Sayaf Hossein, Salimian Niloufar, Mohammadi Mahnaz, Ahmadi Parisa, Gholamzad Amir, Babashah Sadegh, Entezari Maliheh, Farahani Najma, Montazeri Maryam, Hashemi Mehrdad
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.
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.
Mol Cell Probes. 2025 Feb;79:102007. doi: 10.1016/j.mcp.2024.102007. Epub 2025 Jan 6.
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.
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.
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.
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.
乳腺癌相关成纤维细胞（CAF）与转移相关，且对乳腺癌预后不利。由于肉毒杆菌毒素A（Botox-A）对成纤维细胞具有细胞毒性作用，本研究旨在评估Botox-A对正常成纤维细胞和外泌体诱导的CAF的细胞毒性。
分离40例乳腺癌患者和30例健康个体的血清外泌体，采用qRT-PCR法评估lncRNA H19（lnch19）水平。之后，将乳腺癌（BC）外泌体与人包皮成纤维细胞（HFF）共培养，并应用qRT-PCR评估α-SMA、波形蛋白、BCL-2和BAX的表达。正常和恶性HFFs与Botox-A以及负载Botox-A的外泌体共培养24小时和48小时，通过MTT法、Annexin V-FITC和PI染色以及对BCL-2、BAX和细胞周期蛋白D1 mRNA进行qRT-PCR监测其凋亡、细胞增殖和活力。
乳腺癌患者的血清外泌体中lncRNA H19水平显著高于健康个体。MTT分析结果显示，Botox-A以剂量依赖性方式降低人包皮成纤维细胞的活力。BC外泌体显著增加人包皮成纤维细胞中α-SMA、波形蛋白和BCL-2 mRNA水平，另一方面，BAX水平显著降低。外泌体处理的HFF细胞与Botox-A和负载Botox-A的外泌体共培养显著提高了BCL-2 mRNA水平，与BAX和细胞周期蛋白d1的表达完全相反。同时，流式细胞术结果证实，用负载Botox-A的外泌体处理的恶性人包皮成纤维细胞凋亡率很高。
本研究结果表明，外泌体lncRNA H19可能是乳腺癌的诊断标志物，这些乳腺癌外泌体可诱导成纤维细胞出现恶性表型并将其转化为CAF。Botox-A对正常成纤维细胞和CAF均可能有毒性，可诱导它们凋亡并抑制其细胞增殖。
