos fármacos , Condrócitos/metabolismo , Condrócitos/efeitos da radiação , Raios gama , Humanos , Inflamação/patologia , Interleucina-1beta/farmacologia , Doses de Radiação , Transdução de Sinais/efeitos da radiaçãoRESUMO
In a previous study, we showed that induction of ICAM-3 endows radioresistance in cervical cancer [1]. To ascertain whether ICAM-3 also promotes anticancer drug resistance, mock control (H1299/pcDNA3) or ICAM-3-expressing stable transfectants (H1299/ICAM-3) of the non-small cell lung cancer (NSCLC) cell line, NCI-H1299, were generated and treated with the microtubule-damaging agents, paclitaxel (TXL) and vincristine (VCS). TXL-/VCS-treated H1299/ICAM-3 cells showed significantly lower levels of apoptosis, activation of caspases-3, 8 or 9, and decrease in anti-apoptotic protein levels, compared to H1299/pcDNA3 cells. Our data clearly indicate that ICAM-3 promotes drug resistance via inhibition of apoptosis. We additionally showed that Akt, ERK, and CREB-2 are located downstream of ICAM-3, and activation of the ICAM-3-Akt/ERK-CREB-2 pathway induces resistance against TXL and VCS. ICAM-3-expressing stable NCI-H460/ICAM-3 transfectant cells and radioresistant SiHa cells endogenously overexpressing ICAM-3 additionally showed drug resistance against TXL and VCS via activation of the ICAM-3-Akt/ERK-CREB-2 pathway. The finding that ICAM-3 endows drug resistance as well as radioresistance supports its potential utility as a major therapeutic target against cancer.
Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Antígenos CD/fisiologia , Antineoplásicos/farmacologia , Apoptose , Moléculas de Adesão Celular/fisiologia , Resistencia a Medicamentos Antineoplásicos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Moduladores de Tubulina/farmacologia , Antígenos CD/genética , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Humanos , Redes e Vias MetabólicasRESUMO
Biomarkers based on functional signaling have the potential to provide greater insight into the pathogenesis of cancer and may offer additional targets for anticancer therapeutics. Here, we identified hepatoma-derived growth factor-related protein-3 (HRP-3) as a radioresistance-related gene and characterized the molecular mechanism by which its encoded protein regulates the radio- and chemoresistant phenotype of lung cancer-derived A549 cells. Knockdown of HRP-3 promoted apoptosis of A549 cells and potentiated the apoptosis-inducing action of radio- and chemotherapy. This increase in apoptosis was associated with a substantial generation of reactive oxygen species (ROS) that was attributable to inhibition of the Nrf2/HO-1 antioxidant pathway and resulted in enhanced ROS-dependent p53 activation and p53-dependent expression of PUMA (p53 upregulated modulator of apoptosis). Therefore, the HRP-3/Nrf2/HO-1/ROS/p53/PUMA cascade is an essential feature of the A549 cell phenotype and a potential radiotherapy target, extending the range of targets in multimodal therapies against lung cancer.
Assuntos
Anticarcinógenos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/radioterapia , Proteínas Nucleares/genética , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Proteínas do Citoesqueleto , Resistencia a Medicamentos Antineoplásicos , Heme Oxigenase-1/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Pulmão/efeitos da radiação , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Nucleares/metabolismo , Interferência de RNARESUMO
The mechanisms by which activated Ras accelerates malignant transformation of normal cells are not fully understood. Here, we characterized the role and molecular mechanism of γ-catenin in regulating the malignant phenotype of Rat2 cells induced by codon 12-mutant K-Ras (K-Ras12V). Suppression of γ-catenin signaling by K-Ras12V was an early event and played a crucial role in promoting the acquisition of a highly metastatic phenotype of Rat2 cells. Notably, the gene encoding histone deacetylase 4 (HDAC4) was identified as a target of γ-catenin during this process. The transcription factor, lymphoid enhancer-binding factor-1 (Lef1), was involved in the modulation of HDAC4 transcription, and disruption of this pathway was a key event in promoting the invasion and migration of K-Ras12V-transduced Rat2 cells. Thus, our findings extend the range of targets for the development of new drugs for the therapy of oncogenic K-Ras-driven cancer.
Assuntos
Transformação Celular Neoplásica/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes ras , Histona Desacetilases/metabolismo , gama Catenina/metabolismo , Animais , Linhagem Celular , Movimento Celular , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/patologia , Histona Desacetilases/genética , Histona Desacetilases/farmacologia , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Invasividade Neoplásica/patologia , Fenótipo , Mapeamento de Interação de Proteínas , Transporte Proteico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Transdução de Sinais , Transcrição Gênica , beta Catenina/genética , beta Catenina/metabolismo , gama Catenina/genéticaRESUMO
Although much is known about interleukin (IL)-1ß and its role as a key mediator of cartilage destruction in osteoarthritis, only limited information is available on IL-1ß signaling in chondrocyte dedifferentiation. Here, we have characterized the molecular mechanisms leading to the dedifferentiation of primary cultured articular chondrocytes by IL-1ß treatment. IL-1ß or lipopolysaccharide, but not phorbol 12-myristate 13-acetate, retinoic acid, or epidermal growth factor, induced nicotinamide phosphoribosyltransferase (NAMPT) expression, showing the association of inflammatory cytokines with NAMPT regulation. SIRT1, in turn, was activated NAMPT-dependently, without any alteration in the expression level. Activation or inhibition of SIRT1 oppositevely regulates IL-1ß-mediated chondrocyte dedifferentiation, suggesting this protein as a key regulator of chondrocytes phenotype. SIRT1 activation promotes induction of ERK and p38 kinase activities, but not JNK, in response to IL-1ß. Subsequently, ERK and p38 kinase activated by SIRT1 also induce SIRT1 activation, forming a positive feedback loop to sustain downstream signaling of these kinases. Moreover, we found that the SIRT1-ERK complex, but not SIRT1-p38, is engaged in IL-1ß-induced chondrocyte dedifferentiation via a Sox-9-mediated mechanism. JNK is activated by IL-1ß and modulates dedifferentiation of chondrocytes, but this pathway is independent on NAMPT-SIRT1 signaling. Based on these findings, we propose that IL-1ß induces dedifferentiation of articular chondrocytes by up-regulation of SIRT1 activity enhanced by both NAMPT and ERK signaling.