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Antiproliferative effect of c-myc antisense phosphorothioate oligodeoxynucleotides in malignant glioma cells
W C Broaddus1, Z J Chen, S S Prabhu
1Division of Neurosurgery, Medical College of Virginia/Virginia Commonwealth University, Richmond, USA.
Objective:
To improve the prognosis for primary malignant tumors of the central nervous system, new therapeutic strategies are needed. Antisense oligodeoxynucleotides (ODNs) offer the potential to block the expression of specific genes within cells. The proto-oncogene c-myc has long been implicated in the control of normal cell growth and its deregulation in the development of neoplasia. We therefore reasoned that a strategy using ODNs complementary to c-myc messenger ribonucleic acid would be a potent inhibitor of glioma cell proliferation.
Methods:
A variety of antisense, sense, and scrambled (15-mer) phosphorothioate ODNs targeted to rat and human c-myc messenger ribonucleic acid were synthesized and added to the media of cultured RT-2 cells (a rat glioblastoma cell line). Cell growth was assessed by 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide dye assay 1 to 5 days after adding the ODNs. c-Myc protein expression was analyzed by Western blot analysis. The stability of the ODNs was confirmed by gel electrophoresis.
Results:
Compared with cultures containing standard media, two of three antisense ODNs significantly inhibited the growth of glioma cells, whereas sense and scrambled sequence ODNs did not significantly affect cell growth at the concentrations tested. A human c-myc antisense sequence, which differed from the rat sequence by one base substitution, also had an inhibitory effect on RT-2 cells. Western blot analysis demonstrated that expression of immunoreactive c-Myc protein was also greatly reduced in the rat antisense ODN-treated cells (and not in sense-, scrambled-, or control-treated cells). The degree of reduction of c-Myc protein expression correlated well with the decrease in cell growth observed with several antisense ODNs. Phosphorothioate ODNs were stable in cell culture media for at least 5 days.
Conclusion:
These results suggest that c-Myc plays a critical role in glioma cell proliferation and demonstrate that antisense ODNs can suppress proto-oncogene expression and inhibit the proliferation of glioma cells. Our results indicate that the antiproliferative activity of these ODNs was mediated predominantly through sequence-specific antisense mechanisms, but that sequence-specific nonantisense effects may also contribute to the strongest effects demonstrated. These findings support a potential role for antisense strategies designed to inhibit c-myc expression in the treatment of malignant gliomas.
Insights
Antisense oligodeoxynucleotides (ODNs) targeting the c-myc gene significantly inhibited glioma cell growth and reduced c-Myc protein expression. These findings support antisense ODN therapy for malignant gliomas.
Area of Science:
- Neuro-oncology
- Molecular Biology
- Gene Therapy
Background:
- Malignant gliomas require novel therapeutic strategies.
- Antisense oligodeoxynucleotides (ODNs) can inhibit gene expression.
- The proto-oncogene c-myc is implicated in glioma development.
Purpose of the Study:
- To investigate the efficacy of antisense ODNs targeting c-myc messenger RNA (mRNA) in inhibiting glioma cell proliferation.
- To assess the impact of antisense ODNs on c-Myc protein expression in glioma cells.
Main Methods:
- Synthesized antisense, sense, and scrambled phosphorothioate ODNs targeting c-myc mRNA.
- Treated cultured rat glioblastoma cells (RT-2) with ODNs.
- Assessed cell growth using a dye assay and c-Myc protein levels via Western blot.
Main Results:
- Two of three antisense ODNs significantly inhibited glioma cell growth.
- Antisense ODNs reduced c-Myc protein expression in a dose-dependent manner.
- Sense and scrambled ODNs showed no significant effect on cell growth or protein expression.
Conclusions:
- c-Myc plays a crucial role in glioma cell proliferation.
- Antisense ODNs effectively suppress c-myc expression and inhibit glioma cell growth.
- Antisense strategies targeting c-myc show potential for treating malignant gliomas.