Testosterone Modulates the Multiplication and Viability of C2C12 Cells Cultivated In Vitro

Abstract

The use of C2C12 cell cultures is strategic for the study of functional mechanisms of skeletal striated muscle, as well as their possible correlations with other tissues and organ systems, in physiological and pathological situations. The objective of this work was to evaluate the effects of different concentrations of testosterone (T) on the multiplication and viability of C2C12 cells, in the absence and presence of flutamide (F), an androgen receptor inhibitor. The cells were cultured (in triplicates) in Dulbecco's Modified Eagle Medium (DMEM) supplemented with fungicide, streptomycin, penicillin, and 2% equine serum, in 48‐well plates (5000 cells / 200 μL‐day zero). The cells were incubated (37 °C, 5% CO2, 95% atmospheric air) for up to 10 days, in the absence (control) and presence of T (10−8 M and 10−4 M, mimicking normo‐ and hyperandrogenemia), and in the absence and presence of F (100 nM). The medium was exchanged every 48 h. Cell counting was performed with a Bio‐Rad TC20™ cell counter. The MTT (3‐ (4,5‐dimethylthiazol‐2‐yl) ‐2,5‐diphenyltetrazolium) reduction method was used to determine cell viability. Statistical analysis of the data employed ANOVA and Fisher's test. Cell multiplication. After 5 days of culture, cell multiplication was inhibited by T at 10−4 M, in the presence of F. After 10 days of culture, the cell count showed inhibition in the presence of T at 10−4 M, in the absence of F, and stimulation in the presence of T at 10−8 M, in the presence of F. Cell viability. After 5 days of culture, cell viability was stimulated by T at 10−8 M, with and without flutamide, and by T at 10−4 M, in the absence of F. After 10 days of culture, cell viability decreased in the presence of T at 10−8 M, without flutamide, and in the presence of T at 10−4 M, with F. The results suggested that the culture time influenced the cellular responses analyzed. In addition, there was a dual effect of T on multiplication and cell viability, in the absence and presence of flutamide, for both culture times studied. Interestingly, when T, in the absence or presence of flutamide, stimulated or inhibited cell multiplication, the cell viability was unaffected by the treatments, and vice versa. The findings suggested that T directed the cellular mechanisms towards multiplication, and sometimes towards metabolic activity. This cell culture model is shown to be feasible for studies of the effects of T mediated by androgen receptors; however, molecular studies are needed in order to obtain a better understanding of the mechanisms involved.Support or Funding InformationFunding: CNPq (Grant 159893/2017‐8)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.