RESUMEN
BACKGROUND: Newer targeted agents are increasingly used in combination chemotherapy regimens with enhanced survival and improved toxicity profile. Taxols, such as paclitaxel, independently potentiate tumor destruction via apoptosis and are used as first line therapy in patients with advanced non-small cell lung cancer (NSCLC). Procaspase-3-activating compound-1 (PAC-1) is a novel proapoptotic agent that directly activates procaspase-3 (PC-3) to caspase-3, leading to apoptosis in human lung adenocarcinoma cells. Hence, we sought to evaluate the antitumor effects of paclitaxel in combination with PAC-1. METHODS: Human NSCLC cell lines (A-549 and H-322m) were incubated in the presence of PAC-1 and paclitaxel. Tumor cell viability was determined by a tetrazolium-based colorimetric assay (MTT assay). Western blot and flow cytometric analysis were performed to evaluate expression of PC-3 and the proportion of apoptotic cells, respectively. A xenograft murine model of NSCLC was used to study the in vivo antitumor effects of PAC-1. RESULTS: PAC-1 significantly reduced the inhibitory concentration 50% of paclitaxel from 35.3 to 0.33 nM in A-549 and 8.2 to 1.16 nM in H-322m cell lines. Similarly, the apoptotic activity significantly increased to 85.38% and 70.36% in A-549 and H322m, respectively. Significantly enhanced conversion of PC-3 to caspase-3 was observed with PAC-1 paclitaxel combination (P < 0.05). Mice treated with a drug combination demonstrated 60% reduced tumor growth rate compared with those of controls (P < 0.05). CONCLUSIONS: PAC-1 significantly enhances the antitumor activity of paclitaxel against NSCLC. The activation of PC-3 and thus the apoptotic pathway is a potential strategy in the treatment of human lung cancer.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Hidrazonas/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Paclitaxel/uso terapéutico , Piperazinas/uso terapéutico , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular Tumoral , Sinergismo Farmacológico , Quimioterapia Combinada , Femenino , Humanos , Hidrazonas/farmacología , Ratones , Ratones Desnudos , Piperazinas/farmacología , Distribución Aleatoria , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: The hallmark of lung ischemia-reperfusion injury (IRI) is the production of reactive oxygen species (ROS), and the resultant oxidant stress has been implicated in apoptotic cell death as well as subsequent development of inflammation. Dietary flaxseed (FS) is a rich source of naturally occurring antioxidants and has been shown to reduce lung IRI in mice. However, the mechanisms underlying the protective effects of FS in IRI remain to be determined. METHODS: We used a mouse model of IRI with 60 min of ischemia followed by 180 min of reperfusion and evaluated the anti-apoptotic and anti-inflammatory effects of 10% FS dietary supplementation. RESULTS: Mice fed 10% FS undergoing lung IRI had significantly lower levels of caspases and decreased apoptotic activity compared with mice fed 0% FS. Lung homogenates and bronchoalveolar lavage fluid analysis demonstrated significantly reduced inflammatory infiltrate in mice fed with 10% FS diet. Additionally, 10% FS treated mice showed significantly increased expression of antioxidant enzymes and decreased markers of lung injury. CONCLUSIONS: We conclude that dietary FS is protective against lung IRI in a clinically relevant murine model, and this protective effect may in part be mediated by the inhibition of apoptosis and inflammation.
Asunto(s)
Alimentación Animal , Suplementos Dietéticos , Lino , Neumonía/prevención & control , Daño por Reperfusión/prevención & control , Lesión Pulmonar Aguda/dietoterapia , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/prevención & control , Animales , Antioxidantes/metabolismo , Apoptosis/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Caspasa 3/genética , Caspasa 3/metabolismo , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo/inmunología , Neumonía/dietoterapia , Neumonía/metabolismo , ARN Mensajero/metabolismo , Daño por Reperfusión/dietoterapia , Daño por Reperfusión/metabolismoRESUMEN
The resistance of inositol 1,4,5-trisphosphate receptor (IP3R)-deficient cells to multiple forms of apoptosis demonstrates the importance of IP3-gated calcium (Ca2+) release to cellular apoptosis. However, the specific upstream biochemical events leading to IP3-gated Ca2+ release during apoptosis induction are not known. We have shown previously that the cyclin-dependent kinase 1/cyclin B (cdk1/CyB or cdc2/CyB) complex phosphorylates IP3R1 in vitro and in vivo at Ser421 and Thr799. In this study, we show that: 1) the cdc2/CyB complex directly interacts with IP3R1 through Arg391, Arg441, and Arg871; 2) IP3R1 phosphorylation at Thr799 by the cdc2/CyB complex increases IP3 binding; and 3) cdc2/CyB phosphorylation increases IP3-gated Ca2+ release. Taken together, these results demonstrate that cdc2/CyB phosphorylation positively regulates IP3-gated Ca2+ signaling. In addition, identification of a CyB docking site(s) on IP3R1 demonstrates, for the first time, a direct interaction between a cell cycle component and an intracellular calcium release channel. Blocking this phosphorylation event with a specific peptide inhibitor(s) may constitute a new therapy for the treatment of several human immune disorders.
Asunto(s)
Proteína Quinasa CDC2/fisiología , Canales de Calcio/fisiología , Ciclina B/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Secuencia de Aminoácidos , Animales , Apoptosis , Calcio/metabolismo , Señalización del Calcio , Ciclina B1 , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Células Jurkat , Datos de Secuencia Molecular , Fosforilación , RatasRESUMEN
Calcium (Ca2+) release from the endoplasmic reticulum (ER) controls numerous cellular functions including proliferation, and is regulated in part by inositol 1,4,5-trisphosphate receptors (IP3Rs). IP3Rs are ubiquitously expressed intracellular Ca2+-release channels found in many cell types. Although IP3R-mediated Ca2+ release has been implicated in cellular proliferation, the biochemical pathways that modulate intracellular Ca2+ release during cell cycle progression are not known. Sequence analysis of IP3R1 reveals the presence of two putative phosphorylation sites for cyclin-dependent kinases (cdks). In the present study, we show that cdc2/CyB, a critical regulator of eukaryotic cell cycle progression, phosphorylates IP3R1 in vitro and in vivo at both Ser(421) and Thr(799) and that this phosphorylation increases IP3 binding. Taken together, these results indicate that IP3R1 may be a specific target for cdc2/CyB during cell cycle progression.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Canales de Calcio/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Fosfo-Específicos/metabolismo , Calcio/metabolismo , Ciclo Celular/fisiología , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Células Jurkat , Fosforilación , Serina/metabolismo , Treonina/metabolismoRESUMEN
We investigated the role of the orphan nuclear receptor farnesoid X receptor (FXR) in the regulation of cholesterol 7alpha-hydroxylase (CYP7A1), using an in vivo rabbit model, in which the bile acid pool, which includes high affinity ligands for FXR, was eliminated. After 7 days of bile drainage, the enterohepatic bile acid pool, in both New Zealand White and Watanabe heritable hyperlipidemic rabbits, was depleted. CYP7A1 activity and mRNA levels increased while FXR was deactivated as indicated by reduced FXR protein and changes in the expression of target genes that served as surrogate markers of FXR activation in the liver and ileum, respectively. Hepatic bile salt export pump mRNA levels and ileal bile acid-binding protein decreased while sterol 12alpha-hydroxylase and sodium/taurocholate cotransporting polypeptide mRNA levels increased in the liver. In addition, hepatic FXR mRNA levels decreased significantly. The data, taken together, indicate that FXR was deactivated when the bile acid pool was depleted such that CYP7A1 was upregulated. Further, lack of the high affinity ligand supply was associated with downregulation of hepatic FXR mRNA levels.