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Isoenzyme pattern and subcellular localization of hexokinases in human brest cancer and nonpathological breast tissue
Abstract
Subcellular distribution of hexokinase (HK) isoenzymes in 22 human breast cancers (21 primary cancers and 1 axillary metastatic growth) and 7 non-pathological human mammary gland tissue samples was studied with starch gel electrophoresis on isolated cell fractions obtained by differential centrifugation. Fractions used were cytosol, mitochondria and microsomes. A comparison of two methods for detecting HK activity was made, yielding different results regarding HK II and HK III. A method based on the ability of NADPH to fluoresce in UV gave a constant pattern of HK isoenzymes. In non-pathological breast tissue, only HK I was seen, i.e. in the cytosol and the microsomal fractions. HK I was also seen in all fractions of the cancers, but another more anodal band of HK I, as well as HK II and HK III, consistently appeared in the cytosol fractions. The more commonly used staining technique with tetrazolium dye revealed HK II in 45% and HK III in 50% of the samples. The pattern of HK isoenzymes in the cancers was the same irrespective of estrogen and progesterone cytosolic receptor contents and the histology of the tumors. The fluorescence method is, therefore, much more sensitive than the tetrazolium technique for detecting HK activity after electrophoresis and could explain difference in results obtained by various laboratories.
... It is currently accepted that elevated ROS can act as a mitogenic signal in tumors to support proliferation [209,210]. In addition, studies in cancer cell lines and tumor models have shown that different PTPC members are overexpressed, especially TSPO, VDAC, and ANT, possibly to favor their specific metabolic condition [16, [211][212][213][214][215][216][217]. We can, therefore, speculate that tumor cells can survive pressure selection by the highly regulated suppression of MPT, which allows the maintenance of MPT-related features with potential mitogenic effects ( Figure 2). ...
Mitochondrial permeability transition (MPT) is the sudden loss in the permeability of the inner mitochondrial membrane (IMM) to low-molecular-weight solutes. Due to osmotic forces, MPT is paralleled by a massive influx of water into the mitochondrial matrix, eventually leading to the structural collapse of the organelle. Thus, MPT can initiate outer-mitochondrial-membrane permeabilization (MOMP), promoting the activation of the apoptotic caspase cascade and caspase-independent cell-death mechanisms. The induction of MPT is mostly dependent on mitochondrial reactive oxygen species (ROS) and Ca2+, but is also dependent on the metabolic stage of the affected cell and signaling events. Therefore, since its discovery in the late 1970s, the role of MPT in human pathology has been heavily investigated. Here, we summarize the most significant findings corroborating a role for MPT in the etiology of a spectrum of human diseases, including diseases characterized by acute or chronic loss of adult cells and those characterized by neoplastic initiation.
... Among the 4 isoforms found in mammalian tissues, HK2 is reported to be particularly overexpressed in cancer tissues [91]. HK2 was found to be expressed in nearly 80% of 27 untreated primary BC analysed by immunohistochemistry [92], corroborating earlier findings [93]. More recently, HK2 was shown to be required for tumorigenesis in mouse models of ErbB2/Her2driven BC, while HK2 ablation inhibited the malignant phenotype of BC cells both in vitro and in vivo [94]. ...
During tumorigenesis, breast tumour cells undergo metabolic reprogramming, which generally includes enhanced glycolysis, tricarboxylic acid cycle activity, glutaminolysis and fatty acid biosynthesis. However, the extension and functional importance of these metabolic alterations may diverge not only according to breast cancer subtypes, but also depending on the interaction of cancer cells with the complex surrounding microenvironment. This microenvironment comprises a variety of non-cancerous cells, such as immune cells (e.g. macrophages, lymphocytes, natural killer cells), fibroblasts, adipocytes and endothelial cells, together with extracellular matrix components and soluble factors, which influence cancer progression and are predictive of clinical outcome. The continuous interaction between cancer and stromal cells results in metabolic competition and symbiosis, with oncogenic-driven metabolic reprogramming of cancer cells shaping the metabolism of neighbouring cells and vice versa. This review addresses current knowledge on this metabolic crosstalk within the breast tumour microenvironment (TME). Improved understanding of how metabolism in the TME modulates cancer development and evasion of tumour-suppressive mechanisms may provide clues for novel anticancer therapeutics directed to metabolic targets.
... Some tumor cells (e.g., breast cancer [44] along with HK-2 express type-1 hexokinase (HK-1) that like type-2 can bind to VDAC and could mediate their highly glycolytic type [87]. In our prior study, we have shown that human CRC cells are characterized by the presence of mitochondrially-bound HK-2 and that its interaction with VDAC may be responsible for increased rates of aerobic glycolysis in these cancer cells [58]. ...
The aim of the work was to evaluate whether or not there is glycolytic reprogramming in the neighboring cells of colorectal cancer (CRC). Using postoperative material we have compared the functional capacity of oxidative phosphorylation (OXPHOS) in CRC cells, their glycolytic activity and their inclination to aerobic glycolysis, with those of the surrounding and healthy colon tissue cells. Experiments showed that human CRC cannot be considered a hypoxic tumor, since the malignancy itself and cells surrounding it exhibited even higher rates of OXPHOS than healthy large intestine. The absence of acute hypoxia in colorectal carcinomas was also confirmed by their practically equal glucose-phosphorylating capacity as compared with surrounding non-tumorous tissue and by upregulation of VEGF family and their ligands. Studies indicated that human CRC cells in vivo exert a strong distant effect on the energy metabolism of neighboring cells, so that they acquire the bioenergetic parameters specific to the tumor itself. The growth of colorectal carcinomas was associated with potent downregulation of the creatine kinase system. As compared with healthy colon tissue, the tumor surrounding cells display upregulation of OXPHOS and have high values of basal and ADP activated respiration rates. Strong differences between the normal and CRC cells in the affinity of their mitochondria for ADP were revealed; the corresponding K m values were measured as 93.6 7 7.7 mM for CRC cells and 84.9 7 9.9 mM for nearby tissue; both these apparent K m (ADP) values were considerably (by almost 3 times) lower in comparison with healthy colon tissue cells (256 7 34 mM).
... VDAC isoforms are significantly higher in malignant tumor cells (Shinohara et al. 2000); ANT-2 is upregulated in renal tumors and transformed hepatocytes (Faure Vigny et al. 1996). HK2 is upregulated in multiple tumors (Shinohara et al. 1991;Rempel et al. 1996;Azoulay-Zohar et al. 2004;Gudnason et al. 1984), and there is a positive correlation between tumorigenesis and the expression level of TSPO (Beinlich et al. 2000;Maaser et al. 2001). Such alterations in gene expression would lead to the erroneous conclusion that the MPT should be favored in tumors. ...
The mitochondrial permeability transition (MPT) consists of an abrupt increase in the permeability of the inner mitochondrial membrane to low molecular weight solutes, resulting in the osmotic breakout of the organelle. MPT drives cell death and provides an etiological contribution to several human disorders characterized by the acute loss of post-mitotic cells. These conditions include ischemia/reperfusion injury, cancer and neurodegenerative disorders. However, precise knowledge of the structure and regulators of the supramolecular entity that induces MPT, the so-called permeability transition pore complex (PTPC), is lacking and this constitutes a substantial obstacle in the development of MPT-targeting agents with clinical applications. Here we report the current evidences about molecular structure and regulatory components of PTPC. In particular we pay attention on new two proteins which recently were added to the list of PTPC components: the mitochondrial F1FO ATP synthase, particularly and the SPG7 paraplegin matrix AAA peptidase subunit. At least a detailed overview of MPT contribution to pathological condition is provided, focusing on the idea that to develop therapeutic drugs, it will be fundamental to understand the molecular composition of the PTPC.
... This enzyme is located on the cytoplasmic side of the mitochondrial outer membrane, where it interacts with other mPTP regulators, and removing this enzyme from this location causes the mPTP to open (79,80). The expression level of HKII is higher in several tumors (81)(82)(83)(84). However, because of its role in glycolysis, the upregulation of HKII expression may be linked to the Warburg effect, which is generally observed in tumors and provides a metabolic advantage rather than specific resistance to apoptosis. ...
Since its discovery in the 1970s, the mitochondrial permeability transition (MPT) has been proposed to be a strategic regulator of cell death. Intense research efforts have focused on elucidating the molecular components of the MPT because this knowledge may help to better understand and treat various pathologies ranging from neurodegenerative and cardiac diseases to cancer. In the case of cancer, several studies have revealed alterations in the activity of the mitochondrial permeability transition pore (mPTP) and have determined its regulatory mechanism; these studies have also suggested that suppression of the activity of the mPTP, rather than its inactivation, commonly occurs in solid neoplasms. This review focuses on the most recent advances in understanding mPTP regulation in cancer and highlights the ability of the mPTP to impede the mechanisms of cell death.
Metabolism is the main characteristic of bio-organism. Deregulation of metabolism including up or down regulation of enzymes and metabolites result in human disease and in turn, development of the disorders are manifested in metabolism. This concept is observed in breast cancer evidently. In this book, we reviewed more than 4000 articles regarding carbohydrate metabolism in breast cancer in the enzyme by enzyme, metabolite by metabolite and step by step manner. The topics were summarized in 12 chapters that including glycolysis, pyruvate fate, lactate production, transporters, TCA cycle, electron transport chain, hexosamine biosynthetic pathway, glycogen metabolism, gluconeogenesis, pentose phosphate pathway, glucuronic acid metabolism, glycosaminoglycans. Ultimately we discussed the importance of diabetes and other metabolic disorders in breast cancer pathogenesis. In this book, we try to answer several questions; which enzyme, metabolite or metabolic change could be considered as biomarker for diagnosis, classification, and prognosis and patients outcome. What is the role of metabolic shifts in the cancer initiation, progression, metastasis, and chemoresistance and treatment failure? Which enzymes or pathways may be a potential target for breast cancer treatment and how it is possible to modulate these critical points in order to cancer treatment? Finally, this book is recommended for biologist, breast cancer researchers and physicians.
In this study high-refractive-index polyimide/titania hybrid optical thin films were successfully prepared using a sol-gel process combined with spin coating and multistep baking. The hybrid thin films were prepared from a soluble polyimide, a coupling agent, and a titania precursor. Transparent hybrid thin films can be obtained at TiO2 content as high as 40 wt.-%. The FE-SEM results suggest that the TiO2 particles in the hybrid thin films have diameters in the nanometer range. The thermal decomposition temperatures of the prepared hybrid materials are above those of the respective polyimide except for the highest TiO2 content hybrids. The refractive indices at 633 nm of the prepared hybrid thin films increase linearly from 1.66 to 1.82 with increasing TiO2 content. The excellent optical transparency, thermal stability, and tunable refractive index provide the potentials of the polyimide/titania hybrid thin films in optical applications.
The use of FDG-PET imaging in tumour detection is now well established. Many studies have suggested that changes in the uptake of FDG predict tumour response to therapy and that further clinical information regarding tumour grade and proliferative status may also be derived from FDG-uptake scans. More studies are required to verify the role of FDG-PET in patient management. Upregulation of hexokinase and glucose transporters, especially Glut-1, and downregulation of glucose-6-phosphatase are frequently associated with transformation. The extent of these changes has been related to both differentiation and proliferation largely corresponding with FDG-uptake studies.
A thyroid tumor cell line has been established from the metastases of a follicular carcinoma in a female patient. Although the primary tumor released thyroglobulin (Tg) into the circulation (greater than 10,000 ng/ml), the uptake of I131 was less than 2%. After 37 replications the doubling time was 4 days and confluency was reached after 7 days from inoculation of 3 x 10(7) cells. This human thyroid tumor cell line has now been growing in culture for several years. An aneuploid chromosomal pattern was observed (62-82 chromosomes). A pair of X chromosomes was present but no Y chromosome was found which is compatible with the female origin of the cell line. EM studies revealed the presence of microvilli. Immunoperoxidase staining using specific anti-human Tg antisera indicated the presence of Tg within the cells. Nude mice developed solid-cystic tumors within 6 months after injection of the cells. The basal release of immunodetectable Tg, as measured in a perifusion system, increased in response to thyroid stimulating hormone (TSH) (P less than 0.025) or TSH combined with theophylline (P less than 0.001). Unusual isoenzyme patterns for galactose-1-phosphate-uridyltransferase (GALT) and phosphoglucomutase1 (PGM1) were detected in the tumor, compared with normal human fibroblasts and blood cells and isoenzyme patterns from the patient's lymphocytes. Because this malignant human thyroid follicular cell line has retained the ability to synthesize Tg it represents a valuable model for the study of human follicular carcinomas.
High resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in combination with computer-assisted densitometry was used to analyze 800-1000 silver stained postmitochrondrial and 600-800 cytosolic polypeptides extracted from malignant and nonmalignant human breast tissues. The 2D-PAGE patterns of polypeptides from malignant and normal tissues were similar, although both qualitative and quantitative polypeptide differences were noted. Six cytosolic polypeptides (pI/molecular mass x 10(-3), 5.20/80, 5.75/43, 6.20/40, 5.43/35, 5.46/34.5, and 5.50/34 were detected exclusively in malignant tissues. One constitutive polypeptide, p52 (7.25/52), was not detected in tumor samples. Marked quantitative differences in spot density were noted in polypeptides localized mainly in the molecular weight ranges of 22-40 kDa and pI of 5.65-7.00. An overall increase in polypeptide expression was noted in this region of 2D-PAGE gels of malignant tissues as compared to normal. Twenty-two acidic and 19 polypeptides separated under nonequilibrium isoelectric focusing conditions were significantly increased in tumor samples while one polypeptide was decreased. One polypeptide, p24 (6.15/24), was expressed in greatest concentrations in tumors which also expressed the greatest estrogen receptor content. Expression of p24 was markedly reduced in normal tissue and in malignant tissues expressing low levels of estrogen and progesterone receptors. No significant differences in the expression of the Yb and Ya subunits of glutathione-S-transferases (GST)-A, -B and ligandin were observed between normal and malignant breast tissue. None of the Yp subunits of the placental isoform of GST were detected in either normal or malignant breast tissues.
Activities of some enzymes associated with carbohydrate and lipid metabolism were determined in 48 human breast carcinomas and compared with those found in 35 nonmalignant breast tumours and also in 13 normal breast tissues. In fibrocystic disease only the activity of citrate lyase was markedly higher (14-fold) than in normal tissue. The activities of the remaining enzymes did not differ significantly from those in normal tissue. Enzyme activities in breast carcinoma were 4--160 x those determined in normal tissue according to the following sequence : phosphofructokinase less than malate NADP dehydrogenase less than hexokinase less than lactate dehydrogenase less than isocitrate NADP dehydrogenase less than ATP citrate lyase. Activity of citrate lyase, very low in normal breast (0.0017 mumol/min/g of tissue) rose gradually to 0.039, 0.072 and 0.258 mumol/min/g of tissue in localized fibrocystic disease, fibroadenomas and carcinomas respectively. These data support the idea that citrate lyase may play an important role in lipogenesis in hyperplastic human breast tissues.
Electrophoresis of cytosol prepared from normal and malignant tissue samples of uterine cervix and endometrium revealed interesting differences which may be relevant to the characteristic alterations in glucose metabolism associated with tumour development. Hexokinase II was detected in 30% of the cancer material from both sources, but in none of the samples of normal cervix. A duplet band of 6-phosphogluconate dehydrognease was seen in the majority of the cancer samples but in no sample of normal cervix; it appeared to be partly due to ageing of the sample, and is not phenotypically related to the malignant process. Analysis of genetic variance for phosphoglucomutase at the PGM1 locus revealed a highly significant excess of the PGM1-1 phenotype in patients with cancer of the endometrium, which may reflect susceptibility to endometrial cancer in patients with this phenotype. At the PGM2 locus, samples of malignant cervix were deficient in "Band f" compared with normal cervix samples, all of which showed this band. Conversely, gene products of the PGM3 locus were found in most samples of malignant cervix and a small minority of normal cervix samples. Compared with the isomorphic distribution of lactate dehydrogenase enzymes in normal uterine tissue, cancers showed a shift towards either a more anodal or a more cathodal pattern. The former may be associated with tumours enjoying a good oxygen supply, and the latter with tumours which, because of their depth or poor blood supply have to function under less aerobic conditions.
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The complement of enzymes in a differentiated tissue from an adult animal or person is quite characteristic for that tissue. It may change in response to hormonal or dietary influences, but is generally such that tissue metabolism is optimal under a particular set of conditions. While some enzymes are common to all tissues, the quantities may vary from organ to organ. Other enzymes are found predominantly in a particular organ. Amylase, for example, is found in highest concentration in the pancreas; a different form of the enzyme occurs in saliva. Lactose synthase is confined to the lactating mammary gland, whereas the enzymes of gluconeogenesis are limited to the liver and the kidney cortex.
Human infiltrating ductal carcinoma of the breast, fibrocystic disease of the breast, and normal human mammary tissues were assayed for their concentration of nucleic acid, lipids, and 11 selected enzymes involved in carbohydrate, lipid, and amino acid metabolism. Comparing infiltrating ductal carcinomas to normal mammary tissue, striking elevations (p <0.001) were seen in pyruvate kinase (100-fold), glucose-6-phosphate, isocitrate and malate dehydrogenases (10-15-fold), free fatty acids and cholesterol (2-3-fold). The RNA/DNA ratios in the carcinomas were unchanged but triglycerides and α-glycerolphosphate dehydrogenase were reduced. Also studied were samples from eight cases of fibrocystic disease, a common nonmalignant proliferative disease of the human breast. Ranking the levels of nucleic acids and lipids and the activities of the enzymes in the tissues studied clearly showed that normal ≦ fibrocystic disease ≦ infiltrating ductal carcinoma. No significant differences in the biochemical characteristics of the carcinomas were found relative to the size of the lesion or to the menopausal state of the patient. These data on infiltrating ductal carcinomas closely resemble the data obtained from single dose 7,12-dimethylbenz [a] anthracene-induced rat mammary carcinomas, suggesting that the latter experimental tumor may represent a valid metabolic model of human breast cancer.
1. Hexokinase and glucokinase activities were assayed in ultracentrifugally prepared fractions of livers and mammary glands of virgin, pregnant, lactating and 2-days-postlactating mice. 2. Liver contained both enzymes and both were located in the particle-free supernatant fraction. The levels of hepatic glucokinase activities observed in virgin, pregnant and postlactating mice were similar (about 600 mμmoles of TPNH formed per gram tissue), and about one-third of those observed in the lactating mice. 3. In the mammary glands of virgin mice glucose-ATP phosphotransferase activity was present mainly in the particle-free supernatant fraction. But in the glands of pregnant and lactating mice, glucose phosphorylation (solely by hexokinase) was also detected in the mitochondrial fraction. 4. The glands of virgin and pregnant mice contained both enzymes and, in contrast to liver, hexokinase activity exceeded that of glucokinase activity. Glucokinase was not detected in glands of lactating mice. Glucokinase activity in the virgin and pregnant glands probably reflects the presence of large amounts of adipose tissue in these glands. 5. The more than 2-fold increase in glucose phosphorylating activity observed in the mammary glands of lactating mice, as compared to that in the nonlactating mice, is attributed to the increase in activity of hexokinase associated with the mitochondrial fraction. This increase in hexokinase activity is probably related to the increased proportion of secretory elements of the lactating gland.
The activities and isoenzyme distributions of soluble hexokinase were investigated spectrophotometrically and by cellulose acetate membrane electrophoresis in extracts of normal human brain, a series of fetal brains ranging in gestational age between 6 and 42 weeks and in 27 gliomas. The enzyme was first detected at 14 weeks gestational age, at a level approximately half of the normal adult level. Only one isoenzyme (type I) was present. The activity gradually increased until similar levels to those found in the adult were reached at term. The rise in enzyme levels was particularly rapid between 35 and 40 weeks. This rapid rise was associated with the appearance of a second isoenzyme of hexokinase (type II), first detected at the age of 36 weeks gestation. Normal adult brain contained type I hexokinase only. Further work needs to be done to determine when the type II enzyme disappears.Types I and II isoenzymes were detected in fourteen of the gliomas studied, with a high incidence in the poorly differentiated tumours.
The starch gel electrophoretic isozyme patterns of phosphoglucomutase, glucose-6-phosphate dehydrogenase, hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase in the cancerous and in the apparently uninvolved regions of livers from human primary hepatoma patients were compared. The HK, PK, and lactate dehydrogenase isozyme patterns in hepatoma were also compared with those in normal adult tissues (liver, muscle, dura) and fetal tissues (liver, muscle, heart, brain). In addition, other human tumors (esophageal cancer, meningioma, mesothelioma) and cell cultures of tumors (hepatoma, esophageal cancer, HeLa) and fibroblasts were examined. The phosphoglucomutase and glucose-6-phosphate dehydrogenase patterns in hepatoma were similar to those in host livers, except in 1 case in which the glucose-6-phosphate dehydrogenase pattern was altered in the tumor. Hepatoma patterns differed from those of normal liver in that HK II was present, and the proportion of HK III was reduced; the proportion of another form of HK (II(f)), observed in most of the tissues, was increased in hepatoma. In cell lines of hepatoma and esophageal cancer, the proportion of HK II was increased, compared with the respective tissues of origin. The PK patterns in host livers and hepatomas, although often different from normal liver, were variable, and there were no consistent trends. Several host livers and hepatomas had additional bands (possibly hybrids) between PK L and PK M2. All other tumor tissues and cell lines had PK M2. The lactate dehydrogenase patterns in hepatoma differed from the liver patterns in only a few cases, when there appeared to be a slight increase in the proportion of H subunits. The proportion of H subunits was also increased in hepatoma cells.
The mitochondrial-bound hexokinases (adenosine triphosphate:D-hexose 6-phosphotransferase) of mammary adenocarcinoma and of normal gland were compared in lactating C3H mice. Treatment of mitochondria isolated from both the normal and neoplastic tissue with 0.5 m NaCl or 0.1 mM glucose 0-phosphate effected the release of about 50% of the bound hexokinase. In the presence of magnesium ion, enzyme from either source attached to mitochondria from either tissue and in all combinations to the same extent. Identification of the isoenzyme complement in the mitochondrial extract by diethylaminoethylcellulose chromatography revealed only types I and II. In the tumor, the hexokinase activity in both the cytosol and the fraction solubilized from mitochondria was predominantly in the form of type I ( 60%). In contrast, the activity released from mitochondria isolated from normal gland was predominately type II, while the cytosol contained almost equivalent amounts of types I and II. While this difference does not explain differences in glucose utilization between the normal and neoplastic tissue, it may provide a means of distinguishing between the two.
1. A form of hexokinase which appears to correspond to HK III, seen in liver, spleen and lung, has been identified in human polymorphonuclear leucocytes. HK III activity has not been demonstrated in lymphocytes, permanent lymphoblastoid cell lines or in cultured fibroblasts. 2. In fresh leucocyte preparations HK III usually appeared as a single band, but in extracts of post-mortem tissues multibanded patterns were seen. Leucocyte preparations also give multi-banded patterns after storage for more than a few hours. 3. In certain individuals the HK III showed a two-banded pattern even in fresh preparations. Repeat samples and family studies suggested that this pattern was genetically determined and represented a heterozygote for a common allele HK1III and a less common variant HK2III. Out of a total of 330 English people successfully tested 10 probable heterozygotes were found. 4. It is suggested that HK III may be a monomeric enzyme whose genetic determination is independent of that of the other main forms of hexokinase.