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B16 Melanoma Cell Arrest in the Mouse Liver Induces Nitric Oxide Release and Sinusoidal Cytotoxicity: A Natural Hepatic Defense against Metastasis¹

Department of Pathology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R3E OW3 [H. H. W., E. S. R., N. A., D. M. N., F. W. O.], and Faculty of Pharmacy, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2 [A. R. M., B. B. H.]

The formation of liver metastases involves interactions between intravascular cancer cells and the hepatic microvasculature. Here we provide evidence that the arrest of intravascular B16F1 melanoma cells in the liver induces a rapid local release of nitric oxide (NO) that causes apoptosis of the melanoma cells and inhibits their subsequent development into hepatic metastases. B16F1 melanoma cells (5 x 10⁵) labeled with fluorescent microspheres were injected into the portal circulation of C57BL/6 mice. The production of NO in vivo was detected by electron paramagnetic resonance spectroscopy ex vivo using an exogenous NO-trapping agent. A burst of NO was observed in liver samples examined immediately after tumor cell injection. The relative electron paramagnetic resonance signal intensity was 667 ± 143 units in mice injected with tumor cells versus 28 ± 5 units after saline injection (P < 0.001). Two-thirds of cells arrested in the sinusoids compared with the terminal portal venules (TPVs). By double labeling of B16F1 cells with fluorescent microspheres and a TdT-mediated UTP end labeling assay, we determined that the melanoma cells underwent apoptosis from 4–24 h after arrest. The mean rate of apoptosis was 2-fold greater in the sinusoids than in the TPVs at 4, 8, and 24 h after injection (P < 0.05–0.01). Apoptotic cells accounted for 15.9 ± 0.8% of tumor cells located in the sinusoids and 7.1 ± 0.9% of tumor cells in the TPVs. The NO synthase inhibitor N_G-nitro-L-arginine methyl ester completely blocked the NO burst (P < 0.001) and inhibited the apoptosis of B16F1 cells in the sinusoids by 77%. However, the rate of tumor cell apoptosis in the TPVs was not changed. There were 5-fold more metastatic nodules in the livers of N_G-nitro-L-arginine methyl ester-treated mice (P < 0.05). The inactive enantiomer N_G-nitro-D-arginine methyl ester had no effect on the initial NO burst or on apoptosis of tumor cells in vivo. Both annexin V phosphatidylserine plasma membrane labeling and DNA end labeling of apoptotic cells were demonstrated after a 5-min exposure (a time equivalent to the initial transient NO induction in vivo) of B16F1 cells to a NO donor in vitro. These results identify the existence of a natural defense mechanism against cancer metastasis whereby the arrest of tumor cells in the liver induces endogenous NO release, leading to sinusoidal tumor cell killing and reduced hepatic metastasis formation.

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