bluuebunny/crossref_metadata_2025 huggingface dataset:
Histone Deacetylase Inhibitors Suppress the Inducibility of Nuclear Factor-κB by Tumor Necrosis Factor-α Receptor-1 Down-regulation
Authors: Imre Gabriele , Gekeler Volker , Leja Astrid , Beckers Thomas , Boehm Markus
Published: May 2006

Recently, the inhibition of histone deacetylase (HDAC) enzymes has attracted attention in the oncologic community as a new therapeutic opportunity for hematologic and solid tumors including non–small cell lung cancer (NSCLC). In hematologic malignancies, such as diffuse large B-cell lymphoma, the HDAC inhibitor (HDI), suberoylanilide hydroxamic acid (SAHA), has recently entered phase II and III clinical trials. To further advance our understanding of their action on tumor cells, we investigated the possible effect of HDI treatment on the functionality of the nuclear factor-κB (NF-κB) pathway in NSCLC. We found that in the NSCLC cell lines, A549 and NCI-H460, the NF-κB pathway was strongly inducible, for example, by stimulation with tumor necrosis factor-α (TNF-α). Incubation of several NSCLC cell lines with HDIs resulted in greatly reduced gene expression of TNF-α receptor-1. HDI-treated A549 and NCI-H460 cells down-regulated TNF-α receptor-1 mRNA and protein levels as well as surface exposure, and consequently responded to TNF-α treatment with reduced IKK phosphorylation and activation, delayed IκB-α phosphorylation, and attenuated NF-κB nuclear translocation and DNA binding. Accordingly, stimulation of NF-κB target gene expression by TNF-α was strongly decreased. In addition, we observed that SAHA displayed antitumor efficacy in vivo against A549 xenografts grown on nude mice. HDIs, therefore, might beneficially contribute to tumor treatment, possibly by reducing the responsiveness of tumor cells to the TNF-α-mediated activation of the NF-κB pathway. These findings also hint at a possible use of HDIs in inflammatory diseases, which are associated with the overproduction of TNF-α, such as rheumatoid arthritis or Crohn's disease. (Cancer Res 2006; 66(10): 5409-18)
Mammary Gland Tissue Targeted Overexpression of Human Protease-Activated Receptor 1 Reveals a Novel Link to β-Catenin Stabilization
Authors: Yin Yong-Jun , Katz Vered , Salah Zaidoun , Maoz Myriam , Cohen Irit , Uziely Beatrice , Turm Hagit , Grisaru-Granovsky Sorina , Suzuki Hiromu , Bar-Shavit Rachel
Published: May 2006

Protease-activated receptor 1 (PAR1) is emerging with distinct assignments in tumor biology. We show that tissue targeted overexpression of hPar1 in mice mammary glands results in precocious hyperplasia, characterized by a dense network of ductal side branching and accelerated proliferation. These glands exhibit increased levels of wnt-4 and wnt-7b and a striking β-catenin stabilization. Nuclear localization of β-catenin is observed in hPar1 transgenic mouse tissue sections but not in the wild-type, age-matched counterparts. PAR1 induces β-catenin nuclear localization also in established epithelial tumor cell lines of intact β-catenin system (transformed on the background of mismatch repair system; RKO cells). We propose hereby that PAR1-mediated β-catenin stabilization is taking place primarily via the increase of Wnt expression. Enforced expression of a specific Wnt antagonist family member, secreted frizzled receptor protein 5 (SFRP5), efficiently inhibited PAR1-induced β-catenin stabilization. Likewise, application of either SFRP2 or SFRP5 on epithelial tumor cells completely abrogated PAR1-induced β-catenin nuclear accumulation. This takes place most likely via inhibition of Wnt signaling at the level of cell surface (forming a neutralizing complex of “Receptors-SFRP-Wnt”). Furthermore, depletion of hPar1 by small interfering RNA (siRNA) vectors markedly inhibited PAR1-induced Wnt-4. The striking stabilization of β-catenin, inhibited by SFRPs on one hand and Wnt-4 silencing by hPar1 siRNA on the other hand, points to a novel role of hPar1 in Wnt-mediated β-catenin stabilization. This link between PAR1 and β-catenin may bear substantial implications both in developmental and tumor progression processes. (Cancer Res 2006; 66(10): 5224-32)
Microarray-Assisted Pathway Analysis Identifies Mitogen-Activated Protein Kinase Signaling as a Mediator of Resistance to the Green Tea Polyphenol Epigallocatechin 3-Gallate in Her-2/neu–Overexpressing Breast Cancer Cells
Authors: Guo Shangqin , Lu Jun , Subramanian Aravind , Sonenshein Gail E.
Published: May 2006

Overexpression of the epidermal growth factor receptor family member Her-2/neu in breast cancer leads to autophosphorylation of the receptor and induction of multiple downstream signaling pathways, including the Akt kinase to nuclear factor-κB (NF-κB) cascade that is associated with poor prognosis. Previously, we showed that the green tea polyphenol epigallocatechin 3-gallate (EGCG) inhibits growth of NF639 Her-2/neu–driven breast cancer cells via reducing receptor autophosphorylation and downstream Akt and NF-κB activities. Interestingly, upon prolonged culture in the presence of EGCG, cells resistant to the polyphenol could be isolated. Here, we report that resistant cells have lost tyrosine phosphorylation on the Her-2/neu receptor. Surprisingly, they displayed elevated NF-κB activity, and inhibition of this activity sensitized cells to EGCG. Data from microarray studies of the original and resistant NF639 populations of cells were subjected to Gene Set Enrichment Analysis pathway assessment, which revealed that the mitogen activated protein kinase (MAPK) pathway was activated in the resistant cells. Treatment of the resistant cells with the MAPK inhibitor U0216 reduced growth in soft agar and invasive phenotype, whereas the combination of EGCG and U0216 resulted in cells with a cobblestone epithelial phenotype. Thus, activation of the MAPK pathway mediates resistance to EGCG. (Cancer Res 2006; 66(10): 5322-9)
Antiangiogenic Antithrombin Induces Global Changes in the Gene Expression Profile of Endothelial Cells
Authors: Zhang Weiqing , Chuang Yung-Jen , Jin Tianquan , Swanson Richard , Xiong Yan , Leung Lawrence , Olson Steven T.
Published: May 2006

Antithrombin, a serpin family protease inhibitor crucial to hemostasis, acquires antiangiogenic properties on undergoing conformational alterations induced by limited proteolysis or elevated temperature. To better understand the biochemical mechanisms underlying antithrombin antiangiogenic activity, we did genome-wide expression profiling, coupled with quantitative reverse transcription-PCR, Northern blot, and Western blot analyses, to characterize the gene expression patterns that are induced by antiangiogenic antithrombin in cultured primary human umbilical vein endothelial cells. Overall, 35 genes with significantly increased expression and 93 genes with significantly reduced expression (≥2-fold changes) due to antiangiogenic antithrombin treatment were identified. More than half of the down-regulated genes have well-established proangiogenic functions in endothelial cells, including cell-surface and matrix proteoglycans (e.g., perlecan, biglycan, and syndecans 1 and 3) and mitogenesis-related signaling proteins (e.g., mitogen-activated protein kinase 3, signal transducers and activators of transcription 2, 3, and 6, and early growth response factor 1). In contrast, most up-regulated genes (e.g., caspase-3, p21, tissue inhibitor of metalloproteinases 1, 2, and 3, and adenomatosis polyposis coli) are known for their antiangiogenic functions which include the promotion of cell apoptosis and cell cycle arrest and the inhibition of tumor growth and metastasis. These results show that the antiangiogenic activity of antithrombin is mediated at least in part by a global genetic reprogramming of endothelial cells and strongly implicate an endothelial cell ligand-receptor signaling mechanism in this reprogramming. (Cancer Res 2006; 66(10): 5047-55)
Tight Junction Protein Claudin-1 Enhances the Invasive Activity of Oral Squamous Cell Carcinoma Cells by Promoting Cleavage of Laminin-5 γ2 Chain via Matrix Metalloproteinase (MMP)-2 and Membrane-Type MMP-1
Authors: Oku Naohisa , Sasabe Eri , Ueta Eisaku , Yamamoto Tetsuya , Osaki Tokio
Published: May 2006

Although adherent junctions have been extensively studied, the role of tight junctions in cancer cell invasion is not sufficiently explored. We investigated whether claudin-1, a component of tight junctions, regulated invasion activity in oral squamous cell carcinoma (OSC) cells. The expression of claudin-1, activity of matrix metalloproteinase (MMP)-2, and cleavage of laminin-5 γ2 chains were assessed by Western blot analysis, immunohistochemistry, and zymography in OSC cell lines (OSC-4 and NOS-2, highly invasive; OSC-7, weakly invasive) and their xenografts in severe combined immunodeficient (SCID) mice. The influence of claudin-1 small interfering RNA (siRNA) on the invasion activity of the cell lines was also investigated. Compared with OSC-7, both OSC-4 and NOS-2 more strongly expressed claudin-1 and possessed high activities of MMP-2 and MMP-9. Tumors formed in the tongues of SCID mice xenografted with OSC-4, NOS-2, and OSC-7 immunohistochemically revealed strong, moderate, and weak expression of laminin-5 γ2 chains, respectively, and laminin-5 γ2 chains were secreted in the conditioned medium of the cancer cells in parallel with the in vivo results. Claudin-1 siRNA largely suppressed the invasion of OSC-4 and decreased the activation of MMP-2, the expression of membrane-type MMP-1 (MT1-MMP), and the cleavage of laminin-5 γ2. In addition, not only antibodies against MT1-MMP and epidermal growth factor receptor (EGFR) but also MMP-2 and EGFR inhibitors strongly suppressed the invasion activity of OSC-4. These results suggest that claudin-1 up-regulates cancer cell invasion activity through activation of MT1-MMP and MMP-2, which results in enhanced cleavage of laminin-5 γ2 chains. (Cancer Res 2006; 66(10): 5271-7)
Regulation of Microtubule-Dependent Protein Transport by the TSC2/Mammalian Target of Rapamycin Pathway
Authors: Jiang Xiuyun , Yeung Raymond S.
Published: May 2006

Protein transport plays a critical role in the interaction of the cell with its environment. Recent studies have identified TSC1 and TSC2, two tumor suppressor genes involved in tuberous sclerosis complex, as regulators of the mammalian target of rapamycin (mTOR) pathway. Cells deficient in TSC1 or TSC2 possess high levels of Rheb-GTP resulting in constitutive mTOR activation. We have shown previously that the TSC1/TSC2 complex is involved in post-Golgi transport of VSVG and caveolin-1 in mammalian cells. Here, we show that modulation of mTOR activity affects caveolin-1 localization and that this effect is independent of p70S6K. Tsc1- and Tsc2-null cells exhibit abnormal caveolin-1 localization that is accompanied by disorganized microtubules in the subcortical region. Analyses of green fluorescent protein-EB1 and tubulin in live mutant cells suggest a failure of the plus-ends to sense cortical signals and to halt microtubule growth. Down-regulation of CLIP-170, a putative mTOR substrate with microtubule-binding properties, rescued the abnormal microtubule arrangement and caveolin-1 localization in Tsc2−/− cells. Together, these findings highlight a novel role of the TSC2/mTOR pathway in regulating microtubule-dependent protein transport. (Cancer Res 2006; 66(10): 5258-69)
Inhibition of Tumor Growth and Elimination of Multiple Metastases in Human Prostate and Breast Xenografts by Systemic Inoculation of a Host Defense–Like Lytic Peptide
Authors: Papo Niv , Seger Dalia , Makovitzki Arik , Kalchenko Vyacheslav , Eshhar Zelig , Degani Hadassa , Shai Yechiel
Published: May 2006

We report on a short host defense–like peptide that targets and arrests the growth of aggressive and hormone-resistant primary human prostate and breast tumors and prevents their experimental and spontaneous metastases, respectively, when systemically inoculated to immuodeficient mice. These effects are correlated with increased necrosis of the tumor cells and a significant decrease in the overall tumor microvessel density, as well as newly formed capillary tubes and prostate-specific antigen secretion (in prostate tumors). Growth inhibition of orthotopic tumors derived from stably transfected highly fluorescent human breast cancer cells and prevention of their naturally occurring metastases were visualized in real time by using noninvasive whole-body optical imaging. The exclusive selectivity of the peptide towards cancer derives from its specific binding to surface phosphatidylserine and the killing of the cancer cells via cytoplasmic membrane depolarization. These data indicate that membrane disruption can provide a therapeutic means of inhibiting tumor growth and preventing metastases of various cancers. (Cancer Res 2006; 66(10): 5371-8)
Identification of a Novel Germ Line Variant Hotspot Mutant p53-R175L in Pediatric Adrenal Cortical Carcinoma
Authors: West Alina Nico , Ribeiro Raul C. , Jenkins Jesse , Rodriguez-Galindo Carlos , Figueiredo Bonald C. , Kriwacki Richard , Zambetti Gerard P.
Published: May 2006

Hotspot mutations in the p53 tumor suppressor gene result in the disruption of DNA contact points or alter the overall structure of the protein to prevent DNA binding. When inherited, hotspot mutants are associated with Li-Fraumeni syndrome (LFS), a familial cancer predisposition. One of the most common hotspot mutations occurs at codon 175, resulting in an arginine to histidine substitution. We have identified a novel germ line variant of the 175 mutant (Arg to Leu; R175L) in a pediatric patient who developed adrenal cortical carcinoma. Surprisingly, the family is not tumor prone or associated with LFS. In vitro, the R175L mutant displayed an attenuated tumor suppressor activity in the regulation of transcription, colony formation, and apoptosis when compared with wild-type p53 and the R175H mutant. These findings suggest that p53-R175L retains sufficient activity to suppress LFS, but not adrenal cortical carcinoma. Therefore, not all hotspot mutants are functionally equivalent and the biochemical nature of the mutant may significantly influence clinical outcome. The implications of these results for genetic counseling are discussed. (Cancer Res 2006; 66(10): 5056-62)
Selective Expansion of Marginal Zone B Cells in Eμ-API2-MALT1 Mice Is Linked to Enhanced IκB Kinase γ Polyubiquitination
Authors: Baens Mathijs , Fevery Sabine , Sagaert Xavier , Noels Heidi , Hagens Sofie , Broeckx Vicky , Billiau An D. , De Wolf-Peeters Christiane , Marynen Peter
Published: May 2006

The translocation t(11;18)(q21;q21) that generates an API2-MALT1 fusion protein is the most common structural abnormality among the genetic defects reported in mucosa-associated lymphoid tissue (MALT)-type lymphomas, and its presence correlates with the apparent lack of further genetic instability or chromosomal imbalances. Hence, constitutive nuclear factor-κB (NF-κB) activation induced by the API2-MALT1 fusion protein is considered essential for B-cell transformation. To examine its role in B-cell development and lymphomagenesis, Eμ-API2-MALT1 transgenic mice were produced. Our data show that expression of the API2-MALT1 fusion protein alone is not sufficient for the development of lymphoma masses within 50 weeks. Nevertheless, API2-MALT1 expression affected B-cell maturation in the bone marrow and triggered the specific expansion of splenic marginal zone B cells. Polyubiquitination of IκB kinase γ (IKKγ), indicative for enhanced NF-κB activation, was increased in splenic lymphocytes and promoted the survival of B cells ex vivo. In addition, we show that the API2-MALT1 fusion resided in the cholesterol- and sphingolipid-enriched membrane microdomains, termed lipid rafts. We provide evidence that association of the MALT1 COOH terminal with the lipid rafts, which is mediated by the API2 portion, is sufficient to trigger NF-κB activation via enhanced polyubiquitination of IKKγ. Taken together, these data support the hypothesis that the API2-MALT1 fusion protein can contribute to MALT lymphoma formation via increased NF-κB activation. (Cancer Res 2006; 66(10): 5270-7)
Progression-Specific Genes Identified by Expression Profiling of Matched Ductal Carcinomas In situ and Invasive Breast Tumors, Combining Laser Capture Microdissection and Oligonucleotide Microarray Analysis
Authors: Schuetz Christina S. , Bonin Michael , Clare Susan E. , Nieselt Kay , Sotlar Karl , Walter Michael , Fehm Tanja , Solomayer Erich , Riess Olaf , Wallwiener Diethelm , Kurek Raffael , Neubauer Hans J.
Published: May 2006

Becoming invasive is a crucial step in breast cancer oncogenesis. At this point, a lesion carries the potential for spreading and metastasis—a process, whose molecular characteristics still remain poorly understood. In this article, we describe a matched-pair analysis of ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) of nine breast ductal carcinomas to identify novel molecular markers characterizing the transition from DCIS to IDC. The purpose of this study was to better understand the molecular biology of this transition and to identify candidate genes whose products might serve as prognostic markers and/or as molecular targets for treatment. To obtain cellular-based gene expression profiles from epithelial tumor cells, we combined laser capture microdissection with a T7-based two-round RNA amplification and Affymetrix oligonucleotide microarray analysis. Altogether, a set of 24 tumor samples was analyzed, comprised of nine matched DCIS/IDC and replicate DCIS/IDC preparations from three of the nine tumors. Cluster analysis on expression data shows the robustness and reproducibility of the techniques we established. Using multiple statistical methods, 546 significantly differentially expressed probe sets were identified. Eighteen candidate genes were evaluated by RT-PCR. Examples of genes already known to be associated with breast cancer invasion are BPAG1, LRRC15, MMP11, and PLAU. The expression of BPAG1, DACT1, GREM1, MEF2C, SART2, and TNFAIP6 was localized to epithelial tumor cells by in situ hybridization and/or immunohistochemistry, confirming the accuracy of laser capture microdissection sampling and microarray analysis. (Cancer Res 2006; 66(10): 5278-86)
A Prospective Study on Habitual Duration of Sleep and Incidence of Breast Cancer in a Large Cohort of Women
Authors: Pinheiro Simone P. , Schernhammer Eva S. , Tworoger Shelley S. , Michels Karin B.
Published: May 2006

Mounting evidence suggests habitual sleep duration is associated with various health outcomes; both short and long sleep duration have been implicated in increased risk of cardiovascular disease, diabetes, and all-cause mortality. However, data on the relation between sleep duration and cancer risk are sparse and inconclusive. A link between low levels of melatonin, a hormone closely related to sleep, and increased risk of breast cancer has recently been suggested but it is unclear whether duration of sleep may affect breast cancer risk. We explored the association between habitual sleep duration reported in 1986 and subsequent risk of breast cancer in the Nurses' Health Study using Cox proportional hazards models. During 16 years of follow-up, 4,223 incident cases of breast cancer occurred among 77,418 women in this cohort. Compared with women sleeping 7 hours, covariate-adjusted hazard ratios and 95% confidence intervals for those sleeping ≤5, 6, 8, and ≥9 hours were 0.93 (0.79-1.09), 0.98 (0.91-1.06), 1.05 (0.97-1.13), and 0.95 (0.82-1.11), respectively. A moderate trend in risk increase towards longer sleep duration was observed when analyses were restricted to participants who reported same sleep duration in 1986 and 2000 (Ptrend = 0.05). In this prospective study, we found no convincing evidence for an association between sleep duration and the incidence of breast cancer. (Cancer Res 2006; 66(10): 5521-25)
Regulatory T Cells and Toll-Like Receptors in Cancer Therapy
Authors: Wang Rong-Fu
Published: May 2006

T regulatory (Treg) cells that suppress immune responses may limit the efficiency of cancer immunotherapy. Recent findings indicate that Toll-like receptors (TLR) directly regulate the suppressive activity of Treg cells. Linking TLR signaling to the functional control of Treg cells may offer new opportunities to improve the outcome of cancer immunotherapy by coadminstration of certain TLR ligands. (Cancer Res 2006; 66(10): 4987-90)
Quantitative In situ Analysis of β-Catenin Expression in Breast Cancer Shows Decreased Expression Is Associated with Poor Outcome
Authors: Dolled-Filhart Marisa , McCabe Anthony , Giltnane Jennifer , Cregger Melissa , Camp Robert L. , Rimm David L.
Published: May 2006

The role of β-catenin in breast cancer and its prognostic value is controversial. The prognostic value had been assessed previously in a series of nonquantitative immunohistochemical studies with conflicting results. In efforts to clarify the relationship between β-catenin protein expression and breast cancer prognosis, we have assessed a retrospective 600 case cohort of breast cancer tumors from the Yale Pathology archives on tissue microarrays. They were assessed using automated quantitative analysis (AQUA) with a series of array-embedded cell lines for which the β-catenin concentration was standardized by an ELISA assay. The expression levels of the standard clinical markers HER2, estrogen receptor (ER), progesterone receptor (PR), and Ki-67 were also assessed on the same cohort. X-tile software was used to select optimal protein concentration cutpoints and to evaluate the outcome using a training set and a validation set. We found that low-level expression of membranous β-catenin is associated with significantly worse outcome (38% versus 76%, 10-year survival, validation set log-rank P = 0.0016). Multivariate analysis of this marker, assessed in a proportional hazards model with tumor size, age, node status, nuclear grade, ER, PR, HER2, and Ki-67, is still highly significant with a hazard ratio of 6.8 (P < 0.0001, 95% confidence interval, 3.1-15.1). These results suggest that loss of β-catenin expression at the membrane, as assessed by objective quantitative analysis methods, may be useful as a prognostic marker or may be part of a useful algorithm for prognosis in breast cancer. (Cancer Res 2006; 66(10): 5487-94)
MDM2 SNP309 Accelerates Tumor Formation in a Gender-Specific and Hormone-Dependent Manner
Authors: Bond Gareth L. , Hirshfield Kim M. , Kirchhoff Tomas , Alexe Gabriella , Bond Elisabeth E. , Robins Harlan , Bartel Frank , Taubert Helge , Wuerl Peter , Hait William , Toppmeyer Deborah , Offit Kenneth , Levine Arnold J.
Published: May 2006

The importance of the p53 stress response pathway in the suppression of tumor formation is well documented. In a previous report, a single nucleotide polymorphism (SNP309 T/G) was found in the promoter of the MDM2 gene resulting in higher levels of MDM2 RNA and protein and, consequently, in the attenuation of the p53 pathway both in vitro and in vivo. As the SNP309 locus is found in a region of the MDM2 promoter, which is regulated by hormonal signaling pathways, and the G-allele of SNP309 increases the affinity of a well-described cotranscriptional activator of nuclear hormone receptors (i.e., Sp1), the hypothesis that the SNP309 locus could alter the effects of hormones on tumorigenesis was tested in vivo in humans. Data obtained from patients with three different sporadic cancers, from four independent case studies, support this hypothesis, providing an example for the genetic basis of gender differences in cancer and showing that the genotype at a specific locus can affect how hormones, like estrogen, affect tumorigenesis in humans. (Cancer Res 2006; 66(10): 5104-10)
(−)-Epigallocatechin-3-Gallate Prevents Photocarcinogenesis in Mice through Interleukin-12–Dependent DNA Repair
Authors: Meeran Syed M. , Mantena Sudheer K. , Elmets Craig A. , Katiyar Santosh K.
Published: May 2006

We have shown previously that topical application of (−)-epigallocatechin-3-gallate (EGCG), the major polyphenol of green tea, prevents photocarcinogenesis in mice. EGCG prevents UVB-induced immunosuppression by inducing interleukin-12 (IL-12). As immunosuppression is a risk factor for photocarcinogenesis, we investigated the possibility that EGCG also prevents UVB-induced photocarcinogenesis through an IL-12–dependent DNA repair mechanism. To investigate this possibility, we determined the effects of EGCG on photocarcinogenesis in IL-12 knockout (KO) mice using the formation of cyclobutane pyrimidine dimers (CPD) as an indicator of the extent of UVB-induced DNA damage. Topical application of EGCG (1 mg/cm2 skin) prevented photocarcinogenesis in wild-type (C3H/HeN) mice in terms of tumor incidence and tumor multiplicity but did not prevent photocarcinogenesis in IL-12 KO mice. UVB-induced DNA damage, as determined by the formation of CPDs and the number of sunburn cells, was resolved more rapidly in the skin of wild-type mice treated with EGCG than untreated control mice. In contrast, the extent of UVB-induced DNA damage and the numbers of sunburn cells were not significantly different in the EGCG-treated IL-12 KO mice and untreated control mice. In addition, treatment of XPA-proficient human fibroblast cells with EGCG promoted repair of UVB-induced CPDs in a dose-dependent manner but not in an XPA-deficient cells, indicating that the nucleotide excision repair mechanism is involved in EGCG-mediated DNA repair. Taken together, these results indicate for the first time that EGCG can prevent photocarcinogenesis through an EGCG-induced IL-12–dependent DNA repair mechanism. (Cancer Res 2006; 66(10): 5512-20)
c-Jun NH2-Terminal Kinase Signaling Axis Regulates Diallyl Trisulfide–Induced Generation of Reactive Oxygen Species and Cell Cycle Arrest in Human Prostate Cancer Cells
Authors: Antosiewicz Jedrzej , Herman-Antosiewicz Anna , Marynowski Stanley W. , Singh Shivendra V.
Published: May 2006

We have shown previously that generation of reactive oxygen species (ROS) is a critical event in G2-M phase cell cycle arrest caused by diallyl trisulfide (DATS), which is a highly promising anticancer constituent of processed garlic. Using DU145 and PC-3 human prostate cancer cells as a model, we now report a novel mechanism involving c-Jun NH2-terminal kinase (JNK) signaling axis, which is known for its role in regulation of cell survival and apoptosis, in DATS-induced ROS production. The DATS-induced ROS generation, G2-M phase cell cycle arrest and degradation, and hyperphosphorylation of Cdc25C were significantly attenuated in the presence of EUK134, a combined mimetic of superoxide dismutase and catalase. Interestingly, the DATS-induced ROS generation and G2-M phase cell cycle arrest were also inhibited significantly in the presence of desferrioxamine, an iron chelator, but this protection was not observed with iron-saturated desferrioxamine. DATS treatment caused a marked increase in the level of labile iron that was accompanied by degradation of light chain of iron storage protein ferritin. Interestingly, DATS-mediated degradation of ferritin, increase in labile iron pool, ROS generation, and/or cell cycle arrest were significantly attenuated by ectopic expression of a catalytically inactive mutant of JNK kinase 2 and RNA interference of stress-activated protein kinase/extracellular signal-regulated kinase 1 (SEK1), upstream kinases in JNK signal transduction pathway. In conclusion, the present study provides experimental evidence to indicate existence of a novel pathway involving JNK signaling axis in regulation of DATS-induced ROS generation. (Cancer Res 2006; 66(10): 5379-86)
Therapeutic Efficacy of Antigen-Specific Vaccination and Toll-Like Receptor Stimulation against Established Transplanted and Autochthonous Melanoma in Mice
Authors: Tormo Damia , Ferrer Aleix , Bosch Pilar , Gaffal Evelyn , Basner-Tschakarjan Etiena , Wenzel Jörg , Tüting Thomas
Published: May 2006

Malignant melanoma is an attractive model disease for the development of antigen-specific immunotherapy because many antigens recognized by tumor-specific T cells have been identified. In C57BL/6 mice, genetic immunization with recombinant adenovirus encoding xenogeneic human tyrosinase-related protein 2 (Ad-hTRP2) induces protective but not therapeutic cellular immunity against growth of transplanted B16 melanoma cells. Here, we additionally applied CpG DNA and synthetic double-stranded RNA, which activate the innate immune system via Toll-like receptors (TLR). Both adenoviral vaccination and peritumoral injections of TLR ligands were required for rejection of established B16 melanoma in the skin. To more closely mimic the clinical situation in patients with melanoma, we evaluated this combined immunotherapeutic strategy in genetically modified mice, which overexpress hepatocyte growth factor (HGF) and carry an oncogenic mutation in the cyclin-dependent kinase 4 (CDK4)R24C. HGF × CDK4R24C mice rapidly develop multiple invasive melanomas in the skin following neonatal carcinogen treatment, which spontaneously metastasize to lymph nodes and lungs. Vaccination with Ad-hTRP2 followed by injections of TLR ligands resulted in delayed growth of autochthonous primary melanomas in the skin and reduction in the number of spontaneous lung metastases but did not induce tumor regression. Carcinogen-treated HGF × CDK4R24C mice bearing multiple autochthonous melanomas did not reject transplanted B16 melanoma despite treatment with Ad-hTRP2 and TLR ligands, suggesting the development of tumor immunotolerance. Further investigations in our novel genetic melanoma model may help to better understand the role of the immune system in the pathogenesis and treatment of this life-threatening disease. (Cancer Res 2006; 66(10): 5427-35)
CpG Island M`ethylation Status in Gastric Carcinoma with and without Infection of Epstein-Barr Virus
Authors: Chang Moon-Sung , Uozaki Hiroshi , Chong Ja-Mun , Ushiku Tetsuo , Sakuma Kazuya , Ishikawa Shunpei , Hino Rumi , Barua Rita Rani , Iwasaki Yoshiaki , Arai Kuniyoshi , Fujii Hideki , Nagai Hideo , Fukayama Masashi
Published: May 2006

Purpose: EBV-associated gastric carcinoma shows global CpG island methylation of the promoter region of various cancer-related genes. To further clarify the significance of CpG island methylator phenotype (CIMP) status in gastric carcinoma, we investigated methylation profile and clinicopathologic features including overall survival in four subgroups defined by EBV infection and CIMP status: EBV-associated gastric carcinoma and EBV-negative/CIMP-high (H), EBV-intermediate (I), and EBV-negative (N) gastric carcinoma.

Experimental Design: Methylation-specific PCR was applied to 106 gastric carcinoma cases. CIMP-N, CIMP-I, and CIMP-H status was determined by the number (0, 1-3, and 4-5, respectively) of methylated marker genes (LOX, HRASLS, FLNc, HAND1, and TM), that were newly identified as highly methylated in gastric cancer cell lines. The methylation status of 10 other cancer-related genes (p14, p15, p16, p73, TIMP-3, E-cadherin, DAPK, GSTP1, hMLH1, and MGMT) was also evaluated.

Results: Nearly all (14 of 15) of EBV-associated gastric carcinoma exhibited CIMP-H, constituting a homogenous group (14%). EBV-negative gastric carcinoma consisted of CIMP-H (24%), CIMP-I (38%), and CIMP-N (24%). EBV-associated gastric carcinoma showed significantly higher frequencies of methylation of cancer-related genes (mean number ± SD = 6.9 ± 1.5) even if compared with EBV-negative/CIMP-H gastric carcinoma (3.5 ± 1.8). Among EBV-negative gastric carcinoma subgroups, CIMP-H gastric carcinoma showed comparatively higher frequency of methylation than CIMP-I or CIMP-N, especially of p16 and hMLH1. CIMP-N gastric carcinoma predominantly consisted of advanced carcinoma with significantly higher frequency of lymph node metastasis. The prognosis of the patients of CIMP-N was significantly worse compared with other groups overall by univariate analysis (P = 0.0313).

Conclusion: The methylation profile of five representative genes is useful to stratify gastric carcinomas into biologically different subgroups. EBV-associated gastric carcinoma showed global CpG island methylation, comprising a pathogenetically distinct subgroup in CIMP-H gastric carcinoma.
Prognostic Significance of Activated AKT Expression in Soft-Tissue Sarcoma
Authors: Tomita Yasuhiko , Morooka Takaya , Hoshida Yoshihiko , Zhang Binglin , Qiu Ying , Nakamichi Itsuko , Hamada Ken-ichiro , Ueda Takafumi , Naka Norifumi , Kudawara Ikuo , Aozasa Katsuyuki
Published: May 2006

Purpose: AKT is a serine/threonine kinase which is important in tumorigenesis. Several molecules involved in AKT pathway are dysregulated in various kinds of human cancers.

Patients and Methods: Ninety-three patients (53 males and 40 females), ages ranging from 19 to 77 years (median, 57 years), with localized soft-tissue sarcomas arising in the trunk and extremities, were analyzed. Immunoperoxidase procedure (avidin-biotin complex method) was done on paraffin-embedded sections with anti–phosphorylated AKT (Thr308), anti–phosphorylated p44/42 extracellular signal–regulated kinase 1 and 2 (ERK1/2) (Thr202/Tyr204), anti–phosphorylated forkhead in rhabdomyosarcoma (FKHR) (Ser256), and anti-Ki 67 antibodies. Expression levels of phosphorylated AKT (p-AKT), phosphorylated ERK1/2 (p-ERK1/2), and phosphorylated FKHR (p-FKHR) were categorized as either weaker (level 1) or equal to or stronger (level 2) compared with those in the endothelial cells of the same specimens. Percentage of cells showing intranuclear staining with Ki-67 was shown as the Ki-67 labeling index (LI). Cases were divided into two groups: level 1, Ki-67 LI < 20%; level 2, Ki-67 LI ≥ 20%.

Results: Twenty-six (28.0%), 6 (6.5%), and 46 (44.1%) of the tumors showed level 2 expression for p-AKT, p-ERK1/2, and Ki-67 LI, respectively. Tumors with level 2 p-AKT expression showed a higher ratio of level 2 p-FKHR expression (P < 0.01). Multivariate analysis revealed p-AKT expression and Ki-67 LI to be independent prognosticators for overall survival, and p-AKT expression for disease-free survival.

Conclusion: p-AKT expression level is a significant prognosticator in soft-tissue sarcoma.
Clinical Significance ofTROP2Expression in Colorectal Cancer
Authors: Ohmachi Takahiro , Tanaka Fumiaki , Mimori Koshi , Inoue Hiroshi , Yanaga Katsuhiko , Mori Masaki
Published: May 2006

Purpose and Experimental Design: To identify cancer-related genes, the expression profiles of colorectal cancer cells and normal epithelial cells were examined and compared using laser microdissection and cDNA microarray analysis. From these combined techniques, several cancer-related genes, including TROP2, were identified. TROP2 is known as a calcium signal transducer and is highly expressed in several types of tumors. However, no studies have investigated the significance of TROP2 expression in colorectal cancer. Thus, the expression status of TROP2 was investigated in 74 colorectal cancer samples by quantitative real-time reverse transcription-PCR and immunohistochemical studies.

Results: Laser microdissection and cDNA microarray analysis showed that there were 84 overexpressed genes in cancer cells. One of the highly overexpressed genes was TROP2. Quantitative real-time reverse transcription-PCR showed that TROP2 expression in cancer samples was significantly higher than in normal samples (P < 0.001). The samples were divided into high (n = 26) and low (n = 48) TROP2 expression groups. The cases with high TROP2 expression showed a higher frequency of liver metastasis (P = 0.005) and more cancer-related death (P = 0.046). Those cases also had an inclination of deeper depth of invasion (P = 0.064) and more lymph node metastasis (P = 0.125). Interestingly, the patients with high TROP2 expression tumors had poorer prognosis (P = 0.0036). Multivariate analysis showed that TROP2 expression status was an independent prognostic factor (relative risk, 2.38; 95% confidence interval, 1.29-4.74; P < 0.01).

Conclusion: TROP2 is one of the cancer-related genes that correlates with biological aggressiveness and poor prognosis of colorectal cancer. Thus, TROP2 is a possible candidate gene for diagnosis and molecular target therapy of colorectal cancer.
17β-Estradiol Induces Down-Regulation of Cap43/NDRG1/Drg-1, a Putative Differentiation-Related and Metastasis Suppressor Gene, in Human Breast Cancer Cells
Authors: Fotovati Abbas , Fujii Teruhiko , Yamaguchi Miki , Kage Masayoshi , Shirouzu Kazuo , Oie Shinji , Basaki Yuji , Ono Mayumi , Yamana Hideaki , Kuwano Michihiko
Published: May 2006

Purpose: Cap43 is known as a nickel- and calcium-inducible gene. In the present study, we examined whether 17β-estradiol (E2) could affect the expression of Cap43 in breast cancer.

Experimental Design: Real-time PCR, immunoblotting, and immunocytochemistry were used to examine the expression of Cap43 and estrogen receptor-α (ER-α) in breast cancer cell lines. MDA-MB-231 and SK-BR-3 cell lines were transfected with ER-α cDNA to establish cells overexpressing ER-α. Immunohistochemistry was used to evaluate the expression of the Cap43 protein in breast cancer patients (n = 96), and the relationship between Cap43 expression and clinicopathologic findings was examined.

Results: Of the eight cell lines, four expressed higher levels of Cap43 with very low levels of ER-α, whereas the other four expressed lower levels of Cap43 with high ER-α levels. Treatment with E2 decreased the expression of Cap43 dose-dependently in ER-α-positive cell lines but not in ER-α-negative lines. Administration of antiestrogens, tamoxifen and ICI 182780, abrogated the E2-induced down-regulation of Cap43. Overexpression of ER-α in both ER-α-negative cell lines, SK-BR-3 and MDA-MB-231, resulted in down-regulation of Cap43. Immunostaining studies showed a significant correlation between Cap43 expression and the histologic grade of tumors (P = 0.0387). Furthermore, Cap43 expression was inversely correlated with the expression of ER-α (P = 0.0374).

Conclusions: E2-induced down-regulation of Cap43 seems to be mediated through ER-α-dependent pathways in breast cancer cells both in culture and in patients. Cap43 has potential as a molecular marker to determine the therapeutic efficacy of antiestrogenic anticancer agents in breast cancer.
Glutathione S-Transferase P1 Polymorphism (Ile105Val) Predicts Cumulative Neuropathy in Patients Receiving Oxaliplatin-Based Chemotherapy
Authors: Lecomte Thierry , Landi Bruno , Beaune Philippe , Laurent-Puig Pierre , Loriot Marie-Anne
Published: May 2006

Purpose: Glutathione S-transferases (GST) are xenobiotic metabolizing enzymes involved in the detoxification of a variety of chemotherapeutic drugs, including platinum derivatives. Genetic polymorphisms of GSTs have been associated with enzyme activity variations. Thus, a study was done to investigate the relationship between GST polymorphisms and oxaliplatin-related cumulative neuropathy in gastrointestinal cancer patients treated with oxaliplatin-based chemotherapy.

Experimental Design: Ninety patients were included. Clinical neurologic evaluation was done at baseline and before each cycle of treatment. We determined genetic variants for GSTP1 exon 5 (Ile105Val), GSTP1 exon 6 (Ala114Val), GSTM1 (homozygous deletion), and GSTT1 (homozygous deletion). We conducted analyses in a subgroup of 64 patients receiving a minimal cumulative dose of 500 mg/m2 of oxaliplatin to examine whether the GST polymorphisms are associated with oxaliplatin-related cumulative neuropathy.

Results: Among patients receiving a minimal cumulative dose of 500 mg/m2 of oxaliplatin, 15 patients showed clinically evident oxaliplatin-related cumulative neuropathy scored grade 3 according to an oxaliplatin-specific scale. The oxaliplatin-related cumulative neuropathy scored grade 3 was significantly more frequent in patients homozygous for the GSTP1 105Ile allele than in patients homozygous or heterozygous for the GSTP1 105Val allele (odds ratio, 5.75; 95% confidence interval, 1.08-30.74; P = 0.02). No association was found with respect to any of the GSTM1, GSTT1, or GSTP1 exon 6 genotypes.

Conclusions: The results of the current study suggest that the 105Val allele variant of the GSTP1 gene at exon 5 confers a significantly decreased risk of developing severe oxaliplatin-related cumulative neuropathy.
Phosphatidylinositol 3′-Kinase/AKT Signaling Is Activated in Medulloblastoma Cell Proliferation and Is Associated with Reduced Expression of PTEN
Authors: Hartmann Wolfgang , Digon-Söntgerath Boris , Koch Arend , Waha Anke , Endl Elmar , Dani Indra , Denkhaus Dorota , Goodyer Cynthia G. , Sörensen Niels , Wiestler Otmar D. , Pietsch Torsten
Published: May 2006

Purpose: Medulloblastomas represent the most frequent malignant brain tumors of childhood. They are supposed to originate from cerebellar neural precursor cells. Recently, it has been shown that Sonic Hedgehog–induced formation of medulloblastoma in an animal model is significantly enhanced by activation of the phosphatidylinositol 3′-kinase (PI3K) signaling pathway.

Experimental Design: To examine a role for PI3K/AKT signaling in the molecular pathogenesis of human medulloblastoma, we did an immunohistochemical study of the expression of Ser473-phosphorylated (p)-AKT protein in 22 medulloblastoma samples: All samples displayed p-AKT expression. To investigate if an activated PI3K/AKT pathway is required for medulloblastoma cell growth, we treated five human medulloblastoma cell lines with increasing concentrations of the PI3K inhibitor LY294002 and analyzed cellular proliferation and apoptosis. The antiproliferative effect could be antagonized by overexpressing constitutively active AKT. As the activation of PI3K/AKT signaling may be associated with alterations of the PTEN gene located at 10q23.3, a chromosomal region subject to frequent allelic losses in medulloblastoma, we screened PTEN for mutations and mRNA expression.

Results: Proliferation of all of the medulloblastoma cell lines was dependent on PI3K/AKT signaling, whereas apoptosis was not prominently affected. Allelic loss was detected in 16% of the cases. One medulloblastoma cell line was found to carry a truncating mutation in the PTEN coding sequence. Even more important, PTEN mRNA and protein levels were found to be significantly lower in medulloblastomas compared with normal cerebellar tissue of different developmental stages. Reduction of PTEN expression was found to be associated with PTEN promoter hypermethylation in 50% of the tumor samples.

Conclusions: We conclude that activation of the PI3K/AKT pathway constitutes an important step in the molecular pathogenesis of medulloblastoma and that dysregulation of PTEN may play a significant role in this context.
Liposomal Quercetin Efficiently Suppresses Growth of Solid Tumors in Murine Models
Authors: Yuan Zhi-ping , Chen Li-juan , Fan Lin-yu , Tang Ming-hai , Yang Guang-li , Yang Han-su , Du Xiao-bo , Wang Guo-qing , Yao Wen-xiu , Zhao Qu-mei , Ye Bin , Wang Rui , Diao Peng , Zhang Wei , Wu Hong-bin , Zhao Xia , Wei Yu-Quan
Published: May 2006

Purpose: Quercetin is a potent chemotherapeutic drug. Clinical trials exploring different schedules of administration of quercetin have been hampered by its extreme water insolubility. To overcome this limitation, this study is aimed to develop liposomal quercetin and investigate its distribution in vivo and antitumor efficacy in vivo and in vitro.

Experimental Design: Quercetin was encapsulated in polyethylene glycol 4000 liposomes. Biodistribution of liposomal quercetin i.v. at 50 mg/kg in tumor-bearing mice was detected by high-performance liquid chromatography. Induction of apoptosis by liposomal quercetin in vitro was tested. The antitumor activity of liposomal quercetin was evaluated in the immunocompetent C57BL/6N mice bearing LL/2 Lewis lung cancer and in BALB/c mice bearing CT26 colon adenocarcinoma and H22 hepatoma. Tumor volume and survival time were observed. The mechanisms underlying the antitumor effect of quercetin in vivo was investigated by detecting the microvessel density, apoptosis, and heat shock protein 70 expression in tumor tissues.

Results: Liposomal quercetin could be dissolved in i.v. injection and effectively accumulate in tumor tissues. The half-time of liposomal quercetin was 2 hours in plasma. The liposomal quercetin induced apoptosis in vitro and significantly inhibited tumor growth in vivo in a dose-dependent manner. The optimal dose of liposomal quercetin resulted in a 40-day survival rate of 40%. Quantitative real-time PCR showed that liposomal quercetin down-regulated the expression of heat shock protein 70 in tumor tissues. Immunohistochemistry analysis showed that liposomal quercetin inhibited tumor angiogenesis as assessed by CD31 and induced tumor cell apoptosis.

Conclusions: Our data indicated that pegylated liposomal quercetin can significantly improve the solubility and bioavailability of quercetin and can be a potential application in the treatment of tumor.
Peroxisome Proliferator–Activated Receptor α Activation Decreases Metastatic Potential of Melanoma Cells In vitro via Down-Regulation of Akt
Authors: Grabacka Maja , Plonka Przemyslaw M. , Urbanska Krystyna , Reiss Krzysztof
Published: May 2006

Purpose: Peroxisome proliferator-activated receptors (PPAR) regulate lipid and glucose metabolism but their anticancer properties have been recently studied as well. We previously reported the antimetastatic activity of the PPARα ligand, fenofibrate, against melanoma tumors in vivo. Here we investigated possible molecular mechanisms of fenofibrate anti metastatic action.

Experimental Design: Monolayer cultures of mouse (B16F10) and human (SkMell88) melanoma cell lines, soft agar assay, and cell migration assay were used in this study. In addition, we analyzed PPARα expression and its transcriptional activity in response to fenotibrate by using Western blots and liciferase-based reporter system.

Results: Fenofibrate inhibited migration of B16F10 and SkMel188 cells in Transwell chambers and colony formation in soft agar. These effects were reversed by PPAR inhibitor, GW9662. Western blot analysis revealed time-dependent down-regulation of Akt and extracellular signal–regulated kinase l/2 phosphorylation in fenofibrate-treated cells. A B16F10 cell line stably expressing constitutively active Akt mutant was resistant to fenofibrate. In contrast, Akt gene silencing with siRNA mimicked the fenofibrate action and reduced the migratory ability of B16F1O cells. In addition, fenofibrate strongly sensitized BI6FIO cells to the proapoptotic drug staurosporine, further supporting the possibility that fenofibrate-induced down-regulation of Akt function contributes to fenofibrate-mediated inhibition of metastatic potential in this experimental model.

Conclusions: Our results show that the PPAR-dependent antimetastatic activity of fenofibrate involves down-regulation of Akt phosphorylation and suggest that supplementation with this drug may improve the effectiveness of melanoma chemotherapy.