Theses and Dissertations Collection

Cancer is the leading cause of global death, and the magnitude of mortality is even higher than coronary heart disease. Chemotherapy is still considered the preferred treatment in many cases, but it comes with certain challenges such as poor specificity, toxicity to healthy cells, and development of multidrug resistance. Hence, there is an urgent need of developing novel anticancer therapeutics which are biocompatible and least side effects. Medicinal plants are being used for therapeutical purposes for centuries. One such example is cruciferous plants (a type of Brassicaceae family such as mustard seeds, broccoli, cabbage, and brussels sprouts) in which sinigrin is present in significant amounts. Epidemiological studies suggest that the consumption of such vegetables decreases the risk of cancer, and the effect is attributed mainly to allyl isothiocyanate (AITC), a hydrolysis product of sinigrin catalyzed by myrosinase. Anticancer activity of AITC has been previously investigated for several cancer models, but less attention was paid to delivering AITC on the target site. In current work, sinigrin–myrosinase system is purposed as enzyme prodrug therapy to eradicate cancer cells. In the first part, a fusion protein composed of myrosinase and core streptavidin (coreSA) was obtained through bacterial expression and purifying using immobilized metal affinity chromatography (IMAC), and then tethered on the outer membrane of biotinylated A549 lung cancer cells. After treating with sinigrin, the myrosinase decorated on A549 cells produced AITC, which induced cell apoptosis. Next, silver nanoparticles were green synthesized using sinigrin as a reducing and capping agent and tested on A549 cells transfected with myrosinase-encoding plasmid. The overexpression of myrosinase in cancer cells catalyzed the sinigrin into AITC which inhibited the malignant growth along with Ag ion released from silver nanoparticles. Furthermore, for directed-delivery of myrosinase to cancer site using anti-HER2 scFv (single chain variable fragment) antibody, two approaches were adopted. First, the vector encoding gene sequences of anti-HER2 scFv, myrosinase, and coreSA were transformed into lemo21 (DE3) competent E. Coli for expression. Then, the purified chimeric protein was achieved using IMAC and labeled with biotin-FITC and delivered to HER2+ cancer cells. The results showed that after dosing sinigrin, cancer cells underwent apoptosis which caused cell death. During the second approach, gene-directed enzyme prodrug therapy (GDEPT) was adopted. The myrosinase cDNA was packed into a biotinylated lentivirus. The gene sequences of scFv of anti-HER2 and coreSA were cloned in pET-30a(+) plasmid and expressed in lemo21 (DE3) competent E. Coli. The chimeric protein of anti-HER2-coreSA was purified using IMAC and decorated on lentivirus through streptavidin-biotin binding. Th lentiviral vector tethered with anti-HER2 scFv selectively delivered myrosinase to HER2+ cancer cells which were eradicated by treating with sinigrin.