RESUMEN
In breast cancer therapy, Gemcitabine (Gem) is an antineoplastic antimetabolite with greater anticancer efficacy and tolerability. However, effectiveness of Gem is limited by its off-target effects. The synergistic potential of MUC1 (mucin 1) inhibitors and Gem-loaded polymeric nanoparticles (NPs) was discussed in this work in order to reduce dose-related toxicities and enhance the therapeutic efficacy. The double emulsion solvent evaporation method was used to prepare poly(ethylene glycol) methyl ether-block-poly-caprolactone (PEG-PCL)-loaded Gem and MUC 1 inhibitor NPs. The average size of Gem and MUC 1 inhibitor-loaded NPs was 128 nm, with a spherical shape. Twin-loaded NPs containing Gem and the MUC1 inhibitor decreased IC50 and behaved synergistically. Furthermore, in vitro mechanistic studies, that is, loss of MMP, clonogenic assay, Annexin V FITC assay, and Western blotting to confirm apoptosis with simultaneous induction of autophagy using acridine orange (AO) staining were performed in this study. Furthermore, the investigated NPs upon combination exhibited greater loss of MMP and decreased clonogenic potential with simultaneous induction of autophagy in MCF-7 cells. Annexin V FITC clearly showed the percentage of apoptosis while Western blotting protein expression analysis revealed an increase in caspase-3 activity with simultaneous decrease in Bcl-2 protein expression, a hallmark of apoptosis. The effectiveness of the Ehrlich ascites solid (EAT) mice treated with Gem-MUC1 inhibitor NPs was higher than that of the animals treated alone. Overall, the combined administration of Gem and MUC1 inhibitor-loaded NPs was found to be more efficacious than Gem and MUC1 inhibitor delivered separately.
Asunto(s)
Neoplasias de la Mama , Nanopartículas , Animales , Anexina A5/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Femenino , Fluoresceína-5-Isotiocianato , Humanos , Ratones , Mucina-1 , Poliésteres , Polietilenglicoles , GemcitabinaRESUMEN
Lysosomal biogenesis plays a vital role in cell fate. Under certain conditions, excessive lysosomal biogenesis leads to susceptibility for lysosomal membrane permeabilization resulting in various pathological conditions including cell death. In cancer cells apoptosis machinery becomes dysregulated during the course of treatment, thus allows cancer cells to escape apoptosis. So it is therefore imperative to identify cytotoxic agents that exploit non-apoptotic mechanisms of cell death. Our study showed that pancreatic cancer cells treated with SDS-203 triggered an incomplete autophagic response and a nuclear translocation of transcriptional factor TFEB. This resulted in abundant biosynthesis and accumulation of autophagosomes and lysosomes into the cells leading to their death. It was observed that the silencing of autophagy genes didn't alter the cell fate, whereas siRNA-mediated silencing of TFEB subdued SDS-203 mediated lysosomal biogenesis and associated cell death. Further mouse tumors treated with SDS-203 showed a significant reduction in tumor burden and increased expression of lysosomal markers. Taken together this study demonstrates that SDS-203 treatment triggers non-apoptotic cell death in pancreatic cancer cells through a mechanism of lysosome over accumulation.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Neoplasias Pancreáticas , Animales , Autofagia/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Muerte Celular , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismoRESUMEN
Betulinic acid (BA), a pentacyclic lupine-type triterpene, is reported to inhibit cell growth in a variety of cancers. However, its efficacy is limited by its poor aqueous solubility and relatively short half-life. In this study, BA-monomethoxy polyethylene glycol (mPEG) conjugate was synthesized by covalent coupling the C-28 carboxylic acid position of BA with amine groups of mPEG, in order to improve its solubility and anticancer efficacy. mPEG-BA conjugate was characterized using various analytical techniques including NMR, FT-IR and MALDI-MS. The mPEG-BA conjugate was cytotoxic, demonstrated internalization and induced cell apoptosis in Hep3B and Huh7 hepatic cancer cells. The western-blot analysis revealed, marked decrease in Bcl-2/Bax ratio, and increase in cleaved-PARP and cleaved-caspase-3 expressions. In vivo studies in Ehrlich ascites tumor (EAT) model following intravenous administration demonstrated significant reduction in tumor volume in case of PEGylated BA as compare to native BA. Furthermore, PEGylated BA treated EAT mice showed no biochemical and histological toxicities. These findings demonstrate the potential of PEGylated BA in cancer therapy, with improved water solubility and efficacy.
Asunto(s)
Antineoplásicos Fitogénicos/síntesis química , Antineoplásicos Fitogénicos/farmacología , Polietilenglicoles/síntesis química , Triterpenos/síntesis química , Triterpenos/farmacología , Animales , Antineoplásicos Fitogénicos/uso terapéutico , Biomarcadores/metabolismo , Western Blotting , Espectroscopía de Resonancia Magnética con Carbono-13 , Carcinoma de Ehrlich/sangre , Carcinoma de Ehrlich/tratamiento farmacológico , Carcinoma de Ehrlich/patología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Estabilidad de Medicamentos , Hemólisis/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Masculino , Ratones , Triterpenos Pentacíclicos , Polietilenglicoles/química , Espectroscopía de Protones por Resonancia Magnética , Ratas Wistar , Solubilidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectroscopía Infrarroja por Transformada de Fourier , Fracciones Subcelulares/metabolismo , Triterpenos/química , Triterpenos/uso terapéutico , Ácido BetulínicoRESUMEN
The eukaryotic translation initiation factor 4E (eIF4E) is considered as a key survival protein involved in cell cycle progression, transformation and apoptosis resistance. Herein, we demonstrate that medicinal plant derivative 3-AWA (from Withaferin A) suppressed the proliferation and metastasis of CaP cells through abrogation of eIF4E activation and expression via c-FLIP dependent mechanism. This translational attenuation prevents the de novo synthesis of major players of metastatic cascades viz. c-FLIP, c-Myc and cyclin D1. Moreover, the suppression of c-FLIP due to inhibition of translation initiation complex by 3-AWA enhanced FAS trafficking, BID and caspase 8 cleavage. Further ectopically restored c-Myc and GFP-HRas mediated activation of eIF4E was reduced by 3-AWA in transformed NIH3T3 cells. Detailed underlying mechanisms revealed that 3-AWA inhibited Ras-Mnk and PI3-AKT-mTOR, two major pathways through which eIF4E converges upon eIF4F hub. In addition to in vitro studies, we confirmed that 3-AWA efficiently suppressed tumor growth and metastasis in different mouse models. Given that 3-AWA inhibits c-FLIP through abrogation of translation initiation by co-targeting mTOR and Mnk-eIF4E, it (3-AWA) can be exploited as a lead pharmacophore for promising anti-cancer therapeutic development.