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Paper text:
flavone , apigenin , kaempferol , quercetin , genistein were obtained from carl roth gmbh ( karsruhe , germany ) and sigma chemical company ( st .
hela cervical cancer cell line ( dsmz - deutsche sammlung von mikroorganismen und zellkulturen gmbh - german collection of microorganisms and cell cultures , braunschweig , germany ) was cultured as monolayers and maintained in rpmi 1640 ( paa  the cell culture company , germany through immuniq , poland ) containing 10% fetal bovine serum ( paa  the cell culture company , germany through immuniq , poland ) , 100 u / ml penicillin and 100 g / ml streptomycin ( paa  the cell culture company , germany through immuniq , poland ) in a humidified atmosphere with 5% co2 at 37 c. for the experiments the cells were seeded onto 96-well microtiter plates at a density of 1 x 10 cells per well and allowed to adhere for 24 h. after this time the culture medium was removed and hela cells were incubated with rhstrail at the concentrations of 100 ng / ml for 18 hours with or without flavonoids .
the tested polyphenolic compounds were added over a concentration range of 10  50 m . working dilutions were freshly prepared on the day of testing .
after 18 hours of incubation the cell growth rate was evaluated by mtt assays ( sigma chemical company , st .
the spectrophotometric absorbance of each well was measured by microculture plate reader with the test wavelength of 550 nm .
the percent cytotoxicity was calculated by the formula , percent cytotoxicity = ( 1- ( absorbance of experimental wells / absorbance of control wells ) ) x 100 .
the results are expressed as ic50 , which is the concentration necessary for 50% inhibition .
the ic50 values for the combination of each compound with trail are calculated from dose response curves using linear regression analysis by fitting the test concentrations that give percentage of inhibition values above and below the reference value ( 50% ) .
ldh activity was measured using a commercial cytotoxicity assay kit ( roche diagnostics gmbh , mannheim , germany ) , in which released ldh in culture supernatants is measured with a coupled enzymatic assay , resulting in conversion of a tetrazolium salt into red formazan product .
the cells were treated with various concentrations of flavonoids and/or rhstrail for indicated period of time .
the sample solution ( supernatant ) was removed and ldh released from cells was measured in culture medium .
maximal release was obtained after treating control cells with 1% triton x-100 ( sigma chemical company , st .
the necrotic percentage was expressed using the formula , ( sample value / maximal release ) x 100% .
apoptotic cells were quantified by microscopic method using apoptotic & necrotic & healthy cells quantification kit from biotium , inc .
hela cells were washed with pbs and were detached from cell culture wells by trypsin . next
the cells were centrifuged to discard supernatant , washed with pbs and resuspended in binding buffer ( 100 l / sample ) . to each tube
were added 5 l of fitc - annexin v , 5 l of ethidium homodimer iii and 5 l of hoechst 33342 solutions .
the samples were incubated at room temperature for 15 minutes in the dark . after staining the cells
were washed with binding buffer and placed on a glass slide and covered with a glass coverslip .
the stained cells were observed under a fluorescence inverted microscope ix51 ( olympus , tokyo , japan ) using filter set for fitc , tritc and dapi .
the healthy cells emitted blue ( stained by hoechst ) , apoptotic cells emitted green and blue ( stained by fitc - annexin v and hoechst ) and necrotic cells emitted red and blue fluorescence ( stained with ethidium homodimer and hoechst ) . cells stained with triple colors blue , red and green were dead cells progressing from apoptotic cell population .
the microscopic studies were performed at 50 m concentration of flavonoids with or without trail .
the ic50 values for each flavonoid were calculated from dose response curves using linear regression analysis by fitting the test concentrations that gave percentage of inhibition values above and below the reference value ( 50% ) using pharm / pcs  version 4 pharmacologic calculation system based on  manual of paharmacologic calculation with computer programs  , 2 edition by r.j .
the results of microscopic studies are expressed as mean values  sd from three separate experiments .
flavone , apigenin , kaempferol , quercetin , genistein were obtained from carl roth gmbh ( karsruhe , germany ) and sigma chemical company ( st .
hela cervical cancer cell line ( dsmz - deutsche sammlung von mikroorganismen und zellkulturen gmbh - german collection of microorganisms and cell cultures , braunschweig , germany ) was cultured as monolayers and maintained in rpmi 1640 ( paa  the cell culture company , germany through immuniq , poland ) containing 10% fetal bovine serum ( paa  the cell culture company , germany through immuniq , poland ) , 100 u / ml penicillin and 100 g / ml streptomycin ( paa  the cell culture company , germany through immuniq , poland ) in a humidified atmosphere with 5% co2 at 37 c.
for the experiments the cells were seeded onto 96-well microtiter plates at a density of 1 x 10 cells per well and allowed to adhere for 24 h. after this time the culture medium was removed and hela cells were incubated with rhstrail at the concentrations of 100 ng / ml for 18 hours with or without flavonoids .
the tested polyphenolic compounds were added over a concentration range of 10  50 m . working dilutions were freshly prepared on the day of testing .
after 18 hours of incubation the cell growth rate was evaluated by mtt assays ( sigma chemical company , st .
the spectrophotometric absorbance of each well was measured by microculture plate reader with the test wavelength of 550 nm .
the percent cytotoxicity was calculated by the formula , percent cytotoxicity = ( 1- ( absorbance of experimental wells / absorbance of control wells ) ) x 100 .
the results are expressed as ic50 , which is the concentration necessary for 50% inhibition .
the ic50 values for the combination of each compound with trail are calculated from dose response curves using linear regression analysis by fitting the test concentrations that give percentage of inhibition values above and below the reference value ( 50% ) .
for the experiments the cells were seeded onto 96-well microtiter plates at a density of 1 x 10 cells per well and allowed to adhere for 24 h. after this time the culture medium was removed and hela cells were incubated with rhstrail at the concentrations of 100 ng / ml for 18 hours with or without flavonoids .
the tested polyphenolic compounds were added over a concentration range of 10  50 m . working dilutions were freshly prepared on the day of testing .
after 18 hours of incubation the cell growth rate was evaluated by mtt assays ( sigma chemical company , st .
the spectrophotometric absorbance of each well was measured by microculture plate reader with the test wavelength of 550 nm .
the percent cytotoxicity was calculated by the formula , percent cytotoxicity = ( 1- ( absorbance of experimental wells / absorbance of control wells ) ) x 100 .
the results are expressed as ic50 , which is the concentration necessary for 50% inhibition .
the ic50 values for the combination of each compound with trail are calculated from dose response curves using linear regression analysis by fitting the test concentrations that give percentage of inhibition values above and below the reference value ( 50% ) .
ldh activity was measured using a commercial cytotoxicity assay kit ( roche diagnostics gmbh , mannheim , germany ) , in which released ldh in culture supernatants is measured with a coupled enzymatic assay , resulting in conversion of a tetrazolium salt into red formazan product .
the cells were treated with various concentrations of flavonoids and/or rhstrail for indicated period of time . the sample solution ( supernatant ) was removed and ldh released from cells was measured in culture medium .
maximal release was obtained after treating control cells with 1% triton x-100 ( sigma chemical company , st .
the necrotic percentage was expressed using the formula , ( sample value / maximal release ) x 100% .
apoptotic cells were quantified by microscopic method using apoptotic & necrotic & healthy cells quantification kit from biotium , inc .
hela cells were washed with pbs and were detached from cell culture wells by trypsin . next
the cells were centrifuged to discard supernatant , washed with pbs and resuspended in binding buffer ( 100 l / sample ) . to each tube
were added 5 l of fitc - annexin v , 5 l of ethidium homodimer iii and 5 l of hoechst 33342 solutions .
the samples were incubated at room temperature for 15 minutes in the dark . after staining the cells
were washed with binding buffer and placed on a glass slide and covered with a glass coverslip .
the stained cells were observed under a fluorescence inverted microscope ix51 ( olympus , tokyo , japan ) using filter set for fitc , tritc and dapi .
the healthy cells emitted blue ( stained by hoechst ) , apoptotic cells emitted green and blue ( stained by fitc - annexin v and hoechst ) and necrotic cells emitted red and blue fluorescence ( stained with ethidium homodimer and hoechst ) .
cells stained with triple colors blue , red and green were dead cells progressing from apoptotic cell population .
the microscopic studies were performed at 50 m concentration of flavonoids with or without trail .
the ic50 values for each flavonoid were calculated from dose response curves using linear regression analysis by fitting the test concentrations that gave percentage of inhibition values above and below the reference value ( 50% ) using pharm / pcs  version 4 pharmacologic calculation system based on  manual of paharmacologic calculation with computer programs  , 2 edition by r.j .
the results of microscopic studies are expressed as mean values  sd from three separate experiments .
trail is a promising candidate for cancer therapeutics that preferentially induces apoptosis in cancer cells . 
 the combined treatment flavonoids with trail might be promising as a chemoprevention and/or new therapy against malignant tumors . 
 we examined the cytotoxic effect of dietary flavonoids in combination with trail on hela cells . 
 it was found that treatment with noncytotoxic concentration of some flavonoids significantly sensititizes to trail induced death in hela cells . 
 our study demonstrated that flavone , apigenin and genistein markedly augmented trail mediated cytotoxicity against hela , whereas kaempferol and quercetin produced no effect .