bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Cyclooxygenase-1 and -2 Expression by Targeting the Endothelin A Receptor in Human Ovarian Carcinoma Cells
Authors: Spinella Francesca , Rosanò Laura , Di Castro Valeriana , Nicotra Maria Rita , Natali Pier Giorgio , Bagnato Anna
Published: September 2005

Purpose and Experimental Design: New therapies against cancer are based on targeting cyclooxygenase (COX)-2. Activation of the endothelin A receptor (ETAR) by endothelin (ET)-1 is biologically relevant in several malignancies, including ovarian carcinoma. In this tumor, the ET-1/ETAR autocrine pathway promotes mitogenesis, apoptosis protection, invasion, and neoangiogenesis. Because COX-1 and COX-2 are involved in ovarian carcinoma progression, we investigated whether ET-1 induced COX-1 and COX-2 expression through the ETAR at the mRNA and protein level in HEY and OVCA 433 ovarian carcinoma cell lines by Northern blot, reverse transcription-PCR, Western blot, and immunohistochemistry; we also investigated the activity of the COX-2 promoter by luciferase assay and the release of prostaglandin (PG) E2 by ELISA.

Results: ET-1 significantly increases the expression of COX-1 and COX-2, COX-2 promoter activity, and PGE2 production. These effects depend on ETAR activation and involve multiple mitogen-activated protein kinase (MAPK) signaling pathways, including p42/44 MAPK, p38 MAPK, and transactivation of the epidermal growth factor receptor. COX-2 inhibitors and, in part, COX-1 inhibitor blocked ET-1-induced PGE2 and vascular endothelial growth factor release, indicating that both enzymes participate in PGE2 production to a different extent. Moreover, inhibition of human ovarian tumor growth in nude mice after treatment with the potent ETAR-selective antagonist ABT-627 is associated with reduced COX-2 and vascular endothelial growth factor expression.

Conclusions: These results indicate that impairing COX-1 and COX-2 and their downstream effect by targeting ETAR can be therapeutically advantageous in ovarian carcinoma treatment. Pharmacological blockade of the ETAR is an attractive strategy to control COX-2 induction, which has been associated with ovarian carcinoma progression and chemoresistance.
Heteroclitic CD33 Peptide With Enhanced Anti-Acute Myeloid Leukemic Immunogenicity
Authors: Bae Jooeun , Martinson Jeffrey A. , Klingemann Hans G.
Published: September 2005

The goal of these studies was to engineer a synthetic CD33 peptide with enhanced immunogenicity for the induction of acute myeloid leukemia (AML)-specific CTLs. Eight modified CD33 peptides YLISGDSPV,YIGSGDSPV,YIIIGDSPV,YIILGDSPV,YIISGISPV,YIISGDLPV,YIISGDSWV andYIISGDSPL were designed for increased HLA-A2.1 or T cell receptor affinity and compared with the native CD3365–73 peptide, AIISGDSPV, for enhanced immunogenicity. The YLISGDSPV peptide was found to be the most immunogenic epitope producing highly cytolytic CTLs against AML target cells. The CTLs generated withYLISGDSPV peptide showed CD33 peptide-specificity through targeting of both native (AIISGDSPV) and modified (YLISGDSPV) peptide presenting EBV-BLCL. The CTL cultures displayed a distinct phenotype consisting of a high percentage of activated memory (CD69+/CD45RO+)-CD8+and a low percentage of naïve (CD45RA+/CCR7+)-CD8+cells. In addition, T-cell clones specific to theYLISGDSPV peptide were isolated and characterized to target AML cells. The clones exhibited both HLA-A2.1-restricted and AML cell-specific cytotoxicity that was mediated through a granule-dependent pathway. More importantly, the CTL clones did not lyse or inhibit the proliferation of normal CD34+ progenitor cells. In conclusion, we report on the identification of a highly immunogenic heterocliticYLISGDSPV CD33 epitope that is a promising candidate for immunotherapy targeting AML.
Two Proliferation-Related Proteins, TYMS and PGK1, Could Be New Cytotoxic T Lymphocyte-Directed Tumor-Associated Antigens of HLA-A2+ Colon Cancer
Authors: Shichijo Shigeki , Azuma Kouichi , Komatsu Nobukazu , Ito Masaaki , Maeda Yoshiaki , Ishihara Yuki , Itoh Kyogo
Published: September 2005

Purpose: The purpose of this work was to provide a scientific basis for specific immunotherapy of colon cancer.

Experimental Design: This study focused on identification of colon tumor-associated antigens and HLA-A2–restricted and tumor-reactive cytotoxic T lymphocytes (CTLs) generated from tumor-infiltrating lymphocytes of a colon cancer patient. A gene expression cloning method was used to identify genes coding for tumor antigens. Fifty-six peptides with HLA-A2–binding motifs encoded by these proteins were examined for their ability to induce HLA-A2–restricted and tumor-reactive CTLs.

Results: We identified the following three genes coding for proliferation-related proteins: thymidylate synthase (TYMS), which is involved in chemoresistance (5-fluorouracil); 5′-aminoimidazole-4-carboxamide-1-β-d-ribonucleotide transfolmylase/inosinicase (AICRT/I); and phosphoglycerate kinase 1 (PKG1), which was secreted by tumor cells and involved in the angiogenic process. TYMS was preferentially expressed in tumor cells, whereas AICRT/I and PKG1 were equally expressed in both cancer cells and normal tissues at the mRNA level. Among 56 peptides with HLA-A2–binding motifs encoded by these proteins, 8 peptides were recognized by the CTLs, and 5 of 8 peptides were also recognized by the CTL precursors without ex vivo activation in the peripheral blood of colon cancer patients. Furthermore, four of them (one each from TYMS and PKG1 and two from AICRT/1) possessed the ability to induce HLA-A2–restricted and peptide-specific CTLs cytotoxic to colon tumor cells in peripheral blood mononuclear cells of colon cancer patients.

Conclusions: TYMS and PGK1, as well as their epitope peptides, might be appropriate target molecules for specific immunotherapy of HLA-A2+ colon cancer patients because of the positive role of TYMS and PGK1 in chemoresistance (5-fluorouracil) and angiogenesis of tumor cells, respectively.
Effective Photoimmunotherapy of Murine Colon Carcinoma Induced by the Combination of Photodynamic Therapy and Dendritic Cells
Authors: Jalili Ahmad , Makowski Marcin , Świtaj Tomasz , Nowis Dominika , Wilczyński Grzegorz M. , Wilczek Ewa , Chorąży-Massalska Magdalena , Radzikowska Anna , Maśliński Włodzimierz , Biały Łukasz , Sieńko Jacek , Sieroń Aleksander , Adamek Mariusz , Basak Grzegorz , Mróz Paweł , Krasnodębski Ireneusz W. , Jakóbisiak Marek , Gołąb Jakub
Published: September 2005

Purpose: The unique mechanism of tumor destruction by photodynamic therapy (PDT), resulting from apoptotic and necrotic killing of tumor cells accompanied by local inflammatory reaction and induction of heat shock proteins (HSPs), prompted us to investigate the antitumor effectiveness of the combination of PDT with administration of immature dendritic cells (DCs).

Experimental Design: Confocal microscopy and Western blotting were used to investigate the influence of PDT on the induction of apoptosis and expression of HSP expression in C-26 cells. Confocal microscopy and flow cytometry studies were used to examine phagocytosis of PDT-treated C-26 cells by DCs. Secretion of interleukin (IL)-12 was measured with ELISA. Cytotoxic activity of lymph node cells was evaluated in a standard 51Cr-release assay. The antitumor effectiveness of PDT in combination with administration of DCs was investigated in in vivo model.

Results: PDT treatment resulted in the induction of apoptotic and necrotic cell death and expression of HSP27, HSP60, HSP72/73, HSP90, HO-1, and GRP78 in C-26 cells. Immature DCs cocultured with PDT-treated C-26 cells efficiently engulfed killed tumor cells, acquired functional features of maturation, and produced substantial amounts of IL-12. Inoculation of immature DCs into the PDT-treated tumors resulted in effective homing to regional and peripheral lymph nodes and stimulation of cytotoxic activity of T and natural killer cells. The combination treatment with PDT and administration of DCs produced effective antitumor response.

Conclusions: The feasibility and antitumor effectiveness demonstrated in these studies suggest that treatment protocols involving the administration of immature DCs in combination with PDT may have clinical potential.
Invasion and Metastasis of Oral Cancer Cells Require Methylation of E-Cadherin and/or Degradation of Membranous β-Catenin
Authors: Kudo Yasusei , Kitajima Shojiro , Ogawa Ikuko , Hiraoka Masae , Sargolzaei Soodabeh , Keikhaee Mohammad Reza , Sato Sunao , Miyauchi Mutsumi , Takata Takashi
Published: September 2005

The extent of lymph node metastasis is a major determinant in the prognosis of oral squamous cell carcinoma (OSCC). Abnormalities of cell adhesion molecules are known to play an important role in invasion and metastasis of cancer cells through the loss of cell-to-cell adhesion. In this study, we isolated highly invasive clones from an OSCC cell line established from a lymph node metastasis by using an in vitro invasion assay method and compared the abnormalities of cell adhesion molecule E-cadherin and β-catenin in these cells. The isolated, highly invasive clones showed significant invasive capacity and reduction of E-cadherin and membranous β-catenin protein in comparison with parent cells. We found that reduced expression of E-cadherin was due to methylation of its promoter region. In fact, most invasive and metastatic area of OSCCs showed reduced expression and methylation of E-cadherin. Moreover, we found that reduced expression of membranous β-catenin was due to its protein degradation. Reduced expression of membranous β-catenin was also found frequently in invasive and metastatic areas of OSCCs. In summary, invasion and metastasis of OSCC cells require methylation of E-cadherin and/or degradation of membranous β-catenin. In addition, we suggest that the method of isolation of highly invasive clones may be useful for studies aimed at discovering novel genes involved in invasion and metastasis.
Effects of Recombinant Erythropoietin in Palliative Treatment of Unselected Cancer Patients
Authors: Lindholm Elisabet , Daneryd Peter , Körner Ulla , Hyltander Anders , Fouladiun Marita , Lundholm Kent
Published: September 2005

Purpose: The purpose is to evaluate relationships between objectively assessed exercise capacity and subjectively assessed scoring of physical functioning and well-being after erythropoietin treatment in cancer patients on palliative care.

Experimental Design: Unselected cancer patients (n = 108) who experienced progressive cachexia were randomized to receive either anti-inflammatory treatment alone (indomethacin) or recombinant erythropoietin plus indomethacin to prevent the appearance of disease-induced anemia and thereby protect patients’ exercise capacity. Follow-up investigations of nutritional status, exercise capacity, and health-related quality of life assessed by SF-36 and the European Organization for Research and Treatment of Cancer QLQ-C30 were compared.

Results: Effective treatment by erythropoietin on top of basal whole body anti-inflammatory treatment was confirmed and indicated by time course changes of biochemical, physiologic, and nutritional objectives, whereas individual self-reported scoring of physical functioning and general health did not indicate a clear-cut effectiveness, particularly at moderately subnormal hemoglobin levels.

Conclusions: Discrepancies between objective and subjective self-reported measures may be either fundamental or indicate scoring limitations for evaluation of therapeutic results. Present results demonstrate a clinical benefit of erythropoietin treatment in cancer patients with subnormal to normal hemoglobin levels, whereas the patients’ own subjective scoring was insufficient to sense such improvements. The discrepancy may be either fundamental or methodological but emphasizes the importance to document therapeutic outcome in both subjective and objective perspectives in palliative care of cancer patients.
Favorable Neuroblastoma Genes and Molecular Therapeutics of Neuroblastoma
Authors: Tang Xao X. , Robinson Marjorie E. , Riceberg Justin S. , Kim David Y. , Kung Bing , Titus Tracy B. , Hayashi Satoshi , Flake Alan W. , Carpentieri David , Ikegaki Naohiko
Published: September 2005

Purpose and Experimental Design: Neuroblastoma (NB) is a common pediatric solid tumor that exhibits a striking clinical bipolarity: favorable and unfavorable. Favorable NB genes (EPHB6, EFNB2, EFNB3, NTRK1, and CD44) are genes whose high-level expression predicts favorable NB outcome, and forced expression of these genes inhibits growth of unfavorable NB cells. In this study, we investigated whether favorable NB gene expression could be augmented in unfavorable NB cells by chemical compounds and whether an increased expression of these genes was associated with suppression of NB growth and metastasis.

Results: We found that inhibitors of DNA methylation [5-aza-2′-deoxycytidine (5AdC)], histone deacetylase (HDAC) [4-phenylbutyrate (4PB)], and proteasome (MG262) enhanced the expression of favorable NB genes in NB cell lines and inhibited the growth of these cells in vitro (P < 0.0005). The growth-inhibitory effects of 5AdC and 4PB in vitro were in part due to caspase-dependent cell death and inhibition of DNA synthesis. Administration of 5AdC and/or 4PB also suppressed growth of subcutaneous NB xenografts in nude mice (P < 0.001), which was accompanied by enhanced favorable NB gene expression and an increase in apoptosis. Moreover, 4PB suppressed bone marrow and liver metastases of NB cells in severe combined immunodeficient/Beige mice (P = 0.007 and P = 0.008, respectively). The growth-suppressive activity of HDAC inhibitors on NB was further confirmed by the efficacy of trichostatin A, a potent and specific HDAC inhibitor.

Conclusions: Collectively, these observations further emphasize the link between the elevated favorable NB gene expression and a benign phenotype of NB.
Thalidomide Pharmacokinetics and Metabolite Formation in Mice, Rabbits, and Multiple Myeloma Patients
Authors: Chung Francisco , Lu Jun , Palmer Brian D. , Kestell Philip , Browett Peter , Baguley Bruce C. , Tingle Malcolm , Ching Lai-Ming
Published: September 2005

Purpose: Thalidomide has a variety of biological effects that vary considerably according to the species tested. We sought to establish whether differences in pharmacokinetics could form a basis for the species-specific effects of thalidomide.

Experimental Design: Mice and rabbits were administered thalidomide (2 mg/kg) p.o. or i.v., and plasma concentrations of thalidomide were measured after drug administration using high performance liquid chromotography. Plasma samples from five multiple myeloma patients over 24 hours after their first dose of thalidomide (200 mg) were similarly analyzed and all data were fitted to a one-compartment model. Metabolites of thalidomide in plasma were identified simultaneously using liquid chromatography-mass spectrometry.

Results: Plasma concentration-time profiles for the individual patients were very similar to each other, but widely different pharmacokinetic properties were found between patients compared with those in mice or rabbits. Area under the concentration curve values for mice, rabbits, and multiple myeloma patients were 4, 8, and 81 μmol/L · hour, respectively, and corresponding elimination half-lives were 0.5, 2.2, and 7.3 hours, respectively. Large differences were also observed between the metabolite profiles from the three species. Hydrolysis products were detected for all species, and the proportion of hydroxylated metabolites was higher in mice than in rabbits and undetectable in patients.

Conclusions: Our results show major interspecies differences in the pharmacokinetics of thalidomide that are related to the altered degree of metabolism. We suggest that the interspecies differences in biological effects of thalidomide may be attributable, at least in part, to the differences in its metabolism and hence pharmacokinetics.
The Prognostic Value of BCAR1 in Patients with Primary Breast Cancer
Authors: Dorssers Lambert C. J. , Grebenchtchikov Nicolai , Brinkman Arend , Look Maxime P. , van Broekhoven Simone P. J. , de Jong Danielle , Peters Harry A. , Portengen Henk , Meijer-van Gelder Marion E. , Klijn Jan G. M. , van Tienoven Doorlene T. H. , Geurts-Moespot Anneke , Span Paul N. , Foekens John A. , Sweep Fred C. G. J.
Published: September 2005

Purpose: BCAR1, the human homologue of the rat p130Cas protein, was identified in a functional screen for human breast cancer cell proliferation resistant to antiestrogen drugs. Here, we study the prognostic value of quantitative BCAR1 levels in a large series of breast cancer specimens.

Experimental Design: A specific ELISA was developed to measure BCAR1 protein levels in 2593 primary breast tumor cytosols. Tumor levels of BCAR1 were correlated with relapse-free survival (RFS) and overall survival (OS) and compared with collected data on urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1).

Results: In tumor cytosols, BCAR1 protein levels varied between 0.02 and 23 ng/mg protein. BCAR1 levels exhibited a positive correlation with steroid hormone receptor levels, age and menopausal status, and uPA and PAI-1 levels. The level of BCAR1 (continuous or categorized as low, intermediate, or high) was inversely related with RFS and OS time. Multivariate analysis showed that BCAR1 levels contributed independently to a base model containing the traditional prognostic factors for both RFS and OS (both P < 0.0001). When added together with uPA and PAI-1 in the multivariate model, BCAR1 contributed independently of PAI-1 and was favored over uPA. Interaction tests allowed for additional analyses of BCAR1 protein levels in clinically relevant subgroups stratified by nodal and menopausal status.

Conclusions: The quantitative BCAR1 protein level represents a prognostic factor for RFS and OS in primary breast cancer, independent of the traditional prognostic factors and the other novel marker PAI-1.
Induction of Thyroid Cancer Cell Apoptosis by a Novel Nuclear Factor κB Inhibitor, Dehydroxymethylepoxyquinomicin
Authors: Starenki Dmitriy V. , Namba Hiroyuki , Saenko Vladimir A. , Ohtsuru Akira , Maeda Shigeto , Umezawa Kazuo , Yamashita Shunichi
Published: September 2005

Purpose: The objective of the study was to determine the effects of a novel selective nuclear factor κB (NF-κB) inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), in thyroid carcinoma cells in vitro and in vivo and to additionally elucidate the molecular mechanisms underlying the action of this chemotherapeutic agent.

Experimental Design: In the in vitro experiments, the induction of apoptosis by DHMEQ in various human thyroid carcinoma cell types was determined by flow cytometry analysis of annexin-V binding and the caspase activation by Western blotting. For the in vivo study, female nu/nu mice were xenografted with s.c. FRO thyroid tumors. DHMEQ solution was injected i.p. at a dose of 8 mg/kg/day for two weeks. Tumor dimensions were monitored twice weekly, and apoptosis in tumor specimens was determined by terminal deoxynucleotidyl transferase-mediated nick end labeling staining.

Results: Treatment with DHMEQ substantially inhibited the translocation of p65 and p50 NF-κB subunits to the nucleus, the DNA-binding activity of the RelA/p65, NF-κB-dependent expression of the inhibitor of apoptosis (IAP)-family proteins, cIAP-1, cIAP-2, and XIAP, and the de novo synthesis of inhibitor of nuclear factor κB α. At concentration levels ranging from 0.1 to 5 μg/ml, DHMEQ induced a caspase-mediated apoptotic response that could be abrogated by the c-Jun NH2-terminal kinase inhibitor SP600125 but not by either mitogen-activated protein/extracellular signal-regulated kinase kinase or p38 inhibitors. In contrast, normal human thyrocytes were resistant to DHMEQ-induced apoptosis. At higher doses of DHMEQ we observed the necrotic-like killing of both normal and malignant thyrocytes, which was resistant to mitogen-activated protein kinase inhibitors. In nude mice DHMEQ substantially inhibited tumor growth without observable side effects, and increased numbers of apoptotic cells were observed in the histologic sections of tumors treated with DHMEQ.

Conclusions: Our results show the potential usefulness of the novel NF-κB inhibitor, DHMEQ, in future therapeutic strategies for the treatment of thyroid cancers that do not respond to conventional approaches.
Combination Immunotherapy of B-Cell Non-Hodgkin’s Lymphoma with Rituximab and Interleukin-2
Authors: Eisenbeis Charles F. , Grainger Andrew , Fischer Beth , Baiocchi Robert A. , Carrodeguas Lester , Roychowdhury Sameek , Chen Lei , Banks Amy L. , Davis Thomas , Young Donn , Kelbick Nicole , Stephens Julie , Byrd John C. , Grever Michael R. , Caligiuri Michael A. , Porcu Pierluigi
Published: September 2005

Purpose: Cytokine-induced modulation of innate immunity is being explored to enhance the activity of monoclonal antibodies. Severe combined immunodeficient (SCID) mice engrafted with peripheral blood leukocytes (PBLs) from Epstein Barr virus-seropositive donors develop human B-cell non-Hodgkin’s lymphomas [B-NHLs (hu-PBL-SCID mouse model)]. We used this hu-PBL-SCID mouse model to study the synergism between interleukin (IL)-2 and rituximab. We also conducted a phase I trial of IL-2 and rituximab in relapsed B-NHL to study whether expansion of natural killer (NK) cells and enhanced cellular cytotoxicity could be safely accomplished in vivo.

Experimental Design: Hu-PBL-SCID mice were treated with various schedules of rituximab and IL-2, with survival as the end point. Patients with relapsed B-NHL received rituximab (375 mg/m2 weekly × 4) followed by daily low-dose IL-2 (1 MIU/m2/day × 4 weeks) with pulses of intermediate-dose IL-2 (3–15 MIU/m2). Toxicity, NK cell numbers, and cellular cytotoxicity were measured.

Results: In the hu-PBL-SCID mouse, the combination of rituximab and IL-2 showed greater activity against B-NHL than either agent alone. Treatment was most effective when IL-2 was given before rituximab. Twelve patients with heavily pretreated B-NHL entered the phase I trial. Toxicity was manageable, and responses were observed. NK cell expansion and enhanced cellular cytotoxicity against a B-cell lymphoma target were observed but did not correlate with response.

Conclusions: The combination of IL-2 and rituximab is synergistic against B-NHL in the hu-PBL-SCID model. In the phase I trial, a sequential combination of rituximab and IL-2 was well tolerated and achieved biological end points. Responses were observed.
Influence of Casein Kinase II in Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis in Human Rhabdomyosarcoma Cells
Authors: Izeradjene Kamel , Douglas Leslie , Delaney Addison , Houghton Janet A.
Published: September 2005

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis via the death receptors DR4 and DR5 in transformed cells in vitro and exhibits potent antitumor activity in vivo with minor side effects. Protein kinase casein kinase II (CK2) is increased in response to diverse growth stimuli and is aberrantly elevated in a variety of human cancers. Rhabdomyosarcoma tumors are the most common soft-tissue sarcoma in childhood. In this investigation, we demonstrate that CK2 is a key survival factor that protects tumor cells from TRAIL-induced apoptosis. We have demonstrated that inhibition of CK2 phosphorylation events by 5,6-dichlorobenzimidazole (DRB) resulted in dramatic sensitization of tumor cells to TRAIL-induced apoptosis. CK2 inhibition also induced rapid cleavage of caspase-8, -9, and -3, as well as the caspase substrate poly(ADP-ribose) polymerase after TRAIL treatment. Overexpression of Bcl-2 protected cells from TRAIL-induced apoptosis in the presence of the CK2 inhibitor. Death signaling by TRAIL in these cells was Fas-associated death domain and caspase dependent because dominant negative Fas-associated death domain or the cowpox interleukin 1β-converting enzyme inhibitor protein cytokine response modifier A prevented apoptosis in the presence of DRB. Analysis of death-inducing signaling complex (DISC) formation demonstrated that inhibition of CK2 by DRB increased the level of recruitment of procaspase-8 to the DISC and enhanced caspase-8-mediated cleavage of Bid, thereby increasing the release of the proapoptotic factors cytochrome c, HtrA2/Omi, Smac/DIABLO, and apoptosis inducing factor (AIF) from the mitochondria, with subsequent degradation of X-linked inhibitor of apoptosis protein (XIAP). To further interfere with CK2 function, JR1 and Rh30 cells were transfected with either short hairpin RNA targeted to CK2α or kinase-inactive CK2α (K68M) or CK2α′ (K69M). Data show that the CK2 kinase activity was abrogated and that TRAIL sensitivity in both cell lines was increased. Silencing of CK2α expression with short hairpin RNA was also associated with degradation of XIAP. These findings suggest that CK2 regulates TRAIL signaling in rhabdomyosarcoma by modulating TRAIL-induced DISC formation and XIAP expression.
Modulation of DNA RepairIn vitroafter Treatment with Chemotherapeutic Agents by the Epidermal Growth Factor Receptor Inhibitor Gefitinib (ZD1839)
Authors: Friedmann Benjamin , Caplin Martyn , Hartley John A. , Hochhauser Daniel
Published: September 2005

Purpose: The epidermal growth factor receptor (EGFR) is commonly expressed in human tumors and provides a target for therapy. Gefitinib (Iressa, ZD1839) is a quinazoline derivative that inhibits EGFR tyrosine kinase activity. Gefitinib demonstrated anticancer efficacy in vivo, and although experiments in vitro have suggested that inhibition of EGFR modulates the activity of chemotherapeutic agents, the mechanism of this interaction is unclear. We investigated mechanisms for this modulation.

Experimental Design: The antiproliferative effect of gefitinib alone or combined with cisplatin, melphalan, and etoposide was determined in a human breast (MCF-7) cancer cell line. Using the alkaline single-cell gel electrophoresis (comet) assay, we investigated kinetics of DNA damage and repair after treatment with the chemotherapeutic drugs combined with gefitinib. To investigate whether the phosphatidylinositol 3′-kinase pathway was contributing to repair-inhibition produced by gefitinib, cells were exposed to chemotherapy in combination with the phosphatidylinositol 3′-kinase inhibitor LY294002.

Results: A superadditive (synergistic) increase in growth inhibition for combined treatment with gefitinib was found for cisplatin and etoposide, but not with melphalan. There was delayed repair of DNA strand breaks after treatment with etoposide combined with gefitinib, and repair of DNA interstrand cross-links produced by cisplatin is delayed in combination with gefitinib. Inhibition of cell proliferation and DNA repair was identical in cells treated with LY294002. Immunoprecipitation of cell extracts demonstrated that after exposure to gefitinib, there was an association between EGFR and DNA-PKCS.

Conclusion: Gefitinib acts through inhibition of repair of cisplatin and etoposide-induced DNA damage; this effect is mimicked by inhibitors of the phosphatidylinositol 3′-kinase suggesting similar mechanisms of action.
Lung Cancers Detected by Screening with Spiral Computed Tomography Have a Malignant Phenotype when Analyzed by cDNA Microarray
Authors: Bianchi Fabrizio , Hu Jiangting , Pelosi Giuseppe , Cirincione Rosalia , Ferguson Mary , Ratcliffe Cathy , Di Fiore Pier Paolo , Gatter Kevin , Pezzella Francesco , Pastorino Ugo
Published: September 2005

Purpose: Spiral computed tomography (CT) can detect lung cancer at an early stage, but the malignant potential is unknown. The question is, as follows: do these small lesions have the same lethal potential as do symptomatic tumors?

Experimental Design: We used a cDNA microarray platform and compared the gene expression profile of spiral CT-detected lung carcinomas with a matched case-control population of patients presenting with symptomatic lung cancer.

Results: CT-detected and symptomatic tumors have shown a comparable gene expression profile. Correspondence analysis has demonstrated that nine genes were differentially expressed, although with a high variability across the samples that prevented distinguishing the two groups of tumors. Analysis of these nine genes has suggested that early-detected tumors have higher levels of retinoic acid production and higher expression levels of caveolin 2, matrix Gla, and cystatin A, which are already known to be lost during tumor progression.

Conclusions: All of the tumors observed are histologically malignant according to the WHO Classification. Early lung cancers that are detected by screening have a gene expression pattern similar to, but not identical to, that of symptomatic lung carcinomas.
Protein Kinase C θ Is Highly Expressed in Gastrointestinal Stromal Tumors But Not in Other Mesenchymal Neoplasias
Authors: Blay Pilar , Astudillo Aurora , Buesa José M. , Campo Elías , Abad Mar , García-García Juan , Miquel Rosa , Marco Vicente , Sierra Marta , Losa Raquel , Lacave Angel , Braña Alejandro , Balbín Milagros , Freije José M. P.
Published: September 2005

Purpose: Gastrointestinal stromal tumors (GIST) are a distinctive group of mesenchymal neoplasms of the gastrointestinal tract. The oncogene KIT has a central role in the pathogenesis of GIST, with c-kit receptor tyrosine kinase (KIT) protein expression being the gold standard in its diagnosis. The identification of GIST patients has become crucial, because the tyrosine kinase inhibitor Imatinib is effective in the treatment of this malignancy. However, a small set of GISTs remain unrecognized, because KIT protein expression is not always evident. The aim of this study was the identification of new markers for the differential diagnosis of GIST.

Experimental Design: By analyzing publicly available data from transcriptional profiling of sarcomas, we found that protein kinase C θ (PKC-θ), a novel PKC isotype involved in T-cell activation, is highly and specifically expressed in GIST. PKC-θ expression in GIST was confirmed by reverse transcription-PCR and Western blot. PKC-θ was analyzed by immunohistochemistry in a panel of 26 GIST, 12 non-GIST soft-tissue sarcomas, and 35 tumors from other histologies.

Results: We found that all of the GISTs expressed PKC-θ, whereas this protein was undetectable in other mesenchymal or epithelial tumors, including non-GIST KIT-positive tumors. PKC-θ immunoreactivity was also observed in interstitial cells of Cajal.

Conclusions: Our results show that PKC-θ is easily detected by immunohistochemistry in GIST specimens and that it could be a sensitive and specific marker for the diagnosis of this malignancy.
Value of
p16
INK4a and
RASSF1A
Promoter Hypermethylation in Prognosis of Patients with Resectable Non–Small Cell Lung Cancer
Authors: Wang Jie , Lee J. Jack , Wang Luo , Liu Diane D. , Lu Charles , Fan You-Hong , Hong Waun Ki , Mao Li
Published: September 2005

The p16INK4a and RASSF1A are tumor suppressor genes frequently inactivated by de novo promoter hypermethylation in non-small cell lung cancer. We studied 119 patients with non-small cell lung cancer (70 stage I/II and 49 stage IIIA) who had undergone surgery with curative intent. The p16INK4a and RASSF1A promoter methylation statuses were determined by methylation-specific PCR. Statistical analyses, all two-sided, were performed to determine the prognostic effect of hypermethylation on various clinical parameters. Hypermethylation of the p16INK4a and RASSF1A promoters was found in 58 (49%) and 46 (39%) tumors, respectively, and 30 tumors (25%) exhibited hypermethylation of both gene promoters. In patients with stage I/II tumors, only p16INK4a promoter hypermethylation was associated with a poor 5-year overall survival rate (P = 0.002). In patients with stage IIIA disease, however, RASSF1A promoter hypermethylation was a stronger predictor of a poor 5-year overall survival rate (P < 0.0001) than p16INK4a promoter hypermethylation. Among the 49 patients with stage IIIA tumors, 16 (89%) of the 18 patients whose tumors showed RASSF1A promoter hypermethylation died within 3 years after surgery, as compared with only 12 (39%) of the 31 patients whose tumors had no RASSF1A promoter hypermethylation (P < 0.0001). Multivariate analysis indicated that RASSF1A promoter hypermethylation was the stronger independent predictor for survival in patients with locally advanced non-small cell lung cancer. Our results indicate that p16INK4a promoter hypermethylation predicts a poor 5-year survival rates for patients with resectable non-small cell lung cancer, particularly for those with early stage tumors, whereas RASSF1A promoter hypermethylation is a profound prognostic predictor for patients with locally advanced non-small cell lung cancer, suggesting an important role of RASSF1A in non-small cell lung cancer progression.
A Randomized Phase IIb Trial of Pulmicort Turbuhaler (Budesonide) in People with Dysplasia of the Bronchial Epithelium
Authors: Lam Stephen , leRiche Jean C. , McWilliams Annette , MacAulay Calum , Dyachkova Yulia , Szabo Eva , Mayo John , Schellenberg Robert , Coldman Andy , Hawk Ernest , Gazdar Adi
Published: September 2005

Purpose: Preclinical studies suggest that inhaled budesonide may be an effective chemopreventive agent for lung cancer. We conducted a phase IIb study to determine the effects of inhaled budesonide in smokers with bronchial dysplasia.

Experimental Design: A total of 112 smokers with more than or equal to one site of bronchial dysplasia > 1.2 mm in size identified by autofluorescence bronchoscopy-directed biopsy was randomly assigned to receive placebo or budesonide (Pulmicort Turbuhaler) 800 μg twice daily inhalation for 6 months. The primary end point was change in the histopathologic grade on repeat biopsy of the same sites at the end of 6 months.

Results: There were no significant differences in the regression or progression rates of bronchial dysplasia between the two groups. There was a statistically significant but modest decrease in p53 and BclII expression in the bronchial biopsies after 6 months of Pulmicort Turbuhaler versus placebo (P = 0.01 and P = 0.001, respectively). There was a small but statistically significant decrease in the proportion of computed tomography-detected lung nodules after Pulmicort Turbuhaler compared with placebo (P = 0.024).

Conclusions: Our results suggest that in smokers, inhaled budesonide in the dose of 1600 μg daily for 6 months had no effect in regression of bronchial dysplastic lesions or prevention of new lesions. Budesonide treatment resulted in a modest decrease in p53 and BclII protein expression in bronchial biopsies and a slightly higher rate of resolution of computed tomography-detected lung nodules. Whether budesonide truly has an effect in preneoplastic lesions in the peripheral airways and alveoli requires additional investigation.
Rat Sodium Iodide Symporter for Radioiodide Therapy of Cancer
Authors: Mitrofanova Elena , Unfer Robert , Vahanian Nick , Daniels Wayne , Roberson Erica , Seregina Tatiana , Seth Prem , Link Charles
Published: September 2005

Design and development of new approaches for targeted radiotherapy of cancer and improvement of therapeutic index by more local radiation therapy are very important issues. Adenovirus-mediated delivery of the sodium iodide symporter (NIS) gene to cancer cells is a powerful technique to concentrate lethal radiation in tumor cells and eradicate tumors with increased therapeutic index. A replication-defective adenoviral vector expressing the rat NIS gene (Ad-rNIS) was used for in vitro gene delivery and into human prostate cancer xenografts to study antitumor effect. Robust function of the rat symporter was detected in DU145, T47D, and HCT-15 human cancer cell lines transduced with Ad-rNIS. All three cancer cell lines successfully transferred functionally active rat symporter to the plasma membrane, resulting in very high levels of iodine-125 accumulation. Three-dimensional multicellular tumor spheroids derived from DU145 human prostate cancer cells were transduced with Ad-rNIS and incubated with 131I for 24 hours. After treatment, spheroids rapidly decreased in size and disappeared within 10 days. In vivo data revealed an inhibition of tumor growth in athymic nude mice after intratumoral Ad-rNIS injection followed by 131I administration. Eighty-eight percent of experimental mice survived >30 days, whereas control groups had only 18% survival >30 days. This is the first report that demonstrates the rat NIS gene can effectively induce growth arrest of human tumor xenografts after in vivo adenoviral gene delivery and 131I administration. The data confirm our hypothesis that the rat NIS gene is an attractive suicide gene candidate for cancer treatment.
A Phase II Study of Etanercept (Enbrel), a Tumor Necrosis Factor α Inhibitor in Patients with Metastatic Breast Cancer
Authors: Madhusudan Srinivasan , Foster Martin , Muthuramalingam Sethupathi R. , Braybrooke Jeremy P. , Wilner Susan , Kaur Kulwinder , Han Cheng , Hoare Susan , Balkwill Frances , Talbot Denis C. , Ganesan Trivadi S. , Harris Adrian L.
Published: September 2005

Purpose: Tumor necrosis factor (TNF) α is a key player in the tumor microenvironment and is involved in the pathogenesis of breast cancer. Etanercept is a recombinant human soluble p75 TNF receptor that binds to TNF-α and renders it biologically unavailable. In the current study, we sought to determine the toxicity, biological activity, and therapeutic efficacy of Etanercept in metastatic breast cancer.

Experimental Design: We initiated a Phase II, nonrandomized, open-labeled study in patients with progressive metastatic breast cancer refractory to conventional therapy (Phase I toxicity data were available in patients with rheumatoid arthritis). Etanercept was administered subcutaneously at a dose of 25 mg twice weekly until disease progression.

Results: Sixteen patients were recruited [median age 53 years (range, 34 to 74)]. A total of 141.6 weeks of therapy was administered (median of 8.1 weeks). Seven patients received ≥12 weeks of therapy. The most common side effects were injection site reactions (6), fatigue (5), loss of appetite (2), nausea (1), headache (1), and dizziness (1). Brief period of disease stabilization was seen in 1 patient lasting for 16.4 weeks. Immunoreactive TNF-α was elevated within 24 hours of therapy and persisted until the end of treatment (days 7, 28, 56, and 84). Phytohemagglutinin stimulates the production of interleukin-6 and CCL2 in peripheral blood cells, and the ability of Etanercept to modulate this response was assessed in a cytokine release assay. A consistent decrease in interleukin-6 and CCL2 level was seen compared with pretreatment values in serial blood samples (days 1, 7, 28, 56, and 84).

Conclusions: Our study shows the safety and biological activity of Etanercept in breast cancer and provides data to assess pharmacodynamic endpoints of different schedules of Etanercept and combinations with chemotherapy or other biological therapies.
Phase I Trial of Irinotecan, Infusional 5-Fluorouracil, and Leucovorin (FOLFIRI) with Erlotinib (OSI-774)
Authors: Messersmith Wells A. , Laheru Daniel A. , Senzer Neil N. , Donehower Ross C. , Grouleff Paula , Rogers Theresa , Kelley Sean K. , Ramies David A. , Lum Bert L. , Hidalgo Manuel
Published: September 2005

Purpose: This phase I study was conducted to establish the dose-limiting toxicities and maximum-tolerated dose of erlotinib, an oral epidermal growth factor receptor tyrosine kinase inhibitor, in combination with FOLFIRI, a standard regimen of irinotecan, leucovorin, and infusional 5-fluorouracil (5-FU) in patients with advanced colorectal cancer.

Experimental Design: The trial used a dose-escalation design beginning with 100 mg/day erlotinib continuously and dose-reduced FOLFIRI (150 mg/m2 i.v. day 1 irinotecan, 200 mg/m2 i.v. leucovorin, 320 mg/m2 i.v. bolus days 1 to 2 5-FU, and 480 mg/m2 i.v. 5-FU infusion over 22 hours, days 1 to 2) administered in 6-week cycles (three FOLFIRI treatments). Plasma sampling was performed for irinotecan, erlotinib, and 5-FU for pharmacokinetic analysis during cycle 1.

Results: The study was halted after six patients at the lowest dose level due to unexpectedly severe toxicities, including disfiguring grade 2 rash (three patients), grade 3 diarrhea (three patients), and grade ≥ 3 neutropenia (three patients). All patients required some dose interruption or reduction of either erlotinib or FOLFIRI, and only one patient completed two 6-week cycles of therapy. Five patients had stable disease after one cycle, and one patient had a partial response. No plasma pharmacokinetic interaction was observed that could explain the observed increased toxicity.

Conclusions: FOLFIRI combined with erlotinib causes excessive toxicity at reduced doses. These findings contrast with available data regarding the optimal safety profile of trials combining small molecule epidermal growth factor receptor inhibitors with other conventional chemotherapy and highlight the need to perform safety-oriented studies of such combinations.
Effects of Drug Loading on the Antitumor Activity of a Monoclonal Antibody Drug Conjugate
Authors: Hamblett Kevin J. , Senter Peter D. , Chace Dana F. , Sun Michael M. C. , Lenox Joel , Cerveny Charles G. , Kissler Kim M. , Bernhardt Starr X. , Kopcha Anastasia K. , Zabinski Roger F. , Meyer Damon L. , Francisco Joseph A.
Published: September 2005

Purpose: An antibody-drug conjugate consisting of monomethyl auristatin E (MMAE) conjugated to the anti-CD30 monoclonal antibody (mAb) cAC10, with eight drug moieties per mAb, was previously shown to have potent cytotoxic activity against CD30+ malignant cells. To determine the effect of drug loading on antibody-drug conjugate therapeutic potential, we assessed cAC10 antibody-drug conjugates containing different drug-mAb ratios in vitro and in vivo.

Experimental Design: Coupling MMAE to the cysteines that comprise the interchain disulfides of cAC10 created an antibody-drug conjugate population, which was purified using hydrophobic interaction chromatography to yield antibody-drug conjugates with two, four, and eight drugs per antibody (E2, E4, and E8, respectively). Antibody-drug conjugate potency was tested in vitro against CD30+ lines followed by in vivo xenograft models. The maximum-tolerated dose and pharmacokinetic profiles of the antibody-drug conjugates were investigated in mice.

Results: Although antibody-drug conjugate potency in vitro was directly dependent on drug loading (IC50 values E8<E4<E2), the in vivo antitumor activity of E4 was comparable with E8 at equal mAb doses, although the E4 contained half the amount of MMAE per mAb. E2 was also an active antitumor agent but required higher doses. The maximum-tolerated dose of E2 in mice was at least double that of E4, which in turn was twice that of E8. MMAE loading affected plasma clearance, as E8 cleared 3-fold faster than E4 and 5-fold faster than E2.

Conclusions: By decreasing drug loading per antibody, the therapeutic index was increased demonstrating that drug loading is a key design parameter for antibody-drug conjugates.
HMMC-1
Authors: Nozawa Shiro , Aoki Daisuke , Tsukazaki Katsumi , Susumu Nobuyuki , Sakayori Motoko , Suzuki Nao , Suzuki Atsushi , Wakita Rie , Mukai Makio , Egami Yuko , Kojima-Aikawa Kyoko , Ishida Isao , Belot Frederic , Hindsgaul Ole , Fukuda Minoru , Fukuda Michiko N.
Published: September 2005

Purpose: The purpose of this research was to generate a human monoclonal antibody specific to gynecological cancers and to evaluate such an antibody as therapy for gynecological cancers.

Experimental Design: Transchromosomal KM mice were immunized with the human uterine endometrial cancer cell line SNG-S. Hybridomas were constructed between spleen cells from KM mice and mouse myeloma cells. Reactivity of the antibody was evaluated by immunohistochemistry of pathological specimens of gynecological cancers. Cytotoxicity of HMMC-1 against SNG-S cells was tested by in vitro cytotoxicity assays. The epitope of HMMC-1 was determined by transfection with a panel of glycosyltransferase cDNAs and by inhibition assays with chemically synthesized oligosaccharides.

Results: HMMC-1 is a human IgM monoclonal antibody that reacts positively with müllerian duct-related carcinomas with positive rates of 54.6% against uterine endometrial adenocarcinoma, 76.9% against uterine cervical adenocarcinoma, and 75.0% against epithelial ovarian cancer. HMMC-1 does not react with normal endometrium at proliferative or secretory phases, normal uterine cervix, or normal and malignant tissue from other organs, whereas it reacts weakly with the epithelium of the gall bladder and the collecting duct of the kidney. HMMC-1 exhibits antigen-dependent and complement-mediated cytotoxicity. Upon cotransfection with cDNAs encoding two glycosyltransferases required for fucosylated extended core 1 O-glycan, mammalian cells express HMMC-1 antigen. Finally, binding of HMMC-1 to SNG-S cells is inhibited by synthetic Fucα1→2Galβ1→4GlcNAcβ1→3Galβ1→3GalNAcα1-octyl.

Conclusions: These results indicate that HMMC-1 specifically recognizes a novel O-glycan structure. The unique specificity and cytotoxicity of HMMC-1 strongly suggest a therapeutic potential of this antibody.
Ran, a Small GTPase Gene, Encodes Cytotoxic T Lymphocyte (CTL) Epitopes Capable of Inducing HLA-A33–restricted and Tumor-Reactive CTLs in Cancer Patients
Authors: Azuma Koichi , Sasada Tetsuro , Takedatsu Hiroko , Shomura Hiroki , Koga Makoto , Maeda Yoshiaki , Yao Akihisa , Hirai Tatsuya , Takabayashi Arimichi , Shichijo Shigeki , Itoh Kyogo
Published: September 2005

Purpose: The purpose is to identify a gene coding for tumor-associated antigen and peptide capable of inducing CTLs reactive to tumor cells with a HLA-A33–restricted fashion to provide scientific basis for specific immunotherapy to HLA-A33+ cancer patients.

Experimental Design: An expression gene-cloning method was used to identify the tumor-associated antigen gene. Northern blot analysis and immunohistochemistry were used to examine the mRNA and protein expression levels in various cells and tissues, respectively. Synthetic peptides were examined for their ability to induce HLA-A33+ tumor-reactive CTLs in peripheral blood mononuclear cells from cancer patients.

Result: A gene of small GTPase, Ran, which controls the cell cycle through the regulation of nucleocytoplasmic transport, mitotic spindle organization, and nuclear envelope formation, was found to encode epitopes recognized by the HLA-A33–restricted CTLs established from T cells infiltrating into gastric adenocarcinoma. The expression of the Ran gene was increased in most cancer cell lines and cancer tissues at both the mRNA and protein levels. However, it was not enhanced in the surrounding normal cells or tissues. It was also undetectable in normal tissues as far as tested. Ran-derived peptides at positions 48–56 and 87–95 could induce CD8+ peptide-specific CTLs reactive to tumor cells from HLA-A33+ epithelial cancer patients in a HLA class I-restricted manner.

Conclusions: Because of its increased expression in cancer cells and involvement in malignant transformation and/or the enhanced proliferation of cancer cells, the two Ran-directed peptides could be potent candidates in use for specific immunotherapy against HLA-A33+ epithelial cancers.