Anti-Angiogenic and Anti-Metastatic Effects of Biogenic Silver Nanoparticles Synthesized Using <i>Azadirachta indica</i>

Abstract

Background: Nanotechnology symbolizes a broad discipline with enormous potential in cancer treatment bridging one of the bottlenecks of traditional approaches in cancer therapy which is an inability to deliver adequate quantities of anti-cancer drugs to the tumor area. Studies on nanoparticles indicate their importance in cancer angiogenesis and metastasis. Aim: The present study assessed anti-angiogenesis and anti-metastatic effects of biogenic silver nanoparticles (AgNPs) synthesized from neem plant ( Azadirachta indica ). Methods: Chicken chorioallantoic membrane (CAM) and two-dimensional (2D) wound healing assays were used to study anti-angiogenic and anti-metastatic effects of the AgNPs respectively. Twenty-four fertilized eggs were divided into four groups: two biogenic AgNPs treatments at 100 μg/ml and 200 μg/ml; negative control (1% DMSO) and positive control (cyclophosphamide). On day 8 of incubation, filter discs impregnated with different concentration levels of the treatments were placed on the CAM. On day 12 of incubation, the CAMs were imaged using a stereomicroscope, scaled using ImageJ, and different morphometric and spatial parameters computed using AngioTool software. Vessel area, vessel percent area, total number of junctions, total vessel length, average vessel length, mean lacunarity, and junction density were measured. The crown-rump length (CRL) and fetal weight were also recorded on day 16 of incubation. In order to determine relative gene expression profiles of iNOS and VEGF, total RNA was extracted from the C AM, and qRT-PCR was performed with β -Actin as a reference gene. For the 2D wound healing assay, DU145 human prostate cells were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% Fetal Bovine Serum. Results: Biogenic AgNPs demonstrated anti-angiogenic effects in a dose-dependent manner in the parameters generated from the CAM images. Also, qRT-PCR revealed down-regulation of iNOS and VEGF genes. The 2-dimensional wound healing assay showed inhibition of migration and motility of the DU145 cells for the 72-hours of assessment. Conclusion: The present study postulates that the biogenic AgNPs can prevent angiogenesis by inactivation of VEGF-NO and VEGF/VEGF-R pathways while inhibiting cell migration and metastasis.