bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1941: Development of novel NSAID conjugated molecules for lung cancer therapy
Authors: Chan Daniel C. , Gera Lajos , Zhang Zhiyong , Zheng Di , Hodges Robert , Stewart John , Bunn Paul
Published: August 2012

Background: Many promising chemopreventive Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) such as Flurbiprofen have acquired anti-cyclooxygenase 2 (COX2) effects with anti-cancer activities. Recently several phosphorylated-NSAID compounds were shown to improve the therapeutic potency over the parental NSAID molecules (JPET 111.183533, 2011) by 10 to 30 folds. Previously, we also reported several novel and potent molecules by the incorporation of NSAIDs such as aspirin, indomethancin, ketorolac, ketoprofen, and sulindac into the aclyting N-terminal of our lead mimetic molecules B317 (F5C-OC2Y-Atmp) (AACR 45: 476, 2004). Here we report a new series of highly potent molecules by replacing the N-terminal F5C with a flurbiprofen (Fbp). The resultant molecules effectively inhibit many cancer cell lines in vitro and in vivo, with efficacy much better than the unmodified parental NSAID molecules or those phosphorylated NSAID analogs. Methods: Standard MTT assays were used to access growth inhibitory effects of the parental and NSAID conjugated molecules. Effects of drugs on cell cycle and apoptosis were analyzed by flow cytometry. In vivo efficacy was evaluated in subcutaneous lung xenografts in athymic nude mice. Results: 32 lung cancer cell lines including 24 NSCLC and 8 SCLC cell lines were treated with various concentrations of some selected conjugated drugs for 4 days. The best compound had an average IC 50 value of 0.5 uM, with a range of 0.1 to 2 uM, while parental Flurbiprofen had IC50 value of 500 uM, with a range of 200 uM to 800uM. Cell cycle analysis showed that exposure of lung cancer cells to these new conjugates resulted in a significant shift of cells from S and G2 phases into G1 arrest, resulting in high percentage of apoptotic cell deaths in a dose- and time-dependent manner. Finally, our in vivo efficacy studies demonstrated that new NSAID conjugates at 5 mg/kg by daily ip injections significantly suppressed growth of subcutaneous xenografts of human lung adenocarcinoma in athymic nude mice without causing weight loss. Conclusion: Our new NSAID conjugate is a novel class of therapeutic agent with anti-growth effects against lung cancer cells in vitro and in vivo, with a 1000 fold increase in potency than the parental Flurbiprofen.

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 1941. doi:1538-7445.AM2012-1941
Abstract 1949: Receptor mediated delivery of therapeutic Fe3O4 core TiO2 shell nanoparticles
Authors: Yuan Ye , Liu William C. , Wu Aiguo , Paunesku Tatjana , Woloschak Gayle E.
Published: August 2012

Our goal is to target the delivery of therapeutic nanoparticles (NPs) composed of an Fe3O4 core and a TiO2 shell (Fe@Ti NPs) to specific cancer cells and to the appropriate organelles within these cells. Controlled delivery of these therapeutic NPs could lead to the development of more effective therapies for individual types of cancer. In order to control the delivery of these Fe@Ti NPs, we have attached a small peptide that can bind Epidermal Growth Factor Receptor (EGFR) to the TiO2 molecules at the surface of the NPs. EGFR is an ideal target because it is overexpressed in epithelial type cancers, can induce its own endocytosis, and can translocate to the nucleus. We hypothesize that Fe@Ti NPs conjugated with these peptides (Fe@Ti NCs) will be able to bind EGFR resulting in improved internalization and nuclear localization of these NCs by EGFR positive cancer cells. In fact, we have shown that these NCs can indeed bind and extract EGFR from cancer cell protein extracts. Furthermore, we have shown by Flow Cytometry that EGFR positive cancer cells internalize EGFR binding Fe@Ti NCs to a greater degree than NCs displaying a scrambled peptide or bare NPs. Finally, recent experiments utilizing X-ray Fluorescence Microscopy (XFM) indicate that these EGFR binding NCs but not bare NPs can cluster with EGFR at the surface of cancer cells as well as within the cell nucleus.

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 1949. doi:1538-7445.AM2012-1949
Abstract 1951: Targeting cancer by a chimeric nanoparticle formulation of Cisplatin and PI828, a PI3 kinase inhibitor
Authors: Roy Bhaskar , Sengupta Poulomi , Dinulescu Daniela , Muto Katherine , Papa Anne Laure , Basu Sudipta , Sengupta Shiladitya
Published: August 2012

Combination chemotherapy is the mainstay of cancer regimes. However, additive off target effect of the drugs is still a major drawback of combination therapy. Nanoscale drug delivery systems, through enhanced permeation and retention effect, target the tumour more efficiently while reducing side effects. Combination of Cisplatin, a widely used platinum based chemotherapeutic with PI3 kinase/mTOR inhibitor has shown synergism. In this study, we designed a chimeric nanoparticle of Cisplatin and PI828, a PI3 kinase inhibitor. PI828 was chemically conjugated to cholesterol by a carbamate linkage. Cisplatin was coordinated to a cholesterol-succinic acid derivative via a unique monocarboxylato and OαPt bond. The chimeric nanoparticle was formulated by mixing both the conjugates in definite proportions with phosphatidylcholine and DSPE-PEG. The chimeric naoparticle had a consistent size between 100 to 130 nm determined by dynamic light scattering. The in vitro release kinetics experiment showed slow and sustained release of both the drugs over a time period of 96 hours. In vitro cell proliferation assay expressed cytotoxicity of the chimeric nanoformulation in 4T1, a murine breast cancer cell line and in KRAS upregulated and PTEN suppressed ovarian cancer model cells. We have evaluated the activity of chimeric nanoparticle in a K-Ras(LSL/+)/PTEN(fl/fl) ovarian cancer model, with a luciferase reporter system. The mice treated with chimeric nanoparticle exhibited suppressed tumour growth as well as reduced toxicity as compared to the free drug combination and vehicle treated mice. These results indicate that chimeric nanoformulation can better the outcome of Cisplatin and PI828 combination.

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 1951. doi:1538-7445.AM2012-1951
Abstract 1952: Targeting the CCL2/CCR2 chemokine pathway in breast tumors through intratumoral delivery of calcium cross linked TAT peptide: siRNA complexes
Authors: Fang Wei Bin , Berkland Corey , Cheng Nikki
Published: August 2012

Breast tumors exhibit inflammatory responses through recruitment of stromal cells in the primary tumor microenvironment, including fibroblasts and macrophages. Accumulation of these stromal cells correlates with development of invasive breast cancer and poor patient prognosis. These stromal cells express high levels of the inflammatory chemokine CCL2, corresponding to overexpression of its receptor CCR2 in the tumor epithelium. Our laboratory and others have shown that CCL2/CCR2 signaling mediates stromal: tumor interactions to regulate breast cancer cell survival, invasion and metastasis. While the CCL2/CCR2 signaling represents a novel and potentially effective therapeutic target in invasive breast cancer, it is necessary to develop new tools to specifically inhibit CCL2/CCR2 signaling in vivo. siRNA knockdown could specifically target CCL2 or CCR2, but is unstable and toxic when delivered in vivo. The HIV-1 derived TAT peptide penetrates cell plasma membranes at high efficiency and contains nuclear translocation signals to enhance siRNA delivery to the nucleus. Recent studies from our research team have shown that CaCl2 condenses TAT/nucleic acids to nanoparticles facilitating cellular uptake. The goals of these studies are to design TAT nanoparticle formulas to target CCL2 and CCR2 protein expression in breast tumors. Using an in vitro luciferase reporter system, we identified specific N/P ratios and CaCl2 concentrations that would transfect stromal cells and PyVmT mammary tumor cells. TAT/CaCl2 nanocomplexes carrying GFP plasmid or Cy3 labeled siRNAs were then injected into PyVmT mammary tumors. We observed visible GFP expression and Cy3 labeled siRNA uptake in over 50% of the PyVmT mammary tumor at low and high N/P ratios. Normal mammary tissues did not show significant uptake of TAT nanoparticles. Flow cytometry analysis showed that high N/P ratios resulted in efficient TAT/CaCl2/siRNA uptake in breast cancer associated fibroblasts and macrophages. TAT/CaCl2/siRNA nanocomplexes formed at low N/P ratios resulted in efficient siRNA uptake in tumor epithelial cells. Preliminary studies of PyVmT mammary tumors showed a reduction in CCL2 and CCR2 expression upon delivery of TAT/CaCl2 nanoparticles carrying CCL2 or CCR2 siRNAs. These data indicate this nanoparticle strategy represents a novel, feasible approach to target the CCL2/CCR2 signaling in breast cancer, with important implications on therapeutic targeting.

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 1952. doi:1538-7445.AM2012-1952
Abstract 1956: Folate coated platinum nanoparticles: An effective strategy for targeting cancer therapy
Authors: Schneider Evan , Chavali Manideep , Collazo Lourdes , Mironova Tatsiana , Rafailovich Miriam
Published: August 2012

PURPOSE: Platinum nanoparticles (Pt-NPs) are directly toxic to cancer, but also to normal cells. The purpose of this project is to demonstrate that Pt-NPs can selectively kill cancer cells by targeting their delivery through the folate receptor, the expression of which is greatly increased on cancer compared with normal cells. METHODS: Folate coated Pt-NPs were synthesized and used to treat normal (MC-3T3-E1) and malignant (ROS 17/2.8) bone cells in tissue culture. The Pt-NPs were characterized using transmission electron microscopy and zeta potential analysis, which indicated that they had a diameter of 3.5 ± 0.6 nm and were negatively charged. The effects of dose titrated NP preparations and controls, including the addition of free folate, were examined by optical, confocal and transmission electron microscopy. Cell counts were performed using a hemocytometer. RESULTS: Folate coated Pt-NPs were exquisitely toxic to both MC-3T3-E1 and ROS 17/2.8 cell lines causing 100% lethality in the initially tested concentration range (130 - 260 ug/mL). Dose dependent toxicity for both cell lines was evident at lower folate coated Pt-NP concentrations (2 - 22 ug/mL). At the lowest concentrations examined (<5.15 ug/mL), optical microscopy demonstrated selective delivery of the Pt-NPs to malignant ROS 17/2.8 cells as compared with normal MC-3T3-E1 cells. At these concentrations, selective toxicity to the ROS 17/2.8 cells was also demonstrated by confocal microscopic examination of actin elongation and cell alignment patterns as well as direct measures of cell survival. With prolonged exposure to folate coated Pt-NPs at 3.2 ug/mL, 98.5% of normal MC-3T3-E1 cells survived compared with just 32.8% survival of malignant ROS 17/2.8 cells (p < 0.0001). Consistent with the postulated mechanism of folate coated Pt-NP cell entry via the folate receptor, all toxic effects of folate coated Pt-NPs (at 3.2 ug/mL) were completely eliminated by the addition of high doses (10x normal) of free folate, postulated to saturate folate receptor binding sites. CONCLUSION: Folate coated Pt-NPs can be used to directly and selectively kill cancer cells by targeting their delivery through the folate receptor. To our knowledge, this is the first report to demonstrate that targeting a functionally over-expressed cancer cell receptor with its natural ligand linked to toxic NPs can impart selective cancer cytotoxicity.

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 1956. doi:1538-7445.AM2012-1956
Abstract 1957: Targeted polymer-based nanoparticles containing camptothecin: Development and function
Authors: Han Han , Davis Mark E.
Published: August 2012

Camptothecin (CPT) is a naturally occurring broad range anti-cancer alkaloid that acts by inhibiting DNA enzyme topoisomerase I and HIF-1α activities. However, its systemic delivery has been problematic due to its low solubility and toxicity. To this end, we have designed a CPT delivery system that increases the solubility of CPT by more than three orders of magnitude and is capable of directing CPT specifically to cancerous cells. This delivery system is composed of CPT conjugated to a copolymer of mucic acid and poly(ethylene glycol) (MAP). When this conjugate is placed in water it self-assembles into MAP-CPT nanoparticles. The polyol containing backbone of MAP is able to form reversible covalent bonds with boronic acids (BA). Targeting can therefore be achieved through using BA as the linker between the targeting agent and MAP-CPT nanoparticles. MAP-CPT nanoparticles with varying MAP lengths of 20kDa, 65kDa and 100kDa and BA linkers with different binding constants to MAP-CPT nanoparticles are synthesized and characterized. Particle sizes of 40nm in diameter are observed by dynamic light scattering and visualized by particle tracking methods (Nanosight) and cryo-transmission electron microscopy. This particle size takes advantage of the accumulation of nanoscaled entities in tumor tissue via enhanced permeability and retention (EPR) effect. In addition, the slightly negative particle surface charge of −1mV reduces clearance by the reticuloendothelial system (RES) and minimizes undesirable interaction with blood components. A prolonged pharmacokinetic (PK) profile of MAP-CPT system is observed in Balb/C mice with greater than 10% of injected dose remaining in circulation 24 hours post injection for MAP-CPT nanoparticles containing 65kDa or 100kDa MAP. Maximum tolerable dose (MTD) for MAP-CPT nanoparticles containing MAP at 20kDa, 65kDa and 100kDa are found to be 20, 10 and 8mg of CPT/kg respectively in nude mice. Initial anti-tumor data using MAP-CPT nanoparticles in A/J mice bearing Neuro2A (mice neuroblastoma) tumors show tumor regression after two weekly doses of MAP-CPT nanoparticles. We are currently investigating a targeted version of the MAP-CPT nanoparticles through anti-tumor studies comparing targeted (using Herceptin antibodies) versus non-targeted efficacy in nude mice bearing BT-474 (HER2 over-expressing human breast carcinoma) tumors. MAP-CPT, a delivery system for the small hydrophobic drug CPT, is developed and has favorable particle size and surface charge properties, prolonged PK profile and targeting potential. Efforts to define the relationships between targeted and non-targeted versions of the MAP-CPT nanoparticles are in progress.

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 1957. doi:1538-7445.AM2012-1957
Abstract 1980: Dihydroartemisinin inhibits ovarian cancer growth and metastasis via targeting PDGFRα
Authors: Li Xiaoguang , Liu Yanling , Yue Qingxi , Zhang Jianbing , Gong Nuoxi , Chen Peizhan , Liu Hong , Geng Meiyu , Qiang Fulin , Zhang Ruiwen , Wang Hui
Published: August 2012

Artemisinin and its analogs, the well-established clinically used anti-malarial traditional medicine, have been proven to have potent anticancer activities. We have demonstrated that Artemisinin and analogs have significant anti-ovarian cancer effect used alone or in combination with conventional chemotherapeutic agents. However, the mechanisms of action for their anticancer activities are not completely understood. Herein we report that dihydroartemisinin, one of the most active derivatives of artemisinin, directly targets platelet-derived growth factor receptor-alpha (PDGFRα) to repress ovarian cancer growth and metastasis. Dihydroartemisinin directly interacts with PDGFRα and induces dephosphorylated inactivation and ubiquitin-mediated degradation of PDGFRα, which further inactivates downstream phosphoinositide 3-kinase (PI3K) and Mitogen-Activated Protein Kinase (MAPK) pathway and subsequently represses epithelial-mesenchymal transition (EMT) in terms of upregulation of epithelial marker E-cadherin and downregulation of mesenchymal markers N-cadherin and vimentin and transcriptional repressors Snail, Twist and Slug, leading to suppression of cell growth and metastasis in PDGFRα-positive ovarian cancer cells in vitro and in vivo mouse orthotopic ovarian cancer model. These findings may contribute to the development of more effective artemisinin-based targeted compounds as novel target therapy for the treatment of human cancers.

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 1980. doi:1538-7445.AM2012-1980
Abstract 1998: Inhibition of hedgehog signaling in prostate cancer by extracts of Sutherlandia
Authors: Jackson Glenn A. , Lin Hui , Lu Yuan , Drenkhahn Sara K. , Starkey Nicholas J. E. , Huber Christal D. , Lei Wei , Rottinghaus George E. , Folk William , Browning Jim D. , Besch-Williford Cindy L. , Fritsche Kevin L. , Lubahn Dennis B.
Published: August 2012

Prostate cancer possesses long latency periods and is responsive to dietary mediators, making it a target for phytochemoprevention. Many botanical compounds that have been proposed to prevent cancer may potentially work via inhibition of the hedgehog signaling pathway. Here we investigated the potential of Sutherlandia frutescens (also called Lessertia and “cancer bush” in South Africa) to inhibit hedgehog signaling. We hypothesize that the anti-cancer effects of Sutherlandia are due to its inhibition of hedgehog signaling pathway activity. Methods: We evaluated Sutherlandia's effects of growth inhibition in vitro both in the human prostate cancer cell line PC3 and mouse prostate cancer cell line TRAMP-C2 by exposing the cells for 72hrs to plant extracts before protein concentrations were measured to evaluate inhibition of growth. To determine hedgehog pathway inhibitory activity, we treated two different cell lines (an NIH3T3 cell line and a TRAMPC2 cell line) stably transfected with a firefly luciferase reporter having Gli response elements in the firefly luciferase promoter. After 48hr exposure to treatment with multiple doses of Sutherlandia, a firefly substrate was added to the cell lysate and the luciferase signal was measured using a Biotek Synergy plate reader. Finally, Sutherlandia was incorporated into a diet and fed to TRAMP (Transgenic Adenocarcinoma Mouse Prostate) mice to examine whether the plant can delay or even inhibit prostate cancer incidence in the TRAMPmouse model. Results: Sutherlandia extracts inhibited growth of both PC3 and TRAMPC2 cells. In addition, hedgehog signaling was inhibited by Sutherlandia in both NIH3T3 and TRAMPC2 reporter cells. Interestingly, in vivo the lowest concentration of Sutherlandia diet had the greatest effect on inhibition of poorly differentiated adenocarcinoma in the TRAMP mice. Conclusions: Sutherlandia's inhibition of prostate cancer growth may be via inhibition of hedgehog signaling. Future work to identify in Sutherlandia the active compound having this effect, and the ideal concentration for use in vivo may lead to a means whereby dietary intervention for prostate cancer prevention and/or treatment is possible.

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 1998. doi:1538-7445.AM2012-1998
Abstract 2002: OCT-3/4 expression is associated with high levels of the pro-apoptotic BH3 only protein NOXA in testicular germ cell tumors (TGCTs)
Authors: Gutekunst Matthias , Mueller Thomas , Weilbacher Andrea , Dengler Michael A. , Kruck Stephan , Bedke Jens , Aulitzky Walter E. , van der Kuip Heiko
Published: August 2012

Embryonal carcinoma (EC) cells are the pluripotent precursors of differentiated structures of testicular germ cell tumors (TGCT) characterized by expression of the embryonal transcription factor OCT-3/4. Loss of OCT-3/4 during differentiation abrogates the extraordinary high sensitivity to cisplatin in these cells. We previously observed a constitutively high expression level of NOXA and a major role of this pro-apoptotic BH3-only protein for cisplatin hypersensitivity in the EC cell lines NTERA-2D1 and 2102EP. We now investigated if these high NOXA protein levels may be directly linked to the OCT-3/4 status in TGCT cells. Therefore, we analyzed NOXA protein expression in an extended TGCT cell line panel of 5 OCT-3/4 negative and 5 OCT-3/4 positive cell lines. Importantly, NOXA protein was highly correlated with OCT-3/4 levels and also with cisplatin sensitivity. These data indicate that constitutively high NOXA levels might be responsible for the low threshold for cisplatin-induced apoptosis in OCT-3/4 positive pluripotent cells. A direct link between OCT-3/4 and NOXA could also be demonstrated by RNAi mediated silencing experiments performed in NTERA-2D1 and 2102EP cells. Silencing of OCT-3/4 resulted in a significant downregulation of NOXA transcript and an almost complete loss of NOXA protein accompanied by a loss of cisplatin sensitivity. This was observed in both differentiation competent NTERA-2D1 cells as well as in differentiation incompetent 2102EP cells pointing to a direct OCT-3/4 dependent NOXA regulation rather than a bystander effect of differentiation upon loss of OCT-3/4. We further validated our in vitro data by comparing OCT-3/4 and NOXA expression levels in patient samples derived from ECs with other non-seminomatous GCTs, seminomas, and tumor entities from lung, breast, and ovary. In agreement with our in vitro observations, OCT-3/4 and NOXA were found to be highly expressed selectively in seminomas and ECs. This could also be confirmed on protein level in primary tissue samples derived from 5 ECs and 8 seminomas by western blot analysis. In conclusion, our data for the first time demonstrate a close correlation between OCT-3/4 and NOXA protein levels and strengthen the previously hypothesized role of constitutively high NOXA levels for the exquisite sensitivity of TGCTs to cisplatin.

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 2002. doi:1538-7445.AM2012-2002
Abstract 2003: STAT3 is a critical mediator of the pro-survival effect of Bcl-2-Rac1 interaction in human cancer cells
Authors: Kang Jia , Vali Shireen , Kapoor Shweta , Abbasi Taher , Pervaiz Shazib
Published: August 2012

We recently showed that Bcl-2 overexpression induced a pro-oxidant intracellular milieu resulting in an inhibition of apoptotic execution and that reversing the pro-oxidant milieu by inhibiting intracellular superoxide production sensitized Bcl-2-overexpressing cells to apoptotic stimuli. We provide evidence that the small GTPase Rac1 is functionally involved since gene silencing and functional inhibition of Rac1 blocked Bcl-2-mediated increase in intracellular superoxide production in tumor cells. A co-localization and physical interaction between the two proteins were observed, which could be blocked by the BH3 mimetics as well as by synthetic Bcl-2 BH3 domain peptides both in vitro and in vivo. The Bcl-2 BH3 peptides as well as silencing and functional inhibition of Rac1 reversed intracellular superoxide levels and overcame Bcl-2-mediated drug resistance in human leukemia and cervical cancer cells, demonstrating that this interaction is functionally relevant. Notably, the interaction was observed in primary cells from patients diagnosed with B-cell lymphoma with Bcl-2 overexpression but not in noncancerous tissue. Furthermore, computer simulation driven virtual predictive experiments based on the protein pathway dynamic network created by Cellworks Group Inc. were carried out to study the functional implication of this interaction. An HCT116 human colorectal cancer cell line with KRAS over-activation, PI3K overexpression, CDKN2A deletion, β-catenin overexpression and Bcl-2 overexpression was created as a base line. Phenotypic indices of angiogenesis, proliferation, viability, metastasis as well as tumor volume were all found to be amplified in a variant cell line when Rac1 was overexpressed based on the above computer modeled HCT116. Strikingly, the expression levels of both STAT3 and β-catenin were significantly upregulated in the variant cell line; however upon Rac1 or Bcl-2 knockdown in both the base and the variant cell lines, apoptotic markers such as Bax, caspase 3 and cleaved PARP1 were amplified while STAT3 and β-catenin were significantly down-regulated. Intrigued by these predictions, we set out to verify the correlation between STAT3 signaling and Bcl-2-Rac1 interaction. Interestingly, there is indeed a strong correlation between the activation status of STAT3 (pY705) and Bcl-2 expression as well as Rac1 activation status. Furthermore, STAT3 was found to be localized to mitochondria and our preliminary results suggest an association with Bcl-2.

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 2003. doi:1538-7445.AM2012-2003
Abstract 2005: Identification of Bcl2L13 as a novel GBM oncoprotein
Authors: Jensen Samuel A. , Calvert Andrea , Luciano Janina , Stegh Alexander
Published: August 2012

Using in silico analysis of 272 GBM samples in the TGCA data set to find important cell death mechanisms in gliomapathogenesis, we identified differential expression of the novel Bcl2-Like13 protein (Bcl2L13). Bcl2L13 shares significant structural homology with Bcl-2 and Bcl-xL, but contains a unique 250 amino acid sequence that may point to unique, non-classical functions. Several studies have shown that Bcl2L13 expression correlates with increased chemotherapeutic resistance and unfavorable treatment outcome in leukemia and ZIC1-driven liposarcomas. These clinical data suggests that Bcl2L13 likely acts as an onco-protein in multiple cancers and may act in a similar manner in GBM. Using qRT-PCR and immunohistochemical analyses, we found that Bcl2L13 was highly overexpressed in >90% of GBM samples, but absent in adjacent normal brain. Using RNAi-loss- and cDNA complementation studies in glioma cell lines and glioma stem cells (GSCs), we determined that Bcl2L13 potently inhibits therapy-induced apoptosis by inhibiting mitochondrial outer membrane permeabalization (MOMP) and post-mitochondrial caspase-3 and -7 cleavage, likely acting in a similar manner as canonical Bcl-2 family proteins. Bcl2L13 overexpression also greatly increased cellular proliferation, and immunofluorescence and cell fractionalization experiments revealed that Bcl2L13 is localized at the mitochondria. To study the impact of Bcl2L13 on the progression of gliomagenesis in vivo, we orthotopically injected glioma cell lines with enforced expression of Bcl2L13-targeting shRNAs into immunocompromised SCID mice. Strikingly, neutralization of Bcl2L13 signaling increased overall glioma-free survival, which was associated with enhanced intratumoral apoptosis and decreased proliferative indices. To molecularly elucidate the mechanism by which Bcl2L13 exerts its anti-apoptotic effects, we performed a Yeast Two Hybrid screen and identified a select number of tumor suppressors and onco-proteins including ceramide synthase 2 (CerS2), (a regulator of MOMP) and O-6-methylguanine-DNA methyltransferase (MGMT), (an important prognostic indicator of telozolomide effectiveness) as Bcl2L13 interaction partners. Co-immunoprecipitations confirmed the interactions between Bcl2L13 and these proteins. Taken together, these results reveal that Bcl2L13 represents a novel anti-apoptotic, Bcl-2-like GBM oncoprotein that inhibits apoptosis progression and promotes tumor growth by impacting mitochondrial membrane physiology.

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 2005. doi:1538-7445.AM2012-2005
Abstract 2025: Blockade of the Shh pathway by KAAD-Cyclopamine promoted apoptosis in estrogen treated breast cancer cells
Authors: na Kang Han , An Hyun Sook , Kang Myoung Hee , Kim Jung Lim , Kim Jun Suk , Seo Jea Hong
Published: August 2012

The Hedgehog (Hh) signaling pathway functions as an organizer in embryonic development. Recent studies have shown constitutive activation of the hedgehog pathway is important in the development of various carcinomas including breast cancer. However, it remains unclear whether this pathway is activated in human breast cancer. Herein, we investigated the role of Shh signaling pathway in tumorigenesis of estrogen treated breast cancer cells and the molecular mechanisms underlying these effects. We showed that breast cancer cell lines express all the components of Shh signaling, albeit to different extents. Moreover, in breast cancer cells, estrogen supplementation triggered Shh up-regulation. This estrogen treated Shh expression activated the Hh pathway in a ligand-dependent manner, and increased cell proliferation. Cell proliferation was done MTT assay with various concentrations of KAAD-Cyclopamine for 24, 48, or 72 h. As a result, KAAD-Cyclopamine had anti-proliferative effect on estrogen treated breast cancer cells. Also, to determine whether the cytotoxicity induced by KAAD-Cyclopamine was due to apoptosis in estrogen-induced breast cancer cells, we measured cell cycle and the fraction of early apoptosis [Annexin V(+)/PI(-)]. Cell cycle analysis by FACS indicated that Cyclopamine treated cell lines showed an increase in the proportion of cells in sub-G1 phase, compared to untreated cells. Exposure of gastric cancer cells to Cyclopamine also resulted in the increase in the percentage of annexin V- positive and PI-negative cells. These observations demonstrate that KAAD-Cyclopamine can inhibit estrogen-induced breast cancer cell cycle progression and promote cell apoptosis. Furthermore, Western blotting analysis was used to investigate the effect of KAAD-Cyclopamine (a Shh signaling inhibitor) on estroen treated breast cancer cells. As a result, expression of cleaved PARP, caspase-3, and -9, Bax was increased. In addition, phosphorylation of Akt and of glycogensynthase kinase 3β (GSK-3β) was also reduced by treatment with KAAD-Cyclopamine. These results, taken together, suggest that Blockade of the Shh pathway by KAAD-Cyclopamine promoted apoptosis in estrogen treated breast cancer 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 2025. doi:1538-7445.AM2012-2025
Abstract 2039: Human Stn1 protects telomere integrity by promoting efficient lagging strand synthesis at telomeres
Authors: Huang Chenhui , Dai Xueyu , Chai Weihang
Published: August 2012

Telomeres, the chromosomal termini, are critical for maintaining genome stability. The length of telomere is regulated by multiple genes including telomere-specific shelterin proteins and telomere non-specific proteins such as DNA replication proteins. Stn1, a component of the newly identified Ctc1/Stn1/Ten1 complex, is also known as (DNA polymerase) α-accessory factor AAF44 and plays an unclear role in telomere maintenance. In our studies, Stn1 depletion causes elongated G-overhang and persistent ssDNA at G-rich strand in both telomerase-positive and negative cells. Furthermore, Stn1 depletion shows an increase of fragile telomeres in strands replicated by lagging strand synthesis, implicating that Stn1 is important in efficient replication of lagging strand telomeres. Consistently, BrdU incorporation into lagging telomeres is reduced when Stn1 is depleted. In addition, Stn1 depletion leads to rapid telomere shortening. Examination of the cell cycle-regulated G-overhang dynamics showed that deficiency of hStn1 delays and compromises C-strand synthesis that occurs in the late S/G2 phase, leading to persistent lengthening of G-overhangs at lagging daughter telomeres. We also found that Stn1 interacts with Polα throughout the cycle. ChIP experiments showed that Stn1 depletion results in increased and prolonged Polα association with telomeres, but does not affect the association of Pot1 with telomeres. Our data indicate a key role of Stn1 in telomere replication and maintenance.

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 2039. doi:1538-7445.AM2012-2039
Abstract 2043: Deregulated Cdc7/Dbf4 expression promotes DNA repair and checkpoint recovery to enhance survival and associates with poor prognosis in OSCC
Authors: Cheng An Ning , Lo Yu-Kang , Jiang Shih Sheng , Lee Alan Yueh-Luen
Published: August 2012

Cdc7 (DDK, Dbf4-dependent kinase) is a serine-threonine kinase and required to initiate DNA replication from each origin. Although Cdc7 and Dbf4 has been shown overexpressed in many human cancer cell lines and certain primary tumors, only few cancer tissue samples were analyzed. In this report, in light of the biological, prognostic, and therapeutic implications of Cdc7 in tumorigenesis, we investigated and found that Cdc7 is overexpressed specifically in malignant non-small-cell lung cancer (NSCLC), colorectal cancer and oral squamous cell carcinoma (OSCC) tissues and Cdc7 expression level is an independent prognosis factor for patient survival in OSCC. Cdc7 is required for DNA replication and higher Cdc7 level is linked to the development of tumor cells, however, increased Cdc7 expression levels do not always correlate with the proliferative status of the tumor cells. Therefore, the underlying mechanism of overexpressed Cdc7/Dbf4 in promoting tumorigenesis still needs to be clarified. Our data suggest that up-regulation of Cdc7 has several roles in promoting tumorigenesis. First, overexpression of Cdc7 promotes cell survival through inhibiting DNA damage-induced cell apoptosis and inducing HR DNA repair. Second, during the early stage, it causes checkpoint activation as well as slow-down of S phase progression and promotes HR DNA repair that may through DSB-mediated mechanism. Third, during the late stage, upon removal of DNA lesions and completion of DNA repair in S-phase, Cdc7 attenuates checkpoint signaling and reactivates DNA replication by promoting the loading of replication initiation proteins Cdc45 as well as the activation of Plk1, which will be important for checkpoint recovery and replication restart. In summary, we have shown that a highly significant association between Cdc7 expression and overall survival in OSCC patient, rendering the prognostic importance and clinical practicability of Cdc7 expression in OSCC. Our studies also indicate that increased Cdc7 expression promotes HR repair and attenuates ATM/ATR checkpoint signaling for recovery of damaged replication forks to enhance the survival of DNA damaging agent-treated tumor cells. These studies will open an avenue to the biological, prognostic, and therapeutic applications of Cdc7 kinase in cancer therapy.

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 2043. doi:1538-7445.AM2012-2043
Abstract 2051: Inhibitors against CHK1 and WEE1, MK-8776 and MK-1775, strongly synergize in vitro and in vivo to inhibit tumor growth
Authors: Shumway Stuart D. , Guertin Amy D. , Martin Melissa M. , Roberts Brian , Hurd Melissa S. , Qu Xianlu , Miselis Nathan R. , Bloecher Andrew , Toniatti Carlo
Published: August 2012

Inhibitors of the checkpoint kinases CHK1 and WEE1 are under development as sensitizers to chemotherapy or radiation therapy. Disruption of either CHK1 or WEE1 activity compromises the cell cycle arrest that normally accompanies the DNA damage response, forcing cells to prematurely divide before repairing damaged DNA. However, inhibition of either CHK1 or WEE1 alone also disrupts DNA replication, resulting in DNA double strand breaks (DSBs) primarily in S-phase cells. Here we describe the ability of inhibitors against CHK1 and WEE1 to synergize both in vitro and in vivo. MK-8776 and MK-1775 are advancing through early clinical trials as potent and selective inhibitors of CHK1 and WEE1, respectively. MK-8776 increases the cell-based potency of MK-1775 in multiple cancer cell lines by as much as a 10-fold EC50 shift in proliferation assays. Staining for the DSB marker γH2AX at low concentrations of each drug demonstrates that MK-8776 and MK-1775 in combination induce up to 50-fold more DNA damage than either MK-8776 or MK-1775 alone. The ability of the combination to drive premature mitosis, determined by pHH3 quantitation, was variable, suggesting that generation of DSBs rather than G2/M checkpoint abrogation is the mechanism underlying combination cytotoxicity. Induction of DSBs requires cyclin-dependent kinase activity and takes place largely in S-phase cells, implying deregulation of DNA replication. Furthermore, staining for DNA damage response markers γH2AX and pCHK1-ser345 was both more intense and more durable in a LoVo xenograft model of cancer when MK-8776 and MK-1775 were dosed together compared to either compound alone. At tolerated doses, the combination demonstrated significantly greater anti-tumor efficacy than either MK-8776 or MK-1775 alone in LoVo xenografts (91% tumor growth inhibition for the combination versus 28% and 41% for MK-8776 and MK-1775, respectively). Similar observations were made in additional xenograft models. These results demonstrate in vitro and in vivo synergy between MK-8776 and MK-1775, indicating unique and complimentary anti-tumor effects of CHK1 and WEE1 inhibition. Our studies provide preclinical rationale for combining WEE1 and CHK1 inhibitors as a novel cancer treatment regimen.

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 2051. doi:1538-7445.AM2012-2051
Abstract 2069: A novel berbamine derivative induces apoptosis of chemotherapy-resistant human osteosarcoma cells through activation of ROS/JNK signaling
Authors: Yang Fan , Nam Sangkil , Zhao Robin , Tian Yan , Horne David , Jove Richard
Published: August 2012

Osteosarcoma is the most common primary bone tumor in children and adolescence. There is a critical need to find more potent drugs for patients with metastatic or recurrent disease. Berbamine (BBM) is a natural compound derived from the Berberis amurensis plants. BBM and its derivatives have been shown to have anti-tumor effects in several cancers. Here, we report that a new berbamine derivative, BBMD3 (5 µM), induces apoptosis of human osteosarcoma cells, which are resistant or insensitive to conventional chemotherapy including cisplatin, doxorubicin and methotrexate. BBMD3 inhibited cell viability and induced apoptosis of G292, KHOS and MG-63 human osteosarcoma cells in a time- and dose-dependent manner. BBMD3 activated caspase-3, and cleaved poly(ADP-ribose) polymerase (PARP). Caspase inhibitor (Z-VAD-FMK) could rescue tumor cells from the cytotoxic effects of BBMD3 on cell survival. Importantly, BBMD3 increased phosphorylation of c-Jun N-terminal kinase (JNK) in these tumor cells, resulting in activation of its substrates, c-Jun and c-Fos. Moreover, reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC), blocked the phosphorylation of JNK and c-Jun induced by BBMD3. However, Z-VAD-FMK treatment did not inhibit the induction of phosphorylated JNK and c-Jun by BBMD3, suggesting that phosphorylation of JNK and c-Jun occurred before activation of caspases. BBMD3 increased the expression of the pro-apototic gene Bad, associated with apoptosis induction. Finally, BBMD3 also decreased the expression of cyclin D1, a positive cell cycle regulator, which is correlated with growth suppression in osteosarcoma cells. Collectively, these findings indicate that BBMD3 is a potentially promising drug for the treatment of chemotherapy-resistant osteosarcomas.

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 2069. doi:1538-7445.AM2012-2069
Abstract 2077: Modulation of reactive oxygen species (ROS) by a dietary agent for the prevention/ treatment of breast cancer
Authors: Arumugam Arunkumar , Boopalan Thiyagarajan , Suman Suman , Das Trinath P. , Damodaran Chendil , Lakshmanaswamy Rajkumar
Published: August 2012

Breast cancer is one of the most common cancers in women, affecting over 1 million women worldwide (208,000 cases in U.S.) and resulting in 400,000 deaths, yearly. The morbidity associated with breast cancer has been only partially impacted by current treatment modalities including surgery, radiotherapy, and adjuvant chemotherapy and/or hormone therapies. There is an immediate need for effective prevention and/or treatment of breast. Changes in reactive oxygen species (ROS) play a pivotal role in these processes. For example, a moderate increase in ROS promotes cell proliferation, but excessive amounts of ROS can induce apoptosis in cancer cells. Therefore, it is possible to manipulate the oxidative stress (redox) system to selectively kill cancer cells. Neem (Azadirachta indica) is a well-known medicinal plant having a wide spectrum of biological activities. In our studies, Ethanolic fraction of Neem Leaf (EFNL) significantly decreased cell viability and induced apoptosis in both estrogen receptor positive (ER+) and triple negative breast cancer (TNBC) cells without causing toxicity to normal primary breast epithelial cells. Oral administration of EFNL (5.0 mg/kg body weight) significantly inhibited the growth of MNU-induced mammary tumors without causing any toxicity. EFNL treatment not only inhibited the growth of established mammary tumors, but also significantly inhibited the growth of any new mammary tumors. These interesting results led us to investigate the mechanism underlying EFNL actions on breast cancer. Our results suggested that EFNL induced Reactive Oxygen Species (ROS) which lead to inhibition of phosphorylation of the pro-survival signal proteins, Akt and its downstream signaling of mTOR and NF kappa B activation. Over expression of antioxidant enzymes such as superoxide dismutases (SOD1 & SOD2) or pharmacological agent [N-acetylcysteine] abrogated EFNL-induced ROS generation, inhibition of cell survival and Akt inactivation in breast cancer cells. These results clearly demonstrate that EFNL induced ROS regulates pro-survival function in breast cancer cells and provides a novel molecular target for breast cancer treatment. The results may provide a preclinical proof-of-principle for a novel targeted therapy for breast cancer cells using EFNL as a nontoxic dietary agent.

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 2077. doi:1538-7445.AM2012-2077
Abstract 2082: The role of cell-cell contacts in the survival of extracellular matrix detached mammary epithelial cells
Authors: Rayavarapu Raju , Schafer Zachary
Published: August 2012

Normal epithelial cells require attachment to the extracellular matrix (ECM) in order to proliferate and survive. Binding to the ECM facilitates the activation of pathways responsible for ATP production and cell survival. When detached from the ECM, normal cells will upregulate apoptotic pathways and will be systematically removed. This detachment-induced apoptosis is known as anoikis. ECM detachment in normal epithelial cells disrupts cellular signaling derived from integrin-mediated basal attachment as well as cadherin assisted signaling from lateral cell contacts. Cadherins, primarily E-cadherin, have been shown to be classic tumor suppressor genes and can prevent the proliferation of cells that fail to assume normal epithelial morphology and cell-cell contacts. The loss of E-cadherin endows an increased invasion capacity to breast tumors and regulation of E-cadherin levels are directly mediated by factors (i.e. Slug) that promote epithelial-to-mesenchymal transition (EMT) and metastasis. Cadherin molecules have previously been reported to interact with other receptor tyrosine kinases, such as EGFR (epithelial growth factor receptor), in order to suppress the ability of the receptor to bind ligand, dimerize and elicit downstream signaling. HER2 is an orphan member of the EGFR family of tyrosine kinases and is a potent oncogene found in 30% of all breast cancer tissue. Previous studies in our laboratory have found a direct correlation between upregulation of survival pathways in ECM detached cells and expression of HER2. In this study, we find that the overexpression of HER2 in non-malignant mammary epithelial cells (MCF-10A) results in substantial aggregation of detached cells, the inhibition of caspase activation and a rescue of ATP production. These data suggest that HER2 plays a role in protecting detached cells from anoikis and this activity may be linked to cell-cell adhesion in suspension. We have found that disruption of cell-cell contacts in HER2-expressing MCF-10A cells utilizing methylcellulose can induce caspase activity and diminish global ATP levels. Preliminary studies also reveal a previously uncharacterized interaction between HER2 and E-cadherin that takes place when E-cadherin dimers are disrupted. Thus, the disruption of E-cadherin dimers in cellular aggregates may facilitate the binding of E-cadherin to HER2 in a fashion that compromises downstream survival signaling. These data together suggest a potential role for E-cadherin as a modulator of HER2 activity and further suggest that the loss of E-cadherin via mechanisms like EMT may function as a means to promote ECM-detached tumor cell survival.

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 2082. doi:1538-7445.AM2012-2082