Nusrat Chowdhury1, Imran Vhora1, Ketan Patel1,2, Ravi Doddapaneni1,3, Arindam Mondal1, Mandip Singh4. 1. College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, Florida, 32307, USA. 2. College of Pharmacy and Health Sciences, St. John's University, Jamaica, New York, 11439, USA. 3. Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, 33136, USA. 4. College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, Florida, 32307, USA. mandip.sachdeva@famu.edu.
Abstract
PURPOSE: Non-small cell lung cancer is the leading cause of cancer related deaths globally. Considering the side effects and diminishing chemosensitivity to chemotherapy, novel treatment approaches are sought. Hence, we aim to develop a liposomal co-delivery system of pDNA expressing shRNA against PFKFB3 (pshPFKFB3) and docetaxel (DTX). METHODS: Cationic DTX liposomes complexed with pshPFKFB3 (PSH-DL) were developed. In vitro cell line studies were performed to evaluate transfection, PFKFB3 mRNA silencing, cytotoxicity, pGP inhibition, and protein markers expression. In vivo efficacy study was performed in A549 xenograft nude mice model. RESULTS: Cytotoxicity studies showed significantly enhanced anticancer activity of PSH-DL against individual treatment alone confirming the chemoenhancing effect of pshPFKFB3 on DTX activity. Fluorescence microscopy and RT-PCR showed effective transfection and RNAi by pshPFKFB3. pGP inhibition assay and western blotting revealed that PFKFB3 downregulation caused diminution of pGP activity leading to changes in cell cycle (Cdk2), survival (survivin), apoptosis (Bcl2 and cleaved caspase 3) and stress (p-JNK and p-p38) markers so that induces apoptosis by PSH-DL in NSCLC cells. PSH-DL also showed ~3.8-fold reduction in tumor volume in A549 xenograft model which was significantly higher than individual treatments alone. CONCLUSION: Targeting PFKFB3 through shRNA based RNAi is a promising approach for potentiating activity of DTX in NSCLC.
PURPOSE:Non-small cell lung cancer is the leading cause of cancer related deaths globally. Considering the side effects and diminishing chemosensitivity to chemotherapy, novel treatment approaches are sought. Hence, we aim to develop a liposomal co-delivery system of pDNA expressing shRNA against PFKFB3 (pshPFKFB3) and docetaxel (DTX). METHODS: Cationic DTX liposomes complexed with pshPFKFB3 (PSH-DL) were developed. In vitro cell line studies were performed to evaluate transfection, PFKFB3 mRNA silencing, cytotoxicity, pGP inhibition, and protein markers expression. In vivo efficacy study was performed in A549 xenograft nude mice model. RESULTS:Cytotoxicity studies showed significantly enhanced anticancer activity of PSH-DL against individual treatment alone confirming the chemoenhancing effect of pshPFKFB3 on DTX activity. Fluorescence microscopy and RT-PCR showed effective transfection and RNAi by pshPFKFB3. pGP inhibition assay and western blotting revealed that PFKFB3 downregulation caused diminution of pGP activity leading to changes in cell cycle (Cdk2), survival (survivin), apoptosis (Bcl2 and cleaved caspase 3) and stress (p-JNK and p-p38) markers so that induces apoptosis by PSH-DL in NSCLC cells. PSH-DL also showed ~3.8-fold reduction in tumor volume in A549 xenograft model which was significantly higher than individual treatments alone. CONCLUSION: Targeting PFKFB3 through shRNA based RNAi is a promising approach for potentiating activity of DTX in NSCLC.
Authors: David L DiGiusto; Amrita Krishnan; Lijing Li; Haitang Li; Shirley Li; Anitha Rao; Shu Mi; Priscilla Yam; Sherri Stinson; Michael Kalos; Joseph Alvarnas; Simon F Lacey; Jiing-Kuan Yee; Mingjie Li; Larry Couture; David Hsu; Stephen J Forman; John J Rossi; John A Zaia Journal: Sci Transl Med Date: 2010-06-16 Impact factor: 17.956