Gene Expression Profile of Side Population Cells in Human Oral Cancer Cell Line SCC-4
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J Osaka Dent Univ 2015 (October) ; 49 (2) : 205−217. Gene expression profile of side population cells in human oral cancer cell line SCC-4 Rie Nishiitsutsuji1, Tadashige Nozaki2 and Kiyoshi Ohura2 1Graduate School of Dentistry (Department of Pharmacology), 2Department of Pharmacology, Osaka Dental University, 8-1 Kuzuhahanazono-cho, Hirakata-shi, Osaka 573-1121, Japan To define the properties of side population (SP) cells of the oral squamous cell carcinoma cell line SCC-4, we compared their gene expression profile with that of main population (MP) cells. SP cells accounted for approximately 1.5% of the cell population. The gene expression profile in SP cells versus MP cells revealed that 15.8% of genes were up-regulated by more than 10%, and 16.6% were down-regulated by more than 10%. Gene Ontology and KEGG pathway enrichment analyses were carried out on the DAVID online tool for 26,879 distinct probes (p＜0.05). The terms involved in immune/inflammatory responses, cell migration and angiogenesis were detected as up-regulated genes. The terms related to DNA replica- tion, DNA metabolic process, mismatch repair and base excretion repair were detected as down-regulated genes. Moreover, mRNAs encoding the ABC transporters ABCG2/BCRP1 and ABCC2/MRP2, which mediate the excretion of anti-cancer drugs such as cisplatin and gefitinib, respectively, were expressed at higher levels. In contrast, the level of mRNA encod- ing the SLC transporter SLC29A1/ENT1, which mediates the uptake of anti-cancer drugs such as fluorouracil, was decreased. These results indicate that SCC-4 SP cells might be po- tentially resistant to anti-cancer drugs. (J Osaka Dent Univ 2015 ; 49 : 205−217) Key words : Gene expression ; Oral cancer ; Side population ; Anti-cancer drug ; Drug re- sistance resistance to anti-cancer drugs and radiation. Using INTRODUCTION fluorescence-activated cell sorting (FACS), we iso- Cancer stem cells represent a small subset of cancer lated the cells that enhanced excretion of Hoechst cells that self-renew, differentiate into multiple line- 33342 in SCC-4. We performed comprehensive ages, and may generate and maintain the phenotypic analysis to determine the gene expression profile of heterogeneity of cancer cells.1, 2 Evidence indicates these cells. that these cells give rise to tumor cells that are intrinsi- MATERIALS AND METHODS cally resistant to anti-cancer drugs3−5 and oxidative stress.6, 7 Moreover, they promote neoangiogenesis8 Cell culture and maintain a quiescent state by persisting in mi- The human oral squamous cell carcinoma cell line croenvironments (niches).9 Cancer stem cells are pre- SCC-4 was purchased from DS Pharma Biomedical, sent in leukemias,10, 11 breast cancers,12 and colon can- Suita, Japan. Cells were cultured in Gibco DMEM/F12 cers.13−16 A novel cancer therapy that targets cancer (Thermo Fisher Scientific, Waltham, MA, USA) sup- stem cells may serve as a potentiality radical cancer plemented with 10% FBS (MP Biomedicals, Santa cure in the future. Cancer stem cells are identified and Ana, CA, USA), 100 units/mL of Gibco penicillin, 100 isolated according to their phenotypic properties as μg/mL of Gibco streptomycin, 0.25 μg/mL of Gibco follows : cell-surface antigen expression,3, 6, 7, 9−16 drug Fungizone (Thermo Fisher Scientific), and 0.4 μg/mL efflux (ABC transporter),4, 5, 17, 18 sphere formation un- of hydrocortisone (MP Biomedicals) at 37°C in a hu- 8, 19 der specific conditions, proliferative potential, and midified atmosphere containing 5% CO2. 206 R. Nishiitsutsuji et al. Journal of Osaka Dental University, October 2015 Isolation of SP cells Genomes (KEGG) pathway terms by using statistical Hoechst 33342 dye has the capacity to incorporate methods, GO and KEGG pathway enrichment analy- into the A−T base sequence of DNA and to stain live ses were performed on the DAVID v 6.7 online tool cells that have high membrane permeability. When (http : //david.abcc.ncifcrf.gov/).20 cells are stained with Hoechst 33342 and FACS GO describes biological and functional annota- analysis is performed using UV laser, the Hoechst tions, such as biological processes, cellular compo- 33342 is able to excite the cells at two-dimensional nents, and molecular functions. The KEGG pathways wavelengths, 450 nm and 675 nm fluorescence inten- are graphical maps of cellular processes, such as me- sities. A total of 7×107 cells grown to 70% confluence tabolism, membrane transport, signal transduction, were analyzed and separated using FACS. Hoechst and the cell cycle.21−23 The protocol for DAVID Func- 33342 was added to a final concentration of 5 μM, and tional Annotation Bioinformatics Microarray Analysis the cells were incubated for 45 min at 37°C with occa- was used to make gene lists.20 The protocol provides sional agitation. the researcher a detailed account of characteristics Reserpine was added to a final concentration of 5 that create a good gene list. The good gene list meets μM as an inhibitor of ABC transporters. Propidium io- seven requirements in the protocol.20 The list of genes dide was added to a final concentration of 1 μg/mL to was uploaded using UniGene IDs. label nonviable cells, excluding the debris in the Each up- or down-regulated genetic feature was re- Hoechst-blue and Hoechst-red channels. In FACS vealed from the enriched GO and KEGG pathway analysis, SP cells have been identified as Hoechst terms in the DAVID database by specifying a statisti- low cells that exist in a darker fraction than the G0/G1 cally significant threshold of p＜0.05. The p-value phase. MP cells have been identified as Hoechst high was tested by the modified Fisher’s exact test. To con- cells that are present in the diploid nuclear fraction. firm the signaling pathways that were enriched in the Analysis and sorting were performed using a FACS SP cells, the up/down expressed groups of probes Vantage SE (Becton, Dickinson and Company, were classified as either yellow or green using the Franklin Lakes, NJ, USA). SP and MP cells were KEGG Mapper Search & Color Pathway analysis on sorted and harvested by ReproCELL, Yokohama, Ja- the KEGG online tool (http : //www.genome.jp/kegg pan. /).21−23 The list of genes was uploaded in accordance with Entrez Gene IDs that were converted from Uni- Microarray analysis Gene IDs. Total RNA was extracted from SP and MP cells using Invitrogen TRIzol Reagent (Thermo Fisher Scien- Semi-quantitative RT-PCR analysis tific), according to the manufacturer’s instructions and The cDNAs were synthesized from total RNA using purified using the RNeasy MinElute Cleanup Kit Invitrogen SuperScript VILO (Thermo Fisher Scien- (Qiagen, Hilden, Germany). Gene expression pro- tific) according to the manufacturer’s instructions. files of SP and MP cells were analyzed using the PCR amplification of first-strand cDNAs was con- GeneChipⓇ Human Gene 2.0 ST Array (Affymetrix, ducted using KOD FX Neo (Toyobo, Osaka, Japan). Santa Clara, CA, USA) containing 26,879 distinct The primer sets for RT-PCR were as follows : ABCC probes. This procedure was performed at Kurabo In- 2/MRP2, 5’-AGT GAT CAC CAT CGC CCA CA-3’ and dustries, Osaka, Japan. The Database for Annota- 5’-GTT CAC ATT CTC AAT GCC AGC TTC-3’ ; tion, Visualization and Integrated Discovery (DAVID) ABCG2/BCRP1, 5’-AGC TGC AAG GAA AGA TCC bioinformatics resource is made up of integrated bio- AA-3’ and 5’-TCC AGA CAC ACC ACG GAT AA-3’ ; logical knowledge and analytic tools to phylogeneti- SLC29A1/ENT1, 5’-GCC ACT CTA TCA AAG CCA cally extract biological meaning from output gene TCC TG-3’ and 5’-CCT GCG ATG CTG GAC TTG lists.20 In order to extract enrichment Gene Ontology AC-3’ ; Ribosomal protein S18 (RPS18), 5’-TTT (GO) terms and Kyoto Encyclopedia of Genes and GCG AGT ACT CAA CAC CAA CAT C-3’ and 5’-GAG Vol. 49, No. 2 Gene expression profiling of human oral cancer SP cells 207 CAT ATC TTC GGC CCA CAC-3’. RPS18 served as SP cells compared with those of MP cells. an internal control to normalize the semi-quantitative RESULTS data. The products were electrophoresed through 15% (w/v) polyacrylamide gels (e-PAGEL ; ATTO, SP cells existed in SCC-4 Tokyo, Japan) and stained with ethidium bromide. SP and MP cells accounted for 1.48% and 35.4% of The intensities of the amplicons were estimated using the SCC-4 cell population, respectively (Fig. 1 A). Af- ImageJ software (http : //imagej.nih.gov/ij/). The ex- ter adding reserpine, the SP cell fraction decreased to pression levels are presented as the ratio of values of 0.150%, while the MP cell fraction remained about the Fig. 1 FACS analysis of SCC-4 cells stained with Hoechst 33342. Hoechst blue and red fluorescence intensities are plotted on the X- and Y-axes, respectively. Reserpine was used as an ABC transporter inhibitor that suppresses excretion of Hoechst 33342. (A) In the absence of 5 μM reserpine, the side population (SP) cell populations were identified as 1.48% Hoechst negative cells and the main population (MP) cell populations were identified as 35.4% Hoechst positive cells. (B) In the presence of 5 μM reserpine of ABC transporter inhibitor, the SP cell fraction decreased to 0.150%, confirming that it was an SP phenotype. Fig. 2 Scatter-plot analysis. The levels of genes expressed by MP and SP cells are plotted on the X- and Y-axes, respectively. The green lines indicate a 2-fold change in expression level. Genes with more than a 10% difference in the expression by the SP cells were extracted for further analysis. Overall, 15.8% of the genes were up-regulated (Fig.