Supplementary Figure S2 from Preclinical Evaluation of Transcriptional Targeting Strategy for Human Hepatocellular Carcinoma in an Orthotopic Xenograft Mouse Model
Supplementary Figure S2 - PDF file 104K, Cell cycle-dependent gene expression of AH-6CC-L2C in proliferating orthotopic PLC/PRF/5-DsRed2 tumors versus non-proliferating quiescent normal liver cells in SCID mice. A, Cell cycle analysis of primary cells isolated from normal liver of control SCID mouse versus orthotopically implanted PLC/PRF/5-DsRed2 tumors in left lateral liver lobe of SCID mouse. B, Non-invasive imaging of luciferase gene expression of mice after 24 h post intrahepatic or intratumoral injection of 3 � 106 TU of AH-6CC-L2C into left lateral liver lobes or PLC/PRF/5-DsRed2 tumors, respectively. The injection sites were indicated by an asterisk (*). Non-invasive imaging of DsRed2 signal and the white line indicated the PLC/PRF/5-DsRed2 tumor region. C, Corresponding quantification of total flux emitted from the injection sites (left panel). Corresponding luciferase assays on normal livers and PLC/PRF/5-DsRed2 tumors harvested from SCID mice after 24 h post injection (right panel). All data were presented as mean plus-minus SEM, n = 3
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ARTICLE ABSTRACT
Gene regulation of many key cell-cycle players in S-, G2 phase, and mitosis results from transcriptional repression in their respective promoter regions during the G0 and G1 phases of cell cycle. Within these promoter regions are phylogenetically conserved sequences known as the cell-cycle–dependent element (CDE) and cell-cycle genes homology regions (CHR) sites. Thus, we hypothesize that transcriptional regulation of cell-cycle regulation via the CDE/CHR region together with liver-specific apolipoprotein E (apoE)-hAAT promoter could bring about a selective transgene expression in proliferating human hepatocellular carcinoma. We show that the newly generated vector AH-6CC-L2C could mediate hepatocyte-targeted luciferase gene expression in tumor cells and freshly isolated short-term hepatocellular carcinoma cultures from patient biopsy. In contrast, normal murine and human hepatocytes infected with AH-6CC-L2C expressed minimal or low luciferase activities. In the presence of prodrug 5-fluorocytosine (5-FC), AH-6CC-L2C effectively suppressed the growth of orthotopic hepatocellular carcinoma patient-derived xenograft mouse model via the expression of yeast cytosine deaminase (yCD) that converts 5-FC to anticancer metabolite 5-fluoruracil. More importantly, we show that combination treatment of AH-6CC-L2C with an EZH2 inhibitor, DZNep, that targets EpCAM-positive hepatocellular carcinoma, can bring about a greater therapeutic efficacy compared with a single treatment of virus or inhibitor. Our study showed that targeting proliferating human hepatocellular carcinoma cells through the transcriptional control of therapeutic gene could represent a feasible approach against hepatocellular carcinoma. Mol Cancer Ther; 12(8); 1651–64. ©2013 AACR.Usage metrics
