Induction of programmed death and cytoskeletal damage on Trichoplusia ni BTI-Tn-5B1-4 cells by azadirachtin

Abstract

Plenty of insect cells are sensitive to azadirachtin, an ideal biopesticide. However, the mode of action of azadirachtin on insect cells still remains unexplained. In this study, Trichoplusia ni BTI-Tn-5B1-4 cell line was used as the model system to evaluate induction of programmed death and cytoskeletal damage by azadirachtin. The result of colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] (MTT) assay indicated that the effect of azadirachtin on cells growth was time-dependent with different concentrations (0.75, 1.5 and 3 μg/mL). As autophagic vacuoles were observed obviously by transmission electron microscopy, azadirachtin could induce autophagy in cells. Furthermore, lysosomal function was affected after treatment with azadirachtin in time-dependent manner. Aberrant morphological figures appeared in treated cells, such as contraction and turning round, especially the appearance of apoptotic bodies and membrane blebbing, which was the evidence of azadirachtin-induced apoptosis, with apoptosis rate of 13.98% at 1.5 μg/mL for 120 h. As the stress fibers changed after treatment, the cytoskeletal function could be influenced. The effect on cell cycle was also analyzed by flow cytometry, with the result that azadirachtin enhanced cell cycle arrest at the G2/M phase gradually.

Introduction

Azadirachtin, extracted from the seeds of neem tree (Azadirachta indica A. Juss), is an environmentally safe pesticide. It has been proved azadirachtin is low toxic to beneficial organisms and humans through toxic tests against a range of mammalian cell lines, with no apparent effect under high concentrations [1], [2], [3], [4], [5]. Therefore, azadirachtin was used widely as antifeedant, growth regulator and sterilant [6], [7] to control a variety of agricultural pests such as Coleoptera, Hemiptera and Lepidoptera [7], [8], [9], [10].

In recent years, the studies towards the mode of action of azadirachtin at cellular and molecular levels have increased. In 1993, it was firstly found that azadirachtin inhibited the proliferation of Sf9 (derived from the ovary of Spodoptera frugiperda), and reduced protein synthesis [11]. Sf9 cells and C6/36 cells (derived from the mosquito Aedes albopictus) were determined, which inferred that azadirachtin was a potent inhibitor against insect cell replication [4]. Synthesis of neurosecretory proteins and peptides was interfered by azadirachtin, resulting in delaying the molting of insects, which is a manifested aspect of growth regulatory [12], [13], [14]. However, the precise cellular target and mode of action of this complex molecule is still elusive, although it has been shown that azadirachtin preferentially binds to the nuclear extract [15], and possibly to a protein complex [16]. As azadirachtin disrupts the patterning of microtubules into more complex structures like axonemes and mitotic spindles [17], [18], and acts by interfering with the mitosis of cells by polymerization of tubulin, the target is thought to be cellular cytoskeletal elements, especially tubulin, the same as other phytochemicals of similar action (colchicines, vinca alkaloids and taxol) [5]. Subsequently, it has been reported that the disruption of azadirachtin on actin was more significant than on tubulin, inferring that actin was a putative target of azadirachtin [19], [20], [21]. Previous researches also indicated that azadirachtin might affect the metabolism of insects by means of influencing certain functional proteins, which is related with insect growth and reproduction [22], [23]. Besides, the activity profiles of two lysosomal enzymes in the fat body were altered by azadirachtin [12].

Programmed cell death (PCD), a major cellular pathway for the degradation of long-lived proteins and organelles in eukaryotic cells, which include apoptosis (programmed cell death I, PCD I) and autophagy (programmed cell death II, PCD II). PCD could be induced by environmental changes like cytokines, drugs and nutritional factors [24]. As the previous studies, autophagic and apoptotic types of PCD exhibit different fates of cytoskeletal filaments [25]. Cell apoptosis could be induced by azadirachtin [19], [26], and azadirachtin-induced autophagy has not been reported up to now.

In the present work, azadirachtin-induced apoptosis and autophagy on BTI-Tn-5B1-4 cells were cleared through a series of studies, which provide a new clue for the study of mechanism of azadirachtin. In addition, to better understand how azadirachtin inhibits the growth of insects, further make clear the mechanism of azadirachtin, and to establish a link between the cellular and insects responses, we have assessed anti-proliferative, cell cycle and skeleton effects in BTI-Tn-5B1-4 cell line for azadirachtin at different time.