Involvement of Fas (APO-1/CD-95) during photodynamic-therapy-mediated apoptosis in human epidermoid carcinoma A431 cells

Photodynamic therapy is a promising treatment modality for a variety of cut aneous neoplasms and other skin disorders. Studies suggest an involvement o f multiple pathways during photodynamic-therapy-mediated cell death. A comp lete knowledge of the mechanisms involved in photodynamic therapy may lead to an improvement in its therapeutic efficacy. In vitro as well as in vivo studies have shown the involvement of apoptosis during photodynamic-therapy -mediated cell death. The pathways by which photodynamic therapy causes thi s are not fully understood. In this study, employing human epidermoid carci noma (A431) cells and silicon phthalocyanine 4 photodynamic therapy, we sho w that the cell surface death receptor Fas (also known as APO-1 or CD-95) p athway is an important contributor to photodynamic-therapy-mediated apoptos is, Employing flow cytometric analysis and confocal microscopy we first est ablished that silicon phthalocyanine 4 photodynamic therapy results in a si gnificant induction of apoptosis in A431 cells. Immunoblot analysis reveale d a significant time-dependent increase in the protein expression of Fas at 5, 15, 30, and 60 min post-photodynamic therapy followed by a decrease at later time-points (2 and 3 h post-photodynamic therapy). A Fas enzyme-linke d immunosorbent assay demonstrated an increase in this protein in cell cult ure medium starting at Ih post-photodynamic therapy and showing a time-depe ndent response up to 3 h following therapy, suggesting a diffusion of solub le Fas from cells into the medium from Ih after photodynamic therapy. Silic on phthalocyanine 4 photodynamic therapy also resulted in a time-dependent increase in (i) the multimerization of Fas protein, (ii) the protein expres sion of Fas ligand, (iii) FADD, an adapter molecule for Fas, and (iv) the b inding of FADD with Fas, Silicon phthalocyanine 4 photodynamic therapy also caused a significant activation of FLICE, as evident from the appearance o f cleaved products of pro-caspase 8. Further, a pretreatment of cells with rhFas:Fc fusion protein or general caspase inhibitor Z-VAD-FMK followed by silicon phthalocyanine 4 photodynamic therapy resulted in a significantly e nhanced cell survival, Taken together, our data, for the first time, deline ate an involvement of the Fas pathway as an important contributor to photod ynamic-therapy-mediated apoptosis of cancer cells. These observations may b e important for improving the efficacy of photodynamic therapy for the trea tment of skin cancer and possibly other skin disorders.