Relation Between WIF-1 Promoter Methylation And Transitional Cell Carcinoma Of Bladder

Wnt passway widely involves the development of organisms and tumorigenesis. Aberrant activation of Wnt passway has been found in various cancers including tumors of colon, lung, head and neck, melanoma and leukemia. Present researches commonly were focused on the changes of downstream signals of Wnt passway, such as mutation ofβ-catenin,APC,GSK-3β,CTNNB1, axin and so on . Relatively Fewer studies had been done to illuminate the relation between tumor behaviors and upstream signals including Wnt proteins and their inhibitors. Recent studies showed that colon cancer is related with the abnormal changes of upstream signals of this passway. Wnt protein expression increases in bladder urothelium during tumorigenesis, which means it functions on the development of bladder cancer. Previous studies indicated that the genetic mutations of downstream signal molecules, such as APC andβ-catenin, were less common in bladder cancer. Hence, we hypothesized that abnormity of upstream signals may activate the Wnt passway in bladder cancer. Wnt inhibitory factor-1 can block the signal transduction by direct binding to Wnt protein. Immunohistochemistry revealed the down-regulation of WIF-1 in bladder cancer. However the underlying mechanism of down-regulation of WIF-1 and its relative biological influences on bladder cancer remain unclear.1. Objectives:(1) To detect the expression of Wnt genes in bladder cancer.(2) To test whether promoter methylation of WIF-1 inhibits the gene expression and to study the biological effect of WIF-1's re-expression on bladder cancer cells.(3) To explore the relation between WIF-1 re-expression and the cytotoxicity of hydroxycamptothecin, and to explore the potential machinery of synergism of 5-aza-2′-deoxycytidine and HCPT by analyzing their combination effect on bladder cancer cells.(4) To explore the relation between WIF-1 promoter methylation and clinical pathology of bladder cancer for the purpose of demonstrating if WIF-1 can be used as a molecular marker for the diagnosis and prognosis prediction of bladder cancer.2. Methods:(1) RT-PCR was used to determine the expression of Wnt1, Wnt5a, Wnt10b and Wnt13 mRNA in bladder cancer cell lines BIU87 and T24; Immunohistochemistry was performed on normal bladder mucosa and bladder cancer tissues to determine the Wnt1 protein expression.(2) RT-PCR, immunocytochemistry and Western-blot analysis were applied to assess the expression pattern of WIF-1 on bladder cancer cell line BIU87 and T24.(3) Demethylating agent 5-aza-2′-dexoycytidine was used to treat bladder cancer cell line BIU87 and T24 in vitro. Methylation-specific polymerase chain reaction (MSP) assay was performed to detect the change of methylation status of WIF-1 gene in bladder cancer cells after the 5-aza-2′-dexoycytidine treatment.(4) Methyl thiazolyl tetrazolium assay (MTT) was used to evaluate the cytotoxicity, and flow cytometry (AnnexinⅤ/PI staining) and terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL) were employed to detect apoptosis of bladder cancer cells after 5-aza-2′-deoxycytidine treatment in vitro.(5) The cytotoxicity of HCPT in combination with 5-aza-dc on bladder cancer cell lines and primary cultured bladder cancer cells was assessed by MTT assay. Isobologram analysis was used to determine if combination effect was synergistic, or additive or antagonistic.(6) FCM and TUNEL were performed to detect the apoptosis of bladder cancer cell lines and primary cultured bladder cancer cells after treatment of HCPT in combination with 5-aza-dc. Then RT-PCR was performed to assess the expression of WIF-1.(7) MSP was applied to evaluate the methylation status of WIF-1 gene of different bladder tissues, including bladder cancer, normal bladder mucosa and glandular cystitis. WIF-1 protein expressions in the tissues were examined by immunohistochemistry. Statistical analysis was performed using SPSS12.0 software.3. Results:(1) Wnt expression pattern of BIU87 and T24 cell was not identical: no Wnt1 expression in both cells, but Wnt13 expressed in both cells; Wnt5a was only seen in BIU87. Wnt1 did not express in bladder cancer tissues and normal bladder mucosa.(2) WIF-1 did not expressed in bladder cancer cell line BIU87 and T24, but re-expressed after 5-aza-dc treatment in both cell lines.(3) 5-aza-dc showed significant effects of growth inhibition and apoptosis-induction on bladder cancer cells compared with control group (p<0.05).(4) HCPT did not interfere with the expression of WIF-1 mRNA when combined with 5-aza-dc in vitro, and HCPT also did not recover the expression of WIF-1.(5) Dose-dependent cytotoxicity of 5-aza-dc and/or HCPT was observed on bladder cancer cells. 5-aza-dc enhanced synergistically the cytotoxicity of HCPT on bladder cancer cell which was related to WIF-1 re-expression.(6) WIF-1 methylation rates increased with the increase of bladder cancer grade (20.0% in G1, 56.3% in G2,and 86.7% G3) and stage (superficial and invasive) (p<0.05). Total 5-year survival of patients with WIF-1 gene promoter methylation was significantly lower that of patients without WIF-1 gene promoter methylation (60.6% versus 91.7%, p<0.05).(7) Positive WIF-1 protein expression did not keep pace with the methylation status of WIF-1, which was lower than the expected percentage. Total expression rate of WIF-1protein expression was observed in 30.1% of patients while 92.3% in normal mucosa and 85% in glandular cystitis. The difference between neoplasia and non-neoplasia was statistically significant (p<0.01).4. Conclusions: (1) DNA methylation was a potential mechanism for silencing the expression of WIF-1 gene in bladder cancer.(2) WIF-1 was an important anti-oncogene for bladder cancer which showed significant inhibitory effect on bladder cancer. WIF-1 re-expression may potentially involve the synergism of 5-aza-dc and HCPT on bladder cancer cells.(3) The methylation status of WIF-1 gene was significantly associated with bladder cancer grade and stage, and long-term survival of patients. Therefore WIF-1 gene promoter methylation can be used as a good molecular marker for the diagnosis and prognosis prediction for bladder cancer.