| Background:pharyngeal carcinoma（NPC）is one of the most incident and dangerous malignant tumors in southern provinces of China.It has threated and impacted of people’s health and quality of life.At present the clinical radiation therapy is still the preferred method of the treatment of nasopharyngea carcinoma,although in recent years,equipment and technologies in radiation therapy and chemotherapy have been greatly advanced in recent years,the 5-year survival rate of patients with nasopharyngeal cancer remains at 50%,which did not fundamentally improve the treatment effect and radiotherapy in itself brought about by local and systemic side effects also give patients a great physical and psychological harm,which seriously impacted on treatment outeome.Therefore,it is very important to explore the most fundamental cause of nasopharyngeal carcinoma and new effective treatment method to it.Multidrug resistance is a condition enabling a disease-causing organism to resist distinct drugs or chemicals of a wide variety of structure and function targeted at eradicating the organism.Organisms that display multidrug resistance can be pathologic cells,including bacterial and neoplastic（tumor）cells.Congenital or acpuired multidrug resistance of tumor were both the problem in the treatment of tumor.Study molecular the mechanism of MDR is extremely important to improve the survival rate of NPC.ATP-binding cassette transporters（ABC-transporter）are members of a protein superfamily that is one of the largest and most ancient families with representatives in all extant phyla from prokaryotes to humans.There are 48 known ABC transporters present in humans,which are classified into seven families by the Human genome organization.ABC transporters are known to play a crucial role in the development of multidrug resistance（MDR）,especially the member of ABCG2,MRP1 and P-gp.The most-studied member in ABCG family is ABCG2,also known as BCRP（breast cancer resistance protein）.ATP-binding cassette sub-family G member 2 is a protein that in humans is encoded by the ABCG2 gene,which is encoded by 655 amino acids on chromosome 4q21-22,and confers multidrug resistance to mitoxantrone and topotecan.ABCG2 substrates including metabolites,drugs,toxic substances,endogenous lipids,polysaccharides,nucleotides,steroids,etc.Energy-dependent drug pump,and could pump drugs with different structure and mechnisms out of cells,resulting in multidrug resistance.The hypothesis of cancer stem cell（CSC）was proposed that a subpopulation of stem-like cells which were called cancer stem cell were responsible for sustaining tumour growth.So far,research is rapidly advancing in this field,to our knowledge,CSC had various pumps that pump out drugs to protect themselves,and CSC were in the dormancy stage which could generate multidrug resistance.There is evidence in 2009 to obtain CSC cells using a fluorescence-activated cell sorting（FACS）and magnetic activated cell sorting（MACS）techniques to isolate nasopharyngeal cancer stem cells.Megnetic cell sorting method is simple,bu the separetion efficiency and accuracy was slightly lower compared with flow cytometry sorting,and it was fit for cell low-purity insensitive sorting.Flow cytometry sorting had the advantage of higher sorting putity and recovery rate than MACS.FACS was suitable for the sorting of cells at low levels.However,it was more complicated to operate,and more up to the experience of skilled equipment operators.Therefore,MACS was more implied in the research.In this article Magnetic separation was carried out to isolate CD133+carcinoma stem cells in human nasopharyngeal carcinoma.Yang Zhuanyi isolated cancer stem cells（U251-CSC）from the cell line U251 by MACS,CCK8 methods were conducted to determine the drug sensitivity of teniposide（Vm-26）,carmustine（BCNU）and cisplatin（DDP）to the cell lines such as U251-CSC and U251,then Three enzymes of expression such as:LRP、MGMT and Topo Ⅱ αwere detected by Western blotting to detect the resistance of glioma cancer stem cells.The cancer stem cell surface expressions of ABC transporters were related with MDR.To explore the mechanism of them,nasopharyngeal carcinoma cell line CNE-2 was studied,using magnetic cell sorting methods to isolate CSC and identification of the CSC,to detect the expression levels of drug resistance gene ABCG2.Objective:1.To research the expression of CD 133 in human nasopharyngeal carcinoma cell line CNE-2 and explore the biological characteristics and the significance of CD133+carcinoma stem cells in human nasopharyngeal carcinoma.2.This study investigated the expression of ABCG2 in nasopharangeal carcinoma CD133+ carcinoma stem cells and CD133-carcinoma cells,and its relationship with the multidrug resistance in nasopharangeal carcinoma chemotherapy.3.To explore the sensitivity of nasopharyngeal CD133+cells and CD133-cells to all sorts of chemotherapeutic agents such as:Cisplatin（DDP）,etoposide（VP-16）,taxol,Chlorpromazine.The IC50 and resistence folds of the multidrug resistence were detected by WST-8 methods.The chemotherapy resistance between the cell lines.were compared.Methods:1.Detection of expression of CD133 in CNE-2 Cell Line by Flow Cytometry:Counting about 107 CNE-2 Cell Line,CNE-2 Cells were trypsinized by 0.25%trypsin and rinsed in 0.01%PBS,the cells were centrifuged at 1000g for 5minites.Remove supematant,adding 10μL anti-CD133 labeled with phyeoerythr in（phycoerythrim,PE）,20 μL FcR bloeker,80μL buffer,fully fixed,the react ant was incubated in thedark for 10 minutes at the temperature of 4℃,buffer washing,re-suspended,centrifuged and removing supernatant,add500μL buffer t o cells,cells were analyzed on Flow Cytometry.2.Magnetic Cell Sorting:Counting about 107 CNE-2 cells,cells were trypsinized by 0.25%trypsin and rinsed in 0.01%PBS,and were centrifuged at 1000g for 5 minutes,remove supernatant.Cells were then suspended in 300μL of buffer.Adding 100μL FcR blocking reagent,100μL microliters of CD133 MieroBeads to the cells,One hundred microliters of CD 133 MicroBeads（Skedsmokorset,Norway）were added to label the cells,fully fixed,the reactant was incubated in the dark for 30 minutes at the temperature of 4℃,cells were washed by adding a buffer and centrifuging at 300g for 10 minutes,Supernatant was pipetted off,Cell pellets were suspended again in a 500μL buffer.An MS column was placed in the magnetic field of the MACS separator.The column was rinsed with 3 mL of buffer.Cell suspension with 0.5 mL of buffer was applied onto the column,which was then washed with 0.5 mL of buffer three times.The column was then removed from the separator and placed on a collection tube.One milliliter of buffer was pipetted onto the column.The fraction with magnetically labeled cells was firmly flushed out using the plunger supplied with the column.The separation step using MS columns was repeated.CD133+ sorted cell populations were cultured in two different medium.3.Cell proliferation Assays In vitro:The separated CD133+ tumor cells,CD133-tumor cells,and unsorted tumor cells were platned in 96-well microwell plates in 0.2mL of RPMI1640（supplemented with growth factors）at a density of 2,000 cells/well（Medium without cells was added as a control）.Cells were fed with medium every 48 hours.Proliferation assays were performed on hours 12,24,48,,72 and 96（post Plating）using Roche3-（4,5-dimethylthiazol-2-yl）-2,5-diPhenyltetrazolium.Quantification of viable cells through reading of ultraviolet absorption spcetrums at 490 nm was performed on a Versamax microplate reader.The number of days in culture and average data of different groups were used to draw growth curves to compare the proliferation abilities of the three groups.4.Tumor Sphere Culture:CD133+ tumor stem cells were purified by magnetic cell sorting from nasopharyngeal cell line CNE-2,and cultured continuously in serum-free medium with EGF and bFGF to obtain stem cell spheres in vitro.Cultures were fed 0.25 mL of serum-free medium everyday.the cells were incubated in a humidified 5%CO2 incubator at 37℃。5.Clone formation of CD133+and CD133-cells:Freshly sorted CD133+and CD133cells were counted,plated in triplicate at 200 cells per well in six-well plates,and cultured with serum-free medium for 2-3 weeks.After most cell clones had expanded to>50 cells,they were washed twice with PBS,fixed in methanol for 15 min,and dyed with crystal violet for 15 min at room temperature.After washing out the dye,we counted the clone number that contained>50 cells and compared the results.The clone formation efficiency（CFE）was the ratio of the clone number to the planted cell number.6.Tumorigenesis ability of CD133+ and CD133-cells:Freshly sorted CD133+ and CD133-cells suspended in 200 μL Nacl were inoculated into the back of 4-to 6-week-old BALB/C nude mice on the afternoon of the sorting day.The mice were monitored once weekly for palpable tumor formation and euthanized 4 weeks after transplantation to assess tumor formation.Tumors were measured using a Vernier caliper,weighed,and photographed.A portion of the s.c.tumor tissue was collected,fixed in 10%formaldehyde,and embedded in paraffin for H＆E staining to assess tumor pathology.7.Transcription of ABCG2 in CD133+ and CD133-cells:Total cell RNA was extracted from newly sorted CD133+ and CD133-cells using Trizol reagent（In vitrogen,San Diego,CA）and reverse transcriptions were done according to th e manufacturer’s instructions（Invitrogen）.The ABCG2 cDNA primers for PCR were 5’-GGGTTCTCTTCTTCCTGACGACC-3’（forward）,5’-GGTTGTGAGAT TGACCAACAGACC-3’（reverse）and the final product was 398 bp long.The internal reference gene was GAPDH,primer sequences were 5’-CCACCCATGG CAAATTCCATGGCA-3’（forward）and 5’-TCTAGACGGCAGGTCAGGTCCAC C-3’（reverse）,and the product was 185 bp long.Thermal cycle conditions incl uded an initial incubation at 94℃ for 2 min followed by 40 cycles of 95℃ f or 30 s,53℃ for 30 s,and 72℃ for 1 min and maintenance at 72℃ for 7 min.The products were analyzed by electrophoresis on 1.5%agarose.8.ABCG2 expression in the xenograft was tested by immunohistoehemieal methods.9.To explore the sensitivity of nasopharyngeal CD133+ cells and CNE-2 cells to all sorts of chemotherapeutic agents such as:Cisplatin（DDP）,etoposide（VP-16）,taxol,Chlorpromazine.The half maximal inhibitory concentration of a substance（IC50）and resistence folds of the multidrug resistence were detected by CCK-8 methods.The chemotherapy resistance between the cell lines were compared.The drug sensitivity assay:Freshly sorted CD133+cells and nasopharyngeal CNE-2 cells were planted at 1,000 cells per well in 96-well plates.Chemotherapeutic agents such as:Cisplatin（DDP）,etoposide（VP-16）,taxol,Chlorpromazine were added 4-6 hours in a concentration gradient and repeated in three wells.The culture medium was then replaced with serum-free medium.After two days,The half maximal inhibitory concentration of a substance（IC50）was determined using the CCK8 method.IC50 was calculated using the formula IC50=（mean absorbance of the test well/mean absorbance of the control）× 100%.10.Statistical methods.SPSS 13.0 statistical package were used in data processing and data were expressed as the mean±standard viaration（x±s）.The significance between CD133+ cells,CNE-2 cells and CD133-cells in proliferation assay was tested using single repeated measures analysis of variance.Difference between samples in the clone formation and CCK8 method for drug resistance assay were tested in two-sample t test,after Levene’s rest for equality of variances using Satterthwaite t test.Difference between samples in RT-PCR was tested using One-Way ANOVA and LSD/Dunnett’s T3 for multiple comparisions.P values less than 0.05 was considered as significant difference.Before comparision,data homogeneity of variance was first examined using F test.In the case of homogeneity of variance,the approximate variance F test/Welch method was used.Results:1.Detcetion of expression of CD 133 in nasopharyngeal carcinoma cell line by flow cytometer:Expression of CD 133 in nasopharyngeal carcinomacell lines CNE-2 were detceted by Flow cytometry,results show that CD 133 expression in nasopharyngeal carcinoma cell lines ranged from 0.200%to 2.590%.2.After isolation,CD133+ cells were immediately cultured in serum-free medium.Cells grown in these conditions were single,spherical,floating.As days Passed,nonadherent single cells became clusters.The cell population also inereased.Ten days after culture,the size of cell clusters increased.3.CCK8 methods were conducted to determine the cell growth rate on the 2nd day after sorting.On the hour of 12,24,48,72,96,the CD133+,CD133-cells and the CNE-2 cells were cultrued in serum-free medium,fed with growth factor.Single repeated measures analysis of variance found that there was differences between groups（F=446.815,P=0.000）.The A value of the CD133+ cells（0.803±0.480）were higher than that of other groups which indicated that the CD133+cells grew faster than the CD 133-cells and CNE-2 cells.Differences were found that in the different times（F=34.486,P=0.000）.Compared with CD133-cells and the CNE-2 cells,CD133+ cells demonstrated increased proliferation capacity in vitro,especially after hour 72.4.Clone formation ability of the CD133+ and CD 133" cells:Clone formation assays were repeated twice in triplicate.After 2-3 weeks of culture,most clones had reached>50 cells.We counted the clone number and found that the mean CFE was（18.029±3.409）%and（3.864±0.809）%in CD133+ and CD133-cells,respectively Statistical analysis showed significant differences in CFE between them（P=0.000）.5.Mice formed tumors with CD133+ in 7 mice but not CD 133-cells,which could form tumors with 100,000 cells after 4 weeks inoculated.Tumorigenesis ability in vivo in CD133+ and CD133-cells showed significant differences（χ2=12.444,P=0.001）.Pathology results confirmed that the tumors formed by CD133+ cells were typical human NPC cells just like unsorted CNE-2 cells.6.ABCG2 gene expression in CD133+ and CD133-cells:Total cell RNA was extracted from CD133+,CNE-2 cells and CD133-cells,reverse transcription-PCR（RT-PCR）was done to detect ABCG2 gene expression.The level of ABCG2 expressed in CD133+ cells（2.374±10.090）was obviously higher than that of CD133cells（0.687±0.006）and CNE-2 cells（1.005±0.027）.7.The positive expression rate of ABCG2 was 100%in implanted nasopharyngeal carcinoma xenografts in nude mice.The ABCG2 expressed in the cell membrane or cytoplasm as brown positive cells,which surrounded and invaded into vessels in glioma tissues.8.We used CCK8 method to further confirm this gene characteristic by drug resistance assay,After treatment with chemotherapeutic agents such as:Cisplatin（DDP）,etoposide（VP-16）,taxol,Chlorpromazine,The final IR of the chemotheraputic agents to CD 133+cells were（455.278±22.342）μg/ml,（18.240 ±0.226）μg/ml,（596.502±10.021）μg/ml.And the IR of CNE-2 cells were（19.070±0.363）μ g/ml,（3.304±0.356）μg/ml,（105.201±7.218）μg/ml.CD133+ cells showed strong resistance,whereas CNE-2 cells were sensitive（P=0.000,P=0.000,P=0.000）.The final IR of chlorpromazine to CD133+ cells and CNE-2 cells were（219.313±20.197）μg/ml,（237.288±35.453）μg/ml（P=0.488）.Conclusions:1.CD 133 was expressed on a small fraction of cells in nasopharyngeal carcinoma cell line,the percentage of CD133+cells ranged from 0.200%-2.590%.2.CD133+cells could be effectively obtained by MACS,3.The characteristics of CD133+ cells and CD133-cells were greatly different,CD133+ cells displayed stem cell-like properties of self-renewal and differentiation compared with the CD133-cells in vitro.4.The resistance gene of ABCG2 was expressed at a low level in CD133+cells but not in CD133-cells and CNE-2 cells.5.We found that CD133+ cells were much more resistant to chemotherapeutic agents such as:cisplatin（DDP）,etoposide（VP-16）,taxol,Chlorpromazine than CD133-cells,especially to the cisplatin.Whereas,CD133+ and the un sorted CNE-2 cells prevealde the same sensitivity to chlorpromazine,which suggested chlorpromazine could potentially be used as an anti-cancer stem cells durg in cancer patients. |
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