| Especial environment of ocean determines usually novel various structure of the marinechemical substances, reaearch on marine drug has been a hotspot for their pharmacological specificity, excellent activity and diversity. Marine anti-cancer drugs is an important area of the reaearch. kalamycin, a poly ketone type compound, isolated from marine actinomycetes M097. It's has been reported that lactoquinomycin and frenolicin B, analogues of kalamycin, can inhibit AKT for the pyran naphthoquinone skeleton they have. We found that although kalamycin has pyran naphthoquinone skeleton, but did not suppress AKT and its downstream signal system, then we make a sytematic analysis on the in vitro anti-tumor effect and mechanism of kalamycin.Growth inhibition of kalamycin on 10 tumor cell lines was analyzed in vitro by SRB assay, the results showed that kalamycin could inhibit a variety of tissue-derived tumor cell growth except for pulmonary cancer cell line A549. The average IC50 against other nine tumor cell lines is 2.5μM. kalamycin had no significant selectivity against different tissue-derived cell lines, and growth inhibition curve against different cell lines are basically similar. In flow cytometry, kalamycin could dose-dependently induce colon cancer cells HCT-116 and hepatoma cells SMMC-7721 G2/M phase arrest, and induce apoptosis of melanoma A375 cells.Acording to previous reports, influence of kalamycin on AKT pathway was analyzed by Western blot, with dose increasing from 1μM to 16μM, kalamycin could not decrease AKT, mTOR and phosphorylated AKT, mTOR, GSK3β. So we don't think kalamycin is a AKT inhibitor. Apoptosis and cycle arrest are usually related to P53 protein, so effect of kalamycin on P53 pathway was analyzed with wild type and P53 missing HCT-116 cell lines, no significant difference in growth inhibition and apoptosis inducement between the two cell lines was fonnd. We conclude that kalamycin can't influence P53 signal pathway.Non selectivity of kalamycin indicated it is a cytotoxic drug. Effect of kalamycin on topoisomerae was analyzed by in vitro enzyme assay. The results showed that kalamycin could't inhibit Topo I, but could dose-dependly inhibit Topo II. kalamycin supress Topo II evidently at concentration of 20μM, the inhibition activity is about ten times of VP16. By DNA stretching assay and Topo II–deraved suppercoiled pBR322 cleavage assay we found kalamycin is not DNA intercalator or Topo II poison but a catalytic inhibitor. Influence of kalamycin on different Topo II catalytical steps was detected, the result showed that kalamycin suppress Topo II–deraved DNA clavage and ATP hydrolysis but not relingation. From semiquantitative analysis, we found inhibition on pre-strand passage DNA cleavage is stronger than post-strand passage DNA cleavage.Angiogenesis support tumor for it's generation, growth and metastasis. Anti-angiogenesis function of kalamycin was detected by several models, the result showed kalamycin inhibit tube formation dose-dependly. IC50 of kalamycin against HMEC-1 in 12 hours is 4.39μM, the tube formation suppression appear at low concentration than this, so we conclude cytoxity is not the only reason of it's inhibition effect. By in vitro enzyme reaction assay, western blot and dual luciferase reporter gene system, we found kalamycin could't suppress tyrosine kinase and HIF-lαpathway.To sum up, kalamycin is not an AKT inhibitor. It induced tumor cells cycle arrest and apoptosis by specific inhibiting Topo II. kalamycin mainly suppress Topo II-mediated DNA clevage and ATP hydrolysis. At the same time, kalamycin can inhibit tumor angiogenesis, while not through tyrosine kinase HIF-lαpathway.
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