Photodynamic therapy (PDT) is a multicomponent cancer treatment in which tumors are exposed to lethal singlet oxygen ('O2)-mediated photooxidative stress induced by a localized sensitizing drug. Much has been learned about mechanism of tumor cell photokilling by various sensitizers, apoptosis (programmed cell death) occurring in some cases and necrosis (non-programmed death) in others. However, the role of metabolic and environmental factors in PDT-induced apoptotic vs. necrotic cell death are still not well understood. Studies supported by the existing grant have focused on the effects of nitric oxide (NO) in this regard. Using breast tumor cells metabolically sensitized with protoporphyrin IX (PpIX), we found that (i) NO delivered during irradiation (NO-now) protected against necrotic photokilling by inhibiting free radical (chain) peroxidation of plasma membrane (PM) lipids;importantly, residual killing was switched from necrosis to apoptosis;(ii)NO delivered much earlier and no longer present during irradiation (NO-then) inhibited photokilling as well, preliminary data suggesting involvement of an iron signalingmechanism. In a liposome system, NO also protected PpIX from photodegradation, thus prolonging its 'Degenerating lifetime. The proposed studies will delve more deeply into these novel effects of NO with the following hypotheses proposed: (a) NO-now and NO-then generated by neighboring microvascularcells can enhance tumor cell resistance to PDT killing; (b) By inhibiting PM lipid chain peroxidation, NO-now can foster apoptosis by reducing ion pump inactivation and membrane permeabilization, thereby supporting pro-apoptotic energy metabolism;(c) By also protecting membrane- bound sensitizer from free radical-mediated degradation, NO-now can result in a """"""""selection"""""""" for pro-apoptotic !O2 targets. The proposed in vitro studies for testing these hypotheses will involve model membranes, two human breast tumor lines (COH-BR1, MCF-7), PpEX and merocyanine 540 (MC540) as sensitizers, chemical and cellular (macrophage, endothelial) NO donors, and techniques such as fluorescence microscopy, spectrofluorimetry, immunoblotting, electrophoretic mobility shift assays, and high-performanceliquid and thin layer chromatography with electrochemical and phosphorimaging detection, respectively. The specific plan is to investigate (i) sensitizer protection by NO-now with prolonged !O2 photogeneration in model systems and cells;(ii)ability of NO-now to facilitate apoptotic photokilling while inhibiting necrosis;(iii) mechanisms by which NO-now accommodatesapoptosis; (iv) characteristics of NO-then-induced photoresistance;and (v) underlying mechanisms of NO-then-induced resistance. Although significant NO is produced by macrophages and endothelial cells in tumor vascular systems, little isknown about how it might impact PDT efficacy. These studies will provide important new insights along these lines, and in the case of NO-now may suggest novel approaches for accommodating apoptosis in PDT,this end-point beingpreferred over necrosis because inflammation is minimized.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA070823-12
Application #
7770902
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Wong, Rosemary S
Project Start
1996-12-15
Project End
2011-07-31
Budget Start
2010-03-01
Budget End
2011-07-31
Support Year
12
Fiscal Year
2010
Total Cost
$248,114
Indirect Cost
Name
Medical College of Wisconsin
Department
Biochemistry
Type
Schools of Medicine
DUNS #
937639060
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
Fahey, Jonathan M; Girotti, Albert W (2017) Nitric oxide-mediated resistance to photodynamic therapy in a human breast tumor xenograft model: Improved outcome with NOS2 inhibitors. Nitric Oxide 62:52-61
Bazak, Jerzy; Fahey, Jonathan M; Wawak, Katarzyna et al. (2017) Bystander effects of nitric oxide in anti-tumor photodynamic therapy. Cancer Cell Microenviron 4:
Girotti, Albert W; Korytowski, Witold (2016) Cholesterol as a natural probe for free radical-mediated lipid peroxidation in biological membranes and lipoproteins. J Chromatogr B Analyt Technol Biomed Life Sci 1019:202-9
Girotti, Albert W (2016) Modulation of the Anti-Tumor Efficacy of Photodynamic Therapy by Nitric Oxide. Cancers (Basel) 8:
Fahey, Jonathan M; Emmer, Joseph V; Korytowski, Witold et al. (2016) Antagonistic Effects of Endogenous Nitric Oxide in a Glioblastoma Photodynamic Therapy Model. Photochem Photobiol 92:842-853
Girotti, Albert W (2015) Tumor-generated nitric oxide as an antagonist of photodynamic therapy. Photochem Photobiol Sci 14:1425-32
Fahey, Jonathan M; Girotti, Albert W (2015) Accelerated migration and invasion of prostate cancer cells after a photodynamic therapy-like challenge: Role of nitric oxide. Nitric Oxide 49:47-55
Yang, Wan Seok; SriRamaratnam, Rohitha; Welsch, Matthew E et al. (2014) Regulation of ferroptotic cancer cell death by GPX4. Cell 156:317-331
Bhowmick, Reshma; Girotti, Albert W (2014) Pro-survival and pro-growth effects of stress-induced nitric oxide in a prostate cancer photodynamic therapy model. Cancer Lett 343:115-22
Bhowmick, Reshma; Girotti, Albert W (2013) Cytoprotective signaling associated with nitric oxide upregulation in tumor cells subjected to photodynamic therapy-like oxidative stress. Free Radic Biol Med 57:39-48

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