Protection of platinum–DNA adduct formation and reversal of cisplatin resistance by anti‐MRP2 hammerhead ribozymes in human cancer cells

Abstract

Resistance to platinum-containing antineoplastic drugs is the major limitation in their clinical use. To elucidate the role of the ABC transporter MRP2 in platinum drug resistance, its expression was analyzed in human cisplatin-resistant cell lines: the ovarian carcinoma line A2780RCIS, the adrenocortical carcinoma line D43/86RCIS and the melanoma line MeWoCIS1. All these cells showed overexpression of MRP2. For reversal of platinum resistance, 2 anti-MRP2 hammerhead ribozymes were introduced into A2780RCIS cells. Both ribozymes showed gene-silencing activities and reversed the drug-resistant phenotype. Moreover, formation of platinum-induced intrastrand cross-links was measured in DNA. The level of DNA platination corresponded inversely to the level of MRP2 expression and was accompanied by increased caspase-3-dependent apoptosis. Kinetics of formation and elimination of platinum-DNA adducts suggest that the DNA repair capacity was not altered; the decrease in platinum-DNA adduct formation was rather a reflection of the protecting activity of MRP2. In conclusion, functional inhibition of MRP2 might be a promising strategy in the reversal of resistance to platinum-based anticancer drugs. This was reflected by the specific inhibition of MRP2 by ribozyme technology, indicating that this gene therapeutic approach may be applicable as a specific means to overcome platinum resistance in human neoplasms. ' 2005 Wiley-Liss, Inc.Key words: ABC transporter; ribozyme; drug resistance; cisplatin; DNA adduct Cisplatin [cis-diamminedichloroplatinum (II)] is one of the most active and widely used anticancer drugs. It has proven efficacy against various cancers, including ovarian carcinoma, 1 adrenocortical carcinoma 2 and malignant melanoma. 3 The cisplatin analog carboplatin [1,1-cyclobutanedicarboxylate platinum (II)] has also been used increasingly in the last 2 decades. Therapeutic use of these platinum-containing compounds is limited by their dose-dependent side effects, including nephrotoxicity, myelosuppression, neurologic damage and strong nausea. Furthermore, the antineoplastic efficiency of cisplatin and its analogs is often unsatisfactory due to primary nonresponsiveness or the development of a secondary, acquired drug resistance of the platinum-treated neoplasm. Acquired drug resistance of tumor cells occurs rapidly both in vitro and in vivo and is the major reason for failure of platinumbased therapy. 4 After the entry of the neutral square-planar platinum (II) complex cisplatin into the cell, binding of the 2 chloride ligands is destabilized. One of the chlorides is displaced by a water molecule. As a consequence, the drug becomes a monoaquated, charged electrophile that can react with nucleophilic sites of intracellular macromolecules such as DNA. The second reactive site of cisplatin can lead, with lower kinetics, to the formation of several types of bifunctional platinum adduct with DNA bases. Although only about 1% of intracellular cisplatin reacts with nuclear DNA, this is presumably the critical event in ...

Multidrug resistance-associated protein 2 determines the efficacy of cisplatin in patients with hepatocellular carcinoma

“…Of several identified ABC transporters, MRP2 is the principal cisplatin transporter (7)(8)(9). There is a paucity of clinical data regarding any relationship between MRP2 expression and tumor necrosis in patients treated with cisplatin-based neoadjuvant chemotherapy prior to hepatectomy for HCC.…”

“…The intrinsic toxicity of MRP2 inhibitors at doses necessary for their activity and their poor specificity are the major obstacles in applying them in vivo (8). In attempt to develop alternative, less toxic, and more efficient therapy, Meterna et al specifically inhibited MRP2 protein expression by 2 anti-MRP2 hammerhead ribozymes.…”

“…In attempt to develop alternative, less toxic, and more efficient therapy, Meterna et al specifically inhibited MRP2 protein expression by 2 anti-MRP2 hammerhead ribozymes. This gene therapeutic approach may be applicable to overcome cisplatin resistance in tumor cells (8). Folmer et al demonstrated that tumors resulting from MRP2-overexpressing subcutaneously grown hepatoma cells, regressed in size upon antisense MRP2 expression in combination with vincristine (28).…”

“…MRP2 is one of the major transporters of cisplatin (7)(8)(9). In vitro experiments have shown that elevated expression of MRP2 decreases cisplatin accumulation in HCC cells and contributes to cisplatin resistance (9).…”

Reversal of drug resistance of hepatocellular carcinoma cells by adenoviral delivery of anti-ABCC2 antisense constructs

“…18 Reversal of cisplatin resistance by anti-ABCC2 hammerhead ribozymes was demonstrated in human cancer cells. 19 The expression of ABCC2 was specifically reduced by antisense DNA expressed from a CMV expression A 'multitarget multiribozyme' constructed that consisted of three hammerhead motifs directed against transcripts of ABCB1, BCRP and ABCC2 specifically reduced the corresponding transporter ABCB1, BCRP and ABCC2 in all cellular systems. 21 Interestingly, it was shown that bile acid-modified oligodeoxynucleotides are secreted via ABCC2 into bile.…”