恶性肿瘤中泛素化修饰对Warburg效应调控机制的研究进展

泛素化和Warburg效应在肿瘤的发生发展中均发挥重要作用。肿瘤中泛素化修饰过程与糖酵解效应密切相关,即肿瘤细胞中的泛素化修饰作用能够调控Warburg效应相关的底物蛋白的表达水平,进而调控肿瘤进展和治疗耐药。本文着重讨论肿瘤细胞中蛋白质的泛素化修饰作用对Warburg效应的调控机制。     

lncRNA BCYRN1在结外NK/T细胞淋巴瘤糖酵解激活中的作用及其机制研究

目的 探讨长链非编码RNA（long non-coding RNA,lncRNA）BCYRN1在结外NK/T细胞淋巴瘤（extranodal NK/T- cell lymphoma,ENKTCL）糖酵解激活中的作用及其机制。方法 收集2010—2021年于首都医科大学附属北京同仁医院诊治的236例ENKTCL患者信息,分析空腹血糖（fasting blood glucose,FBG）与无疾病进展生存（progression-free survival,PFS）的相关性;采用qRT-PCR检测32例初诊ENKTCL患者组织中的BCYRN1含量并分析其与PFS的相关性。利用质粒转染法构建过表达BCYRN1（OE-BCYRN1组）和干扰BCYRN1（shBCYRN1组）的ENKTCL SNK-6细胞株,采用Screen QuestTM比色法检测葡萄糖摄取能力,用乳酸检测试剂盒检测乳酸的生成能力;采用qRT-PCR和Western blot法检测糖酵解关键分子PKM2、HIF-1α、SLC2A1、LDHA和PDK1的表达水平;分别添加蛋白合成抑制剂、溶酶体抑制剂或激活剂后观察BCYRN1对PKM2稳定性的影响。采用RNA pull-down和RNA结合蛋白免疫沉淀（RIP）实验判断BCYRN1与PKM2相互作用的模式。结果 236例ENKTCL患者中,高血糖组（FBG>5.6 mmol/L）49例,正常血糖组（FBG≤5.6 mmol/L）187例,高血糖组5年PFS率低于低血糖组（32.1% vs 63.7%,P<0.001）;BCYRN1高表达组3年PFS率低于低表达组（26.1% vs 82.5%,P=0.014）。干扰BCYRN1后,ENKTCL SNK-6细胞对葡萄糖的摄取量和乳酸生成水平均显著降低（均P<0.05）;过表达BCYRN1后,糖酵解关键分子PKM2、HIF-1α、SLC2A1、LDHA和PDK1的表达量均显著升高（均P<0.05）。应用蛋白合成抑制剂（放线菌酮）分别处理SNK-6细胞3 h和6 h后,OE-BCYRN1组中PKM2的降解比例均显著低于OE-CTRL组（均P<0.05）,而shBCYRN1组中PKM2的降解比例均显著高于shCTRL组（均P<0.05）。经溶酶体抑制剂（Leupeptin）处理后shBCYRN1+Leupeptin组的PKM2降解比例明显下降（P<0.01）;经溶酶体激活剂（6-Aminonicotinamide,6-AN）处理后OE-BCYRN1+6-AN组的PKM2降解比例显著增加（P<0.001）。RNA pull-down和RIP实验显示BCYRN1基因可与PKM2蛋白发生直接相互作用。结论 BCYRN1可能通过溶酶体途径上调PKM2表达而激活ENKTCL糖酵解途径。     

AKT-mediated enhanced aerobic glycolysis causes acquired radioresistance by human tumor cells

Background and purpose

Cellular radioresistance is a major impediment to effective radiotherapy. Here, we demonstrated that long-term exposure to fractionated radiation conferred acquired radioresistance to tumor cells due to AKT-mediated enhanced aerobic glycolysis.

Material and methods

Two human tumor cell lines with acquired radioresistance were established by long-term exposure to fractionated radiation with 0.5 Gy of X-rays. Glucose uptake was inhibited using 2-deoxy-d-glucose, a non-metabolizable glucose analog. Aerobic glycolysis was assessed by measuring lactate concentrations. Cells were then used for assays of ROS generation, survival, and cell death as assessed by annexin V staining.

Results

Enhanced aerobic glycolysis was shown by increased glucose transporter Glut1 expression and a high lactate production rate in acquired radioresistant cells compared with parental cells. Inhibiting the AKT pathway using the AKT inhibitor API-2 abrogated these phenomena. Moreover, we found that inhibiting glycolysis with 2-deoxy-d-glucose suppressed acquired tumor cell radioresistance.

Conclusions

Long-term fractionated radiation confers acquired radioresistance to tumor cells by AKT-mediated alterations in their glucose metabolic pathway. Thus, tumor cell metabolic pathway is an attractive target to eliminate radioresistant cells and improve radiotherapy efficacy.     

Glucose avidity of carcinomas

The cancer cell phenotype has been summarized in six hallmarks [D. Hanahan, R.A. Weinberg, The hallmarks of cancer, Cell 100 (1) (2000) 57-70]. Following the conceptual trait established in that review towards the comprehension of cancer, herein we summarize the basis of an underlying principle that is fulfilled by cancer cells and tumors: its avidity for glucose. Our purpose is to push forward that the metabolic reprogramming that operates in the cancer cell represents a seventh hallmark of the phenotype that offers a vast array of possibilities for the future treatment of the disease. We summarize the metabolic pathways that extract matter and energy from glucose, paying special attention to the concerted regulation of these pathways by the ATP mass-action ratio. The molecular and functional evidences that support the high glucose uptake and the "abnormal" aerobic glycolysis of the carcinomas are detailed discussing also the role that some oncogenes and tumor suppressors have in these pathways. We overview past and present evidences that sustain that mitochondria of the cancer cell are impaired, supporting the original Warburg's formulation that ascribed the high glucose uptake of cancer cells to a defective mitochondria. A simple proteomic approach designed to assess the metabolic phenotype of cancer, i.e., its bioenergetic signature, molecularly and functionally supports Warburg's hypothesis. Furthermore, we discuss the clinical utility that the bioenergetic signature might provide. Glycolysis is presented as the "selfish" pathway used for cellular proliferation, providing both the metabolic precursors and the energy required for biosynthetic purposes, in the context of a plethora of substrates. The glucose avidity of carcinomas is thus presented as the result of both the installment of glycolysis for cellular proliferation and of the impairment of mitochondrial activity in the cancer cell. At the end, the repression of mitochondrial activity affords the cancer cell with a cell-death resistant phenotype making them prone to malignant growth.     

缺氧对食管癌细胞缺氧诱导因子-1α及己糖激酶表达的影响

目的探讨不同缺氧程度对食管癌细胞株TE1中缺氧诱导因子-1α（Hypoxia-inducible factor-1α,HIF-1α）及糖酵解关键酶表达的影响。方法 TE1细胞分别在正常氧分压和缺氧条件下培养,缺氧时间设定为6、12、24及48h,使用Western blot方法检测缺氧培养不同时间后细胞中HIF-1α及糖酵解关键酶己糖激酶II（Hexokinase-II,HK-II）的蛋白水平表达变化。结果常氧及缺氧条件下TE1细胞中HIF-1α及HK-II均有表达,缺氧后表达均较常氧培养时明显增强,且随缺氧时间不同而呈先增多后减少的动态变化。结论低氧能够增加食管癌细胞中HIF-1α及HK-II表达而促进糖酵解进程,联合抑制HIF-1α和糖酵解酶可能成为治疗食管癌的潜在的靶点。     

The relationship between renal metabolism and proximal tubule transport during ontogeny

The proximal tubules of newborn and adult animals reabsorb a similar fraction of the filtered load of Na⁺ and H₂O (65%–70%). In tubules from adult animals, transcellular, active Na⁺ reabsorption accounts for one-third of the total, while two-thirds occur passively through the paracellular pathway, driven by hydrostatic and oncotic forces (one-third) and by cell-generated effective osmotic and ionic gradients (one-third). Since two-thirds of the Na⁺ is reabsorbed passively and does not require energy, the mature proximal tubule has a high Na⁺/O₂ molar ratio (48 Eq of Na⁺/mol of O₂). Measurements of ouabain-sensitive oxygen consumption in suspensions of proximal tubules indicate that in newborn, aerobic metabolism can support about 50% of the net Na⁺ transport rate compared with the 33% in tubules from adult animals. Independent confirmation of the direct and proportional relationship between active Na⁺ transport and ouabain-sensitive O₂ consumption exists for the adult but not for the newborn. However, measurements of epithelial conductances and of transepithelial hydrostatic and oncotic pressure differences indicate that passive paracellular fluxes can account for the remaining 50% of the proximal Na⁺ reabsorption in newborn. The high permeability of the proximal tubules of newborn animals to small molecular weight solutes suggests that cell-generated osmotic and ionic transepithelial gradients are minimal in the tubules of newborn animals. Yet in the newborn, the osmolality of the end proximal tubule fluid was found to exceed that in plasma. This indicates that osmotic gradients due to differences in reflection coefficients for preferentially reabsorbed solutes and Cl^– do exist across the proximal tubules of the newborn and suggests that these gradients may contribute to Na⁺ and H₂O reabsorption. If this is indeed the case, then the contribution of active and of hydrostatic and oncotic pressure-driven flows to the overall reabsorption of Na⁺ and fluid has been overestimated. Resolution of this discrepancy requires measurements of the reflection coefficients for HCO ₃ ^– and Cl^– in the proximal tubule of the newborn. The metabolic processes by which energy is supplied to renal proximal cells during development are also incompletely characterized. There is evidence that maturation of aerobic metabolism, Krebs cycle enzymes activity, and of the mitochondrial membrane surface area precede the development of net reabsorptive transport (Na⁺, H₂O, HCO₃, glucose). By contrast, maturation of Na⁺–K⁺-ATPase activity at the basolateral cell membrane follows that in reabsorptive transport and does not limit its development. The extent to which age-related changes in reabsorptive fluxes are due to the development of luminal membrane transport systems, to the decrease in paracellular permeability, or both remains to be determined. The high activity of enzymes in the hexosemonophosphate pathway and the high NADH/NAD ratio present during the first few weeks of extrauterine life poise the proximal tubules for high rates of biosynthesis of membrane lipids, glycoproteins, nucleic acids, and transporter proteins necessary for final differentiation.     

有氧糖酵解在非霍奇金淋巴瘤发病及耐药机制中的作用

近年来肿瘤代谢研究日益得到重视,大量研究显示有氧糖酵解在非霍奇金淋巴瘤发病及耐药机制中扮演了重要角色。有氧糖酵解相关信号通路的异常激活可增加淋巴造血细胞的有氧糖酵解水平,而有氧糖酵解相关酶的活性与非霍奇金淋巴瘤（NHL）的发病和预后相关。抑制有氧糖酵解的药物对体外培养的NHL细胞具有杀伤作用,联合常规化疗药物可增加化疗药物的敏感性和预防耐药。本文综述了有氧糖酵解相关信号通路蛋白和调控基因在NHL发病以及耐药中的作用,揭示了有氧糖酵解的深入研究在NHL临床诊断和治疗中的价值。     

干扰乳酸脱氢酶A表达对ErbB2高表达乳腺癌细胞迁移和侵袭的影响

目的 探讨体外干扰乳酸脱氢酶A（LDHA）表达对人类表皮生长因子受体2（ErbB2）高表达乳腺癌SK-BR-3和MDA-MB-453细胞迁移和侵袭的影响及其分子机制。方法 用siLDHA转染（以转染scramble siRNA为阴性对照）SK-BR-3和MDA-MB-453细胞干扰LDHA的表达;Western blot法检测LDHA蛋白表达水平;Transwell小室检测细胞的迁移和侵袭能力;采用乳酸脱氢酶（LDH）试剂盒测定细胞的LDH活性;采用葡萄糖和乳酸检测试剂盒分别测定培养基中的葡萄糖浓度和乳酸浓度,计算细胞对葡萄糖的摄取量和乳酸生成量。结果 与阴性对照组比较,siLDHA下调SK-BR-3和MDA-MB-453细胞LDHA的蛋白水平（ P<0.01）;降低细胞的迁移和侵袭能力（ P<0.001）;下调SK-BR-3细胞的LDH活性,减少两种细胞葡萄糖摄取量和乳酸生成量,差异均有统计学意义（ P<0.05）。结论 干扰LDHA表达通过降低糖酵解从而抑制ErbB2高表达乳腺癌细胞的迁移和侵袭。