RESUMO
Since hepatitis C virus (HCV) core protein is known to possess potential oncogenic activity, we explored whether oncolytic vesicular stomatitis virus (VSV) could efficiently induce cytolysis in hepatocellular carcinoma cells stably expressing HCV core protein (Hep3B-Core). We found that Hep3B-Core cells were more susceptible to VSV as compared to control (Hep3B-Vec) cells owing to core-mediated inactivation of STAT1 and STAT2 proteins. Core expression induced lower phosphorylation levels of type I IFN signaling proteins such as Tyk2 and Jak1, and a reduced response to exogenous IFN-α, which resulted in susceptibility to VSV. Furthermore, as STAT1 acetylation by switching phosphorylation regulated its activity, the role of STAT1 acetylation in susceptibility of Hep3B-Core cells to VSV was investigated. Treatment with trichostatin A, an inhibitor of histone deacetylase (HDAC), increased STAT1 acetylation but blocked IFN-α-induced phosphorylation of STAT1, leading to increase of susceptibility to VSV. Interestingly, the core protein decreased HDCA4 transcript levels, leading to down-regulation of HDAC4 protein. However, ectopic expression of HDAC4 conversely enforced phosphorylation of STAT1 and hindered VSV replication, indicating that core-mediated reduction of HDAC4 provides a suitable intracellular circumstance for VSV replication. Collectively, we suggest that VSV treatment will be a useful therapeutic strategy for HCV-infected hepatocellular carcinoma cells because HCV core protein suppresses the anti-viral threshold by down-regulation of the STAT1-HDAC4 signaling axis.
Assuntos
Carcinoma Hepatocelular/terapia , Hepatite C/metabolismo , Histona Desacetilases/metabolismo , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/fisiologia , Proteínas Repressoras/metabolismo , Vesiculovirus/fisiologia , Proteínas do Core Viral/metabolismo , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Resultado do Tratamento , Estomatite Vesicular/virologiaRESUMO
Pancreatic adenocarcinoma upregulated factor (PAUF), a novel oncogene, plays a crucial role in the development of pancreatic cancer, including its metastasis and proliferation. Therefore, PAUF-expressing pancreatic cancer cells could be important targets for oncolytic virus-mediated treatment. Panc-1 cells expressing PAUF (Panc-PAUF) showed relative resistance to parvovirus H-1 infection compared with Panc-1 cells expressing an empty vector (Panc-Vec). Of interest, expression of type I IFN-α receptor (IFNAR) was higher in Panc-PAUF cells than in Panc-Vec cells. Increased expression of IFNAR in turn increased the activation of Stat1 and Tyk2 in Panc-PAUF cells compared with that in Panc-Vec cells. Suppression of Tyk2 and Stat1, which are important downstream molecules for IFN-α signaling, sensitized pancreatic cancer cells to parvovirus H-1-mediated apoptosis. Further, constitutive suppression of PAUF sensitized Bxpc3 pancreatic cancer cells to parvovirus H-1 infection. Taken together, these results suggested that PAUF conferred resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNAR-mediated signaling.
Assuntos
Parvovirus H-1 , Lectinas/metabolismo , Terapia Viral Oncolítica , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/terapia , Receptor de Interferon alfa e beta/metabolismo , Apoptose , Linhagem Celular Tumoral , Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Lectinas/antagonistas & inibidores , Lectinas/genética , Neoplasias Pancreáticas/genética , Infecções por Parvoviridae/genética , Infecções por Parvoviridae/metabolismo , Infecções por Parvoviridae/patologia , RNA Interferente Pequeno/genética , Receptor de Interferon alfa e beta/genética , Fator de Transcrição STAT1/antagonistas & inibidores , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , TYK2 Quinase/antagonistas & inibidores , TYK2 Quinase/genética , TYK2 Quinase/metabolismoRESUMO
As our previous study revealed that N-benzyl-N-methyldecan-1-amine (BMDA), a new molecule originated from Allium sativum, exhibits anti-neoplastic activities, we herein explored other functions of the compound and its derivative [decyl-(4-methoxy-benzyl)-methyl-amine; DMMA] including anti-inflammatory and anti-oxidative activities. Pretreatment of THP-1 cells with BMDA or DMMA inhibited tumor necrosis factor (TNF)-α and interleukin (IL)-1ß production, and blocked c-jun terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), MAPKAP kinase (MK)2 and NF-κΒ inflammatory signaling during LPS stimulation. Rectal treatment with BMDA or DMMA reduced the severity of colitis in 2,4-dinitrobenzenesulfonic acid (DNBS)-treated rat. Consistently, administration of the compounds decreased myeloperoxidase (MPO) activity (representing neutrophil infiltration in colonic mucosa), production of inflammatory mediators such as cytokine-induced neutrophil chemoattractant (CINC)-3 and TNF-α, and activation of JNK and p38 MAPK in the colon tissues. In addition, oral administration of these compounds ameliorated collagen-induced rheumatoid arthritis (RA) in mice. The treatment diminished the levels of inflammatory cytokine transcripts, and protected connective tissues through the expression of anti-oxidation proteins such as nuclear factor erythroid-related factor (Nrf)2 and heme oxygenase (HO)1. Additionally, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels did not differ between the BMDA- or DMMA-treated and control animals, indicating that the compounds do not possess liver toxicity. Taken together, these findings propose that BMDA and DMMA could be used as new drugs for curing inflammatory bowel disease (IBD) and RA.
RESUMO
We previously showed that SIRT1 deacetylase inhibits proliferation of hepatocellular carcinoma cells expressing hepatitis B virus (HBV) X protein (HBX), by destabilization of ß-catenin. Here, we report another role for SIRT1 in HBX-mediated resistance to oxidative stress. Ectopic expression and enhanced activity of SIRT1 sensitize Hep3B cells stably expressing HBX to oxidative stress-induced apoptosis. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for sensitization of oxidation-mediated apoptosis. Furthermore, ectopic expression of SIRT1 and treatment with resveratrol (a SIRT1 activator) attenuated JNK phosphorylation, which is a prerequisite for resistance to oxidative stress-induced apoptosis. Conversely, suppression of SIRT1 activity with nicotinamide inhibited the effect of resveratrol on JNK phosphorylation, leading to restoration of resistance to oxidation-induced apoptosis. Taken together, these results suggest that up-regulation of SIRT1 under oxidative stress may be a therapeutic strategy for treatment of hepatocellular carcinoma cells related to HBV through inhibition of JNK activation.
Assuntos
Apoptose , Carcinoma Hepatocelular/enzimologia , Neoplasias Hepáticas/enzimologia , Estresse Oxidativo , Sirtuína 1/metabolismo , Transativadores/biossíntese , Regulação para Cima , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Humanos , MAP Quinase Quinase 4/metabolismo , Fosforilação , RNA Interferente Pequeno/genética , Resveratrol , Sirtuína 1/genética , Estilbenos/farmacologia , Proteínas Virais Reguladoras e AcessóriasRESUMO
Because we found in a recent study that pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, induces a rapid proliferation of pancreatic cells by up-regulation of ß-catenin, we postulated that ß-catenin might be a target molecule for pancreatic cancer treatment. We thus speculated whether SIRT1, known to target ß-catenin in a colon cancer model, suppresses ß-catenin in those pancreatic cancer cells that express PAUF (Panc-PAUF). We further evaluated whether such suppression would lead to inhibition of the proliferation of these cells. The ectopic expression of either SIRT1 or resveratrol (an activator of SIRT1) suppressed levels of ß-catenin protein and its transcriptional activity in Panc-PAUF cells. Conversely, suppression of SIRT1 expression by siRNA enhanced ß-catenin expression and transcriptional activity. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for reduction of ß-catenin. Treatment with MG132, a proteasomal inhibitor, restored ß-catenin protein levels, suggesting that SIRT1-mediated degradation of ß-catenin requires proteasomal activity. It was reported that inhibition of GSK-3ß or Siah-1 stabilizes ß-catenin in colon cancer cells, but suppression of GSK-3ß or Siah-1 using siRNA in the presence of resveratrol instead diminished ß-catenin protein levels in Panc-PAUF cells. This suggests that GSK-3ß and Siah-1 are not involved in SIRT1-mediated degradation of ß-catenin in the cells. Finally, activation of SIRT1 inhibited the proliferation of Panc-PAUF cells by down-regulation of cyclin-D1, a target molecule of ß-catenin. These results suggest that SIRT1 activation may be a therapeutic strategy for treatment of pancreatic cancer cells that express PAUF via the down-regulation of ß-catenin.
Assuntos
Proliferação de Células , Ciclina D1/genética , Regulação Neoplásica da Expressão Gênica , Lectinas/genética , Oncogenes , Neoplasias Pancreáticas/patologia , Sirtuína 1/fisiologia , beta Catenina/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Leupeptinas/farmacologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Sirtuína 1/genéticaRESUMO
Increased mRNA levels of cancer upregulated gene (CUG)2 have been detected in many different tumor tissues using Affymetrix microarray. Oncogenic capability of the CUG2 gene has been further reported. However, the mechanism by which CUG2 overexpression promotes cancer stem cell (CSC)-like phenotypes remains unknown. With recent studies showing that pyruvate kinase muscle 2 (PKM2) is overexpressed in clinical tissues from gastric, lung, and cervical cancer patients, we hypothesized that PKM2 might play an important role in CSC-like phenotypes caused by CUG2 overexpression. The present study revealed that PKM2 protein levels and translocation of PKM2 into the nucleus were enhanced in CUG2-overexpressing lung carcinoma A549 and immortalized bronchial BEAS-2B cells than in control cells. Expression levels of c-Myc, CyclinD1, and PKM2 were increased in CUG2-overexpressing cells than in control cells. Furthermore, EGFR and ERK inhibitors as well as suppression of Yap1 and NEK2 expression reduced PKM2 protein levels. Interestingly, knockdown of ß-catenin expression failed to reduce PKM2 protein levels. Furthermore, reduction of PKM2 expression with its siRNA hindered CSC-like phenotypes such as faster wound healing, aggressive transwell migration, and increased size/number of sphere formation. The introduction of mutant S37A PKM2-green fluorescence protein (GFP) into cells without ability to move to the nucleus did not confer CSC-like phenotypes, whereas forced expression of wild-type PKM2 promoted such phenotypes. Overall, CUG2-induced increase in the expression of nuclear PKM2 contributes to CSC-like phenotypes by upregulating c-Myc and CyclinD1 as a co-activator. [BMB Reports 2022;55(2): 98-103].
Assuntos
Proteínas de Transporte/genética , Proteínas Cromossômicas não Histona , Proteínas de Membrana/genética , Neoplasias , Piruvato Quinase , Hormônios Tireóideos/genética , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Musculares/genética , Quinases Relacionadas a NIMA/genética , Quinases Relacionadas a NIMA/metabolismo , Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Transdução de Sinais/genética , Proteínas de Ligação a Hormônio da TireoideRESUMO
Overexpression of cancer upregulated gene (CUG) 2 induces cancer stem cell-like phenotypes, such as enhanced epithelial-mesenchymal transition, sphere formation, and doxorubicin resistance. However, the precise mechanism of CUG2-induced oncogenesis remains unknown. We evaluated the effects of overexpression of CUG2 on microRNA levels using a microRNA microarray. Levels of miR-3656 were decreased when CUG2 was overexpressed; on the basis of this result, we further examined the target proteins of this microRNA. We focused on Jumonji C domain-containing protein 5 (JMJD5), as it has not been previously reported to be targeted by miR-3656. When CUG2 was overexpressed, JMJD5 expression was upregulated compared to that in control cells. A 3' untranslated region (UTR) assay revealed that an miR-3656 mimic targeted the JMJD5 3'UTR, but the miR-3656 mimic failed to target a mutant JMJD5 3'UTR, indicating that miR-3656 targets the JMJD5 transcript. Administration of the miR-3656 mimic decreased the protein levels of JMD5 according to Western blotting. Additionally, the miR-3656 mimic decreased CUG2-induced cell migration, evasion, and sphere formation and sensitized the cells to doxorubicin. Suppression of JMJD5, with its small interfering RNA, impeded CUG2-induced cancer stem cell-like phenotypes. Thus, overexpression of CUG2 decreases miR-3656 levels, leading to upregulation of JMJD5, eventually contributing to cancer stem cell-like phenotypes.
Assuntos
MicroRNAs , Neoplasias , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/metabolismo , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Transdução de SinaisRESUMO
PURPOSE: The mechanism by which cancer upregulated gene 2 (CUG2) overexpression induces cancer stem cell-like phenotypes is not fully understood. Because the increased activity and expression of epidermal growth factor receptor (EGFR) kinase have been reported in A549 cancer cells overexpressing CUG2 (A549-CUG2) compared with control cells (A549-Vec), the Sprouty2 (Spry2) protein has gained attention as the downstream molecule of EGFR signaling. Therefore, we aim to identify the role of Spry2 in CUG2-overexpressing lung cancer cells. MATERIALS AND METHODS: Spry2 expression levels were examined in A549-CUG2 and A549-Vec cells by Western blotting and qRT-PCR. Cell migration, invasion, and sphere formation were examined after Spry2 suppression and overexpression. EGFR-Stat1 and Akt-ERK protein phosphorylation levels were detected via immunoblotting. NEK2 kinase and ß-catenin reporter assay were performed for downstream of Spry2 signaling. RESULTS: Although A549-CUG2 cells showed lower levels of the Spry2 protein than A549-Vec cells, no difference in levels of Spry2 transcript was observed between both cells via qRT-PCR. Furthermore, MG132 treatment enhanced the protein levels and ubiquitination of Spry2, suggesting that Spry2 protein expression can be regulated via the ubiquitin-proteasome pathway. The enforced expression of c-Cbl, known as the binding partner of Spry2, decreased the Spry2 protein levels, whereas its knockdown oppositely increased them. Epithelial-mesenchymal transition (EMT) and sphere formation were increased in A549-Vec cells during Spry2 siRNA treatment, confirming the role of Spry2 in CUG2-induced oncogenesis. Furthermore, EMT and sphere formation were determined by the Spry2 protein levels through the regulation of EGFR-Stat1 and ß-catenin-NEK2-Yap1 signaling pathways. CONCLUSION: CUG2 reduces Spry2 protein levels, the negative signaling molecule of cell proliferation, via c-Cbl, possibly activating the EGFR and ß-catenin signaling pathways and, in turn, contributing to the induction of cancer stem cell-like phenotypes.
RESUMO
Reovirus functions as an oncolytic agent for many types of cancer including colon cancer. Although most studies have emphasized the role of activated Ras signaling in enhancing reoviral oncolysis in susceptible cells, we note that many colon cancers also display elevated beta-catenin. Thus, it is possible that enhanced beta-catenin may augment reoviral susceptibility in colon cancer cells. To explore this hypothesis, HEK293 cells were treated with the glycogen synthase kinase (GSK)-3beta inhibitor LiCl, thereby inducing beta-catenin, followed by reoviral infection. Co-administration with LiCl indeed enhanced cell death compared to reovirus infection alone, but this was not associated with elevated reoviral replication. Similarly, HEK293 cells expressing the Frizzled-1 receptor in Wnt3a-conditioned medium also showed reovirus replication equivalent to that in cells in control medium, further suggesting that up-regulation of beta-catenin does not enhance the replication of reovirus. Instead, we observed that inhibition of GSK-3beta with LiCl decreased reovirus-induced NF-kappaB activation, leading to accelerated apoptosis via caspase 8 activation. We further found that colon cancer HCT116 cells were sensitized to apoptosis by co-treatment with reovirus and a GSK-3beta inhibitor, AR-A014418. Finally, we identified that inhibition of NF-kappaB sensitized apoptosis of HEK293 or HCT 116 cells during reovirus infection. Taken together, we propose that inhibition of GSK-3beta sensitizes reovirus-induced apoptosis of colon cancer cells by down-regulation of NF-kappaB activity, offering a potentially improved therapeutic strategy for the treatment of colon cancer.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/virologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Terapia Viral Oncolítica/métodos , Apoptose/fisiologia , Western Blotting , Linhagem Celular Tumoral , Neoplasias do Colo/terapia , Inibidores Enzimáticos/farmacologia , Glicogênio Sintase Quinase 3 beta , Humanos , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Orthoreovirus de Mamíferos/fisiologia , Infecções por Reoviridae/virologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , beta Catenina/efeitos dos fármacos , beta Catenina/metabolismoRESUMO
Herpesvirus saimiri (HVS), a member of the gamma-herpesvirus family, encodes an oncoprotein called Saimiri Transforming Protein (STP) which is required for lymphoma induction in non-human primates. However, a detailed mechanism of STP-A11-induced oncogenesis has not been revealed yet. We first report that STP-A11 oncoprotein interacts with TNF-alpha receptor-associated factor (TRAF) 6 in vivo and in vitro. Mutagenesis analysis of the TRAF6-binding motif (10)PQENDE(15) in STP-A11 reveals that Glu (E)(12) residue is critical for binding to TRAF6 and NF-kappaB activation. Interestingly, co-expression of E12A mutant, lack of TRAF6 binding, with cellular Src (Src) results in decreased transcriptional activity of Stat3 and AP-1, a novel target of STP-A11 compared to that of wild type. Furthermore, the presence of STP-A11 enhances the association of TRAF6 with Src and induces the translocation of both TRAF6 and Src to a nonionic detergent-insoluble fraction. Taken together, these studies suggest that STP-A11 oncoprotein up-regulates both NF-kappaB and AP-1 transcription activity through TRAF6, which would ultimately contribute cellular transformation.
Assuntos
Herpesvirus Saimiriíneo 2/metabolismo , NF-kappa B/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica , Linhagem Celular , Detergentes , Humanos , Íons , NF-kappa B/agonistas , Ligação Proteica , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Fator de Transcrição STAT3/metabolismo , Solubilidade , Fator de Transcrição AP-1/agonistasRESUMO
Overexpression of HIF-1α, a transcription factor responsive to hypoxia, is frequently observed in malignant tumors, which sometimes show resistance to chemotherapy and radiation therapy. Consequently, decrease of HIF-1α through virotherapy offers a logical strategy for the treatment of aggressive tumors. In this study, we found that infection with the oncolytic H-1 parvovirus decreased HIF-1α protein levels in pancreatic cancer cells under CoCl2 or hypoxia. The H-1 virus-induced decrease of HIF-1α was regulated by a proteasome-mediated pathway. Suppression of VHL, an E3 ligase and a critical regulator of HIF-1α, or enforced expression of UCP, an E2 ubiquitin-conjugating enzyme, failed to inhibit the H-1 virus-induced decrease of HIF-1α. Furthermore, siRNA-mediated suppression of RACK1, another regulator of HIF-1α, did not prevent H-1 viral infection from lowering HIF-1α protein levels. Although decrease of HIF-1α was observed after H-1 viral infection, constitutive expression of HIF-1α limited H-1 viral replication. After combined treatment with H-1 parvovirus and YC-1, an inhibitor of HIF-1α, the apoptosis of pancreatic cancer cells was greater than after treatment with H-1 virus alone or YC-1 alone. Accordingly, we propose that H-1 parvovirus could be used with YC-1 as a potential therapeutic agent against aggressive tumors exhibiting hypoxia and increased levels of HIF-1α.
Assuntos
Antineoplásicos/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Terapia Viral Oncolítica/métodos , Neoplasias Pancreáticas/metabolismo , Western Blotting , Linhagem Celular Tumoral , Proteínas de Ligação ao GTP/metabolismo , Parvovirus H-1 , Humanos , Indazóis/farmacologia , Proteínas de Neoplasias/metabolismo , Infecções por Parvoviridae/metabolismo , Receptores de Quinase C Ativada , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
Since multidrug resistance (MDR) is one of the main reasons for failure in cancer treatment, its suppression may increase the efficacy of cancer therapy. In the present study we attempted to identify a new and effective anticancer drug against MDR cancer cells. We first found that lung cancer A549 cells resistant to etoposide (A549RT-eto) exhibit upregulation of NF-κB and SIRT1 in comparison to A549 parental cells. During a search for anticancer drug candidates from medicinal plant sources, we found that an extract fraction (F14) of Bryophyllum laetivirens leaves downregulated expression of NF-κB and SIRT1, sensitizing the levels of A549RT-eto cells to apoptosis through downregulation of P-glycoprotein (P-gp), which is encoded by the MDR1 gene. To address whether NF-κB is involved in resistance to etoposide through P-gp, we treated A549RT-eto cells with Bay11-7802, an inhibitor of NF-κB. We then observed that Bay11-7802 treatment reduced P-gp expression levels, and furthermore combined treatment with the F14 extract and Bay11-7802 accelerated apoptosis through a decrease in P-gp levels, suggesting that NF-κB is involved in MDR. To address whether upregulation of SIRT1 is involved in resistance to etoposide through P-gp, we treated A549RT-eto cells with SIRT1 siRNA or nicotinamide (NAM), an inhibitor of SIRT1. we found that suppression of SIRT1 did not reduce P-gp levels. furthermore, the combined treatment with the F14 extract, and SIRT1 siRNA or NAM did not accelerate apoptosis, indicating that SIRT1 is not involved in the regulation of P-gp levels in A549RT-eto cells. Taken together, we suggest that upregulation of NF-κB determines etoposide resistance through P-gp expression in human A549 lung cancer cells. We herein demonstrated that B. laetivirens extract reverses etoposide resistance in human A549 lung cancer cells through downregulation of NF-κB.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Etoposídeo/farmacologia , Kalanchoe/química , Extratos Vegetais/farmacologia , Sirtuína 1/genética , Fator de Transcrição RelA/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Regulação para Baixo , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Niacinamida/farmacologia , Nitrilas/farmacologia , Interferência de RNA , RNA Interferente Pequeno , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/biossíntese , Sulfonas/farmacologia , Fator de Transcrição RelA/biossíntese , Transcrição Gênica/efeitos dos fármacosRESUMO
Since it has been known that shikonin derived from a medicinal plant possesses anti-cancer activity, we wonder whether acetylshikonin (ASK), a derivate of shikonin, can be used to treat hepatocellular carcinoma cells expressing hepatitis B virus X protein (HBX), an oncoprotein from hepatitis B virus. When ASK was added to Hep3B cells stably expressing HBX, it induced apoptosis in a dose-dependent manner. ASK induced upregulation and export of Nur77 to the cytoplasm and activation of JNK. Likewise, suppression of Nur77 and JNK inactivation protected the cells from ASK-induced apoptosis, indicating that Nur77 upregulation and JNK activation were required for ASK-mediated apoptosis. Furthermore, ASK increased the expression of Bip and ubiquitination levels of cellular proteins, features of endoplasmic reticulum (ER) stress, via the production of reactive oxygen species in a dose-dependent manner. Suppression of reactive oxygen species with N-acetylcysteine reduced levels of Bip protein and ubiquitination levels of cellular proteins during ASK treatment, leading to protection of cells from apoptosis. Cycloheximide treatment reduced ASK-induced ER stress, suggesting that protein synthesis is involved in ASK-induced ER stress. Moreover, we showed using salubrinal, an ER stress inhibitor that reactive oxygen species production, JNK activation, and Nur77 upregulation and its translocation to cytoplasm are necessary for ER-induced stress. Interestingly, we found that JNK inactivation suppresses ASK-induced ER stress, whereas Nur77 siRNA treatment does not, indicating that JNK is required for ASK-induced ER stress. Accordingly, we report that ASK induces ER stress, which is prerequisite for apoptosis of HBX-expressing hepatocellular carcinoma cells.
Assuntos
Antraquinonas/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático , Transativadores/genética , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , MAP Quinase Quinase 4/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , RNA Interferente Pequeno/genética , Regulação para Cima , Proteínas Virais Reguladoras e AcessóriasRESUMO
We showed in our previous study that cancer upregulated gene (CUG) 2, a novel oncogene, confers resistance to infection of oncolytic vesicular stomatitis virus (VSV) by activating Stat1-mediated signal transduction. Since many studies have reported that autophagy is involved in virus replication, we investigated whether autophagy also plays a role in the antiviral activity in A549 cells overexpressing CUG2 (A549-CUG2). We suppressed Atg5 or Beclin 1 expression using siRNA and examined its effect on the susceptibility of cells to infection by oncolytic VSV. We found that A549-CUG2 cells treated with Atg5 or Beclin 1 siRNA became susceptible to VSV infection, whereas A549-CUG2 cells treated with control siRNA were resistant. This result suggests that autophagy is involved in the antiviral response of A549-CUG2 cells. Further investigation revealed that autophagy impairment enhanced the generation of reactive oxygen species (ROS), which resulted in inactivation of S6 kinase. Under these conditions, the levels of ISG15 transcript and protein decreased, which conferred on A549-CUG2 cell susceptibility to VSV infection. Finally, we found that overloading of H2O2 sensitized control A549-CUG2 cells to VSV-induced apoptosis. Taken together, these results indicate that autophagy impairment induces excessive ROS formation, which decreases S6 kinase activity and ISG15 expression, ultimately rendering the A549-CUG2 cells susceptible to VSV infection. We propose that autophagy impairment is a potential strategy for successful VSV virotherapy of CUG2-overexpressing tumors.
Assuntos
Apoptose/genética , Autofagia/genética , Proteínas Cromossômicas não Histona/genética , Citocinas/genética , Ubiquitinas/genética , Vírus da Estomatite Vesicular Indiana/patogenicidade , Animais , Proteínas Reguladoras de Apoptose/genética , Proteína 5 Relacionada à Autofagia , Proteína Beclina-1 , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/biossíntese , Citocinas/biossíntese , Humanos , Neoplasias Pulmonares/virologia , Proteínas de Membrana/genética , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Vírus Oncolíticos/genética , Vírus Oncolíticos/patogenicidade , Interferência de RNA , RNA Interferente Pequeno , Espécies Reativas de Oxigênio/metabolismo , Infecções por Rhabdoviridae/genética , Infecções por Rhabdoviridae/patologia , Proteínas Quinases S6 Ribossômicas/antagonistas & inibidores , Fator de Transcrição STAT1/genética , Transdução de Sinais , Ubiquitinas/biossíntese , Vírus da Estomatite Vesicular Indiana/genética , Replicação ViralRESUMO
Cancer upregulated gene (CUG) 2, as a novel oncogene, has been predominantly detected in various cancer tissues, such as ovary, liver, lung and colon. We recently showed that CUG2 elevates STAT1 activity, leading to resistance to infection by oncolytic vesicular stomatitis virus. To investigate a possible role for CUG2-induced activation of STAT1 in oncogenesis, we first established a colon cancer cell line stably expressing CUG2 (Colon26L5-CUG2). Colon26L5-CUG2 exhibited higher levels not only in phosphorylation of STAT1, but also phosphorylation of Jak1/Tyk2 compared to that of the control (Colon26L5-Vec) cell line. Inhibition of Akt or ERK activity reduced phosphorylation of STAT1 in Colon26L5-CUG2 cells whereas inhibition of p38 MAPK did not significantly decrease levels of STAT1 phosphorylation, indicating that cell proliferation signals may be involved in CUG2-mediated activation of STAT1. Suppression of STAT1 expression diminished cell migration and wound healing compared to the control cells. In addition, since CUG2 expression conferred resistance to DNA damage caused by doxorubicin treatment, we investigated whether STAT1 is involved in resistance to doxorubicin-induced cell death. We found that STAT1 was not activated in Colon26L5-Vec cells while phosphorylated STAT1 was maintained in Colon26L5-CUG2 cells during doxorubicin treatment. Furthermore, suppression of STAT1 expression sensitized Colon26L5-CUG2 cells to doxorubicin-induced apoptosis whereas the control cells exhibited resistance to doxorubicin. Taken together, our results suggest that CUG2 enhances metastasis and drug resistance through STAT1 activation, which eventually contributes to tumor progression.
Assuntos
Neoplasias do Colo/genética , Metástase Neoplásica/genética , Proteínas Nucleares/genética , Fator de Transcrição STAT1/biossíntese , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular , Proteínas Cromossômicas não Histona , Neoplasias do Colo/patologia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Neoplásica/patologia , Proteínas Nucleares/biossíntese , Fator de Transcrição STAT1/genética , Transdução de Sinais , Cicatrização/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
We have previously shown that hepatitis B virus (HBV) protein X (HBX), a regulatory protein of HBV, activates Stat1, leading to type I interferon (IFN) production. Type I IFN secreted from HBX-expressing hepatic cells enforces antiviral signals through its binding to the cognate type I IFN receptor. We therefore investigated how cells handle this detrimental situation. Interestingly, compared to Chang cells stably expressing an empty vector (Chang-Vec), Chang cells stably expressing HBX (Chang-HBX) showed lower levels of IFN-α receptor 1 (IFNAR1) protein, a subunit of type I IFN receptor. The levels of IFNAR1 transcripts detected in Chang-HBX cells were lower than the levels in Chang-Vec cells, indicating that HBX regulates IFNAR1 at the transcriptional level. Moreover, we observed that HBX induced the translocation of IFNAR1 to the cytoplasm. Consistent with these observations, HBX also downregulated Tyk2, which is required for the stable expression of IFNAR1 on the cell surface. Eventually, Chang-HBX cells consistently maintained a lower level of IFNAR1 expression and displayed no proper response to IFN-α, while Chang-Vec cells exhibited a proper response to IFN-α treatment. Taken together, we propose that HBX downregulates IFNAR1, leading to the avoidance of extracellular IFN-α signal transduction.
Assuntos
Interferon-alfa/metabolismo , Receptor de Interferon alfa e beta/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Linhagem Celular , Regulação para Baixo , Vírus da Hepatite B , Humanos , Interferon-alfa/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor de Interferon alfa e beta/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , TYK2 Quinase/genética , TYK2 Quinase/metabolismo , Transativadores/genética , Transfecção , Proteínas Virais Reguladoras e AcessóriasRESUMO
Hepatitis B virus X (HBX) protein has been reported to induce upregulation of ß-catenin, a known proto-oncogene, in p53-knockout and p53-mutant hepatic cell lines both in a GSK-3ß-dependent manner and via interaction with adenomatous polyposis coli, which results in protection from ß-catenin degradation. In this study, we describe a novel mechanism for HBX-mediated upregulation of ß-catenin. We observed that HBX interacts with SIRT1, a class III histone deacetylase. Furthermore, the presence of HBX attenuated the interaction between SIRT1 and ß-catenin, leading to protection of ß-catenin from the inhibitory action of SIRT1. Reduction of SIRT1 with siRNA or suppression of SIRT1 activity with nicotinamide upregulated ß-catenin protein levels. In contrast, enhancement of SIRT1 activity with resveratrol reduced ß-catenin protein levels. Furthermore, in Hep3B cells stably expressing HBX, overexpression of SIRT1 or treatment with resveratrol enhanced sensitivity to doxorubicin-induced apoptosis, indicating that upregulation of SIRT1 could be a therapeutic strategy for HBV-related hepatocellular carcinoma. Based on these results, we propose that HBX upregulates ß-catenin by sequestering SIRT1, which leads to anticancer drug treatment resistance.
Assuntos
Sirtuína 1/metabolismo , Transativadores/fisiologia , Regulação para Cima , beta Catenina/genética , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Neoplasias Hepáticas , Ligação Proteica , Proto-Oncogene Mas , Transativadores/metabolismo , Proteínas Virais Reguladoras e Acessórias , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
It is not yet understood how the enhanced expression of pancreatic adenocarcinoma up-regulated factor (PAUF; a novel oncogene identified in our recent studies), contributes to the oncogenesis of pancreatic cells. We herein report that PAUF up-regulates the expression and transcriptional activity of ß-catenin while the suppression of PAUF by shRNA down-regulates ß-catenin. The induction of b-catenin by PAUF is mediated by the activities of Akt and GSK-3ß, but inhibition of downstream ERK does not reduce ß-catenin expression. To test whether PAUF emulates either the Wnt3a-mediated or the protein kinase A-mediated signaling pathway for the stabilization of ß-catenin, we examined the phosphorylation status of ß-catenin in the presence of PAUF compared with that of ß-catenin during treatment with Wnt3a or dibutyryl cAMP, a cell permeable cyclic AMP analogue. PAUF expression induces phosphorylation at Ser-33/37/Thr-41 and Ser-675 of ß-catenin but no phosphorylation at Ser-45, indicating that a unique phosphorylation pattern of b-catenin is caused by PAUF. Finally, the expression of PAUF up-regulates both cyclin-D1 and c-Jun, target genes of ß-catenin, leading to a rapid proliferation of pancreatic cells; conversely decreased PAUF expression (by shRNA) results in the reduced proliferation of pancreatic cells. Treatment with hexachlorophene (an inhibitor of ß-catenin) reduces the proliferation of pancreatic cells despite the presence of PAUF. Taken together, we propose that PAUF can up-regulate and stabilize ß-catenin via a novel pattern of phosphorylation, thereby contributing to the rapid proliferation of pancreatic cancer cells.
Assuntos
Adenocarcinoma , Lectinas/metabolismo , Neoplasias Pancreáticas , Regulação para Cima , beta Catenina/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Proliferação de Células , Ciclina D1/metabolismo , Regulação Neoplásica da Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Lectinas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais , beta Catenina/genéticaRESUMO
The hepatitis B virus X (HBX) protein, a regulatory protein of the hepatitis B virus (HBV), has been shown to generate reactive oxygen species (ROS) in human liver cell lines; however, the mechanism by which cells protect themselves under this oxidative stress is poorly understood. Here, we show that HBX induces the up-regulation of Forkhead box class O 4 (Foxo4) not only in Chang cells stably expressing HBX (Chang-HBX) but also in primary hepatic tissues from HBX-transgenic mice. HBX also increased ROS, but reduction of the abundance of ROS using N-acetylcystein (NAC) diminished the levels of Foxo4. Elevated Foxo4 was also detected in nuclei of Chang-HBX cells but not in Chang cells stably expressing the vector (Chang-Vec), suggesting that HBX activates the transcriptional activity of Foxo4. When we examined whether HBX bypasses JNK signaling that targets Foxo4, we found that the activity of JNK but not of ERK is required for the up-regulation of Foxo4 even in the presence of HBX. Furthermore, the reduction of Foxo4 levels using siRNA or a JNK inhibitor rendered Chang-HBX cells sensitive to apoptosis under oxidative stress, suggesting that up-regulation of Foxo4 mediated by HBX enhances resistances to oxidative stress-induced cell death. Accordingly, we propose that Foxo4 may be a useful target for suppression in the treatment of HBV-associated hepatocellular carcinoma cells.
Assuntos
Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Estresse Oxidativo/genética , Transativadores/genética , Transativadores/metabolismo , Regulação para Cima , Animais , Morte Celular , Linhagem Celular , Núcleo Celular/metabolismo , Fatores de Transcrição Forkhead/antagonistas & inibidores , Regulação da Expressão Gênica , Humanos , MAP Quinase Quinase 4/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transporte Proteico/genética , Espécies Reativas de Oxigênio/metabolismo , Proteínas Virais Reguladoras e AcessóriasRESUMO
The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been used to treat a variety of cancer cells. However, since some gastric cancer cells are resistant to TRAIL, we explored whether reovirus induces cytolysis in TRAIL-resistant gastric cancer cells. We found that TRAIL-resistant SNU-216 gastric cancer cells were susceptible to apoptosis by reovirus infection. Furthermore, co-treatment with reovirus and TRAIL accelerated apoptosis of SNU-216 cells by down-regulation of Akt activation as assessed by a very low activation of Akt in TRAIL-sensitive SNU-668 gastric cancer cells. Inhibition of Akt signaling with wortmannin or suppression of Akt expression with sh-Akt lentivirus promoted reovirus-mediated apoptosis of SNU-216 gastric cancer cells. Reovirus infection also down-regulates the activation of signaling molecules such as Ras and ERK involved in cell proliferation and survival but not the activation of p38 MAPK involved in cellular stress. In addition, the co-treatment with reovirus and TRAIL resulted in cleavage of caspase-8, caspase-9 and Bid, leading to a decrease in the mitochondrial membrane potential, indicating that reovirus may utilize the mitochondrial intrinsic apoptotic pathway in TRAIL-resistant SNU-216 gastric cancer cells. Accordingly, we first demonstrate that reovirus infection down-regulates Akt activation, leading to apoptosis of TRAIL-resistant gastric cancer cells.