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Hepatocellular carcinoma (HCC) is one of the most malignant tumors in the world. Inadequate efficacy of 5-fluorouracil (5-FU) on HCC could be related to low expression of human organic anion transporter 2 (OAT2). However, the knowledge of downregulation of OAT2 in HCC remains limited. We explored the underlying mechanism focusing on protein expression regulation and attempted to design a strategy to sensitize HCC cells to 5-FU. In this study, we revealed that the 1 bp to 300 bp region of OAT2 mRNA 3′ untranslated region (UTR) reduced its protein expression and uptake activity in Li-7 and PLC/PRF/5 cells. Mechanistically, it was demonstrated that staphylococcal nuclease and Tudor domain containing 1 (SND1) bound at the 1 bp to 300 bp region of OAT2 mRNA 3′ UTR, leading to a decrease in OAT2 protein expression. Enrichment analysis results indicated reduction of OAT2 might be mediated by translational inhibition. Furthermore, the knockdown of SND1 upregulated OAT2 protein expression and uptake activity. Based on this, decreasing SND1 expression enhanced 5-FU-caused G1/S phase arrest in Li-7 and PLC/PRF/5 cells, resulting in suppression of cell proliferation. Additionally, the knockdown of SND1 augmented the inhibitory effect of 5-FU on PLC/PRF/5 xenograft tumor growth in vivo by increasing OAT2 protein expression and accumulation of 5-FU in the tumor. Collectively, a combination of inhibition of SND1 with 5-FU might be a potential strategy to sensitize HCC cells to 5-FU from the perspective of restoring OAT2 protein level.SIGNIFICANCE STATEMENTWe investigated the regulatory mechanism of OAT2 protein expression in HCC cells and designed a strategy to sensitize them to 5-FU (OAT2 substrate) via restoring OAT2 protein level. It found that SND1, an RNA binding protein, regulated OAT2 protein expression by interacting with OAT2 mRNA 3′ UTR 1-300 bp region. Through decreasing SND1, the antitumor effect of 5-FU on HCC was enhanced in vitro and in vivo, indicating that SND1 could be a potential target for sensitizing HCC cells to 5-FU.

Isoneochamaejasmin A (INCA), a biflavonoid, is one of main active ingredients in the dried root of Stellera chamaejasmeL., a widely used traditional Chinese medicine. In the present study, we identified the glucuronidation metabolite of INCA and characterized the UDP glucuronosyltransferases (UGTs) responsible for INCA glucuronidation. 7-O-glucuronide (M1) and 4′-O-glucuronide (M2) were identified by incubation of INCA with human liver microsomes (HLMs) in the presence of UDP glucuronic acid, and their structures were confirmed by high-resolution mass spectrometry and nuclear magnetic resonance analyses. Although INCA is a single enantiomer molecule, its M1 metabolite showed two equal-size peaks on a πNAP stationary phase but only one peak on a C18stationary phase, indicating that the 7-/7′′- and 4′-/4′′′-hydroxyl groups of INCA were in different spatial configurations relative to each other. Among the recombinant human UGT isoform test and correlation analysis, UGT1A1, UGT1A3, and UGT1A9 were found to mediate M1 formation, whereas only UGT1A3 mediated M2 formation. Kinetic studies showed obvious species differences between human, mouse, rat, dog, and pig liver microsomes. UGT1A1, HLMs, and human intestinal microsomes, but not human kidney microsomes, exhibited substrate inhibition for the formation of M1. UGT1A1-mediated formation of M1 showed a 6- and 11-fold higher Vmaxthan did UGT1A3- and UGT1A9-mediated formation of M1, respectively. The results of the relative activity factor assay showed that UGT1A1 contributed approximately 75% in the formation of M1. These findings collectively indicate that UGT1A1 is the major enzyme in the formation of M1, whereas UGT1A3 is the major enzyme in the formation of M2.