The Effects Of STAT3 Signaling Pathway On The Origin And Development Of Bladder Tumor

Objective: To investigate the expression of signal transducer and activator of transcription 3(STAT3) in different human urothelium tissues and explore the correlation between expression of STAT3 and carcinomatous change of urothelial cell. To explore the action of STAT3 signaling pathway in the regulation of cell proliferation, apoptosis, invasion and metastasis of bladder cancer. To evaluate the role of STAT3 signaling pathway in the origin and development of bladder cancer. Methods: (1) The expression of STAT3 in 66 cases of bladder transitional cell carcinoma(BTCC), 22 cases of papilloma of bladder, 100 cases of squamous metaplasia of the bladder, 40 cases of cystitis glandularis and 20 cases of normal bladder mucosa specimens were studied by immunohistochemistry method, and the results were analyzed statistically. (2) The bladder cancer cell line BIU-87 or T24 was treated by AG490 or decoy oligodeoxynucleotides targeting activated STAT3 that can lead to blockage of STAT3 signaling pathway. The cell vitality and proliferation were detected by Trypan Blue staining rejection, cell counting and MTT assay. Drug sensitivity of bladder cancer stem cells to AG490 was detected by clone formation counting assay. Fluorescence dyestuff Hoechst33258 and PI double-staining was used to investigate the cell apoptosis characteristics. Flow cytometry was applied to analyze the cell cycle and apoptosis. The expressions of phosphorylation-specific JAK2(p-JAK2), STAT3, phosphorylation-specific STAT3(p-STAT3), Cyclin D1 and Bcl-xL were measured by western blot. The DNA-binding activity of STAT3 was detected by EMSA. The mRNA expression of Cyclin D1 and Bcl-xL were measured by RT-PCR. (3) The expression of phosphorylation-specific STAT3(p-STAT3) and VEGF were examined in 66 cases of bladder cancer and 22 cases of papilloma ofbladder specimens by immunohistochemistry method, and microvessel density(MVD) was compared according to different combined expression of p-STAT3 and VEGF in bladder cancer. The results were analyzed statistically. The bladder cancer cell line BIU-87 or T24 was treated by AG490 or decoy oligodeoxynucleotides targeting activated STAT3 that led to blockage of STAT3 signaling pathway. The cell invasiveness and metastasis potency were detected by bladder cancer invasion models in vitro and in vivo. The microvessel density of transplanted neoplasm was detected by immunohistochemistry assay. The expression of phosphorylation-specific JAK2(p-JAK2), STAT3 and phosphorylation-specific STAT3(p-STAT3) were measured by Western blot. The DNA-binding activity of STAT3 was detected by EMSA. The expression of MMP-2, TIMP-2 and VEGF mRNA were measured by RT-PCR.Results: (1) The positive rate of STAT3 staining increased with the ascended malignancy of pathology type and differentiation grade. (2) AG490 could remarkably inhibit the proliferation of bladder cancer BIU-87 cells and the formation of the tumor stem cell clones, block the cell cycle, facilitate the apoptosis of bladder cancer cells, and result in less expression of p-JAK2, STAT3, p-STAT3, Cyclin D1 and Bcl-xL. STAT3 decoy oligodeoxynucleotides remarkably inhibited the proliferation of bladder cancer T24 cells by 75.0%, decreased the cell number of S phase from 29.83% to 16.26%, increased the cell number of G0/G1 phase from 50.82% to 71.20%, facilitated the cell apoptosis of bladder cancer with 50.75% of apoptosis rate, decreased the DNA-binding activity of STAT3, and resulted in less mRNA expression of Cyclin D1 and Bcl-xL. (3) In 66 cases of bladder cancer, 69.7% (46/66) was positive for p-STAT3 expression, and 54.5% (36/66) was positive for VEGF, bigger than control group(27.3% and 18.2% for p-STAT3 and VEGF expression respectively). The differences for both p-STAT3 and VEGF expression between bladder cancer group and control group were all significant (P<0.01) . There was a significant correlation between p-STAT3 and VEGF expression. MVD in bladder cancer was significantly greater when p-STAT3 and VEGF were both positivecompared with other groups(P<0.01). The activation of STAT3 signaling pathway was suppressed by AG490 remarkably. AG490 significantly inhibited invasion and metastasis of bladder cancer BIU-87 cells, and resulted in less expression of MMP-2 and VEGF mRNA. The expression of TIMP-2 mRNA was up-regulated. STAT3 decoy oligodeoxynucleotides blocked the activation of STAT3 signaling pathway by inhibiting the DNA-binding of activated STAT3 protein, remarkably inhibited the invasion and metastasis of bladder cancer T24 cells, attenuated mRNA expression of MMP-2 and VEGF, and increased mRNA expression of TIMP-2. Conclusion: (1) The expression of STAT3 is related to the pathology type and differentiation grade of urothelium tissues. The over-expression of STAT3 may be the early exhibition of carcinomatous change of urothelial cell and be related to malignant transform of urothelial cell. (2) STAT3 signaling pathway plays important role in regulation of cell proliferation and apoptosis of bladder cancer. AG490 and decoy oligodeoxynucleotides targeting activated STAT3 could block constitutive activation of STAT3, inhibit the proliferation of bladder cancer cells and induce their apoptosis. (3) STAT3 signaling pathway plays important role in regulation of cell invasion and metastasis of bladder cancer. The expression of phosphorylation-specific STAT3 protein is related to infiltration of bladder cancer. The over-expression of p-STAT3 protein might promote angiogenesis through pathway of VEGF, and thus induce malignant progression of bladder tumor. The cell invasion and metastasis of bladder cancer could be inhibited through blocking STAT3 signaling pathway by AG490 and decoy oligodeoxynucleotides targeting activated STAT3. (4) The abnormally activated STAT3 signaling pathway might be the key regulation pathway for the origin and development of bladder cancer. The drug AG490 and gene therapy decoy method targeting activated STAT3 have offered new valid strategy for treatment of bladder cancer.