Professor University of Idaho Moscow, Idaho, United States
Introduction:: Sinigrin is one of the major glucosinolates found in cruciferous plants such as Brussels sprouts, broccoli, and mustard seeds. The sinigrin–myrosinase chemical defense system is based on the rapid release of large amounts of cytotoxic allyl isothiocyanate (AITC) through the mixing of inert sinigrin and myrosinase upon plant tissue disruption. The potential of sinigrin to prevent the growth of cancer cells has been well reported. However, sinigrin cytotoxicity to cancer cells requires the addition of myrosinase to hydrolyze sinigrin and produce AITC to develop anticancer activity. In this study, phytochemical sinigrin encapsulated in chitosan (CS)-tripolyphosphate (TPP) ionic gelation nanoparticles (NPs) were fabricated to explore their anticancer effectiveness on myrosinase-expressed adenocarcinoma A549 cells. To our knowledge, the study presented here is the first attempt to examine anticancer effectiveness of delivering CS-TPP NPs encapsulating phytochemical sinigrin into cancer cells transfected with myrosinase gene.
Materials and Methods:: The sinigrin-CS-TPP nanoparticles were prepared as following: first goes as follows: 760 µl of chitosan (medium viscosity, 0.1% wt dissolved in 1% acetic acid, pH = 4) was added into a microcentrifuge tube, and then 10 µl of 1 mM sinigrin was added to the chitosan solution with vortex. After the sinigrin was added, 40 µl of TPP (0.1% wt, pH =4) were added dropwise into the sinigrin-chitosan solution under vertex for 30 mins. The final solution containing sinigrin-CS-TPP NPs was filtered by a 0.45 µm syringe filter. The size and charge of the filtered NPs were measured by a dynamic light scattering device. The release profile of sinigrin from the CS-TPP NPs was determined using the dialysis membrane approach with the spectrometer detection of sinigrin at 227 nm. A549 lung cancer cells were transduced with myrosinase-green fluorescent protein (MYR-GFP) fusion gene-encoded lentivirus. The myrosinase-expressing A549 cells cultured in a 6-well plate with 1 ml of culture medium were treated with sinigrin-CS-TPP NPs in a serial dilution manner. The cell viability was determined by the level of GFP expression over a period of 48 hrs.
Results, Conclusions, and Discussions:: The average size of sinigrin-CS-TPP NPs detected by dynamic light scattering was 489.50 nm and the charge was -29.45 mV. Based on the sinigrin releasing profile, Michaelis-Menton type release was determined to be the best fit model. Fluorescent microscopy was used to confirm the expression of GFP. The transfected A549 cells are expected to express both myrosinase and green fluorescent protein. Since the marker protein (i.e., GFP) revealed its functional feature (i.e., green fluorescence), the heterologous protein (i.e., myrosinase) must be correctly folded as well. Therefore, the level of myrosinase expressed in the transfected A549 cell should be proportional to the degree of GFP expression. After loading sinigrin-CS-TPP NPs for 24 hrs, our result demonstrates that phytochemical sinigrin harnessed like a prodrug could be catalyzed by myrosinase-expressed adenocarcinoma A549 lung cancer cells to the production of AITC which induced cell apoptosis and arrested the growth of cancer cells. After 48-hr treatment, the A549 cancer cells were all dead as indicated without any GFP expression under fluorescent microscopy.
