Screening And Antitumor Action Evaluation Of Topoisomerase Ⅱα Inhibitors

Cancer is one of the most serious threats to human health. Researchers have been searching for promising antitumor agents for decades. In the past ten years, there has been an increasing interest in the development of topoisomerase (Topo) inhibitors as chemotherapy agents. As enzyme that can disentangle the DNA topological problems generated from DNA replication, recombination and transcription, topoisomerase has essential physiological function for cells. Compared to normal cells, the cellular level of topoisomerase is much higher in the rapid proliferating cancer cells, making it an ideal target for cancer treatment. Although lots of topoisomerase inhibitors are among the most widely used clinical agents with promising efficiency, a large part of them showed serious side effects like inducing cardiotoxicity, multidrug resistance and secondary malignancies. Hence, it’s urgent to search for novel inhibitors with higher efficiency and lower toxicity.Our previous work revealed that natural p-terphenyls isolated from marine fungus exhibited antitumor activities and topoisomerase inhibitory activities. Hundreds of molecules were synthesized to improve the biological activity, including 2-phenylnaphthalenoids,2-phenylbenzofurans,3,4-diarylprrole, lignans and others. We aim to find novel topoisomerase inhibitor with higher efficiency and selectivity.In this work,143 compounds were screened for antitumor cytotoxicity and in vitro topoisomerase inhibitory activities. A total of 20 Topo IIa inhibitors and 45 cytotoxicity compounds (IC50< 10 μM) were selected. Among these active compounds, several were selected for further investigation for their strong inhibitory activities or interesting mechanisms. For example, compounds A4 and A6, two 2-phenylbenzofurans with low IC50 values, induced DNA damage and influenced cell cycle-related proteins, are promising compounds for the development of chemotherapy agents; compounds HZ51 and HZ61, two p-terphenyls, although similar in structure and both act as Topo IIα catalytic inhibitors, compound HZ51 showed a much stronger Topo IIα inhibitory activity but weaker cytotoxicity against tumor cells, and this weak cytotoxicity was not due to the up-regulation of cellular protein level of Topo IIα, we suppose this may due to compound’s incapability of entering the cell nuclear; compound K10 with a 3,4-diarylprrole scaffold, induced DNA damage and DNA damage response, arrested cell cycle at G2/M phase. It also induced multi-spindle formation and led cell to enter a mitotic catastrophic cell death; ten lignans showed an interesting structure-activity relationship. The cis-trans isomers have distinct antitumor activity, suggesting a high-selective cellular target existed.Currently, most chemotherapy agents function as apoptosis inducer against cancer cells. However, lots of tumor went through mutation in apoptosis pathway, like glioblastoma. This defects in apoptosis pathway made these tumor resistant to normal cytotoxic chemotherapy agents. There has been an increasing interest in discovering novel agents inducing non-apoptosis cell death to overcome the apoptosis resistance, because increasing evidence showed that cancer could go through a non-apoptosis cell death, like necrosis, senescence, autophagy, mitotic catastrophe and methuosis. Dissecting the detailed cell death mechanisms of non-apoptosis pathway has essential meaning in finding new strategies for cancer therapy.Compound K34 is a Topo IIα catalytic inhibitor with a 3,4-diarylprrole scaffold, and displayed a moderate antiproliferative effect on several cancer cell lines. Interestingly, small vacuoles were observed in cancer cells treated by K34. We also found that apoptosis pathway was not activated in U251 and HCT116 cell lines treated by K34, showing a non-apoptosis pathway included in the vacuolization mechanism. The apoptosis-resistant property of U251 cells could not change the cytotoxicity of K.34, showing a glioblastoma sensitive non-apoptosis pathway existed in K34’s action. We found the vacuoles obtain characteristics of late endosome, but couldn’t merge with lysosome thus showed a non-acid environment. However, using Bafliomycin A1, an H+-ATPase inhibitor, and Amiloride, a macropinocytosis inhibitor, could completely abrogate K34-induced vacuolization. These evidences suggest K34 enhances the maturation and fusion process of macropinosome, also block the acidification process and fusion process of late macropinosome with lysosome, results in the formation of large abnormal vacuoles. The network of mitochondria was badly disrupted, and the fission process was enhanced in K34 treated U251 cells. Compound C, an AMPK inhibitor, can significantly enhance K34’s cytotoxicity. Taken together, these results suggested that AMPK pathway might activate to compensate for cellular energy loss, thus function as a cell survival pathway in K34’s mechanism.We also investigated K34’s mechanism in HCT116 cells. Like in U251 cells, K34 induced vacuoles with characteristics of late endosome, disrupted mitochondria network and enhanced mitochondria fission process. AMPK inhibitor also could enhance K34’s cytotoxicity. We also observed that DNA damage and DNA leakage occured in HCT116 cells treated with K34. However, the detailed mechanism is still unclear and deserves further investigation.