Hammerhead ribozyme against gamma-glutamylcysteine synthetase sensitizes human colonic cancer cells to cisplatin by down-regulating both the glutathione synthesis and the expression of multidrug resistance proteins.

Abstract

Multidrug resistance in cancer cells is often associated with an elevation in the concentration of glutathione (GSH) and the expression of gamma-glutamylcysteine synthetase (gamma-GCS), a rate-limiting enzyme for GSH. We constructed a hammerhead ribozyme against a gamma-GCS heavy subunit (gamma-GCSh) mRNA transcript and transfected it to human colonic cancer cells (HCT8DDP) resistant to cisplatin (CDDP). The effect of the ribozyme transfection on the drug resistance of cancer cells was studied. (a) Transfection of the ribozyme decreased the GSH level and the efflux of CDDP-GSH adduct, resulting in higher sensitivity of the cells to CDDP. (b) The transfection suppressed the expression of ATP-binding cassette (ABC) family of transporters such as MRP1, MRP2, and MDR1, and stimulated the expression of mutant p53. (c) An electrophoretic mobility shift assay showed that mutant p53 suppresses the SP1-DNA binding activity, suggesting that this mutant p53 is functional and it, in turn, suppresses the expression of ABC transporters. Collectively, transfection of anti-gamma-GCSh ribozyme reduced the synthesis of GSH and the expression of ABC transporters, which causes an increase in the sensitivity of cancer cells to anticancer drugs. Suppression of the SP1-DNA binding activity by p53 may be a factor of down-regulation of ABC transporters.

• Authors: T Iida, H Kijima, Y Urata, S Goto, Y Ihara, M Oka, S Kohno, K J Scanlon, T Kondo
• Journal: Cancer gene therapy (Cancer Gene Ther) Vol. 8 Issue 10 Pg. 803-14 (Oct 2001) ISSN: 0929-1903 [Print] England
• PMID: 11687904 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antineoplastic Agents
• DNA Primers
• Multidrug Resistance-Associated Proteins
• RNA, Catalytic
• Tumor Suppressor Protein p53
• hammerhead ribozyme
• Glutamate-Cysteine Ligase
• Glutathione
• Cisplatin
• multidrug resistance-associated protein 1

Topics

• Antineoplastic Agents (therapeutic use)
• Blotting, Northern
• Blotting, Western
• Cisplatin (therapeutic use)
• Colonic Neoplasms (drug therapy, genetics, metabolism)
• DNA Primers (chemistry)
• Down-Regulation
• Drug Resistance, Multiple
• Drug Resistance, Neoplasm
• Electrophoretic Mobility Shift Assay
• Female
• Genetic Vectors
• Glutamate-Cysteine Ligase (genetics)
• Glutathione (metabolism)
• Humans
• Multidrug Resistance-Associated Proteins (metabolism)
• RNA, Catalytic (pharmacology)
• Reverse Transcriptase Polymerase Chain Reaction
• Tumor Cells, Cultured
• Tumor Suppressor Protein p53 (genetics)