?(Fig

?(Fig.2a).2a). a specific inhibitor of either PI3K or ERK, and 2DG-ABT was added to activate the mitochondria. The apoptotic rates resulting from 2DG-ABT treatment were higher in the cells treated with the PI3K inhibitor, while the rates remained approximately the same in the cells treated with the ERK inhibitor. In 2DG-ABT-sensitive cells, a 4-h 2DG treatment caused the dissociation of Mcl-1 from Bak, while ABT treatment alone caused the dissociation of Bcl-xL from Bak without substantially reducing Mcl-1 levels. In 2DG-ABT-resistant cells, Mcl-1 dissociated from Bak only when AKT activity was inhibited during the 4-h 2DG treatment. Thus, in VHL-deficient cells, IGF1R activated AKT and stabilized the Bak-Mcl-1 complex, thereby conferring cell resistance to apoptosis. Electronic supplementary material The online version of this article (doi:10.1007/s13277-016-5260-2) contains supplementary material, which is available to authorized users. tests; values for unpaired test varied from 0.3511 to 0.9513). Furthermore, HIF1a expression did not influence the sensitivity of the cells to apoptosis. For example, the sensitivities of RCC4 + VHL cells under hypoxia, and thus expressing HIF1a, and RCC4 + VHL cells under normoxia, and thus not expressing HIF1a, to 2DG-ABT at 10?M ABT-263 were approximately the same (unpaired test test results were *1p?=?0.0052, *2p?=?0.0024, and *3p?=?0.0003. We noted that RCC4 delIGF1R was even more sensitive than RCC4 + VHL, in which there is some IGF1R expression The absence of VHL stabilized IGF1R expression independently of oxygen concentration and interfered with mitochondria-dependent apoptosis We searched the literature and databases for genes regulated by VHL independent of oxygen concentration and found that IGF1R is up-regulated in Sirt5 the absence of VHL, regardless WHI-P258 of the oxygen concentration. Yuen and colleagues found that IGF1R protein levels are unaffected by hypoxia in clear cell renal carcinoma with or without VHL, but exogenously introduced VHL protein reduces both the WHI-P258 promoter activity of IGF1R and the stability of IGF1R mRNA independent of oxygen concentration [11]. We independently verified that IGF1R protein levels decreased when the VHL protein was introduced into UOK121 and RCC4 cells (Fig. ?(Fig.2a).2a). When we depleted IGF1R from RCC4 using siRNA, we observed an increased sensitivity of the cells to 2DG-ABT (Fig. ?(Fig.2c).2c). Furthermore, IGF1R depletion attenuated AKT phosphorylation (Fig. ?(Fig.2b).2b). The application of 1?M picropodophyllin, a specific inhibitor of IGF1R, also attenuated AKT phosphorylation (Fig. ?(Fig.2b).2b). Thus, in the medium, either IGF1 or insulin activates IGF1R, and its signal is transduced to AKT. Furthermore, the treatment of cells with 2DG up-regulates multiple signal transduction pathways [12], as noted in RCC4 cells (Fig. ?(Fig.2b).2b). Zhou and colleagues suggested that 2DG up-regulates IGF1R by directly binding to its inhibitor, IGFBP3 [12]. However, using purified recombinant proteins, Pollak and colleagues showed that the binding between IGF1R and IGFBP3 is not disrupted by 2DG [13]. Thus, the molecular mechanism by which 2DG up-regulates multiple signaling pathways remains unresolved. What is clear from these data is that IGF1R is the major source of pro-survival signals in cultured RCC4 cells. One model explaining how the absence of VHL confers cell resistance to 2DG-ABT is that in the absence of VHL, IGF1R expression is up-regulated in RCC4 and UOK121 cells, thus generating a pro-survival signal through PI3K-AKT and causing these cells to be resistant to 2DG-ABT. In contrast, in RCC4 + VHL, UOK121 + VHL, and UOK121 + 5Aza cells, VHL interferes with IGF1R expression, attenuating the PI3K-AKT pro-survival signal and making these cells sensitive to 2DG-ABT. This possibility is supported by our observation that there were significant differences in the apoptotic rates of RCC4 cells, which WHI-P258 express IGF1R, RCC4 + VHL cells, which express moderate amounts of IGF1R, WHI-P258 and RCC4 cells depleted of IGF1R, which express almost no IGF1R, after treatment with 10?mM 2DG and 10?M ABT-263 (Figs. ?(Figs.11 and ?and22). IGF1R activated both the Ras-ERK and PI3K-AKT pathways, but only the latter pathway interfered with mitochondria-dependent apoptosis IGF1R, like EGFR and many other receptor tyrosine kinases (RTKs), activates both the Ras-ERK proliferation pathway and the PI3K-AKT pro-survival pathway. Activated Ras generates ERK signals through its association with Raf. Activated Ras also directly activates PI3K. Thus, there is cross-talk between these two pathways in the early stages of IGFR-induced signal transduction. However, after these signals reach PI3K and ERK, we can clearly distinguish between the activation of these two pathways. We were thus able to determine which of these two pathways interferes with the apoptotic activation of mitochondria. To address this question, we used PD98509, a specific inhibitor for ERK1/2, and LY294002, a specific inhibitor for PI3K, in UOK121 cells, because in these cells, both Ras-ERK and PI3K-AKT activities are robust and easily.