Mutagenesis of Odorant Receptor Coreceptor Orco Reveals the Odorant-Detected Behavior of the Predator Eupeodes corollae.

The successful mating of the hoverfly and the search for prey aphids are of great significance for biological control and are usually mediated by chemical cues. The odorant receptor co-receptor (Orco) genes play a crucial role in the process of insect odor perception. However, the function of Orco in the mating and prey-seeking behaviors of the hoverfly remains relatively unexplored. In this study, we characterized the Orco gene from the hoverfly, Eupeodes corollae, a natural enemy insect. We used the CRISPR/Cas9 technique to knock out the Orco gene of E. corollae, and the EcorOrco-/- homozygous mutant was verified by the genotype analysis. Fluorescence in situ hybridization showed that the antennal ORN of EcorOrco-/- mutant lack Orco staining. Electroantennogram (EAG) results showed that the adult mutant almost lost the electrophysiological response to 15 odorants from three types. The two-way choice assay and the glass Y-tube olfactometer indicated that both the larvae and adults of hoverflies lost their behavioral preference to the aphid alarm pheromone (E)-ß-farnesene (EBF). In addition, the mating assay results showed a significant decrease in the mating rate of males following the knock out of the EcorOrco gene. Although the mating of females was not affected, the amount of eggs being laid and the hatching rate of the eggs were significantly reduced. These results indicated that the EcorOrco gene was not only involved in the detection of semiochemicals in hoverflies but also plays a pivotal role in the development of eggs. In conclusion, our results expand the comprehension of the chemoreceptive mechanisms in the hoverflies and offers valuable insights for the advancement of more sophisticated pest management strategies.

Efficient Fabrication of Self-Assembled Polylactic Acid Colloidosomes for Pesticide Encapsulation.

Colloidosomes are microcapsules whose shells are composed of cumulated or fused colloidal particles. When colloidosomes are used for in situ encapsulation, it is still a challenge to achieve a high encapsulation efficiency and controllable release by an effective fabrication method. Herein, we present a highly efficient route for the large-scale preparation of colloidosomes. The biodegradable polylactic acid (PLA) nanoparticles (NPs) as shell materials can be synthesized using an antisolvent precipitation method, and the possible formation mechanism was given through the molecular dynamics (MD) simulation. The theoretical values are basically consistent with the experimental results. Through the use of the modified and unmodified PLA NPs, the colloidosomes with controllable shell porosities can be easily constructed using spray drying technology. We also investigate the mechanism of colloidosomes successfully self-assembled by PLA NPs with various factors of inlet temperature, feed rate, and flow rates of compressed air. Furthermore, avermectin (AVM) was used as a model for in situ encapsulation and a controllable release. The spherical modified colloidosomes encapsulating AVM not only achieve a small mean diameter of 1.57 µm but also realize a high encapsulation efficiency of 89.7% and impermeability, which can be further verified by the MD simulation. AVM molecules gather around and clog the shell pores during the evaporation of water molecules. More importantly, the PLA colloidosomes also reveal excellent UV-shielding properties, which can protect AVM from photodegradation.

Conserved Odorant Receptor, EcorOR4, Mediates Attraction of Mated Female Eupeodes corollae to 1-Octen-3-ol.

Odorant receptors (ORs) in insects are crucial for the detection of chemical signals. However, the functions of the conserved OR genes among insect species are rarely studied. In this study, we analyzed a well-conserved OR clade in Diptera insects and cloned a gene from this clade, EcorOR4, in the hoverfly Eupeodes corollae. Real-time quantitative PCR showed that EcorOR4 was highly expressed in the antennae and upregulated in the mated females, and in vitro functional characterization showed that EcorOR4 was narrowly tuned to 1-octen-3-ol. Electroantennogram assays revealed that the antennal response of mated females to 1-octen-3-ol was significantly higher than that of mated males, but no significant differences were observed between male and female virgins. Finally, a Y-tube olfactometer bioassay showed that 1-octen-3-ol is an attractant for only mated female E. corollae adults. These results demonstrate that EcorOR4 is involved in the detection of 1-octen-3-ol and that this compound may affect the host-finding and oviposition behavior in female E. corollae.

Overexpression of PDGF-BB decreases colorectal and pancreatic cancer growth by increasing tumor pericyte content.

We hypothesized that overexpression of PDGF-BB in colorectal cancer (CRC) and pancreatic cancer cells would result in increased pericyte coverage of ECs in vivo, rendering the tumor vasculature more resistant to antiangiogenic therapy. We stably transfected the cDNA for the PDGF-B into HT-29 human CRC and FG human pancreatic cancer cells. Surprisingly, when HT-29 or FG parental and transfected cells were injected into mice (subcutaneously and orthotopically), we observed marked inhibition of tumor growth in the PDGF-BB-overexpressing clones. In the PDGF-BB-overexpressing tumors, we observed an increase in pericyte coverage of ECs. Treatment of PDGF-BB-overexpressing tumors with imatinib mesylate (PDGFR inhibitor) resulted in increased growth and decreased total pericyte content compared with those in untreated PDGF-BB-overexpressing tumors. In vitro studies demonstrated the ability of VSMCs to inhibit EC proliferation by approximately 50%. These data show that increasing the pericyte content of the tumor microenvironment inhibits the growth of angiogenesis-dependent tumors. Single-agent therapy targeting PDGF receptor must be used with caution in tumors when PDGFR is not the target on the tumor cell itself.

Aromatic Volatiles and Odorant Receptor 25 Mediate Attraction of Eupeodes corollae to Flowers.

Flowering plants attract pollinators with volatile chemicals that include aromatic compounds. Syrphid flies are the largest group of flower visitors in Diptera, but little is known about how they detect floral scents at the molecular level. Here, electroantennogram (EAG) recordings from the antennae of Eupeodes corollae were used to measure responses from 14 aromatic compounds. To identify odorant receptors (ORs) of E. corollae tuned to aromatic volatiles, we analyzed functional profiles of Drosophila melanogaster odorant receptors (ORs), DmelOR46a and DmelOR71a, which are narrowly tuned to phenolic compounds and represent the orthologues of E. corollae OR25 and OR28, respectively. The two genes that are expressed in the antennae of both sexes were functionally characterized. EcorOR25 is narrowly tuned to several structurally related floral scent volatiles, including eugenol, p-cresol, and methyl eugenol. Finally, choice behavior assays showed that eugenol and methyl eugenol were attractants for both sexes of E. corollae adults. This study identified the odorant receptors used by E. corollae to detect aromatic volatiles, suggesting environmentally friendly strategies to attract these beneficial insects.

The development and characterization of a human midgut carcinoid cell line.

PURPOSE: Gastrointestinal neuroendocrine tumors (NET) are rare heterogeneous tumors that hypersecrete neuropeptides. The scarcity of good gastrointestinal NET models has limited the ability to study potential therapeutic agents. We describe and characterize the establishment of a human midgut carcinoid tumor cell line carcinoid tumor 2 (CNDT2). EXPERIMENTAL DESIGN: Tumor cells (CNDT2) were isolated from a liver metastasis from a patient with a primary ileal carcinoid. After 9 weeks in culture, the cells were plated in soft agar, and cells from a single colony were put back in culture (CNDT2.1). Those CNDT2.1 cells were injected s.c. into nude mice. Cells were isolated from a single resultant tumor (CNDT2.5), cultured, and characterized by electron microscopy, reverse transcription-PCR, serotonin enzyme immunoassay, Western blotting, and immunohistochemical analysis for NET markers and potential therapeutic targets. RESULTS: CNDT2 cells grew in monolayers in vitro, formed colonies in soft agar, and formed tumors in mice. Electron microscopy revealed round, pleomorphic, electron-dense neurosecretory granules characteristic of NETs. Tumor xenografts exhibited the appearance of NETs with small "salt-and-pepper" nuclei on H&E staining and chromogranin A, synaptophysin, and CD56 on immunohistochemical staining. CNDT2.5 cells produced serotonin and expressed insulin-like growth factor receptor-I, platelet-derived growth factor receptor-beta, vascular endothelial growth factor receptor-1, cMET, epidermal growth factor receptor, neuropilin-1, and somatostatin receptors 1 to 5. Cytogenetic analysis revealed the presence of deletions at 2p and 6q and numerous translocations. CONCLUSION: The establishment of this human midgut carcinoid tumor cell line may serve as a useful model system for studying cell biology and novel targeted agents in preclinical models.

Vascular endothelial growth factor receptor-1 activation mediates epithelial to mesenchymal transition in human pancreatic carcinoma cells.

Our laboratory has shown that vascular endothelial growth factor receptor-1 (VEGFR-1) expression on human pancreatic cancer cell lines mediates cell migration and invasion. Because epithelial to mesenchymal transition (EMT) also plays a role in cell motility by altering the cell phenotype and morphology, we hypothesized that VEGFR-1 activation induces molecular alterations that mediate EMT. Our treatment of the human pancreatic cancer cell line L3.6pl with the VEGFR-1 ligands VEGF-A and VEGF-B led to morphologic changes characteristic of EMT, including loss of polarity, increased intercellular separation, and the presence of pseudopodia. Immunofluorescent staining with antibodies to E-cadherin and beta-catenin showed that VEGFR-1 activation led to translocation of E-cadherin and beta-catenin from their usual cell membrane-bound location to the cytoplasm and nucleus, respectively. Western blotting showed that VEGFR-1 activation led to decreased expression of the epithelial markers E-cadherin and plakoglobin, increased expression of the mesenchymal markers vimentin and N-cadherin, and increased nuclear expression of beta-catenin. Pretreatment of tumor cells with a VEGFR-1 blocking antibody inhibited the VEGFR-1-induced immunohistochemical and molecular changes in E-cadherin. VEGFR-1 activation led to an increase in expression of the EMT-associated transcription factors Snail, Twist, and Slug. The changes mediated by VEGFR-1 in this pancreatic carcinoma cell line are highly consistent with the changes characteristic of EMT. Given our previous finding of VEGFR-1-mediated tumor cell invasion and migration in pancreatic carcinoma cells, we hypothesize that VEGFR-1 plays a role in tumor progression in pancreatic cancer through the induction of EMT.

Regulation of cyclooxygenase-2 (COX-2) expression in human pancreatic carcinoma cells by the insulin-like growth factor-I receptor (IGF-IR) system.

Both the insulin-like growth factor-I receptor (IGF-IR) and cyclooxygenase-2 (COX-2) are frequently overexpressed in pancreatic cancer. We hypothesized that IGF-IR is directly involved in induction of COX-2 and sought to investigate signaling pathways mediating this effect. Pancreatic cancer cells (L3.6pl) were stably transfected with a dominant-negative receptor (IGF-IR DN) construct or empty vector (pcDNA). Cells were stimulated with IGF-I to determine activated signaling intermediates and induction of COX-2. Signaling pathways mediating COX-2 induction were identified using signaling inhibitors. IGF-I up-regulated COX-2 selectively via the MAPK/(Erk-1/2) pathway. In addition, IGF-IR DN cells showed a marked decrease in constitutive COX-2 and a blunted response to IGF-I. Similarly, treatment with an anti-IGF-IR antibody effectively inhibited IGF-IR and MAPK/Erk activation and decreased COX-2 in parental cells. In conclusion, activation of IGF-IR mediates COX-2 expression in human pancreatic cancer cells.

Roles of nitric oxide synthase inhibition and vascular endothelial growth factor receptor-2 inhibition on vascular morphology and function in an in vivo model of pancreatic cancer.

PURPOSE: Both nitric oxide (NO) and vascular endothelial growth factor (VEGF) mediate tumor vascular function. Because these molecules regulate one another's expression, we hypothesized that NO synthase (NOS) inhibition produces effects comparable to those of anti-VEGF therapy on human pancreatic cancer xenografts. EXPERIMENTAL DESIGN: L3.6pl human pancreatic cancer cells were s.c. implanted in nude mice. On day 6, mice were randomized to receive (a) PBS (control), (b) DC101 [VEGF receptor 2 (VEGFR-2) antibody] by i.p. injection, (c) N-nitro-l-arginine (NNLA; NOS inhibitor) in the drinking water, or (d) both DC101 and NNLA. Mice were killed on day 20. RESULTS: DC101 and NNLA as single agents inhibited tumor growth by approximately 50% to 60% (P < 0.008 for both). Furthermore, combined therapy inhibited mean tumor growth by 89% (P < 0.008). Combined inhibition of VEGFR-2 and NOS also decreased mean vessel counts by 65% (P < 0.03) and vessel area by 80% versus controls (P < 0.001). In contrast to DC101 where vessel diameter was similar to control, NNLA decreased mean vessel diameter by 42% (P < 0.001). NNLA also led to a 54% (P < 0.03) decrease in tumor uptake of the perfusion marker Hoechst 33342 versus controls whereas DC101 decreased Hoechst 33342 staining by 43% (P < 0.03). The combination of inhibitors decreased perfusion by 73% (P < 0.03). CONCLUSIONS: Although VEGFR-2 can mediate NOS activity, the combination of VEGFR-2 and NOS inhibition significantly increased the antivascular effect over single agent therapy. The addition of NOS inhibition led to an even further alteration of tumor vessel morphology and vascular perfusion compared with VEGFR-2 blockade, suggesting that NO and VEGFR-2 have distinct but complementary effects on the tumor vasculature.

Chronic oxaliplatin resistance induces epithelial-to-mesenchymal transition in colorectal cancer cell lines.

PURPOSE: Epithelial-to-mesenchymal transition (EMT) is a process whereby cells acquire molecular alterations that facilitate cell motility and invasion. In preliminary studies, we observed that oxaliplatin-resistant (OxR) colorectal cancer (CRC) cells underwent morphologic changes suggestive of a migratory phenotype, leading us to hypothesize that OxR CRC cells undergo EMT. EXPERIMENTAL DESIGN: The human CRC cell lines KM12L4 and HT29 were exposed to increasing doses of oxaliplatin to establish stable cell lines resistant to oxaliplatin. Migration and invasion were assessed by modified Boyden chamber assays. Morphologic and molecular changes characteristic of EMT were determined by immunofluorescence staining and Western blot analyses. RESULTS: The OxR cells showed phenotypic changes consistent with EMT: spindle-cell shape, loss of polarity, intercellular separation, and pseudopodia formation. KM12L4 and HT29 OxR cells exhibited an approximately 8- to 15-fold increase in migrating and invading cells, respectively (P < 0.005 for both). Immunofluorescence staining of OxR cells revealed translocation of E-cadherin and beta-catenin from their usual membrane-bound complex to the cytoplasm and nucleus, respectively. The OxR cells also had decreased expression of the epithelial adhesion molecules E-cadherin and plakoglobin and an increase in the mesenchymal marker vimentin. The KM12L4 OxR cells exhibited increased nuclear expression of Snail, an EMT-regulatory transcription factor, whereas the HT29 OxR cells exhibited an increase in nuclear expression of the EMT-associated transcription factor nuclear factor kappaB. CONCLUSION: We hypothesize that induction of EMT may contribute to the decreased efficacy of therapy in chemoresistant CRC, as the tumor cells switch from a proliferative to invasive phenotype. Further understanding of the mechanisms of chemoresistance in CRC will enable improvements in chemotherapy for metastatic disease.

Targeting of urokinase plasminogen activator receptor in human pancreatic carcinoma cells inhibits c-Met- and insulin-like growth factor-I receptor-mediated migration and invasion and orthotopic tumor growth in mice.

Pancreatic carcinomas express high levels of urokinase-type plasminogen activator (uPA) and its receptor (uPAR), both of which mediate cell migration and invasion. We investigated the hypotheses that (a) insulin-like growth factor-I (IGF-I)- and hepatocyte growth factor (HGF)-mediated migration and invasion of human pancreatic carcinoma cells require uPA and uPAR function and (b) inhibition of uPAR inhibits tumor growth, retroperitoneal invasion, and hepatic metastasis of human pancreatic carcinomas in mice. Using transwell assays, we investigated the effect of IGF-I and HGF on L3.6pl migration and invasion. We measured the induction of uPA and uPAR following treatment of cells with IGF-I and HGF using immunoprecipitation and Western blot analysis. The importance of uPA and uPAR on L3.6pl cell migration and invasion was studied by inhibiting their activities with amiloride and antibodies before cytokine treatment. In an orthotopic mouse model of human pancreatic carcinoma, we evaluated the effect of anti-uPAR monoclonal antibodies with and without gemcitabine on primary tumor growth, retroperitoneal invasion, and hepatic metastasis. IGF-I and HGF mediated cell migration and invasion in L3.6pl cells. In addition, IGF-I and HGF induced uPA and uPAR expression in L3.6pl cells. In vitro, blockade of uPA and uPAR activity inhibited IGF-I- and HGF-mediated cell migration and invasion. Treatment of mice with anti-uPAR monoclonal antibody significantly decreased pancreatic tumor growth and hepatic metastasis and completely inhibited retroperitoneal invasion. Our study shows the importance of the uPA/uPAR system in pancreatic carcinoma cell migration and invasion. These findings suggest that uPAR is a potential target for therapy in patients with pancreatic cancer.

Regulatory role of c-Met in insulin-like growth factor-I receptor-mediated migration and invasion of human pancreatic carcinoma cells.

Pancreatic carcinoma cells overexpress the insulin-like growth factor-I (IGF-I) receptor (IGF-IR) and the hepatocyte growth factor (HGF) receptor, c-Met, which are both known to mediate tumor cell migration and invasion. We hypothesized that IGF-IR and c-Met cooperate to induce migration and invasion of human pancreatic carcinoma cells and that IGF-I-mediated migration and invasion depend on c-Met. Migration and invasion assays were done with the human pancreatic cancer cell line L3.6pl treated with PBS, IGF-I, HGF, or IGF-I plus HGF. To determine if c-Met is necessary for IGF-IR-mediated migration and invasion, c-Met was down-regulated in L3.6pl cells via adenoviral infection with a c-Met ribozyme before IGF-I treatment. IGF-I and HGF increased cell migration and invasion. Furthermore, IGF-I plus HGF had a greater than additive effect on cell migration and invasion compared with either growth factor alone. Down-regulation of c-Met nearly completely inhibited IGF-I-mediated migration and invasion. Our findings suggest that IGF-IR and c-Met cooperate to induce migration and invasion of human pancreatic carcinoma cells. Furthermore, c-Met is required for both HGF- and IGF-I-mediated migration and invasion. Elucidation of the signaling pathways that contribute to tumor progression and metastasis should provide a foundation for the development of targeted therapies for pancreatic carcinoma.

Expression and function of vascular endothelial growth factor receptor-1 on human colorectal cancer cells.

Vascular endothelial growth factor (VEGF) is associated with tumor angiogenesis and poor prognosis in human colorectal cancer (CRC). VEGF receptor-1 (VEGFR-1 or Flt-1) is a high-affinity receptor for VEGF and is typically considered specific to endothelial cells. Here we report the expression and function of VEGFR-1 in CRC cell lines. VEGFR-1 was expressed in all CRC cell lines studied as determined by RT-PCR, Western blot analysis, FACS, and ELISA. Treatment of the human CRC cell lines HT-29 and SW480 with VEGF-A (a ligand for both VEGFR-1 and -2) or VEGF-B (a ligand specific for VEGFR-1) led to activation of Erk-1/2, SAPK/JNK, and translocation of the p65 subunit of nuclear factor-kappaB into the nucleus. Both VEGF-A and -B led to significant induction of cell motility and invasiveness of CRC cells. Stimulation of cells with VEGF-A or -B also led to larger and more numerous colonies in soft agar. However, activation of VEGFR-1 did not increase CRC cell proliferation. In contrast to the previous paradigm that VEGFRs are not present on tumor cells of epithelial origin, we found that VEGFR-1 is present and functional on CRC cells, and activation by VEGF family ligands can activate processes involved in tumor progression and metastasis.

A small interfering RNA targeting vascular endothelial growth factor inhibits Ewing's sarcoma growth in a xenograft mouse model.

Angiogenesis plays an essential role in tumor growth and metastasis and is a promising therapeutic target for cancer. Vascular endothelial growth factor (VEGF) is a key regulator in vasculogenesis as well as in angiogenesis. TC71 human Ewing's sarcoma cells overexpress VEGF, with a shift in isoform production from membrane-bound VEGF189 to the more soluble VEGF165. Transfection of TC71 cells with a vector-based VEGF targeted small interfering RNA expression system (VEGFsi) inhibited VEGF165 expression by 80% and VEGF165 protein production by 98%, with no alteration in VEGF189 expression. Human microvascular endothelial cell proliferation and migration induced by conditioned medium from VEGFsi-transfected TC71 cells was significantly less than that induced by conditioned medium from TC71 cells and control vector-transfected TC71 cells. Furthermore, after s.c. injection into athymic nu/nu mice, the tumor growth of VEGFsi-expressing TC71 cells was significantly less than that of parental or control vector-transfected cells. Vessel density as assessed by CD31 immunohistochemical analysis and VEGF165 expression as assessed by Northern blotting were also decreased. Intratumor gene therapy with polyethylenimine/VEGFsi also resulted in tumor growth suppression. When inoculated into the tibias of nude mice, VEGFsi-expressing TC71 cells induced osteolytic bone lesions that were less severe than those induced by control groups. These data suggest that targeting VEGF165 may provide a therapeutic option for Ewing's sarcoma.

Molecular mechanisms of resistance to therapies targeting the epidermal growth factor receptor.

Targeted therapies that inhibit the activity of tyrosine kinase receptors such as the epidermal growth factor receptor (EGFR) have shown activity against solid malignancies when used as single agents or in combination with chemotherapy. Although anti-EGFR therapies are active in some patients, eventually disease in nearly all patients will become refractory to therapy. Therefore, a better understanding of the mechanisms of resistance to anti-EGFR therapies is critical to further improve the efficacy of this class of agents. Mechanisms that mediate resistance to anti-EGFR therapies include the presence of redundant tyrosine kinase receptors, increased angiogenesis, and the constitutive activation of downstream mediators. Two recent landmark publications have also shown that specific mutations in the kinase domain of EGFR in some lung carcinomas are associated with markedly improved response rates to an EGFR tyrosine kinase inhibitor. Mutations in the EGFR receptor seem to play a significant role in determining the sensitivity of tumor cells to EGFR inhibitor therapy by altering the conformation and activity of the receptor. As the field of molecular therapeutics continues to evolve, a comprehensive understanding of resistance mechanisms will ultimately lead to refinements in our regimens to provide better care for patients with cancer.

Interleukin-1beta regulates angiopoietin-1 expression in human endothelial cells.

Angiopoietin (Ang)-1 is an important regulator of endothelial cell (EC) survival and stabilization. Ang-1 exerts its biological effects by binding to the EC-specific tyrosine kinase receptor Tie-2, and initiates intracellular signaling in ECs. However, regulatory mechanisms for endothelial Ang-1 expression have not been completely elucidated. In this study, we investigated the effects of angiogenic cytokines and growth factors on Ang-1 expression in human umbilical vein ECs (HUVECs). Northern blot analysis was performed after HUVECs were exposed to interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, platelet-derived growth factor-BB, insulin-like growth factor-1, or vascular endothelial growth factor (VEGF). Both IL-1beta and tumor necrosis factor-alpha caused marked down-regulation of Ang-1 mRNA levels at 4 h with a further decrease observed at 24 h. Using signaling inhibitors, we identified the P38 pathway as the pathway that mediates IL-1beta down-regulation of Ang-1. Furthermore, treatment of cells with IL-1beta indirectly (via down-regulation of Ang-1) led to a decrease in Tie-2 autophosphorylation levels in HUVECs. We previously demonstrated that IL-1beta regulates VEGF expression in tumor cells. This observation was confirmed in ECs in the present study. Because pericytes play a role in regulating EC function, we also determined whether IL-1beta would also down-regulate Ang-1 in human vascular smooth muscle cells. Similar to our findings in HUVECs, we found that IL-1beta decreased Ang-1 expression in human vascular smooth muscle cells. Direct effects of IL-1beta on angiogenesis were investigated by use of an in vivo Gelfoam angiogenesis assay in which IL-1beta produced a significant increase in vessel counts (P = 0.0189). These results suggest that IL-1beta indirectly regulates angiogenesis by modulating the expression of Ang-1. IL-1beta may trigger a proangiogenic response by decreasing Ang-1 levels in ECs and pericytes and up-regulating VEGF in ECs and tumor cells.

Alphavbeta3 integrin antagonist S247 decreases colon cancer metastasis and angiogenesis and improves survival in mice.

Members of the integrin family influence several aspects of tumor progression and metastasis, including cell survival, proliferation, and angiogenesis. Specific integrins such as alpha(v)beta(3) and alpha(v)beta(5) are involved in regulating endothelial cell function, and thus angiogenesis. We evaluated the effect of the alpha(v)beta(3)/alpha(v)beta(5) integrin antagonist S247 on the growth and angiogenesis of colon cancer liver metastases in an orthotopic murine model. Murine colon cancer cells were injected into the spleens of BALB/c mice to produce liver metastases. On day 7, miniature osmotic pumps were implanted into the subcutis to continuously infuse either saline or 70 mg/kg/day S247. All mice were sacrificed when control mice became moribund. Mice that received S247 developed significantly fewer liver metastases than did controls (P < 0.05). Using the same model, a subsequent survival study was performed. Mice were sacrificed when moribund as determined by an observer blinded to the treatment given. Treatment with S247 significantly prolonged overall survival (P < 0.05). Interestingly, primary tumors in the spleen were the cause of death in the S247-treated group as S247 appeared to have little effect on these tumors. Immunohistochemical staining demonstrated a significant reduction of vessels in liver metastases of S247-treated mice (P < 0.001), a significant increase in endothelial cell apoptosis (P < 0.05), and a significant decrease in pericyte coverage (P < 0.0001). To determine the role of S247 on angiogenesis, we examined the effect of S247 in vitro on human umbilical vein endothelial cells (HUVECs) and human vascular smooth muscle cells (hVSMCs). The addition of S247 to HUVECs and hVSMCs growing on vitronectin-coated flasks and in Matrigel significantly impaired cell growth and colony formation, respectively (P < 0.05). Furthermore, S247 completely inhibited the attachment of HUVECs and hVSMCs and increased apoptosis by six- to 9fold compared with controls. In in vitro invasion assays, S247-treated cells demonstrated decreased migration (P < 0.05). In conclusion, S247 demonstrated significant antimetastatic and antiangiogenic activity and impaired both endothelial and hVSMC/pericyte function in vitro and in vivo. The use of agents such as integrin antagonists that target multiple cell types involved in angiogenesis may be a more effective method of inhibiting angiogenesis than agents targeting only the endothelial cells.

Angiopoietin-1 inhibits vascular permeability, angiogenesis, and growth of hepatic colon cancer tumors.

Angiopoietin (Ang)-1 and -2 are critical regulators of embryonic and postnatal neovascularization. Ang-1 activates the endothelial cell-specific tyrosine kinase receptor Tie-2, which in turn leads to enhanced endothelial cell survival and stabilization. The effects of Ang-1 on tumor angiogenesis remain controversial; although we have previously demonstrated that Ang-1 overexpression in colon cancer cells leads to a decrease in s.c. tumor growth, others have shown that Ang-1 may be proangiogenic. Few studies have addressed the role of the Angs in tumors growing in the organ of metastatic growth. We hypothesized that overexpression of Ang-1 may inhibit the growth of colon cancers growing in the liver by inhibition of angiogenesis. We also wanted to investigate the mechanisms by which Ang-1 affects angiogenesis in vivo. Human colon cancer cells (HT29) were stably transfected with an Ang-1 construct or an empty vector (pcDNA) and injected directly into the livers of nude mice. After 37 days, livers were harvested and weighed, and tumor sizes were measured. In an additional experiment, to validate the paracrine effect of Ang-1, various mixtures of control cells and Ang-1-transfected cells were injected into livers, and tumor growth was assessed. Direct effects of recombinant Ang-1 on angiogenesis were studied with an in vivo Gelfoam angiogenesis assay. The impact of Ang-1 on vascular permeability was investigated using an intradermal Miles assay with conditioned media from transfected cells. Liver weights (P < 0.05), tumor volumes (P < 0.05), vessel counts (P < 0.01), and tumor cell proliferation (P < 0.01) in the Ang-1 group were significantly lower than those in the control (pcDNA) group. Tumor vessels in the Ang-1 group developed a significantly higher degree of pericyte coverage (P < 0.02) than vessels in pcDNA tumors. In the cell mixture experiment, even as few as a 1:10 mixture of Ang-1-transfected cells/control cells resulted in a significant reduction of hepatic tumor volumes (P < 0.04). In the angiogenesis assay, vessel counts in Gelfoam implants were significantly decreased by the addition of Ang-1 (P < 0.01). Finally, conditioned medium from Ang-1-transfected cells decreased vascular permeability more than that from control cells (P < 0.05). Our results suggest that Ang-1 is an important regulator of angiogenesis and vascular permeability and that this effect may be secondary to increasing periendothelial support and vessel stabilization. Thus, Ang-1 could potentially serve as an antineoplastic or anti-permeability agent for patients with metastatic colorectal cancer.

Cyclooxygenase-2 is up-regulated by interleukin-1 beta in human colorectal cancer cells via multiple signaling pathways.

Overexpression of cyclooxygenase-2 (COX-2) has been observed in human colorectal cancer. COX-2 expression in human tumors can be induced by growth factors, cytokines, oncogenes, and other factors. The mechanisms regulating COX-2 expression in human colon cancer have not been completely elucidated. We hypothesized that the proinflammatory cytokine interleukin-1 beta (IL-1 beta) mediates COX-2 expression in HT-29 human colon cancer cells. Treatment of HT-29 cells with IL-1 beta induced expression of COX-2 mRNA and protein in a time- and dose-dependent manner. Inhibitors of the extracellular signal-regulated kinase 1/2, c-Jun NH(2)-terminal kinase, P38 mitogen-activated protein kinase, and nuclear factor-kappa B (NF-kappa B) signaling pathways blocked the ability of IL-1 beta to induce COX-2 mRNA. In contrast, Wortmannin, a phosphoinositide 3-kinase inhibitor upstream of protein kinase B/Akt, led to a slight increase in COX-2 mRNA expression after IL-1 beta treatment. Electrophoretic mobility shift assay on nuclear extracts demonstrated that IL-1 beta induced NF-kappa B DNA binding activity in HT-29 cells, and the activated NF-kappa B complex was eliminated after treatment with an inhibitor of NF-kappa B. Supershift assay indicated that the two NF-kappa B subunits, p65 and p50, were involved in activation of NF-kappa B complex by IL-1 beta stimulation. The stability of COX-2 mRNA was not altered by IL-1 beta treatment. These data demonstrate that IL-1 beta induces COX-2 expression in HT-29 cells through multiple signaling pathways and NF-kappa B.

Promises and pitfalls of anti-angiogenic therapy in clinical trials.

A significant body of research has implicated the process of angiogenesis in the growth and spread of tumors. Elucidation of the mechanisms of tumor angiogenesis has led to the development of multiple anti-angiogenic agents. However, the perceived differences between the results of preclinical studies and those of early phases of clinical trials have led to questions being asked regarding the efficacy of these agents. There are many reasons for this discrepancy, including difficulties in the appropriate interpretation of preclinical data and clinical trial design. Further insights into the complex process of angiogenesis are essential for the development of effective anti-angiogenic regimens.