Functional CRISPR and shRNA Screens Identify Involvement of Mitochondrial Electron Transport in the Activation of Evofosfamide.

Evofosfamide (TH-302) is a hypoxia-activated DNA-crosslinking prodrug currently in clinical development for cancer therapy. Oxygen-sensitive activation of evofosfamide depends on one-electron reduction, yet the reductases that catalyze this process in tumors are unknown. We used RNA sequencing, whole-genome CRISPR knockout, and reductase-focused short hairpin RNA screens to interrogate modifiers of evofosfamide activation in cancer cell lines. Involvement of mitochondrial electron transport in the activation of evofosfamide and the related nitroaromatic compounds EF5 and FSL-61 was investigated using 143B ρ 0 (ρ zero) cells devoid of mitochondrial DNA and biochemical assays in UT-SCC-74B cells. The potency of evofosfamide in 30 genetically diverse cancer cell lines correlated with the expression of genes involved in mitochondrial electron transfer. A whole-genome CRISPR screen in KBM-7 cells identified the DNA damage-response factors SLX4IP, C10orf90 (FATS), and SLFN11, in addition to the key regulator of mitochondrial function, YME1L1, and several complex I constituents as modifiers of evofosfamide sensitivity. A reductase-focused shRNA screen in UT-SCC-74B cells similarly identified mitochondrial respiratory chain factors. Surprisingly, 143B ρ 0 cells showed enhanced evofosfamide activation and sensitivity but had global transcriptional changes, including increased expression of nonmitochondrial flavoreductases. In UT-SCC-74B cells, evofosfamide oxidized cytochromes a, b, and c and inhibited respiration at complexes I, II, and IV without quenching reactive oxygen species production. Our results suggest that the mitochondrial electron transport chain contributes to evofosfamide activation and that predicting evofosfamide sensitivity in patients by measuring the expression of canonical bioreductive enzymes such as cytochrome P450 oxidoreductase is likely to be futile.

Antihyperglycemic, antihyperlipidemic and antioxidant activity of phenolic rich extract of Brassica oleraceae var gongylodes on streptozotocin induced Wistar rats.

Cruciferous vegetables, in particular those included into the Brassica genus, are good sources of a variety of nutrients and health-promoting phytochemicals. Phenolic compounds are the major antioxidants of Brassica; hence the contribution of Brassica vegetables to health improvement has largely been associated to their antioxidant capacity. This study aimed to assess anti-diabetic, antilipidemic, and antioxidant activity of phenolic rich extract of Brassica oleraceae var gongylodes (BOvG) in Wistar rats. The findings revealed that the administration of BOvG extract to diabetic rats significantly reduced fasting blood glucose by 64% within 7 days of treatment. Additionally, BOvG extract was also observed to normalize the diabetic rats' lipid profile and HbA1c (Glycated hemoglobin). BOvG extract also showed protection of liver- kidney functions, which was evidenced by the significant decrease in Blood Urea Nitrogen (BUN), Serum glutamic oxaloacetic transaminase (SGOT) and Serum glutamic pyruvic transaminase (SGPT). The treatment also improved the antioxidant status of the diabetic rats where the enzymatic activities of Catalase (CAT) and Super Oxide Dismutase (SOD) were significantly increased. Furthermore, RP-HPLC analysis detected chlorogenic acid, rutin, and sinapic acid against known standards in BOvG extract. Hence, the present investigation suggests that BOvG phenolic rich extract (as a multi-component therapy) exhibited anti-diabetic, antilipidemic and antioxidant properties in STZ-induced diabetic rats.