Tumor multifocality and serum albumin levels can identify groups of patients with hepatocellular carcinoma and portal vein thrombosis having distinct survival outcomes.

BACKGROUND: Macroscopic portal vein thrombosis (PVT) is a major poor prognosis factor in patients with hepatocellular carcinoma (HCC), but constitute a heterogeneous group. AIMS: To examine blood and tumor parameters of 1667 HCC patients who had PVT to identify factors that could differentiate different survival subsets. METHODS: a large HCC database was examined for presence of patients with PVT and analyzed retrospectively for PVT-associated factors and prognosis. RESULTS: A logistic regression model was calculated for presence of PVT. Highest odds ratios were found for tumor multifocality and serum albumin levels, as well as serum alpha-fetoprotein (AFP) and bilirubin levels. A Kaplan-Meier and Cox model on survival also showed the highest hazard ratios for tumor multifocality and serum albumin. A model was constructed on all 4 possible combinations of tumor focality and serum albumin in PVT patients. The longest survival group had <2 tumor nodules plus serum albumin >3.5 g/dL. Conversely, the shortest survival group had >2 tumor nodules plus serum albumin <3.5 g/dL. These 2 patient groups differed in maximum tumor diameter and levels of serum AFP, AST and bilirubin. CONCLUSIONS: Combination low tumor focality and high serum albumin identifies prognostically better PVT patient subgroups that might benefit from aggressive therapies.

Plasma lipids, tumor parameters and survival in HCC patients with HBV and HCV.

INTRODUCTION AND AIMS: Hepatocellular carcinoma (HCC) is a consequence of chronic liver disease, particularly from hepatitis B or C and increasingly from obesity and metabolic syndrome. Since lipids are an important component of cell membranes and are involved in cell signaling and tumor cell growth, we wished to evaluate the relationship between HCC patient plasma lipids and maximum tumor diameter and other indices of HCC human biology. METHODS: We examined prospectively-collected data from a multi-institutional collaborative Turkish HCC working group, from predominantly HBV-based patients, for plasma lipid profiles, consisting of triglycerides, total cholesterol, LDL-cholesterol (LDL) and HDL-cholesterol (HDL) and compared these with the associated patient maximum tumor diameter (MTD), portal vein thrombosis, alpha-fetoprotein (AFP) and also with patient survival. RESULTS: We found that both low HDL (p=0.0002) and high LDL (p=0.003) levels were significantly associated with increased MTD, as well as in a final multiple linear regression model on MTD. The combination of low HDL combined with high HDL levels were significant in a regression model on MTD, PVT and an HCC Aggressiveness Index (Odds Ratio 12.91 compared to an Odds Ratio of 1 for the reference). Furthermore, in a Cox regression model on death, the HDL plus LDL combination had a significantly higher Hazard Ratio than the reference category. CONCLUSIONS: Low plasma HDL, high plasma LDL and especially the combination, were significantly related to more aggressive HCC phenotype and the combination was significantly related to a higher Hazard Ratio for death.

Serum levels of gamma-glutamyl transpeptidase in relation to HCC human biology and prognosis.

BACKGROUND AND AIM: Hepatocellular carcinoma (HCC) biomarkers are limited, as even the best studied, alpha-fetoprotein (AFP), is elevated in no more than 50% of HCC patients. The aim was to evaluate several serum liver function tests in relation to survival and tumor characteristics in a large cohort of Turkish HCC patients. METHODS: We retrospectively examined the serum levels of gamma glutamyl transpeptidase (GGT) in relation to patient survival. RESULTS: Kaplan-Meier analysis showed that only GGT and albumin amongst liver function tests, were significantly associated with survival. Survival worsened with increase in GGT levels semi-quantitatively. Increase in GGT levels was also found to significantly correlate with an increase in maximum tumor diameter from 4.5 to 7 cm, a 20-fold increase in serum alpha-fetoprotein level, an increase in tumor multifocality from 20 to 54% of patients, and a doubling in percent of patients with portal vein thrombosis (PVT) from 20 to 40%. Serum GGT levels also showed significant survival differences for patients with low AFP levels. A doublet combination of serum GGT with albumin levels was associated with higher hazard ratios in a Cox regression analysis, as compared with single parameter GGT. The combination parameter pair was also prognostically useful in the low-AFP patient subcohort and was associated with significant differences in patient tumor characteristics. CONCLUSIONS: Serum GGT levels and especially combination serum GGT plus albumin levels, were significantly associated both with HCC patient survival and tumor aggressiveness characteristics, regardless of AFP levels in a large Turkish cohort. This might be especially useful since the majority of HCC patients do not have elevated levels of AFP.

Portal Vein Thrombosis and Markers of Inflammation in Hepatocellular Carcinoma.

BACKGROUND: Macroscopic portal vein thrombosis (PVT) is a major poor prognosis factor in patients with hepatocellular carcinoma (HCC). Inflammation is increasingly recognized to be part of the hepatocarcinogenic process and its markers are also prognostically useful. AIMS: To examine the relationship of inflammation biomarkers to the presence of PVT and to survival in PVT patients with HCC. METHODS: A large HCC cohort was examined for the presence of PVT and analyzed retrospectively. RESULTS: Blood levels of NLR, PLR, ESR, CRP, AFP and GGTP were significantly related to the presence of PVT, but not the Glasgow Index. For patients with low alpha-fetoprotein levels, blood ESR and GGTP levels were also significantly increased in patients with PVT compared with those in patients without PVT. In a Cox regression model, serum GGTP levels had a significantly increased hazard ratio on death (1.52, p = 0.008). Kaplan-Meier analysis showed that PVT patients with low serum GGTP levels had significantly longer survival than PVT patients with high GGTP levels (p = 0.0041). CONCLUSIONS: Indices of inflammation, especially serum GGTP levels, related significantly to the presence of PVT and to survival in HCC patients with PVT.

Treatment before liver transplantation for HCC.

Liver transplantation (LT) which is currently an established therapy for sma1l, early stage hepatocellular carcinoma (HCC) in patients with cirrhosis requires in most cases long waiting period. Tumor development during the waiting period may be associated with vascular invasion which is a strong factor of postoperative recurrence. Therefore, local treatment of the tumor including trans-arterial chemoembolization (TACE), percutaneous radiofrequency (RF) or partial liver resection can be used before transplantation. In the present paper we reviewed the efficacy of these treatments prior to LT. Although, TACE induced complete tumor necrosis in some patients there is no convincing arguments showing that this treatment reduces the rate of drop out before LT, nor improves the survival after LT. Although, RF can induce complete necrosis in the majority of small tumors (<2.5 cm), there is no data demonstrating that this treatment reduce the rate of drop out before LT, nor improves the survival after LT. It has been showed that both short and long term survival after LT was not compromised by previous partial liver resection of HCC. However, there is no data demonstrating that liver resection before LT, which can be used either as a bridge treatment or as a primary treatment, improves the survival after LT. The current data suggest that there is no role for pre-transplant therapy for HCC within Milano criteria transplanted within six months. On the opposite, if the waiting time is predicted to be prolonged, the risk of tumor progression and either drop-off from the list or interval dissemination with post-transplant tumor recurrence is recognized. In this setting, bridge therapy can reduce that risk but its efficacy has to be determined.

Effects of the hepatocarcinogen 2-acetylaminofluorene on insulin binding to microsomal and Golgi fractions of rat liver cells.

Adult male Fischer rats fed the hepatocarcinogen 2-acetylaminofluorene [(2-AAF) CAS: 53-96-3; N-fluoren-2-yl-acetamide] at 0.02% showed a sharp decrease in the insulin binding to the Golgi fraction of the liver cells: from 10.0% specific binding per 0.1 mg protein in control animals to 5.9% after only 2 days and to 1.5% after 21 days of feeding 2-AAF. A less pronounced and slower decrease was observed in the microsomal fraction: from 19.1% specific binding per 0.5 mg protein in controls to a nadir of 10.8% after 46 days. The low binding of insulin to both fractions was observed for the 85 days of the experiment and persisted also in the animals fed 2-AAF for 90-107 days and then taken off the carcinogen for 30-75 days. The decrease in binding was due to the apparent decrease in the number of receptors. Neither 2-AAF nor its metabolites N-hydroxy-2-AAF (CAS: 53-95-2; N-fluoren-2-yl-acetohydroxamic acid) and N-acetoxy-2-AAF (CAS: 6098-44-8; N,O-diacetyl-N-fluoren-2-yl-hydroxylamine) influenced the insulin binding to the microsomes when added to the reaction mixture in vitro.

Effects of diethylnitrosamine on hepatic receptor binding and autophosphorylation of epidermal growth factor and insulin in rats.

Carcinogen-mediated alterations in hepatic membrane growth factor receptor activity were investigated with the use of the hepatocarcinogen diethylnitrosamine [(DENA) CAS: 55-18-5]. For the separate assessment of carcinogen-induced acute membrane receptor changes from changes associated with carcinogen-mediated alterations in growth, the receptor activity was measured both acutely after, and several months after, a single ip dose of DENA in male F344 rats. An acute dose-dependent decrease was observed in ligand binding and autophosphorylation of the epidermal growth factor (EGF) and insulin receptor. Binding decreased sharply by 36 hours and recovered by 30 days in Golgi fractions and more slowly in plasma membranes. Scatchard analysis revealed a decrease in receptor number but not affinity. Chronic DENA administration also decreased insulin and EGF binding. Hepatocellular carcinomas, induced by single DENA injections 1 year previously, had decreased EGF receptor binding and autophosphorylation compared to those seen in age-matched controls and also less extensive insulin receptor changes. Single DENA doses thus have two effects: an acute reversible receptor change and a delayed, apparently permanent change associated with altered growth.

Pleiotropic drug resistance in hepatocytes induced by carcinogens administered to rats.

The effect of hepatocarcinogen administration in vivo on the induction of pleiotropic drug resistance was studied in primary monolayer cultures of adult rat hepatocytes using a cytotoxicity assay in vitro. Dietary 2-acetylaminofluorene, 3'-methyl-4-dimethylaminoazobenzene, aflatoxin B1, ethionine, and diethylnitrosamine rapidly induced resistance to doses of Adriamycin, methotrexate, cycloheximide, and aflatoxin B1 which were cytocidal to normal hepatocytes from untreated rats. Up to 95% of some hepatocyte preparations became drug resistant before any new hepatocyte phenotypes could proliferate. Drug resistance was measured at 24 h after initiation of 2-acetylaminofluorene feeding and remained stable throughout the 16 wk of carcinogen exposure. When limited carcinogen exposure was followed by a return to a basal non-carcinogen-containing diet for many months, the hepatocytes in the resultant hepatocellular carcinomas also displayed pleiotropic drug resistance, and the cells of the peritumorous liver did so to a lesser extent. Drug resistance was not induced by chronic administration of the tumor promoters phenobarbital, choline-deficient diet, phorbol, nor with 2,3,7,8-tetrachlorodibenzo-p-dioxin, but was induced to a variable extent by three hepatotoxins (ethanol, methotrexate, carbon tetrachloride). Whereas the early appearing drug resistance appears to be an adaptation of the liver to the presence of a toxic carcinogen, the late resistance which does not disappear after withdrawal of the inducing carcinogen may be a constitutive characteristic of chemically induced hepatocellular carcinomas.

Carcinogen-induced drug resistance in rat hepatocytes.

The relative resistance of liver cells in primary monolayer cell culture to the cytocidal effects of methotrexate, Adriamycin, cycloheximide, or aflatoxin B1 was studied using cells derived from normal rats, rats subjected to two-thirds hepatectomy, or rat fed dietary carcinogen. Normal rat liver cells were highly sensitive to the toxic effects of methotrexate. Adriamycin, cycloheximide, and aflatoxin B1. In contrast, liver cells from carcinogen-treated rats were resistant to the toxic effects of these agents. Cells derived from rats at 24 hr post two-thirds hepatectomy were sensitive to Adriamycin but not to cycloheximide or aflatoxin B1.

Cell surface-mediated cytotoxicity of polymer-bound Adriamycin against drug-resistant hepatocytes.

Growth inhibition properties of Adriamycin-coupled polyglutaraldehyde microspheres have been assessed using a highly resistant rat liver cell line, RLC. Covalent attachment of Adriamycin to microspheres increased the cytostatic activity of the drug 1000-fold for this cell line as measured by 50% inhibitory concentration determinations. The effects of Adriamycin-polymer complexes were investigated and compared to free drug, using trypan blue dye exclusion and 51Cr release as indicators of cell viability. When carcinogen-altered drug-resistant rat hepatocytes were tested at an Adriamycin concentration of 10(-6) M, the polymer-bound drug killed 32% of the cells, while the free drug had no detectable cytotoxic effect. However, with normal rat hepatocytes at the same drug concentration, the Adriamycin-coupled microspheres were shown to be less toxic than free drug at 24 hr. It was demonstrated that greater than 99.5% of the drug remains covalently coupled to the microspheres throughout the experiments. Scanning electron micrographs are presented for both cell types, which demonstrate the effects of free and bound Adriamycin on the ultrastructure of the cell surface. The cells lose their microvilli, exhibit numerous blebs, and develop holes and pits in the surface. Transmission electron microscopy demonstrates that less than 1% of the microspheres is internalized by either cell type. Multiple interactions of the drug-polymer complexes with the cell surface are presented as the most probable explanation for the results.

Binding of epidermal growth factor and insulin and the autophosphorylation of their receptors in experimental primary hepatocellular carcinomas.

Experimental chemical hepatocellular carcinomas that were induced in male F344 rats using three different regimens of limited exposure to the carcinogens 2-acetylaminofluorene or diethylnitrosamine were characterized by very low (as compared to peritumorous or normal tissues) binding of epidermal growth factor and decreased autophosphorylation of the epidermal growth factor receptors. Similar changes were also found in the insulin receptors. We suggest that the carcinogens 2-acetylaminofluorene and diethylnitrosamine cause an initial chemical effect on the great majority of cells. Most of them with time recover their receptor function, and only a small minority become truly initiated and retain these changed characteristics up to the tumor stage. The observed changes appear to be associated with the altered growth state induced by chemical carcinogens. Simultaneous changes observed in the two receptors raise the possibility of a common underlying mechanism.

Inhibition of DNA synthesis in rat hepatocytes by platelet-derived type beta transforming growth factor.

Platelet-derived type beta transforming growth factor (TGF beta) is a potent inhibitor of DNA synthesis in primary monolayer cultures of adult rat hepatocytes. TGF beta induced a 50% inhibition of epidermal growth factor (EGF)-mediated DNA synthesis at approximately 5 X 10(12) M. This inhibition did not appear to be due to a delay in the peak of DNA synthesis or a toxic action, nor could it be overcome by increasing concentrations of the mitogens EGF, insulin, or glucagon. Inhibition was observed either when TGF beta and EGF were continuously present together in the culture medium or when TGF beta was added to the hepatocyte cultures after removal of the EGF stimulant. This observation together with a lack of an inhibitory effect of TGF beta on the binding of 125I-labeled EGF to hepatocytes in culture, suggests that the inhibitory action of TGF beta was not caused by a direct competition with EGF at the cell surface. TGF beta could not inhibit DNA synthesis once it had begun; however, the inhibitory action of TGF beta could be partially overcome by increasing amounts of conditioned medium produced by normal hepatocytes. Specific saturable receptors for TGF beta were found on the normal rat hepatocytes, but specific binding could not be detected on hepatocytes from regenerating liver. TGF beta is thus a potent inhibitor of EGF-induced DNA synthesis in adult rat hepatocytes. Its significance for growth control in vivo has yet to be assessed.

Induction of heat shock gene expression without heat shock by hepatocarcinogens and during hepatic regeneration in rat liver.

We investigated the expression of the rat hepatic heat-shock protein (hsp) genes under the influence of hepatocarcinogens and during hepatic regeneration. This was undertaken because of the inducibility of the heat-shock response in rat liver and because heat-shock genes can be expressed with or without heat shock in various cell states in a developmentally regulated manner. We found that acute administration of hepatocarcinogens to rats induced an increased hsp gene transcription in a time- and dose-dependent manner. Chronic exposure of rats to complete hepatocarcinogens induced increased levels of mainly Mr 83,000 heat-shock protein gene transcription and, to a lesser extent, Mr 70,000 heat-shock protein. However, the tumor promoter phenobarbital did not induce increased hsp gene expression. Increased levels of both Mr 83,000 heat-shock protein and Mr 70,000 heat-shock protein gene transcription were found during hepatic regeneration. Thus, increased hsp gene transcription, which correlated with increased heat-shock protein synthesis, was observed under the acute and chronic influence of hepatocarcinogens and during normal hepatic proliferation. These results are similar to those observed for c-H-ras and c-myc expression in rat liver, and they suggest that a coordinate expression of these three genes may occur in hepatic regeneration and in the early stages of experimental chemical hepatocarcinogenesis.

Fetal hemoglobin gene activation in a phase II study of 5,6-dihydro-5-azacytidine for bronchogenic carcinoma.

5-Azacytidine and several of its analogues are known to inhibit DNA methylation, alter gene expression, and inhibit cell growth. We report a Phase II study in which we investigated the antineoplastic activity of 5,6-dihydro-5-azacytidine and its induction of fetal hemoglobin synthesis when given by a 5-day continuous i.v. infusion of 1650 mg/m2/day that was repeated every 21 days. Fetal hemoglobin was measured in all patients; increased synthesis was found in 13 of the 17, in the absence of clinically significant anemia. Of the four patients who did not develop increased fetal hemoglobin, three had only one cycle of therapy. Fourteen patients with bronchogenic carcinoma were treated, and ten were evaluable for disease response. Five patients had disease stability of 2 or more mo, and five progressed on treatment. Three additional patients with mesothelioma were treated, and the two who were evaluable for disease response had stabilization of their disease. Fifteen of the 17 patients who received 5,6-dihydro-5-azacytidine developed a pleuritic-type chest pain, 12 had abnormal electrocardiograms, and four developed positive anti-nuclear antibodies. No significant hemopoietic, hepatic, or renal toxicities were observed. This study demonstrates that 5,6-dihydro-5-azacytidine in the dose and schedule used has no significant therapeutic activity in the treatment of lung cancer but does possess an unusual spectrum of clinical toxicities as well as the property of inducing fetal hemoglobin synthesis.

Expression of rat microsomal epoxide hydrolase gene during liver chemical carcinogenesis.

A complementary DNA library was constructed from mRNA of rat liver induced by an initiating dose of a chemical carcinogen, diethylnitrosamine. Using a differential hybridization technique, a complementary DNA clone which is induced more than 10-fold by an acute single dose of diethylnitrosamine was identified. The DNA sequence of this clone was matched with rat microsomal epoxide hydrolase. This gene may be of great interest, since it was found to be highly expressed in neoplastic nodules and primary hepatocellular carcinomas induced by different carcinogenic regimes. The inducible high level expression of this gene becomes constitutive during the process of hepatocarcinogenesis. The gene was also found to be inducibly expressed during partial hepatectomy in a similar manner as a multidrug-resistant gene (mdr-I). No change in the transcriptional initiation site was observed in the gene expression between induced and uninduced rat livers. The 5' upstream region of the gene was characterized and some potential controlling elements for gene regulation, such as Sp-1, AP-2, and Hepatitis B virus enhancer, were found. Based on our own and published results, we hypothesize that the altered expression of this xenobiotic enzyme in nodules and cancer cells could be a result of constitutive internal stimuli which might be associated with cell growth.

Differential interaction of normal and preneoplastic hamster bronchi with adriamycin.

Advanced bronchogenic carcinoma in humans is notoriously resistant to the cytocidal actions of cancer chemotherapy. The experiments reported here were undertaken as a first step in examining the mechanisms of resistance of carcinogen-altered bronchus to the actions of the commonly used cancerocidal agent Adriamycin. Syrian Golden hamsters were treated with an endobronchial carcinogen in order to produce bronchial neoplasms or with no carcinogen as controls. Hamsters were then given i.v. Adriamycin, and the amounts and metabolism of bronchial Adriamycin were determined. Peak uptake values were found 5 min after Adriamycin administration, and the amounts of Adriamycin in normal and carcinogen-altered bronchi were found to be similar. Whereas no metabolism of Adriamycin was observed in normal bronchi, 40-60% of total Adriamycin fluorescence was found to be due to Adriamycinol and Adriamycin aglycones in bronchi with premalignant changes. In separate experiments, the susceptibility of normal and carcinogen-altered bronchial extracts to drug-induced lipid peroxidation was measured in vitro. A 50% decrease was found in the ability of carcinogen-altered bronchi to act as a substrate for lipid peroxidation mediated by Adriamycin and an approximately 30% decrease for lipid peroxidation induced by t-butyl-hydroperoxide. These results demonstrate two different mechanisms by which bronchogenic carcinomas might become resistant to the chemotherapeutic actions of Adriamycin. These are by the carcinogen induction of metabolism of Adriamycin to less toxic products and by resistance of the bronchi to free radical damage.

Effect of 2-acetylaminofluorene on the binding of epidermal growth factor to microsomal and Golgi fractions of rat liver cells.

The livers of rats fed the hepatocarcinogen 2-acetylaminofluorene (0.02%) with chow showed a sharp decrease in the binding of epidermal growth factor to microsomes and Golgi fractions. The binding to the latter decreased from 15.3% specific binding per 0.1 mg protein in controls to 9.4% after 2 days and reached a nadir of 0.8% after 21 days. The binding to microsomes decreased from 26.3% specific binding per 0.5 mg protein in the controls to 17.4% after 4 days and reached a nadir of 7.5% after 46 days. The low binding which persisted until the end of the experiment (85 days) was due to the apparent decrease in the number of receptors without significant changes in their affinity. Also, there was only partial recovery in rats fed 2-acetylaminofluorene for 90 to 107 days and taken off the carcinogen for 30 to 75 days. In vitro, neither 2-acetylaminofluorene nor its metabolites hydroxy- and acetocy -2-acetylaminofluorene significantly decreased epidermal growth factor binding to the isolated microsomal fraction.

Insulin and epidermal growth factor receptors in rat liver after administration of the hepatocarcinogen 2-acetylaminofluorene: ligand binding and autophosphorylation.

The livers of male F344 rats which were fed 0.02% 2-acetylaminofluorene (2-AAF) for two days or more had decreased binding of insulin and epidermal growth factor (EGF) to their hepatic receptors in microsomal and Golgi fractions. Hepatic receptors which were partially purified from carcinogen-fed rats by Triton X-100 solubilization and wheat germ agglutinin affinity column chromatography also had decreased binding activity compared to receptors from normal rats. Scatchard analysis indicated that the decrease in insulin receptor binding was due to decreased receptor number whereas the change in EGF receptor binding was attributed to decreased receptor affinity. Insulin receptor phosphokinase activity was also decreased in 2-AAF-fed rats and correlated with the decrease in receptor binding. EGF receptor phosphokinase activity was unchanged in 2-AAF-fed rats when stimulated with a high concentration (1 microM) of EGF but was decreased when stimulated with low concentrations (0.01-0.1 microM) of EGF. No EGF or insulin competing activity for receptor binding was found using acid-ethanol extracts of 2-AAF-altered liver. These results suggest that 2-AAF causes different alterations in the insulin and EGF receptors of the rat liver.

Cdc25 inhibition and cell cycle arrest by a synthetic thioalkyl vitamin K analogue.

A synthetic vitamin K analogue, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or compound 5 (Cpd 5), was found previously to be a potent inhibitor of tumor cell growth. We now demonstrate that Cpd 5 arrested cell cycle progression at both G1 and G2-M. Because of the potential arylating activity of Cpd 5, it might inhibit Cdc25 phosphatases, which contain a cysteine in the catalytic site. To test this hypothesis, we examined the inhibitory activity of Cpd 5 against several cell cycle-relevant protein tyrosine phosphatases and found that Cpd 5 was a potent, selective, and partially competitive inhibitor of Cdc25 phosphatases. Furthermore, Cpd 5 caused time-dependent, irreversible enzyme inhibition, consistent with arylation of the catalytic cysteine in Cdc25. Treatment of cells with Cpd 5 blocked dephosphorylation of the Cdc25C substrate, Cdc2, and its kinase activity. Cpd 5 enhanced tyrosine phosphorylation of both potent regulators of G1 transition, ie., Cdk2 and Cdk4, and decreased the phosphorylation of Rb, an endogenous substrate for Cdk4 kinase. Furthermore, close chemical analogues that lacked in vitro Cdc25 inhibitory activity failed to block cell cycle progression and Cdc2 kinase activity. Cpd 5 did not alter the levels of p53 or the endogenous cyclin-dependent kinase inhibitors, p21 and p16. Our results support the hypothesis that the disruption in cell cycle transition caused by Cpd 5 was attributable to intracellular Cdc25 inhibition. This novel thioalkyl K vitamin analogue could be useful for cell cycle control studies and may provide a valuable pharmacophore for the design of future therapeutics.

Involvement of Cdc25A phosphatase in Hep3B hepatoma cell growth inhibition induced by novel K vitamin analogs.

We previously found that K vitamin analogues caused cell growth inhibition in Hep3B hepatoma cells in vitro, which was associated with their inhibitory effects on protein tyrosine-phosphatases. In this study, we show that Cdc25A, a protein phosphatase, was inactivated by novel arylating K vitamin analogues. The inactivation of Cdc25A correlated with their effects on cell growth inhibition. Cyclin-dependent kinase (Cdk) 4, an important regulator for G(1) progression, was found to be tyrosine-phosphorylated by the arylating analogues, and this phosphorylation was correlated with the inhibitory effects of the analogues on Cdc25A activity. Furthermore, Cdk4 dephosphorylation experiments showed that Compound (Cpd) 5, a prototype arylating analogue, inhibited Cdc25A-mediated Cdk4 dephosphorylation, whereas Cpd 26, a nonarylating vitamin K analogue, had no effect on this event. We also examined Cdk4 kinase activity using retinoblastoma protein as a substrate and found that Cpd 5 inhibited retinoblastoma protein phosphorylation in a concentration-dependent manner, indicating that Cdk4 activity was inhibited by Cpd 5 treatment. Moreover, the thiol-antioxidants glutathione and N-acetyl-L-cysteine antagonized the Cpd 5-induced Cdk4 tyrosine phosphorylation, whereas the nonthiol-antioxidants catalase and superoxide dismutase did not. These results suggest that Hep3B cell growth inhibition by these K vitamin analogues may be related in part to inactivation of Cdc25A activity and support the hypothesis that Cdc25A is an attractive target for drugs designed to inhibit cancer cell growth.