232, Artykuły Innej Medycyny
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//-->MOLECULAR & CELLULAR TOXICOLOGY, Vol. 2, No. 3, 159-165, September 2006Amygdalin Modulates Cell Cycle Regulator Genes in HumanChronic Myeloid Leukemia CellsHae Jeong Park1, Haing Woon Baik2,Seong Kyu Lee2, Seo Hyun Yoon1,Long Tai Zheng1, Sung Vin Yim1,Seon-Pyo Hong3& Joo-Ho Chung11microarray, Cell cycle regulator genesKohwang Medical Research Institute, Department ofPharmacology, College of Medicine, Kyung Hee University,Seoul 130-701, Korea2Department of Biochemistry and Molecular Biology, School ofMedicine, Eulji University, Daejeon 301-832, Korea3Department of Oriental Pharmaceutical Sciences, College ofPharmacy, Kyung Hee University, Seoul 130-701, KoreaCorrespondence and requests for materials should be addressedto J.-H. Chung (jhchung@khu.ac.kr)Accepted 9 September 2006AbstractTo determine the anticancer effect of D-amygdalinβ(D-mandelinitrole-β -D-gentiobioside) in humanchronic myeloid leukemia cells K562, we profiled thegene expression between amygdalin treatment andcontrol groups. Through 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, the±cytotoxicity of D-amygdalin was 57.79± 1.83% atthe concentration of 5 mg/mL for 24 h. We performedcDNA microarray analysis and compared the geneexpression profiles between D-amygdalin (5 mg/mL,24 h) treatment and control groups. Among the geneschanged by D-amygdalin, we paid attention to cellcycle-related genes, and particularly cell cycle reg-ulator genes; because arrest of cell cycle processingwas ideal tactic in remedy for cancer. In our data,expressions ofcyclin-dependent kinase inhibitor 1B(p27, Kip1)(CDKN1B),ataxia telangiectasia mutated(includes complementation groups A, C, and D)(ATM),cyclin-dependent kinase inhibitor 1C(p57,Kip2)(CDKN1C), andCHK1 checkpoint homolog(CHEK1, formally known asCHK1)were increased,while expressions ofcyclin-dependent kinase 2(CDK2),cell division cycle 25A(CDC25A), andcyclinE1(CCNE1) were decreased. The pattern of thesegene expressions were confirmed through RT-PCR.Our results showed that D-amygdalin might controlcell cycle regulator genes and arrest S phase of cellcycle in K562 cells as the useful anticancer drug.Keywords:Chronic myeloid leukemia, D-Amygdalin, cDNAChronic myeloid leukemia (CML) is a hematolog-ical malignancy characterized by immature granulo-cytosis, basophilia, thrombocytosis, and anemia1.CML represents about 14% of all leukemias andoccurs with a frequency of about 1 in 100,000. Ioniz-ing radiation has been implicated in some cases ofCML, but in most individual no cause is known. Indespite of high responses of new anticancer drugsuch as Gleevec (imatinib mesylate) on CML, drugresistance occurred ultimately in chronic myeloidleukemia blast crisis2,3. In recent years, the develop-ment of new anticancer drug is a key issue for cancerchemotherapy, and herbal medicine has attracted agreat deal of recent attention due to their low toxicityand costs.Armeniacae semen is a seed ofPrunus armeniacaLinnen var.ansuMaximowicz, which belongs to theRosaceae family. This seed has been widely used totreat asthma, aplastic anemia and several cancers inOriental medicine4. Amygdalin, ingredient of Arm-eniacae semen, has been reviewed as prevention andcontrol of cancers. Amygdalin belonging to a familyof compounds called cyanide would be broken downby an enzyme in cancerous tissue, and toxic cyanideto release from broken amygdalin would kill thecancer. It is further hypothesized that another enzyme,rhodanese, which has the ability to detoxify cyanide,is present in normal tissues but deficient in cancercells. These two factors supposedly combine to effecta selective poisoning of cancer cells by the cyanide,while normal cells remain undamaged5,6. Nevertheless,there have been controversial views on the use ofamygdalin because of its toxicity7-9. However, Kwonet al.10was demonstrated that the toxicity of amyg-dalin was due to inactivate isoform of amygdalin.Furthermore, it was reported that D-amygdalin (D-mandelinitrole-β-D-gentiobioside) used in our studyselectively killed cancer cells at the tumor site with-out systemic toxicity, which is the usual problemwhen using general chemotherapeutic agents4.In this study, we determined whether D-amygdalininduced cell death of human chronic myeloid leukemiacells, K562 and compared the expression profilesbetween D-amygdalin-treated and control groupsusing cDNA microarray analysis. Additionally, the160Mol. Cell. Toxicol. Vol. 2(3), 159-165, 2006(A)2(B)2Absorbance1Absorbance148124812Retention time/minRetention time/minFig. 1.Reverse-phase HPLCseparation of amygdalin in phos-phate buffer. (A) D-amygdalinstandard. (B) D-amygdalin ob-tained by our method. Peak 1,neoamygdalin; peak 2, D-amyg-dalin.Cell viability (% of control)genes selected by cDNA microarray analysis wereconfirmed through RT-PCR.12010080*604020Control0.512.55 (mg/mL)**Cytotoxicity of D-amygdalin in K562 CellsWhen treated with D-amygdalin of 0.5, 1.0, 2.5,and 5 mg/mL concentrations for 24 h, viabilities ofK562 cells were 80.13±3.67%, 71.92±0.78%,63.56±2.63%, and 57.79±1.83%, respectively, com-pared with those of nontreated cells (Fig. 2). MTTassay showed the dose-dependent cytotoxicity of D-amygdalin on K562 cells, and significant at con-centration of 2.5 and 5 mg/mL. To compare the pre-cise effect of D-amygdalin, further experiments werecarried out using 5 mg/mL D-amygdalin for 24 h.Analysis of Microarray Expression DataIn order to assess the expression profiles, D-amygdalin (5 mg/mL, 24 h)-treated group was com-pared to control group. The cDNA microarray thatcontained duplicate cDNA probes from 1 K leukemiacancer clones was used (Digital Genomics, Seoul,Korea). To normalize intensity ratio of each geneexpression pattern, global M method was used in thisstudy. First, the primary data were normalized by thetotal spots of intensity between two groups, and thennormalized by the intensity ratio of reference genes,such as housekeeping genes in both groups. Finally,the expression ratio of D-amygdalin-treated group tocontrol group was converted to log2ratio of eachgene. After normalizing the data, a difference of morethan 2 fold in the normalized intensity ratio wasselected and considered as significance. The expres-sion ratios of 40 genes were upregulated more than 2fold by D-amygdalin, whereas those of 25 genes weredownregulated lower than 2 fold (data not shown).Fig. 2.Cytotoxicity of D-amygdalin. Human chronicmyeloid leukemia, K562 cells were treated with variousconcentrations (0.5, 1.0, 2.5, and 5 mg/mL) of D-amygdalinfor 24 h prior to the determination of cellular viabilitythrough 3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenylte-trazolium bromide (MTT) assay. Independent experimentwas repeated three times. Results are presented as mean±S.E.M. (*p⁄0.05, **p⁄0.01).We paid attention to genes belonging to cell cyclecategory. Particularly, expressions of cell cyclerelated genes such ascyclin-dependent kinase inhibitor1B(p27,Kip1)(CDKN1B),ataxia telangiectasiamutated(includes complementation groups A, C, andD) (ATM),cyclin-dependent kinase inhibitor 1C(p57,Kip2)(CDKN1C), andCHK1 checkpoint homolog(CHEK1, formally known asCHK1)were upregu-lated more than 8-fold (Table 1), while the expressionof the genes such ascyclin-dependent kinase 2(CDK2),cell division cycle 25A(CDC25A), andcyclinE1(CCNE1) were downregulated lower than 4-fold(Table 2).Amygdalin Modulates Cell Cycle Regulator Genes161Table 1.List of cell cycle-related genes upregulated by D-amygdalin.Gene nameCDKN1BATMCDKN1CCHEK1CDKN2CNBS1ACPPMDM2CHES1CORO1ABRCA1CCNA1LIG4MSH2HUS1RGS2GenBanknumberAA455410U67093AI088356AF016582AF041248NM_002485AA613916Z12020U68723X89109U14680U66838X83441AW004683Y16893AI675283Chromosome12p13.1-p1211q22-q2311p15.511q24-q241p328q213q21-q2312q14.3-q1514q24.3-q3116p12.117q2113q12.3-q1313q33-q342p22-p217p13-p121q31Titlecyclin-dependent kinase inhibitor 1B (p27, Kip1)ataxia telangiectasia mutated (includes complementationgroups A, C and D)cyclin-dependent kinase inhibitor 1C (p57, Kip2)CHK1 checkpoint homolog (S. pombe)cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4)Nijmegen breakage syndrome 1 (nibrin)acid phosphatase, prostateMdm2, transformed 3T3 cell double minute 2, p53binding protein (mouse)checkpoint suppressor 1coronin, actin binding protein, 1Abreast cancer 1, early onsetcyclin A1ligase IV, DNA, ATP-dependentmutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli)HUS1 checkpoint homolog (S. pombe)regulator of G-protein signalling 2, 24 kDaGlobal M4.1113.4953.2853.0392.9832.9532.9322.8542.7661.9991.7311.6751.4661.4471.2661.144Table 2.List of cell cycle-related genes downregulated by D-amygdalin.Gene nameCDC2CCNB1MAPK6E2F1BCL2EGFRFGF7TGFAMCCCDK2CDC25ACCNE1GenBanknumberY00272AI972071X80692NM_005225M14745X00588AI075338X70340M62397AA789250M81933M73812Chromosome10q21.15q1215q2120q11.218q21.337p1215q15-q21.12p135q21-q2212q133p2119q12Titlecell division cycle 2, G1 to S and G2 to Mcyclin B1mitogen-activated protein kinase 6E2F transcription factor 1B-cell CLL/lymphoma 2epidermal growth factor receptor (erythroblastic leukemiaviral (v-erb-b) oncogene homolog, avian)fibroblast growth factor 7 (keratinocyte growth factor)transforming growth factor, alphamutated in colorectal cancerscyclin-dependent kinase 2cell division cycle 25Acyclin E1Global M-1.129-1.144-1.256-1.26-1.446-1.463-1.632-1.673-1.838-2.158-2.712-2.817Confirmation of cDNA Microarray Findingsby RT-PCRSelecting upregulated 4 genes (CDKN1B,ATM,CDKN1C,andCHEK1)and downregulated 3 genes(CDK2,CDC25A,andCCNE1)by D-amygdalintreatment among cell cycle related genes, we observedthe mRNA expressions using RT-PCR reproduced theresults of cDNA microarray. The efficiency of thereaction was adjusted by GAPDH amplification. Asshown on Fig. 3, the expressions ofCDKN1B, ATM,CDKN1C,andCHEK1were increased (Fig. 3A),while the levels ofCDK2,CDC25A, andCCNE1were decreased by D-amygdalin treatment (Fig. 3B).DiscussionWe referred to concentration of D-amygdalin obs-erved in study of Kwonet al.10and examined itscytotoxicity in human chronic myeloid leukemiacells, K562. D-Amygdalin induced cell death as adose-dependent manner, and showed significantcytotoxicity at the concentrations of 2.5 and 5 mg/mL.In this study, to determine the anticancer effect of D-amygdalin, we identified either decrease or increaseof gene expression by D-amygdalin treatment inK562 cells using cDNA microarray. One of the advan-162Mol. Cell. Toxicol. Vol. 2(3), 159-165, 2006(A)12CDKN1BATMCDKN1CCHEK1GAPDH(B)12CDK2CDC25ACCNE1GAPDHFig. 3.Confirmation of cDNA microarray results of upreg-ulated and downregulated genes by RT-PCR. (A) Fore genesupregulated by treatment of D-amygdalin,cyclin-dependentkinase inhibitor 1B(p27,Kip1)(CDKN1B),ataxia telang-iectasia mutated(includes complementation groups A, C,and D) (ATM),cyclin-dependent kinase inhibitor 1C(p57,Kip2)(CDKN1C), andCHK1 checkpoint homolog(CHEK1,formally known asCHK1)and (B) three genes downregu-lated by treatment of D-amygdalin,cyclin-dependent kinase2(CDK2),cell division cycle 25A(CDC25A), andcyclin E1(CCNE1) were analyzed by RT-PCR with total RNA fromcontrol and D-amygdalin (5 mg/mL, 24 h) treated humanchronic myeloid leukemia cells. As an internal control,GAPDHwas amplified.tages of cDNA microarray is the possibility to observethe expression pattern of the whole genes and tocompare with different conditions. In our data, wepaid attention to genes belonging to cell cycle cate-gory of which genes showed most deferent expres-sion between two groups. Particularly, the genes suchasCDKN1B, ATM, CDKN1C, CHEK1, CDK2,CDC25A,andCCNE1which were shown the differ-ences of remarkable expression were well known forimportant regulators controlled cell cycle progres-sion.In treatment of cancer, the arrest of cell cycle pro-gression has been known as ideal tactic11,12, becausethe cell cycle is a critical regulator of the processes ofcell proliferation and growth as well as of cell divi-sion after DNA damage13. Progression of a cellthrough the cell cycle is promoted by a number ofCDKs complexed with regulatory proteins calledcyclins. The association of cyclin E (CCNE) withCDK2is active at the G1/S transition and directs entryinto S phase. S phase progression is directed by theCCNA/CDK2complex, and the complex ofCCNAwithCDK1is important in G2.CDK1/CCNBisnecessary for mitosis to occur14. Additionally, at anappropriate time of the cell cycle, these cyclin/CDKcomplexes are dephosphorylated by CDC25 and acti-vated.CDC25Aacting onCCNE/CDK2is primarilyresponsible for S phase progression, whileCDC25Cacting onCCNB/CDK1is responsible for G2/Mprogression15,16. Our study showed that expressionsofCDK2, CDC25A,andCCNE1were decreased lowerthan 4 fold by D-amygdalin treatment. These resultsdemonstrated that D-amygdalin would arrest S phaseof cell cycle in K562 cells. Conversely, the expres-sions ofATMandCHEK1were increased more than8 fold. In arresting mechanism of S phase, prior tothe action ofCDC25A,the upstream factors respon-sible for initiating a checkpoint response are the ATMand ATR protein kinases17. These two enzymes arekey components of the DNA damage response thatactivate theCHEK1andCHEK2protein kinase.Anticancer agents are known to rapidly activated theATM/ATR-CHEK1/2 pathway15,18leading to phos-phorylation ofCDC25A,and thereby resulting in theinactivation of theCCNE/CDK2complex15.In cell cycle regulation, not only CDK/cyclin com-plex but corresponding cell cycle inhibitory (CDKinhibitors [CDKIs]) proteins play an important role,which serve as negative regulators of the cell cycleand stop proceeding to the next phase of the cell cycle.CDKIs have been proposed to act as tumor suppressergenes, and several members have been implicated inthe pathogenesis of a variety of human cancers19-21.Particularly, the kinase inhibitor protein (KIP) group ofCDKIs, p21waf1(CDKN1A), p27kip1(CDKN1B), andp57kip2(CDKN1C), negatively regulate cyclinE/CDK2and cyclinA/CDK2complexes22. Our results revealedthat D-amygdalin was upregulated the expressions ofCDKN1BandCDKN1Cmore than 8 fold. Sheaffetal.23showed that expression ofCCNE1-CDK2resultsAmygdalin Modulates Cell Cycle Regulator Genes163in phosphorylation ofCDKN1B,leading to elimi-nation ofCDKN1Bfrom the cell and progression ofthe cell cycle from G1 to S phase.CDKN1Cis a potenttight-binding inhibitor of several G1 cyclin/CDKcomplexes24.CDKN1Cinhibits cyclin A- and E-asso-ciated CDKs, therefore regulates G1/S transition andcompletion of S phase24.In present study, cDNA microarray revealed that D-amygdalin was regulated genes belonging to cellcycle category in K562 cells. Especially, decrease ofexpressions ofCDK2, CDC25A,andCCNE1,andincrease of levels ofCDKN1B, ATM, CDKN1C,andCHEK1were remarkable, and it was confirmed byRT-PCR. Based on these results, D-amygdalininduced DNA damage and thereby triggered S phasearrest, modulated these cell cycle regulator genes.These results suggest that the treatment of D-amyg-dalin revealed the anticancer effect on human chronicmyeloid leukemia K562 cells, and D-amygdalinmight be used for anticancer drug.bilization solution for 24 h. The viability was mea-sured with a microtiter plate reader (Bio-Tek, VT,USA) at a test wavelength of 595 nm with a referencewavelength of 690 nm. The optical density (O.D.)was calculated as the difference between the refer-ence wavelength and the test wavelength. Percentviability was calculated as (O.D. of drug-treated sam-ple/O.D. of untreated sample)×100.MethodsPreparation of D-amygdalinBoth 500 g of Armeniacae semen hatched from theshell and 10 L of 4% citric acid solution were refluxedfor 2 h. Filtered when it was still hot, the filtrate waspassed through the column packed with HP-20. Thesubstance absorbed within the column was concen-trated after it had been eluted by ethanol. D-amyg-dalin (4.2 g; yield rate, 0.84%) was obtained by recrys-tallizing the extract with ethanol. The amygdalin wasused after it had been determined to be over 95.0% ofpurity, by means of high-pressure liquid chromatog-raphy (HPLC) to measure its purity (Fig. 1).Cell CultureThe K562 cell line was obtained from the KoreanCell Line Bank (KCLB, Seoul, Korea). Cells werecultured in RPMI-1640 medium (Gibco, GrandIsland, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco). Cultureswere maintained in a humidified incubator at 37�½ inCan atmosphere of 5% CO2, 95% air.MTT AssayCell viability was determined by the manufacture’sprotocol using cell proliferation kit (MTT) (Roche,Indianapolis, IN, USA). K562 cells were treated withD-amygdalin at concentrations of 0.5, 1.0, 2.5, and 5mg/mL for 24 h. After MTT labeling reagent wasadded to each group, the cells were incubated for 4 h.Then, they were further incubated with the solu-Microarray Hybridization, Scanning, andData AnalysisTotal RNA was extracted using RNAzolTMB (TEL-TEST, TX, USA) as per the manufacturer’s protocol.The cDNA synthesis was performed with 3DNATMArray 50TMdetection method (Genisphere, PA, USA)as per the manufacturer’s protocols. The cDNAs ofcontrol and D-amygdalin-treated groups (5 mg/mL,24 h) were synthesized from total RNA. The cDNAchip of TwinChipTMLeukemia cancer 1 K (DigitalGenomics) was used. The concentrated cDNA and3DNATMwere hybridized on two identical arrays in aslide for a duplicate experiment. Hybridization, scan-ning, and data analysis were done at Digital Genomics.The hybridized microarray was scanned with aconfocal laser scanning microscope (ScanArray 5000;Packard Inc., CT, USA) at 532 nm for Cy3 and 635nm for Cy5. Image analysis using GenePix (AxonInc., CA, USA) produced quantitative values for eachmicroarray spot. Pixel intensity of the backgroundwas subtracted from those of microarray spots. Spotintensities were normalized using the intensitiesgenerated by intensity/location dependent method25.Normalized spot intensities were calculated into geneexpression ratios between the control and treatmentgroups. Mean data acquired from two identical arraysin a single slide of TwinChipTMwere analyzed.RT-PCRWe selected 4 genes,CDKN1B, ATM, CDKN1C,andCHEK1,upregulated in microarray analysis bytreatment of D-amygdalin. Additionally, 3 genes wereselected,CDK2, CDC25A,andCCNE1,downreg-ulated by treatment of D-amygdalin, and performedRT-PCR. Primer sequences, annealing temperaturesand products size of genes were summarized in Table3. The RT-PCR products were electrophoresed on a1.5% agarose gel and visualized by staining withethidium bromide.Statistical AnalysisResults were expressed as mean±SEM. The datawere analyzed by one-way ANOVA following theDunnett’spost-hocanalysis, using SPSS. Differenceswere considered significant atp⁄0.05. [ Pobierz całość w formacie PDF ]
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//-->MOLECULAR & CELLULAR TOXICOLOGY, Vol. 2, No. 3, 159-165, September 2006Amygdalin Modulates Cell Cycle Regulator Genes in HumanChronic Myeloid Leukemia CellsHae Jeong Park1, Haing Woon Baik2,Seong Kyu Lee2, Seo Hyun Yoon1,Long Tai Zheng1, Sung Vin Yim1,Seon-Pyo Hong3& Joo-Ho Chung11microarray, Cell cycle regulator genesKohwang Medical Research Institute, Department ofPharmacology, College of Medicine, Kyung Hee University,Seoul 130-701, Korea2Department of Biochemistry and Molecular Biology, School ofMedicine, Eulji University, Daejeon 301-832, Korea3Department of Oriental Pharmaceutical Sciences, College ofPharmacy, Kyung Hee University, Seoul 130-701, KoreaCorrespondence and requests for materials should be addressedto J.-H. Chung (jhchung@khu.ac.kr)Accepted 9 September 2006AbstractTo determine the anticancer effect of D-amygdalinβ(D-mandelinitrole-β -D-gentiobioside) in humanchronic myeloid leukemia cells K562, we profiled thegene expression between amygdalin treatment andcontrol groups. Through 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, the±cytotoxicity of D-amygdalin was 57.79± 1.83% atthe concentration of 5 mg/mL for 24 h. We performedcDNA microarray analysis and compared the geneexpression profiles between D-amygdalin (5 mg/mL,24 h) treatment and control groups. Among the geneschanged by D-amygdalin, we paid attention to cellcycle-related genes, and particularly cell cycle reg-ulator genes; because arrest of cell cycle processingwas ideal tactic in remedy for cancer. In our data,expressions ofcyclin-dependent kinase inhibitor 1B(p27, Kip1)(CDKN1B),ataxia telangiectasia mutated(includes complementation groups A, C, and D)(ATM),cyclin-dependent kinase inhibitor 1C(p57,Kip2)(CDKN1C), andCHK1 checkpoint homolog(CHEK1, formally known asCHK1)were increased,while expressions ofcyclin-dependent kinase 2(CDK2),cell division cycle 25A(CDC25A), andcyclinE1(CCNE1) were decreased. The pattern of thesegene expressions were confirmed through RT-PCR.Our results showed that D-amygdalin might controlcell cycle regulator genes and arrest S phase of cellcycle in K562 cells as the useful anticancer drug.Keywords:Chronic myeloid leukemia, D-Amygdalin, cDNAChronic myeloid leukemia (CML) is a hematolog-ical malignancy characterized by immature granulo-cytosis, basophilia, thrombocytosis, and anemia1.CML represents about 14% of all leukemias andoccurs with a frequency of about 1 in 100,000. Ioniz-ing radiation has been implicated in some cases ofCML, but in most individual no cause is known. Indespite of high responses of new anticancer drugsuch as Gleevec (imatinib mesylate) on CML, drugresistance occurred ultimately in chronic myeloidleukemia blast crisis2,3. In recent years, the develop-ment of new anticancer drug is a key issue for cancerchemotherapy, and herbal medicine has attracted agreat deal of recent attention due to their low toxicityand costs.Armeniacae semen is a seed ofPrunus armeniacaLinnen var.ansuMaximowicz, which belongs to theRosaceae family. This seed has been widely used totreat asthma, aplastic anemia and several cancers inOriental medicine4. Amygdalin, ingredient of Arm-eniacae semen, has been reviewed as prevention andcontrol of cancers. Amygdalin belonging to a familyof compounds called cyanide would be broken downby an enzyme in cancerous tissue, and toxic cyanideto release from broken amygdalin would kill thecancer. It is further hypothesized that another enzyme,rhodanese, which has the ability to detoxify cyanide,is present in normal tissues but deficient in cancercells. These two factors supposedly combine to effecta selective poisoning of cancer cells by the cyanide,while normal cells remain undamaged5,6. Nevertheless,there have been controversial views on the use ofamygdalin because of its toxicity7-9. However, Kwonet al.10was demonstrated that the toxicity of amyg-dalin was due to inactivate isoform of amygdalin.Furthermore, it was reported that D-amygdalin (D-mandelinitrole-β-D-gentiobioside) used in our studyselectively killed cancer cells at the tumor site with-out systemic toxicity, which is the usual problemwhen using general chemotherapeutic agents4.In this study, we determined whether D-amygdalininduced cell death of human chronic myeloid leukemiacells, K562 and compared the expression profilesbetween D-amygdalin-treated and control groupsusing cDNA microarray analysis. Additionally, the160Mol. Cell. Toxicol. Vol. 2(3), 159-165, 2006(A)2(B)2Absorbance1Absorbance148124812Retention time/minRetention time/minFig. 1.Reverse-phase HPLCseparation of amygdalin in phos-phate buffer. (A) D-amygdalinstandard. (B) D-amygdalin ob-tained by our method. Peak 1,neoamygdalin; peak 2, D-amyg-dalin.Cell viability (% of control)genes selected by cDNA microarray analysis wereconfirmed through RT-PCR.12010080*604020Control0.512.55 (mg/mL)**Cytotoxicity of D-amygdalin in K562 CellsWhen treated with D-amygdalin of 0.5, 1.0, 2.5,and 5 mg/mL concentrations for 24 h, viabilities ofK562 cells were 80.13±3.67%, 71.92±0.78%,63.56±2.63%, and 57.79±1.83%, respectively, com-pared with those of nontreated cells (Fig. 2). MTTassay showed the dose-dependent cytotoxicity of D-amygdalin on K562 cells, and significant at con-centration of 2.5 and 5 mg/mL. To compare the pre-cise effect of D-amygdalin, further experiments werecarried out using 5 mg/mL D-amygdalin for 24 h.Analysis of Microarray Expression DataIn order to assess the expression profiles, D-amygdalin (5 mg/mL, 24 h)-treated group was com-pared to control group. The cDNA microarray thatcontained duplicate cDNA probes from 1 K leukemiacancer clones was used (Digital Genomics, Seoul,Korea). To normalize intensity ratio of each geneexpression pattern, global M method was used in thisstudy. First, the primary data were normalized by thetotal spots of intensity between two groups, and thennormalized by the intensity ratio of reference genes,such as housekeeping genes in both groups. Finally,the expression ratio of D-amygdalin-treated group tocontrol group was converted to log2ratio of eachgene. After normalizing the data, a difference of morethan 2 fold in the normalized intensity ratio wasselected and considered as significance. The expres-sion ratios of 40 genes were upregulated more than 2fold by D-amygdalin, whereas those of 25 genes weredownregulated lower than 2 fold (data not shown).Fig. 2.Cytotoxicity of D-amygdalin. Human chronicmyeloid leukemia, K562 cells were treated with variousconcentrations (0.5, 1.0, 2.5, and 5 mg/mL) of D-amygdalinfor 24 h prior to the determination of cellular viabilitythrough 3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenylte-trazolium bromide (MTT) assay. Independent experimentwas repeated three times. Results are presented as mean±S.E.M. (*p⁄0.05, **p⁄0.01).We paid attention to genes belonging to cell cyclecategory. Particularly, expressions of cell cyclerelated genes such ascyclin-dependent kinase inhibitor1B(p27,Kip1)(CDKN1B),ataxia telangiectasiamutated(includes complementation groups A, C, andD) (ATM),cyclin-dependent kinase inhibitor 1C(p57,Kip2)(CDKN1C), andCHK1 checkpoint homolog(CHEK1, formally known asCHK1)were upregu-lated more than 8-fold (Table 1), while the expressionof the genes such ascyclin-dependent kinase 2(CDK2),cell division cycle 25A(CDC25A), andcyclinE1(CCNE1) were downregulated lower than 4-fold(Table 2).Amygdalin Modulates Cell Cycle Regulator Genes161Table 1.List of cell cycle-related genes upregulated by D-amygdalin.Gene nameCDKN1BATMCDKN1CCHEK1CDKN2CNBS1ACPPMDM2CHES1CORO1ABRCA1CCNA1LIG4MSH2HUS1RGS2GenBanknumberAA455410U67093AI088356AF016582AF041248NM_002485AA613916Z12020U68723X89109U14680U66838X83441AW004683Y16893AI675283Chromosome12p13.1-p1211q22-q2311p15.511q24-q241p328q213q21-q2312q14.3-q1514q24.3-q3116p12.117q2113q12.3-q1313q33-q342p22-p217p13-p121q31Titlecyclin-dependent kinase inhibitor 1B (p27, Kip1)ataxia telangiectasia mutated (includes complementationgroups A, C and D)cyclin-dependent kinase inhibitor 1C (p57, Kip2)CHK1 checkpoint homolog (S. pombe)cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4)Nijmegen breakage syndrome 1 (nibrin)acid phosphatase, prostateMdm2, transformed 3T3 cell double minute 2, p53binding protein (mouse)checkpoint suppressor 1coronin, actin binding protein, 1Abreast cancer 1, early onsetcyclin A1ligase IV, DNA, ATP-dependentmutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli)HUS1 checkpoint homolog (S. pombe)regulator of G-protein signalling 2, 24 kDaGlobal M4.1113.4953.2853.0392.9832.9532.9322.8542.7661.9991.7311.6751.4661.4471.2661.144Table 2.List of cell cycle-related genes downregulated by D-amygdalin.Gene nameCDC2CCNB1MAPK6E2F1BCL2EGFRFGF7TGFAMCCCDK2CDC25ACCNE1GenBanknumberY00272AI972071X80692NM_005225M14745X00588AI075338X70340M62397AA789250M81933M73812Chromosome10q21.15q1215q2120q11.218q21.337p1215q15-q21.12p135q21-q2212q133p2119q12Titlecell division cycle 2, G1 to S and G2 to Mcyclin B1mitogen-activated protein kinase 6E2F transcription factor 1B-cell CLL/lymphoma 2epidermal growth factor receptor (erythroblastic leukemiaviral (v-erb-b) oncogene homolog, avian)fibroblast growth factor 7 (keratinocyte growth factor)transforming growth factor, alphamutated in colorectal cancerscyclin-dependent kinase 2cell division cycle 25Acyclin E1Global M-1.129-1.144-1.256-1.26-1.446-1.463-1.632-1.673-1.838-2.158-2.712-2.817Confirmation of cDNA Microarray Findingsby RT-PCRSelecting upregulated 4 genes (CDKN1B,ATM,CDKN1C,andCHEK1)and downregulated 3 genes(CDK2,CDC25A,andCCNE1)by D-amygdalintreatment among cell cycle related genes, we observedthe mRNA expressions using RT-PCR reproduced theresults of cDNA microarray. The efficiency of thereaction was adjusted by GAPDH amplification. Asshown on Fig. 3, the expressions ofCDKN1B, ATM,CDKN1C,andCHEK1were increased (Fig. 3A),while the levels ofCDK2,CDC25A, andCCNE1were decreased by D-amygdalin treatment (Fig. 3B).DiscussionWe referred to concentration of D-amygdalin obs-erved in study of Kwonet al.10and examined itscytotoxicity in human chronic myeloid leukemiacells, K562. D-Amygdalin induced cell death as adose-dependent manner, and showed significantcytotoxicity at the concentrations of 2.5 and 5 mg/mL.In this study, to determine the anticancer effect of D-amygdalin, we identified either decrease or increaseof gene expression by D-amygdalin treatment inK562 cells using cDNA microarray. One of the advan-162Mol. Cell. Toxicol. Vol. 2(3), 159-165, 2006(A)12CDKN1BATMCDKN1CCHEK1GAPDH(B)12CDK2CDC25ACCNE1GAPDHFig. 3.Confirmation of cDNA microarray results of upreg-ulated and downregulated genes by RT-PCR. (A) Fore genesupregulated by treatment of D-amygdalin,cyclin-dependentkinase inhibitor 1B(p27,Kip1)(CDKN1B),ataxia telang-iectasia mutated(includes complementation groups A, C,and D) (ATM),cyclin-dependent kinase inhibitor 1C(p57,Kip2)(CDKN1C), andCHK1 checkpoint homolog(CHEK1,formally known asCHK1)and (B) three genes downregu-lated by treatment of D-amygdalin,cyclin-dependent kinase2(CDK2),cell division cycle 25A(CDC25A), andcyclin E1(CCNE1) were analyzed by RT-PCR with total RNA fromcontrol and D-amygdalin (5 mg/mL, 24 h) treated humanchronic myeloid leukemia cells. As an internal control,GAPDHwas amplified.tages of cDNA microarray is the possibility to observethe expression pattern of the whole genes and tocompare with different conditions. In our data, wepaid attention to genes belonging to cell cycle cate-gory of which genes showed most deferent expres-sion between two groups. Particularly, the genes suchasCDKN1B, ATM, CDKN1C, CHEK1, CDK2,CDC25A,andCCNE1which were shown the differ-ences of remarkable expression were well known forimportant regulators controlled cell cycle progres-sion.In treatment of cancer, the arrest of cell cycle pro-gression has been known as ideal tactic11,12, becausethe cell cycle is a critical regulator of the processes ofcell proliferation and growth as well as of cell divi-sion after DNA damage13. Progression of a cellthrough the cell cycle is promoted by a number ofCDKs complexed with regulatory proteins calledcyclins. The association of cyclin E (CCNE) withCDK2is active at the G1/S transition and directs entryinto S phase. S phase progression is directed by theCCNA/CDK2complex, and the complex ofCCNAwithCDK1is important in G2.CDK1/CCNBisnecessary for mitosis to occur14. Additionally, at anappropriate time of the cell cycle, these cyclin/CDKcomplexes are dephosphorylated by CDC25 and acti-vated.CDC25Aacting onCCNE/CDK2is primarilyresponsible for S phase progression, whileCDC25Cacting onCCNB/CDK1is responsible for G2/Mprogression15,16. Our study showed that expressionsofCDK2, CDC25A,andCCNE1were decreased lowerthan 4 fold by D-amygdalin treatment. These resultsdemonstrated that D-amygdalin would arrest S phaseof cell cycle in K562 cells. Conversely, the expres-sions ofATMandCHEK1were increased more than8 fold. In arresting mechanism of S phase, prior tothe action ofCDC25A,the upstream factors respon-sible for initiating a checkpoint response are the ATMand ATR protein kinases17. These two enzymes arekey components of the DNA damage response thatactivate theCHEK1andCHEK2protein kinase.Anticancer agents are known to rapidly activated theATM/ATR-CHEK1/2 pathway15,18leading to phos-phorylation ofCDC25A,and thereby resulting in theinactivation of theCCNE/CDK2complex15.In cell cycle regulation, not only CDK/cyclin com-plex but corresponding cell cycle inhibitory (CDKinhibitors [CDKIs]) proteins play an important role,which serve as negative regulators of the cell cycleand stop proceeding to the next phase of the cell cycle.CDKIs have been proposed to act as tumor suppressergenes, and several members have been implicated inthe pathogenesis of a variety of human cancers19-21.Particularly, the kinase inhibitor protein (KIP) group ofCDKIs, p21waf1(CDKN1A), p27kip1(CDKN1B), andp57kip2(CDKN1C), negatively regulate cyclinE/CDK2and cyclinA/CDK2complexes22. Our results revealedthat D-amygdalin was upregulated the expressions ofCDKN1BandCDKN1Cmore than 8 fold. Sheaffetal.23showed that expression ofCCNE1-CDK2resultsAmygdalin Modulates Cell Cycle Regulator Genes163in phosphorylation ofCDKN1B,leading to elimi-nation ofCDKN1Bfrom the cell and progression ofthe cell cycle from G1 to S phase.CDKN1Cis a potenttight-binding inhibitor of several G1 cyclin/CDKcomplexes24.CDKN1Cinhibits cyclin A- and E-asso-ciated CDKs, therefore regulates G1/S transition andcompletion of S phase24.In present study, cDNA microarray revealed that D-amygdalin was regulated genes belonging to cellcycle category in K562 cells. Especially, decrease ofexpressions ofCDK2, CDC25A,andCCNE1,andincrease of levels ofCDKN1B, ATM, CDKN1C,andCHEK1were remarkable, and it was confirmed byRT-PCR. Based on these results, D-amygdalininduced DNA damage and thereby triggered S phasearrest, modulated these cell cycle regulator genes.These results suggest that the treatment of D-amyg-dalin revealed the anticancer effect on human chronicmyeloid leukemia K562 cells, and D-amygdalinmight be used for anticancer drug.bilization solution for 24 h. The viability was mea-sured with a microtiter plate reader (Bio-Tek, VT,USA) at a test wavelength of 595 nm with a referencewavelength of 690 nm. The optical density (O.D.)was calculated as the difference between the refer-ence wavelength and the test wavelength. Percentviability was calculated as (O.D. of drug-treated sam-ple/O.D. of untreated sample)×100.MethodsPreparation of D-amygdalinBoth 500 g of Armeniacae semen hatched from theshell and 10 L of 4% citric acid solution were refluxedfor 2 h. Filtered when it was still hot, the filtrate waspassed through the column packed with HP-20. Thesubstance absorbed within the column was concen-trated after it had been eluted by ethanol. D-amyg-dalin (4.2 g; yield rate, 0.84%) was obtained by recrys-tallizing the extract with ethanol. The amygdalin wasused after it had been determined to be over 95.0% ofpurity, by means of high-pressure liquid chromatog-raphy (HPLC) to measure its purity (Fig. 1).Cell CultureThe K562 cell line was obtained from the KoreanCell Line Bank (KCLB, Seoul, Korea). Cells werecultured in RPMI-1640 medium (Gibco, GrandIsland, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco). Cultureswere maintained in a humidified incubator at 37�½ inCan atmosphere of 5% CO2, 95% air.MTT AssayCell viability was determined by the manufacture’sprotocol using cell proliferation kit (MTT) (Roche,Indianapolis, IN, USA). K562 cells were treated withD-amygdalin at concentrations of 0.5, 1.0, 2.5, and 5mg/mL for 24 h. After MTT labeling reagent wasadded to each group, the cells were incubated for 4 h.Then, they were further incubated with the solu-Microarray Hybridization, Scanning, andData AnalysisTotal RNA was extracted using RNAzolTMB (TEL-TEST, TX, USA) as per the manufacturer’s protocol.The cDNA synthesis was performed with 3DNATMArray 50TMdetection method (Genisphere, PA, USA)as per the manufacturer’s protocols. The cDNAs ofcontrol and D-amygdalin-treated groups (5 mg/mL,24 h) were synthesized from total RNA. The cDNAchip of TwinChipTMLeukemia cancer 1 K (DigitalGenomics) was used. The concentrated cDNA and3DNATMwere hybridized on two identical arrays in aslide for a duplicate experiment. Hybridization, scan-ning, and data analysis were done at Digital Genomics.The hybridized microarray was scanned with aconfocal laser scanning microscope (ScanArray 5000;Packard Inc., CT, USA) at 532 nm for Cy3 and 635nm for Cy5. Image analysis using GenePix (AxonInc., CA, USA) produced quantitative values for eachmicroarray spot. Pixel intensity of the backgroundwas subtracted from those of microarray spots. Spotintensities were normalized using the intensitiesgenerated by intensity/location dependent method25.Normalized spot intensities were calculated into geneexpression ratios between the control and treatmentgroups. Mean data acquired from two identical arraysin a single slide of TwinChipTMwere analyzed.RT-PCRWe selected 4 genes,CDKN1B, ATM, CDKN1C,andCHEK1,upregulated in microarray analysis bytreatment of D-amygdalin. Additionally, 3 genes wereselected,CDK2, CDC25A,andCCNE1,downreg-ulated by treatment of D-amygdalin, and performedRT-PCR. Primer sequences, annealing temperaturesand products size of genes were summarized in Table3. The RT-PCR products were electrophoresed on a1.5% agarose gel and visualized by staining withethidium bromide.Statistical AnalysisResults were expressed as mean±SEM. The datawere analyzed by one-way ANOVA following theDunnett’spost-hocanalysis, using SPSS. Differenceswere considered significant atp⁄0.05. [ Pobierz całość w formacie PDF ]