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

Published on Tuesday, 20 June 2023

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.

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- The Di Bella Method (A Fixed Part - Melatonin tablets. From 30-40mg/day up to 200mg/day orally in patients with advanced stage of cancer disease and/or patients without respond to traditional treatments);

- Melatonin use in cancer patients have started in 1974, when melatonin prepared according to Prof. Di Bella’s formulation [...]. For 11 days was administered to the patient, admitted to the general medical ward at the Maggiore-Pizzardi Hospital in Bologna, very slowly (over approx. 8 hours) and intravenously administered 1000 mg of melatonin for 11 days. During the course of each day, the patient was intravenously administered 4 saline drips of 500 ml, each containing ten 25 mg bottles of freeze-dried melatonin, lasting 2 hours, totaling 1000 mg per day. No other drug of any kind was administered in order to ascertain the effect of the MLT without interference [...]. From Melatonin with adenosine solubilized in water and stabilized with glycine for oncological treatment - technical preparation, effectivity and clinical findings;

- About Melatonin - In vitro, review and in vivo publications;

- Publication: Melatonin anticancer effects: Review (from Di Bella's Foundation);

- Publication: Key aspects of melatonin physiology: 30 years of research (from Di Bella's Foundation);

- The Di Bella Method (A Fixed Part - Cyclophosphamide 50mg tablets and/or Hydroxyurea 500mg tablets, one or two per day);

- The Di Bella Method (A Variable Part - Chondroitin sulfate, up to 3-4 grams per day, orally);

- The Di Bella Method (A Fixed Part - Somatostatin, Octreotide, Sandostatin LAR, analogues and/or derivatives);

- Somatostatin in oncology, the overlooked evidences - In vitro, review and in vivo publications;

- Publication, 2018 Jul: Over-Expression of GH/GHR in Breast Cancer and Oncosuppressor Role of Somatostatin as a Physiological Inhibitor (from Di Bella's Foundation);

- Publication, 2018 Sep: The over-expression of GH/GHR in tumour tissues with respect to healthy ones confirms its oncogenic role and the consequent oncosuppressor role of its physiological inhibitor, somatostatin: a review of the literature (from Di Bella's Foundation);

- Publication, 2019 Aug: The Entrapment of Somatostatin in a Lipid Formulation: Retarded Release and Free Radical Reactivity (from Di Bella's Foundation);

- Publication, 2019 Sep: Effects of Somatostatin and Vitamin C on the Fatty Acid Profile of Breast Cancer Cell Membranes (from Di Bella's Foundation);

- Publication, 2019 Sep: Effects of somatostatin, curcumin, and quercetin on the fatty acid profile of breast cancer cell membranes (from Di Bella's Foundation);