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Acinar Morphogenesis of Human Mammary Epithelial Cells in 3D Culture Induces EcSOD Expression via Epigenetic Reprogramming
in iron NTA-induced rat renal cell carcinoma. Sarcomatoid MM induced by ferric saccharate obtained the same genetic change. Thus, asbestos-induced carcinogenesis is associated with iron overload and is oxidative stress-dependent. Modulation of iron appears a promising strategy for the prevention of MM in people already exposed to asbestos.
174 Acinar Morphogenesis of Human Mammary Epithelial Cells in 3D Culture Induces EcSOD Expression via Epigenetic Reprogramming Melissa Teoh1, Matthew Fitzgerald1, Weixiong Zhong2, and Frederick Domann1 1 2 University of Iowa, University of Wisconsin-Madison Expression of EcSOD in normal human mammary epithelial cells was not recognized until recently. In fact, neither EcSOD mRNA nor protein expression was detectable in normal mammary epithelial cells cultured in conventional 2D culture conditions. In contrast, our immunohistological studies revealed definitive strong positive staining for EcSOD in normal human breast tissue. Based on accumulating evidence for differential gene expression patterns in 3D cultures compared with 2D monolayer cultures, we hypothesized that EcSOD gene expression was lost when normal mammary tissue organization was disrupted, such as in 2D culture and in breast carcinomas. Laminin enriched matrigel provides a 3D in vitro culture system to mimic the in vivo tissue environment that induces acinar morphogenesis by mammary epithelial cells. Using this system, we tested whether culturing HMEC in a 3D context could reactivate EcSOD gene expression. Indeed, EcSOD mRNA expression was activated in HMEC after 5 days of 3D culture and its expression continued to accumulate up to 10 days in the 3D culture. The 3D cultured HMEC formed organized and polarized acinar structures as revealed by immuno-fluorescence microscopy. This phenotype was not stable since expression of EcSOD in 3D cultured HMEC dramatically decreased after just two passages in 2D culture. We have further shown that EcSOD mRNA expression was strongly induced in the 2D cultured HMEC after treatment with a DNA methyltransferase inhibitor and to a lesser extent with a histone deacetylase inhibitor. As indicated by the COBRA bisulfite analysis, there was a decrease in the degree of CpG methylation in the EcSOD promoter after 10 days of 3D culture compared to the 2D cultured HMEC. Furthermore, diminished expression of EcSOD in 2D cultured HMEC was tightly associated with CpG hypermethylation of the EcSOD promoter suggesting that epigenetic mechanisms may play an important role in regulating EcSOD gene expression. Finally, more than 87% of clinical mammary adenocarcinoma samples showed significantly decreased EcSOD mRNA expression levels compared to adjacent normal mammary tissue. Taken together, these data suggest that the epigenetic silencing of EcSOD may contribute to mammary carciniogenesis and that restoring EcSOD expression could be an effective strategy for breast cancer treatment. doi: 10.1016/j.freeradbiomed.2010.10.178
175 Asbestosinduced Carcinogenesis is Oxidative Stressdependent Shinya Toyokuni1, Li Jiang1, Hirotaka Nagai1, Hiroki Ohara1, Yasumasa Okazaki1, Shinya Akatsuka1, and Yoriko Yamashita1 1 Nagoya University Graduate School of Medicine Respiratory exposure to asbestos is associated with malignant mesothelioma (MM) in humans. We studied the ability of chrysotile, crocidolite and amosite to induce oxidative DNA damage. Crocidolite and amosite fibers induced DNA double strand breaks in plasmids preferentially at C:G and repeat sequences. We then used time-lapse videomicroscopy to find that not only RAW264.7 cells but also MeT-5A and HeLa cells engulfed crocidolite fibers, which reached cytoplasm and nucleus. Intraperitoneal (ip) administration of chrysotile, crocidolite or amosite all induced MM in the majority of rats, with iron deposition nearby and significant promotion with a catalytic iron chelator, nictrilotriacetate (NTA). Most tumors examined (25/27) showed homozygous deletion of CDKN2A/2B, which frequently occurred
S72
doi: 10.1016/j.freeradbiomed.2010.10.179
176 HyperthermiaMediated Cell Death in Murine GT1 7 Hypothalamic Neurons and Human LnCap Prostate Carcinoma Cells Exposed to Nanoparticles of SPIONs@AuCysteamineLHRH Faruq Mohammad1,2, Sainath Babu1, Achuthan C Raghavamenon1, Kumar SSR Challa2, Konstantin G Kousoulas3, and Rao M Uppu1 1 2 Southern University and A&M College Louisiana State 3 University Louisiana State University. Magnetite nanoparticle-induced hyperthermia is gaining momentum in cancer therapy. Binding of luteinizing hormone releasing hormone (LHRH) to these magnetite particles has the advantage of targeted delivery to cancer tissues which generally over express LHRH receptors. In order to further investigate the possibility for enhancement of hyperthermia of magnetite nanoparticles, we synthesized SPIONs coated with gold (i.e., SPIONs@Au) and subsequently bound to LHRH through cysteamine bifunctional linker (the amino group of cysteamine is bound to LHRH through peptide linkage while the thiol group is bound to the gold surface of the SPION particle). Both SPIONs and SPIONs@Au were characterized thoroughly and a 4- to 5fold enhancement in hyperthermia was found on gold coating. The results will appear in a manuscript that is currently in print (Mohammad et al., 2010, J. Phys. Chem. C., in press). When LHRH receptor over expressing non-cancerous murine GT1-7 hypothalamic neurons and human prostate carcinoma cell line (LnCap) were exposed to SPIONs@Au-cysteamine-LHRH (0-500 μg/ml concentration) for 24 h, there were no significant differences in the cytotoxicity of the two cell types. However, when pretreated with SPIONs@Au-cysteamine-LHRH (0-500 μg/ml concentration) for 24 h and then exposed to low frequency magnetic field of 44 Hz, both cell types showed significant increase in cell death, the magnitude of which depended on the period of field exposure (15 min twice for GT1-7 hypothalamic neurons and 15 min once for LnCap cells). When stained with acridine orange and ethidium bromide, the cell death in GT1-7 hypothalamic neurons was found to be apoptotic while in LnCap cells late apoptosis/necrosis was prominent. The observed differences in susceptibility between the two cell types is in agreement with the notion that cancer cells are more prone to heat stress than are normal cells. This aspect is currently being investigated through use of pathway-specific gene expression and heat shock protein PCR arrays. doi: 10.1016/j.freeradbiomed.2010.10.180
177 Predicting Cell Vulnerability to Hydrogen Peroxide Generating Compounds: Possible Use in Anti Cancer Therapies Thomas Joost van 't Erve1, Brett Wagner2, Jordan Witmer2, Weipeng Bian2, and Garry Buettner1,2 1 2 IDGP in Human Toxicology, University of Iowa, Free Radical and Radiation Biology Program, University of Iowa Hydrogen peroxide has been known as a toxin for over a century. This has led to the hypothesis that H2O2 can be used as part of anti-cancer therapies. However, H2O2 cannot be delivered directly to tumor cells in vivo, rather approaches are being
SFRBM/SFRRI 2010
174 Acinar Morphogenesis of Human Mammary Epithelial Cells in 3D Culture Induces EcSOD Expression via Epigenetic Reprogramming Melissa Teoh1, Matthew Fitzgerald1, Weixiong Zhong2, and Frederick Domann1 1 2 University of Iowa, University of Wisconsin-Madison Expression of EcSOD in normal human mammary epithelial cells was not recognized until recently. In fact, neither EcSOD mRNA nor protein expression was detectable in normal mammary epithelial cells cultured in conventional 2D culture conditions. In contrast, our immunohistological studies revealed definitive strong positive staining for EcSOD in normal human breast tissue. Based on accumulating evidence for differential gene expression patterns in 3D cultures compared with 2D monolayer cultures, we hypothesized that EcSOD gene expression was lost when normal mammary tissue organization was disrupted, such as in 2D culture and in breast carcinomas. Laminin enriched matrigel provides a 3D in vitro culture system to mimic the in vivo tissue environment that induces acinar morphogenesis by mammary epithelial cells. Using this system, we tested whether culturing HMEC in a 3D context could reactivate EcSOD gene expression. Indeed, EcSOD mRNA expression was activated in HMEC after 5 days of 3D culture and its expression continued to accumulate up to 10 days in the 3D culture. The 3D cultured HMEC formed organized and polarized acinar structures as revealed by immuno-fluorescence microscopy. This phenotype was not stable since expression of EcSOD in 3D cultured HMEC dramatically decreased after just two passages in 2D culture. We have further shown that EcSOD mRNA expression was strongly induced in the 2D cultured HMEC after treatment with a DNA methyltransferase inhibitor and to a lesser extent with a histone deacetylase inhibitor. As indicated by the COBRA bisulfite analysis, there was a decrease in the degree of CpG methylation in the EcSOD promoter after 10 days of 3D culture compared to the 2D cultured HMEC. Furthermore, diminished expression of EcSOD in 2D cultured HMEC was tightly associated with CpG hypermethylation of the EcSOD promoter suggesting that epigenetic mechanisms may play an important role in regulating EcSOD gene expression. Finally, more than 87% of clinical mammary adenocarcinoma samples showed significantly decreased EcSOD mRNA expression levels compared to adjacent normal mammary tissue. Taken together, these data suggest that the epigenetic silencing of EcSOD may contribute to mammary carciniogenesis and that restoring EcSOD expression could be an effective strategy for breast cancer treatment. doi: 10.1016/j.freeradbiomed.2010.10.178
175 Asbestosinduced Carcinogenesis is Oxidative Stressdependent Shinya Toyokuni1, Li Jiang1, Hirotaka Nagai1, Hiroki Ohara1, Yasumasa Okazaki1, Shinya Akatsuka1, and Yoriko Yamashita1 1 Nagoya University Graduate School of Medicine Respiratory exposure to asbestos is associated with malignant mesothelioma (MM) in humans. We studied the ability of chrysotile, crocidolite and amosite to induce oxidative DNA damage. Crocidolite and amosite fibers induced DNA double strand breaks in plasmids preferentially at C:G and repeat sequences. We then used time-lapse videomicroscopy to find that not only RAW264.7 cells but also MeT-5A and HeLa cells engulfed crocidolite fibers, which reached cytoplasm and nucleus. Intraperitoneal (ip) administration of chrysotile, crocidolite or amosite all induced MM in the majority of rats, with iron deposition nearby and significant promotion with a catalytic iron chelator, nictrilotriacetate (NTA). Most tumors examined (25/27) showed homozygous deletion of CDKN2A/2B, which frequently occurred
S72
doi: 10.1016/j.freeradbiomed.2010.10.179
176 HyperthermiaMediated Cell Death in Murine GT1 7 Hypothalamic Neurons and Human LnCap Prostate Carcinoma Cells Exposed to Nanoparticles of SPIONs@AuCysteamineLHRH Faruq Mohammad1,2, Sainath Babu1, Achuthan C Raghavamenon1, Kumar SSR Challa2, Konstantin G Kousoulas3, and Rao M Uppu1 1 2 Southern University and A&M College Louisiana State 3 University Louisiana State University. Magnetite nanoparticle-induced hyperthermia is gaining momentum in cancer therapy. Binding of luteinizing hormone releasing hormone (LHRH) to these magnetite particles has the advantage of targeted delivery to cancer tissues which generally over express LHRH receptors. In order to further investigate the possibility for enhancement of hyperthermia of magnetite nanoparticles, we synthesized SPIONs coated with gold (i.e., SPIONs@Au) and subsequently bound to LHRH through cysteamine bifunctional linker (the amino group of cysteamine is bound to LHRH through peptide linkage while the thiol group is bound to the gold surface of the SPION particle). Both SPIONs and SPIONs@Au were characterized thoroughly and a 4- to 5fold enhancement in hyperthermia was found on gold coating. The results will appear in a manuscript that is currently in print (Mohammad et al., 2010, J. Phys. Chem. C., in press). When LHRH receptor over expressing non-cancerous murine GT1-7 hypothalamic neurons and human prostate carcinoma cell line (LnCap) were exposed to SPIONs@Au-cysteamine-LHRH (0-500 μg/ml concentration) for 24 h, there were no significant differences in the cytotoxicity of the two cell types. However, when pretreated with SPIONs@Au-cysteamine-LHRH (0-500 μg/ml concentration) for 24 h and then exposed to low frequency magnetic field of 44 Hz, both cell types showed significant increase in cell death, the magnitude of which depended on the period of field exposure (15 min twice for GT1-7 hypothalamic neurons and 15 min once for LnCap cells). When stained with acridine orange and ethidium bromide, the cell death in GT1-7 hypothalamic neurons was found to be apoptotic while in LnCap cells late apoptosis/necrosis was prominent. The observed differences in susceptibility between the two cell types is in agreement with the notion that cancer cells are more prone to heat stress than are normal cells. This aspect is currently being investigated through use of pathway-specific gene expression and heat shock protein PCR arrays. doi: 10.1016/j.freeradbiomed.2010.10.180
177 Predicting Cell Vulnerability to Hydrogen Peroxide Generating Compounds: Possible Use in Anti Cancer Therapies Thomas Joost van 't Erve1, Brett Wagner2, Jordan Witmer2, Weipeng Bian2, and Garry Buettner1,2 1 2 IDGP in Human Toxicology, University of Iowa, Free Radical and Radiation Biology Program, University of Iowa Hydrogen peroxide has been known as a toxin for over a century. This has led to the hypothesis that H2O2 can be used as part of anti-cancer therapies. However, H2O2 cannot be delivered directly to tumor cells in vivo, rather approaches are being
SFRBM/SFRRI 2010