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Combination of vitamin K2 and the angiotensin-converting enzyme inhibitor, perindopril, attenuates the liver enzyme-altered preneoplastic lesions in rats via angiogenesis suppression
Journal of Hepatology 42 (2005) 687–693 www.elsevier.com/locate/jhep
Combination of vitamin K2 and the angiotensin-converting enzyme inhibitor, perindopril, attenuates the liver enzyme-altered preneoplastic lesions in rats via angiogenesis suppression Hitoshi Yoshiji1,*, Shigeki Kuriyama2, Ryuichi Noguchi1, Junichi Yoshii1, Yasuhide Ikenaka1, Koji Yanase1, Tadashi Namisaki1, Mitsuteru Kitade1, Masaharu Yamazaki1, Tsutomu Masaki2, Hiroshi Fukui1 1
Third Department of Internal Medicine, Nara Medical University, Shijo-cho 840, Kashihara, Nara 634-8522, Japan 2 Third Department of Internal Medicine, Kagawa University School of Medicine, Kagawa, Japan
Background/Aims: Chemoprevention should be a promising approach to improve the prognosis of the patients with hepatocellular carcinoma (HCC). Angiogenesis is now recognized as a crucial step not only in tumor growth, but also in early carcinogenesis. The aim of this study was to elucidate the combination effect of the clinically used vitamin K2 (VK) and the angiotensin-converting enzyme inhibitor, perindopril (PE), on hepatocarcinogenesis, especially in conjunction with angiogenesis. Methods: In a diethylnitrosamine-induced rat hepatocarcinogenesis model, the effects of VK and PE on the development of liver enzyme-altered preneoplastic lesions and angiogenesis were examined. Results: Treatment with both VK and PE markedly inhibited the development of preneoplastic lesions in association with suppression of neovascularization in the liver. The combination treatment with VK and PE exerted a more potent inhibitory effect as compared with the single agent treatments. The in vitro study demonstrated that VK and PE inhibited the endothelial cell (EC) tubular formation. VK also suppressed the EC proliferation in a dose-dependent manner. Conclusions: The combination of VK and PE exerted a chemopreventive effect against rat liver carcinogenesis via suppression of angiogenesis. Since both agents are widely used in the clinical practice, this combination therapy may represent a potential new strategy for chemoprevention against HCC in the future. q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Vitamin K; ACE inhibitor; Hepatocellular carcinoma; Chemoprevention; Angiogenesis
1. Introduction Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world with an estimated Received 22 September 2004; received in revised form 21 November 2004; accepted 1 December 2004; available online 2 February 2005 * Corresponding author. Tel.: C81 744 22 3051; fax: C81 744 24 7122. E-mail address: [email protected] (H. Yoshiji). Abbreviations: EC, endothelial cells; ACE, angiotensin-converting enzyme; AT-II, angiotensin-II; HCC, hepatocellular carcinoma; PE, perindopril; VK, vitamin K2; VEGF, vascular endothelial growth factor; DEN, diethylnitrosamine; GST-P, placental form of glutathione S-transferase; GGT, gamma-glutamyltransferase; IFN, interferon.
annual incidence of more than one million new cases per year [1,2]. One of the reasons for the poor prognosis of HCC is the high rate of recurrence. It has been shown that this high recurrence rate, even after curative therapy, is due to intrahepatic metastasis or multicentric development of each respective neoplasm clone. Since the high-risk group of HCC development seems to be clearer as compared with other types of tumors, it is likely that a primary or secondary chemopreventive agent would be beneficial in improving the prognosis of HCC. Because a long-term administration is required and the drug metabolism is usually hypoactive in the patients with liver cirrhosis, the safety-proved agent would be preferable for chemoprevention against HCC.
0168-8278/$30.00 q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2004.12.025
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Angiogenesis is a complex and critical process essential to support the growth of solid tumors [3,4]. It has been recently reported that angiogenesis could also be induced at the early stages of tumor formation-, and carcinogenic procedures [5–8]. We have previously proved that angiogenesis played a pivotal role in the murine hepatocarcinogenesis process [9]. It has been reported that alterations of the hepatic microcirculation in the human liver have already occurred at an early stage of liver carcinogenesis, in association with liver cell change or within the dysplastic nodules, before the emergence of morphologically identifiable HCC [10]. The degree of angiogenesis increased gradually according to the stepwise development of hepatocarcinogenesis from the low-grade dysplastic nodules or even at the stage of chronic hepatitis [11,12]. Recently, a retrospective cohort study on 5207 patients receiving ACE inhibitors or other anti-hypertensive drugs with a 10-year follow-up has showed that the ACE inhibitors decreased the incident cancer and fetal cancer (Glasgow study) [13]. Angiotensin-II (AT-II) is an octapeptide produced mainly by proteolytic cleavage of its precursor AT-I by the angiotensin-converting enzyme (ACE) [14]. AT-II induces angiogenesis in several types of cells, including HCC cells, and the activity of ACE was a tumor marker in the HCC patients [15–17]. We previously reported that the clinically used ACE inhibitor, perindopril (PE), possesses a strong anti-angiogenic activity, and that it inhibited the murine hepatocarcinogenesis at a clinically comparable low dose [16,17]. Vitamin K2 (VK) is widely used for treatment of osteoporosis in the clinical practice without serious side effects. A recent Japanese trial enlisted 121 patients with HCC undergoing conventional therapy in the form of transarterial embolization and/or percutaneous intratumoral ethanol injection. Those patients were given 45 mg/day oral VK, and a decrease in the recurrence rate of HCC and a significant improvement in the overall survival were noticed [18]. However, the effect of VK and its combination effect with other agents on hepatocarcinogenesis and its mechanism have not been examined yet. In the present study, to evaluate the feasibility of future clinical applications, we examined the combination effect of the clinically used VK and PE on the rat hepatocarcinogenesis, and attempted to investigate the possible mechanisms involved, especially in conjunction with angiogenesis.
2. Methods 2.1. Animals A total of 50 male Fisher 344 rats, aged 6 weeks, were purchased from Japan SLC, Inc. (Hamamatsu, Shizuoka, Japan). They were housed in stainless-steel, mesh cages under control conditions of temperature (23G3 8C) and relative humidity (50G20%), with 10–15 air changes per hour and light illumination for 12 h a day. The animals were allowed access
to food and tap water ad libitum throughout the acclimatization and experimental periods.
2.2. Compounds and animal treatment VK and PE were provided by Eisai Co. (Tokyo, Japan), and Daiichi Pharmaceutical Co. (Tokyo, Japan), respectively. It has been reported that VK at a dose of 3 mg/kg exerted an anti-osteoporotic effect in the rat [19]. We have previously shown that PE exerted an anti-tumor effect at a dose of 2 mg/kg, which is compatible to of the dose used in the clinical practice [17,20]. We, therefore, employed these doses for respective agents in our current study. The experimental period in all experiments was 12 weeks. The rats were divided into five groups (nZ10 in each experimental group). The rats in group 1–4 (G1–G4) received an intraperitoneal (i.p.) injection of 200 mg/kg of diethylnitrosamine (DEN) underwent partial hepatectomy (PH) at week 4. The rats in G1 did not receive any additional treatment and G1 was considered as the control group. The rats in G2 and G3 received PE and VK daily by gavage, respectively. PE and VK were administered for 8 weeks starting from week 4. G4 was the group treated with combination of PE and VK. The rats which received phosphate buffer saline (PBS) instead of DEN were examined as a negative control group (G5). All rats were anesthetized, the thoracic cavity was opened and blood samples were withdrawn via cardiac puncture. Alanine aminotransferase (ALT) was assessed by the routine laboratory methods. All animal procedures were performed according to standard protocols and in accordance with the standard recommendations for the proper care and use of laboratory animals.
2.3. Immunohistochemical examinations In all experimental groups, 5-mm-thick sections of formalin-fixed and paraffin-embedded livers were processed routinely for hematoxylin and eosin (H–E). Immunohistochemical staining of two different enzymealtered preneoplastic lesions; namely the placental form of glutathione Stransferase (GST-P) and gamma-glutamyltransferase (GGT) (Medical and Biological Laboratories Co., Nagoya, Japan) was performed as described previously [21]. Computer assisted-quantitative analyses of the preneoplastic foci area were carried out with Fuji-BAS 2000 image analyzing system (Fuji, Tokyo, Japan) as previously described [22].
2.4. mRNA expression of CD31 in the liver The mRNA expression of CD-31, which is used widely as a marker of neovascularization, was evaluated by real-time polymerase chain reaction (PCR) as described previously [23]. Briefly, the liver was immediately snap-frozen for RNA extraction. Real-time PCR was performed with the ABI Prism 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s manual. Relative quantitation of the gene expression was performed as described in the manual by using glyceralaldehyde-3-phosphate dehydrogenase as an internal control. The threshold cycle and the standard curve method were used for calculating the relative amount of the target RNA. To prevent genomic DNA contamination, all RNA samples were subjected to DNase I digestion and checked by 40 cycles of PCR to confirm the absence of amplified DNA.
2.5. In vitro proliferation and angiogenesis assay The human HepG2 HCC cell line and HUVEC were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan). The in vitro proliferation was determined by tetrazolium, 3-(4,5-diethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay after harvest at 24, 48, 72, and 96 h as described elsewhere [23]. The cell proliferation was quantified via conversion of MTT in the presence or absence VK (from 1!10K6 to 1!10K4 M) and/or perindoprilat, which is the pro-drug of PE, at a dose of 1!10K6 M. The absorbancy was read with an ELISA plate reader (nZ6 per group). The in vitro angiogenesis was assessed as formation of capillary-like structures of HUVEC co-cultured with human diploid fibroblasts as described previously [24]. The experimental procedure followed the instructions provided with the angiogenesis kit (Kurabo).
H. Yoshiji et al. / Journal of Hepatology 42 (2005) 687–693 Briefly, the cells were treated with VK (from 1!10K6 to 1!10K4 M) and/ or perindoprilat on day 1 and the medium was replaced on days 4, 7, and 9. On day 11, the cells were fixed and HUVEC were stained using an antihuman CD31 antibody (Kurabo) according to the protocol provided with the kit. Computer-assisted quantitation of tubule formation was performed in the same way as for the in vivo assay.
2.6. Statistical analysis To assess the statistical significance of the inter-group differences in the quantitative data, Bonferroni’s multiple comparison test was used after oneway ANOVA. This was followed by Barlett’s test to determine the homology of variance.
3. Results 3.1. General findings The data for the effective numbers of rats, final body weights, relative liver weights in all experimental groups are shown in Table 1. There were no significant differences in the final body weight and relative liver weight among the five groups. PE and VK administration to the PBS-treated rats did not induce any change of these markers, respectively (data not shown). Neither ascites nor other organ abnormalities were observed at the end of the experiment in all groups. PE and VK treatment did not cause any alteration of the ALT level, either.
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group were markedly low as compared to the PH-performed groups (data not shown). 3.3. Neovascularization in the DEN-treated liver We next examined the effects of VK and PE on neovascularization in the liver to elucidate whether or not the inhibitory effects of these agents on the preneoplastic lesions are associated with alteration of angiogenesis. We performed a preliminary experiment to examine the mRNA expression of CD31 in the GST-P-positive lesions and the adjacent non-neoplastic lesions with a laser-microdissection system using the T7-based RNA amplification method. We found CD31 mRNA up-regulation exclusively in the GSTP-positive preneoplastic lesions (data not shown). As shown in Fig. 2, the mRNA expression of CD31was significantly increased in the DEN-treated control group, and the mRNA expression of CD 31 in the VK- and PE-treated groups was markedly suppressed as compared to the control group. Similar to the preneoplastic lesions, the inhibitory effect of PE was more than that of VK, and the combination treatment of VK and PE exerted a much more potent inhibitory effect as compared with that of either single agent. Noteworthy was the finding that the inhibitory effects of VK and PE on the CD31 expression were almost similar in magnitude to that of suppression of the preneoplastic lesion development. 3.4. Effects of VK on HCC and EC proliferation in vitro
3.2. Effect of VK and PE on the development of preneoplastic lesions Neither histological nor biological changes indicating liver injury were observed except the development of GSTP-positive preneoplastic foci. As shown in Fig. 1A and B, the number and size of the preneoplastic foci were both significantly suppressed by the treatment with VK and PE. The inhibitory effect of PE was more potent than that of VK (P!0.01 and 0.05, respectively). The combination treatment of VK and PE exerted a more potent inhibitory effect than that of PE (P!0.01). No positive foci were found in the PBS-treated group. As with the GGT-positive preneoplastic foci, the results were similar to those of the GST-P (Fig. 1C and D, respectively). As previously reported [25], the GGT and GST-P-positive lesions in the sham-operated
To elucidate the possible mechanism of the inhibitory effect of VK, we first examined whether the inhibitory effects of VK were related to cytotoxicity. As shown in Fig. 3, VK suppressed the in vitro proliferation of both HCC cells and EC in a dose-dependent manner. However, the sensitivities of HCC cells and EC to VK were quite different. VK exerted a significant inhibitory effect on EC even at a dose of 1!10K6 M (P!0.01), which was comparable to the serum concentration of the patients treated with VK [26]. On the other hand, this low dose did not have any effect on HCC proliferation. At a dose of 1!10K4 M, VK finally suppressed the in vitro proliferation of the HCC cells. Similar to our previous report [17], PE in the current study had no effect on HCC cells or EC even at a
Table 1 Experimental group details given DEN with PE and/or vitamin K
DEN DENCPE (2 mg/kg) DENCVit K (3 mg/kg) DENCPECVit K PBS
Effective no of rates
Final body weight (g)
Relative liver weight (g/ 100 g body wt)
ALT (U/L)
10 10 10 10 10
255G16 245G18 261G21 247G20 252G23
2.63G0.24 2.59G0.26 2.57G0.32 2.58G0.23 2.57G0.27
254.4G43.8 244.3G47.3 247.8G46.2 251.7G42.8 250.5G44.2
DEN, diethylnitrosamine; Vit K, vitamin K2; PE, perindopril.
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Fig. 1. Effects of VK and PE on the development of DEN-induced GST-P and GGT-positive preneoplastic foci. The number (A) and size (B) of the GST-P positive foci were both significantly suppressed by the treatment of VK and PE. The inhibitory effect of PE was more potent than that of VK. The combination treatment of VK and PE exerted a much potent inhibitory effect as compared with that of PE. No positive foci were found in the PBStreated group. As with the GGT-positive preneoplastic foci, the results were similar to those of the GST-P (Fig. 1C and D, respectively). No positive foci were found in the PBS-treated group. The results of the GGT-positive lesions were similar to those of the GST-P both in number (C) and size (D). The data represent meansGSD. * and **, Statistically significant differences between the indicated groups (P!0.05 and 0.01), respectively.
dose of 1!10K4 M. Moreover, PE did not show any additional effect to that of VK on HCC cells and EC. We also performed the [H]3 uptake methods for assessment of proliferation, and found similar results (data not shown). 3.5. Effects of VK and PE on in vitro angiogenesis We next examined whether VK also inhibited the EC tubular formation. Similar to the EC proliferation, we found that VK significantly inhibited the EC tubular formation even at a dose of 1!10K6 M (Fig. 4A). Our quantitative analysis showed that the inhibitory effect of VK on the EC tubular formation was a dose dependent (Fig. 4B). We also investigated the combination effect of VK and PE on the EC tubular formation at the dose of 1!10K6 M of both agents. As shown in Fig. 5A, the inhibitory effect of PE seemed to be stronger than that of VK at the same low dose (1!10K6 M). The quantitative analysis showed that the total length of the tubules formed in either VK- or PEtreated cultures was significantly less than in the untreated control culture (P!0.05 and 0.01, respectively), and that the combination treatment of PE and VK resulted in
Fig. 2. The effects of VK and PE on neovascularization in the liver. The CD31 mRNA expression was examined by real-time PCR as described in Section 2. The CD31 gene expression was significantly increased during hepatocarcinogenesis. Treatment with VK and PE significantly attenuated neovascularization in the liver. Suppression of angiogenesis by treatment with VK and PE was of similar magnitude to that of inhibition of development of the preneoplastic lesions. DEN, control DEN-treated rats (G1). PE and VK, PE- and VK-treated rats (G2 and G3, respectively). PECVK, PE and VK combination-treated group (G4). PBS, PBS-injected negative control group (G5). The data represent the meansGSD (nZ15). * and **, statistically significant differences between the indicated groups (P!0.05 and 0.01, respectively).
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Fig. 3. Effects of VK on the HCC (A) and EC (B) cell proliferation in vitro. The cell proliferation was measured by MTT assay after harvest at 24, 48, 72, and 96 h as described in Section 2. VK suppressed the in vitro proliferation of both HCC cell and EC in a dose-dependent manner. VK exerted a significant inhibitory effect on EC even at a dose of 1!10K6 M, whereas this low dose did have any effect on HCC proliferation. At a dose of 1!10K4 M, VK finally suppressed the in vitro proliferation of the HCC cells. PE had no effect on the HCC cells or EC even at a dose of 1!10K4 M. Moreover, PE did not show any additional effect to that of VK on HCC cells and EC. The data represent the meansGSD (nZ6). *, Statistically significant differences as compared with the untreated group (P!0.01). HCC and EC, HepG2 and HUVEC, respectively.
a further inhibition of the EC tubular formation as compared with PE alone (P!0.01) (Fig. 5B). To examine whether the initial concentration of EC affects the effect of these agents on tubular formation, we also examined the EC tubular formation with different initial concentrations of EC on Matrigele as described previously [17], and we found that the PE and VK suppressed the EC tubular formation at a similar magnitude regardless of the initial EC concentrations (data not shown).
4. Discussion In the current study, we found that the treatment with the clinically used VK and PE markedly inhibited the development of preneoplastic lesions associated with suppression of neovascularization in the liver, and that the combination
treatment with VK and PE exerted a more potent inhibitory effect as compared with the single agent treatment. It has been reported that the use of anti-angiogenic agents as monotherapy in treating the patients with advanced cancer has not yet shown a significant efficacy [4,27,28]. The limitations of anti-angiogenic monotherapy in this setting were in fact predicted by preclinical studies using the angiogenesis inhibitors endostatin and angiostatin. It has been reported that the combination treatment of antiangiogenic agents, such as endostatin and angiostatin, revealed a synergistic inhibitory effect on the tumor development and angiogenesis [28,29]. It has been reported that VK and several agents, such as retinoic acid, exerted a synergistic anti-cancerous effect [30,31]. In the current study, we found that the combination treatment with VK and PE showed a more inhibitory effect on hepatocarcinogenesis than the single agent treatment. Moreover, we found that
Fig. 4. Effects of VK on the in vitro EC tubular formation. (A) VK significantly inhibited the EC tubular formation. (B) Computer-assisted quantitative analysis showed that the inhibitory effect of VK on the EC tubular formation was in a dose dependent. The total tubule length was measured by an image analysis system as described in Section 2. The data represent the meanGSD (nZ6). * and **, Statistically significant difference as compared to the untreated control group (P!0.05 and 0.01, respectively). Lane 1, untreated control group. Lanes 2–4, VK-treated group at the doses of 1!10K6, 1!10K5 and, 1!10K4 M, respectively.
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Fig. 5. The combination effect of VK and PE on the in vitro EC tubular formation. The tubules formed in the VK- or PE-treated group were less than in the untreated control group. The inhibitory effect of PE seemed to be stronger than that of VK (A). The quantitative analysis showed that the total length of the tubules formed in the VK- or PE-treated cultures was significantly less than in the untreated control culture, and that the combination treatment of PE and VK resulted in a further inhibition of the EC tubular formation as compared to PE alone (B). The total tubule length was measured by an image analysis system as described in Section 2. The data represent the meanGSD (nZ6). * and **, Statistically significant difference between the indicated groups (P!0.05 and 0.01, respectively). Lane 1, untreated control group. Lanes 2 and 3, PE- and VK-treated group at the doses of 1!10K6, respectively. Lane 4, PE and VK combination-treated group.
VK exerted a direct inhibitory effect on the proliferation and tubular formation of EC even at a low concentration. PE also suppressed the EC tubular formation, whereas it did not inhibit the EC proliferation [16,17]. On the other hand, neither PE nor VK induced apoptosis in the HCC cells and EC in vitro (data not shown). Although we need to examine the in vitro effect on the apoptosis-related genes in the preneoplastic lesions in the future, we assume that the anti-angiogenic effect of both agents were mediated mainly through inhibition of proliferation and migration of EC in this model, but not via induction of apoptosis of EC. It has been reported that VK inhibited the proliferation of HCC cells at doses of 50w100 mM [32]. We also found that VK suppressed the proliferation of HCC cells at a dose of 100 mM. As the authors themselves mentioned in their report, however, this dose is very high as compared with the clinical comparable dose. On the other hand, the proliferation of EC was significantly suppressed even at a 1 mM, which mimics the serum level in the clinical practice. We, therefore, think that inhibitory effect of VK on hepatocarcinogenesis in this study was mainly mediated through EC but not via a direct action on the HCC cells. It has been reported that a potent angiogenic factor, the vascular endothelial growth factor (VEGF), stepwise increased during hepatocarcinogenesis along with neovascularization, and that suppression of the VEGF-receptor interaction significantly attenuated the development of HCC [9]. Consistently, we found the VEGF mRNA up-regulation exclusively in the GST-P-positive preneoplastic lesions using the laser-microdissection system, and the mRNA of VEGF expression in the preneoplastic liver was significantly suppressed by treatment with PE and VK. The suppressive effects of these agents on mRNA of CD31 and
VEGF expressions exerted almost parallel reductions. (data not shown). It is likely that the coordination of these different biological activities of anti-angiogenesis agents produced the in vitro combination effect of suppressing hepatocarcinogenesis. Our knowledge of the multistage nature of hepatocarcinogenesis with initiation, promotion, and progression has been derived from studies using animal models [33]. Although we previously reported that PE suppressed the development of preneoplastic lesions in the DEN-treated liver, that study administered PE from the beginning of the initial stage of experiment [34]. As described, it has been suggested that the transformed preneoplastic clone has already developed even at the stage of chronic hepatitis [10]. Considering the future possible clinical application, a better model may be to administer the tested-agent from the promotion stage rather than from the beginning of the experiment. We, therefore, administered VK and PE after partial hepatectomy, from the promotion stage in the current study. Nevertheless, it is important to perform a long-term experiment in the future to determine whether or not VK and PE really prevent HCC development. Furthermore, it would be important to examine whether these agents attenuated the cell transformation or influenced the preneoplastic lesion growth. In summary, we have shown here that the combination treatment of VK and the ACE inhibitor, PE, significantly inhibited hepatocarcinogenesis along with suppression of angiogenesis. Noteworthy was the fact that these inhibitory effects were achieved at clinically comparable low doses. Since both agents are currently widely used in the clinical practice, this combination regimen may represent a potential new strategy for chemoprevention against HCC in the future.
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Combination of vitamin K2 and the angiotensin-converting enzyme inhibitor, perindopril, attenuates the liver enzyme-altered preneoplastic lesions in rats via angiogenesis suppression Hitoshi Yoshiji1,*, Shigeki Kuriyama2, Ryuichi Noguchi1, Junichi Yoshii1, Yasuhide Ikenaka1, Koji Yanase1, Tadashi Namisaki1, Mitsuteru Kitade1, Masaharu Yamazaki1, Tsutomu Masaki2, Hiroshi Fukui1 1
Third Department of Internal Medicine, Nara Medical University, Shijo-cho 840, Kashihara, Nara 634-8522, Japan 2 Third Department of Internal Medicine, Kagawa University School of Medicine, Kagawa, Japan
Background/Aims: Chemoprevention should be a promising approach to improve the prognosis of the patients with hepatocellular carcinoma (HCC). Angiogenesis is now recognized as a crucial step not only in tumor growth, but also in early carcinogenesis. The aim of this study was to elucidate the combination effect of the clinically used vitamin K2 (VK) and the angiotensin-converting enzyme inhibitor, perindopril (PE), on hepatocarcinogenesis, especially in conjunction with angiogenesis. Methods: In a diethylnitrosamine-induced rat hepatocarcinogenesis model, the effects of VK and PE on the development of liver enzyme-altered preneoplastic lesions and angiogenesis were examined. Results: Treatment with both VK and PE markedly inhibited the development of preneoplastic lesions in association with suppression of neovascularization in the liver. The combination treatment with VK and PE exerted a more potent inhibitory effect as compared with the single agent treatments. The in vitro study demonstrated that VK and PE inhibited the endothelial cell (EC) tubular formation. VK also suppressed the EC proliferation in a dose-dependent manner. Conclusions: The combination of VK and PE exerted a chemopreventive effect against rat liver carcinogenesis via suppression of angiogenesis. Since both agents are widely used in the clinical practice, this combination therapy may represent a potential new strategy for chemoprevention against HCC in the future. q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Vitamin K; ACE inhibitor; Hepatocellular carcinoma; Chemoprevention; Angiogenesis
1. Introduction Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world with an estimated Received 22 September 2004; received in revised form 21 November 2004; accepted 1 December 2004; available online 2 February 2005 * Corresponding author. Tel.: C81 744 22 3051; fax: C81 744 24 7122. E-mail address: [email protected] (H. Yoshiji). Abbreviations: EC, endothelial cells; ACE, angiotensin-converting enzyme; AT-II, angiotensin-II; HCC, hepatocellular carcinoma; PE, perindopril; VK, vitamin K2; VEGF, vascular endothelial growth factor; DEN, diethylnitrosamine; GST-P, placental form of glutathione S-transferase; GGT, gamma-glutamyltransferase; IFN, interferon.
annual incidence of more than one million new cases per year [1,2]. One of the reasons for the poor prognosis of HCC is the high rate of recurrence. It has been shown that this high recurrence rate, even after curative therapy, is due to intrahepatic metastasis or multicentric development of each respective neoplasm clone. Since the high-risk group of HCC development seems to be clearer as compared with other types of tumors, it is likely that a primary or secondary chemopreventive agent would be beneficial in improving the prognosis of HCC. Because a long-term administration is required and the drug metabolism is usually hypoactive in the patients with liver cirrhosis, the safety-proved agent would be preferable for chemoprevention against HCC.
0168-8278/$30.00 q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2004.12.025
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Angiogenesis is a complex and critical process essential to support the growth of solid tumors [3,4]. It has been recently reported that angiogenesis could also be induced at the early stages of tumor formation-, and carcinogenic procedures [5–8]. We have previously proved that angiogenesis played a pivotal role in the murine hepatocarcinogenesis process [9]. It has been reported that alterations of the hepatic microcirculation in the human liver have already occurred at an early stage of liver carcinogenesis, in association with liver cell change or within the dysplastic nodules, before the emergence of morphologically identifiable HCC [10]. The degree of angiogenesis increased gradually according to the stepwise development of hepatocarcinogenesis from the low-grade dysplastic nodules or even at the stage of chronic hepatitis [11,12]. Recently, a retrospective cohort study on 5207 patients receiving ACE inhibitors or other anti-hypertensive drugs with a 10-year follow-up has showed that the ACE inhibitors decreased the incident cancer and fetal cancer (Glasgow study) [13]. Angiotensin-II (AT-II) is an octapeptide produced mainly by proteolytic cleavage of its precursor AT-I by the angiotensin-converting enzyme (ACE) [14]. AT-II induces angiogenesis in several types of cells, including HCC cells, and the activity of ACE was a tumor marker in the HCC patients [15–17]. We previously reported that the clinically used ACE inhibitor, perindopril (PE), possesses a strong anti-angiogenic activity, and that it inhibited the murine hepatocarcinogenesis at a clinically comparable low dose [16,17]. Vitamin K2 (VK) is widely used for treatment of osteoporosis in the clinical practice without serious side effects. A recent Japanese trial enlisted 121 patients with HCC undergoing conventional therapy in the form of transarterial embolization and/or percutaneous intratumoral ethanol injection. Those patients were given 45 mg/day oral VK, and a decrease in the recurrence rate of HCC and a significant improvement in the overall survival were noticed [18]. However, the effect of VK and its combination effect with other agents on hepatocarcinogenesis and its mechanism have not been examined yet. In the present study, to evaluate the feasibility of future clinical applications, we examined the combination effect of the clinically used VK and PE on the rat hepatocarcinogenesis, and attempted to investigate the possible mechanisms involved, especially in conjunction with angiogenesis.
2. Methods 2.1. Animals A total of 50 male Fisher 344 rats, aged 6 weeks, were purchased from Japan SLC, Inc. (Hamamatsu, Shizuoka, Japan). They were housed in stainless-steel, mesh cages under control conditions of temperature (23G3 8C) and relative humidity (50G20%), with 10–15 air changes per hour and light illumination for 12 h a day. The animals were allowed access
to food and tap water ad libitum throughout the acclimatization and experimental periods.
2.2. Compounds and animal treatment VK and PE were provided by Eisai Co. (Tokyo, Japan), and Daiichi Pharmaceutical Co. (Tokyo, Japan), respectively. It has been reported that VK at a dose of 3 mg/kg exerted an anti-osteoporotic effect in the rat [19]. We have previously shown that PE exerted an anti-tumor effect at a dose of 2 mg/kg, which is compatible to of the dose used in the clinical practice [17,20]. We, therefore, employed these doses for respective agents in our current study. The experimental period in all experiments was 12 weeks. The rats were divided into five groups (nZ10 in each experimental group). The rats in group 1–4 (G1–G4) received an intraperitoneal (i.p.) injection of 200 mg/kg of diethylnitrosamine (DEN) underwent partial hepatectomy (PH) at week 4. The rats in G1 did not receive any additional treatment and G1 was considered as the control group. The rats in G2 and G3 received PE and VK daily by gavage, respectively. PE and VK were administered for 8 weeks starting from week 4. G4 was the group treated with combination of PE and VK. The rats which received phosphate buffer saline (PBS) instead of DEN were examined as a negative control group (G5). All rats were anesthetized, the thoracic cavity was opened and blood samples were withdrawn via cardiac puncture. Alanine aminotransferase (ALT) was assessed by the routine laboratory methods. All animal procedures were performed according to standard protocols and in accordance with the standard recommendations for the proper care and use of laboratory animals.
2.3. Immunohistochemical examinations In all experimental groups, 5-mm-thick sections of formalin-fixed and paraffin-embedded livers were processed routinely for hematoxylin and eosin (H–E). Immunohistochemical staining of two different enzymealtered preneoplastic lesions; namely the placental form of glutathione Stransferase (GST-P) and gamma-glutamyltransferase (GGT) (Medical and Biological Laboratories Co., Nagoya, Japan) was performed as described previously [21]. Computer assisted-quantitative analyses of the preneoplastic foci area were carried out with Fuji-BAS 2000 image analyzing system (Fuji, Tokyo, Japan) as previously described [22].
2.4. mRNA expression of CD31 in the liver The mRNA expression of CD-31, which is used widely as a marker of neovascularization, was evaluated by real-time polymerase chain reaction (PCR) as described previously [23]. Briefly, the liver was immediately snap-frozen for RNA extraction. Real-time PCR was performed with the ABI Prism 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s manual. Relative quantitation of the gene expression was performed as described in the manual by using glyceralaldehyde-3-phosphate dehydrogenase as an internal control. The threshold cycle and the standard curve method were used for calculating the relative amount of the target RNA. To prevent genomic DNA contamination, all RNA samples were subjected to DNase I digestion and checked by 40 cycles of PCR to confirm the absence of amplified DNA.
2.5. In vitro proliferation and angiogenesis assay The human HepG2 HCC cell line and HUVEC were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan). The in vitro proliferation was determined by tetrazolium, 3-(4,5-diethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay after harvest at 24, 48, 72, and 96 h as described elsewhere [23]. The cell proliferation was quantified via conversion of MTT in the presence or absence VK (from 1!10K6 to 1!10K4 M) and/or perindoprilat, which is the pro-drug of PE, at a dose of 1!10K6 M. The absorbancy was read with an ELISA plate reader (nZ6 per group). The in vitro angiogenesis was assessed as formation of capillary-like structures of HUVEC co-cultured with human diploid fibroblasts as described previously [24]. The experimental procedure followed the instructions provided with the angiogenesis kit (Kurabo).
H. Yoshiji et al. / Journal of Hepatology 42 (2005) 687–693 Briefly, the cells were treated with VK (from 1!10K6 to 1!10K4 M) and/ or perindoprilat on day 1 and the medium was replaced on days 4, 7, and 9. On day 11, the cells were fixed and HUVEC were stained using an antihuman CD31 antibody (Kurabo) according to the protocol provided with the kit. Computer-assisted quantitation of tubule formation was performed in the same way as for the in vivo assay.
2.6. Statistical analysis To assess the statistical significance of the inter-group differences in the quantitative data, Bonferroni’s multiple comparison test was used after oneway ANOVA. This was followed by Barlett’s test to determine the homology of variance.
3. Results 3.1. General findings The data for the effective numbers of rats, final body weights, relative liver weights in all experimental groups are shown in Table 1. There were no significant differences in the final body weight and relative liver weight among the five groups. PE and VK administration to the PBS-treated rats did not induce any change of these markers, respectively (data not shown). Neither ascites nor other organ abnormalities were observed at the end of the experiment in all groups. PE and VK treatment did not cause any alteration of the ALT level, either.
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group were markedly low as compared to the PH-performed groups (data not shown). 3.3. Neovascularization in the DEN-treated liver We next examined the effects of VK and PE on neovascularization in the liver to elucidate whether or not the inhibitory effects of these agents on the preneoplastic lesions are associated with alteration of angiogenesis. We performed a preliminary experiment to examine the mRNA expression of CD31 in the GST-P-positive lesions and the adjacent non-neoplastic lesions with a laser-microdissection system using the T7-based RNA amplification method. We found CD31 mRNA up-regulation exclusively in the GSTP-positive preneoplastic lesions (data not shown). As shown in Fig. 2, the mRNA expression of CD31was significantly increased in the DEN-treated control group, and the mRNA expression of CD 31 in the VK- and PE-treated groups was markedly suppressed as compared to the control group. Similar to the preneoplastic lesions, the inhibitory effect of PE was more than that of VK, and the combination treatment of VK and PE exerted a much more potent inhibitory effect as compared with that of either single agent. Noteworthy was the finding that the inhibitory effects of VK and PE on the CD31 expression were almost similar in magnitude to that of suppression of the preneoplastic lesion development. 3.4. Effects of VK on HCC and EC proliferation in vitro
3.2. Effect of VK and PE on the development of preneoplastic lesions Neither histological nor biological changes indicating liver injury were observed except the development of GSTP-positive preneoplastic foci. As shown in Fig. 1A and B, the number and size of the preneoplastic foci were both significantly suppressed by the treatment with VK and PE. The inhibitory effect of PE was more potent than that of VK (P!0.01 and 0.05, respectively). The combination treatment of VK and PE exerted a more potent inhibitory effect than that of PE (P!0.01). No positive foci were found in the PBS-treated group. As with the GGT-positive preneoplastic foci, the results were similar to those of the GST-P (Fig. 1C and D, respectively). As previously reported [25], the GGT and GST-P-positive lesions in the sham-operated
To elucidate the possible mechanism of the inhibitory effect of VK, we first examined whether the inhibitory effects of VK were related to cytotoxicity. As shown in Fig. 3, VK suppressed the in vitro proliferation of both HCC cells and EC in a dose-dependent manner. However, the sensitivities of HCC cells and EC to VK were quite different. VK exerted a significant inhibitory effect on EC even at a dose of 1!10K6 M (P!0.01), which was comparable to the serum concentration of the patients treated with VK [26]. On the other hand, this low dose did not have any effect on HCC proliferation. At a dose of 1!10K4 M, VK finally suppressed the in vitro proliferation of the HCC cells. Similar to our previous report [17], PE in the current study had no effect on HCC cells or EC even at a
Table 1 Experimental group details given DEN with PE and/or vitamin K
DEN DENCPE (2 mg/kg) DENCVit K (3 mg/kg) DENCPECVit K PBS
Effective no of rates
Final body weight (g)
Relative liver weight (g/ 100 g body wt)
ALT (U/L)
10 10 10 10 10
255G16 245G18 261G21 247G20 252G23
2.63G0.24 2.59G0.26 2.57G0.32 2.58G0.23 2.57G0.27
254.4G43.8 244.3G47.3 247.8G46.2 251.7G42.8 250.5G44.2
DEN, diethylnitrosamine; Vit K, vitamin K2; PE, perindopril.
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Fig. 1. Effects of VK and PE on the development of DEN-induced GST-P and GGT-positive preneoplastic foci. The number (A) and size (B) of the GST-P positive foci were both significantly suppressed by the treatment of VK and PE. The inhibitory effect of PE was more potent than that of VK. The combination treatment of VK and PE exerted a much potent inhibitory effect as compared with that of PE. No positive foci were found in the PBStreated group. As with the GGT-positive preneoplastic foci, the results were similar to those of the GST-P (Fig. 1C and D, respectively). No positive foci were found in the PBS-treated group. The results of the GGT-positive lesions were similar to those of the GST-P both in number (C) and size (D). The data represent meansGSD. * and **, Statistically significant differences between the indicated groups (P!0.05 and 0.01), respectively.
dose of 1!10K4 M. Moreover, PE did not show any additional effect to that of VK on HCC cells and EC. We also performed the [H]3 uptake methods for assessment of proliferation, and found similar results (data not shown). 3.5. Effects of VK and PE on in vitro angiogenesis We next examined whether VK also inhibited the EC tubular formation. Similar to the EC proliferation, we found that VK significantly inhibited the EC tubular formation even at a dose of 1!10K6 M (Fig. 4A). Our quantitative analysis showed that the inhibitory effect of VK on the EC tubular formation was a dose dependent (Fig. 4B). We also investigated the combination effect of VK and PE on the EC tubular formation at the dose of 1!10K6 M of both agents. As shown in Fig. 5A, the inhibitory effect of PE seemed to be stronger than that of VK at the same low dose (1!10K6 M). The quantitative analysis showed that the total length of the tubules formed in either VK- or PEtreated cultures was significantly less than in the untreated control culture (P!0.05 and 0.01, respectively), and that the combination treatment of PE and VK resulted in
Fig. 2. The effects of VK and PE on neovascularization in the liver. The CD31 mRNA expression was examined by real-time PCR as described in Section 2. The CD31 gene expression was significantly increased during hepatocarcinogenesis. Treatment with VK and PE significantly attenuated neovascularization in the liver. Suppression of angiogenesis by treatment with VK and PE was of similar magnitude to that of inhibition of development of the preneoplastic lesions. DEN, control DEN-treated rats (G1). PE and VK, PE- and VK-treated rats (G2 and G3, respectively). PECVK, PE and VK combination-treated group (G4). PBS, PBS-injected negative control group (G5). The data represent the meansGSD (nZ15). * and **, statistically significant differences between the indicated groups (P!0.05 and 0.01, respectively).
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Fig. 3. Effects of VK on the HCC (A) and EC (B) cell proliferation in vitro. The cell proliferation was measured by MTT assay after harvest at 24, 48, 72, and 96 h as described in Section 2. VK suppressed the in vitro proliferation of both HCC cell and EC in a dose-dependent manner. VK exerted a significant inhibitory effect on EC even at a dose of 1!10K6 M, whereas this low dose did have any effect on HCC proliferation. At a dose of 1!10K4 M, VK finally suppressed the in vitro proliferation of the HCC cells. PE had no effect on the HCC cells or EC even at a dose of 1!10K4 M. Moreover, PE did not show any additional effect to that of VK on HCC cells and EC. The data represent the meansGSD (nZ6). *, Statistically significant differences as compared with the untreated group (P!0.01). HCC and EC, HepG2 and HUVEC, respectively.
a further inhibition of the EC tubular formation as compared with PE alone (P!0.01) (Fig. 5B). To examine whether the initial concentration of EC affects the effect of these agents on tubular formation, we also examined the EC tubular formation with different initial concentrations of EC on Matrigele as described previously [17], and we found that the PE and VK suppressed the EC tubular formation at a similar magnitude regardless of the initial EC concentrations (data not shown).
4. Discussion In the current study, we found that the treatment with the clinically used VK and PE markedly inhibited the development of preneoplastic lesions associated with suppression of neovascularization in the liver, and that the combination
treatment with VK and PE exerted a more potent inhibitory effect as compared with the single agent treatment. It has been reported that the use of anti-angiogenic agents as monotherapy in treating the patients with advanced cancer has not yet shown a significant efficacy [4,27,28]. The limitations of anti-angiogenic monotherapy in this setting were in fact predicted by preclinical studies using the angiogenesis inhibitors endostatin and angiostatin. It has been reported that the combination treatment of antiangiogenic agents, such as endostatin and angiostatin, revealed a synergistic inhibitory effect on the tumor development and angiogenesis [28,29]. It has been reported that VK and several agents, such as retinoic acid, exerted a synergistic anti-cancerous effect [30,31]. In the current study, we found that the combination treatment with VK and PE showed a more inhibitory effect on hepatocarcinogenesis than the single agent treatment. Moreover, we found that
Fig. 4. Effects of VK on the in vitro EC tubular formation. (A) VK significantly inhibited the EC tubular formation. (B) Computer-assisted quantitative analysis showed that the inhibitory effect of VK on the EC tubular formation was in a dose dependent. The total tubule length was measured by an image analysis system as described in Section 2. The data represent the meanGSD (nZ6). * and **, Statistically significant difference as compared to the untreated control group (P!0.05 and 0.01, respectively). Lane 1, untreated control group. Lanes 2–4, VK-treated group at the doses of 1!10K6, 1!10K5 and, 1!10K4 M, respectively.
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Fig. 5. The combination effect of VK and PE on the in vitro EC tubular formation. The tubules formed in the VK- or PE-treated group were less than in the untreated control group. The inhibitory effect of PE seemed to be stronger than that of VK (A). The quantitative analysis showed that the total length of the tubules formed in the VK- or PE-treated cultures was significantly less than in the untreated control culture, and that the combination treatment of PE and VK resulted in a further inhibition of the EC tubular formation as compared to PE alone (B). The total tubule length was measured by an image analysis system as described in Section 2. The data represent the meanGSD (nZ6). * and **, Statistically significant difference between the indicated groups (P!0.05 and 0.01, respectively). Lane 1, untreated control group. Lanes 2 and 3, PE- and VK-treated group at the doses of 1!10K6, respectively. Lane 4, PE and VK combination-treated group.
VK exerted a direct inhibitory effect on the proliferation and tubular formation of EC even at a low concentration. PE also suppressed the EC tubular formation, whereas it did not inhibit the EC proliferation [16,17]. On the other hand, neither PE nor VK induced apoptosis in the HCC cells and EC in vitro (data not shown). Although we need to examine the in vitro effect on the apoptosis-related genes in the preneoplastic lesions in the future, we assume that the anti-angiogenic effect of both agents were mediated mainly through inhibition of proliferation and migration of EC in this model, but not via induction of apoptosis of EC. It has been reported that VK inhibited the proliferation of HCC cells at doses of 50w100 mM [32]. We also found that VK suppressed the proliferation of HCC cells at a dose of 100 mM. As the authors themselves mentioned in their report, however, this dose is very high as compared with the clinical comparable dose. On the other hand, the proliferation of EC was significantly suppressed even at a 1 mM, which mimics the serum level in the clinical practice. We, therefore, think that inhibitory effect of VK on hepatocarcinogenesis in this study was mainly mediated through EC but not via a direct action on the HCC cells. It has been reported that a potent angiogenic factor, the vascular endothelial growth factor (VEGF), stepwise increased during hepatocarcinogenesis along with neovascularization, and that suppression of the VEGF-receptor interaction significantly attenuated the development of HCC [9]. Consistently, we found the VEGF mRNA up-regulation exclusively in the GST-P-positive preneoplastic lesions using the laser-microdissection system, and the mRNA of VEGF expression in the preneoplastic liver was significantly suppressed by treatment with PE and VK. The suppressive effects of these agents on mRNA of CD31 and
VEGF expressions exerted almost parallel reductions. (data not shown). It is likely that the coordination of these different biological activities of anti-angiogenesis agents produced the in vitro combination effect of suppressing hepatocarcinogenesis. Our knowledge of the multistage nature of hepatocarcinogenesis with initiation, promotion, and progression has been derived from studies using animal models [33]. Although we previously reported that PE suppressed the development of preneoplastic lesions in the DEN-treated liver, that study administered PE from the beginning of the initial stage of experiment [34]. As described, it has been suggested that the transformed preneoplastic clone has already developed even at the stage of chronic hepatitis [10]. Considering the future possible clinical application, a better model may be to administer the tested-agent from the promotion stage rather than from the beginning of the experiment. We, therefore, administered VK and PE after partial hepatectomy, from the promotion stage in the current study. Nevertheless, it is important to perform a long-term experiment in the future to determine whether or not VK and PE really prevent HCC development. Furthermore, it would be important to examine whether these agents attenuated the cell transformation or influenced the preneoplastic lesion growth. In summary, we have shown here that the combination treatment of VK and the ACE inhibitor, PE, significantly inhibited hepatocarcinogenesis along with suppression of angiogenesis. Noteworthy was the fact that these inhibitory effects were achieved at clinically comparable low doses. Since both agents are currently widely used in the clinical practice, this combination regimen may represent a potential new strategy for chemoprevention against HCC in the future.
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