title: DAT-230, a novel microtubule inhibitor, exhibits potent anti-tumor activity by inducing G2/M phase arrest, apoptosis in vitro and perfusion decrease in vivo to HT-1080.
abstract: OBJECTIVE
  The anti-mitotic agent, combretastatin A-4 (CA-4), is the lead compound of a new class of anti-cancer drugs that target tumor vasculature. 2-Methoxy-5-(2-(3, 4, 5-trimethoxyphenyl) thiophen-3-yl) aniline (DAT-230) is a structurally novel CA-4 analog with more stability. We investigated its anti-tumor activity and mechanisms in vitro and in vivo for the first time.
  
  
  METHODS
  Cytotoxicity was measured by MTT method. Apoptosis, mitochondria membrane potential (ΔΨm) and NO generation were measured by flow cytometry. Intracellular microtubule network was detected by immunofluorescence experiments. Protein expression was analyzed by Western blotting. In vivo, the anti-tumor activity was assessed using fibrosarcoma xenografts subcutaneously established in BALB/c nude mice. Vasculature perfusion was identified using fluorescent DNA-binding compound Hoechst 33342.
  
  
  RESULTS
  DAT-230 exhibited potent anti-proliferative activity against various cancer cells. DAT-230-treatment in HT-1080 cells resulted in microtubule de-polymerization and G2/M phase arrest preceding apoptosis. Phosphor-cdc2 (thr14/tyr15) reduction, cyclin B1 accumulation and aberrant spindles denoted the cyclin B1-cdc2 complex active and M phase arrest in HT-1080 cells treated with DAT-230. Apoptosis induced by DAT-230 was related with the activation of caspase-9, caspase-3 and PARP cleavage, which were at the downstream of mitochondria. The decrease ratio of Bcl-2/Bax, elevation of NO and disruption of ΔΨm confirmed the causal relationship between DAT-230 and mitochondrial pathway. In vivo, DAT-230 delayed tumor growth, induced tumor perfusion decrease and extensive hemorrhagic-necrosis.
  
  
  CONCLUSIONS
  DAT-230 is a promising microtubule inhibitor that has great potential for the treatment of fibrosarcoma in vitro and in vivo. Its potential to be a candidate of anti-cancer agent is worth being further investigated.
authors: Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, China.
authors: Foxiao
authors: F
authors: Qiao
authors: Daiying
authors: D
authors: Zuo
authors: Xueqi
authors: X
authors: Shen
authors: Huan
authors: H
authors: Qi
authors: Haifeng
authors: H
authors: Wang
authors: Weige
authors: W
authors: Zhang
authors: Yingliang
authors: Y
authors: Wu
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D005354:Fibrosarcoma / Q000098:blood supply / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects*; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008856:Microscopy, Fluorescence; D015394:Molecular Structure; D009569:Nitric Oxide / Q000096:biosynthesis; D017382:Reactive Oxygen Species / Q000378:metabolism; D013876:Thiophenes / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D050257:Tubulin Modulators / Q000494:pharmacology* / Q000627:therapeutic use; D023041:Xenograft Model Antitumor Assays
chemical_list: C575717:2-methoxy-5-(2-(3,4,5-trimethoxyphenyl)thiophen-3-yl)aniline; D000814:Aniline Compounds; D000970:Antineoplastic Agents; D017382:Reactive Oxygen Species; D013876:Thiophenes; D050257:Tubulin Modulators; D009569:Nitric Oxide