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

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.