Effect of Apatinib plus melatonin on vasculogenic mimicry formation by cancer stem cells from breast cancer cell line

Published on Thursday, 30 April 2026

Abstract

Background and aim: Vasculogenic mimicry (VM) is one of the most important causes of breast cancer metastasis and resistance against drugs. The cancer stem cells (CSCs) are known as essential factors for VM formation. In this study, the effects of melatonin, Apatinib, and a combination of Apatinib/melatonin on VM formation were investigated by breast CSCs from breast cancer cell line.

Materials and methods: The percentage of CSCs was determined in two breast cancer cell lines (MCF-7 and MDA-MB-231) by flow cytometry. The effects of Apatinib, melatonin, and a combination of Apatinib/melatonin were evaluated on proliferation and viability, migration and invasion, apoptosis, and VM formation in MDA-MB-231 cells. Moreover, expression levels of the involved proteins in cancer cell proliferation and viability, CSCs, migration and invasion, and VM formation were evaluated by real-time polymerase chain reaction (RT-PCR) and western blotting methods.

Results: Results of the present study showed that melatonin and Apatinib reduced survival rate of CSCs in a dose- and time-dependent manner. Apatinib, melatonin, and a combination of Apatinib/melatonin inhibited proliferation of breast CSCs (P ≤ 0.001). Formation of VM was decreased in the MDA-MB-231 cancer cell line treated with Apatinib and combination of Apatinib/melatonin. Apatinib and combination of Apatinib/melatonin reduced invasion of breast CSCs (P ≤ 0.0001). Expression of vascular endothelial VE-cadherin, ephrinA2 receptor (EPHA2), p-PI3K/phosphoinositide-3 kinase (PI3K) and phospho-AKT (p-AKT)/AKT ratios was decreased under the influence of Apatinib and a combination of Apatinib/melatonin (P ≤ 0.01).

Conclusion: Apatinib or a combination of Apatinib/melatonin may be used to manage patients with breast cancer. However, further studies are needed to identify anti-cancer mechanisms of melatonin and Apatinib for better management of the patients with breast cancer.

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About this publication.

The Di Bella's Method: Use of Melatonin, Retinoids (40mg per day Beta-Carotene, 10mg per day ATRA and 10mg per day Axerophthol palmitate), Estrogen therapy (Anastrozole 1 mg tab per day), pseudo-Metronomic Chemotherapy Cyclophosphamide and/or Hydroxyurea, Somatostatin/Octreotide analogues and/or derivatives with Cabergoline and/or Bromocriptine (together with others chemical compounds) in Breast Cancer:

- A retrospective observational clinical study of triple negative breast cancer cases treated with Di Bella Method: A preliminary data;

- The Synergism of Somatostatin, Melatonin, Vitamins Prolactin and Estrogen Inhibitors Increased Survival, Objective Response and Performance Status In 297 Cases of Breast Cancer;

- Complete objective response, stable for 5 years, with the Di Bella Method, of multiple-metastatic carcinoma of the breast;

- Evaluation of the safety and efficacy of the first-line treatment with somatostatin combined with melatonin, retinoids, vitamin D3, and low doses of cyclophosphamide in 20 cases of breast cancer: a preliminary report;

- The Di Bella Method (DBM) improved survival, objective response and performance status in a retrospective observational clinical study on 122 cases of breast cancer;

- Complete objective response to biological therapy of plurifocal breast carcinoma;

- The Di Bella Method (A Fixed Part - Melatonin tablets. From 30-40mg/day up to 200mg/day and more 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 Fixed Part - Somatostatin, Octreotide, Sandostatin LAR, analogues and/or derivatives since 1977);

- 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);

- Publication, 2020 Sep: Two neuroendocrine G protein-coupled receptor molecules, somatostatin and melatonin: Physiology of signal transduction and therapeutic perspectives (from Di Bella's Foundation);

- The Di Bella Method (A Fixed Part - Bromocriptine and/or Cabergoline);

- Prolactin inhibitors in oncology - In vitro, review and in vivo publications;

The Di Bella's Method: Use of Melatonin since 1974, Somatostatin/Octreotide analogues and/or derivatives since 1977 with Cabergoline and/or Bromocriptine associated with pseudo-Metronomic Chemotherapy Cyclophosphamide and/or Hydroxyurea - together with others chemical compounds - in several Oncological Pathologies:

- A retrospective observational study on cases of anaplastic brain tumors treated with the Di Bella Method: A rationale and effectiveness;

- Recurrent Glioblastoma Multiforme (grade IV – WHO 2007): a case of complete objective response achieved by means of the concomitant administration of Somatostatin and Octreotide – Retinoids – Vitamin E – Vitamin D3 – Vitamin C – Melatonin – D2 R agonists (Di Bella Method – DBM) associated with Temozolomide;

- The Di Bella Method DBM improved survival objective response and performance status in a retrospective observational clinical study on 23 tumours of the head and neck;

- Chronic Lymphocytic Leukemia: Long-Lasting Remission with Combination of Cyclophosphamide, Somatostatin, Bromocriptine, Retinoids, Melatonin, and ACTH;

- Somatostatin, retinoids, melatonin, vitamin D, bromocriptine, and cyclophosphamide in advanced non-small-cell lung cancer patients with low performance status;

- Somatostatin, retinoids, melatonin, vitamin D, bromocriptine, and cyclophosphamide in chemotherapy-pretreated patients with advanced lung adenocarcinoma and low performance status;

- Observations on the Report of a case of pulmonary adenocarcinoma with lymph node, hepatic and osseus metastasis;

- Pleural Mesothelioma: clinical records on 11 patients treated with Di Bella's Method;

- Malignant pleural mesothelioma, stage T3-T4. Consideration of a case study;

- Excellent result in a Mesothelioma case treated exclusively with Di Bella Method for over 4 years and still treatment with positive results;

- A case of advanced Multiple Myeloma treated with Di Bella Method (DBM) into total remission for 13 years;

- Neuroblastoma: Complete objective response to biological treatment;