PCK2 activation mediates an adaptive response to glucose depletion in lung cancer.

Cancer cells are reprogrammed to utilize glycolysis at high rates, which provides metabolic precursors for cell growth. Consequently, glucose levels may decrease substantially in underperfused tumor areas. Gluconeogenesis results in the generation of glucose from smaller carbon substrates such as lactate and amino acids. The key gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK), has been shown to provide metabolites for cell growth. Still, the role of gluconeogenesis in cancer is unknown. Here we show that the mitochondrial isoform of PEPCK (PCK2) is expressed and active in three lung cancer cell lines and in non-small cell lung cancer samples. PCK2 expression and activity were enhanced under low-glucose conditions. PEPCK activity was elevated threefold in lung cancer samples over normal lungs. To track the conversion of metabolites along the gluconeogenesis pathway, lung cancer cell lines were incubated with (13)C3-lactate and label enrichment in the phosphoenolpyruvate (PEP) pool was measured. Under low glucose, all three carbons from (13)C3-lactate appeared in the PEP pool, further supporting a conversion of lactate to pyruvate, via pyruvate carboxylase to oxaloacetate, and via PCK2 to phosphoenolpyruvate. PCK2 small interfering RNA and the pharmacological PEPCK inhibitor 3-mercaptopicolinate significantly enhanced glucose depletion-induced apoptosis in A549 and H23 cells, but not in H1299 cells. The growth of H23 multicellular spheroids was significantly reduced by 3-mercaptopicolinate. The results of this study suggest that lung cancer cells may utilize at least some steps of gluconeogenesis to overcome the detrimental metabolic situation during glucose deprivation and that in human lung cancers this pathway is activated in vivo.

SAHA induces caspase-independent, autophagic cell death of endometrial stromal sarcoma cells by influencing the mTOR pathway.

Endometrial stromal sarcomas are rare and molecular mechanisms involved in their pathogenesis are poorly understood. Covalent modifications of histone proteins, in particular de/acetylation of lysine residues, play an important role in the regulation of gene transcription in normal and neoplastic cells, but there are only limited data about these processes in solid mesenchymal tumours. We treated endometrial stromal sarcoma cells (ESS-1) and non-malignant human endometrial stromal cells (HESCs) with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor. SAHA was able to mediate the cell cycle and expression of genes related to the malignant phenotype of endometrial stromal tumours, eg p21(WAF1) and HDAC7. SAHA led to dose-dependent differentiation and death of ESS-1 cells but not of HESCs. Exposure of HESCs to SAHA resulted only in slightly decreased cell proliferation. SAHA also increased the p21(WAF1) expression and caused significant changes in the cell cycle by inhibiting the G1/S transition in ESS-1 cells. Recovery experiments indicated that these changes became irreversible when the tumour cells were treated with SAHA for longer than 24 h. In our experimental system, not apoptotic but autophagic processes were responsible for the cell death. Monodansyl cadaverine accumulation in treated ESS-1 cells and decreased expression of the mTOR and phospho-S6 ribosomal protein (S6rp) additionally supported this observation. Taken together, our study indicates that HDACs might be considered as potential drug targets in the therapy of stromal sarcomas and that SAHA might be a promising therapeutic agent for endometrial stromal sarcoma.

Inhibition of lung carcinoma cell growth by high density lipoprotein-associated alpha-tocopheryl-succinate.

Alpha-tocopheryl-succinate (alphaTS) is a synthetic, anti-neoplastic derivative of alpha-tocopherol. Here we studied the effects of free and high-density lipoprotein subclass 3 (HDL3)-associated alphaTS on the growth of human (A549) and mouse Lewis (LL2) lung carcinoma cells. Both free and HDL3-associated alphaTS inhibited A549 growth in a time- and concentration-dependent manner. Treatment of A549 cells with alphaTS-enriched HDL3 led to DNA fragmentation and a time-dependent decrease in immunoreactivity of poly(ADP-ribose)polymerase. Uptake experiments revealed a high capacity for selective alphaTS uptake in excess of holoparticle endocytosis. Overexpression of scavenger receptor class B, type I (SR-BI), the prime receptor mediating selective lipid uptake, in A549 cells resulted in significantly increased selective alphaTS uptake, a finding associated with complete cellular growth arrest. The present in vitro findings were verified in an in vivo model: tumor inoculation in C57BL6 was performed with either wild-type, beta-galactosidase- or SR-BI-overexpressing LL2 cells. After tumor inoculation, the animals received six consecutive intravenous injections of alphaTS. This experimental setup resulted in significantly reduced tumor burden in animals that were inoculated with SR-BI-overexpressing LL2 cells but not in animals inoculated with wild-type or beta-galactocidase-transfected cells. Based on our in vitro and in vivo findings, we propose that SR-BI could provide a novel route for HDL3-mediated drug delivery of anti-neoplastic drugs.

Adenovirus-mediated rescue of lipoprotein lipase-deficient mice. Lipolysis of triglyceride-rich lipoproteins is essential for high density lipoprotein maturation in mice.

Lipoprotein lipase (LPL) is the rate-limiting enzyme for the hydrolysis of triglycerides and the subsequent uptake of free fatty acids in extrahepatic tissues. Deficiency of LPL in humans (Type I hyperlipoproteinemia) is associated with massive chylomicronemia, low high density lipoprotein (HDL) cholesterol levels, and recurrent attacks of pancreatitis when not controlled by a strict diet. In contrast to humans, homozygous LPL knock-out mice (L0) do not survive suckling and die between 18 and 24 h after birth. In this study, an adenovirus-based protocol was utilized for the transient expression of LPL during the suckling period in an effort to rescue L0 mice. After a single intraperitoneal injection of 5x10(9) plaque-forming units of LPL-expressing virus immediately after birth, more than 90% of L0 mice survived the first days of life. 3% of L0 mice survived the entire suckling period and lived for up to 20 months, although LPL activity in mouse tissues and postheparin plasma was undetectable in all animals after 6 weeks of age. Adult LPL-deficient mice were smaller than their littermates until 2-3 months of age and exhibited very high triglyceride levels in the fed (4997 +/- 1102 versus 113.4 +/- 18.7 mg/dl) and fasted state (2007 +/- 375 versus 65.5 +/- 7.4 mg/dl). Plasma total cholesterol levels, free fatty acids, and ketone bodies were elevated in L0 mice, whereas plasma glucose was normal. Most strikingly, L0 mice lacked apoA-I-containing prebeta-HDL particles as well as mature HDL resulting in undetectable HDL cholesterol and HDL-apoA-I levels. HDL deficiency in plasma was evident despite normal apoA-I mRNA levels in the liver and normal apoA-I protein levels in plasma, which were predominantly found in the chylomicron fraction. The absence of prebeta-HDL and mature HDL particles supports the concept that the lipolysis of triglyceride-rich lipoproteins is an essential step for HDL maturation.

Role of various tissues in apo(a) fragmentation and excretion of fragments by the kidney.

BACKGROUND: Lipoprotein(a) [Lp(a)] is an atherothrombotic plasma lipoprotein with unknown function. Little is known about the catabolism of this lipoprotein, in particular the steps related to apolipoprotein(a) [apo(a)] fragmentation and excretion by the kidney. MATERIAL AND METHODS: High plasma levels (up to 9 mg dL(-1)) of the N-terminal fragment of apo(a) were expressed in mice by adenovirus mediated gene transfer. Plasma of such N-apo(a) mice was injected into acceptor mice and the fragmentation and urinary secretion of N-apo(a) were followed by immunochemical techniques. RESULTS: Mice transduced with N-Ad expressed apo(a)-fragments with 3-11 kringle-IV (KIV) repeats. Injection of N-apo(a)-plasma from donor mice into acceptor mice resulted in fragmentation of N-apo(a)s with 3-11 KIVs yielding smaller peptides down to 2 KIVs. Secretion of N-apo(a)-fragments with 2 to maximally 6 KIVs into urine occurred as early as 2 min after injection. Immunohistochemical studies of kidney suggested filtration as a mechanism of apo(a)-fragment excretion. When N-apo(a) was incubated in vitro with various tissues from perfused mice, skeletal muscle and kidney followed by liver and spleen contributed to fragmentation. Tissues from unperfused organs, or the addition of normal mouse plasma, caused marked reduction in N-apo(a) fragmentation. EDTA, and not aprotinin or leupeptin, prevented apo(a) cleavage. CONCLUSION: Here we provide evidence that apo(a) is cleaved by metalloproteinases located on skeletal muscle, kidney and other organs. Small apo(a)-fragments up to a size of 6 KIVs are excreted into urine, yet a major portion of apo(a) fragments is removed from circulation extrarenally.

Adenovirus-mediated apo(a)-antisense-RNA expression efficiently inhibits apo(a) synthesis in vitro and in vivo.

Apo(a) is a very atherogenic plasma protein without apparent function, which is highly expressed in humans. The variation in plasma Lp(a) concentration among individuals is considerable. Approximately 10-15% of the white population exhibit plasma Lp(a) concentrations above the atherogenic cut-off value of approximately 30 mg/dl. Since there is currently no safe way of treating those patients with drugs, we have tested the possibility of interfering with apo(a) biosynthesis by adenovirus-mediated expression of antisense apo(a) mRNA comprising the 5' UTR, the signal sequence and the first three kringles of native apo(a). Transduction of rat hepatoma McA RH 7777 cells which stably expressed apo(a) with 18 kringle IV (KIV) domains with apo(a)-antisense adenovirus (AS-Ad) at multiplicity of infection (MOI) of 30 reduced apo(a) synthesis to 23% as compared with control cells. As apo(a) is not synthesized in laboratory animals, we induced biosynthesis of the N-terminal fragments of apo(a) in mice by adenovirus-mediated gene transfer. Cotransduction of these mice with AS-Ad, which expressed up to eight times higher amounts of apo(a) than stable transgenic apo(a) mice, led to an almost complete disappearance of apo(a) from plasma. We conclude that the proposed AS-construct is very efficient in interfering with apo(a) biosynthesis in vivo. The strategy of inducing the synthesis of a nonexpressed protein followed by knocking it out by AS technology may also be applicable to other systems.

Impact of apolipoprotein(a) on in vitro angiogenesis.

Angiostatin, which consists of the kringle I-IV domains of plasminogen and which is secreted into urine, is an efficient inhibitor of angiogenesis and tumor growth. Because N-terminal apolipoprotein(a) [apo(a)] fragments, which also contain several types of kringle IV domains, are found in urine as well, we evaluated the potential angiostatic properties of these urinary apo(a) fragments and of a recombinant form of apo(a) [r-apo(a)]. We used human microvascular endothelial cell (hMVEC)-based in vitro assays of tube formation in 3-dimensional fibrin matrixes. Purified urinary apo(a) fragments or r-apo(a) inhibited the basic fibroblast growth factor/tumor necrosis factor-alpha-induced formation of capillary-like structures. At concentrations varying from 0.2 to 10 microgram/mL, urinary apo(a) fragments inhibited tube formation by as much as 70%, whereas there was complete inhibition by r-apo(a). The highest concentrations of both inhibitors also reduced urokinase plasminogen activator production of basic fibroblast growth factor-induced hMVEC proliferation. The inhibitors had no effect on plasminogen activator inhibitor-1 expression. If our in vitro model for angiogenesis is valid for the in vivo situation as well, our data point toward the possibility that apo(a) may also be physiologically operative in modulating angiogenesis, as the concentration of free apo(a) found in humans exceeds that tested herein.

Scavenger receptor class B, type I is expressed in porcine brain capillary endothelial cells and contributes to selective uptake of HDL-associated vitamin E.

It is clearly established that an efficient supply to the brain of alpha-tocopherol (alphaTocH), the most biologically active member of the vitamin E family, is of the utmost importance for proper neurological functioning. Although the mechanism of uptake of alphaTocH into cells constituting the blood-brain barrier (BBB) is obscure, we previously demonstrated that high-density lipoprotein (HDL) plays a major role in the supply of alphaTocH to porcine brain capillary endothelial cells (pBCECs). Here we studied whether a porcine analogue of human and rodent scavenger receptor class B, type I mediates selective (without concomitant lipoprotein particle internalization) uptake of HDL-associated alphaTocH in a similar manner to that described for HDL-associated cholesteryl esters (CEs). In agreement with this hypothesis we observed that a major proportion of alphaTocH uptake by pBCECs occurred by selective uptake, exceeding HDL3 holoparticle uptake by up to 13-fold. The observation that selective uptake of HDL-associated CE exceeded HDL3 holoparticle up to fourfold suggested that a porcine analogue of SR-BI (pSR-BI) may be involved in lipid uptake at the BBB. In line with the observation of selective lipid uptake, RT-PCR and northern and western blot analyses revealed the presence of pSR-BI in cells constituting the BBB. Adenovirus-mediated overexpression of the human analogue of SR-BI (hSR-BI) in pBCECs resulted in a fourfold increase in selective HDL-associated alphaTocH uptake. In accordance with the proposed function of SR-BI, selective HDL-CE uptake was increased sixfold in Chinese hamster ovary cells stably transfected with murine SR-BI (mSR-BI). Most importantly stable mSR-BI overexpression mediated a twofold increase in HDL-associated [14C]alphaTocH selective uptake in comparison with control cells. In line with tracer experiments, mass transfer studies with unlabelled lipoproteins revealed that mSR-BI overexpression resulted in a twofold increase in endogenous HDL3-associated alphaTocH uptake. The results of this study indicate that SR-BI promotes the uptake of HDL-associated alphaTocH into cells constituting the BBB and plays an important role during the supply of the CNS with this indispensable micronutrient.

Urinary excretion of apolipoprotein(a): relation to other plasma proteins.

The atherogenic lipoprotein Lp(a) consists of an LDL-like core and apo(a), linked to apoB via a thiol bridge. Apo(a) fragments ranging in size from 60 to 220 kDa are excreted into urine and the excretion rate correlates significantly with the plasma levels of Lp(a). In order to study the interrelationship of apo(a) secretion with that of other plasma proteins, urinary apo(a) and protein secretion of five probands were followed for 24 h at different urinary densities. The excretion rate of apo(a) fragments, despite their high molecular weight, was highest, followed by apoD, orosomucoid, albumin and beta(2)-glycoprotein-I (beta2-GI) and plasminogen (1.58, 0.87, 0.095, 0.027, 0.013 and <0.001%/day, respectively). There was a highly significant correlation between apo(a), apoD and beta2-GI concentrations but not with albumin and orosomucoid concentrations in urine. The only protein that was fragmented in urine was apo(a) while the other proteins had molecular weights comparable to those in plasma. We conclude that a previously suggested fragmentation of apo(a) by the kidney is not a rate-limiting step in its excretion. Since plasminogen, another kringle-IV-containing plasma compound, and fragments thereof, are undetectable in urine under identical experimental conditions, it is very unlikely that the characteristic kringle structure is responsible for the high excretion rate of apo(a).

Silent mutations in secondary Shine-Dalgarno sequences in the cDNA of human serum amyloid A4 promotes expression of recombinant protein in Escherichia coli.

The serum amyloid A (SAA) superfamily comprises a number of differentially expressed genes with a high degree of homology in mammalian species. SAA4, an apolipoprotein constitutively expressed only in humans and mice, is associated almost entirely with lipoproteins of the high-density range. The presence of SAA4 mRNA and protein in macrophage-derived foam cells of coronary and carotid arteries suggested a specific role of human SAA4 during inflammation including atherosclerosis. Here we underline the importance of ribosome binding site (rbs)-like sequences (also known as Shine-Dalgarno sequences) in the SAA4 cDNA for expression of recombinant SAA4 protein in Escherichia coli. In contrast to rbs sequences coded by the expression vectors, rbs-like sequences in the cDNA of target protein(s) are known to interfere with protein translation via binding to the small 16S ribosome subunit, yielding low or even no expression. Here we show that PCR mutations of two rbs-like sequences in the human SAA4 cDNA promote expression of considerable amounts of recombinant SAA4 in E.coli.

Apo(a)-kringle IV-type 6: expression in Escherichia coli, purification and in vitro refolding.

Lipoprotein (a) [Lp(a)] belongs to the class of highly thrombo-atherogenic lipoproteins. The assembly of Lp(a) from LDL and the specific apo(a) glycoprotein takes place extracellularly in a two-step process. First, an unstable complex is formed between LDL and apo(a) due to the interaction of the unique kringle (K) IV-type 6 (T6) in apo(a) with amino groups on LDL, and in the second step this complex is stabilized by a disulfide bond between apo(a) KIV-T9 and apoB(100). In order to understand this process better, we overexpressed and purified apo(a) KIV-T6 in Escherichia coli. Recombinant KIV-T6 was expressed as a His-tag fusion protein under control of the T7 promoter in BL21 (DE3) strain. After one-step purification by affinity chromatography the yield was 7 mg/l of bacterial suspension. Expressed fusion apo(a) KIV-T6 was insoluble in physiological buffers and it also lacked the characteristic kringle structure. After refolding using a specific procedure, high-resolution (1)H-NMR spectroscopy revealed kringle structure-specific signals. Refolded KIV-T6 bound to Lys-Sepharose with a significantly lower affinity than recombinant apo(a) (EC(50) with epsilon-ACA 0.47 mM versus 2-11 mM). In competition experiments a 1000-fold molar excess of KIV-T6 was needed to reach 60% inhibition of Lp(a) assembly.

Effect of tranexamic acid and delta-aminovaleric acid on lipoprotein(a) metabolism in transgenic mice.

The assembly of lipoprotein(a) (Lp(a)) is a two-step process which involves the interaction of kringle-4 (K-IV) domains in apolipoprotein(a) (apo(a)) with Lys groups in apoB-100. Lys analogues such as tranexamic acid (TXA) or delta-aminovaleric acid (delta-AVA) proved to prevent the Lp(a) assembly in vitro. In order to study the in vivo effect of Lys analogues, transgenic apo(a) or Lp(a) mice were treated with TXA or delta-AVA and plasma levels of free and low density lipoprotein bound apo(a) were measured. In parallel experiments, McA-RH 7777 cells, stably transfected with apo(a), were also treated with these substances and apo(a) secretion was followed. Treatment of transgenic mice with Lys analogues caused a doubling of plasma Lp(a) levels, while the ratio of free:apoB-100 bound apo(a) remained unchanged. In transgenic apo(a) mice a 1. 5-fold increase in plasma apo(a) levels was noticed. TXA significantly increased Lp(a) half-life from 6 h to 8 h. Incubation of McA-RH 7777 cells with Lys analogues resulted in an up to 1. 4-fold increase in apo(a) in the medium. The amount of intracellular low molecular weight apo(a) precursor remained unchanged. We hypothesize that Lys analogues increase plasma Lp(a) levels by increasing the dissociation of cell bound apo(a) in combination with reducing Lp(a) catabolism.