Transcriptional profiling identifies gene expression changes associated with IFN-α tolerance in hepatitis C-related hepatocellular carcinoma cells

Abstract

Purpose: Treatment with IFN-α therapy has been shown to exhibit antitumor effects on patients with hepatocellular carcinoma (HCC). However, individual responses remained unpredictable because of the frequent presence of intrinsic or acquired IFN-α resistance. Hence, delineation of molecular targets implicated in the resistant pathway holds value in refining the therapeutic benefits of IFN-α. Experimental Design: The current study analyzed the effect of IFN-α in human HCC cells. Three hepatitis C virus (HCV)-related, five hepatitis B virus (HBV)-related and two non-B non-C-related cell lines were subjected to IFN-α treatment and the cytotoxic effect on cell viability was measured. Further analysis by cDNA microarray and quantitative reverse transcription-PCR were conducted to examine the gene expression changes that mediated the IFN-α resistance observed. Results: According to the IC 50 values determined, HCV-related cell lines indicated distinct resistance (IC 50, 389-1468 units/mL) compared with the HBV-related (IC 50, 11-77 units/ mL) and non-B non-C-related cell lines (IC 50, 24-108 units/ mL). Unsupervised hierarchical clustering on array data indicated three HCV-related cell lines to cluster independently from the sensitive cell lines, suggesting discrete features in association with IFN-α tolerance. Moreover, Significance Analysis of Microarrays analysis indicated the differential expression of 149 expressed sequence tags that represented 51 up-regulated and 98 down-regulated genes in the resistant cell lines. Comparing the temporal pattern of gene expression between 6- and 24-hour treatments, candidate genes that were considerably induced with time were further highlighted in the tolerant HCV-related cell lines. These candidates were verified by quantitative reverse transcription-PCR, which confirmed the down-regulation of UBA2, ZNF185, and FOXF1 and up-regulation of UBE4B in the drug-tolerant cells. Conclusions: Our present study showed that the insensitivity to IFN-α therapy in HCC cells is associated with drug-inducible transcriptional alterations. Furthermore, our investigation highlighted potential candidate genes in conferring an anti-apoptotic effect toward IFN-α treatment.