RESUMEN
INTRODUCTION: The DNA-alkylating agent chlormethine (CL, or mechlorethamine) is approved in several countries worldwide as a 0.016% w/w topical CL gel formulation, to treat mycosis fungoides cutaneous T-cell lymphoma, with a positive benefit/risk ratio. METHODS: Release profiles of CL from the gel and a compounded ointment-based 0.016% CL formulation were compared via in vitro release testing (IVRT), utilizing static diffusion cells, a pseudo-infinite dose, and polytetrafluoroethylene membranes, over 5 h. The percutaneous absorption profile of CL gel in ex vivo human skin was also examined, using in vitro permeation testing (IVPT) with flow-through diffusion cells, dermatomed skin (epidermis plus dermis) and epidermal membranes, a finite dose, over 24 h. RESULTS: In IVRT experiments, the mean ± SD CL release rate was significantly higher for the gel versus the ointment (5.70 ± 0.73 versus 2.38 ± 1.03 µg/cm2/âh); the formulations were inequivalent per the US Food and Drug Administration scale-up and postapproval changes for nonsterile semisolid dosage forms (FDA SUPAC-SS) criteria. Mean IVPT cumulative CL (gel) permeating through epidermal membrane was higher than for dermatomed skin (4.6% versus 2.5% of applied dose). Mean residual CL on the epidermal membrane surface was 1.3% of the applied dose. CONCLUSIONS: CL gel (0.016%) and ointment were inequivalent, with an optimized release profile, suggesting minimal passage of CL gel through human epidermal tissue to the dermis.
RESUMEN
H-Prune hydrolyzes short-chain polyphosphates (PPase activity) together with an hitherto cAMP-phosphodiesterase (PDE), the latest influencing different human cancers by its overexpression. H-Prune promotes cell migration in cooperation with glycogen synthase kinase-3 (Gsk-3ß). Gsk-3ß is a negative regulator of canonical WNT/ß-catenin signaling. Here, we investigate the role of Gsk-3ß/h-Prune complex in the regulation of WNT/ß-catenin signaling, demonstrating the h-Prune capability to activate WNT signaling also in a paracrine manner, through Wnt3a secretion. In vivo study demonstrates that h-Prune silencing inhibits lung metastasis formation, increasing mouse survival. We assessed h-Prune levels in peripheral blood of lung cancer patients using ELISA assay, showing that h-Prune is an early diagnostic marker for lung cancer. Our study dissects out the mechanism of action of h-Prune in tumorigenic cells and also sheds light on the identification of a new therapeutic target in non-small-cell lung cancer.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proteínas Portadoras/sangre , Glucógeno Sintasa Quinasa 3/metabolismo , Neoplasias Pulmonares/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas Portadoras/genética , Progresión de la Enfermedad , Femenino , Glucógeno Sintasa Quinasa 3 beta , Xenoinjertos , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Monoéster Fosfórico Hidrolasas , beta Catenina/genéticaRESUMEN
Several genes encoding for proteins involved in proliferation, invasion, and apoptosis are known to be direct miR-34a targets. Here, we used proteomics to screen for targets of miR-34a in neuroblastoma (NBL), a childhood cancer that originates from precursor cells of the sympathetic nervous system. We examined the effect of miR-34a overexpression using a tetracycline inducible system in two NBL cell lines (SHEP and SH-SY5Y) at early time points of expression (6, 12, and 24 h). Proteome analysis using post-metabolic labeling led to the identification of 2,082 proteins, and among these 186 were regulated (112 proteins down-regulated and 74 up-regulated). Prediction of miR-34a targets via bioinformatics showed that 32 transcripts held miR-34a seed sequences in their 3'-UTR. By combining the proteomics data with Kaplan Meier gene-expression studies, we identified seven new gene products (ALG13, TIMM13, TGM2, ABCF2, CTCF, Ki67, and LYAR) that were correlated with worse clinical outcomes. These were further validated in vitro by 3'-UTR seed sequence regulation. In addition, Michigan Molecular Interactions searches indicated that together these proteins affect signaling pathways that regulate cell cycle and proliferation, focal adhesions, and other cellular properties that overall enhance tumor progression (including signaling pathways such as TGF-ß, WNT, MAPK, and FAK). In conclusion, proteome analysis has here identified early targets of miR-34a with relevance to NBL tumorigenesis. Along with the results of previous studies, our data strongly suggest miR-34a as a useful tool for improving the chance of therapeutic success with NBL.
Asunto(s)
Redes y Vías Metabólicas , MicroARNs/genética , Neuroblastoma/metabolismo , Proteómica/métodos , Regiones no Traducidas 3' , Línea Celular Tumoral , Dactinomicina/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , MicroARNs/metabolismo , Neuroblastoma/genética , Tetraciclina/farmacologíaRESUMEN
Get well prune: The C-terminal third domain of h-prune is largely unfolded and involved in relevant protein-protein interactions, particularly with Nm23-H1 (see figure), GSK-3ß and gelsolin. This study shows that protein functions mediated by protein-protein interactions can be accurately followed in cell lysates by using fast NMR spectroscopy, which could be easily used for a very efficient NMR drug-discovery strategy.