Abstract
Background and PurposeNAD(P)H: quinone oxidoreductase 1 (NQO1) mediated quinone reduction and subsequent UDP-glucuronosyltransferases (UGTs) catalyzed glucuronidation is the dominant metabolic pathway of tanshinone IIA (TSA), a promising anti-cancer agent. UGTs are positively expressed in various tumor tissues and play an important role in the metabolic elimination of TSA. This study aims to explore the role of UGT1A in determining the intracellular accumulation and the resultant apoptotic effect of TSA.Experimental ApproachWe examined TSA intracellular accumulation and glucuronidation in HT29 (UGT1A positive) and HCT116 (UGT1A negative) human colon cancer cell lines. We also examined TSA-mediated reactive oxygen species (ROS) production, cytotoxicity and apoptotic effect in HT29 and HCT116 cells to investigate whether UGT1A levels are directly associated with TSA anti-cancer effect. UGT1A siRNA or propofol, a UGT1A9 competitive inhibitor, was used to inhibit UGT1A expression or UGT1A9 activity.Key ResultsMultiple UGT1A isoforms are positively expressed in HT29 but not in HCT116 cells. Cellular S9 fractions prepared from HT29 cells exhibit strong glucuronidation activity towards TSA, which can be inhibited by propofol or UGT1A siRNA interference. TSA intracellular accumulation in HT29 cells is much lower than that in HCT116 cells, which correlates with high expression levels of UGT1A in HT29 cells. Consistently, TSA induces less intracellular ROS, cytotoxicity, and apoptotic effect in HT29 cells than those in HCT116 cells. Pretreatment of HT29 cells with UGT1A siRNA or propofol can decrease TSA glucuronidation and simultaneously improve its intracellular accumulation, as well as enhance TSA anti-cancer effect.Conclusions and ImplicationsUGT1A can compromise TSA cytotoxicity via reducing its intracellular exposure and switching the NQO1-triggered redox cycle to metabolic elimination. Our study may shed a light in understanding the cellular pharmacokinetic and molecular mechanism by which UGTs determine the chemotherapy effects of drugs that are UGTs’ substrates.
Highlights
UDP-glucuronosyltransferases (UGTs) catalyze the glucuronidation of many lipophilic endogenous substrates such as bilirubin and steroid hormones, and xenobiotics including carcinogens and clinical drugs [1,2,3]
On the basis of our recent finding that multiple UGT1A isoforms are involved in tanshinone IIA (TSA) glucuronidation [24], the present study focuses on elucidating the role of these UGTs in determining the intracellular accumulation and apoptotic effect of TSA in human colon cancer cells
We first evaluated the expression levels of UGT1A isoforms which were involved in TSA glucuronidation by real time PCR in both HT29 and HCT116 cell lines
Summary
UDP-glucuronosyltransferases (UGTs) catalyze the glucuronidation of many lipophilic endogenous substrates such as bilirubin and steroid hormones, and xenobiotics including carcinogens and clinical drugs [1,2,3]. The enhanced enzymatic activities of UGTs may represent an important contributor to chemotherapeutic resistance of many drugs that are UGTs’ substrates, such as irinotecan, methotrexate, epirubicin, and tamoxifen [8,9,10,11], implying a crucial role of UGTs in the anti-cancer therapy. UGTs have been claimed as an important cause of chemotherapeutic resistance, little is known about the direct influence of UGTs regarding the intracellular accumulation in the target cancer cells and chemotherapeutic efficacy of drugs. UGTs are positively expressed in various tumor tissues and play an important role in the metabolic elimination of TSA. Experimental Approach: We examined TSA intracellular accumulation and glucuronidation in HT29 (UGT1A positive) and HCT116 (UGT1A negative) human colon cancer cell lines. We examined TSA-mediated reactive oxygen species (ROS) production, cytotoxicity and apoptotic effect in HT29 and HCT116 cells to investigate whether UGT1A levels are directly associated with TSA anti-cancer effect. UGT1A siRNA or propofol, a UGT1A9 competitive inhibitor, was used to inhibit UGT1A expression or UGT1A9 activity
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