Abstract
Camptothecin (CPT) selectively traps topoisomerase 1-DNA cleavable complexes (Top1cc) to promote anticancer activity. Here, we report the design and synthesis of a new class of neutral porphyrin derivative 5,10-bis(4-carboxyphenyl)-15, 20-bis(4-dimethylaminophenyl)porphyrin (compound 8) as a potent catalytic inhibitor of human Top1. In contrast to CPT, compound 8 reversibly binds with the free enzyme and inhibits the formation of Top1cc and promotes reversal of the preformed Top1cc with CPT. Compound 8 induced inhibition of Top1cc formation in live cells was substantiated by fluorescence recovery after photobleaching (FRAP) assays. We established that MCF7 cells treated with compound 8 trigger proteasome-mediated Top1 degradation, accumulate higher levels of reactive oxygen species (ROS), PARP1 cleavage, oxidative DNA fragmentation, and stimulate apoptotic cell death without stabilizing apoptotic Top1-DNA cleavage complexes. Finally, compound 8 shows anticancer activity by targeting cellular Top1 and preventing the enzyme from directly participating in the apoptotic process.
Highlights
DNA topoisomerase I (Top1) is often exploited as an imperative anticancer chemotherapeutic target due to its critical role in DNA supercoil relaxation which involves three main steps: (a) DNA strand cleavage by a transesterification reaction initiated by the active site tyrosine attacking DNA phosphodiester backbone and generating a covalent intermediate of DNA 3′-phosphotyrosyl linkage (Top1cc), (b) DNA relaxation by controlled strand rotation, and (c) DNA religation by a similar transesterification and release of the enzyme from the DNA.[1−5]
We provide evidence that in contrast to CPT, the selected porphyrin derivative 8 binds reversibly to the free enzyme (Figure 3) and effectively inhibits the formation of Top1-DNA cleavage complex (Top1cc) both in vitro and in live cell as determined in fluorescence recovery after photobleaching (FRAP) assays (Figure 2)
Compound 8 abrogates CPT-mediated preformed Top1-DNA covalent cleavage complexes (Top1cc) both as recombinant enzyme (Figure 2A-D) and as an endogenous Top[1] in the human breast adenocarcinoma (MCF7) cells (Figure 4D), suggesting a plausibility to overcome the limitations of CPT resistance
Summary
DNA topoisomerase I (Top1) is often exploited as an imperative anticancer chemotherapeutic target due to its critical role in DNA supercoil relaxation which involves three main steps: (a) DNA strand cleavage by a transesterification reaction initiated by the active site tyrosine attacking DNA phosphodiester backbone and generating a covalent intermediate of DNA 3′-phosphotyrosyl linkage (Top1cc), (b) DNA relaxation by controlled strand rotation, and (c) DNA religation by a similar transesterification and release of the enzyme from the DNA.[1−5]. To investigate the mechanistic link between compound 8 induced cellular Top[1] inhibition (Figure 1), degradation (Figure 4A and Figure 4B), and activation of apoptosis (Figure 4C), we tested reactive oxygen species (ROS) formation and accumulation of oxidative DNA fragmentation[15,17,46] in compound 8 treated MCF7 cells. We conclude compound 8 plausibly suppresses the CPT induced cytotoxicity (Figure 6H) because compound 8 reverses CPT-induced Top1cc formation in cells as well as in cleavage assays (Figure 2) Taken together these data suggest that compound 8 is a potential Top[1] inhibitor and may be an appropriate lead to develop as a potential anticancer agent
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