Studies on the mode of action of azadirachtin from the neem tree, Azadirachta indica using insect cell lines
This project focuses on the cellular response in insects to the presence of azadirachtin. Using the two insect cell lines Sf9, Spodoptera frugiperda and a Drosophila cell line Kc167 it was shown that azadirachtin inhibits cell proliferation at EC50’s of 1.38 x 10-9 M and 1.72 x 10-7 M respectively. In contrast azadirachtin had no effect on the Chinese hamster ovarian cell line RR-CHOKI and in the Chinese hamster lung cell line V79-4 where effects were seen only at 10-3M and above. Cytotoxicity was observed by visual analysis of single strand DNA in the two insect cell lines with 50% damage being observed after 12 h at 2.2 x 10-9 M and 3.09 x 10-8 M for Sf9 and Kc167 cells respectively. The effects of azadirachtin on protein expression resulted in 52% of Sf9 proteins being down regulated, 29% showing increased expression and 19% showing no response to azadirachtin treatment. In addition using phage display techniques a monoclonal antibody to azadirachtin was raised. In terms of functionality the clone selected bound to the conjugated and free forms of azadirachtin but was relatively insensitive with an IC50 of 116 mM. The protein expression yield was also low at 1.43mg/ml. However, in a bid to produce a monoclonal antibody the azadirachtin compound was modified to include a linker arm to which a carrier protein (BSA) was conjugated. The modified azadirachtin compound 22’23 dihydro 3-hemiglutarate 1 hydroxy azadirachtin proved an invaluable tool in the final chapter of this work. Using nuclear extracts from the Kc167 cell line and separating these by size exclusion chromatography and Native electrophoresis the 22’23 dihydro 3-hemiglutarate 1 hydroxy azadirachtin - BSA conjugate was used in alliance with a BSA antibody to detect possible azadirachtin binding proteins when present on nitrocellulose membrane. The characterisation of a protein was established as the Drosophila melanogaster heat shock protein 60 (CG12101-PA).