AS-30D Cells Research Articles

Quantitative Expression of Key Cancer Markers in the AS-30D Hepatocarcinoma Model

Hepatocellular carcinoma is one of the cancers with the highest mortality rate worldwide. HCC is often diagnosed when the disease is already in an advanced stage, making the discovery and implementation of biomarkers for the disease a critical aim in cancer research. In this study, we aim to quantify the transcript levels of key signaling molecules relevant to different pathways known to participate in tumorigenesis, with special emphasis on those related to cancer hallmarks and epithelial-mesenchymal transition, using as a model the murine transplantable hepatocarcinoma AS-30D. Using qPCR to quantify the mRNA levels of genes involved in tumorigenesis, we found elevated levels for Tgfb1 and Spp1, two master regulators of EMT. A mesenchymal signature profile for AS-30D cells is also supported by the overexpression of genes encoding for molecules known to be associated to aggressiveness and metastatic phenotypes such as Foxm1, C-met, and Inppl1. This study supports the use of the AS-30D cells as an efficient and cost-effective model to study gene expression changes in HCC, especially those associated with the EMT process.

Regulatory role of acetylation on enzyme activity and fluxes of energy metabolism pathways

BackgroundMost of the enzymes involved in the central carbon metabolism are acetylated in Lys residues. It has been claimed that this covalent modification represents a novel regulatory mechanism by which both enzyme/transporter activities and pathway fluxes can be modulated. MethodsTo establish which enzymes are regulated by acetylation, a systematic experimental analysis of activities and acetylation profile for several energy metabolism enzymes and pathway fluxes was undertaken in cells and mitochondria. ResultsThe majority of the glycolytic and neighbor enzymes as well as mitochondrial enzymes indeed showed Lys-acetylation, with GLUT1, HPI, CS, ATP synthase displaying comparatively lower acetylation patterns. The incubation of cytosolic and mitochondrial fractions with recombinant Sirt-3 produced lower acetylation signals, whereas incubation with acetyl-CoA promoted protein acetylation. Significant changes in acetylation levels of MDH and IDH-2 from rat liver mitochondria revealed no change in their activities. Similar observations were attained for the cytosolic enzymes from AS-30D and HeLa cells. A minor but significant (23%) increase in the AAT-MDH complex activity induced by acetylation was observed. To examine this question further, AS-30D and HeLa cells were treated with nicotinamide and valproic acid. These compounds promoted changes in the acetylation patterns of glycolytic proteins, although their activities and the glycolytic flux (as well as the OxPhos flux) revealed no clear correlation with acetylation. ConclusionAcetylation seems to play no predominant role in the control of energy metabolism enzyme activities and pathway fluxes. General significanceThe physiological function of protein acetylation on energy metabolism pathways remains to be elucidated.

Kinetic modeling of glucose central metabolism in hepatocytes and hepatoma cells

BackgroundKinetic modeling and control analysis of a metabolic pathway may identify the steps with the highest control in tumor cells, and low control in normal cells, which can be proposed as the best therapeutic targets. MethodsEnzyme kinetic characterization, pathway kinetic modeling and control analysis of the glucose central metabolism were carried out in rat (hepatoma AS-30D) and human (cervix HeLa) cancer cells and normal rat hepatocytes. ResultsThe glycogen metabolism enzymes in AS-30D, HeLa cells and hepatocytes showed similar kinetic properties, except for higher AS-30D glycogen phosphorylase (GP) sensitivity to AMP. Pathway modeling indicated that fluxes of glycogen degradation and PPP were mainly controlled by GP and NADPH consumption, respectively, in both hepatocytes and cancer cells. Likewise, hexose-6-phosphate isomerase (HPI) and phosphoglucomutase (PGM) exerted significant control on glycolysis and glycogen synthesis fluxes in cancer cells but not in hepatocytes. Modeling also indicated that glycolytic and glycogen synthesis fluxes could be strongly decreased when HPI and PGM were simultaneously inhibited in AS-30D cells but not in hepatocytes. Experimental assessment of these predictions showed that both the glycolytic and glycogen synthesis fluxes of AS-30D cells, but not of hepatocytes, were inhibited by oxamate, by inducing increased Fru1,6BP levels, a competitive inhibitor of HPI and PGM. ConclusionHPI and PGM seem suitable targets for decreasing glycolytic and glycogen synthesis fluxes in AS-30D cells but not in hepatocytes. General significanceThe present study identified new therapeutic targets within glucose central metabolism in the analyzed cancer cells, with no effects on non-cancer cells.

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.

Histone deacetylase inhibitors promote ATP2A3 gene expression in hepatocellular carcinoma cells: p300 as a transcriptional regulator.

Sarco(endo)plasmic reticulum Ca2+-ATPases (SERCA) expression is reduced or absent in several types of cancer and cancer cell lines; however, their expression and regulation in hepatocellular carcinoma (HCC) are unknown. Histone deacetylase inhibitors (HDACi) increase SERCA3 mRNA expression in gastric and breast cancer cell lines by increasing H3K9ac and binding of Sp1 and Sp3 transcription factors to the promoter; however, the molecular mechanism is not fully understood. Our results show that ATP2A3 (SERCA3) gene expression is decreased in human HCC samples and rat HCC AS-30D cells compared to normal liver, and HCC patients with high expression of ATP2A3 had longer overall survival than those with low expression. Sodium butyrate (NaB) and trichostatin A (TSA) increase SERCA3 mRNA expression in AS-30D cells, whereas SERCA2b mRNA expression did not change. NaB and TSA increase H3K9ac and H3K27ac in two ATP2A3 promoter regions. Besides, NaB treated cells increased Sp1 and Sp3 occupancy at ATP2A3 promoter; whereas TSA treated cells showed increased p300 levels at ATP2A3 promoter. Inhibition of p300 by C646, a specific inhibitor, mitigates SERCA3 mRNA induction by TSA, and reduces more than 70% of basal SERCA3 mRNA expression, suggesting that p300 is important for ATP2A3 gene transcription in AS-30D cells. Moreover, inhibition of p300 decreases H3K9ac in TSA treated cells. Our results provide evidence of decreased SERCA3 expression in human HCC samples and rat AS-30D cells and a correlation of SERCA3 expression with overall survival in HCC patients. Also, reveal new insights in SERCA3 transcriptional regulation mediated by HDACi.

Respiratory complex II in mitochondrial dysfunction-mediated cytotoxicity: Insight from cadmium

In the present work we studied action of several inhibitors of respiratory complex II (CII) of mitochondrial electron transport chain, namely malonate and thenoyltrifluoroacetone (TTFA) on Cd2+-induced toxicity and cell mortality, using two rat cell lines, pheochromocytoma PC12 and ascites hepatoma AS-30D and isolated rat liver mitochondria (RLM). It was shown that malonate, an endogenous competitive inhibitor of dicarboxylate-binding site of CII, restored in part RLM respiratory function disturbed by Cd2+. In particular, malonate increased both phosphorylating and maximally uncoupled respiration rates in KCl medium in the presence of CI substrates as well as palliated changes in basal and resting state respiration rates produced by the heavy metal on the mitochondria energized by CI or CII substrates. Notably, malonate enhanced Cd2+-induced swelling of the mitochondria energized by CI substrates in KCl and, in a much lesser extent and at higher [Cd2+], in sucrose media but did not influence on the Cd2+ effects in NaCl medium. Besides, malonate did not affect swelling in sucrose media of RLM energized by CIV substrates under using of Cd2+ or Ca2+ whereas it strongly increased the mitochondrial swelling produced by selenite. In addition, malonate produced some protection against Cd2+-promoted necrotic death of AS-30D and PC12 cells and reduced intracellular reactive oxygen species (ROS) formation evoked by Cd2+ in PC12 cells. Importantly, TTFA, an irreversible competitive inhibitor of Q-binding site of CII, per se induced apoptosis of AS-30D cells which was inhibited by co-treatment with Cd2+ as well as decreased the Cd2+-enhanced intracellular ROS formation. In turn, decylubiquinone (dUb) at low μM concentrations did not protect AS-30D cells against the Cd2+-induced necrosis and enhanced the Cd2+-induced apoptosis of the cells. High μM concentrations of dUb were highly toxic for the cells. As consequence, the findings give new evidence indicative of critical involvement of CII in mechanism(s) of Cd2+-produced cytotoxicity and support the notion on CII as a perspective pharmacological target in mitochondria dysfunction-mediated conditions and diseases.

Control of the NADPH supply for oxidative stress handling in cancer cells

It has not been systematically analyzed whether the NADPH supply is a limiting factor for oxidative stress management in cancer cells. In the present work, it was determined in non-cancer and cancer cells the protein contents and kinetomics of (i) the cytosolic enzymes responsible for the NADPH production (i.e., Glc6PDH, 6PGDH, ME, IDH-1); and (ii) the two main enzymes responsible for NADPH/NADP+ and GSH/GSSG recycling (GR, GPx-1) associated to oxidative stress management. With these data, kinetic models were built and further validated. Rat liver and hepatoma AS-30D cytosolic fractions exhibited greater Vmax for IDH-1 than for Glc6PDH and 6PGDH whereas human cancer cells and platelets showed greater Vmax for Glc6PDH than for 6PGDH and IDH-1. The ME activity was comparatively low in all cell types tested. The Km values for the respective specific substrates were all similar among the different cell types. Most activities were lower in AS-30D cells than in liver. In contrast, IDH-1, Glc6PDH and GR activities in human cancer cells were similar or greater to those of platelets, but GPx-1 activity was severely suppressed, despite showing similar GPx-1 protein content vs. platelets. Kinetic analysis and pathway modeling revealed a previously unveiled feedback IDH-1 regulation by GSH. The oxidative stress management in cancer cells (i) was mainly controlled by GPx-1 and the main NADPH provider was Glc6PDH; and (ii) modeling indicated that NADPH supply was not a controlling step. These data suggested that Glc6PDH and GPx-1 are adequate and promising targets for anti-cancer therapeutic intervention.

Assessment of the low inhibitory specificity of oxamate, aminooxyacetate and dichloroacetate on cancer energy metabolism

BackgroundExceedingly high therapeutic/experimental doses of metabolic drugs such as oxamate, aminooxyacetate (AOA) and dichloroacetate (DCA) are required to diminish growth, glycolysis and oxidative phosphorylation (OxPhos) of different cancer cells. To identify the mechanisms of action of these drugs on cancer energy metabolism, a systematic analysis of their specificities was undertaken. MethodsHepatocarcinoma AS-30D cells were treated with the inhibitors and glycolysis and OxPhos enzyme activities, metabolites and fluxes were analyzed. Kinetic modeling of glycolysis was used to identify the regulatory mechanisms. ResultsOxamate (i) not only inhibited LDH, but also PYK and ENO activities inducing an increase in the cytosolic NAD(P)H, Fru1,6BP and DHAP levels in AS-30D cells; (ii) it slightly inhibited HPI, ALD and Glc6PDH; and (iii) it inhibited pyruvate-driven OxPhos in isolated heart mitochondria. AOA (i) strongly inhibited both AAT and AlaT, and 2-OGDH and glutamate-driven OxPhos; and (ii) moderately affected GAPDH and TPI. DCA slightly affected pyruvate-driven OxPhos and Glc6PDH. Kinetic modeling of cancer glycolysis revealed that oxamate inhibition of LDH, PYK and ENO was insufficient to achieve glycolysis flux inhibition. To do so, HK, HPI, TPI and GAPDH have to be also inhibited by the accumulated Fru1,6BP and DHAP induced by oxamate. ConclusionOxamate, AOA, and DCA are not specific drugs since they inhibit several enzymes/transporters of the glycolytic and OxPhos pathways through direct interaction or indirect mechanisms. General significanceThese data explain why oxamate or AOA, through their multisite inhibitory actions on glycolysis or OxPhos, may be able to decrease the proliferation of cancer cells.

The effect of modulators of large-conductance Ca2+-modulated K+ channels on rat AS-30D ascites hepatoma cells and isolated liver mitochondria treated with Cd2+

ATP-producing cell organelles, mitochondria, are a primary target of heavy metals, major environmental pollutants causing a variety of diseases and pathologies. The mechanism of heavy metal toxic effect was established to include changes both in the intracellular production of reactive oxygen species (ROS) and mitochondrial dysfunction due to respiratory chain disorders and activation of the Ca2+-dependent non-selective pore in the mitochondrial inner membrane. Te role of other ion channels including such selective potassium channels as large-conductance Ca2+-activated K+ channels, BK(Ca), supposed to be cytoprotective, remains poorly studied. In the present work conducted on rat AS-30D ascites hepatoma cells and liver mitochondria, we studied the effect of different BK(Ca) effectors in the presence or absence of Cd2+ ions in the incubation medium, specifically of two activators, NS1619 and NS004, and one blocker, paxilline. After 24-h incubation of AS-30D cells with 10 μM of both NS1619 and NS004, the number of apoptized cells was found to increase significantly versus control; moreover, the presence of these BK(Ca) activators in the incubation medium exerted an additive effect on Cd2+-induced apoptosis of AS-30D cells. The same concentration of NS1619 and NS004 did not affect significantly AS-30D cellular respiration after 3, 24 and 48 h of incubation, although increasing after 3 h the intracellular production of ROS. In experiments on isolated rat liver mitochondria, NS1619 and NS004 added at the same concentration to the KCl-containing medium did not affect the rates of respiratory State 3 (after Chance) and maximally uncoupled respiration (both in the presence and absence of Cd2+); concurrently, they induced a weak uncoupling effect by increasing both basal and resting-state (State 3 after Chance) respirations, and also enhanced a high-amplitude mitochondrial swelling induced by Cd2+ in this medium. Paxilline (at 1μM) was shown to reduce the mortality of AS-30D cells after 3-, 24- and 48 h-incubation in the presence of Cd2+ and to increase the intracellular ROS production in control after 3 and 24 h of exposure. Paxilline added at a concentration exerting a long-term protective effect did not affect cellular respiration of rat AS-30D cells and isolated liver mitochondria (both in the presence and absence of Cd2+) and did not reduce mitochondrial swelling observed in the presence of Cd2+ and BK(Ca) activators. Possible molecular action mechanisms of BK(Ca) modulators are discussed.

Mitochondrial free fatty acid β-oxidation supports oxidative phosphorylation and proliferation in cancer cells

Oxidative phosphorylation (OxPhos) is functional and sustains tumor proliferation in several cancer cell types. To establish whether mitochondrial β-oxidation of free fatty acids (FFAs) contributes to cancer OxPhos functioning, its protein contents and enzyme activities, as well as respiratory rates and electrical membrane potential (ΔΨm) driven by FFA oxidation were assessed in rat AS-30D hepatoma and liver (RLM) mitochondria. Higher protein contents (1.4–3 times) of β-oxidation (CPT1, SCAD) as well as proteins and enzyme activities (1.7–13-times) of Krebs cycle (KC: ICD, 2OGDH, PDH, ME, GA), and respiratory chain (RC: COX) were determined in hepatoma mitochondria vs. RLM. Although increased cholesterol content (9-times vs. RLM) was determined in the hepatoma mitochondrial membranes, FFAs and other NAD-linked substrates were oxidized faster (1.6–6.6 times) by hepatoma mitochondria than RLM, maintaining similar ΔΨm values. The contents of β-oxidation, KC and RC enzymes were also assessed in cells. The mitochondrial enzyme levels in human cervix cancer HeLa and AS-30D cells were higher than those observed in rat hepatocytes whereas in human breast cancer biopsies, CPT1 and SCAD contents were lower than in human breast normal tissue. The presence of CPT1 and SCAD in AS-30D mitochondria and HeLa cells correlated with an active FFA utilization in HeLa cells. Furthermore, the β-oxidation inhibitor perhexiline blocked FFA utilization, OxPhos and proliferation in HeLa and other cancer cells. In conclusion, functional mitochondria supported by FFA β-oxidation are essential for the accelerated cancer cell proliferation and hence anti-β-oxidation therapeutics appears as an alternative promising approach to deter malignant tumor growth.

Active oxidation of several NADH-linked substrates and high oxidative phosphorylation protein contents indicate fully functional mitochondrial metabolism in AS-30D and HeLa cancer cells

Active oxidation of several NADH-linked substrates and high oxidative phosphorylation protein contents indicate fully functional mitochondrial metabolism in AS-30D and HeLa cancer cells

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.

Phosphofructokinase type 1 kinetics, isoform expression, and gene polymorphisms in cancer cells

Kinetic analysis of PFK-1 from rodent AS-30D, and human HeLa and MCF-7 carcinomas revealed sigmoidal [fructose 6-phosphate, Fru6P]-rate curves with different V(m) values when varying the allosteric activator fructose 2,6 bisphosphate (Fru2,6BP), AMP, Pi, NH(4)(+), or K(+). The rate equation that accurately predicted this behavior was the exclusive ligand binding concerted transition model together with non-essential hyperbolic activation. PFK-1 from rat liver and heart also exhibited the mixed cooperative-hyperbolic kinetic behavior regarding activators. Lowering pH induced decreased affinity for Fru6P, Fru2,6BP, citrate, and ATP (as inhibitor); as well as decreased V(m) and increased content of inactive (T) enzyme forms. High K(+) prompted increased (Fru6P) or decreased (activators) affinities; increased V(m); and increased content of active (R) enzyme forms. mRNA expression analysis and nucleotide sequencing showed that the three PFK-1 isoforms L, M, and C are transcribed in the three carcinomas. However, proteomic analysis indicated the predominant expression of L in liver, of M in heart and MCF-7 cells, of L>M in AS-30D cells, and of C in HeLa cells. PFK-1M showed the highest affinities for F6P and citrate and the lowest for ATP (substrate) and F2,6BP; PFK-1L showed the lowest affinity for F6P and the highest for F2,6BP; and PFK-1C exhibited the highest affinity for ATP (substrate) and the lowest for citrate. Thus, the present work documents the kinetic signature of each PFK-1 isoform, and facilitates the understanding of why this enzyme exerts significant or negligible glycolysis flux-control in normal or cancer cells, respectively, and how it regulates the onset of the Pasteur effect.

Casiopeina II-gly and bromo-pyruvate inhibition of tumor hexokinase, glycolysis, and oxidative phosphorylation

The copper-based drug Casiopeina II-gly (CasII-gly) shows potent antineoplastic effect and diminishes mitochondrial metabolism on several human and rodent malignant tumors. To elucidate whether CasII-gly also affects glycolysis, (a) the flux through the complete pathway and the initial segment and (b) the activities of several glycolytic enzymes of AS-30D hepatocarcinoma cells were determined. CasII-gly (IC₅₀ = 0.74-6.7 μM) was more effective to inhibit 24-72 h growth of several human carcinomas than 3-bromopyruvate (3BrPyr) (IC₅₀ = 45-100 μM) with no apparent effect on normal human-proliferating lymphocytes and HUVECs. In short-term 60-min experiments, CasII-gly increased tumor cell lactate production and glycogen breakdown. CasII-gly was 1.3-21 times more potent than 3BrPyr and cisplatin to inhibit tumor HK. As CasII-gly inhibited the soluble and mitochondrial HK activities and the flux through the HK-TPI glycolytic segment, whereas PFK-1, GAPDH, PGK, PYK activities and HPI-TPI segment flux were not affected, the data suggested glycogenolysis activation induced by HK inhibition. Accordingly, glycogen-depleted as well as oligomycin-treated cancer cells became more sensitive to CasII-gly. The inhibition time-course of HK by CasII-gly was slower than that of OxPhos in AS-30D cells, indicating that glycolytic toxicity was secondary to mitochondria, the primary CasII-gly target. In long-term 24-h experiments with HeLa cells, 5 μM CasII-gly inhibited OxPhos (80%), glycolysis (40%), and HK (42%). The present data indicated that CasII-gly is an effective multisite anticancer drug simultaneously targeting mitochondria and glycolysis.

Mechanism(s) of Toxic Action of Zn2+and Selenite: A Study on AS-30D Hepatoma Cells and Isolated Mitochondria

Mitochondria of AS-30D rat ascites hepatoma cells are found to be the main target for Zn2+ and sodium selenite (Na2SeO3). High [mu]M concentrations of Zn2+ or selenite were strongly cytotoxic, killing the AS-30D cells by both apoptotic and necrotic ways. Both Zn2+ and selenite produced strong changes in intracellular generation of reactive oxygen species (ROS) and the mitochondrial dysfunction via the mitochondrial electron transport chain (mtETC) disturbance, the membrane potential dissipation, and the mitochondrial permeability transition pore opening. The significant distinctions in toxic action of Zn2+ and selenite on AS-30D cells were found. Selenite induced a much higher intracellular ROS level (the early event) compared to Zn2+ but a lower membrane potential loss and a lower decrease of the uncoupled respiration rate of the cells, whereas the mtETC disturbance was the early and critical event in the mechanism of Zn2+ cytotoxicity. Sequences of events manifested in the mitochondrial dysfunction produced by the metal/metalloid under test are compared with those obtained earlier for Cd2+, Hg2+, and Cu2+ on the same model system.

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.

Kinetics of transport and phosphorylation of glucose in cancer cells

Metabolic control analysis of tumor glycolysis has indicated that hexokinase (HK) and glucose transporter (GLUT) exert the main flux control (71%). To understand why they are the main controlling steps, the GLUT and HK kinetics and the contents of GLUT1, GLUT2, GLUT3, GLUT4, HKI, and HKII were analyzed in rat hepatocarcinoma AS-30D and HeLa human cervix cancer. An improved protocol to determine the kinetic parameters of GLUT was developed with D-[2-(3)H-glucose] as physiological substrate. Kinetic analysis revealed two components at low- and high-glucose concentrations in both tumor cells. At low glucose and 37 degrees C, the V(max) was 55 +/- 20 and 17.2 +/- 6 nmol (min x mg protein)(-1), whereas the K(m) was 0.52 +/- 0.7 and 9.3 +/- 3 mM for hepatoma and HeLa cells, respectively. GLUT activity was partially inhibited by cytochalasin B (IC(50) = 0.44 +/- 0.1; K(i) = 0.3 +/- 0.1 microM) and phloretin (IC(50) = 8.7 microM) in AS-30D hepatocarcinoma. At physiological glucose, GLUT1 and GLUT3 were the predominant active isoforms in HeLa cells and AS-30D cells, respectively. HK activity in HeLa cells was much lower (60 mU/mg protein) than that in AS-30D cells (700 mU/mg protein), but both HKs were strongly inhibited by G6P. HKII was the predominant isoform in AS-30D carcinoma and HeLa cells. The much lower GLUT V(max) and catalytic efficiency (V(max)/K(m)) values in comparison to those of G6P-sensitive HK suggested the transporter exerts higher control on the glycolytic flux than HK in cancer cells. Thus, GLUT seems a more adequate therapeutic target.

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.