Bax is essential for mitochondrion-mediated apoptosis but not for cell death caused by photodynamic therapy

Abstract

The role of Bax in the release of cytochrome c from mitochondria and the induction of apoptosis has been demonstrated in many systems. Using immunocytochemical staining, we observed that photodynamic therapy (PDT) with the photosensitiser Pc 4 induced Bax translocation from the cytosol to mitochondria, and the release of cytochrome c from mitochondria as early signalling for the intrinsic pathway of apoptosis in human breast cancer MCF-7c3 cells. To test the role of Bax in apoptosis, MCF-7c3 cells were treated with Bax antisense oligonucleotides, which resulted in as much as a 50% inhibition of PDT-induced apoptosis. In the second approach, Bax-negative human prostate cancer DU-145 cells were studied. Following PDT, the hallmarks of apoptosis, including the release of cytochrome c from mitochondria, loss of mitochondrial membrane potential, caspase activation, and chromatin condensation and fragmentation, were completely blocked in these cells. Restoration of Bax expression in DU-145 cells restored apoptosis, indicating that the resistance of DU-145 cells to PDT-induced apoptosis is due to the lack of Bax rather than to another defect in the apoptotic machinery. However, despite the inhibition of apoptosis, the Bax-negative DU-145 cells were as photosensitive as Bax-replete MCF7c3 cells, as determined by clonogenic assay. Thus, for Pc 4-PDT, the commitment to cell death occurs prior to Bax activation.

Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation

“…DU-145 cells have been shown earlier to be re-sensitised by the ectopic expression of Bax to stimuli such as over expression or photodynamic therapy. 41,42 Likewise, GFP-Bax has been shown earlier to promote apoptosis similar to endogenous Bax. 3 To evaluate the levels of apoptosis in DU-145 cells and DU-145 cells expressing YFP-Bax, cells were seeded into 24-well plates, treated with 1 mM STS (6 h), 50 ng/ml TRAIL and 1 mg/ml CHX (6 h), or vehicle (DMSO, 6 h).…”

Intracellular signaling mechanisms in photodynamic therapy

“…PDT-treated MCF-7c3 cells showed cytochrome C release from mitochondria and apoptotic nuclei, whereas the latter cell type was not affected [99,100]. Also, treatment of MCF-7c3 cells with Bax antisense oligonucleotides reduced Pc 4-PDT induced apoptotic death and restoration of Bax expression in DU-145 cells restored apoptosis, indicating that the lack of Bax is responsible for the resistance of these cells to PDT-induced apoptosis [100]. However, the fact that Bax-negative DU-145 cells are as sensitive to Pc 4-PDT as MCF-7c3 cells, as determined by the clonogenic assay, indicates that the commitment to cell death occurs at a step prior to Bax activation.…”

Apoptotic and autophagic responses to Bcl-2 inhibition and photodamage

“…The responsiveness of the Bax knockdown is consistent with reports by Oleinick's group showing that absence of Bax, or of procaspase-3, did not protect cells from phototoxic effects of PDT using the phthalocyanine photosensitizer Pc 4. [3][4][5][6] Time sequence of autophagy and apoptosis A typical result after exposure of L1210 cells to an LD 90 PDT dose of CPO is shown in Fig. 5.…”

“…2 It was later proposed that apoptosis was a means for the disposing of dead cells, but was not necessary for expression of the cytotoxic effects of PDT. [3][4][5][6] We originally proposed that the initiation of apoptosis by PDT, using a porphycene photosensitizing agent, derived from direct mitochondrial photodamage. 7 Later work revealed that the initial target of many photosensitizing agents was the anti-apoptotic protein Bcl-2, 8,9 a finding confirmed by Oleinick's group.…”