Integrative Cancer Science and Therapeutics

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Integrative Cancer Science and Therapeutics (ICST) is an open access, peer-reviewed online journal interested in attracting high-quality original research and reviews that present or highlight significant advances in all areas of cancer and related biomedical science. The Journal is concerned with basic, translational and clinical research, across different disciplines and areas, enhancing insight in understanding, prevention, diagnosis and treatment of cancer. The journal also prioritizes clinical trials evaluating new treatments, accompanied by research on pharmacology and molecular alterations or biomarkers that predict response or resistance to treatment.

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Research Article ISSN: 2056-4546 Integrative Cancer Science and Terapeutics Integr Cancer Sci Terap 2017 doi: 10.15761/ICST.1000232 Volume 42: 1-4 examined whether the expression of mRNA HMGB1 and RAGE were associated with the development of EOC and evaluated the correlation of the serum HMGB1 level and EOC. Materials and methods Patients A total of 78 women who underwent surgery for a pelvic mass from May 2009 to February 2011 were included in this study. Teir blood samples were collected before they received surgery and their ovarian tissues were selectively sampled during the operation. All surgical tissues were examined by a gynecologic pathologist and fnal surgical pathology reports were obtained and recorded. Te chemotherapy regimen was 6 cycles of paclitaxel 175 mg/m 2 and carboplatin at the area under curve of 5. Te baseline patient characteristics included patient age body mass index performance status tumor stage and tumor grade. Performance status was defned according to Gynecologic Oncology Group criteria as: 0 normal activity 1 symptomatic fully ambulatory 2 symptomatic in bed less than 50 of the time. Written informed consent was obtained from all patients prior to collection of blood and surgery. Tis research was approved by the Institutional Review Board of Yonsei University Health System. Te expression of HMGB1 and the receptor for advanced glycation end products RAGE in epithelial ovarian cancer Jiheum Paek 12 and Young Tae Kim 3 1 Gynecologic Cancer Center Department of Obstetrics and Gynecology Ajou University School of Medicine Korea 2 Department of Obstetrics and Gynecology Yonsei University Graduate School Korea 3 Institute of Womens Life Medical Science Department of Obstetrics and Gynecology Yonsei University College of Medicine Korea Abstract Te aim of this study was to confrm the expression of high mobility group box 1 HMGB1 and its receptor for advanced glycation end products RAGE in patients with epithelial ovarian cancer EOC. A total of 78 patients with EOC comprised our cohort. Tey received debulking surgery followed by taxane and platinum chemotherapy. Te mRNA levels of HMGB1 and RAGE in ovarian tissues obtained from surgery were detected using reverse transcription-PCR. In addition the serum levels of HMGB1 were evaluated between cancer and non-cancer group. Te mRNA levels of HMGB1 were higher in EOC compared to the non-cancer cohort 0.94 vs. 0.90 and there was no statistically signifcant diference. Similarly the mRNA levels of RAGE did not difer between the EOC and non-cancer cohort. Te serum HMGB1 levels were not greater in the cancer cohort than the non-cancer cohort 0.40 vs. 0.45 ng/mL P0.106. Contrary to expectations as a promising biomarker for EOC the expression of both HMGB1 and RAGE did not show signifcant diferences in EOC group. Further studies are needed to assess potential roles in EOC. Introduction Epithelial ovarian cancer EOC is the fourth most common cause of cancer-related death in women worldwide accounting for approximately 3 of all new cancer patients in 2009 1. Unfortunately most of the patients are diagnosed with an advanced stage of EOC. Currently there is no proven efective method for early detection in EOC and only the serum CA125 level has been used to predict the presence of cancer. However its utility as a screening marker is limited due to its low sensitivity and specifcity 2. Moreover the serum level of CA125 can be elevated in other cancers such as uterine cancer gastrointestinal cancer as well as in benign disease 3. In order to increase the sensitivity and specifcity for the detection of EOC a number of novel markers have been investigated including matrix metalloproteinase-1 osteopontin and human epididimis 4 4- 6. Of these high mobility group box 1 HMGB1 was characterized as a non-histone nuclear DNA-binding protein 7. Te constant release of HMGB1 which functions as a proinfammatory cytokine from necrotic tumor cells creates a microenvironment similar to chronic infammation a condition known to contribute to the development of epithelial malignancies particularly infammation-associated cancer 8. Extracellular HMGB1 induces cancer cell growth mobility invasion and metastasis via binding to specifc membrane receptors including the receptor for advanced glycation end products RAGE 9. RAGE a multi-ligand member of the immunoglobulin superfamily of cell surface molecules interacts with distinct molecules implicated in homeostasis development and infammation 10-12. A lot of studies have already investigated the overexpression of HMGB1 in diferent cancers including breast gastrointestinal prostate and pancreatic cancer 13-17 measuring mRNA levels and HMGB1 would be promising as a biomarker in various cancers. In this study we Correspondence to: Jiheum Paek M.D Gynecologic Cancer Center Department of Obstetrics and Gynecology Ajou University School of Medicine Suwon 164 World Cup-ro Y eongtong-gu Suwon 16499 Korea Tel: +82- 31-219-5253 Fax: +82-31-219-5245 E-mail: paek.mdgmail.com key words: HMGB1 RAGE epithelial ovarian cancer Received: March 10 2017 Accepted: March 30 2017 Published: April 03 2017

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Paek J 2017 Te expression of HMGB1 and the receptor for advanced glycation end products RAGE in epithelial ovarian cancer Integr Cancer Sci Terap 2017 doi: 10.15761/ICST.1000232 Volume 42: 2-4 Expression of mRNA levels of HMGB1 and RAGE For DNA and protein extraction fresh ovarian tumors and normal ovarian tissues were obtained immediately afer surgical excision and were stored at -70°C before use. Total RNA was extracted from tissues using the easy-spin TM DNA free Total RNA Extraction Kit Intron Biotechnology Seongnam Korea according to the manufacturers instructions. As a control reaction for intact RNA and cDNA PCR amplifcation of the housekeeping gene β-actin was performed on all samples to normalize the sample amount. Amplifcation was performed for a predetermined optimal number of cycles. Te PCR products were separated by electrophoresis on 1.8 agarose gels which were stained with 0.5 mg/mL ethidium bromide. Quantifcation of proteins was performed by using a laser densitometer and analysis sofware IMAGE READER LAS-1000 lite Fuji Photo Film Co. Ltd. Tokyo Japan referring to the standard series. Te densitometric integral derived from each sample band the integral of a mean density over a measured area was taken to calculate the amount in each sample according to the known standard values Figure 1. Measurement of serum levels of HMGB1 Approximately 25 ml of whole blood was collected in non- heparinized tubes from patients. Within 4 hours of the collection the blood was centrifuged at 3000 rpm for 15 minutes and the serum fraction was aliquoted and stored at -70°C in microfuge tubes until assayed. HMGB1 was measured by the commercially available Human HMGB1 ELISA Kit USCN LIFE Science Wuhan China. Briefy 100 μl of sample diluent was added to each well and then 10 μl of standard and sample or control was added to the well. Te microtiter plates were incubated for 20–24 hours at 37°C. Afer washing 100 μl/well of biotin- conjugated polyclonal antibody preparation specifc for HMGB1 and avidin conjugated to horseradish peroxidase was added and the plates were incubated at room temperature for 2 hours. Afer washing a 3 3 5 5-tetra-methylbenzidine substrate solution was added to each well. Only those wells that contain HMGB1 biotin-conjugated antibody and enzyme-conjugated avidin will exhibit a change in color. Te enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. Te concentration of HMGB1 in the samples is then determined by comparing the optical density of the samples to the standard curve. Statistical analysis All continuous data are expressed as mean ± standard deviation and categorical data are reported as an absolute number or percentage. Frequency distributions were compared using Chi-square test and mean or median values were compared using Student’s t- and Mann- Whitney U-tests. All the tests were to assess the null hypotheses that the patient characteristics and clinical outcomes were not diferent among groups. All calculated P values were 2 sided and P0.05 was considered statistically signifcant. Data were analyzed using SAS/STAT sofware version 9.4 SAS Institute Inc. NC USA. Results Overall 78 patients were eligible for analysis 45 with EOC and 21 with a benign ovary tumor and 12 with normal ovarian tissue. Te median age for patients with EOC was signifcantly higher compared to that among patients without cancer 52 vs. 45 years old respectively P0.018. However the number of menopausal women did not difer between the EOC and non-cancer group. Te clinicopathological characteristics of analyzed patients are shown in Table 1. Te expressions of HMGB1 and its receptor RAGE in EOC were tested by RT-PCR analysis. Te mRNA levels of HMGB1 were higher in EOC compared to the non-cancer cohort 0.94 vs. 0.90 and there was no statistically signifcant diference Figure 2A. Te median value of mRNA HMGB1 levels of cases with normal ovarian tissue 0.90 was estimated as a cut-of value. Similarly there was no statistical signifcance between the EOC group and non-cancer group. Te mRNA levels of RAGE did not difer between the EOC and non-cancer cohort Figure 2B. As the median value of mRNA RAGE levels of cases with normal ovarian tissue 0.01 was estimated as a cut-of value there was no statistical signifcance between EOC group and non-cancer group Table 2. Te serum levels of HMGB1 were evaluated between the groups. No signifcant diferences were found between the EOC and non-cancer group 0.40 vs. 0.45 ng/mL P0.106. Discussion Lotze and Tracey demonstrated that HMGB1 occupied a central role in mediating the local and systemic responses to necrotic cell death and cancer invasion by pathogens trauma and sepsis and it had diferential tissue-specifc activities 7. Furthermore the expression of HMGB1 has been strongly related to tumor growth and invasion. Taguchi et al. showed that HMGB1 induced several intracellular signaling pathways via binding to its receptor RAGE as an extracellular molecular. Te co-localization of RAGE and HMGB1 indicated their potential contribution to cellular migration and tumor invasion and the suppression of tumor growth and metastasis by blocking RAGE- HMGB1 complex in mice had been reported 18. In this study we demonstrated that there was no statistical diference of mRNA levels of HMGB1 between the cancer and the non-cancer group. Although it may be controversial that HMGB1 is regarded as a signifcant factor in the development and progression of cancers one common tumor-promoting mechanism may involve infammation. Infammatory conditions increase the risk of neoplasm and an infammatory component is present also in the microenvironment of tumors that are not related to infammation 19. In the tumor microenvironment infammation could be related with Figure 1. RT-PCR analysis of protein expression in epithelial ovarian cancer A HMGB1 150bp B RAGE 100bp and C β-actin 320bp D-F Densitometry analysis of each band A B C.

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Paek J 2017 Te expression of HMGB1 and the receptor for advanced glycation end products RAGE in epithelial ovarian cancer Integr Cancer Sci Terap 2017 doi: 10.15761/ICST.1000232 Volume 42: 3-4 Cancer n45 Non-cancer n33 P value Number of patients Mean age years SD 51.1 10.72 44.7 11.36 0.018 Menopause 28 62.2 14 42.4 0.083 Performance status 0 16 35.6 1 24 53.3 2 5 11.1 Histology Serous 37 82.2 2 6.1 Mucinous 4 8.9 2 6.1 Clear cell 3 6.7 Undiferentiated 1 2.2 Teratoma 6 18.2 Simple cyst 7 21.2 Endometrioma 4 12.1 Normal ovary 12 36.4 Tumor grade 1 12 26.7 2 12 26.7 3 21 46.6 Lymph node metastases Negative 29 64.4 Positive 16 35.6 Tumor stage I 10 22.2 II 0 III 34 75.6 IV 1 2.2 Table 1. Patient characteristics n78. SD: Standard Deviation Figure 2. Comparison of the mRNA levels of A HMGB1 and B RAGE between the epithelial ovarian cancer and non-cancer group. proliferation and survival of malignant cells angiogenesis metastasis subversion of adaptive immunity reduced response to hormones and chemotherapeutic agents. Recent eforts have shed new light on molecular and cellular pathways linking infammation and cancer 20. For the mRNA analysis used tissues were not sampled from the tumor mass according to a consistent standard able to explain contained tumor percentage. In addition the number of patients in the non- cancer group n33 including benign ovarian tumors n21 was small compared to the EOC group n45. Tis number of patients would not have had the statistical power to defnitely detect a diference in the mean value between the two groups. A recent study showed that HMGB1 could be detected in the serum of cancer patients because it can be passively released from dying tumor cells or actively released from immune cells into the extra-cellular space or serum 17. In the present study we quantifed serum HMGB1 levels in patients with EOC benign ovarian tumors and normal ovarian tissue. However the serum levels of HMGB1 were not signifcantly diferent between the EOC and non-cancer cohort. In addition contrary to expectations the mRNA RAGE levels did not difer signifcantly between the EOC and non-cancer cohort. Although HMGB1 and its receptor RAGE are expected to be elevated in almost all types of cancer recent studies have revealed that this is not always true 21. Especially for ovarian cancer it is recognized that there are at least two diferent molecular pathways that lead to the development of ovarian cancer and that these result in tumors that have quite distinct biological behaviors and probably diferent cells of origin 22. Kurman et al. demonstrated that ovarian cancers were divided into 2 groups type I and type II. Type I tumors are slow growing generally confned to the ovary at diagnosis and develop from well-established precursor lesions that are termed borderline tumors 23. Type I tumors include low- grade micropapillary serous carcinoma mucinous endometrioid and clear cell carcinomas. In contrast type II tumors are rapidly growing highly aggressive neoplasms including high-grade serous carcinoma malignant mixed müllerian tumor and undiferentiated carcinomas. Most ovarian cancers that belong to the type II tumors have a high level of genetic instability 24. Because of these heterologous patterns of ovarian cancer serum CA125 assays and transvaginal ultrasound which are most popularly used as screening modalities for EOC have not been efective in detecting these tumors at the early stage. Similarly the dubitable mRNA or serologic results of HMGB1 in our study can

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Paek J 2017 Te expression of HMGB1 and the receptor for advanced glycation end products RAGE in epithelial ovarian cancer Integr Cancer Sci Terap 2017 doi: 10.15761/ICST.1000232 Volume 42: 4-4 be explained with this characteristic of ovarian cancer. Terefore individual studies of HMGB1 in each type of EOC and knowledge of the pathogenesis of various types of ovarian cancer are needed to have a more accurate understanding of HMGB1-related mechanisms in cancer development and progress. In conclusion the expression of both HMGB1 and RAGE did not show signifcant diferences in EOC group contrary to expectations as a promising biomarker for EOC. Further studies are needed to assess potential roles in EOC. Acknowledgments Tis study was supported by the Brain Korea BK 21 project for medical sciences Yonsei University and by National Research Foundation of Korea Grant funded by the Korean Government 2009- 0071158. References 1. Siegel RL Miller KD Jemal A 2015 Cancer statistics 2015. CA Cancer J Clin 65: 5-29. Crossref 2. Malkasian GD Jr Knapp RC Lavin PT Zurawski VR Jr Podratz KC et al. 1988 Preoperative evaluation of serum CA 125 levels in premenopausal and postmenopausal patients with pelvic masses: discrimination of benign from malignant disease. Am J Obstet Gynecol 159: 341-346. Crossref 3. Jacobs I Bast RC Jr 1989 The CA 125 tumour-associated antigen: a review of the literature. Hum Reprod 4: 1-12. Crossref 4. Wang L Kong B 2015 Analysis of the Association of Matrix Metalloproteinase-1 Gene Promoter rs1799750 Polymorphism and Risk of Ovarian Cancer. Int J Gynecol Cancer 25: 961-967. Crossref 5. Kim JH Skates SJ Uede T Wong KK Schorge JO et al. 2002 Osteopontin as a potential diagnostic biomarker for ovarian cancer. JAMA 287: 1671-1679. Crossref 6. Wilailak S Chan KK Chen CA Nam JH Ochiai K et al. 2015 Distinguishing benign from malignant pelvic mass utilizing an algorithm with HE4 menopausal status and ultrasound fndings. J Gynecol Oncol 26: 46-53. Crossref 7. Lotze MT Tracey KJ 2005 High-mobility group box 1 protein HMGB1: nuclear weapon in the immune arsenal. Nat Rev Immunol 5: 331-342. Crossref 8. Mignogna MD Fedele S Lo Russo L Lo Muzio L Bucci E 2004 Immune activation and chronic infammation as the cause of malignancy in oral lichen planus: is there any evidence Oral oncol 40: 120-130. Crossref 9. Sims GP Rowe DC Rietdijk ST Herbst R Coyle AJ 2010 HMGB1 and RAGE in infammation and cancer. Annu Rev Immunol 28: 367-388. Crossref 10. Schmidt AM Vianna M Gerlach M Brett J Ryan J et al. 1992 Isolation and characterization of binding proteins for advanced glycosylation endproducts from lung tissue which are present on the endothelial cell surface. J Biol Chem 267: 14987- 14997. Crossref 11. Hori O Brett J Slattery T Cao R Zhang J et al. 1995 The receptor for advanced glycation endproducts RAGE is a cellular binding site for amphoterin: mediation of neurite outgrowth and coexpression of RAGE and amphoterin in the developing nervous system. J Biol Chem 270: 25752-25761. Crossref 12. Wautier JL Zoukourian C Chappey O Wautier MP Guillausseau PJ et al. 1996 Receptor-mediated endothelial cell dysfunction in diabetic vasculopathy: soluble receptor for advanced glycation endproducts blocks hyperpermeability. J Clin Invest 97: 238- 243. Crossref 13. Flohr AM Rogalla P Meiboom M Borrmann L Krohn M et al. 2001 Variation of HMGB1 expression in breast cancer. Anticancer Res 21: 3881-3885. Crossref 14. Kuniyasu H Oue N Wakikawa A Shigeishi H Matsutani N et al. 2002 Expression of receptors for advanced glycation end-products RAGE is closely associated with the invasive and metastatic activity of gastric cancer. J Pathol 196: 163-170. Crossref 15. Kuniyasu H Chihara Y Kondo H Ohmori H Ukai R 2003 Amphoterin induction in prostatic stromal cells by androgen deprivation is associated with metastatic prostate cancer. Oncol Rep 10: 1863-1868. Crossref 16. Takada M Hirata K Ajiki T Suzuki Y Kuroda Y 2004 Expression of receptor for advanced glycation end products RAGE and MMP-9 in human pancreatic cancer cells. Hepatogastroenterology 51: 928-930. Crossref 17. Cheng BQ Jia CQ Liu CT Lu XF Zhong N et al. 2008 Serum high mobility group box chromosomal protein 1 is associated with clinicopathologic features in patients with hepatocellular carcinoma. Dig Liver Dis 40: 446-452. Crossref 18. Taguchi A Blood DC del Toro G Canet A Lee DC et al. 2000 Blockade of RAGE- amphoterin signalling suppresses tumour growth and metastases. Nature 405: 354-360. Crossref 19. Colotta F Allavena P Sica A Garlanda C Mantovani A 2009 Cancer-related infammation the seventh hallmark of cancer: links to genetic instability. Carcinogenesis 30: 1073-1081. Crossref 20. Mantovani A Allavena P Sica A Balkwill F 2008 Cancer-related infammation. Nature 454: 436-444. Crossref 21. Bartling B Hofmann HS Weigle B Silber RE Simm A 2005 Down-regulation of the receptor for advanced glycation end-products RAGE supports non-small cell lung carcinoma. Carcinogenesis 26: 293-301. Crossref 22. Kurman RJ Shih I 2008 Pathogenesis of ovarian cancer: lessons from morphology and molecular biology and their clinical implications. Int J Gynecol Pathol 27: 151- 160. Crossref 23. Gershenson DM Sun CC Lu KH Coleman RL Sood AK et al. 2006 Clinical behavior of stage II-IV low-grade serous carcinoma of the ovary. Obstet Gynecol 108: 361-368. Crossref 24. Shih IeM Kurman RJ 2004 Ovarian tumorigenesis: a proposed model based on morphological and molecular genetic analysis. Am J Pathol 164: 1511-1518. Crossref Cancer n45 Non-cancer n33 P value HMGB1 median range 0.94 0.38-1.70 0.90 0-1.46 0.789 ≤ 0.90 22 18 0.653 0.90 23 15 RAGE median range 0.13 0-2.45 0.20 0-1.25 0.636 ≤ 0.01 22 8 0.053 0.01 23 25 Table 2. The expression of mRNA levels of HMGB1 and RAGE among patients with epithelial ovarian cancer and no cancer. Copyright: ©2017 Paek J. This is an open-access article distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited.

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