(175aj) Apoptosis of Myrosinase Gene Expressed Lung Cancer Cells Treated with Glucosinolate-Containing Extracellular Vesicles Isolated from Arabidopsis thaliana

Due to the limitations of cancer treatment using chemotherapy, there is a need to develop anticancer therapeutics which should be target specific and carry minor side effects. Plants have been used as therapeutical agents for years. Specifically, cruciferous plants which contain glucosinolates, present in plant leaves and roots, have been reported to have properties such as anticancer, antibacterial and antioxidant among others. Though glucosinolates are biologically inactive but can be hydrolyzed by β-thioglucoside hydrolases commonly known as myrosinase to produce isothiocyanates, thiocyanates, nitriles, etc. The effect is due to the isothiocyanate (ITC) considered as a product of glucosinolate which is enzymatically hydrolyzed by myrosinase. Based on our hypothesis, glucosinolates, to be specific glucoraphanin and sinigrin among others, are present in extracellular vesicles (EVs) of cruciferous plants and can act as anticancer carriers. In this study, EVs were isolated from the Arabidopsis thaliana leaves using 800-nm biotinylated polystyrene particles bound with core streptavidin (coreSA) and lactadherin C1C2 fusion protein The morphology of isolated EVs was then characterized using transmission electron microscopy (TEM). The concentration and size of EVs were determined by nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS). As per the TEM results, plant EVs revealed cup-shaped structure which is the typical morphology of exosomes. The results of DLS and NTA revealed EVs size ranged between 116-147 nm and zeta potential as -42 mV. EVs were also characterized by Western blotting using commercially available antibodies including CD9, CD63, CD81, TSG101 and ALIX. The concentration of the EVs determined by NTA was 2×10¹⁰ particles/mL. The glucosinolates entrapped within the collected EVs were characterized by LC-MS. The A549 lung caner cells were transfected with plasmid encoding myrosinase gene and eGFP using Lipofectamine 3000 transfection reagent. The cultured A549 cells expressing myrosinase and eGFP were treated with 1×10⁹, 6×10⁹, and 1.2×10¹⁰ concentrations of EVs to confirm the dose dependent anticancer activity. As per our results, EVs with the highest concentrations proved to be more effective in producing ITC and inducing malignant cell apoptosis.