RESUMO
Notch signaling is a conserved pathway that converts extracellular receptor-ligand interactions into changes in gene expression via a single transcription factor (CBF1/RBPJ in mammals; Su(H) in Drosophila). In humans, RBPJ variants have been linked to Adams-Oliver syndrome (AOS), a rare autosomal dominant disorder characterized by scalp, cranium, and limb defects. Here, we found that a previously described Drosophila Su(H) allele encodes a missense mutation that alters an analogous residue found in an AOS-associated RBPJ variant. Importantly, genetic studies support a model that heterozygous Drosophila with the AOS-like Su(H) allele behave in an opposing manner to heterozygous flies with a Su(H) null allele, due to a dominant activity of sequestering either the Notch co-activator or the antagonistic Hairless co-repressor. Consistent with this model, AOS-like Su(H) and Rbpj variants have decreased DNA binding activity compared to wild type proteins, but these variants do not significantly alter protein binding to the Notch co-activator or the fly and mammalian co-repressors, respectively. Taken together, these data suggest a cofactor sequestration mechanism underlies AOS phenotypes associated with RBPJ variants, whereby the AOS-associated RBPJ allele encodes a protein with compromised DNA binding activity that retains cofactor binding, resulting in Notch target gene dysregulation.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Proteínas Correpressoras , DNA , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Displasia Ectodérmica , Humanos , Deformidades Congênitas dos Membros , Mamíferos/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Couro Cabeludo/metabolismo , Dermatoses do Couro Cabeludo/congênito , Crânio/metabolismoRESUMO
Normal development requires tight regulation of cell proliferation and cell death. Here, we have investigated these control mechanisms in the hyaloid vessels, a temporary vascular network in the mammalian eye that requires a Wnt/ß-catenin response for scheduled regression. We investigated whether the hyaloid Wnt response was linked to the oncogene Myc, and the cyclin-dependent kinase inhibitor CDKN1A (P21), both established regulators of cell cycle progression and cell death. Our analysis showed that the Wnt pathway co-receptors LRP5 and LRP6 have overlapping activities that mediate the Wnt/ß-catenin signaling in hyaloid vascular endothelial cells (VECs). We also showed that both Myc and Cdkn1a are downstream of the Wnt response and are required for hyaloid regression but for different reasons. Conditional deletion of Myc in VECs suppressed both proliferation and cell death. By contrast, conditional deletion of Cdkn1a resulted in VEC overproliferation that countered the effects of cell death on regression. When combined with analysis of MYC and CDKN1A protein levels, this analysis suggests that a Wnt/ß-catenin and MYC-CDKN1A pathway regulates scheduled hyaloid vessel regression.
Assuntos
Apoptose/fisiologia , Proliferação de Células/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Endotélio Vascular/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , beta Catenina/metabolismo , Animais , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21/genética , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Olho/irrigação sanguínea , Células HEK293 , Humanos , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-myc/genética , Via de Sinalização Wnt/fisiologiaRESUMO
Angiogenesis and vascular remodeling are essential for the establishment of vascular networks during organogenesis. Here we show that the Hippo signaling pathway effectors YAP and TAZ are required, in a gene dosage-dependent manner, for the proliferation and migration of vascular endothelial cells (ECs) during retinal angiogenesis. Intriguingly, nuclear translocation of YAP and TAZ induced by Lats1/2-deletion blocked endothelial migration and phenocopied Yap/Taz-deficient mutants. Furthermore, overexpression of a cytoplasmic form of YAP (YAPS127D) partially rescued the migration defects caused by loss of YAP and TAZ function. Finally, we found that cytoplasmic YAP positively regulated the activity of the small GTPase CDC42, deletion of which caused severe defects in endothelial migration. These findings uncover a previously unrecognized role of cytoplasmic YAP/TAZ in promoting cell migration by activating CDC42 and provide insight into how Hippo signaling in ECs regulates angiogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Movimento Celular , Endotélio Vascular/citologia , Neovascularização Fisiológica , Fosfoproteínas/fisiologia , Fatores de Transcrição/fisiologia , Proteína cdc42 de Ligação ao GTP/fisiologia , Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular , Proliferação de Células , Endotélio Vascular/fisiologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Camundongos Knockout , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAPRESUMO
In 2014, ISHAM formed a new working group: "Medical Phycology: Protothecosis and Chlorellosis." The purpose of this working group is to help facilitate collaboration and communication among people interested in the pathogenic algae, to share ideas and work together. Here we present reports on recent work we have done in five areas. 1. The history of medical phycology as a branch of science. 2. Aspects of the genetics of Prototheca. 3. Aspects of the proteins of Prototheca. 4. Human infections caused by Prototheca. 5. Dairy cow mastitis caused by Prototheca.
Assuntos
Chlorella , Prototheca , Animais , Chlorella/genética , Chlorella/patogenicidade , Genótipo , Humanos , Infecções , Tipagem Molecular , Prototheca/genética , Prototheca/patogenicidadeRESUMO
Dihydroartemisinin (DHA), an antimalarial drug, has previously unrecognized anticancer activity, and is in clinical trials as a new anticancer agent for skin, lung, colon and breast cancer treatment. However, the anticancer mechanism is not well understood. Here, we show that DHA inhibited proliferation and induced apoptosis in rhabdomyosarcoma (Rh30 and RD) cells, and concurrently inhibited the signaling pathways mediated by the mammalian target of rapamycin (mTOR), a central controller for cell proliferation and survival, at concentrations (<3 µM) that are pharmacologically achievable. Of interest, in contrast to the effects of conventional mTOR inhibitors (rapalogs), DHA potently inhibited mTORC1-mediated phosphorylation of p70 S6 kinase 1 and eukaryotic initiation factor 4E binding protein 1 but did not obviously affect mTORC2-mediated phosphorylation of Akt. The results suggest that DHA may represent a novel class of mTORC1 inhibitor and may execute its anticancer activity primarily by blocking mTORC1-mediated signaling pathways in the tumor cells.
Assuntos
Antineoplásicos/farmacologia , Artemisininas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Genes p53 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Rabdomiossarcoma/tratamento farmacológico , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
Activating non-inherited mutations in the guanine nucleotide-binding protein G(q) subunit alpha (GNAQ) gene family have been identified in childhood vascular tumors. Patients experience extensive disfigurement, chronic pain and severe complications including a potentially lethal coagulopathy termed Kasabach-Merritt phenomenon. Animal models for this class of vascular tumors do not exist. This has severely hindered the discovery of the molecular consequences of GNAQ mutations in the vasculature and, in turn, the preclinical development of effective targeted therapies. Here we report a mouse model expressing hyperactive mutant GNAQ in endothelial cells. Mutant mice develop vascular and coagulopathy phenotypes similar to those seen in patients. Mechanistically, by transcriptomic analysis we demonstrate increased mitogen activated protein kinase signaling in the mutant endothelial cells. Targeting of this pathway with Trametinib suppresses the tumor growth by reducing vascular cell proliferation and permeability. Trametinib also prevents the development of coagulopathy and improves mouse survival.
Assuntos
Melanoma , Neoplasias Uveais , Neoplasias Vasculares , Animais , Camundongos , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células Endoteliais/metabolismo , Apoptose , Melanoma/genética , Neoplasias Uveais/genética , Mutação , Modelos Animais de Doenças , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Linhagem Celular TumoralRESUMO
Curcumin can induce p53-independent apoptosis. However, the underlying mechanism remains to be defined. Here, we show that curcumin-induced apoptosis in a panel of tumor cells with mutant p53. Curcumin rapidly induced activation of the mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase 1/2 (Erk1/2) and c-Jun N-terminal kinase (JNK). Inhibition of JNK (with SP600125) or Erk1/2 (with U0126) partially prevented curcumin-induced cell death in the cells. Similarly, expression of dominant negative c-Jun or downregulation of Erk1/2 in part attenuated curcumin-induced cell death. It appears that curcumin-induced activation of MAPKs and apoptosis was due to induction of reactive oxygen species (ROS), as pretreatment with N-acetyl-L-cysteine, a ROS scavenger, blocked these events. Furthermore, we found that curcumin-induced activation of MAPK pathways was related to inhibition of the serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5). Overexpression of PP2A or PP5 partially prevented curcumin-induced activation of JNK and Erk1/2 phosphorylation as well as cell death. The results suggest that curcumin induction of ROS activates MAPKs, at least partially by inhibiting PP2A and PP5, thereby leading to p53-independent apoptosis in tumor cells.
Assuntos
Apoptose/efeitos dos fármacos , Curcumina/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Fosfoproteínas Fosfatases/antagonistas & inibidores , Proteína Fosfatase 2/antagonistas & inibidores , Linhagem Celular Tumoral , Ativação Enzimática , HumanosRESUMO
We present a Prototheca wickerhamii wound infection case that failed treatment with ketoconazole but was cured with amphotericin-B plus tetracycline. The patient was immunocompetent but had had local steroid injections. We reviewed another 159 cases from the literature. Prototheca has infected many areas of the human body, but most often skin, olecranon bursa, or wounds. Prior treatment with steroids and immune deficiencies are contributing factors. Itraconazole and fluconazole are reasonable initial treatments for patients with mild infections. For serious infections, or for infections that have failed azole treatment, amphotericin-B is the treatment of choice.
Assuntos
Prototheca/isolamento & purificação , Prototheca/patogenicidade , Infecção dos Ferimentos/tratamento farmacológico , Infecção dos Ferimentos/etiologia , Anfotericina B/administração & dosagem , Anti-Infecciosos/administração & dosagem , Seguimentos , Humanos , Tetraciclina/administração & dosagem , Resultado do TratamentoRESUMO
Dihydroartemisinin (DHA), an anti-malarial drug, has been shown to possess potent anticancer activity, partly by inhibiting the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signaling. However, how DHA inhibits mTORC1 is still unknown. Here, using rhabdomyosarcoma (RMS) as a model, we found that DHA reduced cell proliferation and viability in RMS cells, but not those in normal cells, which was associated with inhibition of mTORC1. Mechanistically, DHA did not bind to mTOR or FK506 binding protein 12 (FKBP12). In addition, DHA neither inhibited insulin-like growth factor-1 receptor (IGF-1R), phosphoinositide 3-kinase (PI3K), and extracellular signal-regulated kinase ½ (Erk1/2), nor activated phosphatase and tensin homolog (PTEN) in the cells. Rather, DHA activated AMP-activated protein kinase (AMPK). Pharmacological inhibition of AMPK, ectopic expression dominant negative or kinase-dead AMPK, or knockdown of AMPKa attenuated the inhibitory effect of DHA on mTORC1 in the cells. Additionally, DHA was able to induce dissociation of regulatory-associated protein of mTOR (raptor) from mTOR and inhibit mTORC1 activity. Moreover, treatment with artesunate, a prodrug of DHA, dose-dependently inhibited tumor growth and concurrently activated AMPK and suppressed mTORC1 in RMS xenografts. The results indicated that DHA inhibits mTORC1 by activating AMPK in tumor cells. Our finding supports that DHA or artesunate has a great potential to be repositioned for treatment of RMS.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Artemisininas/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Neoplasias/metabolismo , Rabdomiossarcoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Rabdomiossarcoma/tratamento farmacológicoRESUMO
Almost all life forms can detect and decode light information for adaptive advantage. Examples include the visual system, in which photoreceptor signals are processed into virtual images, and the circadian system, in which light entrains a physiological clock. Here we describe a light response pathway in mice that employs encephalopsin (OPN3, a 480 nm, blue-light-responsive opsin) to regulate the function of adipocytes. Germline null and adipocyte-specific conditional null mice show a light- and Opn3-dependent deficit in thermogenesis and become hypothermic upon cold exposure. We show that stimulating mouse adipocytes with blue light enhances the lipolysis response and, in particular, phosphorylation of hormone-sensitive lipase. This response is Opn3 dependent. These data establish a key mechanism in which light-dependent, local regulation of the lipolysis response in white adipocytes regulates energy metabolism.
Assuntos
Adipócitos Marrons/metabolismo , Adipócitos Marrons/efeitos da radiação , Adipócitos Brancos/metabolismo , Adipócitos Brancos/efeitos da radiação , Luz , Opsinas de Bastonetes/metabolismo , Termogênese/efeitos da radiação , Animais , Temperatura Baixa , Metabolismo Energético/efeitos da radiação , Perfilação da Expressão Gênica , Lipólise/efeitos da radiação , Camundongos Endogâmicos C57BL , Fenótipo , Fótons , Termogênese/genéticaRESUMO
We previously showed that introduction of transporter associated with antigen processing (TAP) 1 into TAP-negative CMT.64, a major histocompatibility complex class I (MHC-I) down-regulated mouse lung carcinoma cell line, enhanced T-cell immunity against TAP-deficient tumour cells. Here, we have addressed two questions: (1) whether such immunity can be further augmented by co-expression of TAP1 with B7.1 or H-2K(b) genes, and (2) which T-cell priming mechanism (tumour direct priming or dendritic cell cross-priming) plays the major role in inducing an immune response against TAP-deficient tumours. We introduced the B7.1 or H-2K(b) gene into TAP1-expressing CMT.64 cells and determined which gene co-expressed with TAP1 was able to provide greater protective immunity against TAP-deficient tumour cells. Our results show that immunization of mice with B7.1 and TAP1 co-expressing but not H-2K(b) and TAP1 co-expressing CMT.64 cells dramatically augments T-cell-mediated immunity, as shown by an increase in survival of mice inoculated with live CMT.64 cells. In addition, our results suggest that induction of T-cell-mediated immunity against TAP-deficient tumour cells could be mainly through tumour direct priming rather than dendritic cell cross-priming as they show that T cells generated by tumour cell-lysate-loaded dendritic cells recognized TAP-deficient tumour cells much less than TAP-proficient tumour cells. These data suggest that direct priming by TAP1 and B7.1 co-expressing tumour cells is potentially a major mechanism to facilitate immune responses against TAP-deficient tumour cells.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antígeno B7-1/metabolismo , Carcinoma Pulmonar de Lewis/imunologia , Antígenos H-2/metabolismo , Transgenes/genética , Animais , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Apresentação Cruzada , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Antígenos H-2/genética , Antígenos H-2/imunologia , Imunoterapia Adotiva , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , TransfecçãoRESUMO
During mouse postnatal eye development, the embryonic hyaloid vascular network regresses from the vitreous as an adaption for high-acuity vision. This process occurs with precisely controlled timing. Here, we show that opsin 5 (OPN5; also known as neuropsin)-dependent retinal light responses regulate vascular development in the postnatal eye. In Opn5-null mice, hyaloid vessels regress precociously. We demonstrate that 380-nm light stimulation via OPN5 and VGAT (the vesicular GABA/glycine transporter) in retinal ganglion cells enhances the activity of inner retinal DAT (also known as SLC6A3; a dopamine reuptake transporter) and thus suppresses vitreal dopamine. In turn, dopamine acts directly on hyaloid vascular endothelial cells to suppress the activity of vascular endothelial growth factor receptor 2 (VEGFR2) and promote hyaloid vessel regression. With OPN5 loss of function, the vitreous dopamine level is elevated and results in premature hyaloid regression. These investigations identify violet light as a developmental timing cue that, via an OPN5-dopamine pathway, regulates optic axis clearance in preparation for visual function.
Assuntos
Dopamina/metabolismo , Olho/irrigação sanguínea , Luz , Proteínas de Membrana/metabolismo , Opsinas/metabolismo , Animais , Proteínas da Membrana Plasmática de Transporte de Dopamina/antagonistas & inibidores , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Endotélio Vascular/metabolismo , Olho/enzimologia , Olho/crescimento & desenvolvimento , Olho/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Opsinas/genética , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/efeitos da radiação , Treonina/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/fisiologia , Corpo Vítreo/metabolismoRESUMO
A number of groups have developed libraries of siRNAs to identify genes through functional genomics. While these studies have validated the approach of making functional RNAi libraries to understand fundamental cellular mechanisms, they require information and knowledge of existing sequences since the RNAi sequences are generated synthetically. An alternative strategy would be to create an RNAi library from cDNA. Unfortunately, the complexity of such a library of siRNAs would make screening difficult. To reduce the complexity, longer dsRNAs could be used; however, concerns of induction of the interferon response and off-target effects of long dsRNAs have prevented their use. As a first step in creating such libraries, long dsRNA was expressed in mammalian cells. The 250 nt dsRNAs were capable of efficiently silencing a luciferase reporter gene that was stably transfected in MDA-MB-231 cells without inducing the interferon response or off-target effects any more than reported for siRNAs. In addition, a long dsRNA expressed in the same cell line was capable of silencing endogenous c-met expression and inhibited cell migration, whereas the dsRNA against luciferase had no effect on c-met or cell migration. The studies suggest that large dsRNA libraries are feasible and that functional selection of genes will be possible.
Assuntos
Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Genes Reporter , Humanos , Interferon beta/biossíntese , Luciferases/análise , Luciferases/genética , Plasmídeos , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , RNA de Cadeia Dupla/genéticaRESUMO
PURPOSE: Peripherally inserted central venous catheters are some of the most useful devices for vascular access used globally. Peripherally inserted central venous catheters have a low rate of fatal mechanical complications when compared to non-tunnel central venous catheters. However, as peripherally inserted central venous catheter access requires a smaller vein, there is a high risk of thrombosis. The axillary vein (confluence of the basilic and brachial veins) can serve as an access for cannulation. Moreover, as this vein is larger than the basilic or brachial vein, it might be a superior option for preventing thrombosis. The risk of catheter-related bloodstream infection should be considered when the puncture site is at the axillary fossa. The aim of this study was to present our new protocol involving peripherally inserted central venous catheters (non-tunneled/tunneled) and a tunneling technique and assess its feasibility and safety for improving cannulation and preventing thrombosis and infection. METHODS: The study included 20 patients. The axillary vein in the upper arm was used for peripherally inserted central venous catheters in patients with a small-diameter basilic vein (<3 mm). When the puncture site was in the axillary fossa, a subcutaneous tunnel of about 3 cm was constructed easily using a peripheral venous catheter. RESULTS: The observed catheter duration was 645 days (median ± standard deviation, 26 ± 22.22 days). Catheterization was successful in all cases, however, two accidental dislodgements were identified. No fatal or serious complications were observed after catheterization. CONCLUSION: Our new protocol for axillary peripherally inserted central venous catheters/tunneled axillary peripherally inserted central venous catheters use for a small-diameter basilic vein is safe and feasible.
Assuntos
Veia Axilar , Cateterismo Venoso Central/métodos , Cateterismo Periférico/métodos , Idoso , Idoso de 80 Anos ou mais , Veia Axilar/diagnóstico por imagem , Cateterismo Venoso Central/efeitos adversos , Cateterismo Venoso Central/instrumentação , Cateterismo Periférico/efeitos adversos , Cateterismo Periférico/instrumentação , Cateteres de Demora , Cateteres Venosos Centrais , Desenho de Equipamento , Estudos de Viabilidade , Feminino , Humanos , Masculino , Punções , Estudos Retrospectivos , Fatores de Risco , Fatores de Tempo , Ultrassonografia de IntervençãoRESUMO
Ciclopirox olamine (CPX), an off-patent fungicide, has recently been identified as a novel anticancer agent. However, the molecular mechanism underlying its anticancer action remains to be elucidated. Here we show that CPX inhibits cell proliferation in part by downregulating the protein level of Cdc25A in tumor cells. Our studies revealed that CPX did not significantly reduce Cdc25A mRNA level or Cdc25A protein synthesis, but remarkably promoted Cdc25A protein degradation. This resulted in inhibition of G1-cyclin dependent kinases (CDKs), as evidenced by increased inhibitory phosphorylation of G1-CDKs. Since Cdc25A degradation is tightly related to its phosphorylation status, we further examined whether CPX alters Cdc25A phosphorylation. The results showed that CPX treatment increased the phosphorylation of Cdc25A (S76 and S82), but only Cdc25A-S82A mutant was resistant to CPX-induced degradation. Furthermore, ectopic expression of Cdc25A-S82A partially conferred resistance to CPX inhibition of cell proliferation. Therefore, our findings indicate that CPX inhibits cell proliferation at least in part by promoting Cdc25A degradation.
RESUMO
Cryptotanshinone (CPT), a natural compound isolated from the plant Salvia miltiorrhiza Bunge, is a potential anticancer agent. However, the underlying mechanism is not well understood. Here, we show that CPT induced caspase-independent cell death in human tumor cells (Rh30, DU145, and MCF-7). Besides downregulating antiapoptotic protein expression of survivin and Mcl-1, CPT increased phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK), and inhibited phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2). Inhibition of p38 with SB202190 or JNK with SP600125 attenuated CPT-induced cell death. Similarly, silencing p38 or c-Jun also in part prevented CPT-induced cell death. In contrast, expression of constitutively active mitogen-activated protein kinase kinase 1 (MKK1) conferred resistance to CPT inhibition of Erk1/2 phosphorylation and induction of cell death. Furthermore, we found that all of these were attributed to CPT induction of reactive oxygen species (ROS). This is evidenced by the findings that CPT induced ROS in a concentration- and time-dependent manner; CPT induction of ROS was inhibited by N-acetyl-L-cysteine (NAC), a ROS scavenger; and NAC attenuated CPT activation of p38/JNK, inhibition of Erk1/2, and induction of cell death. The results suggested that CPT induction of ROS activates p38/JNK and inhibits Erk1/2, leading to caspase-independent cell death in tumor cells.
Assuntos
Caspases/fisiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias/patologia , Fenantrenos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/uso terapêutico , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Ativação Enzimática/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Fenantrenos/uso terapêutico , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Mammalian target of rapamycin (mTOR) controls lymphangiogenesis. However, the underlying mechanism is not clear. Here we show that rapamycin suppressed insulin-like growth factor 1 (IGF-1)- or fetal bovine serum (FBS)-stimulated lymphatic endothelial cell (LEC) tube formation, an in vitro model of lymphangiogenesis. Expression of a rapamycin-resistant and kinase-active mTOR (S2035T, mTOR-T), but not a rapamycin-resistant and kinase-dead mTOR (S2035T/D2357E, mTOR-TE), conferred resistance to rapamycin inhibition of LEC tube formation, suggesting that rapamycin inhibition of LEC tube formation is mTOR kinase activity dependent. Also, rapamycin inhibited proliferation and motility in the LECs. Furthermore, we found that rapamycin inhibited protein expression of VEGF receptor 3 (VEGFR-3) by inhibiting protein synthesis and promoting protein degradation of VEGFR-3 in the cells. Down-regulation of VEGFR-3 mimicked the effect of rapamycin, inhibiting IGF-1- or FBS-stimulated tube formation, whereas over-expression of VEGFR-3 conferred high resistance to rapamycin inhibition of LEC tube formation. The results indicate that rapamycin inhibits LEC tube formation at least in part by downregulating VEGFR-3 protein expression.
Assuntos
Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Sirolimo/farmacologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Camundongos , Camundongos Transgênicos , Biossíntese de Proteínas/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Tumors deficient in expression of the transporter associated with antigen processing (TAP) usually fail to induce T-cell-mediated immunity and are resistant to T-cell lysis. However, we have found that introduction of the B7.1 gene into TAP-negative (TAP(-)) or TAP1-transfected (TAP1(+)) murine lung carcinoma CMT.64 cells can augment the capacity of the cells to induce a protective immune response against wild-type tumor cells. Differences in the strength of the protective immune responses were observed between TAP(-) and TAP1(+) B7.1 expressing CMT.64 cells depending on the doses of gamma-irradiated cell immunization. While mice immunized with either high or low dose of B7.1-expressing TAP1(+) cells rejected TAP(-) tumors, only high dose immunization with B7.1-expressing TAP(-) cells resulted in tumor rejection. The induced protective immunity was T-cell dependent as demonstrated by dramatically reduced antitumor immunity in mice depleted of CD8 or CD4 cells. Augmentation of T-cell mediated immune response against TAP(-) tumor cells was also observed in a virally infected tumor cell system. When mice were immunized with a high dose of gamma-irradiated CMT.64 cells infected with vaccinia viruses carrying B7.1 and/or TAP1 genes, we found that the cells co-expressing B7.1 and TAP1, but not those expressing B7.1 alone, induced protective immunity against CMT.64 cells. In addition, inoculation with live tumor cells transfected with several different gene(s) revealed that only B7.1- and TAP1-coexpressing tumor cells significantly decreased tumorigenicity. These results indicate that B7.1-provoked antitumor immunity against TAP(-) cancer is facilitated by TAP1-expression, and thus both genes should be considered for cancer therapy in the future.
Assuntos
Transportadores de Cassetes de Ligação de ATP/imunologia , Antígeno B7-1/imunologia , Linfócitos T/imunologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Animais , Linhagem Celular Tumoral , CamundongosRESUMO
BACKGROUND: Translation initiation factor eIF4E unwinds long 5'-untranslated regions of certain tightly regulated mRNAs and, thereby, facilitates their translation into proteins. eIF4E has been shown to be overexpressed in a majority of solid tumors, including head and neck cancers. To exploit this dysregulation, a long 5'-untranslated region was spliced upstream of a thymidine kinase (Tk) gene to enhance translation of this "suicide" gene within cells overexpressing eIF4E. We investigated the efficacy of therapy with an adenovirus incorporating this novel suicide gene (Ad-HSV-UTk) following cytoreductive tumor surgery in improving disease-free and overall survival in a mouse soft-tissue metastasis model for head and neck squamous cell carcinoma. MATERIALS AND METHODS: SCC-7 (orally-derived mouse SCCa) cells were treated with Ad-HSV-Tk, Ad-HSV-UTk, Ad-null, or saline and characterized for eIF4E and Tk levels by Western blot analysis. Cytotoxicities for cells treated with Ad-HSV-Tk, Ad-HSV-UTk, or Ad-null were quantified by MTS assay. Mice bearing SCC-7-induced tumors received cytoreduction followed by Ad-HSV-UTk + ganciclovir (GCV) or control treatment and were followed for disease-free and overall survival. RESULTS: SCC-7 cells showed uniformly high levels of eIF4E but elevated Tk for Ad-HSV-Tk- and Ad-HSV-UTk-treated cells over Ad-null-treated cells. Cytotoxicities for Ad-HSV-Tk- and Ad-HSV-UTk-treated cells were, correspondingly, observed to be 100-fold more sensitive than Ad-null-treated cells to GCV treatment. Cytoreduced mice receiving Ad-HSV-UTk + GCV treatment showed significantly longer disease-free survival (P = 0.0045) than control arm mice. CONCLUSIONS: Ad-HSV-UTk suicide gene therapy prolonged disease-free survival in a mouse minimal residual soft-tissue head and neck squamous cell carcinoma metastasis model.
Assuntos
Carcinoma de Células Escamosas/terapia , Fator de Iniciação 4E em Eucariotos/genética , Terapia Genética/métodos , Neoplasias de Cabeça e Pescoço/terapia , Metástase Neoplásica/terapia , Animais , Linhagem Celular Tumoral , Intervalo Livre de Doença , Genes Transgênicos Suicidas , Vetores Genéticos , Camundongos , Camundongos Endogâmicos C3H , Neoplasias ExperimentaisRESUMO
BACKGROUND: In view of the limited success of available treatment modalities for a wide array of cancer, alternative and complementary therapeutic strategies need to be developed. Virotherapy employing conditionally replicative adenoviruses (CRAds) represents a promising targeted intervention relevant to a wide array of neoplastic diseases. Critical to the realization of an acceptable therapeutic index using virotherapy in clinical trials is the achievement of oncolytic replication in tumor cells, while avoiding non-specific replication in normal tissues. In this report, we exploited cancer-specific control of mRNA translation initiation in order to achieve enhanced replicative specificity of CRAd virotherapy agents. Heretofore, the achievement of replicative specificity of CRAd agents has been accomplished either by viral genome deletions or incorporation of tumor selective promoters. In contrast, control of mRNA translation has not been exploited for the design of tumor specific replicating viruses to date. We show herein, the utility of a novel approach that combines both transcriptional and translational regulation strategies for the key goal of replicative specificity. METHODS: We describe the construction of a CRAd with cancer specific gene transcriptional control using the CXCR4 gene promoter (TSP) and cancer specific mRNA translational control using a 5'-untranslated region (5'-UTR) element from the FGF-2 (Fibroblast Growth Factor-2) mRNA. RESULTS: Both in vitro and in vivo studies demonstrated that our CRAd agent retains anti-tumor potency. Importantly, assessment of replicative specificity using stringent tumor and non-tumor tissue slice systems demonstrated significant improvement in tumor selectivity. CONCLUSIONS: Our study addresses a conceptually new paradigm: dual targeting of transgene expression to cancer cells using both transcriptional and mRNA translational control. Our novel approach addresses the key issue of replicative specificity and can potentially be generalized to a wide array of tumor types, whereby tumor selective patterns of gene expression and mRNA translational control can be exploited.