bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Populations of Regulatory T Cells in Peripheral Blood of Patients with Hepatocellular Carcinoma
Authors: Ormandy Lars A. , Hillemann Tina , Wedemeyer Heiner , Manns Michael P. , Greten Tim F. , Korangy Firouzeh
Published: September 2005

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide with a poor prognosis and one for which immunotherapy remains a viable option. Experimental tumor models have shown that regulatory T cells, a functionally unique subset of T cells, can suppress effective antitumor immune responses. This suppression might explain the poor outcome of some of the immunotherapy protocols currently being used. A better understanding of the role of regulatory T cells in HCC is important for design of future immunotherapy-based clinical protocols. We have studied regulatory T cells from 84 patients with HCC and 74 controls, including healthy donors, patients with chronic hepatitis B virus and hepatitis C virus infection and nonviral liver cirrhosis. Regulatory T cells were identified by fluorescence-activated cell sorting using a panel of antibodies and by real-time PCR analysis for Foxp3 expression. Functional studies were done to analyze their inhibitory role. Finally, regulatory T cells were analyzed in tumors and ascites from patients with HCC. Patients with HCC have increased numbers of CD4+CD25+ regulatory T cells in their peripheral blood, which express high levels of HLA-DR, GITR, and low or no CD45RA. These cells were anergic toward T-cell receptor stimulation and, when cocultured with activated CD4+CD25− cells, potently suppressed their proliferation and cytokine secretion. There were also high numbers of regulatory T cells in tumor-infiltrating lymphocytes of HCC patients comparable with the increase in their peripheral blood. Our data suggest that the increase in frequency of regulatory T cells might play a role in modulation of the immune response against HCC and could be important in design of immunotherapeutic approaches.
Increased Expression of Germinal Center–Associated Nuclear Protein RNA-Primase Is Associated with Lymphomagenesis
Authors: Fujimura Satoru , Xing Yan , Takeya Motohiro , Yamashita Yasuyuki , Ohshima Koichi , Kuwahara Kazuhiko , Sakaguchi Nobuo
Published: September 2005

Lymphomas arise containing abnormalities of various differentiation stage-specific molecules. In the study reported here, we have shown abnormal up-regulation of germinal center B cell–associated GANP in various human lymphomas including mantle cell, diffuse large B cell, and Hodgkin lymphoma, by immunohistochemical analysis. To study the role of GANP in lymphomagenesis, we generated mutant mice (ganp-Tg) that express the transgenic ganp gene under immunoglobulin enhancer and promoter control. Ganp-Tg mice showed a high incidence of lymphomagenesis (29.5%) after aging with a non-B/non-T cell surface phenotype having slight CD45R/B220 expression and Ig transcripts of rearranged VH-DH-JH IgH loci. Lymphomas generated in ganp-Tg mice displayed similar pathologic characteristics to mouse reticulum cell neoplasm or Hodgkin lymphoma–like lesions. The VH sequences of individual mice showed that the tumors proliferated from a single clone or oligoclones, as is found in human diffuse large B-cell lymphomas and Hodgkin lymphoma. These results suggest that GANP overexpression is a causative factor in the generation of B lymphomas.
Synthetic Triterpenoids Cooperate with Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand to Induce Apoptosis of Breast Cancer Cells
Authors: Hyer Marc L. , Croxton Rhonda , Krajewska Maryla , Krajewski Stanislaw , Kress Christina L. , Lu Meiling , Suh Nanjoo , Sporn Michael B. , Cryns Vincent L. , Zapata Juan M. , Reed John C.
Published: September 2005

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL or Apo2L) has been shown to induce apoptosis specifically in cancer cells while sparing normal tissues. Unfortunately not all cancer cells respond to TRAIL; therefore, TRAIL sensitizing agents are currently being explored. We have identified synthetic triterpenoids, including 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its derivative 1-(2-cyano-3,12-dioxooleana-1,9-dien-28-oyl) imidazole (CDDO-Im), which sensitize TRAIL-resistant cancer cells to TRAIL-mediated apoptosis. Here we show that TRAIL-treated T47D and MDA-MB-468 breast cancer cells fail to initiate detectable caspase-8 processing and, consequently, do not initiate TRAIL-mediated apoptosis. Concomitant treatment with CDDO or CDDO-Im reverses the TRAIL-resistant phenotype, promoting robust caspase-8 processing and induction of TRAIL-mediated apoptosis in vitro. The combination of triterpenoids and monoclonal anti-TRAIL receptor-1 (DR4) antibody also induces apoptosis of breast cancer cells in vitro. From a mechanistic standpoint, we show that CDDO and CDDO-Im down-regulate the antiapoptotic protein c-FLIPL, and up-regulate cell surface TRAIL receptors DR4 and DR5. CDDO and CDDO-Im, when used in combination with TRAIL, have no adverse affect on cultured normal human mammary epithelial cells. Moreover, CDDO-Im and TRAIL are well tolerated in mice and the combination of CDDO-Im and TRAIL reduces tumor burden in vivo in an MDA-MB-468 tumor xenograft model. These data suggest that CDDO and CDDO-Im may be useful for selectively reversing the TRAIL-resistant phenotype in cancer but not normal cells.
A Mechanism-Based Antioxidant Approach for the Reduction of Skin Carcinogenesis
Authors: Zhao Yunfeng , Chaiswing Luksana , Oberley Terry D. , Batinic-Haberle Ines , St. Clair William , Epstein Charles J. , St. Clair Daret
Published: September 2005

Studies in our laboratories showed that overexpression of manganese superoxide dismutase (MnSOD) reduced tumor incidence in a multistage skin carcinogenesis mouse model. However, reduction of MnSOD by heterozygous knockout of the MnSOD gene (MnSOD KO) did not lead to an increase in tumor incidence, because a reduction of MnSOD enhanced both cell proliferation and apoptosis. The present study extends our previous studies in the MnSOD KO mice and shows that apoptosis in mouse epidermis occurred prior to cell proliferation (6 versus 24 hours) when treated with tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). To investigate the possibility that a timed administration of SOD following apoptosis but before proliferation may lead to suppression of tumor incidence, we applied a SOD mimetic (MnTE-2-PyP5+) 12 hours after each TPA treatment. Biochemical studies showed that MnTE-2-PyP5+ suppressed the level of protein carbonyls and reduced the activity of activator protein-1 and the level of proliferating cellular nuclear antigen, without reducing the activity of p53 or DNA fragmentation following TPA treatment. Histologic examination confirmed that MnTE-2-PyP5+ suppressed mitosis without interfering with apoptosis. Remarkably, the incidence and multiplicity of skin tumors were reduced in mice that received MnTE-2-PyP5+ before cell proliferation. These results show a novel strategy for an antioxidant approach to cancer intervention.
Enhanced Susceptibility of Irradiated Tumor Vessels to Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibition
Authors: Zips Daniel , Eicheler Wolfgang , Geyer Peter , Hessel Franziska , Dörfler Annegret , Thames Howard D. , Haberey Martin , Baumann Michael
Published: September 2005

Previous experiments with PTK787/ZK222584, a specific inhibitor of vascular endothelial growth factor receptor (VEGFR) tyrosine kinases, using irradiated human FaDu squamous cell carcinoma in nude mice, suggested that radiation-damaged tumor vessels are more sensitive to VEGFR inhibition. To test this hypothesis, the tumor transplantation site (i.e., the right hind leg of nude mice) was irradiated 10 days before transplantation of FaDu to induce radiation damage in the host tissue. FaDu tumors vascularized by radiation-damaged blood vessels appeared later, grew at a slower rate, and showed more necrosis and a smaller vessel area per central tumor section than controls. PTK787/ZK222584 at a daily dose of 50 mg/kg body weight had no impact on growth of control tumors. In contrast, tumors vascularized by radiation-damaged vessels responded to PTK787/ZK222584 with longer latency and slower growth rate than controls, and a trend toward further increase in necrosis, indicating that irradiated tumor vessels are more susceptible to VEGFR inhibition than unirradiated vessels. Although not proving causality, expression analysis of VEGF and VEGFR2 shows that enhanced sensitivity of irradiated vessels to a specific inhibitor of VEGFR tyrosine kinases correlates with increased expression of the molecular target.
The CD44 Receptor Interacts with P-Glycoprotein to Promote Cell Migration and Invasion in Cancer
Authors: Miletti-González Karl E. , Chen Shiling , Muthukumaran Neelakandan , Saglimbeni Giuseppa N. , Wu Xiaohua , Yang Jinming , Apolito Kevin , Shih Weichung J. , Hait William N. , Rodríguez-Rodríguez Lorna
Published: September 2005

Invasion and metastases of cancer cells and the development of resistance to anticancer therapies are the main causes of morbidity and mortality from cancer. For more than two decades, these two important but not clearly related aspects in the biology of cancer have been extensively studied. Specifically, P-glycoprotein and CD44 have been characterized and are known to be determinants of multidrug resistance (MDR) and metastases. Despite this body of knowledge, few reports have linked the two phenotypes and only recently have there been reasons to suspect a direct connection. In this report, we show that a novel physical and genetic interaction between CD44s and P-glycoprotein is in part responsible for the correlation between MDR and invasive potential in cancer cells. P-glycoprotein–specific substrates that interfere with its function reduced in vitro invasion, migration, and the physical colocalization of CD44s and P-glycoprotein. CD44 expression in sensitive cells promoted the expression of P-glycoprotein and the MDR phenotype. RNA interference of MDR1 inhibited the rate of cell migration. These data indicate that there is a close interaction between CD44 and P-glycoprotein that results in the concurrent expression and modulation of two malignant phenotypes, invasion and MDR.
Halogenated Thymidine Analogues Restore the Expression of Silenced Genes without Demethylation
Authors: Fan Jun , Kodama Ei-ichi , Koh Yasuhiro , Nakao Mitsuyoshi , Matsuoka Masao
Published: September 2005

Transcriptional silencing of tumor suppressor genes by aberrant DNA methylation is a characteristic frequently observed in cancer cells. Therefore, reversing this process is a therapeutic target against cancer. In this study, we established a screening system for silencing inhibitors with cell lines transfected by a retroviral vector containing a luciferase gene. More than 100 nucleosides were tested for antisilencing activity with a selected clone in which the silenced expression of luciferase could be recovered by 5-aza-2′-deoxycytidine. A group of halogenated thymidine analogues was found to reactivate transcription of not only the reporter retrovirus vector but also endogenous glutathione-S-transferase 1 gene, without influence to DNA hypermethylation. Gel mobility shift assay showed that 5-bromo-2′-deoxyuridine (BrdUrd) or 5-iodo-2′-deoxyuridine incorporation did not affect the binding of the methyl-CpG binding protein motif to methylated DNA. Finally, in the retroviral promoter, BrdUrd treatment increased the acetylated histone H3 level and decreased methylation of histone H3 Lys9 in accordance with recovered transcription. This study shows that halogenated thymidines have an antisilencing effect without changing DNA methylation status by interfering with step(s) between DNA methylation and histone acetylation.
Large-scale Transcriptome Analyses Reveal New Genetic Marker Candidates of Head, Neck, and Thyroid Cancer
Authors: Reis Eduardo M. , Ojopi Elida P.B. , Alberto Fernando L. , Rahal Paula , Tsukumo Fernando , Mancini Ulises M. , Guimarães Gustavo S. , Thompson Gloria M.A. , Camacho Cleber , Miracca Elisabete , Carvalho André L. , Machado Abimael A. , Paquola Apuã C.M. , Cerutti Janete M. , da Silva Aline M. , Pereira Gonçalo G. , Valentini Sandro R. , Nagai Maria A. , Kowalski Luiz Paulo , Verjovski-Almeida Sergio , Tajara Eloiza H. , Dias-Neto Emmanuel ,   Head and Neck Annotation Consortium
Published: September 2005

A detailed genome mapping analysis of 213,636 expressed sequence tags (EST) derived from nontumor and tumor tissues of the oral cavity, larynx, pharynx, and thyroid was done. Transcripts matching known human genes were identified; potential new splice variants were flagged and subjected to manual curation, pointing to 788 putatively new alternative splicing isoforms, the majority (75%) being insertion events. A subset of 34 new splicing isoforms (5% of 788 events) was selected and 23 (68%) were confirmed by reverse transcription–PCR and DNA sequencing. Putative new genes were revealed, including six transcripts mapped to well-studied chromosomes such as 22, as well as transcripts that mapped to 253 intergenic regions. In addition, 2,251 noncoding intronic RNAs, eventually involved in transcriptional regulation, were found. A set of 250 candidate markers for loss of heterozygosis or gene amplification was selected by identifying transcripts that mapped to genomic regions previously known to be frequently amplified or deleted in head, neck, and thyroid tumors. Three of these markers were evaluated by quantitative reverse transcription–PCR in an independent set of individual samples. Along with detailed clinical data about tumor origin, the information reported here is now publicly available on a dedicated Web site as a resource for further biological investigation. This first in silico reconstruction of the head, neck, and thyroid transcriptomes points to a wealth of new candidate markers that can be used for future studies on the molecular basis of these tumors. Similar analysis is warranted for a number of other tumors for which large EST data sets are available.
Canstatin Acts on Endothelial and Tumor Cells via Mitochondrial Damage Initiated through Interaction with αvβ3 and αvβ5 Integrins
Authors: Magnon Claire , Galaup Ariane , Mullan Brian , Rouffiac Valérie , Bidart Jean-Michel , Griscelli Frank , Opolon Paule , Perricaudet Michel
Published: September 2005

Canstatin, the noncollagenous domain of collagen type IV α-chains, belongs to a series of collagen-derived angiogenic inhibitors. We have elucidated the functional receptors and intracellular signaling induced by canstatin that explain its strong antitumor efficacy in vivo. For this purpose, we generated a canstatin-human serum albumin (CanHSA) fusion protein, employing the HSA moiety as an expression tag. We show that CanHSA triggers a crucial mitochondrial apoptotic mechanism through procaspase-9 cleavage in both endothelial and tumor cells, which is mediated through cross-talk between αvβ3- and αvβ5-integrin receptors. As a point of reference, we employed the first three kringle domains of angiostatin (K1-3), fused with HSA, which, in contrast to CanHSA, act only on endothelial cells through αvβ3-integrin receptor–mediated activation of caspase-8 alone, without ensuing mitochondrial damage. Taken together, these results provide insights into how canstatin might exert its strong anticancer effect.
Prostaglandin E2 Enhances Intestinal Adenoma Growth via Activation of the Ras-Mitogen-Activated Protein Kinase Cascade
Authors: Wang Dingzhi , Buchanan F. Gregory , Wang Haibin , Dey Sudhansu K. , DuBois Raymond N.
Published: September 2005

A large body of clinical, genetic, and biochemical evidence indicates that cyclooxygenase-2 (COX-2), a key enzyme for prostanoid biosynthesis, contributes to the promotion of colorectal cancer. COX-2-derived prostaglandin E2 (PGE2) is the most abundant prostaglandin found in several gastrointestinal malignancies. Although PGE2 enhances intestinal adenoma growth in Apcmin mice, the mechanism(s) by which it accelerates tumor growth is not completely understood. Here we investigated how PGE2 promotes intestinal tumor growth and the signaling pathways responsible for its effects. We observed that PGE2 treatment leads to increased epithelial cell proliferation and induces COX-2 expression in intestinal adenomas. Furthermore, we show that PGE2 regulation of COX-2 expression is mediated by activation of a Ras-mitogen-activated protein kinase signaling cascade. One intriguing finding is that COX-2-derived PGE2 mimics the effects of constitutively active Ras through a self-amplifying loop that allows for a distinct growth advantage.
Selective Insensitivity of ZR-75-1 Human Breast Cancer Cells to 2-Methoxyestradiol: Evidence for Type II 17β-Hydroxysteroid Dehydrogenase as the Underlying Cause
Authors: Liu Zhi-Jian , Lee Won Jun , Zhu Bao Ting
Published: September 2005

2-Methoxyestradiol (2-MeO-E2), a nonpolar endogenous metabolite of 17β-estradiol, has strong antiproliferative, apoptotic, and antiangiogenic actions. Among the four human breast cancer cell lines tested (MCF-7, T-47D, ZR-75-1, and MDA-MB-435s), the ZR-75-1 cells were selectively insensitive to the antiproliferative actions of 2-MeO-E2, although these cells had a similar sensitivity as other cell lines to several other anticancer agents (5-fluorouracil, mitomycin C, doxorubicin, colchicine, vinorelbine, and paclitaxel). Mechanistically, this insensitivity is largely attributable to the presence of high levels of a steroid-selective metabolizing enzyme, the type II 17β-hydroxysteroid dehydrogenase (17β-HSD), in the ZR-75-1 cells, which rapidly converts 2-MeO-E2 to the inactive 2-methoxyestrone, but this enzyme does not metabolically inactivate other nonsteroidal anticancer agents. The type II 17β-HSD–mediated conversion of 2-MeO-E2 to 2-methoxyestrone in ZR-75-1 cells followed the first-order kinetics, with a very short half-life (∼2 hours). In comparison, the T-47D, MCF-7, and MDA-MB-435s human breast cancer cells, which were highly sensitive to 2-MeO-E2, had very low or undetectable catalytic activity for the conversion of 2-MeO-E2 to 2-methoxyestrone. Reverse transcription-PCR analysis of the mRNA levels of three known oxidative 17β-HSD isozymes (types II, IV, and VIII) revealed that only the type II isozyme was selectively expressed in the ZR-75-1 cells, whereas the other two isozymes were expressed in all four cell lines. Taken together, our results showed, for the first time, that the high levels of type II 17β-HSD present in ZR-75-1 cells were largely responsible for the facile conversion of 2-MeO-E2 to 2-methoxyestrone and also for the selective insensitivity to the antiproliferative actions of 2-MeO-E2.
Polymorphisms Associated with Asthma Are Inversely Related to Glioblastoma Multiforme
Authors: Schwartzbaum Judith , Ahlbom Anders , Malmer Beatrice , Lönn Stefan , Brookes Anthony J. , Doss Hani , Debinski Waldemar , Henriksson Roger , Feychting Maria
Published: September 2005

A reduced risk of primary malignant adult brain tumors is observed among people reporting asthma, hay fever, and other allergic conditions; however, findings may be attributed to prediagnostic effects of tumors or recall bias. To determine whether asthma and allergic condition polymorphisms are inversely related to glioblastoma multiforme (GBM) risk, we conducted a population-based case-control study of 111 GBM patients and 422 controls. We identified five single nucleotide polymorphisms on three genes previously associated with asthma [interleukin (IL)-4RA, IL-13, ADAM33] and one gene associated with inflammation (cyclooxygenase-2). Confirming previous literature, we found that self-reported asthma, eczema, and fever are inversely related to GBM [e.g., asthma odds ratio (OR), 0.64; 95% confidence interval (CI), 0.33-1.25]. In addition, IL-4RA Ser478Pro TC, CC, and IL-4RA Gln551Arg AG, AA are positively associated with GBM (OR, 1.64; 95% CI, 1.05-2.55; 1.61; 95% CI, 1.05-2.47), whereas IL-13 −1,112 CT, TT is negatively associated with GBM (0.56; 95% CI, 0.33-0.96). Each of these polymorphism-GBM associations is in the opposite direction of a corresponding polymorphism-asthma association, consistent with previous findings that self-reported asthmatics and people with allergic conditions are less likely to have GBM than are people who do not report these conditions. Because we used germ line polymorphisms as biomarkers of susceptibility to asthma and allergic conditions, our results cannot be attributed to recall bias or effects of GBM on the immune system. However, our findings are also consistent with associations between IL-4RA, IL-13, and GBM that are independent of their role in allergic conditions.
The Synergistic Combination of the Farnesyl Transferase Inhibitor Lonafarnib and Paclitaxel Enhances Tubulin Acetylation and Requires a Functional Tubulin Deacetylase
Authors: Marcus Adam I. , Zhou Jun , O'Brate Aurora , Hamel Ernest , Wong Jason , Nivens Michael , El-Naggar Adel , Yao Tso-Pang , Khuri Fadlo R. , Giannakakou Paraskevi
Published: September 2005

Farnesyl transferase (FT) inhibitors (FTI) are anticancer agents developed to target oncogenic Ras proteins by inhibiting Ras farnesylation. FTIs potently synergize with paclitaxel and other microtubule-stabilizing drugs; however, the mechanistic basis underlying this synergistic interaction remains elusive. Here we show that the FTI lonafarnib affects the microtubule cytoskeleton resulting in microtubule bundle formation, increased microtubule stabilization and acetylation, and suppression of microtubule dynamics. Notably, treatment with the combination of low doses of lonafarnib with paclitaxel markedly enhanced tubulin acetylation (a marker of microtubule stability) as compared with either drug alone. This synergistic effect correlated with FT inhibition and was accompanied by a synergistic increase in mitotic arrest and cell death. Mechanistically, we show that the combination of lonafarnib and paclitaxel inhibits the in vitro deacetylating activity of the only known tubulin deacetylase, histone deacetylase 6 (HDAC6). In addition, the lonafarnib/taxane combination is synergistic only in cells lines expressing the wild-type HDAC6, but not a catalytic-mutant HDAC6, revealing that functional HDAC6 is required for the synergy of lonafarnib with taxanes. Furthermore, tubacin, a specific HDAC6 inhibitor, synergistically enhanced tubulin acetylation in combination with paclitaxel, similar to the combination of lonafarnib and paclitaxel. Taken together, these data suggest a relationship between FT inhibition, HDAC6 function, and cell death, providing insight into the putative molecular basis of the lonafarnib/taxane synergistic antiproliferative combination.
Evidence for Nonautonomous Effect of p53 Tumor Suppressor in Carcinogenesis
Authors: Kiaris Hippokratis , Chatzistamou Ioulia , Trimis George , Frangou-Plemmenou Matrisa , Pafiti-Kondi Agatha , Kalofoutis Anastasios
Published: September 2005

Prostate, breast, and probably other epithelial tumors harbor inactivating mutations in the p53 tumor suppressor gene in the stromal cells, implying the nonautonomous action of p53 in carcinogenesis. We have tested this hypothesis by evaluating the tumorigenicity of MCF7 human breast cancer cells in severe combined immunodeficient mice that differ in their p53 status. Our results showed that, indeed, p53 ablation in the hosts reduced the latency for the development of MCF7 tumors. Furthermore, we show that heterozygous hosts frequently undergo loss of heterozygosity at the p53 locus in the tumor stroma tissue by mechanism that resembles the inactivation of p53 in primary tumors. To evaluate the impact of p53 ablation in the stromal fibroblasts, in tumorigenesis, tumors were reconstituted in mice bearing wild-type p53 alleles, by mixing MCF7 cells with fibroblasts isolated from mutant or wild-type p53 mice. Our results suggest that tumors containing p53-deficient fibroblasts developed faster and were more aggressive than their counterparts with wild-type fibroblasts, although their neoplastic component, namely MCF7 mammary carcinoma cells, was identical in both cases. These data strongly support the notion for the operation of a nonautonomous mechanism for p53 action in primary tumors and provide a mechanistic association between p53 mutations in the stromal component of epithelial tumors and carcinogenesis.
Cyclooxygenase-1 Is a Potential Target for Prevention and Treatment of Ovarian Epithelial Cancer
Authors: Daikoku Takiko , Wang Dingzhi , Tranguch Susanne , Morrow Jason D. , Orsulic Sandra , DuBois Raymond N. , Dey Sudhansu K.
Published: September 2005

The precise genetic and molecular defects underlying epithelial ovarian cancer (EOC) remain largely unknown, and treatment options for patients with advanced disease are limited. Cyclooxygenases (COX-1 and COX-2) catalyze the conversion of arachidonic acid to prostaglandins. Whereas overwhelming evidence suggests a role for COX-2 in a variety of cancers, the contribution of COX-1 remains much less explored. The expression status of COX isoforms in ovarian cancers also remains confusing. We have previously shown that human epithelial ovarian tumors have increased levels of COX-1 but not COX-2. To more carefully examine the role of COXs in ovarian cancer, we used a mouse model of EOC in which genetic and oncogenic modifications were experimentally engineered into ovarian surface epithelial cells (OSE) thought to be the cells of origin for human EOC. These OSE cells produce tumors when allografted into host mice. Using multiple approaches, we observed that OSE cells and the tumors comprised of these cells express high levels of COX-1 but not COX-2. Prostacyclin (PGI2) is the major prostaglandin generated downstream of COX-1 in these cells, and SC-560, a COX-1-selective inhibitor, dramatically inhibits PGI2 production. More importantly, SC-560 reduced the growth of tumors when OSE cells were allografted in nude female mice. In contrast, the COX-2-selective inhibitor celecoxib had little effect on tumor growth. The growth inhibitory effects of SC-560 result from reduced cell proliferation and/or accelerated apoptosis. Our results imply COX-1 as a target for the prevention and/or treatment of EOC.
Human Malignant Melanomas Express Receptors for Luteinizing Hormone Releasing Hormone Allowing Targeted Therapy with Cytotoxic Luteinizing Hormone Releasing Hormone Analogue
Authors: Keller Gunhild , Schally Andrew V. , Gaiser Timo , Nagy Attila , Baker Benjamin , Westphal Gabriela , Halmos Gabor , Engel Jörg B.
Published: September 2005

Cytotoxic analogue of luteinizing hormone releasing hormone (LHRH), AN-207, binds with high affinity to LHRH receptors and can be targeted to tumors expressing these receptors. We investigated the expression of LHRH receptors in surgical specimens of human malignant melanoma and evaluated the effects of AN-207 in models of human melanoma. Human melanoma specimens derived from primary tumors or metastases were examined for LHRH receptor expression by immunohistochemistry. Binding assays, Western immunoblotting, and reverse transcription-PCR analyses were used to investigate LHRH receptors in MRI-H255 and MRI-H187 transplantable human melanoma tumor lines. Antitumor effects of AN-207 and its components were evaluated in vivo in nude mice bearing xenografts of either melanoma tumor line. All 19 human melanoma specimens examined showed positive staining for LHRH receptors. The mRNA for LHRH receptors, receptor protein and binding sites for LHRH were detected in both transplantable melanoma tumor lines. AN-207 significantly inhibited the growth of MRI-H255 and MRI-H187 xenografts in vivo, reducing tumor volume by 59.9% to 79.2% and tumor weight by 61.0% to 76.9% (all P < 0.05). The components of AN-207 (LH-RH analogue carrier and cytotoxic radical AN-201 as single drugs or as an unconjugated mixture) had no significant effects. Blockade of LHRH receptors by an excess of LHRH agonist Decapeptyl suppressed the effects of AN-207. LHRH receptors are expressed in a very high percentage of human malignant melanoma specimens and can be used for targeted chemotherapy with cytotoxic LHRH analogue AN-207.
Human Homologue of Cement Gland Protein, a Novel Metastasis Inducer Associated with Breast Carcinomas
Authors: Liu Dong , Rudland Philip S. , Sibson D. Ross , Platt-Higgins Angela , Barraclough Roger
Published: September 2005

A suppression subtractive cDNA library representing mRNAs expressed at a higher level in the malignant human breast cancer cell line, MCF-7, relative to a benign breast tumor-derived cell line, Huma 123, contained a cDNA, M36, which was expressed in estrogen receptor α (ERα)–positive breast carcinoma cell lines but not in cell lines from normal/benign/ERα-negative malignant breast lesions. M36 cDNA had an identical coding sequence to anterior gradient 2 (AGR2), the human homologue of the cement gland–specific gene (Xenopus laevis). Screening of breast tumor specimens using reverse transcription-PCR and immunocytochemistry with affinity-purified anti-AGR2 antibodies showed that the presence of AGR2 mRNA and protein were both statistically significantly associated with ERα-positive carcinomas (P = 0.007, Fisher's exact test) and with malignancy (P ≤ 0.025). When an expression vector for AGR2 cDNA was introduced into benign nonmetastatic rat mammary tumor cells, and three separate clones and two pools of cells were transferred to the mammary glands of syngeneic hosts, there were no consistent differences in the mean latent periods of tumor formation. However, metastases occurred in the lungs of animals receiving the AGR2 transfectants in 77% to 92% of animals with primary tumors (P = 0.0001) compared with no metastases in the control groups. The AGR2 transfectants exhibited enhanced rates of adhesion to a plastic substratum and extracellular AGR2 enhanced the rate of attachment of AGR2-negative but not AGR2-positive cells. These experiments are the first to link mechanistically the developmental gene product, AGR2, with metastasis in vivo.
Proapoptotic Activity of New Glutathione S-Transferase Inhibitors
Authors: Turella Paola , Cerella Claudia , Filomeni Giuseppe , Bullo Angela , De Maria Francesca , Ghibelli Lina , Ciriolo Maria Rosa , Cianfriglia Maurizio , Mattei Maurizio , Federici Giorgio , Ricci Giorgio , Caccuri Anna Maria
Published: September 2005

Selected 7-nitro-2,1,3-benzoxadiazole derivatives have been recently found very efficient inhibitors of glutathione S-transferase (GST) P1-1,55G. Ricci et al. 7-Nitro-2,1,3-benzoxadiazole derivatives: a new class of suicide inhibitors for glutathione S-transferases, submitted for publication. an enzyme which displays antiapoptotic activity and is also involved in the cellular resistance to anticancer drugs. These new inhibitors are not tripeptide glutathione-peptidomimetic molecules and display lipophylic properties suitable for crossing the plasma membrane. In the present work, we show the strong cytotoxic activity of these compounds in the following four different cell lines: K562 (human myeloid leukemia), HepG2 (human hepatic carcinoma), CCRF-CEM (human T-lymphoblastic leukemia), and GLC-4 (human small cell lung carcinoma). The LC50 values are in the micromolar/submicromolar range and are close to the IC50 values obtained with GSTP1-1, suggesting that the target of these molecules inside the cell is indeed this enzyme. The cytotoxic mechanism of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol, the most effective GSTP1-1 inhibitor, has been carefully investigated in leukemic CCRF-CEM and K562 cell lines. Western blot and immunoprecipitation analyzes have shown that 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol promotes in both cell lines the dissociation of the GSTP1-1 in a complex with c-jun NH2-terminal kinase (JNK). This process triggers a reactive oxygen species (ROS)–independent activation of the JNK-mediated pathway that results in a typical process of apoptosis. Besides this main pathway, in K562 cells, a ROS-mediated apoptosis partially occurs (about 30%) which involves the p38MAPK signal transduction pathway. The low concentration of this new compound needed to trigger cytotoxic effects on tumor cells and the low toxicity on mice indicate that the new 7-nitro-2,1,3-benzoxadiazole derivatives are promising anticancer agents.
Human Epididymis Protein 4 (HE4) Is a Secreted Glycoprotein that Is Overexpressed by Serous and Endometrioid Ovarian Carcinomas
Authors: Drapkin Ronny , von Horsten Hans Henning , Lin Yafang , Mok Samuel C. , Crum Christopher P. , Welch William R. , Hecht Jonathan L.
Published: September 2005

Among the genes most commonly identified in gene expression profiles of epithelial ovarian carcinomas (EOC) is the gene for human epididymis protein 4 (HE4). To ascertain its clinical utility, we did a comprehensive assessment of HE4 protein expression in benign and malignant ovarian and nonovarian tissues by immunohistochemistry. In comparison with normal surface epithelium, which does not express HE4, we found that cortical inclusion cysts lined by metaplastic Mullerian epithelium abundantly express the protein. Its expression in tumors was restricted to certain histologic subtype: 93% of serous and 100% of endometrioid EOCs expressed HE4, whereas only 50% and 0% of clear cell carcinomas and mucinous tumors, respectively, were positive. Tissue microarrays revealed that the majority of nonovarian carcinomas do not express HE4, consistent with our observation that HE4 protein expression is highly restricted in normal tissue to the reproductive tracts and respiratory epithelium. HE4 is predicted to encode a secreted protein. Using reverse transcription-PCR, we identified ovarian cancer cell lines that endogenously overexpress HE4. Cultured medium from these cells revealed a secreted form of HE4 that is N-glycosylated. This observation is consistent with the recent report that HE4 circulates in the bloodstream of patients with EOC. Therefore, HE4 is a secreted glycoprotein that is overexpressed by serous and endometrioid EOCs. Its expression in cortical inclusion cysts suggests that formation of Mullerian epithelium is a prerequisite step in the development of some types of EOCs.
RNA Interference Targeting Aurora Kinase A Suppresses Tumor Growth and Enhances the Taxane Chemosensitivity in Human Pancreatic Cancer Cells
Authors: Hata Tatsuo , Furukawa Toru , Sunamura Makoto , Egawa Shinichi , Motoi Fuyuhiko , Ohmura Noriyuki , Marumoto Tomotoshi , Saya Hideyuki , Horii Akira
Published: September 2005

AURKA/STK15/BTAK, the gene encoding Aurora A kinase that is involved in the regulation of centrosomes and segregation of chromosomes, is frequently amplified and overexpressed in various kinds of human cancers, including pancreatic cancer. To address its possibility as a therapeutic target for pancreatic cancer, we employed the RNA interference technique to knockdown AURKA expression and analyzed its phenotypes. We found that the specific knockdown of AURKA in cultured pancreatic cancer cells strongly suppressed in vitro cell growth and in vivo tumorigenicity. The knockdown induced the accumulation of cells in the G2-M phase and eventual apoptosis. Furthermore, we observed a synergistic enhancement of the cytotoxicity of taxanes, a group of chemotherapeutic agents impairing G2-M transition, by the RNA interference–mediated knockdown of AURKA. These results indicate that inhibition of AURKA expression can result in potent antitumor activity and chemosensitizing activity to taxanes in human pancreatic cancer.
Elucidation of Thioredoxin as a Molecular Target for Antitumor Quinols
Authors: Bradshaw Tracey D. , Matthews Charles S. , Cookson Jennifer , Chew Eng-Hui , Shah Manish , Bailey Kevin , Monks Anne , Harris Erik , Westwell Andrew D. , Wells Geoffrey , Laughton Charles A. , Stevens Malcolm F.G.
Published: September 2005

Heteroaromatic quinols 4-(benzothiazol-2-yl)-4-hydroxycyclohexa-2,5-dienone (1) and 4-(1-benzenesulfonyl-1H-indol-2-yl)-4-hydroxycyclohexa-2,5-dienone (2) exhibit potent and selective antitumor activity against colon, renal, and breast carcinoma cell lines in vitro (GI50 < 500 nmol/L). In vivo growth inhibition of renal, colon, and breast xenografts has been observed. Profound G2-M cell cycle block accompanied down-regulation of cdk1 gene transcription was corroborated by decreased CDK1 protein expression following treatment of HCT 116 cells with growth inhibitory concentrations of 1 or 2. The chemical structure of the quinol pharmacophore 4-(hydroxycyclohexa-2,5-dienone) suggested that these novel agents would readily react with nucleophiles in a double Michael (β-carbon) addition. Indeed, COMPARE analysis within the National Cancer Institute database revealed a number of chemically related quinone derivatives that could potentially react with sulfur nucleophiles in a similar manner and suggested that thioredoxin/thioredoxin reductase signal transduction could be a putative target. Molecular modeling predicted covalent irreversible binding between quinol analogues and cysteine residues 32 and 35 of thioredoxin, thereby inhibiting enzyme activity. Binding has been confirmed, via mass spectrometry, between reduced human thioredoxin and 1. Microarray analyses of untreated HCT 116 cells and those exposed to either 1 (1 μmol/L) or 2 (500 nmol/L and 1 μmol/L) determined that of ≥10,000 cancer-related genes, expression of thioredoxin reductase was up-regulated >3-fold. Furthermore, quinols 1 and 2 inhibited insulin reduction, catalyzed by thioredoxin/thioredoxin reductase signaling in a dose-dependent manner (IC50 < 6 μmol/L). Results are consistent with a mechanism of action of novel antitumor quinols involving inhibition of the small redox protein thioredoxin.
Genotype-Dependent Induction of Transmissible Chromosomal Instability by γ-Radiation and the Benzene Metabolite Hydroquinone
Authors: Gowans I. Duncan , Lorimore Sally A. , McIlrath Joanne M. , Wright Eric G.
Published: September 2005

Although it is well established that ionizing radiation and benzene are epidemiologically linked to acute myeloid leukemia (AML), the underlying mechanisms are not understood. We have shown that γ-radiation can induce a persisting genomic instability in the clonal descendants of hemopoietic stem cells manifested as a high frequency of nonclonal chromosome and chromatid aberrations. A strikingly similar instability is shown after exposure to the benzene metabolite hydroquinone. The CBA/Ca but not the C57BL/6 genotype is susceptible to the induction of instability by both ionizing radiation and hydroquinone and exposure of CBA/Ca, but not C57BL/6, mice to either agent is known to be associated with the development of AML. The results are consistent with the proposal that chromosomal instability induced by either agent may contribute to AML development by increasing the number of genetic lesions in hemopoietic cells. Genotype-dependent chromosomal instability can be induced by hydroquinone doses that are not acutely stem cell toxic and this may have important implications for current assessment of safe levels of exposure to benzene as well as for mechanistic understanding of the hemotoxic and leukemogenic effects.
Nitric Oxide–Induced Apoptosis in Lymphoblastoid and Fibroblast Cells Dependent on the Phosphorylation and Activation of p53
Authors: McLaughlin Laura M. , Demple Bruce
Published: September 2005

When nitric oxide (NO) is produced at micromolar concentrations, as during inflammation, exposure to surrounding cells is potentially cytotoxic. The NO-dependent signaling pathways that initiate cell death are thought to involve the tumor suppressor protein p53, but the degree to which this factor contributes to NO-induced cell death is less clear. Various reports either confirm or negate a role for p53 depending on the cell type and NO donor used. In this study, we have used several pairs of cell lines whose only differences are the presence or absence of p53, and we have treated these cell lines with the same NO donor, spermineNONOate (SPER/NO). Treatment with SPER/NO induced such apoptotic markers as DNA fragmentation, nuclear condensation, poly(ADP-ribose) polymerase cleavage, cytochrome c release, and Annexin V staining. p53 was required for at least 50% of SPER/NO-induced apoptotic cell death in human lymphoblastoid cells and for almost all in primary and E1A-tranformed mouse embryonic fibroblasts, which highlights the possible importance of DNA damage for apoptotic signaling in fibroblasts. In contrast, p53 did not play a significant role in NO-induced necrosis. NO treatment also induced the phosphorylation of p53 at Ser15; pretreatment with phosphoinositide-3 kinase (PI3K) family inhibitors, wortmannin, LY294002, and caffeine, blocked such phosphorylation, but the p38 mitogen-activated protein kinase inhibitor, SB203580, did not. Pretreatment with the PI3K family inhibitors also led to a switch from NO-induced apoptosis to necrosis, which implicates a PI3K-related kinase such as ataxia telangiectasia mutated (ATM) or ATR (ATM and Rad3 related) in p53-dependent NO-induced apoptosis.
Signal Transducer and Activator of Transcription 3 Is a Key Regulator of Keratinocyte Survival and Proliferation following UV Irradiation
Authors: Sano Shigetoshi , Chan Keith Syson , Kira Masahiro , Kataoka Ken , Takagi Satoshi , Tarutani Masahito , Itami Satoshi , Kiguchi Kaoru , Yokoi Masayuki , Sugasawa Kaoru , Mori Toshio , Hanaoka Fumio , Takeda Junji , DiGiovanni John
Published: September 2005

UVB irradiation of signal transducer and activator of transcription 3 (Stat3)–deficient keratinocytes resulted in a high incidence of apoptosis compared with controls. Conversely, forced expression of Stat3 desensitized keratinocytes to UVB-induced apoptosis. Upon UVB exposure, keratinocyte Stat3 was rapidly dephosphorylated, followed by decreases of both Stat3 mRNA and protein levels in a p53-independent manner. Vanadate treatment reversed the UVB-induced down-regulation of Stat3 and generation of apoptotic keratinocytes, suggesting the involvement of a tyrosine phosphatase. Furthermore, Stat3 was required for UVB-induced proliferation of follicular keratinocytes, leading to epidermal thickening. Finally, constitutive activation of Stat3 was observed in UVB-induced squamous cell carcinomas of either mice or human origin. These data suggest that Stat3 is required for survival and proliferation of keratinocytes following UVB exposure and that Stat3 is tightly regulated as part of a novel protective mechanism against UVB-induced skin cancer.
Stromal Cell–Derived Factor-1α and CXCR4 Expression in Hemangioblastoma and Clear Cell-Renal Cell Carcinoma: von Hippel-Lindau Loss-of-Function Induces Expression of a Ligand and Its Receptor
Authors: Zagzag David , Krishnamachary Balaji , Yee Herman , Okuyama Hiroaki , Chiriboga Luis , Ali M. Aktar , Melamed Jonathan , Semenza Gregg L.
Published: September 2005

The genetic hallmark of hemangioblastomas and clear cell-renal cell carcinomas (CC-RCCs) is loss-of-function of the von Hippel-Lindau (VHL) tumor suppressor protein. VHL is required for oxygen-dependent degradation of hypoxia-inducible factor-1α (HIF-1α). In hemangioblastomas and CC-RCCs, HIF-1α is constitutively overexpressed leading to increased transcription of HIF-1–regulated genes, including vascular endothelial growth factor (VEGF). Because loss of VHL function is associated with increased expression of the chemokine receptor CXCR4 in CC-RCCs, we investigated the expression of HIF-1α, CXCR4, and its ligand stromal cell–derived factor-1α (SDF-1α) in hemangioblastomas and CC-RCCs. Immunohistochemistry revealed overexpression of both CXCR4 and SDF-1α within tumor cells and endothelial cells of hemangioblastomas and CC-RCCs. HIF-1α was detected in tumor cell nuclei of both hemangioblastomas and CC-RCCs. A specific ELISA showed that hemangioblastomas and CC-RCCs expressed SDF-1α protein at levels that were significantly higher than those found in normal tissue. Analysis of the VHL-null RCC line 786-0 revealed that SDF-1α mRNA levels were 100-fold higher than in a subclone transfected with the wild-type VHL gene. Expression of CXCR4 and SDF-1α mRNA was significantly decreased in HIF-1α-null compared with wild-type mouse embryo fibroblasts (MEFs). ELISA and Western blot studies for SDF-1α and CXCR4 protein expression confirmed the RNA findings in RCC lines and MEFs. These results suggest that loss-of-function of a single tumor suppressor gene can up-regulate the expression of both a ligand and its receptor, which may establish an autocrine signaling pathway with important roles in the pathogenesis of hemangioblastoma and CC-RCC.
Selective Killing of Adriamycin-Resistant (G2 Checkpoint-Deficient and MRP1-Expressing) Cancer Cells by Docetaxel
Authors: Demidenko Zoya N. , Halicka Dorota , Kunicki Jan , McCubrey James A. , Darzynkiewicz Zbigniew , Blagosklonny Mikhail V.
Published: September 2005

Chemotherapy of cancer is limited by toxicity to normal cells. Drug resistance further limits the therapy. Here, we investigated selective killing of drug-resistant cancer cells by antagonistic drug combinations, which can spare (because of drug antagonism) normal cells. We used paired cell lines that are resistant to Adriamycin due to either expression of MRP1 or lack of G2 checkpoints. The goal was to selectively kill Adriamycin-resistant cancer cells with Docetaxel (Taxotere), while protecting parental (Adriamycin-sensitive) cells, using cytostatic concentrations of Adriamycin. Taxotere kills cells in mitosis. Therefore, by arresting parental cells in G2, 20 to 40 ng/mL of Adriamycin prevented cell death caused by Taxotere. Also, Adriamycin prevented the effects of Taxotere in normal human lymphocytes. In contrast, Taxotere selectively killed MRP1-expressing leukemia cells, which did not undergo G2 arrest in the presence of Adriamycin. Also, in the presence of Adriamycin, HCT116-p21−/− cancer cells with a defective G2 checkpoint entered mitosis and were selectively killed by Taxotere. Finally, 20 ng/mL of Adriamycin protected normal FDC-P1 hematopoietic cells from Taxotere. Whereas parental cells were protected by Adriamycin, the mitogen-activated protein/extracellular signal-regulated kinase inhibitor PD90598 potentiated the cytotoxic effect of Taxotere selectively in Raf-1–transformed FDC-P1 leukemia cells. We propose a therapeutic strategy to prevent normal cells from entering mitosis while increasing apoptosis selectively in mitotic cancer cells.
The PKCα-D294G Mutant Found in Pituitary and Thyroid Tumors Fails to Transduce Extracellular Signals
Authors: Zhu Yimin , Dong Qihan , Tan Bee Jen , Lim Wee Guan , Zhou Shufeng , Duan Wei
Published: September 2005

Protein kinase C (PKC) is a key regulator of cell proliferation, differentiation, and apoptosis and is one of the drug targets of anticancer therapy. Recently, a single point mutation (D294G) in PKCα has been found in pituitary and thyroid tumors with more invasive phenotype. Although the PKCα-D294G mutant is implicated in the progression of endocrine tumors, no apparent biochemical/cell biological abnormalities underlying tumorigenesis with this mutant have been found. We report here that the PKCα-D294G mutant is unable to bind to cellular membranes tightly despite the fact that it translocates to the membrane as efficiently as the wild-type PKCα upon treatment of phorbol ester. The impaired membrane binding is associated with this mutant's inability to transduce several antitumorigenic signals as it fails to mediate phorbol ester–stimulated translocation of myristoylated alanine–rich protein kinase C substrate (MARCKS), to activate mitogen-activated protein kinase and to augment melatonin-stimulated neurite outgrowth. Thus, the PKCα-D294G is a loss-of-function mutation. We propose that the wild-type PKCα may play important antitumorigenic roles in the progression of endocrine tumors. Therefore, developing selective activators instead of inhibitors of PKCα might provide effective pharmacological interventions for the treatment of certain endocrine tumors.