Kappler Matthias, Pabst Ulrike, Rot Swetlana, Taubert Helge, Wichmann Henri, Schubert Johannes, Bache Matthias, Weinholdt Claus, Immel Uta-Dorothee, Grosse Ivo, Vordermark Dirk, Eckert Alexander W
Department of Oral and Maxillofacial Plastic Surgery, Martin Luther University Halle-Wittenberg, Ernst-Grube-Str. 40, 06097, Halle(S), Germany.
Department of Urology, University Hospital Erlangen, Friedrich-Alexander-University of Erlangen-Nürnberg, Erlangen, Germany.
Clin Oral Investig. 2017 Jan;21(1):211-224. doi: 10.1007/s00784-016-1780-9. Epub 2016 Mar 9.
The stabilization of the transcription factor and prognostic tumor marker hypoxia-inducible factor 1α (HIF1α) is considered to be crucial for cellular metabolic adaptations to hypoxia. However, HIF1α has also been shown to accumulate under normoxic conditions, although this phenomenon is poorly understood.
We investigated the conditions for normoxic HIF1α stabilization in different tumor cell lines (e.g., two mammary carcinoma cell lines and three oral squamous cell carcinoma cell lines) via Western blot analysis or immunohistochemical staining. The transcriptional activity of HIF1 was demonstrated by analyzing the messenger RNA (mRNA) expression of the HIF1 target carbonic anhydrase 9 (CA9) via PCR.
Our data demonstrate that the combined incubation of tumor cells with glutamine and growth factors (e.g., EGF, insulin, and serum) mediates the normoxic accumulation of HIF1α in vitro. Consequently, the inhibition of glutaminolysis by a glutaminase inhibitor blocked the normoxic accumulation of HIF1α. Additionally, the normoxic HIF1α protein displayed nuclear translocation and transcriptional activity, which was confirmed by the induction of CA9 mRNA expression. Furthermore, the normoxic accumulation of HIF1α was associated with impaired proliferation of tumor cells. Finally, ammonia, the toxic waste product of glutaminolysis, induced a normoxic accumulation of HIF1α to the same extent as glutamine.
Our study suggests that HIF1α is involved in the regulation of glutamine metabolism and the cellular levels of the toxic metabolic waste product ammonia under normoxia. Hence, our results, together with data presented in the literature, support the hypothesis that HIF1α and its target genes play a crucial role in metabolic pathways, such as glutaminolysis and glycolysis, under both hypoxic and normoxic conditions.
Therefore, the inhibition of HIF1α (and/or HIF1α target genes) could emerge as a promising therapeutic approach that would result in the accumulation of toxic metabolic waste products in tumor cells as well as the reduction of their nutrition and energy supply.
转录因子及预后肿瘤标志物缺氧诱导因子1α（HIF1α）的稳定被认为对于细胞代谢适应缺氧至关重要。然而，HIF1α在常氧条件下也会积累，尽管对此现象了解甚少。
我们通过蛋白质免疫印迹分析或免疫组织化学染色，研究了不同肿瘤细胞系（如两种乳腺癌细胞系和三种口腔鳞状细胞癌细胞系）中常氧下HIF1α稳定的条件。通过PCR分析HIF1靶标碳酸酐酶9（CA9）的信使核糖核酸（mRNA）表达，证明了HIF1的转录活性。
我们的数据表明，肿瘤细胞与谷氨酰胺和生长因子（如表皮生长因子、胰岛素和血清）共同孵育可在体外介导HIF1α的常氧积累。因此，谷氨酰胺酶抑制剂对谷氨酰胺分解的抑制作用可阻断HIF1α的常氧积累。此外，常氧下的HIF1α蛋白表现出核转位和转录活性，这通过CA9 mRNA表达的诱导得到证实。此外，HIF1α的常氧积累与肿瘤细胞增殖受损有关。最后，谷氨酰胺分解的有毒废物产物氨诱导HIF1α常氧积累的程度与谷氨酰胺相同。
我们的研究表明，HIF1α参与常氧下谷氨酰胺代谢及有毒代谢废物产物氨的细胞水平调节。因此，我们的结果与文献中的数据共同支持了这样的假说，即HIF1α及其靶基因在缺氧和常氧条件下的代谢途径（如谷氨酰胺分解和糖酵解）中起关键作用。
因此，抑制HIF1α（和/或HIF1α靶基因）可能成为一种有前景的治疗方法，这将导致肿瘤细胞中有毒代谢废物产物的积累以及其营养和能量供应的减少。
