Arrest of B16 Melanoma Cells in the Mouse Pulmonary Microcirculation Induces Endothelial Nitric Oxide Synthase-Dependent Nitric Oxide Release that Is Cytotoxic to the Tumor Cells

Abstract

Metastatic cancer cells seed the lung via blood vessels. Because endothelial cells generate nitric oxide (NO) in response to shear stress, we postulated that the arrest of cancer cells in the pulmonary microcirculation causes the release of NO in the lung. After intravenous injection of B16F1 melanoma cells, pulmonary NO increased sevenfold throughout 20 minutes and approached basal levels by 4 hours. NO induction was blocked by N G -nitro-L-arginine methyl ester (L-NAME) and was not observed in endothelial nitric oxide synthase (eNOS)-deficient mice. NO production, visualized ex vivo with the fluorescent NO probe diaminofluorescein diacetate, increased rapidly at the site of tumor cell arrest, and continued to increase throughout 20 minutes. Arrested tumor cells underwent apoptosis with apoptotic counts more than threefold over baseline at 8 and 48 hours. Neither the NO signals nor increased apoptosis were seen in eNOS knockout mice or mice pretreated with L-NAME. At 48 hours, 83% of the arrested cells had cleared from the lungs of wild-type mice but only ϳ55% of the cells cleared from eNOS-deficient or L-NAME pretreated mice. eNOS knockout and L-NAME-treated mice had twofold to fivefold more metastases than wild-type mice, measured by the number of surface nodules or by histomorphometry. We conclude that tumor cell arrest in the pulmonary microcirculation induces eNOS-dependent NO release by the endothelium adjacent to the arrested tumor cells and that NO is one factor that causes tumor cell apoptosis, clearance from the lung, and inhibition of metastasis. In the lung, metastatic neoplasms are the most common malignant tumors. Their formation usually involves hematogenous seeding of metastatic cancer cells into the lung from remote primary tumors. Interactions between intravascular cancer cells and the endothelium are important determinants of metastatic outcome. 1,2 For example, the expression of constitutive and inducible microvascular adhesion molecules, and the release of reactive oxygen species (NO, O 2 Ϫ , and H 2 O 2 ) by endothelial cells or cancer cells can regulate the mechanisms that govern the metastatic process, including cancer cell adhesion and arrest, 3 the production of endothelial matrix metalloproteinases, 4 and cancer cell apoptosis. 5 Evidence from in vitro and in vivo studies has shown that reactive oxygen and nitrogen species can be cytotoxic to neoplastic cells 6 -9 and reduced their adhesion to postcapillary venules. 10 In vivo, we have recently demonstrated that the arrest of intravascular B16F1 melanoma cells in the liver induces the rapid local release of nitric oxide (NO) that causes apoptosis of the melanoma cells and inhibits their subsequent development into hepatic metastases. 5 Because pulmonary endothelial cells generate NO in response to shear stress, 11 we have postulated that there is a comparable cytotoxic mechanism in the lung.Here we provide data showing that the arrest of B16F1 melanoma cells in the pulmonary circulation of wild-type C57B1/6 mice (WT mice) induces the ...

Expression of Constitutive Endothelial, Neuronal and Inducible Nitric Oxide Synthase in the Testis and Epididymis of Horse

“…It is postulated that over-activation of eNOS mediates the cell death program through the generation of NO, and may play a role in the cell-selection process that occurs in the seminiferous tubules. Spermatogenesis in the tubules is essentially a result of ongoing cell proliferation, cell selection, and cell death [7,19].…”

Impact of cirrhosis on the development of experimental hepatic metastases by B16F1 melanoma cells in C57BL/6 mice

“…B16F1 murine melanoma cells (ATCC, Rockville, MD) were cultured and labeled as described previously. 8,9,12 Cells used for intravital videomicroscopy were labeled with calcein AM (Molecular Probes, Eugene, OR). Cells used for confocal microscopy were labeled with MitoTracker orange dye (Molecular Probes).…”

“…NO generation was monitored in situ by employing a fluorescent NO probe, 4,5-diaminofluorescein diacetate. 12,13 The procedure was performed as described previously. 10 Briefly, after the liver perfusion was established, the perfusate was replaced by 4,5-diaminofluorescein.…”

“…10,12,13 After 15 minutes perfusion, the basal NO generation, expressed as the intensity of fluorescence signals in the periportal zone (zone 1) area, increased greater than 2-fold in both control groups. In comparison, there was less than one fold increase of fluorescence intensity in the cirrhosis model (P Ͻ .05).…”

Prognostic role of syncytin expression in breast cancer

“…However, some fusions between cancer cells and host cells have been found to result in a more aggressive phenotype-a phenomenon that may reflect loss of tumor suppressor genes from the hybrid cells [9]. In addition, expression of increased amounts of proinflammatory mediators like IL-1b may modulate interactions with the host immune system, and increased production of nitric oxide by breast cancer cells and by endothelial cells is known to affect tumor cell apoptosis and metastasis [14][15][16][17][18]. Accordingly, syncytin is expressed in breast cancer and such expression may be expected to influence tumor growth and progression.…”