Maduramicin Inhibits Proliferation and Induces Apoptosis in Myoblast Cells

Maduramicin, a polyether ionophore antibiotic derived from the bacterium Actinomadura yumaensis, is currently used as a feed additive against coccidiosis in poultry worldwide. It has been clinically observed that maduramicin can cause skeletal muscle and heart cell damage, resulting in skeletal muscle degeneration, heart failure, and even death in animals and humans, if improperly used. However, the mechanism of its toxic action in myoblasts is not well understood. Using mouse myoblasts (C2C12) and human rhabdomyosarcoma (RD and Rh30) cells as an experimental model for myoblasts, here we found that maduramicin inhibited cell proliferation and induced cell death in a concentration-dependent manner. Further studies revealed that maduramicin induced accumulation of the cells at G0/G1 phase of the cell cycle, and induced apoptosis in the cells. Concurrently, maduramicin downregulated protein expression of cyclin D1, cyclin-dependent kinases (CDK4 and CDK6), and CDC25A, and upregulated expression of the CDK inhibitors (p21Cip1 and p27Kip1), resulting in decreased phosphorylation of Rb. Maduramicin also induced expression of BAK, BAD, DR4, TRADD and TRAIL, leading to activation of caspases 8, 9 and 3 as well as cleavage of poly ADP ribose polymerase (PARP). Taken together, our results suggest that maduramicin executes its toxicity in myoblasts at least by inhibiting cell proliferation and inducing apoptotic cell death.马杜霉素（maduramicin）是一种源自约马放线菌（Actinomadura yumaensis）的聚醚类离子载体抗生素，目前在全球范围内被用作防治家禽球虫病的饲料添加剂。临床观察显示，若使用不当，马杜霉素可造成骨骼肌与心肌细胞损伤，进而引发动物及人类出现骨骼肌变性、心力衰竭甚至死亡。然而，其对成肌细胞的毒性作用机制目前尚未完全阐明。本研究以小鼠成肌细胞（C2C12）以及人横纹肌肉瘤（RD与Rh30）细胞作为成肌细胞实验模型，结果发现马杜霉素可通过浓度依赖性方式抑制细胞增殖并诱导细胞死亡。进一步研究表明，马杜霉素可使细胞周期阻滞于G0/G1期，并诱导细胞发生凋亡。与此同时，马杜霉素可下调细胞周期蛋白D1（cyclin D1）、细胞周期蛋白依赖性激酶（cyclin-dependent kinases, CDK）4与6及细胞分裂周期蛋白25A（CDC25A）的蛋白表达水平，并上调细胞周期蛋白依赖性激酶抑制剂p21Cip1与p27Kip1的表达，最终导致视网膜母细胞瘤蛋白（retinoblastoma protein, Rb）的磷酸化水平降低。此外，马杜霉素还可诱导BAK、BAD、死亡受体4（death receptor 4, DR4）、TNF受体相关死亡结构域蛋白（TNF receptor-associated death domain, TRADD）及TNF相关凋亡诱导配体（TNF-related apoptosis-inducing ligand, TRAIL）的表达，进而激活半胱天冬酶（caspases）8、9与3，并引发多聚ADP核糖聚合酶（PARP）的剪切。综上，本研究结果表明，马杜霉素对成肌细胞产生毒性作用的至少部分机制为抑制细胞增殖以及诱导凋亡性细胞死亡。