A COMPREHENSIVE STUDY ON APOPTOSIS INDUCTION BY AZADIRACHTIN IN Spodoptera frugiperda CULTURED CELL LINE Sf9

Abstract

The induction of apoptosis by azadirachtin, a well-known botanical tetranortriterpenoid isolated from the neem tree (Azadirachta indica A. Juss) and other members of the Meliaceae, was investigated in Spodoptera frugiperda cultured cell line (Sf9). Morphological changes in Sf9 cells treated by various concentrations of azadirachtin were observed at different times under light microscopy. Morphological and biochemical analysis indicated that Sf9 cells treated by 1.5 μg/mL azadirachtin showed typical morphological changes, which were indicative of apoptosis and a clear DNA ladder. The flow cytometry analysis showed the apoptosis rate reached a maximum value of 32.66% at 24 h with 1.5 μg/mL azadirachtin in Sf9 cells. The inhibition of Sf9 cell proliferation suggested that the effect of azadirachtin was dose dependent and the EC50 at 48 and 72 h was 2.727 × 10(-6) and 6.348 × 10(-9) μg/mL, respectively. The treatment of azadirachtin in Sf9 cells could significantly increase the activity of Sf caspase-1, but showed no effect on the activity of Topo I, suggesting that the apoptosis induced by azadirachtinin Sf9 cells is through caspase-dependent pathway. These results provided not only a series of morphological, biochemical, and toxicological comprehensive evidences for induction of apoptosis by azadirachtin, but also a reference model for screening insect cell apoptosis inducers from natural compounds.

Cytotoxic effects of β‐asarone on Sf9 insect cells

“…Azadirachtin is categorized as an antimitotic insecticide that targets cell division and cause proliferation inhibition and apoptosis in insect cell lines (Huang, Shui et al, ; Huang et al, ; Rembold & Annadurai, ; Salehzadeh et al, ). Shu et al () showed this compound inhibited Sf9 cell proliferation with the half maximal effective concentration at 48 and 72 h was 2.727 × 10 − 6 and 6.348 × 10 − 9 µg/mL, respectively. Huang et al () revealed that azadirachtin could induce apoptosis in Sf9 cells by the release of cytochrome‐c from the mitochondria.…”

Azadirachtin-induced effects on various life history traits and cellular immune reactions of Galleria mellonella (Lepidoptera: Pyralidae)

“…An alternative explanation could be that azadirachtin decreased the counts of circulating hemocytes due to the induction of autophagic or apoptotic pathways resulting in cell death. It was reported in many studies that azadirachtin leads to apoptosis and autophagy in insect cell lines originated from ovarian tissues [13,14]. To prove this hypothesis, more detailed investigations need to be conducted into the effects of azadirachtin on hemocyte death.…”

“…It is generally accepted that azadirachtin acts as an insect growth regulator by blocking the biosynthesis of insect hormones such as ecdysteroids and inhibiting the development of reproductive organs and vitellogenesis that reduce the fecundity and fertility of insects [10,11]. In addition, recent works demonstrated that azadirachtin has antiproliferative effects by arresting the cell cycle and inducing apoptotic effects in insect cell lines [12][13][14] Insects resist external materials with their highly effective immune system that relies on cellular and humoral components. Humoral immune reactions include the production of antimicrobial peptides, intermediates of nitrogen, oxygen and cytokines, as well as the prophenoloxidase (PO) cascade that regulates melanization of hemolymph [15].…”

Changes in immune responses of Helicoverpa armigera Hübner followed by feeding on Knotgrass, Polygonum persicaria agglutinin

“…Some studies have reported that plant lectins cause cytotoxicity through apoptosis process by activation of caspase enzymes (Rahimi et al, ; Ramzi et al, ; Shahidi‐Noghabi et al, ; Sprawka et al, ). Apoptosis process and autophagy in insect cell lines such as hemocytes following azadirachtin have been reported by Shu et al (). Since the cytotoxic effect of PPA on H .…”