bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1339: Upregulation of the glutathione detoxification pathway protects against cytotoxicity induced by the alkylating agent, 2-chloroethyl ethyl sulfide (CEES)
Authors: Abel Erika L. , McIvor Elizabeth , Bubel Jennifer D. , Simper Melissa S. , Powell Leslie , Fields Tammy Y. , Andreeff Michael , MacLeod Michael C. , DiGiovanni John
Published: June 2012

Sulfur mustard (SM) is a vesicating agent that was first used in chemical warfare over 100 years ago. SM alkylates DNA and is mutagenic in various model systems. Epidemiological studies indicate that chronic exposure increases the risk of cancer in exposed tissues. Due to its incapacitating effects, and the relative ease with which it may be synthesized, mustard gas remains a potential chemical threat to the present day. Currently, there are no antidotes for SM-induced toxicity; therefore, chemopreventive and therapeutic measures are needed. Glutathione (GSH) exerts a protective effect against SM toxicity, and detoxification of SM is believed to occur, in part, via GSH conjugation. The rate-limiting step in GSH synthesis is catalyzed by the glutamate cysteine ligase (GCL) holoenzyme, which is composed of a catalytic and a regulatory subunit (GCLC and GCLM, respectively). Cancer chemopreventive agents are known to modulate expression of GCL and GSH S-transferases (GSTs) via activation of the transcription factor, Nrf2, and may, therefore, have utility in inhibiting SM-induced damage in the skin. We have screened 6 chemopreventive agents for ability to induce GSH synthesis and protect cultured NCTC2544 human keratinocytes against the SM analogue, 2-chloroethyl ethyl sulfide (CEES). We found that sulforaphane and methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me) stimulated nuclear localization of Nrf2 and induced expression of GCLM. Additionally, we found that treatment with CDDO-Me elevated reduced GSH content of NCTC2544 cells (118.0±11.6 nmol/mg protein versus 66.5±7.9 nmol/mg protein) and preserved their viability by ∼3-fold following exposure to CEES. CDDO-Me acted additively with 2,6-dithiopurine (DTP), a nucleophilic scavenging agent, to increase the viability of keratinocytes exposed to CEES by 6-fold (60% in cells treated with a combination of CDDO-Me and DTP vs 10% in cells treated with vehicle only). In vivo, topical application of either sulforaphane or CDDO-Me elevated epidermal GSH and GST levels in the skin of C57BL/6 mice. These results suggest that CDDO-Me and sulforaphane are promising chemopreventive agents for SM toxicity in the skin.

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 1339. doi:10.1158/1538-7445.AM2011-1339
Abstract 1343: Protective effect of catechol-O-methyltransferase in human renal cell carcinogenesis
Authors: Chang Inik , Liu Jan , Majid Shahana , Saini Sharanjot , Zaman Mohd S. , Chen Yi , Singh Kamaldeep , Shahryari Varahram , Sandhu Herman , Yamamura Soichiro , Dahiya Angela V. , Deng Guoren , Tanaka Yuichiro
Published: June 2012

Exposure to carcinogenic catechol estrogens plays a role in the development of kidney cancer. Catechol-O-methyltransferase (COMT) neutralizes the genotoxic effects of these compounds by methoxylation. Although reduced activity of COMT has been suggested as a risk factor for estrogen-associated cancers, its critical role in kidney cancer and underlying mechanisms of renal cell carcinogenesis are largely unknown. To address the role and function of COMT in renal cell carcinomas (RCC), we initially determined levels of COMT in normal kidney and clear cell RCC. The expression of COMT mRNA was down-regulated in clear cell RCC tissues as determined by quantitative PCR. Low expression of COMT protein in clear cell RCC tissues was also confirmed by immunohistochemical counting of staining score (2.5 vs. 0.5 in normal kidney and clear cell RCC, respectively, p<0.001). Since COMT expression is reduced in clear cell RCC compared to normal cells, we explored the function of COMT by transiently over-expressing it in the Caki-1 clear cell RCC cell line with low levels of COMT compared to HK-2 normal renal cells. A significant increase of COMT expression in Caki-1 cells (Caki-1/COMT) compared to parent cells transfected with vector control (Caki-1/pCMV6) was confirmed by both quantitative PCR and Western blot. We investigated the role of COMT on cell proliferation and found that proliferation of Caki-1/COMT cells was decreased by 8 to 15 % in a time dependent manner (from 24 hours to 72 hours, p<0.05). We also assessed the effect of COMT on cell cycle progression by flow cytometry. Over-expression of COMT led to an increase (8 ± 1.6%) in the G0/G1 phase population, whereas the number of cells in S-phase decreased from 5.5 % to 2.6 % (p<0.05). These data indicate that COMT over-expression ameliorate the proliferation of Caki-1 renal cancer cell line by G0/G1 phase arrest. In addition, we analyzed whether COMT expression affects apoptosis of Caki-1 cells by flow cytometry using annexin V-FITC/7-AAD (7-amino actinomycin D) dye. COMT expression resulted in a higher percentage of cells undergoing both early and late apoptosis compared to vector controls (2.3% vs. 0.4 %, respectively for early apoptosis, and 5.5 % vs. 0.8 %, respectively for late apoptosis, p<0.05). Our study indicates that COMT plays a protective role in renal cell carcinogenesis by preventing cellular proliferation and enhancing apoptosis of RCC 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 1343. doi:10.1158/1538-7445.AM2011-1343
Abstract 1345: Bioactivation of luteolin as a prodrug by tyrosinase inhibits human glutathione S-transferase and induces toxicity in SK-MEL-28 melanoma cells
Authors: Balyan Rajiv , Kudugunti Shashi , Vad Nikhil , Moridani Majid
Published: June 2012

Glutathione S-transferase (GST) play significant role in the metabolism and detoxification of drugs used in treatment of melanoma, resulting in a decrease in their efficacy against melanoma. In the present study, we investigated: i) the selective inhibition of GST by luteolin in the presence of tyrosinase, an abundant enzyme found in melanoma, and ii) the induction of apoptosis, change in mitochondrial permeability, cell viability, cell migration and GSH depletion by luteolin in human melanoma SK-MEL-28 cell culture. The inhibition of GST was investigated using CDNB method. DTNB method was used to measure GSH depletion. MTT assay, Annexin V apoptosis assay and TMRM fluorescence were used in the investigation of biochemical mechanism of luteolin toxicity in melanoma cells. Luteolin (40 µM) induced toxicity and death in 50% of cells after 48 h incubation. Cell treatment with luteolin (40 µM) resulted in apoptotic cell death in 70% of cells and caused 45% decrease in TMRM fluorescence indicating change in mitochondrial membrane permeability. At 1 h incubation, luteolin was bioactivated by tyrosinase to luteoline-quinone for 71% as measured by GSH depletion. The luteolin is effective in preventing metastasis of cancerous cells as evident by cell migration assay. At 48 h incubation, luteolin as low as 5 µM effectively prevented the cell migration. In the presence of tyrosinase, luteolin (10 µM) showed about 87% GST inhibition; whereas in the absence of tyrosinase, luteolin led to negligible GST inhibition. Both luteolin-SG conjugate and luteolin-quinone (10 µM) inhibited ≥90% of GST activity via reversible and irreversible competitive mechanisms with Ki of 0.9 and 6.4 µM with respect to GSH, respectively. The luteolin-SG conjugate inhibited GST activity reversibly and non-competitively with Ki of 1.8, whereas luteolin-quinone showed irreversible competitive inhibition of GST activity with Ki of 1.0 µM with respect to CDNB. While luteolin is not a substrate for GST, Luteolin (25 µM) non-competitively inhibited GST with Ki of 39 µM with respect to GSH and competitively with Ki of 61 µM with respect to CDNB. Luteolin, at the concentration range of 5-80 µM, exhibited 78-99% GST inhibition in human SK-MEL-28 cell homogenate. In summary, our results suggest that luteolin was bioactivated by tyrosinase to form a quinone and luteolin-glutathione conjugate, which played major role in the inhibition of GST. For the first time, we demonstrate that luteolin acts as a selective inhibitor of GST in the presence of tyrosinase.

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 1345. doi:10.1158/1538-7445.AM2011-1345
Abstract 136: hsa-miR-20a promotes tumorigenesis in ccRCC cancer cell lines
Authors: Kurisetty Vittal , Kovacs Krisztina , Luongo Tony , Merchan Jaime
Published: June 2012

Objective & Background: We have previously reported specific microRNA profiles in renal cell cancer (RCC) cell lines in vitro (Kurisetty et al. AACR Annual Meeting 2010. A # 3045;). Among the miR signatures identified, miR-20a, a member of 17-92 Oncomir cluster, stood out as a highly upregulated miR in most RCC cell lines, regardless of their VHL status. The objectives of this study are to characterize the biological significance of miR 20A upregulation in RCC. Methods: ccRCC cell lines 786.O, ACHN and KV6 and HREC, were used for cell based biological/phenotypic assays. miRNA-gene target prediction analysis was performed using miRGEN and DIANA lab programs. miR-20a-Mimic (Mi) and miR-20a-Inhibitor (In) were transiently transfected into normal HREC cell line and RCC cell lines, respectively. Cell adhesion, proliferation, softagar colony formation and transwell invasion/migration assays were performed to investigate the influences of miR-20a-Mi/In transfected into normal and ccRCC cell lines. Conditioned medium from transfected cell lines was tested for its ability to induce in vitro angiogenesis in HUVECs. Western blotting was performed to identify the changes in E2F5 protein expression in the miR-20a-Mi/In transfected normal and ccRCC cell lines. Results: In normal HREC cells, upregulation of miR 20a (by miR-20a-Mi), significantly induced cell proliferation, adhesion, migration & invasion and soft agar colony formation. miR-20a knock down in RCC cells (by miR-20a-In) significantly reduced cell proliferation, adhesion, migration & invasion and colony formation to levels similar to non-malignant HREC cells. Prediction analyses through bioinformatics approaches identified E2F5, a transcription factor and a tumor suppressor of the E2F family of transcription factors, as a target of miR-20a. Compared to non-tumor HREC cells, E2F5 was significantly downregulated in RCC cell lines, and miR-20a inhibition enhanced E2F5 expression in cancer cells. miR-20a-Mi enhanced the ability of HREC conditioned medium to induce capillary formation in HUVECs. Conclusion: Our results suggest that miR-20a may promote the tumorigenic properties of RCC in vitro. Moreover, miR 20a may promote angiogenesis. We demonstrated that E2F5 is a target of miR 20a, is reduced in RCC cell lines, and its expression is enhanced by mir-20a inhibition, suggesting that E2F5 may mediate the tumor promoting effects of miR 20a. Molecular mechanisms of miR-20a function are currently being investigated further to understand its tumorigenic 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 136. doi:10.1158/1538-7445.AM2011-136
Abstract 1366: Computer-modeled novel cancer targeting peptides for therapy and imaging
Authors: Wang Sheng-Hung , Chen I-Ju , Chang Nai-Chuan , Wu Han-Chung , Yu Alice L. , Yu John
Published: June 2012

We have recently developed practical software programs for optimization of cancer targeting peptides, including combinatorial construction of 3D peptide library (Buildpep), binding pocket analyzer (PscanMS) and high accuracy protein-ligand scoring program (HotLig). Based on these programs, a strategy of structure-based optimization of cancer targeting peptides in silico is developed to target human GRP78 which consists of 654 amino acids. The homology modeling of human GRP78 was constructed through aligning amino acid sequence and 3D structure with its homologs. This protein contains two major structural domains, the peptide-binding domain and the ATPase. Based on the results from the molecular modeling, new peptide analogs were designed and examined for optimized binding to the targeted GRP78 through in silico analysis. We had thus delineated the pharmacophore description of binding motif targeting peptide-binding domain of GRP78 as Pro-X1-Leu-X2. Peptides possessing this motif can be applied in cancer-targeted drug delivery system. We have thus designed several new tumor-targeted peptides [P-12, P-13, and P-6]. Their ability to bind target protein was confirmed in subsequent in vitro binding through Biacore analysis. We next examined their capacity to target cancer cells in the in vitro binding with various cancer cells, and in vivo tumor imaging and targeted chemotherapeutic studies. The results indicate that these peptides exhibit promising therapeutic potential by their selective binding to cancer cells, but not normal cells. Notably, these peptide analogs can bind to breast cancer stem cell (CSC) population in xenografts of primary breast cancer suggesting that they can target breast CSC enriched subpopulation. The microSPECT/CT molecular imaging of a primary human breast tumors, BC0244, xenograft with 188Re-liposome-PEG-P12 or P13 peptide showed significantly more uptake of radioactivity as compared to 188Re- liposome-PEG-L-peptide in the BC0244 breast tumors in NOD/SCID mice at 6, 24 and 48 h after intravenous injection, and even greater difference was found as compared to that of 188Re-liposome alone. To further compare P12 labeled liposome with L-peptide labeled liposome for therapeutic efficacy, mice bearing BC0244 xenografts were distributed into 4 groups for treatment with P12-Lipo-Dox, L-peptide-Lipo-Dox, Lipo-Dox, and PBS. The tumor growth of P12-lipo-Dox group was 1.57-fold slower than that of the L-peptide group; both of them showed significant increase in the therapeutic efficiency than Lipo-Dox control. These optimized cancer targeting peptides may be useful for imaging and targeted therapy of cancers.

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 1366. doi:10.1158/1538-7445.AM2011-1366
Abstract 1367: Substituted Pyrimido[4,5-b]indoles with single agent combination chemotherapeutic potential
Authors: Gangjee Aleem , Zaware Nilesh , Kisliuk Roy L. , Ihnat Michael A.
Published: June 2012

Combinations of cytotoxic agents with antiangiogenic agents have shown significant promise in cancer treatment, and several such clinical trials are currently underway. We previously identified two compounds with a 5-(thioaryl)-9H-pyrimido[4,5-b]indole-2,4-diamine scaffold that each inhibit vascular endothelial growth factor receptor-2 (VEGFR-2) and platelet derived growth factor receptor-beta (PDGFR-β) for antiangiogenic effects and remarkably also inhibit human thymidylate synthase (hTS) for cytotoxic effects. In a COLO-205 xenograft mouse model one of these analogs demonstrated potent tumor growth inhibition, inhibition of metastasis, and antiangiogenic effects in vivo. Thus, these compounds afford combination chemotherapeutic potential in single agents. In an effort to optimize the structural requirements for antiangiogenic effects and hTS inhibition, six additional compounds were synthesized. Electron withdrawing (3,4-diClPh, 4-ClPh), electron donating (2,5-diOMePh, 4-OMePh) and bulky (1-naphthyl, 2-naphthyl) thioaryl groups in the 5-(thioaryl)-9H-pyrimido[4,5-b]indole-2,4-diamine scaffold were selected to establish optimum activity. The synthesis of these compounds involved the nucleophilic displacement of the common intermediate 5-bromo-9H-pyrimido[4,5-b]indole-2,4-diamine with appropriate benzenethiols. A novel four step synthetic scheme to the common intermediate was developed which is more efficient relative to the previously reported six step synthesis. Biological evaluation of these compounds in various RTKs indicated that in the VEGFR-2 assay the 2,5-diOMePh substituted compound was equipotent to the standard SU5416 used in this assay. In the hTS assay, the 2-naphthyl, 4-OMePh, and 4-ClPh substituted compounds were nanomolar inhibitors and were 3-fold better than or equipotent to the standard TS inhibitor raltitrexed.

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 1367. doi:10.1158/1538-7445.AM2011-1367
Abstract 1368: Fragment-based design, synthesis and biological evaluation of a series of novel PDK1 inhibitors
Authors: Sharma Sunil , Venkataswamy Sorna , Warner Steven L. , Call Lee T. , Bearss David J. , Vankayalapati Hariprasad
Published: June 2012

The PDK1 is a monomeric serine/threonine kinase and a promising oncology target of significant current interest for drug development due to its central role in the PI3K/AKT/mTOR pathway. It phosphorylates highly conserved Ser or Thr residues in the activation loop of several AGC super family kinases including PKC, SGK, PKB/Akt, p70S6K, and PDK-1 itself. Approximately, 40-50% of all tumors involve mutations in the PTEN protein, which results in elevated levels of PIP3 and enhanced activation of PKB/AKT, p70S6K, and SGK. It has been proposed that inhibitors of PDK-1 could provide a valuable therapeutic approach to targeting cancer, particularly those with PTEN deficiencies. Using a fragment-based design strategy, we screened a collection of 1100 low molecular weight (< 250 MW) fragments against the PDK1 kinase and identified 9 fragments with moderate inhibitory activity against PDK1 (IC50 values from 45-82 μM).

Of these fragments, those based off of a 5-Br,4-I-1H-indazol-3-amine scaffold seemed the most promising based on initial activity and in keeping with the “rule of 3” for fragment-based drug design. Subsequent molecular docking studies using a crystal structure of PDK1 allowed for the structural rationalization of how these fragments bound in the ATP-binding pocket (hydrogen bonding to S160/A162 hinge residues) and provided insight for further optimization. Concurrently, we carried out scaffold-hopping searches at 2-site points for hydrophobic and solvent pocket fragments. With the addition of one fused heterocyclic ring, the potency increased to 8.8 and 10.9 μM. Our systematic fragment based workflow led to the preparation of target molecules in 4 steps beginning with the condensation, cyclization, and reduction and finally installing the hydrophobic binding site fragments under normal amide coupling. Subsequent SAR and follow-up screening led to the discovery of HCI-1680, a potent PDK1 inhibitor with an IC50 of 97 nM. Additional productive interactions sites with PDK1 were introduced to further improve both biochemical and cellular activities in panel of cancer cells. In cell-based assays HCI-1680 demonstrated enhanced activity in PTEN-deficient cell lines compared to PTEN-wild type lines. HCI-1680 and additional compounds from this series were also shown to inhibit the activation of AKT and other downstream signaling molecules. Kinase selectivity profiling, additional cell-based assays, and animal studies are ongoing. The details of these studies will be presented.

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 1368. doi:10.1158/1538-7445.AM2011-1368
Abstract 1372: A cell based high throughput screening assay for inhibitors of melanoma with high S100B
Authors: Wilder Paul T. , Gianni Kira , Green Deborah L. , Weber David J.
Published: June 2012

The calcium regulated S100B protein is over-expressed in many cancers including malignant melanoma where high levels of S100B correlate inversely with patient survival. It has been shown that increased S100B is not only a marker for cancer but may promote tumor survival by directly affecting the activity of several cell signaling proteins. For example, S100B has been shown to bind wild-type p53 in a calcium-dependent manner, sequestering it, and resulting in the loss of p53-dependent tumor suppression activities, and transient knockdown of S100B with siRNA has been shown to reverse this affect in melanoma cell lines. Therefore, the discovery of compounds that specifically inhibit the growth of melanoma cells with high levels of S100B may provide a useful therapeutic strategy. We have developed a cell based high throughput screening assay where the effect of small compounds on the growth of MALME-3M and WM115 melanoma cells with a stable shRNA to knockdown S100B is compared to the isogenically matched cell control containing a stably expressing a scrambled shRNA. Out of the greater than 15,000 compounds screened many inhibited growth of the melanoma cells, some had selectivity inhibiting the growth of the S100B high cells, and inversely high S100B was found to overcome the growth inhibition caused by several compounds. Previously, many S100B inhibitors found using an in vitro screening assay were found to function by chemically modifying Cys84 in S100B, so S100B might act as a sink to sequester some of the cysteine reactive compounds protecting cells from these compounds in some cases.

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 1372. doi:10.1158/1538-7445.AM2011-1372
Abstract 1396: Bradykinin enhances cell migration in human prostate carcinoma cells via bradykinin B2 receptor, PKCdelta, c-Src and NF-kappaB signaling pathways
Authors: Yu Hsin-Shan , Tang Chih-Hsin
Published: June 2012

Bradykinin (BK) is an inflammatory mediator, and shows elevated levels in regions of severe injury and inflammatory diseases. In addition, bradykinin has been found in many tumor cell lines and has been shown to be involved in carcinogenesis and cancer progression. Prostate cancer is the most common cancer excluding skin cancer in men and usually metastasizes to the particular organs in the late stages of cancer processes. The matrix metalloproteinase (MMP) play an important role in tissue remodeling associated with various physiological and pathological processes such as morphogenesis, angiogenesis, tissue repair, cirrhosis, arthritis and metastasis. In this study, we found that bradykinin increased the migration and the expression of MMP-9 in human prostate cancer cells (PC3,DU145 and LNCaP). Bradykinin-mediated migration and matrix MMP-9 up-regulation was attenuated by bradykinin B2 receptor antagonist (HOE140), protein kinase Cdelta (PKCdelta) inhibitor (Rottlerin), c-Src inhibitor (PP2), NF-kappaB inhibitor (PDTC), and IkappaB protease inhibitor (TPCK). Transfection of cells with bradykinin B2 receptor and protein kinase Cdelta (PKCdelta), siRNA or dominant-negative mutant of c-Src, IkappaBalpha kinase alpha(IKK alpha) and IkappaBalpha kinase beta(IKK beta), also inhibited the potentiating action of bradykinin. Stimulation of PC3 cells with bradykinin induced PKCdelta, c-Src, IKK alpha/beta, IkappaB and p65 phosphorylation, and kappaB-luciferase activity. In addition, BKR2 PCKdelta and c-Src pathways are involved in bradykinin-induced NFkappaB activation. Taken together, our results suggest that bradykinin enhances the migration of prostate carcinoma cells by increasing MMP9 expression through the bradykinin B2 receptor /PKCdelta/c-Src/NF-kappaB signal transduction pathways.

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 1396. doi:10.1158/1538-7445.AM2011-1396
Abstract 1400: Inhibition of cell migration by the synthetic triterpenoid acetylenic tricyclic bis-(cyano enone)
Authors: Chan Eddie , Guglielmo John Di
Published: June 2012

Triterpenoids are a family of naturally occurring compounds that have multiple anti-tumorigenic effects, including the ability to induce cell differentiation, apoptosis and inhibit proliferation. Research in our lab has demonstrated that a synthetically derived 5 ring triterpenoid, 2-cyano-3, 12-dioxooleana-1,9-dien-28-oic acid-imidazolide (CDDO-Im), inhibits cell migration (To et al., 2008, J. Biol. Chem. 283:11700), which is essential in numerous physiological processes including tumor metastasis. Recently, a smaller 3 ring compound containing the same active functional groups as CDDO-Im has been derived; acetylenic tricyclic bis-(cyano enone) [(+/-)-(4aa,8aa,10ab)-1,2,4a,6,8a,9,10,10a-octahydro-8a-ethynyl-1,1,4a-trimethyl-2,6 dioxophenanthrene-3,7-dicarbonitrile] (TBE-31). Here we examined the effect of TBE-31 on cell migration. Using scratch assays with Rat2 fibroblasts we demonstrated that concentrations of TBE-31 above 0.75 µM were effective in inhibiting fibroblast migration. To elucidate the mechanism(s) for this inhibition we examined different components of the cytoskeleton and the polarity complex, which are both crucial for cell migration. To examine cell polarity, we used immunofluorescence microscopy to assess the localization of two proteins involved in the polarization of migrating cells, IQGAP1 and RAC1. Similar to CDDO-Im, TBE-31 was observed to reduce the elongation and polarity of cells, however unlike CDDO-Im, IQGAP1 and RAC1 in TBE-31 treated cells were localized in punctate fashion around the cell perimeter instead of at the leading edge. Using immunofluorescence imaging, the cytoskeleton of Mv1Lu mink lung cells or COS7 cells treated with TBE-31 were analyzed. The microtubule network of treated cells displayed a unique cross-hatched phenotype and were resistant to nocodazole depolymerization. In addition, the intermediate filament cytoskeleton was observed to retract towards the nucleus. Lastly, in vitro actin polymerization assays revealed that TBE-31 inhibits linear but not branched actin polymerization. We conclude that TBE-31 is an effective inhibitor of cell migration and further research will shed light on the mechanisms that TBE-31 uses to modulate the cell migration machinery.

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 1400. doi:10.1158/1538-7445.AM2011-1400
Abstract 1409: Overexpression of inhibitor of DNA (Id-1) correlates with the invasiveness of colon cancer cells
Authors: Pandey Ambarish , Oh Michael , Sengupta Shiladitya
Published: June 2012

Background: Id-1 gene expression has been shown to be involved in carcinogenesis in several types of human cancers. It belongs to a family of helix loop helix proteins that has been shown to induce cell proliferation, increase DNA synthesis and changes in gene expression to promote tumor growth and metastasis. In this Study we look into the expression of Id-1 in human colon cancer cell lines and its effect on the invasive potential of the cancer cells.

Methods: Id-1 gene expression in human colon cancer cell line HT-29 was analyzed by real time Polymerase chain reaction. The expression of Id-1 protein in colon cancer tissue array samples was examined by immunohistochemical assay and its relationship with the clincopathological staging of the tumor was analyzed. Id-1 gene knockdown was carried out in HT-29 cells using siRNA transfection and the knock down cells were subjected to matrigel invasion assay.

Results: A significant overexpression of id-1 gene was observed in the colon cancer cell line HT-29 as compared to normal epithelial cells. Immunohistochemical analysis showed that id-1 protein expression in malignant tissue was significantly more than the normal variant and there was a significant correlation between the degree of Id-1 expression and the stage of the colon cancer. Knockdown of Id-1 in the cancer cells significantly decreased the invasiveness potential in the matrigel after 72 hours as compared to the controls.

Conclusion: The level of id-1 expression is correlated to the malignant potential of colon cancer cells and its expression seems to be critical for the invasiveness and more aggressive behavior of the 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 1409. doi:10.1158/1538-7445.AM2011-1409
Abstract 1412: Two-photon imaging of cancer cell extravasation in live mice
Authors: Digman Michelle A. , Aguilar Jose S. , Gratton Enrico , Suetsugu Atsushi , Hoffman Robert M.
Published: June 2012

MDA-MB-231 breast cancer cells were engineered to express cytoplasmic paxillin-GFP and nuclear H2B-mCherry. In order to image extravasation, the cancer cells were injected in the blood stream of nude mice. Using 2-photon excitation microscopy we can simultaneously excite the two probes and also visualize the autofluorescence of tissues. A skin flap was opened to visualize blood vessels and recognize the position of the cancer cells. Two-photon imaging showed that after an initial phase in which the cells are non-adherent, some cells spread on the internal surface of the capillaries. Days later some cells started to appear on the external side of the capillary. The extravasated cells extend very long protrusions into the tissue. The goal was to determine if at the end of the long protrusion, if it is possible to observe the formation of focal adhesions by imaging paxillin-GFP. Preliminary results show that when cells start to adhere to the blood vessel wall they form focal adhesions as determined by the characteristic elongated features observed in the paxillin-GFP channel. New approaches will allow the tracking of the tip of the protrusion to determine if focal adhesions are forming there as the cells extravasate. This is important in establishing the mechanism of cell extravasation and migration in tissues.

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 1412. doi:10.1158/1538-7445.AM2011-1412
Abstract 1414: Lysophosphatidic acid-induced Cyr61 is associated with prostate cancer progression
Authors: Terada Naoki , Zeng Yu , Kulkarni Prakash , Getzenberg Robert H.
Published: June 2012

Lysophosphatidic acid (LPA) is a serum phospholipid that has been associated with progression in several types of cancer. Cysteine-rich angiogenic inducer 61 (Cyr61) is an extracellular matrix protein involved in the transduction of growth factor and hormone signaling. We have reported that Cyr61 expression in benign prostate cells is regulated by LPA (Endocrinology 2004). Furthermore that Cyr61 expression is significantly up-regulated in prostate cancer (PCa) and that the expression levels correlate with Gleason score (J Urol 2010). The aim of this study was to determine if LPA-induced Cyr61 up-regulation is associated with PCa progression. BPH1 was utilized as a benign prostate cell line and PC3 as a PCa cell line. In real-time PCR of untreated BPH1 and PC3 cells, Cyr61 expression ratios to TBP were 0.5 and 5.1, and these ratios after 2 hours of 1uM LPA under serum-free medium treatment were 1.8 and 44.3, respectively. These results indicated that the Cyr61 expression levels and their increase by LPA stimulation were higher in PCa cells than in benign prostate cells. To examine whether LPA promotes cell invasion, Matrigel invasion assays were performed for 72 hours with or without 1uM of LPA in the lower chamber. LPA stimulation increased the invaded cell number from 0.3±0.5 to 12.6±2.5 (p<0.001) in BPH1 cells and from 31.9±20.9 to 55.2±14.8 (p=0.005) in PC3 cells. These results indicate that LPA promotes cell invasion in both benign prostate and PCa cells. To examine whether LPA-induced Cyr61 is associated with cell invasion, the Cyr61 was knocked down by siRNA in PC3 cells. Invaded cell numbers were changed by 48 hours of LPA stimulation after scramble siRNA from 2.2±1.3 to 12.3±10.2 (p=0.002) and after Cyr61 targeting siRNA from 1.8±2.6 to 2.7±2.7 (p=0.235), respectively, revealing that Cyr61 down-regulation suppresses LPA induced cell invasion. To investigate the association between Cyr61 up-regulation and prostate carcinogenesis, Cyr61 over-expressing BPH1 cells (BPH1-Cyr61) were established and their cell proliferation was compared with vector only transfected BPH1 cells (BPH1-mock) by MTS assays. The cell proliferation ratio of BPH1-mock and BPH1-Cyr61 cells after 48 hours of incubation in serum-free medium was 1.24±0.05 and 1.40±0.02 (p=0.043), respectively, indicating that Cyr61 over-expression promoted cell proliferation of BPH1 in vitro. In conclusion, LPA induced Cyr61 up-regulation was suggested to be associated with PCa progression.

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 1414. doi:10.1158/1538-7445.AM2011-1414
Abstract 1430: Automation of both sample preparation and analysis of a cell migration assay
Authors: Burroughs Sarah E. , Hulkower Keren I. , Schicktanz Simone , Fronczak Jennifer A. , Boettcher Karin , Cloutier Timothy E.
Published: June 2012

Cell migration is a critical event in the process of angiogenesis. Aberrant cell migration can contribute to metastatic cancer and other diseases. Understanding the signaling pathways involved in regulating migration is crucial for discovering new targets to develop drugs that inhibit migration and angiogenesis. Because cell migration is a phenotypic or whole-cell event, it can be challenging to conduct and quantitatively analyze such cell-based assays in a consistent and efficient manner. Automation of such assays allows researchers to analyze more conditions than would be feasible with manual methods. With advances in migration plate technology and automated liquid handling, it is now possible to increase one's capacity to run these important assays. However, liquid handling automation parameters are critical and must be optimized so as not to interfere with the biology or its analysis. Here we demonstrate a successful automation of the sample preparation the Platypus Oris® Pro 384-Well Cell Migration Assay on a PerkinElmer JANUS® Automated Liquid Handling Workstation. In addition, quantitation of such assays is now possible with multi-mode detection and High Content Screening (HCS) technologies. Analysis of cell migration using the Operetta® / Harmony® HCS platform and the EnSpire™ multilabel plate reader shows the assay to be a robust and reproducible quantification of cell motility. Using these methods the authors demonstrate statistically significant inhibition of this phenotypic cellular process by a variety of compounds, including cytochalasin D and the MEK inhibitor U0126.

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 1430. doi:10.1158/1538-7445.AM2011-1430
Abstract 1448: Interaction between decorin and E-cadherin/beta-catenin signaling attenuates colorectal cancer growth and metastasis in vitro and in decorin knockout mice
Authors: Bi Xiuli , Yang George R. , Gou Yuan , Pohl Nicole , Guzman Grace , Kajdacsy-Balla Andre , Iozzo Renato V. , Yang Wancai
Published: June 2012

Decorin is a small leucine-rich proteoglycan. Previous studies have demonstrated that decorin expression is significantly reduced in colorectal cancer tissues and cancer cells, and genetic deletion of the decorin gene caused intestinal tumor formation in mice, which was linked to a downregulation of p21, p27kip1 and E-cadherin and an upregulation of β-catenin signaling. However, the regulation of E-cadherin by decorin and its implication in cancer formation and metastasis is largely unknown. Using manipulated colorectal cancer cells and decorin knockout mouse model (Dcn-/- mice), we found that increasing decorin expression in colorectal cancer cells delayed cancer cell growth, accelerated apoptosis, and importantly, attenuated cancer cell migration, invasion and wound healing. Mechanistic study revealed that the attenuation of cancer cell malignancy was resulted from the interaction between decorin and E-cadherin, a protein that regulates cell-cell adhesion, epithelial-mesenchymal transition and metastasis. First, overexpression of decorin led to upregulation of E-cadherin through increasing of E-cadherin protein stability, while E-cadherin mRNA and promoter activity were not affected; Second, co-immunoprecipitation assay showed that decorin physically bound E-cadherin. In addition, the upregulation of E-cadherin was associated with reduction of β-catenin, c-myc and CDK4. cDNA microarray analysis also showed that the levels of two proteins involved in metastasis were altered, the extracellular proteinase inhibitor (EXPI) was significantly downregulated and Claudin 2 was upregulated in intestinal epithelial cells of the Dcn-/- mice. Moreover, loss of decorin accelerated murine cancer cell MC38 growth and invasion in the flank of Dcn-/- mice. Taken together, our results provide direct evidence that decorin-mediated inhibition of colorectal cancer formation and metastasis is through the interaction between decorin and E-cadherin/beta-catenin signaling.

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 1448. doi:10.1158/1538-7445.AM2011-1448
Abstract 1460: AKAP12 regulates melanoma tumor growth and metastasis
Authors: Finger Elizabeth C. , Banh Alice , Rankin Erinn B. , Vilalta Marta , Krieg Adam J. , Broome-Powell Marianne , Giaccia Amato J.
Published: June 2012

Skin cancer, which accounts for at least half of all cancers in the United States, is predicted to affects 1 in 5 Americans in their lifetime. Melanoma, the most serious form of skin cancer, is also the leading cause of all skin cancer deaths. This study demonstrates that AKAP12 (SSeCKS, AKAP250, gravin), a hypoxia-inducible scaffold protein, is considerably increased at the protein and RNA level in melanoma cell lines. In addition, there is a significant increase in RNA expression in primary and metastatic tumors when compared to normal specimens, as well as patients with poor prognosis, as measured by diminished 5- year survival. AKAP12 functions to dock many kinases and signaling proteins that regulate cell adhesion, spreading and migration, including: PKC, Src and F-actin. Since these processes are critical for tumor cell metastasis, we hypothesize that AKAP12 could be a central regulator of cancer progression. Stable knockdown of AKAP12 in two melanoma cell lines demonstrate decreases in invasion, migration and clonogenic potential in vitro, as well reduced colony forming abilities in conditions requiring anchorage-independent growth in soft agar. In vivo studies in a metastatic model of melanoma demonstrate a statistically significant decrease in tumors within the lungs of the AKAP12 knockdown groups, highlighting the importance of AKAP12 in metastatic disease. An orthotopic model also demonstrates decreased primary tumor growth and metastatic potential in mice injected with AKAP12 knocked down cells. Additionally, changes in cell morphology characteristic of increased motility and invasiveness are seen in cells containing AKAP12 but not those in which it had been knocked down. While this scaffold protein has been found to act as a tumor suppressor in several tissue types (lung, colon, prostate), we have uncovered its novel oncogenic properties in metastatic melanoma.

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 1460. doi:10.1158/1538-7445.AM2011-1460
Abstract 1472: MRP4 and PGT, members of the COX-2 pathway, may contribute to breast cancer progression
Authors: Kochel Tyler , Kundu Namita , Ma Xinrong , Reader Jocelyn , Brodie Angela , Fulton Amy M.
Published: June 2012

Prostaglandin E2 (PGE2) is the major product of the cyclooxygenase 2 (COX-2) enzyme. PGE2 is a substrate of multiple drug resistance-associated protein 4 (MRP4) which exports it from the cell. PGE2 can then signal through any of its four GPCR-family receptors EP1-4. Each receptor is connected to a different cell function including adenylate cyclase activation, calcium flux, and inositol trisphosphate mobilization. Alternatively, extracellular PGE2 can be imported through prostaglandin transporter (PGT) and inactivated by 15-hydroxy prostaglandin dehydrogenase (15-PGDH). PGE2 plays a role in tumor progression as evidenced by the anti-tumor activity of COX-2 inhibitors. Adverse effects of these inhibitors have forced a search to identify other potential therapeutic targets in the COX-2 pathway (such as the receptors, transporters, and associated metabolic enzymes of PGE2). We hypothesize that disregulation of the COX-2 pathway can lead to increased metastatic potential in breast cancer. High extracellular PGE2 in the tumor microenvironment can stem from high COX-2 expression or elevated eicosanoid export activity. Continued prostaglandin signaling is thought to occur due to the interplay between increased expression of the exporting protein (MRP4) shuttling PGE2 into the pericellular space and decreased expression of the importing protein (PGT) removing PGE2 from the pericellular space. Higher concentrations of PGE2 in the pericellular space allow for increased signaling through PGE2 receptors EP1-4. Using a panel of breast cancer cell lines, we demonstrate that highly metastatic tumor cells can express low levels of COX-2, but still actively signal through PGE2 receptors. MRP4 mRNA and protein expression is increased in tumor cell lines with high metastatic potential while expression of PGT mRNA and protein is decreased in these cells when compared to cells with lower metastatic potential. This should lead to higher concentrations of extracellular PGE2 in the tumor microenvironment. Additionally, we have examined MCF7 cells stably transfected with aromatase and an anastrozole-resistant derivative of this cell line for expression of COX-2, EP4, EP1, MRP4 and PGT. At the mRNA level, COX-2, EP1, MRP4 and PGT decrease in the aromatase inhibitor-resistant cells. EP4 mRNA levels increase in the resistant cells. Examining the relative expression levels of all key members in the COX-2 pathway as they relate to tumor progression could highlight new therapeutic targets under this classification of breast 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 1472. doi:10.1158/1538-7445.AM2011-1472
Abstract 1482: Role of TGF-beta signaling and mechanism of osteoblastic tumorigenesis in prostate cancer
Authors: Mishra Sweta , Tang Yuping , Werner Sherry L , Sun LuZhe
Published: June 2012

Prostate cancer is the second leading cause of cancer death in men with 80% of deaths occurring due to bone metastasis. Transforming growth factor-beta (TGFβ) signaling is important for the regulation of bone mass and matrix properties. The role of TGFβ signaling in prostate cancer-induced bone metastasis is unclear due in part to a lack of prostate cancer cell lines that can efficiently induce bone metastasis and osteoblastic lesions in animal models. We have previously shown that a newly established human prostate cancer cell line, PacMetUT1, produces a large amount of active TGFβ1 and induces extensive bone metastases in calvaria, rib, femur, and tibia when inoculated in the left cardiac ventricle of male nude mice. Experimental data from our lab demonstrated that stable knockdown of TGFβ1 using a short hairpin-RNA resulted in a decreased tumor incidence and bone formation when the cells were directly injected into the right tibias of male nude mice. Systemic administration of either a small inhibitor of TGFβ type I receptor kinase or a pan-TGFβ binding protein also decreased osteoblastic intratibial tumor lesions. However, the mechanism by which prostate cancer cells metastasize to bone and how they induce formation of osteoblastic lesions remains to be intensively investigated. We hypothesize that TGFβ and other growth factors released from tumor cells can induce aromatase gene expression and activity in bone cells leading to an enhanced estradiol synthesis which would lead to an increase in bone formation as estrogen is a known osteogenic factor. We examined the effect of TGFβ1 and BMP-4/7 treatment on aromatase expression in pre-osteoblastic cells in vitro. In vitro treatment with 5ng/ml of TGFβ1 and 25ng/ml of BMP-4/7 resulted in an increase in aromatase expression in 2T3, a mouse mesenchymal stem cell and in 63 year old male mesenchymal stem cells (MSC) at the protein level. Furthermore, TGFβ resulted in an induction of Smad-1/5 signaling pathway in these pre-osteoblastic cells. Transient knockdown of Smad-1/5 expression in male MSC by siRNA, resulted in a decreased aromatase expression. Our results demonstrate TGFβ to be a major driver of osteoblastic tumorigenesis in PacMetUT1 cells, and Smad-1/5-dependent aromatase upregulation by TGFβ superfamily ligands in pre-osteoblastic cells. Future studies will delineate whether the induction of aromatase mediates osteoblastic bone metastasis in vivo, and if combination treatment of TGFβ and aromatase inhibitors can be used as a novel therapeutic strategy for bone metastatic prostate 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 1482. doi:10.1158/1538-7445.AM2011-1482