Cancer cells are highly dependent on glycolysis to supply the energy and intermediates required for cell growth and proliferation. The enzyme 6-phosphofructo-1-kinase (PFK) is critical for ...glycolysis, and its activity is directly correlated with cellular glucose consumption. Resveratrol is a potential anti-tumoral drug that decreases glucose metabolism and viability in cancer cells. However, the mechanism involved in resveratrol-mediated anti-tumor activity is not entirely clear. In this work, it is demonstrated that resveratrol decreases viability, glucose consumption and ATP content in the human breast cancer cell line MCF-7. These effects are directly correlated with PFK inhibition by resveratrol in these cells. Moreover, resveratrol directly inhibits purified PFK, promoting the dissociation of the enzyme from fully active tetramers into less active dimers. This effect is exacerbated by known negative regulators of the enzyme, such as ATP and citrate. On the other hand, positive modulators that stabilize the tetrameric form of the enzyme, such as fructose-2,6-bisphosphate and ADP, prevent the inhibition of PFK activity by resveratrol, an effect not observed with increased pH. In summary, our results provide evidence that resveratrol directly inhibits PFK activity, therefore disrupting glucose metabolism and reducing viability in cancer cells.
► Resveratrol disrupt glucose metabolism and cell viability in MCF-7 cell line. ► Resveratrol inhibits PFK in human breast cancer cell line MCF-7. ► Resveratrol directly inhibit the purified PFK. ► A new target for resveratrol is proposed.
Cancer cells consume more glucose than normal human cells and convert most glucose into lactate. It has been proposed that deregulated glycolysis is triggered by the posttranslational modification of ...85 kDa muscle-type 6-phosphofructo-1-kinase (PFK-M) which is cleaved by a specific protease to form shorter, highly active, feedback-inhibition-resistant PFK-M fragments.
To find the protease involved in PFK-M modification, analyses of the protease target sites on the human PFK-M enzyme yielding 45–47 kDa fragments were performed in silico. The results suggested that an enzyme in the kallikrein (KLK) family may be involved. Kallikreins can be self-activated in the cytosol and are often overexpressed in cancer cells. After incubating the internally quenched FRET peptide with a sequence characteristic of the target site, along with the active KLK6, the cleavage of the peptide was observed. The ability of KLK6 to cleave native PFK-M and form highly active citrate-resistant 45 kDa fragments was further confirmed by enzymatic tests and SDS-PAGE. A role of KLK6 in the posttranslational modification of native PFK-M was ultimately confirmed in vivo. A yeast strain that encoded native human PFK-M as the only PFK1 enzyme was additionally transformed with proKLK6 or KLK6 genes under the control of an inducible promoter. The transformants growth rate was found to increase after the induction of proKLK6 gene expression as compared to the strain with the native PFK-M enzyme.
KLK6 may be the key protease involved in the modification of PFK-M and trigger deregulated glycolytic flux in cancer cells.
•Kallikrein 6 is a candidate to cleave 6-phosphofructo-1-kinase (PFK) in cancers.•KLK 6 cleaves short amino acid sequence of human muscle type PFK-M.•KLK6 modifies human PFK-M to form highly active shorter PFK-M fragments.•The 45 kDa shorter fragments are formed from 85 kDa human PFK-M protein by KLK6.•KLK6 increases growth rate of S. cerevisiae strain encoding native human PFK-M.
Aspergillus terreus is successfully used for industrial production of itaconic acid. The acid is formed from cis-aconitate, an intermediate of the tricarboxylic (TCA) cycle, by catalytic action of ...cis-aconitate decarboxylase. It could be assumed that strong anaplerotic reactions that replenish the pool of the TCA cycle intermediates would enhance the synthesis and excretion rate of itaconic acid. In the phylogenetic close relative Aspergillus niger, upregulated metabolic flux through glycolysis has been described that acted as a strong anaplerotic reaction. Deregulated glycolytic flux was caused by posttranslational modification of 6-phosphofructo-1-kinase (PFK1) that resulted in formation of a highly active, citrate inhibition-resistant shorter form of the enzyme. In order to avoid complex posttranslational modification, the native A. niger pfkA gene has been modified to encode for an active shorter PFK1 fragment. By the insertion of the modified A. niger pfkA genes into the A. terreus strain, increased specific productivities of itaconic acid and final yields were documented by transformants in respect to the parental strain. On the other hand, growth rate of all transformants remained suppressed which is due to the low initial pH value of the medium, one of the prerequisites for the accumulation of itaconic acid by A. terreus mycelium.
Aspergillus terreus was reported as the promising fungal strain for itaconic acid; however, the commercial production suffers from the low yield. Low production yield was claimed as the result of ...completing the tricarboxylic acid (TCA) cycle towards biomass synthesis while under limiting phosphate and nitrogen; TCA cycle was somewhat shunted and consequently, the metabolite fluxes move towards itaconic acid production route. By regulating enzymes in TCA cycle, it is believed that itaconic acid production can be improved. One of the key responsible enzymes involved in itaconic acid production was triggered in this study. Pyruvate carboxylase was allosterically inhibited by L-aspartate. The presence of 10 mM L-aspartate in the production medium directly repressed PC expression in the living A. terreus while the limited malate flux regulated the malate/citrate antiporters resulting in the increasing cis-aconitate decarboxylase activity to simultaneously convert cis-aconitate, citrate isomer, into itaconic acid. The transport of cis-aconitate via the antiporters induced citrate synthase and 6-phosphofructo-1-kinase activities in response to balance the fluxes of TCA intermediates. Successively, itaconic acid production yield and final concentration could be improved by 8.33 and 60.32 %, respectively, compared to those obtained from the control fermentation with the shortened lag time to produce itaconic acid during the production phase.
Abstract Introduction Early detection and/or prediction of metastasis provide more prognostic relevance than local recurrence. Direct spread into the peritoneum is frequently found in pancreatic ...cancer patients, but positron emission tomography (PET) with 2-deoxy-2-fluoro- d -glucose (FDG) is not useful for identifying such metastasis. We investigated a method to enhance FDG accumulation using AsPC-1 human ascites tumor cells. Methods14 C-FDG accumulation was assessed under the following conditions: 1) characteristics of14 C-FDG transport were examined using phloridzin, a Na+ -free buffer, and various hexoses, and 2) accumulation of14 C-FDG was measured in cells that were pretreated with hexose for various time periods, and activity of 6-phosphofructo-1-kinase (PFK-1) was assayed. Results14 C-FDG transport into AsPC-1 cells was mediated primarily by a Na+ -independent transport mechanism. Aldohexoses such as d -glucose, d -mannose, and d -galactose inhibited14 C-FDG transport. Cells pretreated with d -glucose, d -mannose, or d -fructose exhibited augmented14 C-FDG accumulation. Pretreatment with higher concentrations of d -glucose or d -fructose tended to increase PFK-1 activity. Conclusions Very little information has been published about the association between PFK-1 and FDG accumulation, and we confirmed the impacts of various hexoses on the activity of PFK-1 and FDG accumulation in AsPC-1 cells. Clarifying the relevance of PFK-1 in FDG accumulation will contribute to developing new features of FDG-PET, because PFK-1 is the main regulator of glycolysis.
A modified 6-phosphofructo-1-kinase was expressed in a citrate producing
Aspergillus niger
strain in combination with
cis
-aconitate decarboxylase from
Aspergillus terreus
to study the effect on the ...production of itaconic acid. The modified
pfkA
gene was also expressed in combination with the itaconic acid biosynthetic cluster from
A. terreus
, which consists of
cis-
aconitate decarboxylase
cadA
, a putative mitochondrial transporter
mttA
and a putative plasmamembrane transporter
mfsA
. The combined expression of
pfkA
and
cadA
resulted in increased citrate levels, but did not show increased itaconic acid levels. The combined expression of
pfkA
with the itaconic acid biosynthetic cluster resulted in significantly increased itaconic acid production at earlier time points. Also the itaconic acid productivity increased significantly. The maximum itaconic acid productivity that was reached under these conditions was 0.15 g/L/h, which is only a factor 17 lower than the 2.5 g/L/h that according to the US Department of Energy should be achieved to have an economically feasible production process.
In
Aspergillus niger cells spontaneous posttranslational modification of 6-phosphofructo-1-kinase (PFK1) occurs. In a two step process the native enzyme (85
kDa) is first cleaved to an inactive ...fragment (49
kDa) that regains its activity after phosphorylation of the protein. The shorter PFK1 fragment exhibits changed kinetics, such as resistance to citrate inhibition. In order to avoid spontaneous complex posttranslational modification, modified gene was prepared encoding an active shorter PFK1 fragment. Since no appropriate microbial strains with disrupted native
pfkA genes were available,
Aspergillus niger strain with reduced likelihood for spontaneous posttranslational modification of PFK1 has been chosen for
in vivo tests. First, the appropriate length of a truncated gene was defined after a number of enzymes encoded by genes of different lengths had been tested. After adding sodium azide to the medium, phosphorylation was induced in the transformed hyphae to activate the shorter fragments which were subsequently screened for changed PFK1 kinetics. In the second step the responsible threonine residue was replaced with glutamic acid to elude the need for phosphorylation. An active shorter PFK1 fragment, resistant to citrate inhibition and activated to a higher level by fructose-2,6-bisphosphate with respect to the native enzyme was encoded directly from the modified gene.
Cancer cells are characterized by a high rate of glycolysis, which is their primary energy source. Glycolysis is known to be controlled by allosteric regulators, as well as by reversible binding of ...glycolytic enzymes to cytoskeleton. Clotrimazole is an anti-fungal azole derivative recently recognized as a calmodulin antagonist with promising anti-cancer effect. Here, we show that clotrimazole induced morphological and functional alterations on human breast cancer derived cell line, MCF-7. The drug decreased cell viability in a dose- and time-dependent manner, exhibiting an IC
50 of 88.6
±
5.3
μM and a
t
0.5 of 89.7
±
7.2
min, with 50
μM clotrimazole. Morphological changes were evident as observed by scanning electron microscopy, which revealed the completely loss of protrusion responsible for cell adhesion after a 180
min of treatment with 50
μM clotrimazole. Giemsa stained cells observed by optical microscopy show morphological alterations and a marked nuclear condensation. These changes occurred in parallel to the detachment of the glycolytic enzymes, 6-phosphofructo-1-kinase and aldolase, from cytoskeleton. After a 45
min treatment with 50
μM clotrimazole, the remaining activities in a cytoskeleton enriched fraction was 16.4
±
3.6% and 41.0
±
15.6% of control for 6-phosphofructo-1-kinase and aldolase, respectively. Immunocytochemistry experiments revealed a decrease in the co-localization of 6-phosphofructo-1-kinase and F-actin after clotrimazole treatment, suggesting the site of detachment of the enzymes. Altogether, our results support evidence for apoptotic events that might be started by clotrimazole involving inhibition of glycolytic flux in MCF-7 cells and makes this drug a promising agent in the fight against human breast cancer.
6-Phosphofructo-1-kinase (PFK) was purified to homogeneity from liver of gilthead sea bream (Sparus aurata) and kinetic properties of the enzyme were determined. The native enzyme had an apparent ...molecular mass of 510 kDa and was composed of 86 kDa subunits, suggesting homohexameric structure. At pH 7, S. aurata liver PFK (PFKL) showed sigmoidal kinetics for fructose-6-phosphate (fru-6-P) and hyperbolic kinetics for ATP. Fructose-2,6-bisphosphate (fru-2,6-P2) converted saturation curves for fru-6-P to hyperbolic and activated PFKL synergistically with AMP. Fru-2,6-P2 counteracted the inhibition caused by ATP, ADP and citrate. Compared to the S. aurata muscle isozyme, PFKL had lower affinity for fru-6-P, higher cooperativity, hyperbolic kinetics in relation to ATP, increased susceptibility to inhibition by ATP, and was less affected by AMP, ADP and inhibition by 3-phosphoglycerate, phosphoenolpyruvate, 6-phosphogluconate or phosphocreatine. The effect of starvation-refeeding on PFKL expression was studied at the levels of enzyme activity and protein content in the liver of S. aurata. Our findings indicate that short-term recovery of PFKL activity after refeeding previously starved fish, may result from allosteric regulation by fru-2,6-P2, whereas combination of activation by fru-2,6-P2 and increase in protein content may determine the long-term recovery of the enzyme activity.
Increasing heart workload stimulates glycolysis by enhancing glucose transport and fructose-2,6-bisphosphate (Fru-2,6-P
2), the latter resulting from 6-phosphofructo-2-kinase (PFK-2) activation. ...Here, we investigated whether adenosine monophosphate (AMP)-activated protein kinase (AMPK) mediates PFK-2 activation in hearts submitted to increased workload. When heart work was increased, PFK-2 activity, Fru-2,6-P
2 content and glycolysis increased, whereas the AMP:adenosine triphosphate (ATP) and phosphocreatine/creatine (PCr:Cr) ratios, and AMPK activity remained unchanged. Wortmannin, the well-known phosphatidylinositol-3-kinase inhibitor, blocked the activation of protein kinase B and the increase in glycolysis and Fru-2,6-P
2 content induced by increased work. Therefore, the control of heart glycolysis by contraction differs from that in skeletal muscle where AMPK is involved.
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