The anthelmintic drug N,N-diethyl-m-toluamide (DEET) has been reported to inhibit AChE (acetylcholinesterase) and has potential carcinogenic properties due to excessive vascularization. In this paper, we show that DEET specifically stimulates endothelial cells that promote angiogenesis, thereby increasing tumor growth. DEET activates cellular processes leading to angiogenesis, including proliferation, migration, and adhesion. This is associated with increased NO production and VEGF expression in endothelial cells. Silencing of M3 or using pharmacological M3 inhibitors abolished all these effects, suggesting that DEET-induced angiogenesis is M3-sensitive. Experiments involving calcium signaling in endothelial and HEK cells overexpressing M3 receptors, as well as binding and docking studies, indicate that DEET acts as an allosteric modulator of M3 receptors. Furthermore, DEET inhibits AChE, thereby increasing the bioavailability of acetylcholine and its binding to M3 receptors, and enhancing proangiogenic effects through allosteric regulation.
Primary ECs were isolated from the aorta of Swiss mice. The extraction method was adapted from the Kobayashi protocol 26 . Murine ECs were cultured in EBM-2 medium supplemented with 5% heat-inactivated FBS until the fourth passage.
The effect of two concentrations of DEET on the proliferation of HUVEC, U87MG, or BF16F10 was analyzed using the CyQUANT Cell Proliferation Assay Kit (Molecular Probes, C7026). Briefly, 5.103 cells per well were seeded in a 96-well plate, allowed to attach overnight, and then treated with DEET for 24 h. After removing the growth medium, add dye binding solution to each well of the microplate and incubate the cells at 37 °C for 30 min. Fluorescence levels were determined using a Mithras LB940 multimode microplate reader (Berthold Technologies, Bad Wildbad, Germany) equipped with 485 nm excitation filters and 530 nm emission filters.
HUVEC were seeded in 96-well plates at a density of 104 cells per well. Cells were treated with DEET for 24 h. Cell viability was assessed using a colorimetric MTT assay (Sigma-Aldrich, M5655). Optical density values were obtained on a multimode microplate reader (Mithras LB940) at a wavelength of 570 nm.
The effects of DEET were studied using in vitro angiogenesis assays. Treatment with 10-8 M or 10-5 M DEET increased the formation of capillary length in HUVECs (Fig. 1a, b, white bars). Compared with the control group, treatment with DEET concentrations ranging from 10-14 to 10-5 M showed that the capillary length reached a plateau at 10-8 M DEET (Supplementary Fig. S2). No significant difference was found in the in vitro proangiogenic effect of HUVECs treated with DEET in the concentration range of 10-8 M and 10-5 M.
To determine the effect of DEET on neovascularization, we performed in vivo neovascularization studies. After 14 days, mice injected with endothelial cells precultured with 10-8 M or 10-5 M DEET showed a significant increase in hemoglobin content (Fig. 1c, white bars).
Furthermore, DEET-induced neovascularization was studied in U87MG xenograft-bearing mice that were injected daily (i.p.) with DEET at a dose known to induce plasma concentrations of 10-5 M, which is normal in exposed humans. in 23. Detectable tumors (i.e. tumors >100 mm3) were observed 14 days after injection of U87MG cells into mice. At day 28, tumor growth was significantly enhanced in DEET-treated mice compared with control mice (Fig. 1d, squares). Furthermore, CD31 staining of tumors showed that DEET significantly increased capillary area but not microvessel density. (Fig. 1e–g).
To determine the role of muscarinic receptors in DETA-induced proliferation, 10-8 M or 10-5 M DETA in the presence of pFHHSiD (10-7 M, a selective M3 receptor antagonist) was used. Treatment of HUVEC. pFHHSiD completely blocked the proliferative properties of DETA at all concentrations (Table 1).
Under these conditions, we also examined whether DEET would increase capillary length in HUVEC cells. Similarly, pFHHSiD significantly prevented DEET-induced capillary length (Fig. 1a, b, gray bars). Furthermore, similar experiments were performed with M3 siRNA. Although the control siRNA was not effective in promoting capillary formation, silencing of the M3 muscarinic receptor abolished the ability of DEET to increase capillary length (Fig. 1a, b, black bars).
Furthermore, both 10-8 M or 10-5 M DEET-induced vascularization in vitro and neovascularization in vivo were completely blocked by pFHHSiD (Fig. 1c, d, circles). These results indicate that DEET promotes angiogenesis through a pathway sensitive to selective M3 receptor antagonists or M3 siRNA.
AChE is the molecular target of DEET. Drugs such as donepezil, which act as AChE inhibitors, can stimulate EC angiogenesis in vitro and in mouse hindlimb ischemia models14. We tested the effect of two concentrations of DEET on AChE enzyme activity in HUVEC. Low (10-8 M) and high (10-5 M) concentrations of DEET decreased endothelial AChE activity compared to control conditions (Fig. 2).
Both concentrations of DEET (10-8 M and 10-5 M) reduced acetylcholinesterase activity on HUVEC. BW284c51 (10-5 M) was used as a control for acetylcholinesterase inhibitors. Results are expressed as percentage of AChE activity on HUVEC treated with the two concentrations of DEET compared to vehicle-treated cells. Values are expressed as mean ± SEM of six independent experiments. *p < 0.05 compared to control (Kruskal-Wallis and Dunn multiple comparison test).
Nitric oxide (NO) is involved in the angiogenic process 33, therefore, NO production in DEET-stimulated HUVECs was studied. DEET-treated endothelial NO production was increased compared to control cells, but reached significance only at a dose of 10-8 M (Fig. 3c). To determine the molecular changes controlling DEET-induced NO production, eNOS expression and activation were analyzed by Western blotting. Although DEET treatment did not alter eNOS expression, it significantly increased eNOS phosphorylation at its activating site (Ser-1177) while decreasing its inhibitory site (Thr-495) compared to untreated cells in eNOS phosphorylation (Fig. 3d). Furthermore, the ratio of phosphorylated eNOS at the activation site and inhibitory site was calculated after normalizing the amount of phosphorylated eNOS to the total amount of enzyme. This ratio was significantly increased in HUVECs treated with each concentration of DEET compared to untreated cells (Fig. 3d).
Finally, the expression of VEGF, one of the main proangiogenic factors, was analyzed by Western blotting. DEET significantly increased VEGF expression, whereas pFHHSiD completely blocked this expression .
Since the effects of DEET are sensitive to both pharmacological blockade and downregulation of M3 receptors, we tested the hypothesis that DEET might enhance calcium signaling. Surprisingly, DEET failed to increase cytoplasmic calcium in HUVEC (data not shown) and HEK/M3 (Fig. 4a, b) for both concentrations used.
Post time: Dec-30-2024