Effect and Mechanism of Ginkgo Biloba Extract on Hepatocarcinogenesis Induced by Aflatoxin B1 in Rats
|School||Guangxi Medical University|
|Keywords||Primary liver cancer Aflatoxin B1 Ginkgo biloba extract Rats Biomarkers Cell Cycle and Related Factors Apoptosis and associated factors HepG2 cells Apoptosis Bcl-2/Bax|
Background and ObjectivePrimary hepatic carcinoma（PHC）is the fifth most common cancers and the third leading cause of cancer death worldwide.About 18.8%of total fatalities caused by malignant tumor are PHC in China,and PHC is the most common cancer in Guangxi province.Aflatoxin B1（AFB1）is a potent hepatotoxic and hepatocarcinogenic agent. It is produced by mould Aspergillus flavus,and can contaminate cereal grains and nuts in tropical regions.AFB1 has been categorized as one of the human carcinogens by International Agency for Research on Cancer（IARC）.It is thought to be largely responsible to the high incidence of PHC in southeast China and southern Africa.AFB1 is biotransformed by cytochrome P450 into its reactive intermediate AFB1-exo-8,9-epoxide（AFBO）to exert its carcinogenic effect.AFBO is capable of reacting with DNA,resulting in the production of DNA damage.It is generally believed that theγ-glutamyl transpeptidase（γ-GT） positive preneoplastic focus or nodule,the AFB1-DNA and the AFB1-albumin adduct,the alpha-fetoprotein（AFP）and 8-hydroxydeoxyguanosine（8-OHdG） are the most reliable biomarkers related to PHC development.Chemoprevention is a pharmacological approach to arrest or reverse the process of carcinogenesis.Since the complete elimination of exposure to AFB1-producing moulds is not possible,chemoprevention becomes an attractive strategy to protect individuals from the risk of PHC caused by exposure to AFB1.Chemoprotective strategies designed to limit both exposure to and the adverse health effects from AFB1 are important public health goals for reducing the incidence of AFB1-induced neoplastic diseases.Ginkgo is one of the oldest living species of trees on earth,therefor is called as "living fossil".Ginkgo biloba leaf extract,standardized extract from the leaves of the Ginkgo biloba tree and labeled as EGb761,includes 24% flavonoid glycosides and 5%～7%terpene trilactones which is uniquely owned by Ginkgo.The study of EGb761 effect on cardiovascular disease and cerebrovascular disorder has been expanded to many other fields.Recent studies have revealed that EGb761 may have anticancer properties that are related to its antioxidant,anti-angiogenic and gene-regulatory abilities.However,so far the effect of EGb761 as a chemopreventive agent on hepatocarcinogenesis induced by AFB1 has not been evaluated.The present study aims to investigate whether EGb761 has the effect on preventing hepatocarcinogenesis induced by AFB1 in rat,and to explore what is the underling mechanism.MethodsSeventy-one male Wistar rats,aged four weeks,were divided into three groups.In group AFB1,28 rats were injected AFB1 intraperitoneally for 46 weeks.In group EGb761,29 rats were exposed to AFB1 as what done in group AFB1 and fed EGb761-containing foodstuff simultaneously.EGb761 concentration in foodstuff was 2g/Kg.In group control,14 rats were raised normally,neither treated with AFB1 nor with EGb761.Blood and biopsied liver samples were collected from all animals in each group at the experimental week of 14th,28th,42thand 55th.The animal experiment ended at 64th-week when the surviving animals were sacrificed by cervical dislocation.The serum specimens were stored at -40℃for testing AFP by ELISA and AFB1-lysine adduct by HPLC.Fresh liver tissues were used for prepating microsomal and cytosolic fractions which then applied to assay the hepatic drug-metabolizing enzymes. Parts of the remaining liver tissue samples from each animal were snap-frozen in liquid N2 then stored at -80℃for FCM and RT-PCR assay,the other parts were acetone-alcohol-fixed or formalin-fixed and then paraffin-embedded,forγ-GT, HE or immunohistochemistry staining respectively.FCM assay was done on the samples collected at 64th-week to analyze cell-cycle and apoptosis.RT-PCR assay was done to test the mRNA expression levels of C-myc and cyclin D1. The immunohistochemistry staining was done to test the protein expression levels of C-myc,cyclin D1,survivin,Bcl-2,Bax and 8-OhdG.Results1.Carcinogenesis induced by AFB1The first case of malignant tumor was discovered at 46th-week and the first case of PHC was discovered at 51th-week.At the end of experiment(64th-week), 25 rats were valid in group AFB1 among which 20 cases of tumor（80.0%） occurred,including 17 cases of hepatocellular carcinome（HCC）,2 cases of cholangiocarcinoma and 1case of fibrosarcoma.The incidence of PHC in group AFB1 was 76.0%（19/25）.The valid rats in group EGb761 at the end of experiment were 26 among which 8 cases of tumor（30.8%）occurred,including 5 cases of hepatocellular carcinome（HCC）,2 cases of cholangiocarcinoma and 1 case of renal carcinoma.The incidence of PHC in group EGb761 was 26.9% （7/26）.No tumor developed in the control animals.2.Effect of EGb761 on AFB1 inducingγ-GT fociThe size ofγ-GT positive foci（mm2/focus）and the total area ofγ-GT positive foci（mm2/cm2）in the liver samples collected at 42th-week were 7.95±0.30 and 2.54±0.05 in group AFB1,4.65±0.16 and 0.97±0.03 in group EGb761,respectively.In the liver samples collected at 55th-week,they were 17.87±0.71 and 4.68±0.12 in group AFB1,9.03±0.35 and 1.62±0.06 in group EGb761,respectively.All these numbers were significantly lower in group EGb761 than these in group AFB1（all P=0.000）.The amount of positive foci per square centimeter（number/cm2）in group EGb761 was significantly lower than that in group AFB1 at 42th-week（P=0.004）.No focus was found in animals from group control.3.Effect of EGb761 on serum AFPThe level of AFP（ng/ml）in the serum samples collecte at week of 28th,42th and 64thwere 120.63±4.35,242.75±5.93 and 421.66±6.79 in group AFB1, 136.72±3.94,131.15±4.85 and 349.49±5.73 in group EGb761,84.33±6.26, 121.35±6.70 and 245.34±12.61 in group control,respectively.At the week of 42thand 64th,the levels of serum AFP in group EGb761 and group control were significantly lower than that in group AFB1（all P＜0.05）.4.Effect of EGb761 on serum AFB1-lysine adductThe maximum AFB1-lysine adduct level（4356.01 pg/mg albumin）was found at 14th-week in group AFB1.EGb761 strongly inhibited the formation of AFB1-lysine adducts in serum,with the inhibition rates as 13.07%at 14th-week （P=0.033）,73.63%at 42th-week（P=0.002）and 100%at 64th-week（P= 0.038）.5.Effect of EGb761 on liver drug enzyme5.1 Effect of EGb761 on the total content of CYP450Levels of the total content of CYP450（nmoL/mgprot）were significantly increased after AFB1 treatment in group AFB1,which were 0.21±0.01, 0.40±0.01,0.45±0.01 and 0.45±0.01 at week of 14th,28th,42thand 64th, respectively.The maximum level was reached at 42th-week and a constant high level was maintained thereafter.In group EGb761,the contents of CYP450 were 0.21±0.01,0.37±0.01,0.45±0.01 and 0.34±0.01 at the same time points as mentioned above,respectively.The contents of CYP450 were lower in group EGb761 than those in group AFB1,but the differences between the two groups were not statistically significant（P=0.085）.The contents of CYP450 in group control were 0.22±0.01,0.34±0.03,0.21±0.02 and 0.30±0.02 at the four time points,respectively.5.2 Effect of EGb761 on GST activityGST activity（U/mgprot）in rat liver tissues collected at week of 14th,28th, 42thand 64thwere 31.50±0.81,44.13±0.90,21.52±0.40 and 16.91±0.41 in group AFB1,13.33±0.29,29.62±0.52,22.79±0.31 and 19.67±0.40 in group EGb761, 18.03±0.74,24.36±1.10,26.68±0.59 and 22.62±0.87 in goup control, respectively.GST activity in rat liver tissues in group AFB1 was significantly higher those in group EGb761 and group control at week of 14thand 28th（P＜0.01）.There were no statistically significant differences among the three groups at week of 42thand 64th（P＞0.05）.5.3 Effect of EGb761 on GSH-Px activityGSH-Px activity（unit of activity）in rat liver tissues collected at week of 14th,28th,42thand 64thwere 28.13±0.75,70.76±1.36,49.10±0.82 and 27.28±0.46 in group AFB1,14.28±0.28,45.25±0.90,47.23±0.74 and 39.71±0.84 in group EGb761,17.77±0.79,22.06±0.89,34.11±0.87 and 30.23±1.02 in group control,respectively.GSH-Px activity in rat liver tissues in group AFB1 was significantly higher than those in group EGb761 and group control at week of 14thand 28th（P＜0.01）,but significantly lower than that in group EGb761 at 64th-week（P=0.008）.There was no statistically significant difference between group AFB1 and group EGb761 at 42th-week（P＞0.05）.The levels in both group AFB1 and group EGb761 at 64th-week were not singnificanly higher than that in group control（P=0.078）.5.4 Effect of EGb761 on SOD activitySOD activity（U/mgprot）in rat liver tissues collected at week of 14th,28th, 42thand 64thwere 43.85±1.06,36.25±0.69,31.67±0.76 and 19.46±0.35 in group AFB1,21.98±0.42,22.93±0.42,43.41±0.82 and 42.28±0.66 in group EGb761, 28.67±1.05,24.71±0.95,34.57±0.98 and 35.09±0.68 in group control, respectively.SOD activity in rat liver tissues in group AFB1 was significantly higher than those in group EGb761 and group control at week of 14thand 28th（P＜0.05）,and singnificantly lower than those in group EGb761 at week of 42th and 64th,respectively（P＜0.05）.There were no statistically significant differences between group EGb761 and group control at week of 42thand 64th（P＞0.05）.5.5 Effect of EGb761 on formation of MDA in liverThe formation of MDA（nmoL/mgprot）in rat liver tissues collected at week of 14th,28th,42thand 64thwere 0.27±0.01,0.66±0.01,0.56±0.01 and 0.93±0.02 in group AFB1,0.14±0.01,0.24±0.01,0.36±0.01 and 0.60±0.01 in group EGb761,0.16±0.01,0.27±0.01,0.43±0.01 and 0.84±0.03 in group control,respectively.The formation of MDA in rat liver tissues in group EGb761 was significantly lower than that in group AFB1 at any one of the four time points（P＜0.05）,and significantly lower than that in group control at 64th-week（P=0.044）.6 Effect of EGb761 on the expression of 8-OHdG proteinThe expression of 8-OHdG protein in rat liver tissues collected at week of 28th,42thand 64thin group EGb761 were significantly lower than that in group AFB1（P＜0.05）.At these three time poins,the expression levels in group control were also significantly lower than that in group AFB1（P＜0.05）.7 Effect of EGb761 on cell cycle and the related factors7.1 Effect of EGb761 on cell cycle of rat liver tissuesThe cell percentage（%）of rat liver tissues distributed among the three phases of cell cycle（G0/G1,S and G2/M）were 67.08±2.32,22.90±4.62 and 12.06±4.20 in group AFB1,79.44±3.11,14.70±3.04 and 6.10±1.68 in group EGb761,90.02±1.53,5.70±1.86 and 4.28±2.41 in group control,respectively.In group EGb761,the cell percentage in phase G0/G1 was significantly increased than that in group AFB1（P＜0.05）,while the percentage in phase S was significantly lower than that in group AFB1（P＜0.05）.7.2 Effect of EGb761 on the expression of cyclin D1 in rat liver tissuesThe expression of cyclin D1 mRNA in rat liver tissues collected at week of 28th,42thand 64thwere 2.07±1.24,0.76±0.40 and 3.64±2.74 in group AFB1, 0.76±0.22,0.82±0.37 and 1.99±0.99 in group EGb761,0.79±0.27,0.62±0.11 and 0.99±0.49 in group control,respectively.The expression of cyclin D1 mRNA in group EGb761 at week of 28thand 64thwere significantly lower than that in group AFB1（P＜0.05）,but not significantly different from those in group control（P＞0.05）.There were no statistically significant differences among the three groups at 42th-week（P＞0.05）. The expression of cyclin D1 protein determent by immunohistochemistry was increased from the start to the end of experiment,especially in group AFB1. The expressions of cyclin D1 protein in group EGb761 at week of 28th,42thand 64thwere significantly lower than that in group AFB1（P＜0.05）.There was no statistically significant difference between group EGb761 and group control at week of 28th,42thand 64th（P＞0.05）.7.3 Effect of EGb761 on the expression of C-myc gene in rat liver tissuesThe expression of C-myc mRNA in rat liver tissues collected at week of 28th,42thand 64thwere 0.24±0.15,0.35±0.17 and 0.64±0.57 in group AFB1, 0.12±0.08,0.30±0.1 and 0.41±0.20 in group EGb761,0.05±0.02,0.03±0.02 and 0.05±0.03 in group control,respectively.The expressions of C-myc mRNA in group EGb761 at week of 28thand 64thwere significantly lower than those in group AFB1（P＜0.05）,but not significantly different from those in group control（P＞0.05）.At 42th-week,there was no statistically significant difference between group EGb761 and group AFB1（P＞0.05）,but the expressions in these two groups were significatly higher than that in group control（P＜0.05）.The expressions of C-myc protein in group EGb761 at week of 28th,42th and 64thwere significantly lower than that in group AFB1（P＜0.05）.There was no statistically significant difference between group EGb761 and group control at any one of the three time points（P＞0.05）.8 Effect of EGb761 on cell apoptosis of rats liver and the related factors8.1 Effect of EGb761 on cell apoptosis of rats liverAt the end of experiment,the apoptosis ratio（%）of liver cell was 0.36±0.15 in group AFB1,9.26±2.78 in group EGb761,and 1.44±0.58 in group control.The apoptosis ratio of group EGb761 was significantly higher than that in group AFB1 and in group control（P＜0.05）. 8.2 Effect of EGb761 on the expression of Survivin protein in rat liver tissuesThe expressions of survivin protein in group EGb761 at week of 28th,42th and 64thwere significantly lower than those in group AFB1（P＜0.05）.There was no statistically significant difference between group EGb761 and group control at any one of the three time points（P＞0.05）.8.3 Effect of EGb761 on the expression of Bcl-2/Bax protein in rat liver tissuesThe expression of Bcl-2 protein in group EGb761 at 42th-week was significantly lower than that in group AFB1（p=0.000）.There was no statistically significant difference between group EGb761 and group AFB1 at week of 28thand 64th（P＞0.05）.The expressions of Bcl-2 protein in group AFB1 at week of 28thand 64thwere significantly higher than those in group control（P＜0.05）.The expressions of Bax protein in group EGb761 at the week of 42thand 64thwere significantly higher than those in group AFB1（P＜0.05）.The expression of Bax protein in group control at these two time points were also significantly higher than those in group AFB1（P＜0.05）.There was no statistically significant difference among the three groups at 28th-week（P＞0.05）.Conclusions1.EGb761 can reduce the formation ofγ-GT positive foci in rat liver and AFB1-lysine adducts in rat serum,decrease the production of MDA and the formation of 8-OhdG,increase GSH-Px and SOD activity.Finally,EGb761 can prevent or postpone the progress of hepatocarcinogenesis induced by AFB1 in rats.2.EGb761 can inhibit the expression of cyclin D1 and C-myc,at both levels of mRNA and protein.These might be parts of the mechniam underling the anticancer effects of EGb761 on cell proliferation.3.During hepatocarcinogenesis induced by AFB1 in rat,EGb761 singnificantly down-regulated the expressiond of survivin protein and Bcl-2 protein,up-regulated the expression of Bax protein.These results suggest that inducing apoptosis may be one of the mechanism for the anticancer effect of EGb761.4.EGb761 has the antitumor effect.It may be applied as a chemopreventive agent for human to prevent AFB1-related PHC. Objective To investigate the molecular mechanisms by which Ginkgo biloba extract（EGb761）inhibits cell proliferation and induces apoptosis of HepG2 cells.Methods HepG2 cells were incubated with EGb761 at various concentrations（from 0 to 1750μg/ml）.After 24h incubation,growth inhibition was assessed via MTT assay,the IC50was then evaluated and used in the succeeding experiment.The biological characters of HepG2 cell treated with EGb761 were observed cytopathologically and tested by colony formation, acridine orange fluorescence staining,electric microscope and flow cytometry analysis.Finally,the expressions of the genes that related to cell cycle and apoptosis were measured by RT-PCR and Immunocytochemistry.Results EGb761 treatment resulted in significant inhibiting the growth of HepG2 cells in a concentration-dependent manner,detected by MTT assay.The IC50value was 438μg/ml at 24h.The numbers of colony were 40±4.3 and 102.67±4.51 in group EGb761 and group control,respectively（P=0.000）.After 24h exposure to EGb761 at 438μg/ml,the typical morphologic changes of apoptosis were observed by acridine orange fluorescence staining or by electric microscope,which includes prominent chromatin condensation,loss of normal nuclear architecture and accumulation of nuclear debris.In group EGb761 and group control,the cell apoptosis ratios were 10o09%and 0.48%at 12h,11.40% and 1.07%at 24h,13.87%and 0.67%at 48h,respectively.However,between group EGb761 and group control,there were no significant differences in cell cycle distribution and in mRNA or protein expression levels of Cyclin D1. EGb761 significantly decreased mRNA level of C-myc and protein level of Bcl-2,and significant increased protein level of Bax.Conclusion EGb761 can suppress proliferation and induce apoptosis in HepG2 cells.Its anti-tumor effect might relate to inducing apoptosis,in which the path of Bcl-2/Bax might involved.