Gerald A. Soff

Combined effects of angiostatin and ionizing radiation in antitumour therapy

Angiogenesis, the formation of new capillaries from pre-existing vessels, is essential for tumour progression. Angiostatin, a proteolytic fragment of plasminogen that was first isolated from the serum and urine of tumour-bearing mice, inhibits angiogenesis and thereby growth of primary and metastatic,, tumours. Radiotherapy is important in the treatment of many human cancers, but is often unsuccessful because of tumour cell radiation resistance,. Here we combine radiation with angiostatin to target tumour vasculature that is genetically stable and therefore less likely to develop resistance. The results show an antitumour interaction between ionizing radiation and angiostatin for four distinct tumour types, at doses of radiation that are used in radiotherapy. The combination produced no increase in toxicity towards normal tissue. In vitro studies show that radiation and angiostatin have combined cytotoxic effects on endothelial cells, but not tumour cells. In vivo studies show that these agents, in combination, target the tumour vasculature. Our results provide support for combining ionizing radiation with angiostatin to improve tumour eradication without increasing deleterious effects.

Expression of plasminogen activator inhibitor type 1 by human prostate carcinoma cells inhibits primary tumor growth, tumor-associated angiogenesis, and metastasis to lung and liver in an athymic mouse model.

Expression of urokinase-type plasminogen activator (uPA) by malignant cells correlates with an aggressive phenotype, including increased invasiveness, tumor-associated angiogenesis, and metastases. Plasminogen activator inhibitor type 1 (PAI-1) is undetectable in cells of some aggressive malignancies, but present in the stroma of tumor-associated microvasculature. This analysis of an athymic mouse model of prostate carcinoma further defines the role of the uPA/PAI-1/plasmin system in primary growth and metastasis. A marked increase in PAI-1 expression was induced in clones of the aggressive human prostate carcinoma line, PC-3, by stable transfection. Primary PC-3 tumors, in mice, were significantly smaller when derived from PAI-1 expressing versus control cells. PAI-1 expression reduced the density of tumor-associated microvasculature by 22-38%. Microscopic metastases were quantitated using stable expression of the chromogenic label (beta-galactosidase) in control and PAI-1 expressing cells. PAI-1 expression resulted in a significant inhibition of lung metastases, and liver metastases. Expression of PAI-1 by malignant prostate cells resulted in a less aggressive phenotype, presumably by inhibition of uPA activity, suggesting pharmacologic or molecular inhibition of uPA activity as a potential therapeutic target.

Tumor–host interactions in the gallbladder suppress distal angiogenesis and tumor growth: Involvement of transforming growth factor β1

Angiogenesis inhibitors produced by a primary tumor can create a systemic anti-angiogenic environment and maintain metastatic tumor cells in a state of dormancy. We show here that the gallbladder microenvironment modulates the production of transforming growth factor (TGF)-β1, a multifunctional cytokine that functions as an endogenous anti-angiogenic and anti-tumor factor in a cranial window preparation. We found that a wide variety of human gallbladder tumors express TGF-β1 irrespective of histologic type. We implanted a gel impregnated with basic fibroblast growth factor or Mz-ChA-2 tumor in the cranial windows of mice without tumors or mice with subcutaneous or gallbladder tumors to study angiogenesis and tumor growth at a secondary site. Angiogenesis, leukocyte–endothelial interaction in vessels and tumor growth in the cranial window were substantially inhibited in mice with gallbladder tumors. The concentration of TGF-β1 in the plasma of mice with gallbladder tumors was 300% higher than that in the plasma of mice without tumors or with subcutaneous tumors. In contrast, there was no difference in the plasma levels of other anti- and pro-angiogenic factors. Treatment with neutralizing antibody against TGF-β1 reversed both angiogenesis suppression and inhibition of leukocyte rolling induced by gallbladder tumors. TGF-β1 also inhibited Mz-ChA-2 tumor cell proliferation. Our results indicate that the production of anti-angiogenesis/proliferation factors is regulated by tumor–host interactions.

Smooth muscle cell expression of type I cyclic GMP-dependent protein kinase is suppressed by continuous exposure to nitrovasodilators, theophylline, cyclic GMP, and cyclic AMP.

A key component of the nitric oxide-cyclic guanosine monophosphate (cGMP) pathway in smooth muscle cells (SMC) is the type I GMP-dependent protein kinase (PK-G I). Activation of PK-G I mediates the reduction of cytoplasmic calcium concentrations and vasorelaxation. In this manuscript, we demonstrate that continuous exposure of SMC in culture to the nitrovasodilators S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP) results in approximately 75% suppression of PK-G I mRNA by 48 h. PK-G I mRNA and protein were also suppressed by continuous exposure to cGMP analogues 8-bromo- and 8-(4-chlorophenylthio) guanosine-3,5-monophosphate or the cAMP analogue dibutyryl cAMP. These results suggest that activation of one or both of the cyclic nucleotide-dependent protein kinases mediates PK-G I mRNA suppression. Using isoform-specific cDNA probes, only the PK-G I alpha was detected in SMC, either at baseline or after suppression, while PK-G I beta was not detected, indicating that isoform switch was not contributing to the gene regulation. Using the transcription inhibitor actinomycin D, the PK-G I mRNA half-life in bovine SMC was observed to be 5 h. The half-life was not affected by the addition of SNAP to actinomycin D, indicating no effect on PK-G I mRNA stability. Nuclear runoff studies indicated a suppression of PK-G I gene transcription by SNAP. PK-G I suppression was also observed in vivo in rats given isosorbide dinitrate in the drinking water, with a dose-dependent suppression of PK-G I protein in the aorta. PK-G I antigen in whole rat lung extract was also suppressed by administration of isosorbide or theophylline in the drinking water. These data may contribute to our understanding of nitrovasodilator resistance, a phenomenon resulting from continuous exposure to nitroglycerin or other nitrovasodilators.

Regulation of the expression of cyclic GMP-dependent protein kinase by cell density in vascular smooth muscle cells.

Cyclic GMP-dependent protein kinase (cGMP kinase) is the major receptor protein for cGMP in vascular smooth muscle. Vascular smooth muscle cells (VSMC) isolated from the rat aorta express type I cGMP kinase at high levels, but expression decreases markedly upon passage of the cells. In primary or early passage, the expression of cGMP kinase is lowest when cells are plated at low density as assessed by immunological and Northern analyses. Expression increases at confluence and is maintained in postconfluent cultures. With repeated passaging, however, the levels of cGMP kinase decrease even in confluent and postconfluent cultures so that after several passages enzyme levels are undetectable. The decrease in expression in passaged cells is not due to exposure to serum-derived growth factors, but rather on the repeated exposure of cells to conditions in which cell density is reduced (i.e., subculturing). These results indicate that aortic VSMC grown at low density or those repetitively passaged have reduced expression of cGMP kinase, and thus may not represent appropriate cultures with which to investigate the role of nitric oxide and cGMP in VSMC function.

Update on angiogenesis inhibitors.

Purpose of review This review highlights recent developments in the pathophysiology of treatment-induced mucosal barrier injury, outlines the application of new diagnostic tools, focuses on risk factors and complications, and offers an up-to-date overview on treatment options. Recent findings Treatment-induced mucosal damage is now thought to occur in five phases: initiation, up-regulation and message generation, amplification and signaling, ulceration, and healing. It is now possible to assess gut mucosal damage both by sugar permeability tests and serum citrulline. Amifostine reduces the oral mucositis of stem cell transplantation recipients after radiotherapy and high-dose chemotherapy. Palifermin (recombinant human keratinocyte growth factor 1), a trophic growth factor, has been shown to reduce significantly both the incidence and duration of severe mucositis after myeloablative therapy and may have the potential to reduce gut mucosal damage. Summary Treatment-induced mucosal barrier injury is a complex, dynamic pathobiological process manifested not only in the oral cavity but throughout the entire digestive tract, diminishing the quality of life and predisposing the patient to serious clinical complications. Therefore, it is important to detect mucosal damage induced by cytotoxic therapy adequately to be able to test the efficacy of new therapeutic options for preventing or ameliorating this complication.

Alternatively spliced human tissue factor promotes tumor growth and angiogenesis in a pancreatic cancer tumor model

INTRODUCTION Tissue Factor (TF) expression is observed in many types of cancer, associated with more aggressive disease, and thrombosis. Alternatively-spliced human tissue factor (asHTF) has recently been identified in which exon 5 is deleted. asHTF is soluble due to the substitution of the transmembrane and cytoplasmic domains of exon 6 with a unique COOH-terminal domain. MATERIALS AND METHODS We examine the expression and function of asHTF and full-length Tissue Factor ((FL)TF) in six human pancreatic cancer cells. Further, we transfected asHTF, (FL)TF, and control expression vectors into a non-expressing, human pancreatic cancer line (MiaPaCa-2). We studied the procoagulant activity of asHTF and (FL)TF and the effect on tumor growth in mice. RESULTS asHTF is expressed in 5 of 6 human pancreatic cancer cell lines, but not in normal human fibroblasts, nor the MiaPaCa-2 line. (FL)TF conferred procoagulant activity, but asHTF did not. Transfected cells were injected subcutaneously in athymic mice. Interestingly, compared with control transfection, (FL)TF expression was associated with reduced tumor growth (mean 7 mg vs 85 mg), while asHTF-expression was associated with enhanced tumor growth (mean 389 mg vs. 85 mg). asHTF expression resulted in increased mitotic index and microvascular density. CONCLUSIONS These data suggests that asHTF expression promotes tumor growth, and is associated with increased tumor cell proliferation and angiogenesis in vivo. Our results raise a new perspective on the understanding of the relationship between TF expression and cancer growth, by showing a dissociation of the procoagulant activity of (FL)TF and the cancer-promoting activity of asHTF.

Activated-Protein-C Resistance in Cancer Patients

BACKGROUND Resistance to activated protein C (aPC) is usually linked to factor V Leiden, but may occur in other disorders associated with hypercoagulability. In this study, we investigated the frequency of resistance to aPC in patients with advanced cancer and examined the relationship of aPC resistance to other markers of coagulation activation. METHODS Patients (n = 39) had established diagnosis of advanced cancer; controls (n = 20) were healthy persons. aPC resistance was measured as the ratio of activated partial thromboplastin times with and without aPC (aPC-sensitivity ratio, aPC-SR). The factor V Leiden mutation was detected by a polymerase-chain-reaction based technique. Other assays were performed by standard laboratory methods. Data were analyzed using t tests and the Pearson correlation. RESULTS aPC-SR was below 2 SD for 5 of the cancer patients (13%), but none of the controls; only 1 of the 5 had the factor V Leiden mutation. aPC-SR was inversely correlated (p < 0.01) with factor VIII and fibrinogen in patients and with prothrombin activation fragment 1.2 (F1.2) in controls. Patient factor VIII, von Willebrand factor, (vWF), fibrinogen, F1.2 and D dimer were all significantly increased (p < 0.01; antithrombin III, protein C and proteins were similar to controls. Factor VIII correlated with vWF (p < 0.001) and F1.2 with d-dimer (p < 0.001). Other associations (p < 0.05) were observed between factor V and protein C, fibrinogen and protein C, factor V and antithrombin III and protein C and antithrombin III. Four cancer patients had a history of thromboembolism; their aPC-SR was similar to that of patients without thrombosis. Of the several coagulation measures examined, only vWF was higher in the patients with thrombosis (p = 0.01). INTERPRETATION Cancer patients have evidence of intravascular coagulation and increases in procoagulants and may have aPC resistance. The aPC resistance is not due to factor V Leiden, but is rather associated with elevated levels of factor VIII and fibrinogen, and in itself does not predict thrombosis.

Analysis of incidence and clinical outcomes in patients with thromboembolic events and invasive exocrine pancreatic cancer

Pancreatic adenocarcinoma is among the most common malignancies associated with thromboembolic events (TEs); however, reported incidence figures vary significantly and contain small patient cohorts. Pancreatic cancer‐specific thrombosis studies examining the correlation between clinical variables, including thrombosis timing and the impact of thrombosis on survival, have not been reported.

Safe and effective use of rivaroxaban for treatment of cancer-associated venous thromboembolic disease: a prospective cohort study

Low-molecular weight heparin (LMWH) has been the standard of care for treatment of venous thromboembolism (VTE) in patients with cancer. Rivaroxaban was approved in 2012 for the treatment of pulmonary embolism (PE) and deep vein thrombosis (DVT), but no prior studies have been reported specifically evaluating the efficacy and safety of rivaroxaban for cancer-associated thrombosis (CAT). Under a Quality Assessment Initiative (QAI), we established a Clinical Pathway to guide rivaroxaban use for CAT and now report a validation analysis of our first 200 patients. A 200 patient cohort with CAT (PE or symptomatic, proximal DVT), whose full course of anticoagulation was with rivaroxaban, were accrued. In competing risk analysis, primary endpoints at 6 months included new or recurrent PE or symptomatic proximal lower extremity DVT, major bleeding, clinically-relevant non-major bleeding leading to discontinuation of rivaroxaban, or death. In competing risk analysis, the 6 months cumulative incidence of new or recurrent VTE was 4.4 % (95 % CI = 1.4–7.4 %), major bleeding was 2.2 % (95 % CI = 0−4.2 %) and all-cause mortality 17.6 % (95 % CI = 11.7–23.0 %). In this cohort of 200 patients with active cancer and CAT the rates of new or recurrent VTE and major bleeding were comparable to the cancer subgroup analysis from the EINSTEIN studies. The results of our Clinical Pathway provide guidance on Rivaroxaban use for treatment of CAT, and suggest that safety and efficacy is preserved, compared with past-published experience with LMWH.