Evidence that bcl-2 is the Target of Three Photosensitizers that Induce a Rapid Apoptotic Response¶

Abstract

We originally proposed that the subcellular target for one class of photosensitizing agents was the mitochondrion. This classification was based on effects that occur within minutes of irradiation of photosensitized cells: rapid loss of the mitochondrial membrane potential (delta psi m), release of cytochrome c into the cytosol and activation of caspase-3. These effects were followed by the appearance of an apoptotic morphology within 30-90 min. Fluorescence localization studies on three sensitizers initially classified as 'mitochondrial' revealed that these agents bind to a variety of intracellular membranes. The earliest detectable effect of photodamage is the selective loss of the antiapoptotic protein bcl-2 leaving the proapoptotic protein bax undamaged. Bcl-2 photodamage can be detected directly after irradiation of cells at 10 degrees C. Subsequent warming of cultures to 37 degrees C results in loss of delta psi m, release of cytochrome c and activation of caspase-3. The latter appears to amplify the other two effects. Based on results reported here we propose that the apoptotic response to these photosensitizers is derived from selective photodamage to the antiapoptotic protein bcl-2 while leaving the proapoptotic protein bax unaffected.

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

“…This finding is consistent with the conclusion ) that Bax or Bak is needed as an effector of apoptosis, even when antiapoptotic Bcl-2 proteins are neutralised by the overexpression of BH-3 peptide. The requirement that Bax must be activated and migrate to the mitochondria for the release of cytochrome c would predict (a) a delay in the cytochrome c release process after PDT, as we previously observed in mouse lymphoma LY-R cells and in human tumour A431 and MCF-7 cells Lam et al, 2001;Xue et al, 2001b) and (b) temperature dependence of the processes of Bax migration and cytochrome c release (Kessel and Castelli, 2001). In contrast, the prediction is inconsistent with earlier reports of an immediate release of cytochrome c following PDT (Granville et al, 1998;Kessel and Luo, 1998).…”

Apoptotic and autophagic responses to Bcl-2 inhibition and photodamage

“…Localization and other properties of CPO have been described. 9,18 CPO was dissolved in DMSO to yield a 1mMstock solution. This was stored at 4 °C under nitrogen.…”

Death pathways associated with photodynamic therapy

“…In the latter organelle, Pc 4 was also found to bind in the close vicinity of the lipid cardiolipin, a product unique to mitochondria; this may provide a basis for the resulting mitochondrial photodamage [6]. We discovered [7] that three other photosensitizing agents also targeted Bcl-2: the tin etiopurpurin termed SnET2, the porphycene CPO [9-capronyloxy-tetrakis(methoxyethyl) porphycene], and mTHPC (metatetrahydroxyphenyl) chlorin). This report also demonstrated that Bcl-2 photodamage did not result in loss of the mitochondrial membrane potential unless the temperature was raised above 15 °C.…”

“…Our assumption was that more cells would ultimately become apoptotic with time, and that an LD 90 PDT dose would eventually lead to a 90% apoptotic cell population, but this was never clearly established. Another unanswered question was the ability of PDT directed toward ER and/or mitochondria to kill a cell population even when apoptosis was inhibited by caspase inhibitors [7] or where the cell line lacked caspase-3 or Bax [27,28], important elements of the program.…”