Reactive oxygen species (ROS) are mutagenic and may thereby promote cancer1. Normally, ROS levels are tightly controlled by an inducible antioxidant program that responds to cellular stressors and is predominantly regulated by the transcription factor Nrf2 and its repressor protein Keap12-5. In contrast to the acute physiological regulation of Nrf2, in neoplasia there is evidence for increased basal activation of Nrf2. Indeed, somatic mutations that disrupt the Nrf2-Keap1 interaction to stabilize Nrf2 and increase the constitutive transcription of Nrf2 target genes were recently identified, suggesting that enhanced ROS detoxification and additional Nrf2 functions may in fact be pro-tumorigenic6. Here, we investigated ROS metabolism in primary murine cells following the expression of endogenous oncogenic alleles of K-Ras, B-Raf and Myc, and find that ROS are actively suppressed by these oncogenes. K-RasG12D, B-RafV619E and MycERT2 each increased the transcription of Nrf2 to stably elevate the basal Nrf2 antioxidant program and thereby lower intracellular ROS and confer a more reduced intracellular environment. Oncogene-directed increased expression of Nrf2 is a novel mechanism for the activation of the Nrf2 antioxidant program, and is evident in primary cells and tissues of mice expressing K-RasG12D and B-RafV619E, and in human pancreatic cancer. Furthermore, genetic targeting of the Nrf2 pathway impairs K-RasG12D-induced proliferation and tumorigenesis in vivo. Thus, the Nrf2 antioxidant and cellular detoxification program represents a previously unappreciated mediator of oncogenesis.

Background and Purpose Naringenin, a flavonoid compound with strong anti‐inflammatory activity, attenuated non‐alcoholic fatty liver disease (NAFLD) induced by a methionine‐choline deficient (MCD) diet in mice. However, the mechanisms underlying this suppression of inflammation and NAFLD remain unknown. Experimental Approach WT and NLRP3−/− mice were fed with MCD diet for 7 days to induce NAFLD and were given naringenin by gavage at the same time. in vitro experiments used HepG2 cells, primary hepatocytes, and Kupffer cells (KCs) stimulated by LPS or LPS plus oleic acid (OA). Key Results Treating WT mice with naringenin (100 mg·kg−1·day−1) attenuated hepatic lipid accumulation and inflammation in the livers of mice given the MCD diet. NLRP3−/− mice showed less hepatic lipid accumulation than WT mice, but naringenin did not ameliorate hepatic lipid accumulation further in NLRP3−/− mice. Treating the HepG2 cells with naringenin or NLRP3 inhibitor MCC950 reduced lipid accumulation. Naringenin inhibited activation of the NLRP3/NF‐κB pathway stimulated by OA together with LPS. In KCs isolated from WT mice, naringenin inhibited NLRP3 expression. Naringenin also inhibited lipid deposition, NLRP3 and IL‐1β expression in WT hepatocytes but was not effective in NLRP3−/− hepatocytes. After re‐expressing NLRP3 in NLRP3−/− hepatocytes by adenovirus, the anti‐lipid deposition effect of naringenin was restored. Conclusion and Implications Naringenin prevented NAFLD via down‐regulating the NLRP3/NF‐κB signalling pathway both in KCs and in hepatocytes, thus attenuating inflammation in the mice livers.

The formation of 15-oxo-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15-oxo-ETE) as a product from rabbit lung 15-hydroxyprostaglandin dehydrogenase (PGDH)-mediated oxidation of 15(S)-hydroperoxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid was first reported more than 30 years ago. However, the pharmacological significance of 15-oxo-ETE formation has never been established. We have now evaluated 15-lipoxygenase (LO)-1-mediated arachidonic acid (AA) metabolism to 15-oxo-ETE in human monocytes and mouse RAW macrophages that stably express human 15-LO-1 (R15L cells). A targeted lipidomics approach was used to identify and quantify the oxidized lipids that were formed. 15-oxo-ETE was found to be a major AA-derived LO metabolite when AA was given exogenously or released from endogenous esterified lipid stores by calcium ionophore (

The reactive thiol of cysteine is often used for coupling maleimide-containing linker-payloads to antibodies resulting in the generation of antibody drug conjugates (ADCs). Currently, a numbers of ADCs in drug development are made by coupling a linker-payload to native or engineered cysteine residues on the antibody. An ADC conjugated via hinge-cysteines to an auristatin payload was used as a model in this study to understand the impact of the maleimide linkers on ADC stability. The payload was conjugated to trastuzumab by a protease-cleavable linker, maleimido-caproyl-valine-citruline-p-amino-benzyloxy carbonyl (mcVC-PABC). In plasma stability assays, when the ADC (Trastuzumab-mcVC-PABC-Auristatin-0101) was incubated with plasma over a 144-h time-course, a discrepancy was observed between the measured released free payload concentration and the measured loss of drug-to-antibody ratio (DAR), as measured by liquid chromatography-mass spectrometry (LC-MS). We found that an enzymatic release of payload from ADC-depleted human plasma at 144 h was able to account for almost 100% of the DAR loss. Intact protein mass analysis showed that at the 144 h time point, the mass of the major protein in ADC-depleted human plasma had an additional 1347 Da over the native albumin extracted from human plasma, exactly matching the mass of the linker-payload. In addition, protein gel electrophoresis showed that there was only one enriched protein in the 144 h ADC-depleted and antipayload immunoprecipitated plasma sample, as compared to the 0 h plasma immunoprecipitated sample, and the mass of this enriched protein was slightly heavier than the mass of serum albumin. Furthermore, the albumin adduct was also identified in 96 h and 168 h postdose in vivo cynomolgus monkey plasma. These results strongly suggest that the majority of the deconjugated mc-VC-PABC-auristatin ultimately is transferred to serum albumin, forming a long-lived albumin-linker-payload adduct. To our knowledge, this is the first report quantitatively characterizing the extent of linker-payload transfer to serum albumin and the first clear example of in vivo formation of an albumin-linker-payload adduct.