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. 2021 May 6;1(3):100023.
doi: 10.1016/j.xjidi.2021.100023. eCollection 2021 Sep.

Topical Treatment of Human Skin and Cultured Keratinocytes with High-Dose Spironolactone Reduces XPB Expression and Induces Toxicity

M Alexandra Carpenter  1 , Michael G Kemp  1
Affiliations

Topical Treatment of Human Skin and Cultured Keratinocytes with High-Dose Spironolactone Reduces XPB Expression and Induces Toxicity

M Alexandra Carpenter et al. JID Innov. .

Abstract

Spironolactone (SP) is used to treat a variety of disparate disease states ranging from heart failure to acne through antagonism of the mineralocorticoid and androgen receptors. Although normally taken as an oral medication, recent studies have explored the topical application of SP onto the skin. However, because SP induces the proteolytic degradation of the XPB protein, which plays critical roles in DNA repair and transcription, there may be safety concerns with the use of topical SP. In this study, we show that the topical application of a high concentration of either SP or its metabolite canrenone onto human skin ex vivo lowers XPB protein levels and induces toxic responses in the epidermis. Interestingly, although SP and canrenone both inhibit cell proliferation, induce replication stress responses, and stimulate apoptotic signaling at high concentrations in cultured keratinocytes in vitro, these effects were not correlated with XPB protein loss. Thus, high concentrations of SP and canrenone likely inhibit cell proliferation and induce toxicity through additional mechanisms to XPB proteolytic degradation. This work suggests that care may need to be taken when using high concentrations of SP directly on human skin.

Keywords: Can, canrenone; KC, keratinocyte; MR, mineralocorticoid receptor; PCNA, proliferating cell nuclear antigen; SP, spironolactone.

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Figures

Figure 1
Figure 1
Topical treatment of human skin with SP and Can depletes the epidermis of the XPB protein. (a) Human skin biopsies placed in hanging well inserts in a cell culture dish were treated twice over 2 days with either 20 μM SP, Can, or vehicle (DMSO) added to the cell culture medium below the biopsy. Immunoblotting was performed with total epidermal protein, and the relative level of XPB protein was calculated from three to four different skin donors. (b) Human skin from four to five different donors was treated and processed as in a except that 25 μl of 120 mM SP or Can was added to the top of the skin biopsy. The asterisk (∗) above the XPB band denotes a nonspecific band recognized by the anti-XPB antibody. One-way ANOVA was used to determine the statistical significance. Can, canrenone; SP, spironolactone.
Figure 2
Figure 2
SP and Can have different effects of XPB protein depletion on keratinocytes in vitro. (a) HaCaT keratinocytes were treated for 6 hours with the indicated concentrations of SP. Cell lysates were then analyzed by immunoblotting. (b) Cells were treated with increasing concentrations of Can as in (a). (c) XPB protein levels from three independent experiments performed as in (a) and (b) were quantified and graphed. Two-way ANOVA revealed that whereas all concentrations of SP induced a significant decrease in XPB protein levels in comparison with those of the untreated control (P < 0.0001), only the two highest concentrations of Can were associated with a decrease in XPB protein (P < 0.01). Can, canrenone; SP, spironolactone.
Figure 3
Figure 3
High concentrations of SP and Can inhibit cell proliferation in keratinocytes in vitro. (a) HaCaT keratinocytes were treated with increasing concentrations of SP or Can. MTT assays were performed 2 days later. The relative absorbance values were normalized to values for the vehicle (DMSO)-treated cells, and the relative survival was then graphed from three to five independent experiments. IC50 values for each individual experiment were determined by nonlinear regression. The t-tests revealed significant differences between the IC50 values for SP and Can (P = 0.017). (b) HaCaT cells were treated with the indicated concentrations of DMSO for 2 days. MTT assays were performed to monitor cell proliferation and/or survival. Results are the average (and SEM) from five independent experiments. One-way ANOVA showed no significant difference in relative survival. (c) N-TERTs were analyzed as in (a), and the results from two experiments were graphed. (d) HaCaT keratinocytes were treated with increasing concentrations of SP or EP. MTT assays were performed 2 days later. The relative absorbance values were normalized to values for the vehicle (DMSO)-treated cells, and the relative survival was then graphed from three to five independent experiments. Can, canrenone; EP, eplerenone; IC50, half-maximal inhibitory concentration; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; N-TERT, telomerase-immortalized neonatal keratinocyte; SP, spironolactone.
Figure 4
Figure 4
High concentrations of SP and Can induce replication stress responses and apoptosis in keratinocytes in vitro. (a) HaCaT keratinocytes were treated with 1 mM SP for the indicated periods of time. Immunoblotting was performed with cell lysates with antibodies targeting the indicated proteins or -P proteins. (b) Quantitation of protein levels in which signals were normalized to the time point at which the signal was highest. The asterisk (∗) above the XPB band denotes a nonspecific band recognized by the anti-XPB antibody. (c) HaCaT cells were treated with the indicated concentrations of SP for 2 hours and were then analyzed by immunoblotting. (d) HaCaT cells were treated with vehicle (0.83% DMSO), 1 mM SP, 1 mM or Can or exposed to the indicated fluences of UVB radiation. Cell lysates were prepared 2 hours later and examined by immunoblotting. (e) Cells were pretreated with DMSO (0.1% DMSO) or 10 μM of the ATRi VE-821 for 30 minutes before exposure to DMSO or 1 mM SP and/or Can. Cells were harvested 2 hours later. (f) Cells were treated with 1 mM SP and/or Can for the indicated periods of time in which BrdU (10 μg/ml) was added to the media during the final 15 minutes before harvesting. Genomic DNA was purified and analyzed by immunoblotting with an anti-BrdU antibody. Signals were normalized to cells that were not treated with SP or Can, and the average (and SEM) relative DNA synthesis from two independent experiments was graphed. (g) Cells were treated for 12 hours with the indicated concentrations of SP or Can. (h) Cells were pretreated with DMSO (0.2% DMSO) or 20 μM of the pan-caspase inhibitor Z-VAD-FMK along with vehicle or 1 mM SP and/or Can. Cells were harvested 9 hours later. (i) Quantitation of H2AX phosphorylation from three independent experiments performed as in (g). The individual experimental values are indicated by the black squares and circles. Paired t-tests were used to compare the relative changes in H2AX phosphorylation. For all other experiments, the results are representative of at least two independent experiments. ATRi, ATR inhibitor; Can, canrenone; hr, hour; -P, phosphorylated; PCNA, proliferating cell nuclear antigen; SP, spironolactone; -ub, ubiquitin.
Figure 5
Figure 5
Treatment of human skin with high concentrations of SP and Can induces toxicity ex vivo. (a) Human skin explants were treated topically with DMSO, SP, or Can as in Figure 1. Epidermal lysates were probed for H2AX-P (Ser139) and PCNA. Results from replicate biopsies from a representative experiment are shown. (b) The relative level of H2AX-P was quantified from four independent skin samples and then graphed. One-way ANOVA was used to compare the responses between the treatment groups. (c) The relative level of PCNA protein was analyzed as in (b). (d) Effect of adding SP or Can to media below skin punch biopsies on basal H2AX phosphorylation. The black circles refer the individual experimental values. (e) Epidermal lysates from (a) were analyzed for PARP cleavage as a measure of apoptotic signaling. (f) H&E staining was performed with human skin biopsies treated as in (a) and viewed by microscopy at both ×4 magnification (top; bar = 0.5 mm) and ×20 magnification (bottom; bar = 0.1 mm). Note the separation of the epidermis from the dermis. Can, canrenone; H2AX-P, phosphorylated histone H2AX; PCNA, proliferating cell nuclear antigen; SP, spironolactone.
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