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1.
PLoS Pathog ; 19(9): e1011169, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37669313

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several human diseases including Kaposi's sarcoma (KS), a leading cause of cancer in Africa and in patients with AIDS. KS tumor cells harbor KSHV predominantly in a latent form, while typically <5% contain lytic replicating virus. Because both latent and lytic stages likely contribute to cancer initiation and progression, continued dissection of host regulators of this biological switch will provide insights into fundamental pathways controlling the KSHV life cycle and related disease pathogenesis. Several cellular protein kinases have been reported to promote or restrict KSHV reactivation, but our knowledge of these signaling mediators and pathways is incomplete. We employed a polypharmacology-based kinome screen to identify specific kinases that regulate KSHV reactivation. Those identified by the screen and validated by knockdown experiments included several kinases that enhance lytic reactivation: ERBB2 (HER2 or neu), ERBB3 (HER3), ERBB4 (HER4), MKNK2 (MNK2), ITK, TEC, and DSTYK (RIPK5). Conversely, ERBB1 (EGFR1 or HER1), MKNK1 (MNK1) and FRK (PTK5) were found to promote the maintenance of latency. Mechanistic characterization of ERBB2 pro-lytic functions revealed a signaling connection between ERBB2 and the activation of CREB1, a transcription factor that drives KSHV lytic gene expression. These studies provided a proof-of-principle application of a polypharmacology-based kinome screen for the study of KSHV reactivation and enabled the discovery of both kinase inhibitors and specific kinases that regulate the KSHV latent-to-lytic replication switch.


Assuntos
Herpesvirus Humano 8 , Sarcoma de Kaposi , Humanos , Herpesvirus Humano 8/genética , Polifarmacologia , África , Cognição , Proteínas Serina-Treonina Quinases , Peptídeos e Proteínas de Sinalização Intracelular , Proteína Serina-Treonina Quinases de Interação com Receptores
2.
Proc Natl Acad Sci U S A ; 115(13): 3452-3457, 2018 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-29540569

RESUMO

Renewable tissues exhibit heightened sensitivity to DNA damage, which is thought to result from a high level of p53. However, cell proliferation in renewable tissues requires p53 down-regulation, creating an apparent discrepancy between the p53 level and elevated sensitivity to DNA damage. Using a combination of genetic mouse models and pharmacologic inhibitors, we demonstrate that it is p53-regulated MDM2 that functions together with MDMX to regulate DNA damage sensitivity by targeting EZH2 (enhancer of zeste homolog 2) for ubiquitination/degradation. As a methyltransferase, EZH2 promotes H3K27me3, and therefore chromatin compaction, to determine sensitivity to DNA damage. We demonstrate that genetic and pharmacologic interference of the association between MDM2 and MDMX stabilizes EZH2, resulting in protection of renewable tissues from radio-/chemotherapy-induced acute injury. In cells with p53 mutation, there are diminished MDM2 levels, and thus accumulation of EZH2, underpinning the resistant phenotype. Our work uncovers an epigenetic mechanism behind tissue sensitivity to DNA damage, carrying important translation implications.


Assuntos
Cromatina/metabolismo , Dano ao DNA , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Epigênese Genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas/genética , Proteína Supressora de Tumor p53/genética , Animais , Apoptose , Cromatina/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Camundongos , Camundongos Transgênicos , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinação
3.
bioRxiv ; 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36778430

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several human diseases including Kaposi's sarcoma (KS), a leading cause of cancer in Africa and in patients with AIDS. KS tumor cells harbor KSHV predominantly in a latent form, while typically <5% contain lytic replicating virus. Because both latent and lytic stages likely contribute to cancer initiation and progression, continued dissection of host regulators of this biological switch will provide insights into fundamental pathways controlling the KSHV life cycle and related disease pathogenesis. Several cellular protein kinases have been reported to promote or restrict KSHV reactivation, but our knowledge of these signaling mediators and pathways is incomplete. We employed a polypharmacology-based kinome screen to identifiy specific kinases that regulate KSHV reactivation. Those identified by the screen and validated by knockdown experiments included several kinases that enhance lytic reactivation: ERBB2 (HER2 or neu ), ERBB3 (HER3), ERBB4 (HER4), MKNK2 (MNK2), ITK, TEC, and DSTYK (RIPK5). Conversely, ERBB1 (EGFR1 or HER1), MKNK1 (MNK1) and FRK (PTK5) were found to promote the maintenance of latency. Mechanistic characterization of ERBB2 pro-lytic functions revealed a signaling connection between ERBB2 and the activation of CREB1, a transcription factor that drives KSHV lytic gene expression. These studies provided a proof-of-principle application of a polypharmacology-based kinome screen for the study of KSHV reactivation and enabled the discovery of both kinase inhibitors and specific kinases that regulate the KSHV latent-to-lytic replication switch. Author Summary: Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma, a cancer particularly prevalent in Africa. In cancer cells, the virus persists in a quiescent form called latency, in which only a few viral genes are made. Periodically, the virus switches into an active replicative cycle in which most of the viral genes are made and new virus is produced. What controls the switch from latency to active replication is not well understood, but cellular kinases, enzymes that control many cellular processes, have been implicated. Using a cell culture model of KSHV reactivation along with an innovative screening method that probes the effects of many cellular kinases simultaneously, we identified drugs that significantly limit KSHV reactivation, as well as specific kinases that either enhance or restrict KSHV replicative cycle. Among these were the ERBB kinases which are known to regulate growth of cancer cells. Understanding how these and other kinases contribute to the switch leading to production of more infectious virus helps us understand the mediators and mechanisms of KSHV diseases. Additionally, because kinase inhibitors are proving to be effective for treating other diseases including some cancers, identifying ones that restrict KSHV replicative cycle may lead to new approaches to treating KSHV-related diseases.

4.
Cancer Res ; 83(20): 3442-3461, 2023 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-37470810

RESUMO

Although external beam radiotherapy (xRT) is commonly used to treat central nervous system (CNS) tumors in patients of all ages, young children treated with xRT frequently experience life-altering and dose-limiting neurocognitive impairment (NI) while adults do not. The lack of understanding of mechanisms responsible for these differences has impeded the development of neuroprotective treatments. Using a newly developed mouse model of xRT-induced NI, we found that neurocognitive function is impaired by ionizing radiation in a dose- and age-dependent manner, with the youngest animals being most affected. Histologic analysis revealed xRT-driven neuronal degeneration and cell death in neurogenic brain regions in young animals but not adults. BH3 profiling showed that neural stem and progenitor cells, neurons, and astrocytes in young mice are highly primed for apoptosis, rendering them hypersensitive to genotoxic damage. Analysis of single-cell RNA sequencing data revealed that neural cell vulnerability stems from heightened expression of proapoptotic genes including BAX, which is associated with developmental and mitogenic signaling by MYC. xRT induced apoptosis in primed neural cells by triggering a p53- and PUMA-initiated, proapoptotic feedback loop requiring cleavage of BID and culminating in BAX oligomerization and caspase activation. Notably, loss of BAX protected against apoptosis induced by proapoptotic signaling in vitro and prevented xRT-induced apoptosis in neural cells in vivo as well as neurocognitive sequelae. On the basis of these findings, preventing xRT-induced apoptosis specifically in immature neural cells by blocking BAX, BIM, or BID via direct or upstream mechanisms is expected to ameliorate NI in pediatric patients with CNS tumor. SIGNIFICANCE: Age- and differentiation-dependent apoptotic priming plays a pivotal role in driving radiotherapy-induced neurocognitive impairment and can be targeted for neuroprotection in pediatric patients.


Assuntos
Proteínas Reguladoras de Apoptose , Apoptose , Animais , Criança , Pré-Escolar , Humanos , Camundongos , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/metabolismo , Proteína X Associada a bcl-2/metabolismo , Morte Celular , Transdução de Sinais , Proteína Supressora de Tumor p53/genética
5.
BMC Complement Altern Med ; 12: 93, 2012 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-22784363

RESUMO

BACKGROUND: Numerous studies have demonstrated that autophagy plays a vital role in maintaining cellular homeostasis. Interestingly, several anticancer agents were found to exert their anticancer effects by triggering autophagy. Emerging data suggest that autophagy represents a novel mechanism that can be exploited for therapeutic benefit. Pharmacologically active natural compounds such as those from marine, terrestrial plants and animals represent a promising resource for novel anticancer drugs. There are several prominent examples from the past proving the success of natural products and derivatives exhibiting anticancer activity. Helenalin, a sesquiterpene lactone has been demonstrated to have potent anti-inflammatory and antitumor activity. Albeit previous studies demonstrating helenalin's multi modal action on cellular proliferative and apoptosis, the mechanisms underlying its action are largely unexplained. METHODS: To deduce the mechanistic action of helenalin, cancer cells were treated with the drug at various concentrations and time intervals. Using western blot, FACS analysis, overexpression and knockdown studies, cellular signaling pathways were interrogated focusing on apoptosis and autophagy markers. RESULTS: We show here that helenalin induces sub-G1 arrest, apoptosis, caspase cleavage and increases the levels of the autophagic markers. Suppression of caspase cleavage by the pan caspase inhibitor, Z-VAD-fmk, suppressed induction of LC3-B and Atg12 and reduced autophagic cell death, indicating caspase activity was essential for autophagic cell death induced by helenalin. Additionally, helenalin suppressed NF-κB p65 expression in a dose and time dependent manner. Exogenous overexpression of p65 was accompanied by reduced levels of cell death whereas siRNA mediated suppression led to augmented levels of caspase cleavage, autophagic cell death markers and increased cell death. CONCLUSIONS: Taken together, these results show that helenalin mediated autophagic cell death entails inhibition of NF-κB p65, thus providing a promising approach for the treatment of cancers with aberrant activation of the NF-κB pathway.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Arnica/química , Autofagia/efeitos dos fármacos , Neoplasias/metabolismo , Fitoterapia , Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Sesquiterpenos/farmacologia , Clorometilcetonas de Aminoácidos/metabolismo , Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/efeitos dos fármacos , Proteína 12 Relacionada à Autofagia , Caspases/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Fase G1/efeitos dos fármacos , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias/tratamento farmacológico , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , RNA Interferente Pequeno/metabolismo , Sesquiterpenos/uso terapêutico , Sesquiterpenos de Guaiano , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo
6.
J Mol Cell Biol ; 12(9): 713-722, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32706867

RESUMO

A role of tumor-suppressive activity of p53 in the tumor microenvironment (TME) has been implicated but remains fairly understudied. To address this knowledge gap, we leveraged our MdmxS314A mice as recipients to investigate how implanted tumor cells incapacitate host p53 creating a conducive TME for tumor progression. We found that tumor cell-associated stress induced p53 downregulation in peritumor cells via an MDMX-Ser314 phosphorylation-dependent manner. As a result, an immunosuppressive TME was developed, as reflected by diminished immune cell infiltration into tumors and compromised macrophage M1 polarization. Remarkably, ablation of MDMX-Ser314 phosphorylation attenuated p53 decline in peritumor cells, which was associated with mitigation of immunosuppression and significant tumor growth delay. Our data collectively uncover a novel role of p53 in regulating the tumor immune microenvironment, suggesting that p53 restoration in the TME can be exploited as a potential strategy of anticancer therapy.


Assuntos
Regulação para Baixo , Terapia de Imunossupressão , Proteínas Proto-Oncogênicas/metabolismo , Microambiente Tumoral/imunologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular Tumoral , Polaridade Celular , Técnicas de Introdução de Genes , Humanos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Fosforilação , Fosfosserina/metabolismo
7.
Apoptosis ; 14(9): 1095-107, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19544095

RESUMO

Kruppel-like factor 4 (KLF4) belongs to a family of evolutionarily conserved zinc finger-containing transcription factors. It has been shown to mediate self renewal and pluripotency, regulate adipogenesis and play a critical role in monocyte differentiation. KLF4 is also highly expressed in squamous cell carcinomas and in 70% of all primary human breast cancers, suggesting a putative role for KLF4 as being an oncogene and as an antiapoptotic factor. However, the mechanism of this regulation remains unclear. Here, we show that KLF4 is induced during histone deacetylase inhibitor treatment, and regulates the extrinsic apoptosis pathway by inhibiting caspase cleavage. In addition, KLF4 binds to the p57(Kip2) promoter and transcriptionally upregulates its expression, which in turn inhibits the stress activated protein kinase cascade and c-Jun phosphorylation. Our findings indicate that in cancer cells that express high levels of KLF4 may be refractory to HDACi treatment. Results of our study demonstrate an unexpected antiapoptotic function of KLF4, and suggest an important cell fate determinant following histone deacetylase inhibitor induced apoptosis.


Assuntos
Caspases/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/farmacologia , Fatores de Transcrição Kruppel-Like/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Apoptose/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p57/genética , Citoproteção/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Células HCT116 , Humanos , Fator 4 Semelhante a Kruppel , Oligopeptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Vorinostat
8.
Oncogene ; 37(16): 2150-2164, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29371679

RESUMO

Although ΔNp63 is known to promote cancer cell proliferation, the underlying mechanism behind its oncogenic function remains elusive. We report here a functional interplay between ΔNp63 and Δ133p53. These two proteins are co-overexpressed in a subset of human cancers and cooperate to promote cell proliferation. Mechanistically, Δ133p53 binds to ΔNp63 and utilizes its transactivation domain to upregulate GLUT1, GLUT4, and PGM expression driving glycolysis. While increased glycolysis provides cancer cells with anabolic metabolism critical for proliferation and survival, it can be harnessed for selective cancer cell killing. Indeed, we show that tumors overexpressing both ΔNp63 and Δ133p53 exhibit heightened sensitivity to vitamin C that accumulate to a lethal level due to accelerated uptake via overexpressed GLUT1. These observations offer a new therapeutic avenue that could be exploited for clinical applications.


Assuntos
Proliferação de Células/genética , Glicólise/genética , Neoplasias/patologia , Fatores de Transcrição/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Células A549 , Animais , Metabolismo dos Carboidratos/genética , Células Cultivadas , Códon sem Sentido , Feminino , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Proteínas Mutantes/genética , Proteínas Mutantes/fisiologia , Neoplasias/genética , Neoplasias/metabolismo , Isoformas de Proteínas/fisiologia , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética
10.
Oncotarget ; 7(6): 7179-92, 2016 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-26769849

RESUMO

TRAF6 plays a crucial role in the regulation of the innate and adaptive immune responses. Although studies have shown that TRAF6 has oncogenic activity, the role of TRAF6 in melanoma is unclear. Here, we report that TRAF6 is overexpressed in primary as well as metastatic melanoma tumors and melanoma cell lines. Knockdown of TRAF6 with shRNA significantly suppressed malignant phenotypes including cell proliferation, anchorage-independent cell growth and metastasis in vitro and in vivo. Notably, we demonstrated that Basigin (BSG)/CD147, a critical molecule for cancer cell invasion and metastasis, is a novel interacting partner of TRAF6. Furthermore, depletion of TRAF6 by shRNA reduced the recruitment of BSG to the plasma membrane and K63-linked ubiquitination, in turn, which impaired BSG-dependent MMP9 induction. Taken together, our findings indicate that TRAF6 is involved in regulating melanoma invasion and metastasis, suggesting that TRAF6 may be a potential target for therapy or chemo-prevention in melanoma.


Assuntos
Basigina/metabolismo , Movimento Celular , Neoplasias Pulmonares/secundário , Melanócitos/patologia , Melanoma/patologia , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitina/metabolismo , Animais , Apoptose , Western Blotting , Adesão Celular , Proliferação de Células , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Neoplasias Pulmonares/metabolismo , Masculino , Melanócitos/metabolismo , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Fator 6 Associado a Receptor de TNF/genética , Células Tumorais Cultivadas , Ubiquitinação , Cicatrização , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Front Pharmacol ; 6: 113, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26106328

RESUMO

Mesothelial cells are fundamental to the maintenance of serosal integrity and homeostasis and play a critical role in normal serosal repair following injury. However, when normal repair mechanisms breakdown, mesothelial cells take on a profibrotic role, secreting inflammatory, and profibrotic mediators, differentiating and migrating into the injured tissues where they contribute to fibrogenesis. The development of new molecular and cell tracking techniques has made it possible to examine the origin of fibrotic cells within damaged tissues and to elucidate the roles they play in inflammation and fibrosis. In addition to secreting proinflammatory mediators and contributing to both coagulation and fibrinolysis, mesothelial cells undergo mesothelial-to-mesenchymal transition, a process analogous to epithelial-to-mesenchymal transition, and become fibrogenic cells. Fibrogenic mesothelial cells have now been identified in tissues where they have not previously been thought to occur, such as within the parenchyma of the fibrotic lung. These findings show a direct role for mesothelial cells in fibrogenesis and open therapeutic strategies to prevent or reverse the fibrotic process.

12.
Oncotarget ; 6(3): 1519-30, 2015 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-25544756

RESUMO

Gli transcription factors of the Hedgehog (Hh) pathway have been reported to be drivers of malignant mesothelioma (MMe) cell survival. The Gli inhibitor GANT61 induces apoptosis in various cancer cell models, and has been associated directly with Gli inhibition. However various chemotherapeutics can induce cell death through generation of reactive oxygen species (ROS) but whether ROS mediates GANT61-induced apoptosis is unknown. In this study human MMe cells were treated with GANT61 and the mechanisms regulating cell death investigated. Exposure of MMe cells to GANT61 led to G1 phase arrest and apoptosis, which involved ROS but not its purported targets, GLI1 or GLI2. GANT61 triggered ROS generation and quenching of ROS protected MMe cells from GANT61-induced apoptosis. Furthermore, we demonstrated that mitochondria are important in mediating GANT61 effects: (1) ROS production and apoptosis were blocked by mitochondrial inhibitor rotenone; (2) GANT61 promoted superoxide formation in mitochondria; and (3) mitochondrial DNA-deficient LO68 cells failed to induce superoxide, and were more resistant to apoptosis induced by GANT61 than wild-type cells. Our data demonstrate for the first time that GANT61 induces apoptosis by promoting mitochondrial superoxide generation independent of Gli inhibition, and highlights the therapeutic potential of mitochondrial ROS-mediated anticancer drugs in MMe.


Assuntos
Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Mesotelioma/tratamento farmacológico , Mesotelioma/metabolismo , Mitocôndrias/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Fase G1/efeitos dos fármacos , Células HCT116 , Células HT29 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mesotelioma/genética , Mesotelioma/patologia , Mesotelioma Maligno , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Fatores de Transcrição/metabolismo , Proteína GLI1 em Dedos de Zinco
13.
PLoS One ; 9(10): e110955, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25354194

RESUMO

Microtubules are a highly validated target in cancer therapy. However, the clinical development of tubulin binding agents (TBA) has been hampered by toxicity and chemoresistance issues and has necessitated the search for new TBAs. Here, we report the identification of a novel cell permeable, tubulin-destabilizing molecule--4,5,6,7-tetrahydro-1H-indazole-3-carboxylic acid [1p-tolyl-meth-(E)-ylidene]-hydrazide (termed as Suprafenacine, SRF). SRF, identified by in silico screening of annotated chemical libraries, was shown to bind microtubules at the colchicine-binding site and inhibit polymerization. This led to G2/M cell cycle arrest and cell death via a mitochondria-mediated apoptotic pathway. Cell death was preceded by loss of mitochondrial membrane potential, JNK-mediated phosphorylation of Bcl-2 and Bad, and activation of caspase-3. Intriguingly, SRF was found to selectively inhibit cancer cell proliferation and was effective against drug-resistant cancer cells by virtue of its ability to bypass the multidrug resistance transporter P-glycoprotein. Taken together, our results suggest that SRF has potential as a chemotherapeutic agent for cancer treatment and provides an alternate scaffold for the development of improved anti-cancer agents.


Assuntos
Antineoplásicos/farmacologia , Hidrazinas/farmacologia , Indazóis/farmacologia , Microtúbulos/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Apoptose , Sítios de Ligação , Colchicina/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular , Células HeLa , Humanos , Hidrazinas/química , Hidrazinas/isolamento & purificação , Indazóis/química , Indazóis/isolamento & purificação , Potencial da Membrana Mitocondrial , Camundongos , Microtúbulos/química , Microtúbulos/metabolismo , Dados de Sequência Molecular , Células PC12 , Ligação Proteica , Ratos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
14.
PLoS One ; 8(6): e66685, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23826113

RESUMO

BACKGROUND: The Hedgehog (HH) signaling pathway is critical for embryonic development and adult homeostasis. Recent studies have identified regulatory roles for this pathway in certain cancers with mutations in the HH pathway genes. The extent to which mutations of the HH pathway genes are involved in the pathogenesis of malignant mesothelioma (MMe) is unknown. METHODOLOGY/PRINCIPAL FINDINGS: Real-time PCR analysis of HH pathway genes PTCH1, GLI1 and GLI2 were performed on 7 human MMe cell lines. Exon sequencing of 13 HH pathway genes was also performed in cell lines and human MMe tumors. In silico programs were used to predict the likelihood that an amino-acid substitution would have a functional effect. GLI1, GLI2 and PTCH1 were highly expressed in MMe cells, indicative of active HH signaling. PTCH1, SMO and SUFU mutations were found in 2 of 11 MMe cell lines examined. A non-synonymous missense SUFU mutation (p.T411M) was identified in LO68 cells. In silico characterization of the SUFU mutant suggested that the p.T411M mutation might alter protein function. However, we were unable to demonstrate any functional effect of this mutation on Gli activity. Deletion of exons of the PTCH1 gene was found in JU77 cells, resulting in loss of one of two extracellular loops implicated in HH ligand binding and the intracellular C-terminal domain. A 3-bp insertion (69_70insCTG) in SMO, predicting an additional leucine residue in the signal peptide segment of SMO protein was also identified in LO68 cells and a MMe tumour. CONCLUSIONS/SIGNIFICANCE: We identified the first novel mutations in PTCH1, SUFU and SMO associated with MMe. Although HH pathway mutations are relatively rare in MMe, these data suggest a possible role for dysfunctional HH pathway in the pathogenesis of a subgroup of MMe and help rationalize the exploration of HH pathway inhibitors for MMe therapy.


Assuntos
Proteínas Hedgehog/metabolismo , Mesotelioma/metabolismo , Mutação , Transdução de Sinais , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Feminino , Proteínas Hedgehog/química , Proteínas Hedgehog/genética , Humanos , Masculino , Mesotelioma/genética , Mesotelioma/patologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos
15.
Integr Cancer Ther ; 9(1): 36-49, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20150221

RESUMO

An essential oil extract, derived from the rhizome of Curcuma wenyujin (CWE), possesses antioxidative, antimicrobial, and anti-inflammatory properties. However, it remains unknown how exactly CWE inhibits tumor growth. In this study, using human cervical cancer HeLa cells, the authors postulated that CWE has the ability to inhibit tumor growth. The study shows that CWE dose-dependently suppressed colony formation and inhibited the proliferation of HeLa cells through blockade of cell cycle progression at G1 phase and apoptosis. CWE-induced G1 arrest was associated with retinoblastoma protein dephosphorylation and reduced amounts of cyclins D1 and D3, and cyclin-dependent kinase 4 and 6 proteins. CWE treatment resulted in apoptosis in HeLa cells as evidenced by morphological changes, caspase activation and PARP cleavage, which can be reversed by a pan-caspase inhibitor. It was observed that CWE treatment activated the mitochondrial apoptotic pathway indicated by a decrease in Mcl-1 and Bcl-xL levels, resulting in mitochondrial membrane potential loss and caspases 9 activation. CWE-treated cells displayed reduced PTEN, AKT, and STAT3 phosphorylation and downregulation of NFkappaB signaling, providing a mechanism for the G1 arrest and apoptosis observed. Furthermore, CWE inhibited tumor growth of HeLa in a xenograft mouse tumor model, suggesting that CWE inhibited tumorigenesis by inhibiting cell proliferation and inducing apoptosis. These findings are the first to reveal the molecular basis for the anticervical cancer action of CWE. The results suggest that CWE could be developed as a drug for the management of cervical cancer.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Curcuma , Medicamentos de Ervas Chinesas/farmacologia , Neoplasias do Colo do Útero/patologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Curcuma/química , Regulação para Baixo/efeitos dos fármacos , Medicamentos de Ervas Chinesas/uso terapêutico , Feminino , Fase G1/efeitos dos fármacos , Células HeLa , Humanos , Camundongos , Camundongos SCID , Regulação para Cima/efeitos dos fármacos , Neoplasias do Colo do Útero/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Cell Div ; 1: 20, 2006 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-16959044

RESUMO

BACKGROUND: FAT10 is a member of the ubiquitin-like-modifier family of proteins. Over-expression of the FAT10 gene was observed in the tumors of several epithelial cancers. High FAT10 expression was found to lead to increased chromosome instability via the reduction in the kinetochore localization of MAD2 during the prometaphase stage of the cell-cycle. FAT10 expression was also previously reported to be regulated by cytokines and p53. RESULTS: Here, we report that FAT10 expression is regulated at the protein and transcript level during cell-cycle with highest expression observed during the S-phase of the cell-cycle. The distal region between -1997 to -975 bp from the transcription start site of the FAT10 promoter may play a role in the repression of FAT10 expression during G2/M phase of the cell-cycle. CONCLUSION: FAT10 expression is regulated during cell-cycle.

17.
Scand J Infect Dis ; 36(5): 342-9, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15287378

RESUMO

Nosocomial isolates of Pseudomonas aeruginosa exhibit high rates of resistance to antibiotics, and are often multidrug resistant. P. aeruginosa clinical isolates (n = 56) were obtained from ICU patients in a hospital in Pakistan over a 3-y period. Antimicrobial susceptibility of the 56 P. aeruginosa clinical isolates was investigated using 7 antibiotics and the resistance rates were as follows: aztreonam (68% resistant), ceftazidime (67%), imipenem (66%), ofloxacin (59%), amikacin (56%), gentamicin (44%), and piperacillin-tazobactam (27%) (p < 0.01). In addition, 55% of the P. aeruginosa clinical isolates were resistant to 4 or more antibiotics. Imipenem-resistant strains were frequently associated with ceftazidime, ofloxacin, aztreonam, and more strikingly, amikacin resistance (p < 0.05). PCR (using P. aeruginosa-specific primers VIC1 + VIC2 and P1 + P2, respectively) was highly specific and sensitive, and was positive for all 56 P. aeruginosa isolates tested. Automated ribotyping was used to investigate the clonal diversity of the 56 P. aeruginosa isolates. Automated ribotyping indicated that the clinical isolates were clonally related and could be clustered into 4 major ribogroups based on their similarity index, with ribogroup II being the dominant one. The P. aeruginosa isolates in ribogroup II were correlated with their antibiotic resistance pattern and, interestingly, there seemed to be a gradual acquisition of multiple antibiotic resistance associated with the isolates within this group over time. The ribotyping data, together with the antibiotic resistance profile, provide valuable molecular epidemiology information for the control of hospital-acquired P. aeruginosa infections.


Assuntos
Reação em Cadeia da Polimerase/métodos , Infecções por Pseudomonas/diagnóstico , Pseudomonas aeruginosa/isolamento & purificação , Antibacterianos/farmacologia , Automação , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/microbiologia , DNA Bacteriano/análise , Feminino , Humanos , Unidades de Terapia Intensiva , Masculino , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Ribotipagem , Estudos de Amostragem , Sensibilidade e Especificidade
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