Esperamicins, a class of potent antitumor antibiotics: mechanism of action.

The esperamicins represent a class of antitumor antibiotics characterized by an unusual chemical core structure and extremely potent cytotoxicity. The mechanism by which these drugs produce cytotoxicity was investigated and found to be related to the formation of single- and double-strand DNA breaks. Using five structurally related analogs, we defined a structure-activity relationship for cytotoxicity in various eukaryotic and DNA-repair-deficient prokaryotic cell lines, for DNA breakage in a human colon carcinoma cell line, and for DNA breakage in vitro in pBR322 DNA. Mild reducing agents such as dithiothreitol greatly increased the DNA breakage potency of these analogs in vitro. Results suggest that the pendant aromatic chromophore of esperamicin A1 may contribute to the uptake of the drug into cells but may also hinder double-strand DNA break formation. Little DNA breakage specificity was observed for the drug in a 139-base-pair fragment of pBR322 DNA. Evidence supports a previously proposed mechanism whereby esperamicins may produce the observed DNA breaks through reduction of the methyl trisulfide group to a thiolate anion followed by a Michael addition of the anion across the alpha,beta-unsaturated ketone. This addition may result in the saturation of the bridgehead double bond, thus allowing the two triple bonds to approach each other, causing cyclization of the diyn-ene to form a phenylene diradical. It is likely that this diradical is the active form of the drug responsible for single- and double-strand DNA breakage produced by this class of antitumor agents.

DNA breakage in human lung carcinoma cells and nuclei that are naturally sensitive or resistant to etoposide and teniposide.

Evidence suggests that the anticancer agents etoposide (VP16-213) and teniposide (VM26) produce DNA breaks and cytotoxicity by interaction with type II topoisomerase. Therefore, levels of type II topoisomerase may influence sensitivity to VP16-213 and VM26. We have characterized four lung carcinoma-derived cell lines for natural sensitivity or resistance to VP16-213 and VM26. Included in this study were two small cell lung carcinoma lines (SW900 and SW1271), an adenocarcinoma line (A549), and a large cell carcinoma (H157). SW1271 was the most sensitive line with a median inhibitory concentration for cell proliferation of 0.5 microM for VM26 and 2.7 microM for VP16-213, and SW900 was the most resistant with median inhibitory concentration values of 2.0 and 16 microM, respectively. A549 and H157 cells were intermediate in sensitivity to these drugs. Alkaline elution techniques were used to study in vivo formation and repair of single and double strand DNA breaks. Single strand DNA breaks were observed in SW1271 cells exposed to as little as 10 nM VM26 or 100 nM VP16-213 for 1 h, whereas SW900 cells required exposure to 10-fold higher concentrations of VM26 or VP16-213 to produce similar results. Single strand DNA breaks predominated only in SW1271 and A549 cells and then, only at low drug concentrations, whereas the ratios between single and double strand DNA breaks decreased at higher drug concentrations. Plots of cytotoxicity versus single and double strand DNA breakage revealed that cytotoxicity produced by both drugs was more closely related to double strand DNA break formation in all four cell lines. DNA breaks appeared rapidly upon addition of drug, reaching plateaus in DNA breaks within 30 min, and repair of both single and double strand DNA breaks occurred rapidly with time to repair one-half of the DNA breaks of 20 to 60 min in all four cell lines upon removal of drug, arguing against repair as a mechanism for drug resistance. DNA breakage was also observed in nuclei isolated from SW900 and SW1271 cells in similar magnitude to that observed in the respective cells. Results indicate that DNA breakage plateaus may reflect a steady-state equilibrium established between the drug and its nuclear target, possibly type II topoisomerase, and suggest that natural resistance to VP16-213 and VM26 may be due to different enzyme levels in sensitive and naturally resistant cells.

Single- and double-strand DNA breakage and repair in human lung adenocarcinoma cells exposed to etoposide and teniposide.

The anticancer agents 4'-demethylepipodophyllotoxin-4-(4,6-O-ethylidene-beta-D-glucopyra noside (etoposide) (VP16-213) and 4'-demethylepipodophyllotoxin-4-(4,6-O-thenylidene-beta-D-gl ucopyranoside (teniposide) (VM26) produce cytotoxicity by inhibiting type II topoisomerase, resulting in an accumulation of DNA breaks. By using alkaline elution techniques to assess in vivo DNA break frequencies, we have been able to follow formation and repair of both single- and double-strand DNA breaks induced by the exposure of A549 human lung adenocarcinoma cells to VP16-213 and VM26. Single-strand DNA breaks are detectable in cells within 2 min of drug exposure, increase in frequency to a maximum after as little as 15 min of exposure, and remain near maximum levels. Double-strand breaks accumulate more slowly, reaching a maximum after 1 to 2 h, and remaining constant thereafter upon continuous exposure to drug. Single-strand DNA breaks predominate at early incubation times and low drug concentrations, whereas the ratios between single- and double-strand DNA breaks decrease at higher drug concentrations. Changing to drug-free medium after 1-h drug exposure results in rapid exponential repair of both single- and double-strand DNA breaks with a time required for repair of one-half of the DNA breaks of 20 to 60 min. VM26 and VP16-213 have similar kinetics for DNA break formation and repair and similar relationships between DNA breakage and cytotoxicity, but VM26 is five to ten times more potent than VP16-213. Results indicate that DNA breakage plateaus may reflect a steady state equilibrium established between the drug and its nuclear target, possibly type II topoisomerase, and demonstrate unique properties of VP16-213- and VM26-induced DNA breakage.

Comparison of cytotoxicity and DNA breakage activity of congeners of podophyllotoxin including VP16-213 and VM26: a quantitative structure-activity relationship.

Fourteen congeners of podophyllotoxin were evaluated for their abilities to induce DNA breakage and inhibit growth of A549 human lung adenocarcinoma cells. Among the congeners studied were VP16-213, VM26, alpha-peltatin, beta-peltatin, and picropodophyllotoxin. Alkaline elution methods were used to assess DNA break frequencies following 1-h exposure to different concentrations of the congeners. DNA breakage was dependent upon drug concentration and was detectable when cells were exposed for 1 h to concentrations of VM26 as low as 0.05 microM. DNA breaks formed rapidly in cells after addition of drug but increased little after 30 min of continuous exposure. Repair of drug-induced DNA breaks was equally rapid with repair of 90% of the breaks occurring within 1 h following removal of the drug. Relationships between the structures of the congeners and the resulting DNA breakage activities were obtained, which correlated well with the cytotoxicity. The data suggest that a free hydroxyl group at the 4'-position is essential for DNA breakage activity, epimerization at the 4-position of the podophyllotoxin rings enhances activity, glucosylation of the hydroxyl group at the 4-position diminishes activity, aldehyde condensation with the glucose moiety greatly enhances activity, and the structure of the group associated with the resulting acetal linkage influences DNA breakage activity. These studies present quantitative data supporting and expanding upon the structure-activity relationship first proposed by Loike and Horwitz [Loike, J. D., & Horwitz, S. B. (1976) Biochemistry 15, 5443-5448].