AbstractCD133 was used as a cancer stem cells marker to identify, isolation and enrichment the CSCs from many types of tumors. In present study, FTC133 cells were divided into CD133+ FTC133 and CD133 FTC133 cell through magnetic activated cell sorting system (MACS), the CD133 positive cells displayed self-renewal properties and chemo-resistance. The protein level of Bax and p53 in CD133+ FTC133 cells was much lower than FTC133 and CD133 FTC133 cells, the protein level of Bcl-2 and Survivin in CD133+ FTC133 cells was much higher than FTC133 and CD133 FTC133 cells. Moreover, the activity of ?-catenin/Wnt signaling was enhanced in CD133+ FTC133 cells. These results suggest that, in comparison with CD133 FTC133 cells, CD133+ FTC133 exhibited chemo-resistance to doxorubicin (DOX) through increased the activity of ?-catenin/Wnt signaling.Key Words: CD133, FTC133 cell, Human thyroid cancer, ?-catenin, Apoptosis IntroductionOver the past 10 years, thyroid cancer is a common type of malignancy in endocrine organs with a global increasing incidence rates(1-3). Although more than 90% of thyroid cancer is referred to well-differentiated thyroid cancer (DTC) which are commonly diagnosed at an early stage and have high cause-specific survival rates (exceed 95%), including follicular thyroid cancer (FTC) and papillary thyroid cancer (PTC), there also have a subset of tumors that do not respond to traditional therapies and will present or eventually develop resistance to chemotherapy and progress to a worse outcomes(4-6).The glycoprotein CD133 (cluster of differentiation 133) have five transmembrane domains, which was identified from human hematopoietic stem cells and mouse neuro-epithelial stem cells(7). However, CD133 was not only expressed in normal stem cells, but also used as a cancer stem cells marker to identify, isolation and enrichment the CSCs from many types of tumors(8-10). The brain tumor stem cell (BTSC) was exclusively isolated with the cell fraction expressing the neural stem cell surface marker CD133, capacity for proliferation, self-renewal, and differentiation. The increased self-renewal capacity of the brain tumor stem cell (BTSC) was highest from the most aggressive clinical samples of medulloblastoma compared with low-grade gliomas(11). Only the CD133+ brain tumour fraction contains cells that are capable of tumour initiation in NOD-SCID (non-obese diabetic, severe combined immunodeficient) mouse brains(12). CD133+ cells in human breast cancer cells also revealed increased expression of stem cell associated genes, like Oct4, Notch1, Aldh1, Fgfr1, and Sox1(13).Moreover, CD133 also reported to be related with chemo-resistance in many types of tumors. The breast cancer resistance protein (BCRP)/ATP-binding cassette subfamily G member 2 (ABCG2) is contributes to multidrug resistance during chemotherapy and its expression was correlated with CD133+ colorectal carcinoma cells, when ABCG2 expression was down-regulated, the efficacy of chemotherapy induced apoptosis was enhanced in CD133+ colorectal carcinoma cells(14).However, whether the CD133+ FTC133 cells were related with chemo-resistance in human thyroid cancer cell line, FTC133 cell, was less to know. In this paper, the CD133+ FTC133 cells revealed enhanced chemo-resistance ability for doxorubicin (DOX) by interferencing the nucleus location of ?-catenin and regulating the expression of Bax, p53, Bcl-2 and Survivin. MATERIALS AND METHODS Materials and AntibodyCell Lines and CultureHuman thyroid cancer cell line FTC133 was provided from the Shanghai Institute of biology, Chinese Academy of Sciences. The FTC133 cells were cultured in H-DMEM medium with 10% FBS at 37 ? and 5%CO2.Magnetic Activated Cell SortingIn the logarithmic growth phase, discard the culture medium, the separation buffer washed 3 times, each time 2min, 0.25% trypsin digestion, the cells became round but not off, discarding the trypsin, the separation buffer into monoplast suspension, 2000rpm 3min washing out the supernatant, re sorting buffer, trypan blue staining, the cell concentration was adjusted to 1 * 107/80 L, adding 10 L FCR blockers, 4 C were incubated with 15min, then add CD133 magnetic beads 100 L, mixing, in 4 C dark dark incubation 30 min. The buffer solution with 10~20 times cell volume was washed, 1500 r/min centrifuged 5 min, the supernatant was abandoned, and the buffer solution of 500 mu L was added to buffer the heavy suspension. The MS sorting column was installed on the magnetic sorting rack. After adding 0.5 mL buffer, the cell was washed and sorted. The cells were moved into the MS sorting column and added 4 times volume buffer. The cell suspension flowed out (CD133 cells at this time). When the liquid stops outflow, the separation column is removed and the remaining liquid is quickly pressed into the bottle with the supporting plug, and CD133+ cells are obtained at this time.Flow CytometryAfter sorting, the CD133+ and CD133 cells were washed with PBS. The cells were collected in the test tube, and the density was adjusted to 1×105 /mL. The cells were centrifuged for 5 min with 1500 rpm, precooling ethanol at 4?, stored 30 min, and PBS washed for 3 times. Resuspended in PI staining, after incubation on detection by flow cytometry (FACS flow cytometry, BD) to detect the expression rate of CD133+ cells.Tumorsphere FormationThe CD133 FTC133 cells and CD133+ FTC133 cells were cultured in DMEM/F12 medium (adding 20ng/ mL EGF, 20 ng/mL bFGF, 0.4% BSA and 2% B27) for 500 cells/ per well, respectively. The pictures were collected every day to compare the capacity for tumorsphere formation between CD133 FTC133 cells and CD133+ FTC133 cells.MTT AssayThe CD133 FTC133 cells and CD133+ FTC133 cells were cultured in DMEM with 10% FBS in 96-well plate for 5000 cells/per well, respectively. After overnight, the different concentration of doxorubicin (DOX, 25, 12.5, 6.25, 3.12, 1.56, 0 g/mL) was added into each well, after 24h, adding 20 ?l MTT into each well and cultured 4h. Then, be to the culture liquid was absorbed carefully, adding 150 ?l DMSO and measurements the OD at 490nm.Apoptosis AssayThe selected CD133+ and CD133 cells were seeded in 6 well plates, respectively. The cells were treated with different concentrations of DOX to 24h, and the cells were collected to adjust the concentration of cells to be 5 × 105 /ml. 4? centrifugation for 10 minutes, discarding the supernatant. Add 1ml pre-cooled PBS, gently concussion to cell suspension, 1000rpm, 4? centrifuge for 10 minutes, discarding the supernatant. The cells were suspended at 200?l Binding Buffer. Add 10 ?l Annexin V-FITC and 10 ?l PI, gently mix and avoid light room temperature for 15 minutes. 300 ?l Binding Buffer was added to the test by flow cytometry within 1h.ImmunofluorescenceAfter treated with 3.12 g/ml DOX for 24h, the fixative was fixed and the washing solution was washed. The closed solution was closed at 1H at room temperature, the washing solution was washed, and the beta-catenin p65 antibody was added to incubate for 4 hours. Washing liquid washing, adding anti rabbit Cy3, incubated at room temperature for 1h, washing liquid washing, adding proper amount of anti fluorescence quenching by liquid, mounting microscope camera.Real-time PCRAfter the cells were digested, PBS was washed 3 times by centrifugation, the cells were collected, and the total mRNA was extracted by Trizol. cDNA synthe- sis and real-time (RT)-PCR were performed using the first strand cDNA synthesis kit (Fermentas) and SYBR Green Master Mix kit (Roche). FZD1: 5?-CGGTAAAATCTAAGCGCAGG-3?; 5?-AGCTTTGTGTGGGTTGGAAG-3?, MDR1: 5?-TCAGCCTCACCACAGATGAC-3?, 5?-TCACTTCAGGAAGCAACCAG-3?.Western BlottingAfter drug treatment for 24 h, protein content was determined applying the BCA Protein Assay Kit (Life Technologies). Equal amounts of protein were separated by 10% SDS-PAGE and transferred to PVDF membranes (Milipore). The membranes were incubated overnight with primary antibodies against human Bax, p53, Bcl-2 and Survivin. After washing, membranes were incubated and developed with a horseradish peroxidase- conjugated secondary antibody (Life Technologies). ?-actin served as internal control.Statistical analysisAll results are expressed as the mean ± SD and were analyzed with the SPSS18.0 software (SPSS Inc, Chicago, Illinois)using the Student t-tests and one way analysis of variance (ANOVA). In all figures, error bars represent the standard error of the mean. A level of P < 0.05 was accepted as significant (*P < 0.05, **P < 0.01). ResultsCD133+ cells enhanced self-renewal capacity of FTC133 cellsThe CD133 positive cells in FTC133 cells were sorted by using CD133 as the stem cell marker through magnetic activated cell sorting system (MACS), and the cells were divided into CD133+ FTC133 cell and CD133 FTC133 cell.The number of CD133+ cells after sorted was detected by using flow cytometry assay. As shown in Fig.1A, the CD133 positive cells was 1.85% in FTC133 cell, after sorted, the CD133 positive cells was 95.06% in CD133+ FTC133 cells?and the CD133 positive cells was only 0.65% in CD133 FTC133 cells. These results suggested that the magnetic activated cell sorting system could enrich CD133 positive cells from FTC133 cells efficiently.Moreover, tumorsphere formation experiment was performed between CD133+ FTC133 cell and CD133 FTC133 cell. Be consistent with the expectation, almost every CD133+ FTC133 cell formed the tumorsphere in stem cell conditioned medium, comparing with the no tumorsphere formation in CD133 FTC133 cell, suggesting that CD133 expression enhanced self-renewal in FTC133 cells.CD133expression reduced cell death in FTC133 cellsThe MTT assay was used to detect the cell viability between CD133+ FTC133 cells and CD133 FTC133 cells after treating with DOX. As shown in Fig.2A, the cell viability was decreased from 100% (DOX: 0 ?g /mL) to 87.34% (DOX: 0.75 ?g /mL), 79.45% (DOX: 1.5 ?g /mL), 63.89% (DOX: 3 ?g /mL) and 31.08% (DOX: 6 ?g /mL) in DOX treated CD133+ FTC133 cells. And the cell viability was decreased from 100% (DOX: 0 ?g /mL) to 80.65% (DOX: 0.75 ?g /mL), 63.62% (DOX: 1.5 ?g /mL), 40.60% (DOX: 3 ?g /mL) and 14.11% (DOX: 6 ?g /mL) in DOX treated CD133 FTC133 cells. Especially, when the concentration of DOX was higher than 1.5 ?g /mL, the cell viability was much higher in CD133+ FTC133 cells than CD133 FTC133 cells (p<0.05). And the IC50 was also higher in CD133+ FTC133 cells (3.83?g/mL) than CD133 FTC133 cells (2.05?g/mL, p<0.05). These results demonstrated that CD133 expression reduced sensitivity to DOX in CD133+FTC133 cells.Furthermore, cell apoptosis was also detected in CD133+ FTC133 cells and CD133 FTC133 cells by using flow cytometry. After treating with DOX, the apoptosis cell was lower in CD133+FTC133 cells (17.8%, 0.75 ?g /mL and 9.7%, 1.5 ?g /mL) than CD133 FTC133 cells (22.9%, 0.75 ?g /mL and 12.3%, 1.5 ?g /mL), suggesting that CD133 expression prevented cells from apoptosis in CD133+ FTC133 cells.In addition, the protein level of Bax and p53 was much lower in CD133+ FTC133 cells than FTC133 and CD133 FTC133 cells. The protein level of Bcl-2 and Survivin was much higher in CD133+ FTC133 cells than FTC133 and CD133 FTC133 cells. The activity of ?-catenin/wnt signaling was enhanced in CD133+ FTC133 cellsAlthough the expression of ?-catenin in cell nucleus had no difference among FTC133 cells, CD133 FTC133 cells and CD133+FTC133 cells by treating with DOX for 60 min, but the expression of ?-catenin in cell nucleus was much more in CD133+ FTC133 cells than FTC133 cells than CD133 FTC133 cells by treating with DOX for 30 min, suggesting that the transport speed for ?-catenin transferring into cell nucleus was faster in CD133+FTC133 cells than FTC133 cells than CD133 FTC133 cells. Moreover, after adding the inhibitor of ?-catenin (XAV939) into the cells, the expression of ?-catenin in cell nucleus was decreased in FTC133 cells, CD133 FTC133 cells and CD133+FTC133 cells.Furthermore, the mRNA expression of FZD1 and MDR1was detected in FTC133, CD133 FTC133 and CD133+FTC133 cells. The mRNA expression of FZD1 and MDR1 was much higher in CD133+FTC133 cells than FTC133 and CD133 FTC133 cells. After adding the XAV939, the mRNA expression of MDR1 was decreased in the CD133+FTC133 cells, however, the mRNA expression of FZD1 had no changed. These results demonstrated that the activity of ?-catenin/Wnt signaling was enhanced in CD133+ FTC133 cells. DiscussionThyroid cancer is a most common type of malignancy in endocrine organs. Previous study had demostrated that CD133 was involved in tumor cell proliferation, metastasis, tumorigenesis and recurrence, as well as chemo- and radio-resistance. In order to investigate the function of CD133 positive cells in FTC133 cells, the FTC133 cell was divided into two groups (CD133+ FTC133 cell and CD133 FTC133 cell) through magnetic activated cell sorting system (MACS) by using CD133 as the stem cell marker. The results from flow cytometry assay showed that the CD133 positive cell was 1.85% in FTC133 cell (Fig1A), 0.65% in CD133 FTC133 cells (Fig1B) and 95.06% in CD133+ FTC133 cells (Fig1C).Previous study had demostrated that CD133+ cancer cells exhibit significantly higher levels of p-Akt than CD133? cancer cells in several cancer types, CD133+ cancer cells were closely related to tumorigenesis and CSC properties. CD133+ colon cancer cells showed CSC properties both in vitro and in vivo by involving in the Akt and MAPK(6). And CD133 also facilitated CSC-like properties in hepatocellular carcinoma by stabilizing EGFR-AKT signaling(15). therefore, the self-renewal capacity in CD133+ FTC133 cells were detected by tumorsphere formation assay. Consistent with expectations, almost every CD133+ FTC133 cell could form the tumorsphere in stem cell conditioned medium, comparing with the no tumorsphere formation in CD133 FTC133 cell. These results suggested the CD133 positive cells could enrich through MACS efficiently, and CD133 positive cells revealed enhanced self-renewal capacity in FTC133 cells.The chemo-resistance was reported as a charactor for cancer stem cells in many types of tumors(16-18), and CD133 was also correlated with multidrug resistance during tumors chemotherapy(19-22). In neuroblastoma, CD133 expression was associated with chemoresistance via mediated by the AKT pathway(23). In these study, the IC50 to DOX was higher in CD133+ FTC133 cells (3.83?g/mL) than CD133 FTC133 cells (2.05?g/mL, p<0.05). And the cell viability was much higher in CD133+ FTC133 cells than CD133 FTC133 cells when the concentration of DOX was higher than 1.5 ?g /mL. Moreover, cell apoptosis assay revealed that the apoptosis cell was much lower in CD133+ FTC133 cells than CD133 FTC133 cells. Furthermore, the protein level of Bax and p53 was much lower in CD133+ FTC133 cells than FTC133 and CD133 FTC133 cells, and the protein level of Bcl-2 and Survivin was much higher in CD133+ FTC133 cells than FTC133 and CD133 FTC133 cells. All of these results demonstrated that CD133 expression prevented cells from apoptosis and reduced sensitivity to DOX in CD133+FTC133 cells.Previous study showed that CD133 expression contributes to ?-catenin-mediated transcriptional activation and to the self-renewal capacity of sphere-formation in CSCs from brain, colon and lung cancers, and a polarized cell migration-induced CD133/integrin/Src/Akt/GSK3?/?-catenin axis is required for maintenance of CSC properties(24). Upregulated Wnt/?-catenin activity in CD133+ hair follicle dermal papilla cells increases the number of proliferating hair follicle dermal papilla cells and promotes postnatal hair growth(25). In this study, after treating with DOX for 30 min, the expression of ?-catenin was much more in CD133+FTC133 cells than FTC133 cells, and the mRNA expression of FZD1 and MDR1 was much higher in CD133+FTC133 cells than FTC133 and CD133 FTC133 cells. These results demonstrated that the was enhanced in CD133+ FTC133 cells.In conclusion, the present study has demonstrated that CD133+ FTC133 cells display enhanced self-renewal capacity and chemo-resistance in FTC133 cells, and such effect was partly by enhancing the activity of ?-catenin/Wnt signaling in FTC133 cells.LegendsFigure1. CD133+ cells enhanced self-renewal capacity in FTC133 cells. Flow cytometry detected CD133 positive cells in FTC133 (A), CD133 FTC133 cells (B) and CD133+ FTC133 cells (C). (D) The tumorsphere formation efficiency in CD133 FTC133 cells and CD133+ FTC133 cells.Figure 2. CD133expression reduced cell death in FTC133 cells. (A) The MTT assay was used to detect the cell viability between CD133+ FTC133 cells and CD133 FTC133 cells after treating with DOX. (B) Cell apoptosis was detected in CD133+ FTC133 cells and CD133 FTC133 cells by using flow cytometry after treating with DOX, and the quantitative analysis was shown in (C).Figure 3. The activity of ?-catenin/wnt signaling was enhanced in CD133+ FTC133 cells. (A) The expression of ?-catenin was detected by using immunofluorescence after treating with DOX in FTC133, CD133 FTC133 cells , CD133+ FTC133 cells and CD133++XAV939 cells. (B) The mRNA expression of FZD1 and MDR1was detected in FTC133, CD133 FTC133 cells, CD133+ FTC133 cells and CD133++XAV939 cells. (C) The mRNA expression of MDR1was detected in FTC133, CD133 FTC133 cells, CD133+ FTC133 cells and CD133++XAV939 cells.Figure 4. 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