Long Non-Coding RNA KCNQ1OT1 Promotes Cataractogenesis via miR-214 and Activation of the Caspase-1 Pathway

Background/Aims: KCNQ1OT1 regulates the expression of tissue-specific imprinted genes within the Kcnq1 domain. Imprinted genes are positive regulators of apoptosis, one of the forms of cell death related to cataract formation, and thus may provide novel therapeutic targets for cataract treatment. Here, we studied the role of non-coding RNAs(ncRNA) in cataract formation. Methods: Human lens epithelium cells (HLECs) were treated with H2O2, and the expression of KCNQ1OT1 and miR-214 was detected by qRT-PCR. The expression of caspase-1 was detected using qRT-PCR, western blot and immunostaining. To confirm our findings in cell cultures, we analysed KCNQ1OT1, miR-214, and caspase-1 expression in lens anterior capsules of both cataract patients and normal controls by qRT-PCR and western blot analysis. Results: We found that the expression of KCNQ1OT1 was increased in both human cataract lens anterior capsular samples and SRA01/04 cell lines treated with H2O2. Knockdown of KCNQ1OT1 expression significantly suppressed H2O2-induced SRA01/04 cell pyroptosis in vitro, which is the critical step in cataract formation. The expression of microRNA-214 (miR-214) was also decreased in cataract lens anterior capsular tissues and H2O2-induced SRA01/04 cell lines. Knockdown of KCNQ1OT1 significantly increased the expression of miR-214. Conclusions: We demonstrated for the first time that caspase-1 is a functional downstream target of miR-214, and knockdown of KCNQ1OT1 reduced the expression of caspase-1. These results provide evidence that the KCNQ1OT1-miR-214-caspase-1 regulatory network is a novel mechanism for promoting cataract formation.

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Resveratrol Exerts Antiproliferative Effect and Induces Apoptosis in Waldenstrom’s Macroglobulinemia.

Blood ◽

10.1182/blood.v110.11.1383.1383 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1383-1383

Author(s):

Aldo Roccaro ◽

Xavier Leleu ◽

Anne-Sophie Moreau ◽

Antonio Sacco ◽

Lian Xu ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Flow Cytometry ◽

Western Blot ◽

Cell Lines ◽

Expression Analysis ◽

Gene Expression Analysis ◽

Downstream Target ◽

Dose Dependent

Abstract Background: Resveratrol is a polyphenolic natural product, synthesized by a wide variety of plant species including grapes. It has gained considerable attention because of its anti-cancer properties, as demonstrated in solid and haematological malignancies. We therefore examined Resveratrol for its anti-tumor activity in Waldenstrom’s Macroglobulinemia (WM). Methods: We examined the effect of increasing concentrations of resveratrol (5–80 μM) on WM cell lines (BCWM.1), IgM secreting low-grade lymphoma cell lines (WM-WSU, MEC-1, RL), peripheral blood mononuclear cells (PBMCs) isolated from healthy donors, primary CD19+ WM cells and bone marrow stromal cells (BMSCs) isolated from bone marrow of patients with WM. [3H]-thymidine uptake and calcein-AM assay were used to evaluate the effect of resveratrol on proliferation and cytotoxicity respectively. Apoptosis and cell cycle analysis were investigated at 24h by flow cytometry using Annexin V-propidium iodide (PI) staining and PI-staining respectively. Apoptotic and cell signaling pathways targeted by resveratrol were investigated by Western Blot at 24 h and 6 h respectively. Since BMCSc confer growth and resistance to conventional treatments, we also tested the effect of resveratrol on WM cells co-cultured with BMSCs. Gene expression analysis has been performed on BCWM.1 cultured in presence or absence of resveratrol. Results: Resveratrol induced significant cytotoxicity and inhibition of DNA synthesis at 24 and 48 h on BCWM.1 with an IC50 of 10–20μM. Similar data was obtained with primary CD19+ WM cells. In contrast, resveratrol did not trigger significant reduction of proliferation of PBMCs. Resveratrol induced apoptosis in BCWM.1, as demonstrated by flow cytometry. Dose-dependent apoptosis at 24h with induction of JNK followed by caspases 3, 8, 9 and PARP cleavage was also observed. Resveratrol induced reduction of Mcl-1 and increase of p53, p63 and p73, as shown by gene expression analysis and western blot, providing an alternative mechanism of cell growth arrest in absence or mutation of p53. In parallel, resveratrol induced down-regulation of cyclin-D1, -D2, -E1, cdk-2, -4, -6 and up-regulation of p21Cip1 and p27Kip1, demonstrated in terms of transcript by gene expression analysis and protein levels by western blotting. We next observed that resveratrol inhibited ERK and Akt phosphorylation in BCWM.1 in a dose-dependent manner, as well as Akt activity, as shown by the in vitro Akt kinase assay. Phosphorylation of GSK3α/β and ribosomal protein-S6, downstream target proteins of Akt, were also markedly inhibited. Resveratrol also down-regulated Wnt signaling pathway with a reduction of nuclear β-catenin levels and a decrease of myc and survivin, both downstream target proteins of β-catenin. Lastly, adherence to BMSCs did not confer protection to WM cells against resveratrol-induced cytotoxicity Furthermore, resveratrol demonstrated synergistic cytotoxicity when combined with dexamethasone, fludarabine and bortezomib. Conclusion: These in vitro data demonstrated that resveratrol has significant antitumor activity in WM, providing the framework for clinical trials in WM patients.

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Increased Association of Deamidated αA-N101D with Lens Membrane of Transgenic αAN101D vs. Wild Type αA Mice: Potential Effects on Intracellular Ionic Imbalance and Membrane Disorganization

10.21203/rs.2.17769/v4 ◽

2020 ◽

Author(s):

Om Srivast ◽

Kiran Srivast ◽

Roy Joseph ◽

Landon Wilson

Keyword(s):

Epithelial Cells ◽

Western Blot ◽

Immunogold Labeling ◽

Human Lens ◽

Membrane Association ◽

Related Protein ◽

Wild Type ◽

Age Related ◽

Ionic Imbalance

Abstract We have generated two mouse models, in one by inserting the human lens αAN101D transgene in CRYαAN101D mice, and in the other by inserting human wild-type αA-transgene in CRYαAWT mice. The CRYαAN101D mice developed cortical cataract at about 7-months of age relative to CRYαAWT mice. The objective of the study was to determine the following relative changes in the lenses of CRYαAN101D- vs. CRYαAWT mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of αAN101D vs. WTαA proteins, and changes in intracellular ionic imbalance and membrane organization. Methods: Lenses of varying ages from CRYαAWT and CRYαAN101D mice were compared for an age-related protein insolubilization. The relative lens membrane-association of the αAN101D- and WTαA proteins in the two types of mice was determined by immunohistochemical-, immunogold-labeling-, and western blot analyses. The relative levels of membrane-binding of recombinant αAN101D- and WTαA proteins was determined by an in vitro assay, and the levels of intracellular Ca2+ uptake and Na, K-ATPase mRNA were determined in the cultured epithelial cells from lenses of the two types of mice.Results: Compared to the lenses of CRYαAWT, the lenses of CRYαAN101D mice exhibited: (A) An increase in age-related protein insolubilization beginning at about 4-months of age. (B) A greater lens membrane-association of αAN101D- relative to WTαA protein during immunogold-labeling- and western blot analyses, including relatively a greater membrane swelling in the CRYαAN101D lenses. (C) During in vitro assay, the greater levels of binding αAN101D- relative to WTαA protein to membranes was observed. (D) The 75% lower level of Na, K-ATPase mRNA but 1.5X greater Ca2+ uptake were observed in cultured lens epithelial cells of CRYαAN101D- than those of CRYαAWT mice. Conclusions: The results show that an increased lens membrane association of αAN101D--relative WTαA protein in CRYαAN101D mice than CRYαAWT mice occurs, which causes intracellular ionic imbalance, and in turn, membrane swelling that potentially leads to cortical opacity.

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RPNs Levels are Prognostic and Diagnostic Markers for Hepatocellular Carcinoma

10.21203/rs.3.rs-1071357/v1 ◽

2021 ◽

Author(s):

Wangyang zheng ◽

Yuling Zheng ◽

Xue Bai ◽

Yongxu Zhou ◽

Liang Yu ◽

...

Keyword(s):

Western Blot ◽

Cell Lines ◽

Mammalian Cells ◽

Tumor Grade ◽

Ubiquitin Proteasome System ◽

Functional Enrichment ◽

Migration And Invasion ◽

Regulatory Subunits ◽

Hcc Cells

Abstract Background: Ribophorin family (RPNs) are important regulatory subunits of the proteasome. By influencing Ubiquitin-proteasome system activity, RPNs are responsible for almost all processes of physiology and pathology of mammalian cells. Nevertheless, little is known about the role of RPNs in HCC.Methods: In this work, using the online databases Oncomine, UCSC, Kaplan-Meier Plotter, UALCAN, cBioPortal, TIMER2, GeneMANIA,and STRING, we first evaluated the expression, diagnostic, prognostic, genetic alteration, immunity, gene network, and functional enrichment of RPNs in HCC. QPCR and western blot were used to detect RPN6 and RPN9 expressions in HCC tissues and cell lines. Then we performed studies to eveulated their functions in HCC cells proliferation, migration, and invasion in vitro. Results: All RPNs were surprisingly consistently upregulated in HCC tissues. Moreover, RPNs expression pattern is correlated with HCC tumor grade. RPN2, RPN3, RPN6, RPN9, RPN10, RPN11, and RPN12 have robust values in HCC diagnose. Then, survival analysis revealed that high expression of RPN1, RPN2, RPN4, RPN5, RPN6, RPN9, and RPN11were correlated with unfavorable HCC overall survival. Functional enrichment for RPNs, indicated that RPNs have many potential biosynthesis activities expert for UPS functions. Western blot, and qRT-PCR further verified these results in HCC tissues and cell lines. The silencing of RPN6 and RPN9 significantly influenced HCC cells' proliferation, migration, and invasion in vitro.Conclusions: RPN families functions as an important oncogene in HCC. RPN6 and RPN9 have the potential to be potential biomarkers and targets for HCC.

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Identification of Prostaglandin F2 Receptor Negative Regulator (PTGFRN) as an internalizable target in cancer cells for antibody-drug conjugate development

PLoS ONE ◽

10.1371/journal.pone.0246197 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0246197

Author(s):

Jorge Marquez ◽

Jianping Dong ◽

Chun Dong ◽

Changsheng Tian ◽

Ginette Serrero

Keyword(s):

Flow Cytometry ◽

Western Blot ◽

Cell Lines ◽

Cancer Cell ◽

Cancer Cell Lines ◽

Negative Regulator ◽

Target Validation ◽

Antibody Drug

Antibody-drug conjugates (ADC) are effective antibody-based therapeutics for hematopoietic and lymphoid tumors. However, there is need to identify new targets for ADCs, particularly for solid tumors and cancers with unmet needs. From a hybridoma library developed against cancer cells, we selected the mouse monoclonal antibody 33B7, which was able to bind to, and internalize, cancer cell lines. This antibody was used for identification of the target by immunoprecipitation and mass spectrometric analysis, followed by target validation. After target validation, 33B7 binding and target positivity were tested by flow cytometry and western blot analysis in several cancer cell lines. The ability of 33B7 conjugated to saporin to inhibit in vitro proliferation of PTFRN positive cell lines was investigated, as well as the 33B7 ADC in vivo effect on tumor growth in athymic mice. All flow cytometry and in vitro internalization assays were analyzed for statistical significance using a Welsh’s T-test. Animal studies were analyzed using Two-Way Analysis of Variance (ANOVA) utilizing post-hoc Bonferroni analysis, and/or Mixed Effects analysis. The 33B7 cell surface target was identified as Prostaglandin F2 Receptor Negative Regulator (PTGFRN), a transmembrane protein in the Tetraspanin family. This target was confirmed by showing that PTGFRN-expressing cells bound and internalized 33B7, compared to PTGFRN negative cells. Cells able to bind 33B7 were PTGFRN-positive by Western blot analysis. In vitro treatment PTGFRN-positive cancer cell lines with the 33B7-saporin ADC inhibited their proliferation in a dose-dependent fashion. 33B7 conjugated to saporin was also able to block tumor growth in vivo in mouse xenografts when compared to a control ADC. These findings show that screening antibody libraries for internalizing antibodies in cancer cell lines is a good approach to identify new cancer targets for ADC development. These results suggest PTGFRN is a possible therapeutic target via antibody-based approach for certain cancers.

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MiR-27b Promotes Muscle Development by Inhibiting MDFI Expression

Cellular Physiology and Biochemistry ◽

10.1159/000489595 ◽

2018 ◽

Vol 46 (6) ◽

pp. 2271-2283 ◽

Cited By ~ 12

Author(s):

Lianjie Hou ◽

Jian Xu ◽

Yiren Jiao ◽

Huaqin Li ◽

Zhicheng Pan ◽

...

Keyword(s):

Skeletal Muscle ◽

Western Blot ◽

Satellite Cells ◽

Muscle Regeneration ◽

Muscle Injury ◽

Target Gene ◽

Muscle Development ◽

Muscle Satellite Cells ◽

Qrt Pcr

Background/Aims: Skeletal muscle plays an essential role in the body movement. However, injuries to the skeletal muscle are common. Lifelong maintenance of skeletal muscle function largely depends on preserving the regenerative capacity of muscle. Muscle satellite cells proliferation, differentiation, and myoblast fusion play an important role in muscle regeneration after injury. Therefore, understanding of the mechanisms associated with muscle development during muscle regeneration is essential for devising the alternative treatments for muscle injury in the future. Methods: Edu staining, qRT-PCR and western blot were used to evaluate the miR-27b effects on pig muscle satellite cells (PSCs) proliferation and differentiation in vitro. Then, we used bioinformatics analysis and dual-luciferase reporter assay to predict and confirm the miR-27b target gene. Finally, we elucidate the target gene function on muscle development in vitro and in vivo through Edu staining, qRT-PCR, western blot, H&E staining and morphological observation. Result: miR-27b inhibits PSCs proliferation and promotes PSCs differentiation. And the miR-27b target gene, MDFI, promotes PSCs proliferation and inhibits PSCs differentiation in vitro. Furthermore, interfering MDFI expression promotes mice muscle regeneration after injury. Conclusion: our results conclude that miR-27b promotes PSCs myogenesis by targeting MDFI. These results expand our understanding of muscle development mechanism in which miRNAs and genes work collaboratively in regulating skeletal muscle development. Furthermore, this finding has implications for obtaining the alternative treatments for patients with the muscle injury.

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CHEMOTHERAPEUTIC POTENTIAL OF NOVEL XANTHONE SOURCED FROM SWERTIA CHIRATA AGAINST SKIN CARCINOGENESIS

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2020.v13i12.39842 ◽

2020 ◽

pp. 84-88

Author(s):

ATISH BARUA ◽

PRITHA CHOUDHURY ◽

CHINMAY KUMAR PANDA ◽

PROSENJIT SAHA

Keyword(s):

Skin Cancer ◽

Antitumor Activity ◽

Cell Line ◽

Cell Lines ◽

Cancer Cell ◽

Cancer Cell Lines ◽

Self Renewal ◽

Qrt Pcr ◽

Swertia Chirata

Objective: Swertia chirata forms a rich source of bio-active compounds, among which xanthones form an important part. Among the xanthones present in it, 1,5,8 Tri-hydroxy-3-methoxy xanthone (TMX) was found to be the most active. The present study aims to evaluate the chemotherapeutic potential of it against metastatic skin cancer cell lines. Methods: In this study, the antitumor activity of TMX (the active component of chirata plant) was evaluated in A431, SKMEL-5, and A375 cell line by using in-vitro assays such as cell viability assay, cell cycle analysis, caspase 3 activity assay, intracellular reactive oxygen species (ROS) level determination by dichlorofluorescein diacetate, and quantitative real-time polymerase chain reaction (qRT-PCR). Results: In vitro studies showed that TMX from S. chirata exhibited significant antitumor activity by inducing apoptosis and restricting proliferation in both melanoma and non-melanoma skin cancer cell lines, but no such activity was seen in normal skin cancer cell line WS1. The qRT-PCR analysis revealed that in both the melanoma ad non-melanoma cell lines, TMX could exert its antitumor activity by downregulating c-Myc, cyclin-D1, and β-catenin and up-regulating Wnt antagonist gsk-3β, thereby suppressing wnt self-renewal pathway, but such regulation was absent in normal cell line. Conclusions: TMX from chirata could effectively inhibit the proliferation of metastatic skin cancer (both melanoma and non-melanoma) cell lines while being non-toxic to normal cell lines. The chemotherapeutic potential of TMX against metastatic skin cancer cell lines was achieved by downregulating several key regulatory genes enabling the suppression of the self-renewal pathway, the chief reason behind the invasiveness of cancer cells.

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Downregulation of the Ubiquitin-E3 Ligase RNF123 Promotes Upregulation of the NF-κB1 Target SerpinE1 in Aggressive Glioblastoma Tumors

Cancers ◽

10.3390/cancers12051081 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1081 ◽

Cited By ~ 3

Author(s):

Xiaowen Wang ◽

Matias Bustos ◽

Xiaoqing Zhang ◽

Romela Ramos ◽

Cong Tan ◽

...

Keyword(s):

Cell Lines ◽

Multivariable Analysis ◽

E3 Ligase ◽

Isocitrate Dehydrogenase 1 ◽

Poor Overall Survival ◽

Downstream Target ◽

Ubiquitin E3 Ligase ◽

Increased Risk ◽

Poor Outcomes

This study examined the role of the ubiquitin E3-ligase RNF123 in modulating downstream NF-κB1 targets in glioblastoma (GB) tumor progression. Our findings revealed an oncogenic pathway (miR-155-5p-RNF123-NF-κB1-p50-SerpinE1) that may represent a new therapeutic target pathway for GB patients with isocitrate dehydrogenase 1 and 2 (IDH) WT (wild type). Mechanistically, we demonstrated that RNF123 is downregulated in IDH WT GB patients and leads to the reduction of p50 levels. RNA-sequencing, reverse-phase protein arrays, and in vitro functional assays on IDH WT GB cell lines with RNF123 overexpression showed that SerpinE1 was a downstream target that is negatively regulated by RNF123. SERPINE1 knockdown reduced the proliferation and invasion of IDH WT GB cell lines. Both SerpinE1 and miR-155-5p overexpression negatively modulated RNF123 expression. In clinical translational analysis, RNF123, SerpinE1, and miR-155-5p were all associated with poor outcomes in GB patients. Multivariable analysis in IDH WT GB patients showed that concurrent low RNF123 and high SerpinE1 was an independent prognostic factor in predicting poor overall survival (p < 0.001, hazard ratio (HR) = 2.93, 95% confidence interval (CI) 1.7–5.05), and an increased risk of recurrence (p < 0.001, relative risk (RR) = 3.56, 95% CI 1.61–7.83).

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The Oral PKC-β Inhibitor Enzastaurin (LY317615) Suppress Phosphorylation and Induces Apoptosis in Multiple Myeloma Cell Lines by Inhibition of AKT Pathway.

Blood ◽

10.1182/blood.v108.11.1371.1371 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1371-1371

Author(s):

Antonino Neri ◽

Sandra Marmiroli ◽

Pierfrancesco Tassone ◽

Luigia Lombardi ◽

Lucia Nobili ◽

...

Keyword(s):

Multiple Myeloma ◽

Western Blot ◽

Cell Lines ◽

Molecular Mechanisms ◽

Caspase 3 ◽

Time Dependent ◽

Parp Cleavage ◽

Akt Phosphorylation ◽

Downstream Target ◽

Pkc Β

Abstract The PKC pathway has been shown to play a role in the regulation of cell proliferation in several hematologic malignancies. In this study we tested the oral PKC-β inhibitor, Enzastaurin (LY317615 - Eli Lilly) for its therapeutic efficacy in Multiple Myeloma (MM). We first analyzed PKC-β I and II expression by Western blot in a panel of 19 human MM cell lines, showing that 9 cell lines express either 1 or both isoforms. We next examined the growth inhibition effect of Enzastaurin in the same panel of MM cell lines using either WST-1 or MTT assay and cell viability assessment by Tripan Blue exclusion. Eighteen cell lines have IC50 value ranging from 1,2 μM to 12,5 μM. To examine molecular mechanisms whereby Enzastaurin induces cytotoxicity, we performed cell cycle profiling using PI and observed a significant increase of the percentage of cells in the sub G0–G1 fraction. To determine whether Enzastaurin-induced cell death is mediated by apoptosis, we studied by ELISA and Western blot caspase 3 and PARP cleavage. We observed induction of caspase 3 and PARP cleavage in a dose and time dependent fashion. Notably, the broad caspase (Z-VAD-FMK) inhibitor reduced Enzastaurin-induced cytotoxicity. We next determined whether Enzastaurin could inhibit AKT phosphorylation in MM cell lines with constitutive phosphorylation of AKT. Enzastaurin decreased AKT phosphorylation in a dose and time dependent fashion. Phosphorylation of GSK3β, a downstream target protein of AKT, was also markedly inhibited. Phosphorylation of PDK-1, a known upstream activator of AKT, was not affected by Enzastaurin. In conclusion, our results indicate that Enzastaurin-induced cytotoxicity is mediated via activation of caspase. This effect is associated with significant inhibition of AKT activity and its downstream target GSK3 β. Enzastaurin does not alter the phosphorylation of the upstream AKT activator PDK-1. These data suggest that Enzastaurin inhibit AKT signalling pathway and support its evaluation in a murine model of human MM.

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SGI-110, a Novel Hypomethylating Agent, Induces the WNT Inhibitor Secreted Frizzled Related Protein-2 (SFRP2), and Down Regulates β-Catenin in Acute Myeloid Leukemia (AML) Cells

Blood ◽

10.1182/blood.v120.21.1290.1290 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1290-1290

Author(s):

Michelle Golding ◽

Pragya Srivastava ◽

Golda Collamat ◽

Smitha R James ◽

Adam R. Karpf ◽

...

Keyword(s):

Western Blot ◽

Cell Lines ◽

Research Funding ◽

Nuclear Translocation ◽

Similarity Score ◽

Cellular Distribution ◽

U937 Cells ◽

Fold Reduction ◽

Wnt Inhibitor

Abstract Abstract 1290 Introduction: SGI-110 (Astex Pharmaceuticals, Inc.) is a dinucleotide hypomethylating agent whose active metabolite is decitabine (DAC). This drug demonstrates superior pharmacokinetics relative to the parent drug as a result of resistance to modification by cytidine deaminase, and is being investigated in myeloid malignancy in the phase I/II setting. We and others have demonstrated that WNT inhibitory genes including SFRP2 are epigenetically silenced in AML and that exposure to DNA methyltransferase inhibitors such as 5-Azacitidine (AZA) and DAC can re-express these genes and down-regulate β-catenin signaling in AML cell lines. We hypothesized that treatment with SGI-110 would have a similar effect upon the epigenetically silenced WNT inhibitor SFRP2 and further would down-regulate β-catenin signaling in AML cells in vitro. Methods: The AML cell lines HL60 and U937 were cultured in vitro using standard techniques and treated with phosphate buffered saline, 0.1, 1 or 5 μM SGI-110, 2μM AZA or 0.5μM DAC. Results presented are pooled data from a minimum of three biological replicates. Samples were harvested on day 5 and viable cells, DNA, RNA and protein obtained. β-catenin levels and cellular localization were quantified using imaging flow cytometry (ImageStream), DNA was extracted and bisulfite converted for analysis of gene specific and global DNA methylation by pyrosequencing (LINE-1, SFRP2), RNA was converted to cDNA for analysis by RT-PCR, and protein was obtained to confirm ImageStream results by Western blot. Nuclear translocation of β-catenin, indicative of its signaling activity, was assessed in individual cells by ImageStream using a similarity score: a log-transformed Pearson's correlation coefficient between the digitized images of immunostained β-catenin and a nuclear stain (DAPI). Shifts in the population (n=5,000) distributions of this similarity score were assessed by a resolution metric (Fishers discriminant ratio, Rd). Results: Treatment of AML cell lines with 5μM SGI-110 was toxic, and in line with previous experiments in AML cell lines, above the IC90. Treatment at the lowest dose of SGI-110 had minimal effects upon viability, methylation, and mRNA and protein expression in both cell lines tested. Treatment with SGI-110 at the 1μM dose resulted in reductions in LINE-1 methylation in HL60 cells by 21% (from 82% to 61%), compared to 8% with AZA (to 74%) and 20% with DAC (to 62%). In U937 cells, LINE-1 methylation decreased by 40% (from 67% to 27%) after SGI-110 treatment compared to a 25% reduction with AZA (to 42%) and a 30% reduction with DAC (to 36%). SFRP2 methylation in HL60 and U937 decreased from 86 and 88% at baseline to 66 and 60% with SGI-110 at the 1μM dose, compared to 68% with AZA and to 61% with DAC. Expression of SFRP2 mRNA was observed following treatment with 1μM SGI-110 and with DAC, but was limited following AZA treatment. ImageStream analysis of total cellular β-catenin in HL-60 and U937 cells demonstrated 2.4-fold and 1.2-fold reductions in total β-catenin following 1μM SGI-110 treatment. These results were similar to those seen with DAC (1.8-fold and 1.3-fold in HL-60 and U937 cells respectively). AZA treatment appeared to have a greater effect on total β-catenin in U937 cells (1.3-fold reduction) than in HL-60 cells (0.84-fold reduction). Western blot confirmed reductions in β-catenin protein. We also observed decreased nuclear translocation of β-catenin after treatment of HL-60 and U937 cells with 1 μM SGI-110 (Rd = −0.58 and −0.21 respectively; the negative sign indicates a change in cellular distribution from the nucleus to the cytoplasm). Changes were comparable to those observed with DAC (Rd = −0.75 and −0.26 in HL-60 and U937 cells respectively). AZA treatment of U937 cells resulted in a shift in cellular distribution (Rd = −0.20) similar to that for DAC and SGI-110 but had no effect on β-catenin distribution in HL-60 cells (Rd= 0.00). Conclusions: SGI-110 is a novel DNMT inhibitor which demonstrates robust effects on LINE-1 methylation, SFRP2 mRNA expression, and β-catenin level and localization consistent with epigenetically mediated re-expression of the WNT inhibitor SFRP2. Both upregulated β-catenin signaling and SFRP2 methylation have been demonstrated to correlate with inferior survival in patients with myeloid malignancies. Re-expression of epigenetically silenced WNT inhibitory genes such as SFRP2 may abrogate β-catenin signaling in AML cells. Disclosures: Karpf: Astex Pharmaceuticals: Research Funding. Griffiths:Celgene: Honoraria; Astex Pharmaceuticals: Research Funding.

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Capicua homology protein inhibits the progression of gastric cancer through the PI3K/AKT signaling pathway

10.21203/rs.3.rs-29464/v1 ◽

2020 ◽

Author(s):

LU GE ◽

Chang-long Hu ◽

Zheng-hui Ge ◽

Chun-rong Wang ◽

Li Qian ◽

...

Keyword(s):

Gastric Cancer ◽

Western Blot ◽

Signaling Pathway ◽

Cell Lines ◽

Epithelial Mesenchymal Transition ◽

Akt Signaling ◽

Akt Signaling Pathway ◽

Mesenchymal Transition ◽

Matrigel Invasion ◽

Qrt Pcr

Abstract Purpose Capicua homolog protein (CIC) played a broad role in the development of cancer in humans, however, its role in the progression of gastric cancer (GC) specifically has been unclear. This study aimed to explore the expression of CIC and its potential clinical value in patients with GC. Methods The CIC levels in GC tissues and cell lines were examined by quantitative real-time polymerase chain reaction (qRT-PCR). And the in-vitro effects of CIC expression in MGC-803 cells on their proliferation, invasion, and the progression of epithelial-mesenchymal transition were assessed by CCK-8 assays, Matrigel-invasion analysis, qRT-PCR and Western blot assays, separately. In addition, the effects of downregulation of CIC on the activation of PI3K/AKT signaling pathway were measured using Western-blot analysis. Results The results showed CIC levels were lower in GC tissues and GC cell lines, and these lower CIC levels were correlated with tumor differentiation, Helicobacter pylori infection, TNM stage, and patient survival. In addition, CIC overexpression could promote cell proliferation, invasion, and progression of epithelial-mesenchymal transition in MGC-803 cells. Notably, exotic expression of CIC inactivated the phosphoinositide 3-kinase/protein kinase B signaling pathway. Conclusions In conclusion, our finding suggested CIC could serve as a potential diagnostic and prognostic biomarker and a probable therapy target for GC.

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