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Inhibition of type II topoisomerase by fostriecin
T J Boritzki1, T S Wolfard, J A Besserer
1Parke-Davis Pharmaceutical Research Division, Warner-Lambert Co., Ann Arbor, MI 48105.
Abstract:
Fostriecin is a new antitumor antibiotic which is being developed further as an anticancer agent based on its marked activity in murine leukemias. Its mechanism of action, however, has thus far remained unknown. The present study demonstrates that fostriecin inhibits the catalytic activity of partially purified type II topoisomerase from Ehrlich ascites carcinoma. Under the experimental conditions employed, fostriecin completely inhibited the enzyme at 100 microM. A general kinetic analysis showed that fostriecin inhibited topoisomerase in an uncompetitive manner with a Ki,app of 110 microM and produced kinetics that were distinctly different from those of VM-26 which exhibited noncompetitive inhibition. Fostriecin did not cause DNA strand breaks in L1210 cells, suggesting that it did not stabilize a cleavable complex as do other known inhibitors of this enzyme. Fostriecin, however, did partially inhibit DNA strand breaks produced by amsacrine. An analysis by flow cytometry showed that L1210 cells exposed to 5 microM fostriecin for 12 hr caused a block in the G2 phase of the cell cycle. These studies thus suggest that the mechanism by which fostriecin produces its antitumor effects may be through inhibition of topoisomerase II and that the type of inhibition is markedly different from existing antitumor agents which inhibit this enzyme.
Insights
Fostriecin, an antitumor antibiotic, inhibits type II topoisomerase, an enzyme crucial for cancer cell replication. This novel uncompetitive inhibition mechanism differs from existing agents, offering a new avenue for cancer therapy.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Fostriecin exhibits significant antitumor activity, particularly in murine leukemias.
- The precise mechanism of action for fostriecin has remained undetermined.
- Understanding fostriecin's mechanism is crucial for its development as an anticancer agent.
Purpose of the Study:
- To elucidate the mechanism of action of fostriecin.
- To investigate fostriecin's effect on type II topoisomerase activity.
- To compare fostriecin's inhibitory kinetics with known topoisomerase inhibitors.
Main Methods:
- Enzyme inhibition assays using partially purified type II topoisomerase from Ehrlich ascites carcinoma.
- Kinetic analysis to determine the type of inhibition and inhibition constants.
- DNA strand break assays in L1210 cells.
- Flow cytometry to analyze cell cycle progression.
Main Results:
- Fostriecin demonstrated uncompetitive inhibition of type II topoisomerase with a Ki,app of 110 microM.
- Unlike VM-26, fostriecin's inhibition kinetics were distinct.
- Fostriecin did not induce DNA strand breaks but partially inhibited amsacrine-induced breaks.
- Fostriecin caused a G2 phase cell cycle block in L1210 cells.
Conclusions:
- Fostriecin likely exerts its antitumor effects by inhibiting type II topoisomerase.
- The uncompetitive inhibition mechanism of fostriecin is novel and differs from existing topoisomerase inhibitors.
- This unique mechanism may offer new therapeutic strategies for cancer treatment.