The oncometabolite 2-hydroxyglutarate inhibits microglial activation via the AMPK/mTOR/NF-κB pathway.

Microglia, the brain-resident macrophage, is known as the innate immune cell type in the central nervous system. Microglia is also the major cellular component of tumor mass of gliomas that plays a key role in glioma development. Mutations of isocitrate dehydrogenases 1 and 2 (IDH1/2) frequently occur in gliomas, which leads to accumulation of oncometabolic product 2-hydroxyglutarate (2HG). Moreover, IDH1/2 mutations were found to correlate with better prognosis in glioma patients. In the present study, we investigated the effects of the 2HG on microglial inflammatory activation. We showed that the conditioned media (CM) from GL261 glioma cells stimulated the activation of BV-2 microglia cells, evidenced by markedly increased expression of interleukin-6 (IL-6), IL-1ß, tumor necrosis factor-α (TNF-α), CCL2 (C-C motif chemokine ligand 2) and CXCL10 (C-X-C motif chemokine 10). CM-induced expression of proinflammatory genes was significantly suppressed by pretreatment with a synthetic cell-permeable 2HG (1 mM) or a nuclear factor-κB (NF-κB) inhibitor BAY11-7082 (10 µM). In lipopolysaccharide (LPS)- or TNF-α-stimulated BV-2 microglia cells and primary microglia, pretreatment with 2HG (0.25-1 mM) dose-dependently suppressed the expression of proinflammatory genes. We further demonstrated that 2HG significantly suppressed LPS-induced phosphorylation of IκB kinase α/ß (IKKα/ß), IκBα and p65, IκB degradation, and nuclear translocation of p65 subunit of NF-κB, as well as NF-κB transcriptional activity. Similarly, ectopic expression of mutant isocitrate dehydrogenase 1 (IDH1) (R132H) significantly decreased TNF-α-induced activation of NF-κB signaling pathway. Finally, we revealed that activation of adenosine 5'-monophosphate-activated protein kinase (AMPK) and subsequent inhibition of mammalian target of rapamycin (mTOR) signaling contributed to the inhibitory effect of 2HG on NF-κB signaling pathway in BV-2 cells. Taken together, these results, for the first time, show that oncometabolite 2HG inhibits microglial activation through affecting AMPK/mTOR/NF-κB signaling pathway and provide evidence that oncometabolite 2HG may regulate glioma development via modulating microglial activation in tumor microenvironment.

[Growth inhibition and mechanisms of human bladder cancer T24 cells by adenovirus-mediated ING4 gene in vitro].

OBJECTIVE: To explore the inhibitory effect and anti-cancer mechanisms of adenovirus-mediated ING4 gene on the human bladder cancer T24 cells in vitro. METHODS: The methods of reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the expression of ING4 in human bladder urothelial carcinoma T24 line. The influence of Ad-ING4 transfection on cell proliferation was evaluated by MTT assay and cell apoptosis by Hochest33258 staining and flow cytometry. RT-PCR and Western blot were performed to detect the transcriptional level of such apoptosis-related genes as Bcl-2, Bax, p53, HIF-1α and caspase-3. RESULTS: Human ING4 was successfully transcribed in T24 cell. Apoptosis rate of T24 cell in Ad-ING4 group was significant higher than that in control groups (17.2% ± 4.1% vs 4.7% ± 1.2% and 4.8% ± 1.2%, P < 0.05). Ad-ING4 not only up-regulated the expression of p53 and Bax(1.40 ± 0.11 vs 0.27 ± 0.04, 1.50 ± 0.12 vs 0.60 ± 0.05) and down-regulated the expression of Bcl-2 and HIF-1α (0.19 ± 0.02 vs 1.20 ± 0.08, 0.33 ± 0.03 vs 0.98 ± 0.06, all P < 0.05), but also enhanced the caspase-3 activation and eventually led to apoptosis. CONCLUSION: Adenovirus-mediated ING4 gene exhibits anti-tumor capacity in T24 human bladder cancer cell and induces in vitro apoptosis. It may be related to the up-regulation of P53 and Bax/Bcl-2, and the down-regulation of HIF-1α. Thus the caspase-3 activation is enhanced so as to lead to apoptosis.

[In vitro and in vivo inhibitory effect of Ad-ING4 gene on proliferation of human prostate cancer PC-3 cells].

BACKGROUND AND OBJECTIVE: Adenovirus vector has been widely used in tumor gene therapy. ING4 is a member of growth inhibiting factors and a potent anti-tumor gene which could induce apoptosis of many tumor cells. This study was to investigate the inhibitory effects of adenovirus-mediated ING4 (Ad-ING4) gene on the proliferation of human prostate cancer PC-3 cells in vitro and in vivo, and to explore its mechanisms. METHODS: Ad-ING4 was obtained by virus-amplification technique. After transfection of purified Ad-ING4 into PC-3 cells, the expression of ING4 was detected by reverse transcription-polymerase chain reaction(RT-PCR); the influence of Ad-ING4 transfection on cell proliferation was evaluated using MTT assay. Cell apoptosis was assessed using Hoechst33258 staining and flow cytometry. RT-PCR was performed to detect the mRNA levels of the transcription of apoptosis-related genes such as bcl-2, bax, p53, and caspase-3. Athymic nude mice bearing PC-3 tumors were intratumorally injected with Ad-ING4 (100 microL, 1x10(9) pfu/mL). Tumor growth was recorded. All nude mice were killed at the end of the experiment to observe the growth of xenografts. The expressions of Bcl-2, Bax, Caspase-3, and CD34 proteins in tumor tissues were detected by immunohistochemistry. RESULTS: Human ING4 gene was successfully transcribed in PC-3 cells and induced apoptosis by up-regulating p53, bax, caspase-3 expression and down-regulating bcl-2 expression. Inhibition of cell proliferation was significant in PC-3 cells. Tumor growth was significantly inhibited in the Ad-ING4 group as compared with that in the Ad-GFP group and the PBS group (P<0.05). The weight inhibitory rate was 37.0% in the Ad-ING4 group. The expressions of Bax and Caspase-3 were up-regulated, and the expressions of Bcl-2 and CD34 were down-regulated in the Ad-GFP group. CONCLUSIONS: Adenovirus-mediated ING4 gene exhibits anti-tumor ability in human prostate cancer PC-3 cells in vitro and in vivo, and induces apoptosis. This may be related to the up-regulations of p53, bax, Caspase-3 and down-regulation of bcl-2.