bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 131: Mechanisms by which 2,3’, 4,5’ tetramethoxystilbene (TMS), a resveratrol derivative, induces death in breast cancer cells
Authors: Aiyar Sarah E. , Santen Richard J. , Park Hoyong , Aldo Paula B. , Mor Gil
Published: February 2011

We have examined a non-toxic pro-apoptotic agent, TMS (2, 3’,4, 5’ tetramethoxystilbene), which belongs to the Resveratrol family of stilbenes. To learn about the mechanism by which TMS achieved its effects we carried out microarray analysis and found that TMS treatment increased tubulin genes as well as stress response and pro-apoptotic genes. Fractionation studies uncovered that TMS treatment causes cleavage of Bax from the p21 form to a truncated p18 form. Co-localization analysis of immunofluorescent studies showed that Bax moved from the cytosol to the mitochondria. In addition, the pro-apoptotic proteins Noxa and Bim (EL, L, and S) were increased upon TMS treatment. Breast cancer cell lines reduced for Bax, Bim and Noxa are compromised for TMS-mediated cell death. Transmission electron microscopy revealed evidence of nuclear condensation, formation of apoptotic bodies and DAPI staining showed evidence of DNA fragmentation. TMS treatment was able to induce both caspase-independent and caspase-dependent death via the intrinsic death pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 131.
Abstract 1314: Real-time imaging of individual cancer cell nuclear-cytoplasmic dynamics of UV-induced apoptosis of cancer cells in the brain
Authors: Momiyama Masashi , Endo Itaru , Hoffman Robert M.
Published: February 2011

We have developed a novel in vivo mouse model to image cancer cell nuclear-cytoplasmic dynamics in the brain in real-time and to visualize UV-C light killing of these cells. We used dual-color fluorescent cancer cells that express green fluorescent protein (GFP) linked to histone H2B in the nucleus and retroviral red fluorescent protein (RFP) in the cytoplasm. In order to visualize metastatic single cancer cell nuclear-cytoplasmic dynamics in the brain, dual-color Lewis Lung carcinoma (LLC) cells were injected into the right internal carotid artery of nude mice. A craniectomy open window was made over the right parietal bone. Metastatic dual-color LLC cells were irradiated with a 3.6-watt hand held UV-C light source for 60 seconds. The cells were then observed over 24 hours at 3 hour intervals through the open craniectomy window. The cancer cells were observed in the brain with the Olympus OV100 Small Animal Imaging System under high magnification. Metastatic dual-color LLC cells were visualized at the subcellular level in the brain enabling apoptosis to be imaged by nuclear morphology. UV-C light induced apoptosis of the dual-color LLC cells in the brain. Aggregation of chromatin at the nuclear membrane and fragmented nuclei of the cancer cell were followed in real time in the brain. More than 90% of the cancer cells irradiated with UV-C light died and disappeared without overt toxicity of the host. The novel mouse model used here will enable a deeper understanding of cancer cell dynamics in the brain. UV-C light may be a powerful tool for treatment of cancer cells expressing fluorescent reporters which now can be effected in vivo (Proc. Natl. Acad. Sci. USA, 1-6, 14514-14517, 2009 and Molecular Cancer Therapeutics 8, 3001-3008, 2009).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1314.
Abstract 1315: Real-time imaging of nuclear-cytoplasmic dynamics in UV light killing of cancer cells expressing fluorescent proteins
Authors: Kimura Hiroaki , Tsuchiya Hiroyuki , Tomita Katsuro , Hoffman Robert M.
Published: February 2011

Our laboratory pioneered dual-color cancer cells, in which red fluorescent protein (RFP) is expressed in the cytoplasm and green fluorescent protein (GFP) in the nucleus (Cancer Research 64, 4251-4256, 2004; Nature Protocols 1, 928-935, 2006). Total cellular dynamics can be visualized in the living dual-color cells in real time. In this study, we investigated the cancer-cell-killing efficacy of UV light using the dual-color cancer cells. For UV irradiation experiments, a Benchtop 3UV transilluminator, which emits UVC with an emission peak at 254 nm; UVB with an emission peak at 302 nm; and UVA with an emission peak at 365 nm; was used. After exposure to various doses of UVA, UVB, or UVC, apoptotic, necrotic and viable cells were quantitated under fluorescence microscopy using dual-color 143B human osteosarcoma cells (143B). UV-induced cancer cell death was wave-length and dose dependent. After UVA exposure, most cells were viable even if the UV dose was increased up to 200 J/m2. In the UVB group, cell death began to appear when irradiated at 50 J/m2. For UVC, the rate of cell killing was proportional to increased UVC irradiation. 25 J/m2 UVC irradiation killed over 40% of the 143B dual-color cells. However, the rate of cell killing plateaued at 100 J/m2. We also tested 50J/m2 UVC and 100J/m2 of UVB on four types of dual-color cancer cell lines. UV-induced cancer cell death varied among the cell lines. Cell death began about 4 hours after irradiation and continued until 10 hours after irradiation. Real-time movies were made of cells undergoing UV-induced cell death. Most cells died via apoptosis and only a small portion of them died via necrosis. We will develop UV irradiation for treatment of fluorescent-protein-expressing minimal residual cancer remaining after resection.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1315.
Abstract 1317: The 2-phenylbenzimidazole-5-sulfonic acid sunscreen agent photosensitizes oxidized guanines following UVA or UVB exposure
Authors: Bastien Nathalie , Millau Jean-François , Rouabhia Mahmoud , Davies Jerry R. , Drouin Régen
Published: February 2011

The 2-phenylbenzimidazole-5-sulfonic acid (PBSA) is a sunscreen agent widely used in the cosmetic industry because it absorbs strongly at UVB wavelengths providing a good protection against the cyclobutane pyrimidine dimer (CPD) generation. Previous studies have shown that PBSA oxidize guanine bases in vitro (isolated DNA). Due to its potential phototoxic effect, it is of great interest to determine whether PBSA can play the role of a photosensitizer in cellulo. For this purpose, we investigated in vitro and in cellulo (in fibroblasts) the effect of 0 to 4 mM PBSA on DNA damage formation after UVA or UVB irradiation. In order to monitor oxidized bases and CPD formation, DNA was digested with either endonuclease III and FaPy-DNA glycosylase or T4 endonuclease V and photolyase, then analyzed by means of glyoxal gel electrophoresis and ligation-mediated polymerase chain reaction (LMPCR). We found that in cellulo and in vitro, PBSA provided a good protection against CPD formation, but induced oxidative damage and DNA-strand breaks. Moreover, with the LMPCR technology we were able to determine that oxidative damage were preferentially formed at guanine sites. Our results indicate that PBSA has the potential to act as a double-edged sword toward DNA, on one hand it blocks the formation of CPDs after UVB and on the other hand increase the formation of oxidized guanines upon UVA and UVB irradiation. This raises major concern regarding its suitability for sunscreen applications.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1317.
Abstract 132: Cytoprotective induction of nitric oxide synthase in a cellular model of 5-aminolevulinic acid-based photodynamic therapy
Authors: Bhowmick Reshma , Girotti Albert W.
Published: February 2011

Photodynamic therapy (PDT) employs a photosensitizing agent, molecular oxygen, and visible light to produce reactive oxygen species that kill tumor and tumor vasculature cells. Nitric oxide (NO) produced by these cells could be pro-carcinogenic by inhibiting apoptosis or promoting angiogenesis and tumor growth. Our previous studies showed that NO from a chemical donor made COH-BR1 breast tumor cells hyperresistant to apoptotic photokilling induced by photoactivation of 5-aminolevulinic acid (ALA)-generated protoporphyrin IX localized in mitochondria. Hyperresistance was associated with inhibition of proapoptotic JNK and p38α MAP kinase phosphorylation-activation. The purpose of this study was to determine whether tumor cells upregulate NOS-generated NO as a cytoprotective measure during PDT.COH-BR1 cells sensitized in mitochondria with ALA-derived protoporphyrin IX died apoptotically following irradiation as measured by Hoechst staining. Western analysis revealed that inducible nitric oxide synthase (iNOS) was upregulated >3-fold within 4 h after ALA/light treatment, while other NOS isoforms were unaffected. Exposing cells to the iNOS inhibitor, 1400W during photochallenge enhanced caspase-3/7 activation and apoptotic killing up to 2-3-fold while substantially reducing chemiluminescence-assessed NO production, suggesting that this NO was cytoprotective. Consistently, the NO scavenger cPTIO enhanced ALA/light-induced caspase-3/7 activation and apoptotic kill by >2.5-fold. Of added significance, cells could be rescued from 1400W-exacerbated apoptosis by an exogenous NO donor, spermine-NONOate. Using Hoechst and TUNEL staining, we found that short hairpin RNA (shRNA)-induced knockdown of iNOS enhanced the apoptotic kill of ALA/light treated COH-BR1 cells. ALA/light-treated COH-BR1cells exhibited a transient post-irradiation activation of JNK and p38α as measured by Western blot analysis. Consistently, both effects were intensified and prolonged by 1400W. The survival MAP kinase ERK1/2 was deactivated more rapidly when 1400W was present during a PpIX/light challenge. Similar effects on MAP kinase activation/deactivation were observed for iNOS knockdown cells under photostress cinfirming iNOS's protective role. As demonstrated for non-photodynamic stress systems, NO could have interfered with apoptosis by inactivating participating MAP kinases and/or caspases. This is the first reported evidence for increased tumor cell resistance due to iNOS upregulation in a PDT model. Our findings indicate that stress-elicited NO in PDT-treated tumors could compromise therapeutic efficacy, and suggest NOS-based pharmacologic interventions for preventing this. (Supported by NIH Grant CA70823)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 132.
Abstract 1322: Sensitizing radiation-induced integrin β-1 signaling by targeting uPAR in medulloblastoma cells
Authors: Nalla Arun Kumar , Kotipatruni Rama Prasadarao , Ganji Purnachandra Nagaraju , Fassett Daniel , Klopfenstein Jeffrey D. , Dinh Dzung H. , Gujrati Meena , Rao Jasti S.
Published: February 2011

Although radiation is an effective therapy, it has several adverse effects, including the induction of secondary (new) tumor within the irradiated field. Several studies have reported this effect with medulloblastoma despite effective radiotherapy during the initial stages. Here, we attempt to capitalize on the radiation-induced aggressive behavior of medulloblastoma cells by examining migration, cell adhesion and matrigel invasion properties. Comparison at the mRNA levels between the matrigel-invading and non-invading cells (with and without radiation) clearly demonstrates the aggressiveness of irradiated medulloblastoma cells. RT-PCR analysis confirmed the increased expression of uPA, uPAR, focal adhesion kinase (FAK), N-Cadherin, and integrins like α3, α5, β1 and β6 in irradiated cells. Increased expression of uPAR and integrin β-1 in irradiated cells was also confirmed by immunofluorescence and immunoprecipitation assays. The interaction between uPAR and integrin β-1 and subsequent downstream signaling is a key process in malignant progression and metastasis and has been recently reported in mediating resistance to radiation. With this view, we aimed to study the effect of siRNA-mediated knockdown of uPAR on cell migration and adhesion in irradiated and non-irradiated medulloblastoma cells. Downregulation of uPAR not only reduced cell migration and adhesion, but also reduced the increased migration and adhesiveness associated with irradiated cells. Apart from specific knockdown of uPAR in siRNA-transfected cells, the increased expression of uPAR and integrin β-1 upon radiation were also reduced both at the transcript and protein levels. Studies of the uPAR/integrin β-1 downstream signaling cascade have shown that uPAR knockdown reduces both the expression and phosphorylation of FAK, paxillin, p130Cas and Rac-1, which are known to be actively involved in coordinating tyrosine kinase based signaling related to cell migration and adhesion. Our in vitro results demonstrate that targeting uPAR might sensitize the radiation-induced aggressiveness of medulloblastoma cells by affecting the uPAR/integrin β-1 signaling pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1322.
Abstract 1324: Modulation of IR induced NFKB activity by manipulation of NOS coupling
Authors: Rabender Chris , Mikkelsen Ross
Published: February 2011

The transcriptional activity of NFκβ is in part regulated by nitric oxide (NO) and reactive nitrogen species (RNS). Previously we demonstrated that low doses of ionizing radiation (IR) activate constitutive, Ca2+ dependent NO Synthases (NOS) and the generation of RNS, resulting in protein Tyr nitration and/or Cys s-nitrosylation. Previous studies in our lab demonstrated that nitration of IκBα is critical for the IR-induced activation of NFκβ whereas others have shown that s-nitrosylation of the p65 subunit leads to the inhibition of NFκβ. Here we show that exposing MCF-7 cells to 5Gy IR results not only in Tyr nitration of IκBα but also Cys s-nitrosylation of NFκβ both with a maximum at 30min. post IR. One explanation for the discordant results is that NOS activity is uncoupled, resulting in the production of increased levels of superoxide anion as opposed to NO. Tetrahydrobiopterin (BH4) is a necessary cofactor that stabilizes the enzyme and allows the generation of NO through oxidation of L-arginine. However, if BH4 levels become limiting as found in chronic inflammatory or oxidative conditions, electron transfer is instead shuttled to molecular oxygen resulting in the production of superoxide anion, a scenario in which NOS is described as “uncoupled.” When NOS is uncoupled the resulting increase in superoxide can lead to enhanced levels of peroxynitrite formation, resulting in enhanced IκBα Tyr nitration and NFκβ activity. This evidence has led to the hypothesis that by altering cellular levels of BH4, the levels of Tyr nitration and Cys s-nitrosylation can be manipulated, leading to changes in NFκβ activity. To determine the effects of exogenous BH4 on IκBα nitration in MCF-7 cells, cells were treated with varying concentrations of cell permeable sepiapterin (SP), a precursor of BH4, and exposed to 5Gy IR. The results show that between 100nM-500nM IκBα nitration is all but completely eliminated, resulting in decreased NFκβ activity as assessed by luciferase assay. Initial experiments looking at the effects of SP on p65 s-nitrosylation have been variable. At a minimum, levels of s-nitrosylation remain the same as untreated cells; however, enhancement of s-nitrosylation is seen in some experiments. Studies are ongoing to clarify SP's effects on p65 s-nitrosylation as well as studies to determine SP's effects on tumor cell survival post IR.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1324.
Abstract 1332: Pediatric sarcomas are infiltrated with myeloid derived suppressive cells
Authors: Bouchkouj Najat , Qin Haiying , Galli Susana , Buckley John , Meadors Joanna L. , Larabee Shannon , Mackall Crystall L. , Tsokos Maria G. , Fry Terry J.
Published: February 2011

Introduction: Pediatric cancers have been reported to be infiltrated with macrophages. Tumor associated macrophages and myeloid derived suppressive cells (MDSCs) are increasingly recognized to play an important role in cancer immune escape, promote tumor progression by suppression of adaptive immunity, and are associated with poor prognosis in adult cancers. Little is known about the presence and the role of these cells in the pathobiology of pediatric cancers. We sought to analyze pediatric sarcomas for the presence of myeloid cells expressing inducible nitric oxide synthase (iNOS), arginase1 (Arg1), and/or indolamine 2,3 deoxygenase (IDO). Concomitantly, we used a murine model of embryonal rhabdomyosarcoma (ERMS) to further explore MDSCs associated with pediatric sarcomas.

Methods: We conducted immunohistochemical (IHC) studies on 20 paraffin embedded tumor samples of rhabdomyosarcoma, Ewing's sarcoma, desmoplastic small round cell tumor and malignant peripheral nerve sheath tumor, assessing for CD68, CD163, iNOS, Arg1 and IDO immunoreactivity. In the murine model, 1×106 RMS M3-9-M murine ERMS cells were injected intramuscularly into the gastrocnemius muscle of C57BL/6 mice. Tumors and spleens were excised on days 7, 12, 16 and 19 post injection and tumor and spleen myeloid cell were analyzed for CD11b, CD11c, Gr-1 and IL4R alpha expression and for gene expression of iNOS, Arg1 and IDO using RT-PCR.

Results: Pediatric sarcomas demonstrate significant macrophage infiltration with iNOS, Arg1 and IDO present in most of these samples. Murine ERMS also shows substantial numbers of MDSCs both in the tumor and spleen of tumor bearing mice, with increasing numbers as tumor burden increased. Comparisons between different myeloid cells subsets by relative quantitation of gene expression reveal the presence of an expanded population of cells that bear the hallmarks of MDSCs by expressing iNOS and Arg1. Significantly higher IDO expression was seen in MDSCs compared to other myeloid cells as tumors progressed.

Conclusion: Macrophages infiltrating pediatric sarcomas expressed markers associated with MDSCs, which have been shown to contribute to immune evasion and augment tumor growth in several other model systems. Similarly, murine ERMS M3-9-M induced accumulation of MDSCs in spleen and tumor microenvironment, with increasing numbers associated with tumor progression, suggesting that they may play an important role in growth of pediatric sarcomas. Immunomodulation of these cells might be a key step in enhancing the effectiveness of immune-based therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1332.
Abstract 1334: Intratumoral soluble interleukin-6 receptor associated with disease progression in colorectal cancer
Authors: Okugawa Yoshinaga , Miki Chikao , Toiyama Yuji , Yasuda Hiromi , Yokoe Takeshi , Saigusa Susumu , Hiro Junichiro , Tanaka Koji , Inoue Yasuhiro , Nobori Tsutomu , Kusunoki Masato
Published: February 2011

Purpose. Interleukin-6 (IL-6) binds not only to membrane form but also to soluble IL-6 receptor (sIL-6R), derived from the extracellular part of the membrane receptor. Although most soluble receptors act as functional antagonists to their cytokine, sIL-6R plays a role in agonistic activity. The aim of this study was to clarify the relationship between concentration of intra-tumoral soluble interleukin-6 receptor levels and cancer progression in colorectal cancer patients, and to clarify its kinetics with clinical outcome.

Methods. We studied 161 patients undergoing surgery for colorectal cancer. We measured concentrations of sIL-6R in tumors and normal mucosa, and in supernatants from colonic cancer cell lines. Expressions of IL-6, membranous IL-6R and gp130 were evaluated by immunohistochemically

Results. The net balance between the concentration of sIL-6R in cancer tissue and normal mucosa (sIL-6R Ca/N expression ratio: the cancer tissue sIL-6R concentration divided by normal mucosa sIL-6Rntration) was 1.262 ± 1.156. The decreased sIL-6R Ca/N expression ratio was significantly associated with T classification (p=0.0076), distant metastasis (p=0.0102), UICC stage (p=0.0251) and poor prognosis (p=0.0003). In Cox multivariate analysis, distant metastasis and decreased sIL-6R Ca/N expression ratio were independent risk factors for poor prognosis. Colon cancer cell lines produced sIL-6R, and the production was exaggerated by IL-1beta stimulation, and was suppressed by the addition of IL-1 receptor antagonist. Immunohistochemically, IL-6, membranous IL-6R and gp130 were intensely expressed in cancer cell specifically, and IL-6 expression in cancer cytoplasm was associated with poor prognosis (p=0.0266). The decreased levels of sIL-6R Ca/N expression ratio in cancer tissue was inversely correlated with the intense IL-6 immunoreactivity in cancer cytoplasm (p=0.0088). Conclusion: Relative decrease in sIL-6R in the tumor stroma which reflects increased IL-6/sIL-6R affinity may play a key role in the progression of colorectal carcinoma via IL-6 tran-signaling.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1334.
Abstract 1335: Immune regulation in endometriosis and epithelial ovarian cancer
Authors: Mantia-Smaldone Gina M. , Budiu Raluca , Mansuria Suketu , Lee Ted , Zorn Kristin , Elishaev Esther , Edwards Robert P. , Vlad Anda
Published: February 2011

Objective: Examine the expression and immunogenicity of a known ovarian tumor-associated antigen, MUC1, in ectopic endometriosis lesions and epithelial ovarian cancer (EOC). Determine whether an imbalance in CD4 T cell subsets contributes towards endometriosis pathogenesis and ovarian carcinogenesis.

Methods: Biologic specimens were obtained from women with endometriosis, endometriosis-related (endometrioid and clear cell) or serous EOC, and healthy controls, according to University of Pittsburgh IRB protocols. MUC1 and immune cell expression were determined via immunohistochemistry (IHC). Anti-MUC1 IgM and IgG antibody levels were determined via ELISA. Peripheral blood mononuclear cells and cryopreserved cell suspensions from tissue lesions were immunophenotyped using multicolor flow cytometry. Stroma and epithelial cells from tissue biopsies were collected by laser capture microdissection (LCM) and examined with immune-based quantitative RT-PCR arrays.

Results: MUC1 was overexpressed in 67% of endometriosis and 89% of EOC lesions. Women with early or late stages of endometriosis had significantly higher anti-MUC1 IgM responses when compared to those women with endometriosis-related EOC (25.3 % of positive control vs.14.2%, p=0.03; 26.3 vs. 14.2%, p=0.04) and to those with serous EOC (25.3 vs. 14.6%, p=0.04; 26.3 vs.14.6%, p=0.04). Women without disease also had significantly higher anti-MUC1 IgM responses compared to endometriosis-related (29.2 vs.14.2%, p=0.01) and serous EOC (29.2 vs.14.6%, p=0.02). Immune cells, including CD3, CD4, and CD8 T cells, are present in endometriotic lesions and appear more abundant than in eutopic endometrium. Flow cytometric analysis of endometriotic lesion-infiltrating T cells revealed a decrease in the frequency of IFN-γ CD4 and CD8 T cells, similar to that seen in patients with EOC.

Conclusions: MUC1 is present and appears to be overexpressed in ectopic endometriotic and EOC lesions. Women with endometriosis-related EOC or with serous EOC had significant lower anti-MUC1 IgM levels, suggesting that a compression of antigen-specific humoral (i.e., Th2 driven) immunity may play a role in carcinogenesis. Endometriotic and EOC lesions also appear to have diminished Th1 immunity, suggesting a possible role for local immune suppression. Quantitative analyses of several immune genes in the lesion microenvironment are currently ongoing and will potentially contribute to immune biomarker discovery and improvements in the early detection and prevention of ovarian cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1335.
Abstract 1340: High densities of tumor-infiltrating FOXP3+ regulatory T cells in primary tumor or lymph node metastasis are associated with favorable outcome in gastric cancer
Authors: Lee Hee Eun , Chae Seoung Wan , Lee You Jeong , Kim Min A , Lee Hye Seung , Lee Byung Lan , Kim Woo Ho
Published: February 2011

Background: It has been reported that the frequency of FOXP3+ regulatory T cells (Tregs) among tumor-infiltrating lymphocytes is increased in cancer-bearing patients. We designed the study to determine the prognostic significance of FOXP3+ Tregs in gastric cancer.

Methods: Tissue microarray and immunohistochemistry were used to assess the densities of FOXP3+ T regs in primary tumor tissue (n=216) and unmatched metastatic tumor tissue of regional lymph nodes (n=149) from gastric cancer patients. Numbers of FOXP3+ Tregs were counted using an image analysis program. FOXP3+ Treg densities were evaluated for correlation with clinicopathologic characteristics and overall survival.

Results: With respect to primary tumor, low density of FOXP3+ Tregs was associated with advanced T stage (p = 0.028), but not with any other clinicopathologic features. Kaplan-Meier survival analysis revealed that the high density group of FOXP3+ Tregs had longer survival time than the low density group (p = 0.001, log-rank test). In multivariate analysis, FOXP3+ Treg density remained an independent prognostic factor with hazard ratio (95% CI) of 0.651 (0.435-0.975). In addition, it was found that FOXP3+ Treg density in metastatic tumor of lymph nodes was an independent prognostic indicator, i.e., high density of FOXP3+ Tregs was significantly associated with improved survival (hazard ratio (95% CI) = 0.519 (0.318-0.848)).

Conclusions: FOXP3+ Treg densities in not only primary tumor, but also metastatic loci of regional lymph nodes were significantly associated with patient survival in gastric cancer. As our finding that high density of FOXP3+ Tregs was related to favorable prognosis contrasts with several previous studies, further investigation will be needed.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1340.
Abstract 1343: Toll-like Receptor 9 (TLR9) stimulation results in a change in tumor macrophage infiltration associated with an anti tumor effect in a mouse model of lung adenocarcinoma induced by oncogenic K-ras
Authors: Rabbe Nathalie , Belmont Laure , Yu Jing , Poulot Virginie , Gounant Valérie , Antoine Martine , Kurie Jonathan , Cadranel Jacques , Wislez Marie
Published: February 2011

Microenvironment and immune system contribute to tumor escape in cancer. Toll-like Receptor 9 (TLR9) stimulation with a CpG ODN (PF-3512676) was expected to restore antitumor response in a mouse model of lung adenocarcinoma (ADC) induced by oncogenic K-ras (K-rasLA1 mice). In these mice, TLR9 was not expressed by normal lung, nor tumor cells but only by the inflammatory tumor component as shown by TLR9 mRNA quantification by real time quantitative fluorescent PCR and immunohistochemistry. Sixteen week-old K-rasLA1 mice received PF-3512676 (n=13) or vehicle (n=13) through subcutaneous administration for 3 weeks. Mice were killed after completion of treatment. During autopsy procedure, lesions were counted on lung surfaces by investigators blinded to treatment group. Compared to the vehicle group, there was a decrease of tumor cell proliferation (PCNA staining score: 28.5% ± 1 vs 22.4% ± 0.8; p<0.0001, respectively) and an increase of tumor cell apoptosis (CC3 western blot and staining score: 0.058% ± 0.01 vs 0.105% ± 0.01; p=0.0154, respectively) in the PF group. No effect was observed on the autopsy count (26.5 ± 3.7 tumors vs 24 ± 2.3; p=0.85) nor on the histological count (27 ± 3.4 tumors vs 20.7 ± 2.2; p=0.23). Macrophage infiltration studied by F4/80 staining was different between the two groups with a higher number of macrophages inside the tumors in PF group while a higher number in peritumor area in the vehicle group (intra/peritumoral macrophage ratio: 3.35 ± 0.5, PF group vs 1.68 ± 0.1 vehicle, p=0.003). LKR-13, a lung ADC cell line derived from K-rasLA1 mice, does not express TLR9 and was resistant to treatment with PF in vitro. However, LKR-13 cells grown as syngeneic tumors recruited macrophages, and those tumors regressed in response to treatment with PF. These findings provide evidence that TLR9 stimulation might reduced the expansion of lung ADC induced by oncogenic K-ras and suggest that phenotypic modification of macrophages may contribute to ADC regression.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1343.
Abstract 1356: Real-time in vivo color-coded imaging of the interaction between immunocytes and tumor cells during cancer therapy
Authors: Kimura Hiroaki , Hayashi Katsuhiro , Tsuchiya Hiroyuki , Tomita Katsuro , Aki Ryoichi , Kishimoto Hiroyuki , Suetsugu Atsushi , Momiyama Masashi , Bouvet Michael , Hoffman Robert M.
Published: February 2011

We imaged the interaction between osteosarcoma cells and splenocytes using fluorescent proteins during chemotherapy or dendritic-cell (DC) therapy. GFP-expressing splenocytes (5 × 106), derived from GFP transgenic BALB/c mice, were transplanted to BALB/c nude mice. Then, RFP-143B human osteosarcoma cells (5 × 105) were injected in a skin flap raised in these mice. The mice were treated with doxorubicin (5 mg/kg), i.p. three days in a row. The RFP-expressing 143B tumor and the GFP splenocytes accumulation around the tumor were imaged for three days. Dendritic cells (1×106), which were activated with a 143B cells lysate, were injected into the inguinal lymph node of the mice. The tumor grew gradually in the control group, whereas in the chemotherapy group, the tumor became progressively smaller. In the DC group, the tumor regressed and was almost eradicated 12 days after treatment. In the DC group, GFP splenocytes accumulated in large numbers around the tumor three days after DC treatment. The accumulation of DC cells was diminished as the tumor became smaller. In the chemotherapy group, in contrast, accumulation of splenocytes gradually increased even though the tumor became progressively smaller. Future experiments will be on syngeneic models to determine the influence of an intact immune system on the interaction of splenocytes and DC cells with cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1356.
Abstract 1374: Synthetic heparan sulfate oligosaccharides inhibit endothelial cell functions essential for angiogenesis
Authors: Avizienyte Egle , Cole Claire L. , Hansen Steen U. , Barath Marek , Rushton Graham , Gardiner John M. , Jayson Gordon C.
Published: February 2011

Heparan sulfate (HS), a linear glycosaminoglycan, is an essential structural component required for the activity of numerous angiogenic cytokine-receptor signalling complexes. To exploit the potential of inhibiting HS-dependent signalling complexes we synthesized a series of heparan sulfate oligosaccharides containing up to 12 saccharide residues with variable but defined sulfation patterns. The ability of oligosaccharides to compete with HS in FGF2, VEGF and IL-8 binding significantly increased with the length of oligosaccharides and especially with additional sulfate groups incorporated at specific positions in the hexuronic acid and glucosamine. In this competition assay, oligosaccharides exhibited higher levels of inhibition of FGF2 binding to HS than of VEGF and IL-8 binding to HS. Correspondingly, the inhibitory potential of oligosaccharides against FGF2-, VEGF- and IL-8-mediated endothelial cell responses was greater in longer species of oligosaccharides that bore more sulfate residues. Oligosaccharides inhibited cytokine-induced endothelial cell migration to the same extent as kinase inhibitors targeting FGF receptors or VEGF receptor 2 (VEGFR2). In contrast, FGF2-induced endothelial cell proliferation was only inhibited when cells were treated with an oligosaccharide composed of 12 saccharide residues at a concentration that significantly exceeded that of the FGFR inhibitor suggesting that oligosaccharides mainly interfere with the migration, but not proliferation of endothelial cells. The ability of oligosaccharides to target endothelial cell migration correlated with their ability to ablate FGF2- or VEGF-induced accumulation of activated peripheral FAK-pY397 and actin polymerization at the cell periphery. More importantly, there was a strong correlation between oligosaccharide structure and the level of inhibition of endothelial tube formation. Correlating with the biological activity of oligosaccharides, FGF2- and VEGF-induced downstream signalling events were sensitive to the treatment with biologically active oligosaccharides that specifically reduced FRS2, VEGFR2, ERK1/2 and AKT phosphorylation. In conclusion, we have determined the critical number of HS saccharide residues and sulfation pattern required to inhibit endothelial cell responses to key angiogenic cytokines.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1374.
Abstract 1381: The effect of proteasome inhibitor velcade on tumor microenvironment
Authors: He Fuqiu , Sun Xiaorong , Ackerstaff E , Xing Ligang , Minami Akiko , Hsiao Hung Tsung , Carlin Sean , Ling Clifton C. , Koutcher Jason , Li Gloria C.
Published: February 2011

It has been reported that the therapeutic effect of the proteasome inhibitor Velcade is due to its selective interference in the hypoxia pathway. We studied the effect of Velcade on tumor microenvironment and examined the underlying molecular mechanisms. We have generated a human colorectal cancer xenograft model (#C53) in which the hypoxia-inducible dual reporter fusion gene (HSV1-TK and eGFP) was under the control of hypoxia-response-element (HRE). In vitro, #C53 cells were treated with Velcade in normoxic and hypoxic conditions, and the following assays were performed in comparison with controls: eGFP (flow cytometry), CA9 (western blot), VEGF (ELISA), and TK activity (trapping of the marker substrate 14C-FIAU). In vivo, #C53 xenografts were treated with Velcade and various assays performed, including a) Dynamic contrast-enhanced (DCE) MRI pre and post treatment; b) dual hypoxia marker (pimonidazole and EF5) administration pre and post treatment; c) fluorescence microscopy of Hoechst 33342 (perfusion), eGFP, HIF-1α, and CA9; and d) plasma VEGF level (ELISA), Where applicable data from control and treated tumors were compared. Our results showed that in both in vitro and in vivo experiments Velcade treatment increased the level of HIF1α, but decreased those of hypoxia-induced eGFP, TK, CA9 and VEGF. Interestingly, in the dual hypoxia marker study there were significant EF5-positive regions that did not co-localize with pimonidazole-positive regions, suggesting de novo hypoxia and perhaps another novel effect of Velcade on tumor microvasculature. Consistent with these results, DCE MRI demonstrated decrease global tumor blood flow with Velcade treatment. Our data suggest that Velcade suppresses the hypoxia response by disrupting the HIF1 transcriptional activity. In addition, our results suggest a novel function of Velcade in modifying the tumor microenvironment and decreasing tumor perfusion as noninvasively detected by DCE MRI.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1381.
Abstract 1385: Fusion protein containing RGD-endostatin and human Fc of IgG4improves anti-angiogenic and anti-tumor activity
Authors: Jing Yawu , Wu Kailang , Lu Huarui , Subramanian Indira V. , Ramakrishnan Sundaram
Published: February 2011

Inhibiting angiogenesis is a promising strategy for cancer therapy. Endostatin, a C-terminal fragment of collagen XV[[Unsupported Character - Ш]], is an endogenous inhibitor of angiogenesis. We have earlier shown that addition of integrin-targeting sequences to a mutant endostain (P125A-endo) improved localization to tumor vasculature. In the present study, we investigated whether genetically fusing endostatin to Fc region of human IgG can prolong circulatory half-life and improve anti-tumor activity. Two types of constructs were made: a) RGD-endostatin-Fc and b) Endostatin-RGD-Fc. The location of the RGD moiety was either placed at the NH2-terminus or between the carboxyl terminus of endostatin and Fc. Both constructs were expressed in HEK-293 cells and purified using affinity chromatography. Our results show the biological activity of endostatin was significantly enhanced by linking to the Fc region of IgG when compared to P125A-endostatin expressed in yeast. Both RGD-Endo-Fc and Endo-RGD-Fc were able to bind endothelial cells very efficiently and inhibited endothelial cells proliferation, migration in vitro and angiogenesis in vivo. We then investigated the ability of the fusion protein to inhibit ovarian cancer growth in a xenograft model. Once a week administration of the fusion protein into mice bearing established ovarian tumors inhibited tumor growth and increased their survival. Anti-tumor activity of the fusion protein was comparable to Bevacizumab-mediated suppression of tumor growth. Furthermore, when mice were treated with a combination of the fusion protein and Bevacizumab, more than additive inhibition of tumor growth was observed. These data suggest that fusion with Fc-fragment can improve the anti-angiogenic and anti-tumor activity of endostatin. Moreover, our results support a strategy to combine two anti-angiogenic reagents such as anti-VEGF antibody and endostatin-Fc fusion protein to additively inhibit ovarian cancer growth.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1385.
Abstract 139: The identification of potent inhibitor of apoptosis protein (IAP) inhibitors for clinical development
Authors: Morris Stephen J. , Ashdown Helen , Boudreault Alain , Durkin Jon , Gillard John , Hewitt Kim , Jaquith James , Jerome Lori , Laurent Alain , Maltais Stephane , Methot Danielle , Patel Harshila , Romeo Andrea , Devalaraja M , Humphreys R
Published: February 2011

The inhibitor of apoptosis proteins (IAPs) are important regulators of cell death. Over-expression of IAPs, including XIAP, cIAP1 and cIAP2, predicts poor patient outcome in several types of cancer. XIAP binds to and inhibits caspases 3, 7 and 9. Inhibition of IAP function can enhance cell death in tumor cells. Inhibition of IAPs is being explored clinically with XIAP anti-sense (AEG35156) and small molecule inhibitor (HGS1029) strategies.

We describe small molecule inhibitors of the IAPs that have been designed to bind to the homologous BIR3 domains on the IAPs, which is the site of binding of second mitochondria-derived activator of caspase (SMAC). Structure-activity trends of IAP inhibitors were evaluated for binding potency and selectivity towards IAP BIR domains and analyzed for their ability to sensitize cancer cells to death alone or in conjunction with the agonistic TRAIL receptor 1 monoclonal antibody, mapatumumab, or chemotherapeutic agents.

Bridging selected compounds at various sites led to compounds with binding affinities for IAPs in the picomolar range. In cellular assays, IAP inhibitor compounds caused rapid and robust loss of cIAP1, consistent with stimulation of E3 ligase activity. In vitro cytotoxicity assays demonstrated sensitization of tumor cell lines to co-treatment with mapatumumab or chemotherapy at low nanomolar concentrations.

In vitro ADME studies were conducted in multiple species to select stable molecules which were negative in CYP and hERG binding assays. Selected compounds had high protein binding in plasma from multiple species. IV administration in mice resulted in Cmax values in excess of the EC50 required for cancer cell death in vitro.

Efficacy of lead compounds was demonstrated against several xenograft tumor models as single agents and in combination with either mapatumumab or conventional chemotherapeutics. This led to the identification of a highly potent compound series from which a clinical development candidate, HGS1029, was selected.

Solid tumor and lymphoid malignancies Phase 1 cancer trials with HGS1029 have been initiated.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 139.
Abstract 1392: HEDS selectively radiosensitizes human cancer cells in glucose-deprived environments via inhibition of Ku protein function
Authors: Ward Kathleen M. , Li Jie , Zhang Donglan , Dayanandam Eswarkumar , DeNittis Albert S. , Prendergast George C. , Ayene Iramoudi S.
Published: February 2011

There remains great interest in strategies to increase the efficacy of radiation therapy in cancer patients. Glucose depletion occurs commonly in certain regions of bulky solid tumors, owing to high glucose metabolism combined with poor perfusion caused by a disorganized blood vasculature. Evidence suggests that cancer cells located in glucose-deprived regions are resistant to therapeutic killing. Agents that sensitize glucose-deprived cancer cells might offer useful adjuvants for tumor radiotherapy. In an earlier study, we demonstrated that rodent cells which are genetically deficient in the oxidative pentose phosphate cycle (OPPC) display a heightened response to hydroxyethyldisulfide (HEDS), a thiol-specific oxidant that can radiosensitize cells by blocking the redox-dependent function of DNA repair protein Ku. Since glucose is the chief substrate of the OPPC, and the OPPC is responsible for detoxifying HEDS, we reasoned that glucose deprivation should phenocopy the effects of OPPC deficiency in enhancing radiosensitization by HEDS. To test this prediction, we compared the effects of HEDS treatment in human colon cancer cell lines HCT116 or HT29, which are radiation sensitive or resistant, respectively. In both cell lines, HEDS was detoxified by OPPC-mediated conversion to mercaptoethanol (ME) in high-glucose media but not in low-glucose media. Consistent with the decrease in cellular bioreductant activity by glucose depletion, HEDS greatly decreased free glutathione levels under low-glucose conditions. Similarly, HEDS greatly decreased the free thiol content of total proteins and impaired the Ku DNA binding activity in low-glucose conditions. Most significantly, in clonogenic cell growth assay, the cytotoxic effects of HEDS were greatly increased under low glucose conditions, when added by itself to HCT116 cells but particularly when added in combination with irradiation to HCT116 or HT29 cells. Together, our findings indicate that glucose depletion enhances the ability of HEDS to radiosensitize cancer cells. More broadly, our findings suggest the potential to use HEDS in a novel strategy to enhance killing of radioresistant regions of tumors that are characterized by a glucose-depleted state.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1392.