The Role Of Glutathione-S-Transferase In Epithelial Mesenchymal Transition (Emt) Model In Colorectal Cancer And The Enhancement Of Adjuvant Therapy By Targeting Glutathione-S-Transferase Inhibitor

Abstract

Colorectal cancer is the third leading cause of cancer-related deaths worldwide. For colorectal cancers that have not spread to distant sites, surgery is usually the primary or first treatment option. However, chemotherapy (neoadjuvan, adjuvan and advanced-stage chemotherapy) is still widely used. Several chemotherapeutic agents are available for the treatment of CRC, but eventually cancer relapse occurs. A major impediment in the success of available therapies is the recurrent adaptation of cancer cells, leading to metastases, which are often considered as the point of no return, and are associated with the worst outcome. Therefore, understanding the mechanisms that drive resistance of cancer cells bears special importance. In the recent years, epithelial-mesenchymal transition (EMT), naturally occurring transdifferentiation program, is believed to play an important role in resistance toward chemotherapeutics. Although EMT is widely demonstrated to play a critical role in chemoresistance and metastasis, the potential signaling network between EMT and drug resistance is still unclear. Glutathione S-Transferases (GSTs), a family of isozymes that catalyze the reaction of glutathione (GSH) with electrophiles of both endogenous and xenobiotic origins, assist in the development of drug resistance through direct detoxification. The pi (π) and mu (µ) classes of GSTs play a regulatory role in cellular survival and also in development of cancer. Thus, in the present study, in vitro EMT model was created in HT-29 CRC cell line and EMT was demonstrated using immunohistochemical and biochemical methods. Expression and protein levels of GST-π and GST-µ in the epithelial and mesenchymal phenotype of HT-29 CRC cells was determined. Oxidative stress was generated and oxidative damage was detected; correlation between the oxidative damage and GST isoenzymes was determined at gene and protein levels in the epithelial and mesenchymal phenotypes of HT-29 CRC cells. Both phenotypes of HT-29 CRC cells were treated with ethacrynic acid (ETA) that is a FDA approved GST inhibitor. Expressions of the GST isoenzymes and their protein levels were determined following ETA treatment. Assays were repeated for oxidatively damaged epithelial and mesenchymal phenotypes of HT-29 cells in order to investigate the effect of GST inhibitor on EMT in CRC. Epithelial and mesenchymal phenotypes were treated with adjuvant therapy combination and also with the adjuvant therapy combination plus ETA to investigate the efficacy of the new tharapeutic protocol. Blank and ETA loaded PLGA-b-PEG nanoparticles and mPLGA-PEG-SS-ETA nanoconjugates were prepared by nanoprecipitation technique. These nanoformulations were characterized in terms of mean particle size, PDI, zeta potential and morphology. ETA loading capacity and loading efficiency of nanoformulations were determined by validated HPLC method. The optimized nanoformulations were coupled with Vimentin (Vim) monoclonal antibody specific to mesenchymal phenotype of HT-29 CRC cells for targeted delivery. In vitro cytotoxicity of nanoformulations were determined on L929 cells. Epithelial and mesenchymal phenotypes were treated with adjuvant therapy combination plus ETA loaded and mesenchymal phenotype targeted nanoformulations to investigate the efficacy of the new targeted therapeutic protocol. In vivo EMT model was created in the immunosuppressed Wistar rats. Animals were treated with adjuvant therapy combination and adjuvant therapy combination plus ETA loaded and mesenchymal phenotype targeted nanoformulations to investigate the in vivo efficacy of the new targeted therapeutic protocol. In vitro EMT model was successfully established in HT-29 CRC cells. In mesenchymal phenotype of HT-29 cells, elevated expression and protein levels of GST-π were observed, however, no remarkable difference in GST-µ expression patterns was observed. GST inhibitor ETA was successfully loaded in PLGA-b-PEG nanoparticles. ETA was also conjugated to PLGA-b-PEG copolymer via disulphide bond and resulting polymer self-assembled into nanosized particles. ETA loaded nanoformulations in the range of 115 to 130 nm with a narrow size distribution were obtained. Both nanoformulations were successfully targeted to mesenchymal phenotype with Vim mAb conjugation. Targeted formulations had higher cell uptake rates and showed inhibition effect on GST-π isoenzyme activity even at lower concentrations. In vivo EMT model were successfully induced by the injection of HT-29 CRC cells in immuno-suppressed Wistar rats. Increased levels of GST-π isoenzyme expression in animals with EMT formation were observed. ETA-loaded and mesenchymal phenotype targeted nanoformulations increased adjuvant therapy efficacy and reduced formed granuloma sizes.