Yu et al. Journal of Experimental & Clinical Cancer Research 2010, 29:119 http://www.jeccr.com/content/29/1/119 Page 2 of 9 Table 1 Clinical information of patient samples analyzed Epithelial-mesenchymal transition (EMT) has emerged as a critical process during cancer progression in which downregulation or loss of E-cadherin expression (epithe- lial marker) constitute a molecular hallmark [4,5]. The transcriptional factors Snail and Slug (zinc finger proteins) have been described to be direct repressors of E-cadherin [6-11] in vitro and in vivo through an inter- action of their COOH-terminal region with a 5′- CACCTG-3′ sequence in the E-cadherin promoter [12]. Both have been suggested to be involved in the acquisi- tion of resistance to apoptosis, thereby promoting tumor survival. Recently, it has been postulated that Twist, another promoter repressor of CDH1 (E-cadherin gene), may be involved in tumor progression by silencing E- cadherin expression and EMT induction [13,14]. Twist is considered as a promoter of the EMT, which is a key event in the tumoral invasion step. Up-regulation of Twist is associated with malignant transformation of melanoma and T-cell lymphoma [13]. It is possibly involved in E-cadherin conversion during EMT [14]. Studies in other cancers have shown that overexpression of Snail and Slug leads to a reduction of E-cadherin expression. An overexpression of Twist resulted in an a further decrease of E-cadherin expression [15]. Because Snail, Twist and Slug are potential regulators of cell adhesion and migration, this study aimed to determine the levels of expression of Snail, Slug, and Twist in human bladdert cancer tissues and to elucidate whether these levels are clinically significant. Also, to clarify whether the three factors may be used as a novel parameter to predict prognosis in bladder carcinoma. Materials and methods Patients and paraffin-embedded tissue sample The study included 120 patients with a primary bladder tumor and 42 background tissue(paracarcinoma tissue, more than 1.5-2 cm from cancer tissue). The tissues were obtained from patients who had undergone a transurethral resection or a partial/total cystectomy between 1999 and 2002 at the Urology Department, The affiliated hospital of Qingdao medical college, Qingdao university, China. None of the patients had received pre- operative treatment. All patients were classified accord- ing to the 1997 UICC TNM classification for the stage and OMS 2004 for the grade (LMP: low malignant potential; LG: low grade; HG: high grade). Immunos- taining was evaluated by 2 independent pathologists to validate the diagnosis. Each sample was used after writ- ten consent was obtained from the patients. The clinico- pathological data of the tumors are shown in Table 1. Tumor recurrence/progression was defined based on clinical, radiological, or histological diagnoses. The study was approved by the affiliated hospital of Qingdao Variable Background Tumor Tissue type Age - yr (mean) < 70 ≥70 Gender - number of patients Male Female Grade - no. of patients LG HG Stage - number of patients (%) NMIBC: Ta T1 MIBC: T2N0M0 T3 N0M0 T4/Any T N+/M+ Surgical procedure TUR Cystectomy Recurrence Number of patients with NMIBC Progression: Number of patients with NMIBC Number of patients with MIBC Survival Number of patients with MIBC Cancer-specific Overall survival Alive Deceased Alive Deceased n (%) 42 120 64 (53) 56 (47) 87 (72) 33 (28) 41 (34) 79 (66) 31 (26) 45 (37) 23 (19) 19 (16) 2 (1.6) 76(63) 44(37) 23(19) 8 (6.6) 15 (12.5) 27 (22.5) 17(14) 25 (21) 19 (16) medical college Faculty of Medicine Human Investiga- tion Committee. Immunohistochemistry Immunohistochemical staining was done on paraffin- embedded tissue, which had described in detail before [16]. Briefly, three-micrometer-thick sections were cut, using a rotation microtom. The sections were deparaffi- nized in xylene and rehydrated in graded alcohols and distilled water. After antigen retrieval with 0.01% EDTA (pH 8.0), endogenous peroxidase activity was blocked with 1% hydrogen peroxide in distilled water for 25 min followed by washing with distilled water and finally PBS + 0.1% Tween for 5 min. To bind nonspecific antigens, the sections were incubated with 1× Power Block