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C-Met antisense oligodeoxynucleotide inhibits growth of glioma cells
Shenghua Chu1, Xianhou Yuan, Zhiqiang Li
1Department of Neurosurgery, Zhongnan Hospital of Wuhan University, Wuhan 430071, China.
Background:
C-Met, a receptor tyrosine kinase, and its ligand, hepatocyte growth factor, are critical in cellular proliferation, motility, and invasion and are known to be overexpressed in gliomas. The aim of our study was therefore to investigate the effect of transfected caroboxyfluorescein-5-succimidyl ester (FAM)-labeled c-Met antisense oligonucleotide (ASODN) on growth of glioma cells.
Methods:
Conjugated FAM-labeled c-Met ASODN was encapsulated by LIPOFECTAMINE PLUS Reagent and then added into the human glioma cell line U251. Cultured cells were divided into 5 groups: control group, 500 nmol/L nonsense oligonucleotide (NSODN) group, 250 nmol/L ASODN group, 500 nmol/L ASODN group, and 750 nmol/L ASODN group. The intracellular distribution of c-Met ASODN was observed with fluorescence microscopy; cell growth was detected by methyl thiazole tetrazolium assay. The apoptosis of U251 cells was also examined with a flow cytometer. Semiquantitative reverse transcriptase polymerase chain reaction and Western blot examinations were carried for expression of c-Met messenger RNA (mRNA) and protein.
Results:
The blue fluorescence was seen in the cytoplast and nuclei of cells of FAM-labeled c-Met ASODN groups with fluorescence microscopy after the cells were treated with FAM-labeled c-Met ASODN-LIPOFECTAMINE PLUS Reagent complex for 3 hours. Antisense (AS) oligonucleotide caused a statistically significant reduction of cell viability (P < .05), whereas NSODN had no such changes. The cell growth was also significantly inhibited by ASODN (P < .05). After transfection, 250, 500, and 750 nmol/L ASODN induced significant apoptotic response, about 4.67% +/- 2.86%, 8.65% +/- 3.18%, and 12.76% +/- 3.15% for 24 hours (P < .05) and 7.79% +/- 1.92%, 11.43% +/- 1.54%, and 15.78% +/- 1.86% for 48 hours (P < .01), respectively. However, 500 nmol/L NSODN did not induce any significant apoptotic response until 48 hours after transfection (P > .05). A significant loss of c-Met mRNA was presented in ASODN-treated cells, and this was not seen in treatment with NSODN. Protein level was significantly decreased 48 hours after c-Met ASODN transfected.
Conclusions:
Antisense oligonucleotide targeting c-Met can be identified as a most potent AS compound, which can inhibit cell growth and induce cell apoptosis. This provides evidence that c-Met plays a role in tumor progression of glioma by acting as an oncogene and suggests that c-Met ASODN may provide a novel approach to therapy for human glioma.
Insights
C-Met antisense oligonucleotide (ASODN) effectively inhibits human glioma cell growth and induces apoptosis. This study suggests c-Met ASODN as a potential novel therapeutic strategy for glioma treatment.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- C-Met receptor tyrosine kinase and hepatocyte growth factor are overexpressed in gliomas, driving cellular proliferation, motility, and invasion.
- Investigating targeted therapies is crucial for improving glioma treatment outcomes.
Purpose of the Study:
- To evaluate the efficacy of carboxyfluorescein-5-succimidyl ester (FAM)-labeled c-Met antisense oligonucleotide (ASODN) in inhibiting glioma cell growth.
- To determine the impact of c-Met ASODN on glioma cell apoptosis and c-Met expression.
Main Methods:
- Human glioma U251 cells were treated with varying concentrations of FAM-labeled c-Met ASODN and a nonsense oligonucleotide (NSODN).
- Cell viability and growth were assessed using MTT assays, apoptosis by flow cytometry, and c-Met mRNA and protein levels by RT-PCR and Western blot.
Main Results:
- FAM-labeled c-Met ASODN demonstrated significant inhibition of U251 cell viability and growth.
- ASODN treatment led to a dose-dependent increase in apoptosis and a significant reduction in c-Met mRNA and protein expression.
- Nonsense oligonucleotide (NSODN) did not exhibit significant effects on cell viability or apoptosis.
Conclusions:
- Targeting c-Met with antisense oligonucleotide is a potent strategy for inhibiting glioma cell growth and inducing apoptosis.
- c-Met plays a significant role in glioma progression, acting as an oncogene.
- c-Met ASODN represents a promising novel therapeutic approach for human glioma.
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