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1.
J Virol ; 96(21): e0140122, 2022 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-36286481

RESUMO

Macrophages are one of the first innate immune infiltrates in the cornea of mice following ocular infection with herpes simplex virus 1 (HSV-1). Using gamma interferon (IFN-γ) and interleukin-4 (IL-4) injections to polarize macrophages into M1 and M2, respectively, and in M1 and M2 conditional knockout mice, we have shown that M1 macrophages play an important role in suppressing both virus replication in the eye and eye disease in HSV-1-infected mice. Autophagy is also important in controlling HSV infection and integrity of infected cells. To determine if blocking autophagy in M1 and M2 macrophages affects HSV-1 infectivity and eye disease, we generated two transgenic mouse strains expressing the HSV-1 γ34.5 autophagy gene under the M1 promoter (M1-γ34.5) or the M2 promoter (M2-γ34.5). We found that blocking autophagy in M1 macrophages increased both virus replication in the eyes and eye disease in comparison to blocking autophagy in M2 macrophages or wild-type (WT) control mice, but blocked autophagy did not affect latency-reactivation. However, blocking autophagy affected fertility in both M1 and M2 transgenic mice. Analysis of 62 autophagy genes and 32 cytokines/chemokines from infected bone marrow-derived macrophages from M1-γ34.5, M2-γ34.5, and WT mice suggested that upregulation of autophagy-blocking genes (i.e., Hif1a, Mtmr14, mTOR, Mtmr3, Stk11, and ULK2) and the inflammatory tumor necrosis factor alpha (TNF-α) gene in M1-γ34.5 transgenic mice correlated with increased pathogenicity, while upregulation of proautophagy genes (Nrbf2 and Rb1cc1) in M2-γ34.5 macrophages correlated with reduced pathogenicity. The in vivo and in vitro responses of M1-γ34.5 and M2-γ34.5 transgenic mice to HSV-1 infection were independent of the presence of the γ34.5 gene in wild-type HSV-1. Our results suggest that M1 macrophages, but not M2 macrophages, play an important role in autophagy relative to primary virus replication in the eye and eye disease in infected mice. IMPORTANCE Autophagy plays a critical role in clearing, disassembling, and recycling damaged cells, thus limiting inflammation. The HSV-1 γ34.5 gene is involved in neurovirulence and immune evasion by blocking autophagy in infected cells. We found that blocking autophagy in M1 macrophages enhances HSV-1 virus replication in the eye and eye disease in ocularly infected transgenic mice. Our results also show the suppressive effects of γ34.5 on immune responses to infection, suggesting the importance of intact autophagy in M1 but not M2 macrophages in controlling primary infection and eye disease.


Assuntos
Oftalmopatias , Herpes Simples , Herpesvirus Humano 1 , Camundongos , Animais , Camundongos Transgênicos , Herpesvirus Humano 1/fisiologia , Replicação Viral , Macrófagos , Camundongos Knockout , Córnea , Interferon gama/genética , Autofagia , Proteínas Relacionadas à Autofagia , Transativadores , Monoéster Fosfórico Hidrolases
2.
Oncotarget ; 12(18): 1749-1762, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34504648

RESUMO

Triple negative breast cancer (TNBC) is a deadly disease with limited treatment options. Selinexor is a selective inhibitor of nuclear export that binds covalently to exportin 1 thereby reactivating tumor suppressor proteins and downregulating expression of oncogenes and DNA damage repair (DDR) proteins. Olaparib is a poly (ADP-ribose) polymerase (PARP) inhibitor approved for the treatment of patients with breast cancer harboring BRCA mutations. We examined the effects of co-treatment with selinexor and olaparib in TNBC cell lines. BRCA1 wildtype (BRCA1-wt) and BRCA1 mutant (BRCA1-mut) TNBC cell lines were treated with selinexor and/or olaparib and effects on cell viability and cell cycle were evaluated. The effects of treatment were also evaluated in mouse xenograft models generated with BRCA1-wt and BRCA1-mut TNBC cell lines. Treatment with selinexor inhibited cell proliferation and survival of all TNBC cell lines tested in vitro. This effect was enhanced following treatment of the cells with the combination of selinexor and olaparib, which showed synergistic effects on tumor growth inhibition in MDA-MB-468-derived (BRCA1-wt) and MDA-MB-436-derived (BRCA1-mut) xenografts. As co-treatment with selinexor and olaparib exhibits anti-tumor activity regardless of BRCA1 mutation status, the clinical implications of the combination warrant further investigation.

3.
PLoS One ; 14(4): e0215727, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30998796

RESUMO

Complex interactions between HSV-1 and infiltrating immune cells play important roles in establishing localized, acute virus replication as well as chronic latent infection. The extent and duration of initial virus replication are the key determinants of subsequent pathologic inflammatory responses and therefore, the accumulation of immune cell populations at this time point is a key target for prevention. Therefore, we evaluated the role of various immune cell infiltrates between 1 h and 28 days post-infection (PI) using mice infected with virulent HSV-1 strain McKrae without corneal scarification. The effect of corneal scarification on immune cell infiltrates was also determined. We first determined the activation status and origin of macrophage infiltrates as early as 1 h PI. We found a sharp increase in the total macrophage population after 12 h PI, that was primarily due to infiltration of CCR2+ migratory macrophages, mostly in M1 status (MHC II+). The number of CCR2- resident macrophages, mostly unpolarized (M0), increased gradually over time and peaked at 48 h PI. Interestingly, some of the resident macrophages gained an M2-like phenotype (CD206Low), which peaked at 12 h PI, concurrent with M1 macrophage infiltration. From 1-7 days PI, infiltration of various immune cells correlated strongly with HSV-1 replication, with neutrophils showing the biggest increase, and NKT cells the biggest decrease, after infection. The presence of geographical ulcer did not correlate with increased infiltration, while mice with corneal scarring had significantly more immune cell infiltration than those without corneal scarring. Overall, we showed time-dependent infiltration of various immune cells in the eye of HSV-1 infected mice. Initial infiltration of macrophages followed by infiltration of T cells at later times PI demonstrates the importance of targeting macrophages rather than other immune cells type, for therapeutic treatment of HSV-1.


Assuntos
Movimento Celular/imunologia , Córnea/imunologia , Herpesvirus Humano 1/fisiologia , Ceratite Herpética/imunologia , Macrófagos/imunologia , Receptores CCR2/imunologia , Replicação Viral/imunologia , Animais , Linhagem Celular , Córnea/patologia , Feminino , Ceratite Herpética/patologia , Macrófagos/patologia , Camundongos , Coelhos
4.
Biomed Pharmacother ; 99: 543-551, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29902865

RESUMO

PURPOSE: Despite similarities with BRCA-mutated breast cancers, triple-negative breast cancers (TNBC) remain resistant to poly(ADP-ribose) polymerase (PARP) inhibitors as single agents. Histone deacetylase inhibitors (HDACi) can decrease expression of proteins involved in DNA repair. We thus hypothesized that a HDACi (suberoylanilide hydroxamic acid (SAHA) or belinostat) could sensitize TNBC to the PARP inhibitor olaparib. METHODS: Human TNBC cells were co-treated with olaparib and either SAHA or belinostat, and their effects on survival, proliferation, cell cycle, apoptosis and DNA repair pathways were evaluated. Subcutaneous xenografts were used to determine the effect of the combination treatment in vivo. RESULTS: HDACi and olaparib synergistically inhibited proliferation of a panel of 8 TNBC cell lines in vitro and in nude mice harboring TNBC xenografts in vivo. We noted a weaker synergism in PTEN-deficient TNBC cells and a stronger synergism in BRCA1-mutated TNBC cells. In the BRCA1-mutated cell line HCC-1937, we observed a drastic decrease in the expression of proteins involved in homologous recombination (HR), leading to a large imbalance of the ratio P-H2AX/RAD51. In BRCA1 wild type (wt) cell lines, effect of the combination treatment relied on DNA damage-induced cell cycle arrest followed by induction of apoptosis. CONCLUSION: In summary, these results provide a preclinical rationale to combine a HDACi with a PARP inhibitor to reduce HR efficiency in TNBC and sensitize these aggressive tumors to PARP inhibition.


Assuntos
Proteína BRCA1/genética , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/metabolismo , Mutação/genética , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Poli(ADP-Ribose) Polimerases/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Camundongos Nus , Ftalazinas/farmacologia , Ftalazinas/uso terapêutico , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Neoplasias de Mama Triplo Negativas/patologia , Vorinostat , Ensaios Antitumorais Modelo de Xenoenxerto
5.
J Virol ; 92(10)2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29491152

RESUMO

We found previously that altering macrophage polarization toward M2 responses by injection of colony-stimulating factor 1 (CSF-1) was more effective in reducing both primary and latent infections in mice ocularly infected with herpes simplex virus 1 (HSV-1) than M1 polarization by gamma interferon (IFN-γ) injection. Cytokines can coordinately regulate macrophage and T helper (TH) responses, with interleukin-4 (IL-4) inducing type 2 TH (TH2) as well as M2 responses and IFN-γ inducing TH1 as well as M1 responses. We have now differentiated the contributions of these immune compartments to protection against latency reactivation and corneal scarring by comparing the effects of infection with recombinant HSV-1 in which the latency-associated transcript (LAT) gene was replaced with either the IL-4 (HSV-IL-4) or IFN-γ (HSV-IFN-γ) gene using infection with the parental (LAT-negative) virus as a control. Analysis of peritoneal macrophages in vitro established that the replacement of LAT with the IL-4 or IFN-γ gene did not affect virus infectivity and promoted polarization appropriately. Protection against corneal scarring was significantly higher in mice ocularly infected with HSV-IL-4 than in those infected with HSV-IFN-γ or parental virus. Levels of primary virus replication in the eyes and trigeminal ganglia (TG) were similar in the three groups of mice, but the numbers of gC+ cells were lower on day 5 postinfection in the eyes of HSV-IL-4-infected mice than in those infected with HSV-IFN-γ or parental virus. Latency and explant reactivation were lower in both HSV-IL-4- and HSV-IFN-γ-infected mice than in those infected with parental virus, with the lowest level of latency being associated with HSV-IL-4 infection. Higher latency correlated with higher levels of CD8, PD-1, and IFN-γ mRNA, while reduced latency and T-cell exhaustion correlated with lower gC+ expression in the TG. Depletion of macrophages increased the levels of latency in all ocularly infected mice compared with their undepleted counterparts, with macrophage depletion increasing latency in the HSV-IL-4 group greater than 3,000-fold. Our results suggest that shifting the innate macrophage immune responses toward M2, rather than M1, responses in HSV-1 infection would improve protection against establishment of latency, reactivation, and eye disease.IMPORTANCE Ocular HSV-1 infections are among the most frequent serious viral eye infections in the United States and a major cause of virus-induced blindness. As establishment of a latent infection in the trigeminal ganglia results in recurrent infection and is associated with corneal scarring, prevention of latency reactivation is a major therapeutic goal. It is well established that absence of latency-associated transcripts (LATs) reduces latency reactivation. Here we demonstrate that recombinant HSV-1 expressing IL-4 (an inducer of TH2/M2 responses) or IFN-γ (an inducer of TH1/M1 responses) in place of LAT further reduced latency, with HSV-IL-4 showing the highest overall protective efficacy. In naive mice, this higher protective efficacy was mediated by innate rather than adaptive immune responses. Although both M1 and M2 macrophage responses were protective, shifting macrophages toward an M2 response through expression of IL-4 was more effective in curtailing ocular HSV-1 latency reactivation.


Assuntos
Herpes Simples/imunologia , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/fisiologia , Interleucina-4/imunologia , Macrófagos Peritoneais/imunologia , Células Th2/imunologia , Ativação Viral/imunologia , Animais , Células Cultivadas , Lesões da Córnea/imunologia , Lesões da Córnea/prevenção & controle , Lesões da Córnea/virologia , Olho/imunologia , Olho/virologia , Oftalmopatias/virologia , Infecções Oculares/imunologia , Infecções Oculares/virologia , Feminino , Herpes Simples/virologia , Interferon gama/genética , Interleucina-4/biossíntese , Interleucina-4/genética , Macrófagos Peritoneais/classificação , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Coelhos , Gânglio Trigeminal/imunologia , Gânglio Trigeminal/virologia , Latência Viral/fisiologia , Replicação Viral/imunologia
7.
J Virol ; 91(15)2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28490589

RESUMO

Macrophages are the predominant infiltrate in the corneas of mice that have been ocularly infected with herpes simplex virus 1 (HSV-1). However, very little is known about the relative roles of M1 (classically activated or polarized) and M2 (alternatively activated or polarized) macrophages in ocular HSV-1 infection. To better understand these relationships, we assessed the impact of directed M1 or M2 activation of RAW264.7 macrophages and peritoneal macrophages (PM) on subsequent HSV-1 infection. In both the RAW264.7 macrophage and PM in vitro models, HSV-1 replication in M1 macrophages was markedly lower than in M2 macrophages and unstimulated controls. The M1 macrophages expressed significantly higher levels of 28 of the 32 tested cytokines and chemokines than M2 macrophages, with HSV-1 infection significantly increasing the levels of proinflammatory cytokines and chemokines in the M1 versus the M2 macrophages. To examine the effects of shifting the immune response toward either M1 or M2 macrophages in vivo, wild-type mice were injected with gamma interferon (IFN-γ) DNA or colony-stimulating factor 1 (CSF-1) DNA prior to ocular infection with HSV-1. Virus replication in the eye, latency in trigeminal ganglia (TG), and markers of T cell exhaustion in the TG were determined. We found that injection of mice with IFN-γ DNA, which enhances the development of M1 macrophages, increased virus replication in the eye; increased latency; and also increased CD4, CD8, IFN-γ, and PD-1 transcripts in the TG of latently infected mice. Conversely, injection of mice with CSF-1 DNA, which enhances the development of M2 macrophages, was associated with reduced virus replication in the eye and reduced latency and reduced the levels of CD4, CD8, IFN-γ,and PD-1 transcripts in the TG. Collectively, these results suggest that M2 macrophages directly reduce the levels of HSV-1 latency and, thus, T-cell exhaustion in the TG of ocularly infected mice.IMPORTANCE Our findings demonstrate a novel approach to further reducing HSV-1 replication in the eye and latency in the TG by modulating immune components, specifically, by altering the phenotype of macrophages. We suggest that inclusion of CSF-1 as part of any vaccination regimen against HSV infection to coax responses of macrophages toward an M2, rather than an M1, response may further improve vaccine efficacy against ocular HSV-1 replication and latency.


Assuntos
Herpesvirus Humano 1/fisiologia , Ceratite Herpética/imunologia , Ceratite Herpética/virologia , Macrófagos/virologia , Replicação Viral , Animais , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Camundongos , Gânglio Trigeminal/virologia , Latência Viral
8.
PLoS Pathog ; 13(5): e1006401, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28542613

RESUMO

We have established two mouse models of central nervous system (CNS) demyelination that differ from most other available models of multiple sclerosis (MS) in that they represent a mixture of viral and immune triggers. In the first model, ocular infection of different strains of mice with a recombinant HSV-1 that expresses murine IL-2 constitutively (HSV-IL-2) causes CNS demyelination. In the second model, depletion of macrophages causes CNS demyelination in mice that are ocularly infected with wild-type (WT) HSV-1. In the present study, we found that the demyelination in macrophage-intact mice infected with HSV-IL-2 was blocked by depletion of FoxP3-expressing cells, while concurrent depletion of macrophages restored demyelination. In contrast, demyelination was blocked in the macrophage-depleted mice infected with wild-type HSV-1 following depletion of FoxP3-expressing cells. In macrophage-depleted HSV-IL-2-infected mice, demyelination was associated with the activity of both CD4+ and CD8+ T cells, whereas in macrophage-depleted mice infected with WT HSV-1, demyelination was associated with CD4+ T cells. Macrophage depletion or infection with HSV-IL-2 caused an imbalance of T cells and TH1 responses as well as alterations in IL-12p35 and IL-12p40 but not other members of the IL-12 family or their receptors. Demyelination was blocked by adoptive transfer of macrophages that were infected with HSV-IL-12p70 or HSV-IL-12p40 but not by HSV-IL-12p35. These results indicate that suppression of IL-12p70 formation by IL-2 or following macrophage depletion causes T-cell autoreactivity leading to CNS demyelination in HSV-1-infected mice.


Assuntos
Sistema Nervoso Central/imunologia , Herpesvirus Humano 1/fisiologia , Interleucina-12/imunologia , Interleucina-2/imunologia , Macrófagos/citologia , Esclerose Múltipla/imunologia , Bainha de Mielina/metabolismo , Linfócitos T/imunologia , Animais , Sistema Nervoso Central/metabolismo , Modelos Animais de Doenças , Herpesvirus Humano 1/genética , Humanos , Interleucina-12/genética , Interleucina-2/genética , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Esclerose Múltipla/virologia , Bainha de Mielina/imunologia
9.
Int J Oncol ; 50(3): 863-872, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28197627

RESUMO

Osteosarcoma (OS) has a high degree of chromosomal instability and total copy number (CN) changes. We examined 58 human OS samples including 40 primary tumors, 11 explants, and 7 cell lines using single nucleotide polymorphism (SNP) arrays, and revealed that 70% of the samples had one or more recurrent CN-neutral loss of heterozygosity (CNN­LOH) also known as uniparental disomy (UPD). Importantly, 17% of the samples showed prominent homozygous deletion of 3q13.31, suggesting its role in tumorigenesis. We identified and characterized two novel lncRNAs, LOC285194 and BC040587, within this genomic locus, strongly suggesting their tumor suppressor activity. Frequent deletions and UPD suggest that OS often has mutant or non-expressed tumor suppressor genes including two lncRNAs.


Assuntos
Variações do Número de Cópias de DNA/genética , Dosagem de Genes/genética , Genes Supressores de Tumor , Osteossarcoma/genética , RNA Longo não Codificante/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único/genética , Transplante Heterólogo , Dissomia Uniparental/genética
10.
RNA Biol ; 14(3): 305-316, 2017 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-27858503

RESUMO

Extracellular vesicles (EVs) contain a wide range of RNA types with a reported prevalence of non-coding RNA. To date a comprehensive characterization of the protein coding transcripts in EVs is still lacking. We performed RNA-Sequencing (RNA-Seq) of 2 EV populations and identified a small fraction of transcripts that were expressed at significantly different levels in large oncosomes and exosomes, suggesting they may mediate specialized functions. However, these 2 EV populations exhibited a common mRNA signature that, in comparison to their donor cells, was significantly enriched in mRNAs encoding E2F transcriptional targets and histone proteins. These mRNAs are primarily expressed in the S-phase of the cell cycle, suggesting that they may be packaged into EVs during S-phase. In silico analysis using subcellular compartment transcriptome data from the ENCODE cell line compendium revealed that EV mRNAs originate from a cytoplasmic RNA pool. The EV signature was independently identified in plasma of patients with breast cancer by RNA-Seq. Furthermore, several transcripts differentially expressed in EVs from patients versus controls mirrored differential expression between normal and breast cancer tissues. Altogether, this largest high-throughput profiling of EV mRNA demonstrates that EVs carry tumor-specific alterations and can be interrogated as a source of cancer-derived cargo.


Assuntos
Biomarcadores Tumorais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Vesículas Extracelulares/metabolismo , RNA Mensageiro/genética , Neoplasias da Mama/sangue , Ciclo Celular/genética , Linhagem Celular Tumoral , Biologia Computacional/métodos , Citosol/metabolismo , Fator de Transcrição E2F4/metabolismo , Feminino , Perfilação da Expressão Gênica , Ontologia Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Sinais Direcionadores de Proteínas/genética , RNA Mensageiro/sangue , Transcriptoma , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
11.
Oncotarget ; 6(40): 42429-44, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26643872

RESUMO

Liposarcoma (LPS) is the most common type of soft tissue sarcoma accounting for 20% of all adult sarcomas. Due to absence of clinically effective treatment options in inoperable situations and resistance to chemotherapeutics, a critical need exists to identify novel therapeutic targets. We analyzed LPS genomic landscape using SNP arrays, whole exome sequencing and targeted exome sequencing to uncover the genomic information for development of specific anti-cancer targets. SNP array analysis indicated known amplified genes (MDM2, CDK4, HMGA2) and important novel genes (UAP1, MIR557, LAMA4, CPM, IGF2, ERBB3, IGF1R). Carboxypeptidase M (CPM), recurrently amplified gene in well-differentiated/de-differentiated LPS was noted as a putative oncogene involved in the EGFR pathway. Notable deletions were found at chromosome 1p (RUNX3, ARID1A), chromosome 11q (ATM, CHEK1) and chromosome 13q14.2 (MIR15A, MIR16-1). Significantly and recurrently mutated genes (false discovery rate < 0.05) included PLEC (27%), MXRA5 (21%), FAT3 (24%), NF1 (20%), MDC1 (10%), TP53 (7%) and CHEK2 (6%). Further, in vitro and in vivo functional studies provided evidence for the tumor suppressor role for Neurofibromin 1 (NF1) gene in different subtypes of LPS. Pathway analysis of recurrent mutations demonstrated signaling through MAPK, JAK-STAT, Wnt, ErbB, axon guidance, apoptosis, DNA damage repair and cell cycle pathways were involved in liposarcomagenesis. Interestingly, we also found mutational and copy number heterogeneity within a primary LPS tumor signifying the importance of multi-region sequencing for cancer-genome guided therapy. In summary, these findings provide insight into the genomic complexity of LPS and highlight potential druggable pathways for targeted therapeutic approach.


Assuntos
Lipossarcoma/genética , Neoplasias de Tecidos Moles/genética , Animais , Análise Mutacional de DNA , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Xenoenxertos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Transcriptoma
12.
Sci Rep ; 5: 12580, 2015 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-26219418

RESUMO

Dedifferentiated liposarcoma (DDLPS) is a highly malignant subtype of human liposarcoma (LPS), whose genomic profile is characterized by chromosomal amplification at 12q13-q22. miR-26a-2 is one of the most frequently amplified genes in the region, and inhibition of its downstream target genes likely contributes to LPS tumorigenesis. Our previous study of LPS predicted homeobox protein A5 (HOXA5) as a target of miR-26a-2, and here we explored further the function of HOXA5, and its relationship with miR-26a-2 in DDLPS cells. Compared to normal human adipocytes, all LPS cell lines showed significant downregulation of HOXA5 (p = 0.046), and inhibition of miR-26a-2 using anti-miR-26a-2 substantially upregulated HOXA5 expression in these LPS cells. Interestingly, overexpression of HOXA5 alone induced very strong apoptotic response of LPS cells. HOXA5-induced apoptosis was p53-independent and caspase-dependent. Surprisingly, overexpression of HOXA5 induced nuclear translocation of RELA (p65), which was not associated with the transcriptional activity of RELA. Rather, nucleolar sequestration of RELA was observed. Overall, our study demonstrated for the first time that the downregulation of HOXA5 in LPS cells, partly by overexpression of miR-26a-2 in DDLPS, confers LPS cells resistance to apoptotic death. Further studies are required to understand the relationship of HOXA5 and the NFκB pathway in LPS cells.


Assuntos
Apoptose/genética , Expressão Ectópica do Gene/genética , Proteínas de Homeodomínio/genética , Lipossarcoma/genética , Proteína Supressora de Tumor p53/genética , Carcinogênese/genética , Caspases/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Regulação para Baixo/genética , Amplificação de Genes/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , MicroRNAs/genética , NF-kappa B/genética , Ativação Transcricional/genética , Regulação para Cima/genética
13.
Int J Cancer ; 136(9): 2055-64, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25307878

RESUMO

Bromodomain and extra terminal domain (BET) proteins are important epigenetic regulators facilitating the transcription of genes in chromatin areas linked to acetylated histones. JQ1, a BET protein inhibitor, has antiproliferative activity against many cancers, mainly through inhibition of c-MYC and upregulation of p21. In this research, we investigated the use of JQ1 for human osteosarcoma (OS) treatment. JQ1 significantly inhibited the proliferation and survival of OS cells inducing G1 cell cycle arrest, premature senescence, but little effect on apoptosis. Interestingly, c-MYC protein levels in JQ1-treated cells remained unchanged, whereas the upregulation of p21 protein was still observable. Although effective in vitro, JQ1 alone failed to reduce the size of the MNNG/HOS xenografts in immunocompromised mice. To overcome the resistance of OS cells to JQ1 treatment, we combined JQ1 with rapamycin, an mammalian target of rapamycin (mTOR) inhibitor. JQ1 and rapamycin synergistically inhibited the growth and survival of OS cells in vitro and in vivo. We also identified that RUNX2 is a direct target of bromodomain-containing protein 4 (BRD4) inhibition by JQ1 in OS cells. Chromatin immunoprecipitation (ChIP) showed that enrichment of BRD4 protein around RUNX2 transcription start sites diminished with JQ1 treatment in MNNG/HOS cells. Overexpression of RUNX2 protected JQ1-sensitive OS cells from the effect of JQ1, and siRNA-mediated inhibition of RUNX2 sensitized the same cells to JQ1. In conclusion, our findings suggest that JQ1, in combination with rapamycin, is an effective chemotherapeutic option for OS treatment. We also show that inhibition of RUNX2 expression by JQ1 partly explains the antiproliferative activity of JQ1 in OS cells.


Assuntos
Azepinas/farmacologia , Osteossarcoma/tratamento farmacológico , Sirolimo/farmacologia , Triazóis/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Sinergismo Farmacológico , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Genes myc/genética , Humanos , Camundongos , Camundongos Nus , Proteínas Nucleares/metabolismo , Osteossarcoma/metabolismo , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
14.
Mol Carcinog ; 53(9): 722-35, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23475695

RESUMO

Pancreatic ductal adenocarcinoma is a devastating disease with few therapeutic options. Histone deacetylase inhibitors are a novel therapeutic approach to cancer treatment; and two new pan-histone deacetylase inhibitors (HDACi), belinostat and panobinostat, are undergoing clinical trials for advanced hematologic malignancies, non-small cell lung cancers and advanced ovarian epithelial cancers. We found that belinostat and panobinostat potently inhibited, in a dose-dependent manner, the growth of six (AsPc1, BxPc3, Panc0327, Panc0403, Panc1005, MiaPaCa2) of 14 human pancreatic cancer cell lines. Belinostat increased the percentage of apoptotic pancreatic cancer cells and caused prominent G2 /M growth arrest of most pancreatic cancer cells. Belinostat prominently inhibited PI3K-mTOR-4EBP1 signaling with a 50% suppression of phorphorylated 4EBP1 (AsPc1, BxPc3, Panc0327, Panc1005 cells). Surprisingly, belinostat profoundly blocked hypoxia signaling including the suppression of hypoxia response element reporter activity; as well as an approximately 10-fold decreased transcriptional expression of VEGF, adrenomedullin, and HIF1α at 1% compared to 20% O2 . Treatment with this HDACi decreased levels of thioredoxin mRNA associated with increased levels of its endogenous inhibitor thioredoxin binding protein-2. Also, belinostat alone and synergistically with gemcitabine significantly (P = 0.0044) decreased the size of human pancreatic tumors grown in immunodeficiency mice. Taken together, HDACi decreases growth, increases apoptosis, and is associated with blocking the AKT/mTOR pathway. Surprisingly, it blocked hypoxic growth related signals. Our studies of belinostat suggest it may be an effective drug for the treatment of pancreatic cancers when used in combination with other drugs such as gemcitabine.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , NF-kappa B/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Técnicas In Vitro , Indóis/farmacologia , Camundongos , NF-kappa B/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Panobinostat , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina-Treonina Quinases TOR/genética , Células Tumorais Cultivadas
15.
Int J Cancer ; 132(12): 2730-7, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23165325

RESUMO

Despite recent advances in therapy, breast cancer remains the second most common cause of death from malignancy in women. Chemotherapy plays a major role in breast cancer management, and combining chemotherapeutic agents with nonchemotherapeutic agents is of considerable clinical interest. Cucurbitacins are triterpenes compounds found in plants of the Cucurbitaceae family, reported to have anticancer and anti-inflammatory activities. Previously, we have shown antiproliferative activity of cucurbitacin B (CuB) in breast cancer, and we hypothesized that combining CuB with chemotherapeutic agents can augment their antitumor effect. Here, we show that a combination of CuB with either docetaxel (DOC) or gemcitabine (GEM) synergistically inhibited the proliferation of MDA-MB-231 breast cancer cells in vitro. This antiproliferative effect was accompanied by an increase in apoptosis rates. Furthermore, in vivo treatment of human breast cancer orthotopic xenografts in immunodeficient mice with CuB at either low (0.5 mg/kg) or high (1 mg/kg) doses in combination with either DOC (20 mg/kg) or GEM (12.5mg/kg) significantly reduced tumor volume as compared with monotherapy of each drug. Importantly, no significant toxicity was noted with low-dose CuB in combination with either DOC or GEM. In conclusion, combination of CuB at a relatively low concentration with either of the chemotherapeutic agents, DOC or GEM, shows prominent antiproliferative activity against breast cancer cells without increased toxicity. This promising combination should be examined in therapeutic trials of breast cancer.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/patologia , Triterpenos/farmacologia , Animais , Antineoplásicos Fitogênicos/toxicidade , Apoptose/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Docetaxel , Sinergismo Farmacológico , Feminino , Humanos , Camundongos , Taxoides/farmacologia , Triterpenos/toxicidade , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Gencitabina
16.
Cancer Lett ; 306(2): 161-170, 2011 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-21440986

RESUMO

We investigated the use of cucurbitacin B, a plant-derived tetracyclic triterpenoid, as a single agent or in combination with methotrexate (MTX) for human osteosarcoma (OS) treatment. Cucurbitacin B showed antiproliferative activity against seven human OS cell lines in vitro accompanying G2/M cell cycle arrest, apoptosis, and inhibition of ERK, Akt, and mTOR proteins. Cucurbitacin B in combination with MTX synergistically inhibited OS cell growth in vitro. Low-dose cucurbitacin B (LD-CuB, 0.5 mg/kg body weight) or low-dose MTX (LD-MTX, 150 mg/kg) failed to decrease the size of human OS xenografts in nude mice. However, combined therapy at identical concentrations inhibited tumor growth by 62% vs. LD-CuB and 81% vs. LD-MTX (p<0.001). Strikingly, the effect persisted even when the dose of MTX was decreased by two thirds (VLD-MTX, 50 mg/kg). In conclusion, cucurbitacin B alone or in combination with MTX shows promising antiproliferative activity against human OS.


Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Metotrexato/uso terapêutico , Osteossarcoma/tratamento farmacológico , Triterpenos/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
17.
ScientificWorldJournal ; 10: 413-8, 2010 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-20209387

RESUMO

Cucurbitacins and their derivatives are triterpenoids found in medicinal plants known for their diverse pharmacological and biological activities, including anticancer effects, throughout human history. Although initial attention to cucurbitacin as a potential anticancer drug withered for decades, recent discoveries showing that cucurbitacin is a strong STAT3 (Signal Transducers and Activators of Transcription-3) inhibitor have reclaimed the attention of the drug industry one more time. There is increasing evidence showing that some cucurbitacins not only inhibit the JAK-STAT pathway, but also affect other signaling pathways, such as the MAPK pathway, which are also known to be important for cancer cell proliferation and survival. Moreover, some reports have shown the synergistic effect of cucurbitacins with known chemotherapeutic agents, such as doxorubicin and gemcitabine. In this review, we will summarize the recent discoveries regarding molecular mechanisms of action of cucurbitacins in human cancer cells and discuss the possibilities of cucurbitacin as a future anticancer drug in clinical settings.


Assuntos
Antineoplásicos/farmacologia , Cucurbitacinas/farmacologia , Neoplasias/patologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Sistema de Sinalização das MAP Quinases , Fatores de Transcrição/metabolismo
18.
Blood ; 115(14): 2882-90, 2010 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-20068225

RESUMO

Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) including polycythemia vera, essential thrombocythemia, and primary myelofibrosis show an inherent tendency for transformation into leukemia (MPN-blast phase), which is hypothesized to be accompanied by acquisition of additional genomic lesions. We, therefore, examined chromosomal abnormalities by high-resolution single nucleotide polymorphism (SNP) array in 88 MPN patients, as well as 71 cases with MPN-blast phase, and correlated these findings with their clinical parameters. Frequent genomic alterations were found in MPN after leukemic transformation with up to 3-fold more genomic changes per sample compared with samples in chronic phase (P < .001). We identified commonly altered regions involved in disease progression including not only established targets (ETV6, TP53, and RUNX1) but also new candidate genes on 7q, 16q, 19p, and 21q. Moreover, trisomy 8 or amplification of 8q24 (MYC) was almost exclusively detected in JAK2V617F(-) cases with MPN-blast phase. Remarkably, copy number-neutral loss of heterozygosity (CNN-LOH) on either 7q or 9p including homozygous JAK2V617F was related to decreased survival after leukemic transformation (P = .01 and P = .016, respectively). Our high-density SNP-array analysis of MPN genomes in the chronic compared with leukemic stage identified novel target genes and provided prognostic insights associated with the evolution to leukemia.


Assuntos
Crise Blástica/genética , Cromossomos Humanos/genética , Neoplasias Hematológicas/genética , Janus Quinase 2/genética , Perda de Heterozigosidade , Proteínas de Neoplasias/genética , Cromossomo Filadélfia , Idoso , Crise Blástica/metabolismo , Crise Blástica/mortalidade , Cromossomos Humanos/metabolismo , Intervalo Livre de Doença , Feminino , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/mortalidade , Humanos , Janus Quinase 2/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Prevalência , Taxa de Sobrevida
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