invasion is a most important and characteristic step in the complicated process of cancer cells .
the blockage of these biological events by drugs or factors from food and natural resources will prolong the life span of an affected host .
our previous research showed that green , oolong , and black tea extracts , the sera from rats orally given these teas , and related components inhibited the proliferation and invasion of a rat ascites hepatoma cell line of ah109a   through induction of apoptosis and cell cycle arrest   and antioxidative activity .
lignans also suppressed both the proliferation and invasion of ah109a hepatoma cells in vitro , and hence solid tumor growth and metastasis of ah109a cells in vivo .
resveratrol ( 3,5,4-trihydroxy - trans - stilbene ) is known to possess cancer chemopreventive activity and strong antioxidative activity .
hsieh et al . reported that resveratrol showed a greater antiproliferative effect on highly invasive breast carcinoma cells ( mda - mb-435 ) than on minimally invasive cells ( mcf-7 ) .
however , our previous study showed that , in the hepatoma cells , resveratrol as such showed a weaker suppressive effect on the proliferation than on the invasion , and the sera from rats orally given resveratrol completely lost the antiproliferative activity in culture , although its anti - invasive activity was maintained [ 8 , 9 ] .
these findings suggest that the effect of resveratrol on the proliferation may be dependent on cancer cell properties .
piceatannol ( 3,4,3,5-tetrahydroxy - trans - stilbene ) , an analogue of resveratrol , has been reported to induce apoptosis and cell cycle arrest in human melanoma cell ( sk - mel-28 ) .   in the present study
, we examined whether piceatannol or its in vivo metabolite(s ) would have antiproliferative and anti - invasive effects against ah109a and their modes of actions .
, osaka , japan ) and the piceatannol solution was added to the medium at a final ethanol concentration of 0.2% .
the animal experiments in this article were conducted in accordance with guidelines established by the animal care and use committee of tokyo university of agriculture and technology and were approved by this committee .
male donryu rats ( 4 weeks of age ) were purchased from nrc haruna ( gunma , japan ) .
ah109a cells were generously provided by the cell resource center for biomedical research , tohoku university , sendai , japan .
ah109a cells were maintained in peritoneal cavity of male donryu rats and isolated from accumulated ascites and then cultured in eagle 's minimum essential medium ( mem ) ( nissui pharmaceutical co. , tokyo , japan ) containing 10% calf serum ( cs , jrh biosciences , lenaxa , ks , usa ) ( 10% cs / mem ) .
these cells were cultured for at least 2 weeks after isolation to eliminate contaminated macrophages and neutrophils and used for the assays described hereinafter .
effect of piceatannol on ah109a proliferation was examined by measuring the incorporation of [ methyl - h]thymidine ( 0.15  ci / well , specific radioactivity ; 20  ci / mmol , perkinelmer life and analytical sciences , boston , ma , usa ) into dna as described previously [ 8 , 11 ] .
effect of piceatannol on ah109a invasion was estimated by coculturing ah109a cells with rat mesentery - derived mesothelial cell ( m - cell ) monolayers , which were isolated and cultured from donryu rats as described previously   with slight modifications .
briefly , mesothelial cells ( m - cells ) were isolated from mesentery of male donryu rats ( 68 weeks of age ) .
after digestion by trypsin , 1.32.0  10  m - cells were plated in a 60 mm culture dish with 2 mm grids ( corning incorporated , corning , ny , usa ) and cultured for 57 days to attain a confluent state in 10% cs / mem .
then , ah109a cells ( 2.4  10 cells per dish ) were applied on the monolayer of m - cells in 10% cs / mem with piceatannol for 24  h. invaded cells and colonies underneath m - cells were counted with a phase - contrast microscope .
the invasive activity of ah109a cells was expressed as the number of invaded cells and colonies / cm . 

piceatannol was suspended in a 0.3% carboxymethyl cellulose sodium salt ( cmc , wako pure chemical industries , osaka , japan ) aqueous solution at concentrations of 0.25 , 0.5 , and 1  mg / ml , unless otherwise noted .
piceatannol suspension or 0.3% cmc alone ( vehicle control ) ( 1  ml/100  g body weight ) was intubated to male donryu rats ( 5 weeks old ) which had been fasted overnight , and blood was collected 2  h after oral administration as described previously [ 8 , 13 ] .
the sera were prepared by centrifugation , sterilized by filtration , and added to the culture medium at a concentration of 10% instead of cs . for cell cycle analysis , 3.0  10 cells of ah109a per
well were seeded in a 6-well plate in the medium containing various concentrations of piceatannol and cultured for 24  h. cells were collected and washed twice with sterilized , ca- and mg - free , phosphate - buffered saline [ pbs( ) ] .
thereafter , 500  l of propidium iodide ( pi ) solution containing 1  mg of pi ( sigma chemical co. , st .
louis , mo , usa ) in 20  ml of 1% triton x-100 ( sigma ) and 0.1% of sodium citrate ( wako pure chemical industries ltd .
, osaka , japan ) was added and cells were incubated for 30  min on ice .
cells at different cell cycle phases were then analyzed with a flow cytometer ( epics elite eps , beckman - coulter , hialeah , fl , usa ) as previously described .
the effect of piceatannol on apoptosis in ah109a cells was assessed using annexin - v - fitc kit ( immunotech , marseille , france ) according to the manufacturer 's instructions .
phosphatidylserine ( ps ) , which can specifically bind to annexin - v , is one of the phospholipids in the cell membrane and exists predominantly in inner leaflet of the cell membrane of normal cells .
when apoptosis occurs , ps in the cell membrane immediately appears on the outer leaflet of the cell membrane .
the cells with ps on their surface can thus be thought to be early apoptotic cells .
briefly , 5  10 cells of ah109a per well were seeded in a 6-well plate and cultured in the medium containing 0 , 50 , and 100  m piceatannol for 3  h. at the end of culture , cells were labeled with annexin - v - fitc and analyzed with a flow cytometer as described previously .
ah109a cells were cultured for 4  h in the absence or presence of 10  m piceatannol with or without 25  m hydrogen peroxide ( h2o2 , wako pure chemical industries ) .
ah109a cells were then washed once with 10% cs / mem and seeded on the m - cell monolayer in 10% cs / mem without piceatannol and ros . after cultured for 24  h , invaded cells and colonies underneath m - cells
intracellular peroxide levels in ah109a cells were assessed by flow cytometric analysis using a fluorometric probe ( 2,7-dichlorofluorescin diacetate ; dcfh - da , molecular probes , eugene , or )   with a flow cytometer as described previously .
male donryu rats ( 3 weeks of age ) were maintained on a stock pellet diet ( ce-2 , clea japan , tokyo , japan ) for 10 days .
starting 4 days prior to ah109a cell injections , the rats were fed ain-93 g basal diet containing 20% casein ( oriental yeast co. , ltd . , tokyo , japan ) , 39.75%  -corn starch ( nihon nosan kogyo co. ltd . , yokohama , japan ) , 13.2%  -corn starch ( nihon nosan kogyo co. ltd . ) , 10% sucrose ( nihon nosan kogyo co. ltd . ) , 7% corn oil ( miyazawa yakuhin co. ltd . ,
tokyo , japan ) , 5% cellulose powder ( oriental yeast co. , ltd . ) , 3.5% mineral mixture ( ain-93 g composition , nihon nosan kogyo co. ltd . ) , 1% vitamin mixture ( ain-93 composition , nihon nosan kogyo co. ltd . ) , 0.3% l - cystine ( wako pure chemical industries , ltd . ) , and 0.25% choline bitartrate ( wako pure chemical industries , ltd . ) .
piceatannnol was given to rats by adding 0.001% and 0.005% to the basal diet at the expense of -corn starch .
each rat was implanted subcutaneously on one site in the dorsal region with 110 ah109a cells suspended in pbs( ) ( 0.5  ml / rat ) to produce a solid tumor , as described previously .
the tumor size is expressed as the sum of three measured dimensions , that is , height , length , and width . on day 20
, the rats were deprived of their diets at 9:00 but allowed free access to water until they were sacrificed at 13:00 .
metastatic tumor foci in lung and inguinal and axillary lymphatic nodes were macroscopically examined and excised , and the number of metastatic foci was recorded .
multiple comparison was performed by one - way analysis of variance ( anova ) followed by tukey - kramer multiple comparisons test , and p < 0.05 was considered statistically significant .
when added to medium , piceatannol dose - dependently and significantly reduced the proliferation of ah109a hepatoma cells .
it commenced to decrease at 12.5  m and continued to decrease up to 200  m ( figure 1(a(a ) ) ) .
of sera obtained from rats 1 , 2 , 3 , and 6 hours after oral administration of piceatannol at a dose of 10 mg / kg body weight , serum obtained 2 hours after administration was found to show most prominent inhibitory effect against both the proliferation and invasion of ah109a hepatoma cells ( data not shown ) .
thus , we prepared sera obtained from rats 2 hours after oral administration of piceatannol at doses 0 , 2.5 , 5 , and 10  mg / kg body weight .
these sera also reduced dose - dependently and significantly the proliferation of ah109a cells as seen in figure 1(a(b ) ) .
when added to medium , piceatannol was demonstrated to induce cell cycle arrest at g2/m phase in ah109a cells at lower concentrations of 25 and 50  m ( figures 1(b(b ) ) and 1(b(c ) ) ) as compared with the control ( 0  m ) ( figure 1(b(a ) ) ) .
in contrast to the effect on cell cycle , piceatannol failed to induce apoptosis at lower concentration of 50  m as compared with the control ( figures 1(c(a ) ) and 1(c(b ) ) ) .
however , it induced strong apoptosis at higher concentration of 100  m ( figure 1(c(c ) ) ) . when added to medium , piceatannol dose - dependently and
it commenced to decrease at 12.5  m and continued to decrease up to 200  m ( figure 2(a(a ) ) ) .
sera obtained from rats 2 hours after oral administration of piceatannol at doses 0 , 2.5 , 5 , and 10  mg / kg body weight also reduced dose - dependently and significantly the invasion of ah109a cells as shown in figure 2(a(b ) ) . to examine whether or not piceatannol would inhibit the invasion of tumor cells by its antioxidative activity , the invasion assay was performed with ah109a cells precultured in hydrogen peroxide- ( ros- ) containing medium for 4 hours . as shown in figure 2(b(a ) ) , the invasive activity of ah109a cells precultured in ros ( 25  m hydrogen peroxide ) was significantly higher than that of ah109a cells with no treatment .
piceatannol suppressed the ros - potentiated invasive activity of the precultured cells when added to the medium at a concentration of 10  m on preculturing the hepatoma cells with ros . in our preliminary experiment , 12.5  m of piceatannol exposure for 4 hours to ah109a cells exerted no influence on the following ah109a proliferation during 24 hours ( data not shown )
thus , the concentration of 10  m of piceatannol was adopted in this experiment to avoid an influence of the piceatannol - mediated proliferation inhibition on the ah109a invasion . as illustrated in figure 2(b(b ) ) , ah109a cells treated with 100  m hydrogen peroxide for 4 hours contained more intracellular peroxides than did control cells when analyzed with a flow cytometer using dcfh - da as an indicator ( control versus h2o2 ) .
piceatannol ( 10  m ) suppressed the rise in the intracellular peroxide levels of ah109a cells ( h2o2 versus h2o2 and piceatannol ) .
dietary piceatannol ( 0.001% and 0.005% ) tended to suppress the ah109a tumor size dose - dependently , although significant differences were not seen ( figure 3(a ) ) .
accordingly , at the end of the 20-day treatment period , the weights of solid tumors were lower in the piceatannol - treated groups than in the control group ( figure 3(b ) ) .
the solid tumor weight of the 0.005% piceatannol group was significantly reduced from 20.5  4.4 ( control ) to 9.4  2.5 ( 0.005% piceatannol group ) g / rat , indicating that ca .
numbers of metastatic foci were 0.22 , 0.2 , and 0 ( number / rat ) in the control , 0.001% , and 0.005% piceatannol groups , respectively .
in the present study , piceatannol was demonstrated to inhibit both the proliferation and invasion of ah109a hepatoma cells in vitro .
sera obtained from donryu rats orally given piceatannol also inhibited both the proliferation and invasion of ah109a hepatoma cells ex vivo . in our previous experiment ,
the sera obtained from donryu rats orally given resveratrol completely lost the antiproliferative activity ex vivo , although its anti - invasive activity was preserved [ 8 , 9 ] .
these previous and present findings suggest that metabolism of resveratrol by intestinal flora , intestine itself , or in the liver may be different from that of piceatannol .
precise reasons for the difference between two stilbenoids , however , are unclear at present . to elucidate the mechanisms for the inhibition of hepatoma cell proliferation by piceatannol ,
the effect of piceatannol treatment on cell cycle and on the rate of apoptosis was examined by using flow cytometry .
piceatannol was found to suppress the proliferation from lower concentrations ( ~25  m ) by arresting cell cycle of ah109a cells at g2/m phase ( figure 1(b ) ) .
furthermore , piceatannol increased the rate of apoptosis at higher concentrations ( ~100  m ) ( figure 1(c ) ) .
dependencies of cell cycle arrest and apoptosis induction on piceatannol concentrations are similar to those on -gingerol .
although the doses of piceatannol which induced apoptosis were higher than those which induced cell cycle arrest in ah109a hepatoma cells , these results suggest that piceatannol affects both cell proliferation and cell death which at least in part account for the inhibitory effect of piceatannol on the proliferation of ah109a cells .
our previous studies have demonstrated that the invasion of ah109a cells is accelerated by ros [ 16 , 17 ] . in the present study ,
we therefore examined the effect of piceatannol on the ros - potentiated invasive activity using h2o2 .
piceatannol was demonstrated to suppress the ros - induced elevation of the ah109a invasion ( figure 2(b(a ) ) ) .
we have found that ros can induce gene expression of hepatocyte growth factor ( hgf ) , which is known as a cell motility factor , in m - cells as well as ah109a cells [ 9 , 16 ] .
thus , hgf produced by ah109a and m - cells may potentiate the motility of ah109a cells , leading to acceleration of the invasion of ah109a cells .
if piceatannol , like resveratrol , suppresses the production of hgf through antioxidative activity , piceatannol may reduce the motility of ah109a invasion , this leading to the reduction of the ah109a invasion . although preliminarily , we have measured hgf secretion from ah109a cells by elisa .
piceatannol was found to dose - dependently suppress the hgf secretion after exposure of ah109a to piceatannol for 1 hour or 24 hours at the concentration ranges of 25100  m ( data not shown ) , suggesting an involvement of decreased hgf secretion in anti - invasive activity of piceatannol against ah109a hepatoma cells .
although not significant , piceatannol dose - dependently suppressed solid tumor size in hepatoma - bearing rats ( figure 3(a ) ) . however , solid tumor weights , a most simple but reliable index for tumor growth , were significantly reduced by feeding the 0.005% diet ( figure 3(b ) ) .
in addition to in vitro efficacy , piceatannol is demonstrated to be effective in vivo in suppressing tumor growth .
antimetastatic effect of piceatannol was not clear in vivo in the present study , this being probably due to weak metastatic activity in the control group .
selection and use of highly metastatic ah109a cell clone will clarify the in vivo antimetastatic effect of piceatannol . in summary ,
piceatannol was clearly demonstrated to suppress both the proliferation and invasion of ah109a hepatoma cells in vitro and ex vivo .
antiproliferative effect might be due to induction of cell cycle arrest and apoptosis in ah109a , while anti - invasive effect might be due to anti - oxidative property of piceatannol .
as was expected , piceatannol is shown to significantly suppress the solid tumor growth in vivo .
these functions of piceatannol may be of significance from the aspects of both nutritional and pharmacological control of cancers .
 piceatannol is a stilbenoid , a metabolite of resveratrol found in red wine . 
 piceatannol and sera from rats orally given piceatannol were found to dose - dependently suppress both the proliferation and invasion of ah109a hepatoma cells in culture . 
 its antiproliferative effect was based on cell cycle arrest at lower concentration ( 25~50  m ) and on apoptosis induction at higher concentration ( 100  m ) . 
 piceatannol suppressed reactive oxygen species - potentiated invasive capacity by scavenging the intracellular reactive oxygen species . 
 these results suggest that piceatannol , unlike resveratrol , has a potential to suppress the hepatoma proliferation by inducing cell cycle arrest and apoptosis induction . 
 they also suggest that the antioxidative property of piceatannol , like resveratrol , may be involved in its anti - invasive action . 
 subsequently , piceatannol was found to suppress the growth of solid tumor and metastasis in hepatoma - bearing rats . 
 thus , piceatannol may be a useful anticancer natural product . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion