| ObjectiveOur previous studies have found that there were certain correlations between the expression of GLTU-1and the radioresistance of laryngeal carcinoma Hep-2cells. However,the real mechanism of radioresistance of laryngeal carcinoma is unclear, it may be the results of interactions of multiple factors. Abnormal expression of GLUT-1and its activity are regulated by many factors, including oncogenes,hypoxia in an HIF-1-dependent/independent way, signaling pathways such as MAP kinase, and the PI3K/Akt pathway. Recently, the PI3K/Akt pathway was reported to play a key role in control of GLUT-1trafficking and activity. The PI3K-Akt pathway was also implicated in the regulation of GLUT-1localization in T cells. The PI3K/Akt pathway itself is frequently overactive in a variety of tumor types and triggers a cascade of responses, from cell growth and proliferation to cell survival and motility, which drive tumor progression. Activation of the PI3K/Akt pathway may be associated with radioresistance of cancer.Thus, clinical researchers have devoted increasing.The PI3K/Akt signaling pathway was activated by growth factors and receptors. The activation of PI3K/Akt signal pathway could promote cell growth, survival and differentiation. In some malignant tumors, such as pancreatic cancer, head and neck cancers, lung cancer and breast cancer,it was found that the activation of PI3K/Akt signaling pathway was associated with the proliferation of tumor cells, and its activation was closely related to the radiosensitivity. The possible mechanism of radiation resistance caused by PI3K/Akt are hypoxia,intrinsic radiation resistance and external factors including tumor cell proliferation after radiotherapy. However there is no report about inhibition GLUT-1expression and PI3K/Aktl pathway concurrently to enhance the radiosensitivity of laryngeal carcinoma. We first propose a hypothesis that targeting GLUT-1and PI3K/Aktl pathway may improve the radiosensitivity of laryngeal carcinoma. To confirm above hypothesis,we first investigate whether GLUT-1over-expression may induce the radioresistance of laryngeal carcinoma Hep-2cells,and wether PI3K and p-Akt protein involve in GLUT-1-mediated radioresistance in the present study. Next,we testify wether inhibition of GLUT-1expression could enhance the radiosensitivity of laryngeal carcinoma Hep-2cells.Methods1. Design and Transfection of GLUT-1-siRNA: GLUT-1SiRNA was transiently transfected into Hep-2cells to deplete GLUT-1expression. Screening and identification of transfection was determined by real-time reverse transcription polymerase chain reaction (RT2-PCR). Transfection efficiency was determined by Western blotting.2. Clonogenic radiation survival assay:Colonies consisting of>50cells were counted. The surviving fraction at each groip was calculated following the equation:number of colonies/(number of cells seeded).The surviving fraction at each group point was determined in triplicate and the results are shown as means±SD.3. Real-time reverse transcription polymerase chain reaction Transient transfected six groups Hep-2cells:ck, mock, NC,803-SiRNA,1382-SiRNA,1563-SiRNA.Thirty experimental groups:CK(control check),NC(negative control), siRNA(803-siRNA),50μm apigenin,10μm LY294002,5μm Wortmannin,50μm apigenin+siRNA,10μm LY294002+siRNA,5μm Wortmannin+siRNA,4Gy X-ray,6Gy X-ray,8Gy X-ray,4Gy X-ray+50μm apigenin,6Gy X-ray+50μm apigenin,8Gy X-ray+50μm apigenin,4Gy X-ray+10μm LY294002,6Gy X-ray+10μm LY294002,8Gy X-ray+10μm LY294002,4Gy X-ray+5μm Wortmannin,6Gy X-ray+5μm Wortmannin,8Gy X-ray+5μm Wortmannin,4Gy X-ray+50μm apigenin+siRNA,6Gy X-ray+50μm apigenin+siRNA,8Gy X-ray+50μmResultl.The optimal transfection efficiency of SiRNAGLUT-1was siRNA803determined by RT-PCR and Western blotting. 2.Compared with CK,cell clone formation rate (%) in different X-ray radiation was lower alongwith increasing dosage of X-ray (p<0.05). Apigenin, LY294002and wortmannin synergied with SiRNAGLUT-1may enhance the sensitivity of laryngeal carcinoma Hep-2cells (p<0.05).3.SiRNAGLUT-1could Inhibit GLUT-1mRNA expression,apigenin could synergy with SiRNAGLUT-1to enhance the inhibition (p<0.05). After4Gy,6Gy and8Gy X-ray,apigenin could obviously inhibit GLUT-1mRNA expression of laryngeal carcinoma Hep-2cells (p<0.05),apigenin and LY294002could synergy with SiRNAGLUT-1to reduce GLUT-1mRNA expression of laryngeal carcinoma Hep-2cells (p<0.05). No matter in the control group or X-ray groups, wortmannin could’t reduce GLUT-1mRNA expression of laryngeal carcinoma Hep-2cells (p<0.05),in the case of X-ray irradiation,wortmannin could’t synergy with SiRNAGLUT-1to reduce GLUT-1mRNA expression of laryngeal carcinoma Hep-2cells (p>0.05).4.(1)The effect of Various treatments of laryngocarcinoma on the GLUT-1protein expression of Hep-2cells:SiRNA,50μM apigenin,5μM wortmannin and10μM LY294002worked separately on laryngeal carcinoma Hep-2cells, the GLUT-1protein expression had no differences compared with the control group. While50μM apigenin and10μM LY294002separately synergied with SiRNAGLUT-1,the GLUT-1protein expression declined significantly compared with the control group (p value were0.047,0.047),but when5μM wortmannin synergied with SiRNAGLUT-1,the GLUT-1protein expression had no differences compared with the control group(p>0.05).After4Gy,6Gy and8Gy irradiation respectively,the GLUT-1protein expression had no differences compared with the control group (p>0.05). After4Gy,6Gy and8Gy irradiation respectively,the GLUT-1protein expression had no differences between groups (p>0.05).After4Gy,6Gy and8Gy irradiation respectively,50μM apigenin,5μM wortmannin and10μM LY294002separately or synergied separately with SiRNAGLUT-1(respectively compared with4Gy,6Gy and8Gy irradiation groups),there had no obvious inhibitory effects on the expression of GLUT-1(p>0.05).(2)The effect of Various treatments of laryngocarcinoma on the PI3K protein expression of Hep-2cells:SiRNAGLUT-1could Inhibit PI3K protein expression compared with the control group (p=0.012).50μM apigenin synergied with SiRNAGLUT-1could significantly inhibit the PI3K protein expression compared with the control group (p=0.026). While the PI3K protein expression of other groups had no differences compared with the control group (p>0.05).In4Gy X-ray groups,while10μM LY294002worked on laryngeal carcinoma Hep-2cells, the PI3K protein expression had differences compared with the control group (p=0.007),but other groups had not.After6Gy irradiation,50μM apigenin,5μM wortmannin and10μM LY294002separately or synergied separately with SiRNAGLUT-1(respectively compared with6Gy irradiation group),there had no significant inhibition on the PI3K protein expression (p>0.05)(3)The effect of Various treatments of laryngocarcinoma on the p-Akt protein expression of Hep-2cells:while5μM wortmannin could synergy with SiRNAGLUT-1to reduce p-Akt protein expression of laryngeal carcinoma Hep-2cells compared with the control group (p=0.038),but other groups had not (p>0.05)After4Gy,6Gy and8Gy irradiation respectively,50μM apigenin,5μM wortmannin and10μM LY294002separately or synergied separately with SiRNAGLUT-1(respectively compared with4Gy,6Gy and8Gy irradiation groups),there had no obvious inhibitory effects on the p-Akt protein expression (p>0.05).ConclusionApigenin,LY294002and wortmannin synergied with SiRNAGLUT-1may enhance the radiosensitivity of laryngeal carcinoma Hep-2cells. The possible mechanism may be through inhibition of GLUT-1mRNA expression of laryngeal carcinoma Hep-2cells. |
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