Studying mechanisms underlying ER-stress induction by LY294002@PLGA nanodrug

Abstract

The phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (Akt) signaling pathway has been activated and regulated a variety of cellular processes in many cancers and lung cancer as well LY294002 is a potent inhibitor of PI3K activity and also has been demonstrated having antitumor activity in vitro and in vivo While its restrictive bio-application such as poor specificity solubility potential side effects and cytotoxicity offer us the key to an understanding of therapeutic efficacy of LY294002 PLGA is a biodegradable nanomaterial that can be used as a favorable carrier for drug delivery We have developed LY294002-loaded surfactant-free poly lactic-co-glycolic acid nanoparticles (LY294002@PLGA) synthesized by two methods ethanol titration and two emulsion to improve therapeutic effects In our research LY294002@PLGA unexpectedly increases cancer cell death with its profound endoplasmic reticulum (ER) stress However insufficient information is available about ER stress induced and its underlying mechanisms activated by LY294002@PLGA In this study lung cancer cell lines (H460 H157 and H1650) treated with various ER stress-related inducers (tunicamycin thapsigargin brefeldin A and DBeQ) for exploring the underlying mechanisms The cellular responses between these agents free form LY294002 and LY294002@PLGA will be compared and reserved for further study Characterizations of nanoparticles synthesized by two methods were analyzed using dynamic light scattering (DLS) and high-performance liquid chromatography (HPLC) The mean diameter and encapsulation efficiency of LY294002@PLGA synthesized by ethanol titration method was 94 02±5 05 nm and 6 35±0 03% by two emulsion method was 174 5±3 29 nm and 54 66%±3 81% Determined by MTT assay the cell cytotoxicity of LY294002@PLGA was enhanced and no obvious toxicity of PLGA nanoparticles in three lung cancer cell lines In addition LY294002@PLGA treatment sustained suppressed Akt and ERK activations simultaneously Unfolded protein response (UPR) including activating transcription factor 6 (ATF6) PKR-like ER kinase (PERK) and inositol requiring enzyme 1 (IRE1) are activated for cell survival or death in different ER stress conditions After tunicamycin and thapsigargin treatments all of UPR pathways were induced for survival but PERK-ATF4-CHOP pathway was activated for cell death in three lung cancer cell lines Cells treated with brefeldin A were induced to express PERK and IRE1 pathways but not ATF6 pathway and were more susceptible to apoptosis because of CHOP and phosphorylated JNK elevation Only PERK pathway was activated by DBeQ to increase apoptotic cell death Cells treated with LY294002 caused slight ER stress for cell survival By LY294002@PLGA cells were induced profound ER stress and caused apoptotic cell death through PERK-ATF4-CHOP pathway Compared with the tested ER stress-related inducers only DBeQ valosin-containing protein (VCP) inhibitor had similar ER stress-derived mechanism of action with LY294002@PLGA Therefore VCP may play an important role in LY294002@PLGA-derived cell death These findings confirmed that both ER-stress induction and sustained suppression of Akt and ERK by LY294002@PLGA resulted in profound cytotoxic activity and cell apoptosis improved through PERK-ATF4-CHOP pathway LY294002@PLGA may be a potential developed drug for cancer therapy in the future

Date of Award	2015 Feb 9
Original language	English
Supervisor	Wu-Chou Su (Supervisor)