Hepatic Cancer Cell Lines Research Articles

Computational identification and experimental validation of microRNAs binding to the Alzheimer-related gene ADAM10

BackgroundMicroRNAs (miRNAs) are post-transcriptional regulators involved in numerous biological processes including the pathogenesis of Alzheimer’s disease (AD). A key gene of AD, ADAM10, controls the proteolytic processing of APP and the formation of the amyloid plaques and is known to be regulated by miRNA in hepatic cancer cell lines. To predict miRNAs regulating ADAM10 expression concerning AD, we developed a computational approach.MethodsMiRNA binding sites in the human ADAM10 3' untranslated region were predicted using the RNA22, RNAhybrid and miRanda programs and ranked by specific selection criteria with respect to AD such as differential regulation in AD patients and tissue-specific expression. Furthermore, target genes of miR-103, miR-107 and miR-1306 were derived from six publicly available miRNA target site prediction databases. Only target genes predicted in at least four out of six databases in the case of miR-103 and miR-107 were compared to genes listed in the AlzGene database including genes possibly involved in AD. In addition, the target genes were used for Gene Ontology analysis and literature mining. Finally, we used a luciferase assay to verify the potential effect of these three miRNAs on ADAM10 3'UTR in SH-SY5Y cells.ResultsEleven miRNAs were selected, which have evolutionary conserved binding sites. Three of them (miR-103, miR-107, miR-1306) were further analysed as they are linked to AD and most strictly conserved between different species. Predicted target genes of miR-103 (p-value = 0.0065) and miR-107 (p-value = 0.0009) showed significant overlap with the AlzGene database except for miR-1306. Interactions between miR-103 and miR-107 to genes were revealed playing a role in processes leading to AD. ADAM10 expression in the reporter assay was reduced by miR-1306 (28%), miR-103 (45%) and miR-107 (52%).ConclusionsOur approach shows the requirement of incorporating specific, disease-associated selection criteria into the prediction process to reduce the amount of false positive predictions. In summary, our method identified three miRNAs strongly suggested to be involved in AD, which possibly regulate ADAM10 expression and hence offer possibilities for the development of therapeutic treatments of AD.

Abstract 1805: Comparative analysis of 3 rapalogues, everolimus, temsirolimus and sirolimus, in hepatocarcinoma and renal cancer models resistant to VEGFR inhibitors

Abstract Background. In RCC temsirolimus has been approved for first line treatment in advanced disease whereas everolimus has been approved after failure of sunitinib or sorafenib treatment. The aim of our study was to compare everolimus and temsirolimus activity in renal and hepatic cancer cell lines characterized for primary or acquired resistance to sunitinib and sorafenib. Materials and Methods: Parental SK-HEP1 hepatocellular, CAKI1 and 786-0 renal carcinoma cells were exposed to stepwise increasing doses of sunitinib and sorafenib for >6 months until acquisition of resistance. Antiproliferative effects of rapalogues were assessed using MTT assay. mRNA levels of selected genes were evaluated in sensitive and resistant cell lines using q-RT-PCR. Protein phoshorylation was assessed by western-blot. Results. Our panel of HCC and RCC cell lines was characterized for the expression of AKT, Bcl2, PTEN, Cyclin D1 and p27 as well as for the mutation state of KRAS and BRAF that are known to be possible predicting factors of sensitivity to mTOR inhibitors. The HCC cell lines SK-HEP1 and its sunitinib- and sorafenib-resistant counterparts, SK-Suni and SK-Sora, carrying a mutated BRAF gene, displayed high BCL2, PTEN and CCND1 gene expression. Constitutive activation of AKT was found in all sunitinib and sorafenib-resistant cell lines. Rapamycin, everolimus and temsirolimus at concentrations that ranged from 1 to 10µM displayed only low antiproliferative effects (15-20% growth inhibition). In contrast, at 20µM concentration the three rapalogues displayed cytotoxic effects in all cell lines. At this concentration everolimus was more cytotoxic than temsirolimus with a growth inhibition of 80-85% and 55-60%, respectively; both drugs were more active than equimolar rapamycin. Interestingly, SK-Sora and CAKI-Suni cells were more sensitive to the different rapalogues than their parental counterparts. In 786-0, 786-Suni and 786-Sora cells, 72h exposure to rapalogues induced cell cycle modifications with an increase in the G1-phase and a decrease in the S-phase. Exposure of these cells to 1µM rapalogues for 5 and 24h resulted in strong inhibition of phosphorylation of the S6 and P70S6K proteins, two well-established mTORC1 downstream targets. Exposure to 1 µM everolimus also decreased 4EBP-1 phosphorylation (p4EBP-1) whereas temsirolimus and rapamycin effects on p4EBP-1 were less potent. In response to 5h rapalogues exposure, phospho AKT (Ser473) was transiently activated. After 24 hours, AKT activation was inhibited in presence of everolimus and temsirolimus but not rapamycin. Conclusion. Everolimus displayed potent antiproliferative effects that were equal or superior to those observed with temsirolimus and rapamycin in HCC and RCC cancer cell lines with acquired resistance to sunitinib and sorafenib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1805. doi:1538-7445.AM2012-1805

Characterization of P1 promoter activity of the β-galactoside α2,6sialyltransferase I gene (siat 1) in cervical and hepatic cancer cell lines

The level of beta-galactoside alpha2,6-sialyltransferase I (ST6Gal I) mRNA, encoded by the gene siat1, is increased in malignant tissues. Expression is regulated by different promoters - P1, P2 and P3 - generating three mRNA isoforms H, X and YZ. In cervical cancer tissue the mRNA isoform H, which results from P1 promoter activity, is increased. To study the regulation of P1 promoter, different constructs from P1 promoter were evaluated by luciferase assays in cervical and hepatic cell lines. Deletion of a fragment of 1048 bp (-89 to +24 bp) increased 5- and 3-fold the promoter activity in C33A and HepG2 cell lines, respectively. The minimal region with promoter activity was a 37 bp fragment in C33A cells. The activity of this region does not require the presence of an initiator sequence. In HepG2 cells the minimal promoter activity was detected in the 66 bp fragment. Sp1 (-32) mutation increased the promoter activity only in HepG2 cells. HNF1 mutation decreased promoter activity in HepG2 cell line but not in C33A cells. We identified a large region that plays a negative regulation role. The regulation of promoter activity is cell type specific. Our study provides new insights into the complex transcriptional regulation of siat1 gene.

The role of miR‐17‐92 cluster in hepatic carcinogenesis

MiR‐17‐92 cluster is a potent oncogenic miRNA which has been shown to promote cell proliferation, increase angiogenesis and sustain cell survival in several cancer types. However, the potential role of miR‐17‐92 cluster in hepatic carcinogenesis has not been investigated. This study was designed to examine the impact of miR‐17‐92 in hepatic cancer cells. qRT‐PCR analysis showed that the expression of miR‐17‐92 cluster is increased in several human hepatic cancer cell lines (Hep3B, Huh7 and HepG2) compared to the transformed, non‐cancerous hepatic cell line, THLE‐2. The miR‐17‐92 inhibitors (blocking the entire cluster or individual miRNAs within the cluster) prevented hepatic cancer cell growth. In contrast, stable transfection of pri‐miR‐17‐92 cluster increased tumor cell proliferation. Western blot analysis showed decreased PTEN protein in hepatic cancer cells transfected with the pri‐miR‐17‐92 cluster, suggesting that PTEN may represent one of the miR‐17‐92 downstream targets. Experiments are ongoing to document hepatocellular cancer development in transgenic mice with targeted expression of homo miR‐17‐92 cluster gene in the liver (driven by albumin‐cre, with or without diethylnitrosamine treatment). Results of these experiments are expected to provide important in vitro and in vivo evidences to define the role of miR‐17‐92 in hepatic carcinogenesis and tumor progression.

Knockdown of ZNF403 inhibits cell proliferation and induces G2/M arrest by modulating cell-cycle mediators

ZNF403, also known as GGNBP2 (gametogenetin binding protein 2), is a highly conserved gene implicated in spermatogenesis. However, the exact biological function of ZNF403 is not clear. In this study, we identified the role of ZNF403 in cell proliferation and cell-cycle regulation by utilizing short hairpin RNA (shRNA)-mediated knockdown. ZNF403-specific shRNA expressing helper-dependent adenoviral vector (HD-Ad-ZNF403-shRNA) was constructed and transduced human cell lines. ZNF403 mRNA and protein expression levels were inhibited as evidenced by real-time PCR and western blot analyses. Noticeably, we found that knockdown of ZNF403 expression suppressed cell proliferation compared to the non-target shRNA and vector controls. Furthermore, cell-cycle analysis demonstrated that downregulation of ZNF403 promoted G2/M cell-cycle arrest in a dose-dependent manner. Moreover, human cell-cycle real-time PCR array revealed that ZNF403 knockdown influenced the expression profile of genes in cell-cycle regulation. Among these genes, western blot analysis confirmed the protein up-regulation of p21 and down-regulation of MCM2 in response to the ZNF403 knockdown. Additionally, knockdown of ZNF403 also showed an anti-carcinogenetic effect on anchorage-independent growth by colony formation assay and tumor cell migration by wound-healing assay with human laryngeal cancer cell line Hep-2 cells. Altogether, our findings suggest an essential role of ZNF403 in cell proliferation and provide a new insight into the function of ZNF403 in regulating the G2/M cell-cycle transition.

Suppression of Aurora-A by RNA interference inhibits laryngeal cancer Hep-2 cell growth

To investigate the effects of knockdown of Aurora-A by RNA interference on laryngeal cancer Hep-2 cell growth in vitro and in vivo. A plasmid containing siRNA against Aurora-A was constructed and transfected into human laryngeal cancer cell line Hep-2. Measurements included the CCK-8 assay for viability and proliferation, Transwell assay for invasion, colony formation assay for cell anchorage-independent growth. Western blot and immunohistochemistry assay for protein expression. Tumorigenicity was observed in vivo. In Hep-2 cells transfected by Aurora-A siRNA (designated as siRNA-3), protein expression of Aurora-A was suppressed by 52%. In CCK-8 assay, absorbance value of siRNA-3 cells (3.268 ± 0.106, (x(-) ± s)) was lower than that of Hep-2 cells (3.722 ± 0.152, F = 17.634, P < 0.001). In Transwell assay, the average invasive cells per field in siRNA-3 cells (110.0 ± 18.0) was less than that in Hep-2 cells (236.0 ± 26.0, F = 26.462, P < 0.01). In colony formation assay, the average colony number of siRNA-3 cells (31.0 ± 6.6) was lower than that of Hep-2 cells (104.0 ± 14.0). The average tumor size in siRNA-3 group was (127.77 ± 174.83) mm(3), which was less than Hep-2 cell group (837.26 ± 101.80) mm(3), (F = 28.187, P < 0.001). Silencing of Aurora-A decreased the expression of focal adhesion kinase (FAK) and matrix metalloproteinase-2 (MMP-2), key regulators in cell adhesion and invasion. The knockdown of Aurora-A inhibits the growth and invasiveness of Hep-2 cells in vitro and in vivo, which may be a promising therapeutic strategy for LSCC.

Aldehyde dehydrogenase 1 can be used as a new marker of cancer stem cells in laryngeal cancer cells in vitro

To detect the expression of aldehyde dehydrogenase 1 in human laryngeal cancer cells in vitro, and to explore whether it can be used as a marker of stem cells in human laryngeal cancer. Fluorescence staining and flow cytometry were used to detect the expression of ALDH1 in a human laryngeal cancer Hep-2 cell line, and fluorescence activated cells sorting was used to separate ALDH1(br) cells. ALDH1 tumor cells were cultured and their ability of proliferation and differentiation was observed in vitro. The expression of ALDH1 in Hep-2 cells was different. The number of cells highly expressing ALDH1 was 2.9% ± 0.6%. Compared with ALDH1(low) cells and unsorted cells, ALDH1(br) cells exhibited increased proliferation ability. In serum-containing RPM I1640 culture medium, the proportion of ALDH1(br) cells was decreasing as days passed. The percentage of ALDH1(br) cells decreased from 94.2% ± 3.8% to the level before sorting. The ALDH1(br) cells demonstrated enhanced tumorigenic ability in nude mice. In the laryngeal cancer Hep-2 cell line, the highly ALDH1-expressing cells show a strong ability of differentiation, proliferation and tumorigenesis. It indicates that ALDH1 can be used as a new marker of stem cells of laryngeal cancer cells.

Investigation on the substitution effects of the flavonoids as potent anticancer agents: a structure–activity relationships study

Three series of flavonoid analogues substituted with different aminomethyl substitutions at C-6, C-7, and C-8 were designed and synthesized for the structure–activity relationship studies as potent anticancer agents. The prepared analogues were evaluated for their in vitro inhibitory activity against the growth of the hepatic cancer cell lines HepG2 and SMMC-7721. Structure–activity relationships indicated that not only the compounds with amino methyl groups were more active than those without the groups in the same series but also the compounds substituted by aminomethyl groups at position C-8 were more active than those at positions C-6 and C-7.

Synthesis and efficient hepatocyte targeting of galactosylated chitosan as a gene carrier in vitro and in vivo

While chitosan (CS) has been researched widely as a non-viral vector, its usefulness has been limited by its low cell specificity and transfection efficiency. Therefore, we successfully synthesized galactosylated chitosan (GC) and complexed it with an enhanced green fluorescent protein plasmid (pIRES-EGFP) for transfection into cultured H22 cells (murine hepatic cancer cell line) using various GC/EGFP (N/P) charge ratios. Maximal gene transfection rates detected by flow cytometry occurred at an N/P ratio 5:1. Compared with those of lipofectin/EGFP and naked pIRES-EGFP, GC/EGFP complexes show a very efficient cell-selective transfection to hepatocytes. The MTT assay detected relatively low cytotoxicity in cells transfected with GC. A recombinant plasmid granulocyte-macrophage colony-stimulating factor (GM-SCF) and interleukin (IL) 21 (pIRES/GM-CSF-IL21) was successfully constructed and GC/GM-CSF-IL21 nanoparticles (average diameter, 82.1 nm) were administered via the tail vein of mice with liver metastasis of colon cancer model, for 5 consecutive days. The GC/GM-CSF-IL21 nanoparticles exhibited hepatocyte and passive tumor specificity, increased therapeutic efficacy compared to control groups, promoted leukocytes to aggregate in tumor tissues, and activated the cytotoxicity of natural killer (NK) cells and cytolytic T lymphocyte (CTL). Our results indicate that GC can be used in gene therapy to improve transfection efficiency and can be used as an immunological stimulant in vivo.

Interferon-α/β and Anti-Fibroblast Growth Factor Receptor 1 Monoclonal Antibody Suppress Hepatic Cancer Cells In Vitro and In Vivo

BackgroundHepatocellular carcinoma (HCC) is the most commonly occurring primary liver cancer and ranks as the fifth most frequently occurring cancer, overall, and the third leading cause of cancer deaths, worldwide. At present, effective therapeutic options available for HCC are limited; consequently, the prognosis for these patients is poor. Our aim in the present study was to identify a novel target for antibody therapy against HCC.Methodology/Principal FindingsWe used Western blot and flow cytometric and immunocytochemical analyses to investigate the regulation of FGFR1 expression by interferon-α/β in several human hepatic cancer cell lines. In addition, we tested the efficacy of combined treatment with anti-FGFR1 monoclonal antibody and interferon-α/β in a murine xenograft model of human HCC. We found that interferon-α/β induces expression of FGFR1 in human HCC cell lines, and that an anti-FGFR1 monoclonal antibody (mAb) targeting of the induced FGFR1 can effectively inhibit growth and survival of HCC cells in vitro and in vivo. Moreover, the combination of interferon-α, anti-FGFR1 mAb and peripheral blood mononuclear cells (PBMCs) exerted a significant antitumor effect in vitro.ConclusionsOur results suggest that the combined use of an anti-FGFR1 antibody and interferon-α/β is a promising approach to the treatment of HCC.

Bacterial monocultures, propionate, butyrate and H2O2 modulate the expression, secretion and structure of the fasting‐induced adipose factor in gut epithelial cell lines

Previous research showed that an intestinal microbial community represses the fasting-induced adipose factor (FIAF) in the gut epithelium, thereby increasing fat storage in the host. This study was designed to investigate the overall effect of different bacterial species and metabolites on FIAF in intestinal (Caco-2, HT-29 and HCT-116) and hepatic (HepG2) cancer cell lines. First, we showed that FIAF was present in different isoforms, and secreted as N-glycosylated proteins, exclusively at the basal side of the cell monolayer. Second, co-incubation of cell lines with bacterial monocultures and metabolites altered both FIAF production and isoform appearance. Propionate and/or butyrate treatment increased FIAF expression and cleavage in all tested cell lines. In contrast, different bacteria induced cell line-specific FIAF modulation. Clostridium perfringens induced FIAF isoform changes in Caco-2 cells. Enterococcus faecalis and Bacteroides thetaiotaomicron treatment resulted in cell line-specific FIAF increases, whereas Escherichia coli significantly decreased FIAF expression in HCT-116 cells. Treatment with H(2) O(2) and peroxide-producing E. faecalis strains induced FIAF isoform changes in Caco-2 cells. Since bacteria and bacterial metabolites alter both FIAF production and isoform appearance, further investigation may reveal an important role for bacteria in FIAF-regulated physiological processes, such as cell differentiation and fat metabolism.

Biliary epithelial cells inhibit proliferation of co-cultured hepatic cancer cell line HepG2

Objective To investigate the possible effect of mouse intrahepatic biliary epithelial cell line(mIBEC) on co-cultured human hepatoma cell lines HepG2.Methods HepG2 and mIBEC cells were co-cultured in a membrane-separated Transwell system.CellTiter 96 ○R AQueous One Solution Cell Proliferation Assay(Promega Corporation,Madison,WI) was used to examine HepG2 cell proliferation in the system with or without co-cultured mIBEC.Real-time PCR(RT-PCR) was used to determine Ki67 and caspase3 mRNA expression in HepG2 cells in a system with or without co-cultured mIBEC.Western blotting analysis was used to determine caspase3 protein level in HepG2 cells.Results The proliferation of the co-cultured HepG2 cells was significantly lower than those cultured alone(P 0.01).Expression of Ki67,a cell proliferation marker,was also significantly down-regulated in mIBEC co-cultured HepG2 cells(P 0.05).The levels of caspase3 mRNA and protein were significantly up-regulated in mIBEC co-cultured HepG2 cells compared with HepG2 cells cultured alone(P 0.05).Conclusion mIBEC can inhibit the proliferation of co-cultured HepG2,and caspase3 activation might be one of the reasons for the inhibitory effect of mIBEC against HepG2 cells.

Antinociceptive, Free Radical–Scavenging, and Cytotoxic Activities of Acanthus hirsutus Boiss

In vivo antinociceptive and in vitro radical scavenging and cytotoxic activities of Acanthus hirsutus Boiss. aqueous extract were investigated to give a new insight into plant usage in traditional medicine. The extract showed significant antinociceptive activity in acetic acid-induced writhing test in mice after oral application and did not change the hind-leg retraction period in the hot-plate test for any dose applied. In addition, the extract showed radical-scavenging activity against 2,2-diphenyl-1-picryl-hydrazyl, nitric oxide, and superoxide radicals similar to those of standard compounds 3-t-butyl-4-hydroxyanisole, ascorbic acid (vitamin C), and quercetin. The gallic acid equivalent total phenolic content of the plant was found to be 65.4 mg/g dry extract. Cytotoxic activity of the aqueous extract was tested against 3 different cancer cell lines-Hep-2 (human larynx epidermoid carcinoma), RD (human rhabdomyosarcoma), and L20B (transgenic murine L cells)-and 1 noncancerous cell line (VERO) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Through the phytochemical studies, the following compounds were isolated: 3 lignan glucosides [(-)-syringaresinol-di-O-β-glucopyranoside, (-)-medioresinol-di-O-β-D-glucopyranoside, (-)-pinoresinol-4'-O-β-glucopyranoside], 2 benzoxazinoids [2-hydroxy-1,4-benzoxazin-3(4H)-one, (2R)-2-O-β-glucopyranosyl-1,4-benzoxazin-3(4H)-one], 4 phenylethanoid glycosides (acteoside, leucosceptoside A, martynoside, hattushoside), and 2 phenylpropanoid glucosides (sinapyl aldehyde-4-O-β-glucopyranoside, sinapyl alcohol-4-O-β-glucopyranoside). Cytotoxic and radical-scavenging activities of the isolated compounds were also determined. 2-hydroxy-1,4-benzoxazin-3(4H)-one and acteoside were the most active compounds in both experiments.

244 DIAGNOSTIC AND PROGNOSTIC ROLE OF SCCA-IGM IN HEPATOCELLULAR CARCINOMA (HCC)

generation of reactive oxygen species (ROS), phosphorylation/ inactivation of GSK-3b as well as phosphorylation of PI-3K, ERK1/2, JNK1/2, p38MAPK and STAT3. Invasiveness of cancer cells was prevented either by inhibiting ERK1/2, PI3K or JNK1/2 as well as by preventing ROS generation. Finally, OSM was expressed “in vivo” in tumor cells of HCC in areas also positive for hypoxia-related antigens. Conclusions: OSM, which is expressed in human HCC cells and peritumoral cirrhotic tissue, can induce EMT in human hepatic cancer cell lines and stimulate their increased invasiveness through the involvement of redox-dependent activation of EMT-related critical kinases and transcription factors.

Study on synthesis of thalidomide analogues and their bioactivities; inhibition on iNOS pathway and cytotoxic effects

Synthetic thalidomide analogues (compounds 1–35), including phenylphthalimide, pyridylphthalimide, aminobenzylphthalimide, and diphenylazophthalimide, were tested for their cytotoxic effects on human cancer cell lines Hep2 (Human Larynx Carcinoma Cells), HL-60 (Human Myeloid Leukemia Cells), NUGC (Human Gastric Carcinoma Cells), and HONE-1 (Human Nasopharyngeal Carcinoma Cells) because the incidence rate is more prominent in Asian countries than in Western countries. Compounds 17, 27, 28, and 35 were found to have antitumor activity in Hep2 and HL-60 cell lines. Compounds 2, 4, 15, 17, 19, 20, 23, and 27 can inhibit nitric oxide (NO) synthase activity by more than 90%. These thalidomide analogues were found to be potent inducible nitric oxide synthase (iNOS) inhibitors, and the iNOS inhibiting potential of compounds 17 and 27 might be an advantage for anticancer therapy. In conclusion, inhibition of NO synthesis is a new development in cancer therapy for now and in the future. We modified the structures of the thalidomide analogues to have a stronger anticancer effect and a good therapeutic effect.

α4 is highly expressed in carcinogen-transformed human cells and primary human cancers

A regulator of the protein phosphatase 2A (PP2A), α4, has been implicated in a variety of functions that regulate many cellular processes. To explore the role of α4 in human cell transformation and tumorigenesis, we show that α4 is highly expressed in human cells transformed by chemical carcinogens including benzo(a)pyrene, aflatoxin B(1), N-methyl-N'-nitro-N-nitrosoguanidine, nickel sulfate and in several hepatic and lung cancer cell lines. In addition, overexpression of α4 was detected in 87.5% (74/80) of primary hepatocellular carcinomas, 84.0% (21/25) of primary lung cancers and 81.8% (9/11) of primary breast cancers, indicating that α4 is ubiquitously highly expressed in human cancer. Functional studies revealed that elevated α4 expression results in an increase in cell proliferation, promotion of cell survival and decreased PP2A-attributable activity. Importantly, ectopic expression of α4 permits non-transformed human embryonic kidney cells (HEKTER) and L02R cells to form tumors in immunodeficient mice. Furthermore, we show that the highly expressed α4 in transformed cells or human tumors is not regulated by DNA hypomethylation. A microRNA, miR-34b, that suppresses the expression of α4 through specific binding to the 3'-untranslated region of α4 is downregulated in transformed or human lung tumors. Taken together, these observations identify that α4 possesses an oncogenic function. Reduction of PP2A activity due to an enhanced α4-PP2A interaction contributes directly to chemical carcinogen-induced tumorigenesis.

Gardenoins E-H, Cycloartane Triterpenes from the Apical Buds of Gardenia obtusifolia

Four new cycloartane triterpenes, named gardenoins E-H (1-4), were isolated from the apical buds of Gardenia obtusifolia, together with five known cycloartanes. Only compound 1 displayed cytotoxicity against colon, hepatic and lung cancer cell lines.

Cancer/Testis Antigen CAGE Exerts Negative Regulation on p53 Expression through HDAC2 and Confers Resistance to Anti-cancer Drugs

The role of the cancer/testis antigen CAGE in drug resistance was investigated. The drug-resistant human melanoma Malme3M (Malme3M(R)) and the human hepatic cancer cell line SNU387 (SNU387(R)) showed in vivo drug resistance and CAGE induction. Induction of CAGE resulted from decreased expression and thereby displacement of DNA methyltransferase 1(DNMT1) from CAGE promoter sequences. Various drugs induce expression of CAGE by decreasing expression of DNMT1, and hypomethylation of CAGE was correlated with the increased expression of CAGE. Down-regulation of CAGE in these cell lines decreased invasion and enhanced drug sensitivity resulting from increased apoptosis. Down-regulation of CAGE also led to decreased anchorage-independent growth. Down-regulation of CAGE led to increased expression of p53, suggesting that CAGE may act as a negative regulator of p53. Down-regulation of p53 enhanced resistance to drugs and prevented drugs from exerting apoptotic effects. In SNU387(R) cells, CAGE induced the interaction between histone deacetylase 2 (HDAC2) and Snail, which exerted a negative effect on p53 expression. Chromatin immunoprecipitation assay showed that CAGE, through interaction with HDAC2, exerted a negative effect on p53 expression in Malme3M(R) cells. These results suggest that CAGE confers drug resistance by regulating expression of p53 through HDAC2. Taken together, these results show the potential value of CAGE as a target for the development of cancer therapeutics.

Genomics and proteomics approaches to the study of cancer-stroma interactions

BackgroundThe development and progression of cancer depend on its genetic characteristics as well as on the interactions with its microenvironment. Understanding these interactions may contribute to diagnostic and prognostic evaluations and to the development of new cancer therapies. Aiming to investigate potential mechanisms by which the tumor microenvironment might contribute to a cancer phenotype, we evaluated soluble paracrine factors produced by stromal and neoplastic cells which may influence proliferation and gene and protein expression.MethodsThe study was carried out on the epithelial cancer cell line (Hep-2) and fibroblasts isolated from a primary oral cancer. We combined a conditioned-medium technique with subtraction hybridization approach, quantitative PCR and proteomics, in order to evaluate gene and protein expression influenced by soluble paracrine factors produced by stromal and neoplastic cells.ResultsWe observed that conditioned medium from fibroblast cultures (FCM) inhibited proliferation and induced apoptosis in Hep-2 cells. In neoplastic cells, 41 genes and 5 proteins exhibited changes in expression levels in response to FCM and, in fibroblasts, 17 genes and 2 proteins showed down-regulation in response to conditioned medium from Hep-2 cells (HCM). Nine genes were selected and the expression results of 6 down-regulated genes (ARID4A, CALR, GNB2L1, RNF10, SQSTM1, USP9X) were validated by real time PCR.ConclusionsA significant and common denominator in the results was the potential induction of signaling changes associated with immune or inflammatory response in the absence of a specific protein.

Abstract 1055: Character of tumor assocated protien recognized by monoclonal antibody against yunnan gejiu lung cancer

Abstract Character of tumor assocated protien recognized by monoclonal antibody against Yunnan Gejiu lung cancer: about protein N35 WANG Qinqin The Clinical Reseach Center. The First Hospital of Kunming Medial College Kunming, Yunnan, P.R. China 650032 ABSTRACT Objectives: To identify and characterize lung cancer associated protein N35 and attempt to learn the prospective possibility of the clinical application of the protein N35. Methods: Immunoprecipitation, immunoblotting, differential centrifigation and subcellular assay, immunohistochemistry, Nglycanase digestion, mitotic cell immunoflourescence and multiple methods of affinity chromatography have been used with the monoclonal antibody N-35 to detect the distribution of the protein N35 among the various cancer cell lines and normal human tissue, the relationship between the protein N35 and glycoprotein, the location of the subcellular structure and chromosomal domain of the protein N35,the most effective way of purification of tumor associated protein N35. Results: The protein N35 is a glycoprotein, distributes to the human lung cancer cell line GLC-82, human cervical cancer cell line Hela, human hepatic cancer cell line HepG-2 and human breast cancer cell line PMC with different relative molecular mass(Mr), but no expression of the protein ingredient in normal human fresh tissue; concentrates at the nuclei significantly, much more than at the mitochondrail and membrane, locates at the centriole of the chromosomal domain. Conclusions: The lung cancer associated protein N35 might be expressed only by the cancer cells and related with the proliferation of cancer cells as a role of tumor cell growth regulator. Keywords: Lung cancer cells, Tumor associated protein, Glycoprotein, Centriole Qinqin Wang Prof of The Clinical Reseach Center. The First Hospital of Kunming Medial College 295 Xichang Rd. 650032 Kunming Yunnan, P.R. China Tel:868714191985 Fax: 868715336015 Email: zys@public.km.yn.cn Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1055.