Class 3 semaphorins (sema 3) are secreted guidance proteins. Sema 3A expressed by endothelial cells controls vascular morphogenesis through integrin inhibition. Sema 3C is required for normal cardiovascular patterning. Here we examined the potential role of sema 3C as regulator of endothelial cell function in vitro using mouse glomerular endothelial cells (MGEC). We determined that MGEC express sema 3C mRNA and protein and its receptors mRNA. Recombinant sema 3C induced MGEC proliferation 18 +/- 2% above control, as assessed by bromodeoxyuridine (BrdU) incorporation, and reduced starvation-induced apoptosis by 46 +/- 3%, as indicated by an in situ marker of activated caspase 3. Sema 3C increased MGEC adhesion to fibronectin 79 +/- 13% and to collagen 55 +/- 12% as compared with control. Sema 3C-induced MGEC adhesion was prevented by integrin blocking antibodies and involved beta1 integrin serine phosphorylation. Sema 3C-induced MGEC adhesion and proliferation were similar to those induced by vascular endothelial growth factor (VEGF)-A. Sema 3C induced a 44 +/- 11% increase in MGEC directional migration and stimulated MGEC capillary-like network formation on collagen I gels. Collectively, our data indicate that sema 3C promotes glomerular endothelial cell proliferation, adhesion, directional migration, and tube formation in vitro by stimulating integrin phosphorylation and VEGF120 secretion, functions that are similar to VEGF-A and opposite to sema 3A.

Radiotherapy combined with androgen depletion is generally successful for treating locally advanced prostate cancer. However, radioresistance that contributes to recurrence remains a major therapeutic problem in many patients. In this study, we define the high-affinity neurotensin receptor 1 (NTR1) as a tractable new molecular target to radiosensitize prostate cancers. The selective NTR1 antagonist SR48692 sensitized prostate cancer cells in a dose-and time-dependent manner, increasing apoptotic cell death and decreasing clonogenic survival. The observed cancer selectivity for combinations of SR48692 and radiation reflected differential expression of NTR1, which is highly expressed in prostate cancer cells but not in normal prostate epithelial cells. Radiosensitization was not affected by androgen dependence or androgen receptor expression status. NTR1 inhibition in cancer cell-attenuated epidermal growth factor receptor activation and downstream signaling, whether induced by neurotensin or ionizing radiation, establish a molecular mechanism for sensitization. Most notably, SR48692 efficiently radiosensitized PC-3M orthotopic human tumor xenografts in mice, and significantly reduced tumor burden. Taken together, our findings offer preclinical proof of concept for targeting the NTR1 receptor as a strategy to improve efficacy and outcomes of prostate cancer treatments using radiotherapy. Cancer Res; 71(21);

2-Methoxyestradiol (2ME2) is an endogenous estradiol metabolite that inhibits microtubule polymerization, tumor growth, and angiogenesis. Because prostate cancer is often treated with radiotherapy, and 2ME2 has shown efficacy as a single agent against human prostate carcinoma, we evaluated 2ME2 as a potential radiosensitizer in prostate cancer models. A dose-dependent decrease in mitogen-activated protein kinase phosphorylation was observed in human PC3 prostate cancer cells treated with 2ME2 for 18 h. This decrease correlated with in vitro radiosensitization measured by clonogenic assays, and these effects were blocked by the expression of constitutively active MEK. Male nude mice with subcutaneous PC3 xenografts in the hind leg were treated with 2ME2 (75 mg/kg) p.o. for 5 days, and 2 Gy radiation fractions were delivered each day at 4 h after drug treatment. A statistically significant super-additive effect between radiation and 2ME2 was observed in this subcutaneous model, using analysis of within-animal slopes. A PC-3M orthotopic model was also used, with bioluminescence imaging as an end point. PC-3M cells stably expressing the luciferase gene were surgically implanted into the prostates of male nude mice. Mice were given oral doses of 2ME2 (75 mg/kg), with radiation fractions (3 Gy) delivered 4 h later. Mice were then imaged weekly for 4 to 5 weeks with a Xenogen system. A significant super-additive effect was also observed in the orthotopic model. These data show that 2ME2 is an effective radiosensitizing agent against human prostate cancer xenografts, and that the mechanism may involve a decrease in mitogen-activated protein kinase phosphorylation by 2ME2. [Cancer Res 2007;67(17):8316-24]

In 1985 Brinster et al.[1] observed that linearized DNA injected in the cytoplasm of mouse zygotes underwent spontaneous supercoiling within 24 h. This finding suggests that DNA prefers and functions best in tertiary structure. In an effort to improve the efficiency of transgenesis by cytoplasmic injection, DNA was condensed with MgCl 2 to form a three dimensional rod-shaped DNA prior to injection in pronuclear stage murine zygotes. The DNA used was enhanced green fluorescent protein on a cytomegalovirus promoter (CMV-EGFP) and served as a marker for gene integration and protein expression in culture conditions. The condensed CMV-EGFP construct was injected in the cytoplasm at 3 concentrations (100, n = 816; 425, n = 464; and 625 µg/ml, n = 708). For comparison linear CMV-EGFP construct was injected into the pronucleus (5 µg/ml, n = 196) and into the cytoplasm (625 µg/ml, n = 628). In all treatment groups the control and buffer injected embryos developed similarly. Among DNA treatment groups, the highest development of fluorescing embryos was observed in zygotes injected in the cytoplasm with linear CMV-EGFP (625 µg/ml); however, zygotes injected in the cytoplasm with condensed CMV-EGFP (625 µg/ml) had the highest percentage (44%) of fluorescing embryos, the highest percentage (16.7%) of fluorescing morula and blastocysts, and the lowest percentage of fluorescence mosaicism at every stage of embryo development after 4 d in culture; thereby making it the best method for generating transgenic animals.

There is mounting evidence that endothelin-1(ET-1) / endothelin receptor axis contributes to the pathogenesis of urothelial bladder cancer. In a previous study, reduced expression of the metastasis suppressor protein RhoGDI2 in bladder cancer cell lines and tumors was associated with increased expression of ET-1 (Titus B et al, Ca Res 65:7320, 2005). This suggests that ET-1 acts down-stream of repressed RhoGDI2 expression to promote the metastatic phenotype. If so, then blocking activation of endothelin receptor A (ETAR) by ET-1 would suppress this phenotype. To test this hypothesis, Zibotentan, a specific inhibitor of ETAR (IC50 = 21 nM), was evaluated for anti-tumor activity in a xenograft metastasis model. To screen for toxicity, 40 athymic nu/nu female received 12.5 to 50 mg/kg/d of zibotentan by gastric lavage for 97 days. The drug was well tolerated at all dose levels and produced no significant changes in body weight, behavior, or general physical condition. In a separate experiment, T24T bladder cancer cells (106 cells suspended in 0.1 mL of medium) were inoculated into the tail veins of a second group of 40 mice. One week later, 20 of animals were treated with zibotentan 50 mg/Kg/d and 20 controls received vehicle only. After 90 days, the animals were euthanized and the lungs were removed, fixed with Bouin's solution, stained, and examined by microscopy to determine the number and size of metastasis (large ≥3mm, small <3mm). Metastases were found in 12/20 (60%) controls and 3/20 (15%) of treated mice (p=0.024); overall, 89 metastases were identified in the controls and 19 in the treated animals (p=0.012). Although the drug-treated mice had fewer metastases, 13/19 (68%) of these were classified as large tumors as compared to 20/89 (22%) in the control group. These results suggest that inhibition of ETAR signaling by zibotentan reduced the frequency and number of metastases, most likely by suppressing metastatic colonization rather than the growth of established metastases. The latter is consistent with earlier data that inhibition of ETAR does not block the proliferation of T24T cells in vitro. Taken together, these and the previous observations provide a rationale for clinical trials to determine if treatment with zibotentan will prevent or delay the development of metastatic disease in patients with clinically localized but high-risk bladder cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B102.