The Anti-tumor Activity And The Mechanisms Of DAT-230 In Human Fibrosarcoma (HT-1080) And Gastric Adenocarcinoma (SGC-7901)

Purpose: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 analogue 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 (MMP) and NO generation was 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 human fibrosarcoma (HT-1080) or human gastric adenocarconoma (SGC-7901) xengrafts subcutaneously established in BALB/c nude mice. Perfused vasculature was identified using fluorescent DNA-binding compound Hoechst 33342. Apoptosis in tumor tissues was detected by TUNEL analysis. Pathological changes were detected through HE-staining.Results:DAT-230 exhibited potent anti-tumor activity against various cancer cells. DAT-230-treatment in HT-1080 cells resulted in microtubule depolymerization and G2/M 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, Casepase-3 and PARP cleavage, which were at the downstream of mitochondria. The reduction of Bcl-2/Bax ratio, elevation of NO and disruption of MMP confirmed the causal relationship of DAT-230 and mitochondrial pathway. In vivo, DAT-230 delayed tumor growth, induced tumor perfusion decrease and extensive hemorrhagic-necrosis.Anti-proliferative effect of DAT-230 on SGC-7901 cells was associated with microtubule de-polymerization and mitosis arrest, which was mediated by Cyclin B1 accumulation and translocation into nuclei, and activation of cdc2. Mitochondria pathway participated in apoptosis after G2/M arrest in SGC-7901 cells treated with DAT-230, which was characterized by DNA fragmentation, cleavage of PARP, activation of Casepase-3 and Caspase-9, up-regulated of Bcl-2 and down-regulated of Bax, release of Cytochrome c from mitochondria and decrease of mitochondrial membrane potential (MMP). Moreover, DAT-230 suppressed the tumor growth and induced apoptosis in BALB/c nude mice bearing SGC-7901 tumor xenografts.Conclusions:DAT-230 is a promising microtubule inhibitor that has great potential for treatment of human fibrosarcoma (HT-1080) and gastric adenocaicinoma (SGC-7901) in vitro and in vivo. Its potential to be a candidate of anticancer agent is worth being further investigated.