| Objective: To study the expressions of Stat3, p-Stat3, CyclinD1 and Survivin in cervical cancer cell lines. The correlation between p-Stat3, CyclinD1 and Survivin were also observed. After treated by AG490 (a selective inhibitor of JAK2), we studied the relationship between the cervical cancer cell life cycle and the expressions of JAK2, Stat3, p-Stat3, CyclinD1 and Survivin protein. The potential mechanism of Stat3 signal transduction pathway in controlling the cancer cell transformation from G1 to S stage, proliferation and apoptosis was analysed to search for new biologic marker for diagnosis and therapy in human cervical cancer at early process. Finaly, the regulating mechanism of drug to occurre and develop cervical cancer by blocking Stat3 signal pathway was tried to explain.Methods: 1.Hela cells were cultured in vitro. 2. Western-blot technique was used for detecting the protein expressions of Stat3, p-Stat3, and their downstream target gene product CyclinD1 and Survivin in human cervical cancer cell lines. 3. Hela cells were treated with different AG490 as experiment group, and the serum-free medium was used as control group. The blank control group did not plant cells at all. 4. Cell proliferation was detected by MTT assay. 5. Flow cytometry was applied to analyze the cell cycle and apoptosis. 6. The expressions of phosphorylation-JAK2 (JAK2), phosphorylation-Stat3 (p-Stat3), Stat3, CyclinD1 and Survivin were measured by Western blot after different AG490 treating. The correlation between p-Stat3, CyclinD1 and Survivin were also investigated.Results: 1. Constitutively activated Stat3 signal existed in human cervical cancer cell lines. 2. Western Blotting showed that the vary tendency of CyclinD1 and Survivin expressions was similar to that of p-Stat3. 3. Proliferation of Hela cells decreased by concentration-depended manner after treatment with AG490. Detected at different doses after AG490 treating for 48h, the inhibition rate increased when AG490 concentration raised from 25 to 100μmol/L (P < 0.05). Three doses of AG490 treated expenriment groups have statistic meaning compared with control group(P < 0.01). Detected inhibition rate at different time after treatment with AG490 showed that some inhibition effects were found after 24h. The effect became obvious after treatment about 72h and cell survival rate decreased constantly (P < 0.05). 4. Stat3 signal transduction pathway contributes to the change of Hela cell from G1 phase to S phase. It was found that the cell proportion of G0/G1 phase increased from (65.37±1.18)% to (74.67±3.13)% (P < 0.05), and S phase decreased from (22.54±0.78)% to (17.45±1.45)% (P < 0.05). AG490 blocked Hela cell life cycle by concentration-dependent manner. 5. AG490 promoted the apoptosis of Hela cells. AG490 was used to treat Hela cells for 24h, and the percentage of apoptotic cells raised from (1.51±0.03)% to (8.43±2.57)%(P < 0.05). Detected apoptosis at 25μmol/L,50μmol/L,100μmol/L after AG490 treating 48h, the apoptosis rate was determined as (13.58±1.42)%, (23.44±1.55)%, (31.05±2.09)% respectively. Comparing the difference between control group and experiment groups have statistical significance (P < 0.05). Moreover, apoptosis rate differece of Hela cells with various concentration AG490 treating is significant (P < 0.05), which indicate AG490 induce Hela cells apoptosis with a concentration-dependent manner. 6. The expressions of JAK2 and p-Stat3 declined significantly (P < 0.05), but Stat3 had no obvious change after different concentration AG490 treating. 7. The expressions of CyclinD1 and Survivin decreased in Hela cells after AG490 treatment. Pearson's correlation analysis was used to analyse the correlation and variability of expressions of p-Stat3, CyclinD1 and Survivin in cervical cancer cell line. The expressions of p-Stat3 and CyclinD1 exhibit linear correlation (r=0.901, P < 0.01). Moreover, p-Stat3 and Survivin have the similar linear correlation (r=0.844, P < 0.01).Conclusion: 1. The study of protein level confirmed that constitutive activation of Stat3 signal pathway exists in Hela cells. 2. AG490 can block Stat3 signal pathway selectively. 3. Blocking Stat3 pathway by AG490 in Hela cells, the cell cycle were changed, percentage of G0/G1 phase increased and S phase declined. Cell proliferation was also inhibited. The intrinsic mechanism is that AG490 downregulate the expression of CyclinD1. 4. AG490 inhibits Hela cells proliferation and promotes cell apoptosis through declining the expression of Survivin. 5. As targeted gene product, CyclinD1 and Survivin participate in the regulation of cell cycle, proliferation and apoptosis. 6. Stat3 signal pathway could be a novel target of cervical cancer therapy. 7. p-Stat3 can be a biologic marker for diagnosis and therapy in human cervical cancer at early process. |
