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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Among the vitamin K (VK) compounds, VK3 exhibits distinct cytotoxic activity in cancer cells and is thought to affect redox cycling; however, the underlying mechanisms remain unclear. Here we demonstrate that VK3 selectively inhibits DNA polymerase (pol) γ, the key enzyme responsible for mitochondrial DNA replication and repair. VK3 at 30 µM inhibited pol γ by more than 80%, caused impairment of mitochondrial DNA replication and repair, and induced a significant increase in reactive oxygen species (ROS), leading to apoptosis. At a lower concentration (3 µM), VK3 did not cause a significant increase in ROS, but was able to effectively inhibit cell proliferation, which could be reversed by supplementing glycolytic substrates. The cytotoxic action of VK3 was independent of p53 tumor suppressor gene status. Interestingly, VK3 only inhibited pol γ but did not affect other pol including human pol α, pol β, pol δ, and pol ɛ. VK1 and VK2 exhibited no inhibitory effect on any of the pol tested. These data together suggest that the inhibition of pol γ by VK3 is relatively specific, and that this compound seems to exert its anticancer activity by two possible mechanisms in a concentration-dependent manner: (1) induction of ROS-mediated cell death at high concentrations; and (2) inhibition of cell proliferation at lower concentrations likely through the suppression of mitochondrial respiratory function. These findings may explain various cytotoxic actions induced by VK3, and may pave the way for the further use of VK3. (Cancer Sci 2008; 99: 1040–1048)

Abbreviations:
c-DCF

5-carboxy-2′,7′-dichlorodihydrofluorescein diacetate

DMSO

dimethyl sulfoxide

EDTA

ethylenediaminetetraacetic acid

FITC

fluorescein isothiocyanate

HAART

highly active antiretroviral therapy

HE

dihydroethidine

His

histidine

HIV

human immunodeficiency virus

IC50

50% inhibitory concentration

mtDNA

mitochondrial DNA

MTT

3-(4,5-dimethyl thiazol-2)-2,5-diphenyltetrazolium bromide

NAC

N-acetylcysteine

nDNA

nuclear DNA

PBS

phosphate-buffered saline

pol

DNA polymerase

ROS

reactive oxygen species

SOD

superoxide dismutase

VK

vitamin K

Vitamin K is a family of fat-soluble compounds that includes phylloquinone (VK1), menaquinone (VK2), and menadione (VK3). The best-known member of the VK family is phylloquinone, which is found in many higher plants and algae, with the highest concentrations in green leafy vegetables. Menaquinones are produced by bacteria and menadione is a synthetic analog that acts as a provitamin (turns into a vitamin in the body). VK has two important characteristics: it is a critical factor in blood coagulation (clotting) and it inhibits cancerous cell growth.(1)

Although the antitumor effects of VK compounds have been under investigation since 1947(2) it is unfortunate that more progress has not been made. The reason may be that the proposed theories were unconvincing to explain the difference in antitumor actions between VK1, VK2, and VK3. The most popular theory is that VK compounds cause oxidative damage to malignant tumor cells, and other theories include the induction of cell death and inhibition of the cell cycle of the malignant cell so that it cannot continue growing.(1) In general, although VK3 has a higher cytotoxic effect than VK1 and VK2, it is still uncertain how VK3 induces higher cytotoxicity in malignant cells.

DNA polymerases are indispensable for maintaining the integrity of the genome, both through faithful replication of DNA and by repairing damage to DNA. Among the 16 highly specialized mammalian polymerases, 15 are involved in maintaining nuclear genetic information. Replication and maintenance of the mitochondrial genome relies on a unique polymerase, pol g.(3) Pol g is responsible for all aspects of mtDNA synthesis, including all replication, recombination of the mitochondrial genome, and repair of mtDNA damage. It has been reported that mtDNA mutations or impairments of mtDNA-coded protein syntheses may lead to superoxide production,(4–6) and the superoxide itself is the major cause of both nDNA and mtDNA damage. Thus, it remains a matter of investigation whether pol g plays a crucial role in the process of repair after mtDNA damage, or whether inhibition of pol g leads to antitumor effects.

Here, we show novel mechanisms for the cytotoxic actions of VK3, that it selectively inhibits pol g activities in various human cancer cells. Superoxide production and cytotoxicity showed two different effects according to the concentrations of VK3. These findings may integrate several reported cytotoxic actions of VK3 and pave the way for the further use of VK3.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Cell lines and chemicals.  HCT116 colon carcinoma cell lines with wild-type p53 (HCT116 p53+/+) and their isogenic derivatives that lack p53 (HCT116 p53−/–) were kindly gifted by Dr Bert Vogelstein (Johns Hopkins University, Baltimore). The cells were maintained in McCoy's 5A medium containing 10% fetal bovine serum (normal medium), or in McCoy's 5A-based enriched medium in several experiments, adding 10% fetal bovine serum, 2 mM sodium pyruvate, and 50 µg/mL uridine to the normal medium at 37°C with 5% CO2. The immortalized human bronchial epithelial cells (BEAS-2B) described previously(7) were cultured in supplemented Keratinocyte-SFM medium (Invitrogen, Tokyo, Japan). Immortalized human B cells (PSC B cells) were obtained from the Health Science Research Resources Bank (Osaka, Japan) and were cultured in RPMI-1640 medium.(8) Other cells, such as HCT15, SW620, H1299, A549, MCF-7, HeLa, HepG2, PANC-1, LNCaP, PC3, DU145, Raji, HL60, and MRC-5, were obtained from American Type Culture Collection (ATCC) and cultured in RPMI-1640 medium. VK compounds (VK1, VK2, and VK3), rotenone, antimycin A, and NAC were purchased from Sigma (St Louis, MO, USA). Nucleotides and chemically synthesized DNA template primers such as poly(dA), poly(rA), and oligo(dT)12–18, and [3H]-dTTP (1.6 TBq/mmol) were purchased from GE Healthcare Biosciences (Buckinghamshire, UK). HE and c-DCF and Mitotracker green were obtained from Molecular Probes (Eugene, OR, USA). All other reagents were of analytical grade and purchased from Nakalai Tesque (Kyoto, Japan).

Enzymes.  Pol α was purified from calf thymus by immunoaffinity column chromatography as described by Tamai et al.(9) Recombinant rat pol β was purified from Escherichia coli JMpβ5 as described by Date et al.(10) The human pol γ catalytic gene was cloned into pFastBac. Histidine-tagged enzyme was expressed and purified as described by Umeda et al.(11) Human pol δ and pol ɛ were purified by the nuclear fractionation of human peripheral blood cancer cells (Molt-4) using the second subunit of pol δ- and pol ɛ-conjugated affinity column chromatography, respectively.(12) Recombinant human pol δ and pol ɛ tagged with His6 at their C-termini were expressed in SF9 insect cells using the baculovirus expression system, and were purified as described previously.(13,14) A truncated form of pol κ (i.e. hDINB1DC) with His6 attached at the C-terminus was overproduced using the BAC-to-BAC Baculovirus Expression System kit (Gibco, Tokyo, Japan) and purified as described previously.(15) Recombinant human His-pol λ was overexpressed and purified according to a method described previously.(16)

DNA polymerase assays.  The reaction mixtures for pol α, pol β, plant pol, and prokaryotic pol were described previously.(17,18) Those for pol γ, and pol δ and ɛ were as described by Umeda et al.(11) and Ogawa et al.,(19) respectively. The reaction mixtures for pol δ, ɛ, and κ were the same as that for pol α, and the reaction mixture for pol λ was the same as that for pol β. For pol (i.e. DNA-dependent pol), poly(dA)/oligo(dT)12–18 (A/T = 2/1) and dTTP were used as the DNA template primer and nucleotide (i.e. dNTP) substrate, respectively. For RNA-dependent pol γ, poly(rA)/oligo(dT)12–18 (A/T = 2/1) and dTTP were used as the template primer and nucleotide substrate, respectively.

Vitamin K compounds were dissolved in distilled DMSO. Aliquots (4 µL) of sonicated samples were mixed with 16 µL of each enzyme (final amount 0.05 units) in 50 mM Tris-HCl (pH 7.5) containing 1 mM dithiothreitol, 50% glycerol, and 0.1 mM EDTA, and kept at 0°C for 10 min. These inhibitor–enzyme mixtures (8 µL) were added to 16 µL of each of the enzyme standard reaction mixtures, and incubation was carried out at 37°C for 60 min. Activity without the inhibitor was considered 100%, and the remaining activity at each concentration of the inhibitor was determined relative to this value. One unit of pol activity was defined as the amount of enzyme that catalyzed the incorporation of 1 nmol of dNTP (i.e. dTTP) into synthetic DNA template primers in 60 min at 37°C under the normal reaction conditions for each enzyme.(17,18)

Isolation of mitochondria.  Mitochondria were isolated from control and treated cells as described previously.(20) Cells were then harvested and resuspended in three volumes of isolation buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 70 mM sucrose, 210 mM mannitol, and protease inhibitors). After incubating in an ice bath for 15 min, the cell suspension was homogenized with 15 strokes.

Measurement of pol γ inhibitory activity in the mitochondrial extract from human cancer cells.  Both HCT116 p53+/+ and HCT116 p53−/– cells were treated with 0, 3, and 30 µM of VK3 for 24 h and then stored at –80°C. Using the isolated mitochondrial fraction, the pol γ activity of 2 µg of the extract from the mitochondrial fraction was assayed as described previously.(17,18)

Evaluation of mitochondrial mass in each cell.  To investigate whether VK3 has a destructive effect on mitochondria, cells were stained with Mitotracker green, and mitochondrial mass volumes per cell were measured with a flow cytometer as described previously.(21)

Analyses of cellular superoxide and hydrogen peroxide.  Cellular superoxide and hydrogen peroxide were measured by flow cytometer analyses using HE and c-DCF.(4) HE was dissolved in DMSO (100 mg/mL stock) and further diluted with PBS at 1:10 000. The diluted dye was added to the cell culture at a final concentration of 50 ng/mL and incubated at 37°C. During the last 60 min c-DCF was also dissolved in DMSO (20 mM stock) and used for staining in 50 µM at 37°C for 60 min.

Western blot analysis.  Protein lysates were prepared from the control and VK3-treated cells, and separated by 8–15% sodium dodecylsulfate–polyacrylamide gel electrophoresis. The amount of pol γ in the mitochondrial fraction was evaluated using its polyclonal antibodies (Ab-3; NeoMarkers, Fremont, CA, USA). Antibodies for SOD1 (Calbiochem, Tokyo, Japan), SOD2 (Calbiochem), and β-actin (Sigma) were used.(22) Antibodies for cytochrome c (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and cytochrome oxidase subunit IV (Molecular Probes) were purchased for the evaluation of mitochondria-specific proteins. The mitochondrial Hsp60 protein (antibody N-20; Santa Cruz Biotechnology) was blotted to ensure equal loading of mitochondrial protein.(10) Those primary antibodies were diluted at 1:1000–1:5000 and detected using appropriate horseradish peroxidase-conjugated secondary antibodies, followed by detection with a SuperSignal enhanced chemiluminescence kit (Pierce, Rockford, IL, USA).

Cytotoxicity (MTT) assay and long-term survival (colony-forming) assay.  Cytotoxicity was determined by a MTT assay (72 h) as described previously.(4) For a longer-term survival analysis, a colony-forming assay was carried out. VK compounds were added about 18 h (overnight) after seeding, and cells were incubated for 10–14 days. Fixation and staining were according to previously described methods.(23,24)

Mitochondrial DNA fragmentation analysis.  MtDNA fragmentation was evaluated by the following method. The mitochondrial pellets were digested in buffer containing 10 mM Tris-HCl, pH 7.8, 10 mM NaCl, 25 mM EDTA, 0.5% sodium dodecyl sulfate, and 0.1 mg/mL proteinase K for 30 min. For analysis, 10 µg of purified mtDNA was loaded onto a 2% agarose gel containing 10 µg/mL ethidium bromide and electrophoresed with DNA molecular weight markers (λ-HindIII, and φX-174 HaeIII) (Takara, Tokyo, Japan) in 0.5× Tris borate–EDTA buffer (18 mM Tris-HCl pH 8.0, 18 mM boric acid, and 1 mM EDTA) at 100 V for 1 h. After treatment with RNase A (final concentration, 100 µg/mL) overnight, mtDNA bands were visualized under ultraviolet light and photographed.

Sequencing of mtDNA.  Direct mitochondrial sequencing of multiple regions (ND1, ND4, and cytochrome b) was carried out according to previously described methods.(6) Briefly, primer sequences were as follows: 5′-AACATACCCATGGCCAACCT-3′ sense and 5′-GGCAGGAGTAATCAGAGGTG-3′ antisense for NDI (#3304–3836), 5′-GACTCCCTAAAGCCCATGTCG-3′ sense and 5′-TTGATCAGGAGAACGTGGTTAC-3′ antisense for ND4 (#11403–11927), and 5′-AGTCCCACCCTCACACG ATTC-3′ sense and 5′-ACTGGTTGTCCTCCGATTCAGG-3′ for cytochrome b (#15260–15774). The annealing temperatures were 55°C for NDI, 58°C for ND4, and 58°C for cytochrome b. The sequencing chromatograms were further evaluated manually, excluding the unstable first 20–30 portions. Complete replacement of nucleotides was defined as a point mutation. Any nucleotide position with two or more significant peaks of mixed nucleotide signals (heteroplasmy) was estimated for the percentage of each nucleotide, based on the area under the curve of the corresponding nucleotide peak. Cases of heteroplasmy accounting for less than 30% of the total base signal were considered insignificant and were not scored. Only those with greater than 30% heteroplasmic signal were counted.

Assessment of apoptosis by annexin-V and propidium iodide staining.  Cells were stained with annexin-V–FITC for exposure of phosphatidylserine on the cell surface as an indicator of apoptosis, following the manufacturer's instructions (BD Biosciences, Tokyo, Japan).(25)

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Effects of VK compounds on pol activity.  First, inhibition of the activities of mammalian pol by VK was investigated. Of the three VK compounds tested, 30 µM VK3 showed pol γ inhibitory activity, but the same concentration of VK1 and VK2 did not influence the activity of all pol tested, including pol γ. VK3 selectively inhibited pol γ activity, and did not suppress the activities of other mammalian pol such as pol α, β, δ, ɛ, η, ι, κ, and λ (Table 1). Pol γ is the sole pol in animal mitochondria. Biochemical and genetic evidence document a key role for pol γ in mtDNA replication. Pol γ has not only DNA-dependent polymerase activity but also RNA-dependent polymerase activity;(26) therefore, the influence of VK3 on the activities of both polymerases was investigated. The inhibitory activity of VK3 on RNA-dependent pol γ was as strong as that on DNA-dependent pol γ, with 50% inhibition observed at doses of 6.8 and 6.0 µM, respectively (Table 1). When activated DNA (i.e. DNA digested by bovine DNaseI) was used as the template primer instead of a synthesized DNA template primer such as poly(dA)/oligo(dT)12–18, the mode of inhibition by these compounds did not change (data not shown). These results suggest that the longer isoprenoid chains of the benzquinone ring in VK weaken the inhibitory effect on pol γ. VK compounds had no inhibitory effect on fish (cherry salmon, Oncorhynchus masou) pol α and δ, insect (fruit fly, Drosophila melanogaster) pol α, δ, and ɛ, plant (cauliflower inflorescence) pol I (α-like), pol II (β-like), and pol λ, or prokaryotic pol such as the Klenow fragment of E. coli pol I, Taq pol and T4 pol (data not shown).

Table 1. IC50 values of vitamin K (VK) compounds for the activities of various DNA polymerases and other DNA metabolic enzymes
EnzymeIC50 values of VK (µM)
VK1VK2VK3
  1. These compounds were incubated with each enzyme (0.05 units). Enzymatic activity was measured as described in the text. Enzymatic activity in the absence of compounds was taken as 100%, IC50, 50% inhibitory concentration.

Calf DNA polymerase α>200>200>200
Rat DNA polymerase β>200>200>200
Human DNA polymerase γ
  DNA-dependent polymerase activity>200>2006.0
  RNA-dependent polymerase activity>200>2006.8
Human DNA polymerase δ>200>200>200
Human DNA polymerase ∈>200>200>200
Human DNA polymerase η>200>200>200
Human DNA polymerase ι>200>200>200
Human DNA polymerase κ>200>200>200
Human DNA polymerase λ>200>200>200

Inhibitory effect of VK compounds on the activities of other DNA metabolic enzymes.  VK compounds did not inhibit the activities of other DNA metabolic enzymes such as calf primase of pol α, calf terminal deoxynucleotidyltransferase, HIV-1 reverse transcriptase, T7 RNA polymerase, T4 polynucleotide kinase, and bovine deoxyribonuclease I (data not shown). These results suggest that VK3 selectivity inhibited the activity of mitochondrial γ.

Effects of VK compounds on pol  inhibitory activity and growth inhibition in human cancer cells.  Pol activity in the mitochondrial extract of VK compound-treated cells was investigated (Fig. 1a). The pol in the mitochondria fraction was pol γ only, and 2 µg of the extract of human colon cells, HCT116, with or without the p53 gene, had approximately 0.11 and 0.12 units of pol γ activity, respectively. The pol γ activity of VK3-treated cells was significantly lower than that of non-treated cells, and enzyme activity was decreased 26.8–28.8% with 3 µM and 70.6–70.8% with 30 µM. The inhibitory effect of VK3 seemed to be independent of p53. In contrast, VK1- and VK2-treated cells had almost no influence. To investigate the underlying mechanisms of action of VK3, mitochondrial volume was evaluated by flow cytometry using Mitotracker green. As shown in Figure 1b, the mitochondrial mass of each cell first enlarged and then decreased within 8 h of exposure to 30 µM VK3, whereas the mitochondrial mass did not changed with 3 µM VK3. These results indicate that VK3 induced destructive mitochondria-targeted damage with the 30 µM dose. This phenomenon was further supported by the quantitative changes of pol γ. As shown in Figure 1c, using a dose of 30 µM VK3, the amount of pol γ decreased in a time-dependent manner, whereas it did not change using lesser doses (3 or 10 µM) of VK3. Therefore, 30 µM VK3 clearly decreased or damaged pol γ in the mitochondria. Moreover, with the treatment of 30 µM VK3, mitochondria-specific proteins such as SOD2, cytochrome c, and cytochrome oxidase subunit IV, decreased within 6 h. In contrast, SOD1 in the cytoplasm seemed to be unchanged. Cytochrome c seemed to be released from the mitochondrion to the cytoplasm within 3 h and then induced apoptosis. Together, these data show that treatment with 30 µM VK3 induced certain damage to the mitochondria, but not to the cytoplasm. (Fig. 1d).

image

Figure 1. (a) Total DNA polymerase (pol) activity of the extract of mitochondria fraction from HCT116 (p53+/+ and p53−/–) cells incubated with or without vitamin K (VK) compounds for 24 h. One unit of pol activity is defined as the amount that catalyzes the incorporation of 1 nmol of dexyribonucleoside triphosphates (i.e. dTTP) into synthetic template primers (i.e. poly(dA)/oligo(dT)12–18, A/T = 2/1) at 37°C in 60 min. Enzymatic activity in the absence of compound was taken as 100%. Data are shown as the mean ± SEM for three independent experiments. (b) Sequential mitochondrial mass changes after VK3 treatment. Each bar indicates the median size of mitochondrial mass out of 10 000 cells stained with Mitotracker green. The mitochondrial mass was higher with a higher dose (30 µM) of VK3 and then decreased within 8 h, whereas it did not change with a lesser dose (3 µM) of VK3. (c) Western blots analysis of pol γ after 0, 3, and 6 h incubation with 3, 10, and 30 µM VK3. With a 30-µM dose of VK3, the amount of pol γ decreased in a time-dependent manner, whereas it did not change with lesser doses (3 or 10 µM) of VK3. The Hsp60 band showed equal amounts of mitochondrial protein loaded. (d) Western blot analyses of mitochondria-specific proteins with 30 µM VK3. Mitochondria-specific proteins such as superoxide dismutase (SOD) 2, cytochrome c, and cytochrome oxidase subunit IV (COX IV), decreased within 6 h. In contrast, SOD1 in the cytoplasm seemed to be unchanged. Cytochrome c seemed to be released from the mitochodria to the cytoplasm within 6 h.

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As shown in Figure 2, VK3 at a dose of 3 µM had a potent growth-inhibitory effect on HCT116 p53+/+ and p53−/– cells in a colony-forming assay, whereas VK1 and VK2 did not prevent cell growth at the same concentration. To clarify whether the inhibitory effect of VK3 might be observed in other types of tumor, the cytotoxic effects caused by VK compounds were investigated using MTT assays (Table 2). Interestingly, VK3 was the strongest inhibitor of various human cancer cells and the concentration required for IC50 ranged from 6.0 to 12 µM (median 7 µM), whereas VK3 showed lesser inhibitory effects in human normal cells (ranging from 10 to 26 µM, median 13 µM). Because the IC50 values of VK3 were 6.0 µM for human pol γ activities (Table 1), the IC50 values for cell survival obtained by both the colony-forming and MTT assays were consistent with the in vitro IC50 values for the enzyme (Table 1).

image

Figure 2. (a) Growth-inhibitory effect of vitamin K (VK) compounds (3 µM) in both HCT116 p53+/+ and p53−/– cells. Equal amounts of dimethyl sulfoxide with VK compounds were added as a control. (b) The number of colonies after 14 days’ incubation with VK compounds was counted.

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Table 2. IC50 values of the growth-inhibitory effect of vitamin K (VK) compounds evaluating by 3-(4,5-dimethyl thiazol-2)-2,5-diphenyltetrazolium bromide (MTT) assays in human cancer and normal cells
Cell typeTissue originIC50 (µM)
VK1VK2VK3
  1. IC50, 50% inhibitory concentration

HCT116 p53+/+Colon>100>100 6
HCT116 p53−/–Colon>100>100 6
HCT15Colon>100>10010
SW620Colon>10090 5
H1299Lung>1002810
A549Lung>1005512
MCF-7Breast>10025 8
HeLaUterus>100>100 6
HepG2Liver>100>100 8
PANC-1Pancreas>100>100 6
LNCaPProstate>10085 7
PC3Prostate>100>100 6
DU145Prostate>100>10010
RajiB cell lymphoma>100>100 8
HL60Leukemia>100>100 6
Median>100 100 (SD 7) 7 (SD 0.5)
BEASNormal bronchial>100>10010
epithelial cells   
PSC B cellNormal B cell>100>10013
MRC-5Normal fibroblast>100>10026
Median>100>10013 (SD 4.9)

Reactive oxygen species-generating effect of VK compounds.  To assess intracellular events in VK-treated cells, the amount of superoxide was first investigated. With 30 µM of VK3, the superoxide level increased more than 30-fold in p53+/+ cells and more than 40-fold in p53−/– cells in 24 h, whereas the same dose of VK1 and VK2 induced a minimum increase in superoxide generation (less than two-fold) (Fig. 3a). Sequential evaluation of 30 µM VK3 showed that superoxide generation was observed in a time-dependent manner in both p53+/+ and p53−/– cells (Fig. 3b). With 3 µM VK3 for 24 h incubation, the increase in superoxide was similar to those with rotenone and antimycin A (data not shown), whereas the results with 30 µM VK3 were quite different. Therefore, it is possible that the cytotoxic effect with 3 µM VK3 was due to redox cycling modulation without large amounts of superoxide generation. Next, the amounts of hydrogen peroxide were assessed. The amounts of hydrogen peroxide were slightly elevated by VK3 (three-fold in p53+/+ cells, and four-fold in p53−/– cells) for the same incubation time (Fig. 3c), but they were not increased by VK1 or VK2 in either of the cell types (data not shown). To test whether the mode of this free-radical generation was consistent with other types of tumors, the same experiment was carried out using several human cancer and normal cells (Table 3). As a result, superoxide increased 11- to 40-fold (median 20-fold) in cancer cells, whereas hydrogen peroxide did not increase in the same way (range 3- to 16-fold, median 10-fold). The tendency in normal cells was almost similar to those in cancer cells.

image

Figure 3. (a) Superoxide generation after exposure to vitamin K (VK) 1, VK2, and VK3 in both HCT116 p53+/+ and p53−/– cells. (b) Time-dependent increase in superoxide generation caused by 30 µM VK3. (c) Hydrogen peroxide generation after exposure to VK3 (3 or 30 µM) was evaluated.

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Table 3. Superoxide and hydrogen peroxide generation after exposure to vitamin K (VK) 3 in human cancer and normal cells
Cell typeTissue originSuperoxideHydrogen peroxide
(-fold, VK3 30 µM/control)
HCT116 p53+/+Colon24 3
HCT15Colon35 7
SW620Colon4016
H1299Lung1310
MCF-7Breast11 9
PANC-1Pancreas1311
PC3Prostate1412
RajiB cell lymphoma20 3
HL60Leukemia2512
Median20 (SD 3.5)10 (SD 1.3)
BEASNormal bronchial epithelial cells14 4
PSC B cellNormal B cell1913
Median17 (SD 2.5) 9 (SD 4.5)

Effect of mtDNA repair through the inhibition of pol γ by VK3.  The amount of superoxide generated by 30 µM VK3 seemed to be quite different from that generated by 3 µM VK3. As for the mechanism producing a large amount of superoxide, a possible theory has not been proposed previously. Therefore, we hypothesized that VK3 may suppress mtDNA replication or repair the activity of pol γ, and that suppression may lead to superoxide production, because pol γ enzymatic activity was dramatically decreased by 30 µM VK3 (Fig. 1a). To address this hypothesis, the status of mtDNA, especially the length of mtDNA, was first studied by direct electrophoresis of mtDNA, with the result that no fragmentation or deletion was seen in the treatment with VK3 (0, 3, or 30 µM for 24 h) (Fig. 4a). Next, the occurrence of point mutation or heteroplasmy was assessed by direct sequencing in several mtDNA-coded regions. As a result, the frequency of heteroplasmy greatly increased in multiple regions, consisting of a mitochondrial respiratory complex (Fig. 4b,c). However, point mutations were rarely observed (Fig. 4c), seemingly due to the experimental design being carried out within 24 h. These results together indicate that treatment with 30 µM VK3 decreased pol γ mtDNA repair activity, in which the impairment of mtDNA repair might lead to greater amounts of superoxide production.

image

Figure 4. (a) Mitochondrial DNA (mtDNA) fragmentation analysis by direct electrophoresis of mtDNA. Samples and DNA markers were electrophoresed after the indicated vitamin K (VK) 3 treatments of both HCT116 p53+/+ and p53−/– cells for 24 h (control, 3, and 30 µM). No fragmentations or deletions were observed under those conditions. (b) Example of direct sequencing analysis. In the NADH dehydrogenase 1 (ND1) portion, typical heteroplasmic changes were frequently observed in the sample treated with 30 µM VK3 for 24 h (arrows), compared to the control sample. (c) Frequencies of point mutation or heteroplasmy after 3 or 30 µM VK3 in HCT116 p53+/+ cells. Averaged numbers of mutation or heteroplasmy in three independent experiments are shown.

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Link of pol γ inhibition with VK3 and induction of apoptotic cell death through superoxide generation.  To clarify whether superoxide generation was the main factor in VK3-induced cytotoxicity, annexin-V and propidium iodide double-staining apoptosis assays were carried out. The proportion of apoptosis increased in a dose-dependent manner with VK3, and the induction of apoptosis was dramatically suppressed by the ROS scavenger NAC (Fig. 5a). Superoxide-mediated apoptosis caused by 30 µM VK3 occurred at 18 h, which may be independent of p53 (Fig. 5b).

image

Figure 5. (a) Vitamin K (VK) 3 induced reactive oxygen species-mediated apoptosis in HCT116 p53+/+ cells. Cells were incubated with various concentrations (1, 3 µM; 2, 10 µM; and 3, 30 µM) of VK3 alone, and with the antioxidant N-acetylcysteine (NAC) (4, 30 µM VK3 + 1 mM NAC) for 24 h at 37°C. (b) There was a time-dependent increase in apoptosis caused by 30 µM VK3 (control, 1, 4, 18, and 24 h) in HCT116 p53+/+ and p53−/– cells C, control.

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Cell survival by glycolytic pathway after VK3 cytotoxicity.  It has been widely reported that cell-depleted mtDNA could produce lower amounts of ATP by glycolysis and survive in a uridine- and sodium pyruvate-containing medium.(27) Those cells lack the ability to carry out oxidative phosphorylation because of defective mitochondrial respiratory function and were called ‘ρ0 cells’. We then hypothesized that if the cytotoxic mechanism of VK3 may be caused by mitochondrial respiratory dysfunction, cells could be rescued by glycolytic energy production in enriched medium. Our results showed that 3 µM VK3 had high cytotoxicity in both p53+/+ and p53−/– cells, and cells exposed to the same dose of VK3 were successfully rescued by enriched medium (Fig. 6a); however, cells could not survive when 30 µM VK3 was exposed in the same conditions (Fig. 6b). The difference clearly illustrates that VK3 has at least two different cytotoxic actions according to VK3 concentration. VK3, at a concentration of 3 µM, inhibits mitochondrial respiratory function and induces cytotoxicity in human cancer cells. Based on these observations, a model was proposed by illustrating a novel mechanism of VK3 for the inhibition of pol γ activity leading to superoxide generation and mitochondrial respiratory dysfunction (Fig. 7).

image

Figure 6. Cell growth inhibition by vitamin K (VK) 3 and cell proliferation by glycolysis. (a) The 3 µM of VK3 showed growth inhibition in both HCT116 p53+/+ and p53−/– cells; however, it was successfully recovered by adding glycolytic substrates. (b) In contrast, 30 µM VK3 also showed cell growth inhibition, which was not rescued under the same conditions.

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image

Figure 7. A schema of the mechanisms of vitamin K (VK) 3-induced growth inhibition and cytotoxicity in human cancer cells.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The major reason why VK3 has not been widely developed as an antitumor agent may be that the mechanism of toxicity in normal cells has not been clarified. However, it is important to note that most previous investigations have been concerned with cells such as hypatocytes, cardiomyocytes, and neurons, which contain greater amounts of mitochondria, and that cancer cells are also believed to upregulate their mitochondrial biogenesis and to contain larger amounts of mitochondria compared to their normal-cell counterparts.(21,28,29) As for the mechanisms of the cytotoxic effects of VK3, there are several interesting theories demonstrating that VK3 decreases intracellular thiols,(30) modifies intracellular Ca2+ homeostasis,(30) and causes cell death.(31) Interestingly, those findings were closely related to mitochondrial dysfunction, and they might be initiated in mitochondria. Thus, those reports suggested that VK3 penetrates the mitochondrial membrane and has several effects on the components of mitochondria. In the present report, we first identified the inhibitory effect of VK3 on pol γ, which resides in the mitochondrial matrix. As shown in Table 2, the IC50 values of the cancer cells tended to be less than those of normal cells; however, it was quite difficult to conclude whether those differences were significant or not. To clarify the difference, large numbers of normal cells might be evaluated. However, it is possible that there is a small difference in poly γ activity or amount between cancer and normal cells. As far as we investigated, there are no reports demonstrating that the cancerous pol γ is different from the normal one. On the contrary, our group previously reported that number of mitochondria in cancer cells is larger than in normal counterparts.(21) Because each mitochondrion contains mtDNA and pol γ, it could be speculated that the amount of pol γ in cancer cells and the effects of VK3 might be larger than in normal cells. These differences might explain the demonstrated results why VK3 has greater cytotoxic effect on cancer cells than normal cells. These findings also imply that VK3 penetrates both the inner and outer mitochondrial membranes; therefore, these findings may be a key to integrate several reported VK3 actions in the mitochondria, and lead to solving the biological effects of VK3.

Historically, the effectiveness of VK3 against cancer was believed to be due to oxidative stress via redox cycling of quinone to produce ROS, such as the superoxide radical and hydrogen peroxide.(32) From our results in both human cancer and normal cells, the major ROS induced by VK3 was superoxide. When a lesser dose of VK3 (3 µM) was applied, the increase in superoxide production was quite small and similar to the results caused by mitochondrial respiratory chain complex inhibitors. However, the mechanism of superoxide generation with a higher dose of VK3 (30 µM) seemed different. Notably, the two different actions were consistent with the magnitude of inhibition of pol γ activity in vitro and in vivo (Fig. 1a). It was shown that irreversibly greater amounts of superoxide production were caused by impairment of the synthesis of respiratory chain complex components. From the literature, when the coding regions of pol γ were mutated, it was reported that mtDNA might induce mutations in experimental models(33,34) and in clinical observation.(35) In our results, the majority of nucleotides changes were heteroplasmy, whereas the proportion of point mutations was quite small (Fig. 4c). As a single cell contains 100–1000 mitochondria and mitochondria carry 1–10 copies of the mitochondrial genome, it is reasonable that after short-term inhibition (24 h) of pol γ activity, errors of mtDNA repair might be induced to various extents in each mitochondrion. Those changes are mixed and might be detected as heteroplasmic change.

As far as we investigated, VK3 is the second compound with an inhibitory effect on pol γ activity. HAART against HIV, such as zidovudine (3′-azido-2′,3′-deoxythymidine) and other nucleoside analogs, was first reported as having an effect leading to mitochondrial dysfunctions, such as cardiac dysfunction, hepatic failure, skeletal myopathy, and lactic acidosis.(36) Notably, those side effects induced by HAART had quite similar characteristics, manifested by mitochondrial dysfunction. It is also reported that HAART is effective to AIDS-related malignancies.(37) In these points, VK3 and HAART have similar effects and characteristics. Martin and colleagues demonstrated mtDNA mutations in 5 of 26 patients undergoing nucleoside reverse transcription inhibitor (NRTI) therapy in which novel mtDNA sequence variations were found to arise within individuals over a relatively brief period (average treatment duration was 24 months).(38) In addition to these reports, although the chemical structure of VK3 is not similar to that of zidovudine, their actions were quite similar.

Nishikawa and colleagues energetically investigated the growth-inhibitory effects of their originally synthesized VK-related compounds.(39) From their reports, they demonstrated that the potency of growth-inhibitory action correlated with the decreasing length of the side chain. VK-related quinoid compounds with short side chains or without a side chain like VK3 seemed to have greater cytotoxicity against hepatoma cells. A phase I study using a VK3-related compound was conducted and the adverse effects seemed acceptable.(40) Based on these observations, VK3 and its related compounds have the possibility to be applied as antitumor agents. Further investigations are expected in addition to these findings.

In conclusion, novel mechanisms were identified in that VK3 selectively suppressed pol γ activities leading to two different cytotoxic actions that were concentration dependent. These findings may explain various cytotoxic actions by VK3, and may pave the way for the further use of VK3.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The Shinryokukai (Kobe Japan) (R. S.), Hyogo Science and Technology Association (Japan) (R. S.), the Japan Food Chemical Research Foundation (Japan), and Grant-in-aid 17790859, 17501269, and 17591268 for Scientific Research, MEXT (Ministry of Education, Culture, Sports, Science and Technology, Japan) (R. S. and Y. O.); Kobe-Gakuin University Joint Research (A), and ‘Academic Frontier’ Project for Private Universities: matching fund subsidy from MEXT (Japan), 2006-10 (H. Y. and Y. M.); and Grant-in-aid 19680031 for Young Scientists (A) MEXT (Japan), and the Nakashima Foundation (Japan) (Y. M.).

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  5. Discussion
  6. Acknowledgments
  7. References
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