scholarly journals ET-04 MOLECULAR TARGETED THERAPY AGAINST (PRO)RENIN RECEPTOR FOR GLIOBLASTOMA

2019 ◽  
Vol 1 (Supplement_2) ◽  
pp. ii8-ii9
Author(s):  
Takeshi Fujimori ◽  
Daisuke Ogawa ◽  
Kenta Suzuki ◽  
Masaaki Kochi ◽  
Yuki Shibayama ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
Stem Cell ◽  
Wnt Signaling ◽  
Cell Line ◽  
Annexin V ◽  
Stem Cell Markers ◽  
Glioma Stem Cell ◽  
Cell Markers ◽  
Induced Apoptosis

Abstract INTRODUCTION (Pro)renin receptor(PRR) is part of the Wnt receptor complex. Wnt/β-catenin signaling pathway (Wnt signaling) plays important role in pathogenesis and self-renewal of glioblastoma (GBM), or differentiation of glioma stem cell. We previously reported that PRR activate Wnt signaling, PRR expression correlated with malignancy of glioma, and treatment with PRR siRNA reduced the proliferative capacity. This time, we have developed monoclonal antibodies against PRR and examined their effects in GBM. MATERIAL AND METHODS We used GBM cell line (U251MG and U87MG) and primary human glioma stem cell line (MGG23). Glioma stem-like cells were cultured and isolated by neurosphere method from U251MG and U87MG. PRR antibody was made targeting the extracellular domain of the PRR with rat lymph node method. WST-1 assay or MTT assay were performed to determine the cell proliferation. Apoptosis was examined by FITC labeled annexin V and propidium iodide with flow cytometry. We analyzed molecules of Wnt signaling and stem cell markers with qRT-PCR. RESULTS We observed that PRR antibody significantly reduced cell proliferation, decreased sphere formation. Antibody suppressed cell adherent in stem-like cell. Flow cytometry showed that antibody induced apoptosis. Antibody inhibited Wnt signaling and stem cell markers. CONCLUSIONS PRR antibody reduced cell proliferation and induced apoptosis through Wnt signaling. PRR antibody also suppressed stemness. Our results demonstrated that PRR was a potential target for future glioma therapy.

scholarly journals Abalone Collagen Extracts Potentiate Stem Cell Properties of Human Epidermal Keratinocytes

Marine Drugs ◽  
2019 ◽  
Vol 17 (7) ◽  
pp. 424
Author(s):  
Sajee Thaweekitphathanaphakdee ◽  
Pithi Chanvorachote ◽  
Sagaw Prateepchinda ◽  
Mattaka Khongkow ◽  
Apirada Sucontphunt
Keyword(s):  
Cell Proliferation ◽  
Wound Healing ◽  
Stem Cell ◽  
Cell Migration ◽  
Expression Level ◽  
Epidermal Keratinocytes ◽  
Stem Cell Markers ◽  
Spheroid Formation ◽  
Human Epidermal Keratinocytes ◽  
Cell Markers

Stem cell activities in human tissues are critical for tissue integrity and function. Maintaining keratinocyte stem cells (KSCs) stemness helps sustain healthy skin by supporting keratinocyte renewal, involving the formation of epidermal barriers. In this study, abalone collagen (AC) extracts with molecular weights of 3 kDa (AC 1) and 300 kDa (AC 2) were compared to the epidermal growth factor (EGF) for their effects on cell proliferation, cell migration (wound healing), spheroid formation, and the expression level of stem cell markers on human keratinocytes (HaCaT cells). Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell proliferation was quantified by ATP and DNA content analysis and Sulforhodamine B (SRB) assays. Cell migration assay was determined using the scratch wound healing test. Spheroid formation was evaluated and the expression level of stem cell markers was investigated by western blot analysis. The results showed that AC 1 at the concentration of 100 µg/mL could stimulate HaCaT cell proliferation, migration, spheroid formation, and the expression level of stem cell markers (keratin 19, β-catenin, ALDH1A1) compared to the control. In conclusion, a smaller molecular weight of abalone collagen extract exhibits a better effect on keratinocytes proliferation, migration, and stemness, which could be a potential active ingredient in cosmeceutical products.

ID: 22: DIFFERENTIAL ACTIONS OF ESTROGEN RECEPTOR α AND β VIA NON-GENOMIC SIGNALING IN HUMAN PROSTATE STEM-PROGENITOR CELLS

2016 ◽  
Vol 64 (4) ◽  
pp. 933-933
Author(s):  
S Majumdar ◽  
JC Rinaldi ◽  
T Gauntner ◽  
L Xie ◽  
W Hu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Stem Cell ◽  
Cell Line ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Mapk Pathway ◽  
Human Prostate ◽  
Stem Cell Markers ◽  
Self Renewal ◽  
Progenitor Cell Proliferation

Genomic signaling via estrogen receptors (ER) has been widely studied and implicated as the main ER signaling pathway in prostate development and carcinogenesis. Non-genomic ER signaling has also been reported in prostate epithelium although down-stream cascades have not been clarified. Our lab has recently identified ERs in human prostate epithelial stem/progenitor cells and shown that that 17β-estradiol (E2) can stimulate stem cell symmetric self-renewal and progenitor cell proliferation. In this study we interrogate non-genomic membrane initiated ER signaling in this prostate stem/progenitor cell population. Human prostate stem-progenitor cells were enriched from primary prostate epithelial cell cultures (PrEC) of young, disease-free donors using a 3D prostasphere (PS) model as previously described. Cells were labeled using ERα or ERβ antibodies along with prostate stem cell markers CD49f and TROP2 followed by triple channel FACS to quantify ERα+/ERβ+ cell numbers. To explore ERα, the benign human prostate stem cell line WPE with extremely low levels of ERα and ERβ, was stably transfected with a lentiviral-ERα expression vector. The human prostate cancer stem-like cell line HuSLC (ERβ++, ERα−) was utilized to interrogate ERb actions. Cells were exposed to 10 nM estradiol (E2) over a 15 to 60 minute time course +/− ICI 182,870 (ICI), an ERα/β antagonist. FACS analysis of day 7 PS cells labeled for ERα or ERβ revealed 66% of day 7 PS cells as ERα+ and 40% as ERβ+. Among ERα or ERβ positive PS cells, 4% were Trop2+/CD49fhigh (stem-like cells) and 10–12% were Trop2+/CD49fmedium (early stage progenitor cells). PS exposed to 10 nM E2 showed sequential phosphorylation of Src, Erk1/2, p38, Akt and NFκB (p65) over 60 minutes. Phosphorylation of up-and downstream targets (EGFR, Jnk, GSK 3α/β, p70 S6 kinase, PRAS40, MSK1/2) was also seen using a phospho-kinase array. Furthermore, phosphorylation of ERα at S167 was noted over 60 min of E2 exposure enabling enhancement of genomic ERα transactivational activity in a feed-forward manner. ICI attenuated Akt and Erk1/2 phosphorylation, confirming membrane bound ERs are involved in downstream signaling. E2 treatment of HuSLCs showed phosphorylation of Erk1/2 but not Akt, indicating that ERβ signals exclusively through the MAPK pathway in these cells. Conversely, E2 treatment of WPE-stem cells overexpressing ERα resulted in robust phosphorylation of Akt but lower levels of Erk1/2 phosphorylation suggesting that Akt activation may be more reliant on ERα signaling. To identify pathway specific roles, specific inhibitors were added to PS cultures. PS treated with LY294002 (Akt inhibitor) for 7 days attenuated the E2-mediated increase in PS number and size. Inhibition of the NFκB downstream of the Akt pathway by IKK VII (IKK inhibitor) blocked p65 phosphorylation, abrogated the E2-induced increase in stem cell symmetric self-renewal and blunted E2 stimulation of progenitor cell proliferation. Analysis of PS cyclin mRNA levels revealed a G1 arrest of progenitor cells upon IKK inhibition suggesting an essential role of NFκB in progenitor cell amplification. MAPK pathway inhibition with U0126(Erk1/2 inhibitor) resulted in an attenuation of the E2-mediated increase in PS number and size and an increase stem cell symmetric self-renewal suggesting that MAPK pathway activation promotes commitment to stem and progenitor cell expansion. Taken together, the present findings reveal that human prostate stem-progenitor cells express both ERα and ERβ which differentially activate different signaling cascades originating at the membrane. These signaling events may lead to unique downstream actions that influence prostate stem-progenitor cell proliferation as well as lineage commitment decisions.

scholarly journals CBMS-12 Pro renin receptor antibody regulates glioblastoma stemness

2020 ◽  
Vol 2 (Supplement_3) ◽  
pp. ii5-ii5
Author(s):  
Takeshi Fujimori ◽  
Daisuke Oagawa ◽  
Takahiro Kanda ◽  
Kenta Suzuki ◽  
Saki Shibayama ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Stem Cell ◽  
Wnt Signaling ◽  
Glioma Cell ◽  
Glioma Stem Cell ◽  
Human Glioma ◽  
Stem Cell Line ◽  
Glioma Cell Lines ◽  
Sphere Formation ◽  
Signaling Components

Abstract Objective: Glioblastoma multiforme (GBM) is characterized by a strong self-renewal potential and poor differentiated state. We previously reported that (pro)renin receptor (PRR) was a potential target for glioma therapy by silencing the gene of PRR. Here, we have developed the monoclonal antibody of PRR and examined their effects on GBM. Materials and methods: We performed immunohistochemical analysis to detect the protein expression of PRR and SOX-2 in human sample of 56 gliomas. We used human glioma cell lines (U251MG and U87MG) and glioma stem cell line (MGG23) in vitro study. PRR antibody was designed to target the extracellular domain of the PRR with the rat lymph node method. Expression of the Wnt signaling components and stem marker (SOX-2, Oct3/4) in human glioma cell lines and glioma stem cell line treated with PRR antibody were measured using Western blotting. The effects of PRR antibody on cell proliferation, sphere formation, apoptosis, invasion were also examined. Subcutaneous xenografts with U87MG were induced in nude mice. Results: PRR expression showed a positive correlation with SOX-2 expression in glioma samples. Treatment with PRR antibody significantly reduced expression of Wnt signaling components and stem marker. We observed that PRR antibody significantly reduced cell proliferation and decreased sphere formation. Furthermore, PRR antibody suppressed invasion and induced apoptosis. In a subcutaneous U87MG xenograft model, systemic administration of the PRR antibody significantly reduced the size of the tumor volume. Conclusion: PRR has important role for the maintenance of stem cells and contribute to stem cell proliferation. PRR antibody inhibits cell proliferation and cell invasion and induces apoptosis. Treatment with PRR antibody could be an attractive therapeutic strategy for GBM.

scholarly journals Desialylation of Sonic-Hedgehog by Neu2 Inhibits Its Association with Patched1 Reducing Stemness-Like Properties in Pancreatic Cancer Sphere-forming Cells

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1512
Author(s):  
Shalini Nath ◽  
Susmita Mondal ◽  
Ramesh Butti ◽  
Vinoth Prasanna Gunasekaran ◽  
Uttara Chatterjee ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Stem Cell ◽  
Xenograft Model ◽  
Stem Cell Markers ◽  
Pathway Activity ◽  
Cell Markers ◽  
Close Relationship ◽  
The Status ◽  
Induced Apoptosis

Cancer stem cells (CSCs) are crucial regulators of tumor recurrence/progression. The maintenance of CSCs is dependent on aberrant activation of various pathways, including Hedgehog. Prevalent sialylations contribute to aggressiveness in CSCs. Here, we have addressed the role of sialylation in regulating stemness-like properties of pancreatic cancer sphere-forming cells (PCS) through modulation of the Hedgehog (Hh) pathway. The status of CD133/CD44/surface-sialylation was checked by flow cytometry and effects of Neu2 overexpression in PCS were compared using qPCR, immunoblotting, co-immunoprecipitation and also by colony-formation assays. The work was also validated in a xenograft model after Neu2 overexpression. Neu2 and Shh status in patient tissues were examined by immunohistochemistry. PCS showed higher Hh-pathway activity and sialylation with reduced cytosolic-sialidase (Neu2). Neu2 overexpression caused desialylation of Shh, thereby reducing Shh-Patched1 binding thus causing decreased Hh-pathway activity with lower expression of Snail/Slug/CyclinD1 leading to reduction of stemness-like properties. Neu2-overexpression also induced apoptosis in PCS. Additionally, Neu2-overexpressed PCS demonstrated lower mTORC2 formation and inhibitory-phosphorylation of Gsk3β, reflecting a close relationship with reduced Hh pathway. Moreover, both Neu2 and Rictor (a major component of mTORC2) co-transfection reduced stem cell markers and Hh-pathway activity in PCS. Neu2-overexpressed tumors showed reduction in tumor mass with downregulation of stem cell markers/Shh/mTOR and upregulation of Bax/Caspase8/Caspase3. Thus, we established that reduced sialylation by Neu2 overexpression leads to decreased stemness-like properties by desialylation of Shh, which impaired its association with Patched1 thereby inhibiting the Hh pathway. All these may be responsible for enhanced apoptosis in Neu2-overexpressed PCS.

Adamantinomatous craniopharyngiomas express tumor stem cell markers in cells with activated Wnt signaling: further evidence for the existence of a tumor stem cell niche?

Pituitary ◽  
2013 ◽  
Vol 17 (6) ◽  
pp. 546-556 ◽  
Author(s):  
Annett Hölsken ◽  
Christina Stache ◽  
Sven Martin Schlaffer ◽  
Jörg Flitsch ◽  
Rudolf Fahlbusch ◽  
...  
Keyword(s):  
Stem Cell ◽  
Wnt Signaling ◽  
Stem Cell Niche ◽  
Tumor Stem Cell ◽  
Stem Cell Markers ◽  
Cell Markers ◽  
Cell Niche ◽  
In Cells

Abstract 3372: Analysis of putative tumor stem cell markers in the NCI-60 tumor cell line panel

2010 ◽  
Author(s):  
Christina H. Stuelten ◽  
Susan D. Mertins ◽  
Johanna I. Busch ◽  
Meghan Gowens ◽  
Dominic A. Scudiero ◽  
...  
Keyword(s):  
Stem Cell ◽  
Tumor Cell ◽  
Cell Line ◽  
Tumor Cell Line ◽  
Tumor Stem Cell ◽  
Stem Cell Markers ◽  
Cell Markers ◽  
Cell Line Panel

scholarly journals 268.Identifying markers for stromal stem/progenitor cells in human endometrium

2004 ◽  
Vol 16 (9) ◽  
pp. 268 ◽  
Author(s):  
K. E. Schwab ◽  
C. E. Gargett
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Endometrial Tissue ◽  
Cell Populations ◽  
Stem Cell Markers ◽  
Endometrial Stromal Cell ◽  
Cell Markers

The endometrium is divided into upper functionalis, which rapidly grows then differentiates before being shed, and lower basalis, from which cyclical regeneration begins. A small proportion of endometrial stromal cells have been identified with clonogenic activity, a functional property of stem cells (1). We hypothesised that stromal stem/progenitor cells expressing known stem cell markers reside in the basalis. The aims of this study were to: (1) investigate the clonogenic activity of human endometrial stromal cell populations enriched and depleted for known stem cell markers, and (2) identify a marker that will differentiate basalis from functionalis stroma. Endometrial tissue acquired from 23 ovulating women undergoing hysterectomy was digested with collagenase to produce single cell suspensions. Leukocytes and epithelial cells were removed, and stromal cells analysed by flow cytometry, FACS sorted into enriched and depleted populations, and cultured for clonal analysis as described (1). Markers analysed included stem cell markers, STRO-1, CD133, CD45 and CD34, and an endometrial stromal cell marker, CD90 (2). Immunohistochemical analysis of CD90 was performed on full thickness human endometrial tissue. CD45– endometrial stromal cell populations contained 2.13 � 0.65% (n = 13) STRO-1+, and 5.43 � 1.42% (n = 16) CD133+ cells. Stromal cell populations enriched (0.65 � 0.42%) and depleted (0.95 � 0.58%) for STRO-1 showed no significant difference (P = 0.19, n = 5) for clonogenic activity. Surprisingly, clonogenicity of CD133+ stromal cells (0.74 � 0.56%) was lower than CD133– (3.89 � 1.35%) cells (P = 0.03, n = 6). Immunohistochemical staining showed strong CD90 staining in the functionalis, with lighter staining in the basalis. These observations were confirmed by flow cytometric analysis which identified two distinct populations (n = 9), CD90low (19.55 � 4.35%) and CD90hi (74.71 � 5.20%). Clonogenic analysis of these two populations is underway. Interestingly, dual-colour flow cytometry showed the CD133+ cells to be CD90low (n = 7). Further analysis suggests that the CD90lowCD133+ population are CD45–CD34+, suggesting endothelial progenitor cells. This study identified CD90 as a marker that distinguishes basalis and functionalis stroma, and demonstrated that STRO-1 and CD133 are not functional markers for clonogenic endometrial stromal stem/progenitor cells. (1) Chan RW, Schwab KE, Gargett CE (2004) Biol. Reprod. 70, in press. (2) Fernandez-Shaw S, Shorter SC, Naish CE, Barlow DH, Starkey PM (1992) Hum. Reprod. 7,156–161.

scholarly journals Complex Display of Putative Tumor Stem Cell Markers in the NCI60 Tumor Cell Line Panel

Stem Cells ◽  
2010 ◽  
Vol 28 (4) ◽  
pp. 649-660 ◽  
Author(s):  
Christina H. Stuelten ◽  
Susan D. Mertins ◽  
Johanna I. Busch ◽  
Meghan Gowens ◽  
Dominic A. Scudiero ◽  
...  
Keyword(s):  
Stem Cell ◽  
Tumor Cell ◽  
Cell Line ◽  
Tumor Cell Line ◽  
Tumor Stem Cell ◽  
Stem Cell Markers ◽  
Cell Markers ◽  
Cell Line Panel ◽  
Complex Display

scholarly journals CD44 and CD24 cannot act as cancer stem cell markers in human lung adenocarcinoma cell line A549

2014 ◽  
Vol 19 (1) ◽  
Author(s):  
Raheleh Roudi ◽  
Zahra Madjd ◽  
Marzieh Ebrahimi ◽  
Fazel Samani ◽  
Ali Samadikuchaksaraei
Keyword(s):  
Stem Cell ◽  
Growth Factor ◽  
Cell Line ◽  
A549 Cells ◽  
Stem Cell Markers ◽  
Double Positive ◽  
Cell Markers ◽  
Cancer Stem Cell Markers ◽  
Lung Adenocarcinoma Cell Line

AbstractCancer stem cells (CSCs) are subpopulations of tumor cells that are responsible for tumor initiation, maintenance and metastasis. Recent studies suggested that lung cancer arises from CSCs. In this study, the expression of potential CSC markers in cell line A549 was evaluated. We applied flow cytometry to assess the expression of putative stem cell markers, including aldehyde dehydrogenase 1 (ALDH1), CD24, CD44, CD133 and ABCG2. Cells were then sorted according to the expression of CD44 and CD24 markers by fluorescence-activated cell sorting (FACS) Aria II and characterized using their clonogenic and sphere-forming capacity. A549 cells expressed the CSC markers CD44 and CD24 at 68.16% and 54.46%, respectively. The expression of the putative CSC marker ALDH1 was 4.20%, whereas the expression of ABCG2 and CD133 was 0.93%. Double-positive CD44/133 populations were rare. CD44+/24+ and CD44+/CD24−/low subpopulations respectively exhibited 64% and 27.92% expression. The colony-forming potentials in the CD44+/CD24+ and CD44+/CD24−/low subpopulations were 84.37 ± 2.86% and 90 ± 3.06%, respectively, while the parental A549 cells yielded 56.65 ± 2.33% using the colony-formation assay. Both isolated subpopulations formed spheres in serumfree medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). CD44 and CD24 cannot be considered potential markers for isolating lung CSCs in cell line A549, but further investigation using in vivo assays is required.

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