Studies On The Antitumor Activity And Mechanism Of A Citrinin Trimer, Tricitrinol B

To survive in the extreme environment, extremophiles gradually developed special architecture and physiological function in the long evolutionary process. Thus they can produce extremophilic enzyme and many other bioactive substances, which have become one of the most important drug resources. A series of compounds derived from P. citrinum HGY1-5, which come from the settlings in the crater, were screened for searching for the new antitumor compounds. Of those, Tricitrinol B, which is a trimer of citrinin possessing novel structure, exhibits much stronger antitumor activity in vitro. In the present study, we evaluated the antitumor activity of Tricitrinol B in vitro and elucidated the detailed mechanisms of the anti-tumor actions by targeting topoisomerase.To evaluate the antitumor activity of Tricitrinol B in vitro, Sulfurhodamine B assay was adopted. And the results showed that Tricitrinol B possesses broad spectrum antitumor activities in vitro against a panel of 17 human cell lines coming from different tissues with an average IC50 value of 4.77 uM. Meanwhile, the anti-MDR (multidrug resistance) effects on two MDR cell lines (KB/VCR and MCF7/ADR) were assessed. The results demonstrate that Tricitrinol B possesses well anti-MDR effects and the average RF (resistance factor) was nearly 1.0, which was much lower than those of reference drugs, including VCR and ADR. DNA fragmentation assays were further applied to proved that Tricitrinol B induced-cell death was through apoptosis pathway. And Annexin V/PI assay demonstrated that Tricitrinol B can concentration- dependently and time-dependently induces apoptosis. Before 12 h, Tricitrinol B can induce early apoptosis. while at 24h Tricitrinol B induces late apoptosis. JC-1 assay was applied to test the effects of Tricitrinol B on mitochondrial membrane potentials and results revealed that Tricitrinol B concentration-dependently and time-dependently reduces mitochondrial membrane potentials. Western blot assay showed that caspase-8, caspase-9. Bid, caspase-3 and PARP were activated in both HL-60 cells and HCT116 cells after treatment of different dose of Tricitrinol B for 24h, indicating that both death receptor pathway and mitochondria pathway may be activated by Tricitrinol B; Applying three kinds of caspase inhibitors, both caspase-8 and pan-caspase inhibitor could invert the apoptosis, while caspase 9 inhibitor had no effect, suggesting that death receptor pathway may be mainly responsible for the apoptosis induced by Tricitrinol B.Topoisomerase (Topo) is an essential enzyme that plays an important role in DNA replication, repair, transcription and chromosome segregation. Given its critical functions, Topo has been identified as an important antitumor target. Mechanism studies further revealed that Tricitrinol B inhibited Topo I-mediated supercoiled pBR322 relaxation. However, the in vivo complex of enzyme (ICE) assays and DNA cleavage assays showed that Tricitrinol B did not stablize the Topo I-DNA cleavage complex. In contrast, Tricitrinol B inhibited topoisomeraseⅡα(TopoⅡα-mediated kDNA decatenation and relaxation of supercoiled pBR322. ICE assays further confirmed that Tricitrinol B inhibits TopoⅡof by trapping and stabilizing TopoⅡcleavage complexs, which is the hallmark of TopoⅡpoison. And this was further confirmed by the results of comet assays and y-H2AX assays that Tricitrinol B can induce DNA double strand breaks. In agreement with this result, the down regulation of TopoⅡby the use of RNAi could attenuated the ability of Tricitrinol B to induce cell apoptosis. The results above collectively suggest that Tricitrinol B exert antitumor effects by targets to topoisomeraseⅡin cells acting as a novel topoisomeraseⅡpoison.The subsequent unwinding assays and ethidium bromide displacement fluorescence assays exhibited that Tricitrinol B intercalates into DNA helices. DNA migration assay showed that Tricitrinol B reduces the non-covalent binding of TopoⅡαto DNA; TopoⅡα-'mediated cleavage and religation assay showed Tricitrinol B apparently enhances TopoⅡ-mediated DNA cleavage and suppresses TopoⅡα-mediated DNA religation both before and after DNA strand passage. The results indicated that Tricitrinol B can interfere with the Topo Ila-mediated DNA cleavage/religation equilibria, thus increasing the amount of the broken DNA. It is especially to note that Tricitrinol B interfered with the post-strand passage equilibrium more obviously than the pre-strand passage one. making its activity similar to that of the classical TopoⅡinhibitor mAMSA. Taken together, our data collectively indicate that the trimer of citrinin, Tricitrinol B, is a novel TopoⅡpoison.In summary, the present study indicate that the novel trimer of citrinin Tricitrinol B is a DNA intercalator targeting TopoⅡαby interfering with the DNA binding, enhancing TopoⅡα-mediated DNA cleavage and suppressing TopoⅡα-mediated DNA religation both before and after DNA strand passage, and thus produces DNA double strand breaks and induces cell apoptosis. Taken together, as a novel TopoⅡpoison, Tricitrinol B displayed potent in vitro antitumor activities, and the study on the antitumor mechanism of Tricitrinol B provide insights into derivatives of citrinin to develop novel anticancer drugs.