Cytotoxicity of nimbolide, epoxyazadiradione and other limonoids from neem insecticide.

Neem seed preparations contain not only azadirachtin as the active insect antifeedant or growth regulator but also a variety of their limonoids, some of which are cytotoxic to N1E-115 neuroblastoma (mouse), 143B.TK- osteosarcoma (human) and Sf9 (insect) cultured cell lines. The most potent of these limonoids is nimbolide with an IC50 ranging from 4 to 10 microM, and averaging 6 microM for the three cell lines. Other limonoids of decreasing potency and their average IC50 values (microM) are epoxyazadiradione 27 microM, salannin 112 microM, and nimbin, deacetylnimbin and azadirachtin each >200 microM (practically nontoxic). Nimbolide at 10 microM acts rapidly in the neuroblastoma cells to induce blebbing associated with disruption of plasma membranes almost instantaneously and 50% loss of cell viability with 30 min. At 5 microM nimbolide, the cells become elongated and assume a neuronal shape accompanied by spikes and lamellipodia within 1-2 hr followed shortly thereafter by extensive cytological changes and and vacuolization associated with irreversible processess leading to cell death. Calcium is apparently not involved the cytotoxic effect since a calcium-free medium, leading to profound morpholigical changes, does not alter the sensitivity to nimbolide. In contrast, epoxyazadiradione requires higher concentrations and a few hr for 50 % viability loss without major morphological changes, indicating a difference in mode of action for nimbolide and epoxyazadiradione. and epoxyazadiradione.