The standard deviations are represented as error bars from three independent experiments

2DG uptake in the SC cells, with or without insulin. Similar findings were observed in the KD cells under hypoxia. However, under hypoxia, less 2DG was incorporated into the KD cells than in the SC cells, with or without the additional insulin treatment. To assess the mechanism of insulin-dependent glucose uptake, the membranous expression of GLUT1 was analyzed in the KD cells under normoxia and hypoxia. Under normoxia, the membranous GLUT1 expression was elevated with high glucose and/or insulin treatment in comparison to that with no treatment. Compared to that observed under normoxia, under hypoxia, the membranous GLUT1 expression was elevated in all treatments. 10 / 18 HIF-1 Inhibition plus GI Treatment for Gastric Cancer Fig 5. The examination of glucose uptake in SC and KD cells with or without insulin treatment., The 2DG uptake level in SC cells or KD cells with or without insulin treatment was evaluated under normoxia and hypoxia. The Western blot analysis of membranous GLUT1 expression in the KD cells with control, high glucose and/or insulin treatments under both normoxia and hypoxia as indicated.Furthermore, the expression was increased by high glucose and/or insulin treatment, compared with that by no treatment. In particular, the membranous GLUT1 expression was most strongly increased by high glucose and insulin treatment in the hypoxic KD cells. On the other hand, the expression of another GLUT family, GLUT3, was faintly observed in the KD cells, and this finding was not altered among these various treatments. In this study, GLUT2 and GLUT4 were not expressed in the KD cells. HIF-1 knockdown plus GI treatment strongly suppressed the growth of tumor xenografts in nude mice Finally, we determined the in vivo effect of the GI treatment on KD and SC tumor xenografts. Fig 6A demonstrates the experimental design of the xenograft murine model. Ten days after the subcutaneous inoculation of SC or KD cells, xenografts were grown on the backs of nude mice. At this point, a Western blot analysis confirmed the HIF-1 expression in the SC tumors, but not in the KD tumors. Thereafter, three drugs, consisting of PBS, glucose or GI, were intraperitoneally injected into nude mice bearing an SC or KD tumor. The representative images of the tumor-bearing mice that were treated with PBS, glucose or GI are shown in Fig 6C. The KD-Glucose and KD-GI tumors appeared to be smaller than the other tumors. Fig 6D showed the growth curve of the 6 tumors. The sizes of the KD-Glucose and KD-GI tumors were significantly smaller than the KD-PBS tumor on day 12. The KD-GI tumor was the smallest. On the other hand, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19737141 in the SC mice, there was no significant difference in size of the SC-PBS, SC-Glucose and SC-GI tumors. An immunohistochemical analysis of cleaved caspase 3 was performed to assess the apoptosis induced by glucose or GI treatment. The positive expression of cleaved caspase3 was frequently observed in the KD-Glucose and KD-GI tumors. However, all of the KD tumors exhibited some degree of cleaved caspase 3. In contrast, there was no significant difference in expression of the cleaved caspase 3 among the SC-PBS, SC-Glucose and SC-GI tumors. In the KD tumors, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19737141 the positive expression of cleaved caspase3 was significantly higher in the KD-PBS tumor than in the 212141-51-0 site SC-PBS tumor. Moreover, the expression of cleaved caspase3 was significantly higher in the KD-Glucose or KD-GI tumors than in the KD-PBS tumor. The highest expression was observed in the KD-GI tu

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