Mouthwash: An anticancer solution

Commercially clinical

The active ingredients of commercially available mouthwash, the bisbiguanide oral disinfectants chlorhexidine (Chlorhexamed, Chlorhexal, Periogard, Corsodyl, and Chlorohex) and alexidine (Esemdent) have been in widespread use for many years. Now, ¹H,¹⁵N-HSQC NMR spectroscopy has been used in studies to investigate the structural changes these compounds can induce that lead to apoptosis in oral cancer cells.

Thorsten Berg and colleagues have demonstrated that common mouthwash ingredients can interfere with protein-protein interactions between the anti-apoptotic Bcl-2 family protein Bcl-xL and its pro-apoptotic binding partners potentially paving the way to a novel treatment for cancer of the mouth. Berg worked with Martin Graeber, Michael Hell, Corinna Gröst, Anders Friberg, Bianca Sperl, and Michael Sattler at the University of Leipzig, the Max Planck Institute for Biochemistry, the Center for Integrated Protein Science Munich, the Helmholtz Center Munich, the Technical University Munich, and ETH Zurich, Switzerland.

Fresh approach to oral cancer

Many people use disinfectant mouthwash to freshen their breath, reduce the risk of tooth decay and to protect their gums from gingivitis as part of their daily oral hygiene routine. But, it turns out that some of the small molecule additives in mouthwash, which reduce oral bacteria numbers, chlorhexidine and alexidine, can also increase the rate of programmed cell death, apoptosis, in cancers of the mouth and throat through a previously unrecognized protein interaction.

Apoptosis is a normal process among healthy cells in foetal development and throughout life. The process sculpts tissues before birth and keeps a check on cell replication. When it fails or is otherwise decreased as occurs in tumour cells, the replication process is not checked and cancer cells carry on dividing, growing into a potentially life-threatening mass. Part of the problem is the over-expression of apoptosis-inhibiting protein.

The team was initially investigating protein-protein interactions they knew to be relevant to human health. The interactions on which they focused were two proteins that control apoptosis through their interaction. The researchers began by screening a collection of more than 4000 substances in a compound library for activity against the interaction between the two proteins, dubbed Bcl-xL and Bad. Most of the compounds screened are small molecules already being used in one clinical setting or another. They used a binding experiment to find out which of the thousands of substances might inhibit the formation of a link between the two target proteins. They also carried out similar binding experiments against a range of other proteins to home in on only those small molecules that were active specifically against the target.

Clinical screen

From this mass screening, Berg and his colleagues plucked out the two mouthwash ingredients. Both inhibit the binding of the apoptosis inhibitor to the apoptosis trigger. They found that chlorhexidine was very specific, but alexidine had very weak effects on other proteins too. In experiments with cultures of cancer cells from various tongue and throat carcinomas the team saw increased apoptosis compared with the much smaller effect on apoptosis in healthy cells.

The team investigated the binding site of chlorhexidine and alexidine on the protein Bcl-xL using ¹H,¹⁵N-HSQC (Heteronuclear Single Quantum Coherence) NMR experiments with a protein construct originally used in the NMR characterization of the interaction between Bcl-xL and Bak. The protein is engineered to lack the unfolded loop domain at residues 45-84, which, based on earlier experiments was known not to be required for anti-apoptotic function of Bcl-xL. The engineered protein also lacks the C- terminal transmembrane domain, which again is not needed for its anti-apoptotic functionality. The NMR shifts were, the team says, consistent with a small molecules binding to the hydrophobic groove of Bcl-xL.

The team's ongoing works is also investigating other small molecule drugs with prior clinical approval for protein-protein interactions in other areas.