hepatocellular carcinoma ( hcc ) is the fifth most common malignancy worldwide.1 in malaysia , hcc is the thirteenth most common cancer affecting the population.2   the etiology of liver cancer is multi factorial ; some of the well known risk factors include hepatitis b and c viral infection , exposure to chemicals such as aromatic hydrocarbons , and the ingestion of aflatoxin b1.3 - 5   the process of carcinogenesis involves an initiating event which induces genetic damage , followed by survival and progression of selected clones of the transformed cells to form tumors . apoptosis , a form of programmed cell death , has been associated with delaying or inhibiting cancer growth.6,7 much of cancer research over the past two decades has focused on genes that , when mutated , act in either a dominant or recessive manner to enhance proliferation with dysregulation of apoptosis that is responsible for initiating cancer and its progression.8,9 many emerging data in recent years have shown that dietary chemopreventive agents preferentially inhibit growth of cancer cells by targeting signaling molecules , such as caspases , that subsequently lead to the induction of apoptosis.7 some of these dietary agents include epigallocatechin3gallate ( egcg ) , found in green tea,10 curcumin in turmeric,11 genistein in soybeans,12 lycopene in tomatoes,13 anthocyanins in pomegranates14 and isothiocyanates in broccoli.15   there is a large body of evidence that links dna damage and apoptosis.16,17 the tumor suppressor protein p53 provides an important link between dna damage and apoptosis as it has been shown to mediate the upregulation of apoptotic proteins such as bax , caspase3 and 8 , noxa , puma and p53aip.18   chlorella vulgaris , a unicellular green algae , has been widely used as a food supplement and credited with high antioxidant and therapeutic abilities.19,20 the supplement can be taken in the form of tablets , capsules , as a food additive or extracted as a liquid . some health claims and benefits include improvement in the control of hypertension , fibromyalgia and ulcerative colitis.21 in vivo studies have revealed the significant antitumor and antigenotoxic efficacy of c. vulgaris.20 a study by hasegawa et . al showed that a glycoprotein , designated ars2 , found in c. vulgaris extract has an anticancer effect on miceinduced fibrosarcoma.22   to the best of our knowledge , the effect of c. vulgaris on hepatoma cells has not been studied in great detail . in this study , we investigated the anticancer effect of c. vulgaris extract on the hepatoma cell line hepg2 by evaluating changes in proliferation , dna damage and apoptosis . stock of c. vulgaris beijerinck ( strain 072 ) was obtained from the university of malaya algae culture collection ( umacc , malaysia ) and grown in bold 's basal media ( bbm ) with a 12  hours dark 12  hours light cycle . the algae were dried using a freeze dryer and then mixed in distilled water at a concentration of 10% ( w / v ) . the algal suspension was then boiled at 100c for 20  minutes using reflux method followed by centrifugation at 10  000  rpm for 20  min . the supernatant was lyophilized using a freeze dryer to obtain the powdered form of c. vulgaris liver cancer cell line hepg2 and normal liver cell line wrl68 were maintained in eagle 's minimal essential medium ( emem ; flow labaratories , sydney australia ) supplemented with 1 mm sodium pyruvate ( sigma , st louis , mo , usa ) , 2 mm glutamine , 10% fetal calf serum and 100  u / ml penicillin and streptomycin at 37c in humidified 5% co2 incubator . cell proliferation , apoptosis and collection of protein were performed when cells reached 70% confluence density . c. vulgaris extract at various concentrations ( 04  mg / ml ) was added to cell lines 24  hours after incubation . a 96well plate was seeded with hepg2 and wrl68 cells at a uniform density of 2  10 cells per well . twentyfour hours after incubation , cells were treated with the hot water extract of c. vulgaris and incubated for a further 24  hours . cells were labeled with bromodeoxyuridine ( brdu ) during the last 3  hours of c. vulgaris extract treatment . the cells were fixed with denaturing solution and the incorporation of brdu was detected by immunoreaction . after substrate solution was added to each well , the amount of brdu incorporated was determined by measuring the absorbance at dual wavelengths ( 450/690  nm ) using a scanning multiwell spectrophotometer [ enzymelinked immunosorbent assay ( elisa ) reader ] apoptotic cell death was determined by dead end colorimetric tunel system ( promega , madison , wi , usa ) . after 24  hours of treatment with c. vulgaris extract , floating cells and adherent cells in culture were collected in a tube , trypsinized , centrifuged and washed in phosphate buffered saline ( pbs ) . cells were fixed by immersing slides in 4% formalin in pbs for 25  minutes at room temperature . after washing with pbs , cells were permeabilized by immersing the slides in 0.2% triton  100 solution in pbs for 5  minutes at room temperature . cells were then equilibrated with 100  l of equilibration buffer ( 2.5 mm trishcl ph 6.6 , 0.2 m potassium cacodylate , 2.5 mm cocl2 , 0.25 mg / ml bsa . ) for 7  minutes . the equilibrated areas were blotted with tissue paper before adding biotinylated nucleotide and tdt reaction mix ( 100  l ) to the slides . the slides were then covered with coverslips to ensure an even distribution of the reagent before incubating at 37c for 60  minutes in a humidified chamber . coverslips were removed and the slides were immersed in 2 salinesodium citrate ( ssc ; sodium chloride 0.15 m , trisodium citrate 0.015 m ) buffer for 15  minutes at room temperature , and washed twice with pbs . endogenous peroxidases were blocked by immersing the slides in 0.3% hydrogen peroxide for 4  minutes at room temperature and washed again in pbs . streptavidin horseradish peroxidase ( hrp ) solution ( 1500 pbs ) was added to each slide and incubated for 30  minutes at room temperature . after final washing with pbs , diaminobenzedine ( dab ) solution was added to the slides for 20  minutes until light brown staining was observed . finally , each slide was mounted with dpx ( bdh , england ) to be examined under light microscope . the assay was performed as described by singh et al.23 thirty microlitres of the hepg2 cell suspension ( < 400,000 cells / ml ) was mixed with 80  l of 1% low melting point ( lmp ) agarose and added to fully frosted slides that had been covered with a layer of 1% lmp agarose . subsequently , the slides were immersed in lysis solution [ 2.5 m naoh , ph10 ; 0.1 m ethylenediaminetetraacetic acid ( edta ) ; 0.01 m tris ; and 1% triton  100 ] for 1  h at 4c , followed by electrophoresis solution ( 300 mm naoh ; and 1 mm edta , ph13 ) for 20  min to allow dna unwinding , and electrophoresed for 20  min at 25  v and 300  ma . finally , the slides were neutralized with 0.4 m tris buffer ( ph  7.5 ) , stained with ethidium bromide ( 5  mg / ml ) and analyzed using a fluorescence microscope ( carl zeiss , gttingen , germany ) . images of 50 randomly selected cells per experimental point were visually analyzed under the microscope . hepg2 cells ( 110/dish ) were seeded into a 9  cm dish and treated with various concentrations of hot water extract of c. vulgaris for 2 , 6 , 12 , 18 and 24  hours . hcl , 150  mm nacl , 1  mm ethylene glycol tetraacetic acid ( egta ) , 1  mm edta , 20  mm naf , 100  mm na3vo4 , 1% np40 , 1% triton x100 , 1  mm phenylmethylsulfonyl fluoride ( pmsf ) , 10  g / ml aprotinin and 10  g / ml leupeptin ] ( sigma , st louis , mo , usa ) on ice for 30  min to lyse the cells . after centrifugation , total protein was determined using a biorad protein assay kit ( biorad , hercules , ca , usa ) . protein was resolved ( 50 g ) by 1015% sdspage and transferred to polyvinyl difluoride ( pvdf ) membranes . the membrane was blocked with blocking buffer ( 5% skim milk in 1% tween 20 in 20  mm trisbuffered saline , ph7.5 ) by incubating for 1  h at room temperature followed by incubation with the appropriate primary antibody ( p53 , bax , bcl2 , caspase3 and 8 ; chemicon , billerica , ma , usa ) at dilutions of 11000 in blocking buffer for 2  h at room temperature . the membranes were then incubated with the respective secondary antibodies for 1  h and antigens were detected by using the enhanced chemiluminescence blotting detection system ( ge healthcare , piscataway , nj , usa ) . results are expressed as mean  sd with the experiment repeated at least 3 times . stock of c. vulgaris beijerinck ( strain 072 ) was obtained from the university of malaya algae culture collection ( umacc , malaysia ) and grown in bold 's basal media ( bbm ) with a 12  hours dark 12  hours light cycle . the algae were dried using a freeze dryer and then mixed in distilled water at a concentration of 10% ( w / v ) . the algal suspension was then boiled at 100c for 20  minutes using reflux method followed by centrifugation at 10  000  rpm for 20  min . the supernatant was lyophilized using a freeze dryer to obtain the powdered form of c. vulgaris liver cancer cell line hepg2 and normal liver cell line wrl68 were maintained in eagle 's minimal essential medium ( emem ; flow labaratories , sydney australia ) supplemented with 1 mm sodium pyruvate ( sigma , st louis , mo , usa ) , 2 mm glutamine , 10% fetal calf serum and 100  u / ml penicillin and streptomycin at 37c in humidified 5% co2 incubator . cell proliferation , apoptosis and collection of protein were performed when cells reached 70% confluence density . c. vulgaris extract at various concentrations ( 04  mg / ml ) was added to cell lines 24  hours after incubation . a 96well plate was seeded with hepg2 and wrl68 cells at a uniform density of 2  10 cells per well . twentyfour hours after incubation , cells were treated with the hot water extract of c. vulgaris and incubated for a further 24  hours . cells were labeled with bromodeoxyuridine ( brdu ) during the last 3  hours of c. vulgaris extract treatment . the cells were fixed with denaturing solution and the incorporation of brdu was detected by immunoreaction . after substrate solution was added to each well , the amount of brdu incorporated was determined by measuring the absorbance at dual wavelengths ( 450/690  nm ) using a scanning multiwell spectrophotometer [ enzymelinked immunosorbent assay ( elisa ) reader ] apoptotic cell death was determined by dead end colorimetric tunel system ( promega , madison , wi , usa ) . after 24  hours of treatment with c. vulgaris extract , floating cells and adherent cells in culture were collected in a tube , trypsinized , centrifuged and washed in phosphate buffered saline ( pbs ) . cells were fixed by immersing slides in 4% formalin in pbs for 25  minutes at room temperature . after washing with pbs cells were then equilibrated with 100  l of equilibration buffer ( 2.5 mm trishcl ph 6.6 , 0.2 m potassium cacodylate , 2.5 mm cocl2 , 0.25 mg / ml bsa . ) for 7  minutes . the equilibrated areas were blotted with tissue paper before adding biotinylated nucleotide and tdt reaction mix ( 100  l ) to the slides . the slides were then covered with coverslips to ensure an even distribution of the reagent before incubating at 37c for 60  minutes in a humidified chamber . coverslips were removed and the slides were immersed in 2 salinesodium citrate ( ssc ; sodium chloride 0.15 m , trisodium citrate 0.015 m ) buffer for 15  minutes at room temperature , and washed twice with pbs . endogenous peroxidases were blocked by immersing the slides in 0.3% hydrogen peroxide for 4  minutes at room temperature and washed again in pbs . streptavidin horseradish peroxidase ( hrp ) solution ( 1500 pbs ) was added to each slide and incubated for 30  minutes at room temperature . after final washing with pbs , diaminobenzedine ( dab ) solution was added to the slides for 20  minutes until light brown staining was observed . finally , each slide was mounted with dpx ( bdh , england ) to be examined under light microscope . the assay was performed as described by singh et al.23 thirty microlitres of the hepg2 cell suspension ( < 400,000 cells / ml ) was mixed with 80  l of 1% low melting point ( lmp ) agarose and added to fully frosted slides that had been covered with a layer of 1% lmp agarose . subsequently , the slides were immersed in lysis solution [ 2.5 m naoh , ph10 ; 0.1 m ethylenediaminetetraacetic acid ( edta ) ; 0.01 m tris ; and 1% triton  100 ] for 1  h at 4c , followed by electrophoresis solution ( 300 mm naoh ; and 1 mm edta , ph13 ) for 20  min to allow dna unwinding , and electrophoresed for 20  min at 25  v and 300  ma . finally , the slides were neutralized with 0.4 m tris buffer ( ph  7.5 ) , stained with ethidium bromide ( 5  mg / ml ) and analyzed using a fluorescence microscope ( carl zeiss , gttingen , germany ) . images of 50 randomly selected cells per experimental point were visually analyzed under the microscope . hepg2 cells ( 110/dish ) were seeded into a 9  cm dish and treated with various concentrations of hot water extract of c. vulgaris for 2 , 6 , 12 , 18 and 24  hours . hcl , 150  mm nacl , 1  mm ethylene glycol tetraacetic acid ( egta ) , 1  mm edta , 20  mm naf , 100  mm na3vo4 , 1% np40 , 1% triton x100 , 1  mm phenylmethylsulfonyl fluoride ( pmsf ) , 10  g / ml aprotinin and 10  g / ml leupeptin ] ( sigma , st louis , mo , usa ) on ice for 30  min to lyse the cells . after centrifugation , total protein was determined using a biorad protein assay kit ( biorad , hercules , ca , usa ) . protein was resolved ( 50 g ) by 1015% sdspage and transferred to polyvinyl difluoride ( pvdf ) membranes . the membrane was blocked with blocking buffer ( 5% skim milk in 1% tween 20 in 20  mm trisbuffered saline , ph7.5 ) by incubating for 1  h at room temperature followed by incubation with the appropriate primary antibody ( p53 , bax , bcl2 , caspase3 and 8 ; chemicon , billerica , ma , usa ) at dilutions of 11000 in blocking buffer for 2  h at room temperature . the membranes were then incubated with the respective secondary antibodies for 1  h and antigens were detected by using the enhanced chemiluminescence blotting detection system ( ge healthcare , piscataway , nj , usa ) . results are expressed as mean  sd with the experiment repeated at least 3 times . statistical evaluation was done using the student 's ttest . a p value of < 0.05 was considered significant . chlorella vulgaris inhibited the proliferation of human liver cancer cells ( hepg2 ) in a concentrationdependent manner , ranging from 04  mg / ml as shown by brdu proliferation assay with a 50% reduction at 1.6  mg / ml , ( ic50 ) ( fig . the high ic50 is expected as c. vulgaris is classified as a food and not a drug . proliferation of normal liver cells ( wrl68 ) was decreased when treated with c. vulgaris extract , resulting in a 50% reduction at 1.7  mg / ml . one hundred percent inhibition of proliferation of hepg2 cells and wrl68 cells was achieved at approximately 3  mg / ml and 4mg / ml , respectively . it was clearly seen that c. vulgaris induced apoptosis in hepg2 cells with distinct nuclear condensation and blebbing of the plasma membrane , a typical characteristic of apoptotic bodies ( fig . 2).24   the percentange of apoptosis of hepg2 and wrl68 cells as induced by c. vulgaris extract at 2mg / ml is demonstrated in figure 3 ; the rate of apoptosis was significantly higher ( 70% ) in hepg2 cells compared to normal wrl68 cells ( 15% ) . wrl68 cells also underwent apoptosis but at a higher concentration of c. vulgaris ( 34  mg / ml ) , perhaps as a result of the cytotoxicity effects seen at higher concentrations ( fig . figure 4a shows that the level of p53 increased in a dosedependent manner and reached maximum induction at 2  h with 2  mg / ml treatment of c. vulgaris . 4b ) in a timedependent manner . however , the activation of the mitochondrial apoptotic pathway in downstream caspase8 was not significant ( fig . 4e ) , but a significant increase in the active form of caspase3 was observed after 24  h of treatment with c.vulgaris in a timedependent manner ( fig . these results demonstrate that the mitochondrial signaling pathway is involved in c. vulgarisinduced apoptosis of hepg2 cells . dna damage in hepg2 cells was increased by c. vulgaris treatments at all concentrations ( fig . 5 ) . at low doses of c. vulgaris extract , hepg2 and wrl68 cells generated smaller comets that were not significant , whilst at 2  mg / ml , c. vulgaris induced more damage in hepg2 cells ( 80% ) compared to wrl68 ( 50% ) . many chemopreventive agents have been associated with antiproliferative and apoptotic effects on cancer cells because of their high antioxidant activity , targeting signaling molecules , and preventing or protecting cells from further damage or transformation into cancer cells.7 chlorella vulgaris has been shown to exhibit high antioxidant activity compared to other microalgae.25 the antiproliferative effect of c. vulgaris ( fig . 1 ) could be caused by an acidic glycoprotein found to have antitumour and antioxidant properties in tumourbearing mice.22   dna damage is a common event in life following which , repair mechanisms and apoptosis will be activated to maintain genome integrity . dna damage results in cell cycle arrest at checkpoints or at g1 or g2 stage to inhibit cell cycle progression and to induce apoptosis , thus protecting cells against further damage.24 antioxidants are important inhibitors of lipid peroxidation as a defence mechanism against oxidative damage.26 plants and microalgae have developed a protective mechanism by possessing antioxidant compounds mainly with phenolic moieties to eradicate the accumulation of reactive oxygen species ( ros ) produced by ultraviolet ( uv ) radiation or heat from sunlight.26 our study showed that c. vulgaris , with its high antioxidant activity , protected normal wrl68 cells against severe dna damage but induced severe dna damage in hepg2 cells . many chinese herbal remedies such as ganoderma lucidum , rubus coreanum , paeoniae radix and phyllanthus urinaria have been shown to trigger the apoptotic pathway in mcf7 human breast cancer cells , ht29 human colon cancer cells , hepg2 human hepatoma cells and lewis lung carcinoma cells , respectively.27 - 30 apoptosis is modulated by antiapoptotic and proapoptotic effectors , which involve a large number of proteins . the propapoptotic and antiapoptotic members of the bcl2 family act as a rheostat in regulating programmed cell death and as a target of anticancer therapy.31 the ratio of death antagonists ( bcl2 , bclxl ) to agonists ( bax , bclxs , bad , bid ) determines whether a cell will respond to an apoptotic stimulus . downregulation of the death suppressor bcl2 protein could repress tumor growth by promoting programmed cell death.32 - 34 . to investigate the mechanisms of c. vulgarisinduced apoptosis , we assessed the expression and activity of tumor suppressor protein p53 and a host of pro and antiapoptotic proteins : bcl2 , bax , and caspases3 and 8 . our results showed that c. vulgaris mediated apoptosis in a p53dependent manner with increased expression of bax and decreased expression of bcl2 proteins in a timedependent manner . the activation of p53 and related family members can either enforce cell cycle arrest or induce apoptosis . the rules that govern the choice between growth , arrest and apoptosis are likely to be enforced by other proteins that can antagonize or synergize with p53 to regulate apoptosis.35 the maximal increase of bax expression in hepg2 cells after 18  h incubation with c. vulgaris may be the result of the increase of p53 accumulation that peaked after 2  h. as a herbal extract , c. vulgaris contains a variety of compounds including antioxidants and a glycoprotein that may act on different pathways of tumor cell growth and survival , triggering an antagonistic effect of bax and bcl2 . apoptotic signals are generally believed to be mediated through a hierarchy of caspase activation controlled by one of two distinct pathways that are associated with either mitochondrial caspase8 or 9 . although we did not find significant activation of downstream caspase8 there was , however , a significant increase of the active form of caspase3 after 24  h of treatment with c. vulgaris , in a timedependent manner . these results demonstrated that the mitochondrial signaling pathway is involved in c. vulgarisinduced apoptosis of hepg2 cells . it could be postulated that c. vulgaris did not induce the initiator caspase8 but it may have activated the other initiator caspase9 . among the ten different members of caspases identified in mammalian cells , caspase3 may serve as a general mediator of apoptosis . when cells are undergoing apoptosis , executioner or effector caspase3 triggers cellular proteins such as poly ( adpribose ) polymerase and dna fragmentation factor , resulting in the characteristic changes of apoptosis.36 caspase3 is synthesized as a 33  kda inactive proenzyme that requires proteolytic activation . our results showed that a high level of proenzyme of caspase3 was present in untreated tumor cells , and active caspase3 gradually increased after c. vulgaris treatment , suggesting that c. vulgaris induced apoptosis through a caspase3dependent mechanism . the results of the present study suggest that the anticancer mechanism of c. vulgaris in hepatoma cells ( hepg2 ) is by inhibiting dna synthesis , triggering dna damage and inducing apoptosis . this is shown by the reduced incorporation of brdu into replicating dna of hepg2 cells and an increase in the number of dna damageinducing and apoptotic proteins , bax and caspase3 in hepg2 cells treated with c. vulgaris . the possible mechanism is speculated to be an increase in p53 , bax and caspase3 expression that would subsequently lead to apoptosis . this research was supported by a grant from ministry of science , technology and innovation ( mosti )
objectives : the aim of this study was to determine the antiproliferative and apoptotic effects of hot water extracts of chlorella vulgaris on hepatoma cell line hepg2.introduction:the search for food and spices that can induce apoptosis in cancer cells has been a major study interest in the last decade . 
 chlorella vulgaris , a unicellular green algae , has been reported to have antioxidant and anticancer properties . 
 however , its chemopreventive effects in inhibiting the growth of cancer cells have not been studied in great detail.methods:hepg2 liver cancer cells and wrl68 normal liver cells were treated with various concentrations ( 04 mg / ml ) of hot water extract of c. vulgaris after 24 hours incubation . 
 apoptosis rate was evaluated by tunel assay while dna damage was assessed by comet assay . 
 apoptosis proteins were evaluated by western blot analysis.results:chlorella vulgaris decreased the number of viable hepg2 cells in a dose dependent manner ( p < 0.05 ) , with an ic50 of 1.6  mg / ml . dna damage as measured by comet assay was increased in hepg2 cells at all concentrations of chlorella vulgaris tested . 
 evaluation of apoptosis by tunel assay showed that chlorella vulgaris induced a higher apoptotic rate ( 70% ) in hepg2 cells compared to normal liver cells , wrl68 ( 15% ) . 
 western blot analysis showed increased expression of pro apoptotic proteins p53 , bax and caspase3 in the hepg2 cells compared to normal liver cells wrl68 , and decreased expression of the antiapoptotic protein bcl2.conclusions:chlorella vulgaris may have anticancer effects by inducing apoptosis signaling cascades via an increased expression of p53 , bax and caspase3 proteins and through a reduction of bcl2 protein , which subsequently lead to increased dna damage and apoptosis .