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

Multidrug resistance in cancer cells is often associated with an elevation in the concentration of glutathione (GSH) and the expression of gamma -glut amylcysteine synthetase (gamma -GCS), a rate-limiting enzyme for GSH. We co nstructed a hammerhead ribozyme against a- gamma -GCS heavy subunit (gamma -GCSh) mRNA transcript and transfected it to human colonic cancer cells (HC T8DDP) resistant to cisplatin (CDDP). The effect of the ribozyme transfecti on 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, res ulting in higher sensitivity of the cells to CDDP. (b) The transfection sup pressed 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 suppres ses the SP1-DNA binding activity, suggesting that this mutant p53 is functi onal and it, in turn, suppresses the expression of ABC transporters. Collec tively, 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-DN A binding activity by p53 may be a factor of down-regulation of ABC transpo rters.