A synthetic tissue kallikrein inhibitor suppresses cancer cell invasiveness.
Journal: 2001/December - American Journal of Pathology
ISSN: 0002-9440
Abstract:
Serine proteinases modulate the interaction of tumor cells with extracellular matrix components during extravasation and metastasis. The serine proteinase tissue kallikrein has been previously demonstrated in several human adenocarcinomas, and we presently report the localization of immunoreactive kallikrein and its mRNA in pancreatic adenocarcinoma. In addition, a synthetic peptide-based inhibitor specific for tissue kallikrein (FE999024) was used in our studies to explore a possible role for kallikrein in cancer cell invasiveness. Matrigel invasion assays were performed with a human breast-cancer cell line, MDA-MB-231, which expresses tissue kallikrein in culture. In the presence of FE999024 invasion through Matrigel was inhibited in a dose-dependent manner to a maximum of 39%. We also developed a novel ex vivo assay in which breast cancer cells are infused into the pulmonary circulation of artificially ventilated explanted rat lungs. At intervals up to 6 hours after infusion pulmonary invasion was quantified by bronchial alveolar lavage to recover human cancer cells from the airspace. Invading cells in the lung interstitium were also quantified after immunohistochemistry with a monoclonal antibody specific for human cytokeratin 18. The synthetic kallikrein inhibitor attenuates breast cancer cell invasion into the airspace by 33% when quantified by lavage recovery and up to 34% as quantified in the lung interstitium by cytokeratin 18 immunostaining. Our results indicate tissue kallikrein may participate in the invasion and metastasis of human adenocarcinomas. The newly developed explanted rodent lung assay should be useful for the study of cancer cells, neutrophils, or other extravasating cells.
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Am J Pathol 159(5): 1797-1805

A Synthetic Tissue Kallikrein Inhibitor Suppresses Cancer Cell Invasiveness

From the Department of Biochemistry and Molecular Biology,*
Medical University of South Carolina, Charleston, South Carolina; and the Ferring Research Institute,
University of Southampton Research Centre, Southampton, United Kingdom
Accepted 2001 Jul 27.

Abstract

Serine proteinases modulate the interaction of tumor cells with extracellular matrix components during extravasation and metastasis. The serine proteinase tissue kallikrein has been previously demonstrated in several human adenocarcinomas, and we presently report the localization of immunoreactive kallikrein and its mRNA in pancreatic adenocarcinoma. In addition, a synthetic peptide-based inhibitor specific for tissue kallikrein ({"type":"entrez-nucleotide","attrs":{"text":"FE999024","term_id":"207420231"}}FE999024) was used in our studies to explore a possible role for kallikrein in cancer cell invasiveness. Matrigel invasion assays were performed with a human breast-cancer cell line, MDA-MB-231, which expresses tissue kallikrein in culture. In the presence of {"type":"entrez-nucleotide","attrs":{"text":"FE999024","term_id":"207420231"}}FE999024 invasion through Matrigel was inhibited in a dose-dependent manner to a maximum of 39%. We also developed a novel ex vivo assay in which breast cancer cells are infused into the pulmonary circulation of artificially ventilated explanted rat lungs. At intervals up to 6 hours after infusion pulmonary invasion was quantified by bronchial alveolar lavage to recover human cancer cells from the airspace. Invading cells in the lung interstitium were also quantified after immunohistochemistry with a monoclonal antibody specific for human cytokeratin 18. The synthetic kallikrein inhibitor attenuates breast cancer cell invasion into the airspace by 33% when quantified by lavage recovery and up to 34% as quantified in the lung interstitium by cytokeratin 18 immunostaining. Our results indicate tissue kallikrein may participate in the invasion and metastasis of human adenocarcinomas. The newly developed explanted rodent lung assay should be useful for the study of cancer cells, neutrophils, or other extravasating cells.

Abstract

Cancer cells exploit serine proteinases to influence the local blood supply, extravasate into and out of vessels, and to migrate through tissue matrix during metastasis. The serine proteinase tissue kallikrein has been localized in human adenocarcinomas and related cell lines from a number of organs including prostate, breast, pituitary, colon, ovary, endometrium, kidney, and esophagus. 1-6 Our results demonstrate its expression in pancreatic adenocarcinoma as well. The best known function of kallikrein is the cleavage of low-molecular weight kininogen to release the kinin peptides bradykinin and lys-bradykinin. Kinins are locally active hormones that mediate classical inflammatory responses such as increased vascular permeability, vasodilation, and increased local blood flow. 7 By binding to endothelial bradykinin B2 receptors, kinin stimulates release of potent vasodilators such as nitric oxide, prostacydin, and endothelium-derived hyperpolarizing factor. 8 Kinin’s ability to vasodilate and increase permeability could enhance a tumor’s supply of nutrients and growth factors. Recently, enhanced vascular permeability in a murine sarcoma tumor model was investigated with icatibant (HOE140), a bradykinin B2 receptor antagonist. Icatibant not only decreased tumor vascular permeability, it also reduced primary tumor growth by 32%. 9

Another important new finding indicated that the kallikrein-kinin system stimulated angiogenesis in an in vivo model. 10 In this study the femoral artery was removed in mice to induce hindlimb ischemia, and kallikrein gene delivery significantly increased capillary density and blood flow to the affected limb. This effect was reversed by bradykinin B2 and B1 receptor antagonists, demonstrating a role for both receptors in the angiogenic response. An increased expression of tissue kallikrein and bradykinin B2 receptors, as well as kinin’s promotion of growth, have previously been shown in cultured microvascular endothelial cells. 11,12In vivo, the kininogen substrate is abundant in plasma and tissues, 7 so the expression and availability of kallikrein are most likely the rate-limiting factors in kinin production.

Our goal was to investigate whether tissue kallikrein possibly facilitates cancer cell invasiveness. Tissue kallikrein has been localized to neutrophils 13 and has been postulated to be involved in neutrophil extravasation through the vascular endothelium and into tissues. Kinin activation of endothelial B2 receptors has been shown to cause increased intracellular calcium and subsequent endothelial retraction, 14 a critical step in diapedesis. In addition to the enzymatic formation of vasoactive kinins, tissue kallikrein in vitro efficiently activates two matrix-degrading metalloproteases that are important for cancer cell mobility: progelatinase A (72-kd gelatinase, MMP-2) and progelatinase B (92-kd gelatinase, MMP-9). 15,16 Our present results demonstrate that a tissue kallikrein inhibitor suppresses cancer cell invasiveness in in vitro assays and provide evidence indicating tissue kallikrein may enhance cancer cell metastasis. Tissue kallikrein inhibition may be of therapeutic value in the treatment of metastatic disease.

Footnotes

Address reprint requests to Dr. Julie Chao, Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 171 Ashley Ave., Charleston, South Carolina 29425. E-mail: .ude.csum@joahc

Supported by National Institutes of Health grants HL 44083 and HL 29397.

Footnotes

References

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