Glial cell-induced endothelial morphogenesis is inhibited by interfering with extracellular signal-regulated kinase signaling.

PURPOSE: Tumor vasculature provides the infrastructure by which malignant tissue can be nourished; therefore, targeting angiogenesis may be an effective means of treating cancer. We showed previously that SNB19 glioblastoma cells modulate bovine retinal endothelial cells in cocultures to form capillary-like network structures, that matrix metalloproteinase-9 (MMP-9) expression is critical for endothelial morphogenesis, and that MMP-9 expression in glioblastoma cells is regulated by extracellular signal-regulated kinase-1 (ERK-1). In the present study, we investigated whether interfering with the activation of this mitogen-activated protein (MAP) kinase would repress MMP-9 synthesis and inhibit capillary formation. EXPERIMENTAL DESIGN: Cocultures of bovine retinal endothelial and SNB19 cells were analyzed for MMP-9 secretion, and phospho- and total ERK levels. These cocultures were treated with PD98059, a specific inhibitor of MAP/ERK kinase 1, or transfected with dominant-negative ERK-1 mutant containing expression vector. Alterations in capillary-like structure formation, and actin cytoskeleton and secretion of vascular endothelial growth factor (VEGF), MMP-9, and tissue inhibitor of metalloproteinase-1 were determined by immunofluorescence, gelatin zymography, and Western blotting. RESULTS: We found that inhibition of the ERK-1/2 pathway with PD98059 abrogated glial cell-mediated capillary formation by the endothelial cells and reduced the levels of MMP-9 in the coculture. Strikingly, the abrogation of MAP kinase signaling by a dominant-negative ERK-1 mutant inhibited glial-induced capillary network formation by reducing VEGF levels and MMP-9 activity and increasing the levels of tissue inhibitor of metalloproteinase-1. Inhibition of ERK activity also disrupted the formation of the actin cytoskeleton, a prerequisite for endothelial cell migration. CONCLUSION: The mechanism underlying activation of ERK is involved in reorganization of the actin cytoskeleton, and induction of VEGF and MMP-9, thereby stimulating endothelial cell morphogenesis. These studies clearly provide experimental evidence that ERK inhibition diminishes glial-induced endothelial-cell morphogenesis; therefore, interfering with ERK signaling may be a viable approach to target angiogenesis.     

Meeting highlights: updated international expert consensus on the primary therapy of early breast cancer.

This account of the highlights of the eighth St Gallen (Switzerland) meeting in 2003 emphasizes new information that has emerged during the 2 years since the seventh meeting in 2001. This article should be read in conjunction with the report of that earlier meeting. Recommendations for patient care are so critically dependent on assessment of endocrine responsiveness that the importance of high-quality steroid hormone receptor determination and standardized quantitative reporting cannot be overemphasized. The International Consensus Panel modified the risk categories so that only endocrine receptor-absent status was sufficient to reclassify an otherwise low-risk, node-negative disease into the category of average risk. Absence of steroid hormone receptors also was recognized as indicating endocrine nonresponsiveness. Some important areas highlighted at the recent meeting include: (1) recognition of the separate nature of endocrine-nonresponsive breast cancer-both invasive cancers and ductal carcinoma-in-situ; (2) improved understanding of the mechanisms of acquired endocrine resistance, which offer exciting prospects for extending the impact of successful sequential endocrine therapies; (3) presentation of high-quality evidence indicating that chemotherapy and tamoxifen should be used sequentially rather than concurrently; (4) availability of a potential alternative to tamoxifen for treatment of postmenopausal women with endocrine-responsive disease; and (5) the promise of newly defined prognostic and predictive markers.     

Overexpression of the receptor tyrosine kinase Tie-1 intracellular domain in breast cancer.

OBJECTIVE: Tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (Tie-1) is a receptor tyrosine kinase that regulates angiogenesis and antiapoptotic survival signaling. Tie-1 expression is generally associated with endothelial cells and neovascularization. We previously identified Tie-1 in human breast tumor samples using a PCR-based screen for protein kinases expressed in breast tumors. The purpose of this study was to determine the cell types expressing Tie-1, whether Tie-1 is expressed in tumor cells, and to examine the regulation of Tie-1 in breast cancer. METHODS: Tie-1 expression was analyzed by Western blot and immunohistochemistry using an antibody to the carboxy terminus of Tie-1. Tie-1 expression was determined in a variety of cancer cell lines, clinical breast and colon tumor samples, and in corresponding benign tissue from the same patient. Tie-1 expression and distribution in breast tumors was scored by immunohistochemistry. RESULTS: Tie-1 was overexpressed in 14/23 breast tumors compared with 0/9 corresponding normal tissues from the same patients. Immunohistochemistry revealed that Tie-1 was overexpressed in epithelial breast cancer cells and ductal carcinoma in situ. In all breast tumor samples, Tie-1 was expressed as a truncated 40- to 43-kD doublet consisting of the intracellular portion of the protein, which contains the tyrosine kinase catalytic domain. The 40- to 43-kD Tie-1 doublet was expressed in a broad variety of cell lines. CONCLUSIONS: We have shown that breast cancer cells overexpress a cleaved form of the Tie-1 protein. Our results implicate the intracellular domain of Tie-1, which includes the catalytic kinase domain, in breast cancer progression.     

Prostate cancer detection by GSTP1 methylation analysis of postbiopsy urine specimens.

PURPOSE: We assess the feasibility of a urinary test for prostate cancer detection in a high-risk patient cohort based on methylation-specific PCR analysis of the pi class glutathione S-transferase (GSTP1) gene promoter. EXPERIMENTAL DESIGN: A total of 45 men underwent transrectal ultrasound-guided biopsy of the prostate for suspected malignancy. Clean-catch voided urine specimens were prospectively collected from each patient immediately after biopsy. Genomic DNA was isolated from urine specimens and subjected to sodium bisulfite modification. Methylation of the GSTP1 promoter was examined in a blinded manner by methylation-specific PCR analysis and correlated with pathology results, and clinical information was obtained from the patient record. RESULTS: Methylation of GSTP1 in the urine was detected in a total of 18 of 36 (50%) informative cases. A total of 7 of 18 (39%) patients with prostate adenocarcinoma identified on their initial biopsy had detectable urinary GSTP1 methylation (58% sensitivity among informative cases). Abnormal urinary GSTP1 methylation was also detected in 7 of 21 (33%) patients without evidence of cancer on biopsy and in 4 of 6 (67%) patients diagnosed with atypia or high-grade prostatic intraepithelial neoplasia. CONCLUSIONS: We have demonstrated the feasibility of a novel, noninvasive molecular approach for the detection of epigenetic changes associated with prostate cancer. A screening test based on GSTP1 methylation in the urine specimens of patients with suspected prostate malignancy may be a useful adjunct to serum screening tests and digital rectal examination findings for identification of men at increased risk of harboring cancer despite a negative biopsy. This molecular assay has potential application for stratification of patients into low- and high-risk groups for surveillance versus repeat biopsy.     

Intravenous delivery of adenovirus-mediated soluble FLT-1 results in liver toxicity.

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent angiogenic agent and plays a major role in tumor growth and metastases. We have previously reported the locoregional (i.p.) delivery of adenovirus-mediated antiangiogenic soluble FLT-1 (sFLT-1; a naturally encoded potent VEGF antagonist) gene therapy to inhibit VEGF action in a murine ovarian carcinoma model. This study was predicated on the fact that systemic delivery of sFLT-1 might allow an approach for therapy of disseminated tumor. The purpose of this study is to test the effects of i.v. delivered, adenovirus-mediated sFLT-1 on the survival duration in a murine ovarian tumor model and to evaluate the safety of i.v.-delivered versus i.p.-delivered adenovirus-mediated sFLT-1 in non-tumor-bearing mice. EXPERIMENTAL DESIGN: To determine the effects of i.v.-administered adenovirus-mediated sFLT-1 on survival duration of mice bearing i.p. human ovarian tumors, an E1A/B-deleted, (replication-deficient) infectivity-enhanced recombinant adenovirus AdRGDGFPsFLT-1 encoding cDNA for both sFLT-1 and GFP (green fluorescent protein), a control adenovirus AdRGDGFP encoding GFP alone, or PBS was delivered i.v. The therapeutic effect of sFLT-1 was evaluated by survival duration of the mice. Furthermore, the safety of i.v.- or i.p.-delivered adenovirus-mediated sFLT-1 was evaluated by administering AdRGDGFPsFLT-1, AdRGDGFP, or PBS either i.v. or i.p. into non-tumor-bearing mice. Adenovirus-mediated gene expression was determined by determining GFP expression using fluorescent microscopy and by assessing sFLT-1 expression in liver, lungs, spleen, and kidneys by immunohistochemistry using anti-FLT-1 monoclonal antibody. Systemic levels of sFLT-1 were evaluated by ELISA and the toxicity was evaluated by histopathology. RESULTS: The i.v. delivery of AdRGDGFPsFLT-1 in the ovarian tumor model resulted in a shorter duration of survival of the mice as compared with the control group. Furthermore, in the safety evaluation experiment, i.v. administration of AdRGDGFPsFLT-1 in non-tumor-bearing mice principally localized to the liver. This localization lead to sFLT-1 overexpression, mainly in the liver, resulting in hemorrhage and tissue toxicity. However, i.p. delivery of AdRGDGFPsFLT-1 did not localize principally to the liver, leading to negligible expression of sFLT-1, and no intrahepatic hemorrhage or toxicity was observed. The i.v. delivery of the control virus AdRGDGFP also principally localized to the liver, leading to GFP expression mainly in the liver. However, neither hemorrhage nor morphological cytotoxicity was observed. i.p. delivery of AdRGDGFP resulted in ectopic localization to the liver with very little GFP expression and no toxicity. These results suggest that overexpression of sFLT-1 in the liver as a result of i.v. delivery is hepatotoxic. CONCLUSIONS: Our results suggest that i.v. delivery of the sFLT-1 gene via replication-deficient, infectivity-enhanced recombinant adenoviral vectors will result in overexpression of sFLT-1 in the liver leading to unacceptable hepatotoxicity. Tumor-specific targeting of the vectors and tumor-specific expression strategies should be used to ensure a clinically useful antiangiogenesis gene therapy.     

Pharmacodynamic Evaluation of CCI-779, an Inhibitor of mTOR, in Cancer Patients.

CCI-779 is an ester of rapamycin and inhibitor of mammalian target of rapamycin (mTOR) currently in Phase II clinical development for the treatment of patients with cancer. CCI-779 interacts with mTOR and inhibits its kinase activity, resulting in inhibition of the mTOR-regulated translational controllers p70(s6) kinase and 4E-BP1. Ultimately, CCI-779 decreases the translation of mRNAs involved in the control of the cell cycle, resulting in cell cycle arrest. The objective of this study was to develop a method to determine the pharmacodynamic effects of CCI-779 suitable for use in clinical trials. Exposure of Raji lymphoblastoid cells to increasing concentrations of rapamycin resulted in a linear concentration-dependent inhibition of p70(s6) kinase activity, suggesting that p70(s6) kinase activity could be an appropriate marker for mTOR-interacting agents. In subsequent experiments, treatment of nude mice bearing the CCI-779 susceptible breast cancer cell line MDA-468 with a single dose of 10 mg/kg CCI-779 resulted in a >80% inhibition of p70(s6) kinase activity in peripheral blood mononuclear cells (PBMCs) 72 h after treatment. Importantly, the degree of p70(s6) kinase inhibition was identical in PBMCs and simultaneously collected tumor tissue, suggesting that the PBMCs are an adequate surrogate tissue for p70(s6) kinase activity in vivo. The intrasubject coefficient of variation of p70(s6) kinase activity measured in PBMCs collected from five healthy volunteers on days 1, 4, and 8 was 14%, indicating that p70(s6) kinase activity in PBMCs remains relatively stable over time. Finally, p70(s6) kinase activity was measured in PBMCs from nine patients with renal cell cancer treated with a single dose of 25, 75, or 250 mg of CCI-779 i.v. (three patients each). PBMCs were collected on days 2, 4, and 8 after CCI-779 treatment. In this small data set, eight of the nine patients had evidence of p70(s6) kinase activity inhibition after treatment that was independent of the administered dose. There was a significant linear association between time to disease progression and inhibition of p70(s6) kinase activity 24 h after treatment. In conclusion, these results indicate that the pharmacodynamic effects of CCI-779 can be determined using a p70(s6) kinase assay in PBMCs. This assay is currently being incorporated in Phase I and II studies with CCI-779 to determine its relationship with dose and plasma concentration of the agent and its value as a predictor of treatment efficacy.     

Limited access trial using amifostine for protection against cisplatin- and three-hour paclitaxel-induced neurotoxicity: a phase II study of the Gynecologic Oncology Group.

PURPOSE: The purpose of this study was to determine whether amifostine (WR-2721) prevents or ameliorates clinically significant (grade 2 to 4) neurotoxicity associated with cisplatin and 3-hour paclitaxel chemotherapy.Materials and METHODS: The chemotherapy program consisted of intravenous paclitaxel 175 mg/m2 over 3 hours followed by amifostine 740 mg/m2 and cisplatin 75 mg/m2 administered over 90 minutes beginning 15 minutes after amifostine administration. At baseline, before each treatment cycle, and for 3 months after completing chemotherapy, patients were evaluated for evidence of neurotoxicity and other treatment-related adverse effects using three methods: standard clinical evaluation (National Cancer Institute common toxicity criteria [CTC] grading), a neurotoxicity questionnaire to assess symptoms and limitations imposed by peripheral neuropathy, and vibration perception threshold (VPT) testing. RESULTS: Four of 27 assessable patients developed grade 2 to 4 neurotoxicity based on clinical assessments and CTC grading. This number of neuropathic events exceeded the predetermined threshold level for a second stage of accrual and the study was closed. CONCLUSION: Amifostine's level of activity in this trial was insufficient to warrant further study in a phase III trial. Based on the receiver operating characteristic analysis, it would appear that VPT measurements are less sensitive to the development of peripheral neuropathy than the neurotoxicity questionnaire. The questionnaire, referred to as the Functional Assessment of Cancer Therapy/Gynecologic Oncology Group-Neurotoxicity, may be used instead of VPT measurements in future studies of chemotherapy-induced peripheral neuropathy.     

Synergistic activity of recombinant human endostatin in combination with adriamycin: analysis of in vitro activity on endothelial cells and in vivo tumor progression in an orthotopic murine mammary carcinoma model.

PURPOSE: Current combination treatment strategies in malignancy are designed to evaluate the use of cytotoxic drugs and antiangiogenic agents. Endostatin, a fragment of collagen XVIII, specifically inhibits proliferation, migration, and differentiation of endothelial cells in vitro as well as angiogenesis and tumor progression in in vivo models. In this study, we determine the antitumor effect of rhEndostatin administered alone or in combination with Adriamycin against established orthotopic murine mammary carcinoma. EXPERIMENTAL DESIGN: Mice bearing orthotopically established DA-3 mammary adenocarcinoma tumors received varying doses of rhEndostatin alone and in combination with Adriamycin to assess tumor growth inhibition. Additional studies of this in vivo combination included a determination of Adriamycin-induced cardiotoxicity and in vitro effects on human umbilical vein endothelial cell proliferation and cord formation. RESULTS: For single-agent activity, optimal tumor growth inhibition was observed after s.c. administration of 50 mg/kg/day rhEndostatin or 5 mg/kg Adriamycin injected i.v. every 4 days. Combination of Adriamycin with optimal or suboptimal doses of rhEndostatin resulted in synergistic inhibition of DA-3 tumor growth. Importantly, unlike other antiangiogenic agents, rhEndostatin did not exacerbate the cardiotoxicity of Adriamycin. The synergistic interaction between rhEndostatin and Adriamycin was also observed in vitro for inhibition of human umbilical vein endothelial cell proliferation and inhibition of cord formation. CONCLUSIONS: These data suggest that the synergy observed with rhEndostatin in combination with Adriamycin is exerted at the level of the endothelial cell and can result in enhanced tumor growth inhibition. The potential benefit of Adriamycin used in combination with rhEndostatin is being considered for clinical evaluation.     

Distribution of ran-domised controlled trials of drugs for post-operative nausea and vomiting

Purpose: Randomised controlled trials (RCTs) guide therapeutic decisions. But which RCTs are done; which omitted; and which should be done? This study illustrates a method to explore these questions applied to drugs for post-operative nausea and vomiting (PONV). Methods: Review artides listed 18 drugs for PONV. All RCTs of these drugs for PONV were sought. The first drug mentioned in an RCT was counted and tabulated against others in all the arms of the RCT (against itself in a dose-ranging RCT). Additional drugs mentioned in these RCTs were added to the study, for a total of 40 drugs. Results: Drugs involved in the most RCTs were: ondansetron 131 RCTs; propofol 118; droperidol 74; metoclopramide 67; granisetron 52; scopolamine 22; tropisetron 16. Drugs involved in the fewest RCTs: two drugs with 2 RCTs; twelve drugs with one; three with none. Probability that this distribution occurred by chance:P<0.00001; that the distribution of dose-ranging RCTs occurred by chance:P<0.001. Regression of RCT numbers on cost: R=0.86,P<0.0001; on year of drug introduction: R=0.14. Of 1600 possible comparisons of drugs for PONV, (including dose-ranging) 97.8% have never been published. Conclusion: Although some antiemetic drugs for PONV have been studied in large numbers of RCTs, many have not been adequately evaluated. Finding relevant RCTs and tabulating their comparison arms is useful for directing future research, and is applicable to any symptom or disorder.