Hexokinase 2 promoted cell motility and proliferation by activating Akt1/p-Akt1 in human ovarian cancer cells.

BACKGROUND: Recently, increasing evidence has indicated that elevation of Hexokinase 2 (HK2) plays an important role in several cancers on regulating cell motility and growth. However, its role on regulating cell EMT in human ovarian cancer still less to known. METHODS: The transwell and wound-healing assay were used to detect the effective of HK2 on regulating motility of ovarian cancer cells. Real Time PCR and Western Blotting were used to explore the changing of EMT-related proteins in HK2-modified cells. The clonogenic formation, cell growth curves and MTT assays were used to evaluate the effective of HK2 on regulating cell proliferation in HK2-modified cells. The flow cytometry was used to detect the differences in the distribution of cells in the cell cycle between the HK2-modified cells and their control cells. The correlation of HK2 and Akt1/p-Akt1 was explored by using Western Blotting, Akt1 inhibitor (MK2206) and transient transfection of an Akt1 recombinant plasmid. The potential correlation between HK2 and EMT-related proteins in human ovarian cancer tissues and OV (ovarian serous cystadenocarcinoma) was confirmed by using Pearson correlation analysis and TIMER 2.0. RESULTS: In ovarian cancer cells, overexpressing of HK2 enhanced cell motility by inducing of EMT-related proteins, such as CDH2, fibronectin, MMP9, ZEB1, ZEB2 and vimentin. Moreover, overexpressing of HK2 promoted cell growth by reducing p21 and p27 expression in ovarian cancer cells. Further studies demonstrated that this promotion of cell motility and growth by HK2 was probably a result of it activating of Akt1 (p-Akt1) in ovarian cancer cells. Additionally, the positive correlation between HK2 and p-Akt1, fibronectin, MMP9 expression in human ovarian cancer samples was verified by using Pearson correlation analysis. The positive correlation between HK2 and CDH2, fibronectin, MMP9, ZEB1, ZEB2 and vimentin in OV (ovarian serous cystadenocarcinoma) was confirmed by using TIMER 2.0. CONCLUSION: This study demonstrated that HK2 could induce EMT-related proteins and reduce cell cycle inhibitor by activating Akt1 in human ovarian cancer cells, subsequently enhancing cell motility and growth, suggesting that HK2 participate in the malignant process of ovarian cancer by interacting with Akt1.

Increased hexokinase-2 as a novel biomarker for the diagnosis and correlating with disease severity in rheumatoid arthritis.

ABSTRACT: Abnormal glucose metabolism brings out joint inflammation and destruction in rheumatoid arthritis (RA). The aim of this study was to evaluate the potential of circulating hexokinase-2 (HK2) in peripheral blood mononuclear cells (PBMCs) of rheumatoid arthritis (RA) patients.PBMCs were obtained from patients with RA or osteoarthritis (OA) and healthy controls (HCs). The expression of HK2 was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The C-reactive protein (CRP) level, erythrocyte sedimentation rate (ESR), Calprotectin, rheumatoid factor (RF), anti-cyclic citrullinated peptides (anti-CCP) antibody level and 28-joint Disease Activity Score (DAS28), Clinical Disease Activity Index (CDAI) and Simplified Disease Activity Index (SDAI) were measured. Spearman's analysis was performed to determine the association between the level of HK2 and clinical characteristics. A receiver operating characteristic (ROC) curve was employed to evaluate the diagnostic value of HK2 in PBMCs. Logistic regression was used to identify risk factors. Sixty-five RA patients, 35 OA patients, and 40 HCs were included in the study.HK2 was upregulated in RA and OA patients compared with that in HCs (P < .05). The area under the ROC of HK2 for diagnosing RA and OA was 0.808 and 0.640, respectively. In addition, HK2 levels were increased in active RA compared with those in remittent RA (P = .03). Furthermore, HK2 correlated positively with the DAS28-ESR (P < .001), CDAI (P = .02) and SDAI scores (P = .02). Moreover, HK2 was independently associated with an increased risk of disease activity (DAS28-ESR>3.2, P = .02; CDAI score>10, P = .03; SDAI score>11, P = .04). Additionally, HK2 positivity was more frequently detected in patients treated with biologic disease-modifying antirheumatic drugs (bDMARDs) than in those not treated with bDMARDs.HK2 levels in PBMCs can be considered an ideal biomarker for diagnosing RA and involved in disease activity in RA. Dysregulation of HK2 may participate in the molecular mechanism of RA and could be an attractive selective metabolic target for RA treatment.

Enhanced Diffusion and Oligomeric Enzyme Dissociation.

The concept that catalytic enzymes can act as molecular machines transducing chemical activity into motion has conceptual and experimental support, but experimental support has involved oligomeric enzymes, often studied under conditions where the substrate concentration is higher than biologically relevant and accordingly exceeds kM, the Michaelis constant. Urease, a hexamer of subunits, has been considered to be the gold standard demonstrating enhanced diffusion. Here we show that urease and certain other oligomeric enzymes dissociate above kM into their subunits that diffuse more rapidly, thus providing a simple physical mechanism that contributes to enhanced diffusion in this regime of concentrations. Mindful that this conclusion may be controversial, our findings are supported by four independent analytical techniques: static light scattering, dynamic light scattering (DLS), size-exclusion chromatography (SEC), and fluorescence correlation spectroscopy (FCS). Data for urease are emphasized and the conclusion is validated for hexokinase, acetylcholinesterase, and aldolase. For hexokinase and aldolase no enhanced diffusion is observed except under conditions when these oligomeric enzymes dissociate. At substrate concentration regimes below kM at which acetylcholinesterase and urease do not dissociate, our finding showing up to 10% enhancement of the diffusion coefficient is consistent with various theoretical scenarios in the literature.

Rapid radiochemical filter paper assay for determination of hexokinase activity and affinity for glucose-6-phosphate.

Glucose phosphorylation by hexokinase (HK) is a rate-limiting step in glucose metabolism. Regulation of HK includes feedback inhibition by its product glucose-6-phosphate (G6P) and mitochondria binding. HK affinity for G6P is difficult to measure because its natural product (G6P) inhibits enzyme activity. HK phosphorylates several hexoses, and we have taken advantage of the fact that 2-deoxyglucose (2-DG)-6-phosphate does not inhibit HK activity. By this, we have developed a new method for rapid radiochemical analysis of HK activity with 2-DG as a substrate, which allows control of the concentrations of G6P to investigate HK affinity for inhibition by G6P. We verified that 2-DG serves as a substrate for the HK reaction with linear time and concentration dependency as well as expected maximal velocity and KM. This is the first simple assay that evaluates feedback inhibition of HK by its product G6P and provides a unique technique for future research evaluating the regulation of glucose phosphorylation under various physiological conditions.NEW & NOTEWORTHY Traditionally, hexokinase activity has been analyzed spectrophotometrically in which the product formation of glucose-6-phosphate (G6P) is analyzed by an indirect reaction coupled to NADPH formation during conversion of G6P to 6-P gluconolactone. By nature, this assay prevents measurements of hexokinase (HK) affinity for inhibition by G6P. We have developed a rapid radiochemical filter paper assay to study HK affinity for G6P by use of radiolabeled 2-deoxyglucose as substrate to study physiological regulation of HK affinity for G6P-induced inhibition.

AKT2 phosphorylation of hexokinase 2 at T473 promotes tumorigenesis and metastasis in colon cancer cells via NF-κB, HIF1α, MMP2, and MMP9 upregulation.

It has been well-established that AKT2 plays an important role in the development and progression of colon cancer; however, its precise function remains unclear. In the present study, we found that AKT2 can interact with and phosphorylate hexokinase 2 (HK2), the rate-limiting enzyme in glycolysis. Moreover, threonine phosphorylation dramatically increases its catalytic activity and enhances glycolysis. Mechanistically, AKT2 phosphorylation of HK2 at T473 was found to increase hexokinase activity and lactic acid production. A mutation in the AKT2 phosphorylation site of HK2 substantially reduced the stimulating effects of AKT2 on glycolysis, cellular apoptosis, invasion, tumorigenesis, and metastasis. In addition, AKT2 regulated NF-κB, HIF1Α, MMP2, and MMP9 via the phosphorylation of HK2 at the T473 site. Taken together, AKT2 increases the invasion, tumorigenesis, and metastasis of colon cancer cells in vitro and promotes lung metastasis in nude mice in vivo through the phosphorylation of the T473 site of HK2 by upregulating NF-κB, HIF1α, MMP2, and MMP9. In conclusion, our findings highlight a novel mechanism for the AKT2-HK2-NF-κB/HIF1α/MMP2/MMP9 axis in the regulation of colon cancer progression. Moreover, our results suggest that both AKT2 and HK2 may be potential targets for the treatment of colon cancer.

cAMP Receptor Protein of Salmonella enterica Serovar Typhimurium Modulate Glycolysis in Macrophages to Induce Cell Apoptosis.

We studied the role of glycolysis in the mechanism of cAMP receptor protein-induced macrophage cell death of Salmonella enterica serovar Typhimurium (S. Typhimurium). Cell apoptosis, caspase-3, -8, -9 enzyme activity, and pyruvic acid, lactic acid, ATP, and hexokinase (HK) contents were determined after infection of macrophages with S. Typhimurium SL1344 wild-type and a cAMP receptor protein mutant strain. While cell apoptosis, caspase-3, -8, -9 enzyme activity, lactic acid, hexokinase, and ATP levels significantly changed by infection with crp mutants compared to the wild-type strain (P < 0.05). Our data suggest that the cAMP receptor protein of S. Typhimurium can modulate macrophage death by effecting glycolysis levels. This finding may help to elucidate the mechanisms of S. Typhimurium pathogenesis.

Neuron-specific deficits of bioenergetic processes in the dorsolateral prefrontal cortex in schizophrenia.

Schizophrenia is a devastating illness that affects over 2 million people in the United States and costs society billions of dollars annually. New insights into the pathophysiology of schizophrenia are needed to provide the conceptual framework to facilitate development of new treatment strategies. We examined bioenergetic pathways in the dorsolateral prefrontal cortex (DLPFC) of subjects with schizophrenia and control subjects using western blot analysis, quantitative real-time polymerase chain reaction, and enzyme/substrate assays. Laser-capture microdissection-quantitative polymerase chain reaction was used to examine these pathways at the cellular level. We found decreases in hexokinase (HXK) and phosphofructokinase (PFK) activity in the DLPFC, as well as decreased PFK1 mRNA expression. In pyramidal neurons, we found an increase in monocarboxylate transporter 1 mRNA expression, and decreases in HXK1, PFK1, glucose transporter 1 (GLUT1), and GLUT3 mRNA expression. These results suggest abnormal bioenergetic function, as well as a neuron-specific defect in glucose utilization, in the DLPFC in schizophrenia.

Studies on the Tissue Localization of HKDC1, a Putative Novel Fifth Hexokinase, in Humans.

Hexokinase domain component 1 (HKDC1) is a recently discovered novel protein, which is being promoted as a putative fifth hexokinase. Although the exact role HKDC1 plays in physiology is still unclear, it has been shown to be important during pregnancy in the regulation of glucose homeostasis. In this study, we have comprehensively studied the expression pattern of HKDC1 in the human body. Using human tissue sample, immunohistochemistry imaging was performed. Our studies indicate that the tissues with highest HKDC1 expression were the brush border epithelium of the intestines, parts of the pancreas, and lung alveolar macrophages. Future directions will be to understand the role of this fifth hexokinase in these tissues, with a focus on its relative function as compared with other endogenously expressed hexokinases.

Molecular Cloning and Functional Characterization of a Hexokinase from the Oriental River Prawn Macrobrachium nipponense in Response to Hypoxia.

Metabolic adjustment to hypoxia in Macrobrachium nipponense (oriental river prawn) implies a shift to anaerobic metabolism. Hexokinase (HK) is a key glycolytic enzyme in prawns. The involvement of HK in the hypoxia inducible factors (HIFs) pathway is unclear in prawns. In this study, the full-length cDNA for HK (MnHK) was obtained from M. nipponense, and its properties were characterized. The full-length cDNA (2385 bp) with an open reading frame of 1350 bp, encoded a 450-amino acid protein. MnHK contained highly conserved amino acids in the glucose, glucose-6-phosphate, ATP, and Mg+2 binding sites. Quantitative real-time reverse transcription PCR assays revealed the tissue-specific expression pattern of MnHK, with abundant expression in the muscle, and gills. Kinetic studies validated the hexokinase activity of recombinant HK. Silencing of HIF-1α or HIF-1ß subunit genes blocked the induction of HK and its enzyme activities during hypoxia in muscles. The results suggested that MnHK is a key factor that increases the anaerobic rate, and is probably involved in the HIF-1 pathway related to highly active metabolism during hypoxia.

Hexokinase 2 drives glycogen accumulation in equine endometrium at day 12 of diestrus and pregnancy.

BACKGROUND: Secretion of histotroph during the prolonged pre-implantation phase in mares is crucial to pregnancy maintenance, manifested as increased embryonic loss in mares with age-related endometrial degeneration. Glycogen content of uterine histotroph is higher during the progesterone-dominated phase of the estrous cycle in mares, but regulatory mechanisms are not well understood. METHODS: mRNA expression of glycogen-metabolizing enzymes (HK1, HK2, GSK3B, GYS1, PEPCK, PKM, PYGM) in endometrial samples were compared among mares in anestrus, estrus, and at Day 12 of diestrus and pregnancy. In addition, hexokinase 2 (HK2) activity was assessed using a colorimetric assay. RESULTS: HK2 was the key regulator of glycogen accumulation during diestrus and pregnancy; hexokinase transcript abundance and enzyme activity were significantly higher during diestrus and pregnancy than estrus and anestrus. In addition, despite similar relative transcript abundance, hexokinase activity was significantly greater in the pregnant versus diestrous endometrium. Therefore, we inferred there was regulation of hexokinase activity through phosphorylation, in addition to its regulation at the transcriptional level during early pregnancy. Based on immunohistochemistry, HK2 was localized primarily in luminal and glandular epithelial cells, with weaker staining in stromal cells. CONCLUSION: Among glycogen metabolizing enzymes identified, expression of HK2 was significantly greater during the progesterone-dominated phase of the cycle.

Hexokinase and phosphofructokinase activity and intracellular distribution correlate with aggressiveness and invasiveness of human breast carcinoma.

Glycolytic enzymes, such as hexokinase and phosphofructokinase, have been reported to be upregulated in many cancer types. Here, we evaluated these two enzymes in 54 breast cancer samples collected from volunteers subjected to mastectomy, and the results were correlated with the prognosis markers commonly used. We found that both enzymes positively correlate with the major markers for invasiveness and aggressiveness. For invasiveness, the enzymes activities increase in parallel to the tumor size. Moreover, we found augmented activities for both enzymes when the samples were extirpated from patients presenting lymph node involvement or occurrence of metastasis. For aggressiveness, we stained the samples for the estrogen and progesterone receptors, HER-2, p53 and Ki-67. The enzyme activities positively correlated with all markers but Ki-67. Finally, we conclude that these enzymes are good markers for breast cancer prognosis.

System Accuracy Evaluation of Different Blood Glucose Monitoring Systems Following ISO 15197:2013 by Using Two Different Comparison Methods.

BACKGROUND: Adherence to established standards (e.g., International Organization for Standardization [ISO] 15197) is important to ensure comparable and sufficient accuracy of systems for self-monitoring of blood glucose (SMBG). Accuracy evaluation was performed for different SMBG systems available in Europe with three reagent lots each. MATERIALS AND METHODS: Test procedures followed the recently published revision ISO 15197:2013. Comparison measurements were performed with a glucose oxidase (YSI 2300 STAT Plus™ glucose analyzer; YSI Inc., Yellow Springs, OH) and a hexokinase (cobas Integra(®) 400 Plus analyzer; Roche Instrument Center, Rotkreuz, Switzerland) method. Compliance with ISO 15197:2013 accuracy criteria was determined by calculating the percentage of results within ±15% or within ±0.83 mmol/L of the comparison measurement results for glucose concentrations at and above or below 5.55 mmol/L, respectively, and by calculating the percentage of results within consensus error grid Zones A and B. RESULTS: Seven systems showed with all three tested lots that 95-100% of the results were within the accuracy limits of ISO 15197:2013 and that 100% of results were within consensus error grid Zones A and B, irrespective of the comparison method used. Regarding results of individual lots, slight differences between the glucose oxidase method and the hexokinase method were found. Accuracy criteria of ISO 15197:2003 (±20% for concentrations ≥4.2 mmol/L and±0.83 mmol/L for concentrations <4.2 mmol/L) were fulfilled by eight systems with all three lots and by one system with two lots. CONCLUSIONS: In this study, seven systems complied with the accuracy criteria of ISO 15197:2013. The results also indicate that the comparison measurement method/system is important, as it may have a considerable impact on accuracy data obtained for a system.

HK2 is a radiation resistant and independent negative prognostic factor for patients with locally advanced cervical squamous cell carcinoma.

The mechanism by which overexpression of hexokinase 2 (HK2) indicates locally advanced cervical squamous cell carcinoma (LACSCC) with radio-resistance is still unknown despite being an independent biomarker of poor prognosis. Here, we retrospectively analyzed 132 female patients receiving radiotherapy for cervical squamous cell carcinoma including 85 radiation-sensitive cases and 47 radiation-resistant cases. The expression of HK2 was examined by immunohistochemistry. The percentage of high HK2 expression in the radiation-resistant group differed from the radiation-sensitive group with statistical significance (P < 0.001) even if divided into three subgroups including a lower 5-year progression free survival group (PFS) for comparison (P < 0.001). The Kaplan Meier curve analysis showed that there were differences between the two groups (P < 0.001). Therefore, this study proves a close relationship between HK2 expression and radio-resistance. Multivariate Cox regression analysis implied that HK2 was an independent prognostic indicator of cervical squamous carcinoma (HR (95% CI), 2.940 (1.609, 1.609); P = 0.002).

Evidence for heightened hexokinase II immunoexpression in hepatocyte dysplasia and hepatocellular carcinoma.

BACKGROUND: Normal hepatocytes exhibit low-affinity hexokinase (glucokinase [HKIV]), but during oncogenesis, there is a switch from HKIV to HKII expression. The aims of this study were to compare the immunoexpression of HKII in non-dysplastic cirrhosis (NDC), liver cell change/dysplasia in cirrhosis (LCD), HCC, and normal liver control tissues, and to correlate HKII expression with clinical and histopathological parameters. DESIGN: Immunohistochemistry was performed on a liver cancer progression tissue array consisting of specimens from explants with cirrhosis, including 45 tissue samples with HCC, 108 without HCC, 143 with LCD, and 8 normal liver control tissues. HKII expression was quantified as positive pixel counts/square millimeter (ppc/mm(2)) by image analysis. RESULTS: There was a stepwise increase in HKII level from normal liver tissue to NDC, to LCD, and to HCC (p = 0.001). HKII levels were significantly higher in areas of LCD versus NDC (p ≤ 0.001), and in LCD and HCC versus NDC (p = 0.007). HKII levels were similar in LCD and HCC (p = 0.124). HKII levels were higher in grade 2-4 versus grade 1 HCCs (p = 0.044), and in pleomorphic versus non-pleomorphic HCC variants (p = 0.041). Higher levels of HKII expression in LCD and HCC versus NDC and in higher tumor grade remained significant in multivariate analysis. CONCLUSIONS: Higher levels of HKII immunoexpression in LDC and HCC compared with NDC suggest that upregulation of HKII occurs during the process of hepatocarcinogenesis in humans. In HCC, higher levels of HKII are associated with more aggressive histological features.

Expression of hexokinase 2 in epithelial ovarian tumors and its clinical significance in serous ovarian cancer.

"Warburg effect" emphasizes that malignant cells exhibit active glycolysis even under aerobic conditions. Hexokinase 2 (HK2) is a key glycolytic enzyme that helps to exhibit a "Warburg effect". In the present study, the main aim was to detect the expression of HK2 in epithelial ovarian tumor tissues. Immunohistochemistry and qRT-PCR were used to examine the expression of HK2 in different epithelial ovarian tissues. The expression of HK2 in ovarian cancer tissues was significantly higher than that in normal ovarian, benign, and borderline tumors both in protein (p < 0.001) and mRNA (p < 0.05) levels. HK2 expression was significantly higher in Stage III/IV compared to Stage III (p < 0.001). Expression of HK2 in poorly-differentiated carcinoma was higher than that in well-differentiated carcinoma (p = 0.008). The level of HK2 was higher in serous groups than in non-serous groups in both protein (p = 0.008) and mRNA (p < 0.05) level. Collectively, HK2 is highly expressed in epithelial ovarian cancer, especially in serous groups. Its expression is related with clinical stage and histological differentiation.

Expression patterns of three regulation enzymes in glycolysis in esophageal squamous cell carcinoma: association with survival.

Enhanced glycolysis is a common trait of many types of human cancers. This study was to detect the expression pattern of three regulatory enzymes during glycolysis in esophageal squamous cell carcinoma (ESCC) and to investigate their correlation with patients' outcome based on banked pathology material. A total of 141 surgically resected specimens of primary ESCC patients without prior treatments were retrospectively recruited from the First Affiliated Hospital of Wenzhou Medical College Hospital from 2007 to 2009. Expression of HK1, PFKB, and PKM2 in ESCC specimens was analyzed by immunohistochemical staining and Western blotting analysis. HK1-shRNA was used to knock down HK1 expression in ESCC cells, and the functional significance was assessed by CCK8 assay. It was found that the expression of two glycolytic enzymes, HK1 and PKM2, was associated with disease progression, invasion, and poor survival of patients with ESCC. Silence of HK1-inhibited cell proliferation in vitro and suppressed phospho-S6 kinase expression. Our findings suggest that activation of key enzymes in glycolysis might serve as potential therapeutic targets and/or prognostic factors for patients with ESCC.

Techniques to monitor glycolysis.

An increased flux through glycolysis supports the proliferation of cancer cells by providing additional energy in the form of ATP as well as glucose-derived metabolic intermediates for nucleotide, lipid, and protein biosynthesis. Thus, glycolysis and other metabolic pathways that control cell proliferation may represent valuable targets for therapeutic interventions and diagnostic procedures. In this context, the measurement of glucose uptake and lactate excretion by malignant cells may be useful to detect shifts in glucose catabolism, while determining the activity of rate-limiting glycolytic enzymes can provide insights into points of metabolic regulation. Moreover, metabolomic studies can be used to generate large, integrated datasets to track changes in carbon flux through glycolysis and its collateral anabolic pathways. As discussed here, these approaches can reveal and quantify the metabolic alterations that underlie malignant cell proliferation.

Hexokinase 2 overexpression promotes the proliferation and survival of laryngeal squamous cell carcinoma.

Proliferating cancer cells preferentially use anaerobic glycolysis rather than oxidative phosphorylation for energy production. Hexokinase 2 (HK2) is highly expressed in many malignant cells and is necessary for anaerobic glycolysis. The role of HK2 in laryngeal squamous cell carcinoma (LSCC) is unknown. In this study, the expression of HK2 in LSCC was investigated and the effect of inhibiting HK2 expression with small hairpin RNA (shRNA) on tumor growth was investigated. Using immunohistochemistry, HK2 expression was assessed in LSCC tissues. Human laryngeal carcinoma Hep-2 cells were stably transfected with a plasmid expressing HK2 shRNA (pGenesil-1.1-HK2) and were compared to control cells with respect to the cell cycle, cell viability, apoptosis, and their ability to form xenograft tumors. HK2 expression was significantly higher in LSCC than in papilloma or glottis polypus. Tumor samples of higher T, N, and TNM stage often had stronger HK2 staining. HK2 shRNA reduced HK2 mRNA, protein levels, and HK activity in Hep-2 cells. HK2 cells expressing shRNA demonstrated a higher G0-G1 ratio, increased apoptosis, and reduced viability. Xenograft tumors derived from cells expressing HK2 shRNA were smaller and had lower proliferation than those from untransfected or control-plasmid-transfected cells. In conclusion, depletion of HK2 expression resulted in reduced xenograft tumor development likely by reducing proliferation, altering the cell cycle, reducing cell viability and activating apoptosis. These data suggest that HK2 plays an important role in the development of LSCC and represents a potential therapeutic target for LSCC.

Molecular mechanisms of [18F]fluorodeoxyglucose accumulation in liver cancer.

To elucidate the molecular mechanisms underlying the insufficient sensitivity in the detection of hepatocellular carcinoma (HCC) by [18F] 2-fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET), the characteristics of glucose metabolism-related protein expression in HCC were examined in liver metastasis from colorectal cancer (Meta). Thirty-four patients (14 Meta and 20 HCC) who underwent FDG-PET and hepatectomy were studied. The relationships between the maximum standardized uptake value (SUV) in tumors and the mRNA expression of glucose metabolism-related proteins [hexokinase (HK), glucose transporter 1 (GLUT1), and glucose-6-phosphatase (G6Pase)] and proliferating cell nuclear antigen (PCNA) were examined in snap-frozen specimens with quantitative PCR. Tumor detection rates were lower in HCC (15/20) compared to Meta (13/14) patients. HK and GLUT1 expression was lower and G6Pase expression was higher in HCC compared to Meta. In particular, GLUT1 overexpression was 92-fold in Meta and 11-fold in HCC compared to the surrounding liver. The SUV correlated with GLUT1 and PCNA expression in HCC, but not Meta patients. Of note, four cases of poorly differentiated (P/D) HCC compared to moderately differentiated (M/D) HCC produced completely different results for FDG uptake (SUV, 14.4 vs. 4.0) and mRNA expression (G6Pase expression, 0.007 vs. 1.5). Variations in the expression of glucose metabolism-related enzymes between HCC and Meta patients are attributed to origin or degree of differentiation. Low FDG uptake in M/D HCC reflected low GLUT1 and high G6Pase expression, while high FDG accumulation in P/D HCC could reflect increased GLUT1 and decreased G6Pase expression. These results may explain why M/D HCC is not detected as sensitively by FDG-PET.

Development of assays using hexokinase and phosphoglucomutase gene sequences that distinguish strains of Leishmania tropica from different zymodemes and microsatellite clusters and their application to Palestinian foci of cutaneous leishmaniasis.

BACKGROUND/OBJECTIVES: Palestinian strains of L.tropica characterized by multilocus enzyme electrophoresis (MLEE) fall into two zymodemes, either MON-137 or MON-307. METHODOLOGY/PRINCIPLE FINDINGS: Assays employing PCR and subsequent RFLP were applied to sequences found in the Hexokinase (HK) gene, an enzyme that is not used in MLEE, and the Phosphoglucomutase (PGM) gene, an enzyme that is used for MLEE, to see if they would facilitate consigning local strains of L.tropica to either zymodeme MON-137 or zymodeme MON-307. Following amplification and subsequent double digestion with the restriction endonucleases MboI and HaeIII, variation in the restriction patterns of the sequence from the HK gene distinguished strains of L.tropica, L.major and L.infantum and also exposed two genotypes (G) among the strains of L.tropica: HK-LtG1, associated with strains of L.tropica of the zymodemes MON-137 and MON-265, and HK-LtG2, associated with strains of L.tropica of the zymodemes MON-307, MON-288, MON-275 and MON-54. Following amplification and subsequent digestion by the restriction endonuclease MboI, variation in the sequence from the PGM gene also exposed two genotypes among the strains of L.tropica: PGM-G1, associated only with strains of L.tropica of the zymodeme MON-137; and PGM-G2, associated with strains of L.tropica of the zymodemes MON-265, MON-307, MON-288, MON-275 and MON-54, and, also, with six strains of L.major, five of L.infantum and one of L.donovani. The use of the HK and PGM gene sequences enabled distinction the L.tropica strains of the zymodeme MON-137 from those of the zymodeme MON-265. This genotyping system 'correctly' identified reference strains of L.tropica of known zymodemal affiliation and also from clinical samples, with a level of sensitivity down to <1 fg in the case of the former and to 1 pg of DNA in the case of the latter. CONCLUSIONS/SIGNIFICANCE: Both assays proved useful for identifying leishmanial parasites in clinical samples without resource to culture and MLEE.