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 ∼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 μmol/L in FaDu, 42.2 μmol/L in NIH 3T3, 5.3 μmol/L in C666-1, and 17.0 μmol/L in GM05757. In vitro, this compound did not interfere with the effects of cisplatin, 5-fluorouracil, or γ-irradiation. Benzethonium chloride induced apoptosis and activated caspases after 12 hours. Loss of mitochondrial membrane potential (ΔΨM) 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.

[1]  R. Pazdur,et al.  Approval summary: imatinib mesylate in the treatment of metastatic and/or unresectable malignant gastrointestinal stromal tumors. , 2002, Clinical cancer research : an official journal of the American Association for Cancer Research.

[2]  S. Baudet,et al.  Sildenafil and vardenafil, types 5 and 6 phosphodiesterase inhibitors, induce caspase-dependent apoptosis of B-chronic lymphocytic leukemia cells. , 2003, Blood.

[3]  R. Kyle,et al.  Drug therapy: Multiple myeloma , 2004 .

[4]  B. O'Sullivan,et al.  Potential efficacy of p16 gene therapy for EBV‐positive nasopharyngeal carcinoma , 2004, International journal of cancer.

[5]  Ping Chen,et al.  Overriding Imatinib Resistance with a Novel ABL Kinase Inhibitor , 2004, Science.

[6]  S. Canevari,et al.  Alterations of choline phospholipid metabolism in ovarian tumor progression. , 2005, Cancer research.

[7]  P. Flood,et al.  Ketamine and its preservative, benzethonium chloride, both inhibit human recombinant α7 and α4β2 neuronal nicotinic acetylcholine receptors in Xenopus oocytes , 2001 .

[8]  A. D. Russell,et al.  Antiseptics and Disinfectants: Activity, Action, and Resistance , 1999, Clinical Microbiology Reviews.

[9]  M. Krause,et al.  Recovery from sublethal damage during fractionated irradiation of human FaDu SCC. , 2005, Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology.

[10]  Thomas D. Y. Chung,et al.  A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays , 1999, Journal of biomolecular screening.

[11]  Clemencia Pinilla,et al.  Small-molecule antagonists of apoptosis suppressor XIAP exhibit broad antitumor activity. , 2004, Cancer cell.

[12]  B. Murphy,et al.  Randomized phase III evaluation of cisplatin plus fluorouracil versus cisplatin plus paclitaxel in advanced head and neck cancer (E1395): an intergroup trial of the Eastern Cooperative Oncology Group. , 2005, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[13]  R. Gilbert,et al.  Potential utility of BimS as a novel apoptotic therapeutic molecule. , 2004, Molecular therapy : the journal of the American Society of Gene Therapy.

[14]  S. Gupta,et al.  Differential antiproliferative and apoptotic response of sanguinarine for cancer cells versus normal cells. , 2000, Clinical cancer research : an official journal of the American Association for Cancer Research.

[15]  C. Elmets,et al.  Photodynamic therapy of multiple nonmelanoma skin cancers with verteporfin and red light-emitting diodes: two-year results evaluating tumor response and cosmetic outcomes. , 2004, Archives of dermatology.

[16]  B. Druker,et al.  Lessons learned from the development of an abl tyrosine kinase inhibitor for chronic myelogenous leukemia. , 2000, The Journal of clinical investigation.

[17]  Stuart L. Schreiber,et al.  Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes , 1991, Cell.

[18]  M. Minden,et al.  The BH3 domain of BAD fused to the Antennapedia peptide induces apoptosis via its alpha helical structure and independent of Bcl-2 , 2001, Cell Death and Differentiation.

[19]  A. McPhail,et al.  Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. , 1971, Journal of the American Chemical Society.

[20]  D. Blakey,et al.  ZD6126: a novel vascular-targeting agent that causes selective destruction of tumor vasculature. , 2002, Cancer research.

[21]  Gleevec approved for first-line treatment of CML. , 2003, FDA consumer.

[22]  M Crompton,et al.  The mitochondrial permeability transition pore and its role in cell death. , 1999, The Biochemical journal.

[23]  S. Holbeck,et al.  Update on NCI in vitro drug screen utilities. , 2004, European journal of cancer.

[24]  R. Wong,et al.  Identification of benzethonium chloride in commercial grapefruit seed extracts. , 2001, Journal of agricultural and food chemistry.

[25]  William Pao,et al.  Epidermal growth factor receptor mutations, small-molecule kinase inhibitors, and non-small-cell lung cancer: current knowledge and future directions. , 2005, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[26]  L. Kublik,et al.  Antitumor effect of avermectins. , 2004, European journal of pharmacology.

[27]  M. V. Heiden,et al.  Bcl-xL prevents cell death following growth factor withdrawal by facilitating mitochondrial ATP/ADP exchange. , 1999, Molecular cell.

[28]  O. Sanchez,et al.  A conditionally replicating adenovirus for nasopharyngeal carcinoma gene therapy. , 2004, Molecular therapy : the journal of the American Society of Gene Therapy.

[29]  L. Nutt,et al.  Cardiac glycosides stimulate Ca2+ increases and apoptosis in androgen-independent, metastatic human prostate adenocarcinoma cells. , 2000, Cancer research.

[30]  B. Stockwell.  Chemical genetics: ligand-based discovery of gene function , 2000, Nature Reviews Genetics.

[31]  G. Steele,et al.  Cancer: Principles and Practice of Oncology , 1983 .

[32]  T. Liu,et al.  p16 gene therapy: a potentially efficacious modality for nasopharyngeal carcinoma. , 2003, Molecular cancer therapeutics.

[33]  V. Devita,et al.  Cancer : Principles and Practice of Oncology , 1982 .

[34]  A. Markoe,et al.  Phase II Study of Tolerance and Efficacy of Hyperfractionated Radiation Therapy and 5-Fluorouracil, Cisplatin, and Paclitaxel (Taxol) and Amifostine (Ethyol) in Head and Neck Squamous Cell Carcinomas: A-3 Protocol , 2005, American journal of clinical oncology.

[35]  P. Schiff,et al.  Promotion of microtubule assembly in vitro by taxol , 1979, Nature.

[36]  S. Korsmeyer,et al.  An inhibitor of Bcl-2 family proteins induces regression of solid tumours , 2005, Nature.

[37]  D. Hedley,et al.  Respiratory chain-generated oxidative stress following treatment of leukemic blasts with DNA-damaging agents. , 2001, Experimental cell research.

[38]  S. Waldman,et al.  Guanylyl cyclase C agonists regulate progression through the cell cycle of human colon carcinoma cells , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[39]  R. Gilbert,et al.  Combination Bcl-2 Antisense and Radiation Therapy for Nasopharyngeal Cancer , 2005, Clinical Cancer Research.

[40]  T. Brac.  Intracellular polycationic molecules cause reversible swelling of the rough endoplasmic reticulum. , 1983, Tissue & Cell.

[41]  I. Tannock,et al.  Management of advanced prostate cancer after first-line chemotherapy. , 2005, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[42]  Stuart L. Schreiber,et al.  A mammalian protein targeted by G1-arresting rapamycin–receptor complex , 1994, Nature.

[43]  G. Opelz,et al.  Apoptosis-mediated selective killing of malignant cells by cardiac steroids: maintenance of cytotoxicity and loss of cardiac activity of chemically modified derivatives. , 2003, International immunopharmacology.

[44]  T. Brac.  The charge distribution in the rough endoplasmic reticulum/Golgi complex transitional area investigated by microinjection of charged tracers. , 1984, Tissue & cell.

[45]  John Calvin Reed,et al.  Endoplasmic reticulum stress: cell life and death decisions. , 2005, The Journal of clinical investigation.

[46]  O. Cope,et al.  Multiple myeloma. , 1948, The New England journal of medicine.

[47]  Mechthild Krause,et al.  Experimental study on different combination schedules of VEGF-receptor inhibitor PTK787/ZK222584 and fractionated irradiation. , 2003, Anticancer research.

[48]  Fei-Fei Liu.  Novel gene therapy approach for nasopharyngeal carcinoma. , 2002, Seminars in cancer biology.

[49]  L. Dick,et al.  Potent and selective inhibitors of the proteasome: dipeptidyl boronic acids. , 1998, Bioorganic & medicinal chemistry letters.

[50]  N. Spinner,et al.  Homozygous deletion of the death receptor DR4 gene in a nasopharyngeal cancer cell line is associated with TRAIL resistance. , 2000, International journal of oncology.

[51]  R. Bauer,et al.  Molecular modes of action of cantharidin in tumor cells. , 2004, Biochemical pharmacology.

[52]  Peng Huang,et al.  Targeting endoplasmic reticulum protein transport: a novel strategy to kill malignant B cells and overcome fludarabine resistance in CLL. , 2006, Blood.