DNA Cross-linking and Cytotoxicity of the Alkylating Cyanomorpholino Derivative of Doxorubicin in Multidrug-Resistant Cells2

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Abstract
The cyanomorpholino derivative of doxorubicin (MRA-CN) is an anthracycline that is extremely potent and non-cross-resistant with doxorubicin (DOX) in multidrug-resistant cells. MRA-CN binds to and cross-links DNA and thus has been proposed to act as a targeted alkylating agent. In our study, the number of DNA interstrand and DNA-protein Cross-links produced by MRA-CN was identical in multidrug-resistant Dx5 and parental MES-SA Cells, as shown by alkaline elution analysis. The amount of cross-linking was directly proportional to drug concentration at concentrations from 10−11 to 10−7M MRA-CN. Extensive DNA cross-linking was evident within 30 minutes of drug exposure. After 1 hour of drug exposure, the number of DNA cros-links increased for 90 minutes, reached a plateau, and then began to decrease after 120 minutes. Loss of cell viability was also observed as early as 3 hours after exposure to MRA-CN. The finding of the same number of DNA cross-links in MES-SA and Dx5 cells indicates that similar amounts of MRA-CN are likely to enter the nuclei of multidrug-resistant and sensitive cells. Other anthracyclines have major differences in nuclear dis-tribution in sensitive and resistant cells. Several factors may contribute to the non-cross-resistance of MRA-CN in multidrug-resistant cells, (a) The lipophilicity of MRA-CN facilities cell entry. (b) The substitution and loss of basicity at the amino nitrogen may reduce the affinity of the drug for the P-glycoprotein efflux pump, compared with that of DOX. (c) The detoxification function of P-glycoprotein may be less effective for drugs that produce rapid and irreversible cell damage, such as the DNA-targeted alkylation caused by MRA-CN.