| [Objective]Colonrectal cancer(CRC),a most common disease in gastrointestinal tract,was the second major cause of cancer death in Europe and the third in America.Most cases of CRC patient are diagnosed in advanced stage,surgical resection plus chemotherapy is the the most important way to cure these patients.Fluorouracil(5-FU) and Oxaliplatin(L-OHP) are used for CRC chemotherapy as first line drugs.A meta analysis of systemic therapy was reported by Jeffrey A et al.The research datas show FOLFOX regimen based on 5-FU and L-OHP may still be the best choise for advanced CRC therapy.patients receiving FOLFOX regimen had a response rate varing from 45%to 54%,and median overall survival time vary from 19.5 to 20.6 months.Although a lot of new drugs including irinotecan,cetuximab and bevacizumab have been used for CRC therapy in the past decades,the response rate still remains low.Intrinsic drug resistance and acquired drug resistance,which limit the effectiviness of current therapies,are believed to be main cause of treatment failure in over 90%paitients with advanced cancer.Overcoming drug resistance is one of the main challenges of current cancer research.Many drug metabolization associated proteins including thymidylate synthase(TS),thymidine phosphorylase(TP),glutathione-S-transferase-π(GST-π), P-glycoprotein(Mdr-1/pgp),multi-drug resistance protein(MRP) et al are reported to be useful to predict chemosensitivity in CRC.The proteins can reduce the accumulation of agents by increasing drug targets,drug inactivation and drug efflux,which leads tumor cells to escape from cytotoxity of chemical agents.Cancer stem cells(CSCs) are G0 cells which can renew itselves and differentiate to tumor cells.Recent studies have shown the associations between drug resistance and CSCs.It is reported that CSCs participates in drug resistance by overexpressing ATP-binding cassette transporters of multidrug resisitance protein(MDR) and multidrug resistance associated protein(MRP) family.It is supposed that CSCs may be the main source of tumor drug resistance.In the present study,SW1116,SW480,LOVO,HT-29,HCT116 cell lines were used and TS,TP,GST-π,pgp,MRP1 protein expression was detected by western blotting and immunocytochemistry,their mRNA expression was also detected by real time quantative polymase chain reaction(real time PCR).In vitro chemosensitivity of 5-FU and L-OHP are tested to analyse the correlations between IC50 and drug metabolization associated proteins mRNA expression.In the second part of this study,we also detected all five drug metabolization associated proteins mRNA in colon cancer stem cells(CSCs),which were individually obstained by serum free medium cultivation and Magnetic-activated cell sorting from SW480.We are primary aimed to investigate the probable mechanisms existed in CRCs by observing the differences of these proteins mRNA expression comparing to SW480.[Methods]1.ImmunohistochemistryHuman colon cancer tissue specimens were acquired by endoscopic protractor biopsy,then fixed in 4%formalin and enbeded in paraffin.4-μm sections were cut and placed on a glass slide.Antigens retrievals were mounted according to datasheet for first antibody.The slides were subsequently rinsed in PBS and incubated with 3% hydrogen peroxide for 10 min to reduce endogenous peroxidase activity.After rinsing in PBS,they were incubating with mouse anti-human monoclonal antibody(1:50) diluted in PBS overnight at 4℃.The slides were rinsed again in PBS for 5 min three times again and incubated with goat anti-mouse HRP-polymer secondary antibody for 30 min at room tempreture.After a final rinsing in PBS,tissue sections were incubated with diaminobenzidine substrate for 30 s,counterstained in hematoxylin,and mounted with glass coverslips and Permount.2.Cell lines and culture conditionsFive human colon cancer cell lines including SW1116,SW480,LOVO,HT-29 and HCT116 used in this study were preserved in our institute.All cell lines were cultured in RPMI1640 supplemented with fetal bovine serum(10%FBS) at 37℃in a 5%CO2 atmosphere.3.Western blotting analysisTotal cell lysates were prepared by lysing harvested cells in RIPA buffer(20 mM Tris,pH 7.4,140 mM NaC1,0.1%Nonidet P-40,1 mM PMSF,1μg/ml each of pepstatin,leupeptin,and aprotinin),DNA was sheared by sonication.The total amounts of protein were determined by the BCA assay.A total of 50μg of protein from samples were then mixed with 5×sample buffer,denatured at 95℃for 5 min, and separated on 10%or 6%SDS-PAGE.Proteins were transferred to PVDF membranes(Milipore).Membranes were blocked in 5%skim milk for lh at 37℃,washed in TBST,and then incubated with primary first mouse monoclonal antibodies for TS(1:300 dilution,Santa Cruz,USA),TP(1:400 dilution,Santa Cruz, USA),GST-Pi(1:500 dilution,Santa Cruz,USA),Pgp(1:200 dilution,Santa Cruz, USA) and MRP1(1:200 dilution,Santa Cruz,USA)overnight at 4℃.The membrances were washed and incubated with horseradish peroxidase-conjugated secondary antibody(1:3000 dilution) against each IgG for host of primary antibodies for 1 h at 37℃.The membrances were then stained using the detection regment of ECL detection kit(Amersham,USA).4.Immunocytochemistry analysisColon cancer cells LOVO and HT-29 grew on coverslips overnight.The coverslips was subsequently rinsed in PBS and incubated with 3%hydrogen peroxide for 10 min to reduce endogenous peroxidase activity.After rinsing in PBS,they were incubating with mouse anti-human monoclonal antibody(1:50) diluted in PBS 1h at room tempreture.The slides were rinsed in PBS for 5 min three times again and incubated with goat anti-mouse HRP-polymer secondary antibody for 30 min at room tempreture.After a final rinsing in PBS,tissue sections were incubated with diaminobenzidine substrate for 30 s,counterstained in hematoxylin,and mounted with glass coverslips and Permount. 5.WST-8 colorimetric assayCytotoxicity in vitro was evaluated by WST-8(2-(2-methoxy-4nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,monosodium salt) colorimetric assay.5000 cells/well were seed in 96-well cell plates in 100ul of RPMI1640 medium with 10%FBS for 4h prior to drug exposure.5-FU was diluted in cuture medium at a concentration range from 1.6uM to 1000uM.Oxaliplatin was also diluted in cuture medium at a concentration range from 0.16uM to 100uM.Cells were subsequently treated with various concentrations of 5-FU or L-OHP for 48hours.After drug exposure,the medium was discarded and replaced with 90ul of fresh medium followed by 10ul WST-8 reagent solution(cell counting kit-8) and incubated for 4h at 37℃in an incubator.Cell viability was determined according to colrimetric comparison by reading optical density(OD) values with a microplate reader at a absorption wavelength of 450nm.cytotoxity was evaluated using a Cell Counting Kit according to the manufacturer's instructions.6.Cultivation of colon cancer stem cellSW480 was suspended in DMEM/F12 serum free medium supplemented with 20ng/ml EGF,10ng/ml b-FGF,10ng/ml LIF,B27(1:50) and 2mM glutamine and was subsequently cultured in ultra low attachment flask at 37℃in an incubator.Colon cancer stem cells was harvested after SW480 generating tumor cell spheres.7.Magnetic-activated cell sortingAbout 108 SW480 cells were harvested and incubated in MACS CellFix solution at 25℃for 30 min.The supernatant was removed after centrifuge.Then,cells were washed with 20 mL 1×MACS CellStain solution,resuspended in 300 mL 1×MACS Cellstain solution.After adding 100 mL Fc receptor blocking reagent,100 mL CD44 or CD166 immunomagnetic microbeads were added to the samples,then incubated at 25℃for 45 min.A positive separation column was placed into the magnetic field of a MACS separator.The cell suspension was added into the separation column. CD44- or CD166- cells were harvested by rinsing thrice with 500 mL degassed 1×MACS dilution buffer,the separation column was removed from the separator field.Then 1 mL dilution buffer was added to the column.The cells labeled with immunomagnetic microbeads were eluted into another tube quickly using the plunger supplied with the column.The cells was then resuspended in PBS for PCR analysis.8.Real time Polymerase chain ReactionTotal RNA was extracted from 106cells with a TRIZOL reagent according to the manufacture's instruction.The protocol included a DNase digestion step to prevent carry-over of genomic DNA.A two step was employed for PCR analysis.Firstly,total RNA was reverse-transcribed using a 20ul of reverse transcription system including 5 ug RNA,0.5ug Oligo primer,lul Ribolock Ribonuclease inhibitor,2ul 10mM dNTP mix and 1ul RevertAid M-Mulv reverse transcriptase.The constitution of each PCR reaction were 1ul of cDNA,200nM of each primer and 7.5ul PCR mix.Product amplification was peformed up to 60 cycles after polymerase activation(5 min at 95℃).Each two step PCR step cycle comprised denaturing(20s at 94℃),annealing(20s at 58℃),and extending(40s at 72℃).Glyceraldehyde-3 phosphate dehydrogenase(GAPDH) was used as internal reference gene.The housekeeping gene was amplified parallel to the target genes in separate wells.Each amplicon was amplified in a separate reaction to prevent competition among multiple sets of primers,and negative controls with no template was added in every experiment.All assays were run in triplicate.The PCR cycle number that generated the first fluorescence signal above a threshold(threshold cycle,Ct) was determined,and a comparative Ct method was then used to measure relative gene expression.The following formula was used to calculate the relative amount of the transcript in the sample:2-ΔCt,whereACt is the difference in Ct between the target genes and the internal reference gene.9.Statistic analysisAll results were analysed by using a SPSS13.0 software package.Differences of taget genes mRNA expression level and IC50 of drugs among colon cancer cell lines were assessed using one-way ANOVA.Spearman bivariate Correlation was used to evaluate correlations between drug's IC50 and taget genes mRNA expression level. Differences of taget genes mRNA expression level between SW480 and CSCs, CD44+SW480 and CD44-SW480,CD166-SW480 and CD166+SW480 were compared using Independent samples T-test.P value at 0.05 level is of statistic significance.[Results]1.TS,TP,GST-π,Pgp,MRP1 expression were commonly observed in colonrectal cancer tissue.2.HT-29 cell line expresses a high level of TS,TP,GST-π,Pgp,MRP1,whereas only TS and Pgp expressions were detected by western blotting and immunocytochemistry analysis,which predicts HT-29 may be most resistant to chemotherapy,and LOVO could be relative sensitive to chemical agents.3.HT-29 and SW1116 express TS,TP,Pgp at a significant higher level than other cell lines(p＜0.05).The LOVO cell line express the highest level of GST-π(p＜0.05),whereas HCT116 and SW480 express a relative high level of MRP1(p＜0.05). 4.Cytotoxicity test in vitro certified that HT-29 cell was most resistantto 5-FU or L-OHP,while LOVO and SW480 cells were relative sensitive to 5-FU and L-OHP respectively.5.The cytotoxity of 5-FU was inversely correlated to mRNA expression level of TP,Pgp,TS in CRC cell lines.Whereas sensitivity to L-OHP was inversely correlated to GST-π,Pgp mRNA expression level.No relationship between MRP1 expression and cytotoxity of 5-FU or L-OHP was observed.6.Colon cancer stem cell sphere was successfully obstained from SW480 cultured in SFM supplemented with EGF,b-FGK LIF and B27.Analysis of mRNA expression of drug metabolizing associated genes by real time PCR indicated that Pgp and MRP1 expressed at a higher level while TS expressed at a lower level in CSCs than in SW480.No differences of statistic significance in both TP and GST-πexpression were observed.7.CD44+ or CD166+ side populations of colon cancer stem cells was sorted from SW480 cell line with immunomagnetic microbeads labled with CD44 or CD166 monoclonal antibodies.TS and TP mRNA expressions were confirmed significantly higher in CD44+ side population cells than that expressed in CD44-cells by real time PCR analysis,no expression differences of this two genes were found significant between CD166+ cells and CD 166- cells.Meanwhile,a lower level of Pgp and MRP1 mRNA expressed in CD166+ cells was observed than CD166-cells,but their expression differences between CD44+ and CD44- cells were found of no statistic significance.Both CD44+ cells and CD166+ cells expressed GST-πmRNA at a significant higher level than CD44- cells and CD 166- cells individually. [Conclusions]1.TS,TP,GST-π,Pgp,MRP1 proteins expressed commonly in CRC tissue.The inverse correlation between 5-FU chemosensitivity and mRNA expression levels of TP,Pgp,TS was observed in five CRC cell lines in vitro.we also found the inverse correlation between L-OHP sensitivity and GST-π,Pgp,TPmRNA expression level in these cell lines.The results of this study suggests that drug metabolizing associated gene expression in CRC tissue can effectively predict response of CRC to 5-FU or L-OHP based therapy.It is also implied that detection of these genes expression in CRC tissue by IHC or realtime PCR could be a useful method to predict patients response,which will permit clinical physician to select the best and individualized treatment for CRC patients.2.MRP1 mRNA expressed at a higher level and TS mRNA expressed at a lower level in CRC stem cells,Only a higher level of TP mRNA expression was observed in CD44+/SW480 side population.The results implied CRC stem cells was conferred to escape from cytotoxity of chemical agents by express a high level of these proteins,which ultimately causes treatment failure and cancer metastasis in CRC paitients.To explore drug resistance mechanisms existed in cancer stem cells and develope new targeted therapies will contribute to response rate elevation and prognosis improvement of colon cancer. |
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