Experimental Study On Anti-angiogenic Drugs Identified By Zebrafish Chemical Genetic Screen In Treatment Of Prostate Cancer

Part I. Chemical Genetic Screen to Identify Small Molecules Inhibiting Zebrafish AngiogenesisObjective:To identify small molecules compounds with the activity to inhibit zebrafish angiogenesis.Methods:1. Tg(flk1:EGFP) zebrafish husbandry. A transgenic zebrafish line, Tg(flkl:EGFP), with the endothelial cell-specific flk1promoter directing enhanced green fluorescent protein (EGFP) expression was used. The entire vascular network of the fish is marked by EGFP and could be visualized under fluorescence microscopy. For each mating, one Tg(flk1:EGFP) fish of either homozygous or heterozygous genetic makeup was paired with one wild type fish and5-6such pairs were set up. On average, each pair generated100-150embryos, and embryos were incubated in Holtfreter’s solution at28℃.2. Drug screen. The Library of Micro Source Spectrum Collection containing2000compounds was used for this screen. The dechorionated embryos were placed in Holtfreter’s solution within96-well plates with3-5embryos/well at the12-14hpf stage of development. Next, the compounds, diluted in0.5%dimethyl sulfoxide (DMSO), were added to each well at an initial concentration of0.1μM,1μM, and5μM for72h. After24,48and72h of exposure to the compound, the embryos were visually inspected for viability, gross morphological defects, heart rate and circulation. The0.5%DMSO with equal volume and the known angiogenic inhibitor, PD173074, were used as negative and positive control, respectively.Results:A total of seven hits (hit rate0.35%,7/2000) were identified according to the definition aforementioned with the optimal concentrations varying from0.3μM (simvastatin) to10μM (aristolochic acid and rosuvastatin). Interestingly, these lead compounds can be classified into three groups based on their bioactivities:rotenoids (isorotenone, dihydromunduletone), aristolochic acid and statins (simvastatin, mevastatin, lovastatin and rosuvastatin). Furthermore, we found the anti-angiogenic activity of rosuvastain was specific, rather than cytotoxic. Part II. Experimental Study on Anti-angiogenic Mechanisms of Rosuvastatin in vitro.Objective:To explore the anti-angiogenic mechanisms of rosuvastain in vitro..Methods:1. HUVEC Cell proliferation, tube formation and migration assays. The effects of rosuvastain on HUVEC proliferation, tube formation and migration were determined by the Promega CellTiter96(?) non-radioactive cell proliferation assay, the Endothelial Tube Formation Assay kit and the Cultrex(?) Cell Migration Assay, respectively.2. Cell cycle and apoptosis assays. For the cell cycle assay, the cells were stained with propidium iodide (PI) and analyzed using FlowJo software to determine the cell cycle parameters. For the apoptosis assay, cells were double-stained with annexin V and PI and analyzed by FACScan flow cytometer using Cell Quest software.3. Human angiogenesis PCR array. The effect of rosuvastatin on human angiogenesis pathway gene profiling in HUVEC was analyzed with human angiogenesis RT2Profiler(?) PCR array.Results:Rosuvastatin significantly suppressed HUVEC proliferation (IC50,5.87μM) in a dose-dependent manner. Moreover, after treatment with5u.M of rosuvastatin for48h, the average number of HUVECs arrested in the G1phase was significantly increased by12.5%(61.9vs49.4%, P<0.05) and the number of cells in the S-phase was decreased by19.7%(5.4vs25.1%, P<0.05) compared to the control, indicating rosuvastatin arrested HUVEC cell-cycle progression at the G1phrase. Meanwhile, the average number of the double positive annexin V+/PI+cells was increased by8.5%in rosuvastatin-treated HUVECs relative to the control (6.1vs14.6%, P<0.05), indicating that5μM of rosuvastatin could induce apoptosis in HUVECs in vitro. Subsequently, the expression levels in43of the88examined genes in various angiogenesis pathways were significantly changed (>2-fold) in the rosuvastatin-treated HUVECs, including21down-and22up-regulated genes. Of those, most of the proangiogenic genes, such as FGF-1(fibroblast growth factor1), HGF (hepatocyte growth factor), VEGF (vascular endothelial growth factor), and TGF β1(transforming growth factor, beta1) were decreased, whereas numerous anti-angiogenic factors, such as TIMP-1(tissue inhibitor of metalloproteinase1), and STAB1(Stabilin1) were elevated.Part Ⅲ. Experimental Study on the Efficacy of Rosuvastatin in Treatment of Prostate Cancer.Objective:To observe the therapeutic efficacy of rosuvastatin in human prostate cancer.Methods:1. Cell proliferation assay. The effect of rosuvastain on human prostate cancer cell line PPC-1proliferation was determined by the Promega CellTiter96(?) non-radioactive cell proliferation assay.2. Xenograft mouse model. A human prostate cancer cell line PPC-1xenograft mouse model was established in immunodeficient NOD/SCID mice. When tumors were established and of measurable size, mice were randomly assigned to the rosuvastatin treatment group (n=6) or the control group (n=6). Rosuvastatin (40mg/kg) was administered daily by intraperitoneal injection (i.p.). Tumor volume and animal weight were recorded daily.3. Microvessel density assay. Immunohistochemistry method was used to determine MVD in tumor tissue sections stained with anti-mouse CD31.4. Drug toxicity. The heart, liver, colon, lung and kidney tissues of xenograft mice were fixed for HE staining to assess rosuvastatin toxicity.Results:Rosuvastatin effectively inhibited human prostatic cancer cell PPC-1growth in vitro (IC5014μM). At the day15, the average tumor size of the rosuvastatin-treated group was markedly decreased relative to that of the control group (802.87±306.04mm3vs1801.26±395.7mm3, P<0.01). Accordingly, the average tumor weight of the rosuvastatin-treated group was also significantly reduced compared to that of the control group (0.86±0.39g vs1.39±0.88g, P<0.05). Also the reduction in vessel number within tumors of rosuvastatin-treated mice was statistically significant in comparison to that of the control group at day15(13.7±3.5/HPF vs2.1±1.4/HPF, P<0.01), suggesting tumor angiogenesis was markedly inhibited by rosuvastatin. In addition, no significant difference was detected in body weight and histopathologic morphology in important organs tissues between the rosuvastatin-treated group and the control group. Conclusions:Zebrafish chemical genetic screen is an excellent method for antiangiogenic and anticancer drugs discovery; the antiangiogenic mechanisms of rosuvastatin could be associated with the inhibition of endothelial cell functions and the alteration of human angiogenesis pathway gene profiling; our study may offer the preclinical data for the novel therapeutic potential of rosuvastatin in prostate carcinoma by targeting angiogenesis and cell proliferation.