bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4231: A 3′-substituted 7-bromoindirubin induces apoptosis of human melanoma cells via inhibition of STAT3 and AKT signaling
Authors: Liu Lucy , Nam Sangkil , Wu Jun , Wang Yan , Skaltsounis Leandros , Jove Richard
Published: July 2012

Small-molecule inhibitors of STAT3 or AKT signaling represent potential molecular-targeted cancer therapeutic agents. Here, we investigated the role of STAT3 and AKT signaling in growth inhibition of human melanoma cells by a 3′-substituted 7-bromoindirunin derivative (MLS2438). Effects of MLS2438 on cell viability, apoptosis and expression of signaling proteins were evaluated by MTS cell viability assay, Annexin V staining and immunobloting analysis respectively in human melanoma cells. SRC and AKT in vitro kinase assays were conducted using recombinant kinases, substrates and 33P-labeled ATP. Immunodeficient NOD/SCID/IL2Rgamma null (NSG) mice were used in the animal studies. We show that MLS2438 inhibits STAT3 and AKT signaling, and induces apoptosis of human melanoma cells. MLS2438 directly inhibits SRC and AKT kinase activity both in vitro and in intact cancer cells. Moreover, MLS2438-induced apoptosis of human melanoma cells is associated with reduced phosphorylation of SRC, JAK, STAT3 and AKT in both dose- and time-dependent manners. Consistent with inhibition of STAT3 and AKT signaling, the anti-apoptotic protein Mcl-1 and pro-proliferative protein c-Myc were down-regulated. In contrast to the decreased phosphorylation levels of SRC, JAK, STAT3 and AKT, phosphorylation levels of the MAPK (Erk1/2) signaling protein were not inhibited in cells treated with MLS2438. Importantly, MLS2438 suppressed tumor growth in vivo with low toxicity in an A2058 human melanoma xenograft mouse model. Our data demonstrate that MLS2438, a novel natural product derivative, induces apoptosis of human melanoma cells associated with inhibition of STAT3 and AKT signaling. Our findings support further development of MLS2438 as a potential small-molecule inhibitor that targets STAT3 and AKT signaling in tumor cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4231. doi:10.1158/1538-7445.AM2011-4231
Abstract 4232: Naematoloma sublateritium extracts suppresses TNF-alpha-induced expression of adhesion molecules through down-regulation of NF kappa B in human umbilical vein endothelial cells
Authors: Lee Yu Ran , Jang Hyun Jae , Jeon Byeong Hwa , Kim Tae Woong , Kim Ji Hong , Choi Jong Woon , Choi Sunga
Published: July 2012

Inflammation is considered to play an important role in the cancer pathophysiology as a result of chronic reaction in vivo. Chronic inflammation has been involved in carcinogenesis including cellular transformation, proliferation, invasion, angiogenesis and metastasis as well as vascular diseases. During the carcinogenesis, several kinds of pro-inflammatory cytokines and chemokines which were produced by tumor-infiltrating inflammatory cells can activate NFκB transcriptional factor influencing proliferation, differentiation and movement of tumor cells. Therefore, the regulation of NFκB signaling pathway and might be a therapeutically impact diseases associated with cancer. Naematolma (=Hypholoma) is a basidiomycete of the fungus that is known to produce antitumor compound, clavaric acid. In this present study, we report for the first time that Naematoloma sublateritium butanol fraction (NSBF) dose-dependently inhibited TNF alpha-induced monocyte adhesion to endothelial cells. Anti-adhesive activity of NSBF was correlated with the suppressed expression of monocyte chemoattractant protein-1, interleukin-8, vascular cell adhesion molecule-1 and intercellular cell adhesion molecule-1 at both mRNA and protein levels. In addition, NSBF significantly regulated nuclear factor (NF)-kB activation by TNF-alpha in luciferase reporter assay. Interestingly, NSBF also inhibited cyclooxygenase-2 expression in dose-dependent manner. It suggests the down-regulation of cyclooxygenase-2 might be correlated with inhibition of proliferation in human breast cancer cell lines, MCF7 and MDA-MB-231. In conclusion, we propose that NSBF may be a safe and potential therapeutic agent against chronic inflammation such as cancers.

* This study was supported by Forest Sceince & Technology Projects (NO. S120910L110000) KFS (Korea Forest Service)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4232. doi:10.1158/1538-7445.AM2011-4232
Abstract 4234: Ursolic acid induces apoptosis in pancreatic cancer cells associated with inhibition of STAT3 and down-regulation of Bcl-XL
Authors: Leal Ana S. , Salvador Jorge , Jing Yongkui
Published: July 2012

Pancreatic cancer is the fourth leading cause of cancer death and without effective medical therapy. New agents acting on novel targets need to be developed. Natural products provide a promising source for developing effective anticancer agents. Oleanolic acid (OA) and ursolic acid (UA) are two natural triterpenoids with reported antitumor activities in several types of cancer. The antitumor effects of OA, oleanolic acid methyl ester (OAME) and UA were determined in pancreatic cancer Aspc-1 cells. Cell growth inhibition of the three compounds was determined using the MTT assay. The IG50s of OA, OAME and UA after treatment with UA for 4 days are >30, 8.9, 12.5 μM, respectively. OA is a weak cell growth inhibitor and methyl esterification improves its antitumor activity. UA is more effective than OA to inhibit cell growth in Aspc-1 cells and was used to study the mechanism of action. Aspc-1 cells treated with UA at a concentration higher than 25 uM induced apoptosis as determined by PARP cleavage detection. Western blot analysis revealed that the levels of procaspase-3, -9 and Bcl-XL were markedly decreased while the levels of Mcl-1, XIAP and survivin were weakly or not decreased. Correlated with the decrease in the level of Bcl-XL, the level of phosphorylated STAT3 was significantly inhibited. STAT3 is activated in pancreatic cancer cells and contributes to pancreatic tumorigenesis. Activated STAT3 increases the levels of antiapoptotic protein such as Bcl-XL, Mcl-1 and survivin, are being considered as a target for developing agents for pancreatic cancer treatment. Our data suggest that UA is a lead candidate for targeting STAT3 and structural modification of UA may result in a new group of compounds with improved antitumor activity in pancreatic cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4234. doi:10.1158/1538-7445.AM2011-4234
Abstract 4248: Antiangiogenic mechanisms of Aflibercept in mouse hepatoma model
Authors: Torimura Takuji , Ueno Takato , Iwamoto Hideki , Nakamura Toru , Koga Hironori , Yano Hirohisa , Sata Michio
Published: July 2012

[Background/Aim] VEGF plays a critical role in vascular development in tumor tissues. A new angiogenic inhibitor, aflibercept is a soluble decoy VEGF receptor created by fusing the Ig domains of VEGF receptor1 and 2 and captures VEGF and PlGF in the blood stream. We have reported that aflibercept suppresses tumor growth by inhibiting vascular development in mouse hepatoma model. In the present study, we investigated the precise antiangiogenic mechanisms of aflibercept in mouse hepatoma model.

[Materials and methods] in vitro study: The inhibitory effects of aflibercept on cell proliferation of HUVEC and on phosphorylation of VEGF receptor 1 and 2 were evaluated. Influence of VEGF, PlGF and aflibercept on the phosphorylation of VEGF receptor 1 and 2, PI3K, AKT, and mTOR and the expression of VEGF by murine hepatoma cell line (Hepa1-6) were evaluated. The inhibitory effect of aflibercept on endothelial progenitor cells (EPC) differentiation was evaluated by colony-forming units (CFU) of EPC. I n vivo study: Aflibercept was injected intraperitoneally twice a week into tumor bearing nude mice of hepatoma cells (HuH-7, HAK1-B, KYN-2, Hepa1-6). Microvessel density in tumor tissue was counted. Hepa1-6 was implanted to GFP-bone marrow transplanted (BMT) mice to investigate the inhibitory effect of aflibercept on homing of EPC to tumor tissues. The expression of PlGF, VEGF in tumor tissue and Hepa1-6 cells was evaluated.

[Results] in vitro study: Although VEGF stimulated the cell proliferation of HUVEC, PlGF did not upregulate the cell proliferation. Aflibercept suppressed the phosphorylation of VEGF receptor-1 and 2 and also inhibited the cell proliferation of HUVEC. In Hepa1-6 cells, VEGF phosphorylated VEGF receptor-1 and 2. PlGF phosphorylated VEGF receptor-1. VEGF and PlGF phosphorylated PI3K, AKT and mTOR. Aflibercept suppressed the phosphorylation of VEGF receptor-1, 2, PI3K, AKT and mTOR. Aflibercept suppressed the upregulated VEGF production by PlGF in Hepa1-6 cells. in vivo study: Aflibercept suppressed the homing of bone marrow cells to tumor tissues, the differentiation of bone marrow cell to EPC and also reduced microvessel density. The expression of VEGF in tumor tissue was suppressed by aflibercept administration.

[Conclusion] Aflibercept suppresses the vascular development in hepatoma by inhibiting endothelial cells proliferation, VEGFavailability, homing of bone marrow cells to tumor tissue and EPC differentiation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4248. doi:10.1158/1538-7445.AM2011-4248
Abstract 4249: NGR-TNF, a novel direct-acting vascular targeting agent, does not induce “cytokine-rebound” recruitment of pro-angiogenic bone-marrow derived cells
Authors: Matteo Paola Di , Hackl Christina , Jedeszko Christopher , Valentinis Barbara , Bordignon Claudio , Traversari Catia , Kerbel Robert S. , Rizzardi Gian-Paolo
Published: July 2012

NGR-TNF, presently in advanced clinical development, is a direct-acting vascular targeting agent coupling the CNGRCG peptide (NGR), homing to angiogenic blood vessels, and tumour necrosis factor alpha (TNF). In order to further elucidate the mechanism of action, we investigated whether NGR-TNF mobilizes bone-marrow derived cells (BMDCs) and growth factors. Herein, treatment with either certain chemotherapy drugs at maximum tolerated dose (e.g. paclitaxel, cyclophosphamide, 5-FU), vascular-disrupting agents (VDAs; e.g. fosbretabulin, Oxi4503), or local irradiation can rapidly induce mobilization and subsequent tumor homing of proangiogenic BMDCs, such as circulating endothelial progenitor cells (CEPs), CD11b/Tie-2-expressing monocytes (TEMs), CD11b+Gr1+ neutrophils and myeloid-derived suppressor cells (MDSCs), and CD11b+F4/80+ macrophages. Increase in several growth factors and chemokines, such as SDF-1, G-CSF as well as osteopontin, contribute to the mobilization of BMDCs that colonize the tumour site and induce angiogenesis and rapid tumor regrowth. Lewis lung carcinoma (LLC) cells were subcutaneously implanted into immunocompetent C57BL/6J mice or into GFP+ bone marrow-chimeric mice, obtained by transplanting into lethally irradiated C57BL/6J mice bone marrow cells from UBI-GFP/BL6 donors. Blood was obtained 4 or 24 hours after treatment with either NGR-TNF, VDA, an anti-VEGFR2/flk-1 antibody or saline, by either cardiac puncture or retro-orbital sinus bleeding. Using flow cytometry (LSRII), CEPs were defined as CD45-/CD13+/flk-1+/CD117+/7AAD-, TEMs as CD45+/CD11b+/Tie2+/flk-1+ and MDSCs as CD45+/CD11b+/Gr1+ cells. Levels of circulating growth factors were assessed by ELISAs. When administrated at low doses (optimal biological dose), comparable to doses being used in clinical trials, NGR-TNF causes a decrease of tumor blood vessels density and induces apoptosis of tumor cells and tumor endothelial cells in vivo. It is worth noting that, unlike the above-mentioned treatments, NGR-TNF fails to mobilize or recruit to the tumor site different subtypes of pro-angiogenic BMDCs nor induces growth factors. Therefore, low doses of NGR-TNF exert an antitumor activity without inducing reactive a pro-angiogenic host response, which is likely an important feature for preventing/overcoming resistance and for designing combination therapeutic strategies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4249. doi:10.1158/1538-7445.AM2011-4249
Abstract 425: Alteration of ADAM9 and integrin interaction regulated cancer invasion and metastasis potential
Authors: He Yun-Chi , Liu Jer-Ming , Sung Shian-Ying
Published: July 2012

Alteration of integrin composition is highly correlated with cancer malignancy and metastasis. Recent evidence demonstrated the metastasis activities involved the interaction of integrin and ADAM gene family, such as ADAM8, ADAM9, ADAM10 and ADAM17. These may either stabilized or cleavage of integrin-matrice interaction at the lamellipodia of cancer cell. Mahimakar et al., recently demonstrated the interaction of ADAM9 with integrins alpha3, alpha6 and beta1 by pull down assay, suggests ADAM9 might work together with β1 class integrins. To further analyze their roles and possible downstream regulatory proteins, we perform knockdown of ADAM9 using shRNA and determine its role in cancer metastasis both in vitro and in vivo. Western blotting confirmed the knockdown of ADAM9 in prostate cancer, and oral cell carcinoma. Cell migration assay was performed using micro-slide Chemotaxis chambers either with or without collagen coating. Time-Lapse microscope analysis of cell migration was plotted and analyzed. Interestingly, we observed the opposite cell migration behavior in which faster and longer cell migration activities was observed in cancer cell with knockdown of ADAM9 expression in the chamber coated with collagen, whereas less migration activities was observed in chamber without collagen coating. This may reflect the possible role of ADAM9 in alteration of integrin composition or function. Flow Cytometry analyses of integrin composition on the surface of prostate cancer cells revealed no integrin composition change in knockdown strain compared to parental. We also notice highly expression of integrin alpha2beta1 and alpha6beta4 in C4-2 and PC3 cells. Analyses of signaling pathways demonstrated no significant changes in p-FAK, p-Erk and p-Akt, suggesting not through beta1 downstream signal regulation. Immunoprecipitation of ADAM9 demonstrated the highly associated with integrin beta4 but not beta1. Intra-cardiac injection of prostate cancer cell line, PC3-pSM2C, PC3-siADAM9 demonstrated 100% metastasis activities in PC3-pSM2C, and no any metastasis in mice with PC3-siADAM9. In summary, we notice ADAM9 may associated with the activation/stabilization of integrin complex. Knockdown of ADAM9 demonstrated decreased of cell migration on dish and metastasis when inoculated in mice, suggests hemidesmosome-ADAM9 interaction may involved in cancer migration and metastasis potential.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 425. doi:10.1158/1538-7445.AM2011-425
Abstract 4259: Preclinical anti-tumor study of henatinib, a novel and selective inhibitor of VEGFR-2 in phase I clinical trials
Authors: Quan Haitian , Chengying Xie , Xu Yongping , Liguang Lou
Published: July 2012

Inhibition of VEGF/VEGFR-2 signaling represents a promising therapeutic approach to solid malignancies. Here, we demonstrated that henatinib, a selective VEGFR-2/KDR inhibitor, exhibited potent anti-angiogenic and anti-tumor activity. Henatinib inhibited the activity of VEGFR-2, c-kit, PDGFRα and PDGFRβ with IC50 values of 0.6 nM, 3.3 nM and 41.5 nM, respectively. Henatinib significantly inhibited VEGFR-2 phosphorylation and its downstream signal pathway in human umbilical vein endothelial cells (HUVECs), and consistently inhibited VEGF-stimulated HUVEC proliferation, migration and tubule formation, and micro-vessels spouting from matrigel-embedded rat aortic rings. In vivo, henatinib exhibited broad and potent anti-tumor activity, leading to regression or growth arrest of various established xenografts derived from human tumor cell lines. In summary, preclinical data indicate that henatinib is a novel and selective VEGFR-2 inhibitor with potent in vivo anti-tumor activity. Henatinib is currently in phase I clinical trials in China.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4259. doi:10.1158/1538-7445.AM2011-4259
Abstract 426: The molecular microenvironment critically affects the formation of isoDGR-dependent integrin binding sites in fibronectin
Authors: Curnis Flavio , Sacchi Angela , Longhi Renato , Corti Angelo
Published: July 2012

Fibronectin (FN) is an adhesive protein present in most body fluids and in the extracellular matrix of many tissues. This protein mediates a variety of cellular interactions with extracellular matrix and play important roles in haemostasis, thrombosis, inflammation, wound repair and angiogenesis. FN is constituted of three types of repeating homologous modules termed FN-I, FN-II and FN-III. Each module contains binding sites for molecules of the extracellular matrix and for cell adhesion receptors, such as integrins. Moreover, FN contains four NGR sites located in the: 5th type I, 2nd type II, 7th type I repeat, 9th type III repeats. We have previously shown that the NGR site of the 5th type I repeat (FN-I5) rapidly undergoes deamidation leading to formation of isoDGR. A deamidated FN fragment corresponding to this domain binds αvβ3, inhibits endothelial cell adhesion and proliferation and inhibits tumor growth in animal models. In the present study we have investigated the molecular mechanisms that regulate isoDGR formation in FN in vivo. We provide evidence to suggest that FN purified from plasma contains very low amounts of isoDGR (<3%) despite its long half life (48h). In contrast, using anti-isoDGR antibodies, we observed that isoDGR is formed in significant amounts in small FN fragments injected in mice and left to circulate for 4 or 6 h (>17% in 4 h). In vitro cell adhesion assays, antibody binding assays and integrin binding studies showed that FN deamidation can rapidly occur when FN is bound to microtiter plates, a condition known to cause conformational changes in this protein, but not with FN in solution. Rapid deamidation was observed to occur also with short fragments (FN-I5), either adsorbed on microtiter plates or in solution. These results suggest that the kinetics of NGR-to-isoDGR transition in plasma FN is slower than its clearance rate. Furthermore, the finding that conformational changes and/or protein fragmentation increase the kinetics of NGR-to-isoDGR transition suggest that changes in the molecular microenvironment of NGR sites, potentially occurring after FN deposition in tissues, could be critical for the formation of isoDGR integrin binding sites.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 426. doi:10.1158/1538-7445.AM2011-426
Abstract 4262: Regorafenib (BAY 73-4506): A broad spectrum tumor deactivator with high combinability potential and antimetastasis activity
Authors: Zopf Dieter , Scholz Arne , Fichtner Iduna , Becker Michael , Müller Tina , Kissel Maria
Published: July 2012

Regorafenib is an oral multi-kinase inhibitor which is in clinical development for the treatment of patients with metastatic and/or unresectable gastrointestinal stromal tumors who have failed imatinib and sunitinib therapy and patients with advanced colorectal tumors who have failed standard therapies.

Regorafenib is a tumor deactivation agent that addresses all three mechanisms of tumor growth and progression (angiogenesis, oncogenesis, and stromal interactions) by potently blocking multiple RTKs. Inhibition of angiogenic RTKs (e.g., VEGFR1-3, TIE2) by regorafenib results in breakdown of tumor vascular structure, which disrupts the tumor's oxygen and nutrient supply. Furthermore, regorafenib targets oncogenic RTKs (e.g., KIT, Ret), which control tumor growth, and stromal RTKs (e.g., PDGFR, FGFR), which are implicated not only in the establishment of tumor infrastructure but also in tumor progression.

Regorafenib was evaluated for additional tumor indications in several human cancer xenograft and syngeneic tumor preclinical models as single agent or in combination with standards of care or targeted agents in early clinical development. Regorafenib effectively inhibited tumor growth in a broad spectrum (e.g. liver, stomach, ovarian, breast and colon) of subcutaneous and orthotopic models of human xenograft and murine syngeneic tumors at an oral daily dose of 10mg/kg. Regorafenib inhibited various patient-derived oxaliplatin insensitive colorectal xenografts as single agent and in combination with concomitant irinotecan dosing. Finally, regorafenib was shown to exert potent antimetastasis activity in orthotopic syngeneic 4T1 and triple-negative MDA-MB 231 human xenograft breast cancer models by prevention of the formation of lung or lymph node metastasis, respectively.

In summary, by blocking multiple abnormal angiogenic and oncogenic signalling pathways, regorafenib deactivates tumors and prevents their continued growth and metastatic potential. In preclinical models regorafenib has shown a high potential as a combination partner with other therapeutic agents such as irinotecan, which may make it suitable as a future backbone for treatment of multiple cancer types.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4262. doi:10.1158/1538-7445.AM2011-4262
Abstract 4263: Dopamine ameliorates hematological complications in tumor bearing animals
Authors: Sarkar Chandrani , Chakroborty Debanjan , Basu Sujit
Published: July 2012

One of the major complications of anti-VEGF-A (vascular endothelial growth factor-A) agents are their serious hematological toxicities. We had earlier shown that the catecholamine neurotransmitter dopamine is a potent inhibitor of VEGF-A induced tumor angiogenesis and thereby, tumor growth. Therefore, it is prudent to investigate the effects of dopamine on important hematological parameters in preclinical cancer models. Our study shows that in addition to inhibiting tumor angiogenesis and growth of colon and prostate cancer bearing animals, dopamine can also significantly improve hemoglobin concentration, RBC and platelet counts in these animals. Anemia and thrombocytopenia are the two most common hematological toxicities induced by anti-VEGF-A drugs, which in turn increases the morbidity in cancer patients treated with these agents. Our report here shows that the anti-angiogenic action of dopamine inhibits tumor angiogenesis and growth in preclinical models without adversely affecting hematopiesis; rather it ameliorates anemia associated with cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4263. doi:10.1158/1538-7445.AM2011-4263
Abstract 4265: Bithionol as an inhibitor of tumor angiogenesis
Authors: Zhai Ling , Roy Anshu M. , Zamani Ali , Liu Ximeng , Wang Yimin , Li Rongbao , Qu Zhican
Published: July 2012

Tumor progression depends on angiogenesis. The clinical successes in recent years have demonstrated that antiangiogenic agents can be developed as effective treatment for cancer patients. However, the resistance to the current FDA approved antiangiogenic therapies is emerging, which presents challenges in cancer research and urgent needs for novel angiogenesis inhibitors with different mechanisms of drug actions to overcome the resistance. In our previous studies, we have identified a small molecule bithionol as a potential antiangiogenic agent through a chemical diversity library screening with a cell-based angiogenesis assay. Bithionol is a FDA approved and current clinically used anthelmintics, which has been found to directly inhibit activity of autotaxin, a secreted enzyme that catalyzes lysophosphatidic acid (LPA) production. LPA, as a lipid signaling molecule, is a potent angiogenic and cancer cell motility stimulating factor. Therefore, autotaxin plays important role in tumor angiogenesis and metastasis and has been recently identified as an attractive angiogenesis and cancer target. Our recent studies have shown that autotaxin is expressed at high levels in human endothelial and number of human cancer cell lines. Bithionol has demonstrated inhibitory activities against human endothelial cell proliferation, migration, and tube-formation as the three key cellular steps in angiogenesis process. The effect of bithionol on inhibition of tumor angiogenesis was also evaluated with an ex vivo xenograft chicken embryo chorioallantoic membrane (xenograft-CAM) model system. Bithionol is able to inhibit tumor-induced new blood vessel formation in a dose dependent manner and has demonstrated an additive inhibitory activity when it is applied in combination with Sutent, a current clinically used antiangiogenic drug. The results of this research suggest that bithionol provide a unique promising opportunity in developing new combinational treatment to increase the anticancer efficacy and minimize the resistance in cancer patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4265. doi:10.1158/1538-7445.AM2011-4265
Abstract 4267: Biomimetic anti-angiogenic peptide as therapeutic agent for breast cancer
Authors: Rosca Elena V. , Penet Marie-France , Koskimaki Jacob E. , Pandey Niranjan B. , Bhujwalla Zaver M. , Popel Aleksander S.
Published: July 2012

Despite the continuous research in novel therapeutics, globally each year more people succumb to cancer than HIV, TB and malaria combined. Angiogenesis, the development of neovasculature, has been a major focus of cancer research, and it resulted in the development, approval and clinical use of bevacizumab, an anti-VEGF monoclonal antibody. However, in the case of breast cancer, the results show gains in progression-free survival but not in overall survival. Other agents targeting VEGF receptors are under investigation with the premise that a cocktail of agents could be more effective.

Recently, there has been great interest in the development of peptides as therapeutic agents due to their high specificity and low toxicity, which are conducive for the development of new therapeutic cocktails. Identification of pharmacophores (minimal information content) allows for the introduction of non-natural amino acids in the peptide sequence to increase its stability while maintaining or improving its activity, thus creating optimized peptides for clinical applications. We present the application of a 20-mer biomimetic peptide derived from the α5 fibril of collagen IV in which a cysteine is replaced by a non-natural amino acid, i.e., amino-butyric acid, and investigate its mechanism of action as an anti-angiogenic agent in treatment of breast cancer. We demonstrated its activity in suppressing tumor growth in an orthotopic triple negative breast tumor model. In vivo magnetic resonance imaging (MRI) demonstrated reduction in vascular permeability and vascular volume. In vitro studies of receptor pulldown and cellular surface proximity staining demonstrate that this peptide interacts specifically with the αVβ1 integrin. This biomimetic stable peptide is a novel non-VEGF targeting agent active as a single agent, or that could be used in combination with chemotherapy or other therapeutic peptide-based cocktails.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4267. doi:10.1158/1538-7445.AM2011-4267
Abstract 4268: Interleukin-27 inhibits angiogenesis in non-small cell lung cancer
Authors: Kachroo Puja , Zhang Ling , Walser Tonya C. , Cui Xiaoyan , Hazra Saswati , Lin Qiang Ying , Minna John D. , Dubinett Steven M. , Lee Jay M.
Published: July 2012

Interleukin-27 (IL-27), the newest member of the IL-6/IL-12 family, is secreted by antigen presenting cells and has anti-tumor activity through its activation of the JAK-STAT pathway. Transcriptional factors STAT1 and STAT3 have been implicated in the balance of tumor suppressor and oncogenic functions. Angiogenesis is a critical process during carcinogenesis. We investigated the role of IL-27 in tumor angiogenesis in lung cancer. Human bronchial epithelial cells (HBECs), genetically modified HBEC to over express Snail and K-ras (HBEC-Snail, HBEC-K-ras), and human non-small cell lung cancer (NSCLC) lines (A549, H2122) were treated with IL-27 at multiple time points. Phosphorylation of STAT1 and STAT3 measured by Western Blot after IL-27 exposure demonstrated activation of both pathways in these cell lines. Protein production of an important pro-angiogenic factor, vascular endothelial growth factor (VEGF), was measured by ELISA and Western analysis. After exposure to IL-27, there was a 60-80% decrease in VEGF protein expression of VEGF by ELISA (confirmed by Western analysis in the NSCLC lines but not in the HBECs or their genetically modified derivatives). The IL-27 down regulation of VEGF was not increased by the addition of a STAT3 inhibitor. In summary, IL-27 inhibits VEGF production in NSCLC through a mechanism independent of STAT3, suggesting that STAT1 may play a dominant role in IL-27 mediated angiogenesis suppression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4268. doi:10.1158/1538-7445.AM2011-4268
Abstract 4272: Hexastatin is a potent therapeutic agent for targeting tumor angiogenesis and tumor growth
Authors: Akulapalli Sudhakar , Boosani Chandra Shekhar , Laknaur Archana , Gunda Venugopal
Published: July 2012

Peptides or short protein domains derived from the extracellular matrix are reported to possess anti-tumor properties. In the present study, one such molecule “Hexastatin or [α6(IV)NC1]” a non-collagenous domain of α6 chain of type (IV) collagen was derived from the carboxy terminal and was found to inhibit tumor growth, but the mechanism by which it inhibits the growth of solid tumors has not been reported yet. In the present study, we identified that the biological functions of hexastatin are attributed to its binding to different cell surface integrins. We identified that hexastatin binds to α3β1, αVβ3 and α5β1 integrins and inhibits p38-MAPK signaling. Hexastatin competes with human vascular endothelial cells in binding to α1β1 integrins on type IV collagen, and or with α5β1/αVβ3 integrins on fibronectin/vitronectin matrix, thus inhibiting endothelial migration and tube formation. Interestingly, p38-MAPK phosphorylation was not inhibited in α3 and β3 integrin null endothelial cells upon treatment with Hexastatin which confirms that the antiangiogenic functions of Hexastatin are possibly mediated through α3β1 and αVβ3 integrins. While both the integrins (α3β1 and α5β1) are required for the inhibition of tube formation by Hexastatin in endothelial cells, only α3β1 integrin was found to regulate endothelial cells migration. In addition we also demonstrate that Hexastatin inhibits tumor growth, tumor angiogenesis and circulating endothelial cells in-vivo. These in-vitro and in-vivo findings indicate that α3β1 and αVβ3 integrins are critical for Hexastatin mediated inhibition of angiogenic signaling and tumor progression. Collectively, our findings demonstrate that hexastatin is a potent therapeutic agent for targeting tumor angiogenesis and tumor growth.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4272. doi:10.1158/1538-7445.AM2011-4272
Abstract 4274: Characterization and drug sensitivity testing of HTS compatible cancer microtissues
Authors: Helbling Marianne M. , Drewitz Maren , Bieri Manuela , Lotz Carina , Wyder Lorenza , Lehembre Francois , Lichtenberg Jan , Moritz Wolfgang , Regenass Urs , Kelm Jens M.
Published: July 2012

The tumor microenvironment strongly effects the drug response. Although the advantages of organotypic 3D-cell-culture models are known for years, complex production and readout processes impede the implementation in an industrial setting. This study, presents for the first time a novel multi-well platform in a standard 96-well format to produce organotypic, scaffold-free cancer microtissues models and its implementation in an automated screening process. This technology allows to produce either homotypic or heterotypic tumor microtissues in a hanging-drop culture by liquid top-loading similar to standard 96-well plates. The core piece of the well is a microfluidic channel connecting an inlet funnel at the top and an outlet funnel at the bottom of the plate, where a hanging drop is formed and stabilized by a combination of capillary and surface-tension forces. The use of this technology enables the automated production of microtissues with a very narrow size distribution below 10% on plate and batch-to-batch.

3 cancer microtissue models were developed from two colon cancer and one breast cancer cell lines and characterized in terms of their proliferative capacity and growth characteristics. Process parameters for media exchange, addition of compounds, microtissue-size variances and implementation of cell-based viability assays (ATP, LDH and size) were established. As a proof of concept, a whole screening process using four reference compounds was performed in comparison to classical monolayer cultures underlining the different behavior of both cell-culture models. Finally, four novel compounds that inhibit tumor cell proliferation in monolayer cultures, were tested for their potential in a 3-D system.

Overall, we could demonstrate that organotypic microtissues can be implemented into an automated screening process using standard liquid-handling robotics. The use of this system in drug development may strengthen the predictive force of the in-vitro evaluation of novel drug candidates and reduce drug failure in clinics.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4274. doi:10.1158/1538-7445.AM2011-4274
Abstract 4275: A prostate cancer spheroid related gene and chemotherapy
Authors: Watanabe Masatoshi , Takagi Akimitsu , Hirokawa Yoshifumi , Shiraishi Taizo
Published: July 2012

Spheroid culture is one of the three-dimensional (3-D) culture models. We have constructed a spheroid culture model for prostate cancer therapy and characterized this model. The change of global gene expression profiles in spheroids and solid tumor tissues were also compared with monolayer cultures, leading to extraction of candidate genes in contexts that more closely mimic tumor growth in vitro under different in vitro growth conditions. It is possible that these genes may be specific to the construction of spheroids and reveal the possibilities of new mechanisms of acquiring anticancer drug resistance or sensitivity. The plk-2 was extracted as a candidate

e gene related with spheroid formation. A member of the polo-like kinase (PLK) family, PLK2 is one of highly conserved serine/threonine protein kinases. In this study, we analyzed effects of the PLK2 on the spheroid characterization and anticancer drug resistance in DU-145 spheroids by suppressing plk2 with RNAi. When docetaxel (DTX) was exposed for siRNA-transfected spheroids, the viability changed, the volume decreased at each concentration of DTX, and spheroids were collapsed at last. The result indicates that the plk2 may be involved in acquiring anticancer drug resistance.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4275. doi:10.1158/1538-7445.AM2011-4275
Abstract 4276: Characterization of an ex vivo primary multicellular renal cell culture as a potential model system for renal cell carcinoma tumorigenesis
Authors: Arreola Alexandra , Rathmell W. Kimryn
Published: July 2012

Sporadic and germline mutations or loss of the von Hippel-Lindau (VHL) tumor suppressor gene have been linked to varying levels of risk for Renal Cell Carcinoma (RCC). RCC is a highly prevalent malignancy that affects roughly 56,000 people annually in the United States. With no true murine model for RCC, patient derived renal carcinoma cell lines and mouse embryonic stem cell lines have been largely used for the study of this malignancy. The purpose of this study is to generate an ex vivo model for Renal Cell Carcinoma using primary neonatal epithelial kidney cells grown as multicellular cultures. To better understand the genetic changes associated with RCC tumor formation following VHL loss or inactivation, primary epithelial kidney cells from newborn conditional Vhl null and Vhl G518A mutant genetically engineered mouse lines are undergoing investigation. Analysis of primary neonatal epithelial kidney cells cultured under mild hypoxia show that these cells preferentially grow in an organized multicellular spherical culture. Additionally, these multicellular cultures, termed “renospheres”, display focal expression of progenitor cell marker CD133, and also globally express Oct4 while select cells in the renospheres express renal cell type markers as seen by immunohistochemistry. Current work is aimed at identifying the cellular makeup of these spheres and determining any role individual cellular components may have in tumorigenesis. Future plans will compare growth characteristics and cellular components of renospheres in the setting of VHL mutation or loss with those with an intact VHL network. This ex vivo system can provide insight into both the cellular characteristics and molecular events that contribute substantially to RCC tumor formation as growth conditions of the renospheres are meant to mimic as close to a physiological state as possible, a factor not currently being addressed by common practices in the field.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4276. doi:10.1158/1538-7445.AM2011-4276
Abstract 4281: Real-time imaging of the cell-cycle in vivo
Authors: Suetsugu Atsushi , Moriwaki Hisataka , Saji Shigetoyo , Pinney Emmett , Naughton Gail , Yano Shuya , Fujiwara Toshiyoshi , Hoffman Robert M.
Published: July 2012

FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator) cells are labeled orange in the G1 phase nuclei (monomeric Kusabira-Orange2: mKO2) and labeled green in the S/G2/M phases (monomeric Azami-Green1: mAG1) (Cell 132, 487-498, 2008). FUCCI-expressing MKN45 gastric cancer cells were used for imaging cell-cycle dynamics in vivo and in vitro. When a confluent monolayer of MKN45-FUCCI was scratched, the edge of the wound turned green within 24hr, indicating that healing of the wound required proliferation of MKN45 cells. FUCCI-expressing MKN45 cells were injected subcutaneously in the nude mice. The injected cells gradually grew and emitted both green and red fluorescence for 14 days, indicating cell cycle progression. In the chorioallantonic membrane (CAM) chick embryo model, green and red fluorescence FUCCI cells and blood vessels were observed for 5 days. FUCCI cells of the edge in the tumor migrated into the transparent egg membrane. Mouse and CAM FUCCI tumor models will be used to study the role of cell cycle dynamics in metastasis including dormancy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4281. doi:10.1158/1538-7445.AM2011-4281
Abstract 4282: Real-time subcellular imaging of cancer cell nuclear dynamics in the brain of live mice
Authors: Momiyama Masashi , Suetsugu Atsushi , Tome Yasunori , Bouvet Michael , Chishima Takashi , Endo Itaru , Hoffman Robert M.
Published: July 2012

In order to visualize single cancer cell nuclear dynamics in the brain, U87 human glioma cells were injected into the brain stereotactically, or Lewis lung mouse carcinoma (LLC) cells were injected into the carotid artery of nude mice. Both cancer cells types express GFP in the nucleus and RFP in the cytoplasm. A craniotomy open window was made over the right parietal bone for real-time imaging of cancer cells on the brain. Mitotic and apoptotic cancer cells in the brain were imaged in real time. Temozolomide (TMZ) and UVC light were used for induction of cancer cell apoptosis. Imaging showed that approximately 86% of Lewis lung carcinoma cells irradiated with UVC light underwent apoptosis and died in contrast to U87 cells which were resistant to UVC. Real-time subcellular imaging in the brain showed TMZ-induced apoptosis of both U87 and LLC cells. Imaging could be carried out for at least two weeks through the craniotomy window enabling treatment to be evaluated on cancer cells in the brain at the subcellular level. The subcellular real-time imaging model of cancer cells in the brain will be of important use for discovery and evaluation novel drugs active against brain cancer and brain metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4282. doi:10.1158/1538-7445.AM2011-4282
Abstract 4284: Combined magnetic resonance and fluorescence-protein imaging to simultaneously assess the anti-tumor and anti-angiogenesis efficacy of selenium
Authors: Bhattacharya Arup , Turowski Steve G. , Martin Ivan Dominguez San , Rajput Ashwani , Rustum Youcef M. , Hoffman Robert M. , Seshadri Mukund
Published: July 2012

In this report, we determined the feasibility to combine fluorescent protein imaging (FPI) and magnetic resonance imaging (MRI) to demonstrate the efficacy of selenium on tumor progression and angiogenesis of colon cancer in an orthotopic model. GEO human colon carcinoma cells expressing green fluorescent protein (GFP), were implanted orthotopically into the colon of athymic nude mice. Beginning 5 days post implantation, whole body FPI was performed to monitor tumor growth in vivo. Upon visualization of tumor growth by FPI, animals were randomly assigned to either a control group or a treatment group. Treatment consisted of daily oral administration of the organoselenium compound, methyselenocysteine (MSC; 0.2 mg/day × 5 weeks). Longitudinal monitoring of tumor growth was performed using FPI and MRI. In addition, dynamic contrast-enhanced MRI was performed to examine the change in tumor blood volume following treatment. Changes in tumor vascularity visualized by imaging were correlated with immunohistochemical determination of microvessel density. Both FPI and MRI allowed non-invasive visualization of the longitudinal growth of orthotopic colon tumors. While T1- and T2-weighted MRI provided adequate contrast and volumetric assessment of GEO tumor growth, GFP imaging allowed for high-throughput visualization of tumor progression. Selenium treatment resulted in a significant reduction in blood volume and microvessel density of GEO tumors. A significant inhibition of tumor growth was also observed in selenium-treated animals compared to untreated control animals. Together, these results highlight the usefulness of multimodal imaging approaches to demonstrate antitumor and anti-angiogenesis efficacy and the potential of selenium treatment of colon cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4284. doi:10.1158/1538-7445.AM2011-4284
Abstract 4287: Ets-related gene (Erg) expression in developing and adult mouse tissues
Authors: Sreenath Taduru , Tan Shyh-Han , Mikhalkevich Natallia , Ponniah Sathibalan , Vasioukhin Valeri , Bieberich Charles J. , Sesterhenn Isabell A. , Dobi Albert , Srivastava Shiv , Mohamed Ahmed
Published: July 2012

Oncogenic activation of the ETS Related Gene (ERG) in human neoplasms was originally identified in subsets of Ewing sarcomas, myeloid leukemias and, recently, in the majority of prostate cancers. Expression of human ERG protein and consequently its functions in normal and disease states needs to be better understood in light of its suggested role in cell differentiation and proliferation.

In this study, we analyzed temporal and spatial expression of the Erg protein by immunohistochemical analysis during mouse embryonic and adult organogenesis using a highly specific ERG monoclonal antibody (CPDR ERG-MAb) developed by our group. Initially, we evaluated the specificity of three recently available ERG monoclonal antibodies (Epitomics EPR 3864 and EPR 3863, and CPDR ERG-MAb) using ERG expressing MOLT4, KG1, COLO 320, VCaP tumor cell lines and LNCaP, MCF7, Jurkat cell lines that do not express ERG. Unlike, rabbit monoclonal antibodies to ERG obtained from Epitomics, the ERG MAb did not show cross reactivity to FLI-1 or to the other ETS related proteins. Our comprehensive evaluation of mouse tissues established a widespread immunolocalization of Erg protein in endothelial cells and restricted expression in precartilage and hematopoietic tissues. Intriguingly, Erg is not expressed in any epithelial tissue including prostate epithelium, a common site of tumors with ERG rearrangements and unscheduled ERG expression. Further, use of two different monoclonal antibodies revealed that infiltrating lymphocytes that are occasionally seen in the prostate environment were positive for Fli-1, did not exhibit any Erg expression.

These findings will further aid in investigations of Erg functions in normal and disease conditions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4287. doi:10.1158/1538-7445.AM2011-4287