Xenobiotic metabolism, cancer chemoprevention and cancer biomarkers

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

Project 1, Podophyllotoxin and hepatotoxicity: Introduction. Since the early 19th century, extracts of Podophyllum peltatum, Podophyllum emodi, also known as podophyllin, were used to treat a variety of diseases and conditions such as scrofula, syphilis, gonorrhea, and coughing. Podophyllotoxin (POD) was first isolated from this herb and the planar structure and configuration of POD was established, and its synthesis achieved in the 1960s. POD can inhibit the growth of epithelial cells infected with human papilloma virus (HPV), and thus, it was initially used to treat genital warts. As the primary compound, POD was then revealed to have antimitotic and antitumor activities and showed more potency than podophyllin extracts. However, clinical development of POD was impeded due to its severe side effects. Because of its poor selectivity against tumor cells and narrow therapeutic window, POD has been implicated in many poisoning cases as a result of either overdose or accidental ingestion of herbs containing POD. Major toxicities induced by POD include clinical symptoms in the gastrointestinal tract such as vomiting, diarrhea, abdominal pain, and abnormal hepatic functions, and sometimes even neurological disorders. Etoposide and teniposide, two semi-synthetic derivatives of POD and DNA topoisomerase-II inhibitors, were approved by the FDA and are presently used as anticancer agents. POD is mainly used as a first-line treatment for condyloma acuminate as an externally applied agent. Drug metabolism, an important contributor to the clearance of drugs and determination of safe and effective dosage, can detoxify toxic compounds or activate drugs to chemically reactive electrophilic derivatives that may be potentially toxic or produce oxidative stress. Drug-induced toxicity is one of the main reasons limiting the clinical use of many drugs. Whether the toxicity of POD is due to the parent compound or its metabolites is still not clear. A previous study showed that POD possesses strong inhibitory effects on CYP2C9 and CYP3A4 in a concentration-dependent manner. However, there are no comprehensive studies on POD metabolism and the metabolic enzymes involved in its metabolism, which is a prerequisite for a better understanding of the mechanism of POD-induced toxicity. Results. The purpose of the present study was to determine the metabolic map of POD in vitro and in vivo. Mouse and human liver microsomes were employed to identify POD metabolites in vitro and recombinant drug-metabolizing enzymes were used to identify the mono-oxygenase enzymes involved in POD metabolism. All in vitro incubation mixtures and bile samples from mice treated with POD were analyzed with ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry. A total of 38 metabolites, including six phase-I metabolites and 32 phase-II metabolites, of POD were identified from bile and feces samples after oral administration, and their structures were elucidated through interpreting MS/MS fragmentation patterns. Nine metabolites, including two phase-I metabolites, five glucuronide conjugates, and two GSH conjugates were detected in both human and mouse liver microsome incubation systems and the generation of all metabolites were NADPH-dependent. The main phase-I enzymes involved in metabolism of POD in vitro include CYP2C9, CYP2C19, CYP3A4, and CYP3A5.POD administration to mice caused hepatic and intestinal toxicity, and the cellular damage was exacerbated when 1-aminobenzotriazole, a broad-spectrum inhibitor of CYPs, was administered with POD, indicating that POD, but not its metabolites, induced hepatic and intestinal toxicities.This study elucidated the metabolic map and provides important reference basis for the safety evaluation and rational for the clinical application of POD. Project 2, Urinary diacetylspermine as a biomarker of doxorubicin effectiveness in triple negative breast cancer: Introduction. Women have a 1-in-8 chance of developing breast cancer, which is the second leading cause of cancer-related death. Triple negative breast cancer (TNBC) accounts for 10-20% of breast cancers. In the absence of targets to exploit with therapy, TNBC patients are treated with aggressive chemotherapy as the primary systemic treatment. TNBC chemotherapy regimens typically contain a combination of taxanes, cyclophosphamide, and anthracyclines such as doxorubicin, to maximize therapeutic response. Only 30-40% of patients with TNBC who receive taxane- and anthracycline-based therapy will achieve a pathological complete response. TNBC has the worst prognosis of all breast cancer subtypes, with the highest 5-year mortality across all disease stages. Recurrence is frequent in TNBC patients with most events occurring within 3 years of the disease diagnosis. Current techniques for detecting TNBC recurrence rely on imaging and are limited by many factors including tumor size, individual breast characteristics, skill of the examiner, and follow-up to minimize false negatives. Consequently, a recurrence can only be detected months after primary therapy. Therefore, identification of clinical biomarkers that can be used to evaluate drug response during treatment would be beneficial to patients with TNBC. Metabolomics was sed to identify diacetylspermine as cancer biomarker Results. TNBC patients receive chemotherapy treatment, including doxorubicin, due to the lack of targeted therapies. Drug resistance is a major cause of treatment failure in TNBC and therefore, there is a need to identify biomarkers that determine effective drug response. A pharmacometabolomics study was performed using doxorubicin sensitive and resistant TNBC patient-derived xenograft (PDX) models to detect urinary metabolic biomarkers of treatment effectiveness. Evaluation of metabolite production was assessed by directly studying tumor levels in TNBC-PDX mice and human subjects. Metabolic flux leading to biomarker production was determined using stable isotope labelled tracers in TNBC-PDX ex vivo tissue slices. Findings were validated in 12-hour urine samples from control (n=200), ER+/PR+(n=200), ER+/PR+/HER2+ (n=36), HER2+ (n=81) and TNBC (n=200) subjects. Diacetylspermine was identified as a urine metabolite that robustly changed in response to effective doxorubicin treatment, which persisted after the final dose. Urine diacetylspermine was produced by the tumor and correlated with tumor volume. Ex vivo tumor slices revealed that doxorubicin directly increases diacetylspermine production by increasing tumor spermidine/spermine N1-acetyltransferase 1 expression and activity, which was corroborated by elevated polyamine flux. In breast cancer patients, tumor diacetylspermine was elevated compared to matched non-cancerous tissue and increased in HER2+ and TNBC compared to ER+ subtypes. Urine diacetylspermine was associated with breast cancer tumor volume and poor tumor grade. This study describes a pharmacometabolomics strategy for identifying cancer metabolic biomarkers that indicate drug response. Our findings characterize urine diacetylspermine as a non-invasive biomarker of doxorubicin effectiveness in TNBC.