Abstract A154: Screening of the compounds that modulate cellular bilirubin level in hepatocellular carcinoma HepG2 cells

Abstract

Abstract Background: Bilirubin (BR), the end product of heme metabolism, arises from heme degradation by heme oxygenase and biliverdin reductase. BR level in serum or urine is widely used as biomarker to diagnose liver disorders such as liver cancer. Also, BR is a potent oxidant scavenger that can protect biomolecules such as lipids from oxidative stress. In spite of its clinical importance, there are few studies about cellular level of BR. Therefore, we constructed fluorescence-based high-throughput BR measurement system, and using this system, we screened the compound that modulate cytosolic BR levels. Methods: UnaG, a BR-inducible fluorescent protein from eel muscle, binds to unconjugated bilirubin and subsequently emits green fluorescence. We designed bicistronic plasmid vector pcDNA3-Flag/UnaG-2A-mCherry by linking UnaG cDNA to mCherry cDNA with 2A peptide sequence derived from picornaviruses using recombinant PCR. Since 2A peptide mediates cleavage between two proteins, the cells expressing this vector produce equimolar amounts of UnaG (bilirubin-dependent) and mCherry (bilirubin-independent). Using this construct, change of cellular bilirubin level is estimated by calculating ratio of UnaG/mCherry fluorescence intensity. Then we cloned cells stably expressing this construct by transfection to HepG2 hepatocellular carcinoma cells. Fluorescence microscopy or micro-plate reader measurement of HepG2/UnaG-2A-mCherry cells showed that treatment of BR or biliverdin, a precursor of BR, increased UnaG/mCherry ratio in a dose-dependent manner. Thus, we developed simple and objective measurement system of cellular BR based on ratiometric analysis. Results: Oxidative stress is implicated in the initiation and progression of cancer. Under oxidative stress, BR may protect tumor cells from cellular damages by its antioxidative properties. Therefore, we screened 400 chemical inhibitors of signal transduction to identify compounds that modulate cytosolic BR level. As a result, we found that rotenone induced the decrease in the BR levels. Furthermore, heme oxygenase-1 (HO-1), a primal enzyme of heme degradation, was downregulated by rotenone treatment, indicating that downregulation of HO-1 by rotenone is responsible for the decrease in cellular BR levels. Moreover, PI3k/Akt or MEK inhibitors were also found to reduce the cellular BR levels. HO-1 inhibition has been reported to enhance anticancer effect of several drugs. Taken together, screening compounds that induce the decrease in cellular BR level caused by HO-1 suppression may lead to identify novel drug seeds to overcome anticancer drug resistance. Acknowledgement: We thank Dr. A. Miyawaki (RIKEN) for kindly providing us with the pcDNA3-Flag/UnaG plasmid. We also thank the Screening Committee of Anticancer Drugs supported by Grant-in-Aid for Scientific Research on Innovative Areas, Scientific Support Programs for Cancer Research (MEXT, Japan) for providing us with chemical inhibitors in SCADS Inhibitor Kits. No conflict of interest. Citation Format: Tetsushi Kataura, Masaya Imoto. Screening of the compounds that modulate cellular bilirubin level in hepatocellular carcinoma HepG2 cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A154.