The objective of this research proposal is to determine whether G6PD protects breast cancer cells from PDT-induced apoptosis and clonogenic cell kill in vitro and in vivo. Our preliminary data demonstrate a dramatic increase in PDT-induced apoptosis in CHO cell lines deficient in glucose-6-phosphate dehydrogenase (G6PD) enzyme activity. This enhanced incidence of PDT-induced apoptosis was observed when either Photofrin II(TM) or LeutrinR were used. G6PD- cells also are more susceptible to PDT-induced clonogenic cell kill. The increase observed in PDT-induced apoptosis accounted for the observed difference in surviving fraction between the wild type and G6PD- cells. These results represent a paradigm shift in our view of G6PD, suggesting that it functions as an anti-apoptotic enzyme in response to the oxidative damage induced by PDT Notably, the G6PD- cells also exhibit an increased apoptotic response to ionizing radiation.
In specific aim 1, R3230Ac and MCF7 breast cancer cell lines will be transfected with a cDNA encoding an anti-G6PD antibody fragment, cloned in a mammalian expression vector. The transfectants will be used to demonstrate that inhibiting G6PD activity, in a highly specific manner, makes breast cancer cells more sensitive to PDT-induced apoptosis.
In specific aim 2, we will determine if chemical inhibitors of G6PD, such as, dehydroepiandrosterone (DHEA), increase the sensitivity of R3230Ac and MCF7 breast cancer cells to LeutrinR-based PDT, in vitro. We will examine how DHEA, and other inhibitors, effect G6PD activity, and 02 utilization in R3230Ac tumors. Ultimately the goal of this last specific aim will be to demonstrate that DHEA can sensitize R3230Ac tumors to LeutrinR -based PDT, in vivo, without affecting normal skin. These experiments will predict if LeutrinR -based PDT can be used to treat locally recurrent breast cancers. We also plan to determine the molecular targets that play a role in regulating PDT-induced apoptosis, in a G6PD dependent manner (specific aim 4). We propose that PDT activates pro-apoptotic signaling proteins by oxidizing key protein thiols. Alternately, we propose that PDT-induced peroxidation of cell membranes, could account for the enhanced sensitivity of the G6PD- cell line.
| Tuttle, Stephen W; Maity, Amit; Oprysko, Patricia R et al. (2007) Detection of reactive oxygen species via endogenous oxidative pentose phosphate cycle activity in response to oxygen concentration: implications for the mechanism of HIF-1alpha stabilization under moderate hypoxia. J Biol Chem 282:36790-6 |
