Cyclosporine A suppresses keratinocyte cell death through MPTP inhibition in a model for skin cancer in organ transplant recipients

Abstract

Transplant recipients have an elevated risk of skin cancer, with a 65- to 250-fold increase in squamous cell carcinoma. Usage of the immunosuppressant cyclosporine A (CsA) is associated with the development of skin cancer. We hypothesized that the increased incidence of skin cancer was due to the action of CsA within keratinocyte mitochondria where it can inhibit mitochondrial permeability transition pore (MPTP) opening. Normally, MPTP opening is induced by oxidative stress such as that caused by UV light and leads to cell death, thereby eliminating a cell that has been exposed to genotoxic insult. However, in the presence of CsA, damaged cells may survive and consequently form tumors. To test this hypothesis, we treated keratinocytes with levels of CsA used therapeutically in transplant patients and assessed their viability following UVA-irradiation. CsA prevented cell death by inhibiting MPTP opening, even though the levels of oxidative stress were increased markedly. Nim811, a non-immunosuppressive drug that can block the MPTP had a similar effect while the immunosuppressive drug tacrolimus that does not interact with the mitochondria had no effect. These findings suggest that CsA may promote skin cancer in transplant patients by allowing keratinocyte survival under conditions of increased genotoxic stress.

Fig. 1

CsA represses UVA-induced cell death. HaCaT cells treated with CsA at 125nM and 250nM or vehicle (95% ethanol) were subjected to repetitive UVA irradiation (3 irradiations at 12 J/cm² at 12 hour intervals) and analyzed by flow cytometry. (a) Viable cells excluded Ann V-PE and 7-AAD. (b) Apoptotic cells stained with Ann V-PE and, (c), necrotic cells were stained with Ann V-PE and 7-AAD or 7-AAD. Data represent means ± s.e.m. n=3. Asterisks indicate significant differences compared to vehicle treated cells, *P < 0.05.

Fig. 2

CypD-binding drugs repress UVA-induced cell death. HaCaT cells were subjected to repetitive UVA irradiation in the presence of vehicle, CsA, tacrolimus (Tac), or NIM811 (NIM) at 125nM and 250nM and analyzed by flow cytometry. The data are expressed as values relative to vehicle, which is set at 0. (a) Viable cells excluded Ann V-PE and 7-AAD. (b) Apoptotic cells stained with Ann V-PE and, (c), necrotic cells stained with Ann V-PE and 7-AAD or 7-AAD. Data represent means ± s.e.m. n=3. Asterisks indicate significant differences compared to vehicle treated cells, *P < 0.05.

Fig. 3

Mitochondrial membrane potential is maintained in UVA-irradiated cells treated with CypD-binding drugs. HaCaT cells were treated with vehicle and CsA, Tac, or NIM at 125 nM and repetitively irradiated with UVA. Mitochondrial depolarization was assessed as the fluorescence shift of JC-1 from red to green by flow cytometry analysis. Data are represented as a ratio of red to green fluorescence. Data represent means ± s.e.m. n=3. Asterisks directly above error bars indicate significant differences compared to vehicle treated cells, asterisks above brackets indicate significant differences between drug groups, *P < 0.05.

Fig. 4

Mitochondrial Permeability Transition Pore activity is diminished in UVA-irradiated cells treated with CypD-binding drugs. The MPTP was monitored by quantifying the fluorescence of calcein in the mitochondria of HaCaT cells by flow cytometry. Cells were treated with vehicle, CsA, Tac, or NIM at 125 nM and irradiated with a single dose of 12 J/cm² of UVA. The cells were then loaded with calcein AM (cAM) and CoCl₂ (cytosolic calcein quencher) to determine the calcein fluorescence in the mitochondria. Control cells were loaded with cAM alone cAM, CoCl₂, and ionomycin (iono) which triggers pore opening and loss of mitochondrial calcein fluorescence. Data represent means ± s.e.m. n=3. Asterisks indicate significant differences compared to vehicle treated cells, *P < 0.05.

Fig. 5

Oxidative stress is elevated in UVA-irradiated cells treated with CypD-binding drugs. (a) HaCaT cells were treated with vehicle and CsA, Tac, or NIM811 at 125 nM and irradiated with a single dose of 12 J/cm² of UVA. Control cells were not treated with UVA or drug. Data represent the amount of mitochondrial superoxide as measured by the linear mean of MitoSox Red fluorescence by flow cytometry analysis. (b) F₂-IsoPs were quantified in HaCaT cells treated with vehicle and CsA, Tac, and NIM at 125 nM and subjected to repetitive UVA irradiation as described in the methods section. Control cells were not treated with UVA or drug. Data represent means ± s.e.m. n=3. Asterisks directly above error bars indicate significant differences compared to vehicle treated cells, asterisks above brackets indicate significant differences between drug groups, *P < 0.05.