bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5259: Non-invasive determination of in vivo myeloperoxidase activity in ovarian cancer mouse model
Authors: Papineni Rao V. , Craveiro Vinicius , Pizzonia John , Holmberg Jennie , Mor Gil
Published: August 2012

Myeloperoxidase (MPO) is an inflammatory heme protein present in myeloid cells- neutrophils, microglia, and macrophages. The enzyme activity of MPO utilizes hydrogen peroxide in the process of reactive oxygen species generation. Myeloperoxidase has been suggested recently as a likely marker in ovarian carcinoma. This relationship between the MPO and ovarian cancer however was deduced primarily from data obtained from in vitro studies using cancer cell lines and from immunostaining of ovarian carcinoma paraffin blocks. Therefore, a real-time determination of in vivo MPO activity in the tumor and during the progression of ovarian cancer will be valuable to assess the role of innate immune process in ovarian cancer progression, in cancer treatment, and the proper management of current therapies. Here, we developed non invasive methodologies to determine the MPO activity in intraperitoneal ovarian cancer tumor mouse model. Tri-modal non invasive imaging of the metastatic ovarian cancer in athymic mice model were performed to obtain fluorescence, luminescence, and X-ray images at different time points during the progression of the intraperitoneal tumor metastasis. Progression of the tumor metastasis was monitored using fluorescence; simultaneously MPO activity was obtained by luminescence imaging. The MPO activity was detected by i.p injection of Luminol (5-amino-2,3-dihydro-1,4-phthalazine-dione) in physiological buffered saline (5 mg / 25gm mice). Luminol a redox-sensitive compound emits blue luminescence upon exposure to oxidizing agents, and in vivo, and has been established to have unique specificity to MPO activity. The fluorescence and luminescence images were overlaid on planar X-ray images for anatomical coregistration. The results show a robust increase in MPO activity in the mouse with the tumor development as compared to the control mice of the same cohort. Increase in MPO activity as a result of neutrophil activation was observed in the early phase of the ovarian cancer progression, and this increase subsides in the latter stages. However, the lumiscence signal intensities vary between the tumors, and near negligible MPO activity was observed in some tumors indicating a differential tumor microenvironment within the mouse. Further, the activity is far greater, at least in the initial stages, at the lung, nasal and other distal regions when compared to the intraperitoneal tumors. These real-time monitoring methodologies of MPO activity by in vivo imaging will greatly enhance the understanding of the tumor microenvironment during ovarian cancer progression, and contribute significantly in the development of better cancer treatments.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5259. doi:1538-7445.AM2012-5259
Abstract 5320: Sonic hedgehog in non-small cell lung cancer lymph node metastasis; ROS and PI3kinase/Akt signaling pathway
Authors: Cheon Seungwoo , Kang Myounghee , Kang Hanna , Kim Junglim , An Hyunsuk , Kim Hyunkoo , Yoo Younga , Kim Junsuk , Oh Sangcheul
Published: August 2012

Sonic hedgehog signaling pathway plays an important role in the metastasis of various organs. Recently, we found that increased shh producing cells correlate with enhanced migration, invasiveness and increased lymphangiogenesis in Non-small-cell lung cancer (NSCLC), but the mechanism is unknown. In this study, we investigated the function of shh pathway in human NSCLC. We performed immunohistochemistry on lung tumor biopsies and the result showed the expressions of shh, Gli1, VEGF-D and LYVE1 were enhanced in lymph node metastasis specimens compared to lymph node negative lung cancer specimens. We also investigated the role of shh signaling in the invasiveness and the motility of NSCLC. We found that Shh N-terminal peptide (N-shh) enhanced cell motility and invasiveness in NSCLC, and this process was inhibited by AAD-cyclopamine (SMO inhibitor). We observed that the mRNA expressions of shh, Gli1, VEGF-D and LYVE1 were enhanced by N-shh treatment, whereas no increase of these factors was observed when KAAD-Cyclopamine (a Shh signaling inhibitor) was treated together. In addition, we confirmed that Shh-induced lymphangiogenesis and motility/invasiveness of NSCLC are also reduced by LY294002, PI-3 Kinase/Akt inhibitor meaning that shh pathway and PI-3 Kinase/Akt are strongly related. Thus, we investigated the functional relationship between Shh and Akt signaling in the metastatic progression in NSCLC. Phosphorylation of Akt was enhanced by N-Shh, but regulated by KAAD-cyclopamine or LY294002. We further analyzed the effects of shh signaling in vitro by stimulating a HMVEC with N-shh and we observed N-shh enhanced the tube formation on matrigel, but the enhancement was inhibited upon cyclopamine or LY294002 treatment. The ROS production was increased in shh-overexpressing cells. These results indicate that shh signaling pathway can contribute to ROS generation enhancing the metastatic and lymphangiogenetic potential of tumor cells. Our findings suggest that Shh-Akt signaling processes through ROS and the activation of these pathway increases the metastatic potential of non-small cell lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5320. doi:1538-7445.AM2012-5320
Abstract 5321: Tyrosine kinase activation by the metastasis promoting protein S100A4
Authors: Grotterød Ida , Boye Kjetil , Mælandsmo Gunhild Mari
Published: August 2012

S100A4 is a protein linked to increased invasion and metastatic potential of cancer cells and the significance of S100A4 as a prognostic marker has been reported for different tumor types. It has no enzymatic activity and exerts its functions through interaction with other proteins, both intracellularly and in the extracellular space. Many functions and biological effects of S100A4 have been elucidated, but the detailed knowledge on receptor(s) affected and signaling pathways induced are not completely revealed. Induction of metastasis resembles changes occurring during epithelial-to-mesenchymal transition (EMT) and it has been suggested that several of the metastases promoting properties of S100A4 is caused by involvement in this process. In a previous study we identified a set of 136 genes affected by extracellular S100A4. Induced expression of some of these genes was validated and found to be dependent on NF-κB signaling. In the osteosarcoma cell line used, S100A4 activated NF-κB through IKK and IκBα phosphorylation, and the induced signal transduction was found independent of the previously suggested S100-receptor RAGE (receptor for advanced glycation end products). In the present study, tyrosine kinase arrays (PamGene) were utilized to screen for substrates phosphorylated by S100A4-activated kinases. Comparing levels of substrate phosphorylation, S100A4 stimulation significantly affected 32 substrates, including EGFR and PDGFR. EGFR and PDGFR phosphorylation has been validated using western immunoblotting and immunoprecipitation. The EGFR inhibitors AG 1478 and gefitinib reduced the level of basal and S100A4-induced EGFR phosphorylation. Interestingly, reduced levels of S100A4-induced IκBα phosphorylation and NF-κB activation were also observed. The mechanisms of EGFR activation are under further investigation. Of particular interest, EGFR could be activated by ligands cleaved from the cell membrane in a process involving ADAM activity, referred to as ectodomain shedding. Thus, we are in the process of testing the effect of ADAM inhibitors in our cell system and characterize the expression of specific ADAMs. Insight into the signaling mechanisms induced by S100A4 is important to understand how this protein is involved in the biological processes constituting the metastatic cascade and will provide valuable information for identification of novel therapeutic targets.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5321. doi:1538-7445.AM2012-5321
Abstract 5323: Fractalkine receptor CX3CR1 regulates peritoneal dissemination of ovarian carcinoma
Authors: Barbolina Maria V. , Xie Jia , Burdette Joanna E.
Published: August 2012

Epithelial ovarian carcinoma (EOC) is the deadliest gynecologic malignancy largely due to the metastatic disease. EOC metastases spread by shedding the malignant cells off of the ovarian surface and seeding tissues and organs of the peritoneal cavity. Currently used therapies, a combination of chemotherapy and surgery, fail to keep most patients in the remission. Mechanistic understanding of the biology of EOC metastasis will facilitate development of new therapeutic approaches. Our previous data demonstrated that fractalkine receptor (CX3CR1) is expressed by EOC cells and can support cell migration and proliferation. Moreover, our previous data suggested that CX3CR1-positive EOC cells can adhere to the CX3CL1-postive peritoneal mesothelial cells in in vitro assay. Thus, we hypothesized that CX3CL1/CX3CR1 axis could be important for peritoneal dissemination of metastatic EOC. To test this hypothesis, we performed short-term ex vivo and in vivo adhesion assay, as well as long-term metastasis formation assay. Expression of CX3CR1 in EOC cells was downregulated in SKOV-3 using shRNA. Both CX3CR1 shRNA-expressing and scrambled shRNA-expressing EOC cells were seeded atop of mouse peritoneum and omentum tissues in the ex vivo assay and allowed to adhere for up to 24 h. CX3CR1 shRNA-expressing and scrambled shRNA-expressing EOC cells were intraperitoneally injected into abdomens of athymic nude mice for 4 h following sacrifice of the animals and examination of the peritoneal organs and tissues for green fluorescence. Data analysis has demonstrated that downregulation of CX3CR1 in EOC cells led to a significant decrease of adhesion in these assays. Furthermore, parental SKOV-3 as well as CX3CR1 shRNA-, scrambled shRNA-, and vector shRNA-expressing cells were intraperitoneally injected into athymic nude mice and allowed to form metastasis for 10 weeks. As a result, we observed very small omental metastatic lesions in three out of six animals injected with CX3CR1 shRNA-expressing cells. In opposite, all of the control groups developed gross intraperitoneal metastasis in many organs and tissues. Our data suggest that CX3CR1 may be one of the key factors that determine peritoneal dissemination of EOC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5323. doi:1538-7445.AM2012-5323
Abstract 5330: Androgen independent promotion of prostate cancer cell invasion by HOXB13-mediated zinc regulation
Authors: Kim Young-Rang , Kim In-Je , Kang Taek Won , Choi Chan , Kim Min Soo , Nam Kwang Il , Jung Chaeyong
Published: August 2012

A healthy prostate maintains the high level of zinc while intracellular zinc is diminished during prostate cancer (PCa) developments. However, down-regulation of intracellular zinc in PCa are not fully understood, particularly in development of hormone refractory PCa (HRPC). Our previous studies showed that HOXB13 was overexpressed in HRPC. In our attempt to profile androgen-independent HOXB13 target genes in LNCaP PCa cells by DNA microarray, most strikingly regulated gene was ZnTs, a group of zinc output transporters. HOXB13 drove PCa cells to decrease intracellular zinc concentration. In the absence of androgen, HOXB13 dramatically promoted NF-κB-mediated signaling by reduction of inhibitor of NF-κB alpha (IκBα) and correspondingly stimulated nuclear translocation of RelA/p65. HOXB13-mediated NF-κB activation was nearly eliminated by suppression of ZnT4. At the same time, prostate tumors strongly indicated inverse correlation of HOXB13 and IκBα expression. Correspondingly, HOXB13 stimulated an invasive property of LNCaP cells, which can be negated by suppression of ZnT4 in an androgen-free environment. Taken together, these results demonstrate that HOXB13 promoted PCa cell invasion in an androgen-deprived condition by decreasing intracellular zinc concentration to further stimulate NF-κB-mediated transactivation, indicating that HOXB13 overexpression contributes to the malignant biological behavior of HRPC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5330. doi:1538-7445.AM2012-5330
Abstract 5337: Laminin alpha 2 enables glioblastoma stem cell growth
Authors: LI Meizhang
Published: August 2012

Glioblastomas (GBMs) are lethal cancers that display a cellular hierarchy parallel to the normal brain and are resistant to conventional therapies. It has been suggested that interaction between GSCs and their adjacent perivascular niche plays the critical roles to maintain their malignancy and self-renewal. Extracellular matrix (ECM) cues instruct stem cell behavior in many niches and the objective of our study was to elucidate its composition and contribution to GSC maintenance in the perivascular niche. We utilized human tumor tissue for immunofluorescence analysis and derived GSC for subsequent functional studies from tumor tissues. We find that laminin family of ECM proteins are located in the perivascular GBM niche and are negative prognostic indicators of patient survival. To identify the source of laminins, we characterized cellular elements within the niche and found that key laminins were expressed by non-stem tumor cells and tumor-derived endothelial cells (ECs). Targeting the laminin alpha 2 chain by RNA interference inhibited GSC growth and self-renewal. In co-culture studies of GSCs and non-stem tumor cells, laminin alpha 2 knockdown in non-stem tumor cells resulted in decreased GSC growth. These findings indicate that growth/proliferation of GSCs depends on the direct connection between extrinsic laminin alpha 2 and environmental matrix. Our studies highlight the contribution of non-stem tumor cell-derived laminin juxtracrine signaling. As laminin alpha 2 has recently been identified as a molecular marker of aggressive ependymoma, we propose that the brain vascular ECM promotes tumor malignancy through maintenance of the GSC compartment providing not only a molecular fingerprint but also a possible therapeutic target.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5337. doi:1538-7445.AM2012-5337
Abstract 5349: Cancer stem cells isolation and characterization from breast tumor of a germline carrier of TP53 p.R337H (Brazilian founder) mutation
Authors: Mambelli Lisley I. , Kerkis Irina , Hainaut Pierre , Achatz Maria Isabel W.
Published: August 2012

Li-Fraumeni syndrome (LFS), an inherited cancer predisposition syndrome, is associated with germ line mutations in TP53. It is characterized by high risk of multiple, early cancers. In Brazil, a variant form of LFS is exceedingly frequent due to a widespread founder TP53 mutation, p.R337H, detected in about 0.3% of the general population in Southern Brazil. This mutation occurs in p53 oligomerization domain and its effect on p53 oligomerization is supposed to be dependent upon pH conditions. We have isolated and characterized Cancer Stem Cells from tumors of p.R337H mutation carriers. After informed consent and surgical resection fragments were dissociated and brought in culture. Adherent cells and spheroids were derived from different tumor types. Spheroids derived from a breast cancer (BC) were further analyzed to demonstrate positive immunolabeling for CD44, CD24, Oct4 and Sox2 antibodies. Time-lapse videomicroscopy showed rapid growth, frequent asymmetric division and absence of senescent phenotypes for least 17 passages. At confluence, some cells spontaneously differentiated into adipogenic and muscle-like cells. Floating and adherent spheroids contained self-renewing cells capable of long-term proliferation without losing their primary characteristics. These properties are similar to those of stem cells with inactivated p53 protein. However, treatment of adherent cells with the DNA damaging agent doxorubicin elicited p53 accumulation, p21 induction, cell cycle arrest at low doses of Dox and cell death/apoptosis at higher doses. These results are consistent with the notion that p.R337H may retain a quasi-normal p53 function in response to stress while expressing properties similar to mutant p53 in long-term CSC culture. These p.R337H CSC may provide a very useful model for studying the defects underlying inherited cancer in p.R337H carriers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5349. doi:1538-7445.AM2012-5349
Abstract 5360: Towards identification of cancer stem cells in esophageal adenocarcinoma
Authors: Honing Judith , Meijer Coby , Plukker John T.M. , Kruyt Frank A.E.
Published: August 2012

Introduction: Cancer stem cells (CSC) are characterized by stem cell properties such as self-renewal potential and multilineage differentiation capacity. CSC's are thought to be the main drivers of tumor formation and progression and the main cause of therapy resistance and metastatic spread. In contrast to other cancer types such as glioblastoma and colon cancer, attempts thus far have not resulted in the identification of a proven CSC population in esophageal cancer. Therefore in this study we aim to identify and characterize CSC's in esophageal adenocarcinoma (EAC). Methods and Results: To address this goal, we attempted to culture CSC's from primary untreated EAC biopsy material using serum-free medium formulations that, however, have not been successful thus far. As an alternative model we employed two EAC cell lines, OE19 and OE33, which we were able to maintain as spheroids in serum-free Neural Basal Medium thought to enrich for stem cell growth. Dissociation of spheres and limiting dilution assays showed enhanced spheroid forming potential in OE19 but not in OE33 when compared to cells obtained from normal 10% FCS monolayer cultured counterparts. Next, the expression of several known CSC markers was determined by FACS analysis to determine self-renewal potential of sorted fractions in subsequent limited dilution assays. Side population, ALDH1, CD44/CD24 fractions were tested of which only CD44+ /CD24+ cells showed enhanced capacity to form spheroids. Tumor growths of these populations are currently being evaluated on immune-compromised mice. Other stem cell markers such as OCT4 and SOX2 were enhanced in OE19 spheroids compared to 10% FCS cultured cells. However, in OE33 spheroids expression of these markers were unchanged or downregulated. Since EAC derives from dysplastic epithelia with intestinal characteristics in Barrett's esophagus, we examined the status of the Wnt pathway known to be involved in maintenance of intestinal/ colon (cancer) stem cells. In the EAC cells β-catenin expression was present that could be enhanced by LiCl. Reporter assays to determine Wnt- activity are in progress. Finally, the sensitivity of spheroid versus monolayer cultured OE33 cells to 5-FU, cisplatin and paclitaxel was evaluated. Sensitivity did not differ much, although monolayer cultured OE33 cells were more resistant for 5-FU. Conclusion: Given the differences in the two spheroid models regarding spheroid forming potential, CSC-marker expression and chemosensitivity, caution is warranted when using esophageal adenocarcinoma cell line derived spheroids as model for CSC's. However, CD44 and CD24 might have a potential as CSC-markers in EAC, which is currently further corroborated.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5360. doi:1538-7445.AM2012-5360
Abstract 5361: Enriching and characterizing cancer stem cell grown on collagen type IV-coated plate in primary pediatric brain tumors
Authors: Kim Dong Ok , Park Jun Bum , Ra Young Shin
Published: August 2012

There is accumulating evidence that cancer stem cells (CSC) in adherent culture maintain and enrich their characteristics by extracellular matrix (ECM). This study focused on differential expression of not only CSC markers and differentiation markers, but also embryonic signaling pathways when cultured on various ECM-coated plates. Four cells from pediatric patients with brain tumor were grown in DMEM/F12 medium supplemented with 10ng/ml FGF, bFGF and 0.5x B-27. After forming of neurosphere, the cells were cultivated on each three kinds of ECM-coated plate including collagen type IV, fibronectin and poly-D-lysine. And the expression level of CD133, CD15, Nestin, Oct3/4, SOX2 as CSC markers, GFAP, β-tubuliln III for differentiation markers, Wnt, Notch, Shh, their transcription factors like β-catenin, Hes1, Gli1 respectively for embryonic signaling pathways was assessed by reverse transcriptase (RT)-PCR. As a result, two among four brain tumor cells grown on collagen type IV-coated plate expressed higher expression level of CD133, CD15, Nestin, SOX2, Oct3/4, Wnt5a and Notch than in the cells cultured on fibronectin or poly-D-lysine-coated plate. And residual two cells cultivated on collagen type IV and fibronectin, not on poly-D-lysine were shown similar expression level of almost all genes. There is no difference of Shh, Gli1, GFAP and β-tubuliln III expressions in all cells grown on each of three ECM-coated plates. In conclusion, collagen type IV is required for enriching and maintaining of CSC characteristics in primary pediatric brain tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5361. doi:1538-7445.AM2012-5361
Abstract 5367: Long-term survival in a phase II atudy of belagenpumatucel-L (TGF-β antisense modified tumor cell vaccine) in non-small cell lung cancer (NSCLC).
Authors: Bazhenova Lyudmila , Carrier Ewa , Shawler Daniel , Fakhrai Habib
Published: August 2012

Background: Cancer therapeutics vaccines continue to be investigated in a variety of solid malignancies. Belagenpumatucel-L (Lucanix®), a therapeutic vaccine comprised of 4 TGF-β2 antisense gene-modified allogeneic NSCLC cell lines. Phase II trial finished its enrollment in March of 2004 and was published in 2006. We now report an updated survival analysis with over 5 years of follow up. Methods: Three arms, open label clinical trial enrolled seventy-five subjects. 2 stage II, 12 stage IIIA, 15 stage IIIB, and 46 stage IV patients were randomized into three dose cohorts of 1.25, 2.5, or 5 107 cells/injection. Several cellular (ELISPOT and cytoplasmic cytokine expression) and humoral (antibody ELISA) immunity assays were also performed and correlated with survival. Results: Median follow up for all patients was 14.5 months and for patients with stable disease 44 months. Median survival for all subjects was 14.5 months and one-year, two-years and five-year survival were respectively 55%, 35% and 20%. Stages IIIB/IV subjects enrolled into cohorts 2 and 3 (N=40) had a median survival of 15.9 months and one-year, two-year and five-year survivals were respectively 61%, 41% and 18%. For stage IIIB/IV patients with non progressive disease following frontline chemotherapy, median survival was 44.4 months and five-year survival was 50%. For subjects who progressed following frontline chemotherapy, median survival was 14.1 months and five-year survival was 9.1%. Subjects who demonstrated an increase in both cellular and humoral immune reactivity following treatment had a significant survival advantage over subjects who showed an increase in only one measure of immunity with a median survival of 32.5 months vs. 11.6 months (p = 0.015). Conclusion: Long term follow up of a phase II clinical trial confirmed encouraging survival for patients with stage IIIB/IV disease. Based on these data, we have instituted an international, randomized, pivotal Phase III trial to evaluate the efficacy of belagenpumatucel-L in a maintenance setting in stage III/IV NSCLC patients who have stable disease or better following frontline chemotherapy. The trial is designed to enroll 506 patients and is powered to measure a 3.5 month survival difference. There are two planned interim analyses. To date, 285 patients have been enrolled in 8 countries. Confirmation of the phase II data in a randomized, phase III setting would provide an important improvement for the treatment of non-small cell lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5367. doi:1538-7445.AM2012-5367
Abstract 5406: Glioblastoma derived exosomes induce apoptosis in T cells
Authors: Sabin Keith Z. , Winn Robert J.
Published: August 2012

Intro: Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and is associated with an average lifespan of 15 months after diagnosis. One contributing factor to this poor prognosis is the immune protection afforded by the tumor microenvironment. One method of immune evasion poorly characterized is the effect of tumor released exosomes. Methods: FasL gene expression was determined using reverse transcription polymerase chain reaction (RT-PCR) in three GBM cells lines: LN-229, U-138 MG and T98G. Exosomes were isolated from serum free supernatants by differential ultracentrifugation. Exosomes were quantified using a protein concentration assay. A3 Jurkat T cells were treated with quantified exosomes, recombinant FasL or untreated. After 24 hours a TUNEL assay was performed to investigate the potential apoptotic effects of GBM-derived exosomes. Results: FasL is expressed in LN-229, U-138 MG and T98G cell lines as determined by RT-PCR. After treating A3 Jurkat T cells with exosomes it appeared apoptosis was induced as compared to an untreated control as determined by DNA fragmentation. Discussion: Studies using prostate and colon cancer cell lines showed that FasL was expressed in tumor derived exosomes. When T cells were co-cultured with exosomes apoptosis was induced in a FasL-dependant manner. Our lab previously demonstrated that GBM-derived exosomes are able to inhibit T cell proliferation. These current data suggests that GBM-derived exosomes induce apoptosis in T cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5406. doi:1538-7445.AM2012-5406
Abstract 5409: Cancer immunosuppression: The role of human leukocyte antigen G expression
Authors: Elliott Robert L. , Jiang Xian P. , Phillips Jeffery T. , Head Jonathan F.
Published: August 2012

Human leukocyte antigen G (HLA-G) is an immunotolerant nonclassical major histocompatibility complex class Ib molecule. During pregnancy it is expressed by trophoblastic placental cells to protect the fetus from maternal alloreactivity. HLA-G is involved in cancer immune evasion and is overexpressed by many tumors. Reverse transcription-polymerase chain reaction and immunohistochemistry were used to examine HLA-G expression in human normal breast and breast cancer cell lines, and human normal breast and breast cancer tissues. Reverse transcription-polymerase chain reaction confirmed that normal epithelial MCF-12A cells had no HLA-G mRNA, but cancer cell lines MCF-7, T47D, and MDA-MB-231 and NCI/Adr-Res had various levels of HLA-G mRNA expression. Thirty-eight breast cancers and 12 normal breast tissues were examined by immunohistochemistry. Fifty-eight percent (22/38) of cancers had medium to strong staining for HLA-G, whereas only 8% (1/12) of normal breast tissues had medium to strong staining, and the difference was statistically significant (p<0.05). HLA-G staining was found in the cytoplasm and membranes of cancer cells. In conclusion breast cancer cells overexpress HLA-G mRNA and protein, which probably contributes to immune evasion and treatment resistance. We believe HLA-G should be monitored in breast and other epithelial cancers as it may be both a prognostic indicator and a therapeutic target.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5409. doi:1538-7445.AM2012-5409
Abstract 5410: Tim-3 expression and function in natural killer cells from metastatic melanoma patients
Authors: da Silva Ines P. , Jimenez-Baranda Sonia , Gallois Anne , Kuchroo Vijay K. , Osman Iman , Bhardwaj Nina
Published: August 2012

BACKGROUND: Tim-3 (T-cell immunoglobulin domain and mucin domain 3) is a type-I glycoprotein receptor with pivotal roles in immune regulation and tolerance induction. Tim-3 has opposing roles in innate and adaptive immunity. It synergizes with TLR signaling in DCs inducing the production of inflammatory cytokines, while it acts as a negative regulator of Th1/Tc1 cell function by triggering T cell apoptosis upon interaction with its ligand, galectin-9. Tim-3 is also responsible for the functional exhaustion of T cells observed in some chronic infectious diseases and tumors, such as metastatic melanoma (MM). Natural Killer (NK) cells, innate immune cells which eliminate tumors and infected cells through cytotoxicity and IFNγ production, constitutively express Tim-3. Moreover, it has been described that some tumor cells (including melanoma cells) express and secrete galectin-9. However, little is known about the role of Tim-3 on NK cells, particularly in the presence of galectin-9, in healthy or melanoma patients. METHODS: Purified NK cells from PBMCs of 20 melanoma donors (MD) and 40 healthy donors (HD) were characterized according to the expression of Tim-3 by flow cytometry. Lamp-1 expression on NK cell surface was used to measure NK cell cytotoxicity against the following target cells: i) K562 cells which don't express Galectin-9 and ii) Gmel galectin-9+ and Gmel galectin-9- sorted melanoma cells. IFNγ production was assessed by intracellular staining after 4 hours culture in presence of IL-12. RESULTS: MD NK cells express higher levels of Tim-3 compared to HD NK cells (p<0.05). Moreover, MD NK cells have a defect in cytotoxicity, proliferation and IFNγ production, but it still remains unclear if Tim-3 is involved in this exhausted phenotype. So far, we found that Tim-3 expression by itself (without engagement of specific ligands) does not negatively affect NK cell cytotoxicity. However, the expression of Galectin-9 by the target cells induces a decrease in NK cell cytotoxicity (Gmel Gal-9+ vs Gmel Gal-9-). CONCLUSION: These data suggest that when engaged with Galectin-9, Tim-3 expression down-modulates NK cell cytotoxicity. Therefore, galectin-9 expression may be a possible mechanism for tumors to evade immune surveillance. Given that an increasing body of data supports an important role for Tim-3/Galectin-9 interaction in immune regulation in infectious diseases and cancer, deciphering the function of Tim-3 in different cell types will be critical for potentially targeting it therapeutically.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5410. doi:1538-7445.AM2012-5410
Abstract 5415: Expression of immunosuppressive leukocyte inhibitory immunoglobulin like transcript 3(ILT3) receptors increases with the development and progression of ovarian tumors
Authors: Khan Mohammad F. , Yellapa Aparna , Bahr Janice , Bitterman Pincas , Abramowicz Jacques , Basu Sanjib , Edassery Seby , Barua Animesh
Published: August 2012

Background: Immunosuppressive tumor microenvironment has been suggested as one of the barriers to anti-tumor immune response in patient with ovarian cancer (OVCA). Leukocyte Inhibitory Receptor Immunoglobulin-like Transcript 3 (ILT3) has been proposed as a factor involved in the suppression of immune response in several malignancies. However, the expression and role of ILT3 in the development and progression of ovarian tumors has not yet been studied. Objective: The goal of this study was to examine the expression and association of ILT3 in ovarian tumors in laying hen, a preclinical model of OVCA in humans. Methods: A cohort of mature White Leghorn laying hens with normal or low egg laying rates were evaluated by transvaginal ultrasound scan (15 normal hens and 30 hens with OVCA).All hens were euthanized and serum and ovarian tissues from all hens were collected and processed for immunohistochemistry (IHC), Western blotting (WB) and RT-PCR. Presence of tumors and tumor staging were confirmed by gross morphology and histology. Expression of ILT3 was examined by IHC, WB and RT-PCR. Results: Immune cell like cells in the ovarian stroma as well as epithelium of the normal ovaries and ovarian tumors expressed ILT3. The intensity of ILT3 expression was significantly higher in hens with OVCA than normal hens. Moreover, the intensity of ILT3 expression varied with histological subtypes of ovarian tumors and was significantly higher in serous OVCA than endometrioid OVCA. A band of approximately 55kDa of ILT3 protein was detected in the homogenates of normal ovarian surface epithelium or tumor epithelium as well as whole normal or ovarian tumor extracts. Similar to IHC observations, protein expression was stronger in hens with OVCA than in normal hens. Conclusion: This is the first study to show that immunosuppressive ILT3 is expressed by ovarian tumor epithelium and the intensity of expression increases as the disease progresses. Thus the results are consistent with the hypothesis that ILT3 may be involved in the suppression of anti-tumor immunity in OVCA. The laying hens may be a useful model to understand the OVCA-associated expression of ILT3. This animal model also offers the opportunity to develop and test anti-ILT3 therapy to enhance anti-tumor immunity against OVCA in humans. Support: Department of Defense (OC#93303) and Elmer Sylvia and Sramek Foundation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5415. doi:1538-7445.AM2012-5415
Abstract 5424: Docosahexaenoic acid (DHA) induces gene expression of RASSF1A through epigenetics mechanisms in breast cancer cells
Authors: de Cassia Borges de Castro Rita , Almeida Danielle Fontes de , Ravacci Graziela Rosa , Roela Rosimeire Aparecida , Mazzotti Tatiane Katsue Furuya , Brentani Maria Mitzi , Conti Aline De , Ong Thomas Prates , Moreno Fernando Salvador , Waitzberg Dan Linetzky
Published: August 2012

Background: Epigenetic changes, such as DNA methylation and post-translational histones modifications have an important role in mammary tumorigenesis. Changes in DNA methylation include global hypomethylation and hypermethylation of promoter regions of specific genes. Several genes are hypermethylated in breast cancer, especially the tumor suppressor gene RASSF1A, that have potential prognostic value. Epigenetic events are important as therapeutic targets due to its reversible feature. Dietary compounds can modulate the epigenetic processes in cancer. Experimentally, docosahexaenoic acid (DHA), a member of the omega-3 fatty acids, can reduce proliferation, induce apoptosis and decrease the invasive potential of breast tumor cells. However, the antitumor mechanisms of DHA and its ability to modulate epigenetic events are not yet fully elucidated. Objectives: To verify DHA action on RASSF1A expression and promoter region methylation in different human breast cancer cell lines. Material and Methods: Three different breast cell lines (MCF-7, MDA-MB-231, SKBR-3) were treated or not with 100 μM of DHA for 72 hours. Real time quantitative PCR were performed for gene expression quantification of RASSF1A. DNA methylation of the promoter region of RASSF1A was evaluated by methylation specific PCR. The western blot will be held for analyze of the histone 3 lysine 9 acetylated (H3K9ac) protein. Moreover, we will evaluate the phases of the cell cycle and apoptosis by flow cytometry. Results and Conclusion: Compared to control cells (not treated), DHA increased RASSF1A expression on MCF-7, but not in MDA-MB and in SKBR-3. These three transformed breast cells lines show methylation in specific region of RASSF1A promoter, indicating its role as a tumour supressing gene. DHA treatment increased RASSF1A promoter region hypomethylation in MCF-7, but not in MDA-MB and in SKBR-3. From this perspective, we can suggest that DHA could act by epigenetic mechanisms and reactivate RASSF1A, previously silenced. This action occured specifically in MCF-7, a less aggressive breast tumor cell line. It is expected that the present results promote better understanding of DHA role as a potential anti-oncogenic compound of breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5424. doi:1538-7445.AM2012-5424
Abstract 5435: Tolcapone inhibits catechol-O-methyl transferase-mediated methylation of (-)-epigallocatechin-3-gallate in vivo
Authors: Forester Sarah C. , Lambert Joshua D.
Published: August 2012

Green tea polyphenols (GTP) have been shown to exert anti-cancer effects in vitro and in vivo. Studies suggest that (-)-epigallocatechin-3-gallate (EGCG) is largely responsible for these anti-cancer effects. EGCG reduces lung cancer cell viability in vitro and reduces tumor incidence, multiplicity, and size in vivo. EGCG, however, is rapidly methylated by catechol-O-methyl transferase (COMT), a transformation that may limit its effectiveness in lung cancer prevention. The nitrocatechol drug tolcapone is a potent inhibitor of COMT and is used to mitigate the symptoms of Parkinson's disease. In this study, we examined the effect of tolcapone on the bioavailability of EGCG in male CF-1 mice. Plasma and tissue levels of EGCG and its major methyl metabolites in mice (n = 6) were determined by LC-MS following oral administration of EGCG (100 mg/kg b.w.), tolcapone (30 mg/kg b.w.), or the combination (100 mg/kg b.w. + 30 mg/kg b.w., respectively). In mice treated with EGCG for 720 min, plasma EGCG accounted for 34.6 % of the total (EGCG + methylated metabolites), whereas in mice treated with EGCG plus tolcapone this increased to 65.7 %. In urine, EGCG accounted for 9.4 % of the total, whereas in mice treated with EGCG plus tolcapone this increased to 100 % (metabolites not detected). Similar effects were observed in the organ samples. For example, tolcapone extensively inhibited COMT- mediated methylation of EGCG in the lungs of mice. In conclusion, tolcapone effectively increased the bioavailability of unmethylated EGCG in vivo. The combination, may therefore increase the cancer preventive effects of EGCG. Future work will test the effect of tolcapone on the bioavailability of other GTP, and also determine the effect of tolcapone/GTP combination in animal models of cancer. Funded by NIH grant AT004678.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5435. doi:1538-7445.AM2012-5435
Abstract 5437: Blueberries: Conventional cancer prevention
Authors: Kraus Ryan D. , Martinez Andres , Measom Kylie , Gill Matthew , Gaytri Gupta , Garrett Andrew , Robison Richard , O'Neill Kim L.
Published: August 2012

Coronary heart disease and cancer, besides remaining the two leading causes of death in the United States, share another common trait: they can both be attributed to lifestyle choices that increase their risk. Epidemiological studies suggest that the consumption of both an antioxidant and phytochemical-rich diet contribute towards the reduction of heart disease and several cancer types. There has been a substantial amount of interest in Vaccinium corymbosum, also known as blueberries, due to their high antioxidant levels which have been show to help prevent cancer of the colon, breast, and prostate. Blueberries exhibit a high content of both phytochemicals such as flavonoids and tannins; and antioxidants such as phenolic acid and resveratrol. Antioxidants provide protection against oxidative stress, a condition that has been suggested to contribute to a variety of diseases, including atherosclerosis, Parkinson's disease, and cancer. The purpose of this study was to explore the health benefits of organic vs. conventional blueberry consumption by comparing the antioxidant content of their individual components, including pulp, skin, seed, and juice using both the Hydrophilic Oxygen Radical Absorbance Capacity (H-ORAC) and the Lipophilic Oxygen Radical Absorbance Capacity (L-ORAC) assay. High-Performance Liquid Chromatography (HPLC) was also used to identify and quantify the amount of Vitamin C in both the organic and conventional blueberries. Blueberry samples were obtained from local distributors and the assays were replicated with five different lots of blueberries. Results indicated that conventional blueberries (33,388 TE/100g pulp, seed, skin and 607,700 TE/L juice) have higher antioxidant levels than organic blueberries (25,800 TE/100g pulp, seed, skin and 415,608 TE/L juice) across all samples. Conversely, HPLC determined a slightly higher Vitamin C content in the organically grown blueberries (592.879 μg/g) compared to conventional blueberries (386.947 μg/g). These results suggest that conventional blueberries may have a higher health benefit potential in preventing cancer and many other degenerative diseases that plague the world.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5437. doi:1538-7445.AM2012-5437
Abstract 5443: Prevention of breast cancer by spices: Involvement of miRNA and other molecular targets
Authors: Aqil Farrukh , Jeyabalan Jeyaprakash , Munagala Radha , Ravoori Srivani , Vadhanam Manicka V. , Gupta Ramesh C.
Published: August 2012

We have previously demonstrated the chemoprotective effects of dietary Apiaceae spices against estrogen induced mammary tumorigenesis in ACI rats by inhibiting cell proliferation and few cellular and molecular markers as early as three weeks of estrogen administration. Here we report inhibition of 17ß-estradiol-mediated mammary carcinogenicity by fennel, cumin and caraway and potential mechanisms. Groups of female ACI rats were given either AIN-93M diet or diet supplemented with fennel, cumin and caraway (7.5% each, w/w) and then treated with subcutaneous 17ß-estradiol silastic implants (1.2 cm; 9 mg). Four additional groups were maintained on control or experimental diets. Rats received diet and water ad libitum. Animals were euthanized when estrogen-treated group on control diet reached over 90% tumor incidence. Tumor palpation from 12 wks revealed first tumor incidence by 92 days in control group that was significantly delayed by 22, 37 and 29 days by fennel, cumin and caraway, respectively. At the end of the study (26 wks), the tumor incidence was found to be 96% in control group whereas only 83%, 55% and 90% animals had tumors in fennel, cumin and caraway groups, respectively. Significant reduction in tumor volume (661±123 vs 196±64, 138±50 and 313±81 mm3) and tumor multiplicity (4.2±0.4 vs 2.2±0.4, 1.1±0.3 and 2.5±0.5 tumors/animal) was also observed by fennel, cumin and caraway, respectively compared to control. All experimental diets significantly offset estrogen-related growth of pituitary prolactinomas, the plasma prolactin levels, and the cell proliferation marker, PCNA in the mammary tissues. The spice intervention had no significant effects on any of these markers in the absence of estrogen treatment. Mechanistically, all the experimental diets resulted in significant downregulation of estrogen-associated increase in the expression of the ERα and cyclin D1. We have recently reported ‘miRNA signature’ associated with estrogen treatment in the ACI rat model. Of the four candidate miRNAs, miR-182 and miR-375 which were overexpressed by 4- and 7-fold and miR-127 and miR-206 were downregulated by 17- and 35-fold, respectively with estrogen treatment. These miRNAs were significantly modulated favorably by dietary cumin analyzed thus far; molecules downstream of these miRNAs remain to be explored. Together, our data show that apiaceae spices can significantly delay and prevent estrogen-mediated mammary tumorigenesis, with maximal protective effects observed with cumin, by modulating different cellular and molecular targets, including key dysregulated miRNAs. (Supported by CA-125152 and Agnes Brown Duggan Endowment).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5443. doi:1538-7445.AM2012-5443