Danazol and Stanozolol Induce Expression of Pro-Apoptotic Genes

Androgens (danazol and stanozolol) have been used to treat bone marrow failure (BMF) syndromes due to their multifaceted effects on erythropoiesis, telomere maintenance and immune homeostasis. In low and middle-income countries, androgens are used to treat aplastic anaemia, particularly in individuals who cannot afford immunosuppressive therapy and HSCT. However, patient responses to these treatments vary, with previous studies showing a 50-70% response to inherited bone marrow failures and a 40-50% response to acquired AA and MDS. There is uncertainty surrounding how androgens work, with only a few elucidated effects on the stimulation of erythropoiesis, stem cell telomere elongations and immune cell modulation. This study aims to understand the mechanisms of androgen action in hepatic cell lines since they are metabolised there and extrapolate the findings to HSCs.

HepG2 cell lines at passage 6 were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum. Mobilised peripheral blood from stem cell donors was used to enrich CD34+ cells cultured in StemSpan SFEM Hematopoietic cell culture medium. The purity and percentage of CD34+ cells were assessed by flow cytometry, and > 90% purity was maintained. MTT assays determined the drug concentration in HepG2 and CD34+positive cells. HepG2 and CD34 cells were treated with danazol and stanozolol at different concentrations (100nM, 1uM and 10uM) for 24, 48, and 72 hours.

Cell proliferation rates were monitored by staining the cells with CFSE dye, incubating them with the respective concentrations for 24, 48 and 72 hours and analysing the proliferation in a flow cytometer. Further, the drug-treated cells were stained with propidium iodide to examine the effects of drugs in the cell cycle at 24 hours post-drug treatment.

RNA was extracted from treated cells, and expression of apoptotic (CASP8, CASP9, Fas, and BAX), senescent genes (TERF1, SIRT1, ARID1a), and androgen receptor gene was analysed using quantitative real-time PCR using relative quantification. The influence of androgens on genes involved in iron metabolism is being investigated, and protein data is awaited.

Based on the MTT assay on CD34+ and HepG2 cells, 100nM and 1uM danazol and stanozolol were used for further experiments due to increased viability at 24 and 48 hrs. HepG2 cells showed a maximum proliferation at 24 hours at varying concentrations of danazol and stanozolol. We further observed an arrest of cells at the G1 phase post-drug treatment at 24 hrs.

Both danazol and stanozolol, the peak significant androgen receptor expression was observed at 6 hours (fold change > 3.3; p = 0.0004). Pro-apoptotic gene CASP8 increased significantly with danazol over 24 hours (p = 0.025 and 0.0009, and fold change >1.5 and 1.4, respectively), but stanozolol caused a minor increase in expression levels. Danazol and stanozolol (100 nM conc.) significantly enhanced the expression level of CASP9 (p = 0.0253, 0.0009, and 0.007; fold change > 1.5 and 1.8 respectively), while FAS9 levels were significantly upregulated with stanozolol (1 uM) and danazol (p = 0.0001, 0.0001, and 0.004; fold change 2.3, 2.39, and 1.7).

The mechanism of action of androgens appears complex. Genes involved in apoptotic processes are upregulated with no difference in the expression of senescent genes with both Danazol and Stanozolol. Danazol is known to have immunosuppressive effects on immune cells in AA, and this may be due to the induction of apoptotic processes in activated cells. Results from CD34 cell experiments and protein analysis may shed light on these pathways and their mechanisms.

No relevant conflicts of interest to declare.