Transformation of the Anticancer Drug Doxorubicin ... - ACS Publications

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Transformation of the anticancer drug doxorubicin in the human gut microbiome Austin Yan, Elizabeth Culp, Julie Perry, Jennifer Lau, Lesley MacNeil, Michael G. Surette, and Gerard D Wright ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00166 • Publication Date (Web): 21 Nov 2017 Downloaded from http://pubs.acs.org on November 26, 2017

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ACS Infectious Diseases

Transformation of the anticancer drug doxorubicin in the human gut microbiome Austin Yan1,3, Elizabeth Culp1,3, Julie Perry1,3, Jennifer T. Lau2,3, Lesley T. MacNeil1,2,3,4, Michael G. Surette1,2,3, Gerard D. Wright1,3* 1 - M. G. DeGroote Institute for Infectious Disease Research, McMaster University, 1280 Main Street West, Hamilton, ON, L8S 4K1, Canada 2 - Farncombe Family Digestive Health Research Institute, McMaster University, 1280 Main Street West, Hamilton, ON, L8S 4K1, Canada 3 - Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main Street West, Hamilton, ON, L8S 4K1, Canada 4 - Department of Medicine, McMaster University, 1280 Main Street West, Hamilton, ON, L8S 4K1, Canada

*Correspondence to:

[email protected]

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Bacteria living in the human gut are implicated in the etiology of several diseases. Moreover, dozens of drugs are metabolized by elements of the gut microbiome, which may have further implications for human health. Here we screened a collection of gut isolates for their ability to inactivate the widely used antineoplastic drug doxorubicin and identified a strain of Raoultella planticola as a potent inactivator under anaerobic conditions. We demonstrate that R. planticola deglycosylates doxorubicin to metabolites 7-deoxydoxorubicinol and 7deoxydoxorubicinolone via a reductive deglycosylation mechanism. We further show that doxorubicin is degraded anaerobically by Klebsiella pneumoniae and Escherichia coli BW25113, and present evidence that this phenotype is dependent on molybdopterin-dependent enzyme(s). Deglycosylation of doxorubicin by R. planticola under anaerobic conditions is found to reduce toxicity to the model species Caenorhabditis elegans, providing a model to begin understanding the role of doxorubicin metabolism by microbes in the human gut. Understanding the in vivo metabolism of important therapeutics like doxorubicin by the gut microbiome has the potential to guide clinical dosing to maximize therapeutic benefit while limiting undesirable side effects.

Key words: microbiome, drug inactivation, pharmacology, chemotherapy

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For Table of Contents Use Only:

Transformation of the anticancer drug doxorubicin in the human gut microbiome Austin Yan, Elizabeth Culp, Julie Perry, Jennifer T. Lau, Lesley T. MacNeil, Michael G. Surette, Gerard D. Wright

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The human gastrointestinal tract is colonized by a diverse consortium of microorganisms, many of which are implicated in host pathophysiology.1,2 An altered microbiota has been observed in inflammatory bowel disease, irritable bowel syndrome, obesity, type-1 diabetes, colon carcinoma, autism, and cardiovascular disease.2,3 The impact of the microbiome on host health is not surprising given that the collective genome size of gut-resident microbes exceeds the size of the human genome. There is interest in ‘mining’ these genes for potential therapeutic benefit in a variety of areas including manipulation of nutritional homeostasis, antibiotic discovery, and immune regulation, along with treating and preventing diseases outside of the GI tract.4-7 Importantly, the gut microbiome has metabolic capacity that could influence the effectiveness of drugs used to treat human disease. More than 40 drugs are known to be metabolized by the gut microbiome;8,9 the best characterized are the cardiac drug digoxin,8,10 the anti-cancer drug irinotecan,11-13 and the non-steroidal anti-inflammatory drug diclofenac.14-16 The influence of bacterial metabolism on these drugs and the subsequent impact on host health is only beginning to be understood, with the use of model organisms such as the nematode Caenorhabditis elegans offering a simplified system to study these interactions.17 Doxorubicin is an anti-cancer drug commonly used to treat carcinomas, soft tissue sarcomas, and haematological malignancies, and is the most commonly prescribed drug of the anthracycline family. It is thought to inhibit the growth of both cancer cells and bacteria by multiple mechanisms, including generation of free radicals, DNA intercalation, alkylation and cross-linking, interference with DNA unwinding and topoisomerase II, and direct membrane damage(reviewed in Gewirtz18). Due to the general mechanism of action, the use of any anthracycline drug is limited due to cumulative toxicity in non-tumour tissues. The heart is particularly sensitive where the drug can be converted into toxic metabolites that lack the

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daunosamine sugar, which is essential for anticancer activity. Doxorubicin causes typical chemotherapy-induced side effects including diarrhoea, vomiting, and hair loss, but may also cause life-threatening cardiotoxicity weeks to years after completion of treatment.19 Cardiac toxicity is cumulative, dose-dependent, and thought to be linked to the reduced production of reactive-oxygen species detoxifying enzymes in cardiac tissues and mitochondrial iron accumulation.20,21 Damage to the gut epithelium by induced apoptosis of epithelial cells in the jejunum and damage to mucosal tissues is also known.22 Detoxifying doxorubicin is therefore an area of great interest to extend its clinical utility. Doxorubicin is excreted primarily in feces (40-50%) and is encountered by bacteria in the gastrointestinal tract where it causes cell and tissue damage through both bacteria-dependent and independent mechanisms.23 In a previous study, we have shown that doxorubicin is aerobically inactivated by deglycosylation by soil Streptomyces through interaction with complex 1 of the oxidative phosphorylation pathway.20 As this mechanism of deglycosylation is the very same by which doxorubicin leads to the generation of reactive oxygen species linked to toxic effects, we wondered whether alternate mechanisms exist and what role these may play in the anaerobic environment of the gut. In this work, we show that doxorubicin can be detoxified by a variety of Enterobacteriaceae, isolated from the gut of a healthy donor and of laboratory origin, under strict anaerobic conditions and by a mechanism independent to that in Streptomyces. Genetic studies implicate molybdopterin-dependent enzymes that are required for anaerobic growth. We further investigate the effects of doxorubicin degradation on host fitness using C. elegans as a model, and show that metabolism of the drug by the gut isolate Raoultella planticola detoxifies it and improves animal survival. These findings have potential implications for patients undergoing chemotherapy with doxorubicin and related anthracycline anticancer drugs.

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RESULTS AND DISCUSSION Screen of human gut isolates identifies doxorubicin-resistant strains Motivated by a lack of understanding of how doxorubicin is metabolized in the human gut and the possible utility in discovering novel degradation mechanisms, we established a screen of human gut isolates for the ability to degrade doxorubicin. First, stool from a healthy donor was cultured aerobically and anaerobically on five different media types to capture a diverse collection of bacteria from the gut. To identify strains that may be able to degrade doxorubicin, we next screened for isolates resistant to doxorubicin, which itself has antimicrobial activity. A total of 190 gut isolates were screened for doxorubicin resistance by pinning strains onto Brain Heart Infusion (BHI) agar containing 150 µg/ml doxorubicin, under both aerobic and anaerobic conditions. No aerobically grown strains visibly inactivated doxorubicin as judged by monitoring the conditioned medium for intact drug. In contrast, 29 isolates (15%) were resistant to 150 µg/ml doxorubicin under anaerobic conditions. Most resistant strains (23/29) were derived from primary isolation on MacConkey agar. Moreover, 13 strains were able to decolourize doxorubicin, which has a deep red colour, under anaerobic conditions. Doxorubicin consists of a quinone-containing, rigid, planar, anthracycline ring system linked via a glycosyl bond to a daunosamine aminosugar. It is bright red in appearance (λmax 480 nm), and photosensitive. Decolourization may indicate that resistance to doxorubicin is due to modification/degradation of the anthracycline core tetracycle rather than efflux or deglycosylation, which maintain the chromophore. Since this intact tetracycle is known to be a source of oxygen radical damage in tissues, we chose to follow up on strains displaying this phenotype. Two of the 13 isolates capable of decolourization under anaerobic conditions were Grampositive and identified by 16S rDNA sequencing as Coprococcus and Ruminococcus. Six of the

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Gram negative isolates had similar macroscopic morphology and were confirmed to be the same organism using BOX-PCR-based genomic fingerprinting (Figure S1), and identified by 16S rDNA sequencing as Raoultella planticola. This organism served as our major focus for drug degradation studies. Members of the genus Raoultella are Gram negative, capsulate bacilli that belong to the family Enterobacteriaceae, and were classified within Klebsiella spp. until 2001.24 Raoultella is a common but low abundance organism in 16S rDNA profiles of human stool microbiota. MetaQuery, which provides abundance and prevalence data over 2000 publicly available human gut metagenomes, found Raoultella to have a mean abundance of 0.00295%.25 R. planticola can also be a rare opportunistic pathogen causing bacteremia, pneumonia, intraabdominal infections (including gastroenteritis), in addition to skin and soft tissue infections. It can carry important antibiotic resistance genes including the carbapenemases NDM-1 and IMP-8 (reviewed in Xu et al.26, Tseng et al.27, and Li et al.28). In addition to doxorubicin, our primary isolate of R. planticola was resistant to ampicillin (>64 µg/ml) but sensitive to ciprofloxacin (

Transformation of the Anticancer Drug Doxorubicin ... - ACS Publications

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