Benzethonium chloride: a novel anticancer agent identified by using a cell-based small-molecule screen

Purpose: This study aims to identify a novel therapeutic agent for head and neck Cancer and to evaluate its antitumor efficacy.

Experimental design: A cell-based and phenotype-driven high-throughput screening of approximately 2,400 biologically active or clinically used compounds was done using a tetrazolium-based assay on FaDu (hypopharyngeal squamous Cancer) and NIH 3T3 (untransformed mouse embryonic fibroblast) cells, with secondary screening done on C666-1 (nasopharyngeal Cancer) and GM05757 (primary normal human fibroblast) lines. The "hit" compound was assayed for efficacy in combination with standard therapeutics on a panel of human Cancer cell lines. Furthermore, its mode of action (using transmission electron microscopy and flow cytometry) and its in vivo efficacy (using xenograft models) were evaluated.

Results: Benzethonium chloride was identified as a novel cancer-specific compound. For benzethonium (48-hour incubation), the dose required to reduce cell viability by 50% was 3.8 micromol/L in FaDu, 42.2 micromol/L in NIH 3T3, 5.3 micromol/L in C666-1, and 17.0 micromol/L in GM05757. In vitro, this compound did not interfere with the effects of cisplatin, 5-fluorouracil, or gamma-irradiation. Benzethonium chloride induced Apoptosis and activated caspases after 12 hours. Loss of mitochondrial membrane potential (DeltaPsiM) preceded cytosolic Ca2+ increase and cell death. In vivo, benzethonium chloride ablated the tumor-forming ability of FaDu cells, delayed the growth of xenograft tumors, and combined additively with local tumor radiation therapy. Evaluation of benzethonium chloride on the National Cancer Institute/NIH Developmental Therapeutics Program 60 human Cancer cell lines revealed broad-range antitumor activity.

Conclusions: This high-throughput screening identified a novel antimicrobial compound with significant broad-spectrum Anticancer activity.