RESUMO
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.
Assuntos
Neoplasias da Mama , Nanopartículas , Animais , Anexina A5/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Feminino , Fluoresceína-5-Isotiocianato , Humanos , Camundongos , Mucina-1 , Poliésteres , Polietilenoglicóis , GencitabinaRESUMO
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.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Neoplasias Pancreáticas , Animais , Autofagia/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Morte Celular , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismoRESUMO
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.
Assuntos
Antineoplásicos Fitogênicos/síntese química , Antineoplásicos Fitogênicos/farmacologia , Polietilenoglicóis/síntese química , Triterpenos/síntese química , Triterpenos/farmacologia , Animais , Antineoplásicos Fitogênicos/uso terapêutico , Biomarcadores/metabolismo , Western Blotting , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Carcinoma de Ehrlich/sangue , Carcinoma de Ehrlich/tratamento farmacológico , Carcinoma de Ehrlich/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Hemólise/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Triterpenos Pentacíclicos , Polietilenoglicóis/química , Espectroscopia de Prótons por Ressonância Magnética , Ratos Wistar , Solubilidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectroscopia de Infravermelho com Transformada de Fourier , Frações Subcelulares/metabolismo , Triterpenos/química , Triterpenos/uso terapêutico , Ácido BetulínicoRESUMO
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.