| Objective: Signal transducer and activator of transcription3(STAT3) isactivated in many kinds of human cancers, which promotes tumorproliferation, apoptosis, invasion and metastasis. Hypoxia-inducible factor-1α(HIF-1α） is a transcription factor which is produced in the hypoxicenvironments, and modulates the transcription of several hypoxic responsegenes. In the study, we investigated the effects of single and combinedinhibition of STAT3and HIF-1α in the enhancement of sensitivity of laryngealsquamous cell carcinoma to radiation therapy and chemotherapy.Methods: Human laryngeal squamous carcinoma Hep-2cells and Hep-2cells transfected with HIF-1α siRNA (Hep-2HIF-1α-/-cells) were cultured in anincubator conditioned at37℃,5%CO2,20%O2and95%humidity. Cells in thelogarithmic growth phase were used in the following experiment.1Hep-2cells and Hep-2HIF-1α-/-cells were cultured in normoxia andhypoxia environments. Western blot was used to observe the expression ofHIF-1α.2Hep-2cells and Hep-2HIF-1α-/-cells were cultured in hypoxiaenvironments, and treated with different concentrations（0μg/ml,30μg/ml,50μg/ml,100μg/ml）of AG490for24hours. MTT assay was used to evaluatethe influence of AG490on the proliferation of Hep-2cells and Hep-2HIF-1α-/-cells.3Cells were treated with50μg/ml AG490for24hours. Western blot wasused to observe the expression of P-STAT3and HIF-1α.4Cells were treated with different radiation dosage（0Gy,5Gy,10Gy,15Gy,20Gy）or different concentrations（0μg/ml,0.5μg/ml,1.0μg/ml,2.0μg/ml,5.0μg/ml）of cisplatin together with or without50μg/ml AG490. Cell proliferation was examined using MTT assay at the time of24hours aftertreatment.Results:1AG490induced significant proliferation inhibition on Hep-2cells andHep-2HIF-1α-/-cells in a dose-dependent manner. Randomized block designANOVA analysis showed that the proliferation rate of each concentrationgroup varied significantly difference from the control group (F=572.884,P<0.01); the proliferation rate of Hep-2HIF-1α-/-group revealed a significantdifference compared to that of Hep-2group (F=160.114，P<0.01). Moreover,at the same concentration, AG490had a stronger effect of inhibition onHep-2HIF-1α-/-cells than on Hep-2cells.2The result of Western blot show that AG490(50μg/ml) can inhibit theexpression of P-STAT3obviously both in Hep-2cells (t=6.0927, P<0.01) andin Hep-2HIF-1α-/-cells (t=8.6536, P<0.01). However, the differences ofexpression of P-STAT3in Hep-2cells and Hep-2HIF-1α-/-cells are not ofstatistical significance (t=2.1421, P>0.05). HIF-1α protein levels in Hep-2cells treated with AG490are lower than that without AG490(t=3.5626,P<0.05).3The MTT results of the proliferation of cells intervened withchemotherapy or radiotherapy in the presence or absence of AG490:(1)Radiotherapy can inhibit the proliferation of Hep-2cells and Hep-2HIF-1α-/-cells.The inhibition rates between Hep-2cells and Hep-2HIF-1α-/-cells are verydifferent in that radiotherapy combined with AG490(50μg/ml) had a strongerproliferation inhibition effects than radiotherapy alone.(2) Cisplatin caneffectively inhibit the proliferation of Hep-2cells and Hep-2HIF-1α-/-cells underthe hypoxic condition in a dose-dependent manner（F=10.646, P<0.05）. Theinhibition rates between Hep-2cells and Hep-2HIF-1α-/-cells are different（F=402.043，P<0.01）. When Hep-2cells were treated withAG490(50μg/ml)combined with cisplatin (0.5μg/ml), the q value was less than1.15. However,it was more than1.15in other experimental groups. These findings suggestthat there is a cooperative effect between50μg/ml AG490and0.5μg/ml cisplatin on the proliferation inhibition of Hep-2cells, but a synergistic effectin cells treated with50μg/ml AG490and1.0μg/ml cisplatin,2.0μg/ml cisplatin,and5.0μg/ml cisplatin.Conclusions:1Suppressing expression of STAT3and/or HIF-1α can inhibit theproliferation of laryngeal carcinoma cells.2Combined inhibition of STAT3and HIF-1α can enhance radio-and chemo-sensitivity in laryngeal squamous carcinoma cells under hypixia. |
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