Photodynamic therapy (PDT), a cancer treatment utilizing a photosensitizer, visible light, and oxygen, is a unique oxidative stress that generates singlet oxygen, extranuclear cytotoxic damage, and a rapid induction of apoptosis in vitro and in vivo. PDT also produces ceramide, a lipid second messenger that is associated with stress responses and induces apoptosis. The proposes research will test the hypothesis that ceramide mediates apoptosis in response to PDT. A431 human epidermoid carcinoma cells, that respond to PDT by an increased ceramide accumulation and apoptosis, will be studied, as well as EBV-transformed normal or Niemann-Pick human lymphoblasts, the latter lacking acidic sphingomyelinase (SMase). The photosensitizers to be used, the phthalocyanine Pc 4 and merocyanine-540, differ in subcellular localization and in their ability to causes apoptosis. The hypothesis will be tested in four Specific Aims.
For Aim 1, ceramide and sphingomyelin levels, SMase activities, cell survival, and apoptosis will be measured to define the response to PDT and to assess the importance of survival, and apoptosis will be measured to define the response to PDT and to assess the importance of acidic SMase deficiency. In order to determine if an increase in reactive oxygen species is linked to PDT-induced apoptosis via ceramide, for Aim 2, the cellular and mitochondrial levels of superoxide anion and ceramide will be measured after PDT, as well as the influence of superoxide dismutase mimics and the anti-apoptotic oncoprotein Bcl-2 on ceramide generation and apoptosis.
In Aim 3, the activity of stress- activated protein kinases will be determined and the roles of c-Jun, apoptosis-associated caspases, and Bcl-2 will be assessed with respect to their coupling to ceramide-mediated PDT-induced apoptosis.
In Aim 4, the influence of tumor necrosis factor as a mediator in ceramide and apoptotic responses to PDT will be evaluated. The proposed studies should elucidate mechanisms of apoptosis initiating in specific membrane sites, identify early biochemical responses driven by PDT-associated ceramide and suggest methods to improve the therapeutic efficacy of PDT.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29CA077475-05
Application #
6624672
Study Section
Radiation Study Section (RAD)
Program Officer
Stone, Helen B
Project Start
1998-12-02
Project End
2004-11-30
Budget Start
2003-01-10
Budget End
2004-11-30
Support Year
5
Fiscal Year
2003
Total Cost
$111,383
Indirect Cost
Name
Wayne State University
Department
Public Health & Prev Medicine
Type
Schools of Medicine
DUNS #
001962224
City
Detroit
State
MI
Country
United States
Zip Code
48202
Dolgachev, Vladislav; Oberley, Larry W; Huang, Ting-Ting et al. (2005) A role for manganese superoxide dismutase in apoptosis after photosensitization. Biochem Biophys Res Commun 332:411-7
Dolgachev, Vladislav; Farooqui, M Sharjeel; Kulaeva, Olga I et al. (2004) De novo ceramide accumulation due to inhibition of its conversion to complex sphingolipids in apoptotic photosensitized cells. J Biol Chem 279:23238-49
Dolgachev, Vladislav; Nagy, Biserka; Taffe, Bonita et al. (2003) Reactive oxygen species generation is independent of de novo sphingolipids in apoptotic photosensitized cells. Exp Cell Res 288:425-36
Wispriyono, Bambang; Schmelz, Eva; Pelayo, Homer et al. (2002) A role for the de novo sphingolipids in apoptosis of photosensitized cells. Exp Cell Res 279:153-65
Nagy, B; Yeh, W C; Mak, T W et al. (2001) FADD null mouse embryonic fibroblasts undergo apoptosis after photosensitization with the silicon phthalocyanine Pc 4. Arch Biochem Biophys 385:194-202
Belichenko, I; Morishima, N; Separovic, D (2001) Caspase-resistant vimentin suppresses apoptosis after photodynamic treatment with a silicon phthalocyanine in Jurkat cells. Arch Biochem Biophys 390:57-63
Azizuddin, K; Kalka, K; Chiu, S M et al. (2001) Recombinant human tumor necrosis factor alpha does not potentiate cell killing after photodynamic therapy with a silicon phthalocyanine in A431 human epidermoid carcinoma cells. Int J Oncol 18:411-5
Nagy, B; Chiu, S M; Separovic, D (2000) Fumonisin B1 does not prevent apoptosis in A431 human epidermoid carcinoma cells after photosensitization with a silicon phthalocyanine. J Photochem Photobiol B 57:132-41
Chiu, S M; Davis, T W; Meyers, M et al. (2000) Phthalocyanine 4-photodynamic therapy induces ceramide generation and apoptosis in acid sphingomyelinase-deficient mouse embryonic fibroblasts. Int J Oncol 16:423-7
Separovic, D; Pink, J J; Oleinick, N A et al. (1999) Niemann-Pick human lymphoblasts are resistant to phthalocyanine 4-photodynamic therapy-induced apoptosis. Biochem Biophys Res Commun 258:506-12