Transcriptional profiling identifies gene expression changes associated with IFN-alpha tolerance in hepatitis C-related hepatocellular carcinoma cells.
OBJECTIVE
Treatment with IFN-alpha 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-alpha resistance. Hence, delineation of molecular targets implicated in the resistant pathway holds value in refining the therapeutic benefits of IFN-alpha.


METHODS
The current study analyzed the effect of IFN-alpha 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-alpha 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-alpha 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-alpha 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-alpha 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-alpha treatment.
Wong
Nathalie
N
Department of Anatomical and Cellular Pathology, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, N.T., Hong Kong. natwong@cuhk.edu.hk
Chan
Kathy Y-Y
KY
Macgregor
Pascale F
PF
Lai
Paul B-S
PB
Squire
Jeremy A
JA
Beheshti
Ben
B
Albert
Monique
M
Leung
Thomas W-T
TW
D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology / Q000821:virology; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D016000:Cluster Analysis; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006526:Hepatitis C / Q000473:pathology / Q000821:virology; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology / Q000821:virology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D014158:Transcription, Genetic / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D014409:Tumor Necrosis Factor-alpha