Introduction: Targeted multimodal approaches need to be strategically developed to control tumour growth and prevent metastatic burden successfully. Breast cancer presents a unique clinical problem because of the variety of cellular subtypes that arise. The tumour stage and cellular subtypes often dictate the appropriate clinical treatment regimen. Also, the development of chemoresistance is a common clinical challenge with breast cancer. Higher doses and additional drug agents can produce additional adverse effects leading to a more aggressive malignancy. Acetylsalicylic acid (ASA), metformin (Met), and oseltamivir phosphate (OP) were investigated for their efficacy to sensitize MDA-MB-231 triple-negative breast cancer and its tamoxifen (Tmx) resistant variant (MDA-MB-231-TmxR) together in combination with Tmx treatment.
Methods: Microscopic imaging, the formation of 3D multicellular tumour spheroids, immunocytochemistry, flow cytometry, Annexin V Assay, Caspase 3/7 Apoptosis Assay, tube formation assay and analysis, and WST-1 cell viability assay evaluated the formation of MCTS, morphologic changes, cell viability, apoptosis activity and the expression levels of ALDH1A1, CD44 and CD24 on the cell surface, MDA-MB231 triple-negative breast cancer, tamoxifen (Tmx) resistant variant (MDA-MB-231-TmxR).
Results: The results using a triple combination of ASA, Met and OP on MDA-MB-231 and MDA-MB-231-TmxR cells and their matrix-free 3D multicellular tumour spheroids (MCTS) formed by using the cyclic Arg-Gly-Asp-D-Phe-Lys peptide modified with 4-carboxybutyl-triphenylphosphonium bromide (cyclo-RGDfK(TPP)) peptide method demonstrate a consistent and significant decrease in cell and tumour spheroid viability and volume with increased apoptotic activity, and increased sensitivity to Tmx therapy. Tmx treatment of MDA-MB-231 cells in combination with ASA, Met and OP markedly reduced the CD44/CD24 ratio by 6.5-fold compared to the untreated control group. Tmx treatment of MDA-MB-231-TmxR cells in combination with ASA, Met and OP markedly reduced the ALDH1A1 by 134-fold compared to the same treatment for the parental cell line. Also, the triple combination treatment of ASA, Met, and OP inhibited vasculogenic endothelial cell tube formation and induced endothelial cell apoptosis.
Conclusion: For the first time, the findings demonstrate that repurposing ASA, Met, and OP provides a novel and promising targeted multimodal approach in the treatment of triple-negative breast cancer and its chemoresistant variant.

Keywords: MDA-MB-231, MDA-MB-231-TmxR, human umbilical vein endothelial cell lines, multicellular tumour spheroids, MCTS, endothelial cell tube formation, apoptosis


中文翻译：

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二甲双胍、乙酰水杨酸和磷酸奥司他韦的三重组合会影响肿瘤球体的活力并颠覆三阴性乳腺癌的化疗耐药性。

简介：需要战略性地开发有针对性的多模式方法，以成功控制肿瘤生长和预防转移负担。由于出现了多种细胞亚型，乳腺癌呈现出独特的临床问题。肿瘤分期和细胞亚型通常决定了适当的临床治疗方案。此外，化学抗性的发展是乳腺癌常见的临床挑战。更高的剂量和额外的药物会产生额外的副作用，导致更具侵袭性的恶性肿瘤。研究了乙酰水杨酸 (ASA)、二甲双胍 (Met) 和磷酸奥司他韦 (OP) 对 MDA-MB-231 三阴性乳腺癌及其他莫昔芬 (Tmx) 耐药变体 (MDA-MB-231-TmxR) 的敏感性) 与 Tmx 治疗相结合。
方法：显微成像、3D 多细胞肿瘤球体的形成、免疫细胞化学、流式细胞术、Annexin V Assay、Caspase 3/7 Apoptosis Assay、管形成测定和分析以及 WST-1 细胞活力测定评估 MCTS 的形成、形态学变化、细胞活力、凋亡活性及细胞表面ALDH1A1、CD44和CD24的表达水平，MDA-MB231三阴性乳腺癌，他莫昔芬（Tmx）耐药变异体（MDA-MB-231-TmxR）。
结果：使用 ASA、Met 和 OP 三重组合对 MDA-MB-231 和 MDA-MB-231-TmxR 细胞及其使用环状 Arg-Gly-Asp- 形成的无基质 3D 多细胞肿瘤球体 (MCTS) 的结果用 4-羧基丁基-三苯基溴化鏻 (环-RGDfK(TPP)) 肽法修饰的 D-Phe-Lys 肽显示细胞和肿瘤球体活力和体积持续显着降低，细胞凋亡活性增加，对 Tmx 治疗的敏感性增加。与未处理的对照组相比，Tmx 处理 MDA-MB-231 细胞与 ASA、Met 和 OP 的组合显着降低了 CD44/CD24 比率 6.5 倍。与亲本细胞系的相同处理相比，Tmx 处理与 ASA、Met 和 OP 组合的 MDA-MB-231-TmxR 细胞显着降低了 ALDH1A1 134 倍。还，
结论：研究结果首次表明，重新利用 ASA、Met 和 OP 为治疗三阴性乳腺癌及其化疗耐药变异提供了一种新颖且有前景的靶向多模式方法。

关键词： MDA-MB-231, MDA-MB-231-TmxR, 人脐静脉内皮细胞系, 多细胞肿瘤球体, MCTS, 内皮细胞管形成, 细胞凋亡