| Objective: To study the growth inhibitory effect of quercetin on bladder cancer EJ cell line and to evaluate the role of STAT3 signaling pathway in bladder cancer.Methods: The human bladder cancer EJ cell line was treated with 100μM quercetin, then the cell proliferation and the cell cycle were determined by Trypan Blue counts, HE staining and flow cytometry (FCM). The expression of STAT3 protein in human bladder cancer tissues was detected by paraffin embedded tissue array and the immunohistochemical (IHC). The effect of quercetin and AG490 (JAK2 inhibitor) on STAT3 signaling pathway was evaluated by HE staining, FCM and immunocytochemical (ICC), then the protein expression of STAT3 and PIAS3 (protein inhibitor of activated STAT3) was detected by ICC.Results:1. HE staining results showed that EJ cells didn't change significantly in morphology after being treated with 100μM quercetin for 48h, but the nucleus of EJ cells become round in shape and the cell numbers became fewer and the morphology scattered after being treated with 60μM AG490,2. Trypan Blue assay revealed that after 60μM AG490 treatment for 0, 12, 24, 36, and 48h, the percentages of unviable cells were 0.01%,1.2%,25.63%,29.72%, and 45.55%, respectively. The tesults showed that not qucercein but AG490 inhibited the growth of bladder cancer EJ cell line.3. Flow cytometry analyses demonstrated that the G1, G2 and S fractions were 24.48%, 26.88% and 48.25% in normally cultured EJ cells, and changed to 35.99%, 19.73% and 44.27% in the cells treated with 100μM quercetin for 48h; but changed to 56.46%, 12.86% and 30.68% after 60μM AG490 treatment. The FCM results showed that the EJ cell cycle was blocked at G1 phase after 48h treatment with 60μM AG490.4. Immunohistochemical staining showed that the STAT3 was constitutively expressed in 87 cases human bladder cancer tissue. The positive rate of STAT3 expression was 93.1%. And the negative, weakly positive, positive and strongly positive were 6(6.9%), 19 (21.8%), 23 (26.4%) and 39 cases (44.8%), respectively.5. Immunocytochemistry (ICC) assay showed that the STAT3 expression in EJ cells was strong, but the expression level of STAT3 was decreased after treatment with AG490 compared with normal cells. But both control and quercetin groups showed a similar level in STAT3 protein expression.6 ICC assay showed that the PIAS3 expression in EJ cells was positive in the cytoplasm, but negative in the nucleus. After treated with quercetin, PIAS3 didn't appear the phenomenon of the nuclear translocation, and was mainly expressed in the cytoplasm. But AG490 treatment group showed significant phenomenon of the nuclear translocation, and the expression of PIAS3 was positive in the cytoplasm and nucleus, correspondingly the EJ cell numbers was reduced, and showed apoptotic tendency.Conclusions:1. Qucercein couldn't inhibit the growth of human bladder cancer EJ cell line.2. STAT3 was constitutively expressed in human bladder cancer tissue.3. Qucercein didn't show the inhibitory effect on STAT3 signaling pathway in human EJ cell line.4 AG490 and PIAS3 could inhibit EJ cells proliferation by inhibiting the STAT3 activation. The results indicated that STAT3 signaling pathway play an important role in the tumor proliferation, which suggestted that inhibiting STAT3 signaling pathway maybe was an important drug target to treat human bladder cancer. |
PDF Full Text Request