| Objective: Signal transducer and activator of transcription 3(Stat3) signal pathway as a hot spot is constantly activated and abnormally modulated in great quantities of human tumors. It can influence the proliferation, apoptosis, infilitration and metastasis of cancer cell. In this experimentation, Hep-2 cells were treated with Stat3 antisense oligonucleotides and DDP in hypoxic condition which is similar to the solid tumor in vivo. We detected the apoptosis, cell cycle and the expression of p-Stat3, HIF-1α(hypoxia-inducible factor-1α), VEGF (vascular endothelial growth factor) protein. We approach the relationship between activation of Stat3 signal pathway and hypoxic condition; analyze the molecular mechanism of chemotherapy sensitization of hypoxia cell transfected by Stat3 AS-ON. This will be helpful for the development of new anticancer drugs and chemotherapy ancillary drugs with high performance and low toxicity.Methods:1 The culture environment: the culture environment of hypoxia group is 37℃, 5%CO2, 2%O2, 93% N2, and saturated humidity. The environment of normoxia control group is 37℃, 5%CO2, 20%O2, 75%N2 and saturated humidity. Hep-2 cells were divided into five groups as hypoxia 3h, 6h, 12h, 24h, and normoxia group. Expression of p-Stat3 (activated state of Stat3), HIF-1α(active subunit of hypoxia-inducible factor-1), VEGF were detected by flow cytometry.2 The Hep-2 cells in both normoxia and hypoxia groups were treated with DDP at the concentrations including 0μg/ml, 1μg/ml, 3μg/ml and 5μg/ml for 6h. Then we detected the cell apoptosis rate by flow cytometry.3 Stat3 antisense oligonucleotides (Stat3 AS-ON) and Stat3 sense oligonucleotides (Stat3 S-ON) packaged with lipid body were transfected into Hep-2 cells. Group treated with lipid body was taken as non-transfection control. The Hep-2 cells of both transfection group and control group were treated with different concentration (100nmol/L, 200nmol/L and 400nmol/L) of Stat3 AS-ON or Stat3 S-ON. Then we detected the survival rate at 24h by MTT.4 The Hep-2 cells were transfected by Stat3 AS-ON of 200nmol/L for 24h, and then treated with DDP of 3μg/ml in hypoxic environment for 6h. Finally we detected the expression of p-Stat3, HIF-1α, VEGF, cell cycle and apoptosis by flow cytometry.Results:1 The expressions of p-Stat3, HIF-1αand VEGF in Hep-2 cells were up-regulated in hypoxic environment. In respective calculation, the FI of p-Stat3 at 3h, 6h, 12h and 24h group in hypoxia is 1.04±0.03, 1.12±0.03, 1.16±0.05 and 1.12±0.05. The FI of HIF-1αis 0.91±0.321, 1.87±0.83, 3.91±0.67 and 5.04±0.51. The FI of VEGF is 1.06±0.14, 1.08±0.14, 1.71±0.26 and 2.20±0.75. The expression of HIF-1αis positive correlated to p-Stat3 (r=0.589 p<0.05). Furthermore the up-regulation of p-Stat3 expression is earlier than that of HIF-1α. These results indicate that p-Stat3 modulate the expression of HIF-1α.2 The apoptosis increase with increasing doses (0~5μg/ml) of DDP, either under hypoxia or normoxia. The apoptosis of Hep-2 cells treated with 1μg/ml in hypoxia have no statistics discrepancy vs. control group. Hep-2 cells in hypoxia group treated with DDP of 3μg/ml or 5μg/ml showed lower apoptosis rate than that of normoxia group. Hypoxia reduced the sensitivity of chemotherapeutics.3 Green fluorescence could be seen in all Stat3 oligonucleotide groups under fluorescence microscope. Lipid body-oligonucleotide compound was transfected into cytoplasm 30min after transfection. Fluorescence can be visualized in the cell nuclease with about 80~90% of all the cells showing strong fluorescence in the cytoplasm 1h after transfection and above 90% after 2h. The survival rate of Hep-2 cell decreased with increasing dose (100nmol/L~400nmol/L) of Stat3 AS-ON transfection for 24h detected by MTT (p<0.01). The survival rates of Hep-2 cells treated with Stat3 S-ON of 100nmol/L or 200nmol/L did not change obviously but that of 400nmol/L, which inhibited cell hyperplasia (p<0.01).4 The expressions of p-Stat3, HIF-1αand VEGF proteins decrease after transfected by 200nmol/L Stat3 AS-ON for 24h. The degressions have significant difference vs. control group (p<0.01).5 The cell cycle of Hep-2 cells is blocked at G0/G1 after treated with Stat3 AS-ON (200nmol/L) for 24h and DDP (3μg/ml) in hypoxic environment for 6h, the percentage of S and G2/M degrade, the FI of p-Stat3, HIF-1α, VEGF down-regulated. The percentage of G0/G1 also increased after transfected by Stat3 AS-ON cultured under hypoxia for 6h.6 After treated with Stat3 AS-ON (200nmol/L) for 24h and DDP (3μg/ml) under hypoxia for 6h, the expression of p-Stat3 HIF-1αand VEGF protein decreased obviously, the apoptosis increased vs. control group (p<0.01). Pearson correlation analysis showed positive correlation between p-Stat3 and HIF-1α(r=0.884, p<0.01), between HIF-1αand VEGF (r=0.667, p<0.01), between p-Stat3 and VEGF (r=0.783, p<0.01), and negative correlation between p-Stat3 and apoptosis (r=0.-0.909, p<0.01).Conclusion:1 Study in vitro shows that the expression of p-stat3 protein increase as hypoxia continues for a short time. Hypoxia active Stat3 signal pathway in Hep-2 laryngeal carcinoma cells. We also confirmed that the expression of HIF-1αis later than Stat3 signaling. This indicates that Stat3 plays an important part in HIF-1αactivation.2 In the hypoxic environment similar to the solid tumor in vivo, Stat3 AS-ON could suppress activation of Stat3 and the expression of HIF-1αand VEGF significantly. This certificate that p-stat3 is absolutely necessary for the hypoxia-induced expression of HIF-1α. The expression of VEGF is reduced without activated Stat3.3 Transfection of Stat3 AS-ON can repress the cytoactive of Hep-2 cells. Hep-2 cell survival rates decrease because of the induced cell apoptosis. However Stat3 AS-ON transfection can't completely suppress the tumor cell multiplication and its induction of the apoptosis of tumor cells is also limited. High concentration of Stat3 S-ON can suppress the Hep-2 cells. Above all, we demonstrate that effects of lower cytotoxicity and highest specificity of Stat AS-ON are limited. We could not enhance the tumoricidal effect of Stat AS-ON by simply up-regulating the concentration of AS-ON randomly.4 The apoptosis induced by DDP under hypoxia is lower than that under normoxia. The cell cycle of Hep-2 cells cultured in hypoxic environment was blocked in G0/G1, which occurred obviously after transfected by Stat AS-ON in our study. Apoptosis rate of Hep-2 cells transfected by Stat3 AS-ON is up-regulated in hypoxic condition without increasing the dose of DDP. Combined application of Stat AS-ON and DDP can induce tumor cell apoptosis significantly; increase the chemotherapy sensitivity of tumor cell. The effect of chemo-sensitization may be a result of Stat3 pathway blockage and down-regulation of downstream anti-apoptosis genes such as Bcl-xl, Mcl-1 and survivin. This result indicates that Stat3 AS-ON interference targeting Stat3 could be a new method of gene therapy for laryngeal carcinoma, which provides a new way to increase the sensitization of chemotherapy in clinical.
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