Endogenous Expression Of Sodium Iodide Symporter In Hepatocellular Carcinoma Cells

3113 Background: Effective treatment of cancers using radioactive iodine is contingent upon constitutive and strong expression of NIS. Doxorubicin has been shown to induce expression of NIS in hepatocellular cancer (HCA) by activation of p63 and p73 binding to NIS promoter, leading to an increased expression of NIS mRNA and protein in HCA cells but not in primary human hepatocytes. Purpose: To demonstrate that NIS expression in HCA cells following doxorubicin treatment can be studied using immunofluorescence imaging.\n Methods: HepG2 cells were seeded at 1 x 105 cells per well and treated with 1 µM doxorubicin hydrochloride for 24hrs. Cells were washed in 1xPBS, fixed for 10 minutes with 4% paraformaldehyde (PFA), permeabilized for 10 mins in 0.2% Triton X-100 in PBS and blocked with 1% BSA, 22.52mg/mL glycine in PBST for 30 mins. Samples were not incubated (control) or incubated overnight at 4 deg C with rabbit anti-NIS primary antibody (Imanis Life Sciences) at 1:1000, washed 3 times with 1X PBST, and incubated with goat anti-rabbit IgG Alexa Fluor® 488 labeled secondary antibody (Santa Cruz Biotechnology) for 1 hour in dark. Coverslips were counterstained with 1 μg/mL Hoechst, mounted on slides using Vectashield medium (Vector labs, Ca) and sealed with nail polish. Fluorescence imaging was accomplished using a confocal microscope (inverted Nikon Eclipse Ti2 microscope equipped with a 60X apochromat oil-immersion OFN25 objective).\n Results: Our studies demonstrate that: 1) HCA cells can be viably treated with Doxorubicin. 2) NIS expression in HCA cells are recognized by anti-NIS antibodies and imaged with immunofluorescence. 3) NIS protein is expressed on the surface of HCA cells following treatment with Doxorubicin at 1μM.\n Conclusions: NaI symporter is expressed on the surface of hepatocellular cancer cells following doxorubicin treatment. These findings can be exploited in the use of I-131 radioisotope as a theranostic tool for hepatocellular cancers.\nFig.1. Imaging of Sodium Iodide Symporter Expression Following Treatment with Doxorubicin in HepG2 Hepatocellular Cancer Monolayer Cells. HepG2 cells were treated with 1µM doxorubicin hydrochloride for 24 hours and fluorescent imaging was performed without (left) or with (right) anti-NIS 10 antibody, followed by Alexa Fluor® 488 20 antibody and Hoechst.\nFig. 2. Sodium Iodide Symporter is expressed on the surface of HepG2 Hepatocellular Caner Cell Following Doxorubicin Treatment. Representative slices of Z-stack confocal imaging of NaI symporter expression in HepG2 cell following 1µM doxorubicin hydrochloride treatment. A single cell was imaged at 20X (left) and 60X (right) with anti-NIS 10 antibody, Alexa Fluor® 488 20 antibody, and Hoechst.