A Novel Benzoylurea Derivative F13: Anticancer Activity And Mechanism

3-Bromoacetamino-4-methoxy-benzoylurea(F13) is a benzoylurea derivative selected from the library of small molecule tubulin ligands.Previous data showed that F13 lost the ability of interrupting microtubular dynamics while reserved anticancer activity.In this study,we tried to explore the anticancer effects and underlying mechanisms of F13.1.To explore the underlying anticancer effects of F13MTT assay showed that F13 significantly in vitro inhibited cell proliferation in eight cancer cell lines representing five types of cancers,including fibrosarcoma,hepatoma, colon carcinoma,breast and lung cancer.The IC₅₀ values were in the range of 1.11～4.517μg/ml for various human cancer cells and 4.65μg/ml for human normal liver L02 cells. The highly mobile fibrosarcoma HT-1080 cells also sensitized to F13(IC₅₀=2.51μg/ml).In vivo experiments showed that F13 at 40 mg/kg significantly inhibited the growth of H22 cells by 44.4%in tumor weight,while mitomycin c inhibited by 30.4%.Gene chip technology revealed that the expressions of 310 genes were altered up to 1.5 folds in F13-treated MCF7 cells.These genes are involved in a series of signal transduction,including cytoskeleton regulation,focal adhesion,ECM-receptor interaction,MAPK signaling,cell communication,tight junction and so on.Considering that the pathways are greatly related to cell migration and adhesion,we hypothesized that F13 might affect cell migration and invasion.Therefore we chose the highly mobile fibrosarcoma cancer cell HT-1080 to investigate the anticancer effects of F13.2.The anticancer effects of F13 on huaman fibrosarcoma HT-1080 cellsOur previous data have shown that F13 lost the ability of interrupting microtubular dynamics.In the present study,Flow cytometry showed that F13 treatment did not obviously affect cell cycle distribution,consistent with our previous data.In the experiment,we found that F13 over 5μg/ml resulted in shrinkage of cell volume at 2 h after treatment.The similar effect was observed after F13 at 2.5μg/ml given for 4 h.No obvious morphological changes were induced by 1μg/ml,indicating that F13 over 2.5μg/ml had an immediate cytotoxicity.In addition,compared with persistent treatments,treatments with F13 for a short time resulted in the same inhibition of HT-1080 cell growth,indicating that F13-induced cytotoxicity was not reversible.It has become clear that cellular Ca²⁺ overload,or perturbation of intracellular Ca²⁺ compartmentalization,can cause cytotoxicity and trigger either apoptotic or necrotic cell death¹.We then detected the concentration of cellular Ca²⁺.Flow cytometry assay revealed that F13 of 5μg/ml or 10μg/ml caused Ca²⁺ overload at 1 h,4 h,8 h,and 24 h after treatment,and the same effect was observed in 2.5μg/ml-treated cells starting from 4 h.And F13 at 1μg/ml had no effect on the cellular Ca²⁺.The variation trend of cellular Ca²⁺ was in agreement with F13-induced morphological changes,indicating that the cellular Ca²⁺ overload might involved in F13-induced cytotoxicity of HT-1080 cells.We further investigated the effect of F13 on HT-1080 cell growth.After treatment,F13 inhibited the proliferation of HT-1080 cells in time- and dose-dependent manners.At the concentrations of 2.5～5μg/ml,F13 markedly inhibited cell growth and there was no significant effect until day 6 when F13 was given at 0.5～1μg/ml compared with control.For apoptosis analysis,Annexin-V/PI staining and TUNEL staining revealed that F13 of 2.5～5μg/ml resulted in a significantly increase of HT-1080 cell apoptosis in a dose-dependent manner,while no obvious apoptosis was induced by 1μg/ml.And this effect was further demonstrated by western blot,F13 up to 2.5μg/ml caused decrease of caspase 3 protein level and consequent cleavage of PARP to its characteristic 85 kDa.Due to the fact that F13 could reduce cell viability and induce cell apoptosis at the concentrations of over 1μg/ml,we investigated whether F13 inhibited cell migration, invasion and adhesion at the concentratior/s of≤1μg/ml.The results of wound-healing assay and Transwell migration models showed that F13 was able dose-dependently (0.25～1μg/ml) to significantly inhibit HT-1080 cell migration.In addition, Matrigel-based Transwell assay revealed that F13 was able to significantly inhibit cell invasion.Since cell adhesion to extracellular matrix(ECM) is a critical step of tumor cell invasion,we further determined the effect of F13 on cell adhesion to ECM molecule fibronectin(FN),and the adhesion was inhibited by F13 in a dose-dependent manner.A key step in the invasive progress is the degradation of ECM by matrix metalloproteinases(MMPs).Gelatin zymography revealed that the activities of MMP-9 and MMP-2 in the culture supernatant were down-regulated by F13 with an absolute suppression at 1μg/ml.Interesttingly,the expression of MMP-2/9 mRNA appeared a slight increase,detected by qRT-PCR.Taken together,it was suggested that the down-regulations of MMP-2/9 activities might be a result of post-transcriptional event.We next examined the expression of the reversion-inducing cysteine-rich protein with Kazal motifs(RECK).RECK is a novel MMP inhibitor.Studies have shown that stimulated-expression of RECK significantly caused down-regulation of MMP-2/9 activities^2-6.In this study,RT-PCR showed that HT-1080 cells expressed a low level of RECK.However,the expression was dose-dependently up-regulated after F13 treatment. Previous studies have implied that ERK1/2 pathway negatively regulate RECK expression^7-9.In this study,phosphorylations of ERK1/2 were dramatically decreased by F13 in a dose-dependent manner.Furthermore,highly selective MEK1/2 inhibitor U0126 led to the inhibition of phosphorylated activations of ERK1/2 in HT-1080 cells.U0126 also efficiently increased the expression of RECK.These results indirectly demonstrate that F13 up-regulated the expression of RECK via ERK1/2 signaling pathway.In addition,we detected the effect of F13 on integrin signaling.Western blot and RT-PCR revealed that the expressions ofβ1,β5,andβ6 integrin were markedly inhibited. For the downstream molecules FAK and Akt,the phosphorylations were also dramatically decreased,indicating that the integrin/FAK/Akt pathway may be involved in response to F13 in HT-1080 cells,which needs further investigation.Also,western blot revealed that F13 inhibited the activation of NF-κB,an important transcription factor.Gene chip technology revealed that the expressions of 457 genes were altered up to 2 folds in F13-treated HT-1080cells.These genes are involved in a series of signal transduction,including MAPK signaling,cytoskeleton regulation,cell communication, focal adhesion,ECM-receptor interaction,cell adhesion molecules,and so on.In conclusion,benzoylurea derivative F13 dose-dependently exerts multiple anticancer effects:induction of apoptosis and inhibitions of cell proliferation,migration,adhesion and invasion.F13-mediated inhibition was associated with inactivation of MMP-2/9 by induction of RECK expression,which was regulated by down-regulation of ERK1/2.F 13 might be a lead compound of RECK inducer for development of new anticancer agents.