RESUMEN
BACKGROUND: Tumors of the head and neck present aggressive pathological behavior in patients due to high expression of CDK/CCND1 proteins. P276-00, a novel CDK inhibitor currently being tested in clinic, inhibits growth of several cancers in vitro and in vivo. The pre clinical activity of P276-00 in head and neck cancer and its potential mechanisms of action at molecular level are the focus of the current studies. METHOD: We have investigated the anti-cancer activity of P276-00 in head and neck tumors in vitro and in vivo. Candidate gene expression profiling and cell based proteomic approaches were taken to understand the pathways affected by P276-00 treatment. RESULTS: It was observed that P276-00 is cytotoxic across various HNSCC cell lines with an IC50 ranging from 1.0-1.5 µmoles/L and culminated in significant cell-cycle arrest in G1/S phase followed by apoptosis. P276-00 treatment suppressed cell proliferation through inhibition of CCND1 expression, reduced phosphorylation of retinoblastoma protein and abrogative transcription of E2F1 gene targets. Further, we observed that apoptosis was mediated through P53 activation leading to higher BAX/BCL-2 ratio and cleaved caspase-3 levels. It was also seen that P276-00 treatment reduced expression of tumor micro-environment proteins such as IL-6, secreted EGFR and HSPA8. Finally, P276-00 treatment resulted in significant tumor growth inhibition in xenograft tumor models via lowered proliferative activity of E2F1 and aggravated P53 mediated apoptosis. CONCLUSION: In summary, we have observed that P276-00 inhibits cyclin-D/CDK4/P16/pRB/E2F axis and induces apoptosis by increased P53 phosphorylation in HNSCC cells. These results suggest a novel indication for P276-00 in head and neck cancer with a potential role for IL-6 and HSPA8 as candidate serum biomarkers.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Células Escamosas/tratamiento farmacológico , Flavonas/farmacología , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Ciclina D/metabolismo , Ciclina D1/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Factor de Transcripción E2F1/metabolismo , Receptores ErbB/metabolismo , Perfilación de la Expresión Génica , Proteínas del Choque Térmico HSC70/metabolismo , Humanos , Concentración 50 Inhibidora , Interleucina-6/metabolismo , Ratones , Ratones SCID , Trasplante de Neoplasias , Fosforilación , Proteína de Retinoblastoma/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Several studies have characterized drug-induced toxicity in liver and kidney. However, the majority of these studies have been performed with 'individual' organs in isolation. Separately, little is known about the role of whole blood as a surrogate tissue in drug-induced toxicity. Accordingly, we investigated the 'concurrent' response of liver, kidney and whole blood during a toxic assault. Rats were acutely treated with therapeutics (acetaminophen, rosiglitazone, fluconazole, isoniazid, cyclophosphamide, amphotericin B, gentamicin and cisplatin) reported for their liver and/or kidney toxicity. Changes in clinical chemistry parameters (e.g. AST, urea) and/or observed microscopic tissue damage confirmed induced hepatotoxicity and/or nephrotoxicity by all drugs. Drug-induced toxicity was not confined to an 'individual' organ. Not all drugs elicited significant alterations in phenotypic parameters of toxicity (e.g. ALT, creatinine). Accordingly, the transcriptional profile of the organs was studied using a toxicity panel of 30 genes derived from literature. Each of the test drugs generated specific gene expression patterns which were unique for all three organs. Hierarchical cluster analyses of purported hepatotoxicants and nephrotoxicants each led to characteristic 'fingerprints' (e.g. decrease in Cyp3a1 indicative of hepatotoxicity; increase in Spp1 and decrease in Gstp1 indicative of nephrotoxicity). In whole blood cells, a set of genes was derived which closely correlated with individual drug-induced concomitant changes in liver or kidney. Collectively, these data demonstrate drug-induced multi-organ toxicity. Furthermore, our findings underscore the importance of transcriptional profiling during inadequate phenotypic anchorage and suggest that whole blood may be judiciously used as a surrogate for drug-induced extra-hematological organ toxicity.
Asunto(s)
Células Sanguíneas/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Riñón/efectos de los fármacos , Animales , Biomarcadores/sangre , Biomarcadores/metabolismo , Células Sanguíneas/metabolismo , Células Sanguíneas/patología , Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/sangre , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/metabolismo , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/patología , Femenino , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Especificidad de Órganos , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Pruebas de Toxicidad Aguda/métodosRESUMEN
Studying peripheral blood transcriptome in the quest for translational markers of toxicity is considered to be an attractive offshoot in the field of toxicogenomics. Moreover, it is acknowledged that, xenobiotics which cause a toxic response through similar mechanisms lead to distinctive gene expression patterns. The current study was undertaken to gauge the response of an accessible surrogate tissue, such as blood, to drug-induced perturbations aimed at deriving gene expression patterns. Human peripheral blood mononuclear cells (hPBMC) were exposed to conventional drugs, with reported kidney and/or liver injury, in order to determine their transcriptomic response. Test drugs were divided into two classes viz., drugs affecting kidney (cyclophosphamide, amphotericin B, gentamicin and cisplatin) and liver (acetaminophen, rosiglitazone, fluconazole and isoniazid). After performing gene expression analysis and hierarchical clustering, signature patterns for the two classes were obtained, with a set of 365 genes that can discriminate the two classes of drugs. Our results imply that transcriptional profile of hPBMC get altered as a consequence of drug exposure and unique patterns indicative of specific organ toxicity can hence be deduced. These signature patterns obtained for drugs could be studied for their qualification to identify drug-induced toxicity.
Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Perfilación de la Expresión Génica , Riñón/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Hígado/efectos de los fármacos , Células Cultivadas , Femenino , Humanos , Masculino , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Microarray technology can be used to study the molecular mechanisms of new chemical entities with the aim to develop effective therapeutics. 7-Hydroxyfrullanolide (7HF) is a sesquiterpene lactone that was found to be efficacious in multiple animal models of inflammation by suppression of pro-inflammatory cytokines; however, its molecular mechanism of action remains unclear. We investigated the effects of 7HF on lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells using microarray-based gene expression studies and explored the molecular targets affected. Gene expression profiles and pathway analysis revealed that 7HF potently suppressed multiple inflammatory pathways induced by LPS. More importantly, 7HF was found to inhibit NF-κB related transcripts. These transcripts were further validated using freshly isolated synovial cells from rheumatoid arthritis patients, thus clinically validating our findings. Cell-based imaging and subsequent Western blot analysis demonstrated that 7HF inhibited the translocation of NF-κB into the nucleus by directly inhibiting the phosphorylation of IKK-ß. Since the transcription of adhesion molecules is regulated by NF-κB, further investigation showed that 7HF dose-dependently suppressed ICAM-1, VCAM-1 and E-selectin expression on LPS-stimulated endothelial cells as well as inhibited the adhesion of monocytes to LPS-stimulated endothelial cells. Taken together, our results reveal that 7HF possesses NF-κB inhibitory potential and suggest a likely molecular mechanism of its anti-inflammatory activity.