苯乙双胍联合己糖激酶抑制剂诱导三阴性乳腺癌细胞凋亡

目的 探讨降糖药物苯乙双胍联合己糖激酶抑制剂2-脱氧葡萄糖（2-DG）对三阴性乳腺癌细胞4T1和MDA-MB-231凋亡作用的影响。方法 苯乙双胍单独或联合2-DG处理4T1与MDA-MB-231细胞48 h,用SRB法检测细胞的增殖,流式细胞术检测细胞凋亡,试剂盒检测培养上清液中葡萄糖消耗量和乳酸含量,多功能化学发光仪检测线粒体呼吸链复合物I活性,海马能量检测仪测定细胞线粒体耗氧量(OCR)。结果 苯乙双胍组的4T1与MDA-MB-231细胞上清液己糖激酶表达量（4.6±0.17,3.73±0.21）,葡萄糖消耗量（356±31,397±42）μg/10⁵个细胞,乳酸浓度（5.59±0.52,7.83±0.78）μmol/L均高于空白组的已糖激酶表达量（1±0.15,1±0.12）,葡萄糖消耗量（289±25,301±32）μg/10⁵个细胞,乳酸浓度（2.37±0.18,4.01±0.45）μmol/L（P < 0.01）;苯乙双胍联用2-DG组的细胞存活率（64.63±2.28,51.97±2.29）% ,即使降低90%剂量,仍高于苯乙双胍组（86.70±1.83,85.53±1.46）%（P<0.001）,两药联用极大地促进了4T1与MDA-MB-231细胞的凋亡,此外,相比于苯乙双胍组（5.59±0.52,7.83±0.78）μmol/L,苯乙双胍与2-DG联用组（3.46±0.37,5.18±0.62）μmol/L细胞的乳酸产量也大大下降（P<0.01）;与苯乙双胍或2-DG单药组相比,苯乙双胍联合2-DG组可显著抑制荷瘤小鼠体内肿瘤的生长速度（P<0.01）;苯乙双胍联合2-DG组荷瘤小鼠中位生存时间72.5 d,高于苯乙双胍组57 d、2-DG组55.5 d（P<0.01）,苯乙双胍联合2-DG可以延长荷瘤小鼠生存时间。结论 己糖激酶抑制剂2-DG显著增强了苯乙双胍对三阴性乳腺癌细胞的治疗作用。     

Cancer metabolism: The Warburg effect today

One of the first studies on the energy metabolism of a tumour was carried out, in 1922, in the laboratory of Otto Warburg. He established that cancer cells exhibited a specific metabolic pattern, characterized by a shift from respiration to fermentation, which has been later named the Warburg effect. Considerable work has been done since then, deepening our understanding of the process, with consequences for diagnosis and therapy. This review presents facts and perspectives on the Warburg effect for the 21st century.     

Anaerobic function of CNS white matter declines with age

The mammalian central nervous system (CNS) is generally believed to be completely dependent on the presence of oxygen (O₂) to maintain energy levels necessary for excitability. However, previous studies on CNS white matter (WM) have shown that a large subset of CNS-myelinated axons of mice aged 4 to 6 weeks remains excitable in the absence of O₂. We investigated whether this surprising WM tolerance to anoxia varied with age. Acutely isolated mouse optic nerve (MON), a purely myelinated WM tract, was studied electrophysiologically. Excitability in the MONs from 1-month-, 4-month-, and 8-month-old mice was assessed quantitatively as the area under the supramaximal compound action potential (CAP). Anoxia-resistant WM function declined with age. After 60 minutes of anoxia, ∼23% of the CAP remained in 1-month-old mice, 8% in 4-month-old mice, and ∼0 in the 8-month-old group. Our results indicated that although some CNS axons function anaerobically in young adult animals, they lose this ability in later adulthood. This finding may help explain the clinical impression that favorable outcome after stroke and other brain injuries declines with age.     

Hypoxia-induced lncRNA CASC9 enhances glycolysis and the epithelial-mesenchymal transition of pancreatic cancer by a positive feedback loop with AKT/HIF-1α signaling

Increasing evidence indicates the dysregulations and pivotal roles of lncRNAs in the development and progression of various cancers, including pancreatic cancer. Enhanced glycolytic flux and epithelial-to-mesenchymal transition (EMT) have been considered as important factors in driving the malignance of pancreatic cancer. Here, we sought to evaluate the biological role and involved mechanism of lncRNA CASC9 (CASC9) in pancreatic cancer. Our present study showed that CASC9 was upregulated in various pancreatic cancer cell lines. Loss- and gain-of function of CASC9 demonstrated its critical roles in promoting the glycolysis and EMT phenotypes of pancreatic cancer. Moreover, knockdown of CASC9 inhibited the tumorigenicity and metastasis in vivo. Additionally, our findings showed that hypoxia induced the expression of CASC9 and enhanced the binding of HIF-1α to its promoter. We also demonstrated that the positive feedback loop of CASC9 and the AKT/HIF-1α signaling cascade partially mediated this biological process. Altogether, our results suggest that CASC9 promotes the glycolysis and EMT of pancreatic cancer by a positive feedback loop with AKT/HIF-1α signaling, which is synergistically enhanced by the tumor hypoxic niche. Our study will provide potential therapeutic targets for treating pancreatic cancer.     

circPVT1 promotes osteosarcoma glycolysis and metastasis by sponging miR-423-5p to activate Wnt5a/Ror2 signaling

Osteosarcoma (OS) is the most prevalent form of bone cancer. It has a high metastatic potential and progresses rapidly. The molecular mechanisms of OS remain unclear and this study aims to examine the functional role of circPVT1 and miR-423-5p in OS. Quantitative RT-PCR (qRT-PCR) and western blotting were used to examine levels of miR-423-5p, circPVT1, Wnt5a, Ror2, and glycolysis-related proteins, including HK2, PKM2, GLUT1, and LDHA. Colony formation and transwell assays were used to test the roles of miR-423-5p, circPVT1, and Wnt5a/Ror2 in OS cell proliferation, migration, and invasion. Dual luciferase assay and Ago2-RIP were used to validate the interactions of miR-423-5p/Wnt5a, miR-423-5p/Ror2, and circPVT1/miR-423-5p. Glucose uptake assay and measurement of lactate production were performed to assess the glycolysis process. A nude mouse xenograft model was used to evaluate the effects of sh-circPVT1 and miR-423-5p mimics on tumor growth and metastasis in vivo. miR-423-5p was reduced in both OS tissues and OS cell lines, while Wnt5a/Ror2 and circPVT1 were elevated. miR-423-5p bound to 3′-UTR of Wnt5a and Ror2 mRNA, and inhibited glycolysis and OS cell proliferation, migration, and invasion by targeting Wnt5a and Ror2. circPVT1 interacted with miR-423-5p and activated Wnt5a/Ror2 signaling by sponging miR-423-5p. Knockdown of circPVT1 or overexpression of miR-423-5p suppressed OS tumor growth and metastasis in vivo. miR-423-5p inhibited OS glycolysis, proliferation, migration, and metastasis by targeting and suppressing Wnt5a/Ror2 signaling pathway, while circPVT1 promoted those processes by acting as a sponge of miR-423-5p.     

Metabolic wiring of murine T cell and intraepithelial lymphocyte maintenance and activation

Adaptive immunity critically depends on cell migration combined with clonal selection and rapid expansion of rare lymphocytes recognising their cognate antigen in secondary lymphoid organs. It has since become apparent that large populations of T cells are maintained in tissues, which do not migrate throughout the body and do not require clonal expansion. Murine intraepithelial lymphocytes (IELs), located in the skin and small intestines, are maintained in a state of semi‐activation, in marked contrast to the quiescent condition naive and memory lymphocytes are kept in. The poised activation state of IELs, their location in the top layers of barrier organs and close bidirectional interactions with epithelial cells suggests IELs are part of a sophisticated strategy of immune‐surveillance and compartmentalisation of immune responses. Recent murine studies have reemphasised the influence of metabolism in T‐cell activation and differentiation, with different metabolic make up of naive, effector and memory T cells. Here we highlight and discuss some of the current insights on immunometabolism of IELs, with emphasis on novel data contrasting how IELs may be maintained in a semi‐activated state and may become fully functional compared with conventional T cells.     

AMP-activated protein kinase is dispensable for maintaining ATP levels and for survival following inhibition of glycolysis,but promotes tumour engraftment of Ras-transformed fibroblasts

Lactic acid generated by highly glycolytic tumours is exported by the MonoCarboxylate Transporters, MCT1 and MCT4, to maintain pHi and energy homeostasis. We report that MCT1 inhibition combined with Mct4 gene disruption severely reduced glycolysis and tumour growth without affecting ATP levels. Because of the key role of the 5′-AMP-activated protein kinase (AMPK) in energy homeostasis, we hypothesized that targeting glycolysis (MCT-blockade) in AMPK-null (Ampk^−/−) cells should kill tumour cells from ‘ATP crisis’. We show that Ampk^−/−-Ras-transformed mouse embryonic fibroblasts (MEFs) maintained ATP levels and viability when glycolysis was inhibited. In MCT-inhibited MEFs treated with OXPHOS inhibitors the ATP level and viability collapsed in both Ampk^+/+ and Ampk^−/− cells. We therefore propose that the intracellular acidification resulting from lactic acid sequestration mimicks AMPK by blocking mTORC1, a major component of an ATP consuming pathway, thereby preventing ‘ATP crisis’. Finally we showed that genetic disruption of Mct4 and/or Ampk dramatically reduced tumourigenicity in a xenograft mouse model suggesting a crucialrolefor these two actors in establishment of tumours in a nutrient-deprived environment. These findings demonstrated that blockade of lactate transport is an efficient anti-cancer strategy that highlights the potential in targeting Mct4 in a context of impaired AMPK activity.     

Distinct von Hippel-Lindau gene and hypoxia-regulated alterations in gene and protein expression patterns of renal cell carcinoma and their effects on metabolism

During the last decade the knowledge about the molecular mechanisms of the cellular adaption to hypoxia and the function of the “von Hippel Lindau” (VHL) protein in renal cell carcinoma (RCC) has increased, but there exists little information about the overlap and differences in gene/protein expression of both processes. Therefore the aim of this study was to dissect VHL- and hypoxia-regulated alterations in the metabolism of human RCC using ome-based strategies. The effect of the VHL- and hypoxia-regulated altered gene/protein expression pattern on the cellular metabolism was analyzed by determination of glucose uptake, lactate secretion, extracellular pH, lactate dehydrogenase activity, amino acid content and ATP levels. By employing VHL⁻/VHL⁺ RCC cells cultured under normoxic and hypoxic conditions, VHL-dependent, HIF-dependent as well as VHL-/HIF-independent alterations in the gene and protein expression patterns were identified and further validated in other RCC cell lines. The genes/proteins differentially expressed under these distinct conditions were mainly involved in the cellular metabolism, which was accompanied by an altered metabolism as well as changes in the abundance of amino acids in VHL-deficient cells. In conclusion, the study reveals similarities, but also differences in the genes and proteins controlled by VHL functionality and hypoxia thereby demonstrating differences in the metabolic switch of RCC under these conditions.