Srinagarind Med J 2011: 26 (Suppl)

Wnt Expression Profile in Human Cholangiocarcinoma

Pornpan Bungkanjana1, 2, Anchalee Techasen1, 2, Nisana Namwat1, 2, Puangrat Yongvanit1, 2, Watcharin Loilome1, 2 1Department of Biochemistry, 2Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand E-mail: [email protected]

Keywords: Cholangiocarcinoma, Wnt

Introduction Objective The Wnt molecules are a family of cysteine-rich The present study aimed to determine the secreted glycoproteins that act as ligands to activate expression of four human Wnt including Wnt1, a -mediated signaling pathway through the Wnt2, Wnt8b and Wnt10b which have been reported (Fz)/low density lipoprotein (LDL) receptor-related to be associated with human cancers in human CCA protein (LRP). Activation of Wnt pathway leads to tissues and CCA cell lines. the accumulation of cytoplasmic β-catenin, which then enters the nucleus where β-catenin converts Materials and methods Tissue samples the TCF/LEF complex from transcriptional repression CCA tissues were collected from CCA patients to a transcriptional activating complex, modulating admitted at surgical ward of Srinagarind Hospital, the expression of several genes involved in a variety Khon Kaen University. Normal livers from accidentally of biological functions including cell proliferation, 1 dead persons were donated in agreement of their differentiation, migration and apoptosis . In humans, families. The protocol of collection and study are the Wnt family is composed of 19 structurally related approved by Ethic Committee for Human Research, molecules. The members exhibit unique expression Khon Kaen University. patterns and distinct functions in development2. Accumulated evidence has demonstrated Cell lines a significant role of Wnt/β-catenin pathway in the Human CCA cell lines including KKU-M055, development and progression of human malignancies1. KKU-M156, KKU-M213 and KKU-M214 were There are many studies have shown aberrant expression established from CCA patients of Srinagarind Hospital, of Wnt molecules in different types of cancer such as Khon Kaen University. All cell lines were cultured in lung cancer3, colorectal cancer4, and gastric cancer5, 6, Ham’s F12 medium containing 100 U/ml penicillin and respectively. Recently, our group had identified the 100 µg/ml streptomycin with 10% fetal bovine serum at activated protein kinase profiles by using Human 37°C under and atmosphere of 5% CO2. phospho- RTKs and phospho- kinases array methods in both CCA cell lines and human CCA tissues. We found RNA extraction and quantitative RT-PCR that β-catenin, a downstream signaling molecule in Wnt/ Total RNA was isolated from liver tissues (approximately Proceeding β-catenin signaling pathway is prominently activated 100 mg) or cell lines following the manufacturer’s protocol in both CCA cell lines and CCA tissues. Thus, a better (Invitrogen, Carlsbad, CA). Reverse transcription understanding the molecular mechanism by which reaction consists of 5 µg total RNA and random this particular pathway involved in CCA development, hexamer (0.5 µg) which were mixed together and then o we sought to investigate the expression profile of Wnt incubated at 70 C for 5 min. After incubation, reaction genes in human CCA tissues and human CCA cell lines. buffer containing 5x RT buffer, 20U RNase inhibitors,

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10mM dNTP mix, 200 units reverse transcriptase Results and discussions (Promega), RNase free water was added in to RNA. In this study, expression levels of four Wnt Reverse transcription was carried using a DNA thermal transcripts including Wnt1, Wnt2, Wnt8b, and Wnt10b cycler (GeneAmp PCR system 2400, Perkin-Elmer were investigated in adjacent non-tumor and tumor Applied Biosystems). The thermal conditions were tissue of 48 CCA patients and nine healthy controls. 25 °C for 10 min, 42 °C for 1 hr, and 70°C for 10 min. The relative Wnt mRNAs expression determined using ® Real time PCR was performed using Taqman Gene real-time RT-PCR is shown in Figure 1. There was no expression assay kit and the ABI 7500 real time PCR differential expression of Wnt1 among the studied system (Applied Biosystems, Foster City, CA). Each 20 groups, whereas the expression of Wnt2 was significantly µl/reaction contained 10 µl of 2X Taqmanmaster mix, increased in adjacent non-tumor (P<0.0001) and tumor 1 µl of Taqman probe and 9 µl of 1:6 diluted cDNA. tissues (P=0.0105) when compared with cadaveric o The PCR cycling conditions are 50°C for 2 min, 95 C donor. The Wnt8b mRNA level in the tumor was significantly o o for 10 min, then 50 cycles of 95 C for 15 sec and 60 C increased compared to adjacent tissues (P=0.0038). for 1 min. PCR reactions were performed in the relative Furthermore, the expression of Wnt10b in adjacent quantification of Wnt genes expression was done using tissues was significantly increased when compared the comparative cycle threshold (CT) method. GAPDH with liver tissues from a cadaveric donor (P=0.0275). expression was used as the endogenous control. All In addition, Wnt1, Wnt2, Wnt8b and Wnt10b mRNA data are analyzed using 7500 system SDS software. could also be detected in four CCA cell lines, although in difference level as shown in Figure 2. The increased Statistical analysis expression of Wnts mRNA in human CCA tissues and Statistical analyses are performed using SPSS cell lines observed in the present study indicated the version 15.0 software. Difference expression between involvement of Wnt/b-catenin signaling pathway in CCA groups was examined using Mann-Whitney U-test development. Therefore, the details of the molecular and Wilcoxon signed rank test. A P-value <0.05 was mechanism by which Wnt/b-catenin signaling pathway considered statistically significant. are involved in CCA need to be further elucidated. Proceeding Figure 1 Wnts mRNA expression in cadaveric donor, adjacent non-tumor tissue, and CCA tissue. GAPDH was use as an internal control. * P<0.05, ** P<0.001.

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Figure 2 Wnts mRNA expression in human CCA cell lines

Conclusion 2. Maiese K, Li F, Chong ZZ, Shang YC. The Wnt signaling In the present study, we found up-regulation pathway: aging gracefully as a protectionist? Pharmacol of Wnt2, Wnt8b, and Wnt10b mRNA in human CCA Ther 2008; 118:58-81. tissues. Moreover, four CCA cell lines showed the 3. Huang CL, Liu D, Ishikawa S, et al. Wnt1 overexpression different levels of Wnt mRNA expression. Further study promotes tumour progression in non-small cell lung is required to elucidate the significant role of this protein cancer. Eur J Cancer 2008; 44:2680-8. family in CCA development. 4. Ma XR, Edmund Sim UH, Pauline B, Patricia L, Rahman J. Overexpression of WNT2 and TSG101 genes in Acknowledgement colorectal carcinoma. Trop Biomed 2008; 25:46-57. This work is supported by the Thailand Research 5. Saitoh T, Mine T, Katoh M. Up-regulation of WNT8B Fund (Grant No. MRG5480034) to WL, PB is supported mRNA in human gastric cancer. Int J Oncol 2002; by the Research Strengthening Grant from BIOTEC- 20:343-8. NSTDA, Thailand. 6. Saitoh T, Kirikoshi H, Mine T, Katoh M. Proto-oncogene WNT10B is up-regulated by tumor necrosis factor alpha References in human gastric cancer cell line MKN45. Int J Oncol 1. Logan CY, Nusse R. The in 2001; 19:1187-92. development and disease. Annu Rev Cell Dev Biol 2004; 20:781-810. Proceeding

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