Novel approaches to reversing anti-cancer drug resistance using gene-specific therapeutics.
One of the underlying mechanisms of multidrug resistance (MDR) is cellular overproduction of P-glycoprotein (P-gp), which acts as an efflux pump for various anti-cancer drugs. P-gp is encoded by a group of related genes termed MDR; only MDR1 is known to confer the drug resistance, and its overexpression in cancer cells has been a therapeutic target to circumvent the resistance. To overcome P-gp-mediated drug resistance, we have developed six anti-MDR1 hammerhead ribozymes and delivered them to P-gp-overproducing human leukemia cell line by a retroviral vector containing RNA polymerase III promoter. These ribozyme-transduced cells became vincristine-sensitive, concomitant with the decreases in MDR1 expression, P-gp amount and efflux pump function. Among the ribozymes tested, the anti-MDR1 ribozyme against the translation-initiation site exhibited the highest efficacy. The retrovirus-mediated transfer of this most potent anti-MDR1 ribozyme into a human lymphoma cell line, which was made resistant by infection of pHaMDR1/A retroviral vector and thus possessed a low degree of MDR due to P-gp expression relevant to clinical MDR, resulted in a complete reversal of MDR phenotype. In addition to retrovirus-mediated transfer of ribozymes, we evaluated the efficacy of cationic liposome-mediated transfer of ribozyme. Treatment of a P-gp-producing human breast cancer cell line with the liposome-ribozyme complex resulted in reversal of resistance, concomitant with the decreases in both MDR1 expression and P-gp amount. Confocal microscopic imaging of the cells after treatment with liposome/FITC-dextran showed cytoplasmic fluorescence that was abolished by cytochalasin B, indicating a high endocytotic activity in these cells. The endocytotic activity was well correlated with the success of cationic liposome-mediated transfer of MDR1 ribozyme. These distinct approaches using either retrovirus- or liposome-mediated transfer of anti-MDR1 ribozyme may be selectively applicable to the treatment of MDR cells with different properties such as endocytotic activity as a specific means to reverse resistance.
Kobayashi
H
H
Department of Pharmacology, Tokai University School of Medicine. hkobayas@is.icc.u-tokai.ac.jp
Takemura
Y
Y
Miyachi
H
H
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000502:physiology; D000818:Animals; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D018014:Gene Transfer Techniques; D019450:Genes, MDR; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D008081:Liposomes; D009369:Neoplasms / Q000235:genetics / Q000628:therapy; D016337:RNA, Catalytic / Q000235:genetics; D012190:Retroviridae; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008081:Liposomes; D016337:RNA, Catalytic; C427204:hammerhead ribozyme