Frank Reiher

Inducer-stimulated Fas targets activated endothelium for destruction by anti-angiogenic thrombospondin-1 and pigment epithelium–derived factor

Natural inhibitors of angiogenesis are able to block pathological neovascularization without harming the preexisting vasculature. Here we show that two such inhibitors, thrombospondin-1 and pigment epithelium–derived factor, derive specificity for remodeling vessels from their dependence on Fas/Fas ligand (FasL)-mediated apoptosis to block angiogenesis. Both inhibitors upregulated FasL on endothelial cells. Expression of the essential partner of FasL, Fas/CD95 receptor, was low on quiescent endothelial cells and vessels but greatly enhanced by inducers of angiogenesis, thereby specifically sensitizing the stimulated cells to apoptosis by inhibitor-generated FasL. The anti-angiogenic activity of thrombospondin-1 and pigment epithelium–derived factor both in vitro and in vivo was dependent on this dual induction of Fas and FasL and the resulting apoptosis. This example of cooperation between pro- and anti-angiogenic factors in the inhibition of angiogenesis provides one explanation for the ability of inhibitors to select remodeling capillaries for destruction.

Inhibition of tumor growth by systemic treatment with thrombospondin-1 peptide mimetics

Many normal human cells produce thrombospondin‐1 (TSP‐1), a potent antiangiogenic protein that promotes vascular quiescence. In various organ systems, including the brain, breast and bladder and in fibroblasts, TSP‐1 secretion is reduced during tumorigenesis, thereby allowing induction of the vigorous neovascularization required for tumor growth and metastasis. Full‐length and short TSP‐1–derived peptides inhibit angiogenesis by inducing endothelial cell apoptosis and thus disrupting the vasculature of the growing tumor. CD36 expressed on the surface of endothelial cells functions as the primary antiangiogenic receptor for TSP‐1. A D‐isoleucyl enantiomer of a TSP‐1 heptapeptide specifically inhibits the proliferation and migration of capillary endothelial cells. DI‐TSP, an approximately 1 kDa capped version of this peptide, is also antiangiogenic in vitro, with a specific activity approaching that of the 450 kDa parental molecule. Here, we show that DI‐TSP delivered systemically dose‐dependently inhibits the growth of murine melanoma metastases in syngeneic animals and that its more soluble isomer, DI‐TSPa, similarly blocks the progression of primary human bladder tumors in an orthotopic model in immune‐deficient mice. Like intact TSP‐1, these peptide mimetics had no effect on cancer cells growing in vitro but markedly suppressed the growth of endothelial cells by inducing receptor‐dependent apoptosis. Antibodies raised against CD36 blocked the ability of peptides to induce apoptosis in endothelial cells but had no effect on tumor necrosis factor‐α–induced apoptosis. In vivo, the peptide mimetics were associated with a significantly reduced microvessel density and increased apoptotic indices in both the endothelial and tumor cell compartments. Such short peptides targeted to a specific antiangiogenic receptor, potent and easy to synthesize, show great promise as lead compounds in clinical antiangiogenic strategies.

c-Jun N-terminal kinase activation is required for the inhibition of neovascularization by thrombospondin-1

Thrombospondin-1 (TSP-1) is a potent inhibitor of angiogenesis that acts directly on endothelial cells via the CD36 surface receptor molecule to halt their migration, proliferation, and morphogenesis in vitro and to block neovascularization in vivo. Here we show that inhibitory signals elicited by TSP-1 did not alter the ability of inducers of angiogenesis to activate p42 and p44 mitogen-activated protein kinase (MAPK). Rather, TSP-1 induced a rapid and transient activation of c-Jun N-terminal kinases (JNK). JNK activation by TSP-1 required engagement of CD36, as it was blocked by antagonistic CD36 antibodies and stimulated by short anti-angiogenic peptides derived from TSP-1 that act exclusively via CD36. TSP-1 inhibition of corneal neovascularization induced by bFGF was severely impaired in mice null for JNK-1, pointing to a critical role for this stress-activated kinase in the inhibition of neovascularization by TSP-1.

p53 AND MICROVESSEL DENSITY IN PRIMARY RESECTION SPECIMENS OF SUPERFICIAL BLADDER CANCER

PURPOSE p53 Regulates angiogenesis in fibrosarcoma and correlative studies suggest a similar role for muscle invasive bladder cancer. We evaluated the associations of p53 status and microvessel density with pathological features and clinical outcomes in a large population of patients with superficial bladder cancer. In addition, we assessed the correlation of p53 status with microvessel density, which would suggest the regulation of angiogenesis by p53. MATERIALS AND METHODS We stained 84 primary bladder resection specimens, including 55 stage pTa, 29 stage pT1, 27 grade 1, 35 grade 2 and 22 grade 3 samples, for p53, CD31 and CD34. The relationships of p53 or microvessel density and tumor stage-grade or clinical recurrence-progression were analyzed by analysis of variance and pairwise comparison analysis for least significant difference, and Pearson correlation coefficients. Only patients with no previous biopsy were included in analysis to preclude interference by granulation tissue related neovascularization. The 4 samples with significant inflammation were also excluded from study. RESULTS At a mean followup of 33 months (range 1 to 93) 34 of 84 patients (40.4%) experienced 1 or more tumor recurrences and 10 (11.9%) had stage and/or grade progression. Statistically significant associations were observed of p53 immunostaining and microvessel density with tumor stage and grade (p <0.05). However, the association of p53 status with microvessel density was weak and not statistically significant. Similar results were observed for the CD31 and CD34 based estimates of microvessel density. Neither p53 status nor microvessel density correlated with recurrence or progression. CONCLUSIONS Our study confirms the strong association of p53 and microvessel density with the well established prognostic factors of grade and stage in superficial bladder cancer, supporting other evidence of an important role for p53 and angiogenesis in the tumor biology of this disease. However, our data argue against a primary role of p53 in the regulation of angiogenesis in superficial bladder cancer. This study, which to our knowledge is the first to focus on primary resection specimens, suggests that other genetic or environmental factors may contribute to the regulation of angiogenesis in superficial bladder cancer.

The role of hypoxia and p53 in the regulation of angiogenesis in bladder cancer.

PURPOSE Our previous studies defined thrombospondin-1 (TSP-1) and vascular endothelial growth factor (VEGF) as the primary mediators of angiogenesis in the bladder and the loss of inhibitory TSP-1 as a key event in the transition to an angiogenic phenotype during bladder cancer development. We evaluated the role of p53, which is commonly inactivated in bladder cancer, and hypoxia in the regulation of angiogenesis in the bladder. MATERIALS AND METHODS The p53 status was modulated in normal urothelial and bladder cancer cells, and conditioned media was collected under normal oxygen or hypoxic (0.5% O2) conditions. Angiogenic activity was evaluated with the endothelial cell migration assay, and the levels of secreted TSP-1 and VEGF were determined by Western blot analysis and enzyme-linked immunosorbent assay, respectively. RESULTS Retroviral mediated expression of the E6 oncoprotein reduced wild-type p53 levels in normal urothelial cells by greater than 90% but did not significantly alter TSP-1 or VEGF levels, while total inductive and inhibitory activities remained unchanged. Adenoviral mediated expression of wild-type p53 was confirmed in 4 bladder cancer cell lines by Western blot analysis for p53 and its downstream effector protein p21 (2.5 to 5.0-fold increase). TSP-1 levels remained unchanged but the levels of secreted VEGF in the high grade UMUC-3 and 253J cell lines were significantly decreased 5 to 50-fold and a corresponding decrease in net angiogenic activity was observed. However, (increased expression) of p53 had no effect on the angiogenic activity of the low grade RT4 or high grade HT1376 bladder cancer cells. Hypoxia converted normal urothelial cell derived conditioned media from anti-angiogenic to angiogenic and increased the angiogenic activity of bladder cancer cell derived conditioned media. This change was due to 2.5 to 6-fold hypoxic up-regulation of VEGF because the expression of inhibitory TSP-1 was not significantly altered. CONCLUSIONS Our results suggest that p53 does not regulate angiogenesis in the bladder in the setting of an otherwise normal genome and gene therapy with wild-type p53, which is currently being studied for this cancer, may have only limited effects on angiogenesis. In contrast, hypoxia regulates angiogenesis in this system, primarily through its effects on VEGF.

Management and outcome of bilateral testicular germ cell tumors : A 25-year single center experience

Objectives:  To analyze risk factors, management, histology, and outcome of bilateral testicular germ cell tumors (TGCT) based on a 25‐year single center experience.

Results of Immunochemotherapy with Interleukin-2, Interferon-α2 and 5-Fluorouracil in the Treatment of Metastatic Renal Cell Cancer

Objective: In patients with advanced metastatic renal cell carcinoma (RCC) seen at a single institution, the toxicity and long-term clinical effects of a combination therapy with recombinant interleukin-2 (rIL-2), recombinant interferon-α2 (rIFN-α2) and 5-fluorouracil (5-FU) were evaluated. Method: From August 1992 through August 1997, 47 consecutive patients (38 men) with metastatic RCC were treated using rIL-2 and rIFN-α2 subcutaneously in combination with intravenous 5-FU. An average of 2.4 cycles/patient (range 1–9) was administered. Results: Toxicity grades II and III (World Health Organization) were observed in 24 and 17 patients, respectively. We achieved 9 major responses (7 complete responses (CR) and 2 partial responses (PR)) for an objective response rate of 19.1% (95% confidence interval 9.1–33.3%). A further 13 patients (27.7%) had a stabilization of disease. After a mean follow-up of 17.9 (2–53) months, 4 patients are alive with no evidence of disease. The 1- and 3-year survival probability was 70 and 37%, respectively. In an univariate analysis, two prognostic factors were correlated with disease outcome: Karnofsky performance index (p = 0.01) and the presence of bone metastases (p = 0.023). Conclusion: This triple-drug combination therapy was effective in the treatment of progressive RCC in almost every fifth patient.

Antiangiogenic Approaches to Renal Cell Carcinoma

Angiogenesis-the formation of new blood vessels from preexisting ones-is a complex process regulated by a number of soluble factors as well as important interactions between endothelial cells, extracellular matrix components, and adjacent cells (1-5). Activation of the endothelial cell, which occurs when the balance between proangiogenic and antiangiogenic signals within a given microenvironment tilts in a positive direction, leads initially to increased expression of proteases, allowing the endothelial cell to mobilize itself and release inducers sequestered within the matrix (1,6,7). This is followed by endothelial-cell proliferation and migration and culminates in reorganization of the endothelial cell plexus to form tubules and eventually capillary structures that can conduct blood. Most proangiogenic factors-such as VEGF and basic fibroblast growth factor (bFGF)-are peptide growth factors that bind to transmembrane-receptor tyrosine kinases on the surface of the endothelial cell, initiating intracellular transduction pathways resulting in cellular activation (8,9). Other important angiogenic factors-the angiopoietins-further modulate this process by stabilizing or destabilizing interactions between small blood vessels and adjacent pericytes (10 ). Expression of angiopoietin 2 results in dissociation of pericytes, which can lead to endothelial-cell activation or vascular regression, depending on whether angioinductive or angioinhibitory signals predominate (10,11). In contrast, angiopoietin 1 stabilizes interactions with pericytes and promotes vascular quiescence (10,12).

RANDOMISED STUDY OF DOCETAXEL (D) AND DEXAMETHASONE (DX) WITH LOW OR HIGH DOSE ESTRAMUSTINE (E) FOR PATIENTS WITH ADVANCED HORMONE-REFRACTORY PROSTATE CANCER (HRPC)

4653 Background: D-based chemotherapy is a burgeoning option for men with advanced HRPC. Alone or in combination with E, D has been shown to improve median survival. In this study we tested the combination of D with two different doses of E in patients (pts) with HRPC to improve response rates and to lower side effects. METHODS 72 metastatic HRPC pts were randomly assigned to receive D (70 mg/m2 IV, d2, q3w) and E (3 × 280 mg/d PO starting 1 day prior to D, for 5 consecutive days) for arm A or E (3 x 140 mg/d PO starting 1 day prior to D, for 3 consecutive days) for arm B. Premedication with oral DX at a total daily dose of 16 mg, in divided doses two times a day was administered in arm A on day 1 to 5 and in arm B on day 1 to 3. Initially, 6 cycles were administered and repeated after significant PSA rise. Pts were monitored for PSA response, time to progression (TTP), survival and toxicity. RESULTS PSA declines of ≥75%, ≥50% and ≤50% were 36.8%, 55.3% and 44.7% in arm A and 38.2%, 67.6% and 32.4% in arm B, respectively (P = .442). TTP in Arm A and Arm B were 11 months (95% CI, 7-14) versus 14 months (95% CI, 8-19), P = .6911) and overall survival 21 months (95% CI, 6-35) versus 22 months (95% CI, 18-27), respectively, (P = .4149). The primary treatment-related side effects observed in arm A and arm B were granulocytopenia (34% and 29%, P = .663) and thrombotic complications caused by E (four pts (11%) and one pt (3%), respectively, P = .206). Associated baseline factors with overall survival in univariate analysis were ECOG performance status (P < .001), hemoglobin level (P < .001), bone pain (P < .001), and PSA (P < .097) and in multivariate analysis ECOG performance status (95% CI, 2.9-13.9) and bone pain (95% CI, 3.2-20.1), (P < .001). CONCLUSIONS In this randomized phase II study the combination of D and E had substantial activity in HRPC. We did not find a statistically significant difference of higher dose of E in combination with D compared to a lower dose of E and D regarding PSA response, TTP and survival. However, there was a tendency of higher toxicity in the high dose E group. These treatment-related toxicities were mainly hematologic and manageable. The results of this study support the assertion that estramustine is not necessary in docetaxel-based treatment regimens. No significant financial relationships to disclose.