Mitochondria-targeted hydrogen sulfide donors potentiate methyl aminolevulinate (MAL)-induced photodynamic therapy killing of skin cancer cells

Daniel Ferguson1, Alexis Perry2, Gary Smerdon3, Michael Murphy4, Alison Curnow1, Mark Wood2, Matthew Whiteman1, PG Winyard1. 1University of Exeter Medical School, Exeter, UK, 2Biosciences, University of Exeter, Exeter, UK, 3Diving Diseases Research Centre, Plymouth, UK, 4MRC Mitochondrial Biology Unit, Cambridge, UK

Protoporphyrin IX photodynamic therapy (PpIX-PDT) involves the treatment of skin cancer with an inert pro-drug (e.g. methyl aminolevulinate; MAL), leading to the mitochondrial accumulation of PpIX. Irradiation of cancer cells with red light (630 nm) induces the production “reactive oxygen species” from photosensitised PpIX resulting in cell killing. In this study we evaluated the effects of novel mitochondria-targeted compounds on MAL-induced PDT cell killing in human A431 skin epidermoid carcinoma cells. To do this we used mitochondria-targeted antioxidants (MitoQ and MitoE2) and a series of novel mitochondria-targeted H2S donors (MtH2SD) containing triphenylphosphonium (TPP+), a variable length aliphatic linker (C6-C12, for structure-activity determination) and either anethole dithiolethione (ADT-OH) or hydroxythiobenzamide (HTB) as the source of H2S [1,2]. GYY4137 was used as a control for non-targeted H2S delivery. Cell death was assessed by flow cytometry with Annexin V-FITC and propidium iodide. Data are given as mean ± SD of four or more separate experiments performed in at least duplicate and analysis was performed by unpaired Student’s T-test. MAL-induced cell killing (27.5 ± 5.5%, p < 0.001 c.f. untreated control) was significantly increased by treatment of cells with 100 nmol/L of all TPP+-containing H2S donors (ADT-OH: C8 - 46.3 ± 2.1%, C10 - 45.3 ± 1.9%, C12 - 51.6 ± 3.2%. HTB: C8 - 67.5 ± 6.4% C10 - 50.9 ± 6.5% C12 - 72.5 ± 2.0%. p < 0.001 c.f. MAL+PDT treatment). In each case, MAL-induced PDT cell killing in the presence of MtHDs was overwhelmingly apoptotic. Control experiments using (i) MtH2SDs alone, (ii) MAL+MtHDs in the absence of red light, or (iii) red light+ MtHDs without MAL, did not result in significant cell death. Using the H2S fluorogenic probe WSP-1, H2S generation from MtHDs was increased only in the presence of PpIX. In sharp contrast, MAL-induced cell death was inhibited by MitoQ (100 nmol/L) and MitoE2 (100 nmol L-1) and no effect (cell killing or cell protection) was observed with GYY4137 (100 µmol/L), HTB (100 nmol/L) or ADT-OH (100 nmol/L) indicating that mitochondria-targeting of H2S is required for cell death. This study strongly suggests that mitochondria-targeted H2S donors are a useful adjunct to PDT in skin cancer and highlights important differences between mitochondria-targeted antioxidants (e.g. MitoQ and MitoE2) and mitochondria-targeted and non-targeted H2S donors.

[1] Le Trionnaire et al.,(2014). Med. Chem. Commun. 5: 728-736.

[2] Szceszny et al., (2014). Nitric Oxide 41: 120-130.