Maduramicin Inhibits Proliferation and Induces Apoptosis in Myoblast Cells

<div><p>Maduramicin, a polyether ionophore antibiotic derived from the bacterium <i>Actinomadura yumaensis</i>, 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 (p21^Cip1 and p27^Kip1), 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.</p></div

Maduramicin upregulates expression of DR4, TRADD, TRAIL, BAK and BAD, leading to activation of caspases 8, 9 and 3 as well as cleavage of PARP in C2C12 cells.

<p>C2C12 cells were treated with maduramicin for 24 h at indicated concentrations, followed by Western blotting with indicated antibodies. β-Tubulin was used for loading control. Representative blots are shown (A, C and E). Blots for indicated proteins were semi-quantified using NIH image J (B, D and F). Results are presented as means ± SE (n = 3, corresponding to three independent experiments). *<i>P</i><0.05, **<i>P</i><0.01, difference with the control group.</p

Maduramicin arrests C2C12 cells at G₀/G₁ phase of the cell cycle.

<p>C2C12 cells were treated with maduramicin for 24 h at indicated concentrations (A), or for indicated time at 0.5 µg/ml (B, C), followed by staining with PI and flow cytometry. (A, B) Results are presented as means ± SE (n = 3, corresponding to three independent experiments). *<i>P</i><0.05, **<i>P</i><0.01, difference with the control group. (C) Histograms from a representative experiment show the time-course effect of maduramicin on cell cycle profile in C2C12 cells. Note: Maduramicin increased sub-G₁ in a time-dependent manner.</p

Maduramicin induces caspase-dependent apoptosis in C2C12 cells.

<p>C2C12 cells (plated in triplicates), pretreated with or without z-VAD-fmk (20 µM) for 1 h, were treated with maduramicin for 24 h at indicated concentrations, followed by Western blotting with indicated antibodies (A), or for 48 h at indicated concentrations, followed by trypan blue exclusion assay (B). Data represents mean ± SE (n = 3, corresponding to three independent experiments). ^*<i>P</i><0.05, ^**<i>P</i><0.01, difference with the control group. ^#<i>P</i><0.05, difference with z-VAD-fmk group.</p

Maduramicin inhibits cell proliferation in myoblast cells.

<p>C2C12 and RD cells (plated in triplicates) were exposed to maduramicin at indicated concentrations for 24, 48 or 72 h, followed by one solution assay. Data represents mean ± SE (n = 6, corresponding to six independent experiments). *<i>P</i><0.05, **<i>P</i><0.01, difference with the control group.</p

Maduramicin downregulates protein expression of cyclin D1, CDK4, CDK6, and CDC25A, and upregulates expression of p21^Cip1 and p27^Kip1, resulting in hypophosphorylation of Rb in C2C12 cells.

<p>C2C12 cells were treated with maduramicin for 24 h at indicated concentrations, followed by Western blotting with indicated antibodies. β-Tubulin was used for loading control. Representative blots are shown (A). Blots for indicated proteins were semi-quantified using NIH image J (B). Results are presented as means ± SE (n = 3, corresponding to three independent experiments). *<i>P</i><0.05, **<i>P</i><0.01, difference with the control group.</p

Maduramicin induces cell death in myoblast cells.

<p>C2C12 and RD cells (plated in triplicates) were exposed to maduramicin at indicated concentrations for 24, 48 or 72 h, followed by trypan blue exclusion assay. Data represents mean ± SE (n = 3, corresponding to three independent experiments). *<i>P</i><0.05, **<i>P</i><0.01, difference with the control group.</p

FC101 induces caspase-dependent apoptosis.

<p>(A–D) COS7 cells, pretreated with or without Z-VAD-FMK (10 µM) for 1 h, were incubated with FC101 at indicated concentrations for 24 h, followed by caspase 3/7 activity assay (A), for 48 h, followed by trypan blue exclusion assay (B), or for 72 h, followed by Annexin V-FITC/PI staining and flow cytometry (C, D). (C) Histograms from a representative experiment show the effect of Z-VAD-FMK on FC101-induced apoptosis of COS7 cells. The percentages of necrotic, late apoptotic, viable, and early apoptotic cells are displayed in Q1, Q2, Q3 and Q4, respectively. (D) Bar graphs show that Z-VAD-FMK partially prevented FC101-induced apoptosis of COS7 cells. Quantitative results (Q2+Q4) were displayed as fold change compared with control. For (A), (B), and (D), data represents mean ± SE (n = 3). ^a<i>P</i><0.05, ^b<i>P</i><0.01, ^c<i>P</i><0.001, difference with the control group (FC101 = 0 µM). ^d<i>P</i><0.01, difference with Z-VAD-FMK group.</p

FC101 inhibits cell proliferation and reduces cell viability.

<p>COS7 and HEK 293 cells were treated with FC101 (0–5 µM) for 6 days (for COS7) or 4 days (for HEK 293) (A, B), or 48 h (C, D), followed by cell number counting (A), morphological analysis (B), one solution assay (C), and trypan blue exclusion assay (D). For (A), (C), and (D), data represents mean ± SE (n = 6). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, difference with the control group (FC101 = 0 µM).</p

FC101 induces apoptosis. COS7 cells were treated with FC101 (0–5 µM) for 72 h.

<p>The cells were harvested and processed for apoptosis assay using the Annexin V-FITC Apoptosis Detection Kit. The cell distribution was analyzed by flow cytometry. (A) Histograms from a representative experiment show the apoptotic effect of FC101 on COS7 cells. The percentages of necrotic, late apoptotic, viable, and early apoptotic cells are displayed in Q1, Q2, Q3 and Q4, respectively. (B) Bar graphs show that FC101 induced apoptosis of COS7 cells in a concentration-dependent manner. Quantitative results (Q2+Q4) are displayed as fold change compared with control. Data represents mean ± SE (n = 3). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, difference with the control group (FC101 = 0 µM).</p