Methylation and Expression of Genes in Ovarian Clear Cell Carcinoma: A multicenter cooperative study

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1 Research Volume 17 Issue 7 - March 11, 2011 [ ] Methylation and Expression of Genes in Ovarian Clear Cell Carcinoma: A multicenter cooperative study Cheng-Yang Chou Department of Obstetrics and Gynecology, College of Medicine, National Cheng Kung University chougyn@mail.ncku.edu.tw International Journal of Cancer 127, (2010) NCKU Landmark Project I Introduction Ovarian cancer is the second most common gynecological malignancy and the fifth leading cause of cancer-related death among women in Taiwan; nearly 60% of women with ovarian cancer eventually succumb to the disease. Chemoresistance almost always occurs in recurrent patients, and the mechanism responsible for chemoresistance of ovarian cancer remains unclear. Ovarian clear cell carcinoma (OCCA) is a distinct disease entity of ovarian carcinoma that has unpredictable clinical course. In addition, the tumor biology and molecular signature of OCCA remain to be determined. OCCA occurs more frequent in Japan and Taiwan, compared with its lower incidence in western countries (5%). As a result, we initiated a multicenter cooperative study to address this problem. 2. Experimental results and discussion A multicenter study of OCCA is initiated to collect and analyzed this type of cancer through the gene expression and methylation approach. A total of 389 cases of OCCA are reviewed and frozen tumor tissues and bio-fluids from 79 patients are collected. The promoter methylation status of certain candidate genes was analyzed. Those genes include SFRP5, DLEC1, E-cadherin, RASSF1A, Stat1 etc. We have found that SFRP5, DLEC1, E-cadherin, RASSF1A and Stat1 promoter are frequently methylated in ovarian cancer (Fig. 1). Among them, SFRP5 and Stat1 are related to overall survival of OCCA patients (Fig. 2). Moreover, SFRP5 is related to malignant phenotype and chemo-resistance. SFRP5 belongs to a family of five secreted glycoproteins, are extracellular signaling molecules that antagonize the Wnt signaling pathway. We also examined the methylation status of SFRP5 in ovarian cancer cell lines, and the role of DNA methylation in gene silencing was confirmed using demethylation agents. Restoration and knockdown of SFRP5 in different ovarian cell lines were used to test the effects of SFRP5 on tumor suppression and drug resistance. We found SFRP5 suppresses anchorage-independent growth, invasion phenotype and tumor growth (Fig. 3). Silencing of SFRP5 significantly increased the chemoresistance to cisplatin, taxol, and etoposide. Re-expression of SFRP5 markedly decreased invasion phenotype in SKOV3 and OVCAR3 cells. 1 of 2

2 Fig 1. Promoter methylation status of ovarian clear cell carcinoma. Fig 2. Promoter methylation of SFRP5 and Stat in OCCC. 3. Conclusions Our observations provide evidence that epigenetic silencing of SFRP5 might upregulate the cisplatin-based chemoresistance of ovarian cancer and that this change is associated with Wnt signaling and the EMT process and occurs partly through the TWIST/Akt signaling pathway. Clarifying the relationship between the epigenetic modification of SFRP5 and ovarian tumors will help us understand the context of ovarian tumors. The development of Wnt signaling inhibitors may lead to new ovarian cancer treatments. Taken together, identification of specific OCCA biomarkers will lead to a better understanding of molecular mechanism and a better treatment of OCCA. Fig 3. The effects of SFRP5 on invasion assay in SKOV3 and OVCAR3. Acknowledgement The authors would like to thank NCKU Landmark project for financially supporting this research. References 1. International Journal of Cancer 127, (2010) 2. European Journal of Clinical Investigation 40, (2010) 2 of 2

3 Research Volume 17 Issue 7 - March 11, 2011 [ ] Study on Historical Transition in East Asia:Gender, Body and Multiculture Wing-Sheung Cheng Department of history, N.C.K.U. Chairperson em52301@ .ncku.edu.tw NCKU Landmark Project R002 A Integration Research on East Asia History Development has been set as a devoted research direction for the History Department of National Cheng Kung University (NCKU) since We meant to set a core-topic for all the research teams which major in different fields, in attempt to braoden our visions and to integrate all the resources devoted to this study. Besides, it is hoped that directed by this project, the research team could exchange points of views with scholars from different institutions through holding international symposiums, or conferences. The research team devoted to turn the NCKU History Department into an important research center and a platform for research exchanging of East Asia History Development, thus consolidate our international academic status in this field. The NCKU History Department has begun the process by: 1. Constantly holding biennial international symposiums on East Asia History Development thus make NCKU History Department a regular research base on this topic. The second international symposium The Spreading and Conflict of Different Religions: A Symposium on Historical Development in East Asia (Ⅱ) which was held in November 2009 had invited foreign scholars from Hong Kong, China, Japan, Korea, Vietnam, Malaysia. 2. Holding annual scholar workshop and doctoral student workshop on each year's topic. The scholar workshop invites scholars at home and abroad to give lectures and lead discussions on the yearly topic. In December 2009, we held "Gender, Body and Multiculture" scholar workshop in which invited Professor Jen-Der Lee (Institute of History and Philology, SINICA), Professor Yu-Chen Lee (Department of Chinese Literature, NTHU) and Professor Fang-Chi Yang (Department of Taiwanese Literature, NCKU) to give speeches. The doctoral student workshop invites doctoral students at home and abord to present papers on the yearly topic, and invites the experts to comment, in order to enhance the young scholar's research. In May 2010, we held "Gender, Body and Multiculture" doctoral student workshop in which invited experts, doctoral students at home and abord to attend this seminar, and the achievement is plentiful. 3. To build an academic website in East Asia History Development. Thus, NCKU History Deaprtment research team can make scholars in the same field who are from Taiwan, China, and other North-East and South- East Asia countries cohere successfully. By field inspection, we try to exchange experiences with different academic organizations at the surrounding countries. Through this we may to comprehend the vicissitudes, similarities and differences between bilateral cultures, besides, we can enlarge the academic platform of East Asia historical studies by such interaction. 4. The research team members submit articles on the yearly topic every year, set to understand the history of East Asia ethnic cultures, of how they exchange, alter and develope. This year (2009), the topic is "Gender, 1 of 2

4 Body and Multiculture." While looking back, the research team can also re-evaluate East Asis Civilizaion in the 21th century, at the same time; the East Asia History can be given a new reinterpretation. The research team members presented twelve articles in " Historical Development in East Asia--The Spreading and Conflict of Different Religions: Research result presentation from August 2008 to July 2009" which held in September These drafts of research articles will also be published in academic journals after being revised. 2 of 2

5 Research Volume 17 Issue 7 - March 11, 2011 [ ] Study of the live cell membrane interacted with biomolecules by nanoplasmonic biosensing and molecular imaging Shean-Jen Chen Department of Engineering Science, National Cheng Kung University, Tainan 701, Taiwan sheanjen@mail.ncku.edu.tw This research reports the first combination of high NA oil-immersion objective-based NCKU Landmark Project B0161 wide-field Surface Plasmon (SP)-enhanced fluorescence microscopy and SP phase microscopy. According to theoretical investigations, the decrease of thin silver film thickness not only decreases the propagation length of SPs, so that more detailed intensity and phase features can be recognized owing to improved lateral resolution, but also decreases the surface plasmon resonance (SPR) angle so as to resolve the restriction associated with the maximum angle imposed by finite NA value of the objective. Through a moderate spacer to adjust the metal-fluorophore distance, the SPs obviously contribute to the increase in the quantum yield for achieving brighter a fluorescent signal and a reduction in the fluorescence lifetime for attaining better photostability. A candidate tumor suppressor WW domain-containing oxidoreductase, known as marine WOX1 or WWOX, and monkey kidney COS7 fibroblasts are used to demonstrate the imaging capabilities of the developed microscopy. WOX1 proteins play an important role in the regulation of a wide variety of cellular functions. We also attempt to identify a suitable spacer between the fluorophore and the metal surface as a trade-off between the fluorescence enhancement and quenching in two-photon excited TIRF microscopy. The local electric field enhancement, fluorescence lifetime and quantum yield, and fluorescence emission coupling yield by the SPs with a spacer between the dye and the metal surface are theoretically studied by using the Fresnel equation and classical dipole radiation modeling. Also, a two-photon TIRF microscopy with a time-correlated single photon counting module has been developed to observe the fluorescence lifetime, photostability, and enhancements. From the simulations and the experiments, the trend of the enhancement and the fluorescence lifetimes based on the surface plasmon-total internal reflection fluorescence setup are consistent. We can find that the maximum fluorescence enhancement has been increased up to 30 fold with suitable SiO2 spacers. The experimental results demonstrate that the configuration with an optimal SiO2 spacer not only clearly reveals a brighter fluorescent signal compared to that of conventional TIRF, but also improves fluorescence photostability compared to that of a less quenching setup with a thicker spacer. Finally, we utilized a developed SP phase microscopy to observe cell-biosubstrate contacts. The developed SP phase microscopy is highly sensitive to cell membrane contact with biosubstrates and also provides long-term phase stability to achieve time-lapse living cell observation. As such, an SP intensity and phase sensitivity comparison demonstrates that the sensitivity of the phase measurement is 160-fold greater than that of the intensity measurement. Also, a more than 2-hour cell apoptosis observation via the SP phase microscopy is presented. To implement the incident angle from 70 to 78, cell-biosubstrate contact images corresponding to the SPR angles are obtained by utilizing the SP phase measurement. According to the information of the corresponding SPR angle image and a multilayer simulation, the contact distances between a living melanoma cell and a bovine serum 1 of 3

6 albumin substrate at four different locations have been estimated. Fig. 1. Live melanoma-gfp-tagged thrombomodulin SP-enhanced fluorescence images by utilizing (a) the 1.49 NA oil-immersion objective and (b) the 1.0 NA water-immersion objective, both with an exposure time of 0.5 sec at an incident angle of Fig. 2. The overall enhancement factor of the fluorescence intensity as function of spacer thickness. 2 of 3

7 Fig. 3. (a) 3D image and (b) 2D image of the corresponding SPR angles at different locations based on the SPP phase measurement. The color scales indicate the SPR angle in degrees. 3 of 3

8 Research Volume 17 Issue 7 - March 11, 2011 [ ] Acquisition of stops in Taiwanese Li-mei Chen 1*, Ya-Yu Hsu 1, Eun Jong Kong 2, Jan Edwards 2, and Mary Beckman 3 1 Department of Foreign Languages & Literature, National Cheng Kung University 2 Waisman Center, University of Wisconsin-Madison, USA 3 Department of Linguistics, Ohio State University, USA leemay@mail.ncku.edu.tw Paper to present at the 31 st annual Symposium on Research in Child Language Disorders in Madison, Wisconsin, June 3-5, (2010). NCKU Landmark Project I004 This study investigates three-way voicing contrasts in Taiwanese word-initial stops (aspirated stops /p h, t h, k h /, unaspirated stops /p, t, k/, and voiced stops /b, g/) in both adults' and children's productions. Phonological impairment is one of the most frequently encountered communication disorders in the preschool years. However, the developmental speech disorders cannot be attributed to identifiable organic pathology, such as hearing impairment, neurological deficit, or structural abnormality of the vocal tract (Leonard, 1995). It is estimated that approximately 10% of preschool children cannot develop intelligible speech, and 80% of this group require speech and language therapy services (Weiss, Gordon, & Lillywhite, 1987). Therefore, studies of early phonetic development are crucial to establish developmental normality and to develop criteria for the assessment of early speech disorders. This study investigates three-way voicing contrasts in Taiwanese word-initial stops (aspirated stops /p h, t h, k h /, unaspirated stops /p, t, k/, and voiced stops /b, g/) in both adults' and children's productions. This study based on the same methodology as the paidologos project (a cross-linguistic study of phonological development, Edwards and Beckman, ), and the long-term purpose is to examine universal and language-specific characteristics in the order and rate of sound acquisition. Data from 2 year olds and 3 year olds were analyzed. All children were reported to have typical language development according to parents and teachers, and had passed a hearing screening test (otoacoustic emissions). Data from 10 adults were also included for comparison. Target consonants were elicited in word-initial positions with color photograph and the associated sound files for children to repeat. 1 of 3

9 Figure 1. Distribution of VOT values in real words (left) and non-words (right) of adult males and females Figure 2. Distribution of VOT values in real words (left) and non-words (right) of 2 year olds and 3 year olds Major findings in this preliminary report are: 1) Children at age 2 follow the rule of the further back the articulation, the longer VOTs when producing real words. 2) In real word production, voiced labial stops have shorter VOT than voiced velar stops in both adults and children at both ages 2 and 3. 3) However, aveolar unaspirated stops have shorter VOTs than bilabial unaspirated stops in adults' productions. This language-specific pattern is also found in 3 year olds. 4) Aspiration distinction in VOT values is found at 2 years old, and children at 2 of 3

10 age 3 have better ability to distinguish aspiration contrasts. 5) Production of voiceless stops of 3 year olds is more accurate 2 year olds. 6) Lead VOTs for voiced stops are found in both adults and 3 year olds. 7) The idiosyncratic characteristics are consistently found in the production of voiced stops. Moreover, women tend to use lead VOTs for voiced stops more often. This project is part of the paidologos project (a cross-linguistic investigation of phonological development, Edwards and Beckman, ). 3 of 3