Does interferon therapy prevent hepatocellular carcinoma in patients with chronic viral hepatitis?

Chronic hepatitis C and B are well-recognized and potentially preventable risk factors for hepatocellular carcinoma (HCC) development. Clinical and epidemiological studies suggest that therapy with interferon-α may reduce the overall risk of HCC development in patients with chronic hepatitis C, who achieve sustained virological response, but even in those who fail to eradicate the infection. In chronic hepatitis B, interferon therapy reduces the risk of HCC development in HBeAg-positive and cirrhotic patients who achieve persistent suppression of viral replication, while in HBeAg-negative patients the beneficial effect of interferon-α is not definitively confirmed. The preventive role of interferon-α after potentially curative treatment for HCC in both chronic hepatitis B and C is uncertain due to methodological flaws of the existing studies and prospective randomized controlled trials with pegylated interferon-α are needed to clarify this issue.     

Antiproliferative effects of interferonγ in combination withα-difluoromethylornithine on human carcinoma cell cultures

The antiproliferative effects of human recombinant interferon (IFN) in combination with -difluoromethylornithine (DFMO) or as single agents were assessed on human cell cultures derived from carcinomas of the breast (MCF-7), the ovary (EFO-27) or the kidneys (EGI-4). Results were obtained in proliferation assays by direct cell counting. The cell lines differed considerably in their sensitivities to the antiproliferative effect of IFN as compared by the 50% inhibition doses of the growth (ID₅₀). In contrast to the findings with IFN, similar antiproliferative effects resulted from the application of comparable doses of DFMO. While IFN induced cytotoxic effects in EGI-4 cells, DFMO produced only cytostatic actions in the cell lines analyzed. Synergistic growth inhibition resulted from the combined application of IFN and DFMO in EFO-27 cell cultures. This finding was most pronounced after treatment with IFN or DFMO doses below the respective ID₅₀ values. However, antagonistic effects occurred in cells of the line EGI-4 after DFMO had been combined with IFN at concentrations below the cytotoxic dose range. Within the sensitivity of our proliferation assay, no synergistic interactions were found in MCF-7 cell cultures. In the cell lines tested, no relation between the sensitivity for the single agents and the effectivity of the drug combination was identified. Despite promising synergistic effects in the moderately IFN-sensitive ovarian carcinoma cell line EFO-27, the efficacy of the IFN/DFMO combination was restrained by possible antagonistic effects as demonstrated in the highly IFN-sensitive EGI-4 renal carcinoma cell cultures. We conclude that the differential interaction patterns in the cell cultures analyzed preclude general suggestions for clinical studies using IFN and DFMO.Abbreviations IFN interferon - DFMO difluoromethylornithine This work was part of the doctoral thesis of Mariam Klouche     

Construction of retroviral vectors to induce a strong expression of human class Ⅰ interferon gene in human hepatocellular carcinoma cells in vitro

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Immunological predictors of different responses to combination therapy with interferon α and ribavirin in patients with chronic hepatitis C

Background: This study aimed to investigate peripheral blood CD4+ T-helper (Th) and CD8+ cytotoxic T-lymphocyte (CTL) responses to combination treatment with interferon (IFN) α and ribavirin in 59 patients with chronic hepatitis C, and to correlate the results with the therapy outcome. Methods: The expression of activation molecules on the surface of CD8+ T cells and cytokine production by in-vitro activated CTLs and Th lymphocytes were examined before and at the end of the therapy, using flow cytometry. Results: There were 36 complete responders to the treatment and 23 transient responders who relapsed after withdrawal of the therapy. A significant increase in the production of Th1-type cytokines [IFNγ, interleukin 2 (IL2), and tumor necrosis factor-α (TNFα)] was found at the end of the treatment in complete responders compared with baseline values (P < 0.001). In contrast, transient responders had a marked decrease in the percentage of activated CD8+ T cells expressing CD28 or HLA-DR costimulatory molecules in peripheral blood, and a lower production of TNFα by CTLs and Th cells at the end of the therapy with respect to pretreatment values (P < 0.001). Conclusions: The efficacy of IFNα and ribavirin combination therapy for chronic hepatitis C is associated with a vigorous response of peripheral blood Th1 cells, whereas weak CTL responses at the end of the therapy might predict a further relapse of the disease. Received: March 26, 2002 / Accepted: August 30, 2002 Acknowledgments. This work was supported by grants MZd CzR 5740-3, 6015-3/00. Reprint requests to: R. Amaraa Editorial on page 302     

Abnormal β-catenin gene expression with invasiveness of primary hepatocellular carcinoma in China

AIM To study the abnormal expression of β-catenin gene and its relationship with invasiveness of primary hepatocellular carcinoma among Chinese people. METHODS Thirty-four hepatocellular carcinoma (HCC) specimens and adjacent para-cancerous tissues, 4 normal liver tissues were immunohistochemically stained to study subcellular distribution of β-catenin. Semiquantitive analysis of expression of β-catenin gene exon 3 mRNA was examined by RT-PCR and in situ hybridization. The relationship between expressions of both β-catenin protein, mRNA and clinicopathological characteristics of HCC was also analyzed. RESULTS Immunohistochemistry showed that all normal liver tissues and para-cancerous tissues examined displayed membranous type staining for β-catenin protein,occasionally with weak expression in the cytoplasm.While 21 cases (61.8%) of HCC examined showed accumulated type in cytoplasms or nuclei. The accumuled type Labling Index (LI) of cancer tissue and paracancarous tissue was (59.9 ± 26.3) and (18.3 ± 9.7)respectively (P＜0.01). Higher accumulated type LI was closely related with invasiveness of HCC. Results of RTPCR showed the β-catenin gene exon 3 mRNA Expression Index (El) of 34 HCCs was higher than that of paracancerous tissue and normal liver tissue. Using in situ hybridization, the signal corresponding to β-catenin gene exon 3 mRNA was particularly strong in cytoplasm of HCC when compared with those of para-cancerous and normal liver tissues. Over expression of β-catenin exon 3 was also found to be correlated with high metastatic potential of HCC. CONCLUSION Abnormal expression of β-catenin gene may contribute importantly to the invasiveness of HCC among Chinese people.     

KIAA0008 gene is associated with invasive phenotype of human hepatocellular carcinoma—a functional analysis

Purpose To investigate the function of the KIAA0008 gene, one of the leading genes in the signature associated with hepatocellular carcinoma (HCC) metastasis selected by cDNA microarray, and especially its possible roles in invasion and metastasis of hepatocellular carcinoma.Methods Expression levels of KIAA0008 in 27 primary tumors and 23 matched non-tumor liver tissues from HCC patients, and four HCC cell lines with different metastatic potentials were detected by semi-quantitative RT-PCR and real-time RT-PCR. Recombinant expression plasmid vectors of the KIAA0008 gene were constructed and transfected into HCC cells. The subcellular localization of the KIAA0008 gene product and in vitro effects of KIAA0008 overexpression on proliferation and invasion of HCC cell line were also investigated.Results Expression levels of KIAA0008 in HCC tissues were statistically higher than those of paired non-tumorous liver tissues (P<0.001, paired Wilcoxon test), and in HCCs with high invasiveness these were statistically higher than those with low invasiveness (P=0.002, Mann-Whitney test). In the four HCC cell lines with an identical genetic background and stepwise higher invasiveness potentials, its expression was consistent with their invasiveness potential. The KIAA0008 gene product was concentrated on the nucleus and cell membrane of HCC cells, without any distribution in the cytoplasm. Overexpression of KIAA0008 in the MHCC97L cell line resulted in increased cell proliferation, colony formation, and invasion.Conclusions KIAA0008 expression is associated with invasiveness of HCC; overexpression of KIAA0008 leads to a more invasive phenotype of HCC cell lines.This work was supported in part by the National Foundation for the Excellent Younger Researcher of China (30325041), the 863 R & D High-tech Key Project (2002BA711A02–4), the 973 State Key Basic Research Program Grant of China (G1998051211), the Fund for the Innovative Research Group in Shanghai, and the Fund for Outstanding Scholars of the New Era of the Ministry of Education in China (2003)     

Sensitivity of Ph1+ CFU-GM to human recombinant interferon α and γ alone and in combination

Summary The in vitro effect of human recombinant interferon (IFN) alone and in combination were studied on granulomonocytic colony forming units (CFU-GM) from the peripheral blood of 10 Ph1+ chronic myeloid leukemia (CML) patients and from the marrow of 5 normal or non-leukemic subjects. - and -IFN alone determined a slight inhibition on colony growth with a preferential effect on pure macrophagic colonies. At maximum concentration (10⁴ U/ml) leukemic colony inhibition was 46±34% for IFN and 43±19% for IFN. Culture growth with + IFN in combination were significantly inhibited (up to 96±4%) with a concentration-related effect. Similar results were obtained with normal CFU-GM. The synergism that was found in vitro is probably relevant for the in vivo therapeutic effects of these compounds in CML and suggest that the combination is worth testing in vivo.This work was supported by CNR, Oncology Finalized Project, contract n. 8701169.44. and n. 8600603.44. and by AIRC     

S-phase delay in human hepatocellular carcinoma cells induced by overexpression of integrin β1

AIM: To clarify the mechanisms of integrin overexpression in negatively regulalting the cell cyde control of hepatocellular carcinoma cells SMMC-7721.METHODS: The cell cycle pattern was determined by flow cytometry. The mRNA and protein expression levels were assayed by RT-PCR and Western blot, respectively. Stable transfection was performed by Upofectamine 2000 reagent,and cells were screened by G418.RESULTS: Overexpression of α5β1 or β1 integrin induced S-phase delay in SMMC-7721 cells, and this delay was possibly due to the accumulaltion of cydin-dependent kinase inhibitors (CKIs) p21^cip1 and p27^kip1. The decrease of protein kinase B (PKB) phosphorylation was present in this signaling pathway, but focal adhesion kinase (FAK) was not involved.When phosphorylation of PKB was solely blocked by wortmannin, p27^kip1 protein level was increased. Moreover,S-phase delay was recurred when attachment of the parental SMMC-7721 cells was inhibited by the preparation of poly-HEME, and this cell cycle pattern was similar to that of β1-7721 or α5β1-7721 cells.CONCLUSION: S-phase delay induced by overexpression of integdn 151 subunit is attributed to the decrease of PKB phosphorylation and subsequent increases of p21^cip1 and p27^kip1 proteins, and may be involved in the unoccupied α5β1 because of lack of its ligands.     

Abnormal expression of HSP gp96 associated with HBV replication in human hepatocellular carcinoma

BACKGROUND: Heat shock protein (HSP) gp96 is a member of the HSP90 family and presumably overexpresses as a result of stimulation by mutated or abnormal proteins. Its abnormal expression correlates with carcinogenesis, progression and prognosis of hepatocellular carcinoma (HCC). In this study, we investigated the pathological characteristics of liver gp96 expression and its relationship with hepatitis B virus (HBV) replication in HCC patients. METHODS: Tumor specimens were prospectively collected from 30 HCC patients undergoing liver resection. Total RNAs were extracted from HCC or their noncancerous tissues. The distribution of gp96 expression in hepatocytes was investigated by streptavidin peroxidase (S-P) immunohistochemistry and tissue HBV-DNA was detected by the in situ molecular hybridization technique. The association of gp96 expression with HBV replication, and the histopathological characteristics of HCC were analyzed. RESULTS: The gp96 was strongly expressed in HCC (73.3%, 22 of 30) and weakly (46.7%, 14 of 30) in non-cancerous tissues. The gp96 expression in HCC tissues was correlated with degree of tumor differentiation and tumor size, but not with tumor number (P>0.05). Immunohistochemical analysis showed that 17 of 19 HCC patients with HBVDNA -positive were strongly expressed for gp96, whereas only 5 of 11 patients with HBV-DNA-negative were positive for gp96. A significant difference was found between the two groups (89.5% vs. 45.5%, P<0.05). CONCLUSIONS: The abnormal expressions of HSP gp96 in HCC tissues are associated with HBV replication. This finding indicates that HBV infection plays an important role in the development of HCC.     

Expression of cytochrome P4502E1 gene in hepatocellular carcinoma

AIM: To investigate cytpchrome P4502E1 (CYP2E1) geneexpression in occurrence and progression of hepatocellularcarcinoma (HCC).METHODS: The human liver arrayed library was spottedonto the nylon membranes to make cDNA array.Hybridizationof cDNA array was performed with labeled probes synthesizedfrom RNA isolated from HCC and adjacent liver tissues.Sprague-Dawley rats were administrated diethylnitrosamine(DENA) to induce HCC.CYP2E1 expression was detected bythe method of RT-PCR and Northern blot analysis.RESULTS: CYP2E1 was found by cDNA array hybridizationto express differently between HCC and liver tissues.CYP2E1only expressed in liver,but did not express in HCC tissuesand expressed lowly in cirrhotic tissues.In the progressionof cirrhosis and HCC,the expression level of CYP2E1 wasgradually decreased and hardly detected until the late stageof HCC.CONCLUSION: Using arrayed library to make cDNA arraysis an effective method to find differential expression genes.CYP2E1 is a unique gene expressing in liver but did notexpress in HCC.CYP2E1 expression descended along withthe initiation and progression of HCC,which is noteworthyfurther investigations in its significance in the developmentof HCC.     

Hemizygous deletion and hypermethylation of RUNX3 gene in hepatocellular carcinoma

AIM:To analyze the genetic and epigenetic alterations ofRUNX3 gene,a potential putative tumor suppressor gene,in hepatocellular carcinoma (HCC).METHODS:PCR-based loss of heterozygosity (LOH)detection,analysis of mutation with PCR-single strandconformational polymorphism (SSCP) and sequencing,andmethylation study with methylation specific PCR (MSP) wereperformed on RUNX3 gene in a series of 62 HCCs alongwith their matched normal tissues.RESULTS:Mutation of RUNX3 gene was not found,butone single nucleotide polymorphism with T to A transversionat the second nucleotide of the 18th condon was found.Nine of 26 informative cases (34.6%) showed allelic losson the polymorphic site and 30 cases (48.4%) revealedhypermethylation of RUNX3 gene in promoter CpG islands.Furthermore,of the 9 cases with LOH,8 (88.9%) also hadhypermethylation.CONCLUSION:Our findings indicate that inactivation ofRUNX3 gene through allelic loss and promoter hypermethylationmight be one of the major mechanisms in hepatocellualrcarcinogenesis.     

Significance of cyclooxygenase—2 expression in human primary hepatocellular carcinoma

AIM：To clarife the significance of cyclooxygenase-2(COX-2)expression in human primary hepatcellular carcinoma(HCC)and adjacent nontumorous tissues.METHODS;TheCOX-2protein and mRNA were investigated in 27HCC tissues with adjacent nontumorous tissues,and 5histologically normal liver tissues,using immunohistochemistry and in situ hybridization.RESULTS:The well-differentiated HCC expressed COX-2protein(5.68&#177;1.19)more strongly than moderated HCC(3.43&#177;1.98)and poor differentiated HCC(3.33&#177;1.50)(P&lt;0.05 respectively),adjacent nontumorous tissues(4.93&#177;1.05)and normal live tissues(3.20&#177;1.92)(P&lt;0.01 respectively);More intensive staining of COX-2in adjacent nontumorous tissues was observed than that in normal liver tissues(P&lt;0.05).There was no significant difference among adjacent nontumorous tissues,moderately differentiated HCC and poorly differentiated HCC(P&gt;0.05).The expression of COX-2mRNA was observed in the cytoplasm of the cells of HCC and of gtthe hepatocytes in adjacent nontumorous tissues in which COX-2 protein was positive.CONCLUSION:The overexpression of COX-2 in well-differentiated HCsuggets that COX-2 may play a role in the early stages of hepatocarcinogensis.     

Expression of pituitary tumor transforming gene in human gastric carcinoma

AIM:Pituitary tumor transforming gene (PTIG1) isoverexpressed in a variety of tumors,including carcinomasof the lung,breast,colon,as well as in leukemia,lymphomaand pituitary adenomas.However,there is little informationon its expression in gastric carcinoma.We sought toinvestigate the expression of PTTG1 in gastric carcinomaand to explore the relationship between its expression andclinicopathological factors.METHODS:We studied 75 primary human gastricadenocarcinomas,including 17 mucosal carcinomas,21submucosal infiltrative carcinomas,12 carcinomas invadingproprial muscle layers,6 carcinomas reaching the subserosa,and 19 carcinomas penetrating the serosal surface.Immunohistochemical analysis was performed using paraffin-embedded sections of gastric adenocarcinomas.RESULTS:PTTG1 was expressed heterogeneously incarcinomas. Positive PTTG1 staining was observed in65.3% of the carcinomas (49 of 75).Its expression did notcorrelate significantly with either the histological type orthe depth of infiltration of the gastric carcinomas.However,a statistical analysis showed significant differences betweenthe primary adenocarcinomas and the associated metastaticlymph nodes.CONCLUSION:The results of this study demonstrate thatPTTG1 expression is enhanced in metastatic lymph nodesin comparison to that in primary carcinomas.We suggest that PTTG1 may contribute to lymph node metastases ingastric carcinoma.     

Inhaled interleukin-2 in combination with low-dose systemic interleukin-2 and interferon α in patients with pulmonary metastatic renal-cell carcinoma: effectiveness and toxicity of mainly local treatment

We describe here a mainly topical interleukin-2 (IL-2) application in pulmonary metastatic renal-cell carcinoma: a high-dose long-term inhalation of IL-2 (90% of IL-2 dose) and low-dose systemic subcutaneous IL-2 (10% of IL-2 dose) and systemic subcutaneous interferon (IFN ). The effectiveness of this treatment is remarkable. No pulmonary metastases progressed during treatment. One complete response, 8 partial responses, and 6 cases of stable disease were achieved in the lungs of the 15 patients. In addition, 3 of 7 patients had partial responses and 1 of 7 had stabilization of non-pulmonary metastases. Overall response according to WHO criteria was 1 complete response, 6 partial responses, 2 mixed responses, 5 stable diseases and 1 progressive disease. Toxicity was low. Only WHO grade I toxicity occurred, except for a single grade II event (bronchospasm). This allowed long-term ambulatory treatment (1–23 months) inclusion of high-risk patients, and inclusion of patients with advanced disease. The expected mean survival of patients was 9.9 months, the actual mean survival is now 19.1 months, and 11 of 15 patients are still alive. Quality of life during treatment was good. Inhalation of IL-2 serves as a clinical model for high effectiveness and low toxicity of long-term local IL-2 application. We conclude that mainly local treatment might be the key to successful nontoxic use of IL-2 in cancer patients.