| BackgroundNasopharyngeal carcinoma (NPC) is a rare form of epithelial cancer that occurs in most parts of the world. However, its occurrence is particularly high in Southeast Asia and southern China, where its incidence rate is approximately25to50cases per100,000individuals, which is25-fold higher than that in Western countries. Surgical approaches to treat NPC are limited by the inaccessibility of the anatomic location. However, NPC is sensitive to radiation and, therefore, treatments primarily rely on radiotherapy. However, approximately30%of patients presenting with localized tumors develop recurrent disease, and30%to60%of patients with metastatic NPC die within5years of diagnosis. In recent years, the concept of cancer stem cell (CSC) has been proposed, which is defined as a cell within a tumor that possesses the capacity to self-renew and to generate the heterogeneous lineages of cancer cells that comprise the tumor. This concept has been extensively investigated following the identification of CSCs in diverse cancers including breast, brain, lung, and liver. Analyses are based on the exploitation of CSC surface markers, elevated levels of aldehyde dehydrogenase (ALDH1+), and enhanced PKH26dye-retaining capacity. This has raised fie prospect of human CSC isolation. Recent data suggest that CSCs are more resistant to chemo-and radiotherapy than non-stem cells. These key properties enable CSCs to initiate tumors and promote cancer progression and may account for the failure of current therapies to eradicate malignant tumors. A recent study showed that CSCs contribute to radioresistance through preferential activation of the CHK.1and CHK2checkpoint response and an increase in DNA-repair capacity.The c-MYC oncoproteih is a well-characterized transcription factor, and deregulation of c-MYC contributes to the genesis of most human tumors. As a result, c-MYC is considered to play an important role in carcinogenesis and tumor progression due to its influence on all basic cellular processes. MYC can activate cyclin E/Cdk2in quiescent fibroblasts by inhibiting the cdk2inhibitor p27. MYC was reported for cooperative actions of p53and PTEN in the regulation of normal and malignant stem/progenitor cell differentiation, self-renewal, and tumorigenic potential. Previous research has also indicated that c-MYC network accounts for similarities between embryonic stem and cancer cell transcription programs. The aim of this study is to determine the capacity of PKH26dye retention to successfully identify slow-cycling PKH26+cells.Wedetermined that these cells were radioresistant. It has also been shown that c-MYC can directly regulate CHK1/2. Here, we explore the role of c-MYC in the radioresistance of nasopharyngeal carcinoma, and the molecular mechanisms of DNA-damage repair that are implicated in this process.Materials and Methods1. Cells and culture conditionsAll cell lines used in this study were maintained in RPMI-1640medium with10%FBS,100U/mL penicillin,100mg/mL streptomycin, and incubated at5%CO2at37℃. PKH26labeling of cells and sorting of cell populations CNE1and CNE2cells were labeled using PKH26Red Fluorescent Cell Linker Kit before cell culture, Labeled CNE2cells were cultured for about4weeks. PKH26-and PKH26+fractions were identified by fluorescence-activated cell sorting (FACS). Freshly PKH26-labeled cells and unlabeled cells were used as positive and negative controls, respectively.2. Tumor spheroid assaySingle cells were plated in6-well ultralow attachment plates. At a density of1,000cells/mL in tumorspheric culture medium Dulbecco’s Modified Eagle’s Medium, DMEM-F12supplemented with1%N2Supplement,2%B27Supplement,20ng/mL human platelet growth factor,100ng/mL epidermal growth factor, and1%antibiotic-antimycotic at37℃in a humidifiedatmosphere of95%air and5%CO2.3. Immunofluorescent staining.For immunofluorescent staining, adherent or semi-differentiated spheroid cells were grown on the surface of cover slides. Spheroids staining was performed in96-well microplates. The cells were fixed with4%paraformaldehyde. After rehydration in KB, cells were incubated with respective primary antibodies at37℃for45min. Slides or spheroids were then washed with KB for15min and secondary antibodies were incubated at37℃for45min. The nuclei were stained with DAPI. Sections were examined with a confocal microscopy.4. Cell-cycle analysiscells were trypsinized, washed with PBS, resuspended in70%ethanol, and stored at-20’C overnight. Cells were subsequently centrifuged, washed in PBS, resuspended in450mL PBS and10mL10mg/mL DNase-free RNase, and incubated at37℃for45minutes. Following RNase treatment,50mL of propidium iodide was added, and cells were incubated at room temperature for10minutes protected from light. Cell aggregates were removed by filtration before analysis. Cell-cycle analysis was carried out using the BD FACSDiva.5. SP cells assayCells obtained from adherent or spheroid cells were suspended in DMEM/2%fetal bovine serum at1×106/ml cells and stained with Hoechst33342dyefor90min at37℃. Following this incubation, cells were washed with ice-cold PBS, stained with propidium iodide label and exclude dead cells. Cells were maintained at4℃for flow cytometry analyses and for sorting of SP traction using a FACSAria Flow cytometer6. Antibodies, Western blotting,Western blot analyses were conducted according to standard protocols, Cells were lysed and equal amount of protein were subjected to electrophoresis on a SDS-PAGE gel. The separated proteins were transferred to PVDF membranes and probed with appropriate primary antibodies. After extensive washing, the membranes were incubated with peroxidase-conjugated secondary antibodies and protein bands were detected by enhanced chemiluminescence reagents according to manufacturer’s instructions.using the following antibodies:mouse anti-human cyclin D1, cyclin A, and cyclin B (1:300), mouse anti-human ABCG2, CD-44, CHK1, CHK2pCHK1, pCHK2GAPDH (1:1,000), and g-H2AX (1:1,000). Peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) secondary antibody (1:500) was used as the secondary detection antibody.7. Clonogenic survival assayFreshly sorted PKH26+and PKH26-cells were counted and plated into6-well culture plates. Radiation (300cGy/min) was delivered at room temperature with a Linear accelerator (2100EX, Varian) and cultured for14days. Subsequently, the number of surviving colonies (defined as a colony with more than50cells) was counted. 8. ImmunohistochemistryParaffin sections were dewaxed, ethanol gradient hydration, antigen retrieval, the inactivation of endogenous peroxidase4℃, an anti-incubated overnight, secondary antibody25℃incubated for10min DAB chromogenic color microscope, hematoxylin nuclear hydrochloric acid ethanol differentiation, dehydrated, and transparent.9. Quantitative real-time (qRT)-PCRExpression of CHK1, CHK2, c-MYC, and ataxia telangiectasia mutated (ATM) was measured by qRT-PCR using a Taq-Man(?) MicroRNA Reverse Transcription Kit and TaqMan Universal PCR Master Mix (Applied Biosystems) in a two-step real-time RT-PCR assay according to the protocol provided by the manufacturer. Expression was normalized to that of the endogenous reference control gene GAPDH. The primer sequences used in this study are shown in Supplemental Table S3.10. Luciferase assayThe pGL4-hRluc/TK vectors were obtained from Promega. Promoters of selected CHK1and CHK2transporter genes were amplified by PCR and cloned into the basic pGL4vector. The activity of firefly or Renilla luciferase was measured with a dual luciferase assay kit (Promega) according to the instructions provided by the manufacturer. Mutagenesis of c-MYC binding sites in the CHK1and CHK2promoters was performed using the QuikChange multi-site-directed mutagenesis kit (Stratagene) according to the instructions provided by the manufacturer. The oligonucleotides used for mutagenesis are listed in Supplemental Table S3. Relative luciferase activity was calculated as the ratio between the firefly luciferase and the control Renilla luciferase activity.11. c-MycBS prediction and ChIP assayPutative c-MycBSs were predicted by Patch software (http://www.biobase-international.com/gene-regulation). The DNA from CNE2cells, was prepared and digested with EZ-Zyme Chromatin Prep kit (Millipore). The digested sample was subjected to ChIP assay using EZ-Magna ChIP Kit (Millipore). All procedures were done as per the manufacturer’s instructions. Anti-c-Myc (Santa Cruz Biotechnology, Inc.) was used for the assay. After ChIP assay was performed, the DNA sample was amplified by PCR using the primers listed in Table S3.12. Xenograft experimentsAnimal studies were conducted in strict accordance with the principles and procedures approved by the Committee on the Ethics of Animal Experiments of Southern Medical University. Nude mice were fed autoclaved water and laboratory rodent chow. Freshly sorted PKH26+and PKH26-cells suspended in200mL PBS were inoculated into the flanks of6-to7-week-old nude female mice on the a on the day of sorting. Tumors were measured using a Vernier caliper, weighed, and photographed. A portion of the subcutaneous (s.c.) tumor tissue was collected, fixed in10%formaldehyde, and embedded in paraffin for hematoxylin&eosin (H&E) staining to assess tumor pathology. To analyze the effect of ionizing radiation (IR) and si-c-MYC as combination therapy,5×103PKH26+derived from CNE2cells were subcutaneously injected into athymic nude mice, and tumor volume was monitored every5days as calculated by the equation V(mm3)1/4(a×b2)/2. When the tumors reached a size of70mm3, mice were randomly distributed to6groups (4mice per group) and treated with IR (8Gy and2*4Gy), si-c-MYC, or the combination of IR and si-c-MYC. In addition, cells obtained from the treated tumors were analyzed for the percentage of PKH26+and CD44+by flow cytometry.Result1. Cell sorting and PKH+cell characterizationwe used flow cytometry to isolate healthy PKH-negtive (PKH-) and PHK-positive(PKH+) cells. We sorted the top2%from the total cell population in CNE1and CNE2cells. Then, we used FACS to determine cell-cycle distribution. PKH+cells showed higher fractions of cells in the G1phase and lower fractions of cells in S and G2-M phase. We next examined the expression of cell cycle-related proteins. Sorted CNE1cells, the PKH26+population contained32.1-5.2%SP cells, whereas the PKH26-population contained0.5-0.3%SP cells. We sorted CNE2cells and found that the PKH26+population contained37.1-7.1%SP cells, and the PKH26-population contained only0.7-0.5%SP cells. Western blotting analysis of ABCG2and CD44, revealed expression of these proteins in all PKH26-and PKH26+cells. PKH26+cells gave a rise to significantly more colonies than PKH26-cells. Tumor spheroid assays using single-cell suspensions derived from sorted PKH26-and PKH26+cells. PKH26+cells formed spheres that were significantly greater in number and larger than those formed by PKH26-cells.2. PKH26+cells promote radioresistance through c-MYC overexpressionWe irradiated (8Gy) PKH26-and PKH26+cells and measured protein expression of c-MYC, CHK1, pCHK1, CHKk2, and pCHK2. Irradiation increased levels of CHK1and CHK2phosphorylation in both PKH26-and PKH26+cells. Immunofluorescence staining revealed that c-MYC, pCHK1, and pCHK2were also localized to the nucleus in PKH26+cells. As mentioned above, c-MYC is highly expressed in PKH26+NPC cells. DNA-damage-related protein g-H2AX in the c-MYC overexpressing group is greatly reduced. Using comet assays, we found that the percentage of cells displaying DNA damage decreased by approximately2.8-fold in both CNE1and CNE2cells overexpressing c-MYC. EDU assay showed that following irradiation, cells overexpressing c-MYC showed improved proliferative capacity. we used the Gene Regulation website and Patch software http://www.biobase-intemational.com/gene-regulation found3potential c-MYCc binding sites (c-MYCBS) within the2,000bp promoter upstream of the CHK1and CHK2genes, respectively. The ChIP results suggest that c-MYC is most significantly bound to sites C and E within the CHK1and CHK2promoters. Knockdown of c-MYC diminished the amount of DNA c-MYCBSs C and E that could be immunoprecipitated with the c-MYC antibody. overexpressed in NPC cells, c-MYC increases lueiferase expression from the promoter reporter. Next, we mutated the c-MYCBSs C and E in CHK1and CHK2luciferase reporter gene vectors. The results showed that mutated c-MYCBSs did not display increased luciferase activity after c-MYC overexpression, thus, confirming that c-MYC acts through CHK1c-MYCBSs C and CHK2c-MycBSs E.3.c-MYC overexpression can enhance PHK26+cell DNA repair capacity and stemnessSilence c-MYC gene in nasopharyngeal carcinoma cells, detected after irradiation, c-MYC expression and DNA damage response. Next, the inhibition of c-MYC side population cells reduce the proportion of PKH26+cells, CNE1the PKH26+cell side population increased from32.8%to2.4%, CNE2of PKH26+cell side population ratio decreased from35.67%to2.0%. To further confirm our. hypothesis, we to collect PKH26+cells from CNE1and CNE2cell lines, tumor sphere formation assay found that inhibition of c-MYC can reduce the number of tumors ball. Alkaline comet assay, we shc-MYC processing PKH26+cells compared with the control group, the damage increased significantly.4. C-MYC overexpression can enhance PHK26+cell DNA repair capacity and stemnessWe inhibited CHK1/2expression in PKH26+cells with shRNA. The percentage of cells with comet tails increased3to4.5times more rapidly in vector control PKH26-cells than in CHK1/2shRNA infected PKH26+and PKH26-cells. Next, PKH26+cells to the c-MYC and CHK1/2co-transfection, by colony formation assay, Western blot found PKH26+cells resistant to radiotherapy. To determine the basis for why combinatorial therapy of IR with si-c-MYC was more effective than IR alone, we examined the populations of cells from tumors. After treatment, the PKH26+and CD44+population were nearly absent from mice subjected to combinatorial therapy, while they were easily observed in tumors from mice treated with IR alone, we conducted a correlation analysis of c-MYC, CHK1/2, and CD44protein expression levels using IHC, respectively, in primary nasopharyngeal carcinoma tissue consisting of62primary nasopharyngeal carcinoma samples. Consistently, high expression of c-M Y C positively correlated withincreased CHK1/2and CD44expression levels. Expression of c-MYC and CHK1/2also positively correlated with poor differentiation in high-grade tumors.Statistical analysisUnless stated otherwise, all experiments were conducted in triplicate. Data are expressed as the mean|SD of at least3independent experiments. The significance of differences between mean values was determined using2-way ANOVA. P less than0.05was considered significant. |
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