5 - fluorouracil , ccnu , methotrexate , etoposide , vinblastine , oxaliplatin , dimethyl - sulfoxide ( dmso ), the β - actin specific antibody , species - specific igg conjugated with horseradish peroxidase , and 2 ′, 7 ′- dichlorofluorescin diacetate ( dcf - da ) were purchased from sigma - aldrich ( st . louis , mo ., usa ). dulbecco &# 39 ; s modified eagle &# 39 ; s medium with f - 12 nutrient mixture ( dmem / f - 12 ) and fetal bovine serum ( fbs ) were obtained from hyclone ( logan , utah , usa ). l - glutamine , 0 . 25 % trypsin , g418 , hygromycin , lipofectamine 2000 ™, an hmsh6 - specific antibody and pcr primers with or without fluorescent labeling were purchased from invitrogen ( grand island , n . y ., usa ). the easypure genomic dna mini kit was purchased from bioman scientific ( taipei , taiwan ). the polyvinylidene difluoride membrane and the chemiluminescent detection kit were from millipore ( billerica , mass ., usa ), and antibodies specific for hmlh1 and hmsh2 came from bd ( franklin lakes , n . j ., usa ). the hct116 cell line from american type culture collection ( atcc , manassas , va ., usa ) was maintained at 5 % co 2 and 37 ° c . in the growth medium ( dmem / f - 12 containing 10 % fbs and 2 mm l - glutamine ). derived from hct116 , the hct116 -( ca ) 13 and hct116 -( n ) 16 stable transfectants harbor a dual - fluorescent reporter containing the ( ca ) 13 microsatellite and a random ( n ) 16 sequence respectively [ li et al ., 2014 ]. these transfectants were cultured in the growth medium supplemented with 200 μg / ml hygromycin . hct116 - derived and mmr - proficient hct116 + chr3 cells [ koi et al ., 1994 ], kindly provided by c . r . boland , harboring a ( ca ) 13 microsatellite in the dual - fluorescent reporter were cultured in the growth medium containing 200 μg / ml hygromycin and 400 μg / ml g418 . all cells were evaluated by their morphology and by western blot analysis of mmr gene products , as well as by str dna profiling ( performed by the ncku dna sequencing core ). specified cells were seeded at 1 × 10 4 or 1 × 10 5 cells per well in 96 - well or 12 - well plates respectively . one day after seeding , the cells were treated in triplicates for 3 days with a tested drug in the presence or absence of an antioxidant at indicated concentrations . subsequently , the drug and / or antioxidant were removed and cells were washed and recovered in fresh growth medium for 3 days before being subjected to analyses . solvents used for making the stock solution of drugs include ethanol for 5 mm ccnu stock , 0 . 1 mm naoh for 1 mm methotrexate , saline ( 0 . 9 % nacl ) for 50 mm 5 - fu , 5 % dextrose for 5 mm oxaliplatin , and dmso for 42 . 5 mm etoposide and 5 . 5 mm vinblastine stocks . on the other hand , pbs was used to make 0 . 5 m vitamin c stock , saline for making 0 . 5 m nac and 0 . 1 m gsh stocks , and dmso for making 10 mm curcumin and 10 mm eugenol . prior to the experiments , pbs was used for diluting the drugs and antioxidants and the final solvent concentration was kept the same for all tested compounds , including untreated controls . the redox library ( cat # bml - 2835 , enzo , usa ) is composed of 84 compounds that were supplied at 10 mm in dmso . hct116 -( ca ) 13 cells were seeded at 1 × 10 4 cells per well in 96 - well glass plates containing 100 μl growth medium . one day after seeding , the cells were co - treated with 1 μm oxaliplatin and an antioxidant at specified concentrations in triplicate for three days , followed by a 3 - day recovery in fresh growth medium without the drug and antioxidants . subsequently , the cells were fixed with 4 % paraformaldehyde and nuclei were stained with 1 μg / ml hoechst 33258 before being analyzed for msi and cell number by high - content fluorescence microscopy . cell viability was determined by the mtt assay . briefly , the optical density of colored formazan converted from mtt by viable cells was determined as previously described [ chang et al ., 2002 ] at 595 nm with an elisa reader ( thermo labsystems ). cell viability is expressed as the optical density value of drug - treated cells relative to that of solvent - treated cells , after subtracting the background from the medium . at the end of treatment , adherent cells in 12 - well plates were trypsinized and resuspended in pbs containing 1 mm edta , and filtered through a 40 - μm strainer , and then subjected to flow cytometry ( quanta ™ sc - mpl , beckman coulter ). as detailed previously [ li et al ., 2014 ], a minimum of 1 × 10 4 gfp + cells were analyzed per sample and displayed on green fluorescence ( fl1 ) versus red fluorescence ( fl2 ) axes using quanta sc mpl analysis software . the frequency of frameshift mutations is expressed as the percentage of dsred + gfp + subpopulation in the gfp + cell population . after treatment , hct116 -( ca ) 13 cells in 96 - well glass plates ( corning - costar ®) were fixed with 4 % paraformaldehyde and nuclei were stained with 1 μg / ml hoechst 33258 . fluorescent images of the cells were acquired at 100 × magnification by imagexpress micro system ( molecular devices ) and analyzed with metaxpress ® v3 . 1 software as described previously [ li et al ., 2014 ]. the frequency of frameshift mutations of the ( ca ) 13 microsatellite is expressed as the percentage of the dsred + gfp + hoechst + subpopulation in the gfp + hoechst + population . genomic dna was isolated from hct116 derivatives in 12 - well plates with easypure genomic dna mini kit . the isolated dna were amplified with fluoresceinated primers specific for the bethesda panel of microsatellite [ boland et al ., 1998 ] and selected coding microsatellites as described previously [ li et al ., 2014 ]. fluoresceinated pcr products were analyzed by the abi 310 genetic analyzer , and electropherograms were generated with genescan collection software ( abi ). * indicates a specified fluorescent dye that end - labeled each forward primer , including fluorescein - ce phosphoramidite ( fam ), hexachloro - fluorescein - ce phosphoramidite ( hex ), and tetrachloro - fluorescein - ce phosphoramidite ( tet ). intracellular ros levels were measured using an oxidation - sensitive 2 ′, 7 ′- dichlorodihydrofluorescein diacetate ( dcdhf - da ) probe , which can be oxidized to the fluorescent 2 ′, 7 ′- dichloro - fluorescein ( dcf ) product . after treatment , hct116 derivatives in 12 - well plates were harvested , washed and resuspended in pbs containing 10 μm dcdhf - da , followed by 30 - min incubation at 37 ° c . in the dark before being subjected to flow cytometry ( quanta sc - mpl ). for a ros curve of h 2 o 2 , 2 × 10 5 hct116 derivatives were detached , washed , and incubated with 10 μm dcdhf - da for 30 min at 37 ° c ., followed by flow cytometric analysis after 30 - min exposure to different concentrations of h 2 o 2 . after excluding cell debris on the basis of electronic volume and side scatter , the fluorescence intensity of dcf was measured in fluorescence channel 1 of the flow cytometer ( λ ex : 488 nm ; λ em : 525 nm ). the ros level is expressed as the mean fluorescence intensity ( mfi ) of dcf from 10 , 000 cells per sample . equal amounts of total proteins in cell lysate were resolved by 8 % sds - polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene difluoride membrane and mmr proteins were immunochemically and chemiluminescently detected as previously described [ chang et al ., 2002 ]. all experiments were performed in triplicate and repeated at least three times , and data are presented as means ± sd . a difference between any treated sample and the control was assessed by 2 - tailed student &# 39 ; s t - test . p & lt ; 0 . 05 was considered significant . 2 . 1 chemotherapeutic agents preferentially induce mutations in the ( ca ) 13 reporter microsatellite . previously we developed a sensitive and reliable in - vivo dual - fluorescent reporter system in mmr - deficient human colorectal cancer hct116 cells , yielding hct116 -( ca ) 13 and hct116 -( n ) 16 stable transfectants that harbor a ( ca ) 13 reporter microsatellite or ( n ) 16 random sequence , respectively , in the dsred coding region [ li et al ., 2014 ]. in these cells we examined the mutation - inducing ability of five chemotherapeutic agents (“ tested drugs ”, or “ drug ”), namely 5 - fu and methotrexate ( anti - metabolites ), ccnu ( an alkylating agent ), etoposide ( a topoisomerase ii poison ) vinblastine ( a microtubule disruptor ) and oxaliplatin ( a platinum - based alkylating - like agent ). we first determined sub - lethal dose ranges of tested drugs after a 3 - day treatment in hct116 derivatives using the mtt assay ( fig1 a - fig . 1 f ). between hct116 -( ca ) 13 and hct116 -( n ) 16 cells , tested drugs at specified concentrations did not show significant differences in the viability . to determine the mutation - inducing ability of tested drugs , the cells were treated with each drug at sub - lethal doses for three days followed by 3 - day recovery to allow mutations to accumulate . based on flow cytometric analysis , 2 . 5 , 5 and 10 μm 5 - fu increased the mutation frequency from 0 . 46 ± 0 . 04 % to 5 . 91 ± 0 . 49 %, 6 . 37 ± 0 . 96 % and 8 . 05 ± 2 . 01 % respectively in hct116 -( ca ) 13 cells ( fig1 g ). compared to the ( n ) 16 random sequence , the ( ca ) 13 microsatellite was 1 . 2 - 2 . 8 fold more susceptible to 5 - fu - induced mutations ( fig1 g ). the other tested drugs similarly increased the mutation ( i . e ., msi ) frequency in hct116 -( ca ) 13 cells in a dose - dependent fashion as analyzed by flow cytometry ( fig1 h - fig . 1 l ). at the highest doses tested , etoposide , vinblastine and oxaliplatin induced 3 - 5 fold higher msi frequency than 5 - fu , ccnu and methotrexate . relative to the ( n ) 16 random sequence , the ( ca ) 13 microsatellite was 1 . 7 - 9 . 1 fold more vulnerable to 50 μm ccnu , 25 nm methotrexate , 5 μm etoposide , 25 nm vinblastine and 1 μm oxaliplatin ( fig1 g - fig . 1 l ). in sum , all the tested chemotherapeutic agents from different drug classes individually increased the mutation frequency , especially in the ( ca ) 13 microsatellite sequence , in hct116 derivatives . the bethesda panel of microsatellite markers includes three dinucleotide repeats ( d2s123 , d5s346 and d17s250 ) and two mononucleotide repeats ( bat25 and bat26 ) [ boland et al ., 1998 ]. in a fluorescinated pcr - based assay , 10 μm 5 - fu induced the instability of the bat25 microsatellite , whereas 5 μm etoposide or 25 nm vinblastine destabilized the bat26 microsatellite in hct116 cells after a 3 - day drug treatment and a 3 - day recovery ( fig1 m ). however , we failed to detect alterations in these microsatellite markers after the cells were treated with 50 μm ccnu or 25 nm methotrexate for three days followed by a 3 - day recovery ( data not shown ). on the other hand , a 1 - day treatment with 100 μm ccnu or 100 nm methotrexate followed by a 3 - day recovery caused the instability of bat25 and / or bat26 microsatellites ( fig1 m ). in addition , 100 μm ccnu also caused a 1 - bp insertion in a non - repetitive region of d5s346 thereby shifting all the peaks in the dinucleotide repeats correspondingly ( fig1 m ). a 3 - day treatment with 1 μm oxaliplatin followed by a 3 - day recovery resulted in alterations of four out of five microsatellite markers in the bethesda panel ( fig1 m ). collectively , the tested drugs caused the instability of endogenous microsatellites in hct116 cells that lack a functional mmr system . to test whether a functional mmr system protects cells from drug - induced msi , drug effects on mmr - proficient hct116 + chr3 -( ca ) 13 [ li et al ., 2014 ] were compared with that on mmr - deficient hct116 -( ca ) 13 cells . based on the mtt assay , all tested drugs had similar effects on the viability of mmr - deficient and mmr - proficient cells ( fig2 a ). using high - content microscopy , we found that ccnu , etoposide and vinblastine significantly increased the msi frequency from a base line of 0 . 35 % to 1 . 26 % ( p = 0 . 0001 ), 3 . 74 % ( p = 0 . 0039 ) and 2 . 10 % ( p = 0 . 0194 ), respectively , in mmr - proficient hct116 + chr3 -( ca ) 13 cells ( fig2 b ). in hct116 -( ca ) 13 cells treated with tested drugs , except anti - metabolites and oxaliplatin , the msi frequency determined by high - content microscopy was 31 - 50 % lower than that determined by flow cytometry . this is similar to what we previously observed in h 2 o 2 - treated cells , which is due to cell loss during the fixation and staining steps required for high - content microscopic analysis of msi [ li et al ., 2014 ]. nevertheless , our findings indicate that a functional mmr system in hct116 + chr3 cells prevented 65 - 96 % of drug - induced msi seen in mmr - deficient hct116 cells . in addition to using the bethesda panel of microsatellites , immunocytochemical analysis of mmr protein levels is also frequently used for diagnosing the mmr status of colon cancer patients [ boland et al ., 1998 ; umar et al ., 2004 ]. based on western blot analysis , we observed that each of the tested drugs decreased steady - state levels of hmsh2 and hmsh6 proteins in mmr - proficient and mmr - deficient cells ( fig2 c ). while hct116 cells do not express hmlh1 , the tested drugs also slightly decreased the hmlh1 protein level in hct116 + chr3 cells ( fig2 c ). by decreasing steady - state levels of mmr proteins , the tested drugs likely attenuated the mmr function . certain chemotherapeutic agents , such as 5 - fu , methotrexate and etoposide , are known to generate intracellular reactive oxygen species ( ros ) [ martin et al ., 2009 ; hwang et al ., 2001 ; oh et al ., 2007 ]. we therefore measured drug - generated ros levels in hct116 cells . after a 3 - day recovery from a 3 - day drug treatment , 10 μm 5 - fu but not 25 nm methotrexate increased intracellular ros levels ( fig3 ). among the tested drugs , 50 μm ccnu generated the highest ros level ( fig3 ). next , antioxidants were utilized to interrogate possible contributions of ros to drug - induced msi and cytotoxicity in hct116 -( ca ) 13 cells by high - content microscopy and the mtt assay respectively . the doses for selected antioxidants were chosen because they exerted minimal effects on the viability and msi of hct116 -( ca ) 13 cells ( data not shown ), [ li et al ., 2014 ]. after a 3 - day recovery from a 3 - day treatment with 10 μm 5 - fu , in the presence or absence of an antioxidant , 150 μm eugenol was the only antioxidant that suppressed 44 % of drug - induced msi without affecting 5 - fu - mediated cytotoxicity ( fig4 a and fig4 b ). all tested antioxidants individually suppressed msi induced by 50 μm ccnu , but exerted different effects on the drug cytotoxicity ( fig4 c and fig4 d ). notably , 2 . 5 mm nac and 5 mm gsh suppressed 56 - 66 % msi induced by ccnu , while adversely decreasing ccnu - mediated cytotoxicity by approximately 25 % ( fig4 c and fig4 d ). vitamin c , at 250 μm , decreased ccnu - induced msi by ˜ 24 % without significantly affecting drug cytotoxicity . on the other hand , 1 . 5 μm curcumin or 150 μm eugenol suppressed ccnu - induced msi by approximately 55 % while positively enhancing ccnu - mediated cytotoxicity by 11 - 14 %. methotrexate - induced msi was suppressed by co - treatment with nac , gsh or vitamin c by 34 %, 13 % or 12 % respectively without affecting drug cytotoxicity ( fig4 e and fig4 f ). although 1 . 5 μm curcumin did not affect methotrexate - induced msi , it positively enhanced methotrexate - mediated cytotoxicity by approximately 15 % ( fig4 e and fig4 f ). only vitamin c suppressed etoposide - induced msi , by ˜ 40 %, while enhancing etoposide - mediated cytotoxicity by ˜ 10 % ( fig4 g and fig4 h ). similarly , only eugenol suppressed vinblastine - induced msi by 40 % but had no effects on drug cytotoxicity ( fig4 i and fig4 j ). all tested antioxidants dramatically suppressed msi induced by 1 μm oxaliplatin ( fig4 k and fig4 l ). notably , 2 . 5 mm nac and 5 mm gsh totally abolished oxaliplatin - mediated cytotoxicity 25 % ( fig4 k and fig4 l ). also , 150 μm eugenol slightly compromised oxaliplatin - mediated cytotoxicity . collectively , the antioxidants examined appear to differentially affect msi and the cytotoxicity mediated by the tested drugs . chemotherapy is generally given in a combination of drugs . for example , the folfox regimen , consisting leucovorin , 5 - fu and oxaliplatin , has become a standard regimen for treating patients with high risk stage ii and stage iii crc [ andre et al ., 2004 ; grothey and sargent , 2005 ]. although mmr - deficient cancer cells do not develop resistance to oxaliplatin [ vaisman et al ., 1998 ; ahmad , 2010 ], this drug caused mmr deficiency since it displayed strong msi - inducing and intermediate ros - generating abilities among tested drugs ( fig1 a to 1m , fig2 a to 2c and fig3 ). we therefore screened a redox library in hct116 -( ca ) 13 cells to identify antioxidants that can suppress oxaliplatin - induced msi but do not decrease oxaliplatin - mediated cytotoxicity in hct116 -( ca ) 13 cells by high - content microscopy . based on known functions , 42 . 3 % of 84 compounds in the redox library are phenolic antioxidants , 11 % are metal chelators and 7 . 7 % are flavone antioxidants among others ( fig5 a ). hct116 -( ca ) 13 cells were manually co - treated in 96 - well plates with 1 μm oxaliplatin and each of compounds at its ic 50 or 10 % of ic 50 values , if available in the literature . after a 3 - day co - treatment , followed by a 3 - day recovery , the cells were fixed , stained , and analyzed for the msi frequency and cell numbers simultaneously by high - content microscopy . if msi was analysis only , ˜ 90 % of compounds in the library suppressed oxaliplatin - induced msi ( fig5 b ). if only cell numbers were analyzed , ˜ 34 % of compounds enhanced oxaliplatin - mediated cytotoxicity ( fig5 c ). to determine the assay quality , the z ′- factor was calculated and was 0 . 76 for the msi assay and 0 . 48 for cell number counting by high - content microscopy . it is considered as an excellent assay , when 1 & gt ; z ′- factor ≧ 0 . 5 [ zhang et al ., 1999 ]. we further plotted both msi frequency and cell numbers together . at a low dose , such as 10 % of ic 50 , the majority of compounds in the library suppressed oxaliplatin - induced msi while adversely decreasing oxaliplatin - mediated cytotoxicity ( fig5 d ). at a 10 - fold higher dose , such as ic 50 , more compounds suppressed oxaliplatin - induced msi while enhancing oxaliplatin - mediated cytotoxicity ( fig5 e ). on the other hand , some compounds dramatically enhanced oxaliplatin - mediated cytotoxicity at the expense of increasing oxaliplatin - induced msi ( fig5 e ). these findings indicate that a clinical value of an antioxidant relies on simultaneously evaluate both drug - induced msi and drug - mediated cytotoxicity . from a list of potential candidates identified in the primary screen , we performed a secondary screen by including additional concentrations . the most promising antioxidants include cdc ( fig6 a ), ciclopirox ethanolamine ( fig6 b ), gossypol ( fig6 c ), n - octyl caffeate ( fig6 d ), baicalein ( fig6 e ) and curcumin ( fig6 f ). ciclopirox ethanolamine is a hydroxyl radical scavenger , baicalein is a flavone antioxidant and the rest candidates are phenolic antioxidants . 2 . 8 effects of gossypol or curcumin on oxaliplatin - mediated cytotoxicity of mmr - deficient and mmr - deficient cells we further investigated whether the mmr status affects the effect of gossypol or curcumin on oxaliplatin - mediated cytotoxicity . mmr - deficient hct116 cells and isogenic mmr - proficient hct116 + chr3 cells were co - treated with 1 μm oxaliplatin and 2 - 10 μm gossypol or curcumin . the cytotoxicity was determined by the mtt assay after a 3 - day co - treatment with ( dotted lines ) or without ( solid lines ) a 3 - day recovery . as shown in fig7 , gossypol similarly enhanced oxaliplatin - mediated cytotoxicity in a concentration - dependent manner in both mmr - deficient cells ( a ) and mmr - deficient cells ( b ). three days after recovery from the co - treatment , there was no new proliferation occurred (( a ) and ( b )). in contrast , curcumin did not show a significant effect on oxaliplatin - mediated cytotoxicity in both mmr - deficient and mmr - deficient cells (( c ) and ( d )). although the present invention has been explained in relation to its preferred embodiment , it is to be understood that many other possible modifications and variations can be made without departing from the spirit and scope of the invention as hereinafter claimed .
a therapeutic approach to prevent drug resistance and chemotherapy - related secondary cancer associated with dna mismatch repair deficiency is disclosed based on screening antioxidants for reducing microsatellite instability while enhancing the cytotoxicity of chemotherapeutic agents . the work is based on experiments using antioxidants to target reactive oxygen species generated by oxaliplatin , a commonly used chemotherapeutic agent , and is applicable to other chemotherapeutic agent , and in particular 5 - fluorouracil , methotrexate , ccnu , etoposide and vinblastine . in particular oxaliplatin is co - treated with an antioxidant , including cdc , cape , ciclopirox ethanolamine , hinokitiol , gossypol , n - octyl caffeate , baicalein , or curcumin .