apoptosis since the yield of apoptotic nucleus after microinjection with this mutant form (15%) was similar to that obtained with 5.0 mM sodium phosphate buffer pH 7.4. The test of the caspase-3 activation was realized using cytosolic extract obtained of the same cellular lineage and Z-DEVD-AMC as fluorescent substrate. The addition of mutant forms indicated that H26N/H33N is capable to activate caspase-3, similar to wild type cytochrome c, but the H26N/H33N/K79A is less efficient to do that. The process of caspase-3 activation in the absence of K79 is slower than other forms and incomplete in the same incubation time suggesting its importance in the apoptotic process. Supported by FAPESP, CAPES, CNPq and FAEP.

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Masaki Nagane1, Hironobu Yasui1, Tohru Yamamori1, Songji Zhao1, Yuji Kuge1, Nagara Tamaki1, Hiromi Kameya2, Hideo Nakamura3, and Osamu Inanami1 1Hokkaido University,Japan 2National Food Research Institute,Japan, 3Hokkaido University of Education, Japan When solid tumors are irradiated with ionizing radiation (IR), it leads to the reduction of hypoxic fraction in tumors, known as reoxygenation. Although various physiological factors were reported to affect this process, the precise mechanisms of reoxygenation are not fully understood. In this study, we characterized the changes of oxygenation status after IR in murine SCCVII tumors. By monitoring the changes in hypoxic regions and pO2 in tumors with immunohistochemistry and ESR oxymetry, we found that IR induced biphasic reoxygenation. Hoechst33342 perfusion assay revealed that the increase of intratumoral circulation after IR. When the effect of a NO synthase inhibitor, L-NAME, on tumor reoxygenation was tested, it abolished the early-phase of reoxygenation without affecting the late-phase of it. These results suggested that the radiation-induced reoxygenation occurs in a biphasic manner through the NO-dependent and -independent mechanisms.

99

Michael R Weaver1, Sonu Venkataraman1, Sujatha Venkataraman2, and Rebecca E Oberley-Deegan1 1National Jewish Health, 2University of Colorado-Anschutz Medical Campus Roughly 1.5 million US men are prostate cancer survivors due to improvements in radiation therapy. However, the majority of these survivors will suffer significant reduction in quality of life stemming from the long term side effect(s) of pelvic irradiation exposure, including erectile dysfunction. Currently there are no effective clinical treatments used to protect normal tissues from pelvic radiation induced damage. Our laboratory has shown that the superoxide dismutase (SOD) mimetic, MnTE-2-PyP, is a potent radio-protector against radiation induced damage to urogenital tissues. We have shown that the administration of MnTE-2-PyP during radiation therapy protects from developing erectile dysfunction in rats. However, in order for MnTE-2-PyP to work as a radio-protector, MnTE-2-PyP cannot protect the prostate tumor

from radiation damage. The purpose of this study was to determine if MnTE-2-PyP has anti-tumor properties. We found that MnTE-2-PyP (1-30 μM), in a dose dependant fashion, significantly inhibited growth of three prostate cancer cells: PC3 (98.2%±0.9%), Du145 (94.3%±0.7%) and LnCAP (100%±0%) cells using colony focus assays. MnTE-2-PyP further reduced the growth of tumor cells when added in combination with radiation (5 Gy). We measured the effect that MnTE-2-PyP had on proliferation of the tumor cells and found that MnTE-2-PyP does not inhibit the proliferation of the prostate tumor cells. MnTE-2-PyP did significantly inhibit the invasiveness of PC3 cells on matrigel (67%±3.1) and significantly inhibited Du145 cells (56.7%±5.6%) ability to migrate across a filter. MnTE-2-PyP reduces NF- B activity in prostate tumor cells during irradiation and this could be one mechanism as to how MnTE-2-PyP is inhibiting tumor colony formation. MnTE-2-PyP reduces the phosphorylation of the focal adhesion kinase, which is involved in breaking down the extracellular matrix to allow cells to migrate. Finally, MnTE-2-PyP reduces VEGFA in hypoxic prostate cells, another protein involved in the growth migration of tumor cells. In combination with our earlier findings, we conclude that MnTE-2-PyP can be used as an effective radio-protector, since MnTE-2-PyP also acts as an anti-tumor agent.

100

Adam Franklin Prasanphanich1,2, Michael Butler1, and Melissa Lambeth Kemp1,2 1Georgia Institute of Technology, 2Emory University The cellular trans-differentiation process of epithelial-mesenchymal transition (EMT) leads to loss of an epithelial phenotype and acquisition of mesenchymal features such as increased migratory capacity and degradation of the extracellular matrix. EMT imbues carcinomas with an aggressive phenotype and a capacity to seed metastases. Reactive oxygen species (ROS) have been shown to induce EMT in A549 lung carcinoma cells in a Transforming Growth Factor (TGF )-dependent manner. The objective of this study was to characterize the short and long-term changes in cellular oxidation during TGF treatment that facilitates cellular trans-differentiation to the mesenchymal phenotype. Upon TGF treatment, activation the transcription factor Smad3 is responsible for the gene expression changes necessary to shift cellular phenotype. To monitor how ROS influence transcriptional regulation, we perturbed the cellular redox environment through co-administration of N-acetylcysteine (NAC). Phospho-Smad3 levels and a Smad (truncated Pai-1) promoter luciferase reporter activity were attenuated in the presence of NAC, demonstrating redox-mediated changes in TGF induced signaling on the 1-6 hour timeframe. Phospho-Smad3 is sustained over the course of EMT as cellular reprogramming to a mesenchymal phenotype. We observe NADPH oxidase 4 protein levels being up-regulated concurrently with the loss of epithelial phenotype. EMT was confirmed over a 4-day window by quantifying phenotypic markers by western blot: E-cadherin down-regulation, N-cadherin up-regulation, and vimentin up-regulation. Our findings suggest that the cellular redox state can tune Smad3 phosphorylation in A549 cells and that the redox environment shifts toward increasing oxidation during TGF treatment. Based upon these preliminary results we hypothesize that ROS and TGF possess reciprocal positive feedback during EMT.

SFRBM 2012S48

doi:10.1016/j.freeradbiomed.2012.10.126

doi:10.1016/j.freeradbiomed.2012.10.127

doi:10.1016/j.freeradbiomed.2012.10.128