Abstract
Since the incidence of hepatocellular carcinoma in chronic viral hepatitis patients with cirrhosis is very high, it is valuable to develop effective methods for its prevention. In the present study, the effect of a carotenoid mixture on hepatocel- lular carcinoma development was examined.
Patients were randomly divided into two groups and treated with a carotenoid mixture in addi- tion to conventional antisymptomatic treatment, or antisymptomatic treatment alone. Cumula- tive incidence of hepatocellular carcinoma de- velopment was periodically analyzed using the Kaplan-Meier method. Significantly lower in- cidence was observed in the carotenoid-treated group compared with the control group in the analysis at year 4.
Introduction
Infection with hepatitis virus is well recognized as one of the important risk factors for liver can- cer. In Japan, hepatocellular carcinoma associated with hepatitis C virus infection is continuously increasing, and its prevention is now an urgent social problem. Average incidence of hepatocel- lular carcinoma in a year has been reported to be about 7% in chronic hepatitis C patients with cirrhosis. In the case of hepatitis B infection, Southeast Asia and Africa are known as severely invaded areas, and in fact, the incidence of liver cancer is very high in these countries. Therefore,
we have tried to develop effective methods for prevention against hepatitis virus infection-re- lated liver cancer.
Our previous clinical investigation showed that the serum level of hydrocarbon carotenoids such as beta-carotene, alpha-carotene, and lyco- pene is lower in liver cancer patients with viral hepatitis than in healthy individuals.
In addition to this clinical observation, we have also found that β-carotene and α-carotene suppressed liver carcinogenesis in animal experi- ments (Murakoshi et al. 1992). It is particularly interesting that treatment with a mixture of these carotenoids resulted in more effective inhibition than each carotenoid alone. For example, palm fruit carotene mixture, which consisted of β-caro- tene, α-carotene, and a small portion of other carotenoids such as lycopene showed the highest suppressive effect on liver carcinogenesis in mice compared with β-carotene alone or α-carotene alone at the same dose. Therefore, it may be pos- sible that combined use of various carotenoids improves the efficacy of cancer prevention. It is also possible that a small portion of other carot- enoids has potent activity in suppressing liver carcinogenesis. In fact, lycopene was proven to have very high potency in preventing liver can- cer in animal experiments (Nishino 1997).
These clinical and experimental observations prompted us to evaluate the effect of a carotenoid mixture on hepatocellular carcinoma develop- ment in viral hepatitis patients.
6 Prevention of Hepatocellular Carcinoma in Chronic Viral Hepatitis Patients
with Cirrhosis by Carotenoid Mixture
Hoyoku Nishino
Recent Results in Cancer Research, Vol. 174
© Springer-Verlag Berlin Heidelberg 2007
Protocol and Results of the Clinical Trial
The study protocol was approved by the review board for research on human subjects at National Shikoku Cancer Center, Japan. Written informed consent was obtained from each patient.
Patients of viral hepatitis with cirrhosis (more than 90% of these individuals were hepatitis C virus-infected patients) were randomly divided into two groups (Table 1). Patients in group 1 (46 patients) were administered the carotenoid mix- ture in addition to conventional antisymptom- atic treatment. The daily dose of carotenoids was 20 mg in total. The carotenoid mixture contained lycopene 10 mg, β-carotene 6 mg, α-carotene 3 mg, and other small portions of carotenoids such as phytoene and phytofluene, 1 mg. These carotenoids were packed into capsules with α-to- copherol (daily dose, 50 mg). Patients in group 2 (46 patients as the control group) were treated with antisymptomatic treatment alone. Placebo was not used in this study.
These groups were followed up for 2–5 years (3.4 years on average), and hepatocellular carci- noma development was clinically analyzed and recorded. Cumulative incidence of hepatocellu- lar carcinoma development was plotted using the Kaplan-Meier method and compared between the two groups. In this plotting, one patient in group 2 was omitted, since this patient devel- oped liver cancer within 6 months after the entry in the clinical trial.
In the 4th annual analysis, a significant reduc- tion of hepatocellular carcinoma development was observed in group 1 (carotenoid group), as shown in Fig. 1. A greater than 50% reduction of hepatocellular carcinoma incidence was found at 4 years in the group receiving carotenoids, i.e., cumulative incidence of hepatocellular carci- noma in group 1 was 12.3%, while that in group 2 was 34.6% (p<0.02).
Since the effect of the carotenoid mixture treatment was proven to be significant in the annual analysis at year 4, the clinical trial was fi- nalized at this time point, although the duration of the carotenoid administration in the original protocol was scheduled to last an additional 3 years.
Improvement of Cancer Preventive Efficacy by Additional Use of Other Food Factors
Besides hydrocarbon carotenoids, some xantho- phylls such as β-cryptoxanthin are also found to be lower in the blood of liver cancer patients with viral hepatitis than in healthy individuals (Jinno et al. 1997). Therefore, additional use of β- cryptoxanthin may improve the efficacy for liver cancer prevention.
We have also found experimentally that myo- inositol may be useful for the prevention of liver carcinogenesis. Since myo-inositol has been known to prevent fatty liver as well as cancer in several organs, such as colon, we evaluated the effect of oral administration of myo-inositol us- ing the experimental model of spontaneous liver carcinogenesis in C3H/He male mice (Nishino et al. 1999). The mean number of liver tumors was significantly decreased by myo-inositol treat- ment as compared with that in the control group;
the control group developed 7.82 tumors/mouse, whereas the 1% myo-inositol-treated group had 0.77 tumors/mouse (p<0.01, Student’s t-test).
Based on these clinical and experimental data, we have planned a new clinical trial. Since β-cryptoxanthin is rich in Japanese tangerine oranges, we decided to use them as a form of juice for a clinical trial. The experimental sample contained 3 mg of β-cryptoxanthin and 1 g of myo-inositol in 190 ml canned juice. Hepatitis C virus-infected patients with cirrhosis were ad-
Table 1 Subjects for clinical trial
Group N Age AST (GOT)
IU/l
ALT (GPT) IU/l
Platelet(x104/μl) Observation period (years)
1 46 61.4 ± 9.7 75 ± 44 79 ± 51 10.1 ± 4.1 3.4 ± 1.5
2 46 59.9 ± 8.9 70 ± 37 67 ± 46 10.4 ± 4.9 3.3 ± 1.4
Total 92 60.6 ± 9.3 73 ± 41 73 ± 41 10.3 ± 4.5 3.4 ± 1.5
Mean ± SD
ministered this preparation (one can per day), in addition to capsules of a carotenoid/ α-tocoph- erol mixture. In the annual analysis at year 1, none from treated group (n=30) had developed hepatocellular carcinoma. This clinical trial is now on-going.
Analysis of the Mechanism of Action
Carotenoids are well known as natural antioxi- dants and have potent activity to scavenge free radicals. Since inflammation is accompanied
with excess production of free radicals, the scavenging activity of carotenoids against free radicals seems to play an important role in pre- vention of hepatitis virus infection-related liver carcinogenesis. In addition to the free radical scavenging activity, carotenoid-induced modula- tion of the expression of various genes also seems to be important. For example, lycopene has been found to increase the promoter activity of anti- oncogenes, such as the RB gene and the Waf1 gene (Table 2).
Discussion
Interferon is well known as an effective treat- ment for hepatitis patients and has been proven to prevent hepatocellular carcinoma. However, large numbers of patients are nonresponders for interferon treatment. Although repeating admin- istration of interferon is recommended for these cases, it is not always possible to continue. These patients are a high-risk group for future devel- opment of hepatocellular carcinoma, and alter- native treatments should be tried. In such cases, administration of a carotenoid mixture seems to be valuable.
6 0 4
8 3
6 2
4 1
2 0
0 1 0 2 0 3 0 4 0 5 0
Time (month) (%)
Control group Carotenoid group
Fig. 1 Effect of carot- enoids/ α-tocopherol mixture on hepatocellular carcinoma development in chronic viral hepatitis patients with cirrhosis.
Cumulative incidence (%) of hepatocellular carcinoma was plotted using the Kaplan-Meier method and compared between two groups: the carotenoid-treated group and the control group
Table 2 Effect of lycopene on promoter activity of anti- oncogenes
Anti-oncogene Concentration of lycopene
Induction (fold increased)
RB 2 μM 1.42
5 μM 3.32
10 μM 4.04
20 μM 4.81
Waf1 10 μM 3.49
Lycopene was treated for 24 h.
At present we have selected lycopene, β-caro- tene, α-carotene, and β-cryptoxanthin as the major components for the carotenoid mixture.
In addition to these elements, other natural ca- rotenoids such as zeaxanthin, astaxanthin, and fucoxanthin may also be valuable for clinical use, since these carotenoids showed potent activity in reducing liver carcinogenesis in experimental studies, as shown in Table 3.
In the present study, mixture of carotenoids was packed into capsules with α-tocopherol. α- Tocopherol, a natural antioxidant, was used as a stabilizer for the carotenoid mixture, which is highly sensitive to oxidative stress. In addi- tion, α-tocopherol itself has been proven to have some preventive activity for liver carcinogenesis (Tsuda et al. 1997). Recently, it was also found
that tocotrienols, the family of natural vitamin E distributed in various foods with tocopherols, showed potent preventive activity against liver carcinogenesis (Wada et al. 2005). Therefore, combinational use of tocotrienols may be useful for the improvement of efficacy.
Herbal medicine, Sho-saiko-to (TJ-9), has also been reported to prevent hepatocellular carcinoma (Oka et al. 1995). It is of interest that glycyrrhizin, an active component in Sho-saiko- to, was proven experimentally to have preventive activity against liver carcinogenesis (Table 4).
For the mechanism of action analysis, genom- ics and proteomics seem to be valuable. In fact, comprehensive analysis using DNA array and protein-antibody array has shown the network- like modulation of cellular regulatory systems.
Table 3 Effect of carotenoid on spontaneous liver carcinogenesis in C3H/He male mice
Experiment n Percentage of Tumor-
bearing mice
Mean number of tumors/mouse
(Inhibition %)
Exp. 1
Control 14 35.7 1.75
+Zeaxanthin 12 8.3 0.08 (95.4)
Exp. 2
Control 15 53.3 0.87
+Astaxanthin 15 26.7 0.27 (69.0)
Exp. 3
Control 15 100 5.93
+Fucoxanthin 15 86.7 3.07 (48.2)
Exp. 1: Zeaxanthin (0.005% in drinking water) was given during the entire experimental period (40 weeks).
Exp. 2: Astaxanthin (0.2 mg suspended in 0.2 ml of corn oil, intragastric gavage, three times per week) was given during the entire experimental period (40 weeks).
Exp. 3: Fucoxanthin (0.001% in drinking water) was given during the entire experimental period (40 weeks).
Table 4 Effect of glycyrrhizin on spontaneous liver carcinogenesis in C3H/He male mice
Exp. n Percentage of tumor-
bearing mice
Mean number of tumors/mouse
(Inhibition %)
Control 15 80.0 3.07
+Glycyrrhizin 15 73.3 1.40 (54.4)
Glycyrrhizin (0.005% in drinking water) was given during the entire experimental period (40 weeks).
For example, DNA array analysis for β-crypto- xanthin showed the significant induction of ex- pression in various important genes, such as p16 and p73, and proteome analysis for tocotrienol revealed the increase in pRB2 protein induction (data, not shown). It is of interest that the pat- tern of modulation for DNA and protein expres- sion has been proven to be unique and specific for each carotenoid. These results support the combined administration of these carotenoids against cancer as a reasonable strategy.
Conclusion
In conclusion, a mixture of various carotenoids seems to be promising for the prevention of liver cancer in hepatitis virus-infected patients with cirrhosis, although a further extended clinical trial is needed to confirm and improve the effi- cacy of this method.
Acknowledgements
This study was carried out in collaboration with Dr. K. Jinno, Dr. H. Ishikawa and the research groups of Kyoto Prefectural University of Medi- cine, National Cancer Center Research Institute, National Shikoku Cancer Center, Fruit Tree Research Institute, and Meiji Pharmaceutical University. This work was supported in part by grants from the Program for Promotion of Basic Research Activities for Innovative Biosciences (ProBRAIN), the Ministry of Agriculture, For- estry, and Fisheries, the Ministry of Education, Culture, Sports, Science and Technology, and the Institute of Free Radical Control (IFRC), Japan.
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