| Objective:This study was designed to explore the anti-tumor characteristics and the possible molecular mechanisms of GLA in human bladder cancer cells.Methods:1.To study the inhibitory effect of GLA on the proliferation of human bladder cancer cells1.1 The proliferation of human bladder cancer cells and the growth curve were examined by Cell Counting Kit-8(CCK-8).1.2 Colony formation assay was used to assess whether GLA significantly suppressed the colony formation in UMUC3 cells.2.To explore the anti-tumor mechanism of GLA on UMUC3 cells2.1 Flow cytometry(FCM)was used to detect the cell cycle progression of UMUC3 cells after GLA treatment.2.2 Annexin V-FITC/PI assay was used to detect the apoptosis of UMUC3 cells induced by GLA.2.3 Western blotting analysis was performed to detect the related proteins of cell cycle,apoptosis and the PI3K/Akt pathway in order to verify whether GLA induced G2/M cycle arrest and apoptosis in UMUC3 cells through the PI3K/Akt pathway.2.4 Immunofluorescence staining was further performed to confirm the cellular localization and expression of p-Akt and Cleaved Caspase-3.3.To evaluate the anti-tumor effect of GLA in a mouse model3.1 We established the UMUC3 tumor xenograft NOD/SCID mice model to evaluate the anti-tumor effect of GLA in vivo.3.2 In subcutaneous tumor tissues,cell proliferation and microvessel density were evaluated through Ki-67 and CD31 by immunohistochemistry.Results:1.GLA inhibits cell proliferation and colony formation in human bladder cancer cells.1.1 Inhibition was observed in a time-dependent inhibition manner following treatment with 10 ?M GLA in bladder cancer cell lines UMUC3,HT1197,T24 and J82.But in the SV-HUC-1 cells,no statistical change was observed after each treatment.GLA inhibited the viability of UMUC3 cells in a time-and dose-dependent manner.The IC50 values of GLA on UMUC3 cells at 24,48,72 and 96 h were 18.87,9.77,6.75,and 5.40 ?M,respectively.1.2 GLA significantly suppressed colony formation with a decreased number and size of cell colonies in a dose-dependent manner compared with the control group.2.GLA triggers cell cycle arrest at the G2/M phase in UMUC3 cells.2.1 After 24 hours,we observed that with higher concentration,more cells stayed in G2/M phase rather than G0/G1 and no significant difference in S phase,which indicates GLA seems to cause a G2/M phase arrest in UMUC3 cells.2.2 GLA upregulated the expression level of p21Waf1/Cip1 but downregulated Cyclin B1,CDK1 and Cdc25C.These results above indicate that GLA triggered G2/M phase arrest of UMUC3 cells in a dose-dependent manner.3.GLA induces mitochondrial-mediated apoptosis of UMUC3 cells.3.1 UMUC3 cells were treated with different concentrations of GLA(0,5,10,and 20?M)for 24 h and were analyzed by flow cytometry.The apoptotic rates in GLA groups were(10.16 ± 1.10)%,(29.99 ± 8.55)%and(53.61 ± 5.11)%of the total cells respectively,compared with only(4.37 ± 1.17)%in control group.3.2 Western blotting analysis demonstrated that GLA increased Bax levels but decreased Bcl-2 levels as compared to control cells,leading to upregulation of the Bax/Bcl-2 ratio in a dose-dependent manner.Furthermore,the levels of Caspase-9,Caspase-3 and PARP decreased following GLA treatment with a corresponding significant accumulation of Cleaved Caspase-9,Cleaved Caspase-3 and Cleaved PARP.3.3 Meanwhile,immunofluorescence staining indicated that both cytoplasmic and nuclear expressions of Cleaved Caspase-3 were significantly enhanced after treatment with GLA.4.GLA inhibits the PI3K/Akt pathway in UMUC3 cells.4.1 GLA significantly decreased the levels of PI3K p85 subunit and the phosphorylation of Akt at Ser473 site,while the levels of PTEN in a dose-dependent manner.There was no significant difference in the expression of total Akt.4.2 Furthermore,decreased expressions of p-Akt(Ser473)were confirmed both in the cytoplasm and nuclear by immunofluorescence staining.5.GLA exhibits the anti-tumor effect on UMUC3 cells in vivo.5.1 To evaluate the anti-tumor effect of GLA in vivo,we established the UMUC3 tumor xenograft NOD/SCID mice model.The representative change of tumor size in mice treated with GLA(20 mg/kg)was significantly lower than that of vehicle group.Compared with the vehicle group,the tumor volume grew slowly in GLA group.5.2 The average tumor weight was(3.73 ± 0.68)g for the control group and(1.71 ±0.42)g for GLA(20 mg/kg)group.5.3 In the histological level,cell proliferation and microvessel density were evaluated through Ki-67 and CD31,respectively.GLA markedly suppressed the expressions of Ki67 and CD31 in subcutaneous tumor tissue.Conclusion:1.GLA inhibits the proliferation of human bladder cancer cell lines in a time-and dose-dependent manner.2.GLA triggers cell cycle arrest at G2/M phase through Cdc25C/Cyclin B1/CDK1 pathway.3.GLA induces apoptosis in UMUC3 cells via the mitochondrial-mediated pathway.4.Inhibition of the PI3K/Akt pathway is involved in GLA-induced apoptosis and G2/M arrest.5.GLA may be developed into a promising therapeutic agent against bladder cancer by targeting the PI3K/Akt pathway. |
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