Regardless of the significant global consumption of dietary or medicinal mushrooms, only four epidemiological studies suggested chemopreventive effects of mushrooms against gastric, gastrointestinal and breast cancer, respectively 
. In the present study, we evaluated anti-cancer and anti-inflammatory activities of a triterpene extract from medicinal mushroom Ganoderma lucidum
(GLT) in an animal model of the food-borne carcinogen (PhIP) and inflammation (DSS) induced colon carcinogenesis. Our data clearly supports our hypothesis that GLT prevents and suppresses both colon carcinogenesis and colon inflammation in ICR mice exposed to PhIP and DSS. Previous study using uncharacterized water soluble extract from cultured medium of G. lucidum
mycelia (MAK), demonstrated that MAK in the diet reduced the size but not the amount of colon tumors induced by N,N′-dimethylhydrazine (DMH) in ICR mice 
. Therefore, our study is the first to demonstrate that chemically characterized extract from mushroom G. lucidum
, GLT, suppresses the number of colon tumors induced by the food-borne carcinogen and inflammation.
As recently demonstrated, an induction of preneoplastic lesions in rat colon by PhIP was not preceded or accompanied by an inflammatory process 
, whereas the incidence and multiplicity of dysplastic lesions were promoted by the addition of DSS 
. Indeed, here we demonstrate that focal hyperplasia induced by PhIP was further accelerated by DSS. Most importantly, GLT suppressed colonic hyperplasia induced by the combination of PhIP and DSS in a dose response manner, further confirming the role of GLT in the prevention of colon carcinogenesis. The inhibition of hyperplasia was also confirmed by the suppression of the proliferative marker Ki-67 in colonic tissue in mice treated with GLT. In addition, we have found that although PhIP induced ACF, DSS-initiated inflammation further increased the amount of ACF in mice treated with PhIP and DSS. In agreement with the study using an extract from G. lucidum
mycelia MAK 
, GLT inhibited ACF formation in ICR mice in a dose-responsive manner.
Increased mortality, in our pilot and preventive studies, could be caused by the toxicity of DSS and by the additional effect of GLT. Because DSS (inflammation) itself induces mice mortality 
, and all death animals (but 1 in the pilot and 2 in the preventive control groups) were exposed to DSS, inflammation could be responsible for this increased mortality. Moreover, COX-2 knock-out mice, specifically in myeloid and endothelial cells but not epithelial cells, are more sensitive to DSS 
, and GLT treatment markedly decreased expression of COX-2 in mice treated with DSS and PhIP in our study. Therefore, it is possible that GLT inhibition of COX-2 further sensitize mice to DSS, resulting in the increased mortality in the PhIP/DSS and PhIP/DSS+GLT treatment groups. In addition, as in our both studies, we have observed an increased aggressive behavior in the gavaged mice.
Here, we also demonstrate anti-inflammatory effects of GLT in the PhIP/DSS-model of colon carcinogenesis. DSS-induced inflammation/colitis is associated with the shortening of the large intestine in DSS treated animals 
, and we observed the same effect in the PhIP/DSS treated animals. However, GLT prevented the shortening of colon in PhIP/DSS treated animals to the colon length comparable to control animals, suggesting anti-inflammatory effect of GLT in the PhIP/DSS model of colon carcinogenesis.
The tumor microenvironment contains stromal cells such as fibroblasts, endothelial cells, and macrophages. An association of chronic inflammation, which involves macrophages, with cancer initiation and promotion and the reduction of cancer risk by treatment with anti-inflammatory drugs was recently described 
. As mentioned above, increased infiltration of macrophages was previously detected in the DSS-dependent colon inflammation 
. Our data demonstrate that PhIP/DSS induce the amount of infiltrating macrophages whereas GLT treatment significantly reduced their amount. Therefore, in addition to the inhibition of colon carcinogenesis, our data suggest that GLT exerts its effect through the inhibition of inflammation and by the depletion of infiltrating macrophages from colon tissue. Moreover, we have recently demonstrated direct anti-inflammatory activity of GLT in LPS-stimulated macrophages in vitro
and in LPS-challenged mice in vivo
. Hence, our data are in agreement with previous study by Loddenkemper et al.
demonstrating that ursodeoxycholic acid, another triterpene with anti-inflammatory properties, prevented colitis-associated carcinogenesis in mice 
The overexpression of cyclin D1 and COX-2 in colon tissues in animals treated with PhIP/DSS was markedly reduced by the GLT treatment. We have previously demonstrated that an extract of mushroom G. lucidum
, which contains 6% of GLT, suppressed cyclin D1 expression and NF-κB activity in human breast cancer cells 
. Moreover, GLT down-regulated expression of COX-2 and suppressed activation of AP-1 and NF-κB in stimulated macrophages 
. Therefore, it is plausible to hypothesize that GLT down-regulates the expression of cyclin D-1 and COX-2 in vivo
by inhibiting AP-1 and NF-κB activity. Indeed, recent study demonstrated that euphol, a natural triterpene from the Euphorbia tirucalli
plant, inhibits NF-κB activity in colon tissue from mice the DSS-induced colitis 
As previously demonstrated, polysaccharides isolated from G. lucidum
, induced total P450 levels in rat liver, whereas suppressed the activity of CYP1A2, CYP3A and CYP2E1 in hepatic microsomes 
. Our results indicate that GLT is able to induce XRE/AhR and PXR which regulate expression of CYP1A2 and CYP3A, respectively. Because CYP1A2 is involved in the metabolism of PhIP 
, it is possible that GLT-induced increase in the expression of CYP1A2, could contribute to the elimination of this carcinogen. Moreover, PXR can be also responsible for the GLT-induced down-regulation of expression of cyclin D1. Since the ligand bound PXR heterodimerizes with the retinoid X receptor (RXR) and this interaction is required for an induction of the gene expression 
, the depletion of the RXR resulted in a decrease in cyclin D1 expression 
. Therefore, the decrease in cyclin D1 expression by GLT treatment could be caused by the interaction between PXR, GLT and RXR. Moreover, the expression of PXR regulated genes are decreased in inflammatory bowel disease 
, suggesting that GLT induced PXR could account of decreased inflammation seen in our study. It is not unusual that GLT is able to activate both PXR and AhR mediated gene expression since Omeprazole (a dyspeptic drug) is able to activate both PXR and AhR (J. Lamb, personal communication), and a “cross-talk” between PXR and AhR has been described 
. The PhIP/DSS-induced expression of CYP1A2 in mice is an agreement with previous study demonstrating increased expression of this enzyme by PhIP in rat 
. Although the metabolism of PhIP by CYP1A2 differs between human and rodents, the inhibition of a PhIP/DSS-dependent expression of CYP1A2 by GLT can reduce the amount of carcinogenic PhIP metabolite N-hydroxy-PhIP in colon tissue 
. Since, Curcuma drugs suppressed activity and expression of CYP3A4 in intestinal epithelial cells 
, our observation that GLT down-regulates PhiP/DSS-dependent expression of CYP3A4 in vivo
, further confirms cancer preventative activities of GLT.
In our studies we used GLT concentrations up to 500 mg/kg of body weight 3 times per week, which would correspond 90–120 g of GLT for 60–80 kg per person per week (12.9–17.1 g/day). Since the amount of biologically active mushroom triterpenes (GLT) depends on the source and extraction procedures, the consumption of 1–2 servings (100–200 grams) of fresh or dried mushrooms could be sufficient to reach these concentrations. In addition, daily intake of fresh (≥10 g) or dried (≥4 g) mushrooms significantly decreased the risk of breast cancer 
. In summary, our data demonstrate, for the first time, that GLT prevented and inhibited colon carcinogenesis in mice which was induced by the food-borne carcinogen and inflammation. Moreover, GLT also suppressed colon inflammation, reduced the amount of infiltrating macrophages and down-regulated expression of cyclin D1 and COX-2, and suppressed PhIP/DSS-induced expression of CYP1A and CYP3A in colon tissue. Taken together, GLT could be considered as potential natural agent for the prevention and suppression of colitis-associated colon carcinogenesis.