Role of Amblyomin-X on microcirculatory vessels, endothelial cell migration and tumor growth

s / Toxicology Letters 196S (2010) S37–S351 S179 Gadd45a siRNA-treated cells which implies that Gadd45a affects the APE1 function. We also observed that the decreased interaction with PCNA by the point mutation of PCNA binding site on Gadd45a induced the reduction of APE1 activity as well as interaction with APE1. We concluded that the predicted PCNA binding site on Gadd45a might play a crucial role in modulating APE1 function inBER. These results provide anovel insight into themechanisms of modulation the BER activity, although the interaction of Gadd45a andAPE1will need to be clarified in the future. (Thiswork was supported by grants from the Ministry of Environment (200909001-0083-0), and by a Korea Research Foundation (KRF) grant funded by the Korean government (MEST) (No. E00062).) doi:10.1016/j.toxlet.2010.03.609 P203-020 Identification of ionizing radiation (IR) responsive mRNA in p53-deficient human non-small cell lung cancer (NSCLC) grown in three-dimensional cell culture system J.Y. Kwon1, J.S. Kim2, Y.R. Seo1 1 Department of Pharmacology, Biomedical Science Institute, School of Medicine, Kyung Hee University, Seoul, Republic of Korea, 2 Department of Biochemistry, Chonbuk National University Medical School, Jeonju, Republic of Korea Non-small cell lung cancer (NSCLC) has been known as the most common human malignancy. The p53 tumor suppressor gene is mutated in approximately 50% of NSCLC. For treatment of lung cancer, radiotherapy using -ray is general treatment. However, many patients suffer from recurred disease. Therefore, the definite mechanism of radiation based on in vivo mimic system is required for effective remedical value to NSCLC. Most of radiation biology studies were performed in conventional monolayer culture system which cannot represent in vivo tumor environment. Hence, study applying in vivo mimic three-dimensional culture system is necessary to elucidate the molecular mechanism of solid tumor. Here, we analyzed gene expression profile induced by ionizing radiation (IR) in H1299 cells grown as spheroid in three-dimensional cell culture system. Our data showed that different gene expression profiles among monolayer culture system, three-dimensional small spheroid and three-dimensional large spheroid system. Particularly, genes related with microenvironment including adhesion junction and hypoxia had different expression levels in three-dimensional spheroid system compared with monolayer culture system. This study suggested that in vivo mimic three-dimensional cell culture engineering approach might provide critical clues for understanding of cellular responses in p53 mutated or deleted lung cancer cells against IR. (This work was supported by Nuclear Research Development Program of the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean government (MEST) (grant codes: 2007-2001431, 2008-2001694, 2009-0078295) from Korea Science and Engineering Foundation.) doi:10.1016/j.toxlet.2010.03.610 P203-021 Role of Amblyomin-X on angiogenesis and endothelial cell proliferation C.C. Drewes1, R.Y.S. Dias1, C.B. Hebeda1, S.M. Simons2, A.M. Chudzinski-Tavassi2, S.H.P. Farsky1 1 University of Sao Paulo, Brazil, 2 Institute Butantan, Brazil Introduction: Amblyomin-X is a recombinant protein inhibitor of serinoprotease, initially isolated from the salivary gland of Amblyoma cajennense. It inhibits in vivo tumors development, by unknown mechanisms. Our previous data have demonstrated that Amblyomin-X reduces the number of vessels in the microcirculatory beds. Therefore, we investigated the role of this toxin on in vivo VGEF-new vessels formation and on in vitro endothelial cell proliferation. Methods: In vivo angiogenesis was studied using dorsal chambers implanted on Male Swissmice anaesthetized by ketamine/xylazine solution. PBS (control), Amblyomin-X (10 or 100ng/10 L) and VEGF (10ng/10 L) treatment was topically applied each 48h. Numbers of vessels were quantified in images obtained before and at 8th day after beginning of treatments. Microcirculatory endothelial cell lineage (T-end lineage) was incubated with fluorophore CFSE (5 M) and Amblyomin-X (1, 10,100 or 1000ng/well) or VEGF (50ng/well) was subsequently added. Cell proliferation was monitored at 24, 48 and 72h after the treatments by flow cytometer. Results: Topical application of Amblyomin-X reduced the VEGFinduced new vessels in the dorsal subcutaneous microcirculation (10ng/10 L =26% and 100ng/10 L =32% vs number of vessels before treatments). In control animals, the number of vessels was not modified by topic application of PBS during all period of treatment. Amblyomin-X reduced the endothelial cell proliferation at 48h (1ng/well = 80±8% and 1000ng/well = 121±40%) and 72h (10ng/well = 107±10%, 100ng/well = 107±10% and 1000ng/well = 108±7%) after incubations with/without VEGF (72h, 1ng/well = 11±6% and 10ng/well = 111±7% of reduction vs control). Discussion: Here we show that Amblyomin-X, a recently isolated protein, reduces in vivo new vessels formation, whichmay be dependent, at least in part, on impairment of endothelial cell proliferation. Future investigations will be carried out to clarify themechanisms involved in this process. Financial support: FAPESP (Project 08/57850-8; 08/56072-1). doi:10.1016/j.toxlet.2010.03.611 P203-022 Role of Amblyomin-X on microcirculatory vessels, endothelial cell migration and tumor growth R. Dias1, C. Drewes1, C. Hebeda1, S. Simons2, A.M. Chudzinski-Tavassi2, S. Farsky1 1 Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of Sao Paulo, Brazil, 2 Laboratory of Biochemistry, Institute Butantan, Brazil Introduction: Amblyomin-X is a recombinant protein inhibitor of serinoprotease, isolated from the salivary gland ofAmblyoma cajennense. Systemic administration decreases the in vivo formation of tumors, by unknown mechanism. Here we aimed to investigate the role of Amblyomin-X on in vivo new vessels formation, tumor growth and in vitro endothelial cell migration and secretion. Methods: In vivo angiogenesis was studied using dorsal chambers implanted on Male Swiss mice after anesthesia. Saline or S180 Abstracts / Toxicology Letters 196S (2010) S37–S351 Amblyomin-X treatment (10or 100ng/10 L)was topically applied each 48h in the absence or presence of B16F10 lineage, which was injected intomicrocirculatory beds of the subcutaneous tissue. Numbers of vessels and tumor growth were quantified in images obtained before and at 8th day after beginning of treatments. After microcirculatory endothelial cell lineage (T-end lineage) confluence, amechanic lesion in the culturewasdoneby a cell scraper and Amblyomin-X (10 or 100ng/well) or/and VEGF (100ng/mL) were added to the wells. Cell migration was monitored at 12h by the number of cells migrating into the lesion and PGE2 and nitric oxide (NO) were quantified in the supernatant. Results: Amblyomin-X significantly reduced the number of vessels in the subcutaneous microcirculation (10ng=31.7% and 100ng/10 L =42.7% in comparison to the first day of treatment) and the tumor growth. Only in the absence of VEGF, Amblyomin-X reduced endothelial cell migration into the lesion focus during all period of time monitored, independently of PGE2 and NO secretion (10ng=16.4% and 100ng/10 L =23.1% in comparison to the control). Discussion: Local application of Amblyomin-X reduces, in vivo, the number of microcirculatory vessels and tumor growth. Impairment of endothelial cell migration may suggest an effect on new vessels formation. Future investigations will be carried out to clarify the mechanisms involved in this process. Financial support: CAPES, FAPESP grant 08/57850-8. doi:10.1016/j.toxlet.2010.03.612 P203-023 Effects of the environmental pesticide DDT and its metabolites on the human breast cancer cell line MCF-7 D. Pestana1, D. Teixeira1, A. Faria1,2, V. Domingues3, R. Monteiro1, C. Calhau1 1 Department of Biochemistry (U38-FCT), Faculty of Medicine, University of Porto, Portugal, 2 Chemistry Investigation Centre (CIQ), Faculty of Sciences, University of Porto, Portugal, 3 Requimte – Instituto Superior de Engenharia, Instituto Politecnico do Porto,