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1.  Clinicopathological Significance and Prognostic Value of Lactate Dehydrogenase A Expression in Gastric Cancer Patients 
PLoS ONE  2014;9(3):e91068.
LDH-A, the enzyme responsible for transforming pyruvate into lactate, has been demonstrated to be up-regulated in many types of cancer and to give rise to more aggressive behavior by regulating proliferation and anti-apoptosis. However, its expression in gastric cancer (GC) has not been characterized thoroughly. The purpose of this study was to clarify the expression and potential impact of LDH-A in GC.
We examined LDH-A expression by immunohistochemistry on GC tissue microarray (TMA) and using Western blot on fresh GC tissues and cell lines. Prognostic value and correlation with other clinicopathologic factors were evaluated. We transfected siRNA into GC cells against LDH-A. LDH-A was analyzed by Western blotting and real-time RT-PCR. Cell growth was evaluated in vitro and in vivo. Lactate and ATP production by cells were determined.
There was significantly higher LDH-A expression in carcinoma than in non-neoplastic mucosa (NNM). There was a positive correlation of LDH-A expression with age, histological type and Lymph node metastases. Survival analysis demonstrated that high expression of LDH-A in GC was associated with lower overall survival (OS). When stratified by Lauren grade and histological classification, significance appeared in diffuse type and undifferentiated type GC. In multivariate analysis, the LDH-A expression in GC was an independent prognostic risk factor for OS (hazard ratio = 1.829, 95%CI 1.375–2.433,P<0.0001). Specific siRNA against LDH-A in GC cell line retarded cell growth both in vitro and in mouse models. LDH-A knockdown also reduced lactate and ATP production in GC cells.
Our study indicated the oncogenic role of LDH-A in GC. LDH-A expression is an independent prognostic risk factor in GC patients and up-regulated expression of LDH-A could be predictive of poor outcomes in diffuse type and undifferentiated type GC. Our results suggested that LDH-A might be a potential therapeutic target in gastric cancer.
PMCID: PMC3946663  PMID: 24608789
2.  TKTL1 is overexpressed in a large portion of non-small cell lung cancer specimens 
Diagnostic Pathology  2008;3:35.
In several tumors the transketolase activity, controlled inter alia by enzymes of the pentose phosphate pathway which is an alternative, energy generating reaction-cascade to glycolysis, has been correlated with proliferation. The increase of thiamine-dependant transketolase enzyme reactions is induced especially through upregulated transketolase-like enzyme 1 (TKTL1)-activity; that shows TKTL1 to be a causative enzyme for tumors enhanced, anaerobic glucose degradation. We investigated TKTL1-expression in 88 human, formalin-fixed non-small cell lung cancer tissues and 24 carcinomas of the breast by immunohistochemical stainings applying a 0 to 3 staining-score system (3 = strongest expression). For means of validation we additionally stained 40 NSCLC fixed and paraffin-embedded utilizing the HOPE-technique; showing comparable results to the formalin-fixed, paraffin-embedded specimens (not shown). Potential correlations with age, sex, TNM-classification parameters and tumor grading as well as tumor transcription factor 1 (TTF1) and surfactant protein A (SPA) expression were investigated. 40.9% of the analyzed lung tumors expressed TKTL1 weakly (Score 1), 38.6% moderately (score 2) and 17.1% strongly (score 3). 3 tumors were diagnosed TKTL1-negative (3.4%; score 0). All Breast cancer specimen stainings were positive and scored 1: 32%; scored 2: 36%; scored 3: 32%. Alveolar macrophages and Alveolar Epithelial Cells Type II were also found to be TKTL1-positive.
None of the listed clinical parameters could be found to show a significant correlation to TKTL1 signal appearance.
Although we describe the expression of TKTL1 in lung cancers, we need to state that up till now there is no scientific indication for any treatment regimens based upon these findings.
PMCID: PMC2526982  PMID: 18700018
3.  TKTL1 is activated by promoter hypomethylation and contributes to head and neck squamous cell carcinoma carcinogenesis via increased aerobic glycolysis and HIF1α stabilization 
This study aims to investigate the role of aberrant expression of TKTL1 (Transkelolase-like 1) in Head and Neck Squamous Cell Carcinoma (HNSCC) tumorigenesis and to characterize TKTL1 contribution to HNSCC tumorigenesis via aerobic glycolysis and HIF1α stabilization.
Experimental design
TKTL1 promoter hypomethylation and mRNA/protein aberrant expression were studied in human HNSCC tumor samples and normal mucosas. Oncogenic functions of TKTL1 were examined in HNSCC cell line panels and tumor xenograft models with TKTL1 expression construct. The metabolite levels of fructose-6-phosphate, glyceraldehydes-3-phosphate, pyruvate, lactate, and the levels of HIF1α protein and its downsteam glycolytic targets were compared between the TKTL1-expressing and vehicle-expressing HNSCC cells. Meanwhile, the effects of HIF1α/glycolytic inhibitors were evaluated on the TKTL1 transfectants.
TKTL1 exhibits high frequency of promoter hypomethylation in HNSCC tumors compared with the normal mucosas, correlating with its overexpression in HNSCC. Overexpression of TKTL1 in HNSCC cells promoted cellular proliferation and enhanced tumor growth in vitro and in vivo. Overexpression of TKTL1 increased the production of fructose-6-phosphate and glyceraldehyde-3-phosphate, in turn elevating the production of pyruvate and lactate, resulting in the normoxic stabilization of the malignancy-promoting transcription factor HIF1α and the upregulation of downstream glycolytic enzymes. Notably, reduction of TKTL1 expression decreased HIF1α accumulation and inhibition with HIF1α and/or glycolysis inhibitor could abrogate the growth effects mediated by TKTL1 overexpression.
TKTL1 is a novel candidate oncogene that is epigenetically activated by aberrant hypomethlation and contributes to a malignant phenotype via altered glycolytic metabolism and HIF1α accumulation.
PMCID: PMC2824550  PMID: 20103683
TKTL1; hypomethylation; Warburg effect; HIF1α
4.  A study of salivary lactate dehydrogenase isoenzyme levels in patients with oral leukoplakia and squamous cell carcinoma by gel electrophoresis method 
The enzyme lactate dehydrogenase (LDH), which is found in almost all the cells of body tissues, can be separated into five fractions and the isoenzyme pattern is believed to vary according to the metabolic requirement of each tissue. LDH concentration in saliva, as an expression of cellular necrosis, could be considered to be a specific indicator for oral lesions that affect the integrity of the oral mucosa.
The present study was designed to evaluate salivary LDH isoenzyme pattern in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) and to correlate between LDH isoenzyme levels and histopathologic grading in selected cases of OL and OSCC.
Materials and Methods:
Clinically diagnosed 30 cases each of OL and OSCC were selected for the study and 30 healthy individuals of comparable age served as control. Unstimulated whole saliva was aseptically collected and was processed immediately for LDH isoenzymes measurement by agarose gel electrophoresis. Biopsy specimen obtained was processed and stained by hematoxylin and eosin. Sections of OL and OSCC cases were scrutinized histopathologically and appropriately graded for epithelial dysplasia and differentiation of carcinoma respectively.
Statistical Analysis Used:
Two sample t test for testing the significance of difference between two group means was used.
Results and Conclusion:
The present salivary analysis for LDH isoenzyme reveals an overall increased salivary LDH isoenzyme level in OL and OSCC cases and a significant correlation between levels of salivary LDH isoenzymes and histopathologic grades of dysplasia in OL and OSCC. Salivary analysis of LDH will definitely provide the clinician and/or the patient himself with an efficient, non invasive and friendly new tool for diagnosis and monitoring of oral precancer and cancer.
PMCID: PMC4211236  PMID: 25364177
Lactate dehydrogenase enzyme; oral leukoplakia; oral squamous cell carcinoma; saliva
5.  Overexpression of lactate dehydrogenase-A in human intrahepatic cholangiocarcinoma: its implication for treatment 
Previous studies have shown that lactate dehydrogenase-A (LDH-A) is strongly expressed in several malignancies, that LDH-A expression is associated with poor prognosis, and that LDH-A inhibition severely diminishes tumorigenicity. However, little is known about the implications of LDH-A expression in intrahepatic cholangiocarcinoma. The purpose of this study was to investigate the expression of LDH-A and to clarify its effect on intrahepatic cholangiocarcinoma.
We studied the expression of LDH-A in tissue samples from patients with intrahepatic cholangiocarcinoma (n = 54) using the ultrasensitive surfactant protein (S-P) immunohistochemical method. We then inhibited LDH-A using small hairpin RNA (shRNA) in the cholangiocarcinoma cell line HuCCT-1 in vitro to study the role it plays in promoting growth and escaping apoptosis.
We report that LDH-A was overexpressed in 52 of 54 (96%) paraffin-embedded cancer tissue samples and 0 of 54 para-carcinoma tissue samples. Reduction of LDH-A by RNA interference (RNAi) inhibited cell growth and induced apoptosis in HuCCT-1 cells. This result correlated with the elevation of cytoplasmic reactive oxygen species (ROS) levels.
LDH-A expression is closely correlated with histopathological variables of intrahepatic cholangiocarcinoma, indicating that LDH-A may serve as a new treatment target.
PMCID: PMC4230420  PMID: 24679073
Intrahepatic cholangiocarcinoma; Lactate dehydrogenase-A; RNA interference
6.  Lactate Dehydrogenase 5 Expression in Non-Hodgkin Lymphoma Is Associated with the Induced Hypoxia Regulated Protein and Poor Prognosis 
PLoS ONE  2013;8(9):e74853.
Lactate dehydrogenase 5 (LDH-5) is one of the major isoenzymes catalyzing the biochemical process of pyruvate to lactate. The purpose of this study was to investigate the expression of serum LDH-5 and test whether this enzyme is regulated by tumor hypoxia and represents a prognostic marker in patients with Non-Hodgkin’s lymphoma (NHL). In this study, LDH-5 levels were detected using agarose gel electrophoresis in NHL patients (n = 266) and non-NHL controls including benign lymphadenectasis (n = 30) and healthy cohorts (n = 233). We also explored the expression of LDH-5 and hypoxia-inducible factor (HIF) 1α in NHL and benign controls by immunohistochemistry and immunofluorescence staining, respectively. Moreover, the role of LDH-5 in the progression of NHL was assessed by multivariate Cox analyses and Kaplan-Meier survival estimates. Serum concentrations of LDH-5 were significantly higher in NHL patients (9.3%) than in benign patients and healthy controls (7.5% and 7.2%, respectively, P<0.01). Application of LDH-5 detection increased the sensitivity of NHL detection, identifying 53.4% of NHL patients as positive, compared with the measurement of total LDH levels (36.5% sensitivity). LDH-5 concentrations increased with clinical stage, extra-nodal site involvement, and WHO performance status of patients with NHL. Exposure to a hypoxic environment induced the expression of LDH-5 and its overexpression correlated with HIF1α cytoplasmic accumulation in NHL cells. In multivariate analyses, LDH-5 was an independent marker for progression-free survival in patients with NHL (P<0.001). Overall, the expression of LDH-5 was elevated in NHL, showing an association with tumor hypoxia and unfavorable prognosis. Thus, LDH-5 emerges as a promising prognostic predictor for NHL patients.
PMCID: PMC3781153  PMID: 24086384
7.  Expression of transketolase TKTL1 predicts colon and urothelial cancer patient survival: Warburg effect reinterpreted 
British Journal of Cancer  2006;94(4):578-585.
Tumours ferment glucose to lactate even in the presence of oxygen (aerobic glycolysis; Warburg effect). The pentose phosphate pathway (PPP) allows glucose conversion to ribose for nucleic acid synthesis and glucose degradation to lactate. The nonoxidative part of the PPP is controlled by transketolase enzyme reactions. We have detected upregulation of a mutated transketolase transcript (TKTL1) in human malignancies, whereas transketolase (TKT) and transketolase-like-2 (TKTL2) transcripts were not upregulated. Strong TKTL1 protein expression was correlated to invasive colon and urothelial tumours and to poor patients outcome. TKTL1 encodes a transketolase with unusual enzymatic properties, which are likely to be caused by the internal deletion of conserved residues. We propose that TKTL1 upregulation in tumours leads to enhanced, oxygen-independent glucose usage and a lactate-based matrix degradation. As inhibition of transketolase enzyme reactions suppresses tumour growth and metastasis, TKTL1 could be the relevant target for novel anti-transketolase cancer therapies. We suggest an individualised cancer therapy based on the determination of metabolic changes in tumours that might enable the targeted inhibition of invasion and metastasis.
PMCID: PMC2361175  PMID: 16465194
pentose phosphate pathway (PPP); transketolase (TKT); transketolase-like-1 (TKTL1); aerobic glycolysis; Warburg effect; pharmacodiagnostic marker
8.  A biomarker based detection and characterization of carcinomas exploiting two fundamental biophysical mechanisms in mammalian cells 
BMC Cancer  2013;13:569.
Biomarkers allowing the characterization of malignancy and therapy response of oral squamous cell carcinomas (OSCC) or other types of carcinomas are still outstanding. The biochemical suicide molecule endonuclease DNaseX (DNaseI-like 1) has been used to identify the Apo10 protein epitope that marks tumor cells with abnormal apoptosis and proliferation. The transketolase-like protein 1 (TKTL1) represents the enzymatic basis for an anaerobic glucose metabolism even in the presence of oxygen (aerobic glycolysis/Warburg effect), which is concomitant with a more malignant phenotype due to invasive growth/metastasis and resistance to radical and apoptosis inducing therapies.
Expression of Apo10 and TKTL1 was analysed retrospectively in OSCC specimen (n = 161) by immunohistochemistry. Both markers represent independent markers for poor survival. Furthermore Apo10 and TKTL1 have been used prospectively for epitope detection in monocytes (EDIM)-blood test in patients with OSCC (n = 50), breast cancer (n = 48), prostate cancer (n = 115), and blood donors/controls (n = 74).
Positive Apo10 and TKTL1 expression were associated with recurrence of the tumor. Multivariate analysis demonstrated Apo10 and TKTL1 expression as an independent prognostic factor for reduced tumor-specific survival. Apo10+/TKTL1+ subgroup showed the worst disease-free survival rate in OSCC.
EDIM-Apo10 and EDIM-TKTL1 blood tests allowed a sensitive and specific detection of patients with OSCC, breast cancer and prostate cancer before surgery and in after care. A combined score of Apo10+/TKTL1+ led to a sensitivity of 95.8% and a specificity of 97.3% for the detection of carcinomas independent of the tumor entity.
The combined detection of two independent fundamental biophysical processes by the two biomarkers Apo10 and TKTL1 allows a sensitive and specific detection of neoplasia in a noninvasive and cost-effective way. Further prospective trials are warranted to validate this new concept for the diagnosis of neoplasia and tumor recurrence.
PMCID: PMC4235042  PMID: 24304513
Biomarker; DNaseX; Apo10; TKTL1; EDIM (epitope detection in monocytes); EDIM-blood test; Early detection and diagnosis
9.  Identification of LDH-A as a therapeutic target for cancer cell killing via (i) p53/NAD(H)-dependent and (ii) p53-independent pathways 
Oncogenesis  2014;3(5):e102-.
Most cancer cells use aerobic glycolysis to fuel their growth. The enzyme lactate dehydrogenase-A (LDH-A) is key to cancer's glycolytic phenotype, catalysing the regeneration of nicotinamide adenine dinucleotide (NAD+) from reduced nicotinamide adenine dinucleotide (NADH) necessary to sustain glycolysis. As such, LDH-A is a promising target for anticancer therapy. Here we ask if the tumour suppressor p53, a major regulator of cellular metabolism, influences the response of cancer cells to LDH-A suppression. LDH-A knockdown by RNA interference (RNAi) induced cancer cell death in p53 wild-type, mutant and p53-null human cancer cell lines, indicating that endogenous LDH-A promotes cancer cell survival irrespective of cancer cell p53 status. Unexpectedly, however, we uncovered a novel role for p53 in the regulation of cancer cell NAD+ and its reduced form NADH. Thus, LDH-A silencing by RNAi, or its inhibition using a small-molecule inhibitor, resulted in a p53-dependent increase in the cancer cell ratio of NADH:NAD+. This effect was specific for p53+/+ cancer cells and correlated with (i) reduced activity of NAD+-dependent deacetylase sirtuin 1 (SIRT1) and (ii) an increase in acetylated p53, a known target of SIRT1 deacetylation activity. In addition, activation of the redox-sensitive anticancer drug EO9 was enhanced selectively in p53+/+ cancer cells, attributable to increased activity of NAD(P)H-dependent oxidoreductase NQO1 (NAD(P)H quinone oxidoreductase 1). Suppressing LDH-A increased EO9-induced DNA damage in p53+/+ cancer cells, but importantly had no additive effect in non-cancer cells. Our results identify a unique strategy by which the NADH/NAD+ cellular redox status can be modulated in a cancer-specific, p53-dependent manner and we show that this can impact upon the activity of important NAD(H)-dependent enzymes. To summarise, this work indicates two distinct mechanisms by which suppressing LDH-A could potentially be used to kill cancer cells selectively, (i) through induction of apoptosis, irrespective of cancer cell p53 status and (ii) as a part of a combinatorial approach with redox-sensitive anticancer drugs via a novel p53/NAD(H)-dependent mechanism.
PMCID: PMC4035693  PMID: 24819061
cancer metabolism; LDH-A; NAD+/NADH; p53; apoptosis; combinatorial anticancer therapy
10.  Safety and outcome of treatment of metastatic melanoma using 3-bromopyruvate: a concise literature review and case study 
Chinese Journal of Cancer  2014;33(7):356-364.
3-Bromopyruvate (3BP) is a new, promising anticancer alkylating agent with several notable functions. In addition to inhibiting key glycolysis enzymes including hexokinase II and lactate dehydrogenase (LDH), 3BP also selectively inhibits mitochondrial oxidative phosphorylation, angiogenesis, and energy production in cancer cells. Moreover, 3BP induces hydrogen peroxide generation in cancer cells (oxidative stress effect) and competes with the LDH substrates pyruvate and lactate. There is only one published human clinical study showing that 3BP was effective in treating fibrolamellar hepatocellular carcinoma. LDH is a good measure for tumor evaluation and predicts the outcome of treatment better than the presence of a residual tumor mass. According to the Warburg effect, LDH is responsible for lactate synthesis, which facilitates cancer cell survival, progression, aggressiveness, metastasis, and angiogenesis. Lactate produced through LDH activity fuels aerobic cell populations inside tumors via metabolic symbiosis. In melanoma, the most deadly skin cancer, 3BP induced necrotic cell death in sensitive cells, whereas high glutathione (GSH) content made other melanoma cells resistant to 3BP. Concurrent use of a GSH depletor with 3BP killed resistant melanoma cells. Survival of melanoma patients was inversely associated with high serum LDH levels, which was reported to be highly predictive of melanoma treatment in randomized clinical trials. Here, we report a 28-year-old man presented with stage IV metastatic melanoma affecting the back, left pleura, and lung. The disease caused total destruction of the left lung and a high serum LDH level (4,283 U/L). After ethics committee approval and written patient consent, the patient received 3BP intravenous infusions (1-2.2 mg/kg), but the anticancer effect was minimal as indicated by a high serum LDH level. This may have been due to high tumor GSH content. On combining oral paracetamol, which depletes tumor GSH, with 3BP treatment, serum LDH level dropped maximally. Although a slow intravenous infusion of 3BP appeared to have minimal cytotoxicity, its anticancer efficacy via this delivery method was low. This was possibly due to high tumor GSH content, which was increased after concurrent use of the GSH depletor paracetamol. If the anticancer effectiveness of 3BP is less than expected, the combination with paracetamol may be needed to sensitize cancer cells to 3BP-induced effects.
PMCID: PMC4110469  PMID: 24636230
Melanoma; Warburg effect; lactate dehydrogenase; 3-bromopyruvate; paracetamol
11.  Tyrosine Phosphorylation of Lactate Dehydrogenase A Is Important for NADH/NAD+ Redox Homeostasis in Cancer Cells ▿  
Molecular and Cellular Biology  2011;31(24):4938-4950.
The Warburg effect describes an increase in aerobic glycolysis and enhanced lactate production in cancer cells. Lactate dehydrogenase A (LDH-A) regulates the last step of glycolysis that generates lactate and permits the regeneration of NAD+. LDH-A gene expression is believed to be upregulated by both HIF and Myc in cancer cells to achieve increased lactate production. However, how oncogenic signals activate LDH-A to regulate cancer cell metabolism remains unclear. We found that the oncogenic receptor tyrosine kinase FGFR1 directly phosphorylates LDH-A. Phosphorylation at Y10 and Y83 enhances LDH-A activity by enhancing the formation of active, tetrameric LDH-A and the binding of LDH-A substrate NADH, respectively. Moreover, Y10 phosphorylation of LDH-A is common in diverse human cancer cells, which correlates with activation of multiple oncogenic tyrosine kinases. Interestingly, cancer cells with stable knockdown of endogenous LDH-A and rescue expression of a catalytic hypomorph LDH-A mutant, Y10F, demonstrate increased respiration through mitochondrial complex I to sustain glycolysis by providing NAD+. However, such a compensatory increase in mitochondrial respiration in Y10F cells is insufficient to fully sustain glycolysis. Y10 rescue cells show decreased cell proliferation and ATP levels under hypoxia and reduced tumor growth in xenograft nude mice. Our findings suggest that tyrosine phosphorylation enhances LDH-A enzyme activity to promote the Warburg effect and tumor growth by regulating the NADH/NAD+ redox homeostasis, representing an acute molecular mechanism underlying the enhanced lactate production in cancer cells.
PMCID: PMC3233034  PMID: 21969607
12.  Lactate Dehydrogenase-B Is Silenced by Promoter Methylation in a High Frequency of Human Breast Cancers 
PLoS ONE  2013;8(2):e57697.
Under normoxia, non-malignant cells rely on oxidative phosphorylation for their ATP production, whereas cancer cells rely on Glycolysis; a phenomenon known as the Warburg effect. We aimed to elucidate the mechanisms contributing to the Warburg effect in human breast cancer.
Experimental design
Lactate Dehydrogenase (LDH) isoenzymes were profiled using zymography. LDH-B subunit expression was assessed by reverse transcription PCR in cells, and by Immunohistochemistry in breast tissues. LDH-B promoter methylation was assessed by sequencing bisulfite modified DNA.
Absent or decreased expression of LDH isoenzymes 1-4, were seen in T-47D and MCF7 cells. Absence of LDH-B mRNA was seen in T-47D cells, and its expression was restored following treatment with the demethylating agent 5'Azacytadine. LDH-B promoter methylation was identified in T-47D and MCF7 cells, and in 25/ 25 cases of breast cancer tissues, but not in 5/ 5 cases of normal breast tissues. Absent immuno-expression of LDH-B protein (<10% cells stained), was seen in 23/ 26 (88%) breast cancer cases, and in 4/8 cases of adjacent ductal carcinoma in situ lesions. Exposure of breast cancer cells to hypoxia (1% O2), for 48 hours resulted in significant increases in lactate levels in both MCF7 (14.0 fold, p = 0.002), and T-47D cells (2.9 fold, p = 0.009), but not in MDA-MB-436 (-0.9 fold, p = 0.229), or MCF10AT (1.2 fold, p = 0.09) cells.
Loss of LDH-B expression is an early and frequent event in human breast cancer occurring due to promoter methylation, and is likely to contribute to an enhanced glycolysis of cancer cells under hypoxia.
PMCID: PMC3578788  PMID: 23437403
13.  Kinetic characterization of lactate dehydrogenase in normal and malignant human breast tissues 
Aerobic glycolysis rate is higher in breast cancer tissues than adjacent normal tissues which providethe ATP, lactate and anabolic precursors required for tumourgenesis and metastasis. Lactate dehydrogenase (LDH) is a critical enzyme during aerobic glycolysis as it is typically responsible for the production of lactate and regeneration of NAD+, which allows for the continued functioning of glycolysis even in the absence of oxygen. LDH has been found to be highly expressed in breast tumors. Enzyme kinetic characteristics is related to environmentinvolving the enzyme, and tumor microenvironment has distinct features relative to adjacent normal tissues, thus we hypothesized that LDH should have different kinetic characteristics in breast tumors compared to normal breast tissues.
LDH was partially purifiedfrom human breast tumors and normal tissues, which were obtained directly from operating room. TheMichaelis-Menten constant (Km), maximum velocity (Vmax), activation energy (Ea) and enzyme efficiency in breast tumors and normal tissueswere determined.
It was found that tumor LDH affinity in forward reaction was the same as normal LDH but Vmax of cancerous LDH was higher relative to normal LDH. In reverse reaction, affinity of tumor LDH for lactate and NAD+ was lower than normal LDH, also enzyme efficiency for lactate and NAD+ was higher in normal samples. The Ea of reverse reaction was higher in cancerous tissues.
It was concluded that thelow LDH affinity for lactate and NAD+ is a valuable tool for preserving lactate by cancer cells. We also conclude that increasing of LDH affinity may be a valid molecular target to abolish lactate dependent tumor growth and kinetic characteristics of LDH could be a novel diagnostic parameter for human breast cancer.
PMCID: PMC4334850
Breast cancer; Aerobic glycolysis; Lactate dehydrogenase; Enzyme kinetic
14.  The influence of the pituitary tumor transforming gene-1 (PTTG-1) on survival of patients with small cell lung cancer and non-small cell lung cancer 
PTTG-1 (pituitary tumor transforming gene) is a novel oncogene that is overexpressed in tumors, such as pituitary adenoma, breast and gastrointestinal cancers as well as in leukemia. In this study, we examined the role of PTTG-1 expression in lung cancer with regard to histological subtype, the correlation of PTTG-1 to clinical parameters and relation on patients' survival.
Expression of PTTG-1 was examined immunohistochemically on formalin-fixed, paraffin-embedded tissue sections of 136 patients with small cell lung cancer (SCLC) and 91 patients with non-small cell lung cancer (NSCLC), retrospectively. The intensity of PTTG-1 expression as well as the proportion of PTTG-1 positive cells within a tumor was used for univariate and multivariate analysis.
PTTG-1 expression was observed in 64% of SCLC tumors and in 97.8% of NSCLC tumors. In patients with SCLC, negative or low PTTG-1 expression was associated with a shorter mean survival time compared with patients with strong PTTG-1 expression (265 ± 18 days vs. 379 ± 66 days; p = 0.0291). Using the Cox regression model for multivariate analysis, PTTG-1 expression was a significant predictor for survival next to performance status, tumor stage, LDH and hemoglobin.
In contrast, in patients with NSCLC an inverse correlation between survival and PTTG-1 expression was seen. Strong PTTG-1 expression was associated with a shorter mean survival of 306 ± 58 days compared with 463 ± 55 days for those patients with no or low PTTG-1 intensities (p = 0.0386). Further, PTTG-1 expression was associated with a more aggressive NSCLC phenotype with an advanced pathological stage, extensive lymph node metastases, distant metastases and increased LDH level. Multivariate analysis using Cox regression confirmed the prognostic relevance of PTTG-1 expression next to performance status and tumor stage in patients with NSCLC.
Lung cancers belong to the group of tumors expressing PTTG-1. Dependent on the histological subtype of lung cancer, PTTG-1 expression was associated with a better outcome in patients with SCLC and a rather unfavourable outcome for patients with NSCLCs. These results may reflect the varying role of PTTG-1 in the pathophysiology of the different histological subtypes of lung cancer.
PMCID: PMC1360069  PMID: 16426442
15.  The Role of Glucose Metabolism and Glucose-Associated Signalling in Cancer 
Aggressive carcinomas ferment glucose to lactate even in the presence of oxygen. This particular metabolism, termed aerobic glycolysis, the glycolytic phenotype, or the Warburg effect, was discovered by Nobel laureate Otto Warburg in the 1920s. Since these times, controversial discussions about the relevance of the fermentation of glucose by tumours took place; however, a majority of cancer researchers considered the Warburg effect as a non-causative epiphenomenon. Recent research demonstrated, that several common oncogenic events favour the expression of the glycolytic phenotype. Moreover, a suppression of the phenotypic features by either substrate limitation, pharmacological intervention, or genetic manipulation was found to mediate potent tumour-suppressive effects. The discovery of the transketolase-like 1 (TKTL1) enzyme in aggressive cancers may deliver a missing link in the interpretation of the Warburg effect. TKTL1-activity could be the basis for a rapid fermentation of glucose in aggressive carcinoma cells via the pentose phosphate pathway, which leads to matrix acidification, invasive growth, and ultimately metastasis. TKTL1 expression in certain non-cancerous tissues correlates with aerobic formation of lactate and rapid fermentation of glucose, which may be required for the prevention of advanced glycation end products and the suppression of reactive oxygen species. There is evidence, that the activity of this enzyme and the Warburg effect can be both protective or destructive for the organism. These results place glucose metabolism to the centre of pathogenesis of several civilisation related diseases and raise concerns about the high glycaemic index of various food components commonly consumed in western diets.
PMCID: PMC2754915  PMID: 19812737
Warburg effect; glucose metabolism; cancer; TKTL1 transketolase; pentose phosphate pathway; western diet
16.  M-LDH physically associated with sarcolemmal KATP channels mediates cytoprotection in heart embryonic H9C2 cells 
Muscle form of lactate dehydrogenase (M-LDH) physically associate with KATP channel subunits, Kir6.2 and SUR2A, and is an integral part of the ATP-sensitive K+ (KATP) channel protein complex in the heart. Here, we have shown that concomitant introduction of viral constructs containing truncated and mutated forms of M-LDH (ΔM-LDH) and 193gly-M-LDH respectively, generate a phenotype of rat heart embryonic H9C2 cells that do not contain functional M-LDH as a part of the KATP channel protein complex. The K+ current was increased in wild type cells, but not in cells expressing ΔM-LDH/193gly-M-LDH, when they were exposed to chemical hypoxia induced by 2,4 dinitrophenol (DNP; 10 mM). At the same time, the outcome of chemical hypoxia was much worse in ΔM-LDH/193gly-M-LDH phenotype than in the control one, and that was associated with increased loss of intracellular ATP in cells infected with ΔM-LDH/193gly-M-LDH. On the other hand, cells expressing Kir6.2AFA, a Kir6.2 mutant that abolishes KATP channel conductance without affecting intracellular ATP levels, survived chemical hypoxia much better than cells expressing ΔM-LDH/193gly-M-LDH. Based on the obtained results, we conclude that M-LDH physically associated with Kir6.2/SUR2A regulates the activity of sarcolemmal KATP channels as well as an intracellular ATP production during metabolic stress, both of which are important for cell survival.
PMCID: PMC2758067  PMID: 19464385
M-LDH; KATP channels; Chemical hypoxia; ATP; H9C2 cells
17.  M-LDH physically associated with sarcolemmal KATP channels mediates cytoprotection in heart embryonic H9C2 cells 
Muscle form of lactate dehydrogenase (M-LDH) physically associate with KATP channel subunits, Kir6.2 and SUR2A, and is an integral part of the ATP-sensitive K+ (KATP) channel protein complex in the heart. Here, we have shown that concomitant introduction of viral constructs containing truncated and mutated forms of M-LDH (ΔM-LDH) and 193gly-M-LDH respectively, generate a phenotype of rat heart embryonic H9C2 cells that do not contain functional M-LDH as a part of the KATP channel protein complex. The K+ current was increased in wild type cells, but not in cells expressing ΔM-LDH/193gly-M-LDH, when they were exposed to chemical hypoxia induced by 2,4 dinitrophenol (DNP; 10 mM). At the same time, the outcome of chemical hypoxia was much worse in ΔM-LDH/193gly-M-LDH phenotype than in the control one, and that was associated with increased loss of intracellular ATP in cells infected with ΔM-LDH/193gly-M-LDH. On the other hand, cells expressing Kir6.2AFA, a Kir6.2 mutant that abolishes KATP channel conductance without affecting intracellular ATP levels, survived chemical hypoxia much better than cells expressing ΔM-LDH/193gly-M-LDH. Based on the obtained results, we conclude that M-LDH physically associated with Kir6.2/SUR2A regulates the activity of sarcolemmal KATP channels as well as an intracellular ATP production during metabolic stress, both of which are important for cell survival.
PMCID: PMC2758067  PMID: 19464385
M-LDH; KATP channels; Chemical hypoxia; ATP; H9C2 cells
18.  Warburg effect in chemosensitivity: Targeting lactate dehydrogenase-A re-sensitizes Taxol-resistant cancer cells to Taxol 
Molecular Cancer  2010;9:33.
Taxol is one of the most effective chemotherapeutic agents for the treatment of patients with breast cancer. Despite impressive clinical responses initially, the majority of patients eventually develop resistance to Taxol. Lactate dehydrogenase-A (LDH-A) is one of the predominant isoforms of LDH expressed in breast tissue, which controls the conversion of pyruvate to lactate and plays an important role in glucose metabolism. In this study we investigated the role of LDH-A in mediating Taxol resistance in human breast cancer cells.
Taxol-resistant subclones, derived from the cancer cell line MDA-MB-435, sustained continuous growth in high concentrations of Taxol while the Taxol-sensitive cells could not. The increased expression and activity of LDH-A were detected in Taxol-resistant cells when compared with their parental cells. The downregulation of LDH-A by siRNA significantly increased the sensitivity of Taxol-resistant cells to Taxol. A higher sensitivity to the specific LDH inhibitor, oxamate, was found in the Taxol-resistant cells. Furthermore, treating cells with the combination of Taxol and oxamate showed a synergistical inhibitory effect on Taxol-resistant breast cancer cells by promoting apoptosis in these cells.
LDH-A plays an important role in Taxol resistance and inhibition of LDH-A re-sensitizes Taxol-resistant cells to Taxol. This supports that Warburg effect is a property of Taxol resistant cancer cells and may play an important role in the development of Taxol resistance. To our knowledge, this is the first report showing that the increased expression of LDH-A plays an important role in Taxol resistance of human breast cancer cells. This study provides valuable information for the future development and use of targeted therapies, such as oxamate, for the treatment of patients with Taxol-resistant breast cancer.
PMCID: PMC2829492  PMID: 20144215
19.  Lactic Acid Is Elevated in Idiopathic Pulmonary Fibrosis and Induces Myofibroblast Differentiation via pH-Dependent Activation of Transforming Growth Factor-β 
Rationale: Idiopathic pulmonary fibrosis (IPF) is a complex disease for which the pathogenesis is poorly understood. In this study, we identified lactic acid as a metabolite that is elevated in the lung tissue of patients with IPF.
Objectives: This study examines the effect of lactic acid on myofibroblast differentiation and pulmonary fibrosis.
Methods: We used metabolomic analysis to examine cellular metabolism in lung tissue from patients with IPF and determined the effects of lactic acid and lactate dehydrogenase-5 (LDH5) overexpression on myofibroblast differentiation and transforming growth factor (TGF)-β activation in vitro.
Measurements and Main Results: Lactic acid concentrations from healthy and IPF lung tissue were determined by nuclear magnetic resonance spectroscopy; α-smooth muscle actin, calponin, and LDH5 expression were assessed by Western blot of cell culture lysates. Lactic acid and LDH5 were significantly elevated in IPF lung tissue compared with controls. Physiologic concentrations of lactic acid induced myofibroblast differentiation via activation of TGF-β. TGF-β induced expression of LDH5 via hypoxia-inducible factor 1α (HIF1α). Importantly, overexpression of both HIF1α and LDH5 in human lung fibroblasts induced myofibroblast differentiation and synergized with low-dose TGF-β to induce differentiation. Furthermore, inhibition of both HIF1α and LDH5 inhibited TGF-β–induced myofibroblast differentiation.
Conclusions: We have identified the metabolite lactic acid as an important mediator of myofibroblast differentiation via a pH-dependent activation of TGF-β. We propose that the metabolic milieu of the lung, and potentially other tissues, is an important driving force behind myofibroblast differentiation and potentially the initiation and progression of fibrotic disorders.
PMCID: PMC3480515  PMID: 22923663
lactate; idiopathic pulmonary fibrosis; myofibroblast; lactate dehydrogenase; hypoxia-inducible factor 1α
20.  Different effects of LDH-A inhibition by oxamate in non-small cell lung cancer cells 
Oncotarget  2014;5(23):11886-11896.
Higher rate of glycolysis has been long observed in cancer cells, as a vital enzyme in glycolysis, lactate dehydrogenase A (LDH-A) has been shown with great potential as an anti-cancer target. Accumulating evidence indicates that inhibition of LDH-A induces apoptosis mediated by oxidative stress in cancer cells. To date, it's still unclear that whether autophagy can be induced by LDH-A inhibition. Here, we investigated the effects of oxamate, one classic inhibitor of LDH-A in non-small cell lung cancer (NSCLC) cells as well as normal lung epithelial cells. The results showed that oxamate significantly suppressed the proliferation of NSCLC cells, while it exerted a much lower toxicity in normal cells. As previous studies reported, LDH-A inhibition resulted in ATP reduction and ROS (reactive oxygen species) burst in cancer cells, which lead to apoptosis and G2/M arrest in H1395 cells. However, when being exposed to oxamate, A549 cells underwent autophagy as a protective mechanism against apoptosis. Furthermore, we found evidence that LDH-A inhibition induced G0/G1 arrest dependent on the activation of GSK-3β in A549 cells. Taken together, our results provide useful clues for targeting LDH-A in NSCLC treatment and shed light on the discovery of molecular predictors for the sensitivity of LDH-A inhibitors.
PMCID: PMC4323009  PMID: 25361010
lactate dehydrogenase A; Warburg effect; G0/G1 arrest; autophagy; apoptosis; Akt/mTOR
21.  A Δ38 Deletion Variant of Human Transketolase as a Model of Transketolase-Like Protein 1 Exhibits No Enzymatic Activity 
PLoS ONE  2012;7(10):e48321.
Besides transketolase (TKT), a thiamin-dependent enzyme of the pentose phosphate pathway, the human genome encodes for two closely related transketolase-like proteins, which share a high sequence identity with TKT. Transketolase-like protein 1 (TKTL1) has been implicated in cancerogenesis as its cellular expression levels were reported to directly correlate with invasion efficiency of cancer cells and patient mortality. It has been proposed that TKTL1 exerts its function by catalyzing an unusual enzymatic reaction, a hypothesis that has been the subject of recent controversy. The most striking difference between TKTL1 and TKT is a deletion of 38 consecutive amino acids in the N-terminal domain of the former, which constitute part of the active site in authentic TKT. Our structural and sequence analysis suggested that TKTL1 might not possess transketolase activity. In order to test this hypothesis in the absence of a recombinant expression system for TKTL1 and resilient data on its biochemical properties, we have engineered and biochemically characterized a “pseudo-TKTL1” Δ38 deletion variant of human TKT (TKTΔ38) as a viable model of TKTL1. Although the isolated protein is properly folded under in vitro conditions, both thermal stability as well as stability of the TKT-specific homodimeric assembly are markedly reduced. Circular dichroism and NMR spectroscopic analysis further indicates that TKTΔ38 is unable to bind the thiamin cofactor in a specific manner, even at superphysiological concentrations. No transketolase activity of TKTΔ38 can be detected for conversion of physiological sugar substrates thus arguing against an intrinsically encoded enzymatic function of TKTL1 in tumor cell metabolism.
PMCID: PMC3485151  PMID: 23118983
22.  Renal Cortical Lactate Dehydrogenase: A Useful, Accurate, Quantitative Marker of In Vivo Tubular Injury and Acute Renal Failure 
PLoS ONE  2013;8(6):e66776.
Studies of experimental acute kidney injury (AKI) are critically dependent on having precise methods for assessing the extent of tubular cell death. However, the most widely used techniques either provide indirect assessments (e.g., BUN, creatinine), suffer from the need for semi-quantitative grading (renal histology), or reflect the status of residual viable, not the number of lost, renal tubular cells (e.g., NGAL content). Lactate dehydrogenase (LDH) release is a highly reliable test for assessing degrees of in vitro cell death. However, its utility as an in vivo AKI marker has not been defined. Towards this end, CD-1 mice were subjected to graded renal ischemia (0, 15, 22, 30, 40, or 60 min) or to nephrotoxic (glycerol; maleate) AKI. Sham operated mice, or mice with AKI in the absence of acute tubular necrosis (ureteral obstruction; endotoxemia), served as negative controls. Renal cortical LDH or NGAL levels were assayed 2 or 24 hrs later. Ischemic, glycerol, and maleate-induced AKI were each associated with striking, steep, inverse correlations (r, −0.89) between renal injury severity and renal LDH content. With severe AKI, >65% LDH declines were observed. Corresponding prompt plasma and urinary LDH increases were observed. These observations, coupled with the maintenance of normal cortical LDH mRNA levels, indicated the renal LDH efflux, not decreased LDH synthesis, caused the falling cortical LDH levels. Renal LDH content was well maintained with sham surgery, ureteral obstruction or endotoxemic AKI. In contrast to LDH, renal cortical NGAL levels did not correlate with AKI severity. In sum, the above results indicate that renal cortical LDH assay is a highly accurate quantitative technique for gauging the extent of experimental acute ischemic and toxic renal injury. That it avoids the limitations of more traditional AKI markers implies great potential utility in experimental studies that require precise quantitation of tubule cell death.
PMCID: PMC3689004  PMID: 23825563
23.  Lactate Dehydrogenase A Promotes Communication between Carbohydrate Catabolism and Virulence in Bacillus cereus▿ †  
Journal of Bacteriology  2011;193(7):1757-1766.
The diarrheal potential of a Bacillus cereus strain is essentially dictated by the amount of secreted nonhemolytic enterotoxin (Nhe). Expression of genes encoding Nhe is regulated by several factors, including the metabolic state of the cells. To identify metabolic sensors that could promote communication between central metabolism and nhe expression, we compared four strains of the B. cereus group in terms of metabolic and nhe expression capacities. We performed growth performance measurements, metabolite analysis, and mRNA measurements of strains F4430/73, F4810/72, F837/76, and PA cultured under anoxic and fully oxic conditions. The results showed that expression levels of nhe and ldhA, which encodes lactate dehydrogenase A (LdhA), were correlated in both aerobically and anaerobically grown cells. We examined the role of LdhA in the F4430/73 strain by constructing an ldhA mutant. The ldhA mutation was more deleterious to anaerobically grown cells than to aerobically grown cells, causing growth limitation and strong deregulation of key fermentative genes. More importantly, the ldhA mutation downregulated enterotoxin gene expression under both anaerobiosis and aerobiosis, with a more pronounced effect under anaerobiosis. Therefore, LdhA was found to exert a major control on both fermentative growth and enterotoxin expression, and it is concluded that there is a direct link between fermentative metabolism and virulence in B. cereus. The data presented also provide evidence that LdhA-dependent regulation of enterotoxin gene expression is oxygen independent. This study is the first report to describe a role of a fermentative enzyme in virulence in B. cereus.
PMCID: PMC3067659  PMID: 21296961
24.  Transketolase-Like 1 Expression Is Modulated during Colorectal Cancer Progression and Metastasis Formation 
PLoS ONE  2011;6(9):e25323.
Transketolase-like 1 (TKTL1) induces glucose degradation through anaerobic pathways, even in presence of oxygen, favoring the malignant aerobic glycolytic phenotype characteristic of tumor cells. As TKTL1 appears to be a valid biomarker for cancer prognosis, the aim of the current study was to correlate its expression with tumor stage, probability of tumor recurrence and survival, in a series of colorectal cancer patients.
Methodolody/Principal Findings
Tumor tissues from 63 patients diagnosed with colorectal cancer at different stages of progression were analyzed for TKTL1 by immunohistochemistry. Staining was quantified by computational image analysis, and correlations between enzyme expression, local growth, lymph-node involvement and metastasis were assessed. The highest values for TKTL1 expression were detected in the group of stage III tumors, which showed significant differences from the other groups (Kruskal-Wallis test, P = 0.000008). Deeper analyses of T, N and M classifications revealed a weak correlation between local tumor growth and enzyme expression (Mann-Whitney test, P = 0.029), a significant association of the enzyme expression with lymph-node involvement (Mann-Whitney test, P = 0.0014) and a significant decrease in TKTL1 expression associated with metastasis (Mann-Whitney test, P = 0.0004).
To our knowledge, few studies have explored the association between variations in TKTL1 expression in the primary tumor and metastasis formation. Here we report downregulation of enzyme expression when metastasis appears, and a correlation between enzyme expression and regional lymph-node involvement in colon cancer. This finding may improve our understanding of metastasis and lead to new and more efficient therapies against cancer.
PMCID: PMC3181277  PMID: 21980427
25.  Prognostic and predictive role of lactate dehydrogenase 5 ( LDH5) expression in colorectal cancer patients treated with PTK787/ZK 222584 (Vatalanib) anti-angiogenic therapy 
The CONFIRM randomized trials, investigating the role of the VEGF-receptor inhibitor PTK787/ZK 222584 (vatalanib) in colorectal cancer (FOLFOX 4 ± vatalanib), showed some benefit in patients with high serum LDH levels. Here we investigated the expression of LDH5 (encoded entirely by the LDHA gene, regulated by the hypoxia inducible factors) in cancer tissues from patients recruited in the CONFIRM trials and relationship to response.
Experimental Design
Paraffin embedded materials from 179 patients recruited in the CONFIRM trials were analyzed by immunohistochemistry for the expression of the LDH5 protein. Correlations with serum LDH, response and survival were assessed.
A significant association of tumor burden and of poor Performance Status (PS) with serum LDH was noted. Poor PS and high tumor LDH5 expression predicted for poor response rates. High tissue LDH5 was related to poor progression free survival (PFS) only in the placebo group of patients, while the addition of Vatalanib seemed to improved response and PFS in this subgroup. High serum LDH levels were linked with significantly poorer overall survival, which however was not sustained in multivariate analysis.
Serum LDH and tissue LDH5 levels are complementary features that help to characterize the activity of lactate dehydrogenase in colorectal cancer and have a potent value in predicting response to chemotherapy. The addition of vatalanib diminished the impact of LDH expression on the prognosis of patients.
PMCID: PMC3145151  PMID: 21632858
LDH; PTK/ZK; vatalanib; colorectal cancer; CONFIRM

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