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Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions
Published on: December 20, 2017
β-Catenin knockdown in liver tumor cells by a cell permeable gamma guanidine-based peptide nucleic acid
Evan Delgado1, Raman Bahal, Jing Yang
1Endowed Chair for Experimental Pathology, Director- Division of Experimental Pathology (EP), Professor of Pathology (EP) & Medicine (GI, Hepatology and Nutrition), University of Pittsburgh School of Medicine, 200 Lothrop Street S-422 BST, Pittsburgh, PA 15261, USA. smonga@pitt.edu.
Abstract:
Hepatocellular cancer (HCC) is the third cause of death by cancer worldwide. In the current study we target β- catenin, an oncogene mutated and constitutively active in 20-30% of HCCs, via a novel, cell permeable gamma guanidine-based peptide nucleic acid (γGPNA) antisense oligonucleotide designed against either the transcription or the translation start site of the human β-catenin gene. Using TOPflash, a luciferase reporter assay, we show that γGPNA targeting the transcription start site showed more robust activity against β-catenin activity in liver tumor cells that harbor β-catenin gene mutations (HepG2 & Snu-449). We identified concomitant suppression of β-catenin expression and of various Wnt targets including glutamine synthetase (GS) and cyclin-D1. Concurrently, γGPNA treatment reduced proliferation, survival and viability of HCC cells. Intriguingly, an angiogenesis quantitative Real-Time-PCR array identified decreased expression of several pro-angiogenic secreted factors such as EphrinA1, FGF-2, and VEGF-A upon β-catenin inhibition in liver tumor cells. Conversely, transfection of stabilized-β-catenin mutants enhanced the expression of angiogenic factors like VEGF-A. Conditioned media from HepG2 cells treated with β-catenin but not the mismatch γGPNA significantly diminished spheroid and tubule formation by SK-Hep1 cells, an HCC-associated endothelial cell line. Thus, we report a novel class of cell permeable and efficacious γGPNAs that effectively targets β-catenin, a known oncogene in the liver. Our study also identifies a novel role of β-catenin in liver tumor angiogenesis through paracrine mechanisms in addition to its roles in proliferation, survival, metabolism and cancer stem cell biology, thus further strengthening its effectiveness as a therapeutic target in HCC.
Insights
This study introduces novel gamma guanidine-based peptide nucleic acid (γGPNA) molecules that effectively inhibit β-catenin activity in liver cancer cells. These γGPNAs reduce tumor cell proliferation and angiogenesis, highlighting a new therapeutic strategy for hepatocellular cancer (HCC).
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Hepatocellular cancer (HCC) is a leading cause of cancer-related death globally.
- β-catenin is a key oncogene frequently mutated in HCC, driving tumor growth and progression.
- Targeting oncogenic pathways like Wnt/β-catenin offers a promising therapeutic avenue for HCC.
Purpose of the Study:
- To develop and evaluate a novel, cell-permeable gamma guanidine-based peptide nucleic acid (γGPNA) antisense oligonucleotide targeting the human β-catenin gene.
- To assess the efficacy of γGPNA in inhibiting β-catenin activity and its downstream targets in HCC cells.
- To investigate the role of β-catenin inhibition in HCC cell proliferation, survival, and angiogenesis.
Main Methods:
- Design of γGPNA antisense oligonucleotides targeting the transcription or translation start sites of the β-catenin gene.
- Utilized TOPflash luciferase reporter assay to measure β-catenin transcriptional activity.
- Employed quantitative Real-Time-PCR arrays to analyze gene expression related to angiogenesis and Wnt targets.
- Assessed HCC cell proliferation, survival, and viability.
- Evaluated the impact of conditioned media on endothelial cell tube and spheroid formation.
Main Results:
- γGPNA targeting the transcription start site demonstrated potent inhibition of β-catenin activity in HCC cell lines (HepG2, Snu-449).
- Concomitant suppression of β-catenin, glutamine synthetase (GS), and cyclin-D1 expression was observed.
- γGPNA treatment significantly reduced HCC cell proliferation, survival, and viability.
- Inhibition of β-catenin led to decreased expression of pro-angiogenic factors (EphrinA1, FGF-2, VEGF-A).
- Conditioned media from treated HCC cells impaired angiogenesis in an HCC-associated endothelial cell line.
Conclusions:
- A novel class of cell-permeable and effective γGPNAs has been developed for targeting β-catenin in liver cancer.
- β-catenin inhibition by γGPNA reduces HCC cell proliferation and survival.
- β-catenin plays a significant role in liver tumor angiogenesis via paracrine mechanisms, in addition to its established roles.
- These findings strengthen the rationale for targeting β-catenin as a therapeutic strategy in HCC.
