Botox-A induced apoptosis and suppressed cell proliferation in fibroblasts pre-treated with breast cancer exosomes

Hossein Sayaf; Niloufar Salimian; Mahnaz Mohammadi; Parisa Ahmadi; Amir Gholamzad; Sadegh Babashah; Maliheh Entezari; Najma Farahani; Maryam Montazeri; Mehrdad Hashemi

doi:10.1016/j.mcp.2024.102007

Botox-A induced apoptosis and suppressed cell proliferation in fibroblasts pre-treated with breast cancer exosomes

Mol Cell Probes. 2025 Jan 6:79:102007. doi: 10.1016/j.mcp.2024.102007. Online ahead of print.

Authors

Affiliations

¹ Department of Biotechnology, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran; Farhikhtegan Medical Convergence Sciences Research Center, Farhikhtegan Hospital Tehran Medical Sciences, Islamic Azad University, Tehran, Iran.
² Farhikhtegan Medical Convergence Sciences Research Center, Farhikhtegan Hospital Tehran Medical Sciences, Islamic Azad University, Tehran, Iran; Department of Biology, Faculty of Basic Sciences, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran.
³ Farhikhtegan Medical Convergence Sciences Research Center, Farhikhtegan Hospital Tehran Medical Sciences, Islamic Azad University, Tehran, Iran; Department of Biology, Faculty of Basic Sciences, Islamshahr Branch, Islamic Azad University, Islamshar, Tehran, Iran.
⁴ Farhikhtegan Medical Convergence Sciences Research Center, Farhikhtegan Hospital Tehran Medical Sciences, Islamic Azad University, Tehran, Iran; Immunology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
⁵ Farhikhtegan Medical Convergence Sciences Research Center, Farhikhtegan Hospital Tehran Medical Sciences, Islamic Azad University, Tehran, Iran; Department of Laboratory Medicine, Faculty of Paramedical Sciences, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran.
⁶ Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
⁷ Farhikhtegan Medical Convergence Sciences Research Center, Farhikhtegan Hospital Tehran Medical Sciences, Islamic Azad University, Tehran, Iran; Department of Genetics, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran. Electronic address: [email protected].
⁸ Farhikhtegan Medical Convergence Sciences Research Center, Farhikhtegan Hospital Tehran Medical Sciences, Islamic Azad University, Tehran, Iran. Electronic address: [email protected].
⁹ Department of Biotechnology, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran; Farhikhtegan Medical Convergence Sciences Research Center, Farhikhtegan Hospital Tehran Medical Sciences, Islamic Azad University, Tehran, Iran. Electronic address: [email protected].
¹⁰ Farhikhtegan Medical Convergence Sciences Research Center, Farhikhtegan Hospital Tehran Medical Sciences, Islamic Azad University, Tehran, Iran; Department of Genetics, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran. Electronic address: [email protected].

PMID: 39732179
DOI: 10.1016/j.mcp.2024.102007

Abstract

Background: breast cancer-associated fibroblast (CAF) is linked to metastasis and is poor for breast cancer prognosis. Since Clostridium Toxin A (Botox-A) had represented a cytotoxic effect on fibroblasts, this study aims to assess Botox-A cytotoxicity in both normal fibroblasts and exosome-induced CAFs.

Material and method: the serum exosomes of 40 BC patients and 30 healthy individuals were isolated and lncRNA H19 (lnch19) levels were assessed by qRT-PCR method. After that, Breast Cancer (BC) exosomes co-cultured with Human foreskin fibroblasts (HFF) and qRT-PCR were applied to evaluate α-SMA, Vimentin, BCL-2, and BAX expression. Both Normal and malignant HFFs co-cultured with Botox-A, and Botox-A loaded exosome for 24 and 48 h and their apoptosis, Cell proliferation, and viability were monitored by MTT assay, Annexin V-FITC and PI staining and qRT-PCR for BCL-2, BAX, and cyclin D1 mRNAs.

Results: Serum exosomes of BC patients had significantly higher levels of lncRNA H19 than healthy individuals. MTT assay results showed Botox-A decreased vital Human foreskin fibroblasts in a dose-dependent manner. BC exosomes significantly increased α-SMA, Vimentin, and BCL-2 mRNA levels in Human foreskin fibroblasts, on the other hand, BAX decreased meaningfully. Co-culture of exosome-treated HFF cells with both Botox-A and Botox-A loaded exosomes significantly boosted BCL-2 mRNA levels, completely contrary to BAX and cyclid d1 expression. Meanwhile, flow cytometry results confirmed a high rate of apoptosis in malignant Human foreskin fibroblasts treated with Botox-A loaded exosome.

Conclusion: The findings of this study indicate that exosomal lncRNA H19 could be a diagnostic marker for Breast Cancer and these Breast cancer exosomes can induce malignant phenotype in fibroblasts and turn them into CAFs. Botox-A could be toxic for both normal fibroblasts and CAFs, inducing apoptosis and suppressing cell proliferation among them.

Keywords: Apoptosis; Botulinum; Breast cancer; Epithelial-mesenchymal transition; lncRNA.