Differential cytotoxicity of MEX: a component of Neem oil whose action is exerted at the cell membrane level

Abstract

Neem oil is obtained from the seeds of the tree Azadirachta indica. Its chemical composition is very complex, being rich in terpenoids and limonoids, as well as volatile sulphur modified compounds. This work focused on the evaluation of a component of the whole Neem oil obtained by methanolic extraction and defined as MEX. Cytotoxicity was assessed on two different cell populations: a stabilized murine fibroblast line (3T6) and a tumor cell line (HeLa). The data presented here suggest a differential sensitivity of these two populations, the tumor line exhibiting a significantly higher sensitivity to MEX. The data strongly suggest that its toxic target is the cell membrane. In addition the results presented here imply that MEX may contain one or more agents that could find a potential use in anti-proliferative therapy.

Figure 1

Vitality of 3T6 cell (blue bars) compared to HeLa cells (green bars) after treatment with MEX at different concentrations (central red area). Cell survival is reported as percent with respect to untreated cultures. All samples were in 2% ethanol (final concentration) except for the first two bars to the left where ethanol was absent. Error bars represent the standard error of the mean (mean ± SEM).

Figure 2

A) Expression of the HLM gene (lane 2) which is absent in 3T6 cells (lane 3). The upper band is the internal reference standard (actin) in HeLa (lane 1) and in 3T6 (lane 3). B) Amplification of the HLM gene in co-cultured 3T6 and Hela cells. Upper band as in Figure 3A. M is a commercial molecular weight marker (100 bp multimer). C) Quantitative analysis of the amplification products reported in B) normalized to the actin amplification product. Error bars (panel C) represent the standard error of the mean (mean ± SEM).

Figure 3

Concentration of malonaldialdehyde [MDA, μM] after treatment with MEX (central bar). MDA concentration is about 25-fold higher as compared to untreated controls (left bar). The effect of treatment with H₂O₂ is shown by the right bar. Error bars represent the standard error of the (mean ± SEM).

Figure 4

Reduction of the production of MDA in 3t6 cells after treatment with MEX (red central area) in the presence of three different antioxidant curcumin (C), trolox (Tr) and resveratrol (Rv) shown respectively in the third, fourth and fifth bar. These compounds reduce drastically the oxidative stress also after treatment with H₂O₂ in the absence of MEX (bars with white central area). Error bars represent the standard error of the standard of the mean (mean ± SEM).

Figure 5

Production of MDA plotted vs. absorbance with the formation of a standard reference curve.