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High-Throughput Screening Identifies Cardiac Glycosides as Potent Inhibitors of Human Tissue Kallikrein Expression: Implications for Cancer Therapies

Authors' Affiliations: ¹ Department of Laboratory Medicine and Pathobiology, University of Toronto; ² Samuel Lunenfeld Research Institute and Department of Pathology and Laboratory Medicine, Mount Sinai Hospital; ³ Sinai-McLaughlin Assay and Robotic Technologies Facility, Samuel Lunenfeld Research Institute, Toronto, Ontario, Canada

Requests for reprints: Eleftherios P. Diamandis, FRCPC, Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 6th Floor, 60 Murray Street, Toronto, Ontario, M5T 3L9. Phone: 416-586-8443; Fax: 416-586-8628; E-mail: ediamandis{at}mtsinai.on.ca.

Purpose: Human tissue kallikreins (KLK) comprise a subgroup of 15 homologous secreted serine proteases. Primarily known for their clinical use as cancer biomarkers (e.g., PSA), KLKs have recently been directly implicated in cancer-related processes, including invasion, angiogenesis, and tumor growth regulation. Therefore, the identification of compounds that would modulate expression of KLKs might be of considerable therapeutic value.

Experimental Design: A cell-based high-throughput screening (HTS) of three small molecule libraries (4,500 compounds) was undertaken; KLK expression in the breast cancer cell line MDA-MB-468 was assessed with sensitive ELISAs.

Results: The initial screening resulted in 66 "putative hits" that decreased KLK5 expression by at least 50% over control. Secondary screening and mini-dose-response assays resulted in 21 "validated hits." These 21 compounds were clustered in only three distinct functional families and were further analyzed in vitro to determine their effectiveness (IC₅₀s). Hits that failed to show dose-responsiveness or interfered with the viability of the cells were excluded. Multiple members of the cardiac glycoside family were found to be novel inhibitors of KLK expression, acting at low concentrations (10-50 nmol/L). Furthermore, members of the same family induced marked decreases in c-MYC and c-FOS expression, in a dose-dependent manner that correlated the KLK inhibition, suggesting a transcriptional mechanism of regulation of KLK expression.

Conclusions: We conclude that cardiac glycosides can dramatically suppress the transcription of KLKs and that these effects may be linked to proto-oncogene (c-myc/fos) expression. These findings may partially explain the recently realized antineoplastic actions of cardiac glycosides.