Since the discovery of cis
-platinum, many transition metal complexes have been synthesized and assayed for antineoplastic activity. In recent years, vanadium-based molecules have emerged as promising anticancer and antimetastatic agents with potential application in platinum-resistant tumors or as alternatives to platinum [14
]. In our screening model for inhibitor of DNA topoisomerases, we discover that Van-7, one kind of vanadium compound, has a strongly inhibitory activity to Topo I, not to Topo II.
Topo I inhibitors include Topo I poison and Topo I suppressor. Both of them are agents designed to interfere with the action of topoisomerase enzymes, but the mechanisms are different. HCPT, as a kind of camptothecin derivative, is a typical Topo I poison. HCPT can stabilize DNA Topo I, forming drug DNA Topo I complex and inhibiting Topo I activity [28
]. However, suppressor is DNA conjugant (such as Hoechst33258) or intercalator (such as aclacinomycin A). Topo I suppressor combines with DNA or deforms the structure of DNA to inhibit the catalytic activity of Topo I to result in cell death. To exert this effect, most Topo I suppressors must be in relatively high concentration, and the activity of DNA conjugant depends on its closely binding with DNA. In the present paper, Van-7 was found to inhibit the activity of Topo I obviously; however, in the test of drug DNA Topo I complex with gel electrophoresis analysis, Van-7 was not found to form cleavable complex even up to 100μ
M. In order to further evaluate Van-7 a direct involvement in the inhibition of topoisomerase in nucleus independently on some potential interference, freshly isolated cell nuclei were used, and the probable occurrence of DNA breaks was assessed by comet assay. We found that HCPT treatment showed obvious comet tail while Van-7 did not. From the above two results, we confirm that inhibitory effect of Van-7 on Topo I activity is different from that of HCPT, and the fact that Van-7 cannot form Drug-DNA Topo I complex indicates that Van-7 is not a poison. Therefore, we presume that Van-7 acts on the upstream of catalytic reaction and probably disturbs the combination of Topo I with DNA.
DNA mobility shift assay (EMSA), also called gel retardation assay, can be used to detect DNA-protein interaction in vitro. In electric field, DNA fragments binding with protein migrate more slowly to positive pole than free DNA fragments. We further evaluate the effect of the compound on the binding of DNA with Topo I. In our tests, we found that Van-7 at 40μ
M obviously inhibited the binding of Topo I to DNA with free DNA fragments band in gel electrophoresis. But this phenomenon was not observed in the present of HCPT. In additional, Van-7 significantly reduced the fluorescence intensity by displaceing ethidium bromide from DNA strand. All these results indicate that Van-7 intercalates to DNA and exert its inhibitory effect to Topo I.
DNase I is a nuclease that cleaves DNA preferentially at phosphodiester linkages adjacent to a pyrimidine nucleotide, yielding 5′-phosphate-terminated polynucleotide with a free hydroxyl group on position 3′, which is similar to topoisomerases cutting the phosphate backbone of the DNA; however, Van-7 was not found to affect the activity of Dnase I, which indicates that Van-7 inhibits activity of Topo I with some selectivity.
Topo I has significant consequences for cancer and cancer chemotherapy via their antiproliferative or cell-differentiating action. From the results of MTT tests, Van-7 was found to strongly inhibit the growth of tumor cells, such as BEL-740A549 and Hela cells, but not normal cells. As the specific activity of Topo I was about 4-fold greater in proliferating (log phase) cells than in nonproliferating (confluent) cells, and in contrast to the changes in Topo I levels, the specific activity of Topo II showed no detectable difference in proliferating versus non-proliferating cells [29
], therefore it is reasonable that Van-7 is more selective to cancer cells with proliferation rate much higher than normal cells. It is reported that the compounds exhibit a different inhibitory mechanism from camptothecin that may induce different phase cell cycle arrest, a novel Topo I inhibitor with repressing the catalytic cleavage activity of Topo I instead of forming the drug-enzyme-DNA covalent ternary complex arrested cell cycle at G2/M phase to K562 Cells [30
]. Our FACS analysis result that Van-7 in the concentration of 100μ
M could block the cell cycle to G2-M other than S phase is consistent with this report.
On the whole, we consider that Van-7 is a potential Topo I inhibitor as a kind of Topo I suppressor. Van-7 can insert into DNA base pairs, resulting in DNA structure distortion, and then inhibiting the binding of DNA to Topo 1, finally affecting the catalytic activity of Topo I. To the best of our knowledge, it is the first report about clarifying inhibitory ability of a vanadium metal compounds on Topo-I catalytic activity and its mechanism. However, the anticancer activity in vivo and detail mechanisms of Van-7 requires further investigation.