Li Z-Z, Wang Y-L, Yu Y-H, Xing Y-L, Ji X-F
Department of Orthopedics, China-Japan Union Hospital of Jilin University, Changchun, P.R. China.
Eur Rev Med Pharmacol Sci. 2019 Jan;23(1):105-112. doi: 10.26355/eurrev_201901_16754.
Osteosarcoma is recognized as the most common primary malignant bone tumor, the 5-year disease-free survival rate in patients with metastatic or recurrent disease is below than 30%. Drug resistance and toxic side effects limit the therapeutic efficacy of osteosarcoma. Therefore, it is urgent to develop new drugs for osteosarcoma treatment. Muscarinic 3 (M3) acetylcholine receptor (AChR) has been demonstrated in nonneurocrest-derived malignancies such as colon, prostate, lung, and ovarian carcinomas. Hence, targeted regulation of M3 AChR may be a possible mechanism for treating tumors. Aclidinium bromide has anti-tumoral properties in several tumors, namely gastric cancer and glioma. In this study, we intended to investigate whether aclidinium bromide, a novel M3 AChR antagonist, had effects on osteosarcoma cells proliferation and migration.
The viability of U2 OS cells was detected by cell counting kit-8 (CCK-8) assay. The migration and invasion capabilities were measured by transwell invasion and migration assays. The cell apoptosis rate was tested by Annexin V-fluorescein isothiocyanate (FITC)/Propidum iodide (PI) staining and flow cytometry. Key apoptosis-related and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway-associated were assessed by Western blot analysis.
Aclidinium bromide markedly decreased the OD value of U2 OS cells 48 h and 72 h after treatment. The number of positive crystal violet staining cells significantly decreased after treatment with aclidinium bromide. Treatment with aclidinium bromide significantly increased cell apoptosis rate, accompanied by the expression of anti-apoptotic protein Bcl-2 decreased, the expression of pro-apoptotic protein Active caspase-3 and Bax significantly increased in U2 OS cells treated with aclidinium bromide. Additionally, aclidinium bromide suppressed the PI3K/AKT signaling pathway in U2 OS cells.
Therefore, the current study reveals that aclidinium bromide might inhibit osteosarcoma cell growth by regulating the PI3K/AKT signaling pathway, which suggests aclidinium bromide is a potential chemotherapeutic agent for osteosarcoma.
骨肉瘤是最常见的原发性恶性骨肿瘤，转移性或复发性骨肉瘤患者的5年无病生存率低于30%。耐药性和毒副作用限制了骨肉瘤的治疗效果。因此，迫切需要开发治疗骨肉瘤的新药。毒蕈碱3（M3）型乙酰胆碱受体（AChR）已在结肠、前列腺、肺和卵巢癌等非神经嵴来源的恶性肿瘤中得到证实。因此，靶向调控M3 AChR可能是治疗肿瘤的一种潜在机制。阿地溴铵在几种肿瘤（即胃癌和神经胶质瘤）中具有抗肿瘤特性。在本研究中，我们旨在探讨新型M3 AChR拮抗剂阿地溴铵是否对骨肉瘤细胞的增殖和迁移有影响。
采用细胞计数试剂盒-8（CCK-8）法检测U2 OS细胞的活力。通过Transwell侵袭和迁移实验检测细胞的迁移和侵袭能力。采用膜联蛋白V-异硫氰酸荧光素（FITC）/碘化丙啶（PI）染色和流式细胞术检测细胞凋亡率。通过蛋白质免疫印迹分析评估关键的凋亡相关蛋白以及磷脂酰肌醇-3-激酶（PI3K）/蛋白激酶B（AKT）信号通路相关蛋白。
阿地溴铵处理后48小时和72小时，显著降低了U2 OS细胞的OD值。阿地溴铵处理后，结晶紫染色阳性细胞数量显著减少。阿地溴铵处理显著提高了细胞凋亡率，同时U2 OS细胞中抗凋亡蛋白Bcl-2的表达降低，促凋亡蛋白活化的半胱天冬酶-3和Bax的表达显著增加。此外，阿地溴铵抑制了U2 OS细胞中的PI3K/AKT信号通路。
因此，本研究表明阿地溴铵可能通过调控PI3K/AKT信号通路抑制骨肉瘤细胞生长，这表明阿地溴铵是一种潜在的骨肉瘤化疗药物。
