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Slagter, Margrita H., Andreas Scorilas, Louis JG Gooren, et al. "Effect of Testosterone Administration on Serum and Urine Kallikrein Concentrations in Female-to-Male Transsexuals." Clinical Chemistry 52, no. 8 (2006): 1546–51. http://dx.doi.org/10.1373/clinchem.2006.067041.
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Abstract Background: Concentrations of human tissue kallikreins (hKs), a group of 15 secreted serine proteases found in many tissues, are modulated by steroid hormones in cancer cell lines. To gain insight into in vivo kallikrein regulation we measured kallikrein concentrations in serum and urinary tissue in female-to-male transsexuals before and after testosterone administration. Methods: We collected blood and urine samples before treatment and after 4 and 12 months from 28 female-to-male transsexuals who received 250 mg of testosterone esters intramuscularly every 2 weeks. We used ELISA assays to measure multiple kallikreins in serum and urine. Results: After testosterone administration, serum testosterone concentrations increased by ∼15-fold. Serum kallikrein concentrations increased dramatically for hK3 (prostate-specific antigen) and increased moderately for hK2, hK5, hK6, hK7, hK8, hK10, and hK11. In urine, we noted major increases for hK3 and hK2 only. For all other kallikrein concentrations, we observed no considerable changes. Conclusions: We conclude that, in serum and urine of female-to-male transsexuals after testosterone administration, hK3 (prostate-specific antigen) and to a lesser extent hK2 concentrations increase dramatically, but concentration of other kallikreins increase either moderately in serum (hK5, hK6, hK7, hK8, hK10, and hK11) or not at all in either serum (hK4, hK13, hK14) or urine (hK4, hK5, hK6, hK7, hK8, hK10, hK11, hK13, hK14).
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Danno, Katsunori, and Tsunenobu Kimoto. "Deep Hole Traps in As-Grown 4H-SiC Epilayers Investigated by Deep Level Transient Spectroscopy." Materials Science Forum 527-529 (October 2006): 501–4. http://dx.doi.org/10.4028/www.scientific.net/msf.527-529.501.
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Deep levels in as-grown p-type 4H-SiC epilayers have been investigated by DLTS. Three deep hole traps (HK2, HK3 and HK4) can be detected by DLTS in the temperature range from 350K to 700K. They are energetically located at 0.84 eV (HK2), 1.27 eV (HK3) and 1.44 eV (HK4) above the valence band edge. The activation energy of the traps does not show any meaningful change regardless of applied electric field, indicating that the charge state of the deep hole traps may be neutral after hole emission (donor-like). By the low-energy electron irradiation, the HK3 and HK4 concentrations are significantly increased, suggesting that the origins of the HK3 and HK4 may be related to carbon displacement. Study on the thermal stability of these hole traps has revealed that the trap concentrations of HK3 and HK4 are reduced to below the detection limit (1-2 × 1011 cm-3) by annealing at 1350°C. The HK2 is thermally more stable than HK3 and HK4, and becomes lower than the detection limit by annealing at 1550°C.
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Chee, Jessica, Jasmine Singh, Anupam Naran, Neil L. Misso, Philip J. Thompson, and Kanti D. Bhoola. "Novel expression of kallikreins, kallikrein-related peptidases and kinin receptors in human pleural mesothelioma." Biological Chemistry 388, no. 11 (2007): 1235–42. http://dx.doi.org/10.1515/bc.2007.139.
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Abstract Malignant mesothelioma is an aggressive cancer of the pleura that is causally related to exposure to asbestos fibres. The kallikrein serine proteases [tissue (hK1) and plasma (hKB1) kallikreins, and kallikrein-related peptidases (KRP/hK2–15)] and the mitogenic kinin peptides may have a role in tumourigenesis. However, it is not known whether hK1, hKB1, KRP/hK proteins or kinin receptors are expressed in pleural mesotheliomas. The expression of hK1, hKB1, KRP/hK2, 5, 6, 7, 8 and 9, and kinin B1 and B2 receptors was assessed in archived selected normal tissue and mesothelioma tumour sections by immunoperoxidase and immunofluorescence labelling. hK1, hKB1 and kinin B1 and B2 receptors were expressed in malignant cells of the epithelioid and sarcomatoid components of biphasic mesothelioma tumour cells. The percentage of cells with cytoplasmic and nuclear labelling and the intensity of labelling were similar for hK1, hKB1 and the kinin receptors. KRP/hK2, 6, 8 and 9 were also expressed in the cytoplasm and nuclei of mesothelioma cells, whereas KRP/hK5 and hK7 showed predominantly cytoplasmic localisation. This is a first report, but further studies are required to determine whether these proteins have a functional role in the pathogenesis of mesothelioma and/or may be potential biomarkers for pleural mesothelioma.
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Marshall, Aaron J., Edgard Mejia, Sen Hou, Affan Sher, Grant Hatch, and Michel Aliani. "PI3K-dependent Reprogramming of Hexokinase Isoforms Regulates B Lymphocyte Metabolism." Journal of Immunology 204, no. 1_Supplement (2020): 151.30. http://dx.doi.org/10.4049/jimmunol.204.supp.151.30.
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Abstract The PI3K signalling pathway is known to regulate B cell metabolic programming upon activation, but the mechanisms involved are not well understood. Here we find that the PI3K pathway controls reprogramming of hexokinases (HKs), the enzymes that convert glucose into glucose-6-phosphate as a key rate-limiting step of glycolysis and other metabolic pathways. In primary mouse B cells, PI3K pathway inhibition substantially impaired the activation-induced increase in extracellular acidification rates (ECAR), a measure of glycolysis. In contrast, B cells isolated from PI3Kdelta gain-of-function mutant mice exhibit elevated ECAR. We find that B cell activation substantially elevates protein levels of HK2 and HK3 isoforms, but not HK1, in a PI3K-dependant manner. PI3K or mTOR inhibition significantly reduced induction of HK2 and HK3 expression, whereas Akt inhibition did not affect HK isoform expression. In human B lymphoma cells, HK isoforms differ significantly in their degree of mitochondrial localization, with HK1 being mitochondrial, HK3 being cytoplasmic and HK2 present in both mitochondria and cytoplasm. To assess whether HK isoforms have unique non-redundant functions, HK2-deficient B lymphoma cells were generated and were found to exhibit decreased ECAR as well as significant changes in metabolomic profile, despite normal expression and localization of HK1 and HK3. Taken together, our study reveals that PI3K-dependant reprogramming of hexokinase isoforms can impact on B cell glycolysis and other metabolic pathways. Studies in progress are examining the functional importance of HK2 in antibody responses using mice with B cell-specific deletion of this enzyme.
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Bustamante, Marta F., Patricia G. Oliveira, Ricard Garcia-Carbonell, et al. "Hexokinase 2 as a novel selective metabolic target for rheumatoid arthritis." Annals of the Rheumatic Diseases 77, no. 11 (2018): 1636–43. http://dx.doi.org/10.1136/annrheumdis-2018-213103.
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ObjectivesRecent studies indicate that glucose metabolism is altered in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). Hexokinases (HKs) catalyse the first step in glucose metabolism, and HK2 constitutes the principal HK inducible isoform. We hypothesise that HK2 contributes to the synovial lining hypertrophy and plays a critical role in bone and cartilage damage.MethodsHK1 and HK2 expression were determined in RA and osteoarthritis (OA) synovial tissue by immunohistochemistry. RA FLS were transfected with either HK1 or HK2 siRNA, or infected with either adenovirus (ad)-GFP, ad-HK1 or ad-HK2. FLS migration and invasion were assessed. To study the role of HK2 in vivo, 108 particles of ad-HK2 or ad-GFP were injected into the knee of wild-type mice. K/BxN serum transfer arthritis was induced in HK2F/F mice harbouring Col1a1-Cre (HK2Col1), to delete HK2 in non-haematopoietic cells.ResultsHK2 is particular of RA histopathology (9/9 RA; 1/8 OA) and colocalises with FLS markers. Silencing HK2 in RA FLS resulted in a less invasive and migratory phenotype. Consistently, overexpression of HK2 resulted in an increased ability to migrate and invade. It also increased extracellular lactate production. Intra-articular injection of ad-HK2 in normal knees dramatically increased synovial lining thickness, FLS activation and proliferation. HK2 was highly expressed in the synovial lining after K/BxN serum transfer arthritis. HK2Col1 mice significantly showed decreased arthritis severity, bone and cartilage damage.ConclusionHK2 is specifically expressed in RA synovial lining and regulates FLS aggressive functions. HK2 might be an attractive selective metabolic target safer than global glycolysis for RA treatment.
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Seiler, Kristina, Petra Minder, Iris Mashimo, et al. "Hexokinase Proteins Impart Distinct Functions in Myeloid Development and Cell Death." Blood 132, Supplement 1 (2018): 5088. http://dx.doi.org/10.1182/blood-2018-99-112145.
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Abstract Many key oncogenic pathways converge to adapt tumor cell metabolism to requirements of cancer cells. Aberrant proliferation that is frequently associated with cancer cells is also linked to an adjustment of metabolism in order to fuel cell growth and division. Cancer cells prefer utilizing glycolysis for energy production and providing essential building blocks for a variety of macromolecules. Hexokinases (HKs) are rate-limiting enzymes that catalyze the first and irreversible step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. Four HK isoforms are expressed in mammalian cells, HK1, HK2, HK3 and HK4 (also known as glucokinase). HKs promote and sustain a concentration gradient that facilitates glucose entry, which ensures the initiation of glucose dependent pathways. In general, HKs have a cytoprotective role that was highlighted by enhanced sensitivity of cancer cells to drugs when HKs were inhibited. Previously we have reported that HK3 was transcriptionally regulated by PU.1 (SPI-1) in myeloid cells. Further, HK3 expression was significantly reduced in patient acute myeloid leukemia (AML) cells, particularly in acute promyelocytic leukemia (APL) cells expressing the PML-RARA oncofusion protein. We now report on the expression and regulatory function of HKs, particularly HK3, during myeloid differentiation and granulocyte associated cell death. First, we analyzed mRNA HK levels in human CD34+ hematopoietic progenitors cells differentiated towards granulocytes or macrophages by qPCR. Interestingly, while HK1 and HK2 levels remain stable during all stages of myeloid differentiation, HK3 mRNA levels significantly increased. The same pattern of HK mRNA expression was seen in NB4 APL and HL60 AML cell lines differentiated towards granulocytes and monocytes using all-trans retinoic acid (ATRA) and vitamin D3, respectively. To determine a specific role for HK1-3 function in myeloid cells, HK1-3 knockdowns (KD) and knockouts (KO) in NB4 and HL60 cell lines, using shRNA or gRNAs (Cas9/CRISPR technology), respectively, were generated. NB4 HK KD AML cells were tested for their differentiation upon ATRA treatment. Knockdown of HKs generally resulted in a decreased differentiation response of about 20% as assessed by the differentiation marker CD11b. We next determined energy metabolism of HK altered KD and KO cells, relative to parental cells, using a seahorse analyzer. While lowering HK1 or HK3 levels in NB4 and HL60 AML cells did not affect glycolytic capacity at steady state, HK2 inhibition significantly reduced steady state glycolytic capacity. In contrast, knocking down or knocking out HK3 resulted in a higher sensitivity to ATRA-induced cell death during differentiation, which was coupled with higher glycolytic capacity. Together, our findings suggest that HK2 has an important role in steady state metabolism of AML cells while HK3 appears to be a metabolic switch for cell survival during myeloid differentiation. Disclosures No relevant conflicts of interest to declare.
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Shrivastava, Rashmi, Ananta Kumar Ghosh, and Amit Kumar Das. "Intra- and intermolecular domain interactions among novel two-component system proteins coded by Rv0600c, Rv0601c and Rv0602c of Mycobacterium tuberculosis." Microbiology 155, no. 3 (2009): 772–79. http://dx.doi.org/10.1099/mic.0.019059-0.
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Two-component signal transduction pathways comprising a histidine kinase and its cognate response regulator play a dominant role in the adaptation of Mycobacterium tuberculosis to its host, and its virulence, pathogenicity and latency. Autophosphorylation occurs at a conserved histidine of the histidine kinase and subsequently the phosphoryl group is transferred to the conserved aspartate of its cognate response regulator. Among the twelve two-component systems of M. tuberculosis, Rv0600c (HK1), Rv0601c (HK2) and Rv0602c (TcrA) are annotated as a unique three-protein two-component system. HK1 contains an ATP-binding domain, and HK2, a novel Hpt mono-domain protein, contains the conserved phosphorylable histidine residue. HK1 and HK2 complement each other's functions. Interactions among different domains of the HK1, HK2 and TcrA proteins were studied using a yeast two-hybrid system. Self-interaction was observed for HK2 but not for HK1 or TcrA. HK2 was found to interact reasonably well with both HK1 and TcrA, but HK1 interacted weakly with TcrA. The conserved aspartate-containing receiver domain of TcrA interacted well with HK2 but not with HK1. These results suggest the existence of a novel signalling mechanism amongst HK1–HK2–TcrA, and a model for this mechanism is proposed.
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Agnihotri, S., M. Li, M. R. Wilson, et al. "PS2 - 174 Hexokinase 2 Drives Radio-Resistance through ERK Signaling and Sensitizes Cells to Azole Compounds." Canadian Journal of Neurological Sciences / Journal Canadien des Sciences Neurologiques 43, S4 (2016): S14. http://dx.doi.org/10.1017/cjn.2016.368.
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Our ongoing work has demonstrated that hexokinase 2 (HK2) but not HK1 or HK3 is a critical mediator of tumour glycolysis and mitochondrial metabolism in Glioblastoma (GB). Furthermore, HK2 is highly expressed in GB but not in normal brain making it an attractive therapeutic target. Our current findings now support that loss of HK2 alters tumor vasculature, increases sensitivity to radiation, and confers a significant survival benefit in several GB xenograft-bearing mice. Using a genome wide transcript analysis, we identified that loss of HK2 attenuates several pro-growth signaling pathways in GB including ERK signaling. Mechanistically, ERK rescue experiments in HK2 depleted cells rescues cell sensitivity to radiation and reduces DNA damage. Furthermore using a systems biology approach and a rationale drug screen we identified several antifungal agents in the azole class as to inhibit tumor metabolism and growth in HK2 expressing GB cells. Loss of HK2 in GB cells dampened the effect of several azoles suggesting that the mechanism of action is mediated in part through HK2. Furthermore, we tested several azole compounds known to cross the blood brain barrier in vivo. Clinically achievable doses of azoles as single agents increased survival in several orthotopic xenograft GB mouse models. In summary, HK2 drives several oncogenic pathways associated with GB including ERK signaling and sensitizes tumour cells to the azole class of antifungals. Future work will determine whether azoles work synergistically with radiation and temozolomide and elucidate the mechanisms by which they inhibit GB growth in HK2 expressing cells.
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Wolf, Amparo, Sameer Agnihotri, Johann Micallef, et al. "Hexokinase 2 is a key mediator of aerobic glycolysis and promotes tumor growth in human glioblastoma multiforme." Journal of Experimental Medicine 208, no. 2 (2011): 313–26. http://dx.doi.org/10.1084/jem.20101470.
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Proliferating embryonic and cancer cells preferentially use aerobic glycolysis to support growth, a metabolic alteration commonly referred to as the “Warburg effect.” Here, we show that the glycolytic enzyme hexokinase 2 (HK2) is crucial for the Warburg effect in human glioblastoma multiforme (GBM), the most common malignant brain tumor. In contrast to normal brain and low-grade gliomas, which express predominantly HK1, GBMs show increased HK2 expression. HK2 expression correlates with worse overall survival of GBM patients. Depletion of HK2, but neither HK1 nor pyruvate kinase M2, in GBM cells restored oxidative glucose metabolism and increased sensitivity to cell death inducers such as radiation and temozolomide. Intracranial xenografts of HK2-depleted GBM cells showed decreased proliferation and angiogenesis, but increased invasion, as well as diminished expression of hypoxia inducible factor 1α and vascular endothelial growth factor. In contrast, exogenous HK2 expression in GBM cells led to increased proliferation, therapeutic resistance, and intracranial growth. Growth was dependent on both glucose phosphorylation and mitochondrial translocation mediated by AKT signaling, which is often aberrantly activated in GBMs. Collectively, these findings suggest that therapeutic strategies to modulate the Warburg effect, such as targeting of HK2, may interfere with growth and therapeutic sensitivity of some GBMs.
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Federzoni, Elena A., Peter Valk, Bob Löwenberg, Martin F. Fey, and Mario P. Tschan. "Linking the Glycolytic Enzyme HK3 to Neutrophil Differentiation of APL Cells Via PU.1." Blood 118, no. 21 (2011): 2425. http://dx.doi.org/10.1182/blood.v118.21.2425.2425.
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Abstract Abstract 2425 Objectives: Identification and characterization of hexokinase 3 (HK3), as novel transcriptional target of the myeloid master regulator PU.1, with a role in APL cell differentiation and survival. Materials: HK3 and PU.1 quantitative RT-PCR in primary AML patient samples (n=170) and granulocytes isolated from healthy donors (n=20). Gene expression profiling in PU.1-knockout and PU.1-restored myeloid cell lines. Generation of NB4 and HT93 PU.1 as well as HK3 knockdown cell lines. ATRA-induced differentiation of these cell lines was assessed by CD11b and CEBPE expression. In vivo binding of PU.1 and PML-RARA to the HK3 promoter was shown by ChIP assays. Viability of NB4 cell lines was assessed by Alamar Blue staining. Results: We identified HK3 as putative transcriptional target of PU.1 by gene expression profiling in PU.1-knockout and PU.1-restored myeloid cell lines. Unlike the other three hexokinase family members (HK1, HK2 and HK4), HK3 expression is limited to hematopoietic cells, where it is mainly expressed in the myeloid lineage. We found 50-times lower HK3 mRNA levels in primary non-APL patients as compared to granulocytes from healthy donors. Most interestingly, we found that HK3 expression in APL patients with the t(15;17) translocation was 400-times lower (p<0.0001) than compared to granulocytes. Given the particularly low HK3 and PU.1 expression in APL patients, we decided to investigate the role of HK3 and its possible regulation by PU.1 during all-trans retinoic acid (ATRA)-induced neutrophil differentiation of NB4 and HT93 APL cell lines. Firstly, we observed a 5500- and 1100-fold increase of HK3 mRNA levels during neutrophil differentiation of NB4 and HT93, respectively. This was paralleled by markedly increased HK3 protein levels. No increased HK3 levels were observed in ATRA-resistant NB4-R2 cell lines. HK1 and HK2 mRNA expression was not markedly increased during neutrophil differentiation pointing to a particular role for HK3 in neutrophil differentiation. Regulation of the liver specific HK4 was not investigated. Next, we observed significantly decreased HK3 expression, both at the mRNA and protein levels, in all PU.1 knockdown cell lines upon ATRA-induced differentiation. In vivo PU.1 binding to three different regions of the HK3 promoter was found. Given our findings in primary APL patients, we decided to test if PML-RARa binds to the HK3 promoter, and indeed, PML-RARA binds to the same promoter regions as PU.1. Knocking down HK3 in APL cell lines resulted in significantly reduced neutrophil differentiation as measured by CD11b and CEBPE expression. Also, we observed that NB4 HK3 knockdown cell lines treated with ATRA were less viable compared to control cells. Conclusions: Our results strongly suggest that HK3 is a novel PU.1 transcriptional target functionally involved in neutrophil differentiation and cell viability of APL cells. Disclosures: No relevant conflicts of interest to declare.
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Shi, Xuetao, Cheng Ye, Xiangfeng Qin, et al. "Novel Pituitary Actions of TAC4 Gene Products in Teleost." International Journal of Molecular Sciences 22, no. 23 (2021): 12893. http://dx.doi.org/10.3390/ijms222312893.
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Tachykinin 4 (TAC4) is the latest member of the tachykinin family involved in several physiological functions in mammals. However, little information is available about TAC4 in teleost. In the present study, we firstly isolated TAC4 and six neurokinin receptors (NKRs) from grass carp brain and pituitary. Sequence analysis showed that grass carp TAC4 could encode two mature peptides (namely hemokinin 1 (HK1) and hemokinin 2 (HK2)), in which HK2 retained the typical FXGLM motif in C-terminal of tachyinin, while HK1 contained a mutant VFGLM motif. The ligand-receptor selectivity showed that HK2 could activate all 6 NKRs but with the highest activity for the neurokinin receptor 2 (NK2R). Interestingly, HK1 displayed a very weak activation for each NKR isoform. In grass carp pituitary cells, HK2 could induce prolactin (PRL), somatolactin α (SLα), urotensin 1 (UTS1), neuromedin-B 1 (NMB1), cocaine- and amphetamine-regulated transcript 2 (CART2) mRNA expression mediated by NK2R and neurokinin receptor 3 (NK3R) via activation cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), phospholipase C (PLC)/inositol 1,4,5-triphosphate (IP3)/protein kinase C (PKC) and calcium2+ (Ca2+)/calmodulin (CaM)/calmodulin kinase-II (CaMK II) cascades. However, the corresponding stimulatory effects triggered by HK1 were found to be notably weaker. Furthermore, based on the structural base for HK1, our data suggested that a phenylalanine (F) to valine (V) substitution in the signature motif of HK1 might have contributed to its weak agonistic actions on NKRs and pituitary genes regulation.
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Garcia, Sara N., Rita C. Guedes, and M. Matilde Marques. "Unlocking the Potential of HK2 in Cancer Metabolism and Therapeutics." Current Medicinal Chemistry 26, no. 41 (2020): 7285–322. http://dx.doi.org/10.2174/0929867326666181213092652.
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:Glycolysis is a tightly regulated process in which several enzymes, such as Hexokinases (HKs), play crucial roles. Cancer cells are characterized by specific expression levels of several isoenzymes in different metabolic pathways and these features offer possibilities for therapeutic interventions. Overexpression of HKs (mostly of the HK2 isoform) have been consistently reported in numerous types of cancer. Moreover, deletion of HK2 has been shown to decrease cancer cell proliferation without explicit side effects in animal models, which suggests that targeting HK2 is a viable strategy for cancer therapy. HK2 inhibition causes a substantial decrease of glycolysis that affects multiple pathways of central metabolism and also destabilizes the mitochondrial outer membrane, ultimately enhancing cell death. Although glycolysis inhibition has met limited success, partly due to low selectivity for specific isoforms and excessive side effects of the reported HK inhibitors, there is ample ground for progress.:The current review is focused on HK2 inhibition, envisaging the development of potent and selective anticancer agents. The information on function, expression, and activity of HKs is presented, along with their structures, known inhibitors, and reported effects of HK2 ablation/inhibition. The structural features of the different isozymes are discussed, aiming to stimulate a more rational approach to the design of selective HK2 inhibitors with appropriate drug-like properties. Particular attention is dedicated to a structural and sequence comparison of the structurally similar HK1 and HK2 isoforms, aiming to unveil differences that could be explored therapeutically. Finally, several additional catalytic- and non-catalytic roles on different pathways and diseases, recently attributed to HK2, are reviewed and their implications briefly discussed.
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Xu, Shili, Tianyuan Zhou, Hanna M. Doh, et al. "An HK2 Antisense Oligonucleotide Induces Synthetic Lethality in HK1−HK2+ Multiple Myeloma." Cancer Research 79, no. 10 (2019): 2748–60. http://dx.doi.org/10.1158/0008-5472.can-18-2799.
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Veveris-Lowe, T. L., M. G. Lawrence, R. L. Collard, et al. "Kallikrein 4 (hK4) and prostate-specific antigen (PSA) are associated with the loss of E-cadherin and an epithelial-mesenchymal transition (EMT)-like effect in prostate cancer cells." Endocrine-Related Cancer 12, no. 3 (2005): 631–43. http://dx.doi.org/10.1677/erc.1.00958.
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Prostate-specific antigen (PSA) and the related kallikrein family of serine proteases are current or emerging biomarkers for prostate cancer detection and progression. Kallikrein 4 (KLK4/hK4) is of particular interest, as KLK4 mRNA has been shown to be elevated in prostate cancer. In this study, we now show that the comparative expression of hK4 protein in prostate cancer tissues, compared with benign glands, is greater than that of PSA and kallikrein 2 (KLK2/hK2), suggesting that hK4 may play an important functional role in prostate cancer progression in addition to its biomarker potential. To examine the roles that hK4, as well as PSA and hK2, play in processes associated with progression, these kallikreins were separately transfected into the PC-3 prostate cancer cell line, and the consequence of their stable transfection was investigated. PC-3 cells expressing hK4 had a decreased growth rate, but no changes in cell proliferation were observed in the cells expressing PSA or hK2. hK4 and PSA, but not hK2, induced a 2.4-fold and 1.7-fold respective increase, in cellular migration, but not invasion, through Matrigel, a synthetic extracellular matrix. We hypothesised that this increase in motility displayed by the hK4 and PSA-expressing PC-3 cells may be related to the observed change in structure in these cells from a typical rounded epithelial-like cell to a spindle-shaped, more mesenchymal-like cell, with compromised adhesion to the culture surface. Thus, the expression of E-cadherin and vimentin, both associated with an epithelial-mesenchymal transition (EMT), was investigated. E-cadherin protein was lost and mRNA levels were significantly decreased in PC-3 cells expressing hK4 and PSA (10-fold and 7-fold respectively), suggesting transcriptional repression of E-cadherin, while the expression of vimentin was increased in these cells. The loss of E-cadherin and associated increase in vimentin are indicative of EMT and provides compelling evidence that hK4, in particular, and PSA have a functional role in the progression of prostate cancer through their promotion of tumour cell migration.
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Pan, Minglin, Ying Han, Aninda Basu, et al. "Overexpression of hexokinase 2 reduces mitochondrial calcium overload in coronary endothelial cells of type 2 diabetic mice." American Journal of Physiology-Cell Physiology 314, no. 6 (2018): C732—C740. http://dx.doi.org/10.1152/ajpcell.00350.2017.
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Coronary microvascular rarefaction, due to endothelial cell (EC) dysfunction, is one of the causes of increased morbidity and mortality in diabetes. Coronary ECs in diabetes are more apoptotic due partly to mitochondrial calcium overload. This study was designed to investigate the role of hexokinase 2 (HK2, an endogenous inhibitor of voltage-dependent anion channel) in coronary endothelial dysfunction in type 2 diabetes. We used mouse coronary ECs (MCECs) isolated from type 2 diabetic mice and human coronary ECs (HCECs) from type 2 diabetic patients to examine protein levels and mitochondrial function. ECs were more apoptotic and capillary density was lower in the left ventricle of diabetic mice than the control. MCECs from diabetic mice exhibited significant increase in mitochondrial Ca2+ concentration ([Ca2+]mito) compared with the control. Among several regulatory proteins for [Ca2+]mito, hexokinase 1 (HK1) and HK2 were significantly lower in MCECs from diabetic mice than control MCECs. We also found that the level of HK2 ubiquitination was higher in MCECs from diabetic mice than in control MCECs. In line with the data from MCECs, HCECs from diabetic patients showed lower HK2 protein levels than HCECs from nondiabetic patients. High-glucose treatment, but not high-fat treatment, significantly decreased HK2 protein levels in MCECs. HK2 overexpression in MCECs of diabetic mice not only lowered the level of [Ca2+]mito, but also reduced mitochondrial reactive oxygen species production toward the level seen in control MCECs. These data suggest that HK2 is a potential therapeutic target for coronary microvascular disease in diabetes by restoring mitochondrial function in coronary ECs.
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Prezas, Panagiotis, Matthias J. E. Arlt, Petar Viktorov, et al. "Overexpression of the human tissue kallikrein genes KLK4, 5, 6, and 7 increases the malignant phenotype of ovarian cancer cells." Biological Chemistry 387, no. 6 (2006): 807–11. http://dx.doi.org/10.1515/bc.2006.102.
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Abstract The human tissue kallikrein family of serine proteases (hK1–hK15 encoded by the genes KLK1–KLK15) is involved in several cancer-related processes. Accumulating evidence suggests that certain tissue kallikreins are part of an enzymatic cascade pathway that is activated in ovarian cancer and other malignant diseases. In the present study, OV-MZ-6 ovarian cancer cells were stably co-transfected with plasmids expressing hK4, hK5, hK6, and hK7. These cells displayed similar proliferative capacity as the vector-transfected control cells (which do not express any of the four tissue kallikreins), but showed significantly increased invasive behavior in an in vitro Matrigel invasion assay (p<0.01; Mann-Whitney U-test). For in vivo analysis, the cancer cells were inoculated into the peritoneum of nude mice. Simultaneous expression of hK4, hK5, hK6, and hK7 resulted in a remarkable 92% mean increase in tumor burden compared to the vector-control cell line. Five out of 14 mice in the ‘tissue kallikrein overexpressing’ group displayed a tumor/situs ratio greater than 0.198, while this weight limit was not exceeded at all in the vector control group consisting of 13 mice (p=0.017; χ2 test). Our results strongly support the view that tumor-associated overexpression of tissue kallikreins contributes to ovarian cancer progression.
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Väisänen, Ville, Susann Eriksson, Kaisa K. Ivaska, Hans Lilja, Martti Nurmi, and Kim Pettersson. "Development of Sensitive Immunoassays for Free and Total Human Glandular Kallikrein 2." Clinical Chemistry 50, no. 9 (2004): 1607–17. http://dx.doi.org/10.1373/clinchem.2004.035253.
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Abstract Background: Free and total human kallikrein 2 (hK2) might improve the discrimination between prostate cancer and benign prostatic hyperplasia. Concentrations of hK2 are 100-fold lower than concentrations of prostate-specific antigen (PSA); therefore, an hK2 assay must have a low detection limit and good specificity. Methods: PSA- and hK2-specific monoclonal antibodies were used in solid-phase, two-site immunofluorometric assays to detect free and total hK2. The total hK2 assay used PSA-specific antibodies to block nonspecific signal. The capture antibody of the free hK2 assay did not cross-react with PSA. To determine the hK2 concentrations in the male bloodstream, total hK2 was measured in a control group consisting of 426 noncharacterized serum samples. Free and total hK2 were measured in plasma from 103 patients with confirmed prostate cancer. Results: All 426 males in the control group had a total hK2 concentration above the detection limit of 0.0008 μg/L. The median total hK2 concentration was 0.022 μg/L (range, 0.0015–0.37 μg/L). hK2 concentrations were 0.1–58% of total PSA (median, 3.6%). hK2 concentrations were similar in men 41–50 and 51–60 years of age. The ratio of hK2 to PSA steadily decreased from 5–30% at PSA &lt;1 μg/L to 1–2% at higher PSA concentrations. In 103 patients with prostate cancer, the median hK2 concentration in plasma was 0.079 μg/L (range, 0.0015–16.2 μg/L). The median free hK2 concentration was 0.070 (range, 0.005–12.2) μg/L. The proportion of free to total hK2 varied from 17% to 131% (mean, 85%). Conclusions: The wide variation in the free-to-total hK2 ratio suggests that hK2 in blood plasma is not consistently in the free, noncomplexed form in patients with prostate cancer. The new assay is sufficiently sensitive to be used to study the diagnostic accuracies of free and total hK2 for prostate cancer.
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Contreras-Galindo, Rafael, Mark H. Kaplan, Derek Dube, et al. "Human Endogenous Retrovirus Type K (HERV-K) Particles Package and Transmit HERV-K–Related Sequences." Journal of Virology 89, no. 14 (2015): 7187–201. http://dx.doi.org/10.1128/jvi.00544-15.
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ABSTRACTHuman endogenous retroviruses (HERV) make up 8% of the human genome. While the youngest of these retroviruses, HERV-K(HML-2), termed HK2, is able to code for all viral proteins and produce virus-like particles, it is not known if these virus particles package and transmit HK2-related sequences. Here, we analyzed the capacity of HK2 for packaging and transmitting HK2 sequences. We created an HK2 probe, termed Bogota, which can be packaged into HK2 viruses, and transfected it into cells that make HK2 particles. Supernatants of the transfected cells, which contained HK2 viral particles, then were added to target cells, and the transmissibility of the HK2 Bogota reporter was tracked by G418 resistance. Our studies revealed that contemporary HK2 virions produced by some teratocarcinoma and breast cancer cell lines, as well as by peripheral blood lymphocytes from lymphoma patients, can package HK2 Bogota probes, and these viruses transmitted these probes to other cells. After transmission, HK2 Bogota transcripts undergo reverse transcription, a step impaired by antiretroviral agents or by introduction of mutations into the probe sequences required for reverse transcription. HK2 viruses were more efficiently transmitted in the presence of HK2 Rec or HIV-1 Tat and Vif. Transmitted Bogota probes formed episomes but did not integrate into the cellular genome. Resistance to integration might explain the relatively low number of HK2 insertions that were acquired during the last 25 million years of evolution. Whether transient transmission of modern HK2 sequences, which encode two putative oncoproteins, can lead to disease remains to be studied.IMPORTANCERetroviruses invaded the genome of human ancestors over the course of millions of years, yet these viruses generally have been inactivated during evolution, with only remnants of these infectious sequences remaining in the human genome. One of these viruses, termed HK2, still is capable of producing virus particles, although these particles have been regarded as being noninfectious. Using a genetic probe derived from HK2, we have discovered that HK2 viruses produced in modern humans can package HK2 sequences and transmit them to various other cells. Furthermore, the genetic sequences packaged in HK2 undergo reverse transcription. The transmitted probe circularized in the cell and failed to integrate into the cellular genome. These findings suggest that modern HK2 viruses can package viral RNA and transmit it to other cells. Contrary to previous views, we provide evidence of an extracellular viral phase of modern HK2 viruses. We have no evidence of sustained, spreading infection.
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Piironen, T., J. Lövgren, M. Karp, et al. "Immunofluorometric assay for sensitive and specific measurement of human prostatic glandular kallikrein (hK2) in serum." Clinical Chemistry 42, no. 7 (1996): 1034–41. http://dx.doi.org/10.1093/clinchem/42.7.1034.
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Abstract Prostate-specific antigen (PSA) and human prostatic glandular kallikrein (hK2) have 79% identity with the primary structure. When we used recombinant hK2 protein, only 7 of 23 monoclonal anti-PSA IgGs (monoclonal antibodies, MAbs) cross-reacted with hK2, which enabled us to design a novel immunofluorometric MAb-MAb assay for the specific detection of hK2. In the first incubation, an excess of MAb 2H11, which does not cross-react with hK2, is added to prevent both free and complexed PSA from reacting in subsequent immunoreactions. In the second incubation, biotinylated MAb H50, which cross-reacts with hK2 by an epitope overlapping with MAb 2H11, served to bind only hK2 to the microtitration wells coated with streptavidin. In the third step, Eu-labeled MAb H117, which cross-reacts with hK2, detected the immobilized hK2. The hK2 assay was calibrated with recombinant hK2. The detection limit of the assay was 0.1 microgram/L, and the cross-reactivity with recombinant PSA was &lt; or = 0.7%. The concentration of hK2 was measured in serum samples from 334 males with total PSA concentrations ranging from 1 to 3400 microgram/L. Most of the samples (57%) had hK2 concentrations below the detection limit. The proportions of hK2 relative to total PSA were 0-2% in 79%, 2-5% in 14%, 5-10% in 4%, and &gt;10% in 3% of the samples. Gel filtration of 10 serum samples with increased hK2 concentrations showed a single peak of hK2 immunoreactivity with an apparent molecular size of approximately 30 kDa, corresponding to that of recombinant hK2 and free PSA.
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Black, Margot H., Angeliki Magklara, Christina V. Obiezu, Dimitrios N. Melegos, and Eleftherios P. Diamandis. "Development of an Ultrasensitive Immunoassay for Human Glandular Kallikrein with No Cross-Reactivity from Prostate-specific Antigen." Clinical Chemistry 45, no. 6 (1999): 790–99. http://dx.doi.org/10.1093/clinchem/45.6.790.
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Abstract Background: Studies demonstrating that human glandular kallikrein (hK2) is increased in prostate cancer patients have prompted speculation that this marker may of use in addition to prostate-specific antigen (PSA). Methods: An ultrasensitive hK2 sandwich immunoassay was developed, and its detection limit, cross-reactivity, analytical recovery, precision, and linearity of dilution were evaluated. hK2 was measured in seminal plasma and sera from healthy males, females, and prostatectomized patients. Results: Our assay has an excellent detection limit (6 ng/L) and precision (&gt;90%). Recovery studies indicated that hK2 binds to serum protease inhibitors. All sera from healthy males had measurable hK2 concentrations (median, 402 ng/L). Almost all female sera had undetectable hK2. Serum hK2 and PSA in males correlated positively (r = 0.44), but hK2 was present at concentrations ∼2.5-fold lower than PSA. The PSA/hK2 ratio in male sera was 0.1–34, with a median of 2.6. In seminal plasma, this ratio was 100–500. More than 94% of immunoreactive hK2 in serum was in the free form (∼30 kDa); traces of hK2 complexed to α1-antichymotrypsin were present. Conclusions: The limit of detection of the method for hK2 measurement described here (∼20-fold lower than any other reported assay for hK2) allows the generation of new clinical information. When combined with a previously described method for PSA measurement that has no cross-reactivity from hK2, this methods allows the relative proportions of hK2 and PSA in biological fluids to be measured.
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Becker, Charlotte, Timo Piironen, Johanna Kiviniemi, Hans Lilja, and Kim Pettersson. "Sensitive and Specific Immunodetection of Human Glandular Kallikrein 2 in Serum." Clinical Chemistry 46, no. 2 (2000): 198–206. http://dx.doi.org/10.1093/clinchem/46.2.198.
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Abstract Background: Human glandular kallikrein 2 (hK2) is expressed in the prostate and is present in serum from men with prostate cancer. Specific detection in serum is difficult mainly because of low concentrations and immunological cross-reactivity with prostate-specific antigen (PSA). Our objectives were to design an assay with improved analytical detection and functional sensitivity and nonsignificant cross-reactivity with PSA, and to characterize different immunoreactive forms of hK2. Methods: In the assay, critical PSA epitopes were blocked with four monoclonal antibodies (MAbs) specific for PSA. Subsequently, hK2 was captured using a MAb against hK2 (5% cross-reactivity with PSA), and after washing, hK2 was detected by a europium-labeled MAb with identical affinity for hK2 and PSA. Results: The analytical detection limit was &lt;10 ng/L, and functional sensitivity was 30 ng/L. Cross-reaction with PSA was &lt;0.01%. Between-assay imprecision was 3.1% for 1600 ng/L hK2 and 4.8% for 160 ng/L hK2; corresponding values for within-assay precision were 1.9% and 4.5%, respectively. Complexes of hK2-α1-antichymotrypsin (ACT) were detected in vitro with −6% bias compared with the free form of hK2. Gel filtration of patient samples showed that hK2 correlated in size mainly with free hK2; only 4–19% corresponded to hK2 possibly complexed with ACT or protein C inhibitor. Conclusions: Our assay had extremely low cross-reactivity with PSA, provided a very low detection limit, and allowed close to equimolar detection of the free and complexed forms of hK2. Moreover, we found that free hK2 is the predominant immunoreactive form of hK2 in serum.
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Finlay, Judith A., John R. Day, Cindy L. Evans, et al. "Development of a Dual Monoclonal Antibody Immunoassay for Total Human Kallikrein 2." Clinical Chemistry 47, no. 7 (2001): 1218–24. http://dx.doi.org/10.1093/clinchem/47.7.1218.
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Abstract Background: Human kallikrein 2 (hK2) shares 80% sequence identity with prostate-specific antigen (PSA). Because both hK2 and hK2-α1-antichymotrypsin (hK2-ACT) complexes have been identified in patient sera, we devised an immunoassay for total hK2 [(thK2); hK2 and hK2-ACT] and evaluated it in healthy subjects and patients with prostate disease. Methods: We developed monoclonal antibodies (mAbs) with high specificity for hK2 and hK2-ACT and minimal cross-reactivity to PSA. Using these mAbs, a sandwich assay was developed and its specificity for forms of hK2 was assessed. Serum samples (n = 1035) from healthy volunteers, patients with increased PSA, and men who had undergone radical prostatectomy were assayed for thK2. We also measured thK2 in samples before and after storage under common laboratory conditions. Results: The minimum detectable concentration in the thK2 assay was 0.008 μg/L, and PSA cross-reactivity was &lt;0.001%. The assay detected prohK2 and three different hK2–serum protease complexes. The median serum concentration of thK2 in control samples (0.013 μg/L) was significantly lower than the median in samples from patients with increased PSA concentrations (0.085 μg/L). Immunoreactive hK2 changed little in samples stored for up to 1 month at −70 °C. Conclusions: The thK2 assay recognizes all forms of hK2 that have been found in bodily fluids to date.
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Klee, George G., Marcia K. Goodmanson, Steven J. Jacobsen, et al. "Highly Sensitive Automated Chemiluminometric Assay for Measuring Free Human Glandular Kallikrein-2." Clinical Chemistry 45, no. 6 (1999): 800–806. http://dx.doi.org/10.1093/clinchem/45.6.800.
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Abstract Background: Human glandular kallikrein (hK2) is a serine protease that has 79% amino acid identity with prostate-specific antigen (PSA). Both free hK2 and hK2 complexed to α1-antichymotrypsin (ACT) are present in the blood in low concentrations. We wished to measure hK2 in serum with limited contribution from hK2-ACT for the results. Methods: We developed an automated assay for hK2 with use of a select pair of monoclonal antibodies. The prototype assay was implemented on a Beckman Coulter ACCESS® analyzer. Results: The detection limit of the assay was 1.5 ng/L, the “functional sensitivity” (day-to-day CV &lt;15%) was &lt;4 ng/L, cross-reactivity with PSA and PSA-ACT was negligible, and cross-reactivity with hK2-ACT was 2%. After surgical removal of prostate glands, serum hK2 was &lt;7 ng/L and was &lt;15 ng/L in most healthy women. The median serum concentration of hK2 in healthy men without prostate cancer was 26 ng/L. The median concentration of hK2 was 72 ng/L for men having prostate cancer with lower Gleason scores compared with 116 ng/L for men with more advanced cancer. The concentration of hK2 correlated weakly with PSA, with the mean hK2 concentrations generally 30- to 80-fold lower than PSA concentrations. Conclusion: The availability of a robust, high sensitivity automated assay for hK2 should facilitate further investigations of the role of hK2 measurements in the management of patients with prostate disease.
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Engelmann, Bodil E., Tina Binderup, Andreas Kjær, et al. "Quantitative gene expression underlying 18f-fluorodeoxyglucose uptake in colon cancer." Journal of Clinical Oncology 33, no. 3_suppl (2015): 653. http://dx.doi.org/10.1200/jco.2015.33.3_suppl.653.
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653 Background: Positron emission tomography (PET) with the glucose analogue 18F-fluorodeoxyglucose (FDG) is widely used in oncologic imaging. This study examines the molecular mechanism underlying the detection of colon cancer (CC) by FDG-PET. Methods: Pre-operative PET/CT scans and tissue samples from primary CC and surrounding normal mucosa were obtained from 42 patients. FDG uptake was quantified using maximal standardized uptake value (SUVmax). The expression of ki67, glucose transporter 1 (GLUT-1), hexokinase 1 (HK1), hexokinase 2 (HK2), vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1α (HIF1α) and carbonic anhydrase IX (CaIX) mRNA was examined by quantitative real time reverse transcriptase-polymerase chain reaction. Results: All primary tumours showed increased uptake of FDG. The mean SUVmax was 15.0 (range 5.3 – 37.8). No correlation was found between tumour size and SUVmax. Mean gene expression levels of GLUT1, HK2, ki67, HIF1α, VEGF and CaIX, but not HK1, were significantly higher in primary tumours than in surrounding normal colonic mucosa. Linear regressions pairing tumour SUVmax with gene expression levels showed significant correlations between SUVmax and HK2, ki67 and CaIX, respectively. Conclusions: These results confirm FDG PET/CT as a functional imaging method in CC, and that FDG accumulation reflects molecular events related to glycolysis, cell proliferation, hypoxia, but not angiogenesis.
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He, Ming, Jun-Jie Zhang, Meiping Ye, Yongliang Lou, and X. Frank Yang. "Cyclic Di-GMP Receptor PlzA Controls Virulence Gene Expression through RpoS in Borrelia burgdorferi." Infection and Immunity 82, no. 1 (2013): 445–52. http://dx.doi.org/10.1128/iai.01238-13.
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ABSTRACTAs an obligate pathogen, the Lyme disease spirocheteBorrelia burgdorferihas a streamlined genome that encodes only two two-component signal transduction systems, Hk1-Rrp1 and Hk2-Rrp2 (in addition to CheA-CheY systems). The output of Hk1-Rrp1 is the production of the second messenger cyclic di-GMP (c-di-GMP), which is indispensable forB. burgdorferito survive in the tick vector. The output of Hk2-Rrp2 is the transcriptional activation of the global regulator RpoS, which is essential for the pathogen to accomplish its tick-mouse transmission and to establish mammalian infection. Although evidence indicates that these two systems communicate with each other, how they are connected is not fully understood. In this study, we showed that the c-di-GMP-binding protein PlzA, a downstream effector of Rrp1, positively modulates the production of RpoS, a global regulator and downstream target of Rrp2. Thus, PlzA functions as a connector that links Hk1-Rrp1 with Hk2-Rrp2. We further showed that PlzA regulatesrpoSexpression through modulation of another regulator, BosR, at both the transcriptional and the posttranscriptional levels. In addition, PlzA was also capable of regulatingrpoSexpression independently of Rrp1, suggesting that besides being a c-di-GMP-binding protein, PlzA has other functions. Along with the previous finding of PlzA controlling motility, these studies demonstrate that PlzA is a multifunctional protein. These findings further reinforce the notion thatB. burgdorferiutilizes its limited signaling systems and regulators to govern multiple cellular processes during its complex enzootic cycle between ticks and mammals.
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Thomas, Geethu Emily, Grace Egan, Laura Garcia Prat, et al. "The Metabolic Enzyme Hexokinase 2 Localizes to the Nucleus in AML and Normal Hematopoietic Stem/Progenitor Cells to Maintain Stemness." Blood 136, Supplement 1 (2020): 1–2. http://dx.doi.org/10.1182/blood-2020-135858.
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Abstract Mitochondrial metabolites affect epigenetic marks, but it is largely unknown whether mitochondrial metabolic enzymes can directly localize to the nucleus to regulate stem cell function in AML. Here, we discovered that the mitochondrial enzyme, Hexokinase 2 (HK2), localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stem cell function. We searched for mitochondrial enzymes moonlighting in the nucleus using 8227 AML cells, a low passage primary AML culture model arranged in a hierarchy with functionally defined stem cells in the CD34+CD38-fraction. By immunoblotting and confocal microscopy, we detected HK2 in the nucleus of 8227 cells with higher expression in the nucleus of stem cells vs bulk cells. HK2 is the first and rate-limiting enzyme in glycolysis and phosphorylates glucose. In contrast, other metabolic enzymes including phosphofructokinase, fumarase, pyruvate kinase 2, glucose phosphate isomerase, enolase1, citrate synthase, aconitase 2, and succinate dehydrogenase were not detected in the nucleus of these cells. We also detected HK2, but not these other metabolic enzymes, in the nucleus of OCI-AML2, U937, NB4 and TEX leukemia as well as 8 of 9 primary AML samples. Next, we tested whether nuclear HK2 was functionally important to maintain stem cell function in AML. We over-expressed HK2 tagged with nuclear localizing signals (PKKKRKV and PAAKRVKLD) in 8227 and NB4 leukemia cells. We confirmed selective over-expression of HK2 in the nucleus of these cells without increasing levels in the cytoplasm or mitochondria. Over-expression of nuclear HK2 increased clonogenic growth and inhibited retinoic acid-mediated cell differentiation without changing basal proliferation. Over expression of HK2 also increased engraftment of 8227 cells into mouse marrow. We evaluated the selective inhibition of nuclear HK2 by over-expressing HK2 with an outer mitochondrial localization signal while knocking down total endogenous HK2 with shRNA targeting the 3'UTR of HK2. Selective depletion of nuclear HK2 reduced clonogenic growth, increased AML differentiation after treatment with retinoic, and decreased the percentage of CD34+CD38- 8227 stem cells without changing basal proliferation. To determine whether nuclear HK2 maintains stemness through its kinase activity, we over-expressed a kinase dead double mutant of nuclear HK2(D209A D657A). Nuclear kinase dead HK2 increased clonogenic growth and inhibited differentiation after retinoic acid treatment, demonstrating that HK2 maintains stemness independent of its kinase function. To understand nuclear functions of HK2, we used proximity-dependent biotin labeling (BioID) and mass spectrometry to identify proteins that interact with nuclear HK2 and identified proteins related to chromatin organization and regulation. Therefore, we examined the impact of nuclear HK2 on chromatin accessibility using ATAC-seq. Over expression of nuclear HK2 enhanced chromatin accessibility, whereas the selective knockdown of nuclear HK2 compacted chromatin. In summary, we discovered that HK2 localizes to nucleus of AML cells and functions independent of its kinase activity to maintain the stem/progenitor state of AML. Thus, we define a new role for mitochondrial enzymes in the regulation of leukemic stemness and differentiation. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Schimmer:Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock .
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Magklara, Angeliki, Andreas Scorilas, Carlos López-Otín, Francisco Vizoso, Alvaro Ruibal, and Eleftherios P. Diamandis. "Human Glandular Kallikrein in Breast Milk, Amniotic Fluid, and Breast Cyst Fluid." Clinical Chemistry 45, no. 10 (1999): 1774–80. http://dx.doi.org/10.1093/clinchem/45.10.1774.
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Abstract Background: Human glandular kallikrein (hK2) belongs to the serine protease family of enzymes and has high sequence homology with prostate-specific antigen (PSA). The physiological role of hK2 has not as yet been determined, but there is evidence that it can regulate the proteolytic activity of PSA through processing and activating pro-PSA, an inactive precursor. Thus, it is conceivable that these two secreted proteins may coexist in biological fluids. Currently, hK2 is considered an androgen-regulated and prostate-specific protein. Recently, it has been demonstrated that hK2 is expressed in the breast cancer cell line T-47D after stimulation by steroid hormones, and we reported that hK2 can be detected in a subset of breast tumor extracts. These data suggest that hK2 may be expressed in tissues other than the prostate, such as those in which PSA has already been detected. Because hK2 is a secreted protein, it may be present in various biological fluids. Methods: We analyzed milk samples from lactating women, amniotic fluid from pregnant women, and breast cyst fluid from patients with gross breast cystic disease, using a highly sensitive and specific immunoassay for hK2. Results: hK2 was present in all three biological fluids. We suggest that the female breast may produce hK2 and provide evidence that hK2 may have value as an additional marker for the discrimination between type I and type II breast cysts. Conclusions: The female breast produces hK2 in addition to PSA. More studies are necessary to establish the role of this kallikrein in nondiseased breast, gross breast cystic disease, and breast cancer.
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Thomas, Geethu Emily, Grace Egan, Laura Garcia Prat, et al. "The Metabolic Enzyme Hexokinase 2 Localizes to the Nucleus in AML and Normal Hematopoietic Stem/Progenitor Cells to Maintain Stemness." Blood 134, Supplement_1 (2019): 2532. http://dx.doi.org/10.1182/blood-2019-123121.
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Hematopoietic cells are arranged in a hierarchy where mature blood cells arise from stem and progenitor precursors. AML is also hierarchical with differentiated blasts arising from leukemic stem/progenitor cells. Recent studies show that metabolites can affect epigenetic marks; however, it is unknown whether metabolic enzymes can directly localize to the nucleus to regulate stemness in AML and normal hematopoietic cells. Here, we discovered that the mitochondrial enzyme, Hexokinase 2, localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stemness. Metabolic enzymes that localize to nucleus of stem cells were identified by evaluating stem and bulk fractions of OCI-AML-8227 leukemia cells, which are arranged in a hierarchy with functionally defined stem cells. We separated OCI-AML-8227 cells into CD34+38- and CD34-38+ populations by FACS and prepared nuclear and cytoplasmic lysates. Immunoblotting of the lysates revealed that the metabolic enzyme Hexokinase 2 (HK2) was increased in the nuclear fraction of 8227 stem cells compared to bulk cells. In contrast, other mitochondrial enzymes such as Enolase1, Aconitase2, and Succinate Dehydrogenase A & B, were not detected in the nuclear lysates. HK2 is an outer mitochondrial membrane protein that phosphorylates glucose to glucose-6-phosphate, initiating glycolysis. We confirmed nuclear HK2 in OCI-AML-8227 stem cells by confocal microscopy and also demonstrated nuclear HK2 in AML cell lines (OCI-AML2, NB4, K563, and MV411) and in 7 of 9 primary AML samples. We FACS sorted normal cord blood into populations of stem/progenitor (HSC, MPP, MLP, CMP, GMP and MEP) and differentiated (Monocytes, Granulocytes, B, T, and NK) cells. The localization of HK2 in these cells was analysed and quantified by immunofluorescence. Nuclear HK2 was detected in the stem/progenitor cells and progressively declined to minimal levels as cells matured. Next, we explored mechanisms that regulate nuclear localization of HK2. AKT-mediated phosphorylation of HK2 promoted localization to mitochondria while inhibition of phosphorylation increased its nuclear levels. Moreover, the nuclear import of HK2 was dependent on IPO5, a member of b-importin family that imports protein to the nucleus; CRM1 was responsible for HK2 nuclear export. We tested whether the nuclear localization of HK2 was functionally important to maintain stemness. We overexpressed HK2 tagged with nuclear localizing signals (PKKKRKV or PAAKRVKLD) in 8227 and NB4 leukemia cells. Selective overexpression of HK2 in the nucleus did not alter the rate of proliferation of the cells, however there was enhanced clonogenic growth and inhibition of retinoic acid-mediated cell differentiation. Conversely, we selectively reduced nuclear HK2 by expressing HK2 with an outer mitochondrial localization signal while knocking down endogenous HK2 with shRNA targeting the 3'UTR of HK2. Selective depletion of nuclear HK2 in AML cells did not alter growth rate, but did reduce clonogenic growth and increased differentiation after treatment with retinoic acid. To determine whether nuclear HK2 maintains stemness through its kinase activity, we over-expressed a kinase dead double mutant of nuclear HK2(D209A D657A). Nuclear kinase dead HK2 increased clonogenic growth and inhibited differentiation after retinoic acid treatment, demonstrating that HK2 maintains stemness independent of kinase function. To understand nuclear functions of HK2, we used proximity-dependent biotin labeling (BioID) and mass spectrometry to identify proteins that interact with nuclear HK2. A top hit in our screen was Exonuclease 3'-5' domain containing 2 (EXD2), involved in DNA repair. Of note, DNA damage induces differentiation of AML cells. In 8227 cells, nuclear EXD2 was higher in the stem cell fraction compared to the bulk fraction. Moreover, knockdown of EXD2 reduced AML growth, clonogenic growth and decreased nuclear HK2 levels. Finally, nuclear HK2 overexpression conferred resistance to the PARP inhibitor, olaparib. In summary, we discovered that unphosphorylated HK2 localizes to the nucleus in malignant and normal hematopoietic stem cells. Through mechanisms independent of its kinase function, nuclear HK2 maintains AML cells in their stem/progenitor state potentially by regulating DNA damage and repair. Thus, we define a new role for a mitochondrial enzyme in the regulation of stemness and differentiation. Disclosures Minden: Trillium Therapetuics: Other: licensing agreement. Schimmer:Medivir Pharmaceuticals: Research Funding; Otsuka Pharmaceuticals: Consultancy; Novartis Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy.
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Kim, Dong Jun, Mai-Tram Vo, Seong Hee Choi, et al. "Tristetraprolin-mediated hexokinase 2 expression regulation contributes to glycolysis in cancer cells." Molecular Biology of the Cell 30, no. 5 (2019): 542–53. http://dx.doi.org/10.1091/mbc.e18-09-0606.
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Hexokinase 2 (HK2) catalyzes the first step of glycolysis and is up-regulated in cancer cells. The mechanism has not been fully elucidated. Tristetraprolin (TTP) is an AU-rich element (ARE)-binding protein that inhibits the expression of ARE-containing genes by enhancing mRNA degradation. TTP expression is down-regulated in cancer cells. We demonstrated that TTP is critical for down-regulation of HK2 expression in cancer cells. HK2 mRNA contains an ARE within its 3′-UTR. TTP binds to HK2 3′-UTR and enhances degradation of HK2 mRNA. TTP overexpression decreased HK2 expression and suppressed the glycolytic capacity of cancer cells, measured as glucose uptake and production of glucose-6-phosphate, pyruvate, and lactate. TTP overexpression reduced both the extracellular acidification rate (ECAR) and the oxygen consumption rate (OCR) of cancer cells. Ectopic expression of HK2 in cancer cells attenuated the reduction in glycolytic capacity, ECAR, and OCR from TTP. Taken together, these findings suggest that TTP acts as a negative regulator of HK2 expression and glucose metabolism in cancer cells.
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Kim, Kwan Wook, Sang Woo Kim, Soyeon Lim, Kyung-Jong Yoo, Ki-Chul Hwang, and Seahyoung Lee. "Neutralization of hexokinase 2-targeting miRNA attenuates the oxidative stress-induced cardiomyocyte apoptosis." Clinical Hemorheology and Microcirculation 78, no. 1 (2021): 57–68. http://dx.doi.org/10.3233/ch-200924.
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Hexokinase 2 (HK2) is a metabolic sensor that couples glycolysis and oxidative phosphorylation of mitochondria by binding to the outer mitochondrial membrane (OMM), and it also has been implicated in induction of apoptotic process by regulating the integrity of OMM. When HK2 detaches from the mitochondria, it triggers permeability increase of the OMM and subsequently facilitates the cytosolic release of cytochrome c, a major apoptosis-inducing factor. According to previous studies, a harsh microenvironment created by ischemic heart disease such as low tissue oxygen and nutrients, and increased reactive oxygen species (ROS) can cause cardiomyocyte apoptosis. Under these conditions, the expression of HK2 in heart significantly decrease and such down-regulation of HK2 was correlated to the increased apoptosis of cardiomyocytes. Therefore, prevention of HK2 down-regulation may salvage cardiomyocytes from apoptosis. MicroRNAs are short, non-coding RNAs that either inhibit transcription of target mRNAs or degrade the targeted mRNAs via complementary binding to the 3’UTR (untranslated region) of the targeted mRNAs. Since miRNAs are known to be involved in virtually every biological processes, it is reasonable to assume that the expression of HK2 is also regulated by miRNAs. Currently, to my best knowledge, there is no previous study examined the miRNA-mediated regulation of HK2 in cardiomyocytes. Thus, in the present study, miRNA-mediated modulation of HK2 during ROS (H2O2)-induced cardiomyocyte apoptosis was investigated. First, the expression of HK2 in cardiomyocytes exposed to H2O2 was evaluated. H2O2 (500 μM) induced cardiomyocyte apoptosis and it also decreased the mitochondrial expression of HK2. Based on miRNA-target prediction databases and empirical data, miR-181a was identified as a HK2-targeting miRNA. To further examine the effect of negative regulation of the selected HK2-targeting miRNA on cardiomyocyte apoptosis, anti-miR-181a, which neutralizes endogenous miR-181a, was utilized. Delivery of anti-miR-181a significantly abrogated the H2O2-induced suppression of HK2 expression and subsequent disruption of mitochondrial membrane potential, improving the survival of cardiomyocytes exposed to H2O2. These findings suggest that miR-181a-mediated down-regulation of HK2 contributes to the apoptosis of cardiomyocytes exposed to ROS. Neutralizing miR-181a can be a viable and effective means to prevent cardiomyocyte from apoptosis in ischemic heart disease.
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He, Wanwan, Nengyan Fang, Ruisen Wang, et al. "Fine Mapping of a New Race-Specific Blast Resistance Gene, Pi-hk2, in Japonica Heikezijing from Taihu Region of China." Phytopathology® 107, no. 1 (2017): 84–91. http://dx.doi.org/10.1094/phyto-03-16-0151-r.
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Heikezijing, a japonica rice landrace from the Taihu region of China, exhibited broad-spectrum resistance to more than 300 isolates of the blast pathogen (Magnaporthe oryzae). In our previous research, we fine mapped a broad-spectrum resistance gene, Pi-hk1, in chromosome 11. In this research, 2010-9(G1), one of the predominant races of blast in the Taihu Lake region of China, was inoculated into 162 recombinant inbred lines (RIL) and two parents, Heikezijing and Suyunuo, for mapping the resistance-blast quantitative trait loci (QTL). Three QTL (Lsqtl4-1, Lsqtl9-1, and Lsqtl11-1) associated with lesion scores were detected on chromosomes 4, 9, and 11 and two QTL (Lnqtl1-1 and Lnqtl9-1) associated with average lesion numbers were detected on chromosomes 1 and 9. The QTL Lsqtl9-1 conferring race-specific resistance to 2010-9(G1) at seedling stages showed logarithm of the odds scores of 9.10 and phenotypic variance of 46.19% and might be a major QTL, named Pi-hk2. The line RIL84 with Pi-hk2 derived from a cross between Heikezijing and Suyunuo was selected as Pi-hk2 gene donor for developing fine mapping populations. According to the resistance evaluation of recombinants of three generations (BC1F2, BC1F3, and BC1F4), Pi-hk2 was finally mapped to a 143-kb region between ILP-19 and RM24048, and 18 candidate genes were predicted, including genes that encode pleiotropic drug resistance protein 4 (n = 2), WRKY74 (n = 1), cytochrome b5-like heme/steroid-binding domain containing protein (n = 1), protein kinase (n = 1), and ankyrin repeat family protein (n = 1). These results provide essential information for cloning of Pi-hk2 and its potential utility in breeding resistant rice cultivars by marker-assisted selection.
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Ciscato, Francesco, Lavinia Ferrone, Ionica Masgras, Claudio Laquatra, and Andrea Rasola. "Hexokinase 2 in Cancer: A Prima Donna Playing Multiple Characters." International Journal of Molecular Sciences 22, no. 9 (2021): 4716. http://dx.doi.org/10.3390/ijms22094716.
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Hexokinases are a family of ubiquitous exose-phosphorylating enzymes that prime glucose for intracellular utilization. Hexokinase 2 (HK2) is the most active isozyme of the family, mainly expressed in insulin-sensitive tissues. HK2 induction in most neoplastic cells contributes to their metabolic rewiring towards aerobic glycolysis, and its genetic ablation inhibits malignant growth in mouse models. HK2 can dock to mitochondria, where it performs additional functions in autophagy regulation and cell death inhibition that are independent of its enzymatic activity. The recent definition of HK2 localization to contact points between mitochondria and endoplasmic reticulum called Mitochondria Associated Membranes (MAMs) has unveiled a novel HK2 role in regulating intracellular Ca2+ fluxes. Here, we propose that HK2 localization in MAMs of tumor cells is key in sustaining neoplastic progression, as it acts as an intersection node between metabolic and survival pathways. Disrupting these functions by targeting HK2 subcellular localization can constitute a promising anti-tumor strategy.
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Herrala, Annakaisa, Riitta Kurkela, Katja Porvari, Ritva Isomäki, Pirkko Henttu, and Pirkko Vihko. "Human prostate-specific glandular kallikrein is expressed as an active and an inactive protein." Clinical Chemistry 43, no. 2 (1997): 279–84. http://dx.doi.org/10.1093/clinchem/43.2.279.
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Abstract A polymorphism in the human prostate-specific glandular kallikrein (hKLK2) gene was described by direct sequencing (by PCR) of genomic DNAs isolated from prostatic cancer tissue, benign prostatic hyperplasia tissue, and blood leukocyte specimens. Results showed two forms of human prostate-specific glandular kallikrein protein (hK2), a consequence of a change from C to T at base 792 in the hK2 coding region. Producing the two forms as recombinant proteins in insect cells demonstrated that Arg226-hK2 (CC genotype) is an active protein and Trp226-hK2 (TT genotype) is inactive. Polymorphism studies of 36 patients with prostatic diseases identified only 1 with the TT genotype. The same kind of polymorphism was not detected in the human prostate-specific antigen (hKLK3) gene. Arg226-hK2 possessed only trypsin-like enzyme activity, whereas recombinant human prostate-specific antigen (hPSA) had only chymotrypsin-like activity. Monoclonal and polyclonal antibodies raised against hPSA purified from seminal plasma detected both active and inactive hK2. Thus, because inactive as well as stable hK2 protein may be present, a lack of trypsin-like activity in hPSA standards is not enough to confirm that the materials are free of hK2 contamination.
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Nam, Robert K., Eleftherios P. Diamandis, Ants Toi, et al. "Serum Human Glandular Kallikrein-2 Protease Levels Predict the Presence of Prostate Cancer Among Men With Elevated Prostate-Specific Antigen." Journal of Clinical Oncology 18, no. 5 (2000): 1036. http://dx.doi.org/10.1200/jco.2000.18.5.1036.
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PURPOSE: We hypothesize that serum human glandular kallikrein-2 (hK2) levels predict the presence of prostate cancer among men prescreened by prostate-specific antigen (PSA).PATIENTS AND METHODS: We conducted a cross-sectional study of 324 men who had no history of prostate cancer and who were referred for prostate biopsy. PSA and hK2 levels were measured using specific nonisotopic immunometric techniques. Cases were patients who were diagnosed with adenocarcinoma of the prostate from biopsy, and controls were patients who had no evidence of cancer from biopsy. The odds ratio for detection of prostate cancer was determined for hK2 measurements, controlling for age, total-PSA level, digital rectal examination, and symptoms of urinary obstruction.RESULTS: Of 324 men, 159 (49.1%) had cancer. Mean hK2 levels and hK2:free-PSA ratios were significantly higher in cases than in controls (1.18 v 0.53 ng/mL, respectively, for hK2, P = .0001; 1.17 v 0.62 for hK2:free-PSA ratio, P = .0001). The crude odds ratio for prostate cancer detection for patients in the highest quartile of hK2 level was 5.83 (95% confidence interval [CI], 2.8 to 12.1; P = .0001) compared with patients in the lowest quartile. The adjusted odds ratio was 6.72 (95% CI, 2.9 to 15.6; P = .0001). Similarly, the crude and adjusted odds ratios for prostate cancer detection using the hK2:free-PSA ratio were 7.36 (95% CI, 3.6 to 15.1; P = .0001) and 8.06 (95% CI, 3.7 to 17.4; P = .0001), respectively. These odds ratios were higher than that observed for prostate cancer detection by total-PSA level (2.73; P = .03).CONCLUSION: Among men prescreened with PSA for prostate cancer, patients with high hK2 measurements have a five- to eight-fold increase in risk for prostate cancer, adjusting for PSA level and other established risk factors. hK2 measurements may be a useful adjunct to PSA in improving patient selection for prostate biopsy.
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Thomas, Geethu, Laura Garcia Prat, Marcela Gronda, et al. "The Metabolic Enzyme Hexokinase 2 Localizes to the Nucleus in AML and Normal Hematopoietic Stem/Progenitor Cells to Maintain Stemness." Blood 132, Supplement 1 (2018): 2795. http://dx.doi.org/10.1182/blood-2018-99-110021.
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Abstract Hematopoietic cells are arranged in a hierarchy where stem and progenitor cells differentiate into mature blood cells. Likewise, AML (Acute Myeloid Leukemia) is also hierarchical with leukemic stem and progenitor cells giving rise to more mature and differentiated blasts. Recent studies have shown that mitochondrial enzymes such as IDH2 can regulate AML stemness by altering metabolites that affect epigenetic marks. However, it is unknown whether mitochondrial metabolic enzymes can directly localize to the nucleus to regulate stemness in AML and normal hematopoietic cells. Here, we show that the mitochondrial enzyme, Hexokinase 2, localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stemness. We sought to identify mitochondrial metabolic enzymes that localize to the nucleus of stem cells, by evaluating the stem and bulk fractions from 8227 leukemia cells. 8227 leukemia cells are arranged in a hierarchy with functionally defined stem cells present in the CD34+CD38- fraction. We separated 8227 cells into CD34+CD38- and CD34-CD38+ populations by FACS sorting and prepared lysates of the nuclear and cytoplasmic fractions from each population. Using immunoblotting, we measured levels of mitochondrial enzymes in the subcellular fractions of each population. We discovered that the metabolic enzyme Hexokinase 2 (HK2) was increased in the nuclear fraction of 8227 stem cells compared to bulk cells. In contrast, other mitochondrial enzymes such as Aconitase 2 and Succinate Dehydrogenase B were not detected in the nuclear fractions. HK2 is an outer mitochondrial membrane protein that phosphorylates glucose to glucose-6-phosphate, thereby initiating glycolysis and the entry of glucose metabolites into the TCA cycle in the mitochondria. The nuclear localization of HK2 in mammalian cells has not been previous reported. We confirmed that 8227 cells have nuclear HK2 by confocal fluorescent microscopy and also demonstrated nuclear HK2 in AML cell lines (OCI-AML2, NB4, K562, and MV411) and primary AML samples. We also FACS sorted normal cord blood into populations of stem/progenitor (HSC, MPP, MLP, CMP, GMP and MEP) and differentiated (B cells, T cells, NK cells, Monocytes and Granulocytes) cells. The localization of HK2 in these cell fractions was measured by immunofluorescence and quantified by Metamorph and Imaris. Nuclear HK2 was detected in the stem/progenitor cells and progressively declined to minimal levels as the cells matured (Fig 1A). The mitochondrial localization of HK2 is dependent on AKT-mediated phosphorylation of Thr-473 and inhibited by dephosphorylation by the phosphatase PHLPP1. We asked whether phosphorylation of HK2 regulates the nuclear abundance of HK2. Using AML2 cells, we showed that knockdown of PHLPP1 decreased the abundance of nuclear HK2, while inhibition of AKT increased HK2 in the nucleus. Finally, we tested whether the nuclear localization of HK2 was functionally important to maintain stemness. We over-expressed HK2 tagged with nuclear localizing signals (PKKKRKV and PAAKRVKLD) in 8227 and NB4 leukemia cells. We confirmed the selective over-expression of HK2 in the nucleus of these cells by immunoblotting and immunofluorescence. Increasing nuclear HK2 did not alter the proliferation of the cells under basal conditions. However, increasing nuclear HK2 enhanced clonogenic growth and blocked retinoic acid-mediated cell differentiation. In summary, we discovered that the unphosphorylated form of the metabolic enzyme HK2 localizes to the nucleus in malignant and normal hematopoietic stem cells and is functionally important to maintain stem/progenitor state. Thus, we define a new role for mitochondrial enzymes in the regulation of stemness and differentiation. Disclosures Schimmer: Medivir AB: Research Funding; Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka Pharmaceuticals: Consultancy.
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Siu, Michelle K. Y., Yu-Xin Jiang, Jing-Jing Wang, et al. "Hexokinase 2 Regulates Ovarian Cancer Cell Migration, Invasion and Stemness via FAK/ERK1/2/MMP9/NANOG/SOX9 Signaling Cascades." Cancers 11, no. 6 (2019): 813. http://dx.doi.org/10.3390/cancers11060813.
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Metabolic reprogramming is a common phenomenon in cancers. Thus, glycolytic enzymes could be exploited to selectively target cancer cells in cancer therapy. Hexokinase 2 (HK2) converts glucose to glucose-6-phosphate, the first committed step in glucose metabolism. Here, we demonstrated that HK2 was overexpressed in ovarian cancer and displayed significantly higher expression in ascites and metastatic foci. HK2 expression was significantly associated with advanced stage and high-grade cancers, and was an independent prognostic factor. Functionally, knockdown of HK2 in ovarian cancer cell lines and ascites-derived tumor cells hindered lactate production, cell migration and invasion, and cell stemness properties, along with reduced FAK/ERK1/2 activation and metastasis- and stemness-related genes. 2-DG, a glycolysis inhibitor, retarded cell migration and invasion and reduced stemness properties. Inversely, overexpression of HK2 promoted cell migration and invasion through the FAK/ERK1/2/MMP9 pathway, and enhanced stemness properties via the FAK/ERK1/2/NANOG/SOX9 cascade. HK2 abrogation impeded in vivo tumor growth and dissemination. Notably, ovarian cancer-associated fibroblast-derived IL-6 contributed to its up-regulation. In conclusion, HK2, which is regulated by the tumor microenvironment, controls lactate production and contributes to ovarian cancer metastasis and stemness regulation via FAK/ERK1/2 signaling pathway-mediated MMP9/NANOG/SOX9 expression. HK2 could be a potential prognostic marker and therapeutic target for ovarian cancer.
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An, Ming-Xin, Si Li, Han-Bing Yao, et al. "BAG3 directly stabilizes Hexokinase 2 mRNA and promotes aerobic glycolysis in pancreatic cancer cells." Journal of Cell Biology 216, no. 12 (2017): 4091–105. http://dx.doi.org/10.1083/jcb.201701064.
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Aerobic glycolysis, a phenomenon known historically as the Warburg effect, is one of the hallmarks of cancer cells. In this study, we characterized the role of BAG3 in aerobic glycolysis of pancreatic ductal adenocarcinoma (PDAC) and its molecular mechanisms. Our data show that aberrant expression of BAG3 significantly contributes to the reprogramming of glucose metabolism in PDAC cells. Mechanistically, BAG3 increased Hexokinase 2 (HK2) expression, the first key enzyme involved in glycolysis, at the posttranscriptional level. BAG3 interacted with HK2 mRNA, and the degree of BAG3 expression altered recruitment of the RNA-binding proteins Roquin and IMP3 to the HK2 mRNA. BAG3 knockdown destabilized HK2 mRNA via promotion of Roquin recruitment, whereas BAG3 overexpression stabilized HK2 mRNA via promotion of IMP3 recruitment. Collectively, our results show that BAG3 promotes reprogramming of glucose metabolism via interaction with HK2 mRNA in PDAC cells, suggesting that BAG3 may be a potential target in the aerobic glycolysis pathway for developing novel anticancer agents.
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Janssen, Samuel, Carsten M. Jakobsen, D. Marc Rosen, et al. "Screening a combinatorial peptide library to develop a human glandular kallikrein 2–activated prodrug as targeted therapy for prostate cancer." Molecular Cancer Therapeutics 3, no. 11 (2004): 1439–50. http://dx.doi.org/10.1158/1535-7163.1439.3.11.
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Abstract Objective: Prostate cancer cells secrete the unique protease human glandular kallikrein 2 (hK2) that represents a target for proteolytic activation of cytotoxic prodrugs. The objective of this study was to identify hK2-selective peptide substrates that could be coupled to a cytotoxic analogue of thapsigargin, a potent inhibitor of the sarcoplasmic/endoplasmic reticulum calcium ATPase pump that induces cell proliferation–independent apoptosis through dysregulation of intracellular calcium levels. Methods: To identify peptide sequence requirements for hK2, a combination of membrane-bound peptides (SPOT analysis) and combinatorial chemistry using fluorescence-quenched peptide substrates was used. Peptide substrates were then coupled to 8-O-(12[l-leucinoylamino]dodecanoyl)-8-O-debutanoylthapsigargin (L12ADT), a potent analogue of thapsigargin, to produce a prodrug that was then characterized for hK2 hydrolysis, plasma stability, and in vitro cytotoxicity. Results: Both techniques indicated that a peptide with two arginines NH2-terminal of the scissile bond produced the highest rates of hydrolysis. A lead peptide substrate with the sequence Gly-Lys-Ala-Phe-Arg-Arg (GKAFRR) was hydrolyzed by hK2 with a Km of 26.5 μmol/L, kcat of 1.09 s−1, and a kcat/Km ratio of 41,132 s−1 mol/L−1. The GKAFRR-L12ADT prodrug was rapidly hydrolyzed by hK2 and was stable in plasma, whereas the GKAFRR-L peptide substrate was unstable in human plasma. The hK2-activated thapsigargin prodrug was not activated by cathepsin B, cathepsin D, and urokinase but was an excellent substrate for plasmin. The GKAFRR-L12ADT was selectively cytotoxic in vitro to cancer cells in the presence of enzymatically active hK2. Conclusion: The hK2-activated thapsigargin prodrug represents potential novel targeted therapy for prostate cancer.
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Shariat, Shahrokh F., Michael W. Kattan, Sibel Erdamar, et al. "Detection of Clinically Significant, Occult Prostate Cancer Metastases in Lymph Nodes Using a Splice Variant-Specific RT-PCR Assay for Human Glandular Kallikrein." Journal of Clinical Oncology 21, no. 7 (2003): 1223–31. http://dx.doi.org/10.1200/jco.2003.08.142.
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Purpose: To compare the detection of human glandular kallikrein 2 (hK2) mRNA expression in archival lymph nodes with disease progression, the development of prostate cancer metastases, and mortality in patients undergoing radical prostatectomy for locally advanced nonmetastatic prostate cancer.Patients and Methods: We evaluated total RNA extracted from fixed, paraffin-embedded, histopathologically normal pelvic lymph nodes, removed at radical prostatectomy, from 199 pT3N0 prostate cancer patients (150 extraprostatic extension only; 49 seminal vesicle involvement) for hK2-expressing cells using a novel reverse transcriptase polymerase chain reaction (RT-PCR)/hK2 assay. Cumulative incidence functions and Cox proportional hazards analyses were performed.Results: Forty patients (20%) had positive results, 80 patients (40%) had negative results, and 79 patients (40%) had equivocal results. RT-PCR/hK2 status was not associated with any pathologic characteristics (P > .05). In postoperative multivariable models, the RT-PCR/hK2 result was associated with prostate cancer progression (P = .001), development of distant metastases (P = .001), and prostate cancer–specific survival (P = .005). In patients experiencing biochemical progression (n = 33), RT-PCR/hK2 status was a predictor of failure to respond to salvage radiotherapy (P = .002).Conclusion: RT-PCR/hK2 can detect biologically and clinically significant occult prostate cancer metastases in histopathologically normal lymph nodes. In patients with locally advanced prostate cancer, RT-PCR/hK2 is strongly associated with prostate cancer progression, failure following salvage radiation therapy, development of clinically evident metastases, and prostate cancer–specific mortality after surgery.
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Jia, Ke-Gang, Gang Feng, Yu-Suo Tong, Guang-Zhou Tao, and Lian Xu. "miR-206 regulates non-small-cell lung cancer cell aerobic glycolysis by targeting hexokinase 2." Journal of Biochemistry 167, no. 4 (2019): 365–70. http://dx.doi.org/10.1093/jb/mvz099.
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Abstract Aerobic glycolysis was closely associated with the malignant transformation and prognosis of tumours. miR-206 was found to be downregulated in several cancers. However, whether miR-206 functions in non-small-cell lung cancers (NSCLCs) via the process of aerobic glycolysis remains poorly characterized. Quantitative real-time PCR was performed to detect miR-206 level in NSCLC cells and tissues. The effect of miR-206 on hexokinase 2 (HK2) expression was examined through miR-206 overexpression or miR-206 knockdown. CCK-8 assay and colony formation assay were carried out to explore the role of miR-206 on cell proliferation and colony formation, respectively. The relationship between miR-206 and HK2 was measured by dual-luciferase reporter assay. Glucose consumption, lactate production assay and ATP generation were performed in NSCLC cells following miR-206 and HK2 overexpression. We found that miR-206 was downregulated in NSCLC tissues and cells. miR-206 overexpression downregulated the expression of HK2 via targeting HK2 3′UTR in NSCLC cells. In addition, miR-206 decreased the cell viability and colony formation in NSCLC cells. Furthermore, miR-206 reduced glucose uptake, lactate production and ATP generation in NSCLC cells via HK2 repression. In conclusion, these findings suggested that miR-206 regulated NSCLC cell aerobic glycolysis by targeting HK2.
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Ahmed, Shanzay, Peter John, Rehan Zafar Paracha, Attya Bhatti, and Monica Guma. "Docking and Molecular Dynamics Study to Identify Novel Phytobiologics from Dracaena trifasciata against Metabolic Reprogramming in Rheumatoid Arthritis." Life 12, no. 8 (2022): 1148. http://dx.doi.org/10.3390/life12081148.
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Enhancement of glycolysis and glutaminolysis are the two most common modalities associated with metabolic reprogramming in rheumatoid arthritis (RA). This enhancement is concomitant to the upregulation of hexokinase 2 (HK2) and glutaminase 1 (GLS1). Hence, the current study was undertaken to identify potential phytobiological inhibitors against HK2 and GLS1, from Dracaena (Sansevieria) trifasciata, an indigenous ethnomedicinal plant found in Pakistan, using computational analysis. Phytobiologics from Dracaena trifasciata were assessed for their ability to co-inhibit HK2 and GLS1 via molecular docking and molecular dynamics simulations. The results underscored seven phytobiologics with promising binding affinities for both HK2 and GLS1. Molecular dynamics simulations further elucidated that all seven identified phytobiologics inhibited HK2 by forming stable complexes but only five amongst the seven had the potential to form stable complexes with GLS1 in real time, thereby implying the potential of co-inhibition for these five compounds. Compound 28MS exhibited an equally strong binding profile for both HK2 (−8.19 kcal/mol) and GLS1 (−8.99 kcal/mol). Furthermore, it exhibited a similar trend in stability during simulation for both targets. Our results serve as a primer for a more lucid understanding towards co-inhibition of HK2 and GLS1 using multiple computational approaches. The identified phytobiologics should undergo in-vitro and in-vivo validation to corroborate their therapeutic potential in RA.
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Ma, Yiqiong, Duilio Michele Potenza, Guillaume Ajalbert та ін. "Paracrine Effects of Renal Proximal Tubular Epithelial Cells on Podocyte Injury under Hypoxic Conditions Are Mediated by Arginase-II and TGF-β1". International Journal of Molecular Sciences 24, № 4 (2023): 3587. http://dx.doi.org/10.3390/ijms24043587.
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Hypoxia is an important risk for renal disease. The mitochondrial enzyme arginase-II (Arg-II) is expressed and/or induced by hypoxia in proximal tubular epithelial cells (PTECs) and in podocytes, leading to cellular damage. Because PTECs are vulnerable to hypoxia and located in proximity to podocytes, we examined the role of Arg-II in the crosstalk of PTECs under hypoxic conditions with podocytes. A human PTEC cell line (HK2) and a human podocyte cell line (AB8/13) were cultured. Arg-ii gene was ablated by CRISPR/Case9 in both cell types. HK2 cells were exposed to normoxia (21% O2) or hypoxia (1% O2) for 48 h. Conditioned medium (CM) was collected and transferred to the podocytes. Podocyte injuries were then analyzed. Hypoxic (not normoxic) HK2-CM caused cytoskeletal derangement, cell apoptosis, and increased Arg-II levels in differentiated podocytes. These effects were absent when arg-ii in HK2 was ablated. The detrimental effects of the hypoxic HK2-CM were prevented by TGF-β1 type-I receptor blocker SB431542. Indeed, TGF-β1 levels in hypoxic HK2-CM (but not arg-ii−/−-HK2-CM) were increased. Furthermore, the detrimental effects of TGF-β1 on podocytes were prevented in arg-ii−/−-podocytes. This study demonstrates crosstalk between PTECs and podocytes through the Arg-II-TGF-β1 cascade, which may contribute to hypoxia-induced podocyte damage.
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Liu, Chunhui, Huibing Li, Hua Huang, Pengyi Zheng, and Zhijun Li. "The Correlation of HK2 Gene Expression with the Occurrence, Immune Cell Infiltration, and Prognosis of Renal Cell Carcinoma." Disease Markers 2022 (February 27, 2022): 1–11. http://dx.doi.org/10.1155/2022/1452861.
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Objectives. Hexokinase 2 (HK2) is one of the key factors involved in the development of several human cancers. However, its role in immune cell infiltration (ICI) and tumor development in renal cell carcinoma is not yet known. Thus, we aimed to explore its relationship with ICI, overall survival, and prognosis of renal cell carcinoma. Methods. In this study, RNA-seq data from renal cancer and normal tissues were extracted from TCGA and the relationship between HK2 expression and pathological features of RCC patients was analyzed using the GEPIA and UALCAN databases. Subsequently, Western blot and qRT-PCR were performed to analyze the protein and mRNA expression of HK2 in renal cell carcinoma tissues and cell lines. Lastly, various bioinformatics tools were applied to determine the immune cell infiltration, survival, and developing prediction model. Results. The analysis of RNA-seq data revealed a high expression of HK2 in renal cell carcinoma; furthermore, Western blot and qRT-PCR also showed high expression of HK2 in renal cancer tissues and cell lines. The high expression of HK2 showed a significant positive correlation with the advanced stage of the tumor, lymph node metastasis, and worst survival in renal carcinoma patients. The high expression of HK2 was further identified as an independent risk factor of RCC patients; it also showed a significant positive immune cell infiltration RCC tumor microenvironment including macrophages, B cells, neutrophils, dendritic cells, and CD8+ T cells. Conclusion. the expression of HK2 is positively correlated with the immune cell infiltration and prognosis of renal cell carcinoma patients, thus playing an important role in renal cancer development.
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Obiezu, Chrisitna V., Erik J. Giltay, Angeliki Magklara, et al. "Serum and Urinary Prostate-specific Antigen and Urinary Human Glandular Kallikrein Concentrations Are Significantly Increased after Testosterone Administration in Female-to-Male Transsexuals." Clinical Chemistry 46, no. 6 (2000): 859–62. http://dx.doi.org/10.1093/clinchem/46.6.859.
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Abstract Background: The genes that encode prostate-specific antigen (PSA) and human glandular kallikrein (hK2) are up-regulated by androgens and progestins in cultured cells, but no published studies have described the effect of androgen administration in women on serum and urinary PSA or hK2. Methods: We measured serum and urinary PSA and hK2 before, and 4 and 12 months post testosterone treatment by immunofluorometric methods in 32 female-to-male transsexuals. Results: Mean serum PSA increased from 1.1 ng/L to 11.1 ng/L and then to 22 ng/L by 4 and 12 months post treatment, respectively; the corresponding mean values in urine were 17, 1420, and 18 130 ng/L, respectively. Serum hK2, another kallikrein closely related to PSA, remained undetectable at the three time points. However, urinary hK2 concentration rose from below the detection limit (&lt;6 ng/L) before treatment to 18 and 179 ng/L by the 4th and the 12th month of treatment, respectively. All changes were statistically significant (P &lt;0.001) at 4 months. Conclusions: Testosterone administration increases serum and urinary PSA and urinary hK2 in women. These measurements may be useful as indicators of androgenic stimulation in women.
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Yin, Xueqian, Malay Choudhury, Jeong-Han Kang та ін. "Hexokinase 2 couples glycolysis with the profibrotic actions of TGF-β". Science Signaling 12, № 612 (2019): eaax4067. http://dx.doi.org/10.1126/scisignal.aax4067.
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Metabolic dysregulation in fibroblasts is implicated in the profibrotic actions of transforming growth factor–β (TGF-β). Here, we present evidence that hexokinase 2 (HK2) is important for mediating the fibroproliferative activity of TGF-β both in vitro and in vivo. Both Smad-dependent and Smad-independent TGF-β signaling induced HK2 accumulation in murine and human lung fibroblasts through induction of the transcription factor c-Myc. Knockdown of HK2 or pharmacological inhibition of HK2 activity with Lonidamine decreased TGF-β–stimulated fibrogenic processes, including profibrotic gene expression, cell migration, colony formation, and activation of the transcription factors YAP and TAZ, with no apparent effect on cellular viability. Fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibited an increased abundance of HK2. In a mouse model of bleomycin-induced lung fibrosis, Lonidamine reduced the expression of genes encoding profibrotic markers (collagenΙα1, EDA-fibronectin, α smooth muscle actin, and connective tissue growth factor) and stabilized or improved lung function as assessed by measurement of peripheral blood oxygenation. These findings provide evidence of how metabolic dysregulation through HK2 can be integrated within the context of profibrotic TGF-β signaling.
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Ikeda, Sho, Fumito Abe, Matsuda Yuka, et al. "Hexokinase-2 Regulates Hypoxia-Inducible Autophagy, Leading to Enhance Anti-Apoptotic Capability of Refractory Multiple Myeloma." Blood 134, Supplement_1 (2019): 1787. http://dx.doi.org/10.1182/blood-2019-123360.
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(background) The drug resistance of multiple myeloma (MM) cells is thought to be induced by various factors of the bone marrow microenvironment. Of these factors, hypoxic stress may be associated with drug resistance in various hematologic malignancies, including MM. Hypoxic stress lead MM cells to induce distinct gene expressions. It has been reported that oncogenic transcription factors such as IRF4 and Myc are suppressed under hypoxia. Instead, accumulation of another transcription factor, HIF-1α upregulates anti-apoptotic proteins, increases glycolysis, and enhances neovascularization leading MM cells to represent anti-apoptotic phenotype. Autophagy is an intracellular process that encapsulates cytoplasmic components, which are directed to the lysosome for degradation. Autophagy and proteasomal degradation prevent apoptosis caused by endoplasmic reticulum (ER) stress. Although proteasome inhibitor such as bortezomib, is a key drug for MM, it may induce treatment resistance. This might be because autophagy is induced in hypoxic microenvironment. Autophagy associated molecules might be therapeutic target in MM cells adapted to hypoxia. (Aim and methods) To clarify the association of hypoxia inducible genes and autophagy, and to obtain rational basis for a new therapeutic strategy against MM, we performed following experiments in vitro using myeloma cell lines (MM.1S, KMS-12-PE, KMS-11, and H929) and primary samples (n=6) that were subjected to hypoxia (1% O2). (Results) First, we examined volcano plot analysis on our cDNA microarray data (GSE80545) of patient samples incubated in normoxia or hypoxia for 48 hours. 546 probes were significantly elevated in hypoxia (fold change > 2.0, p < 0.05). Gene ontology analysis revealed that "Glycolytic Process" contained 13 genes such as PFKFB4, ENO2, ALDOC, PFKFB3, HK2, PFKP, GPI, PGK1, LDHA, ALDOA, ENO1, PKM, and GAPDH. We focused on hexokinase-2 (HK2) because it has been reported that HK2 activates autophagy under stress conditions. Western blot analysis for patient samples revealed that HK2 expression was remarkably upregulated under hypoxia. Apoptosis assay showed that viable cells of HK2 knockdowned cell lines were significantly lower than that of control cells under hypoxia, but not under normoxia. Also, in hypoxia, we found that number of 3-bromopyruvate (3-BrPA, a HK2 inhibitor) subjected viable cells were significantly lower than that of normoxia. This suggested that HK2 contributes to anti-apoptotic phenotype of MM cells under hypoxia. Next, we examined the role of HK2 in autophagy under hypoxia. Because degradation of p62 and increase of LC3-II/LC3-I ratio is considered to be useful for autophagy detection, we examined these factors by Western blot analysis. We found that hypoxic stress decreased expression of p62 and increased the ratio of LC3-II/LC3-I in myeloma cell lines, indicating that hypoxia activates autophagy. However, under hypoxia, these changes were canceled by HK2 knockdown. We confirmed that the number of autophagosome were significantly decreased in HK2-knockdowned myeloma cells by electron microscopy analysis. These data suggested that HK2 is required for hypoxia-inducible autophagy in MM. Finally, we examined the effect of combined inhibition of HK2 and proteasome. In hypoxia, apoptosis by bortezomib was significantly increased in HK2-knockdowned myeloma cells when compared with control. Moreover, we found that the combination of 3-BrPA and bortezomib increased apoptotic cells in both normoxia and hypoxia. These results suggested that HK2-inhibition can induce apoptosis against MM cells with enhancement of sensitivity to proteasome inhibitors. (Conclusion) These results suggest that hypoxia induced HK2 promotes autophagy and inhibits apoptosis. Thus, the combination of proteasome inhibitors and HK2 inhibition may bring about a deep response against treatment resistant MM. Disclosures Ikeda: Nippon Shinyaku Research Grant: Research Funding. Takahashi:Bristol-Myers Squibb: Speakers Bureau; Eisai Pharmaceuticals: Research Funding; Pfizer: Research Funding, Speakers Bureau; Otsuka Pharmaceutical: Research Funding, Speakers Bureau; Kyowa Hakko Kirin: Research Funding; Chug Pharmaceuticals: Research Funding; Ono Pharmaceutical: Research Funding; Novartis Pharmaceuticals: Research Funding, Speakers Bureau; Astellas Pharma: Research Funding; Asahi Kasei Pharma: Research Funding.
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Slagter, Margrita H., Louis JG Gooren, Willem de Ronde, et al. "Serum and Urine Tissue Kallikrein Concentrations in Male-to-Female Transsexuals Treated with Antiandrogens and Estrogens." Clinical Chemistry 52, no. 7 (2006): 1356–65. http://dx.doi.org/10.1373/clinchem.2006.068932.
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Abstract Background: The expression of human tissue kallikrein genes is regulated by steroid hormones, but most studies have been conducted with cancer cell lines. Our purpose was to examine serum and urinary tissue kallikrein concentration changes in male-to-female transsexuals before and after treatment with antiandrogens and estrogens. Methods: Thirty-five male-to-female transsexuals receiving cyproterone acetate and estrogens (orally or transdermally) were included in this study. Serum and urine samples were collected before initiation of therapy and 4 and 12 months post therapy. ELISAs were used to measure multiple kallikreins in serum and urine. Results: After antiandrogen and estrogen therapy, serum testosterone concentrations decreased dramatically, as did serum and urinary concentrations of human glandular kallikrein (hK2) and prostate-specific antigen (PSA; hK3). Statistically significant but relatively small changes in serum and urinary concentrations of many other kallikreins were also seen. Kallikreins in serum and urine were correlated before and after treatment. Conclusions: The concentrations of hK2 and hK3, but not of any other kallikreins, decrease dramatically after combined antiandrogen and estrogen treatment in male-to-female transsexuals. The smaller responses of the other kallikreins presumably reflect their expression in multiple tissues.
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Haese, Alexander, Ville Vaisanen, Judith A. Finlay, et al. "Standardization of Two Immunoassays for Human Glandular Kallikrein 2." Clinical Chemistry 49, no. 4 (2003): 601–10. http://dx.doi.org/10.1373/49.4.601.
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Abstract Background: Measurement of human kallikrein 2 (hK2) has improved early detection and staging of prostate cancer. However, reported concentrations of hK2 among currently used assays have not been standardized in any way. We compared two hK2 assays and five different recombinant hK2 variants (rhK2) and suggest a common calibrator as an important step and putative reference substance in hK2 assay standardization. Methods: We measured 146 sera by two hK2 assays, using assay-specific calibrators to assess the difference between the two assays. Serial dilutions of five rhK2 preparations were measured repeatedly, with one preparation assigned as calibrator and the others as unknowns to define which variant provided the closest match between the two assays. This rhK2 variant was used to recalibrate both assays. We measured hK2 concentrations in the same 146 patients to evaluate the change in the difference. Results: Use of assay-specific calibrators for comparison of the two assays yielded a Deming regression equation of: y = 0.789 (95% confidence interval, 0.674–0.922)x + 0.014 (0.004–0.025) μg/L; R2 = 0.667. Analysis of five rhK2 variants revealed that the enterokinase (ek)-rhK2 form provided the best match between both assays. Using the ek-rhK2 as a common calibrator, we observed a change in the slope of the regression curve to: y = 1.106 (0.872–1.340)x + 0.006 (−0.002 to 0.016) μg/L; R2 = 0.648, suggesting an increase in the mean estimate of agreement between the two assays. Conclusion: Calibration with a common calibrator substantially increased agreement between the assays. The ek-rhK2 variant provided the best performance of all tested rhK2 variants and should undergo mass spectrometry and amino acid analysis for exact mass determination and value assignment to evaluate its potential as a reference material for immunoassays for hK2.
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Zhan, Shanqiang, and Baoliang Ni. "hsa-miR-9-5p Down-Regulates HK2 and Confers Radiosensitivity to Nasopharyngeal Carcinoma." Technology in Cancer Research & Treatment 20 (January 1, 2021): 153303382199782. http://dx.doi.org/10.1177/1533033821997822.
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Background: This study was designed to explore the effects of hsa-miR-9-5p on radiotherapy sensitivity of nasopharyngeal carcinoma (NPC) by targeting hexokinase 2 (HK2). Methods: The levels of hsa-miR-9-5 and HK2 in NPC patients and radiosensitive and resistant cells were determined using qRT-PCR. The dual luciferase reporter gene system was used to determine hsa-miR-9-5p targeting HK2. The level of HK2 expression in NPC were determined using qRT-PCR and western blotting after the administration of hsa-miR-9-5p agomir. The effects of hsa-miR-9-5p on proliferation and apoptosis with or without irradiation (IR) were examined using CCK-8, flow cytometry and colony formation assays. (18F)-Flourodeoxyglucose uptake was used to evaluate the growth of tumor with or without radiation therapy in vivo. Results: hsa-miR-9-5p target to inhibit HK2. Moreover, the cell proliferation was seen in a decreased trend while the cell apoptosis increased in the hsa-miR-9-5p group following radiation therapy hsa-miR-9-5p also showed a significant inhibitory effect on the growth of tumor in vivo with radiation therapy. Conclusions: hsa-miR-9-5p improved the radiosensitivity of NPC by targeting HK2.
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Liu, Hai-E., Hao-Hong Shi, and Xing-Jing Luo. "Upregulated Long Noncoding RNA UCA1 Enhances Warburg Effect via miR-203/HK2 Axis in Esophagal Cancer." Journal of Oncology 2020 (November 4, 2020): 1–11. http://dx.doi.org/10.1155/2020/8847687.
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Reprogrammed glucose metabolism of enhanced aerobic glycolysis, also known as Warburg effect, which exerts a significant contributor to cancer progression, is regarded as a hallmark of cancer. The roles of long noncoding RNAs (lncRNA) in regulating cancer via metabolic reprogramming are mostly unknown, including esophagal cancer (EC). Here, we showed that how the lncRNA urothelial carcinoma associated 1 (UCA1) exerts pro-oncogene in regulating EC glucose metabolism. Firstly, we found that upregulated UCA1 expression enhances the malignant phenotypes of EC, including poor outcome, larger tumor size, positive lymphatic invasion, and advanced pathological stages. UCA1 silencing could suppress EC cell proliferation and metastasis. Following, bioinformatics analyses revealed that UCA1 regulated the HK2 expression through functioning as a competing endogenous RNA (ceRNA). Mechanistically, UCA1 overexpression could elevate the activation of HK2 oncogenes via inhibition of miR-203 activity, as evidenced by the positive correlation of UCA1 with HK2 and inverse correlation with miR-203 expression. Luciferase activity assay further verified the targeting relationship between UCA1, miR-203, and HK2. Upregulated UCA1 in EC cells significantly suppressed the degradation of HK2 by miR-203. Further research showed that upregulated UCA1 effectively increased the rate of glucose uptake, lactate output, and ECAR value, all of which can be attenuate by HK2 interference and 2-DG, whereas knockdown of UCA1 had the opposite effect. In sum, our findings suggest that the UCA1/miR-203/HK2 axis contributes to EC development by reprogramming tumor glucose metabolism, providing new insight into the management of EC patients.