Human tumour necrosis factor-α inhibits glucose transport in cultured ehrlich ascites tumour cells

Human recombinant tumour necrosis factor-α (rhTNF-α) arrested the growth of Ehrlich ascites tumour (EAT) cells in vitro. It suppressed cellular glucose uptake and decreased the membrane density of glucose transporters as measured by glucose-reversible cytochalasin B binding. The glucose transporters' affinity for substrate was also reduced. However, rhTNF-α treatment exerted no effect on the phosphoribosyl pyrophosphate level in EAT cells. The role of rhTNF-α on the inhibition of glucose transport of tumour cells is discussed.     

The role of the α-glycerophosphate shuttle in the reoxidation of cytosolic NADH in ehrlich ascites tumour cells

1. Addition of glucose to Ehrlich ascites tumour cells is able to release partially the inhibition by rotenone of the endogenous respiration in the hyperdiploid strain, but has almost no effect on the rotenone-blocked respiration of the hyperdiploid Lettrémutant strain.2. Measurements of redox changes of cytochromes b, c and nicotinamide-adenine nucleotides in the rotenone-inhibited cells indicate that, upon addition of glucose, (a) a translocation of glycolytic reducing equivalents to the mitochondrial cytochromes does occur in the hyperdiploid strain and (b) a reversal of mitochondrial electron transport is stimulated by the increase in the glycolytic phosphate potential in the Lettrémutant.3. Analysis of α-glycerophosphate dehydrogenase activity in the two strains shows that the level of this enzyme is low in the hyperdiploid Lettrécells and much higher (about 20 times) in the hyperdiploid cells. Consequently, upon addition of glucose to rotenone-treated cells, α-glycerophosphate is formed in quantities adequate for the operation of the α-glycerophosphate shuttle only in the latter cells. These findings suggest that the operation of the α-glycerophosphate shuttle is mainly responsible for the aerobic oxidation of glycolytic reducing equivalents found in the wild strain. On the other hand, during glucose breakdown in the presence of rotenone only slight differences are found in the lactate accumulation between the two strains.4. It is concluded that the high rate of aerobic glycolysis in ascites cells cannot be dependent, as suggested by other authors, on the lack of a system for the transfer of glycolytic NADH to the respiratory chain beyond the first phosphorylation site.     

Membrane-bound 5′-nucleotidase inhibitor in Ehrlich ascites tumour cells and newborn mouse liver

5′-Nucleotidase activity in Ehrlich ascites tumour cells was undetectable. The cell homogenate, when mixed with adult mouse liver homogenate, inhibited the 5′-nucleotidase activity of the latter, without affecting is p-nitrophenyl phosphate-hydrolysing activity. The inhibitor activity was enriched (6.8-fold) in a membrane fraction which was enriched in (Na⁺ + K⁺)-ATPase (14-fold) and alkaline phosphatase (8-fold). 5′-Nucleotidase activity in this membrane fraction could be detected only after separating the inhibitor activity from the enzyme on Sephadex G-50. The inhibitor activity was decreased by 27% when heat-treated, 33% when treated with 6 M urea and was almost completely lost when treated with trypsin. It was dialysable from a tubing with a molecular exclusion limit of 10 000, but was retained in a tubing with an exclusion limit of 3000. From these results we conclude that a small molecular weight protein inhibitor(s) of 5′-nucleotidase is present in the plasma membrane of Ehrlich ascites tumour cells. Also, the presence of such an inhibitor in the newborn mouse liver but not in the adult liver suggests that it may have some role in cellular ageing and cancer.     

Synthesis,anti-HIV and cytostatic evaluation of 3′-deoxy-3′-fluorothymidine (FLT) pro-nucleotides

A series of pro-nucleotide phosphoramidates and phosphorodiamidates of the antiviral lead compound 3′-deoxy-3′-fluorothymidine (FLT) have been designed and synthesized. In vitro antiretroviral and cytostatic studies revealed potent (sub-micromolar) inhibition of HIV-1 and HIV-2 replication, with retention of activity in thymidine kinase-negative cell models, as predicted by the ProTide concept.     

Uridine 5′-diphosphate glucose 4-epimerase from ehrlich ascites carcinoma cells

Uridine 5′-diphosphate glucose 4-epimerase (EC 5.1.3.2) from Ehrlich ascites carcinoma cells was purified to apparent homogeneity using conventional procedures and NAD-hexane-agarose affinity chromatography. The protein had a molecular weight of 96,000. The ascites enzyme had an absolute requirement for exogenously added NAD (10 ΜM) for stability. This appears to be a unique feature of ascites epimerase since epimerase from other mammalian sources did not exhibit such a dependence. Exogenously added NAD was also needed for catalysis with an apparentK _m value of 2.5 ΜM. NADH was a very potent competitive inhibitor (K _i = 0.11 ΜM with respect to NAD) of the enzyme activity at pH values close to intracellular pH. The dependence of the enzyme on NAD for stability and its inhibition by NADH may have some potential significance in tumor metabolism     

Selective targeting of 2′-deoxy-5-fluorouridine to urokinase positive malignant cells in vitro

A urokinase targeting conjugate of 2′-deoxy-5-fluorouridine (5-FUdr) was synthesized and tested for tumor-cell selective cytotoxicity in vitro. The 5-FUdr prodrug 2′-deoxy-5-fluoro-3′-O-(3-carboxypropanoyl)uridine (5-FUdrsuccOH) containing an ester-labile succinate linker was attached to the specific urokinase inhibitor plasminogen activator inhibitor type II (PAI-2) and was found to preferentially kill urokinase-over expressing cancer cells. Up to 7 molecules of 5-FUdr were incorporated per PAI-2 molecule without affecting protein activity. This is the first time a small organic cytotoxin has been conjugated to PAI-2.     

Synthesis and anti-HIV activity of 2′-deoxy-2′-fluoro-4′-C-ethynyl nucleoside analogs

Based on the favorable antiviral profiles of 4′-substituted nucleosides, novel 1-(2′-deoxy-2′-fluoro-4′-C-ethynyl-β-d-arabinofuranosyl)-uracil (1a), -thymine (1b), and -cytosine (2) analogs were synthesized. Compounds 1b and 2 exhibited potent anti-HIV-1 activity with IC₅₀ values of 86 and 1.34 nM, respectively, without significant cytotoxicity. Compound 2 was 35-fold more potent than AZT against wild-type virus, and also retained nanomolar antiviral activity against resistant strains, NL4-3 (K101E) and RTMDR. Thus, 2 merits further development as a novel NRTI drug.     

Comparison of glutathione reductase activity and the intracellular glutathione reducing effects of 13 derivatives of 1′-acetoxychavicol acetate in Ehrlich ascites tumor cells

In a previous study, we showed that (1′S)-acetoxychavicol acetate ((S)-ACA) caused a rapid decrease in glutathione (GSH) levels less than 15 min after exposure. (S)-ACA-induced cell death was reversed by the addition of N-acetylcysteine. In the current study, we investigated the inhibitory activities of 13 derivatives of (S)-ACA on tumor cell viability, intracellular GSH level and GR activity. Correlations were found among a decrease in cell viability, intracellular GSH levels and the activity of GR in Ehrlich ascites tumor cells treated with the various ACA analogues. A test of the 13 derivatives revealed that the structural factors regulating activity were as follows: (1) the para or 1′-position of acetoxyl group (or other acyl group) was essential, (2) the presence of a C2′–C3′ double or triple bond was essential, and (3) the S configuration of the 1′-acetoxyl group was preferable.     

Modulation of diphthamide synthesis by 5′-deoxy-5′-methylthioadenosine in murine lymphoma cells

The histidine derivative diphthamide occurs uniquely in eukaryotic elongation factor 2 (EF-2), and is the specific target for the diphtheria toxin mono(ADP-ribosyl)transferase. The first step in diphthamide biosynthesis may involve the transfer of an aminocarboxypropyl moiety from S-adenosylmethionine to the imidazole ring of histidine in EF-2, to yield 2-(3-carboxy-3-aminopropyl)histidine and 5′-deoxy-5′-methylthioadenosine (MeSAdo). As the possible nucleoside product of the initial reaction in the diphthamide biosynthetic pathway, MeSAdo could be an inhibitor of diphthamide formation. In the present experiments, we have analyzed the effects of MeSAdo on diphthamide synthesis in a MeSAdo phosphorylase-deficient mutant murine lymphoma cell line (R1.1, clone H3). As measured by susceptibility to diphtheria toxin-induced ADP-ribosylation, MeSAdo inhibited the formation of diphthamide in EF-2. The inhibition was not due to a nonspecific effect on protein synthesis. Indeed, exogenous MeSAdo substantially protected the lymphoma cells from the lethal effects of diphtheria toxin. These results suggest that MeSAdo can specifically modulate the biosynthesis of diphthamide in EF-2 in murine malignant lymphoma cells.     

Syn- and anti-conformations of 5′-deoxy- and 5′-O-methyl-uridine 2′,3′-cyclic monophosphate

Two uridine 2′,3′-cyclic monophosphate (cUMP) derivatives, 5′-deoxy (DcUMP) and 5′-O-methyl (McUMP), were studied by means of quantum chemical methods. Aqueous solvent effects were estimated based on the isodensity-surface polarized-continuum model (IPCM). Gas phase calculations revealed only slight energy differences between the syn- and anti-conformers of both compounds: the relative energies of the syn-structure are −0.9 and 0.2 kcal mol^-1 for DcUMP and McUMP, respectively. According to the results from the IPCM calculations, however, both syn-conformers become about 14 kcal mol^-1 more stable in aqueous solution than their corresponding anti-structures. Additionally, the effects of a countercation and protonation on DcUMP were studied, revealing that the syn-structure is also favored over the anti-one for these systems.     

7,7′-Diazaindirubin—A small molecule inhibitor of casein kinase 2 in vitro and in cells

Aza- and diaza-bisindoles were synthesized by coupling of 7-azaisatin, 7-azaoxindol, 7-azaindoxyl acetate, and their non-aza counterparts, respectively. Whereas 7,7′-diazaindigo (10) and 7,7′-diazaisoindigo (11) did not show antiproliferative activity in several human tumor cell lines up to 100 μM, 7-azaindirubin (12) and 7′-azaindirubin (13) were more active than the parent molecule, indirubin, in LXFL529L cells (human large cell lung tumor xenograft), and 7,7′-diazaindirubin (14) was exhibiting substantially enhanced growth inhibitory activity in these cells. In the NCI 60 cell line panel, 14 displayed antiproliferative activity preferentially in certain melanoma and non-small cell lung cancer cells. In contrast to the potent serine/threonine/tyrosine kinase inhibition observed for indirubins, kinase inhibition profiling of 14 in 220 kinases revealed largely a loss of kinase inhibitory activity towards most kinases, with retained inhibitory activity for just a few kinases. At 1 μM concentration, especially casein kinases CK1γ3, CK2α, CK2α2, and SIK were inhibited by more than 50%. In cell-based assays, 14 markedly affected CK2-mediated signaling in various human tumor cells. In MCF7 cells, 14 induced cell cycle arrest at G1 and G2/M and apoptosis, whereas CK2-deficient MCF7 cells were resistant. These findings reveal a novel key mechanism of action for 14, suggesting primarily CK2 inhibition to be causally related to growth inhibition of human tumor cells.     

Localization of 3′, 5′-cyclic nucleotide phosphodiesterase activity in isolated thyroid cells and intact thyroid tissue

Summary Cytochemical localization of 3,5-cyclic nucleotide phosphodiesterase (cPDEase) has been investigated by light and electron microscopy in dissociated bovine thyroid cells and in intact bovine thyroid tissue. By light microscopy in isolated thyroid cells reaction product deposition associated with cPDEase activity was localized at the level of the plasma membrane. In intact cryostat cut thyroid tissue, the activity was primarily observed in the cytoplasm and to a lesser extent at the level of the plasma membrane. By electron microscopy, cPDEase activity in isolated cells was found on the plasma membrane and was also encountered on the inner surface of membrane bound vacuoles, presumably pinocytic in origin. In intact tissue, cPDEase activity appeared mostly localized on the apical and lateral plasma membranes and was also present on the outer surface of the endoplasmic reticulum (ER).Even though cPDEase and 5-AMPase did share the same plasma membrane localization, the inhibitory response to theophylline and stimulation with Imidazole permitted the dissociation of their respective activities. 5-AMPase failed to respond to either theophylline or Imidazole suggesting absence of cross reactivity between 5-AMP and cyclic AMP. Thyrotropin (TSH) had no effect on cPDEase activity.We conclude that: (1) regardless of the nature of the material used, the cytomembranes of thyroid cells possess cPDEase activity; and that (2) the variability in distribution as well as in staining intensity recorded by light and electron microscopy between isolated thyroid cells and cryostat cut thyroid tissue is probably inherent to the methodology used.This paper was presented, in part, at the 60th Annual Meeting of the International Academy of Pathology, Montreal, Canada, March 1971 and was initiated in the Department of Pathology, Rhode Island Hospital, Providence, R.I., supported from a Grant-in-Aid of the American Cancer Society, Rhode Island Division, Inc. and the Brown-Hazen Fund.     

Zinc downregulates HIF-1α and inhibits its activity in tumor cells in vitro and in vivo

Background

Hypoxia inducible factor-1α (HIF-1α) is responsible for the majority of HIF-1-induced gene expression changes under hypoxia and for the “angiogenic switch” during tumor progression. HIF-1α is often upregulated in tumors leading to more aggressive tumor growth and chemoresistance, therefore representing an important target for antitumor intervention. We previously reported that zinc downregulated HIF-1α levels. Here, we evaluated the molecular mechanisms of zinc-induced HIF-1α downregulation and whether zinc affected HIF-1α also in vivo.

Methodology/Principal Findings

Here we report that zinc downregulated HIF-1α protein levels in human prostate cancer and glioblastoma cells under hypoxia, whether induced or constitutive. Investigations into the molecular mechanisms showed that zinc induced HIF-1α proteasomal degradation that was prevented by treatment with proteasomal inhibitor MG132. HIF-1α downregulation induced by zinc was ineffective in human RCC4 VHL-null renal carcinoma cell line; likewise, the HIF-1αP402/P564A mutant was resistant to zinc treatment. Similarly to HIF-1α, zinc downregulated also hypoxia-induced HIF-2α whereas the HIF-1β subunit remained unchanged. Zinc inhibited HIF-1α recruitment onto VEGF promoter and the zinc-induced suppression of HIF-1-dependent activation of VEGF correlated with reduction of glioblastoma and prostate cancer cell invasiveness in vitro. Finally, zinc administration downregulated HIF-1α levels in vivo, by bioluminescence imaging, and suppressed intratumoral VEGF expression.

Conclusions/Significance

These findings, by demonstrating that zinc induces HIF-1α proteasomal degradation, indicate that zinc could be useful as an inhibitor of HIF-1α in human tumors to repress important pathways involved in tumor progression, such as those induced by VEGF, MDR1, and Bcl2 target genes, and hopefully potentiate the anticancer therapies.