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

Abstract

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 (f4,500 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 (IC 50 s). 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.

High-Throughput Cell-Based Screening of 4910 Known Drugs and Drug-like Small Molecules Identifies Disulfiram as an Inhibitor of Prostate Cancer Cell Growth

“…The other HTS hit compounds already in ongoing clinical trials 5 in prostate cancer included doxorubicin, Taxol/paclitaxel, mitoxantrone, suramin, camptothecin, staurosporine, ixabepilone, 17-AAG, epothilone B, MS-275, and vinblastine sulfate salt. Also, cardiac glycosides, previously identified as hits inhibiting KLK expression in a recent HTS study with breast cancer cells, and reported to have antiproliferative and apoptotic effects also in PC-3, LNCaP, and DU145 cells (30,31), were identified among antiproliferative compounds in our screen, supporting the overall functionality of our assay. Only a few cancer cell selective growth inhibitory compounds were identified in the HTS experiment.…”

Targeting Calcium Signaling Induces Epigenetic Reactivation of Tumor Suppressor Genes in Cancer

“…These drugs are relevant for cancer treatment since epidemiologic studies have shown that patients treated with cardiac glycosides for heart failure exhibit a reduced cancer rate and less aggressive cancers (11,33,34). Moreover, in vitro studies have previously shown selective cancer killing for some of these cardiac glycosides which we now relate to Ca 2+ signaling and TSGs reactivation (35). Our data suggest that their chemopreventive or chemotherapeutic activity may be in part due to their ability to target Ca 2+ fluxes and reactivate epigenetically silenced TSGs.…”

Progress in Small Molecule Therapeutics for the Treatment of Retinoblastoma

“…However, it is worth noting that there has been no mechanistic explanation reported for efficacy of cardenolide glycosides in retinoblastoma, and these compounds are common hits in cellular screens, particularly as cancer therapies. [129–132] Digoxin and ouabain may simply be promiscuous compounds, [133] and considerably less attractive than therapeutic candidates which kill selectively based on mechanisms specific to retinoblastoma.…”