AS-30D Hepatoma Cells Research Articles

Mitigating effect of paxilline against injury produced by Cd2+ in rat pheochromocytoma PC12 and ascites hepatoma AS-30D cells.

On two rat cell lines, pheochromocytoma PC12 and ascites hepatoma AS-30D, and on rat liver mitochondria we studied action of paxilline (lipophilic mycotoxin from fungus Penicillium paxilli which is blocker of large-conductance potassium channels) against harmful effects of Cd(II) - one of the most dangerous toxic metals and environmental pollutants. We investigated an influence of paxilline on cell viability and mitochondrial function in the presence and in the absence of Cd2+. As found, paxilline protected partially from the Cd2+-induced cytotoxicity, namely taken in concentration of 1μM it decreased the Cd2+-induced cell necrosis in average by 10-14 or 13-23% for AS-30D and PC12cells, respectively. Nevertheless, paxilline did not affect the Cd2+-induced apoptosis of AS-30D cells. The alleviating concentration of paxilline reduced an intracellular production of reactive oxygen species (ROS) in PC12cells intoxicated by Cd2+ and enhanced the ROS production in control AS-30D cells; however, it weakly affected mitochondrial membrane potential of the cells in the absence and in the presence of Cd2+. The ameliorative concentration of paxilline decreased the maximal respiration rates of control cells of both types after short-term (3-5h) treatment with it while the rates reached their control levels after long-term (24-48h) incubation with the drug. Paxilline was not protective against the Cd2+-induced membrane permeability and respiration rate changes in isolated rat liver mitochondria. As result, the mitochondrial electron transport chain was concluded to contribute in the mitigating effect of paxilline against the Cd2+-produced cell injury.

Inhibition of Non-flux-Controlling Enzymes Deters Cancer Glycolysis by Accumulation of Regulatory Metabolites of Controlling Steps.

Glycolysis provides precursors for the synthesis of macromolecules and may contribute to the ATP supply required for the constant and accelerated cellular duplication in cancer cells. In consequence, inhibition of glycolysis has been reiteratively considered as an anti-cancer therapeutic option. In previous studies, kinetic modeling of glycolysis in cancer cells allowed the identification of the main steps that control the glycolytic flux: glucose transporter, hexokinase (HK), hexose phosphate isomerase (HPI), and glycogen degradation in human cervix HeLa cancer cells and rat AS-30D ascites hepatocarcinoma. It was also previously experimentally determined that simultaneous inhibition of the non-controlling enzymes lactate dehydrogenase (LDH), pyruvate kinase (PYK), and enolase (ENO) brings about significant decrease in the glycolytic flux of cancer cells and accumulation of intermediate metabolites, mainly fructose-1,6-bisphosphate (Fru1,6BP), and dihydroxyacetone phosphate (DHAP), which are inhibitors of HK and HPI, respectively. Here it was found by kinetic modeling that inhibition of cancer glycolysis can be attained by blocking downstream non flux-controlling steps as long as Fru1,6BP and DHAP, regulatory metabolites of flux-controlling enzymes, are accumulated. Furthermore, experimental results and further modeling showed that oxamate and iodoacetate inhibitions of PYK, ENO, and glyceraldehyde3-phosphate dehydrogenase (GAPDH), but not of LDH and phosphoglycerate kinase, induced accumulation of Fru1,6BP and DHAP in AS-30D hepatoma cells. Indeed, PYK, ENO, and GAPDH exerted the highest control on the Fru1,6BP and DHAP concentrations. The high levels of these metabolites inhibited HK and HPI and led to glycolytic flux inhibition, ATP diminution, and accumulation of toxic methylglyoxal. Hence, the anticancer effects of downstream glycolytic inhibitors are very likely mediated by this mechanism. In parallel, it was also found that uncompetitive inhibition of the flux-controlling steps is a more potent mechanism than competitive and mixed-type inhibition to efficiently perturb cancer glycolysis.

AS-30D hepatoma as a model to study on insulin resistance in vitro.

Studies on insulin resistance of liver cells are often performed with the use of various hepatoma cell lines. Such an approach allows investigating selected biochemical pathways at the cellular level. However, possible modifications of metabolic processes due to the neoplastic nature of such cells must be considered. Expanding the diversity of hepatoma cell lines used in metabolic studies could deliver new data for comparison with those obtained for other cell lines and should reduce the risk of misleading conclusions. In this study rat hepatoma AS-30D cells were tested as a potential model for studies on palmitate-induced insulin resistance. It was found that insulin-induced Akt kinase phosphorylation was substantially reduced in cells incubated with palmitate at a concentration as low as 75 µM. This effect was not accompanied by excessive reactive oxygen species (ROS) generation or increased Jun N-terminal kinase (JNK) phosphorylation. Moreover, preincubation of AS-30D cells with rosiglitazone, an antidiabetic agonist of peroxisome proliferator-activated receptor gamma (PPARγ), efficiently prevented the palmitate-induced insulin resistance. We conclude that AS-30D hepatoma cells may be used as a model sensitive to insulin and vulnerable to palmitate-induced insulin resistance.

Cd(2 +)-induced injury of rat ascites hepatoma AS-30D cells: A possible involvement of Ca(2 +)-activated large-conductance potassium channels

Cd(2 +)-induced injury of rat ascites hepatoma AS-30D cells: A possible involvement of Ca(2 +)-activated large-conductance potassium channels

Effect of diazoxide on AS-30D rat ascites hepatoma cells treated by Cd2+

For the last years, in several investigations, including ours, it was shown that oxidative stress and mitochondrial dysfunction mediated by disturbance of the mitochondrial respiratory chain and by induction of Ca2+ -dependent nonselective high-conductance pore of the inner mitochondrial membrane are involved in mechanism(s) of cytotoxic action of heavy metals. At the same time, possible interaction of heavy metals with other channels, in particular, with selective potassium channels, such as ATP-dependent potassium channels, which are generally considered to be protective for the cells, have not been studied. The aim of the present work was to examine the effect of diazoxide, an activator of mitochondrial ATP-dependent potassium channels, on mitochondrial physiology and cell survival in the presence of Cd2+. As a model system, we used AS-30D rat ascites hepatoma cells and isolated rat liver mitochondria. We found that diazoxide enhanced intracellular production of reactive oxygen species and induced significant stimulation of the resting respiration rate of the cells. Besides, diazoxide had a protective effect on AS-30D cells by increasing their survival that was substantially decreased in the presence of the tested concentrations of Cd2+ (50 and 100 microM). The protective effect of diazoxide was completely suppressed by increasing duration of incubation of the cells with Cd2+, and partially by addition to the assay medium of 5-hydroxyde- canoic acid (100 or 300 microM), a blocker of mitochondrial ATP-dependent potassium channels. In isolated rat liver mitochondria we found that diazoxide did not prevent the toxic action of Cd2+, since it produced no significant effects on the mitochondrial swelling and the respiration changes evoked by the heavy metal in the KCl assay media. Possible molecular mechanisms of the cytoprotective action of diazoxide are discussed.

Modeling cancer glycolysis

Most cancer cells exhibit an accelerated glycolysis rate compared to normal cells. This metabolic change is associated with the over-expression of all the pathway enzymes and transporters (as induced by HIF-1α and other oncogenes), and with the expression of hexokinase (HK) and phosphofructokinase type 1 (PFK-1) isoenzymes with different regulatory properties. Hence, a control distribution of tumor glycolysis, modified from that observed in normal cells, can be expected. To define the control distribution and to understand the underlying control mechanisms, kinetic models of glycolysis of rodent AS-30D hepatoma and human cervix HeLa cells were constructed with experimental data obtained here for each pathway step (enzyme kinetics; steady-state pathway metabolite concentrations and fluxes). The models predicted with high accuracy the fluxes and metabolite concentrations found in living cancer cells under physiological O 2 and glucose concentrations as well as under hypoxic and hypoglycemic conditions prevailing during tumor progression. The results indicated that HK ≥ HPI > GLUT in AS-30D whereas glycogen degradation ≥ GLUT > HK in HeLa were the main flux- and ATP concentration-control steps. Modeling also revealed that, in order to diminish the glycolytic flux or the ATP concentration by 50%, it was required to decrease GLUT or HK or HPI by 76% (AS-30D), and GLUT or glycogen degradation by 87–99% (HeLa), or decreasing simultaneously the mentioned steps by 47%. Thus, these proteins are proposed to be the foremost therapeutic targets because their simultaneous inhibition will have greater antagonistic effects on tumor energy metabolism than inhibition of all other glycolytic, non-controlling, enzymes. This article is part of a Special Issue entitled Bioenergetics of Cancer.

Arachidonic acid-induced apoptosis in rat hepatoma AS-30D cells is mediated by reactive oxygen species.

Arachidonic acid at micromolar concentrations produced a drastic increase of the generation of reactive oxygen species (ROS) in rat hepatoma AS-30D cells cultivated in vitro along with an increase in the incidence of apoptotic cell death. Both processes were prevented by trolox, a water-soluble tocopherol derivative, and tempol, a known antioxidative agent. A synthetic hybrid of lipoic acid and trolox or preincubation with N-acetylcysteine were less effective. Preincubation of the cells with etomoxir, a known highly specific irreversible inhibitor of carnitine-palmitoyltransferase I, partly decreased the ROS formation induced by arachidonic acid but it did not affect the increase in apoptosis. Cumulatively, these results indicate that apoptosis induced in hepatoma cells by arachidonic acid is mediated by ROS. They also suggest that this effect is due to arachidonic acid as such and not to its mitochondrial oxidative metabolites.

Mitochondria as an important target in heavy metal toxicity in rat hepatoma AS-30D cells

The mechanisms of toxic effects of divalent cations of three heavy metals Hg, Cd and Cu in rat ascites hepatoma AS-30D cells cultivated in vitro were compared. It was found that the toxicity of these ions, applied in the micromolar range (10–500 μM), decreased from Hg 2+ (most toxic) to Cu 2+ (least toxic). Hg 2+ and Cd 2+ produced a high percentage of cell death by both necrosis and apoptosis, whereas Cu 2+ at concentrations up to 500 μM was weakly effective. Hg 2+ at concentration of 10 μM appeared slightly uncoupling (i.e., stimulated resting state respiration and decreased the mitochondrial transmembrane potential), whereas it exerted a strong inhibitory effect on the respiratory chain and rapid dissipation of the membrane potential at higher concentrations. Cu 2+ had inhibitory effect on cell respiration only at 500 μM concentration and after incubation of 48 h but produced a significant uncoupling effect at lower concentrations. Cu 2+ induced an early and sharp increase of intracellular production of reactive oxygen species (ROS). The action of Hg 2+ and Cd 2+ on ROS generation was biphasic. They stimulated ROS generation within the cells at low concentrations and at short incubation times but decreased ROS generation at higher concentrations and at longer incubation. It is concluded that mitochondria are an important target for toxic effects of Hg 2+, Cd 2+ and Cu 2+ in AS-30D rat hepatoma cells.

Reactive oxygen species produced by the mitochondrial respiratory chain are involved in Cd 2+-induced injury of rat ascites hepatoma AS-30D cells

Using AS-30D rat ascites hepatoma cells, we studied the modulating action of various antioxidants, inhibitors of mitochondrial permeability transition pore and inhibitors of the respiratory chain on Cd 2+-produced cytotoxicity. It was found that Cd 2+ induced both necrosis and apoptosis in a time- and dose-dependent way. This cell injury involved dissipation of the mitochondrial transmembrane potential, respiratory dysfunction and initial increase of the generation of reactive oxygen species (ROS), followed by its decrease after prolonged incubation. Inhibitors of the mitochondrial permeability transition pore, cyclosporin A and bongkrekic acid, and inhibitors of respiratory complex III, stigmatellin and antimycin A, but not inhibitor of complex I, rotenone, partly prevented necrosis evoked by exposure of the cells to Cd 2+. Apoptosis of the cells was partly prevented by free radical scavengers and by preincubation with N-acetylcysteine. Stigmatellin, antimycin A and cyclosporin A also abolished Cd 2+-induced increase in ROS generation. It is concluded that Cd 2+ toxicity in AS-30D rat ascites hepatoma, manifested by cell necrosis and/or apoptosis, involves ROS generation, most likely at the level of respiratory complex III, and is related to opening of the mitochondrial permeability transition pore.

Determining and understanding the control of glycolysis in fast‐growth tumor cells

Control analysis of the glycolytic flux was carried out in two fast-growth tumor cell types of human and rodent origin (HeLa and AS-30D, respectively). Determination of the maximal velocity (V(max)) of the 10 glycolytic enzymes from hexokinase to lactate dehydrogenase revealed that hexokinase (153-306 times) and phosphofructokinase-1 (PFK-1) (22-56 times) had higher over-expression in rat AS-30D hepatoma cells than in normal freshly isolated rat hepatocytes. Moreover, the steady-state concentrations of the glycolytic metabolites, particularly those of the products of hexokinase and PFK-1, were increased compared with hepatocytes. In HeLa cells, V(max) values and metabolite concentrations for the 10 glycolytic enzyme were also significantly increased, but to a much lesser extent (6-9 times for both hexokinase and PFK-1). Elasticity-based analysis of the glycolytic flux in AS-30D cells showed that the block of enzymes producing Fru(1,6)P2 (i.e. glucose transporter, hexokinase, hexosephosphate isomerase, PFK-1, and the Glc6P branches) exerted most of the flux control (70-75%), whereas the consuming block (from aldolase to lactate dehydrogenase) exhibited the remaining control. The Glc6P-producing block (glucose transporter and hexokinase) also showed high flux control (70%), which indicated low flux control by PFK-1. Kinetic analysis of PFK-1 showed low sensitivity towards its allosteric inhibitors citrate and ATP, at physiological concentrations of the activator Fru(2,6)P2. On the other hand, hexokinase activity was strongly inhibited by high, but physiological, concentrations of Glc6P. Therefore, the enhanced glycolytic flux in fast-growth tumor cells was still controlled by an over-produced, but Glc6P-inhibited hexokinase.

Short-term and long-term effects of fatty acids in rat hepatoma AS-30D cells: The way to apoptosis

Arachidonic acid and, to a smaller extent, oleic acid at micromolar concentrations decreased the mitochondrial membrane potential within AS-30D rat hepatoma cells cultivated in vitro and increased cell respiration. The uncoupling effect of both fatty acids on cell respiration was partly prevented by cyclosporin A, blocker of the mitochondrial permeability transition pore. Arachidonic acid increased the rate of reactive oxygen species (ROS) production, while oleic acid decreased it. Both fatty acids induced apoptotic cell death of AS-30D cells, accompanied by the release of cytochrome c from mitochondria to the cytosol, activation of caspase-3 and association of proapoptotic Bax protein with mitochondria; arachidonic acid being a more potent inducer than oleic acid. Trolox, a potent antioxidant, prevented ROS increase induced by arachidonic acid and protected the cells against apoptosis produced by this fatty acid. It is concluded that arachidonic and oleic acids induce apoptosis of AS-30D hepatoma cells by the mitochondrial pathway but differ in the mechanism of their action: Arachidonic acid induces apoptosis mainly by stimulating ROS production, whereas oleic acid may contribute to programmed cell death by activation of the mitochondrial permeability transition pore.

Mitochondrial branched chain aminotransferase gene expression in AS-30D hepatoma rat cells and during liver regeneration after partial hepatectomy in rat

Branched chain aminotransferase (BCAT) is the first enzyme in the catabolism of branched chain amino acids (BCAA). Unlike other amino acid degrading enzymes present in liver, BCAT is only expressed in extrahepatic tissues, and is not regulated by dietary protein, glucagon or glucocorticoids. However, the mitochondrial (m) isoform of BCAT is highly expressed in the fetal liver and rapidly decays after birth. The purpose of the present work was to establish if liver cells under conditions of rapid cell proliferation such as in hepatoma AS30D cells or during liver regeneration after partial hepatectomy were associated with an increase in the activity and expression of BCATm. BCAT activity in mitochondria of AS30D cells was 18.6 mU/mg protein. Western, Northern blot, and immunohistochemical analysis revealed that AS30D hepatoma cells expressed only BCATm. The apparent Km of BCATm in isolated AS30D cells mitochondria for leucine, isoleucine and valine was 1.0 ± 0.02, 1.3 ± 0.1 and 2.1 ± 0.1 mM, respectively. The regenerated liver showed BCAT activity from day 3 to day 6, and the maximal BCAT activity (7.0 mU/mg protein) was on day 5. By day 14 after partial hepatectomy BCAT activity and expression was almost undetectable. Interestingly, there was a relationship between BCAT activity and the Mr. of the immunoreactive band of BCATm. The presence of a 41 kDa band was associated with BCAT activity, whereas the 43 kDa band with undetectable activity. The results of this study indicate that BCATm activity is required in liver cells under conditions of rapid cell proliferation.

Toxic effects of copper-based antineoplastic drugs (Casiopeinas ®) on mitochondrial functions

To elucidate some of the subcellular and biochemical mechanisms of toxicity of metal-based antineoplastic drugs, mitochondria and cells were exposed to Casiopeinas ®, a new class of copper-based compounds with high antineoplastic activity. The rates of respiration and swelling, the H + gradient, and the activities of succinate (SDH) and 2-oxoglutarate dehydrogenases (2-OGDH) and ATPase were measured in mitochondria isolated from rat liver, kidney, heart, and hepatoma AS-30D. Also, oligomycin-sensitive respiration and ATP content in hepatoma AS-30D cells were determined. Casiopeinas ® (CS) II-gly and III-i inhibited the rates of state 3 and uncoupled respiration in mitochondria. CS II was 10 times more potent than CS III. The sensitivity to CS II was 4–5-fold higher in mitochondria incubated with 2-OG than with succinate. Thus, at low concentrations (≤10 nmol (mg protein) −1; 10 μM), CS II disturbed mitochondrial functions only when 2-OG was present, due to a specific inhibition of 2-OGDH. At high concentrations (≥15 nmol (mg protein) −1), CS II-induced stimulation of basal respiration, followed by a strong inhibition, which correlated with K +-dependent swelling and cytochrome c release, respectively; K +-channel openers induce a similar mitochondrial response. Mitochondria from liver, kidney and hepatoma showed a similar sensitivity towards CS II, whereas heart mitochondria were more resistant. Oxidative phosphorylation and ATP content were also decreased in tumor cells by CS II. The data suggested that CS affected several different mitochondrial sites, bringing about inhibition of respiration and ATP synthesis, which could compromise energy-dependent processes such as cellular duplication.

Uptake of 153Sm-DTPA-bis-biotin and 99mTc-DTPA-bis-biotin in rat as-30D-hepatoma cells

Labeled biotin has been used mainly for pretargeted therapy, an approach for increasing the amount of radioactivity delivered to a cancer cell. The aim of this investigation was to prepare 153Sm-DTPA-bis-biotin and 99mTc-DTPA-bis-biotin in order to study their in vitro and in vivo uptake in rat AS-30D hepatoma cells found in ascites and in implanted tumor. DTPA-bis-biotin (pH 8) was 153Sm labeled with 153SmCl 3 and 99mTc-DTPA-bis-biotin was prepared via SnCl 2 reduction. Radiochemical purity was >98% in both cases. AS-30D hepatoma cells were obtained from ascites of a rat with hepatoma and were propagated in the peritoneum cavity of normal rats. In vitro ascites cell 153Sm-DTPA-bis-biotin uptake was compared with 153SmCl 3 cell uptake. The ratio cell 153Sm-DTPA-bis-biotin/ 153 SmCl 3 was 39.6 and when avidin was added it increased to 50. The ratio 99mTc-DTPA-bis-biotin/TcO 4Na was 8.7. Concentration of 153Sm-DTPA-bis-biotin in tumor 2, 3 and 24 h after administration, was 5, 15 and 3 times higher than in normal muscle (T/nT). Biodistribution in a 0.083-24 h time period showed that 153Sm-DTPA-bis-biotin was taken up only by ascites tumor cells and hepatoma cells. Two and 3 h ratio ascites/liver (As/Lv) was 6.4 and 6.0. For 99mTc-DTPA-bis-biotin 2 and 3 h T/nT was 15.7 and 4.7 and 2 h As/Lv was 1.4. In conclusion, both radiopharmaceuticals show high uptake in rat AS-30D hepatoma cells in ascites and in implanted tumor. Since lung, thyroid, kidney, liver or pancreas carcinomas are ascites producing cancers 153Sm-DTPA-bis-biotin would be an adequate therapeutic radiopharmaceutical for these patients whose life quality would be enhanced with control of ascites, and a reduction of the primary tumor and its metastases.

Multisite control of the Crabtree effect in ascites hepatoma cells.

AS-30D hepatoma cells, a highly oxidative and fast-growing tumor line, showed glucose-induced and fructose-induced inhibition of oxidative phosphorylation (the Crabtree effect) of 54% and 34%, respectively. To advance the understanding of the underlying mechanism of this process, the effect of 5 mM glucose or 10 mM fructose on the intracellular concentration of several metabolites was determined. The addition of glucose or fructose lowered intracellular Pi (40%), and ATP (53%) concentrations, and decreased cytosolic pH (from 7.2 to 6.8). Glucose and fructose increased the content of AMP (30%), glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate (15, 13 and 50 times, respectively). The cytosolic concentrations of Ca2+ and Mg2+ were not modified. The addition of galactose or glycerol did not modify the concentrations of the metabolites. Mitochondria isolated from AS-30D cells, incubated in media with low Pi (0.6 mM) at pH 6.8, exhibited a 40% inhibition of oxidative phosphorylation. The data suggest that the Crabtree effect is the result of several small metabolic changes promoted by addition of exogenous glucose or fructose.

Substrate Oxidation and ATP Supply in AS-30D Hepatoma Cells

The oxidation of several metabolites in AS-30D tumor cells was determined. Glucose and glycogen consumption and lactic acid production showed high rates, indicating a high glycolytic activity. The utilization of ketone bodies, oxidation of endogenous glutamate, and oxidative phosphorylation were also very active: tumor cells showed a high respiration rate (100 ng atoms oxygen (min × 107 cells)−1), which was 90% oligomycin-sensitive. AS-30D tumor cells underwent significant intracellular volume changes, which preserved high concentrations of several metabolites. A high O2 concentration, but a low glucose concentration were found in the cell-free ascites liquid. Glutamine was the oxidizable substrate found at the highest concentration in the ascites liquid. We estimated that cellular ATP was mainly provided by oxidative phosphorylation. These data indicated that AS-30D hepatoma cells had a predominantly oxidative and not a glycolytic type of metabolism. The NADH-ubiquinol oxido reductase and the enzyme block for ATP utilization were the sites that exerted most of the control of oxidative phosphorylation (flux control coefficient = 0.3–0.42).

Differences in Cholesterol Incorporation into Mitochondria from Hepatoma AS-30D and Human Term Placenta

Cholesterol transport for steroidogenesis in the human placental mitochondria is an enigma as, contrary to other steroidogenic tissues, the human placenta does not express steroidogenic acute regulatory protein (StAR), a protein known to be required for efficient utilization of cholesterol by adrenal and gonadal mitochondria. These observations suggest the possibility that cholesterol transport in human placental mitochondria involves a similar system to that present in other non-steroidogenic tissues. We studied cholesterol incorporation into mitochondria isolated from AS-30D hepatoma cells and the human placenta. Mitochondria from both sources incorporated cholesterol in vitro. There were no differences in cholesterol incorporation into hepatoma mitochondria treated with or without trypsin. In contrast, the human placental mitochondria treated with trypsin did not incorporate exogenous cholesterol. The presence of ATP increased the uptake of cholesterol by human placental mitochondria. This increase was inhibited by vanadate. These results suggest that cholesterol incorporation into human placental mitochondria is mediated by protein(s).

Poly(ethylene glycol) modification of beta-glucuronidase-antibody conjugates for solid-tumor therapy by targeted activation of glucuronide prodrugs.

Methoxypoly(ethylene glycol) (PEG) modification of Escherichia coli beta-glucuronidase (betaG) was examined as a method to improve the stability and pharmacokinetics of antibody-betaG conjugates for the targeted activation of glucuronide prodrugs at tumor cells. Introduction of 3 PEG molecules did not affect betaG activity whereas higher degrees of PEG modification produced progressively greater loss of enzymatic activity. The enzyme was found to be stable in serum regardless of PEG modification. PEG-modified betaG was coupled via a thioether bond to mAb RH1, an IgG2a antibody that binds to the surface of AS-30D hepatoma cells, to produce conjugates with 3 (RH1-betaG-3PEG), 5.2 (RH1-betaG-5PEG) or 9.8 (RH1-betaG-10PEG) PEG molecules per betaG with retention of 75%, 45% and 40% of the combined antigen-binding and enzymatic activity of the unmodified conjugate RH1-betaG. In contrast to the rapid serum clearance of RH1-betaG observed in mice, the PEG-modified conjugates displayed extended serum half-lives. RH1-betaG-3PEG and RH1-betaG-5PEG also exhibited reduced spleen uptake and greater tumor accumulation than RH1-betaG. BHAMG, the glucuronide prodrug of p-hydroxyaniline mustard (pHAM), was relatively nontoxic in vivo. Injection of 6 mg/kg or 12 mg/kg pHAM i.v. depressed white blood cell numbers by 46% and 71% whereas 80 mg/kg BHAMG reduced these levels by 22%. Although the tumor/blood ratio of RH1-betaG-5PEG was adversely affected by slow clearance from serum, combined therapy of small solid hepatoma tumors with this conjugate, followed 4 and 5 days later with i.v. injections of BHAMG, cured all of seven mice with severe combined immunodeficiency. Combined treatment with a control antibody-betaG conjugate and BHAMG delayed tumor growth and cured two of six mice while treatment with pHAM or BHAMG alone was ineffective.

Quantitative analysis of acetoacetate metabolism in AS-30D hepatoma cells with 13C and 14C isotopic techniques.

Experimental hepatoma cells utilize acetoacetate as an oxidative energy source and as a precursor for lipid synthesis. The significance of ketone body metabolism in tumors lies in the study of tumor-host metabolism and the ketoneMic condition that is often present in cancer patients. The quantitative importance of acetoacetate and glucose was investigated in AS-30D cells with use of 13C and 14C isotopic methods. In addition, the effects of acetoacetate were compared with those of dichloroacetic acid (DCA), an activator of pyruvate dehydrogenase (PDH). The 14CO2 ratio method evaluated the entry of pyruvate into the tricarboxylic acid (TCA) cycle and revealed that acetoacetate diverted pyruvate from PDH to pyruvate carboxylation. In contrast, DCA increased the oxidation of glucose largely through PDH, indicating that PDH is not maximally active in the absence of DCA. Isotopomer spectral analysis of lipid synthesis demonstrated that, in the absence of acetoacetate, glucose supplied 65% of the acetyl-CoA used for de novo lipogenesis. When 5 mM acetoacetate was included in the incubation, glucose was displaced as a lipogenic precursor and acetoacetate supplied 85% of the acetyl-CoA for lipogenesis vs. only 2% for glucose. Thus AS-30D cells have a large capacity for acetoacetate utilization for de novo lipogenesis.

Glucose metabolism in cancer cells: immunogold localization of hexokinase to the outer mitochondrial membrane

Hexokinase (ATP: D-hexose 6-phophotransferase EC 2.7.1.1) is the first enzyme of the glycolytic pathway which commits glucose to catabolism by catalyzing the phosphorylation of glucose with ATP. Previous studies have shown diat hexokinase activity is markedly elevated in rapidly growing tumor cells exhibiting high glucose catabolic rates. A large fraction (50-80%) of this enzyme activity is bound to the mitochondrial fraction (1,2) where it has preferred access to ATP (3). In contrast,the hexokinase activity of normal tissues is quite low, with one exception being brain which is a glucose-utilizing tissue (4). Biochemical evidence involving rigorous subfractionation studies have revealed striking differences between the subcellular distribution of hexokinase in normal and tumor cells [See review by Aroraet al(4)].In the present report, we have utilized immunogold labeling techniques to evaluate die subcellular localization of hexokinase in highly glycolytic AS-30D hepatoma cells and in the tissue of its origin, i.e., rat liver.