(Received for publication, July 19, 1995; and in revised form, August 31, 1995)
From the
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. 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
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.
Vitamin K (VK) ()is a generic term for compounds that
include phylloquinone (VK
), menaquinone series
(VK
), and menadione (VK
) (1) .
Physiologically, the natural K vitamins, VK
and
VK
, 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 has a long phytol side chain, whereas VK
has
an unsaturated side chain composed of 4-13 isoprene units.
VK
lacks the side chain. VK
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
-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
and VK
also have been shown to have cell growth
inhibitory effects in vitro, but these effects are much weaker
than those of VK
(11, 24) . VK
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
(26) , and a recent phase I/II trial is in
progress for VK
in human hepatoma(27) .
Figure 1:
Chemical structures of K vitamins and
analogs used in this study. K, vitamin K
(phylloquinone, 2-methyl-3-phythyl-1,4-naphthoquinone); K
, vitamin K
(menaquinone 4); K
, vitamin K
(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, 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
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.
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 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 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 and 9 (Fig. 4). However, some of
the more potent growth inhibitory structures such as VK
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.
Figure 5:
Effect of reduced glutathione and L-cysteine on growth inhibition of Hep3B cells by VK 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 and the quinoid analogs. Catalase antagonized the effect of
VK
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
, 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 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).
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 C;
, vitamin K
;
, vitamin K
;
,
vitamin K
;
, 3;
,
4.
K vitamins, especially VK, 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
to VK
(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, 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
, 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 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
and the
thioether analogs. Similar protection of VK
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
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
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 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
and its thioether derivatives may
inactivate protein-tyrosine phosphatase by adduct
formation(36) . Talcott et al.(52) suggested
that sulfhydryl-reactive derivatives of VK
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 and may be useful in future testing in animal studies for
possible effects on tumor growth inhibition.