alpha-fetoprotein-producing hepatoma cell lines share common expression profiles of genes in various categories demonstrated by cDNA microarray analysis

Liver carcinogenesis is a multistep process involving various genetic alterations. cDNA microarray containing 1,080 elements (930 unique genes) was used to comprehensively analyze the genetic alterations in hepatoma cell lines, and clustering analysis was used to analyze the relatedness of the gene-expression profiles. Among 7 hepatoma cell lines analyzed, 5-alpha-fetoprotein (AFP)-producing hepatoma cell lines (HepG2, Huh7, Hep3B, PLC/PRF/5, and Huh6) were shown to have common gene-expression profiles compared with those of AFP-negative hepatoma cell lines (HLE and SK-Hep1) and cancer cell lines of nonhepatocyte origin (HeLa and KMBC). Furthermore, HepG2, Huh7, and Hep3B had higher expressions of AFP and shared a common gene-expression profile even when compared with other AFP-producing cells. Analysis of the genes with a common expression profile among these 3 AFP-positive cells revealed 254 genes across various categories. We found that 18 of these genes consistently showed altered levels of expression (more than 3-fold changes) in the 3 AFP-producing hepatoma cell lines (11 up-regulated and 7 down-regulated). In these 18 genes, 5 genes, including that for AFP, were previously reported to be involved in HCC and 6 genes involved only in other types of cancer. Our study showed that AFP-producing hepatoma cell lines shared a distinct expression profile of genes in various categories. An understanding of a causal relationship of this particular expression profile of genes to AFP-positive and AFP-negative hepatocellular carcinoma (HCC) may contribute to more rational therapy in future.     

转录因子Foxp3在肝癌细胞中的表达及意义

目的研究转录因子Foxp3在肝癌细胞中的表达及其对肿瘤免疫微环境的作用。方法用常规RT-PCR和基因表达谱芯片检测Foxp3在10种肝癌细胞系中的表达,进一步用Western blot、免疫组化和流式细胞术验证其蛋白水平的表达;将表达Foxp3的肿瘤细胞及用Foxp3 shRNA沉默后的肿瘤细胞分别与CD4＋CD2-5T细胞共培养,CFSE评价免疫效应细胞增殖情况。结果 Foxp3在所有的肝癌细胞系中均出现表达;肝癌细胞与CD4＋T细胞进行共培养,可显著抑制共培养体系中CD＋4T细胞增殖及其表面标记的表达;肝癌细胞Foxp3表达沉默后,可显著逆转共培养体系中肝癌细胞对CD4＋T细胞增殖的表达抑制。结论在肝细胞癌（HCC）细胞系及部分HCC患者组织中均发现Foxp3的表达,肝癌细胞Foxp3的表达参与对肿瘤微环境中效应性T细胞增殖的抑制。     

Gene therapy for alpha-fetoprotein-producing human hepatoma cells by adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene

We have developed a recombinant replication-defective adenovirus containing human alpha-fetoprotein (AFP) promoter/enhancer to direct cell type-specific expression of the herpes simplex virus thymidine kinase (HSVtk) gene to AFP-producing hepatocellular carcinoma (HCC) cells. After an in vitro infection by a recombinant adenovirus carrying the lacZ gene under the control of human AFP promoter/enhancer (AdAFPlacZ), an expression of the lacZ gene was demonstrated efficiently in AFP-producing HuH-7 and HepG2 cell lines, but not in AFP-nonproducing HLE and HLF cell lines, although lacZ gene expression was demonstrated in all these cell lines when infected with adenovirus vector carrying lacZ gene driven by the beta-actin-based promoter. Expression of the HSVtk gene by adenovirus, from AFP promoter/enhancer (AdAFPtk) induced the cells sensitive to ganciclovir (GCV) in the AFP-producing cell line efficiently, but not in AFP-nonproducing HLF hepatoma cells. An in vitro bystander effect was observed when only 10% of the cells were infected with AdAFPtk. These findings suggest that the AFP promoter/enhancer sequence can provide the tumor-specific activity for the therapeutic gene expression, and that the AdAFPtk vector induces the selective growth inhibition by GCV in the adenovirus-infected human hepatoma cells in vitro. Recombinant adenovirus transfer of the HSVtk gene under the control of tumor-specific promoter followed by GCV may have promise as a targeted in situ treatment for solid neoplasms. (Hepatology 1996 Jun;23(6):1359-68)     

Suppression of hepatocellular carcinoma growth in mice via leptin, is associated with inhibition of tumor cell growth and natural killer cell activation

BACKGROUND/AIMS: Leptin exerts potent immune modulatory properties. The aim of this study was to determine leptin's anti-tumor effect in a murine model of hepatocellular carcinoma (HCC). METHODS: In vivo, athymic nude mice were transplanted with Hep3B cells, followed by daily leptin administration for 6 weeks. RESULTS: Leptin administration induced a significant reduction in tumor size, improved survival rate, and was associated with a significant increase in peripheral natural killer (NK) cell number. Splenocytes from leptin-treated mice featured decreased expression of CIS mRNA. SCID mice featured a similar leptin-associated tumor suppression. In contrast, NK-deficient SCID-beige mice developed larger tumors which were unresponsive to leptin. NK cells incubated in vitro with increasing doses of leptin demonstrated increased cytotoxicity and proliferation. Incubation of leptin with hepatoma cells induced a dose-dependent reduction in proliferation, suggesting a direct anti-tumor effect. Leptin induced increased mRNA expression of STAT2 and SOCS1 on HCC cell lines. CONCLUSIONS: Leptin administration induces a significant suppression of human HCC. This effect is mediated by induction of natural killer cell proliferation and activation, along with direct inhibition of tumor growth. Decreased NK expression of inhibitory CIS and over-expression of the antiproliferative STAT2 and SOCS1 proteins in HCC lines may underline the anti-cancerous effects of leptin.     

肝细胞癌Autotaxin cDNA部分序列测定及其意义

进行肝癌Ａｕｔｏｔａｘｉｎ（ＡＴＸ）ｃＤＮＡ部分序测测定,观察ＡＴＸ ｍＰＲＮＡ在肝癌组织中的表达程序,以探讨ＡＴＸ在肝癌转移机理中的作用。方法 提取肝癌７７２１细胞株,５例正常肝组织,３２肝癌组织ＲＮＡ,参照黑色素瘤ＡＴＸ ｍＲＮＡ表达程序分析,同时７７２１细胞株ＲＴ－ＰＣＲ产物进行ｃＤＮＡ序列测定显示与黑色素具有９９。７％的同样性;癌组织ＡＴＸ ｍＲＮＡ表达显著高于正常肝组织。结论 肝癌及正常     

The <Emphasis Type="Italic">PMAIP1</Emphasis> Gene on Chromosome 18 is a Candidate Tumor Suppressor Gene in Human Pancreatic Cancer

Frequent loss of heterozygosity on the long arm of chromosome 18 is observed in pancreatic cancer. Previous studies suggested the existence of one or more tumor-suppressor genes other than SMAD4 on chromosome 18. To identify the candidate tumor-suppressor gene(s), we compared gene expression by cDNA microarray analyses using a pancreatic cancer cell line Panc-1 and its hybrid cell lines showing suppressed cell growth after introduction of one normal copy of chromosome 18. The microarray analyses identified 38 genes on chromosome 18 that showed differential expressional levels. Among these genes, phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1/APR/NOXA) was identified as one of the candidates for tumor suppressor. Expression vector-mediated introduction of PMAIP1 suppressed cell proliferation, and RNAi-mediated knockdown of PMAIP1 induced recovery of cell growth. These results suggest that PMAIP1 may play an important role in the progression of pancreatic cancer.     

Modulation of gene expression in MHCC97 cells by interferon alpha

AIM: To elucidate the molecular mechanisms of the inhibitory effects of IFN-α on tumor growth and metastasis in MHCC97 xenografts. METHODS: Three thousand international units per milliliter of IFN-α-treated and -untreated MHCC97 cells were enrolled for gene expression analysis using cDNA microarray. The mRNA levels of several differentially expressed genes in cDNA microarray were further identified by Northern blot and RT-PCR. RESULTS: A total of 190 differentially expressed genes including 151 IFN-α-repressed and 39 -stimulated genes or expressed sequence tags from 8 464 known human genes were found to be regulated by IFN-α in MHCC97. With a few exceptions, mRNA levels of the selected genes in RT-PCR and Northern blot were in good agreement with those in cDNA microarray. CONCLUSION: IFN-α might exert its complicated anti-tumor effects on MHCC97 xenografts by regulating the expression of functional genes involved in cell metabolism, proliferation, morphogenesis, angiogenesis, and signaling.     

Very low density lipoprotein receptor subtype II silencing by RNA interference inhibits cell proliferation in hepatoma cell lines

Very low density lipoprotein receptor (VLDLR) belongs to the low density lipoprotein receptor family, it is divided into two subtypes according to forms with an absence (type II) or a presence (type I) of the O-linked sugar domain. VLDLR have been detected in kinds of cancers so far; however, the subtype of VLDLR in hepatocellular carcinoma (HCC) tissues and hepatoma cell lines has yet to be reported. We detected the VLDLR expression in 39 cases of hepatocellular carcinoma and in three kinds of hepatoma cell lines: HepG2, HBV transfected HepG2.2.15, SMMC-7721 and normal human fetal liver cell line LO2 using RT-PCR and western blotting. The results showed that both type I and type II VLDLR were detected in HCC tissues and hepatoma cell lines, and the type II VLDLR expression was significantly higher than that of type I in cell lines. We inhibited the type II VLDLR expression by shRNA-mediated RNA interference in HepG2, SMMC-7721 cell and then subsequently found the cell proliferation slowed down. The cyclinD1 expression confirmed the cell cycle was arrested at the G0/G1 phase, suggesting that inhibiting the type II VLDLR expression may have a positive impact on carcinogenesis of HCC.     

Downregulation of alpha-fetoprotein siRNA inhibits proliferation of SMMC-7721 cells

AIM: To study the function of α-fetoprotein (AFP) in SMMC-7721 hepatoma cells. METHODS: A hairpin siRNA expressing plasmid pSilencer3.0-H1-afp was constructed and transfected into SMMC-7721 cells with Lipofectamine 2000. The expression of AFP was monitored by real-time RT-PCR and immunoassays, its effect on SMMC-7721 cell proliferation and cell death was detected by MTT and fluorescenceactivated cell sorter (FACS). RESULTS: The AFP-siRNA expressing plasmid downregulated the expression of AFP obviously (about 34%), and inhibited SMMC-7721 cell proliferation, but did not induce apoptosis. CONCLUSION: Downregulation of AFP siRNA inhibits proliferation of SMMC-7721 cells, but cannot cause apoptosis.     

Tissue factor pathway inhibitor-2 as a frequently silenced tumor suppressor gene in hepatocellular carcinoma

In HCC, inactivation of tumor suppressor genes plays a significant role in carcinogenesis. Apart from deletions and mutations, growing evidence has indicated that epigenetic alterations including aberrant promoter methylation and histone deacetylation are also implicated in inactivation of tumor suppressor genes. The goal of this study was to identify epigenetically silenced candidate tumor suppressor genes in human HCC by comparing the changes in oligonucleotide microarray gene expression profiles in HCC cell lines upon pharmacological treatment with the demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-dC). By analyzing the gene expression profiles, we selected tissue factor pathway inhibitor-2 (TFPI-2), a Kunitz-type serine protease inhibitor, for validation and further characterization. Our results showed that TFPI-2 was frequently silenced in human HCC and HCC cell lines. TFPI-2 was significantly underexpressed in approximately 90% of primary HCCs when compared with their corresponding nontumorous livers. TFPI-2 promoter methylation was detected in 80% of HCC cell lines and 47% of human HCCs and was accompanied by reduced TFPI-2 messenger RNA expression. In addition, TFPI-2 expression in HCC cell lines can be robustly restored by combined treatment with 5-Aza-dC and histone deacetylase inhibitor trichostatin A. These findings indicate that TFPI-2 is frequently silenced in human HCC via epigenetic alterations, including promoter methylation and histone deacetylation. Moreover, ectopic overexpression of TFPI-2 significantly suppressed the proliferation and invasiveness of HCC cells. CONCLUSION: Our findings suggest that TFPI-2 is a candidate tumor suppressor gene in human HCC.