Bleomycin: cancer drug with a hidden flaw

Rohanah Hussain is a Senior Beamline Scientist, working on the B23 beamline at Diamond

While screening compounds produced by bacteria in the 1960s, scientists made a serendipitous discovery. They stumbled upon a molecule called bleomycin with anticancer properties. Since then, this life-saving drug has been used to treat a variety of tumours from squamous cell carcinomas to lymphomas. The drug works by chopping up DNA in cancer cells, and this DNA-drug interaction has been characterised in the past. However, when bleomycin enters the bloodstream, it may interact with plasma proteins and less is known about how this impacts the drug’s effectiveness. Bleomycin shows promising outcomes when tested on cancer cells grown in the lab, but the serum extracts used in lab cultures have a different mix of proteins to the sera of cancer patients, so it’s worth exploring whether plasma proteins in patients could sequester the drug and reduce its effectiveness.

Beamline scientists at B23 were determined to explore this overlooked issue. Led by Rohanah Hussain, they harnessed ultraviolet light from synchrotron radiation to explore how the drug binds plasma proteins using a technique called circular dichroism.

Circular dichroism is the differential absorption of left- and right-circularly polarised ultraviolet light passing through a liquid solution containing biomolecules, in this case proteins with drugs The CD measurement is displayed as a curve (spectrum) of which shape reflects the architecture on  how the protein in solution is folded in helical, ribbon, turn and unordered segments.  Drug binding to protein can affect such a folding that is used to identify and quantify drug binding interaction, in this case bleomycin with the two major blood proteins. Another unique experiment carried out at B23 beamline is the use of the powerful synchrotron beamlight to irradiate multiple of times the protein-drug mixtures for photostability assessment, which varies depending upon the strength of the drug binding interactions.

Hussain explained:

First, the team assessed whether Blenoxane^®, a commercial preparation of bleomycin, could bind to two common plasma proteins: one was human serum albumin (HSA), an abundant serum protein that facilitates the delivery of drugs around the body through the bloodstream. The other was α1-acid glycoprotein (AGP), a protein produced by the liver in response to inflammation that is found in cancer patients at ten times the normal level.

To explore binding interactions with these two proteins, the team examined the circular dichroism curve for each protein across a spectrum of ultraviolet light, and then they observed whether addition of Blenoxane^® altered the protein curve. Sizeable differences were observed with AGP, suggesting the drug binds and induced marked changes to the protein’s shape, but the curve didn’t shift for HSA. This doesn’t indicate that the drug doesn’t bind HSA, only that it doesn’t alter its shape upon interaction. The team adapted their circular dichroism experiments to confirm that Blenoxane^® did bind to HSA by heating the sample to unfold (denature) the proteins’ architecture and then observing spectral changes with and without the drug present.

Having shown that the drug binds to both proteins, the team set out to compare how tightly it was. For tight binding, plasma proteins might sequester the drug away from cancer cells. To answer this question, a complementary spectroscopic technique was used together with circular dichroism. Bleomycin fluoresces in ultraviolet light, which can alter the intensity of the output beam that produces the signature curve. By monitoring the curve and its intensity, the team gathered enough data to quantify how readily bleomycin dissociates from either protein. It was found that bleomycin bound more tightly to AGP by an order of magnitude than to HSA, suggesting the drug may be usurped in the blood of cancer patients carrying ten times the normal level of this plasma protein.

To explore the strength of this binding interaction further, the researchers assessed how different varieties of bleomycin bind to these two proteins. The Blenoxane^® preparation is largely made up of two versions called A2 and B2 with slightly different chemical structures. They heated the proteins to different temperatures in the presence of each bleomycin version. By monitoring when the circular dichroism curves changed shape, they could determine how A2 and B2 alter the melting temperature of the proteins. The most pronounced results were seen with A2, the more abundant form in Blenoxane^®. It raised the melting temperature of AGP by 8 °C, suggesting this binding interaction is highly stable, but it didn’t exert the same effect on HSA. This heat stability may partly explain why the drug binds more tightly to AGP.

Taking research at Diamond to the clinic

Will bleomycin be as effective in practice as previously thought? These findings raise concern about its interaction with elevated concentration of AGP plasma protein in cancer patients that could potentially lower the effective dose of the drug.

Hussain said;

Hussain and her colleagues caution that care should be taken to adjust the ratio of A2 and B2 to optimize effectiveness while minimizing the association with AGP.

Moving forward, the team at B23 aim to study whether bleomycin binds to haemoglobin, the blood  protein that transports oxygen around the body, and whether this interaction could be linked to lung damage in patients receiving bleomycin.

To find out more about B23 or discuss potential applications, please contact Senior Beamline Scientist Rohanah Hussain: rohanah.hussain@diamond.ac.uk