Anticancer Activity of Ω-6 Fatty Acids through Increased 4-HNE in Breast Cancer Cells

Her2-amplified breast cancers resistant to available Her2-targeted therapeutics continue to be a challenge in breast cancer therapy. Dox is the mainstay of chemotherapy of all types of breast cancer, but its usefulness is limited by cumulative cardiotoxicity. Because oxidative stress caused by dox generates the pro-apoptotic Ω-6 PUFA metabolite 4-hydroxynonenal (4-HNE), we surmised that Ω-6 PUFAs would increase the effectiveness of dox chemotherapy. Since the mercapturic acid pathway enzyme RALBP1 (also known as RLIP76 or Rlip) that limits cellular accumulation of 4-HNE also mediates dox resistance, the combination of Ω-6 PUFAs and Rlip depletion could synergistically improve the efficacy of dox. Thus, we studied the effects of the Ω-6 PUFA arachidonic acid (AA) and Rlip knockdown on the antineoplastic activity of dox towards Her2-amplified breast cancer cell lines SK-BR-3, which is sensitive to Her2 inhibitors, and AU565, which is resistant. AA increased lipid peroxidation, 4-HNE generation, apoptosis, cellular dox concentration and dox cytotoxicity in both cell lines while sparing cultured immortalized cardiomyocyte cells. The known functions of Rlip including clathrin-dependent endocytosis and dox efflux were inhibited by AA. Our results support a model in which 4-HNE generated by AA overwhelms the capacity of Rlip to defend against apoptosis caused by dox or 4-HNE. We propose that Ω-6 PUFA supplementation could improve the efficacy of dox or Rlip inhibitors for treating Her2-amplified breast cancer.

Biotransformation Capacity of Zebrafish (Danio rerio) Early Life Stages: Functionality of the Mercapturic Acid Pathway

Zebrafish (Danio rerio) early life stages offer a versatile model system to study the efficacy and safety of drugs or other chemicals with regard to human and environmental health. This is because, aside from the well-characterized genome of zebrafish and the availability of a broad range of experimental and computational research tools, they are exceptionally well suited for high-throughput approaches. Yet, one important pharmacokinetic aspect is thus far only poorly understood in zebrafish embryo and early larvae: their biotransformation capacity. Especially, biotransformation of electrophilic compounds is a critical pathway because they easily react with nucleophile molecules, such as DNA or proteins, potentially inducing adverse health effects. To combat such adverse effects, conjugation reactions with glutathione and further processing within the mercapturic acid pathway have evolved. We here explore the functionality of this pathway in zebrafish early life stages using a reference substrate (1-chloro-2,4-dinitrobenzene, CDNB). With this work, we show that zebrafish embryos can biotransform CDNB to the respective glutathione conjugate as early as 4 h postfertilization. At all examined life stages, the glutathione conjugate is further biotransformed to the last metabolite of the mercapturic acid pathway, the mercapturate, which is slowly excreted. Being able to biotransform electrophiles within the mercapturic acid pathway shows that zebrafish early life stages possess the potential to process xenobiotic compounds through glutathione conjugation and the formation of mercapturates. The presence of this chemical biotransformation and clearance route in zebrafish early life stages supports the application of this model in toxicology and chemical hazard assessment.

Identification Of N-Acetyl-S-(3-Cyano-2-(Methylsulfanyl)Propyl-Cysteine as a Major Human Urine Metabolite from the Epithionitrile 1-Cyano-2,3-Epithiopropane, the Main Glucosinolate Hydrolysis Product from Cabbage

Brassica vegetables such as cabbage or pak choi contain alkenyl glucosinolates which can release epithionitriles and to a lesser degree isothiocyanates upon enzymatic hydrolysis. Here, for the first time, the metabolism of an epithionitrile was investigated in humans, namely 1-cyano-2,3-epithiopropane (CETP). After consumption of Brassica oleracea var. capitata f. alba and Brassica carinata sprouts, the main urinary metabolite of CETP was identified as N-acetyl-S-(3-cyano-2-(methylsulfanyl)propyl-cysteine using an UHPLC-ESI-QToF-MS approach and synthesis of the metabolite. This urinary epithionitrile metabolite is an S-methylated mercapturic acid. No other metabolites were detected. Then, in a preliminary pilot experiment the excretion kinetics of CETP were investigated in three volunteers. After consumption of a B. carinata sprout preparation containing 50.8 µmol of CETP, urinary N-acetyl-S-(3-cyano-2-(methylsulfanyl)propyl-cysteine concentrations were the highest three hours after consumption, ranging from 23.9 to 37.2 µM, and declined thereafter. Thus, epithionitriles are bioavailable compounds that are metabolized similarly to isothiocyanates by the mercapturic acid pathway. In the future, more epithionitrile metabolites should be identified and the pharmacokinetics of these important class of dietary compounds should be assessed in more detail

Rlip depletion prevents spontaneous neoplasia in TP53 null mice

TP53 (p53) is a tumor suppressor whose functions are lost or altered in most malignancies. p53 homozygous knockout (p53−/−) mice uniformly die of spontaneous malignancy, typically T-cell lymphoma. RALBP1 (RLIP76, Rlip) is a stress-protective, mercapturic acid pathway transporter protein that also functions as a Ral effector involved in clathrin-dependent endocytosis. In stark contrast to p53−/− mice, Rlip−/− mice are highly resistant to carcinogenesis. We report here that partial Rlip deficiency induced by weekly administration of an Rlip-specific phosphorothioate antisense oligonucleotide, R508, strongly inhibited spontaneous as well as benzo(a)pyrene-induced carcinogenesis in p53−/− mice. This treatment effectively prevented large-scale methylomic and transcriptomic abnormalities suggestive of inflammation found in cancer-bearing p53−/− mice. The remarkable efficiency with which Rlip deficiency suppresses spontaneous malignancy in p53−/− mice has not been observed with any previously reported pharmacologic or genetic intervention. These findings are supported by cross-breeding experiments demonstrating that hemizygous Rlip deficiency also reduces the spontaneous malignancy phenotype of p53+/− mice. Rlip is found on the cell surface, and antibodies directed against Rlip were found to inhibit growth and promote apoptosis of cell lines as effectively as Rlip siRNA. The work presented here investigates several features, including oxidative DNA damage of the Rlip–p53 association in malignant transformation, and offers a paradigm for the mechanisms of tumor suppression by p53 and the prospects of suppressing spontaneous malignancy in hereditary cancer syndromes such as Li-Fraumeni.