Testosterone Propionate Promotes Proliferation and Viability of Bone Marrow Mesenchymal Stem Cells while Preserving Their Characteristics and Inducing Their Anti-Cancer Efficacy

Abstract

Background: Various studies have reported the effects of testosterone on different cell types, yet bone marrow-derived mesenchymal stem cells’ cellular responses to testosterone remain unknown.

Aims: To investigate the effects of testosterone propionate, an oil-soluble short-acting form of testosterone, on human bone marrow-derived mesenchymal stem cells’ proliferation and viability after 24 hours of incubation. We also investigated the impact of testosterone propionate on bone marrow-derived mesenchymal stem cell’s polarization and cytotoxicity on K562 leukemia cell line.

Study design: In vitro study.

Methods: We expanded commercially available bone marrow derived mesenchymal stem cells in vitro and treated them with testosterone propionate at concentrations ranging from 10^-6-10^-10 M for 24 hours. Ideal concentration was determined by evaluating cellular viability and proliferation with Annexin V/Propidium Iodide assay and carboxyfluorescein succinimidyl ester staining. The characteristic features of bone marrow-derived mesenchymal stem cells were evaluated by immunophenotyping and investigating their differentiation capacities. Bone marrow-derived mesenchymal stem cells’ cytotoxic properties upon testosterone propionate treatment were determined by co-culturing the cells with K562 cells and with confocal imaging investigating polarization.

Results: Testosterone propionate promoted proliferation and maintained the viability of bone marrow-derived mesenchymal stem at 10^-8 M concentration. Further evaluations were conducted with the determined dose. The results showed that, apart from promoting mesenchymal stem cells’ polarization and increasing their cytotoxicity on K562 cells, testosterone propionate did not alter differentiation capacities of bone marrow-derived mesenchymal stem cells and certain cell surface markers, but led to a significant increase in HLA-DR expression.

Conclusion: The findings reveal that testosterone propionate promotes the proliferation and survival of bone marrow-derived mesenchymal stem cells in a dose-dependent manner without hampering their differentiation capacities, induces their polarization to the pro-inflammatory phenotype, and increases their cytotoxicity on the K562 cell line.

Figure 1

Testosterone propionate promoted cell division on BM-MSCs while the highest increase was observed in the 10^-8 M TP group. (a) Bar graphic indicating differences regarding cell division. (b) Representative overlay histogram plot comparing the control (red) and the 10-8 M TP (blue) groups in terms of cell division. *denotes significant difference between the treatment and control groups; #denotes significant difference between the treatment groups. P values lower than 0.05 were considered statistically significant. **p < 0.01, ***p < 0.001, and ****p < 0.0001

Figure 2

Testosterone propionate promoted apoptosis in a dose-dependent manner in BM-MSCs. Bar graphics indicates (a) apoptosis, (b) early apoptosis, (c) late apoptosis, and (d) necrosis. * denotes significant difference between the treatment and control groups; # denotes significant difference between the treatment groups. P values lower than 0.05 were considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.000.1

Figure 3

Testosterone propionate does not alter cell surface markers’ expressions on BM-MSCs. Bar graphics indicating (a) CD73, (b) CD90, (c) CD105, (d) CD11b, (e) CD34, (f) CD45, and (g) HLA-DR levels; (i) Representative micrographs indicating chondrogenic, osteogenic, and adipogenic differentiation on the 21^st day of control (upper row) or 10^-8 M TP (lower row) groups; (h) Representative flow cytometry dot plots regarding immunophenotyping of control (upper row) or 10-8 M TP (lower row) groups. P values lower than 0.05 were considered statistically significant; ****p < 0.0001.

Figure 4

Testosterone propionate treatment promoted MSC-1 polarization and enhanced cytotoxic features of BM-MSCs. (a) Bar graphics indicating dead cell ratios upon co-culturing BM-MSCs with K562 cells for 24 hours. * denotes significance between treatment and the control group, while # denotes significance between treatment groups. (b) Confocal microscopy images indicating either CXCL5 (red) and CXCL9 (green) protein expression or cellular morphology indicated with F-actin labeling, where TP at 10^-8 M concentration led to increased CXCL9 protein expression and broadened cell surface area. (c) Representative flow cytometry dot plots indicating cell death upon co-culturing BM-MSCs with K562 cells at 1:10 ratio.