In this study, we have demonstrated that BOP induced multiple tumours in the liver. The incidence (100%) and pattern of tumours in male BOP-treated livers were consistent with those described previously (22
). The survival rate of the BOP-alone group was 100%. We attribute this phenomenon to the fact that because the BOP-induced HCC is designed to study the early stage, not the late stage of carcinogenesis, all animals in the BOP-alone group lived with the disease (foci) until sacrifice. Sil, Sel as well as the Sb/Bs remedy reduced the number of tumour foci and tumour areas in such a model. To our knowledge, this is the first evidence indicating that the herbal medicine with or without Sel has chemopreventive effects by reducing tumour foci and inducing cell apoptosis. Although the incidence of HBV-related HCC is high in Taiwan, the in vivo
HBV-related HCC model needs 1 year or longer to produce liver tumours compared with the BOP-induced model time scale of 2 months. As a result of this, we used the BOP model to study the chemoprevention effect of herbal medicines on hepatocarcinogenesis. Strong nuclear staining, indicative of a high level of DNA alkylation, was observed at all time points in the livers of BOP-induced carcinogenesis, which has been shown to be related to a high tumour incidence (17
). It is of note that no obvious hepatocyte necrosis was noticed in our model; we attribute this phenomenon to BOP being an ROS-inducing agent that causes free radical damage rather than ischaemic or reperfusion injury. Furthermore, 8OH-dG, a free-radical damaging indicator, was detectable in BOP-induced livers. The Sb/Bs remedy, in combination with Sel, ameliorated 8-OHdG peroxidant production in the nuclei of GST-P-positive foci, validating the methods we used.
Placental-type glutathione S
-transferase has been reported to be unexpressed in normal hepatocytes, while it is strongly expressed in hepatoma cells and initiated cells that occur at a very early stage of chemical hepatocarcinogenesis (23
). The medicinal herbs with or without Sel reduced GST-P expression in BOP-induced HCC. Besides, the Sb/Bs remedy, combined with Sel increased caspase-3 and, hence, the apoptotic change of BOP-induced liver tumours. Taken together, the results suggested that Sb/Bs remedy, combined with Sel, may have chemopreventive effects on BOP-induced hepatocarcinogenesis.
Selenium in the form of selenocysteine plays an important role in many biological functions ranging from antioxidant protection and metabolism to proper reproductive performance (25
). A study of the preventive role of Sel has attributed its effect to immune modulation, but other molecular mechanisms may also be involved in achieving the treatment outcome, including a significant reduction in the ROS (10
). Sel, combined with Sb/Bs remedy, significantly reduced the number of tumour foci compared with selenium alone, indicating that the combination therapy might have a synergic effect on chemoprevention and immunoprevention. Although the flavonoids in medical herbs have anticancer effects and anti-oxidant activity, chemoprevention properties have scarcely been investigated till now (26
Although Shirai et al.
) reported that serum TGF-β1 in patients with HCC was higher than that in chronic hepatitis and liver cirrhosis, there is evidence indicating that serum TGF-β1 level is not well correlated with HCC. For example, Ali et al.
) reported that serum TGF-β1 was significantly increased in chronic hepatitis and liver cirrhosis groups as compared with that in HCC and control groups (P
<0.001), while there was no significant difference between TGF-β1 in HCC and control groups (P
> 0.05). Furthermore, TGF-β1 is shown to induce apoptosis in several human HCC cell lines (30
). Chabicovsky et al.
) suggested that during chemically induced liver carcinogenesis in B6C3F1 mice, basal rates of apoptosis in adenoma and carcinoma are higher than that in normal liver and can be further increased by direct injection of TGF-β1 into the tail vein. Carillo et al.
demonstrated that IFN-α2b administration significantly decreased both the number and the volume percentage of altered hepatitis foci by an induced programmed cell death in the foci. This apoptotic effect of IFN-α2b on preneoplastic liver foci was mediated by the production of endogenous TGF-β1 from hepatocytes acting by a paracrine/autocrine way (32
). Taken together, we suggested that Sb, Bs and their combination could significantly increase serum TGF-β1 level and cell apoptosis in tumour foci.
Significantly decreased body weight, daily activity and ascites were observed in the Sb-treated groups compared with the normal and vehicle groups. We attributed the above-mentioned findings to the fact that S. baicalensis
could induce TNF-α secretion, a strong factor inducing cachexia in the animals (33
). The chemical ingredients of Sb, baicalein, baicalin and wogonin, might be effective candidates for inducing apoptosis or inhibiting proliferation in various human HCC cell lines (34
). Previously, Sb has been found to inhibit cancer cell growth in vitro
and in vivo
and could be an effective chemotherapeutic agent (35
). In addition, baicalein decreases the 8-OH-dG content, which acts as a DNA damage marker, suggesting that the protective effect of baicalein against the cytotoxicity and genotoxicity of hepatocytes is because of its ability to quench free radicals (36
). Baicalin exhibits an anti-inflammatory effect in vivo
and in vitro
, markedly reducing serum aminotransferase activities, protecting hepatoycte apoptosis (37
). Furthermore, Saikosaponin-a and Saikosaponin-d, ingredients of Bs, have been reported to induce cell apoptosis through the caspase-3-dependent and -independent pathways in HCC cells. Besides, Saikosaponin-c exhibits anti-HBV activity and saikosaponin-d possesses potent cytotoxicity against human HCC cells (38
Herbal combinations are made because of their properties of increasing apoptosis of tumour foci and decreasing the side effect of each herb alone or Sel alone. The concept of herbal combination may be applicable in clinical aspects, low-dose Sel with lessened Sb (30%) and Bs (70%) proportional dose in the Sb/Bs remedy, to elevate the survival rate and chemopreventive tumorigenesis effect of the HCC-treated group. Meanwhile, similar to chemotherapy in clinical settings, the data in this study form a good basis for a chronic study of the single compound/herb or combination therapy. It is possible that in a chronic study, the survival rate of the untreated group will be significantly lower than that in the treated groups. A long-term use of a proper ratio and dose of chemopreventive supplement combination must be sufficient to support the safe use of the therapy in a clinical scenario.
Although the model used in this study does not completely represent the human HCC development, both cancers share a common mechanism, oxidative DNA damage, which is the therapeutic target of the present study. Thus, the results of the present study may provide some clues regarding human HCC development and prevention. Besides, the agents used in the therapy groups could induce apoptosis of tumour foci and might elicit a drug effect similar to those observed in chemotherapy clinically. Accordingly, the preventive strategy has to be investigated in other HCC models before study in humans, so that the potential harmful effects of such a remedy could be expected or prevented. We used the mixture formula composed of herbal medicines that could increase the effect of treatment and decrease the side effect of a single herb as the basis of cocktail therapy. Consequently, this concept may be applicable for Sel with an Sb/Bs remedy combination and could be advantageous in the chemoprevention effect during the progression of liver carcinogenesis.