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Constitutive omega-3 fatty acid production in fat-1 transgenic mice protects against UVB-induced skin inflammation and carcinogenesis
Abstracts / Drug Metabolism and Pharmacokinetics 32 (2017) S27eS107
Annexin A5, a member of annexin protein family, is a calcium-dependent phospholipid binding protein. Previous studies showed that the biological function of annexin A5 is related to apoptotic features. But the role of annexin A5 in cell proliferation is not yet clear. In this study, we explored the role of annexin A5 in PC-3 human prostate cancer cells. To clarify whether annexin A5 regulates cell proliferation, we examined the cell viability with annexin A5-knockdowned cells and it showed that annexin A5 inhibits cell proliferation. To identify the detailed mechanism of annexin A5 further, we performed microarray with annexin A5-knockdowned PC-3 cells and selected a number of genes that significantly changed by alteration of annexin A5 expression. Of these, PAI-2, plasminogen activator inhibitor-2 increased about 1.6-fold in annexin A5knockdowned cells and decreased about 0.8-fold when cells were overexpressed with annexin A5. To investigate whether PAI-2 is involved in anti-proliferative mechanism induced by annexin A5, we suppressed PAI2 with specific siRNA and found that PAI-2 regulates cell proliferation. In conclusion, we suggest that PAI-2 is downregulated by annexin A5 and may play an important role in annexin A5-mediated cellular function. P111 CONSTITUTIVE OMEGA-3 FATTY ACID PRODUCTION IN TRANSGENIC MICE PROTECTS AGAINST UVB-INDUCED INFLAMMATION AND CARCINOGENESIS
FAT-1 SKIN
Hye-Won Yum 1, Joydeb Kumar Kundu 2, Yong-Yeon Cho 3, Young-Joon Surh 1. 1 Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul, South Korea; 2 College of Pharmacy, Keimyung University, Daegu, South Korea; 3 College of Pharmacy, The Catholic University of Korea, Bucheon, South Korea Exposure to solar radiation, especially ultraviolet B (UVB), is the most prevalent cause of skin photocarcinogenesis. Omega-6 (u-6) and omega3 (u-3) polyunsaturated fatty acids (PUFAs) are known to have opposite effects on the inflammatory and carcinogenesis processes. In general, u6 PUFAs promote inflammation and tumor development, whereas u-3 PUFAs possess anti-inflammatory as well as antioxidative activity and enhance host immune response. In the present study, we investigated the protective effects of u-3 PUFAs on UVB-induced skin inflammation and carcinogenesis using fat-1 transgenic mice harboring u-3 desaturase gene capable of converting u-6 to u-3 PUFAs. The u-3/u-6 PUFA ratio was significantly higher in the dorsal skin of fat-1 mice than that in the wild-type (WT) C57BL/6 mice. fat-1 mice expressed heme oxygenase-1 and NAD(P)H:quinone oxidoreductase-1 and their mRNA transcripts to a greater extent than did WT mice, which was attributable to the elevated activation of nuclear factor erythroid-related factor-2 (Nrf2) responsible for upregulation of cytoprotective and stress-responsive proteins. Upon single exposure to UVB (500 mJ/cm2) irradiation, fat1 mice exhibited a significantly lower degree of skin inflammation and phototoxicity and the expression of the pro-inflammatory enzyme, cyclooxygenases-2 (COX-2) as compared to those observed in WT mouse skin. The protection of fat-1 mice from UVB-induced skin inflammation was associated with decreased phosphorylation of signal transducer and activator of transcription 3 (STAT3), one of the major proinflammatory transcription factors. To determine whether the elevated u-3 PUFA level also protects against UVB-induced skin papillomagenesis, hairless fat-1 / albino and fat-1+/ albino mice were generated by cross-breeding of male fat-1+/ transgenic mice with female SKH-1 hairless albino mice. After repeated UVB irradiation (180 mJ/cm2) thrice a week for 23 weeks, the incidence and the multiplicity of papillomas were markedly reduced in the fat-1+/ albino group as compared to the fat-1 / albino group. Moreover, the expression of COX-2 and activation of STAT3 in papillomas from fat-1+/ albino mice were significantly lower than those found in UVB-irradiated fat-1 / albino mice. Taken together, our results indicate that functional fat-1 potentiates cellular defense against UVB-induced skin inflammation and tumor development through elevated activation of Nrf2 and upregulation of cytoprotective gene expression.
S59
P112 ABSTRACT WITHDRAWN P113 HUMAN STEROID SULFATASE INDUCES AEROBIC GLYCOLYSIS THROUGH HIF1A INDUCTION IN HELA CELLS Sangyun Shin, Yeo-Jung Kwon, Dong-Jin Ye, Hyoung-Seok Baek, YoungJin Chun. College of Pharmacy, Chung-Ang University, Seoul, South Korea Human steroid sulfatase (STS) is known as an important target enzyme for suppressing estrogen-mediated carcinogenesis. Although high level of STS expression is known to involve in carcinogenesis, the precise mechanism of STS-induced cancer progression is still not clear. STS converts estrone sulfate to estrone and dehydroepiandrosterone sulfate (DHEA-S) to dehydroepiandrosterone (DHEA). On the contrast to normal cells, cancer cells generate cellular energy by inefficient way, aerobic glycolysis. To elucidate whether STS is able to regulate energy production of cancer cells, the effects on aerobic glycolysis were determined. Oxygen consumption rate (OCR) which represents mitochondria respiration is significantly suppressed by STS overexpression, STS specific inhibitor STX064 enhanced STS-induced OCR repression. DHEA also suppressed OCR level, whereas inactive form DHEA-S did not affect mitochondria respiration of cells. Rapid production of lactic acid, which is a characteristic of tumorigenesis, was up-regulated by STS. Treatment of DHEA also enhanced lactic acid production level, while DHEA-S had no effect on production of lactic acid. To understand molecular mechanism of STSinduced cancer metabolism, we investigated glycolytic enzymes. Rate limit enzymes of glycolysis such as hexokinase 2 (HK-2) and pyruvate kinase M2 (PKM2) were highly increased by STS overexpression and DHEA treatment in both protein and mRNA levels. Inhibition of STS reduced increased glycolytic proteins such as HK-2 and PKM2. One of pivotal regulator of aerobic glycolysis, HIF1a was enhanced by STS and DHEA in protein, mRNA, and promoter activity level in HeLa cells. Inhibition of HIF1a with siRNA treatment, glycolytic enzymes STS-induced GLUT-1, HK-2, and PKM2 were suppressed. In conclusion, we suggested that STS and the major product DHEA regulate aerobic glycolysis through HIF1a induction. P114 HUMAN STEROID SULFATASE INDUCES THE WNT/BETA-CATENIN SIGNALING AND EPITHELIAL-MESENCHYMAL TRANSITION IN HUMAN PROSTATE CANCER CELLS Sangyun Shin, Yeo-Jung Kwon, Dong-Jin Ye, Hyoung-Seok Baek, Young-Jin Chun. College of Pharmacy, Chung-Ang University, Seoul, South Korea Although steroid sulfatase (STS) is considered as a therapeutic target for estrogen-dependent diseases, the cellular functions of STS are still not clear. We found STS expression induced the Wnt/b-catenin signaling in PC-3 human prostate cancer cells and showed that STS overexpression increased levels of b-catenin, phospho-b-catenin, and phospho-GSK3b. Enhanced translocation of b-catenin to the nucleus by STS may activate transcription of target genes such as cyclin D1, c-myc, and MMP-7. STS knockdown by siRNA resulted in downregulation of the Wnt/b-catenin signaling. b-Catenin/TCF-mediated transcription was enhanced by STS expression. STS induced epithelial-mesenchymal transition (EMT) program because STS expression reduced the level of E-cadherin, whereas levels of mesenchymal markers such as N-cadherin and vimentin were enhanced. Moreover, EMT-inducing transcription factors such as Zeb1, Zeb2, Snai1, Twist1, and FOXC2 were significantly increased by STS expression. Treatment with a STS inhibitor prevented STS-mediated Wnt/b-catenin signaling and EMT program. However, STS metabolite DHEA was able to induce EMT program. Interestingly, cell migration and invasion were also induced when STS was expressed in PC-3 cells. Taken together, these data strongly suggest that STS expression induces the Wnt/b-catenin signaling and EMT program by upregulating EMT-inducing transcription factors. The ability of STS to induce the Wnt/b-catenin signaling and the EMT program has profound
Annexin A5, a member of annexin protein family, is a calcium-dependent phospholipid binding protein. Previous studies showed that the biological function of annexin A5 is related to apoptotic features. But the role of annexin A5 in cell proliferation is not yet clear. In this study, we explored the role of annexin A5 in PC-3 human prostate cancer cells. To clarify whether annexin A5 regulates cell proliferation, we examined the cell viability with annexin A5-knockdowned cells and it showed that annexin A5 inhibits cell proliferation. To identify the detailed mechanism of annexin A5 further, we performed microarray with annexin A5-knockdowned PC-3 cells and selected a number of genes that significantly changed by alteration of annexin A5 expression. Of these, PAI-2, plasminogen activator inhibitor-2 increased about 1.6-fold in annexin A5knockdowned cells and decreased about 0.8-fold when cells were overexpressed with annexin A5. To investigate whether PAI-2 is involved in anti-proliferative mechanism induced by annexin A5, we suppressed PAI2 with specific siRNA and found that PAI-2 regulates cell proliferation. In conclusion, we suggest that PAI-2 is downregulated by annexin A5 and may play an important role in annexin A5-mediated cellular function. P111 CONSTITUTIVE OMEGA-3 FATTY ACID PRODUCTION IN TRANSGENIC MICE PROTECTS AGAINST UVB-INDUCED INFLAMMATION AND CARCINOGENESIS
FAT-1 SKIN
Hye-Won Yum 1, Joydeb Kumar Kundu 2, Yong-Yeon Cho 3, Young-Joon Surh 1. 1 Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul, South Korea; 2 College of Pharmacy, Keimyung University, Daegu, South Korea; 3 College of Pharmacy, The Catholic University of Korea, Bucheon, South Korea Exposure to solar radiation, especially ultraviolet B (UVB), is the most prevalent cause of skin photocarcinogenesis. Omega-6 (u-6) and omega3 (u-3) polyunsaturated fatty acids (PUFAs) are known to have opposite effects on the inflammatory and carcinogenesis processes. In general, u6 PUFAs promote inflammation and tumor development, whereas u-3 PUFAs possess anti-inflammatory as well as antioxidative activity and enhance host immune response. In the present study, we investigated the protective effects of u-3 PUFAs on UVB-induced skin inflammation and carcinogenesis using fat-1 transgenic mice harboring u-3 desaturase gene capable of converting u-6 to u-3 PUFAs. The u-3/u-6 PUFA ratio was significantly higher in the dorsal skin of fat-1 mice than that in the wild-type (WT) C57BL/6 mice. fat-1 mice expressed heme oxygenase-1 and NAD(P)H:quinone oxidoreductase-1 and their mRNA transcripts to a greater extent than did WT mice, which was attributable to the elevated activation of nuclear factor erythroid-related factor-2 (Nrf2) responsible for upregulation of cytoprotective and stress-responsive proteins. Upon single exposure to UVB (500 mJ/cm2) irradiation, fat1 mice exhibited a significantly lower degree of skin inflammation and phototoxicity and the expression of the pro-inflammatory enzyme, cyclooxygenases-2 (COX-2) as compared to those observed in WT mouse skin. The protection of fat-1 mice from UVB-induced skin inflammation was associated with decreased phosphorylation of signal transducer and activator of transcription 3 (STAT3), one of the major proinflammatory transcription factors. To determine whether the elevated u-3 PUFA level also protects against UVB-induced skin papillomagenesis, hairless fat-1 / albino and fat-1+/ albino mice were generated by cross-breeding of male fat-1+/ transgenic mice with female SKH-1 hairless albino mice. After repeated UVB irradiation (180 mJ/cm2) thrice a week for 23 weeks, the incidence and the multiplicity of papillomas were markedly reduced in the fat-1+/ albino group as compared to the fat-1 / albino group. Moreover, the expression of COX-2 and activation of STAT3 in papillomas from fat-1+/ albino mice were significantly lower than those found in UVB-irradiated fat-1 / albino mice. Taken together, our results indicate that functional fat-1 potentiates cellular defense against UVB-induced skin inflammation and tumor development through elevated activation of Nrf2 and upregulation of cytoprotective gene expression.
S59
P112 ABSTRACT WITHDRAWN P113 HUMAN STEROID SULFATASE INDUCES AEROBIC GLYCOLYSIS THROUGH HIF1A INDUCTION IN HELA CELLS Sangyun Shin, Yeo-Jung Kwon, Dong-Jin Ye, Hyoung-Seok Baek, YoungJin Chun. College of Pharmacy, Chung-Ang University, Seoul, South Korea Human steroid sulfatase (STS) is known as an important target enzyme for suppressing estrogen-mediated carcinogenesis. Although high level of STS expression is known to involve in carcinogenesis, the precise mechanism of STS-induced cancer progression is still not clear. STS converts estrone sulfate to estrone and dehydroepiandrosterone sulfate (DHEA-S) to dehydroepiandrosterone (DHEA). On the contrast to normal cells, cancer cells generate cellular energy by inefficient way, aerobic glycolysis. To elucidate whether STS is able to regulate energy production of cancer cells, the effects on aerobic glycolysis were determined. Oxygen consumption rate (OCR) which represents mitochondria respiration is significantly suppressed by STS overexpression, STS specific inhibitor STX064 enhanced STS-induced OCR repression. DHEA also suppressed OCR level, whereas inactive form DHEA-S did not affect mitochondria respiration of cells. Rapid production of lactic acid, which is a characteristic of tumorigenesis, was up-regulated by STS. Treatment of DHEA also enhanced lactic acid production level, while DHEA-S had no effect on production of lactic acid. To understand molecular mechanism of STSinduced cancer metabolism, we investigated glycolytic enzymes. Rate limit enzymes of glycolysis such as hexokinase 2 (HK-2) and pyruvate kinase M2 (PKM2) were highly increased by STS overexpression and DHEA treatment in both protein and mRNA levels. Inhibition of STS reduced increased glycolytic proteins such as HK-2 and PKM2. One of pivotal regulator of aerobic glycolysis, HIF1a was enhanced by STS and DHEA in protein, mRNA, and promoter activity level in HeLa cells. Inhibition of HIF1a with siRNA treatment, glycolytic enzymes STS-induced GLUT-1, HK-2, and PKM2 were suppressed. In conclusion, we suggested that STS and the major product DHEA regulate aerobic glycolysis through HIF1a induction. P114 HUMAN STEROID SULFATASE INDUCES THE WNT/BETA-CATENIN SIGNALING AND EPITHELIAL-MESENCHYMAL TRANSITION IN HUMAN PROSTATE CANCER CELLS Sangyun Shin, Yeo-Jung Kwon, Dong-Jin Ye, Hyoung-Seok Baek, Young-Jin Chun. College of Pharmacy, Chung-Ang University, Seoul, South Korea Although steroid sulfatase (STS) is considered as a therapeutic target for estrogen-dependent diseases, the cellular functions of STS are still not clear. We found STS expression induced the Wnt/b-catenin signaling in PC-3 human prostate cancer cells and showed that STS overexpression increased levels of b-catenin, phospho-b-catenin, and phospho-GSK3b. Enhanced translocation of b-catenin to the nucleus by STS may activate transcription of target genes such as cyclin D1, c-myc, and MMP-7. STS knockdown by siRNA resulted in downregulation of the Wnt/b-catenin signaling. b-Catenin/TCF-mediated transcription was enhanced by STS expression. STS induced epithelial-mesenchymal transition (EMT) program because STS expression reduced the level of E-cadherin, whereas levels of mesenchymal markers such as N-cadherin and vimentin were enhanced. Moreover, EMT-inducing transcription factors such as Zeb1, Zeb2, Snai1, Twist1, and FOXC2 were significantly increased by STS expression. Treatment with a STS inhibitor prevented STS-mediated Wnt/b-catenin signaling and EMT program. However, STS metabolite DHEA was able to induce EMT program. Interestingly, cell migration and invasion were also induced when STS was expressed in PC-3 cells. Taken together, these data strongly suggest that STS expression induces the Wnt/b-catenin signaling and EMT program by upregulating EMT-inducing transcription factors. The ability of STS to induce the Wnt/b-catenin signaling and the EMT program has profound