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The aim of this study was to develop an accurate predictive system for prognosis of hepatocellular carcinoma (HCC) patients after hepatectomy. We pooled data of clinicopathological features of 234 HCC patients who underwent curative hepatectomy. On the basis of the pooled data, we established a simple predictive staging system (PS score) scored by the mathematical product of tumor number and size, and degree of liver function. We compared the prognostic abilities of the PS score (score 0–3) with those of six well-known clinical staging systems. Then, we found that there were significant differences (P

Downregulation of inhibitor of DNA binding 2 (ID2) is associated with poor prognosis in cases of hepatocellular carcinoma (HCC). Therefore, to search for effective antitumor drugs for the treatment of HCC exhibiting poor prognostic indicators, we used two HCC-derived cell lines (HuH-7 and HLE) to alter ID2 levels. Specifically, ID2 expression was knocked down in HuH-7 cells via transfection with ID2-specific small interfering RNAs and separately ID2 was overexpressed in HLE cells via an ID2 expression plasmid vector. To assess the effect of antitumor drugs, MTS assay was performed. Annexin V staining was used to evaluate apoptosis and real-time RT-PCR was used to measure mRNA levels. ID2 knockdown cells were more susceptible to histone deacethylase (HDAC) inhibitors including sodium butyrate (NaB), sodium 4-phenyl-butyrate, tricostatin A, suberoylanilide hydroxamic acid, MS-275, apicidin and HC-toxin. Conversely, cells that overexpressed ID2 were less susceptible than control cells to HDAC inhibitors. NaB-induced apoptosis was inversely correlated with ID2 expression. Expression of the anti-apoptotic mRNA BCL2 was induced by NaB in control cells, but this induction of BCL2 was inhibited by ID2 knockdown and strengthened by ID2 overexpression. Expression of another anti-apoptotic mRNA, BCL2L1, was decreased by NaB administration and then partially recovered. However, in ID2 knockdown cells, BCL2L1 levels did not recover from NaB-induced suppression. ID2 affected the susceptibility of two HCC-derived cell lines to an HDAC inhibitor by regulating the expression of anti-apoptotic genes. Therefore, HDAC inhibitors may be effective for the treatment of HCC for which the prognosis is poor based on ID2 downregulation and ID2 could serve as a marker that is predictive of the clinical response to HDAC inhibitors.

Pancreatic cancer (PC) is one of the most deadly malignant tumors. The aim of this study was to identify potential biomarkers for PC. Using two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry, the proteomic profiles of pancreatic cancerous and non-cancerous tissues from ten patients with PC were compared. One of the numerous spots that showed stronger intensity in cancerous compared to non-cancerous tissues was identified as non-muscle cofilin (cofilin-1). This up-regulation was validated by Western blot analysis. It is noteworthy that Western blot analysis showed significantly lower expression of muscle cofilin (cofilin-2) in pancreatic cancerous tissues compared to non-cancerous tissues. This is the first time that cofilin isoforms (cofilin-1/2) have been identified to be differentially expressed in pancreatic cancerous tissues. Therefore, cofilin isoforms may serve as candidates for clinically useful biomarkers or therapeutic targets for PC.

Heat shock protein-binding protein 1 (HspBP1) is a co-chaperone that inhibits heat shock 70-kDa protein (Hsp70) activity. In mouse neuroblastomas and lung tumors, the protein levels of HspBP1 and Hsp70 are elevated by a similar amount compared to non-tumor tissues. However, no studies have been reported regarding the levels of HspBP1 in human cancer tissues. Our previous proteomic study demonstrated that the expression of Hsp70 was increased in human hepatitis C virus-related hepatocellular carcinoma (HCV-HCC) tissues. Here, we investigated the expression of HspBP1 in human HCV-HCC. Immunoblotting analysis of HspBP1 and Hsp70 was performed in human HCV-HCC tissues from 20 patients. In 80% of the patients, Hsp70 increased an average of 3.55-fold, and in 50% of the patients, HspBP1 increased an average of 2.02-fold. Comparison and analysis of expression and clinical data revealed a significant difference between moderately-differentiated HCC and non-tumor tissues. In addition, there was a significant difference between the ratio of HspBP1 to Hsp70 levels and tumor size (

Although gemcitabine (GEM) is widely used in the treatment of pancreatic cancers, the molecular mechanisms that underlie its anti-tumor effects are not fully understood. To clarify the anti-tumor mechanism(s) of GEM, we studied a human pancreatic cancer cell line, YPK-1, that showed a 50% inhibitory concentration (IC50) of GEM of 6.3 x 10(-3) microg/ml after 72 h of exposure. Cell proliferation was perturbed by 6 to 72 h of exposure to GEM concentrations equal to one-half or one-quarter of the IC50. We used cDNA microarrays containing 2976 genes to identify genes with expression affected by exposure to GEM. The self-organizing map identified nine clusters, including 85 and 87 genes, that showed differential expression in response to exposure to one half and one quarter IC50 GEM, respectively. Of these, 24 genes were common to cells exposed to the two different concentrations of GEM. Most are signal transduction or transcription-related genes. The microarray data for two of these genes, SPARC and RPS8, were validated by RT-PCR. Although further studies are needed to examine whether the changes in expression profiles of these genes are specific to cells exposed to GEM, the present data provide insights into the anti-tumor effects of GEM on pancreatic cancers.

Recent genome-wide screens have identified genes associated with the metastatic potential of hepatocellular carcinoma (HCC); however, there is little overlap between the identified genes, and interpretations of the results remain controversial. These inconsistencies may be related to differences in the sample populations, use of distinct microarray platforms and algorithms, and the complicated modes of HCC recurrence. We investigated the gene expression profiles of extrahepatic recurrence (EHR) and early intrahepatic recurrence (IHR), which are two representative modes of recurrence of HCC attributable to metastasis. We used DNA microarray analysis and identified 46 signature genes for EHR in 35 HCCs in a supervised learning manner. The obtained gene expression profile was compared with that for early IHR that was determined previously in the same manner. The 46 signature genes for EHR included many cell adhesion-related genes (ITGA6, SPP1, DNMBP, CD44 and POSTN), which all showed higher expression in HCC with EHR than in HCC without EHR. The 46 signature genes for early IHR included 10 immune response-related genes, which all showed lower expression in HCC with early IHR than in HCC without early IHR. The signature genes for EHR included only two immune response-related genes (P=0.013). These results suggest that alteration of the cell adhesion system plays a central role in EHR and that reduction of the immune response is a specific step in early IHR. These results indicate that the metastatic processes in EHR and early IHR involve different molecular pathways.

Many reports suggest that hepatic steatosis leads to hepatocellular carcinoma (HCC), including hepatitis C virus or non-alcoholic steatohepatitis. Proteomic study of tumor tissues from HCC patients, focusing on apolipoprotein (apo) of apoA1, apoB100 and apoE, was performed by immunoblotting. Although the significant changes of apoA1 or apoB100 could not be shown statistically, the immunoblotting showed the increase in protein level of apoE in the tumor tissues of 88% of patients without increase of apoE gene expression and serum level. These results suggest the accumulation of apoE by impaired secretion. Moreover, immunoblot analysis on two-dimensional electrophoresis showed a strong possibility that sialylated forms of apoE also were increased in tumorous tissues of HCC. ApoE level in tumorous tissues is frequently elevated and may be a good histological marker for HCC.

It has been suggested that sex affects not only the incidence of hepatocellular carcinoma (HCC) but also the outcome after treatment. However, no sex-specific therapeutic targets for HCC have been identified. Identification of sex-specific genes will allow for the development of more personalized therapies. To this end, we investigated the expression of approximately 6000 genes in 50 samples of hepatitis C virus (HCV)-related HCC by oligonucleotide microarray. Our supervised learning method and subsequent random permutation test identified 27 genes that were differentially expressed in samples from male (n=34) and female (n=16) patients. Our gene selection was validated by a false discovery rate of only 0.5%. For the 27 genes, expression levels of 12 were higher and expression levels of 15 were lower in HCC samples from men than in HCC samples from women. For the cell proliferation-related genes identified, expression levels of PRDX1 were relatively high in HCC samples from men, and expression levels of PRDX3 were relatively high in HCC samples from women. The DNA microarray data for PRDX1 and PRDX3 were reproduced by reverse transcription-PCR analysis. Our results suggest that these 27 genes may serve as molecular targets or markers for sex-specific treatment of HCV-related HCC. Further studies are needed to elucidate their possible roles in male and female patients with HCV-related HCC.

Chronic infection with hepatitis B or C virus (HBV or HCV) is the most clearly established risk factor for hepato-cellular carcinoma (HCC). One type of HCC (non-B, non-C HCC) also appears to develop in patients negative for both HBV and HCV. Using a supervised learning method, we investigated gene expression in 11 non-B, non-C HCCs with high-density oligonucleotide microarrays, and compared the patterns of gene expression with those of HBV-infected HCCs (B-type HCCs) and HCV-infected HCCs (C-type HCCs) in the previous dataset. Our gene selection identified 112 and 64 genes that were differentially expressed in non-B, non-C HCC in comparison with B- and C-type HCCs, respectively. In both gene selections, we found that the false discovery rate, the percentage of genes identified by chance, was less than 5%. Additionally, in combination with the previous data, our present data revealed a set of genes specific to each type of B- and C-type HCCs and non-B, non-C HCC. Among these, an interferon-induced gene, IFI27, was differentially expressed among all three types of HCCs, and this result was confirmed by RT-PCR. Thus, our present study provides a framework to characterize the molecular features in the three subtypes of HCC with different viral origin.

Our study revealed that the level of circulating cell-free DNA (cfDNA) is increased in the serum of patients with hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). To gain insight into the mechanism underlying this phenomenon, we examined the association between cfDNA levels and various clinicopathological factors in 96 patients with HCV-related HCC and 99 non-HCC patients with HCV. Using pooled DNA microarray data, we profiled the expression patterns of inflammatory cytokine genes in 14 primary tumors from the group of HCC patients. We found that there were positive associations between the cfDNA level, aspartate aminotransferase levels and the number of leukocytes and neutrophils in patients with HCV-related HCC but not in non-HCC patients with HCV. The serum cfDNA level was not associated with other clinicopathological factors in HCC or non-HCC patients. A cluster analysis based on the inflammatory cytokine gene data revealed that HCCs with a high serum cfDNA level had increased levels of several inflammatory cytokine genes, suggesting that the serum cfDNA level is associated with the inflammatory status in primary tumors in HCV-related HCC.

An increased level of glycolysis, an intracellular hallmark of neoplasms, enables cancer cells to survive under various conditions. To elucidate the role of increased glycolysis in the progression of hepatocellular carcinoma (HCC), we investigated the associations between the expression patterns of 14 glycolysis-related genes and clinicopathologic factors in 60 HCCs by using pooled transcriptome data. We then evaluated the therapeutic efficacy of the knockdown of ENO1, which is encoded by a glycolysis-related gene, in HCC cells. Among the 14 genes, levels of 8 genes (GPI, ALDOA, TPI1, GAPD, PGK, PGAM, ENO1 and PKM), all of which can be transcriptionally activated by hypoxia-inducible factor 1alpha (HIF-1alpha), were significantly higher in HCC with venous invasion (VI) than in HCC without VI. Our cluster analysis showed that HCC patients with activation of the 8 HIF-1alpha-regulated genes had significantly shorter overall survival (P=0.023) than did HCC patients without increased expression levels of these genes. The association between the levels of ENO1 and VI was confirmed in an independent sample set of 49 HCCs by real-time reverse-transcription PCR. The knockdown of ENO1 by small-interfering RNA significantly inhibited the proliferation of an HCC cell line (HLE cells) in both the glucose-rich and glucose-free conditions, accompanied by a decreased S phase and increased G2/M phase of the cell cycle. Collectively, these data suggest that activation of an HIF-1alpha-regulated glycolysis module is closely related to the aggressive phenotype of HCC, and that ENO1, a glycolysis module gene, might serve as a new target to circumvent HCC metastasis.