2-PhenyInaphthalenoids Inhibited The Growth Of Human Tumor By Targeting Topoisomerase

Topoisomerases are essential enzymes involved in managing the topological state of DNA, they paly important roles in different DNA processes including replication, transcription, recombination, chromosome condensation and segregation. Topoisomerases have been identified as important molecular targets of various anticancer drugs in clinical. However, the frequent use of these Topoisomerase targeted anticancer drugs displayed some side effects. Except for gastrointestinal toxicities and dermatosis, there are some several secondary effects such as adriamycin induced cardiotoxicity, etoposide induced myelosuppression and leucopoenia, and multidrug resistance. For these reasons, the search for novel small molecules targeted topoisomerase with higher activity and lower toxicity remains an active domain in oncopharmacology.Natural P-terphenyl-type chemical entities, mainly found in mycomycetes, have displayed various biological activities including cytotoxicity, antioxygenation and antipests. In our previous study, the synthetic P-terphenyls H1-H8were found showing potent cytotoxicity against human breast carcinoma MDA-MB-435cells through cell cycle arrest, apoptosis and topoisomerase inhibition. Fourthermore, five P-terphenyls bearing glucuronic acid isolated from sp. LZ35also inhibited topoisomerase. But those compounds showed weak antitumor activity in vivo and the antitumor mechanism remains unclear. To find more potent topoisomerase inhibitors,482-phenylnaphthalenoid were designed and synthesized. In this study, we evaluated the in vitro antitumor activity and topoisomerases inhibition activity, CS1was picked up for further study.First, SRB assay was applied to evaluate the antitumor activity of2-phenylnaphthalenoids against human breast cancer cells MDA-MB-231, human lung cancer cells A549and human cervical cancer cells HeLa Eleven compounds showed potent cytotoxicity with IC50value below5μM. Then, DNA relaxation assay was employed to detect the topoisomerases inhibition of those eleven potent compounds. Five compounds completely inhibited the topoisomerase Ⅱα mediated DNA relaxation and kDNA decatenation at the concentration of100μM. CSl, which showed potent cytotoxicity and selectivity against topoisomerase I and topoisomerase II, was chosen for further study.To investigated the topoisomerase inhibition activity of CS1, pBR322DNA relaxation, kDNA decatenation, DNA intercalation and stabilization of the cleavage complex were employed. The results showed that CS1worked as an nonintercalating topoisomerase II poison by stabilizing the enzyme-DNA cleavable complex. Then, we tested the cytotoxicity of CS1against nine human cancer cell lines and two nontumor cell lines HUVEC and HL7702. CS1showed potent cytotoxicity against tumor cell lines but low toxicity against nontumor cells. Meanwhile, CS1displayed identical cytotoxicity against sensitive cell MCF-7and multidrug resistant cell MCF-7/ADR. Moreover, CS1had no effect on the degradation of topoisomerase Ⅱα and topoisomerase Ⅱβ, which indicated that CS1might not be likely to induce drug resistance. Further study indicated that CS1induced G2/M arrest, the accumulation of sub-G1and DNA fragments proved that CS1induced apopotosis. Annexin V/PI double stain further confirmed that CS1induced dose-dependent apoptosis.As topoisomerase poison could stabilize the enzyme-DNA complex and accumulate DNA breaks. Comet assay, immunofluorescence and western blotting were adopted to investigated whether CS1induced DNA damage and activated DNA damage response. CS1induced DNA damage evidenced by the formation of comet tails and phosphorylation of histone H2AX. Interestingly, CS1selectively activated cell signal checkpoint kinase ATR but not ATM. Which suggested that CS1might induced DNA damage but didn’t activated the typical DNA damage response.To further study the antitumor mechanism, we observed the morphology change of CS1treated cells. Multinuclears were observed in CS1treated cells by Hoechst33342staining. Immunofluorescence and western blotting showed that CS1induced the abnormal formation of spindle and abnormal chromosome segregation, which indicated that CS1trigger cells undergoing mitotic catastrophe. Moreover, CS1exhibited contiguous in vivo antitumor activity with tumor weight inhibition of62.3%and lower toxicity compared with etoposide in nude mice.In summary, CS1, a novel2-phenylnaphthalene, functions as a highly active Topo Ⅱ poison that induces DNA damage. It exhibited potent, broad-spectrum and multidrug resistance irrelevant anticancer activity. Due to its unique feature of inducing mitotic catastrophe and apoptosis, this drug is slightly less cytotoxic than etoposide but more secure against normal cells. This study provides a strong rationale for clinical development of CS1and further exploit the topoisomerase inhibitors with distinct mechanism.