RESUMO
Primary effusion lymphoma (PEL), a subtype of non-Hodgkin's B-lymphoma, is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients. Endoplasmic reticulum (ER) stress induces activation of the unfolded protein response (UPR), which induces expression of ER chaperones, which in turn decrease ER stress, leading to ER homeostasis. The UPR is necessary for not only ER homeostasis but also persistent infection by, and replication of, many viruses. However, the precise roles and regulation of the UPR in KSHV infection remain poorly understood. Here, we found that IRE1α and PERK were significantly downregulated in PEL cells cultured under normal conditions, compared with KSHV-uninfected B-lymphoma cells. IRE1α and PERK mRNA levels were decreased in PEL cells, and KSHV-encoded LANA and v-cyclin D led to suppressed IRE1α transcription. Thapsigargin-induced ER stress activated the UPR and increased the mRNA levels of UPR-related molecules, including IRE1α and PERK, in PEL cells. However the IRE1α and PERK mRNA levels in PEL cells were lower than those in KSHV-uninfected cells. Furthermore, ER stress induced by brefeldin A and thapsigargin dramatically reduced the viability of PEL cells, compared with KSHV-uninfected cells, and induced apoptosis of PEL cells via the pro-apoptotic UPR through expression of CHOP and activation of caspase-9. In addition to the pro-apoptotic UPR, thapsigargin-induced ER stress enhanced transcription of lytic genes, including RTA, K-bZIP and K8.1, and viral production in PEL cells resulted in induction of the lytic cycle. Thus, we demonstrated downregulation of IRE1α and PERK in PEL cells, transcriptional suppression of IRE1α by LANA and v-cyclin D, apoptosis induction in PEL cells by ER stress, and potentiation of lytic replication by ER stress.
Assuntos
Estresse do Retículo Endoplasmático , Herpesvirus Humano 8/fisiologia , Linfoma de Efusão Primária/fisiopatologia , Linfoma de Efusão Primária/virologia , Resposta a Proteínas não Dobradas , Replicação Viral/fisiologia , Sobrevivência Celular , Células HeLa , Humanos , Linfoma de Efusão Primária/patologiaRESUMO
The Sonic hedgehog (Shh) signaling pathway plays a crucial role in cell proliferation and differentiation via Patched1 (Ptc1), a 12-pass transmembrane receptor protein. The C-terminal cytoplasmic tail of Ptc1 can be cleaved to release the 7th intracellular domain (ICD7), whose function is still unclear. In this study, we found that the ICD7 fragment of Ptc1 associates with polyubiquitinated species. Using mass spectrometry, we identified a cluster of E3 ubiquitin ligase complex as novel Ptc1 ICD7-binding proteins. In particular, Ptc1 ICD7 interacted with most components of the Cullin-2 (CUL2)-based E3 ligase complex, including TCEB1 (EloC), TCEB2 (EloB), ZYG11B, and CUL2 itself. To address the significance of CUL2-based E3 ligase in Ptc1 function, we examined the effects of CUL2 knockdown on Shh-induced osteoblast differentiation in the mesenchymal stem cell line C3H10T1/2. Indeed, knockdown of CUL2 abolished the Shh-induced stem cell differentiation. These results suggest that CUL2-based E3 ligase complex may play a role in Shh- and Ptc1-dependent signaling pathways.
Assuntos
Citoplasma/metabolismo , Receptor Patched-1/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Diferenciação Celular/fisiologia , Células HEK293 , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C3H , Células-Tronco/citologia , Ubiquitina/metabolismoRESUMO
Sangivamycin, a structural analog of adenosine and antibiotic exhibiting antitumor and antivirus activities, inhibits protein kinase C and the synthesis of both DNA and RNA. Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients and HIV-infected homosexual males. PEL cells are derived from post-germinal center B cells, and are infected with KSHV. Herein, we asked if sangivamycin might be useful to treat PEL. We found that sangivamycin killed PEL cells, and we explored the underlying mechanism. Sangivamycin treatment drastically decreased the viability of PEL cell lines compared to KSHV-uninfected B lymphoma cell lines. Sangivamycin induced the apoptosis of PEL cells by activating caspase-7 and -9. Further, sangivamycin suppressed the phosphorylation of Erk1/2 and Akt, thus inhibiting activation of the proteins. Inhibitors of Akt and MEK suppressed the proliferation of PEL cells compared to KSHV-uninfected cells. It is known that activation of Erk and Akt signaling inhibits apoptosis and promotes proliferation in PEL cells. Our data therefore suggest that sangivamycin induces apoptosis by inhibiting Erk and Akt signaling in such cells. We next investigated whether sangivamycin, in combination with an HSP90 inhibitor geldanamycin (GA) or valproate (valproic acid), potentiated the cytotoxic effects of the latter drugs on PEL cells. Compared to treatment with GA or valproate alone, the addition of sangivamycin enhanced cytotoxic activity. Our data thus indicate that sangivamycin may find clinical utility as a novel anti-cancer agent targeting PEL.
Assuntos
Apoptose/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Nucleosídeos de Pirimidina/farmacologia , Western Blotting , Caspase 7/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Linfoma de Efusão Primária/metabolismo , Linfoma de Efusão Primária/patologia , Linfoma de Efusão Primária/virologia , Masculino , Fosforilação/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Primary effusion lymphoma (PEL) is a subtype of non-Hodgkin's B-cell lymphoma and is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients. In general, PEL cells are derived from post-germinal center B-cells and are infected with KSHV. To evaluate potential novel anti-tumor compounds against KSHV-associated PEL, seven water-soluble fullerene derivatives were evaluated as potential drug candidates for the treatment of PEL. Herein, we discovered a pyrrolidinium fullerene derivative, 1,1,1',1'-tetramethyl [60]fullerenodipyrrolidinium diiodide, which induced apoptosis of PEL cells via a novel mechanism, the caspase-9 activation by suppressing the caspase-9 phosphorylation, causing caspase-9 inactivation. Pyrrolidinium fullerene treatment reduced significantly the viability of PEL cells compared with KSHV-uninfected lymphoma cells, and induced the apoptosis of PEL cells by activating caspase-9 via procaspase-9 cleavage. Pyrrolidinium fullerene additionally reduced the Ser473 phosphorylation of Akt and Ser196 of procaspase-9. Ser473-phosphorylated Akt (i.e., activated Akt) phosphorylates Ser196 in procaspase-9, causing inactivation of procaspase-9. We also demonstrated that Akt inhibitors suppressed the proliferation of PEL cells compared with KSHV-uninfected cells. Our data therefore suggest that Akt activation is essential for cell survival in PEL and a pyrrolidinium fullerene derivative induced apoptosis by activating caspase-9 via suppression of Akt in PEL cells. In addition, we evaluated whether pyrrolidinium fullerene in combination with the HSP90 inhibitor (geldanamycin; GA) or valproate, potentiated the cytotoxic effects on PEL cells. Compared to treatment with pyrrolidinium fullerene alone, the addition of low-concentration GA or valproate enhanced the cytotoxic activity of pyrrolidinium fullerene. These results indicate that pyrrolidinium fullerene could be used as a novel therapy for the treatment of PEL.
Assuntos
Antineoplásicos/farmacologia , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Linfoma de Efusão Primária/tratamento farmacológico , Linfoma de Efusão Primária/patologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Pirrolidinas/farmacologia , Antineoplásicos/química , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Benzoquinonas/administração & dosagem , Benzoquinonas/farmacologia , Caspase 9/metabolismo , Inibidores de Caspase/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Fulerenos/química , Fulerenos/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Herpesvirus Humano 8/patogenicidade , Humanos , Lactamas Macrocíclicas/administração & dosagem , Lactamas Macrocíclicas/farmacologia , Linfoma de Efusão Primária/metabolismo , Linfoma de Efusão Primária/virologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina/metabolismo , Vírion/efeitos dos fármacosRESUMO
BACKGROUND: Although it has received a degree of notoriety as a cause for antibiotic-associated enterocolitis (AAE), the role of methicillin resistant Staphylococcus aureus (MRSA) in the pathogenesis of this disease remains enigmatic despite a multitude of efforts, and previous studies have failed to conclude whether MRSA can cause AAE. Numerous cases of AAE caused by MRSA have been reported from Japan; however, due to the fact that these reports were written in the Japanese language and a good portion lacked scientific rigor, many of these reports went unnoticed. METHODS: We conducted a systematic review of pertinent literatures to verify the existence of AAE caused by MRSA. We modified and applied methods in common use today and used a total of 9 criteria to prove the existence of AAE caused by Klebsiella oxytoca. MEDLINE/Pubmed, Excerpta Medica Database (EMBASE), the Cochrane Database of Systematic Reviews, and the Japan Medical Abstract Society database were searched for studies published prior to March 2013. RESULTS: A total of 1,999 articles were retrieved for evaluation. Forty-five case reports/series and 9 basic studies were reviewed in detail. We successfully identified articles reporting AAE with pathological and microscopic findings supporting MRSA as the etiological agent. We also found comparative studies involving the use of healthy subjects, and studies detecting probable toxins. In addition, we found animal models in which enteritis was induced by introducing MRSA from patients. Although we were unable to identify a single study that encompasses all of the defined criteria, we were able to fulfill all 9 elements of the criteria by collectively analyzing multiple studies. CONCLUSIONS: AAE caused by MRSA-although likely to be rarer than previous Japanese literatures have suggested-most likely does exist.
Assuntos
Antibacterianos/efeitos adversos , Enterocolite Pseudomembranosa/microbiologia , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas/microbiologia , Animais , Enterocolite Pseudomembranosa/induzido quimicamente , Humanos , Meticilina , Resistência a Meticilina , Infecções Estafilocócicas/induzido quimicamenteRESUMO
A 42-year-old woman was admitted to our hospital because of lower abdominal pain and diarrhea. Although the initial symptoms and imaging findings were similar to those of acute enteritis, blood and ascites cultures led to the diagnosis of primary peritonitis caused by group A Streptococcus. In many cases, the disease rapidly deteriorates, and laparotomy is performed for the early diagnosis and to reduce the number of bacteria in the abdominal cavity. In the present case, intraperitoneal drainage was effective for avoiding surgery. We suggest that intraperitoneal drainage is effective for the treatment of this disease.
RESUMO
Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients and human immunodeficiency virus (HIV)-infected homosexual males. We evaluated the cytotoxic effects of heat shock protein 90 (HSP90) inhibitors on PEL cells. The HSP90 inhibitors geldanamycin (GA), 17(allylamino)-17-demethoxygeldanamycin (17-AAG), and radicicol dramatically inhibited cell proliferation and induced apoptosis of PEL cells through caspase activation. Furthermore, GA induced the stabilization of inhibitor of κB (IκB)α and reduced the phosphorylation of IκBα in PEL cells. HSP90 inhibitors suppressed the transcriptional activity of nuclear factor-kappa B (NF-κB) in PEL cells. It is known that the constitutive activation of NF-κB signaling is essential for the survival of PEL cells and HSP90 contributes to promote activation of NF-κB signaling. The suppression of NF-κB signaling by HSP90 inhibitors may contribute to the induction of apoptosis in PEL cells. In addition, HSP90 activity is required for KSHV replication in KSHV latently infected PEL cells. GA, 17-AAG and radicicol reduced the production of progeny virus from PEL cells at low concentrations, which do not affect PEL cell growth. Our results suggest that HSP90 activity is required for both the survival of PEL cells and viral replication in PEL cells, and that pharmacologic inhibition of HSP90 may be an effective treatment for PEL and KSHV-related diseases.
Assuntos
Apoptose/efeitos dos fármacos , Benzoquinonas/uso terapêutico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Herpesvirus Humano 8/efeitos dos fármacos , Lactamas Macrocíclicas/uso terapêutico , Linfoma de Efusão Primária/tratamento farmacológico , Macrolídeos/uso terapêutico , Replicação Viral/efeitos dos fármacos , Antibióticos Antineoplásicos/farmacologia , Antibióticos Antineoplásicos/uso terapêutico , Benzoquinonas/farmacologia , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Herpesvirus Humano 8/fisiologia , Humanos , Proteínas I-kappa B/metabolismo , Lactamas Macrocíclicas/farmacologia , Linfoma de Efusão Primária/metabolismo , Linfoma de Efusão Primária/virologia , Macrolídeos/farmacologia , NF-kappa B/metabolismo , Fosforilação , Sarcoma de Kaposi/complicações , Sarcoma de Kaposi/virologia , Transdução de Sinais , Transcrição Gênica/efeitos dos fármacosRESUMO
Selenium compounds such as methylseleninic acid (MSA) and sodium selenite (SS) have been widely evaluated as potential anti-cancer agents in the clinical setting. Primary effusion lymphoma (PEL) is a non-Hodgkin's B-cell lymphoma, associated with immunosuppressed individuals, such as post-transplant or AIDS patients. Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of PEL and Kaposi's sarcoma. Here, we found that MSA and SS markedly inhibited the growth of PEL cells compared with KSHV-uninfected B cells. MSA and SS caused ER stress, inducing the unfolded protein response (UPR) pathway in PEL cells that resulted in pro-apoptotic UPR, and finally apoptosis. The expression of UPR-related molecules (GRP78 and GADD34) and pro-apoptotic UPR molecules (CHOP, Bim, or Puma) were augmented in PEL cells treated with MSA or SS. In addition, these compounds induced the activation of caspase-4, an ER stress specific caspase, as well as caspase-3,-7, and -9 in PEL cells. We confirmed that thapsigargin which is an inducer of ER stress, dramatically decreased the viability of PEL cells, compared with KSHV-uninfected Ramos cells. We also investigated whether MSA or SS caused oxidization of cellular proteins in PEL cells. MSA and SS increased the levels of oxidative proteins in PEL cells, and the anti-oxidant agent (N-acetyl-l-cysteine) restored cell viability and suppressed caspase-7 activation in PEL cells treated with MSA or SS. Finally, we confirmed that MSA and SS induced neither lytic replication nor viral production in PEL cells. Taken together, MSA and SS could serve as lead compounds for the development of novel and effective drugs against PEL without the risk of de novo KSHV production.
Assuntos
Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Compostos Organosselênicos/farmacologia , Selenito de Sódio/farmacologia , Resposta a Proteínas não Dobradas , Apoptose/genética , Linhagem Celular Tumoral , Chaperona BiP do Retículo Endoplasmático , Humanos , Estresse Oxidativo/efeitos dos fármacosRESUMO
The allyl sulfides, including diallyl sulfide (DAS), diallyl disulfide (DAD), and diallyl trisulfide (DAT), contained in garlic and members of the Allium family, have a variety of pharmacological activities. Therefore, allyl sulfides have been evaluated as potential novel chemotherapeutic agents. Here, we found that DAT inhibited nuclear factor-κB (NF-κB) signaling and induced apoptosis in primary effusion lymphoma (PEL), a subtype of non-Hodgkin's B-cell lymphoma caused by Kaposi's sarcoma-associated herpesvirus (KSHV). We examined the cytotoxic effects of DAS, DAD and DAT on PEL cells. DAT significantly reduced the viability of PEL cells compared with uninfected B-lymphoma cells, and induced the apoptosis of PEL cells by activating caspase-9. DAT induced stabilization of IκBα, and suppressed NF-κB transcriptional activity in PEL cells. We examined the mechanism underlying DAT-mediated IκBα stabilization. The results indicated that DAT stabilized IκBα by inhibiting the phosphorylation of IκBα by the IκB kinase (IKK) complex. Furthermore, DAT induced proteasomal degradation of TRAF6, and DAT suppressed IKKß-phosphorylation through downregulation of TRAF6. It is known that activation of NF-κB is essential for survival of PEL cells. In fact, the NF-κB inhibitor BAY11-7082 induced apoptosis in PEL cells. In addition, DAT suppressed the production of progeny virus from PEL cells. The administration of DAT suppressed the development of PEL cells and ascites in SCID mice xenografted with PEL cells. These findings provide evidence that DAT has antitumor activity against PEL cells in vitro and in vivo, suggesting it to be a novel therapeutic agent for the treatment of PEL.
Assuntos
Compostos Alílicos/administração & dosagem , Quinase I-kappa B/metabolismo , Proteínas I-kappa B/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Linfoma de Efusão Primária/tratamento farmacológico , NF-kappa B/genética , Proteínas de Neoplasias/biossíntese , Sulfetos/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Quinase I-kappa B/genética , Proteínas I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Linfoma de Efusão Primária/genética , Linfoma de Efusão Primária/patologia , Camundongos , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Proteínas de Neoplasias/genética , Nitrilas/administração & dosagem , Fosforilação , Proteólise , Transdução de Sinais/efeitos dos fármacos , Sulfonas/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Antibacterianos/administração & dosagem , Pneumonia Associada à Ventilação Mecânica/tratamento farmacológico , APACHE , Comorbidade , Estado Terminal/epidemiologia , Mortalidade Hospitalar , Humanos , Pneumonia Associada à Ventilação Mecânica/epidemiologia , Recidiva , Projetos de Pesquisa , Procedimentos Cirúrgicos OperatóriosRESUMO
BACKGROUND: Patched 1 (Ptc1) is a polytopic receptor protein that is essential for growth and differentiation. Its extracellular domains accept its ligand, Sonic Hedgehog, while the function of its C-terminal intracellular domain is largely obscure. PRINCIPAL FINDINGS: In this study, we stably expressed human Ptc1 protein in HeLa cells and found that it is subjected to proteolytic cleavage at the C-terminus, resulting in the generation of soluble C-terminal fragments. These fragments accumulated in the nucleus, while the N-terminal region of Ptc1 remained in the cytoplasmic membrane fractions. Using an anti-Ptc1 C-terminal domain antibody, we provide conclusive evidence that C-terminal fragments of endogenous Ptc1 accumulate in the nucleus of C3H10T1/2 cells. Similar nuclear accumulation of endogenous C-terminal fragments was observed not only in C3H10T1/2 cells but also in mouse embryonic primary cells. Importantly, the C-terminal fragments of Ptc1 modulate transcriptional activity of Gli1. CONCLUSIONS: Although Ptc1 protein was originally thought to be restricted to cell membrane fractions, our findings suggest that its C-terminal fragments can function as an alternative signal transducer that is directly transported to the cell nucleus.
Assuntos
Núcleo Celular/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Células HeLa , Humanos , Fatores de Transcrição Kruppel-Like/fisiologia , Camundongos , Camundongos Endogâmicos C3H , Dados de Sequência Molecular , Receptores Patched , Receptor Patched-1 , Receptores de Superfície Celular/química , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismo , Transcrição Gênica , Proteína GLI1 em Dedos de ZincoRESUMO
BAG-6/Scythe/BAT3 is a ubiquitin-like protein that was originally reported to be the product of a novel gene located within the human major histocompatibility complex, although the mechanisms of its function remain largely obscure. Here, we demonstrate the involvement of BAG-6 in the degradation of a CL1 model defective protein substrate in mammalian cells. We show that BAG-6 is essential for not only model substrate degradation but also the ubiquitin-mediated metabolism of newly synthesized defective polypeptides. Furthermore, our in vivo and in vitro analysis shows that BAG-6 interacts physically with puromycin-labeled nascent chain polypeptides and regulates their proteasome-mediated degradation. Finally, we show that knockdown of BAG-6 results in the suppressed presentation of MHC class I on the cell surface, a procedure known to be affected by the efficiency of metabolism of defective ribosomal products. Therefore, we propose that BAG-6 is necessary for ubiquitin-mediated degradation of newly synthesized defective polypeptides.