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Double-stranded ribonucleic acid decreases C6 rat glioma cell numbers: effects on insulin-like growth factor I gene
1Department of Biochemistry, University of Texas Health Science Center, San Antonio 78229-3900, USA.
Abstract:
Poly(IC), a synthetic double-stranded RNA copolymer of inosinic and cytidilic acids, decreases the growth of normal and tumorigenic cells. We tested the hypothesis that Poly(IC) decreases C6 glioma cell growth by disrupting an autocrine insulin-like growth factor I (IGF-I) growth loop. Addition of Poly(IC) decreased C6 cell number in confluent and sparse cultures in a dose-dependent manner. Addition of exogenous IGF-I partially compensated for the decrease in cell number caused by Poly(IC) in confluent and subconfluent cultures of C6 cells, suggesting that one mechanism of Poly(IC) action is through down-regulation of IGF-I gene expression and/or action. Treatment of confluent C6 cells with 10 and 200 microg/ml Poly(IC) for 24 h decreased IGF-I messenger RNA (mRNA) levels to 50% and 25% of the control value, respectively. Treatment of C6 cells with 200 microg/ml Poly(IC) for 24 h reduced IGF-I receptor mRNA levels to 50% of the control level. IGF-binding protein-1 (IGFBP-1), -2, and -6 mRNAs were not expressed in the C6 cells used in this study. Treatment of C6 cells with 200 microg/ml Poly(IC) for 24 h reduced IGFBP-4 mRNA and IGFBP-5 mRNA levels to 26% and 29% of the control level, respectively. There was no significant change in IGFBP-3, insulin receptor, or actin mRNA levels with Poly(IC) treatment. Treatment of confluent C6 cells with 200 microg/ml Poly(IC) for 24 h decreased levels of immunoreactive IGF-I in conditioned medium (CM) to 55% of the control value, decreased IGF-I receptor beta-subunit levels to 28% of the control value, and decreased levels of IGFBP-3, IGFBP-4, and IGFBP-5 protein in CM to 45%, 50%, and 30% of the control values, respectively. There was no significant change in actin and tubulin protein levels with Poly(IC) treatment. These results suggest that IGF-I gene expression is down-regulated by Poly(IC) treatment and that IGF-I bioavailability and action in C6 cells are also altered due to decreases in IGF-I receptor and binding protein levels.
Insights
Poly(IC) reduces C6 glioma cell growth by disrupting the insulin-like growth factor I (IGF-I) pathway. This synthetic RNA down-regulates IGF-I gene expression and alters IGF-I bioavailability, impacting cell proliferation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Poly(IC), a synthetic double-stranded RNA, inhibits normal and tumor cell growth.
- C6 glioma cells rely on an autocrine insulin-like growth factor I (IGF-I) loop for growth.
- Understanding Poly(IC)'s mechanism in glioma is crucial for therapeutic development.
Purpose of the Study:
- To investigate if Poly(IC) inhibits C6 glioma cell growth by disrupting the IGF-I autocrine loop.
- To determine the effects of Poly(IC) on IGF-I gene expression, receptor levels, and binding protein expression in C6 cells.
Main Methods:
- C6 glioma cells were treated with varying concentrations of Poly(IC).
- Cell proliferation was assessed in sparse and confluent cultures.
- Quantitative real-time PCR was used to measure mRNA levels of IGF-I, IGF-I receptor, and IGF-binding proteins.
- ELISA and Western blotting were employed to quantify protein levels of IGF-I, IGF-I receptor subunits, and IGF-binding proteins in conditioned medium.
Main Results:
- Poly(IC) treatment decreased C6 cell number in a dose-dependent manner.
- Exogenous IGF-I partially rescued the Poly(IC)-induced decrease in cell number.
- Poly(IC) significantly reduced IGF-I, IGF-I receptor, IGFBP-4, and IGFBP-5 mRNA and protein levels.
- Levels of IGF-I, IGF-I receptor beta-subunit, and IGFBP-3, -4, -5 proteins in conditioned medium were decreased by Poly(IC) treatment.
Conclusions:
- Poly(IC) inhibits C6 glioma cell growth by down-regulating IGF-I gene expression.
- Poly(IC) treatment alters IGF-I bioavailability and action by reducing IGF-I receptor and IGF-binding protein levels.
- These findings elucidate a key mechanism by which Poly(IC) exerts its anti-proliferative effects on glioma cells.