Combination of serum human satellite RNA and miR-21-5p levels as a biomarker for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis due to the difficulty of its diagnosis. Because human satellite II (HSATII) RNA, a satellite repeat RNA, is highly and specifically expressed in human PDAC, the serum HSATII RNA level may be a biomarker of PDAC. To measure the serum HSATII RNA level with high sensitivity and reproducibility, we previously developed a convenient method, tandem repeat amplification by nuclease protection (TRAP) combined with droplet digital PCR (ddPCR). Here, we refined the original method by simultaneously measuring the serum miR-21-5p level to enhance the detection of PDAC. The resulting PDAC-Index, constructed using serum HSATII RNA and miR-21-5p levels, discriminated patients with PDAC with high accuracy. We verified the clinical usefulness of the PDAC-Index as a supportive test in difficult-to-diagnose cases. The PDAC-Index has satisfactory diagnostic performance and may routinely be applied for detecting PDAC.

Aberrant expression of a novel circular RNA in pancreatic cancer.

Circular RNAs (circRNAs) are single-stranded, covalently closed RNA molecules that are produced from pre-mRNAs through a process known as back-splicing. Although circRNAs are expressed under specific conditions, current understanding of their comprehensive expression status is still limited. Here, we performed a large-scale circRNA profiling analysis in human pancreatic ductal adenocarcinoma (PDAC) tissues, using circular RNA-specific RNA sequencing. We identified more than 40,000 previously unknown circRNAs, some of which were upregulated in PDAC tissues, compared with normal pancreatic tissues. We determined the full-length sequence of a circRNA upregulated in PDAC, which was derived from two noncoding RNA loci on chromosome 12. The novel circRNA, named circPDAC RNA, was not expressed in normal human cells, but was expressed in PDAC and other carcinoma cells. While postulated biological functions, such as peptide production from the circPDAC RNA, were not detected, its aberrant expression was confirmed in other PDAC tissues and in serum from a PDAC patient. These results demonstrate that comprehensive studies are necessary to reveal the expression status of circRNAs and that the circPDAC RNA identified here might serve as a novel biomarker for cancers, including PDAC.

Gene expressions associated with chemosensitivity in human hepatoma cells.

BACKGROUND/AIMS: Only limited patients with hepatoma benefit from chemotherapy without a clear explanation. We aimed to identify genes associated with chemosensitivity using transcriptional profiles. METHODOLOGY: In 8 hepatoma cells (HLE, HLF, Huh7, Hep3B, PLC/PRF/5, SK-Hep1, Huh6, and HepG2) transcriptional profiles were obtained using cDNA microarray including 2300 genes. Chemosensitivities to 8 anticancer drugs (nimustine, mitomycin C, cisplatin, carboplatin, doxorubicin, epirubicin, mitoxantrone, and 5-fluorouracil) were measured by obtaining 50% growth inhibitory concentrations (GI50) using MTT assay. Genes having drug-specific association with chemosensitivity were selected. RESULTS: Up-regulation of topoisomerase II beta was associated with chemo-resistance, the target of doxorubicin. Platinum-specific resistance was associated with superoxide dismutase 2 expression. Antigen peptide transporter 1 expression correlated with nimustine and mitoxantrone-specific susceptibility. These results were verified by semi-quantitative RT-PCR. Drug inactivators reported in non-liver cancers such as multidrug transporters and drug metabolizers showed less diversity of chemosensitivity in hepatoma cells. CONCLUSIONS: To evaluate these gene expressions may be useful to select anticancer drugs, and possibly to consider new therapeutic target to modify drug action.

Interferon-beta is activated by hepatitis C virus NS5B and inhibited by NS4A, NS4B, and NS5A.

Innate immunity is part of the antiviral response. Interferon (IFN)-beta plays a leading role in this system. To investigate the influence of hepatitis C virus (HCV) on innate immunity, we examined the effect of viral proteins on IFN-beta induction. HepG2 cells were co-transfected with plasmids for seven HCV proteins (core protein, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) and the IFN-beta promoter luciferase. Toll-like receptor (TLR) 3 and Toll/IL-1 receptor domain-containing adapter inducing IFN-beta (TRIF) play key roles in dsRNA-mediated activation of interferon regulatory factor (IRF)-3 and IFN-beta; therefore, the participation of TLR3/TRIF in NS5B-mediated IFN induction was examined. Among seven HCV proteins, only NS5B, a viral RNA-dependent RNA polymerase (RdRp), activated the IFN-beta promoter. However, mutant NS5B without RdRp activity or template/primer association did not activate the IFN-beta promoter. Activation of the IFN-beta promoter by NS5B required the positive regulatory domain III, a binding sequence for IRF-3. Moreover, IRF-3 was phosphorylated by NS5B. Both inhibition of TLR3 expression by small interfering RNA and expression of the dominant negative form of TRIF significantly reduced NS5B-induced activation of IFN-beta. Of the six other HCV proteins, NS4A, NS4B, and NS5A efficiently inhibited this activation. HCV NS5B is a potent activator of the host innate immune system, possibly through TLR3/TRIF and synthesis of dsRNA. Meanwhile, NS4A, NS4B, and NS5A block IFN-beta induction by NS5B, which may contribute toward the persistence of this virus.

Nucleotide change of codon 38 in the X gene of hepatitis B virus genotype C is associated with an increased risk of hepatocellular carcinoma.

BACKGROUND/AIMS: The hepatitis B virus (HBV) genotype C is associated with the development of hepatocellular carcinoma (HCC). In addition, the HBV X gene, which encodes the pleiotropic transactivator HBx, has also been associated with the development of HCC. In this study, we investigated whether nucleotide changes in the X gene of genotype C are associated with the development of HCC. METHODS/RESULTS: We sequenced the X gene in age- and sex-matched 39 HBV-infected patients with HCC and 36 HBV-infected patients without HCC. A novel nucleotide change that resulted in a proline to serine substitution at codon 38 in HBx (codon-38 change) was preferentially found in patients with HCC. Then, sera were collected from a new group of age- and sex-matched 52 patients with HCC and 51 patients without HCC. In this cohort also, the codon-38 change was associated with HCC. Multiple logistic regression analysis showed the prevalence of the codon-38 change was significantly associated with HCC in all patients (P=0.001, odds ratio: 4.89). CONCLUSION: The codon-38 change in genotype C is an independent risk factor for the development of HCC and may serve as a useful molecular marker for predicting the clinical outcomes in patients infected with HBV.

MDM2 promoter SNP309 is associated with the risk of hepatocellular carcinoma in patients with chronic hepatitis C.

PURPOSE: A single nucleotide polymorphism (SNP) in the promoter region of MDM2 gene, SNP309, has recently been shown to be associated with accelerated tumor formation in both hereditary and sporadic cancers in humans. However, the association of SNP309 with hepatocellular carcinoma is unknown. We evaluated the association of SNP309 with the risk of hepatocellular carcinoma development among Japanese patients with chronic hepatitis C virus infection. EXPERIMENTAL DESIGN: We genotyped the SNP309 at the MDM2 promoter in 435 Japanese patients with chronic hepatitis C virus infection, including 187 patients with hepatocellular carcinoma and 48 healthy subjects, using a fluorogenic PCR. Presence of SNP was also confirmed by direct sequencing of the MDM2 promoter region. RESULTS: The proportion of G/G genotype of the SNP309 in patients with hepatocellular carcinoma (33%) was significantly higher than that in patients without hepatocellular carcinoma (23%), with an odds ratio (95% confidence interval) of 2.28 (1.30-3.98). A multivariate analysis revealed that MDM2 SNP309 (G/G versus T/T), age >60 years, male gender, presence of cirrhosis, serum alpha-fetoprotein >20 mug/L, and serum albumin <3.2 g/dL were independently associated with the hepatocellular carcinoma development at odds ratio of 2.27, 2.46, 3.08, 4.15, 4.87, and 6.33, respectively. CONCLUSIONS: The MDM2 promoter SNP309 is associated with the presence of hepatocellular carcinoma in Japanese patients with chronic hepatitis C.

Large-scale search of single nucleotide polymorphisms for hepatocellular carcinoma susceptibility genes in patients with hepatitis C.

Hepatitis C virus (HCV) infection is a major risk factor for developing hepatocellular carcinoma (HCC). The host genetic factors that are involved in the development of HCC in patients with HCV infection remain to be investigated. To search for single nucleotide polymorphisms (SNPs) in HCC susceptibility genes, 393 SNPs in 171 candidate genes were examined in 188 Japanese patients with chronic HCV infection, including 77 patients with HCC. HCC-related SNPs were then examined in another 188 patients (including 93 patients with HCC) with chronic HCV infection. Haplotype analyses of HCC-related genes were performed in a total of 376 patients. Of the 393 SNPs, 31 SNPs in 29 genes were significantly associated with HCC based on an initial screening (P < .05). Of these 31 SNPs, 3 SNPs of 3 genes (SCYB14, GFRA1, and CRHR2) were significantly associated with HCC in a secondary screening. Haplotype analyses of these 3 genes identified 2 haplotype blocks associated with HCC. In conclusion, these SNPs and haplotypes located in the SCBY14, CRHR2, and GFRA1 genes will be used as markers to identify a subgroup of Japanese patients with chronic HCV infection who are at high risk of developing HCC.

Hepatitis C virus core protein and hepatitis activity are associated through transactivation of interleukin-8.

BACKGROUND: We evaluated the association between variations in hepatitis C virus (HCV) core protein and hepatitis severity in patients with chronic HCV infection who achieved remission without viral eradication and had a biochemical response to interferon (IFN) therapy, to evaluate the effect of HCV core sequence in the absence of the influence of host factors. METHODS. Using serum from 10 patients with a biochemical response and 10 patients with no response, we measured serum levels of interleukin (IL)-1 beta , IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFN- gamma , and tumor necrosis factor- alpha before and after IFN therapy. Expression vectors with the core region were transfected into Huh7 cells, and cytokine induction was evaluated by reporter assay. RESULTS: In biochemical responders, only IL-8 levels decreased after IFN therapy (P=.04). Changes in the C-terminal hydrophobic region were observed more frequently in biochemical responders. Activation of the IL-8 promoter by HCV core protein was significantly decreased in biochemical responders after IFN therapy (P=.04). When 69 C-terminal amino acids from before IFN therapy were replaced with those from after IFN therapy in 3 biochemical responders, their ability to transactivate IL-8 decreased. CONCLUSIONS: Differences in amino acids in the HCV core protein correlates with hepatitis activity through the modulation of IL-8 induction in HCV-infected patients.

Hepatic gene expression profiles associated with fibrosis progression and hepatocarcinogenesis in hepatitis C patients.

AIM: To determine fibrosis progression and hepatocellular carcinoma (HCC), using simultaneous gene expression analysis. METHODS: Total RNA samples were extracted from liver biopsies from 19 patients with hepatitis C virus (HCV) infection and 3 patients without HCV infection. Among the 19 HCV-infected patients, 7 and 12 patients had grade F1-2 and F3-4 fibrosis, respectively. Of the 12 patients with F3-4 fibrosis, 8 had HCC. Gene expression in the liver samples was determined using an oligonucleotide microarray. The following comparisons were performed: normal livers vs HCV-infected livers; F1-2 vs F3-4; and F3-4 with HCC vs F3-4 without HCC. Genes that were differentially expressed between these groups were identified based on signal-to-noise ratios. RESULTS: In the HCV-infected livers, genes involved in immune responses were highly expressed. Expression levels of genes for plasma proteins and drug-metabolizing enzymes were decreased and those of genes involved in the cell cycle and oncogenesis were increased in the F3-4 cases as compared to the F1-2 cases. Among the F3-4 cases, genes involved in carbohydrate metabolism tended to be more highly expressed in patients with HCC than in patients without HCC. CONCLUSION: We identified genes that are associated with fibrosis progression and hepatocarcinogenesis. This information may be used to detect increased carcinogenic potential in the livers of patients with HCV infection.

Interaction between the HCV NS3 protein and the host TBK1 protein leads to inhibition of cellular antiviral responses.

The persistent nature of hepatitis C virus (HCV) infection suggests that HCV encodes proteins that enable it to overcome host antiviral responses. Toll-like receptor 3 (TLR3)-mediated signaling, which recognizes the double-stranded RNA that is produced during viral replication and induces type I interferons, including interferon beta (IFN-beta), is crucial to the host defense against viruses. Recent studies suggest that a TIR domain-containing adaptor protein, TRIF, and two protein kinases, TANK-binding kinase-1 (TBK1) and IkappaB kinase-epsilon (IKKepsilon), play essential roles in TLR3-mediated IFN-beta production through the activation of the transcriptional factor interferon regulatory factor 3 (IRF-3). We report that the HCV NS3 protein interacts directly with TBK1, and that this binding results in the inhibition of the association between TBK1 and IRF-3, which leads to the inhibition of IRF-3 activation. In conclusion, these results suggest the mechanisms of the inhibition of the innate immune responses of HCV infection by NS3 protein.

Vitamin K2 binds 17beta-hydroxysteroid dehydrogenase 4 and modulates estrogen metabolism.

Vitamin K is a cofactor for gamma-glutamyl carboxylase, an enzyme that is important for blood coagulation. Recent studies have shown that vitamin K has other roles, in addition to post-transcriptional modification, such as bone metabolism and antitumoral actions; these findings have indicated that there might be unknown intracellular binding proteins that are specific for vitamin K. In this study, vitamin K-binding proteins were characterized by pull-down experiment using a chemically synthesized biotynylated vitamin K followed by mass spectrometric identification of the pull-downed components. The results indicated that 17beta hydroxy steroid dehydrogenase 4, apolipoportein E, and 40S ribosomal proteins S7 and S13 might be the candidates of the vitamin K-binding proteins. Subsequent experiments showed that vitamin K2 binds 17beta hydroxysteroid dehydrogenase 4 and decreases the intracellular estradiol:estrone ratio, which resulted in the inhibition of the amount of estrogen receptor alpha-binding to its target DNA. These results suggest a possible novel role for vitamin K in modulating estrogen function.

Acyclic retinoid inhibits human hepatoma cell growth by suppressing fibroblast growth factor-mediated signaling pathways.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is one of the most common human malignancies. Its high mortality rate is mainly a result of high intrahepatic recurrence. The novel synthetic retinoid acyclic retinoid (ACR) has been reported to prevent the recurrence of human HCC after surgical resection of primary tumors, but the molecular mechanisms underlying its effects remain to be elucidated. In this study, we clarified the molecular targets of ACR. METHODS: The inhibitory effects by ACR on growth were examined. Intracellular signaling induced by ACR was comprehensively studied by a reporter assay. Gene expression changes by ACR were examined using a microarray. From these results, a candidate signaling pathway modulated by ACR was determined and whether antagonizing this pathway reverses the effect was examined. RESULTS: We show that ACR inhibits the growth of HCC cells through the down-regulation of fibroblast growth factor (FGF) receptor 3 expression and FGF-mediated signaling, which in turn suppresses the activity of Rho and serum response factor-mediated transcription. Conversely, overexpression of the active form of FGF receptor 3 or the addition of FGF reverses the ACR-mediated inhibition of growth. In addition, silencing the FGF receptor 3 gene by RNA interference inhibits cell growth. CONCLUSIONS: These studies show that ACR is a potent inhibitor of FGF signaling and that selective blocking of the FGF-mediated pathway could be a promising therapeutic approach for the management of patients with HCC.

A simple combination of serum type IV collagen and prothrombin time to diagnose cirrhosis in patients with chronic active hepatitis C.

BACKGROUND/AIM:: Cirrhosis in chronic hepatitis C is a major cause of mortality. The components of reported diagnostic indices of cirrhosis based on biochemical markers may be modified by therapies for hepatic inflammation. We aimed to construct index of cirrhosis in patients treated for chronic active hepatitis. METHODS:: Using sera of consecutive 140 patients with chronic hepatitis C, routine blood tests including fibrosis markers, type IV collagen and procollagen type III peptide (PIIIP), were performed. Diagnosis of cirrhosis was determined by biopsy. Using multivariate analyses, diagnostic indices of cirrhosis were constructed. RESULTS:: Fifty-eight patients were diagnosed to have cirrhosis. Platelet count, prothrombin time, and albumin were lower, and type IV collagen and PIIIP were higher in patients with cirrhosis (p<0.05). There was no difference in aspartate and alanine aminotransferases (AST, ALT) and gamma-glutamyl-transpeptidase (GGT) (p>0.3). Our diagnostic indices I (prothrombin time and platelet count) and II (prothrombin time and type IV collagen) of cirrhosis showed the area under the ROC curves (AUC) of 0.77 and 0.81, respectively. The index II was relatively superior to the index I. CONCLUSIONS:: Using combination of type IV collagen and prothrombin time, efficient diagnosis of cirrhosis can be performed in patients with chronic active hepatitis C.

Genes associated with human hepatocellular carcinoma cell chemosensitivity to 5-fluorouracil plus interferon-alpha combination chemotherapy.

Recently, combined chemotherapy with 5-fluorouracil (5-FU) and interferon (IFN)-alpha has been reported to show marked effects in patients with advanced hepatocellular carcinoma. We investigated the genes associated with susceptibility to this combination therapy. The gene expression profiles of eight human hepatocellular carcinoma cells (HepG2, Hep3B, Huh7, Huh6, PLC/PRF/5, HLE, HLF, and SK-Hep1) were evaluated using an oligonucleotide microarray that consisted of 3,800 genes. The 50% growth inhibitory concentration (GI50) values for 5-FU, IFN-alpha, and the combination of 5-FU plus IFN-alpha were determined by the MTT assay. We selected genes that were expressed differentially between the cells with increased susceptibility to the combination therapy and the remaining cells. Relevance networks of the gene expression patterns and GI50 values of the susceptible cells were constructed to find genes associated with susceptibility to the combination therapy. Of the eight cells tested, five showed increased susceptibility to 5-FU plus IFN-alpha compared with 5-FU treatment alone. Among the 3,800 genes, 25 were expressed differentially between susceptible cells and resistant cells. The relevance networks revealed that sensitivity to 5-FU plus IFN-alpha involved the expression of 27 independent genes, which included 10 genes that are commonly associated with sensitivity to 5-FU alone. We selected a set of genes to predict susceptibility to 5-FU plus IFN-alpha combination therapy. We also selected genes that play key roles in the synergistic effect of this combination therapy. These gene sets should prove useful in evaluations of the efficacy and underlying molecular mechanisms of this combination therapy.

Vitamin K2 inhibits the growth and invasiveness of hepatocellular carcinoma cells via protein kinase A activation.

Hepatocellular carcinoma (HCC) is a common human malignancy. Its high mortality rate is mainly a result of high intrahepatic recurrence and portal venous invasion (PVI). We previously reported that the development of PVI is related to levels of des-gamma-carboxy prothrombin (DCP), a serum protein that increases at a notably higher rate in patients with HCC. Because DCP is produced by a vitamin K shortage, we examined the biological effects of extrinsic supplementation of vitamin K(2) in HCC cells in vitro and in vivo. Consequently, vitamin K(2) inhibits the growth and invasion of HCC cells through the activation of protein kinase A, which modulates the activities of several transcriptional factors and inhibits the small GTPase Rho, independent of suppression of DCP. In addition, administration of vitamin K(2) to nude mice inoculated with liver tumor cells reduced both tumor growth and body weight loss. In conclusion, similar to an acyclic retinoid--which was previously reported to prevent the recurrence of HCC--vitamin K(2), another lipid-soluble vitamin, may be a promising therapeutic means for the management of HCC.

UDP-glucuronosyltransferase 1A7 genetic polymorphisms are associated with hepatocellular carcinoma in japanese patients with hepatitis C virus infection.

PURPOSE: Genetic polymorphisms of UDP-glucuronosyltransferase 1A7 (UGT1A7), which detoxifies endogenous and environmental carcinogens, have been reported to be associated with hepatocellular carcinoma (HCC) in German populations. On the other hand, we reported that interleukin-1 beta (IL-1 beta) gene polymorphisms were associated with hepatitis C virus (HCV)-related HCC. In this study, we evaluated the association of both genes with the risk of HCC in Japanese HCV-infected patients. EXPERIMENTAL DESIGN: Genetic polymorphisms of UGT1A7 and IL-1 beta were investigated in 280 Japanese patients (122 with HCC and 158 without HCC) with chronic HCV infections, by use of standard PCR-based genotyping techniques. RESULTS: We designated the UGT1A7*1 allele (a haplotype conferring higher activity) as H and the *2, *3, and *4 alleles (haplotypes conferring lower activity) as L. The proportions of UGT1A7 L/L and H/L alleles (genotypes) in patients with HCC (25% and 45%, respectively) were higher than those in patients without HCC (15% and 39%, respectively) with odds ratios of 2.73 (95% confidence interval, 1.40-5.35) and 1.80 (95% confidence interval, 1.05-3.09), respectively, compared with the UGT1A7 H/H alleles. Multivariate analyses revealed that UGT1A7 L/L and IL-1 beta/-31T/T-511C/C genotypes, the presence of cirrhosis, age >60 years, male sex, and alpha-fetoprotein >20 microg/ml were associated with the presence of HCC (odds ratios, 2.33, 2.67, 4.20, 3.12, 3.09, and 2.90, respectively). CONCLUSION: The UGT1A7 polymorphisms together with IL-1 beta were associated with the presence of HCC in Japanese HCV-infected patients.

Liver chip and gene shaving.

A comprehensive profile of genes expressed at the mRNA level in various human tissues is considered to be important for understanding the molecular mechanisms of the tissue-specific function and the pathogenesis of related diseases. Here, the gene expression profiling in three human digestive tissues, liver, stomach, and pancreas, was catalogued by generating a large number of expressed sequence tags, and clarified how quantitatively the gene expressions are different. After assembling the redundant clones among three tissues, the results showed that only 1.7% among the assembled genes was expressed commonly in the investigated tissues. These results suggest that the significant functional divergences in different tissues must be related to the divergence of the gene expression profiles. Recently, microarray technologies are widely used. Considering the results that different genes express in different tissues, however, it is important to spot the cDNAs derived from the same tissues or cells examined to acquire information efficiently. For the study of digestive diseases, we constructed an in-house microarray by using the cDNA sets derived from the digestive tissues (liver and gastric chip). In addition, because the amount of information acquired by the microarray analyses is huge, the power of bioinformatics for unifying the obtained data is indispensable. Some examples of the strategies for handling the microarray data obtained by our in-house microarrays are shown in this article.

Synergistic activation of the serum response element-dependent pathway by hepatitis B virus x protein and large-isoform hepatitis delta antigen.

Hepatitis delta virus (HDV) is a naturally occurring satellite of hepatitis B virus (HBV). There are few studies of the effects of the combination of HBV and HDV proteins (HDV antigens [HDAgs]) on intracellular signaling pathways. To understand the influence of HBV and HDV coinfection on hepatocytes, we investigated the effect of HBV proteins and HDAgs on the serum response element (SRE)-dependent pathway. Reporter assays revealed that only HBV X protein (HBx), alone or with the large isoform of HDAg (LHDAg), synergistically activated the SRE-dependent pathway. The effect of HBx and LHDAg on Elk1 or serum response factor (SRF) was examined, because both proteins bind to the SRE. HBx activated the transcriptional ability of Elk1, whereas LHDAg activated the transcriptional ability of SRF. Thus, HBx and LHDAg synergistically activated the SRE-dependent pathway. These results may help us understand clinical phenomena in patients coinfected with HBV and HDV.

Relevance network between chemosensitivity and transcriptome in human hepatoma cells.

Generally, hepatoma is not a chemosensitive tumor, and the mechanism of resistance to anticancer drugs is not fully elucidated. We aimed to comprehensively evaluate the relationship between chemosensitivity and gene expression profile in human hepatoma cells, by using microarray analysis, and analyze the data by constructing relevance networks. In eight hepatoma cell lines (HLE, HLF, Huh7, Hep3B, PLC/PRF/5, SK-Hep1, Huh6, and HepG2), the baseline expression levels of 2300 genes were measured by cDNA microarray. The concentrations of eight anticancer drugs (nimustine, mitomycin C, cisplatin, carboplatin, doxorubicin, epirubicin, mitoxantrone, and 5-fluorouracil) needed for 50% growth inhibition were examined and used as a measure of chemosensitivity. These data were combined and comprehensive pair-wise correlations between gene expression levels and the 50% growth inhibition values were calculated. Significant correlations with significance were used to construct networks of similarity. Fifty-two relations, including 42 genes, were selected. Among them, nearly 20% were various types of transporters, and most of them negatively correlated with chemosensitivity. Transporter associated with antigen processing 1 was associated with resistance to mitoxantrone, consistent with previous reports. Other transporters were not reported previously to associate with chemosensitivity. Resistance to doxorubicin and its analogue, epirubicin, were positively correlated with topoisomerase II beta expression, whereas it negatively correlated with expression of carboxypeptidases A3 and Z. Response to nimustine was associated with expression of superoxide dismutase 2. Relevance networks identified several negative correlations between gene expression and resistance, which were missed by hierarchical clustering. Our results suggested the necessity of systematically evaluating the transporting systems that may play a major role in resistance in hepatoma. This may provide useful information to modify anticancer drug action in hepatoma.