Enhanced apoptotic response to photodynamic therapy after bcl-2 transfection

Abstract

Apoptosis is a cellular death process involving the sequential activation of a series of caspases, endonucleases, and other enzymes. The initiation of apoptosis can be inhibited by overexpression of bcl-2 and certain other members of a related family of proteins. We examined the effects of bcl-2 overexpression on the apoptotic response to photodynamic therapy (PDT), using aluminum phthalocyanine as the photosensitizing agent. In this study, we compared the immortalized human breast epithelial cell line MCF10A with a subline (MCF10A/bcl-2) transfected with the human bcl-2 gene. The latter was approximately 2-fold more sensitive to the phototoxic effects of PDT. At a 50 mJ/cm2 light dose, photodamage to MCF-10A/bcl-2 resulted in a greater loss of the mitochondrial membrane potential (delta(psi)m), enhanced release of mitochondrial cytochrome c, a more rapid and greater activation of caspase-3, and a greater apoptotic response. Western blot analysis revealed that the transfected cell line showed overexpression of both bcl-2 and bax, and that PDT caused selective destruction of bcl-2, leaving bax unaffected. The greater apoptotic response by the transfected line is, therefore, attributed to the higher bax:bcl-2 ratio after photodamage.

Fig. 1

Loss of viability of MCF10A (○) and MCF10A/bcl-2 (●) after photodynamic therapy. Cells were incubated with 0.3 μM AlPc and then irradiated with the specified light dose. Survival is expressed as a percentage of control (cells exposed to drug in the dark) using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay 96 h after irradiation. Data are reported as means of three experiments; bars, SD.

Fig. 2

Effects of photodynamic therapy with AlPc (light dose, 50 mJ/cm²) on MCF10A (top) and MCF10A/bcl2 (bottom) labeled with HO342 or MTO. A, HO342 control; B, MTO control; C, MTO label directly after irradiation; D, HO342 label 4 h after irradiation; E, MTO label 4 h after irradiation. Bar, 10 μm.

Fig. 3

Western blot analysis of actin, bcl-2 (bcl2), bax, and cytochrome c (cyto c) in control cells and in cells directly after PDT was carried out at 10°C. Cells were photosensitized with AlPc and then irradiated with a light dose of 100 mJ/cm². 1 = control, 2 = transfected, 3 = control + PDT, 4 = transfected + PDT