©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Growth Inhibition of Hepatoma Cells Induced by Vitamin K and Its Analogs (*)

(Received for publication, July 19, 1995; and in revised form, August 31, 1995)

Yuji Nishikawa (1) Brian I. Carr (1)(§) Meifang Wang (1) Siddhartha Kar (1) Frances Finn (2) Paul Dowd (3) Zhizhen B. Zheng (3) Jeffrey Kerns (3) Sriram Naganathan (3)

From the  (1)Pittsburgh Transplantation Institute, the (2)Protein Research Laboratory, and the (3)Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15213

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Congeners of vitamin K are known to inhibit cell growth, although the precise mechanisms of growth inhibition are not well understood. To investigate the mechanisms involved, we synthesized several vitamin K analogs and examined their growth inhibitory activities for a human hepatoma cell line (Hep3B). The analogs included 2-methyl-1,4-naphthoquinone and trimethylbenzoquinone, with and without aliphatic side chains at position 3. The side chains were all-carbon, thioethers, or O-ethers. Growth inhibition was potent in the compounds with short chains. The presence of a sulfur (thio-ether) or oxygen atom (O-ether) at the site of attachment of the side chain to the ring potentiated the activity. Apoptotic cell death was induced by the potent growth inhibitory compounds at low concentrations (20-60 µM), whereas necrotic cell death followed treatment with the same compounds at high concentrations. Expression of c-myc, which is thought to be associated with apoptosis, was increased by most of the compounds tested. Both reduced glutathione and cysteine almost completely abrogated the growth inhibitory effects of the thioether analogs as well as of vitamin K(3). The effect of glutathione was less prominent for the all-carbon and O-ether analogs, and cysteine had no effect on these analogs. Catalase and deferoxamine mesylate had no significant effect on the thioether analogs, although they showed partial antagonistic effects on the growth inhibition of vitamin K(3) and the all-carbon and O-ether analogs. Other non-thiol antioxidants tested had no effect on any of the analogs. Our results indicated that vitamin K-related quinoid compounds cause growth inhibition and both apoptotic and necrotic cell death and that the effects may be mediated by interaction at position 3 of their quinoid nuclei with cellular thiols.


INTRODUCTION

Vitamin K (VK) (^1)is a generic term for compounds that include phylloquinone (VK(1)), menaquinone series (VK(2)), and menadione (VK(3)) (1) . Physiologically, the natural K vitamins, VK(1) and VK(2), are known to act as cofactors for -carboxylation of selected glutamates in the N termini of prothrombin and other VK-dependent coagulation factors (2, 3, 4) . Patients with vitamin K deficiency, those receiving warfarin anticoagulation therapy, and those with various liver disorders, particularly hepatoma, produce under-carboxylated or immature prothrombin (des--carboxy prothrombin) that is secreted into the plasma(5) . Des--carboxy prothrombin has been found to be one of the most reliable markers for hepatoma(6, 7, 8, 9, 10) .

The congeners of VK share a common chemical structure consisting of a naphthoquinone nucleus capable of redox cycling (see Fig. 1). VK(1) has a long phytol side chain, whereas VK(2) has an unsaturated side chain composed of 4-13 isoprene units. VK(3) lacks the side chain. VK(3) has previously been shown to have growth inhibitory effects and to induce cell death in several cell types both in vitro(11, 12, 13, 14, 15, 16) and in vivo(17, 18) . Several investigators have demonstrated that VK(3)-induced cell death is associated with apoptosis (14, 19, 20) and overexpression of c-myc gene(14) , which is considered to be closely related to apoptosis(21, 22, 23) . VK(1) and VK(2) also have been shown to have cell growth inhibitory effects in vitro, but these effects are much weaker than those of VK(3)(11, 24) . VK(3) has been used in experimental animal chemotherapy combined with ascorbic acid (25) and methotrexate(18) . Phase I and phase II clinical trials have been carried out for VK(3)(26) , and a recent phase I/II trial is in progress for VK(1) in human hepatoma(27) .


Figure 1: Chemical structures of K vitamins and analogs used in this study. K(1), vitamin K(1) (phylloquinone, 2-methyl-3-phythyl-1,4-naphthoquinone); K(2), vitamin K(2) (menaquinone 4); K(3), vitamin K(3) (menadione, 2-methyl-1,4-naphthoquinone); pBQ, p-benzoquinone (1,4-benzoquinone); 1, 2-methyl-3-butyl-1,4-naphthoquinone; 2, 2-methyl-3-(1-thiopropyl)-1,4-naphthoquinone; 3, 2-methyl-3-(1-thiobutyl)-1,4-naphthoquinone; 4, 2-methyl-(1-thiooctyl)-1,4-naphthoquinone; 5, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone; 6, 2-butoxy-3-methyl-1,4-naphthoquinone; 7, 2-methyl-3-(1-oxyoctyl)-1,4-naphthoquinone; 8, trimethylbenzoquinone; 9, 2-phytyl-trimethylbenzoquinone; 10, 2-(1-thiopropyl)-trimethylbenzoquinone; 11, 2-(1-thiobutyl)-trimethylbenzoquinone; 12, 2-(1-thiododecyl)-trimethylbenzoquinone.



The mechanism of growth inhibition and cell killing by VK is not well understood. Oxidative stress is considered to be a mechanism of action of quinoid compounds such as VK(3), because toxic oxygen species can be generated during redox cycling involving the quinoid structures(28, 29, 30, 31) . Another possible mechanism of toxicity of VK(3) and related quinones is the direct arylation of cellular thiols resulting in depletion of glutathione and inhibition of sulfhydryl-dependent proteins(32, 33, 34, 35, 36) .

To address the mechanism of VK-induced growth inhibition, we investigated the structural requirements for their actions. We synthesized several VK-related quinoid compounds, including those with a benzoquinone nucleus or naphthoquinone nucleus, with and without aliphatic side chains, and with and without modifications by an additional sulfur, oxygen, or hydroxyl group. We then examined the relationship between the chemical structure and the effects on growth inhibition and cell killing for a human hepatoma cell line (Hep3B). Furthermore, we studied several possible mechanisms, including -carboxylation, oxidative stress, and arylation of cellular thiols.


EXPERIMENTAL PROCEDURES

Materials

VK(1), VK(2), and VK(3) were purchased from Sigma. The chemicals used in the preparation of analogs were purchased from Aldrich. The analogs synthesized included quinoid compounds containing one or two benzene rings with aliphatic side chains, some of which had an additional sulfur (thioether analogs) or oxygen (O-ether analogs). The chemical structures of these compounds are shown in Fig. 1. Trimethyl-1,4-benzoquinone (8) was generated from trimethylhydroquinone by oxidation with AgNO(3). The thioethers were prepared by the addition of 1,8-diazabicyclo[5.4.0]undec-7-ene to an ether solution of VK(3) (2-methyl-1,4-naphthoquinone) or 8 and the corresponding thiols (1-propanethiol for 2 and 10, 1-buthanethiol for 3 and 11, 1-octanethiol for 4, 1-dodecanethiol for 12, and 2-mercaptoethanol for 5) to give the intermediate hydroquinone and then air-oxidized to the quinone(36, 37) . The O-ether analogs were prepared from phthiocol, KH, 18-crown-6, and the corresponding alkyl iodide (1-iodobutane for 6 and 1-iodooctane for 7) in refluxing tetrahydrofuran. Phthiocol (2-hydroxy-3-methyl-1,4-naphthoquinone) was generated from VK(3) by the reaction with Na(2)CO(3) and hydrogen peroxide. The all-carbon derivative 1 was prepared by the free radical method (38) from VK(3) and n-butanolic acid. Another all-carbon derivative 9 was generated from 8 and phytol using a boron-trifluoride-etherate complex. Hep3B cells (a human hepatoma cell line, ATCC HG8064) were maintained in Eagle's minimum essential medium supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and penicillin/streptomycin.

Cell Growth Inhibition Assay

Monolayer cell growth was assayed after cells were plated at 5 times 10^4 cells/well of 6-well plates (Corning, Inc., Science Products Div., Corning, NY). After 24 h, the medium was replaced with a medium containing K vitamins and analogs at various concentrations. After treatment for 3 days, cells were trypsinized and suspended in 1 ml of phosphate-buffered saline with 5% calf serum. Absorbance at 660 nm was measured spectrophotometrically. Control experiments demonstrated a linear correlation between Hep3B cell density and absorbance at 660 nm (39) . The ID values were estimated from the dose response curves. The same assay was used to examine the effects of thiols and non-thiol antioxidants on the growth inhibition by VK and the analogs. We treated cells with the quinoid compounds at 60-90% growth inhibitory doses in the presence or absence of thiols or non-thiol antioxidants in the media. A nonredox cycling quinone, p-benzoquinone (pBQ), and a pure oxidant, tert-butyl hydroperoxide (tBHP), were included to assess the effects of thiols and non-thiol antioxidants. We used reduced glutathione (GSH) (2 mM), L-cysteine (2 mM), D-cysteine (2 mM), and N-acetyl-L-cysteine (NAC) (2 mM) as thiols, and we used catalase (40 and 80 units/ml), deferoxamine mesylate (12 mM pretreatment for 1 h, then 1 mM continuously), superoxide dismutase (500 units/ml), L-ascorbic acid sodium salt (200 µM), butylated hydroxyanisole (150 µM), and nordihydroguaiaretic acid (20 µM) as non-thiol antioxidants. We also tested the effects of oxidized glutathione (2 mM), the glutathione fragment lacking cysteine (-Glu-Gly) (2 mM), L-glutamic acid (2 mM), and glycine (2 mM).

Assay for DNA Fragmentation

To measure cell damage, we assayed the extent of DNA fragmentation by the method described by McCarthy et al.(40) . Subconfluent Hep3B cells were treated with test compounds at various concentrations for 24 h. The treated cells were washed with phosphate-buffered saline and lysed by a buffer containing Triton X-100 (0.5% Triton X-100, 5 mM TrisbulletHCl, 1 mM EDTA, pH 8.0). The lysates were then centrifuged, the pellet (intact DNA) and the supernatant (fragmented DNA) were separated, and then the pellet was resuspended with buffer containing Triton X-100. After sonication of the pellet and supernatant solutions, they were serially diluted on 96-well microplates (Dynatech Laboratories, Inc., Chantilly, VA) separately, and 4`,6-diamidino-2-phenylindole was added. Fluorescence of 4`,6-diamidino-2-phenylindole was quantified by a microplate reader (Dynatech), and titration curves were made. The percentage of DNA fragmentation (100 times [DNA in supernatant]/([DNA in supernatant] + [DNA in pellet])) was determined from the titration curves.

Detection of DNA Laddering

DNA fragmentation was evaluated qualitatively by agarose gel electrophoresis. Subconfluent Hep3B cells were treated with test compounds at 20 µM for 18 h and lysed by digestion buffer (0.5% SDS, 0.1 mg/ml proteinase K, 100 mM NaCl, 10 mM TrisbulletHCl, 25 mM EDTA, pH 8.0) and incubated at 50 °C for 12-18 h. The lysates were extracted with phenol/chloroform (1:1) and then precipitated with ethanol/ammonium acetate at -20 °C. After rinsing the resulting pellets with 70% ethanol, they were air-dried and dissolved into Tris-EDTA buffer containing RNase (8 µg/ml). DNA samples were labeled with [P]dCTP by Klenow fragment of DNA polymerase I, electrophoresed on agarose gel, and exposed to x-ray film (XAR-5, Eastman Kodak Co.) for autoradiography(41) .

In Situ End Labeling of Fragmented DNA

Cells were cultured on chamber slides (Nunc, Inc., Naperville, IL) and treated with test compounds at various concentrations for 24 h. They were then fixed with 10% buffered formalin for several hours at room temperature, washed with phosphate-buffered saline three times, and air-dried. Fragmented DNA was detected by an in situ end labeling technique(42) . We used ApopTag kit (Oncor, Gaithersburg, MD) according to the manufacturer's protocol. Briefly, digoxigenin-labeled dUTP was incorporated at the 3`-OH ends of the fragmented DNA by terminal deoxynucleotidyl transferase, the anti-digoxigenin antibody conjugated with peroxidase was applied, and the peroxidase activity was revealed by 3-amino-9-ethylcarbazol. Nuclei were counterstained lightly with hematoxylin.

Northern Blot Analysis for c-myc Gene Expression

Hep3B cells were treated with test compounds at various concentrations for 24 h. Total RNA was prepared by using Trizol reagent (Life Technologies, Inc.), and RNA samples (20 µg) were resolved by denaturing glyoxal-agarose gel electrophoresis. The separated RNAs were transferred to a Hybond N+ membrane (Amersham Corp.) and cross-linked with ultraviolet light (short wave). The membrane was hybridized to either a [P]dCTP-labeled c-myc cDNA probe (purchased from ATCC) or 18 S ribosomal RNA probe and washed by standard protocols. Autoradiography was performed at -70 °C using intensifying screens.

Carboxylase Assay

Assay solutions contained ammonium sulfate (0.8 M), dithiothreitol (2 mM), Phe-Leu-Glu-Glu-Leu (4 mM), NaCl (48 mM), VK or its analogs (1.8 mM) unless otherwise indicated, and [^14C]sodium bicarbonate (1.6 mM, 1 µCi) in TrisbulletHCl buffer (6.4 mM), pH 7.5. The total reaction volume was 125 µl, of which 50 µl was rat liver microsomal fraction. The reaction was started by the addition of VK or its analogs. The assay mixtures, in 500-µl polypropylene tubes, were incubated with mixing by rotation for 30 min at 20 °C, and the reaction was stopped by addition of 75 µl of 1 N NaOH. A portion of the assay mixture (160 µl) was then transferred to tubes containing 1 ml of 5% trichloroacetic acid, and unincorporated CO(2) was removed by flushing the mixture with N(2) at 90 °C for 3 min. A portion of the mixture (0.8 ml) was transferred to counting vials containing 3 ml of Ultima Gold (Packard Instrument Co., Inc., Meriden, CT) and counted in a scintillation counter.


RESULTS

Growth Inhibitory Effects of K Vitamins and Analogs

We examined the growth of Hep3B cells in the presence of K vitamins and analogs and estimated ID. Marked growth inhibition was observed after treatment with all compounds carrying short side chains. Growth inhibitory activity decreased with increasing side-chain length (Table 1). The presence of a sulfur or an oxygen atom at the start of the aliphatic side chain potentiated the growth inhibitory effect of the compounds as evident in comparison of 1 (all-carbon, ID = 78 µM) with 3 (thioether, ID = 15 µM) or 6 (O-ether, ID = 55 µM). The thioethanol derivative (5) was the most potent analog among the compounds tested.



Effects of K Vitamins and Analogs on Cell Death and c-myc Expression

To test the cell killing effect of K vitamins and analogs, we added the compounds at various concentrations to subconfluent cultures of Hep3B cells and examined cell morphology and DNA damage after 24 h. Compounds with a short or no side chain had strong cell killing effects. Two types of cell changes were distinguished: (a) cell shrinkage followed by detachment and (b) ghost-like necrosis without shrinkage and detachment.

Cell death induced by potent compounds was associated with marked DNA fragmentation (Fig. 2). DNA laddering of nucleosomal size (180-200 bp) was revealed in the cells treated with several potent growth inhibitory compounds, suggesting that a population of the cells underwent apoptotic cell death (Fig. 3).


Figure 2: DNA fragmentation of Hep3B cells induced by VK(3) and the analogs. Cells were treated with test compounds at various concentrations for 24 h and lysed by Triton X-100 containing buffer. The percentage of DNA fragmentation was estimated as described under ``Experimental Procedures.'' The results are shown as the means of two separate experiments.




Figure 3: Nucleosomal DNA fragmentation of Hep3B cells induced by VK(3) and several potent growth inhibitory analogs. Cells were treated with test compounds at 20 µM for 18 h, and their DNA were extracted and labeled with [P]dCTP by Klenow fragment of DNA polymerase I. The labeled samples were electrophoresed in an agarose gel (2%) and visualized by autoradiography. Cont., control.



Microscopically, a small number of typical apoptotic cells, namely shrunken cells with fragmented nuclei (apoptotic bodies), were observed among the cells treated with potent growth inhibitory compounds, and most of them were positively stained by in situ end labeling techniques (data not shown). We counted the number of apoptotic cells that appeared 24 h after treatment and found that it was largely parallel to the potency of growth inhibition of the compounds (Table 2). However, ghost-like necrosis predominated when the more potent compounds were used at higher concentrations (Table 2).



We examined c-myc gene expression, which has been reported to be closely related to apoptosis. c-myc mRNA was induced by all compounds in a dose-dependent manner, except by VK(1) and 9 (Fig. 4). However, some of the more potent growth inhibitory structures such as VK(3) and 5 were not strong c-myc inducers. The presence or the length of the side chain, which affected the growth inhibitory potency, did not influence the extent of c-myc induction (Fig. 4).


Figure 4: Northern blot analysis of c-myc gene expression in Hep3B cells after treatment of K vitamins and analogs. Cells were treated with test compounds at various concentrations for 24 h, and RNA was isolated. The samples (20 µg of total RNA) were denatured by glyoxal and electrophoresed in 1% agarose gels. After the gels were blotted onto nylon membranes, the blotted RNA were hybridized to a P-labeled c-myc or 18 S ribosomal RNA probe and visualized by autoradiography.



Effects of Thiol and Non-thiol Antioxidants on Growth Inhibition

We tested the effect of exogenous thiols on growth inhibition by VK(3) and the analogs. The presence of GSH or NAC in the medium completely abrogated the growth inhibition by most compounds, except 1 and 6, on which the effect was partial (GSH shown in Fig. 5a; NAC, data not shown). L-Cysteine and D-cysteine had a similar strong antagonistic effect, although they did not alter the effect of 1 and 6 (L-cysteine shown in Fig. 5b; D-cysteine, data not shown). Oxidized glutathione, the glutathione fragment lacking cysteine (-Glu-Gly), L-glutamic acid, and glycine had no antagonistic effect (data not shown). The growth inhibition of pBQ was antagonized by GSH, NAC, and both isomers of cysteine, but that of tBHP was antagonized only by GSH (Fig. 5, a and b).


Figure 5: Effect of reduced glutathione and L-cysteine on growth inhibition of Hep3B cells by VK(3) and the analogs. a, GSH (2 mM); b, L-cysteine (2 mM). - and + denote the absence and presence of the antioxidants, respectively. 1 day after plating of Hep3B cells, various drugs were added to the medium, and the culture was continued for 3 days. The cell number was estimated by the absorbance at 660 nm and represented as the percentage of controls (mean ± S.D.) from three separate experiments. Statistical analysis was performed using an unpaired t test (double tails) between non-treated(-) and treated (+) groups. The presence of significant differences is indicated by * (p < 0.05) or** (p < 0.01).



We also examined the effect of non-thiol antioxidants on growth inhibition by VK(3) and the quinoid analogs. Catalase antagonized the effect of VK(3) and 6, but it did not affect the growth inhibition of other potent analogs (Fig. 6a). Deferoxamine mesylate completely abrogated the effect of tBHP, which has been shown to exert its effect by generation of hydroxyl radicals (Fig. 6b). Although it partially antagonized the effect of VK(3), 1, and 6, it did not alter the effect of the other potent analogs (Fig. 6b). Superoxide dismutase had no protective effect at all on any compounds (Fig. 6c). The growth inhibitory action of pBQ, which is known to lack redox cycling activity, was not affected by catalase (Fig. 6a) or deferoxamine mesylate (Fig. 6b) and was slightly potentiated by superoxide dismutase (Fig. 6c). The other non-thiol antioxidants, L-ascorbic acid, butylated hydroxyanisole, and nordihydroguaiaretic acid, had no effect on any of the compounds (data not shown).


Figure 6: Effect of catalase, deferoxamine mesylate, and superoxide dismutase (SOD) on growth inhibition of Hep3B cells by VK(3) and the analogs. a, catalase (80 units/ml); b, deferoxamine mesylate (1 mM following 12 mM pretreatment for 1 h); c, superoxide dismutase (500 units/ml). - and + denote the absence and presence of the antioxidants, respectively. The methods were the same as those of the experiments in Fig. 5. The numbers of experiments were 4, 5, and 4 in a, b, and c, respectively. Statistical analysis was performed using an unpaired t test (double tails) between non-treated(-) and treated (+) groups. The presence of significant differences was indicated by * (p < 0.05) or** (p < 0.01).



In Vitro Carboxylation

The major physiological action of VK is to support glutamine residue carboxylation reactions, especially for the VK-dependent coagulation factors. To assess whether growth inhibition might correlate with carboxylation, we measured the extent of carboxylation by various K vitamin compounds. We found that the natural K vitamins (VK(1) and VK(2)) showed activity as cofactors for -carboxylation; however, VK(3), 2, 3, 4, and 5 had no activity (Fig. 7; data for 2 and 5, not shown).


Figure 7: Carboxylase activity of K vitamins and selected analogs. Carboxylase activity was measured by the carboxylation of a synthetic substrate, Phe-Leu-Glu-Glu-Leu, in the presence of liver microsomal fraction as described under ``Experimental Procedures.'' Abscissa, incubation time (min); ordinate, cpm of incorporated ^14C; box, vitamin K(1); , vitamin K(2); circle, vitamin K(3); up triangle, 3; , 4.




DISCUSSION

K vitamins, especially VK(3), have been reported to inhibit the growth of various tumor cell lines(11, 12, 13, 14, 15, 16) . The synthesis of novel VK compounds in the present study was undertaken with two aims: first, to synthesize more potent growth inhibitory structures that might have future therapeutic potential and second, to identify the structural requirements and the mechanism of the growth inhibitory actions of compounds of the vitamin K class. We tested the relative growth inhibitory effects of several VK-related quinoid compounds on a well described human hepatoma cell line. In general, we found that potency of growth inhibitory action correlated with decreasing length of the side chain. In addition, the potency was increased by the presence of a sulfur (thioether) or oxygen atom (O-ether) at the site of attachment of the side chain to the ring. A short thioethanol side chain produced the most potent compound (5).

Hepatomas have a loss of the ability to carboxylate prothrombin at the -glutamyl position, causing elevated plasma levels of des--carboxy prothrombin(6, 7, 8, 9, 10) . Recent clinical studies have suggested that supra-physiological doses of VK, when given to patients with hepatoma, result in a depression in plasma des--carboxy prothrombin levels (27, 43, 44) and possibly a decrease in tumor growth (27) . This led us to consider the possibility that supra-physiological doses of vitamin K might alter the metabolism of hepatoma cells and may also have growth inhibitory effects. But -carboxylation itself did not appear to be involved in the growth inhibition induced by the VK analogs, because no correlation was found between the ability of various VK compounds to support carboxylation in microsomal preparations and their activity as growth inhibitors on cultured cells. However, some metabolic interconversion among the K vitamins has previously been shown in vivo, such as the conversion of VK(3) to VK(2)(45) , suggesting the possible effects of VK analogs on -carboxylation in vivo. Interestingly, a -carboxylation-dependent protein with significant homology with protein S, an anticoagulant, has been shown to be a ligand for a tyrosine kinase that is involved in cell transformation(46) . Further investigation is needed to clarify the relationship between -carboxylation and cell growth.

Other possible mechanisms of the growth inhibitory actions of several of the potent VK and related compounds were examined. In general, quinones such as VK are thought to undergo redox cycling and to generate reactive oxygen species(28, 29, 30, 31) . In the experiments reported here and reported previously by others(12, 31, 47) , catalase antagonized the growth inhibitory action of VK(3), suggesting that hydrogen peroxide may be important for mediating the toxicity of this compound. However, catalase did not antagonize the effects of growth inhibitory analogs, although it exerted only a minimal effect on an O-ether analog (6), indicating that the growth inhibitory effect of these compounds is not due to the generation of hydrogen peroxide. Deferoxamine mesylate, an inhibitor of hydroxyl radical generation, completely blocked the growth inhibition of tBHP as reported previously(48) . However, it did not antagonize the effects of the growth inhibitory thioether analogs, although it partially antagonized VK(3), 1 (all-carbon), and 6 (O-ether). This suggests that hydroxyl radical generation is not the major cause of their growth inhibitory effects. Superoxide dismutase, which catalyzes the conversion of superoxide anion to hydrogen peroxide, did not antagonize the growth inhibitory effects of any of the compounds. Furthermore, low molecular weight non-thiol antioxidants (L-ascorbic acid, butylated hydroxyanisole, and nordihydroguaiaretic acid) had no antagonistic effects. These results suggest that the growth inhibition of these VK-related compounds are not mainly due to generation of reactive oxygen species.

Direct arylation of cellular thiols has been investigated as another important mechanism of quinone toxicity(32, 33, 34, 35, 36) . This has been shown to be the mechanism in the toxicity of benzoquinone derivatives, which lack redox cycling activity(49) , using rat hepatocytes(33) . This is consistent with our findings that the growth inhibitory action of pBQ was not antagonized by the non-thiol antioxidants but was significantly antagonized by thiols. In the present study, co-incubation with GSH completely abrogated the growth inhibitory action of VK(3) and most analogs, in particular the potent thioether analogs. Furthermore, both isomers of cysteine and NAC, which were ineffective on the growth inhibition by the oxidant tBHP, had a strong antagonistic effect on VK(3) and the thioether analogs. Similar protection of VK(3) action by exogenous thiols has been reported previously(15, 50) . These data suggest that the main reason for toxicity of our compounds is probably due to adduct formation with cellular thiols by arylation, rather than oxidative stress. VK(3) and its derivatives have been demonstrated to form adducts with thiols at position 3 of their naphthoquinone nucleus(32, 34) . A simple addition mechanism is involved in the interaction of VK(3) and thiols, whereas an addition-elimination mechanism is likely to be involved in the interaction of the thioether derivatives and thiols (36) .

As cells contain physiologically important thiols in the forms of reduced glutathione and sulfhydryl residues in many proteins, direct arylation of cellular thiols will impede cell function and may cause cell death. VK(3) has been shown to induce suppression of protein-tyrosine phosphatase and a hyperphosphorylation state of p34 kinase in a hepatoma cell line(16) . Because protein-tyrosine phosphatase contains a cysteine in the active site (51) , VK(3) and its thioether derivatives may inactivate protein-tyrosine phosphatase by adduct formation(36) . Talcott et al.(52) suggested that sulfhydryl-reactive derivatives of VK(3) may inactivate thiol-containing microsomal NADPH-cytochrome c reductase. Similarly, Lee et al.(53) showed that a polyketide and its derivatives inhibited pp60 protein tyrosine kinase, probably by direct arylation.

Our study also showed that VK-related quinoid compounds induced both apoptotic and necrotic cell death. The frequency of apoptosis was largely parallel to the potency of growth inhibition, although ghost-like necrotic cell death became predominant when the more potent compounds were used at higher concentrations. This observation is consistent with recent reports (54, 55) showing that apoptosis is induced at low levels of noxious stimuli, whereas necrosis occurs at higher levels of the same stimuli. In addition, our findings suggest that quinoid compounds may induce apoptosis not only by oxidative stress (20) but also by direct arylation of cellular thiols causing depletion of glutathione and inactivation of sulfhydryl-dependent proteins. Recent evidence has also shown that reactive oxygen species are not always required for apoptosis(56, 57) . Overexpression of the c-myc gene, which has been reported to be associated with apoptosis(14, 21, 22, 23) , was induced by almost all of our compounds in a dose-dependent manner. However, no direct correlation could be discerned between the potency of growth inhibition and the degree of c-myc induction, consistent with other reports(58, 59) .

In summary, several novel VK compounds exhibited structural requirements for growth inhibitory and cell killing activities. We also demonstrated the importance of direct interaction between the compounds and cellular thiols. Thioether derivatives, such as 2, 3, and 5, were found to be as potent as the benchmark VK(3) and may be useful in future testing in animal studies for possible effects on tumor growth inhibition.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant CA 57371 (to B. I. C.) and NHLBI Grant HL 50667 (to P. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Pittsburgh Transplantation Inst., Dept. of Surgery, University of Pittsburgh, BST E1552, 200 Lothrop St., Pittsburgh, PA 15213. Tel.: 412-624-6684; Fax: 412-624-1172.

(^1)
The abbreviations used are: VK, vitamin K; VK(1), phylloquinone; VK(2), menaquinone series; VK(3), menadione; pBQ, p-benzoquinone; tBHP, tert-butyl hydroperoxide; NAC, N-acetyl-L-cysteine; GSH, glutathione.


ACKNOWLEDGEMENTS

We gratefully acknowledge the general support and help of Dr. Ziqiu Wang (Department of Surgery, University of Pittsburgh).


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