Professor University of Idaho Moscow, Idaho, United States
Introduction: Plant-derived extracellular vesicles (EVs), containing a diverse array of bioactive cargos such as proteins, microRNAs, small RNAs, lipids, and metabolites, have recently become the focus of rising interest due to their important roles in various applications. Particularly in cancer therapy, the use of natural EVs sourced from plants are preferred over synthetic compounds for drug delivery due to their physiological and immunological compatibility. Among plant families, Brassicaceae, encompassing cabbage, broccoli, mustard, and Arabidopsis thaliana, emerges as a notable reservoir of glucosinolates, myrosinases, and isothiocyanates, exhibiting potential cancer chemopreventive properties. Recent studies have highlighted the remarkable suppression of human breast cancer cell growth following direct incubation of leaf explants from non-medicinal Arabidopsis thaliana pre-treated with phytohormone jasmonate. Building upon this, our investigation focuses on isolating EVs from Arabidopsis thaliana induced by methyl jasmonate, aiming to elucidate their antineoplastic potency on various cancer cell lines in comparison with non-cancerous cells.
Materials and
Methods: Arabidopsis thaliana seeds were planted by sprinkling uniformly over moist soil in plastic pots and exposed to light intensity of 100 µmol/m2/s with a cycle of 16 h on and 8 h off. After treating with various concentrations of methyl jasmonate (0, 5, 50, and 250 uM) for 24 hr, fifty leaves of 4-week-old Arabidopsis were cut respectively at petioles, and then placed inside a syringe and infiltrated with vesicle infiltration buffer by centrifugation at 900× g for 30 min at 4°C. The collected apoplastic washing fluids (AWFs) from Arabidopsis leaves with and without the induction of phytohormone methyl jasmonate were used for the isolation of EVs. The lactC1C2-SA encoding plasmid was constructed by inserting the gene sequences of lactadherin C1C2 domain into the multiple cloning sites from EcoRV and EcoRI in the established pET-30a(+)-SA plasmid. The constructed plasmid was transformed into competent E. coli cells and have lactC1C2-SA fusion protein expressed using the protocols described previously. Expressed lactC1C2-SA fusion protein was purified by affinity chromatography and characterized by SDS‐PAGE. The isolated lactC1C2-SA was tethered on biotinylated gold nanoparticles (AuNPs) and then further mixed with EV-containing AWFs collected from Arabidopsis leaves. The release of EVs from the bound AuNPs was achieved by adding divalent ion solution. The purified EVs were characterized by TEM and immunoblotting assay. Cytotoxic effects of purified EVs on cancer cells (A549 and PC-3) and non-cancer cells (HEK293T and J774A.1) were assessed by photomicrographs of cell confluence before and 48-hour after the treatment.
Results, Conclusions, and Discussions: As shown in Figure 1a, the gene sequences encoding lactC1C2 and SA from the constructed pET-30a(+)-lactC1C2-SA cloned by PCR were verified by DNA gel electrophoresis. Expressed lactC1C2-SA fusion protein was purified by affinity chromatography and characterized by SDS‐PAGE shown as Figure 1b. Figure 1c show two pots of Arabidopsis thaliana post-4-week growth. As illustrated in Figure 1d, the purified plant EVs were shown by TEM images with average size in the range of 100 nm. The surface marker of purified plant EVs confirmed by the dot blot assay was CD63 widely-reported expressing on mammalian exosomes; on the contrary, the AuNPs used to facilitate the separation of Arabidopsis leave EVs were not detected any CD63 signal (Figure 1e). For antineoplastic potency of EVs collected from jasmonate-induced Arabidopsis leaves, the growth of A549 lung cancer cells and PC-3 prostate cancer cells was arrested for the EVs isolated from the groups with 24-hr treatment of methyl jasmonate up to 250 microM, in comparison with the non-treatment control groups (shown in Figure 2). It is important to confirm that non-cancer cells (HEK293T cells and J774A.1 macrophages) treated with EVs collected from Arabidopsis leaves induced by phytohormone methyl jasmonate were able to proliferate like the non-treatment control groups (illustrated in Figure 2). This study alludes to antineoplastic bioactive agents are boosted in jasmonate-induced Arabidopsis leaves and transported into EV cargos which have implications in developing anticancer treatments.
