In situ allicin generation using targeted alliinase delivery for inhibition of MIA PaCa-2 cells via epigenetic changes, oxidative stress and cyclin-dependent kinase inhibitor (CDKI) expression

摘要

Allicin, an extremely active constituent of freshly crushed garlic, is produced upon reaction of substrate alliin with the enzyme alliinase (EC 4.4.1.4). Allicin has been shown to be toxic to several mammalian cells in vitro in a dose-dependent manner. In the present study this cytotoxicity was taken to advantage to develop a novel approach to cancer treatment, based on site directed generation of allicin. Alliinase was chemically conjugated to a monoclonal antibody (mAb) which was directed against a specific pancreatic cancer marker, CA19-9. After the CA19-9 mAb-alliinase conjugate was bound to targeted pancreatic cancer cells (MIA PaCa-2 cells), on addition of alliin, the cancer cell-localized alliinase produced allicin, which effectively induced apoptosis in MIA PaCa-2 cells. Specificity of anticancer activity of in situ generated allicin was demonstrated using a novel in vitro system-integrated discrete multiple organ co-culture technique. Further, allicin-induced caspase-3 expression, DNA fragmentation, cell cycle arrest, p21(Waf1/Cip1) cyclin-dependent kinase inhibitor expression, ROS generation, GSH depletion, and led to various epigenetic modifications which resulted in stimulation of apoptosis. This approach offers a new therapeutic strategy, wherein alliin and alliinase-bound antibody work together to produce allicin at targeted locations which would reverse gene silencing and suppress cancer cell growth, suggesting that combination of these targeted agents may improve pancreatic cancer therapy.