Description of Research Program
Rapid division of tumor cells results in an imbalance between oxygen demand and blood supply. Although hypoxia can cause tumor cells to die, hypoxia can also allow cancer cells to resist the oxygen-dependent toxicity of ionizing radiation and chemotherapy. Thus, resistance to hypoxia is a selective factor favoring proliferation of tumor cells. Strategies to sensitize tumor cells to hypoxic cell death could prove effective in cancer treatment. We propose that survival of hypoxic tumor cells depends on reactive oxygen species-mediated NFkB activation leading to expression of anti-apoptotic genes, such as X chromosome-linked inhibitor-of apoptosis proteins. Death of the hypoxic tumor cells is mediated by inactivation of NFkB through formation of NFkB-SSG (NFkB glutathionylation). The mechanisms responsible for NFkB-SSG formation are currently under investigation. We are particularly interested how NFkB glutathionylation and subsequent inactivation of NFkB may play a role in killing of pancreatic cancer cells. Pancreatic cancer is known for a very poor prognosis and therefore any manipulations to sensitize the pancreatic cancer cells to die will ultimately have a great impact on the treatment of this devastating disease. This project utilizes techniques of molecular and cell biology, biochemical assays, confocal microscopy, and mass spectrometry.

Photodynamic therapy (PDT) is a novel treatment for cancer that involves exposure of tissues to a photosensitizing drug followed by irradiation with light of appropriate wavelength, typically red or near infrared light. USFDA has approved PDT for advanced esophageal and lung cancer. PDT is currently in clinical trials for a number of other cancer types, including cancers of the prostate, head-and-neck, brain, skin, and breast cancer metastatic to skin or spine. PDT requires a photosensitizer (often a porphyrin-related macrocycle, that accumulates in tumors), non-thermal visible light, generally tissue-penetrating red light; and molecular oxygen. Absorption of a photon activates the photosensitizer to an excited singlet state that can then undergo intersystem crossing to the triplet state. The triplet transfers energy to molecular oxygen to generate singlet oxygen, or it undergoes oxidation-reduction reactions to generate other reactive oxygen species. With adequate oxygen and light intensity, the site of initial photodamage depends on the location of the photosensitizer, generally intracellular membranes, culminating in tumor cell death. Both lipids and proteins can be molecular targets of PDT. Apoptosis is a common mode of cell death following PDT both in vitro and in vivo, especially for photosensitizers localized to mitochondria. We have shown that PDT with phthalocyanine photosensitizer Pc4 causes accelerated generation of reactive oxygen species (ROS), cross-linking of mitochondrial proteins, mitochondrial inner membrane permeabilization, depolarization and swelling, release of cytochrome c, and activation of both necrosis and caspase-dependent apoptosis. We hypothesize that ROS originating with singlet oxygen after PDT initiates an attack on mitochondrial membrane proteins, leading to misfolding and formation of permeability transition (PT) pores, whose opening then initiates bioenergetic failure and cytochrome c release that culminate in necrotic and apoptotic cell death. However, Pc 4 and the analogs of Pc 4 also localize to endoplasmic reticulum (ER) and lysosomes, and we hypothesize that damage to these organelles leads to additional perturbations, such as calcium, iron and protease release, that ultimately promote MPT-dependent cell killing after PDT. Our goal is to further characterize the role of the mitochondrial permeability transition in PDT-induced killing of cancer cells with Pc 4 and its analogs, and to determine the interactions of damage to ER and lysosomes in promotion of death pathways. Live cell imaging with confocal/multiphoton microscopy is an integral part of the experimental design in this project. The link is www.musc.edu/ccdir

Selected Publications
Qanungo, S., Starke, D.W., Pai, H., Mieyal, J.J., and Nieminen, A.-L. (2007) Glutathione supplementation potentiates hypoxic apoptosis by S-glutathionylation of p65-NFkB.  J. Biol. Chem. 282, 18427-18436.

Oleinick, N.L., Morris, R.L., and Nieminen, A.-L. (2007) Photodynamic Therapy-Induced Apoptosis.  In Apoptosis, Senescence, and Cancer, D.A. Gewritz, S.E. Holt and S. Grant, Eds., Series on Cancer Drug Development, Humana Press, Inc., Totowa, NJ, 555-576.

Nieminen, A.-L., Qanungo, S., Schneider, E.A., Jiang, B.-H., and Agani, F.H. (2005) Mdm2 stimulates HIF-1 during hypoxia.  J. Cell Physiol. 204, 364-369.

Wang, M. Qanungo, S., Crow, M., Watanabe, M., and Nieminen, A.-L. (2005) Apoptosis repressor with caspase recruitment domain is expressed in cancer cells and localizes to nuclei.  FEBS Lett. 579, 2411-2415.

Qanungo, S., Wang, M., and Nieminen, A.-L. (2004) N-Acetyl-L-cysteine enhances apoptosis through inhibition of NFkB in mouse embryonic fibroblasts.  J Biol Chem. 279, 50455-50464

Morris, R.L., Azizuddin, K., Lam, M., Berlin, J., Nieminen, A.-L., Kenney, M.E., Samia, A.C.S., Burda, C., and Oleinick, N.L.  (2003) Fluorescence Resonance Energy Transfer (FRET) between the cardiolipin probe nonyl-acridine orange and the photosensitizer Pc 4.  Cancer Res. 63, 5194-5197.

Lam, M., Oleinick, N.L., and Nieminen, A.-L.  (2001) Photodynamic therapy-induced apoptosis in epidermoid carcinoma cells: Reactive oxygen species and mitochondrial inner membrane permeability.  J. Biol. Chem. 276, 47379-47386.

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