OBJECTIVE: To explore the protective effect of NANOG against hydrogen peroxide (H2O2) -induced cell damage in the human hair follicle mesenchymal stem cells (hHF-MSCs). METHODS: NANOG was expressed from a lentiviral vector, pLVX-IRES-ZsGreen. NANOG hHF-MSCs and vector hHF-MSCs were treated with 400 μmol/L hydrogen peroxide (H2O2) for 2 h, the cell survival rate, cell morphology, ROS production, apoptosis and expression of AKT, ERK, and p21 were determined and compared. RESULTS: Our results showed that NANOG could activate AKT and upregulate the expression of p-AKT, but not p-ERK. When treated with 400 μmol/L H2O2, NANOG hHF-MSCs showed higher cell survival rate, lower ROS production and apoptosis, higher expression of p-AKT, higher ratio of p-AKT/AKT. CONCLUSION Our results suggest that NANOG could protect hHF-MSCs against cell damage caused by H2O2 through activating AKT signaling pathway.
Cell Survival ; Drug Evaluation, Preclinical ; Hair Follicle ; cytology ; Humans ; Hydrogen Peroxide ; Lentivirus ; Mesenchymal Stem Cells ; drug effects ; metabolism ; Nanog Homeobox Protein ; metabolism ; pharmacology ; Oxidative Stress ; drug effects ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction

PURPOSE: To investigate the association of cancer stem-cell markers [octamer-binding transcription factor 4 (OCT4), sex determining region Y-box 2 (SOX2), and Nanog homebox (NANOG)] expression with clinicopathological properties and overall survival (OS) in operative rectal cancer (RC) patients receiving adjuvant therapy. MATERIALS AND METHODS: 153 patients with primary RC receiving surgery were enrolled. Tumor tissue and paired adjacent normal tissue sample were collected, and OCT4, SOX2, and NANOG expressions were assessed by immunofluorescent staining. The median follow-up duration was 5.2 years, and the last follow-up date was August 2016. RESULTS: Tumor tissue OCT4 (p < 0.001), SOX2 (p=0.003), and NANOG (p < 0.001) expressions were higher than those in adjacent tissue. OCT4 expression was positively correlated with pathological grade (R=0.185, p=0.022), tumor size (R=0.224, p=0.005), and N stage (R=0.170, p=0.036). NANOG expression was positively associated with tumor size (R=0.169, p=0.036). Kaplan-Meier suggested that OCT4+ was associated with worse OS compared with OCT4− (p < 0.001), while no association of SOX2 (p=0.121) and NANOG expressions (p=0.195) with OS was uncovered. Compared with one or no positive marker, at least two positive markers were associated with shorter OS (p < 0.001), while all three positive markers were correlated with worse OS compared with two or less positive markers (p < 0.001). Multivariate Cox's analysis revealed that OCT4+ (p < 0.001) and N stage (p=0.046) were independent factors for shorter OS. CONCLUSION: Tumor tissue OCT4 expression was correlated with poor differentiation, tumor size, and N stage, and it can serve as an independent prognostic biomarker in operative patients with RC receiving adjuvant therapy.
Aged ; Biomarkers, Tumor/metabolism ; Female ; Humans ; Male ; Multivariate Analysis ; Nanog Homeobox Protein/metabolism ; Neoplastic Stem Cells/metabolism ; Octamer Transcription Factor-3/metabolism ; Prognosis ; Rectal Neoplasms/metabolism ; Rectal Neoplasms/pathology ; Rectal Neoplasms/surgery ; SOXB1 Transcription Factors/metabolism ; Survival Analysis

Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naïve pluripotent gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.
Animals ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Doxycycline ; pharmacology ; Gene Expression Regulation ; drug effects ; Human Embryonic Stem Cells ; metabolism ; Humans ; Mice ; Nanog Homeobox Protein ; biosynthesis ; genetics ; Pluripotent Stem Cells ; metabolism

Thymosin β4 (Tβ4) is a key factor in cardiac development, growth, disease, epicardial integrity, blood vessel formation and has cardio-protective properties. However, its role in murine embryonic stem cells (mESCs) proliferation and cardiovascular differentiation remains unclear. Thus we aimed to elucidate the influence of Tβ4 on mESCs. Target genes during mESCs proliferation and differentiation were detected by real-time PCR or Western blotting, and patch clamp was applied to characterize the mESCs-derived cardiomyocytes. It was found that Tβ4 decreased mESCs proliferation in a partial dose-dependent manner and the expression of cell cycle regulatory genes c-myc, c-fos and c-jun. However, mESCs self-renewal markers Oct4 and Nanog were elevated, indicating the maintenance of self-renewal ability in these mESCs. Phosphorylation of STAT3 and Akt was inhibited by Tβ4 while the expression of RAS and phosphorylation of ERK were enhanced. No significant difference was found in BMP2/BMP4 or their downstream protein smad. Wnt3 and Wnt11 were remarkably decreased by Tβ4 with upregulation of Tcf3 and constant β-catenin. Under mESCs differentiation, Tβ4 treatment did not change the expression of cardiovascular cell markers α-MHC, PECAM, and α-SMA. Neither the electrophysiological properties of mESCs-derived cardiomyocytes nor the hormonal regulation by Iso/Cch was affected by Tβ4. In conclusion, Tβ4 suppressed mESCs proliferation by affecting the activity of STAT3, Akt, ERK and Wnt pathways. However, Tβ4 did not influence the in vitro cardiovascular differentiation.
Animals ; Cell Cycle ; drug effects ; genetics ; Cell Differentiation ; drug effects ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Extracellular Signal-Regulated MAP Kinases ; genetics ; metabolism ; Gene Expression Regulation ; drug effects ; JNK Mitogen-Activated Protein Kinases ; genetics ; metabolism ; Mice ; Mouse Embryonic Stem Cells ; cytology ; drug effects ; metabolism ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Nanog Homeobox Protein ; genetics ; metabolism ; Octamer Transcription Factor-3 ; genetics ; metabolism ; Patch-Clamp Techniques ; Primary Cell Culture ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Proto-Oncogene Proteins c-fos ; genetics ; metabolism ; Proto-Oncogene Proteins c-myc ; genetics ; metabolism ; STAT3 Transcription Factor ; genetics ; metabolism ; Signal Transduction ; Thymosin ; pharmacology

OBJECTIVETo study the relationship between Nanog-promoted metastasis of breast cancer and ezrin(T567) phosphorylation, and explore the possible mechanism by which Nanog regulates ezrin(T567) phosphorylation.

METHODSA siRNA construct targeting Nanog was transfected in breast cancer cells to knock down Nanog expression, and the changes in the cell invasion was detected using Transwell assay. The expression levels of Nanog and PKC and the phosphorylation level of ezrin(T567) were detected using Western blotting and immunofluorescent staining; the protein interaction between PKCε and ezrin was assayed by co-immunoprecipitation and Western blotting.

RESULTSNanog knockdown significantly decreased the expression of PKCε protein, phosphorylation level of ezrin(T567) and the invasion ability of breast cancer cells. PKCε knockdown obviously decreased the phosphorylation level of ezrin(T567) in the cells, and PKCε and ezrin were co-immunoprecipitated.

CONCLUDIONSNanogcan can upregulate the expression of PKCε to promote the phosphorylation of ezrin(T567), which can be a new mechanism by which Nanog promotes tumor metastasis.

Blotting, Western ; Breast Neoplasms ; metabolism ; Cytoskeletal Proteins ; metabolism ; Gene Knockdown Techniques ; Homeodomain Proteins ; metabolism ; Humans ; Nanog Homeobox Protein ; Neoplasm Invasiveness ; Phosphorylation ; Protein Kinase C-epsilon ; metabolism ; RNA, Small Interfering ; Transfection ; Tumor Cells, Cultured ; Up-Regulation

OBJECTIVETo observe the effect of rapamycin on the MG-63 osteosarcoma cells (OC), osteosarcoma stem cells (OSC) and on mTOR signaling pathway, and explore the feasibility of rapamycin as a novel therapeutic measure in osteosarcoma chemotherapy regimens.

METHODSOC and OSC were cultured in vitro. Immunofluorescence assay was used to detect the expression of Nanog and Oct4 in OC and OSC. OC and OSC were treated with rapamycin in concentrations of 0, 20, 50 and 100 nmol/L. Semi-quantitative PCR and RT-PCR were used to detect the mTOR mRNA and CCK-8 assay was used to detect cell proliferation, and the cell morphology was observed under an inverted microscope.

RESULTSThe cores of MG-63 cellular spheres exhibited embryonic stem cell characteristics such as Nanog and Oct4 expession. The mTOR pathway was activated in the OSC and the expression of mTOR mRNA was higher in OSC (0.761 ± 0.080) than that in OS (0.406 ± 0.090, P < 0.05) by semi-quantitative PCR. RT-PCR showed that the expression of mTOR mRNA was lower in OSCs treated with 100 nmol/L rapamycin (0.961 ± 0.060) than that with 0 nmol/L rapamycin (1.654 ± 0.246, P < 0.05). Cell counting kit-8 (CCK-8) assay showed that the proliferation of OC treated with 20, 50 and 100 nmol/L rapamycin was significantly inhibited, compared with that with 0 nmol/L rapamycin (P < 0.05). Compared with 0 nmol/L rapamycin, the proliferation of OSC treated with 20 and 50 nmol/L rapamycin was not significantly inhibited (P > 0.05), but that with 100 nmol/L rapamycin was significantly inhibited (P < 0.05). The invert microscopic observation revealed that rapamycin inhibited the formation of OSC spheres.

CONCLUSIONSRapamycin can effectively inhibit cell proliferation and the ability of sphere formation of OSCs. It will provide a basis for a novel therapeutic approach in osteosarcoma chemotherapy regimens.

Antibiotics, Antineoplastic ; administration & dosage ; pharmacology ; Bone Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Homeodomain Proteins ; metabolism ; Humans ; Nanog Homeobox Protein ; Neoplastic Stem Cells ; metabolism ; pathology ; Octamer Transcription Factor-3 ; metabolism ; Osteosarcoma ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Signal Transduction ; Sirolimus ; administration & dosage ; pharmacology ; TOR Serine-Threonine Kinases ; genetics ; metabolism

OBJECTIVETo investigate the effect of tricostantin A (TSA) on self-renewal of breast cancer stem cells and explore the mechanisms.

METHODSBreast cancer cell lines MDA-MB-468, MDA-MB-231, MCF-7 and SKBR3 were cultured in suspension and treated with different concentrations of TSA for 7 days, using 0.1% DMSO as the control. Secondary mammosphere formation efficiency and percentage of CD44(+)/CD24(-) sub-population in the primary mammospheres were used to evaluate the effects of TSA on self-renewal of breast cancer stem cells. The breast cancer stem cell surface marker CD44(+)/CD24(-) and the percentage of apoptosis in the primary mammospheres were assayed using flow cytometry. The mRNA expressions of Nanog, Sox2 and Oct4 in the primary mammospheres were assayed with quantitative PCR.

RESULTSTSA at both 100 and 500 nmol/L, but not at 10 nmol/L, partially inhibited the self-renewal of breast cancer stem cells from the 4 cell lines. TSA at 500 nmol/L induced cell apoptosis in the primary mammospheres. TSA down-regulated the mRNA expression of Nanog and Sox2 in the primary mammospheres.

CONCLUSIONTSA can partially inhibit the self-renewal of breast cancer stem cells through a mechanism involving the down-regulation of Nanog and Sox2 expression, indicating the value of combined treatments with low-dose TSA and other anticancer drugs to achieve maximum inhibition of breast cancer stem cell self-renewal. The core transcriptional factor of embryonic stem cells Nanog and Sox2 can be potential targets of anticancer therapy.

Antineoplastic Agents ; administration & dosage ; pharmacology ; Apoptosis ; drug effects ; Breast Neoplasms ; metabolism ; pathology ; CD24 Antigen ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Down-Regulation ; Female ; Histone Deacetylase Inhibitors ; administration & dosage ; pharmacology ; Homeodomain Proteins ; genetics ; metabolism ; Humans ; Hyaluronan Receptors ; metabolism ; Hydroxamic Acids ; administration & dosage ; pharmacology ; Nanog Homeobox Protein ; Neoplastic Stem Cells ; metabolism ; pathology ; RNA, Messenger ; metabolism ; SOXB1 Transcription Factors ; genetics ; metabolism

Cancer stem cells (CSCs) are thought to drive uncontrolled tumor growth, and the existence of CSCs has recently been proven by direct experimental evidence, including tracing cell lineages within a growing tumor. However, CSCs must be analyzed in additional cancer types. Cancer stem cell-like cells (CSCLCs) are a good alternative system for the study of CSCs, which hold great promise for clinical applications. OCT4, NANOG, and SOX2 are three basic transcription factors that are expressed in both CSCLCs and embryonic stem cells (ESCs). These transcription factors play critical roles in maintaining the pluripotence and self-renewal characteristics of CSCLCs and ESCs. In this review, we discuss the aberrant expression, isoforms, and pseudogenes of OCT4, NANOG, and SOX2 in the CSCLC niche, which contribute to the major differences between CSCLCs and ESCs. We also highlight an anticancer therapy that involves killing specific cancer cells directly by repressing the expression of OCT4, NANOG, or SOX2. Importantly, OCT4, NANOG, and SOX2 provide great promise for clinical applications because reducing their expression or blocking the pathways in which they function may inhibit tumor growth and turn-off the cancer "switch." In the future, a clear understanding of transcription factor regulation will be essential for elucidating the roles of OCT4, NANOG, and SOX2 in tumorigenesis, as well as exploring their use for diagnostic and therapeutic purposes.
Animals ; Embryonic Stem Cells ; metabolism ; Homeodomain Proteins ; metabolism ; Humans ; Nanog Homeobox Protein ; Neoplasms ; metabolism ; pathology ; Neoplastic Stem Cells ; metabolism ; Octamer Transcription Factor-3 ; metabolism ; SOXB1 Transcription Factors ; metabolism ; Signal Transduction

OBJECTIVE: To investigate the involvement of transcription factor Foxa2 in cardiac differentiation in P19 embryonal carcinoma cells and its molecular mechanism. METHODS: P19 cells were induced to differentiate into cardiomyocytes by adding dimethyl sulfoxide (DMSO) into the culture medium of their embryoid bodies (EBs). The mRNA levels of pluripotency markers of embryonic pluripotent stem cells, cardiac differentiation related genes, and Foxa2 in the cell samples at different time points of cardiac differentiation were detected by reverse transcription PCR (RT-PCR). Differentiated and mature cardiomyocytes were identified by immunofluorescence. Eukaryotic expression plasmid pCMV-rFoxa2 (rat Foxa2) was transfected into P19 cells, and clonal populations of P19 cells that stably expressed green fluorescence protein (GFP)-rFoxa2 were isolated to enhance the expression levels of Foxa2 in P19 cells. The mRNA and protein levels of pluripotency markers and cardiac differentiation related genes in the above cell samples were detected by RT-PCR and Western blot. The mRNA levels of cardiac differentiation related genes in EBs differentiation system were also examined. RESULTS: P19 cells differentiated into cardiomyocytes in the presence of DMSO, accompanied by stimulated expression of Foxa2. Transfection of pCMV-rFoxa2 plasmids into P19 cells upregulated rFoxa2 expression transiently and activated the transcription of its downstream cardiac inducer Cerberus1 (Cer1). The expression of pluripotency marker Nanog was suppressed and the expression of cardiac inducer Sonic Hedgehog (Shh) was elevated in GFP-rFoxa2 P19 cells. The expression of Cer1 and cardiac muscle marker actin, alpha cardiac muscle 1 (Actc1) was upregulated in EBs of GFP-rFoxa2 P19 cells. CONCLUSION Foxa2 participates in cardiac differentiation in P19 embryonal carcinoma cells. Foxa2 may inhibit Nanog expression and stimulate the expression of Cer1 and Shh directly during cardiac differentiation in P19 cells in the presence of DMSO.
Animals ; Cell Differentiation ; drug effects ; Cell Line ; Cytokines ; Dimethyl Sulfoxide ; pharmacology ; Embryonal Carcinoma Stem Cells ; pathology ; Hedgehog Proteins ; metabolism ; Hepatocyte Nuclear Factor 3-beta ; physiology ; Homeodomain Proteins ; metabolism ; Mice ; Myocytes, Cardiac ; cytology ; Nanog Homeobox Protein ; Proteins ; metabolism ; Transfection

BACKGROUNDKeratinocyte serum-free medium (K-SFM) is a defined medium used to support the growth of primary keratinocytes and embryonic stem cell. The aim of this research was to optimize enrichment of breast cancer stem cells (CSCs) using K-SFM.

METHODSA K-SFM was used to enrich CSCs from two breast cancer cell lines and a primary culture of breast cancer. RPMI-1640 supplemented with 10% fetal calf serum (FCS) was used as a control. CSCs were identified with flow cytometry using CD44(+)/CD24(-) as molecular markers. The expression of a variety of CSC markers (Oct-4, ABCG2, Nanog, N-cadherin, and E-cadherin) was analyzed with real-time PCR.

RESULTSMuch higher percentage of CSCs was achieved with K-SFM: 17.3% for MCF-7 cells, 17.4% for SKBR-3, and 20.0% for primary breast cancer culture. Less than 1% CSC was achieved using RPMI-1640 supplemented with 10% FCS. In comparison to the CSCs obtained with RPMI-1640, CSCs in the K-SFM expressed higher levels of Oct-4, ABCG2, Nanog and N-cadherin, and lower level of E-cadherin.

CONCLUSIONK-SFM is an optimal culture medium to maintain and to enrich breast CSCs.

ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; genetics ; Cadherins ; genetics ; Cell Culture Techniques ; methods ; Cell Line, Tumor ; Culture Media, Serum-Free ; Female ; Homeodomain Proteins ; genetics ; Humans ; Keratinocytes ; cytology ; Nanog Homeobox Protein ; Neoplasm Proteins ; genetics ; Neoplastic Stem Cells ; cytology ; metabolism ; Octamer Transcription Factor-3 ; genetics ; Real-Time Polymerase Chain Reaction