RESUMEN
Heat shock protein 90 (HSP90) expression is upregulated in numerous types of cancer. However, its role as a candidate for molecular targeted therapy in oral squamous cell carcinoma (OSCC) cells is poorly understood. In the present study, a common upstream search was performed using molecular network analysis software for proteins with expression abnormalities that were found in a proteomic analysis of six OSCC cell lines. HSP90 was identified as a target protein. In clinical samples, high frequencies of HSP90high expression were detected via immunohistochemistry (26/58; 45%). Furthermore, the HSP90 expression status was associated with cervical lymph node metastasis (P=0.015). Furthermore, the potential of HSP90 as a candidate for molecular targeted therapy in OSCC cells was investigated using the HSP90 inhibitors 17dimethylaminoethylamino17demethoxygeldanamycin (17DMAG) and ganetespib. KON cells, which strongly express HSP90, were treated with the HSP90 inhibitors. The numbers of living cells in the 17DMAG and ganetespibtreated groups were lower than those in the nontreated group. The cells treated with the inhibitors demonstrated reduced cell viability and migration, and this was associated with markedly decreased levels of the HSP90 target proteins EGFR, phosphoEGFR, phosphoMEK and phosphoMAPK in the treated groups compared with the nontreated group. To the best of our knowledge, this was the first study to investigate the effects of 17DMAG and ganetespib on OSCC cells. The present results indicated the potential of HSP90 as a useful candidate for molecular targeted therapy in OSCC. However, additional studies with larger sample sizes are required to confirm these findings.
Asunto(s)
Benzoquinonas/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/farmacología , Neoplasias de la Boca/terapia , Carcinoma de Células Escamosas de Cabeza y Cuello/terapia , Triazoles/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Benzoquinonas/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quimioterapia Adyuvante/métodos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Lactamas Macrocíclicas/uso terapéutico , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida/métodos , Mucosa Bucal/patología , Mucosa Bucal/cirugía , Neoplasias de la Boca/mortalidad , Neoplasias de la Boca/patología , Pronóstico , Carcinoma de Células Escamosas de Cabeza y Cuello/mortalidad , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Tasa de Supervivencia , Triazoles/uso terapéuticoRESUMEN
Intimal hyperplasia (IH) is a pathologic process that leads to restenosis after treatment for peripheral arterial disease. Heat shock protein 90 (HSP90) is a molecular chaperone that regulates protein maturation. Activation of HSP90 results in increased cell migration and proliferation. 17Nallylamino17demethoxygeldanamycin (17AAG) and 17dimethylaminoethylamino17demethoxygeldanamycin (17DMAG) are low toxicity Food and Drug Association approved HSP90 inhibitors. The current study hypothesized that HSP90 inhibition was predicted to reduce vascular smooth muscle cell (VSMC) migration and proliferation. In addition, localized HSP90 inhibition may inhibit postangioplasty IH formation. For proliferation, VSMCs were treated with serumfree media (SFM), 17DMAG or 17AAG. The selected proliferative agents were SFM, platelet derived growth factor (PDGF) or fibronectin. After three days, proliferation was measured. For migration, VSMCs were treated with SFM, 17AAG or 17DMAG with SFM, PDGF or fibronectin as chemoattractants. Balloon injury to the carotid artery was performed in rats. The groups included in the present study were the control, saline control, 17DMAG in 20% pluronic gel delivered topically to the adventitia or intraluminal delivery of 17DMAG. After 14 days, arteries were fixed and sectioned for morphometric analysis. Data was analyzed using ANOVA or a student's ttest. P<0.05 was considered to indicate a statistically significant difference. The results revealed that 17AAG and 17DMAG had no effect on cell viability. PDGF and fibronectin also increased VSMC proliferation and migration. Furthermore, both 17AAG and 17DMAG decreased cell migration and proliferation in all agonists. Topical adventitial treatment with 17DMAG after balloon arterial injury reduced IH. HSP90 inhibitors suppressed VSMC proliferation and migration without affecting cell viability. Topical treatment with a HSP90 inhibitor (DMAG) decreased IH formation after arterial injury. It was concluded that 17DMAG may be utilized as an effective therapy to prevent restenosis after revascularization.
Asunto(s)
Angioplastia/efectos adversos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Túnica Íntima/patología , Animales , Benzoquinonas/farmacología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fibronectinas/farmacología , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Hiperplasia , Lactamas Macrocíclicas/farmacología , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Ratas Sprague-DawleyRESUMEN
BACKGROUND/AIMS: Hepatitis B viral protein X (HBx) is implicated in the pathogenesis of hepatocellular carcinoma (HCC) as well as the elevation of heat shock proteins (HSPs) after hepatitis B virus (HBV) infection. We thus investigated the anticancer effects of an HSP90 inhibitor 17-Dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) in HBx-transfected hepatocellular carcinoma cells. METHODS: pcDNA-HBx was made by inserting the HBx gene derived from the HBV-infected patient into pcDNA3.1 using the restriction enzymes (XbaI/HindIII). HBx-expressing HepG2 cells were then generated by transfecting HepG2 cells with pcDNA containing HBx gene. To compare the anticancer effects of 17-DMAG between pcDNA-HBx transfected HepG2 cells and the control cells (pcDNA-transfected HepG2 cells), we performed various molecular studies, including Ez-cytox proliferation assay, Western blot analysis, and flow cytometry. RESULTS: 17-DMAG inhibited the proliferation of pcDNA-HBx transfected HepG2 cells better than control cells (P<0.05). After treating with a various concentration of 17-DMAG (50–1,000 nM), pcDNA-HBx transfected HepG2 cells exhibited higher expression of pro-apoptotic proteins (c-caspase-3, c-caspase-8, and c-caspase-9) than did control cells (P<0.05). pcDNA-HBx transfected HepG2 cells showed higher activities of caspase-3, caspase-8, and caspase-9 than did control cells (P<0.05). Finally, we found that the expression of pro-apoptotic proteins (PARP and c-caspase-3) was considerably decreased by the use of a caspase inhibitor suggesting that 17-DMAG induces the cell death of HepG2 cells caspase-dependently. CONCLUSIONS: Our study strongly suggests that 17-DMAG has antiviral effects against HBV as well as anticancer effects against HepG2 cells. Thus, the application of 17-DMAG appears to be particularly advantageous to the HCC patients related with HBV infection.
Asunto(s)
Humanos , Apoptosis , Proteínas Reguladoras de la Apoptosis , Western Blotting , Carcinoma Hepatocelular , Caspasa 3 , Caspasa 8 , Caspasa 9 , Caspasas , Muerte Celular , Citometría de Flujo , Proteínas de Choque Térmico , Células Hep G2 , Virus de la Hepatitis B , Hepatitis B , Hepatitis , TransfecciónRESUMEN
PURPOSE: Posterior urethral valves are the most common cause of partial bladder outlet obstruction in the pediatric population. However, to our knowledge the etiology and the detailed mechanisms underlying pathological changes in the bladder following partial bladder outlet obstruction remain to be elucidated. Recent findings suggest that hypoxia and associated up-regulation of HIFs (hypoxia-inducible factors) have a key role in partial bladder outlet obstruction induced pathology in the bladder. We examined the effects of pharmacological inhibition of HIF pathways by 17-DMAG (17-(dimethylaminoethylamino)-17-demethoxygeldanamycin) in pathophysiological phenotypes after partial bladder outlet obstruction. MATERIALS AND METHODS: Partial bladder outlet obstruction was surgically created in male C57BL/6J mice. The animals received oral administration of 17-DMAG or vehicle daily starting from the initiation of obstruction up to 5 days. Sham operated mice served as controls. Bladders were harvested from each group 2, 4 and 7 days postoperatively, and analyzed for histological and biochemical changes. Bladder function was assessed by in vitro muscle contractility recordings. RESULTS: Partial bladder outlet obstruction caused a significant increase in the bladder mass accompanying enhanced collagen deposition in the bladder wall while 17-DMAG treatment suppressed those increases. Treatment with 17-DMAG attenuated the degree of up-regulation of HIFs and their target genes involving the development of tissue fibrosis in obstructed bladders. Treatment with 17-DMAG improved the decreased responses of obstructed bladder strips to electrical field stimulation and KCl. CONCLUSIONS: In vivo 17-DMAG treatment decreased partial bladder outlet obstruction induced pathophysiological changes in the bladder. HIF pathway inhibition has a potential clinical implication for the development of novel pharmacological therapies to treat bladder pathology associated with partial bladder outlet obstruction.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Benzoquinonas/farmacología , Benzoquinonas/uso terapéutico , Lactamas Macrocíclicas/farmacología , Lactamas Macrocíclicas/uso terapéutico , Obstrucción del Cuello de la Vejiga Urinaria/prevención & control , Vejiga Urinaria/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Hipertrofia/prevención & control , Masculino , Ratones , Ratones Endogámicos C57BL , Vejiga Urinaria/patología , Vejiga Urinaria/fisiopatología , Obstrucción del Cuello de la Vejiga Urinaria/patología , Obstrucción del Cuello de la Vejiga Urinaria/fisiopatologíaRESUMEN
The aryl hydrocarbon receptor is a member of the nuclear receptor superfamily that associates with the molecular chaperone HSP90 in the cytoplasm. The activation mechanism of the AhR is not yet fully understood. It has been proposed that after binding of ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3methylcholanthrene (3-MC), or ß-naphthoflavone (ß-NF), the AhR dissociates from HSP90 and translocates to the nucleus. It has also been hypothesized that the AhR translocates to the nucleus and forms a complex with HSP90 and other co-chaperones. There are a few reports about the direct association or dissociation of AhR and HSP90 due to difficulties in purifying AhR. We constructed and purified the PAS domain from AhR. Binding of the AhR-PAS domain to ß-NF affinity resin suggested that it possesses ligand-binding affinity. We demonstrated that the AhR-PAS domain binds to HSP90 and the association is not affected by ligand binding. The ligand 17-DMAG inhibited binding of HSP90 to GST-PAS. In an immunoprecipitation assay, HSP90 was co-immunoprecipitated with AhR both in the presence or absence of ligand. Endogenous AhR decreased in the cytoplasm and increased in the nucleus of HeLa cells 15 min after treatment with ligand. These results suggested that the ligand-bound AhR is translocated to nucleus while in complex with HSP90. We used an in situ proximity ligation assay to confirm whether AhR was translocated to the nucleus alone or together with HSP90. HSP90 was co-localized with AhR after the nuclear translocation. It has been suggested that the ligand-bound AhR was translocated to the nucleus with HSP90. Activated AhR acts as a transcription factor, as shown by the transcription induction of the gene CYP1A1 8 h after treatment with ß-NF.