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Targeting ERβ1-Positive Triple-Negative Breast Cancer: Molecular Effects of Calcitriol and 17β-Estradiol

Published on Thursday, 03 July 2025

Abstract

Background: Breast cancer is the most common malignancy in women. Triple-negative breast cancer (TNBC) is characterized by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, contributing to its aggressive nature, limited treatment options, and poor prognosis. Emerging evidence highlights estrogen receptor beta 1 (ERβ1) as a potential tumor suppressor in TNBC, influencing key oncogenic pathways such as cell proliferation, survival, angiogenesis, and apoptosis. In this regard, calcitriol (active vitamin D) and 17β-estradiol have been identified as key regulators of tumor behavior. Calcitriol shows strong anti-proliferative and pro-apoptotic effects, while the ability of 17β-estradiol to modulate tumor progression through ERβ1 signaling is context-dependent. This study aims to investigate the individual and combined effects of calcitriol and 17β-estradiol in ERβ1-expressing MDA-MB-468 TNBC cells, with a focus on their role in regulation of tumor progression, angiogenesis, and apoptosis. The findings provide novel insights into the potential therapeutic utility of targeting ERβ1 in TNBC.

Methodology: The MDA-MB-468 TNBC cells were treated with calcitriol (1-5 µM) and/or 17β-estradiol (100-500 nM). The effect on cell viability was assayed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. At the same time, immunoblot analysis investigated the time-dependent manner of ERβ1, epidermal growth factor receptor (EGFR), vascular epithelial growth factor (VEGF), and caspase-3.

Results: Both calcitriol and 17β-estradiol substantially decreased TNBC cell viability, with the highest level of cytotoxicity observed at 24 and 32 hours, respectively. The combination increased the amount of cell death. Immunoblots revealed lasting downregulation of ERβ1, EGFR, VEGF, and caspase-3 after calcitriol treatment. In comparison, 17β-estradiol demonstrated biphasic regulatory behavior for ERβ1, where ERβ1 was first downregulated, then partially recovered. The combination therapies produced more significant ERβ1 downregulation and heightened suppression of EGFR and VEGF, further enhancing their effects on TNBC progression.

Conclusions: This study aims to investigate the individual and combined effects of calcitriol and 17β-estradiol in ERβ1-expressing MDA-MB-468 TNBC cells, with a focus on their role in the regulation of tumor progression, angiogenesis, and apoptosis.

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About this publication.

The Di Bella's Method: Use of Vitamin D analogues and/or derivatives (together with others chemical compounds) in TNBC/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 - Dihydrotachysterol, Alfacalcidol, synthetic Vitamin D3);

- Vitamin D (analogues and/or derivatives) and cancer - In vitro, review and in vivo publications;

- 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);

- 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 Vitamin D analogues and/or derivatives - 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;