The discovery of new biomarkers for early detection of drug-induced acute kidney injury (AKI) is clinically important. In this study, sensitive metabolomic biomarkers identified in the urine of rats were used to detect cisplatin-induced AKI. Cisplatin (10 mg kg(-1), i.p.) was administered to Sprague-Dawley rats, which were subsequently euthanized after 1, 3 or 5 days. In cisplatin-treated rats, mild histopathological alterations were noted at day 1, and these changes were severe at days 3 and 5. Blood urea nitrogen (BUN) and serum creatinine (SCr) levels were significantly increased at days 3 and 5. The levels of new urinary protein-based biomarkers, including kidney injury molecule-1 (KIM-1), glutathione S-transferase-α (GST-α), tissue inhibitor of metalloproteinase-1 (TIMP-1), vascular endothelial growth factor (VEGF), calbindin, clusterin, neutrophil, neutrophil gelatinase-associated lipocalin (NGAL), and osteopontin, were significantly elevated at days 3 and 5. Among urinary metabolites, trigonelline and 3-indoxylsulfate (3-IS) levels were significantly decreased in urine collected from cisplatin-treated rats prior to histological kidney damage. However, carbon tetrachloride (CCl4), a hepatotoxicant, did not affect these urinary biomarkers. Trigonelline is closely associated with GSH depletion and results in insufficient antioxidant capacity against cisplatin-induced AKI. The predominant cisplatin-induced AKI marker appeared to be reduced in urinary 3-IS levels. Because 3-IS is predominantly excreted via active secretion in proximal tubules, a decrease is indicative of tubular damage. Further, urinary excretion of 3-IS levels was markedly reduced in patients with AKI compared to normal subjects. The area under the curve receiver operating characteristics (AUC-ROC) for 3-IS was higher than for SCr, BUN, lactate dehydrogenase (LDH), total protein, and glucose. Therefore, low urinary or high serum 3-IS levels may be more useful for early detection of AKI than conventional biomarkers.

The sirtuins (SIRTs), a family of NAD+-dependent class III histone deacetylase, are involved in various biological processes including cell survival, division, senescence, and metabolism via activation of the stress-response pathway. Recently, inhibition of SIRTs has been considered a promising anticancer strategy, but their precise mechanisms of action are not well understood. In particular, the relevance of p53 to SIRT-induced effects has not been fully elucidated. We investigated the anticancer effects of a novel SIRT inhibitor, MHY2256, and its efficacy was compared to that of salermide in MCF-7 (wild-type p53) and SKOV-3 (null-type p53) cells. Cell viability, SIRT1 enzyme activity, cell cycle regulation, apoptosis, and autophagic cell death were measured. We compared sensitivity to cytotoxicity in MCF-7 and SKOV-3 cells. MHY2256 significantly decreased the viability of MCF-7 (IC50, 4.8 μM) and SKOV-3 (IC50, 5.6 μM) cells after a 48 h treatment period. MHY2256 showed potent inhibition (IC50, 0.27 mM) against SIRT1 enzyme activity compared with nicotinamide (IC50, >1 mM). Moreover, expression of SIRT (1, 2, or 3) protein levels was significantly reduced by MHY2256 treatment in both MCF-7 and SKOV-3 cells. Flow cytometry analysis revealed that MHY2256 significantly induced cell cycle arrest in the G1 phase, leading to an effective increase in apoptotic cell death in MCF-7 and SKOV-3 cells. A significant increase in acetylated p53, a target protein of SIRT, was observed in MCF-7 cells after MHY2256 treatment. MHY2256 up-regulated LC3-II and induced autophagic cell death in MCF-7 cells. Furthermore, MHY2256 markedly inhibited tumor growth in a tumor xenograft model of MCF-7 cells. These results suggest that a new SIRT inhibitor, MHY2256, has anticancer activity through p53 acetylation in MCF-7 human breast cancer cells.

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