Expression of CD44V6 in parotid pleomorphic adenoma and carcinoma in pleomorphic adenoma

Free Papers—Poster Presentations Results: Our data demonstrate that siRNA targeting u-PAR markedly reduced cell proliferation, and increased cell apoptosis, and suppressed tumour growth, accompanying with the efficient and specific inhibition of endogenous u-PAR expression in pU group than that in Mock, EV, and pUc groups. Conclusion: These findings suggest that RNA interference targeting u-PAR could effectively inhibit cell proliferation and tumour growth of OSCC in vivo. Thus, it may be used as a potent and specific therapy for oral cancer. doi:10.1016/j.ijom.2009.03.479

P7 Characteristics of cancer stem like side population cell in oral squamous cell carcinoma S. Yanamoto ∗ , G. Kawasaki, S. Yamada, I. Yoshitomi, A. Mizuno Department of Oral and Maxillofacial Surgery, Nagasaki University, Sakamoto, Nagasaki, Japan

Background and Objectives: Recent studies suggest that cancer stem cell may be responsible for tumourigenesis and contribute to some individuals’ resistance to cancer therapy. Some studies demonstrate that side population (SP) cells isolated from diverse cancer cell lines harbour stem cell like properties. However, there are few reports examining the role of SP cells in human oral squamous cell carcinoma (OSCC). Methods: To determine whether OSCCs contain a SP cell fraction, we performed flow cytometry analysis and sorting using SCC25 tongue cancer cell line. Differential expression profile of stem cell markers (ABCG2, Oct-4 and EpCAM) was examined by reverse transcriptasepolymerase chain reaction in either SP cells or non-SP cells. Growth inhibition by 5- fluorouracil (5-FU) was determined by the MTT assay. Clonogenic ability was evaluated by colony formation assay. Results: The SP cell fraction comprised 0.23% of the total cell population, totally disappeared after treatment with the selective ABC transporter inhibitor verapamil. The SP cell fraction showed higher mRNA expression of stem cell markers (ABCG2, Oct-4 and EpCAM) as compared with the non-SP cell fraction. Moreover, the SP cell fraction demonstrated more drug resistance to 5-FU, as compared with the non-SP cell fraction. The clone formation efficiency of SP cells was significantly

higher than non-SP cells at an equal cell number (P < 0.01). Conclusion: We isolated cancer stem like SP cell from oral squamous cell carcinoma cell line SCC25. The SP cell possessed the characteristics of cancer stem cell, and chemo-resistance. Further characterisation of cancer stem like SP cell may provide new insights for novel therapeutic targets. doi:10.1016/j.ijom.2009.03.480

P8 Expression of peroxisome proliferator-activated receptor gamma and cyclooxygenase 2 in oral squamous cell carcinoma S.J. Li 1,∗ , J.D. Cha 1 , H.J. Kim 2 , W. Nam 2 , I.H. Cha 2 1 Oral Cancer Research Institute, College of Dentistry, Yonsei University, Seoul, Korea 2 Department of Oral and Maxillofacial Surgery, College of Dentistry, Yonsei University, Seoul, Korea

Background and Objectives: Peroxisome proliferator-activated receptor gamma (PPAR␥) activators indicate many anticancer effects in vitro that suggest use of a potential PPAR␥ targeting anticancer therapy in several types of cancer. Moreover, cyclooxygenase 2 (COX2) is overexpressed in cancer cells such as colorectal cancer. So we studied PPAR␥ and COX2 expression in oral squamous cell carcinoma (OSCC) tissues and overall and disease-free survival in patients with OSCC. Methods: Surgical tissues of primary oral squamous cell carcinoma from 86 cases were individually performed immunohistochemical staining for PPAR␥ and COX2. The results were scored according to intensity (0: no stained, 1: weakly stained, 2: moderately stained, 3: strongly stained) and extent (0: no stained, 1: 1–25%, 2: 26–50%, 3: > 50%) of staining and confirmed in the immunoreactivity score (IRS). IRS results of PPAR␥ and COX2 were divided into low expression group (LEG 0-3) and high expression group (HEG 4-6). Statistical analysis was performed by using SPSS 13.0 system. Results: PPAR␥ was expressed in the cytoplasm and nuclear OSCC cells and nuclear expression was mainly in differentiated cancer cells. It was highly expressed 74 cases of 86 (86%) in the cytoplasm and 38 cases of 86 (44.2%) in the nuclear of OSCC cancer cells. COX2 was expressed in the cytoplasm of OSCC cells. It was highly expressed 13

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cases of 86 (15.1%). There was a correlation between COX2 expression and tumour stage (P = 0.032) and clinical stage (P = 0.018) of patients with OSCC. There was a correlation between PPAR␥ nuclear expression and tumour stage (p = 0.032) of patients with OSCC. There also has relationship between PPAR␥ and COX2 expression (P = 0.010). However, expression of both two proteins was not related to overall and disease-free survival of patients. Conclusion: The results suggest that PPAR␥ and COX2 were not prognostic factors in OSCC. PPAR␥ expression relates to cell differentiation and apoptosis. PPAR␥ can be a potential therapeutic target gene for OSCC. Overexpression of COX2 may relate to OSCC progression. Moreover, PPAR␥ and COX2 may interact through unknown pathway. Acknowledgement. This work was supported by the Korea Research Foundation Grant funded by the Korean Government (KRF-2008-005-J00803), in which main calculations were performed by using the supercomputing resource of the Korea Institute of Science and Technology Information. doi:10.1016/j.ijom.2009.03.481

P9 Expression of CD44V6 in parotid pleomorphic adenoma and carcinoma in pleomorphic adenoma X.Y. Wang ∗ , S. Yang, H.P. Wang, L.J. Guo, X.F. Tang, Q.H. Gao, M. Xuan Department of Head and Neck Tumour Surgery, West China College of Stomatology, Sichuan University, Chengdu, Sichuan Province, China

Background and Objectives: To investigate the expression differences of CD44v6 in the parotid pleomorphic adenoma (PPA) before and after the recurrence, between the non-recurrent PPA, recurrent PPA, and PPA with canceration so as to identify whether the expression differences have existed before the recurrence and its significance for predicting the recurrence. Methods: The expression differences of CD44v6 were detected by the method of SP (streptavidin-peroxidase) immunohistochemistry in samples of non-recurrent PPA, PPA before and after the recurrence, carcinoma in pleomorphic adenoma (CPA) and normal parotid. Results: The expression of CD44V6 was significantly higher in the group before

534 Free Papers—Poster Presentations the recurrence than in the group after the recurrence (P < 0.05). The expression of CD44v6 was lower in the group after the recurrence than the non-recurrent group, and a significant difference was observed (P < 0.05). The expression of CD44v6 in the group after recurrence was significantly lower than that in the non-recurrent group with a significant difference (P < 0.05). The CD44v6 expression rocketed in the nonrecurrent group, while it decreased in the group with CPA, and a significant difference was noted between the two groups (P < 0.05). And the expression of CD44v6 decreased in both the group after the recurrence and the group with CPA. However, no significant difference was found between them and the decreasing trend in them was consistent. Conclusion: The decrease of CD44v6 expression promoted the recurrence and canceration of PPA, and in turn decreased further in the process. The expression differences of CD44v6 had appeared before the recurrence and had important clinical significance to predict the recurrence and canceration of PPA. doi:10.1016/j.ijom.2009.03.482

P10 Correlation between the expression of thrombospondin-1 and neovascularisation in mucoepidermoid carcinoma S. Yang ∗ , X.Y. Wang, L.J. Guo, X.F. Tang, Q.H. Gao, M. Xuan State Key Laboratory of Oral Diseases, Sichuan University, Chengdu, Sichuan Province, China

Background and Objectives: Researchers have recently demonstrated that thrombospondin-1 (TSP-1) has an important function in regulating neovascularisation. Whether it inhibits or accelerates neovascularisation, however, is still controversial. We found few reports about the correlation between TSP-1 and vascularisation in mucoepidermoid carcinoma. In this research, the distribution and expression of TSP-1 in mucoepidermoid carcinoma were investigated. We also analysed (1) the correlation between the expression of TSP-1 and microvessel density (MVD) as an indicator of neovascularisation activity, and (2) the effect of TSP-1 on neovascularisation and tumour growth in the subcutaneous xenotransplanted model of mucoepidermoid carcinoma. Methods: (1) The sites and intensity of expression of TSP-1 and the MVD were

analysed in 45 cases of mucoepidermoid carcinoma after surgery by the method of streptavidin-peroxidase immunohistochemistry; and (2) recombinant human thrombospondin-1 (rhTSP-1) was injected twice a week for five consecutive weeks around the tumour in the subcutaneous xenotransplanted tumour model of mucoepidermoid carcinoma in nude mice. Each week, the tumour size was measured, in order to draw the growth curve of the xenotransplanted tumour model of mucoepidermoid carcinoma, and MVD was measured. Results: (1) The positive expression of TSP-1 protein was 57.78% (26/45). Most positive staining for TSP-1 was found in the cytoplasm of the cancer cells, while some staining occurred in the extracellular matrix. The mean MVD in 45 cases of mucoepidermoid carcinoma was 58.17 ± 19.77 per 100 visual fields. Tumours with a high expression of TSP-1 showed a low MVD value, and the TSP-1 immunocompetence and microvessel density showed a significant negative correlation (rs = –0.947, P < 0.001). (2) The xenotransplanted tumours with the injection doses of 1.25, 0.75 and 0.25 ␮g/mL respectively were 36.97%, 53.36% and 73.61% of the size of the control group ([451 ± 92], [651 ± 113], [898 ± 86] and [1220 ± 157] mm3 , respectively; F = 53.167, P < 0.001), and their weights were respectively 35.14%, 51.35% and 70.27% of the control group ([1.3 ± 0.5], [1.9 ± 0.5], [2.6 ± 0.3], and [3.7 ± 0.7] g, respectively; F = 62.669, P < 0.001). Their MVDs were 25.00%, 45.93%, and 72.20% respectively of the control group and concentration dependent (15.43 ± 3.45, 28.35 ± 4.24, 44.57 ± 3.35 and 61.73 ± 5.43 per 100 visual fields respectively, F = 54.582, P < 0.001). Conclusions: The TSP-1 has a higher expression in mucoepidermoid carcinoma and the expression has a significant negative correlation with neovascularisation. The TSP-1 inhibits neovascularisation and tumour growth, and it might be a new biological therapy for treatment of patients with mucoepidermoid carcinoma. doi:10.1016/j.ijom.2009.03.483

P11 Reaction oxygen species mediated sulforaphane inducedapoptosis in squamous cell carcinoma-9 human tongue carcinoma cells H. Yao ∗ , X. Gu, G. Wang, J. Wang, H. Wang

Center for Stomatology, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang, China

The present study was undertaken to investigate the oral cancer chemopreventive effects and molecular mechanisms by sulforaphane, which is a naturally isothiocyanate with promising anti-cancer activity. Cell viability and apoptosis assays were done by MTT and annexin V/PI, respectively. Apoptosis related proteins such as caspase-3, 7, 8, 9, Bcl-2, Bax and Bim were determined by Western blot assay. Reaction oxygen species (ROS) was examined by fluorescence microscope and confocal microscope. We demonstrated that sulforaphane significantly induced apoptosis in squamous cell carcinoma (SCC)-9 human tongue carcinoma cells, the apoptosis correlated with the activation of caspase-3, caspase-9. Sulforaphane strongly induced the expression of Bax, Bim, inhibited the expression of BclxL, Bcl-2. ROS mediated the inhibition effect of sulforaphane on SCC-9 cells. Generation of ROS was blocked in the presence of a combined mimetic of superoxide dismutase and catalase. Antioxidant N-acetyl-L-cysteine could significantly inhibit sulforaphane-induced generation of ROS and apoptosis in SCC-9 cells. The activation of NF-kappaB was also associated with inhibition by sulforaphane in SCC-9 cells. In conclusion, the study indicated that sulforaphane was a potent natural dietary compound for chemoprevention of human tongue cancer. ROS played an important role in regulation of sulforaphane-induced apoptosis in SCC-9 human carcinoma cells. doi:10.1016/j.ijom.2009.03.484

P12 Targeted molecular therapy in the murine models of oral squamous cell carcinoma Y.W. Park Department of Oral and Maxillofacial Surgery, Kangnung National University, Gangneung, Gangwon-do, Korea

Background and Objectives: We determined the therapeutic effects of blockade of receptor tyrosine kinases on the growth of oral squamous cell carcinoma (OSCC) xenografted in athymic nude mice. Methods: We investigated the in vivo antitumour effects of a monoclonal antibody for epidermal growth factor receptor (EGFR), IMC-C225 (Erbitux) and a dual inhibitor of the EGFR and vascular endothelial growth factor receptor