RIN YAMAGUCHI, HIROHISA YANO, AKIHIRO IEMURA, SACHIKO OGASAWARA, MAKOTO HARAMAKI, AND MASAMICHI KOJIRO Vascular endothelial growth factor (VEGF) is thought to take an important role in tumor angiogenesis. The present study examined VEGF expression immunohistochemically in hepatocellular carcinomas (HCCs) in various histological grades and sizes. In HCCs that were composed of cancerous tissues of single histological grade, VEGF expression was the highest in well-differentiated HCCs, followed by moderately differentiated HCCs, and then poorly differentiated HCCs. VEGF positivity gradually decreased with the increase in tumor size. In the nodules larger than 3.0 cm, 36.8% were VEGF-negative. In HCCs consisting of cancerous tissues of two different histological grades, the expression was less intensive in the higher-grade HCC component. VEGF was not expressed in sarcomatous areas, while VEGF was expressed in the surrounding HCC tissues. The expression was also remarkable in the noncancerous tissues in which inflammatory cell infiltration was apparent. VEGF expression was also examined in six HCC cell lines. 5,6 and angiogenin 7 are known to promote tumor angiogenesis, and growth factors are thought to be the most important. Vascular endothelial growth factor (VEGF) was first described by Senger et al. 8 and Ferrara et al. 9 VEGF is the most intriguing factor in regard to tumor angiogenesis. 8,10 It selectively acts on the endothelial cells that express VEGF receptor, i.e., fms-like tyrosine kinase 1 (flt-1) or KDR/flk-1, 11,12 whereas the other angiogenesis factors, such as basic fibroblast growth factor (bFGF), which does not possess signal peptide, show no specificity. 13 There are four known splicing variants of human VEGF, i.e., VEGF 121 , VEGF 165 , VEGF 189 , and VEGF 206 . They possess signal peptide. VEGF 121 and VEGF 165 are exported from the cells, while VEGF 189 and VEGF 206 are predominantly cell-associated. 14,15 VEGF has been reported to possess various biological activities, e.g., it increases vascular permeability, 8,15 exerts mitogenic effects on endothelial cells, 9,15 stimulates the proliferation and migration of endothelial cells, 16,17 induces the expression of interstitial collagenase, 16 and promotes macrophage migration. 18 Its expression in mRNA level has been confirmed in various organs, such as the lung, kidney, adrenal gland, heart, liver, and gastric mucosa; and in malignant tumors, brain tumor, bladder cancer, kidney cancer, ovarian cancer, gastric cancer, colon cancer, breast cancer, and hepatocellular carcinoma (HCC). [19][20][21][22][23][24][25][26][27] HCC is generally considered as a hypervascular tumor, but its vascularity varies widely according to tumor size and histological grade. In small-sized and well-differentiated HCCs, artery-like vessels are not well developed, 28,29 and capillarization of blood space (sinusoid of HCC) is incomplete. [30][31][32] These HCCs are often undetectable by angiography. [32][33][34] On the other hand, in moderately or poorly differentiated HCC...

Interferon alfa (IFN-␣) has been shown to possess antiviral activity, antiproliferative activity, and various immunoregulatory activities including: 1) stimulation of cytotoxic activities of lymphocytes and macrophages, and of natural killer cell activity; and 2) induction of class I major histocompatibility complex antigens. 1 The effects of IFN-␣ are mediated through interaction with the specific cell-surface receptor, type I IFN receptor. This receptor consists of two chains, Hu-IFN-␣R1 and Hu-IFN-␣R2, which can be present in different forms. [2][3][4][5][6] The Hu-IFN-␣R1 chain is present as either the full chain (Hu-IFN-␣R1) or a splice-variant (Hu-IFN-␣R1s) lacking exons 4 and 5. Hu-IFN-␣R2 chain exists in soluble, short, and long forms (Hu-IFN-␣R2a, Hu-IFN-␣R2b, and Hu-IFN␣R2c, respectively). [2][3][4] Most likely, the Hu-IFN-␣R1 and Hu-IFN-␣R2c chains represent the predominantly active form. 2 Binding of the receptor and IFN-␣ induce transcription of IFN-inducible genes through the activation of the Jak/signal transducer and activator of transcription (STAT) signaling pathway. 7-9 Interferon regulatory factor (IRF)-1, a transcriptional activator, and its antagonistic repressor, IRF-2, have been identified as regulators of type I IFN (mainly IFN-␣ and IFN-␤) and IFN-inducible genes. 10-12 IRF-1 has recently been shown to inhibit cell proliferation, induce apoptosis, and manifest antioncogenic activities, 10,13-17 while IRF-2 has the oncogenic potential. 10 The IRF-1 gene itself is IFN-inducible and may thus be one of the critical target genes mediating IFN action. 11 Antivirus activity of IFN-␣ has attracted a great deal of attention, and IFN-␣ has been applied in treatment for hepatitis B virus (HBV)-and hepatitis C virus (HCV)-related chronic hepatitis in several countries (reviewed in Gutterman 18 ). In the liver of HCV-infected patients, expressions of Hu-IFN-␣R1 and Hu-IFN-␣R2 chains were investigated in terms of mRNA level, and the relationship between their expression levels and response to IFN-␣ therapy was reported. 19,20 Although IFN-␣ has been proven to have a curative potential in treatment of HBV-and HCV-associated chronic liver diseases, its effect on hepatocellular carcinoma (HCC), which is a common and often fatal complication of HBV-and HCV-related chronic liver diseases, 21 is not well known. Clinical trials of IFN-␣ in treatment of HCC did not achieve consistent results: one study showed beneficial effects, 22 and the other studies did not show significant antitumor effects. 23,24 In contrast, IFN-␣ has been shown to be useful for the treatment of several malignant diseases, including hairy-cell leukemia and chronic myelogenous leukemia (reviewed in Gutterman 18 ).Experimental studies showed that IFN-␣ can inhibit the growth of various normal and malignant cells in vitro by inducing cell-cycle changes (e.g., induction of G 0 /G 1 arrest and prolongation of the S phase) 25-34 and/or apoptoAbbreviations: IFN-␣, interferon alfa; STAT, signal transducer and activator of transcription; IRF, ...

Hepatocellular carcinomas often contain tumor cells of more than one histological grade. The clonal relationship and biological behavior of hepatocellular carcinoma cells in histologically heterogeneous areas have not been fully explored. We established two distinct human hepatocellular carcinoma cell lines (HAK-1A and 1B) from a single nodule showing a three-layered structure with a different histological grade in each layer. Morphologically, HAK-1A and 1B resembled well-differentiated hepatocellular carcinoma cells in the outer layer of the original tumor and poorly differentiated ones in the inner layer, respectively. HAK-1B appeared less differentiated morphologically and more aggressive biologically than HAK-1A; HAK-1B had a shorter doubling time, higher tumorigenicity and an aneuploid DNA index. Chromosome analysis revealed many different abnormalities in the two cell lines, in which, however, two identical structural abnormalities (2q+ and 17p+) were identified. Moreover, sequence analysis of the p53 gene showed identical mutations at codon 242 in both cell lines. These findings suggest that the two cell lines are of clonal origin and that hepatocellular carcinomas consisting of cancerous tissues of more than one histological grade may reflect clonal dedifferentiation in the tumor. Furthermore, we predict that a clonal, morphologically less differentiated subpopulation such as HAK-1B is more aggressive in proliferation and may be closely related to subsequent tumor progression in hepatocellular carcinoma.

It is known that there is a very high incidence of hepatocellular carcinoma (HCC) among patients with type C chronic hepatitis and cirrhosis, and alpha -fetoprotein (AFP) has been widely used as a diagnostic marker for HCC. However, there are some patients showing continuous high AFP values but no evidence of HCC, and some studies have defined such patients as a high-risk group for HCC. In vitro study has shown that interferon (IFN) inhibits cell proliferation and enhances apoptosis as well as specific cytotoxic T lymphocytes against HCC, resulting in direct anticancer actions. In this study, we investigated the effect of IFN on AFP changes in chronic hepatitis C patients. Of 40 patients with chronic hepatitis C in whom diagnostic imaging confirmed the absence of HCC, 24 patients showed high pretreatment AFP values (high AFP group: AFP level > 10 ng/dl; mean +/- SD, 46.3 +/- 41.5 ng/dl) and 16 showed low pretreatment AFP values (low AFP group: pretreatment AFP level < or = 10 ng/dl; mean +/- SD, 5.3 +/- 2.2 ng/dl). Pretreatment clinical parameters were statistically evaluated in relation to the AFP value. In the high AFP group, the platelet count, albumin level, and prothrombin (%) were significantly lower (P = 0.047, P = 0.0002, and P = 0.044, respectively), suggesting that AFP value increases with advancing liver disease. Subsequently 27 patients were administered IFN (IFN group), and the remaining 13 patients were administered Stronger Neo-minophagen C (SNMC), a glycyrrhizin preparation (SNMC group), as a control group receiving liver-protective therapy. Alanine aminotransferase was reduced in both the IFN and the SNMC group (mean, 132.56 to 60.07 mg/ml [P < 0001] and 147.85 to 56.23 mg/ml [P = 0.0240], respectively). AFP was significantly reduced in the IFN group (mean, 30.03 to 12.65 ng/ml; P = 0.0034), but there was no significant change in AFP in the SNMC group (mean, 29.70 to 39.17 ng/ml). AFP is useful for diagnosing HCC; however, some patients show a persistently high AFP level in the absence of HCC, and these patients have been described as a high-risk group for HCC. In this study, we found that IFN therapy but not SNMC universally reduced the AFP baseline. Since AFP is a significant predictor for HCC, therapeutic strategies for hepatitis C, e.g., long-term low-dose IFN treatment, may reduce hepatocarcinogenesis.

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