Characterization of glycolysis-related gene expression in malignant melanoma

Malignant melanoma exhibits a distinct metabolic phenotype with high glycolytic activity. Previously, we have shown that glucose transporter isoform 1 (GLUT1) favors growth and metastasis of malignant melanoma. In this study, we investigated the expression of GLUT1 and the further glycolysis-related genes hexokinase 1 and 2 (HK1, HK2), lactate dehydrogenase A (LDH-A) and monocarboxylate transporters 1 and 4 (MCT1, MCT4) in eleven human melanoma cell lines under normoxic and hypoxic conditions. Furthermore, a set of 25 human malignant melanoma tissue samples was analyzed.Under hypoxic conditions, we could observe a significant upregulation of hypoxia-inducible factor 1 alpha (HIF-1a) target genes GLUT1, HK2 and LDH-A, but not MCT4. While under normoxic conditions the expression of glycolysis-related genes showed no correlation with origin or BRAF mutation status, GLUT1 expression was significantly elevated in metastatic and BRAF-V600E mutated melanoma cell lines under hypoxic conditions. Furthermore, GLUT1 expression in human melanoma tissue samples correlated significantly with HK1, LDH-A and MCT1 expression, confirming a glycolytic phenotype. Notably, Cyclin D1 expression, which is used as a prognostic marker for the outcome of melanoma patients, as it is associated with proliferation and invasiveness of melanoma, significantly correlated with GLUT1, HK1, LDH-A and MCT1 expression. In summary, our findings provide further evidence that enhanced glycolytic activity in melanoma favors disease progression and is an attractive therapeutic target for this highly aggressive tumor.     

磷酸甘油酸脱氢酶基因与肿瘤

磷酸甘油酸脱氢酶(phosphoglycerate dehydrogenase,PHGDH)基因编码3-磷酸甘油酸脱氢酶,是糖酵解-丝氨酸生物合成途径中的第一个分支酶.PHGDH氧化糖酵解中间产物3-磷酸甘油酸为磷酸羟基丙酮酸,后通过一系列酶的作用最终合成丝氨酸.丝氨酸在蛋白和细胞增殖所需其他生物分子(如核苷酸、磷脂丝氨酸、鞘氨醇)的合成中起着重要的作用.最新研究发现PHGDH高表达于一系列肿瘤中,且与肿瘤细胞生长、凋亡相关.该文就PHGDH基因结构、功能及与肿瘤的关系作一综述.     

Silencing of NADPH Oxidase 4 Attenuates Hypoxia Resistance in Neuroblastoma Cells SH-SY5Y by Inhibiting PI3K/Akt-Dependent Glycolysis

Hypoxia-induced chemoresistance is a major obstacle in the development of effective cancer therapy. In our study, the reversal abilities of NADPH oxidase 4 (NOX4) silence on hypoxia resistance and the potential mechanism were investigated. Our data showed that the expression of NOX4 was upregulated in human neuroblastoma cells SH-SY5Y under hypoxia condition time dependently. Knockdown of NOX4 expression by siRNA inhibited glycolysis induced by hypoxia through decreasing the expression of glycolysis-related proteins (HIF-1 , LDHA, and PDK1), decreasing glucose uptake, lactate production, and ROS production, while increasing mitochondria membrane potential. Moreover, NOX4 silence inhibited cell growth under hypoxia condition through suppressing cell proliferation and proliferation-related proteins (Ki-67 and PCNA) compared with the hypoxia 24 h+siRNA NC group. Further, Western blot experiments exhibited that NOX4 siRNA could downregulate the rate of p-Akt/Akt. Treatment with PI3K/Akt signaling activator IGF-1 blocked, while treatment with Akt inhibitor perifosine enhanced the inhibitory effect of si-NOX4 on glycolysis and cell growth. In summary, knockdown of NOX4 had the ability of reversing hypoxia resistance, and the major mechanism is considered to be the inhibition of glycolysis and cell growth via the PI3K/Akt signaling pathway. Therefore, NOX4 could be a novel target against hypoxia resistance in neuroblastoma.     

紫檀芪对卵巢癌SKOV3细胞凋亡和糖酵解的影响及机制研究

目的 探讨紫檀芪对卵巢癌SKOV3细胞凋亡和糖酵解的影响及机制研究。方法 分别采用0、25、50、100、150 μmol/L的紫檀芪处理SKOV3细胞24、48、72 h,用CCK-8检测紫檀芪对SKOV3细胞增殖的影响,根据CCK-8结果选择后续实验组紫檀芪浓度;采用流式细胞术检测紫檀芪对细胞凋亡的影响;采用葡萄糖氧化酶法和化学比色法检测紫檀芪对SKOV3细胞葡萄糖消耗和乳酸生成的影响;Western blot法检测信号转导与转录激活因子3(STAT3)、磷酸化的STAT3(p-STAT3)、己糖激酶2(HK2)蛋白的表达;qRT-PCR法检测葡萄糖转运蛋白1(GLUT1)、M2型丙酮酸激酶(PKM2)mRNA的表达。结果 CCK-8结果显示,紫檀芪对SKOV3细胞的增殖有抑制作用,且呈时间剂量依赖性,根据CCK-8结果,选择100 μmol/L紫檀芪处理组作为后续实验组,0 μmol/L为对照组;流式细胞术结果显示,紫檀芪可明显促进SKOV3的凋亡,浓度越大,凋亡作用越明显,差异有统计学意义(P＜0.05)。此外,100 μmol/L组紫檀芪作用SKOV3细胞后,葡萄糖消耗和乳酸生成水平均较0 μmol/L组降低(P＜0.01)。Western blot结果显示,与0 μmol/L组相比,100 μmol/L组p-STAT3、HK2蛋白的表达明显降低(P＜0.001)。qRT-PCR结果显示,100 μmol/L组GLUT1、PKM2 mRNA的表达水平也较0 μmol/L组降低(P＜0.01)。结论 紫檀芪可抑制SKOV3细胞的增殖,促进凋亡,并可能通过STAT3/HK2途径抑制卵巢癌的糖酵解。     

Loss of function of PTEN alters the relationship between glucose concentration and cell proliferation,increases glycolysis,and sensitizes cells to 2-deoxyglucose

PTEN loss of function enhances proliferation, but effects on cellular energy metabolism are less well characterized. We used an inducible PTEN expression vector in a PTEN-null glioma cell line to examine this issue. While proliferation of PTEN-positive cells was insensitive to increases in glucose concentration beyond 2.5 mM, PTEN-null cells significantly increased proliferation with increasing glucose concentration across the normal physiologic range to ∼10 mM, coinciding with a shift to glycolysis and “glucose addiction”. This demonstrates that the impact of loss of function of PTEN is modified by glucose concentration, and may be relevant to epidemiologic results linking hyperglycemia to cancer risk and cancer mortality.     

Mitochondrial and glycolytic metabolic compartmentalization in diffuse large B-cell lymphoma

Metabolic heterogeneity between neoplastic cells and surrounding stroma has been described in several epithelial malignancies; however, the metabolic phenotypes of neoplastic lymphocytes and neighboring stroma in diffuse large B-cell lymphoma (DLBCL) is unknown. We investigated the metabolic phenotypes of human DLBCL tumors by using immunohistochemical markers of glycolytic and mitochondrial oxidative phosphorylation (OXPHOS) metabolism. The lactate importer MCT4 is a marker of glycolysis, whereas the lactate importer MCT1 and TOMM20 are markers of OXPHOS metabolism. Staining patterns were assessed in 33 DLBCL samples as well as 18 control samples (non-neoplastic lymph nodes). TOMM20 and MCT1 were highly expressed in neoplastic lymphocytes, indicating an OXPHOS phenotype, whereas non-neoplastic lymphocytes in the control samples did not express these markers. Stromal cells in DLBCL samples strongly expressed MCT4, displaying a glycolytic phenotype, a feature not seen in stromal elements of non-neoplastic lymphatic tissue. Furthermore, the differential expression of lactate exporters (MCT4) on tumor-associated stroma and lactate importers (MCT1) on neoplastic lymphocytes support the hypothesis that neoplastic cells are metabolically linked to the stroma likely via mutually beneficial reprogramming. MCT4 is a marker of tumor-associated stroma in neoplastic tissue. Our findings suggest that disruption of neoplastic-stromal cell metabolic heterogeneity including MCT1 and MCT4 blockade should be studied to determine if it could represent a novel treatment target in DLBCL.     

过度疲劳状态下运动性猝死模型大鼠甲状腺及心脏功能变化

BACKGROUND: Exercise fatigue can lead to lactic acid accumulation, metabolism decrease and energy intake disturbance following by overtraining. Further, constant development of negative superposition after overtraining will contribute to karoshi and exercise-induced sudden death. OBJECTIVE:To investigate the changes of rat thyroid function under excessive fatigue state and to further explore the relationship between the exercise-induced sudden cardiac death and thyroid function. METHODS: Seven rats were randomly selected from 30 male Sprague-Dawley rats as blank control group. The remaining rats were trained continuously with exhaustive loaded-swimming exercises to achieve the excessive fatigue state, and seven as fatigue group, seven as sudden death group. Rat samples were collected immediately after exercise to observe the morphology, structure and intercellular substance changes of thyroid follicular epithelial cells and cardiac muscle fibers by hematoxylin-eosin staining;   levels of serum triiodothyronine (T3), tetraiodothyronine (T4), thyroid stimulating hormone (TSH) and lactic dehydrogenase 2 (LDH-2) were detected by ELISA. RESULTS AND CONCLUSION:(1) In the sudden death group, intermyocardiac vessels expanded extremely, some thin myocardial fibers ruptured, and severe congestion and hemorrhage occurred; thyroid follicles were in a hypochromatism with loose intercellular substance, and extensive visible vacuolization in the follicular colloid. (2) The levels of serum LDH-2 and T3 in the sudden death group were significantly higher than those in the blank control group (P < 0.05). (3) The levels of serum T4 and TSH were the highest in the fatigue group, followed by sudden death group, and lowest in the blank control group (P < 0.05). (4) These results suggest that excessive fatigue caused by long-term high-intensity training will lead to thyroid morphological abnormality and myocardial impairment. Increasing serum LDH-2 following excessive fatigue aggravates myocardial damages. Fatigue accumulation causing thyroid dysfunction, abnormal secretion of TSH, T3 and T4, may be one of the causes of exercise-induced sudden death. Exercise-induced sudden death belongs to a multiple organ dysfunction syndrome, and turbulence of thyroid causing cardiac structural and functional alteration may induce sudden cardiac death. 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程     

miR-125b-5p对喉鳞状细胞癌细胞能量代谢及增殖的影响

目的  探讨miR-125b-5p对喉鳞状细胞癌（laryngeal squamous cell carcinoma,LSCC）细胞能量代谢和增殖的影响及其可能的作用机制。方法 实验分为miR-125b-5p转染组（miR-125b-5p模拟物转染LSCC）和对照组（空质粒转染LSCC细胞）。采用RT-qPCR 检测miR-125b-5p在正常支气管上皮细胞和LSCC细胞中的表达,CCK-8和流式细胞术检测LSCC细胞增殖、凋亡情况,Western blot检测miR-125b-5p过表达后LSCC中HK2的表达,3H-2DG法和乳酸盐比色测定实验分别检测LSCC细胞葡萄糖消耗和乳酸产生的情况。结果 RT-qPCR实验结果显示,与正常支气管上皮细胞相比,LSCC细胞中miR-125b-5p的表达降低（0.68±0.03 vs 0.22±0.05,t=7.025,P=0.001）。CCK-8实验结果显示,转染72 h和96 h后,miR-125b-5p转染组LSCC细胞的增殖能力均较对照组明显降低（P<0.05）。流式细胞术检测结果显示,miR-125b-5p转染组LSCC细胞凋亡率较对照组升高 ［（37.52±2.34）% vs （12.46±3.52）%,t=7.025,P<0.001）］,但细胞集落形成能力降低（0.29±0.02 vs 1.02±0.03,t=5.689,P=0.005）;荧光素酶报告基因测定实验结果显示,miR-125b-5p转染组的荧光素酶活性低于对照组（0.32±0.03 vs 1.01±0.02,t=7.543,P=0.001）;Western blot法实验结果显示,miR-125b-5p转染组HK2蛋白表达水平较对照组降低（0.12±0.02 vs 0.75±0.03,t=5.875,P=0.023）;miR-125b-5p转染组葡萄糖消耗量［（3.85±0.86） dpm/mg vs （10.52±1.34） dpm/mg,t=6.118,P=0.005］以及乳酸产生量［（4.23±1.36） dpm/mg vs （10.96±2.45） dpm/mg,t=5.907,P=0.002］亦较对照组降低。结论 miR-125b-5p可能通过下调HK2表达降低喉鳞状细胞癌细胞的能量代谢和增殖能力,诱导细胞凋亡。     

转化生长因子β1增强糖酵解促进舌鳞状细胞癌细胞上皮间充质转化及迁移与侵袭

目的研究转化生长因子β1（TGF-β1）对舌鳞状细胞癌（TSCC）糖酵解活性和上皮间充质转化（EMT）及迁移与侵袭的影响。 方法利用10 ng/mL TGF-β1处理TSCC SCC9细胞1、2、3 d,收集细胞上清液检测葡萄糖和乳酸表达变化;蛋白免疫印迹法（Western blot）检测TGF-β1处理1、2、3 d后糖酵解关键酶表达变化;应用10 ng/mL TGF-β1处理TSCC SCC9细胞2、4、6 d,在倒置显微镜下定时观察细胞形态;Western blot检测TGF-β1诱导2、4、6 d后EMT上皮标记蛋白E-cadherin、间质标记蛋白Vimentin、Snail和Slug的表达变化;Transwell小室检测细胞迁移和侵袭能力;同等培养条件下未经TGF-β1处理的为对照组。SPSS 13.0统计软件进行数据分析。 结果在TGF-β1诱导条件下,TSCC SCC9细胞葡萄糖摄取量在2和3 d时[（34.1 ± 1.2）、（47.1 ± 2.3）mmol]较对照组显著升高（t_{2 d}= 17.941,P_{2 d}= 0.003;t_{3 d}= 24.430,P_{3 d}= 0.002）,同时SCC9细胞乳酸生成量在2和3 d时[（46.4 ± 1.0）、（60.2 ± 2.0）mmol]较对照组明显增加（t_{2 d}= 50.230,P_{2 d}= 0.005;t_{3 d}= 26.883,P_{3 d}= 0.004）;TGF-β1处理后,糖酵解关键酶HK2表达在2和3 d时（1.21 ± 0.04、1.30 ± 0.06）均高于对照组,差异有统计学意义（t_2d= 6.111,P_{2 d}= 0.026;t_{3 d}= 6.423,P_{3 d}= 0.023）;糖酵解关键酶PKM2表达在2和3 d时（1.048 ± 0.002、1.071 ± 0.010）与对照组相比,差异无统计学意义（t_{2 d}= 20.693,P_{2 d}= 0.072;t_{3 d}= 9.875,P_{3 d}= 0.081）;糖酵解关键酶PFKP在2和3 d时（0.820 ± 0.010、0.839 ± 0.036）表达较对照组明显升高（t_{2 d}= 21.829,P_{2 d}= 0.020;t_{3 d}= 9.853,P_{3 d}= 0.022）;糖酵解关键酶GLUT1表达在2和3 d时（0.503 ± 0.007、0.589 ± 0.019）均高于对照,差异具有统计学意义（t_{2 d}= 30.693,P_{2 d}= 0.015;t_{3 d}= 21.173,P_{3 d}= 0.012）。在TGF-β1诱导下,与对照组相比,TSCC SCC9细胞从鹅卵石状变为长梭形,同时EMT上皮标记蛋白E-cadherin表达在2、4和6 d时（0.69 ± 0.03、0.67 ± 0.04、0.65 ± 0.04）较对照组降低,差异有统计学意义（t_{2 d}= 7.187,P_{2 d}= 0.019;t_{4 d}= 6.631,P_{4 d}= 0.022;t_{6 d}= 6.690,P_{6 d}= 0.022）,间质标记蛋白Vimentin（1.089 ± 0.134、0.706 ± 0.025、0.620 ± 0.010）表达在2、4、6 d处与对照组相比表达升高（t_{2 d}= 6.948,P_{2 d}= 0.020;t_{4 d}= 16.710,P_{4 d}= 0.004;t_{6 d}= 6.157,P_{6 d}= 0.025）,EMT转录因子snail在2 d时（1.14 ± 0.17）表达与对照组（0.77 ± 0.10）相比表达升高（t= 3.794,P= 0.015）,EMT转录因子slug在2 d时（1.85 ± 0.11）表达与对照组（0.93 ± 0.02）相比表达升高（t= 15.385,P= 0.014）;与对照组（20.0 ± 2.0）相比,TGF-β1处理后细胞迁移能力（45.7 ± 11.6）显著增加（t= 4.529,P= 0.017）,细胞侵袭能力（58.7 ± 5.0）较对照组（22.3 ± 1.5）明显升高（t= 15.571,P= 0.015）。 结论TGF-β1增强糖酵解,并促进TSCC细胞EMT及迁移和侵袭。     

Real-time Analysis of Glucose Metabolism by Microscopy

Glucose metabolism has traditionally been assayed via biochemical means. Fluorescence monitoring of NAD(P)H levels has provided a non-invasive method to assay glucose metabolism in cells and tissues. However, these measurements have traditionally been of low resolution (no subcellular information) because of limitations imposed by optical and cellular photodamage problems. The recent advent of two-photon excitation microscopy as a dependable tool for biological research has opened the possibility of real-time, high-resolution analysis of glucose metabolism in living cells. Such measurements have the potential to provide subcellular information from intact tissue that cannot be obtained by other techniques.