Transketolase‐like protein 1 (TKTL1) is required for rapid cell growth and full viability of human tumor cells

Cancer cells display high rates of aerobic glycolysis, a phenomenon known as the Warburg effect. Lactate and pyruvate, the end products of glycolysis, are overproduced by cancer cells even in the presence of oxygen. The pentose phosphate pathway (PPP) allows glucose conversion to ribose for nucleic acid synthesis, glucose degradation to lactate, and regeneration of redox equivalents. The nonoxidative part of the PPP is controlled by transketolase (TKT) enzymes. One TKT isoform, the transketolase‐like protein 1 (TKTL1) is specifically upregulated in different human cancers and its overexpression predicts a poor patient's survival. This finding implicates that an increased TKTL1 expression may activate the PPP leading to enhanced cancer cell growth and survival. To analyze the functional role of TKTL1 in malignant progression, we inhibited TKTL1 by RNAi technologies in human HCT116 colon carcinoma cells. TKTL1 suppression resulted in a significantly slowed cell growth, glucose consumption and lactate production. In TKTL1 knockdown‐cells, the intracellular reactive oxygen species levels were not significantly increased, whereas the sensitivity towards oxidative stress‐induced apoptosis was clearly enhanced. These data provide new clues on the importance of TKTL1 dys‐regulation in tumor cells and indicate that TKTL1 overexpression may be considered not only as a new tumor marker but also as a good target for anticancer therapy. © 2008 Wiley‐Liss, Inc.     

Modulation of pentose phosphate pathway during cell cycle progression in human colon adenocarcinoma cell line HT29

Cell cycle regulation is dependent on multiple cellular and molecular events. Cell proliferation requires metabolic sources for the duplication of DNA and cell size. However, nucleotide reservoirs are not sufficient to support cell duplication and, therefore, biosynthetic pathways should be upregulated during cell cycle. Here, we reveal that glucose‐6‐phosphate dehydrogenase (G6PDH) and transketolase (TKT), the 2 key enzymes of oxidative and nonoxidative branches of the pentose phosphate pathway (PPP), respectively, which is necessary for nucleotide synthesis, are enhanced during cell cycle progression of the human colon cancer cell line HT29. These enhanced enzyme activities coincide with an increased ratio of pentose monophosphate to hexose monophosphate pool during late G1 and S phase, suggesting a potential role for pentose phosphates in proliferating signaling. Isotopomeric analysis distribution of nucleotide ribose synthesized from 1,2‐¹³C₂‐glucose confirms the activation of the PPP during late G1 and S phase and reveals specific upregulation of the oxidative branch. Our data sustain the idea of a critical oxidative and nonoxidative balance in cancer cells, which is consistent with a late G1 metabolic check point. The distinctive modulation of these enzymes during cell cycle progression may represent a new strategy to inhibit proliferation in anticancer treatments. © 2009 UICC     

Overexpression of transketolase protein TKTL1 is associated with occurrence and progression in nasopharyngeal carcinoma: a potential therapeutic target in nasopharyngeal carcinoma

Over 80 years ago, Warburg identified a particular metabolic pathway in carcinomas characterised by the anaerobic degradation of glucose even in the presence of oxygen that leads to the production of large amounts of lactate (known as the Warburg effect). Now, widespread clinical use of positron-emission tomography (PET) has confirmed that there exists enhanced glucose degradation in tumors. Recent research demonstrated that pentose phosphate pathway (PPP) was augmented in some tumors, especially non-oxidative part of PPP. The non-oxidative part of PPP is controlled by transketolase enzyme reactions. The present study designed to evaluate the effect of transketolase activity on nasopharyngeal carcinoma. It was found that the transketolase activity was significantly stronger in human nasopharyngeal carcinoma tissues than those in human chronic nasopharyngitis tissues. There is a strong upregulation of the transketolase-like-1 (TKTL1) in human nasopharyngeal carcinoma tissues and cell line (CNE), whereas transketolase (TKT) and transketolase-like-2 (TKTL2) were not upregulated. After inhibited the expression of (TKTL1) by RNAi, we found that total transketolase activity was dramatically downregulated and the proliferation of cancer cells was significantly inhibited in CNE cells. These results indicate that TKTL1 gene influences total transketolase activity and cell proliferation in human nasopharyngeal carcinoma cells, suggesting that TKTL1 gene plays an important role on glycometabolism in tumors and it might become a novel target for tumor gene therapy.     

Transketolase protein TKTL1 overexpression: A potential biomarker and therapeutic target in breast cancer

Malignant tumors degrade glucose to lactate even in the presence of oxygen via the pentose phosphate pathway (ppp). The non-oxidative part of the ppp is controlled by thiamine-dependant transketolase enzyme reactions. Overexpression of the transketolase-like-1-gene (TKTL1) in urothelial and colorectal cancer is associated with poor patient outcome. We analyzed the expression of the TKTL1 protein in a retrospective institution-based patient cohort with invasive breast cancer by immunohistochemical analysis of 124 paraffin-embedded breast cancer tissues. Our study revealed TKTL1 expression in 86% of breast cancer specimens with 45% showing high expression levels. In contrast, only 29% of corresponding non-neoplastic breast tissues were TKTL1 immunopositive, including 9% with high expression levels. High expression levels of TKTL1 correlated significantly to Her2/neu overexpression (p=0.015). However, TKTL1 expression failed to reach statistical significance for other common prognostic parameters. In contrast to recent data for e.g. colorectal cancer TKTL1 overexpression did not correlate to patient outcome and survival. However, in the context of novel insights into TKTL1-related tumor metabolism and the high proportion of TKTL1 overexpressing breast cancers, this enzyme represents a potential candidate for targeted inhibition of tumor growth in this tumor entity.     

Transketolase-like protein 1 expression predicts poor prognosis in colorectal cancer

Background: Transketolase-like protein 1 (TKTL1) is an isoform of tranketolase, a key protein in a cancer cell's glucose metabolism that causes rapid cell growth and controls the non-oxidative part of the pentose phosphate pathway (PPP). Its overexpression occurs in several human cancer types. Our purpose was to study whether TKTL1 expression in colorectal cancer tissue associates with these patients’ prognosis. Methods: We collected retrospectively patient data and tissue samples from 840 colorectal cancer patients treated at Helsinki University Hospital, then stained tumor tissue microarrays for TKTL1 by immunohistochemistry, and compared immunohistochemical tissue expression with clinico-pathological parameters and survival. Results: High expression of TKTL1 associated with high Dukes stage, non-mucinous adenocarcinoma, and left-sided disease. Patients with high TKTL1 expression had poorer prognosis than those with low expression, with a 5-year disease-specific survival of 55.7% vs. 62.7%. Conclusion: We show that high TKTL1 in tumor tissue can lead to poor survival in colorectal cancer. TKTL1 thus can serve as a candidate marker for identifying patients at risk of recurrent disease.     

Roles of the hexosamine biosynthetic pathway and pentose phosphate pathway in bile acid‐induced cancer development

Esophageal squamous cell carcinomas (ESCCs) as well as adenocarcinomas (EACs) were developed in rat duodenal contents reflux models (reflux model). The present study aimed to shed light on the mechanism by which bile acid stimulation causes cancer onset and progression. Metabolomics analyses were performed on samples of neoplastic and nonneoplastic tissues from reflux models, and K14D, cultivated from a nonmetastatic, primary ESCC, and ESCC‐DR, established from a metastatic thoracic lesion. ESCC‐DRtca2M was prepared by treating ESCC‐DR cells with taurocholic acid (TCA) to accelerate cancer progression. The lines were subjected to comprehensive genomic analyses. In addition , protein expression levels of glucose‐6‐phosphate dehydrogenase (G6PD), nuclear factor kappa B (NF‐κB) (p65) and O‐linked N‐Acetylglucosamine (O‐GlcNAc) were compared among lines. Cancers developed in the reflux models exhibited greater hexosamine biosynthesis pathway (HBP) activation compared with the nonneoplastic tissues. Expression of O‐GlcNAc transferase (OGT) increased considerably in both ESCC and EAC compared with nonneoplastic squamous epithelium. Conversely, cell line‐based experiments revealed the greater activation of the pentose phosphate pathway (PPP) at higher degrees of malignancy. G6PD overexpression in response to TCA exposure was observed. Both NF‐κB (p65) and O‐GlcNAc were expressed more highly in ESCC‐DRtca2M than in the other cell lines. Moreover, ESCC‐DRtca2M cells had additional chromosomal abnormalities in excess of ESCC‐DR cells. Overall, glucose metabolism was upregulated in both esophageal cancer tissue and cell lines. While bile acids are not mutagenic, chronic exposure seems to trigger NF‐κB(p65) activation, potentially inducing genetic mutations as well as facilitating carcinogenesis and cancer progression. Glucose metabolism was upregulated in both esophageal cancer tissue and cell lines, and the HBP was activated in the former. The cell line‐based experiments demonstrated upregulation of the pentose phosphate pathway (PPP) at higher degrees of malignancy. While bile acids are not mutagenic, chronic exposure seems to trigger G6PD overexpression and NF‐κB (p65) activation, potentially inducing genetic mutations as well as facilitating carcinogenesis and cancer progression.     

BAG3 elevation inhibits cell proliferation via direct interaction with G6PD in hepatocellular carcinomas

Bcl-2 associated athanogene 3 (BAG3) contains multiple protein-binding motifs to mediate potential interactions with chaperons and/or other proteins, which is possibly ascribed to the multifaceted functions assigned to BAG3. The current study demonstrated that BAG3 directly interacted with glucose 6 phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway (PPP). BAG3 suppressed the PPP flux, de novo DNA synthesis and cell growth in hepatocellular carcinomas (HCCs). The growth defect of HCCs with forced BAG3 expression can be rescued by enforced G6PD expression. However, BAG3 elevation did not cause a reduction in cellular NADPH concentrations, another main product of G6PD. In addition, supplement of nucleosides alone was sufficient to recover the growth defect mediated by BAG3 elevation. Collectively, the current study established a tumor suppressor-like function of BAG3 via direct interaction with G6PD in HCCs at the cellular level.     

SIRT5通过调控G6PD促进结肠癌细胞的糖代谢及增殖

目的:探讨SIRT5对结肠癌细胞糖代谢和增殖的作用及其可能的作用机制,以及SIRT5、G6PD在结肠癌组织中的表达情况.方法:Western blot法检测SIRT5蛋白在结肠癌细胞系HCT116、HT29中的表达水平,构建SIRT5基因过表达及siRNA沉默载体分别转染到SIRT5低表达与SIRT5高表达结肠癌细胞系...     

Transketolase-like protein 1 confers resistance to serum withdrawal in vitro

Transketolase-like protein 1 (TKTL1) is a member of the family of transketolase enzymes of which the founder member transketolase (TKT) is known to play a central role in the non-oxidative part of the pentose phosphate pathway. According to several publications TKTL1 is the only family member, whose expression is substantially de-regulated in a variety of solid tumours. Over-expression of TKTL1 correlates with poor prognosis of cancer patients and TKTL1 itself represents a potential therapeutic target owing to its possible involvement in the regulation of the proliferation and metabolism of cancer cells. We show that exogenously expressed TKTL1 provides HEK293 cells with moderate growth advantages under standard culture conditions, while protecting cells from growth factor withdrawal-induced apoptosis. Importantly, we identified TKTL1 with the JFC12T10 antibody as a 65kDa protein, which was however absent in most tumour cell lines tested. Primary head and neck squamous cell carcinomas of various localisations were characterised by a focal pattern with single cells strongly expressing TKTL1, rather than by a homogeneous expression pattern within the tumour mass.     

G6PD对大肠癌细胞生长侵袭的影响及其与HKⅡ的相关性

目的 探讨G6PD对大肠癌HCT116和SW480细胞生长侵袭的影响,以及G6PD与HKⅡ在大肠癌及其癌旁上皮组织中的表达和两者的相关性。方法 将体外培养的大肠癌HCT116和SW480细胞进行G6PD过表达和干扰处理。Western blot法检测细胞中G6PD和HKⅡ的蛋白表达水平;流式细胞术分析细胞周期进程;葡萄糖氧化酶法检测培养液中葡萄糖的含量;划痕实验检测细胞侵袭能力;免疫组织化学法检测大肠癌及癌旁组织中G6PD与HKⅡ蛋白的表达水平。结果 过表达实验中,与Flag转染组相比,Flag-G6PD转染组HCT116和SW480两种细胞培养液中葡萄糖浓度、S期所占比例及侵袭能力均无明显变化;干扰实验中,与阴性对照转染组相比,G6PD-Homo-1504转染组两种细胞的葡萄糖浓度显著升高,S期所占比例和侵袭能力显著降低。G6PD与HKⅡ表达水平呈正相关。结论 干扰G6PD减少了大肠癌细胞葡萄糖的消耗,抑制了细胞的增殖和侵袭能力;同时G6PD对HKⅡ可能存在调控作用。     

G6PD activity and gene expression in leukemic cells from G6PD-deficient subjects

In the present study we examined gene expression and glucose-6-phosphate dehydrogenase (G6PD) activity in leukemic cells isolated from G6PD normal and deficient subjects. The results have shown that G6PD activity strongly increases in G6PD normal leukemic cells as well as in G6PD deficient leukemic cells when compared to peripheral blood mononuclear cells (PBMC). Higher levels of G6PD gene expression were observed in leukemic cells from G6PD deficient patients compared to G6PD normal. A similar pattern of gene expression was also observed for 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase. These results support the hypothesis that G6PD deficient cell, in order to sustain their growth, must respond to the low activity of their mutant enzyme with an increase in quantity through an induction of gene expression.     

Poor prognosis in nasopharyngeal cancer patients with low glucose-6-phosphate-dehydrogenase activity.

Nasopharyngeal carcinoma (NPC) is endemic among well-defined ethnic groups in several world regions, such as Southeastern China and Taiwan. Glucose-6-phosphate-dehydrogenase (G6PD)- deficiency, a sex-linked disorder, is one of the most common enzymopathies in Taiwan. The major role of G6PD is to generate NADPH to protect cells from oxidative damage, which is a major contributing factor to certain degenerative diseases, such as aging and cancer. In view of the coincidence of epidemic distribution of NPC and G6PD deficiency, as well as the house-keeping function of G6PD in cellular oxidative defense, we investigated the correlation of G6PD activity with NPC. The stage of NPC was classified by AJCC (1997) criteria. G6PD levels were determined in 108 NPC male patients and 75 healthy male individuals. The mean G6PD level of NPC patients was 218.9 U/10(12) RBC or 7.53 U/g hemoglobin (Hb), being much lower than in normal individuals (260.6 U/10(12) erythrocytes (RBC) or 8.92 U / gHb). The level of G6PD activity had no correlation with tumor stage or lymph node or distant metastasis, but was significantly correlated with tumor recurrence (P = 0.004 when using G6PD = 130 U/10(12) RBC as cutoff value). These results indicated that low G6PD activity in patients with NPC is associated with poor prognosis.     

Poor Prognosis in Nasopharyngeal Cancer Patients with Low Glucose-6-phosphate-dehydrogenase Activity

Nasopharyngeal carcinoma (NPC) is endemic among well-defined ethnic groups in several world regions, such as Southeastern China and Taiwan. Glucose-6-phosphate-dehydrogenase (G6PD)-deficiency, a sex-linked disorder, is one of the most common enzymopathies in Taiwan. The major role of G6PD is to generate NADPH to protect cells from oxidative damage, which is a major contributing factor to certain degenerative diseases, such as aging and cancer. In view of the coincidence of epidemic distribution of NPC and G6PD deficiency, as well as the house-keeping function of G6PD in cellular oxidative defense, we investigated the correlation of G6PD activity with NPC. The stage of NPC was classified by AJCC (1997) criteria. G6PD levels were determined in 108 NPC male patients and 75 healthy male individuals. The mean G6PD level of NPC patients was 218.9 U/10¹²RBC or 7.53 U/g hemoglobin (Hb), being much lower than in normal individuals (260.6 U/10¹²erythrocytes (RBC) or 8.92 U/gHb). The level of G6PD activity had no correlation with tumor stage or lymph node or distant metastasis, but was significantly correlated with tumor recurrence ( P =0.004 when using G6PD=130 U/10¹² RBC as cutoff value). These results indicated that low G6PD activity in patients with NPC is associated with poor prognosis.     

Metabolic phenotype of bladder cancer

Metabolism of bladder cancer represents a key issue for cancer research. Several metabolic altered pathways are involved in bladder tumorigenesis, representing therefore interesting targets for therapy.Tumor cells, including urothelial cancer cells, rely on a peculiar shift to aerobic glycolysis-dependent metabolism (the Warburg-effect) as the main energy source to sustain their uncontrolled growth and proliferation. Therefore, the high glycolytic flux depends on the overexpression of glycolysis-related genes (SRC-3, glucose transporter type 1 [GLUT1], GLUT3, lactic dehydrogenase A [LDHA], LDHB, hexokinase 1 [HK1], HK2, pyruvate kinase type M [PKM], and hypoxia-inducible factor 1-alpha [HIF-1α]), resulting in an overproduction of pyruvate, alanine and lactate. Concurrently, bladder cancer metabolism displays an increased expression of genes favoring the pentose phosphate pathway (glucose-6-phosphate dehydrogenase [G6PD]) and the fatty-acid synthesis (fatty acid synthase [FASN]), along with a decrease of AMP-activated protein kinase (AMPK) and Krebs cycle activities. Moreover, the PTEN/PI3K/AKT/mTOR pathway, hyper-activated in bladder cancer, acts as central regulator of aerobic glycolysis, hence contributing to cancer metabolic switch and tumor cell proliferation.Besides glycolysis, glycogen metabolism pathway plays a robust role in bladder cancer development. In particular, the overexpression of GLUT-1, the loss of the tumor suppressor glycogen debranching enzyme amylo-α-1,6-glucosidase, 4-α-glucanotransferase (AGL), and the increased activity of the tumor promoter enzyme glycogen phosphorylase impair glycogen metabolism. An increase in glucose uptake, decrease in normal cellular glycogen storage, and overproduction of lactate are consequences of decreased oxidative phosphorylation and inability to reuse glucose into the pentose phosphate and de novo fatty acid synthesis pathways. Moreover, AGL loss determines augmented levels of the serine-to-glycine enzyme serine hydroxymethyltransferase-2 (SHMT2), resulting in an increased glycine and purine ring of nucleotides synthesis, thus supporting cells proliferation.A deep understanding of the metabolic phenotype of bladder cancer will provide novel opportunities for targeted therapeutic strategies.     

TKTL1 mRNA、RBM6 mRNA在喉癌患者病灶组织中的表达及对预后的影响

目的 探讨类转酮醇酶1(TKTL1)mRNA、RNA结合基序蛋白6(RBM6)mRNA在喉癌患者病灶组织中的表达及对预后的影响.方法 选取喉癌患者102例作为研究对象,其中癌组织作为观察组,癌旁正常组织作为对照组.比较两组TKTL1 mRNA、RBM6 mRNA水平,分析TKTL1 mRNA与RBM6 mRNA相关性,...