Effects of BAPTA‐AM and Forskolin on Apoptosis and Cytochrome c Release in Photosensitized Chinese Hamster V79 Cells

Abstract

The mechanism of caspase-3-dependent apoptosis induced by photodynamic therapy (PDT) of cultured Chinese hamster V79 cells with pheophorbide a (PPa) was investigated. The PPa-PDT induced rapid apoptosis within 30 min after irradiation of cells. This apoptosis was inhibited by the 1O2 quencher N3- and caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde, suggesting that 1O2 activated caspase-3 and then caused apoptosis. The intracellular calcium [Ca2+]i chelator (acetoxymethyl)-1,2-bis(o-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA-AM) and the cyclic adenosine monophosphate (cAMP)-increasing agent forskolin also inhibited not only the PPa-PDT-induced activation of caspase-3 but also apoptosis in V79 cells. Furthermore, PPa-PDT-induced cytochrome c release from mitochondria was found to be inhibited by the treatment with BAPTA-AM but not forskolin. These results indicated that [Ca2+]i and cAMP independently serve as regulators for PPa-PDT-induced apoptosis in the upstream of caspase-3.

Intracellular signaling mechanisms in photodynamic therapy

“…The calcium chelator BAPTA inhibited the release of cytochrome C, caspase-3 activation and apoptotic death of Chinese hamster V79 cells photosensitized with pheophorbide, indicating that Ca 2 + indeed plays a role in PDT-induced apoptosis [63]. Chelation of intracellular Ca 2 + with BAPTA also reduced apoptotic death of HeLa cells (human cervix carcinoma cell line) photosensitized with TBR, which is incorporated into the Golgi apparatus [87].…”

“…These results indicate that the cytoprotective effect of PGE 2 in these cells is mediated by an increase in the intracellular cAMP content. In Chinese hamster V79 cells photosensitized with pheophorbide a, the protective effect of cAMP against apoptotic death was found to occur at a level downstream of cytochrome C release from the mitochondria, and upstream of caspase-3 activation [63].…”

“…Studies using different cell models and various sensitizers have shown that photosensitization induces a rapid release of cytochrome C from the mitochondria ( Table 1). The intracellular Ca 2 + chelator BAPTA inhibited PDTinduced cytochrome C release and caspase-3 activation in photosensitized Chinese hamster V79 cells, suggesting that the increase in the intracellular Ca 2 + levels may be a trigger for cytochrome C release from mitochondria [63]. In L5178Y-R mouse lymphoma (LY-R) cells subjected to PDT with the Pc 4, the rapid release of a fraction of the mitochondrial cytochrome C was followed by a partial inhibition of respiration [64].…”

“…Photodynamic treatment of various cell types has been shown to raise the [Ca 2 + ] i [103 -117], which may even-tually lead to cell death [63,109,114,118]. The calcium chelator BAPTA inhibited the release of cytochrome C, caspase-3 activation and apoptotic death of Chinese hamster V79 cells photosensitized with pheophorbide, indicating that Ca 2 + indeed plays a role in PDT-induced apoptosis [63].…”

Chemotherapeutic Approaches for Targeting Cell Death Pathways

“…However, the majority of the PDT lethality appears to result from loss of mitochondrial inner membrane potential [151]. The mPT inhibitor cyclosporine A showed a protective effect during PDT treatment [152,153].…”