Yu et al. Journal of Experimental & Clinical Cancer Research 2010, 29:119 http://www.jeccr.com/content/29/1/119 Page 4 of 9 /recurrence or date of last follow-up. The curves were done using the Kaplan-Meier method with the log-rank test to assess the statistical significance. Cox propor- tional hazards analysis was used to determine the rela- tive contribution of various factors to the risk of death, recurrence, and progression. P < 0.05 was considered as statistically significant. Analyses were performed with SPSS 10.00 software (SPSS, Chicago, IL). Results Expression of Snail, Slug, Twist and E-cadherin in human bladder cancer cell lines The expression of Snail, Slug, Twist and E-cadherin was analyzed at the mRNA and protein level by semiquanti- tative RT-PCR(Fig. 1A) and western blot (Fig. 1B) in the human bladder cancer cell lines T24, HTB-3, HTB-1, HTB-2 and HTB-9. Slug was expressed with different intensities in all five cancer cell lines. The undifferen- tiated HTB-1 and T24 cells had a strong mRNA and protein expression of Slug, whereas the other 3 cell lines showed only weak expression levels. Twist mRNA and protein was detected in HTB-1 and T24 cells, no appearant Twist mRNA and protein expression was found in other 3 cell lines. E-cadherin was detected in HTB-2, HTB-9 and HTB-3 cell lines. The most undiffer- entiated cell line HTB-1 and T24 cells showed no E- cadherin expression. Snail was not detectable in all five cancer cell lines. To verify intact RNA and protein, b- actin was used as a positive control. Expression of Snail, Slug, and Twist in correlation with E- cadherin in human bladder cancer tissue Slug(A), Twist(B, F), Snail (Fig. 2C and 2G) in primary bladder cancer tissue were identified in the cytoplasm as well as in the nucleus of cancer cells. In general, stain- ing for Slug and Twist was more intense than for Snail. E-cadherin expression in cancer cells was characterized by patterns with variable degrees of cytoplasmatic and membrane staining (Fig. 2D). Figure 1 Expression of Snail, Slug and Twist in five bladder cancer cell lines T24, HTB-1, HTB-2, HTB-3 and HTB-9. The analysis of the relative mRNA and protein intensity of Slug, Snail and Twist compared with E-cadherin showed that bladder cancer cells with a high Slug and Twist expression had no or only low E- cadherin expression. In contrast, cells with low Slug and Twist expression had high expression levels of E-cadherin. Figure 2 Expression of Slug, Twist, Snail and E-cadherin in human bladder cancer and bankground tissue was determined by immunohistochemistry. Staining of Snail(A), Slug(B), and Twist (C) was found in the cytoplasm as well as in the nucleus of tumor cells. Magnification, ×200. E-cadherin (D)expression was identified in the cell membrane and intensive in the cytoplasm. Magnification, ×200. No expression of Slug in bankground tissue(E), strong of Twist and Snail expression in bankground tissue (F-G). Immunohistochemistry showed that 44.2% (53/120) of human bladder carcinoma tissues and 38%(16/42) back- ground tissue(G) expressed Twist(P = 0.156);62.5%(75/ 120) of human bladder Carcinoma tissues and 40%(17/ 42) background tissue(Fig. 2E) expressed Slug(P = 0.044); 15.8% (19/120) of human bladder carcinoma tis- sues and 76%(32/42) background tissue(Fig. 2F) expressed Snail(P = 0.016) and 25.8% (31/120) cases were low for E-cadherin expression in carcinoma tissues (Table 2). More patients with high Slug and Twist expression displayed low E-cadherin expression. Statisti- cally significant correlations were found between Twist, Slug, and E-cadherin expression. No statistically signifi- cant correlations were found between Snail and E-cad- herin expression(Table 3). Correlation between Snail, Slug, Twist and E-cadherin and clinicopathological parameters There was a significant correlation between Twist over- expression and the tumor stage (P = 0.000)and grade (P = 0.000): superficial BT (Ta-1) (19 out of 76: 25%)