Discrimination Of Human Hepatoma HepG2Cells From Healthy Hepatocytes Using A COX2-specific Fluorescent Probe

Background：Early diagnosis and efficient interference is essential for preventing theprogression of HCC. COX-2plays an important role in the development of HCC, and itis overexpressed in HCC. It is said that COX-2has an important influence in the earlystages of hepatocellular carcinoma, and COX-2is a predictive biomarker of HCCmalignant transformation. According to this feature of COX-2, it is essential and helpfulthat we could find a timely, accurate, safe and intuitive method or tool to diagnosis HCCin an early stage.Objective: Hepatocellular carcinoma (HCC) is a common malignancy, but earlydiagnosis is made difficult by lack of an efficient molecular probe. We investigated thepotential of the fluorescent probe ANQ-IMC-6for its ability to discriminate humanhepatoma cells from healthy hepatocytes.Methods: Human liver carcinoma cells (HepG2) were pretreated or not with theCOX-2-specific inhibitor NS-398. These cells and human normal hepatocytes (L02)were stained with ANQ-IMC-6and examined under a confocal scanning microscope.The presence of COX-2in HepG2and L02cells was assessed by western blotting.Because COX-2is primarily localized in the Golgi of cancer cells, to determine ifANQ-IMC-6-labeled COX-2could be visualized in the Golgi, HepG2cells wereco-stained with ANQ-IMC-6and the Golgi apparatus-specific fluorescent probeBODIPY TR C5-ceramide. To determine if ANQ-IMC-6staining could discriminatetumor from normal tissues, primary hepatic carcinoma and matched adjacent normaltissue from an HCC patient were stained withANQ-IMC-6.Results: The COX-2was specifically expressed by HepG2cells but not L02cells.HepG2cells stained with ANQ-IMC-6exhibited strong green fluorescence in thecytoplasm, but no fluorescence was detected in L02cells. In stained HepG2cells pretreated with NS-398, the intensity of fluorescence was greatly reduced. Co-stainingof HepG2cells with ANQ-IMC-6and BODIPY TR C5-ceramide revealed COX-2in theGolgi (Pearson coefficient value=0.89). ANQ-IMC-6staining could discriminatehepatic carcinoma tissues from normal tissues.Conclusions: These findings indicate that ANQ-IMC-6may serve as a valuablefluorescent probe for accurately discriminating HCC cells from normal hepatocytes.