Oxidative stress-based cytotoxicity of delphinidin and cyanidin in colon cancer cells
Jovana Cvorovic a ,1,Federica Tramer a ,Marilena Granzotto b ,Luigi Candussio a ,Giuliana Decorti a ,Sabina Passamonti a ,*
a Department of Life Sciences,University of Trieste,Via Giorgieri 1,34127Trieste,Italy b
Laboratory of Immunology,IRCCS Burlo Garofolo,Via dell’Istria 65/1,34137Trieste,Italy
a r t i c l e i n f o Article history:
Received 31January 2010
and in revised form 6May 2010Available online 28May 2010Keywords:
Anthocyanidins Colon cancer cells Cytotoxicity Apoptosis
Oxidative stress Glutathione
a b s t r a c t
Colorectal cancer is the second most frequent cause of cancer death in the western world.Although the prognosis has improved after the introduction of newer anticancer drugs,the treatment of metastatic colorectal cancer still remains a challenge due to a high percentage of drug-resistant tumor forms.We aimed at testing whether anthocyanidins exerted cytotoxicity in primary (Caco-2)and metastatic (LoVo and LoVo/ADR)colorectal cancer cell lines.Both cyanidin and delphinidin,though neither pelargonidin nor malvidin,were cytotoxic in metastatic cells only.The cell line most sensitive to anthocyanidins was the drug-resistant LoVo/ADR.There,cellular ROS accumulation,inhibition of glutathione reductase,and depletion of glutathione could be observed.This suggests that anthocyanidins may be used as sen-sitizing agents in metastatic colorectal cancer therapy.
Ó2010Elsevier Inc.All rights reserved.
Introduction
Colorectal cancer is the second leading cause of cancer-related death in the ‘developed’world [1].About 50%of patients who un-dergo potentially curative surgery alone ultimately experience dis-ease recurrence [2]and is therefore treated with combination chemotherapy.Drug resistance develops however in nearly all pa-tients [3],calling for finding novel agents capable of killing drug-resistant colorectal cancer cells.
Anthocyanidins,a sub-class of flavonoids,have been suggested as useful agents for chemoprevention [4].Recent studies show that they can trigger apoptosis in human leukemia cell lines through induction of oxidative stress [5,6],and their potential role in cancer therapy has been evaluated [7,8].
With regard to that,the aim of this study was to evaluate whether anthocyanidins are cytotoxic in different colorectal carci-noma cell lines,including drug-resistant cells.
Anthocyanidins were selected for this study,because,in the co-lon,they can be released from dietary
anthocyanins by the action of glycosidases of the colonic microflora [9];though they are less stable than their glycosylated precursors at neutral pH yielding
phenolic aldehydes and phenolic acids [10],they seem to mimic in vivo conditions.
Indeed,we found that delphinidin and cyanidin were cytotoxic in the metastatic human colorectal cancer cell lines,LoVo and LoVo/ADR,where they inactivated the glutathione antioxidant sys-tem and promoted oxidative stress.
Materials and methods Chemicals
All components for cell culture,camptothecin,3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT),20,70-dichlo-rofluorescin diacetate (DCFH-DA),2,20-azobis (2-amidinopropane)dihydrochloride (ABAP 2),Hank’s buffered salt solution (HBSS),glu-tathione reductase (GR),NADPH,glutathione (GSH),glutathione disulfide (GSSG)and 40,6-diamidino-2-phenylindole (DAPI)dilactate were from Sigma–Aldrich (Milano,Italy).Stock solutions of delphin-idin,cyanidin,malvidin and pelargonidin chloride,(Extrasynthèse,Genay,France)were prepared in dimethyl sulfoxide (DMSO)and stored at À20°C.The final content of DMSO in all experiments was
tek-0810003-9861/$-see front matter Ó2010Elsevier Inc.All rights reserved.doi:10.1016/j.abb.2010.05.019
*Corresponding author.Fax:+390405583691.
E-mail addresses:jcvorovic@cicbiogune.es (J.Cvorovic),ftramer@units.it (F.Tramer),ieste.it (M.Granzotto),candussi@units.it (L.Candussio),decorti@units.it (G.Decorti),spassamonti@units.it (S.Passamonti).1
Present address:CICbioGUNE,Cell Biology and Stem Cell Unit,Parque Tecnológico de Vizcaya,Edif.801A,Derio 48160,Vizcaya,Spain.
2
Abbreviations used:ABAP,2,20-azobis (2-amidinopropane)dihydrochloride;CAA,cellular antioxidant activity;DAPI,40,6-diamidino-2-phenylindole;DCFH-DA:20,70-dichlorofluorescin diacetate;DMSO,dimethyl sulfoxide;FBS,fetal bovine serum;GR,glutathione reductase;GSH,glutathione;GSSG,glutathione disulfide;HBSS,Hank’s buffered salt solution;MTT,3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide;PBS,phosphate buffered saline;PI,propidium iodide.
kept under0.2%.Annexin V was from Immunostep(Salamanca, Spain).
Cell lines
The human colorectal carcinoma cell line,Caco-2,was obtained from the Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna,Brescia,Italy.Cells were used between pas-sages35–45and cultured in Minimum Essential Medium Eagle containing10%fetal bovine serum(FBS),1%penicillin–streptomy-cin and sodium pyruvate(1mMfinal concentration).LoVo cells, isolated from human colorectal adenocarcinoma metastasis,and LoVo/ADR cells,a doxorubicin-resistant metastatic human colorec-tal adenocarcinoma cell line,were kindly provided by Dr.C.Gam-bacorti,Milan,Italy.LoVo cells were routinely grown in RPMI-1640 supplemented with10%FBS and1%penicillin–streptomycin.LoVo/ ADR cells were maintained in RPMI-1640supplemented with10% FBS,1%penicillin–streptomycin,and doxorubicin(200ng/ml).Se-ven days before each experiment doxorubicin was removed from the medium.Cell cultures were incubated at37°C in a humidified atmosphere with5%CO2.
MTT assay for cellular viability
Cells(4Â103)were seeded in96-well plates in complete cul-ture medium and treated with camptothecin(0.001–50l M)or anthocyanidins(0.78–100l M),for68h at37°C in a humidified 5%CO2at
mosphere.Each experiment was performed using eight replicate wells for each drug concentration.Control wells con-tained cells without drug and,in some cases with0.2%of DMSO (v/v).After incubation for specified times,20l l of sterile MTT (5mg/ml)were added in each well for additional4h at37°C [11].The culture medium was then removed and formazan crystals were dissolved with200l l of DMSO.Absorbance was recorded on an automated microplate reader EL311s(Bio-Tek Instruments, Winooski,VT)at540nm with a reference wavelength of630nm. The IC50(concentration required for50%inhibition of cell viability) was defined as the drug concentration required to reduce the opti-cal density in each test to50%of controls.All IC50values were determined from three independent experiments.
Determination of apoptosis
Morphological analysis
LoVo and LoVo/ADR cells were seeded at a density of 15Â104cells/well in a six-well plate onto sterilized microscope glasses,left to attach for few hours after which they were treated with delphinidin or cyanidin(100l M)for24h.The control wells contained only medium.After the indicated period,DAPI staining was performed as reported.The medium was aspirated and the glasses were w
ashed with phosphate buffered saline(PBS);the cells werefixed with2%paraformaldehyde(diluted in PBS)at room temperature for30min,and then permeabilized with0.2%Triton X-100(diluted in PBS).After few new washes,the cells were incu-bated with5l g/ml DAPI,for5–7min in the dark at room temper-ature,and washed again.Finally,they were dehydrated with70%, 90%,100%ethanol,coverslipped and observed under afluorescence microscope(Leica Stereoscan430i).
Flow cytometric analysis
Cells were treated with anthocyanidins in the same manner as described above.Apoptosis was measured using FITC-labeled re-combinant human Annexin V assay,according to the manufac-turer’s instructions.Briefly,after washing with PBS,the cells were resuspended in binding buffer with5l l of Annexin V FITC and5l l of propidium iodide(PI)(1mg/ml)for15min at room temperature,washed with PBS and resuspended in200l l of bind-ing buffer.For each measurement50,000events were acquired with a standard FACSCalibur(Becton Dickinson,San Jose,CA)flow cytometer and analysis of data were performed with FlowJo soft-ware(Tree Star Inc.,Ashland,OR,USA).
Cellular antioxidant activity(CAA)assay
The intracellular formation of peroxyl radical was detected by the method of Wolfe and Liu[12].Caco-2,LoVo and LoVo/ADR cells were seeded at a density of1Â104cells/well on a96-well micro-plate in100l l of growth medium/well.The outside wells of the plate were not used.Twenty-four hours after seeding,the growth medium was removed,the wells were washed with PBS,and the cells were treated for1h with delphinidin or cyanidin(25l M, 50l M,100l M)plus25l M DCFH-DA dissolved in treatment med-ium.After the indicated period,cells were washed and600l M ABAP dissolved in HBSS was added to the cells.Fluorescence was measured(k ex=485nm,k em=538nm)every5min for1h at 37°C on a microplate reader(Plate Chameleon,HIDEX).Each con-centration of each substance was repeated in six wells.Each plate also included six control and six blank wells:control wells con-tained cells treated with the dye(DCFH-DA)and the oxidant (ABAP);blank wells contained cells treated with DCFH-DA and HBSS without the oxidant.
Quantification of CAA
After blank subtraction fromfluorescence readings,the area un-der the curve offluorescence versus time was integrated to calcu-late the CAA value at each concentration of delphinidin and cyanidin as follows:CAA unit=100À(
R
SA/
R
CA)Â100,where R
SA is the integrated area under the samplefluorescence versus time curve and
R
CA is the integrated area from the control curve [12].
Glutathione reductase(GR)(EC.1.8.1.7)activity
The cells were seeded on a six-well plate at a density of 2.5Â105/well.Upon reaching confluence,the growth medium was removed,the wells were washed with PBS,and cells were treated for1h with delphinidin or cyandin(25l M,50l M, 100l M),washed again,scraped with a rubber policeman,centri-fuged,resuspended in1ml PBS,counted and sonicated.After these steps,samples were assayed spectrophotometrically(k=340nm) for GR activity.The assay mix contained:0.3ml of the sample;
0.1M phosphate buffer,pH7.0;1mM EDTA;0.26%Triton X-100 (‘‘peroxide free”);0.15mM NADPH,and H2O to 1.5ml,at t=37°C.The baseline was recorded for5min,then2mM GSSG was added and the signal was recorded for further10min.GR activity was calculated from the net change of A340and expressed as nmol NADPH minÀ1Â10À6cells.
Quantitative determination of GSH and GSSG levels
Cells were treated with anthocyanidins in the same manner as for the determination of GR activity,harvested,counted,centri-fuged,and resuspended in1ml of ice-cold extraction buffer(0.1% Triton X-100and0.6%sulfosalicylic acid in0.1M potassium phos-phate buffer with5mM EDTA disodium salt,pH7.5).The cell sus-pension was sonicated and centrifuged.The supernatant(0.180ml) was put into a96-well plate.Then,0.020ml5mM DTNB were added and A412was recorded from a microplate reader(Plate Cha-meleon,HIDEX).The rate of formation of the chromophore TNB is proportional to the concentration of GSH in the sample[13],calcu-lated on the basis of a GSH calibration curve.To assess cellular GSSG levels,the cell extracts werefirst treated with2-vinylpyri-
152J.Cvorovic et al./Archives of Biochemistry and Biophysics501(2010)151–157
dine,which covalently reacts with GSH(but not GSSG)and the ex-cess of2-vinylpyridine was neutraliz
ed with triethanolamine.ANOVA followed by Bonferroni post hoc test.All data analyses were performed using the Prism software version4(GraphPad Software,San Diego,CA).p<0.05were considered statistically
Delphinidin and cyanidin induce apoptosis in LoVo and LoVo/ADR cells.(A)LoVo and LoVo/ADR cells were treated with delphinidin and cyanidin(100l with DAPI for the morphological analysis of nuclei(40Â)and(B)cell death was analyzed cytofluorimetricaly by Annexin V and PI staining;results
increase of apoptotic LoVo and LoVo/ADR cells after the treatment with delphinidin and cyanidin(100l M for24h)as compared to controls.
J.Cvorovic et al./Archives of Biochemistry and Biophysics501(2010)151–157153
Effect of delphidin and cyanidin on ROS levels in Caco-2,LoVo and LoVo/ADR cells.Cells were incubated with delphinidin and cyanidin at the designated concentration control,x25l M,.50l M and N100l M)for1h in the presence of DCFH-DA;the anti/pro-oxidant activity of the anthocyanidins was determined using CAA assay described in Materials and methods.(A)In Caco-2cells delphinidin and cyanidin behave as antioxidants inhibiting DCFH oxidation induced by ABAP(p<0.001,two ANOVA);in LoVo cells only delphinidin at100l M increases the oxidative effect(p<0.001,two way ANOVA)whereas cyanidin at25l M acts as an antioxidant;in LoVo/ADR delphinidin and cyanidin additionally augment the ROS levels induced by ABAP(p<0.001,two way ANOVA).(B,C and D)dose–response curves for inhibition/increase peroxyl radical-induced DCFH oxidation by delphinidin and cyanidin in Caco-2(B),LoVo(C)and LoVo/ADR cells(D)(j delphinidin and N cyanidin).
3.Effect of delphinidin and cyanidin on GR activity.Cells were treated delphinidin and cyanidin for1h at the indicated concentrations and the GR activity measured by changes in NADPH absorbance.No significant changes observed in Caco-2(A)and LoVo(B)cells.NADPH absorbance levels
significantly reduced in LoVo/ADR(C)(ÃÃp<0.01,ÃÃÃp<0.001;one way ANOVA Dunnet’s post test).
Modulation of intracellular GSH levels by delphinidin and cyanidin.
treated for1h with delphinidin and cyanidin at the indicated concentrations GSH levels were determined as described in Materials and methods. significant changes were observed in Caco-2(A)and LoVo(B)cells.GSH levels
significantly reduced in LoVo/ADR cells(C).Intracellular GSH levels were calculated 10À6cells and presented as percentage as compared to controls(ÃÃp
0.001;one way ANOVA with Dunnet’s post test).
J.Cvorovic et al./Archives of Biochemistry and Biophysics501(2010)151–157155
豫公网安备41160202000603
站长QQ:729038198
关于我们
投诉建议