Maduramicin Inhibits Proliferation and Induces Apoptosis in Myoblast Cells

December Maduramicin Inhibits Proliferation and Induces Apoptosis in Myoblast Cells Xin Chen 0 1 2 3 Ying Gu 0 1 2 3 Karnika Singh 0 1 3 Chaowei Shang 0 1 3 4 Mansoureh Barzegar 0 1 3 Shanxiang Jiang 0 1 2 Shile Huang 0 1 3 4 its Supporting Information files. 0 1 0 Funding: This work was supported in part by the grants from National Natural Science Foundation of China (No. 090600253, S.J.), NIH (NCI CA115414 1 Editor: Yi-Hsien Hsieh, Institute of Biochemistry and Biotechnology , Taiwan 2 Laboratory of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Nanjing Agricultural University , Nanjing, Jiangsu Province , P. R. China, 3 Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center , Shreveport, Louisiana , United States of America, 4 Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center , Shreveport, Louisiana , United States of America 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 called Yumamycin) is a monovalent glycoside polyether ionophore antibiotic produced through aerobic fermentation by the bacterium Actinomadura yumaensis, which was originally isolated from a soil sample from Yuma County, Arizona, USA [13]. Maduramicin possesses moderate activity against many Gram-positive bacteria, and exhibits a broad spectrum of anticoccidial activity against the most frequently occurring Eimeria species in chickens and turkeys [4, 5]. Thus, currently it is primarily used to control coccidiosis in chickens and turkeys (so-called target animals) for fattening [4, 5]. A dose of 57 ppm (mg/kg) of maduramicin in feed is recommended in the USA, the European Union, and many other countries, with a withdrawal period of 5 days before slaughter [4, 5]. Higher doses (.10 ppm) of maduramicin in feed can be toxic in both chickens and turkeys [46]. Besides, since maduramicin is excreted rapidly and mainly as unchanged form in broilers [4, 7], 2.56.1 mg/kg of maduramicin in the broiler litter has been noticed [8]. As cattle, sheep and pigs (so-called non-target animals) are more sensitive to maduramicin [4], clinically maduramicin toxicity has been more frequently observed in these animals when fed with the broiler litter as a source of protein and minerals [813]. Furthermore, some cases of accidental poisoning with maduramicin in humans have been reported [14, 15]. Histopathologically, maduramicin can induce severe myocardial and skeletal muscle lesions [814]. It has been proposed that the polyether ionophores (including maduramicin, monensin, narasin, salinomycin, semduramicin, and lasalocid) may form lipophilic complexes with cations (particularly Na+, K+ and Ca2+), thereby promoting their transport across the cell membrane and increasing the osmotic pressure in the coccidia, which inhibits certain mitochondrial functions such as substrate oxidation and ATP hydrolysis, eventually leading to cell death in the protozoa [5, 16]. In general, myoblast cells have more mitochondria. It is not clear whether this is related to maduramicins higher toxicity to skeletal muscle cells. Nevertheless, to our knowledge, the toxic mechanism of maduramicin in myoblast cells of animals and humans remains largely unknown. Cell division or cell proliferation is essential for growth, development and regeneration of eukaryotic organisms [17]. In animals (including humans), cell proliferation is directly determined by the progression of the cell cycle, which is divided into G0/G1, S, and G2/M phases, and is driven by various cyclindependent kinases (CDKs) [17, 18]. A CDK (catalytic subunit) has to bind to a regulatory subunit, cyclin, to become active [18]. Also, Wee1 phosphorylates specific residues (Tyr15 and Thr14) of CDKs, inhibiting CDKs, which is counteracted by CDC25 through dephosphorylation [18]. However, cyclin activating kinase (CAK) phosphorylates CDKs (Thr161), activating CDKs [18]. Furthermore, p21Cip1 and p27Kip1, two universal CDK inhibitors, can bind a CDK, inhibiting the CDK activity and the cell cycle progression [19]. Cyclin D-CDK4/6 and cyclin E-CDK2 complexes control G1 cell cycle progression, whereas cyclin A CDK2 and cyclin B-CDK1 regulate S and G2/M cell cycle progression, respectively [18]. Therefore, disturbing expression of CDKs and/or the regulatory proteins, such as cyclins, CDC25 and CDK inhibitors, may affect cell cycle progression. Apoptosis is a type of programmed cell death and occurs actively in multicellular organisms under physiological and pathological conditions [20]. Under physiological conditions, it plays an essential role in regulating growth, development and immune response, and maintaining tissue homeostasis [20]. Under pathological conditions (such as viral infection, toxins, etc.), when cells are damaged too severely to repair, they will also undergo apoptosis via caspasedependent and -independent mechanisms [20]. In response to apoptotic insults, activation of caspases can be initiated through the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway [21]. The death receptors are members of the tumor necrosis factor (TNF) receptor gene superfamily, which share similar cyteine-rich extracellular domains and have a cytoplasmic death domain of about 80 amino acids [22]. Ligands, such as FasL, TNFa, Apo3L, and Apo2L (also named TRAIL), bind to corresponding death receptors, including Fas (also named CD95), TNFR1, DR3, and DR4/DR5, resulting in receptor oligomerization, which in turn leads to the recruitment of specialized adaptor proteins and activation of caspases 8/10, triggering apoptosis [21, 22]. Furthermore, Bcl-2 family members, including anti-apoptotic (e.g. Bcl-2, Bcl-xL, and Mcl-1) and pro-apoptotic proteins (e.g. BAD, BAK, and BAX), are key players in the regulation of mitochondrial-dependent apoptosis [22, 23]. They work together and with other proteins to maintain a dynamic (...truncated)