Melatonin blunted the angiogenic activity in 3D colon cancer tumoroids by the reduction of endocan

Abstract

Abstract Background Complexity and heterogeneity of the tumor niche are closely associated with the failure of therapeutic protocols. Unfortunately, most data have been obtained from conventional 2D culture systems which are not completely comparable to in vivo microenvironments. Reconstructed 3D cultures composed of multiple cells are valid cell-based tumor models to recapitulate in vivo-like interaction between the cancer cells and stromal cells and the oncostatic properties of therapeutics. Here, we aimed to assess the tumoricidal properties of melatonin on close-to-real colon cancer tumoroids in in vitro conditions. Methods Using the hanging drop method, colon cancer tumoroids composed of three cell lines, including adenocarcinoma HT-29 cells, fibroblasts (HFFF2), and endothelial cells (HUVECs) at a ratio of 2: 1: 1, respectively were developed using 2.5% methylcellulose. Tumoroids were exposed to different concentrations of melatonin, from 0.005 to 0.8 mM and 4 to 10 mM, for 48 h. The survival rate was measured by MTT and LDH leakage assays. Protein levels of endocan and VEGF were assessed using western blotting. Using histological examination (H & E) staining, the integrity of cells within the tumoroid parenchyma was monitored. Results Despite the reduction of viability rate in lower doses, the structure of tumoroids remained unchanged. In contrast, treatment of tumoroids with higher doses of melatonin, 4 and 10 mM, led to disaggregation of cells and reduction of tumoroid diameter compared to the non-treated control tumoroids (p < 0.05). By increasing melatonin concentration from 4 to 10 mM, the number of necrotic cells increased. Data showed the significant suppression of endocan in melatonin-treated tumoroids related to the non-treated controls (p < 0.05). According to our data, melatonin in higher doses did not alter protein levels of VEGF (p > 0.05). Conclusions Melatonin can exert its tumoricidal properties on colon cancer tumoroids via the reduction of tumor cell viability and inhibition of the specific pro-angiogenesis factor.