STUDY ON ANTIANGIOGENENIC EFFECT OF ACANTHOPANAX SENTICOSUS IN HL60 CELL LINES

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4857-4857
Author(s):  
Chen Fangyuan ◽  
Zhang Minyue ◽  
Cai Jiayi ◽  
Shen Lijing
Keyword(s):  
Cell Proliferation ◽  
Signaling Pathway ◽  
Concentration Dependence ◽  
Cell Lines ◽  
Western Blotting ◽  
Leukemia Cell ◽  
Hl60 Cell ◽  
Vegf Receptor ◽  
Acanthopanax Senticosus ◽  
Hl60 Cells

Abstract Introduction Many studies have been confirmed that neovascularization, the formation of new blood vessels from existing vasculature, plays an essential role in growth, development and metastasis of acute leukemia. At present, antiangiogenic therapy of leukemia become the new hot spot. Acanthopanax senticosus(Chianese name Ci Wu Jia ,CWJ) is a kind of Chinese herb, which contain natural flavonoid compounds and have been proven to inhibit leukemia cell proliferation. But there is no detailed report about the relationship with the inhibition of leukemia cells and the inhibition of angiogenesis effect. In this study, we should demonstrate the inhibition of leukemia cell growth and antiangiogenic mechanism through HL60 cell lines, further confirm the inhibitory effect on leukemia and antiangiogenic effect of Acanthopanax senticosus, Methods HL60 cells were treated with different concentrations of Acanthopanax senticosus (25°¢50°¢75°¢100°¢200µg/ml). Cell proliferation were detected using Cell Counting Kit-8. Kinds of transcription factors in Dll4/Notch (Delta-like 4 is the only ligand of Notch expressing in endothelium) and VEGF(R) signaling pathway were evaluated using quantitative real-time PCR (qRT-PCR) and Western blotting. Results Acanthopanax senticosus inhibited the growth of HL60 cells, and the time and concentration dependence(Fig.1). We extracted RNA and protein from these cells at 12hr, 24hr and 48hr respectively, found that Acanthopanax senticosus remarkably results in VEGF, VEGFR2(VEGF Receptor 2), DLL4 down-regulation based on the time and the concentration dependence, and mild inhibit VEGFR1(VEGF Receptor 1) and Notch1 factors gene expression(Fig. 2). Western blotting also showed a significant inhibition protein of VEGFR2, DLL4 and Notch1, mild inhibited the expression of VEGF and VEGFR1 protein, and with time and concentration dependence (Fig. 3). Summary Acanthopanax senticosus can inhibited proliferation of HL60 cells in vitro and anti-angiogenesis effect mainly via inhibition of VEGFR2-mediated signaling. It has an instantaneous effect on Dll4/ Notch signaling pathway. The data have elucidated the potential roles of several key signaling pathways in angiogenesis. Disclosures: No relevant conflicts of interest to declare.

Downregulation of the Cell-Cycle Regulating Ubiquitin-Ligase APC/CCdh1 May Contribute to the Differentiation Block of AML1/Eto Positive AML.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5045-5045
Author(s):  
Manuel Hein ◽  
Dominik Schnerch ◽  
Andrea Schmidts ◽  
Julia Felthaus ◽  
Dagmar Wider ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Ubiquitin Ligase ◽  
Potential Role ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Hl60 Cell ◽  
Cd11b Expression ◽  
Hl60 Cells ◽  
Differentiation Block

Abstract Abstract 5045 Introduction The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase regulating cell cycle progression by targeting various cell cycle regulators for proteasomal degradation. It is activated by the adaptor proteins Cdc20 in mitosis and by Cdh1 in late mitosis and G1/G0. Thereby, Cdh1 establishes a stable G1 phase enabling the cell to either exit the cell cycle and differentiate or to prepare for a new round of cell division. It has also been shown that Cdh1 plays a role in the differentiation of various cell types, such as neurons, myocytes, hepatocytes and lens epithelial cells. Methods and Results We have examined the regulation of Cdh1 in several acute myeloid leukemia (AML) cell lines. We found that in the AML1/Eto positive leukemia cell lines SKNO-1 and Kasumi-1, Cdh1 protein and RNA levels are lower than in AML1/Eto negative cell lines KG-1 and HL-60. In addition, Cdh1 protein level in an AML1/Eto positive primary blast sample was lower than in AML1/Eto negative patient samples. The translocation t(8;21) is one of the most frequent chromosomal rearrangement in AML and results in an AML1/Eto fusion protein, which can act as a transcriptional repressor. Thus, our results are consistent with AML1/Eto mediated downregulation of Cdh1. To evaluate the potential role of APC/CCdh1 in myeloid differentiation, we established a stable Cdh1 knockdown (kd) in the AML1/Eto negative HL60 cell line with high Cdh1 expression by lentiviral vector mediated RNA interference. HL60 cells harbouring either a Cdh1 shRNA or a control shRNA against GFP were established simultaneously. We used PMA at concentrations of 0.5, 1, 2 and 50 nM to differentiate these cells into CD11b positive macrophage-like cells over 48h. Protein isolation and analysis of CD11b expression by flow cytometry were performed at 0, 6h, 12h, 24h and 48h to examine differentiation kinetics. Cdh1 and target proteins with a potential role in cell cycle arrest and differentiation, such as Skp2 (an activator of the SCF-ubiquitin ligase targeting p21 and p27) and ID2 (inhibitor of differentiation 2), were analyzed by Western blotting. We observed that kd of Cdh1 in HL60 cells resulted in 10% to 20% lower CD11b expression at any time, when PMA was used at concentrations 0, 0.5, 1nM over 48h. ID2 and Skp2 were stabilized in these Cdh1 kd cells compared to the control correlating with the less differentiated state. In addition, HL60 cells with a stable Skp2 kd showed a higher CD11b expression indicating a more differentiated status compared to the control. Conclusion This is the first report that indicates a role for APC/CCdh1 in the differentiation of myeloid cells with SCFSkp2 being one of the relevant targets. Downregulation of Cdh1 may contribute to the differentiation block of AML1/Eto postive AML. Disclosures No relevant conflicts of interest to declare.

PI3K/Akt Signaling Interacts With Wnt/β-Catenin Signaling But Does Not Induce An Accumulation Of β-Catenin In The Nucleus Of Acute Lymphoblastic Leukemia Cell Lines

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4886-4886 ◽  
Author(s):  
Tina-Susann Langhammer ◽  
Catrin Roolf ◽  
Saskia Krohn ◽  
Christin Kretzschmar ◽  
Rayk Huebner ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
Signaling Pathways ◽  
Cell Line ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Akt Signaling ◽  
Stimulation Of

Abstract Signaling pathways play essential roles in biological processes as development, cell proliferation and homeostasis. The accurate modulation of signaling pathways, their adapted interaction and their time- and tissue-specific adjusted regulation are required for normal cell development. PI3K/Akt and Wnt/β-Catenin signaling pathways act as key regulators in cell proliferation, differentiation and growth. Both signaling pathways include GSK3β as a common protein, which may mediate an interaction and cross-talk between the pathways. Aberrant activation of PI3K/Akt signaling has been linked to different types of leukemia while Wnt/β-Catenin signaling is known to be deregulated in some solid tumors. However, a potential role of Wnt/β-Catenin signaling for pathogenesis of acute lymphoblastic leukemia (ALL) has not yet been analyzed. In our study we analyzed both signaling pathways in different B- and T-ALL cell lines (RS4;11, SEM, REH, CEM, Jurkat, MOLT-4), thereby focusing mainly on their potential interaction via the protein GSK3β. Western Blot experiments were performed to evaluate the expression of specific PI3K/Akt and Wnt/β-Catenin key proteins. To evaluate the activation status of Wnt signaling immunofluorescence and protein fractionation experiments were performed, analyzing the activation linked nucleic localization of β-Catenin. The effect of pathway activation and inhibition on cell proliferation via chemical compounds was analyzed by WST-1 test. High pAkt levels were detected in B-ALL cell line SEM and T-ALL cell line CEM, indicating a hyperactive PI3K/Akt signaling, whereas other analyzed cell lines diplayed lower pAkt status. Among all cell lines analyzed SEM and CEM also showed the highest cytoplasmic β-Catenin levels, indicating a direct interaction of both signaling pathways. However, immunofluorescence and fractionation experiments revealed that a translocation of β-Catenin into the nucleus did not occur. To further investigate the role and interaction of PI3K/Akt and Wnt/β-Catenin signaling, pathway inhibiting and stimulating experiments were performed. Treatment of cells with Wnt3a led to activation of the Wnt/β-Catenin signaling cascade, characterized by nuclear β-Catenin accumulation. Inhibition of cell proliferation was detected after treatment with high concentrations Wnt3a (≥ 500 ng/ml). PI3K inhibition by LY294002 led to decreased phosphorylation of GSK3β at Ser9 and an increased decay of β-Catenin. Stimulation of PI3K/Akt signaling using activating ligand FLT3L induced GSK3β phosphorylation at Ser9 and accumulation of cytoplasmic β-Catenin. However a translocation of β-Catenin into the nucleus seems not to occur. In summary our results indicate that PI3K/Akt and Wnt/β-Catenin signaling can interact through their common protein GSK3β, but stimulation of the PI3K/Akt signaling pathway by addition of PI3K/Akt specific activators does not fully activate Wnt/β-Catenin signaling in ALL cells. Complete activation of the Wnt cascade characterized by translocation of β-Catenin into the nucleus can only be induced by use of specific Wnt effectors. Disclosures: No relevant conflicts of interest to declare.

Knockdown of Adhesion-Regulating Molecule 1 Inhibits Proliferation in HL60 Cells

2015 ◽  
Vol 134 (2) ◽  
pp. 88-100 ◽  
Author(s):  
Xiaohui Zheng ◽  
Yafei Guo ◽  
Yingying Chen ◽  
Meilin Chen ◽  
Zhenxin Lin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Colony Formation ◽  
Nuclear Translocation ◽  
Leukemia Cell ◽  
26S Proteasome ◽  
Biological Behavior ◽  
Hl60 Cell ◽  
Hl60 Cells ◽  
Leukemia Cell Lines

Background/Aims: Adhesion-regulating molecule 1 (ADRM1), a receptor located on the 26S proteasome, is upregulated in many solid cancers. However, little is known about its role in acute leukemia (AL). Methods: We determined ADRM1 expression levels in both untreated AL samples and leukemia cell lines using real-time polymerase chain reaction or Western blot analysis. Growth curves, colony formation assays, cell cycle and apoptosis analyses, cell migration and invasion assays and NF-κB p65 nuclear translocation assays via Western blotting were used to examine the biological behavior of HL60 cells and the underlying mechanism. Results: ADRM1 was upregulated in both untreated AL samples and leukemia cell lines. ADRM1 knockdown significantly suppressed HL60 cell proliferation (48.82 ± 12.58%) and colony formation and caused cell cycle arrest in the G0/G1 phase. Furthermore, we confirmed that ADRM1 knockdown suppressed p65 nuclear translocation. Conclusion: Our study revealed that ADRM1 was overexpressed in AL, especially in CD34+ leukemia stem and progenitor cells. ADRM1 may play a role in AL via the proteasome-ubiquitin pathway by potentially sustaining the activation of NF-κB signaling.

Downregulation of Sulfiredoxin (Srx) Suppressed Cell Proliferation Migration and Invasion Through Inhibiting Extracellular Signal-Regulated Kinase/Nrf2 Pathway in Hepatocellular Carcinoma

2021 ◽  
Vol 11 (11) ◽  
pp. 2137-2145
Author(s):  
Xuejuan Zhu ◽  
Danqian Lu
Keyword(s):  
Cell Proliferation ◽  
Western Blot ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Colony Formation ◽  
Migration And Invasion ◽  
Related Factor ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Nrf2 Signaling Pathway

Background: Sulfiredoxin (Srx) has been identified to play important roles in the development of various cancers. However, the precise effects and underlying mechanism of Srx on the progression of HCC are far from being fully understood. Materials and Methods: The abundances of Srx in THLE-2 cell and HCC cell lines were determined by western blot and RT-qPCR. Next, SK-Hep-1 cells were transfected with shRNA-Srx or shRNA-NC and treated with TBHQ (an extracellular signal-regulated kinase (ERK) activator) for functional experiments. Then, CCK8 and colony formation assays were used to determine cell proliferation and clone-forming abilities in vitro. Cell migration and invasion were assessed via wound healing and transwell assays. The expression of MMP2, MMP9 and key members in ERK/nuclear factor E2 related factor (Nrf2) signaling pathway was detected by performing western blot analysis. Results: We reported evidence that Srx was frequently up-regulated in HCC cell lines. Srx interference constrained cell proliferation, colony formation rate, migration and invasion of SK-Hep-1 cells. Moreover, mechanistic investigations indicated that Srx interference significantly inhibited the activation of ERK/Nrf2 signaling pathway, and ERK activator TBHQ can reverse the functions of Srx interference in SK-Hep-1 cells. Conclusion: Overall, Downregulation of Srx might impede HCC progression by suppressing ERK/Nrf2 signaling pathway. Findings in the current study reported the functional involvement and molecular mechanism of Srx in HCC, suggesting that Srx might have a potential therapeutic value in HCC treatment.

scholarly journals Capn4 Enhances Osteopontin Expression through Activation of the Wnt/β-Catenin Pathway to Promote Epithelial Ovarian Carcinoma Metastasis

2017 ◽  
Vol 42 (1) ◽  
pp. 185-197 ◽  
Author(s):  
Xiaoming Yang ◽  
Jing Sun ◽  
Dandan Xia ◽  
Xupei Can ◽  
Lei Liu ◽  
...  
Keyword(s):  
Ovarian Carcinoma ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Western Blotting ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Epithelial Ovarian Carcinoma ◽  
Regulatory Subunit ◽  
Epithelial Tissue

Background and Aim: Increasing evidence shows that the calpain regulatory subunit Capn4 can modulate the proliferation and metastasis of cancer cells, and plays an important role in the development of malignant tumors. However, there is no information on the clinical significance of Capn4 in epithelial ovarian carcinoma (EOC) or the molecular mechanisms by which Capn4 promotes the growth and metastasis of EOC. Therefore, the aim of this study was to clarify the role of Capn4 in EOC. Methods: We evaluated Capn4 and osteopontin (OPN) expression in EOC cell lines and tissues from patients with ovarian cancer by western blotting and immunohistochemical analysis. We then created cell lines with downregulated and upregulated Capn4 expression, using Capn4-targeting small interfering RNA and a pcDNA3.1-Capn4 overexpression vector, respectively, to investigate its function in EOC in vitro. In addition, we investigated the potential mechanism underlying the function of Capn4 by examining the effect of modifying Capn4 expression on Wnt/β-catenin signaling pathway-related genes by western blotting. Results: Capn4 was overexpressed in clinical EOC tissues compared with that in normal ovarian epithelial tissue, and was associated with poor clinical outcomes. Upon silencing or overexpressing Capn4 in EOC cells, we concluded that Capn4 promotes cell proliferation and migration in vitro. Furthermore, Capn4 promoted EOC metastasis by interacting with the Wnt/β-catenin signaling pathway to upregulate OPN expression. Conclusion: Our study indicates that Capn4 plays a critical role in the progression and metastasis of EOC, and could be a potential therapeutic target for EOC management.

scholarly journals Expression of Notch–Hif-1α signaling pathway in liver regeneration of rats

2020 ◽  
Vol 48 (9) ◽  
pp. 030006052094379
Author(s):  
Yanshan Li ◽  
Yunxiuxiu Xu ◽  
Ruomei Wang ◽  
Wenxin Li ◽  
Wenguang He ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Body Weight ◽  
Protein Expression ◽  
Liver Regeneration ◽  
Signaling Pathway ◽  
Western Blotting ◽  
Saline Control ◽  
Control Group ◽  
Mrna Levels ◽  
Ki 67

Objective To investigate whether the Notch–Hif-1α signaling pathway is involved in liver regeneration. Methods Rats were divided into two groups and treated with daily intraperitoneal injections of saline (control) or the gamma-secretase inhibitor, Fli-06, for 2 days. Two-thirds of the rat livers were resected and rats were later euthanized at specific time points post-resection to analyze the remnant livers. Each group's liver/body weight ratio was calculated, and immunostaining and western blotting were used to determine the cell proliferation marker, PCNA and Ki-67 expression. Real-time PCR and western blotting were used to compare the mRNA expression of Notch homolog-1 ( Notch1), hairy and enhancer of split-1 ( Hes1), and vascular endothelial growth factor ( Vegf), and the protein expression of NICD and HIF-1α, respectively. Results The liver/body weight ratios and number of Ki-67- and PCNA-positive cells were significantly lower in the experimental group than the control group, indicating lower levels of liver regeneration following the disruption of Notch signaling by Fli-06. The Hes1 and Vegf mRNA levels and NICD and HIF-1α protein expression levels were all down-regulated by Fli-06 treatment. Conclusion Notch–Hif-α signaling pathway activation plays an important role in liver regeneration, where it may contribute toward liver cell proliferation.

scholarly journals LncRNA HOTAIR is a Prognostic Biomarker for the Proliferation and Chemoresistance of Colorectal Cancer via MiR-203a-3p-Mediated Wnt/ß-Catenin Signaling Pathway

2018 ◽  
Vol 46 (3) ◽  
pp. 1275-1285 ◽  
Author(s):  
Zhigang Xiao ◽  
Zhan Qu ◽  
Zhikang Chen ◽  
Zhixue Fang ◽  
Ke Zhou ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Antisense Rna ◽  
Vital Role ◽  
Loss Of Function ◽  
Human Colorectal Cancer ◽  
Expression Levels ◽  
Lncrna Hotair

Background/Aims: HOX transcript antisense RNA (HOTAIR) plays a vital role in carcinogenesis. However, its functional and regulatory roles remain unclear. In this study, we aimed to investigate its biological function and clinical significance in human colorectal cancer (CRC). Methods: We examined the expression levels of lncRNA HOTAIR and miR-203a-3p in CRC tissues and CRC cell lines by qRT-PCR. Gain and loss-of-function assays were performed to examine the effects of HOTAIR and miR-203a-3p on the proliferation and chemoresistance of CRC cells. The possible mechanisms of HOTAIR were also explored by fluorescence reporter assay and Western blot. Results: The expressions of HOTAIR were upregulated in CRC tissue tissues compared to adjacent control tissues. We also found HOTAIR was downregulated by miR-203a-3p in CRC cell lines. Both HOTAIR knockdown and miR-203a-3p overexpression in CRC cell lines led to inhibited cell proliferation and reduced chemoresistance. We also determined that β-catenin and GRG5 were inhibitory targets of miR-203a-3p, and that Wnt/β-catenin signaling was inhibited by both HOTAIR knockdown and miR-203a-3p overexpression. Significantly, we found that increased expression of miR-203a-3p is essential for cell proliferation repression, chemoresistance reduction, and Wnt/β-catenin signaling inhibition induced by HOTAIR knockdown. Conclusions: Our study demonstrated that the lncRNA HOTAIR could regulate the progression and chemoresistance of CRC via modulating the expression levels of miR-203a-3p and the activity of Wnt/β-catenin signaling pathway.

scholarly journals The Long Non-Coding RNA CRNDE Promotes Colorectal Carcinoma Progression by Competitively Binding miR-217 with TCF7L2 and Enhancing the Wnt/β-Catenin Signaling Pathway

2017 ◽  
Vol 41 (6) ◽  
pp. 2489-2502 ◽  
Author(s):  
Bo Yu ◽  
Xuan Ye ◽  
Qiong Du ◽  
Bin Zhu ◽  
Qing Zhai
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Signaling Pathway ◽  
Western Blotting ◽  
Migration And Invasion ◽  
Down Regulation ◽  
Over Expression ◽  
Non Coding Rna ◽  
Colorectal Cancer Cells ◽  
Long Non Coding Rna

Background/Aims: The long non-coding RNA colorectal neoplasia differentially expressed (CRNDE) contributes to the proliferation and migration of tumors. However, its molecular mechanism underlying gastric cancer remains unknown. In the present study, we investigated whether CRNDE was involved in the development of colorectal cancer via the binding of microRNA (miR)-217 with transcription factor 7-like 2 (TCF7L2) to enhance the Wnt signaling pathway. Methods: Quantitative polymerase chain reaction was used to detect CRNDE, miR-217 and TCF7L2 in colorectal cancer tissues and cells. The CCK-8 assay, wound healing assay, and Transwell assay were used to detect cell proliferation, migration and invasion, respectively. Western blotting and luciferase activity assays were used to identify CRNDE and TCF7L2 as one of the direct targets of miR-217. The activity of the Wnt/β-catenin signaling pathway was analyzed by the TOPflash assay, and the subcellular localization of β-catenin and TCF7L2 was analyzed by western blotting and confocal microscopy. Results: In this study, we found that high expression of CRNDE is negatively correlated with low expression of miR-217 in colorectal cancer tissue and colorectal cancer cells. The dual luciferase reporter analysis showed that miR-217 is bound to CRNDE and TCF7L2 and negatively regulate their expression. CRNDE down-regulation inhibited the cell proliferation, migration and invasion in vitro and in vivo and the inhibitions were both completely blocked after miR-217 inhibition or TCF7L2 overexpression. Finally, TOPflash analysis showed that the activity of Wnt/β-catenin signaling is inhibited by CRNDE down-regulation and rescued by TCF7L2 over-expression. Consistently immunostaining and western blotting analysis showed that the expression of b-catenin and TCF7L2 in the nucleus was significantly decreased by CRNDE down-regulation and was rescued by TCF7L2 over-expression. Conclusions: The present study suggest that CRNDE involves in the cell proliferation, migration and invasion of colorectal cancer cells via increasing the expression of TCF7L2 and activity of Wnt/β-catenin signaling through binding miR-217 competitively.

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