Yonago Acta medica 2008;51:95–99

Vitamin K2 Has No Preventive Effect on Recurrence of
Hepatocellular Carcinoma after Effective Treatment
Keiko Hosho, Jun-ichi Okano, Masahiko Koda and Yoshikazu Murawaki
Division of Medicine and Clinical Science, Department of Multidisciplinary Internal Medicine,
School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8504 Japan

Hepatocellular carcinoma (HCC) has a poor prognosis because of its high recurrence
rate. Recently, vitamin K2 has been reported to inhibit the growth of HCC cell lines. To
clarify the preventive effect of vitamin K2 on HCC recurrence, we studied 72 HCC patients who had been treated with surgical resection, local ablation or transarterial embolization: their etiologies were hepatitis B virus (n = 21), hepatitis C virus (n = 47), both B
and C viruses (n = 2) and non-B or non-C virus (n = 2). We divided them into 2 groups:
in one group, patients were treated with 45-mg/day vitamin K2 [K2-treated group (n =23)], and in another, patients were not given vitamin K2 or a placebo [non-treated control group (n = 49)]. The obtained results between the 2 groups were compared. HCC
recurred in 12 (52.2%) of the 23 K2-treated patients, and 22 (44.9%) of the 49 control
patients. The differences in cumulative recurrence-free rate and cumulative survival
rate between both groups were not significant (P = 0.92 and P = 0.08, respectively). As
observed, chemopreventive effects of vitamin K2 at a clinically relevant dose on HCC recurrence were ineffective after effective treatment for HCC. Different regimens such as
higher doses of vitamin K2 or combination therapy with other drugs may be worth testing to further explore the preventive effect on HCC recurrence.
Key words: chemoprevention; hepatocellular carcinoma; vitamin K2

Hepatocellular carcinoma (HCC) is one of themost common malignancies worldwide. In Japan,
primary liver cancer, 95% of which is HCC, isranked 3rd in men and 5th in women as a causeof death from malignant neoplasm. Chronic viral hepatitis associated with hepatitis B virus andhepatitis C virus (HCV) is a major cause of HCC.
In spite of curative treatments, including surgicalresection and local ablation therapy, the recurrence
rate in the remnant liver is high (Araii et al., 2000).
Therefore, chemoprevention for the recurrence of
HCC after curative treatment is important.
For chemopreventive therapy in patientswith chronic HCV infection, interferon has been

administrated and promising results have beenobtained (Ikeda et al., 2000; Suou et al., 2001).
Acyclic retinoid has been shown to prevent therecurrence of HCC after surgical resection or local ablation therapy (Muto et al., 1996). Vitamin
K2 is known to inhibit the growth of a variety oftumor cells including hepatoma cells by inducing apoptosis or differentiation (Miyazaki et al.,2001). Recently, Mizuta et al. reported the benefitof vitamin K2 on the recurrence and survival inpatients with HCC after curative therapy (Mizuta
et al., 2006). To re-confirm their findings, we
conducted a similar study after local ablation
therapy or curative resection.

Abbreviations: CT, computed tomography; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; JIS, Japan Integrated
Staging; MRI, magnetic resonance imaging; PIVKA II, protein induced by vitamin K absence or antagonist

95

K. Hosho et al.

after curative treatment. Examinations were continued until the detection of recurrent HCC. Alfafetoprotein and PIVKA II were assayed monthly.
The present study conformed to the ethical guideline of the Declaration of Helsinki. Informed
consent was verbally obtained from all patients.

Materials and Methods
Patients and study protocol
The study included 72 patients with HCC who
were admitted to Tottori University Hospital between August 2002 and June 2006. Diagnosis
of HCC was confirmed by a combination of histopathological examination from biopsy specimens, serological examination and findings withdiagnostic imagings including contrast-enhanced
computed tomography (CT), magnetic resonanceimaging (MRI) and transhepatic arteriography.
All patients underwent curative treatment including surgical resection, percutaneous ethanol injection or percutaneous radiofrequency ablation
with or without transarterial chemoembolization.
After 1 month of treatment, curative treatment
was defined as no residual HCC visualized withany of the above described imagings. In total, 72
patients were enrolled in the present study. The
patients were assigned to a group treated withor another not treated with vitamin K2 (control
group) based on the judgments by their physician
in charge. Vitamin K2, Glakay (Eisai, Tokyo,
Japan), was given at a fixed dose of 45 mg/day
during the follow-up periods. Patients of thecontrol group were not given a placebo. Serumalfa-fetoprotein levels were measured with a commercially available enzyme immunoassay kit(Abbott Japan, Tokyo), and protein induced byvitamin K absence or antagonist (PIVKA II) wastested with an ELISA kit (Picolumi PIVKA II
kit, Sanko Junyaku, Tokyo). Liver functions were
estimated according to the Japan Integrated Staging (JIS) score (Kudo et al., 2003). The JIS scoreis a new score system that includes 2 previous
classifications, the TNM endorsed by the Union
Internationale Contre le Cancer and the ChildPugh classification (Henderson et al., 2003). The
1st endpoint was the time to recurrence of HCC,
which was diagnosed with imagings. The 2nd
endpoint was overall survival. Contrast-enhanced
CT scans or MRI was performed every 3 months

Statistical methods
Statistical analysis was performed by standard
methods. The cumulative incidence of HCC recurrence and deaths were plotted using the KaplanMeier method, and the statistical significance ofdifferences was determined using the log-rank test
(Cox regression analysis was used for univariate
and multivariate analyses). Differences with a Pvalue < 0.05 were considered significant. The data
are shown as the mean ± SD.

Results
Compliance with vitamin K2 in the K2-treatedgroup was good. No adverse effects were observed and no patients dropped out of the study.
Clinical characteristics are shown in Table 1.
Twenty-three patients were enrolled in the K2treated group, and 49 patients were in the nontreated control group. The number of patientstreated with surgical resection, local ablation
therapy and transcatheter arterial chemoembolization was 2, 7 and 14 in the K2-treated group,and 7, 18 and 24 in the control group, respectively.
Differences in age, gender, observation periods,
etiologies and HCC-related findings were not significant between groups. The JIS score showed
no difference between groups. The mean AFPlevel was 382.6 ± 22.4 ng/dL in the K2-treatedgroup and 2267.5 ± 1747.8 ng/dL in the control
group. The mean PIVKA II level was 1182.9 ±960.9 mAU/mL, and 2421.3 ± 1413.4 mAU/mL,
respectively. The mean follow-up period was 23.8
± 14.1 months (range, 0.5–45.6 months) for theK2-treated group, and 26.9 ± 10.9 months (range,
3.3–43.7 months) for the control group.
96Vitamin K2 and hepatocellular carcinoma

Table 1. Characterstics of the patients enrolled in the present study
		
		
		

Vitamin K2treated group[23]

Non-treatedcontrol group[49]

P

Age 		
(year)
67 ± 969 ± 9Sex		
(male/female)
11/12
27/22
Observation period after HCC therapy(month)23.8 ± 14.1
26.9 ± 10.9
Etiology									Hepatitis virus B		
615
		
Hepatitis virus C		
14
33
		Hepatitis viruses B and C		20		
Non-hepatitis virus B or C		
1
1
JIS score					
		
0		
1
8
		
1		
10
18
		
2		
9
14
		
3		
3
9
Characteristics of HCC		Maximum diameter of tumor(cm)
2.3 ± 0.9
2.8 ± 1.7
		
Number of nodules 		
1.3 ± 0.6
1.4 ± 0.9

NS
NS
NS
NS

NS

NS
NS

[ ], number of subjects.
HCC, hepatocellular carcinoma; mo, months; JIS, Japan Integrated Staging; NS, not significant.

The mean time to tumor recurrence was 16.3
± 10.1 months in the K2-treated group and 10.4
± 6.6 months in the control group, respectively.
During the follow-up period, recurrent HCC
was observed in 22 patients in the control groupand in 12 patients in the K2-treated group. The
cumulative recurrence-free rates were 73.9% at
12 months, 67.2% at 24 months and 21.9% at 36
months in the K2-treated group, and were 50.6%,a38.3% and 32.4% in the control group, respectively (Fig. 1a). The difference in cumulative recurrence-free rate was not significant between the 2groups (log-rank test; P = 0.92). In HCV patients,
the cumulative recurrence-free rates were 77.4%
at 12 months, 46.4% at 24 months and 31.0% at
36 months in the K2-treated group, and were
47.9%, 40.5% and 36.0% in the control group, respectively (log-rank test; P = 0.58, Fig. 1b).

b

0
0.8
0.6
0.4

P = 0.92

0.2
0.0
0

12

1.0

Recurrence free rate

Recurrence free rate

1.0

24

36

(month)

0.8
0.6
0.4

P = 0.58

0.2
0.0
0

12

24

36

(month)Fig. 1. Cumulative recurrence-free rates in patients.
a: HCC patients after curative treatment:, K2-treated group (n = 23); , control group (n = 49).
b: HCV-positive HCC patients after curative treatment:, K2-treated group (n = 15); , control group (n = 33).
Differences between groups are not significant both in a and b. HCC, hepatocellular carcinoma; HCV, hepatitis C virus.

97

K. Hosho et al.

a

b

1.0
0.8
0.6
0.4

1.0

Survival rate

Survival rate0

P = 0.08

0.2
0.0

0.8
0.6

P = 0.10

0.4
0.2
0.0

0

12

24

36

48 (month)

0

12

24

36

48 (month)Fig. 2. Cumulative survival rates in patients.
a: HCC patients after curative treatment:, K2-treated group (n = 23); , control group (n = 49).
b: HCV-positive HCC patients after curative treatment:, K2-treated group (n = 15); , control group (n = 33).
Differences between groups are not significant both in a and b. HCC, hepatocellular carcinoma; HCV, hepatitis C virus.

synthetic form of vitamin K3 (menadione). This
vitamin K family has tumor inhibition properties
in vitro (Lamson et al., 2003). Recently, it has
been shown that vitamin K, specifically vitamin
K2, may have antiproliferative effects toward avariety of cancers including HCC (Nishikawa
et al., 1995; Otsuka et al., 2004), and that it may
cause differentiation in various human myeloid
leukemia cell lines (Miyazawa et al., 2001), although the mechanism of cell growth inhibitory
effects has not been explained. Mastumoto et al.
reported that vitamin K2 inhibits the proliferationof HCC cells in vitro through a cell cycle arrestat the G1 phase and apoptosis (Mastumoto et al.,2006).
Clinically, vitamin K2 treatment has been
widely used for the treatment of osteoporosis inJapan. Recently, Habu et al. revealed that vitamin
K2 exerted a suppressive effect against the development of HCC in women with HCV cirrhosis,
who took vitamin K2 for osteoporosis (Habu etal., 2004). Furthermore, Mizuta et al. reported
that vitamin K2 treatment significantly decreased
the recurrence rate of HCC and achieved a significant improvement in the overall survival rate
(Mizuta et al., 2006). In the present study, we
did not find preventive effects of vitamin K2 on
HCC recurrence or improvement in the survival
rate after curative treatment. Although our study
was not double-blinded or controlled, there wereDuring the follow-up period, 2 K2-treatedpatients and 16 control patients died. The 2 K2treated patients and 12 of the 16 control patientsdied of cancer progression: among the rest 4 control patients, 1 died of hepatic failure and 3 died of
non-hepatic disease. The cumulative survival rates
for the K2-treated group were 100% at 12 months,
93.3% at 24 months and 85.6% at 36 months. The
corresponding rates for the control group were
93.3%, 79.1% and 56.5%, respectively. The difference in survival rate was not significant between
both groups (log-rank test; P = 0.08, Fig. 2a). The
cumulative survival rates of HCV patients were100% at 12 months, 100% at 24 months and 87.5%
at 36 months in the K2-treated group; and 93.3%,
72.6% and 56.9% in the control group, respectively (log-rank test; P = 0.10, Fig. 2b). Vitamin
K2 treatment immediately decreased the PIVKA
II level, which was kept within normal ranges (datanot shown).

Discussion
Vitamin K is a fat-soluble essential vitamin that
activates blood coagulation factors by converting glutamic residues to -carboxy glutaminic
residues. The vitamin K family of molecules
comprises the natural forms vitamin K1 (phylloquinone) and vitamin K2 (menaquinones) and the98Vitamin K2 and hepatocellular carcinoma

no significant differences in age, gender, etiology, observation period and characteristics of
HCC between groups with and without vitamin
K2 treatment. In addition, the patient number,tumor characteristics and etiology were not different from the previous study (Mizuta et al., 2006).
More recently, Hotta et al. also reported that vitamin K2 appeared not to be able to inhibit or delay
the recurrence of HCC after curative treatment(Hotta et al., 2007), which our results agreed with.
In the future, a large-scale study with a longer observation period is required to clarify the preventive effect of vitamin K2 on recurrence of HCC.

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Received June 30, 2008; accepted September 16, 2008
Corresponding author: Keiko Hosho, MD99