bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2482: Effect of L-isoleucine and L-leucine at early stage of rat bladder carcinogenesis
Authors: Kanagawa Akihiro , Xie Xiaoli , Yamano Shyotaro , Wei Min , Wanibuchi Hideki
Published: February 2011

We previously demonstrated the promotion effects of L-isoleucine and L-leucine on N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced rat bladder carcinogenesis when supplemented in AIN-93G diet in a 29-week bioassay. However, little is known about the mechanism underlying the promotion effects. The aim of the present study is to evaluate the effects of L-isoleucine and L-leucine at early stage of rat bladder carcinogenesis. In experiment 1, rats were fed AIN-93G basal diet, 2% L-leucine or L-isoleucine in basal diet for 2 weeks. In experiment 2, rats were administered 0.05% BBN in drinking water for the first 2 weeks, and then fed basal diet, 2% L-leucine or L-isoleucine diet for 4 weeks. No treatment-related histopathologic changes in bladder were observed in both experiments. There were also no differences in proliferative activities and expressions of 12 genes that were previously found to be up-regulated in BBN-treated bladder urothelium from week 2 between basal diet group and L-leucine or L-isoleucine group regardless of BBN initiation. These findings indicated the possibility of lack of effects of L-isoleucine and L-leucine at early stage of rat bladder carcinogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2482.
Abstract 2485: Inhibitory effects of the water extract of Nelumbo Nucifera leaf on human breast carcinoma MCF-7 cells in vitro and in vivo
Authors: Chang Yun-Ching , Yang Mon-Yuan , Wang Chau-Jong
Published: February 2011

Over the passed decade, advances in understanding carcinogenesis have made possible the identification of the candidates of chemopreventive agents to be developed in targeting the key molecules. Lotus (Nelumbo nucifera Gaertn) is widely distributed in central and southern Taiwan. Its leaves are becoming popular as an ingredient of healthy beverages. Nelumbo nucifera Leaf extract (NLE) has been reported to exhibit different therapeutic effects, such as anti-oxidant and reduction of risks for cardiovascular disease. This study was aimed to investigate the antitumor effect of NLE on human MCF-7 cell line in vitro and in vivo. Qualitative analysis showed that the NLE contains several compounds using nine kinds of standard polyphenols by high-performance liquid chromatography (HPLC) method. The polyphenols present in identified compound of NLE were mainly gallic acid, rutin and quercetin. The MTT data showed no significant inhibition on growth of MCF-7 cells following NLE treatment, whereas flow cytometry revealed that the cell cycle was arrested at G0/G1 phase with a dose of 3 mg/ml in MCF-7 cells (approximately 86.54%). With western blotting, we found that treatment with NLE particularly induced p53 phosphorylation and p21 expression, and down-regulated expression of cyclins (cyclin D, cyclin E) and cyclin-dependent kinases (cdk2, cdk4) in a dose-dependent manner. The increased p21 also correlated with the decrease of cyclin D/cdk4 and cyclin E/cdk2 complexes, which was responsible for the high protein expression of Rb/E2F. Inactivation of fatty acid synthase (FAS) was shown in vitro and in vivo, implying that FAS was a potent regulator as well of NLE- mediated anti-carcinogenesis. The tumoricidal effect of NLE was further measured using a xenograft nude mice model. Treatment with NLE (0.5 and 1%) effectively reduced the tumor volume and tumor weight in the nude mice inoculated with MCF-7 cells when compared to the control tissue samples. These results confirmed that cell cycle arrest is sufficient to elicit tumor regression after NLE treatment. In conclusion, our studies indicate that NLE offers an anti-tumor activity and potentially can act as a chemotherapeutic agent.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2485.
Abstract 2486: A possible mechanism for plagiochiline A activity: Inhibition of cytokinesis
Authors: Stivers Nicole S. , Islam Ashraful , Casson Lavona K. , Aponte Jose C. , Hammond Gerald B. , Vaisberg Abraham J. , Bates Paula J.
Published: February 2011

We recently reported on the isolation of plagiochiline A, a 2,3-secoaromadendrane-type sesquiterpenoid, from the Peruvian medicinal plant Plagiochila distichia. This compound was found to have antiproliferative activity against a variety of solid tumor cell lines, including those derived from prostate, colon, breast, lung, and cervical cancers, as well as several leukemia cell lines. Other researchers have also noted the cytotoxicity of plagiochiline A (isolated from other plant species), but the mechanism for this activity was unknown. Here, we describe new research where we have investigated the mechanism of plagiochiline A using a variety of experimental techniques. First of all, we evaluated cell cycle parameters in DU145 prostate cancer cells treated for 24 h with 4 µg/mL (11.4 µM) plagiochiline A using flow cytometry based analysis of propidium iodide-stained cells. We found that plagiochiline A caused a statistically significant increase in the percentage of cells in the G2/M phase compared to vehicle-treated cells (41.7% compared to 24.8%, based on three independent experiments). We next examined similarly treated cells stained with DAPI (a DNA intercalator) and anti-tubulin antibodies (microtubule marker) using fluorescence microscopy to evaluate the mitotic status of the cells. These experiments revealed a large number of binucleate cells, many of which had intercellular bridges evident, specifically in the plagiochiline A-treated samples. Only a few cells were observed in earlier stages of mitosis, suggesting that the accumulation of cells in G2/M is due to a defect in cytokinesis. We are currently carrying out additional research in order to test this hypothesis. We also plan to determine the effects on cells following longer treatments with plagiochiline A and to investigate possible molecular target(s) of plagiochiline A. Although many anti-mitotic compounds are in current use as anticancer agents, there are apparently only a few agents in development that specifically target cytokinesis. Therefore, we anticipate that this research may ultimately lead to novel anticancer agents and new tools to study cytokinesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2486.
Abstract 2489: Dramatically increased microtubule shortening resulting from overexpression of β5-tubulin is reversed by paclitaxel
Authors: Bhattacharya Rajat , Yang Hailing , Cabral Fernando
Published: February 2011

Vertebrate cells express 6-7 isotypes of both α and β-tubulin, all of which coassemble into microtubules. Our previous studies demonstrated that β3 and β5 (but not β1, β2, and β4b) tubulin overexpression destabilizes microtubules, inhibits cell growth, and confers paclitaxel resistance (Blade, K., et al. (1999). J Cell Sci 112: 2213-21; Bhattacharya, R. and F. Cabral (2004). Mol Biol Cell 15: 3123-31; Hari, M., et al. (2003). Cell Motil Cytoskeleton 56: 45-56.). Overexpression of β3 does not appear to significantly alter microtubule dynamics, but dynamic behavior is relatively resistant to suppression by paclitaxel (Kamath, K., et al. (2005). J Biol Chem 280: 12902-7.). We now report the effects of β5 overexpression on microtubule dynamics. Microtubules were visualized in wild type (WT) or β5 overexpressing CHO cells using eGPF-MAP4. Growth and shrinkage of individual microtubules were measured using ImageJ and dynamic parameters were calculated by analyzing life history plots. We found that microtubules with high β5 content had significantly increased shortening rates and very long depolymerization distances compared to wild type. Addition of paclitaxel at a concentration that rescues cell growth, completely reversed these effects; but the dynamic parameters in such microtubules were significantly different compared to WT microtubules and resembled the inhibited microtubule dynamics of WT cells treated with paclitaxel. We conclude that suppression of microtubule dynamics in presence of paclitaxel is insufficient to explain why β5 overexpressing cells become resistant to the drug.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2489.
Abstract 2492: Microtubule-interacting agents increase Class I HLA and HLA-A2 molecules surface expression in ovarian cancer cells
Authors: Pellicciotta Ilenia , Goldberg Gary L. , Shahabi Shohreh
Published: February 2011

Objective: Ovarian cancer is the leading cause of death from gynecologic cancers in the United States. Standard first line therapy for ovarian cancer includes paclitaxel and platinum based agents. Epothilone B, Taxol and Vinblastin belong to a family of anti-neoplastic agents that interfere with microtubules and arrest cells in the G2/M phase of the cell cycle. Little is known about the effect of these drugs on the immune response to tumors. There is compelling evidence that cancer cells use multiple strategies to evade immune recognition. One of these mechanisms is the down-regulation of human leukocyte antigen (HLA) Class I molecule expression on the tumor cell surface, allowing the cancer cell to escape immune recognition and destruction.

Data will be presented to illustrate the effect of microtubule affecting agents on HLA class I molecule expression in ovarian cancer cells. The effect on the expression of HLA-A2, the most common class I allele in the Caucasian population, has been evaluated as well. We also investigated the relationship between surface HLA expression and transcriptional modification of HLA induced by the drugs.

Methods: IC50 was determined for each compound based on a 72 h cytotoxicity assay, using ovarian cancer cell line Hey. Cells were treated with different concentrations ranging from 1-fold IC50 to 100-fold IC50, and incubated at 37°C for 24 h, 48 h and 72 h. Cell surface expression of HLA class I molecules and HLA-A2 was examined by flow cytometry, using anti-HLA-A,B,C antibody (G46-2.6 clone) and HLA-A2 (BB7.2 clone). RT-PCR and real time PCR were used to amplify HLA genes, using Platinum Taq DNA Polymerase and Power SYBR Green master mix respectively.

Results: Low dose Epothilone B, Taxol and Vinblastin (10-fold IC50) increased cell surface expression of HLA Class I up to four-fold in Hey ovarian cancer cells. HLA-A2 molecules were increased up to 2-fold. The increase of HLA Class I molecule expression was dose- and time-dependent.

Conclusions: Nanomolar concentrations of microtubule-interacting agents increase the expression of HLA class I molecules in Hey ovarian cancer cells. Our data suggest that low doses of microtubule-interacting agents can mediate modulation of surface HLA expression and may enhance immune recognition of tumor cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2492.
Abstract 2493: Inhibition of phosphoinositide 3-kinase suppressed growth of cancer cells with PIK3CA mutations or loss of PTEN expression via G0/G1 arrest of cell cycle but not apoptosis
Authors: Dan Shingo , Okamura Mutsumi , Yoshimi Hisashi , Mukai Yumiko , Yamazaki Kanami , Inoue Yasumichi , Hanyu Aki , Imamura Takeshi , Sakaue-Sawano Asako , Miyawaki Atsushi , Yamori Takao
Published: February 2011

Background: The phosphoinositide 3-kinase (PI3K) pathway is frequently activated in cancer by gain-of-function mutation of PIK3CA and loss of PTEN expression, and mediates growth- and anti-apoptotic signals. Therefore, PI3K is considered as a promising therapeutic target to inhibit growth and to induce apoptosis in such cancers. We previously developed a novel PI3K-specific inhibitor ZSTK474 with antitumor efficacy (Yaguchi et al, J. Natl. Cancer Inst. 98: 545-56, 2006; Kong and Yamori, Curr. Med. Chem. 16: 2839-54, 2009). In this study, we have examined the cellular mechanism by which ZSTK474 exerts its antitumor efficacy in human cancer cells with or without PIK3CA/PTEN aberrations.

Methods: We examined the antitumor effect of ZSTK474 on various human cancer cell lines with or without PIK3CA/PTEN aberration in vitro and in vivo. Protein expression and apoptosis in ZSTK474-treated and untreated tumor sections were analyzed by immunohistochemistry and TUNEL assay, respectively. Analysis of cell cycle in vitro was determined by flow cytometry and by Fucci (Fluorescent Ubiquitination-based Cell Cycle Indicator) system introduced to cancer cells. Induction of apoptosis in vitro was estimated by caspase activation using a substrate cleavage assay and by detection of subdiploid cells using flow cytometry.

Results: In vivo, ZSTK474 effectively inhibited the growth of human tumor xenograft derived from a PTEN-null prostate cancer cell line PC3. In parallel, ZSTK474 treatment suppressed the expression of phospho-Akt, suggesting effective PI3K inhibition, and also suppressed the expression of nuclear cyclin D1 and Ki67, both of which are hallmarks of proliferation. However, ZSTK474 treatment did not increase TUNEL-positive apoptotic cells. Similar result was obtained in another PTEN-negative cell line KM-12 and three PIK3CA-mutated cell lines HCT-15 (E545K), MKN1 (E545K) and SK-OV3 (H1047R). In vitro, ZSTK474 induced marked G0/G1 arrest accompanied by a decrease of nuclear cyclin D1, induction of p27kip1 and p15ink4b, and hypophosphorylation of pRB in PC-3 cells. However, ZSTK474 did not increase the subdiploid cells or activate caspase, both of which are hallmarks of apoptosis. Similar result was obtained other cell lines with or without PIK3CA /PTEN aberrations.

Conclusion: These results clearly indicated that ZSTK474 did not induce apoptosis but rather induced strong G0/G1 arrest in cancer cells with or without PIK3CA/PTEN aberrations, which might cause its therapeutic efficacy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2493.
Abstract 2510: Efficacy of the Chk1 inhibitor SCH900776 at abrogating cell cycle arrest and enhancing DNA damage-induced cytotoxicity: Comparison with UCN-01
Authors: Eastman Alan , Montano Ryan E. , Garner Kristen M. , Chung Injae , Parry David A.
Published: February 2011

Many anticancer agents damage DNA and arrest cell cycle progression primarily in S or G2 phase of the cell cycle depending on drug concentration. Previous studies with the topoisomerase I inhibitor SN38 have demonstrated the efficacy of the Chk1 inhibitor UCN-01 to overcome arrest forcing cells through S, G2 and lethal mitosis. UCN-01 was limited in clinical trials by high plasma protein binding; as a consequence many alternate Chk1 inhibitors have now been developed. SCH900776 is a novel Chk1 inhibitor entering Phase II clinical trials. SCH900776 inhibits Chk1 and overcomes SN38-mediated S and G2 arrest at 300 nM in several carcinoma cell lines which is comparable to UCN-01 at 50 nM. While both Chk1 inhibitors accelerated the rate of cell killing induced by SN38, neither enhanced the overall cell killing. Similarly, no overall sensitization to cisplatin was observed. In contrast, both Chk1 inhibitors induced dramatic sensitization of cells to antimetabolite-induced damage. For example, inhibition of Chk1 sensitized MDA-MB-231 cells to hydroxyurea with the 50% growth inhibitory concentration decreasing from 3 mM to 30 µM, a 100 fold sensitization. Importantly, concentrations of hydroxyurea that exhibited no activation of a checkpoint and no growth inhibition alone were sensitized by inhibition of Chk1. SCH900776 also induced a 50-fold sensitization to cytarabine, 10 fold sensitization to gemcitabine, but no sensitization to 5-fluorouracil. Hence, this sensitization does not extend to all antimetabolites. The 100-fold sensitization to hydroxyurea appeared to require complete inhibition of Chk1 (2 µM 776), although 10-fold sensitization to hydroxyurea was still observed at 200 nM SCH900776. In an MDA-MB-231 derivative expressing shRNA that resulted in almost undetectable levels of Chk1, the full level of sensitization to hydroxyurea could be achieved at 200 nM SCH900776 demonstrating that the response of tumors may vary with the endogenous level of Chk1. We are currently comparing the differential response of a panel of carcinoma cell lines and find some that are sensitized to hydroxyurea up to 100-fold at 200 nM SCH900776, although there appear to be determinants other than the Chk1 levels. These results can be extrapolated to suggest that marked differences in efficacy of SCH900776 can be expected in patients. It will be important to define the parameters of response as these may facilitate selection of patients most likely to respond to combination therapy with Chk1 inhibitors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2510.
Abstract 2512: Epothilone B enhances surface EpCAM expression in ovarian cancer cells
Authors: Shahabi Shohreh , Yang Chia-Ping H. , Goldberg Gary , Horwitz Susan B.
Published: February 2011

OBJECTIVES

Epothilone B (EpoB), like Taxol, induces tubulin polymerization and microtubule stabilization resulting in an inhibition of microtubule dynamic instability. The drug is presently being evaluated in phase III clinical trials. An EpoB analog Ixabepilone, has been approved by the FDA for the treatment of metastatic breast cancer. Epithelial cell adhesion antigen (EpCAM) expression is significantly higher in epithelial ovarian cancer cells compared to normal cells.

METHODS

We used biochemical methods, immunofluorescence and flow cytometry to identify EpCAM expression on the surface of the ovarian cancer cell line, Hey, after exposure to EpoB. We also investigated the relationship between EpoB-mediated surface EpCAM expression and EpoB-induced tubulin acetylation in Hey cells.

RESULTS

We investigated the effect of EpoB and other microtubule-interacting agents on surface EpCAM expression and found that nanomolar concentrations of EpoB, Taxol, discodermolide or vinblastine caused a marked increase in surface EpCAM expression in an ovarian cancer cell line, Hey. Alpha-tubulin acetylation, a surrogate marker for stable microtubules, was increased following treatment with Taxol, EpoB and discodermolide, but not with vinblastine, indicating that drug-enhanced surface EpCAM expression does not correlate with tubulin acetylation or stabilization. Unexpectedly, EpoB did not have a significant effect on EpCAM mRNA expression, nor did it alter the level of total cellular EpCAM in the human ovarian cancer cell line.

CONCLUSIONS

Our results suggest that disruption of the microtubule cytoskeleton is associated with the redistribution of cell surface antigens in ovarian cancer cells. The increase in cell surface EpCAM antigen density may facilitate the antibody targeting of EpCAM-positive ovarian cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2512.
Abstract 2518: TAK-733 is a Potent and Selective Inhibitor of Mammalian MEK Kinases Capable of Inducing Apoptosis in BRAF Driven Cells Both in vitro and in vivo
Authors: Fabrey Robyn , Shawn O'Connell , Tsuchiya Shuntarou , Hori Akira , Vincent Patrick
Published: February 2011

Mutations in BRAF a serine/threonine protein kinase are frequently observed in human cancers. A valine to glutamic acid mutation at amino acid position 600 accounts for nearly 80% of all the activating mutations found for this kinase. This mutation (V600E) forces BRAF to become constitutively active and phosphorylate its substrates regardless of upstream signaling. Numerous cancers in tissues such as skin, colon, thyroid, and ovary have been attributed to uncontrolled proliferation driven by this mutation. TAK-733 represents a novel and distinct chemotype which is capable of allosteric inhibition of the BRAF substrates MEK-1 and MEK-2 at low nanomolar concentrations. Viability assays in established human cancer cell lines which bear the BRAF V600E mutation demonstrate this compound was also capable of lowering NADH levels with single digit nanomolar EC50s. Loss of NADH production can be attributed to the induction of programmed cell death pathways involving Caspases 3 and 7. Induction of these apoptosis markers was confirmed in vitro by a Caspase-GLO assay. TAK-733 induces apoptosis more potently in COLO 205 (BRAF V600E) human colo-rectal carcinoma cells when compared to other MEK inhibitors. Pharmacodynamic analysis of COLO 205 tumor bearing mice confirmed TAK-733, unlike other MEK inhibitors tested, is capable of inducing apoptosis 36 hours after a single oral administration. To determine whether the anti-tumor effect of TAK-733 is associated with apoptosis, A375 (BRAF V600E) human melanoma tumors were harvested at sacrifice and protein extracted to determine cleavage products of PARP. A single oral treatment with 1, 10, and 30 mg/kg TAK-733 increased protein expression of cleaved PARP in a dose and time dependent manner. Elevated cleaved PARP protein expression was detected as early as 0.5-hours in tumors following 30 mg/kg dose and detectable at all groups by 4-hours. The increase cleaved PARP protein levels persisted for at least 24-hours and returned to control expression levels by 48-hours. Daily oral administration of 1, 10, and 30 mg/kg/day TAK-733 for 14 days potently inhibited tumor growth resulting in tumor growth delay of 3.4, 10.8, and 26.6 days in A375 cell implanted mice. A 60% partial regression response rate (tumor volume reduced by more than 50% of initial volume) was observed in mice that received 30 mg/kg/day TAK-733. These data suggest a possible explanation as to why TAK-733 is capable of inducing regressions in COLO 205 and A375 tumor growth inhibition studies, and may represent a differentiation point from other MEK inhibitors currently in clinical trials.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2518.
Abstract 2522: Identification and characterization of new highly selective and potent BRAF inhibitors
Authors: Pulici Maurizio , Amboldi Nadia , Biondaro Sonia , Caprera Francesco , Casale Elena , Gasparri Fabio , Leone Antonella , Lupi Rosita , Marchionni Chiara , Modugno Michele , Pastori Wilma , Traquandi Gabriella , Zuccotto Fabio , Colotta Francesco , Donati Daniele , Felder Eduard R. , Galvani Arturo , Isacchi Antonella , Pesenti Enrico , Ciomei Marina
Published: February 2011

Aberrant activation of the MAPK-mediated pathway components, RAF-MEK-ERK, is frequently found in human cancers and clearly contributes to oncogenesis. In particular, one of the three isoforms of RAF, BRAF, presents activating somatic mutations in 70% of melanomas, 50% of thyroid cancers, 10% of colon and 20% ovarian carcinomas. The most common BRAF mutation, substitution of glutamic acid for valine at position 600 within the activation segment of the kinase domain, accounts for 90% of mutated BRAF cases, and results in elevated kinase activity with consequent enhanced promotion of cell survival and proliferation. Here we describe the in vitro and in vivo properties of a novel class of potent and selective BRAF inhibitors. Starting from a series of arylpyrazole derivatives that were found to be active against wild-type and mutated BRAF, an expansion and optimization program was undertaken. This led to the identification of both inhibitors of the active conformation and inhibitors of the inactive conformation of the enzyme, as confirmed by co-crystallography studies. Among the former category, a sub-series endowed with high potency and selectivity was identified, with compounds possessing low nM affinity for BRAFV600E and negligible cross-reactivity against a panel of 44 kinases. Members of this sub-series inhibited 72-hour proliferation of cell lines harboring the BRAFV600E mutation with IC50s below 100 nM and with greater than 100-fold selectivity compared to lines with wild-type BRAF. Consistent with this observation, inhibition of the MAPK signal transduction pathway was highly selective for cell lines with mutated BRAF. Biochemical profile, potency in cell proliferation assays, and cellular mechanism of action compared favorably among the sub-series to a reference BRAF inhibitor, PLX-4720, tested in parallel. The most potent compounds (e.g. NMS-P730 and NMS-P383) were selected for in vivo evaluation. In vivo, potent tumor growth inhibition was observed in the A375 xenograft model following oral administration, with no overt toxicity, and with corresponding modulation of the MAPK pathway, consistent with expected mechanism of action. Selection of candidates most suitable for further development is underway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2522.
Abstract 2527: Potent, highly selective peptidomimetic prodrugs targeting the SH2 domain of Stat3 decrease vasculogenic mimicry, invasion, and anchorage independent growth of cancer cells
Authors: Mandal Pijus K. , Liao Warren S. , Gao Fengqin , Robertson Fredika M. , Thudi Nanda K. , Ekmekcioglu Suhendan , Chattopadhyay Chandrani , Ren Zhiyong , Chen Xiaomin , Lu Zhen , Bast Robert C. , Ramesh Rajagopal , McMurray John S.
Published: February 2011

Signal transducer and activator of transcription 3 (Stat3) transmits signals from IL-6 family cytokines, EGF and VEGF. Stat3 is constitutively activated in lung, head and neck, breast, and prostate cancers, AML and other malignancies, providing a target for cancer drug design. To uncouple Stat3 from its roles in proliferation, survival, angiogenesis, and invasion, we are targeting its SH2 domain with phosphopeptide mimetics derived from the recognition sequence, pTyr-Leu-Pro-Gln. Molecular modeling indicated that a methyl group attached to the β-position of a pTyr mimic would enhance affinity due to hydrophobic contact. This modification increased affinity 2-3 fold in a series of peptidomimetics based on pCinn-Xxx-Yyy-Gln-NHBn (pCinn = 4-phosphorylcinnamic acid), resulting in IC50 values of 35 − 112 nM in a biochemical fluorescence polarization assay. Conversion of these to bis-pivaloyloxymethyl (POM) protected difluoromethylphosphonate prodrugs resulted in complete inhibition of the phosphorylation of Tyr705 of Stat3 at concentrations of 0.5 − 1 μM in breast, ovarian, and melanoma cells. Prodrugs were selective for Stat3 over other SH2 domain-containing proteins such as Stat1, Stat5, Src, and PI3K p85. Quantitative RT-PCR showed that prodrugs inhibited Stat3-targeted gene expression. Prodrugs were not cytotoxic to cells grown in 2D cultures on plastic dishes. However, potent inhibition of Stat3-driven processes such as vasculogenic mimicry, invasion, and anchorage independent growth in soft agar was observed. Conclusions: 1. peptidomimetic prodrugs are highly potent and selective inhibitors of Stat3; 2. cultured tumor cells do not depend on phosphorylation of Tyr705 of Stat3 for survival; 3. highly selective inhibitors of Stat3 phosphorylation are likely to be effective antitumor agents due to inhibition of invasion and angiogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2527.
Abstract 2528: Enhanced sensitivity of pancreatic cancer cells to the concurrent inhibition of aberrant Stat3 and EGFR or Src
Authors: Soumya Jaganathan , Yue Pei Bin , Turkson James
Published: February 2011

Pancreatic cancer is a lethal disease with little understanding of the etiology and no effective therapies. While aberrant epidermal growth factor receptor (EGFR), Src and Stat3 are detected in pancreatic cancer and implicated in the disease, their exact roles in the support of the disease phenotype remain poorly defined. Moreover, the potential for a cross-talk between EGFR, Src and Stat3 poses a challenge to any therapy that targets only one of these entities. In this study, we sought to define the EGFR, Src and Stat3 signaling integration and to investigate the contributory roles of the three entities to the pancreatic cancer phenotype. We found in Panc-1 and Colo-357 lines that phospho-Y845EGFR, pY1068EGFR and pY1086EGFR levels are responsive to c-Src inhibition, in contrast to pY1173EGFR that is solely EGFR kinase-dependent. Treatment of Panc-1 and Colo-357 cells with the EGFR inhibitor, Iressa (ZD 1839) or the Src inhibitor, Dasatinib (Das) suppressed the constitutively-active Stat3 activity, suggesting that both EGFR and Src activities promote aberrant Stat3 activation in pancreatic cancer cells. However, the early suppression of aberrantly-active Stat3 by the EGFR and Src inhibition in Panc-1 cells is countered by a Janus kinase (Jaks)-dependent re-activation, suggesting that Jaks activity could represent a compensatory mechanism for Stat3 induction. The abrogation of c-Src activity by either Das treatment or the over-expression of a kinase-dead c-Src mutant suppressed the levels of phospho-FAK, phospho-p130Cas, phospho-cortactin, phospho-p120catenin, and phospho-paxillin, suggesting that Src activity induces the mediators of motility and migration in pancreatic cancer cells. By contrast, neither EGFR nor Src inhibition modulated the baseline enhanced phospho-ErkMAPK or phospho-Akt levels in Panc-1 and Colo-357 cells, consistent with the observation that the inhibition of EGFR or Src alone only weakly suppressed cell growth and survival of pancreatic cancer cells. By contrast, the concurrent inhibition of Stat3 and EGFR or Src induced greater viability loss and apoptosis, and diminished the migration/invasion of pancreatic cancer cells in vitro. Of therapeutic significance, the concurrent inhibition of Stat3 and EGFR or Src, as compared to mono-targeting modality, induced a stronger human pancreatic tumor growth inhibition in xenografts models. We infer that the large tumor growth inhibition in vivo is due to the simultaneous suppression of the abnormal functions of Stat3 and EGFR or Src. These studies strongly suggest that the concurrent targeting of Stat3 and EGFR or Src could be a beneficial therapeutic approach for pancreatic cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2528.
Abstract 2536: Therapeutic testing of a novel PKC inhibitor GAP-107B8 on ovarian cancer cells
Authors: Yan Fu J. , Steffensen Isabella , Garson Kenneth , Vanderhyden Barbara C.
Published: February 2011

Background: Ovarian cancer is the most fatal gynaecologic disease in the western world. In 2009 in North America, an estimated 23,896 women will develop ovarian cancer and an estimated 15,24050 women will die from this disease. Current treatments are limited to surgery or chemotherapy, but the disease often recurs. Thus, the development of novel cancer therapeutics remains important. The protein kinase C (PKC) family of serine/threonine kinases is involved in cellular proliferation, differentiation, apoptosis and cell polarity. One PKC isoform, PKC iota, has recently been identified as a human oncogene and has been shown to be overexpressed in serous epithelial ovarian cancers and is thus a potential therapeutic target for ovarian cancer. Objective: We have tested a novel PKC inhibitor GAP-107B8 (PharmaGap Inc., Ottawa) in vitro on a panel of nine ovarian cancer cell lines to determine its potential to inhibit cell proliferation, proliferation in soft agar, and migration. Methods: Nine ovarian cancer cell lines were treated with three different concentrations of GAP-107B8 and then screened using high throughput assays to measure the proliferation of cells in adherent and anchorage independent (soft agar) cultures. The ability of cells to migrate in the presence of GAP-107B8 was also determined. Results: We observed significant reduction in cell proliferation in 6 of 9 ovarian cancer cell lines tested, including two cell lines resistant to the standard chemotherapy. GAP-107B8 inhibited cell proliferation by 30% to 79% compared with untreated cells, with more than 50% inhibition in 4 of 7 cell lines. Treatment with GAP-107B caused a reduction in growth in soft agar in 7 of the 9 cell lines tested in vitro. GAP-107B8 inhibited growth in soft agar by 50% to 94% compared with untreated cells, with 80% or greater inhibition in 6 of 7 cell lines. Finally, 5 of 8 cell lines tested showed significant inhibition of mobility following treatment with GAP-107B8. There was 50% or greater inhibition in all 5 cell lines compared with untreated cells. Conclusion: The novel PKC inhibitor GAP-107B8 displays good efficacy in vitro in suppressing several cancer cell characteristics in a variety of ovarian cancer cell lines. Further experiments are underway to investigate the therapeutic potential of GAP-107B8 in xenograft models of ovarian cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2536.
Abstract 2547: Caveolin-1 inhibits survivin and increases sensitivity to paclitaxel in breast cancer cells
Authors: Jafar Nadia , Shajahan Ayesha N. , Clarke Robert
Published: February 2011

Breast cancer is the second leading cause of death due to cancer in women and approximately 194,280 new cases of breast cancer were diagnosed in 2009. The chemotherapeutic drugs known as taxanes disrupt the microtubule function and arrest cell cycle. Paclitaxel (Taxol) is a taxane that is commonly used to treat patients with breast cancer as well as other types of cancer. However, resistance to paclitaxel remains a hindrance to successful treatment. Previously, we have shown that signaling associated with caveolin-1 (CAV1), a structural protein, is an essential determinant of paclitaxel sensitivity in MCF-7 breast cancer cells. CAV1 is part of this family of proteins that has multiple functions in different cancers but it has been proposed to function as a tumor suppressor in breast cancer. Here we show that CAV1 may promote cell death in response to paclitaxel by inhibiting the function of a pro-survival protein called survivin. Survivin is encoded by the BIRC5 gene and is part of the inhibitors of apoptosis (IAP) family. This protein has numerous functions including inhibition of apoptosis and regulation of the cell cycle. Evidence shows that survivin inhibits cell apoptosis by preventing the activation of caspases, which are proteases associated with cell-mediated apoptosis. Up-regulation of survivin is associated with cancer and usually indicates a poor prognosis. We show that while over-expression of CAV1 down-regulates survivin, over-expression of survivin down-regulates CAV1 in MCF-7 cells. Moreover, MCF-7 cells that over-express survivin show decreased inhibition of cell proliferation in response to 10 nM paclitaxel compared to MCF-7 cells expressing the empty vector (EV, control). Thus, our work highlights the role of CAV1 expression in breast cancer cells as an essential determinant of paclitaxel sensitivity. Findings from this research can help researchers and clinicians to evaluate whether CAV1 expression in breast tumors can help predict its responsiveness to taxanes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2547.
Abstract 2549: Histone deacetylase regulation of hypoxia-inducible factor 1α may play a key role in cancer drug resistance
Authors: Lautz Timothy B. , Holub Janette L. , Clark Sandra H. , Qiu Yi-Yong , Madonna Mary Beth , Chu Fei
Published: February 2011

Hypoxia-inducible factor 1α (HIF-1α) is a transcription factor that mediates the downstream expression of genes controlling the cellular response to hypoxic conditions. Overexpression of HIF-1α has been implicated in promoting angiogenesis and a more invasive phenotype in a variety of cancer cells. HIF-1α expression may also promote chemoresistance and increased expression of the MDR gene. Previous work from our laboratory has implicated class II histone deacetylases (HDACs) as key mediators of chemoresistance, however the mechanism for this observation remains unclear. It is known that HDACs decrease HIF-1α acetylation, preventing proteosomal degradation and increasing HIF-1α activity. We hypothesized that class II HDAC modulation of HIF-1α activity may contribute to the development of resistance to anti-cancer therapy. In the present study, HDAC4 and HDAC6 expression were upregulated in doxorubicin-resistant human breast (MCF-7-DoxR), human osteosarcoma (SaOS2-DoxR) and human neuroblastoma (SK-N-SH-DoxR) cells compared to their parental cells by western blot and RT-PCR. Likewise, after treatment with deferoxamine mesylate (DFO) to prevent HIF-1α degradation under normoxic conditions, HIF-1α expression was found to be upregulated in MCF-7-DoxR and SaOS2-DoxR cells. Inhibition of HDACs with suberoylanilide hydroxamic acid (SAHA), sodium butyrate (NaB), or siRNA against HDAC4 or HDAC6 resulted in decreased HIF-1α expression and restoration of sensitivity to anti-cancer agents in both drug resistant cell lines. The percentage of viable drug-resistant cells was lower after treatment with doxorubicin plus SAHA or NaB, compared to doxorubicin, SAHA or NaB alone. In summary, HDAC regulation of HIF-1α expression may be an important mediator of tumor resistance to anti-cancer agents.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2549.
Abstract 2553: Characterization of the UDP-Glucuronosyltransferases in melanocytes and melanoma
Authors: Dellinger Ryan W. , Matundan Harry H. , Meyskens Frank L.
Published: February 2011

The UDP-glucuronosyltransferase (UGT) family of enzymes catalyzes the glucuronidation of a variety of endogenous compounds such as bilirubin and steroid hormones, as well as xenobiotics including chemotherapeutic agents and environmental carcinogens. Glucuronidation of these substrates inactivates them by making them more hydrophilic and easily excreted. UGTs are expressed primarily in the liver, but are also expressed in several extra-hepatic tissues including prostate, breast, colon, lung, brain, kidney, bladder and ovary. However, there have been no reports measuring UGTs in melanocytes or melanoma.

We screened several primary melanocyte cultures from neonatal foreskin as well as several primary and metastatic melanoma cell lines for UGT expression by RT-PCR. Three UGT family members, UGT2B7, UGT2B10 and UGT2B15, were found to be expressed in melanocytes. Interestingly, of all the melanoma cell lines screened only WM115, a primary melanoma, had UGT expression. Again, only UGT2B7, UGT2B10 and UGT2B15 were detected. UGT expression was not detected in another primary melanoma cell line, WM3211, or in any metastatic melanoma cell line assayed suggesting that loss of UGT expression occurs during melanoma progression.

Treatment of WM3211 cells with temozolomide, currently indicated for treatment of metastatic melanoma, induced expression of UGT2B7, UGT2B10 and UGT2B15. Similar induction of these UGTs was observed in response to adriamycin or epirubicin treatment of WM3211 cells. Furthermore, induction of UGT2B7, UGT2B10 and UGT2B15 in response to epirubicin was also observed in the metastatic melanoma cell line SKmel28. Since the UGTs are Phase II xenobiotic metabolism enzymes, we hypothesize that the upregulation of the UGTs in melanoma cells in response to chemotherapeutic agents is a potential mechanism for the well documented drug resistance observed in this malignancy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2553.
Abstract 2556: Granulin-epithelin precursor modulates chemo-resistance in liver cancer
Authors: Wong Chung Lim , Cheung Fung Yi , Cheung Siu Tim
Published: February 2011

Background and Objective: Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and is responsible for 80% of the primary liver tumors in adults. HCC is found to be resistant to most current chemo-therapeutic agents, making the disease as one of the most mortal in the world. There is therefore an urgent demand for better therapeutic approaches. Granulin-epithelin precursor (GEP) was identified as a potential therapeutic target from our genome-wide expression profiles study for HCC. It was an autocrine growth factor with over-expression in most HCCs, and involved in HCCs proliferation, invasion and tumorigenicity. The aim of the current study is to examine the role and signaling mechanism of GEP in chemo-resistance, and if chemo-resistant HCC would response to GEP targeted therapy using GEP monoclonal antibody (mAb).

Methods: HCC cells Hep3B was stably transfected with GEP cDNA for overexpression, and antisense fragment for suppression studies, respectively. Chemo-resistant HCC populations were obtained by selection and expansion of Hep3B cells under different class of chemo-therapeutic agents, including cisplatin, doxorubicin and fluorouracil. The cells were examined for chemotherapy induced apoptosis with chemo-therapeutic agents alone, or in combination with GEP targeted therapy using GEP mAb. The ERK signaling molecules were examined by western blot, and caspases activity by the Caspase-Glo luminescent assay.

Results: The Hep3B transfectants with GEP overexpression demonstrated increased resistance to apoptosis under different class of chemo-therapeutic agents with reference to the parental population. Hep3B cells with GEP suppression resulted in enhanced sensitivity to the chemotherapy induced apoptosis. The Hep3B cells with acquired resistance demonstrated an increment of 8-16 folds in IC50 to cisplatin, doxorubicin and fluorouracil, respectively, with reference to the parental population. The chemoresistant Hep3B cells were responsive to GEP targeted therapy, and combined chemotherapy with GEP targeted therapy demonstrated synergistic effect on apoptosis induction. GEP regulated the ERK pathway including the activation of p-p38 and p-p44/42. GEP targeted therapy alone did not affect the caspases activities, however, combination treatment using the GEP mAb and chemo-therapeutic agents provided a synergistic effect on activation of caspase 3, and magnified the apoptotic effect.

Summary: The present study demonstrated that GEP regulated chemoresistance, and GEP mAb could sensitize both the parental and chemoresistant liver cancer cells to chemo-therapeutic agents. The GEP targeted therapy indicated a new therapeutic approach for chemo-resistant liver cancer patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2556.
Abstract 2558: CEBPD induces SOD1 and cisplatin resistance in urothelial carcinoma
Authors: Huang A-Mei , Lai Yan-Liang , Kuan Ching-I , Sterneck Esta , Wu Wen-Jeng , Pu Yeong-Shiau , Hour Tzyh-Chyuan
Published: February 2011

Bladder cancer is the fourth most common type of cancer in men (ninth in women) in the United States. Cisplatin is an effective agent against the most common subtype, urothelial carcinoma. However, the development of chemotherapy resistance is a severe clinical problem for the successful treatment of cancers. A better understanding of the cellular and molecular events in response to cisplatin treatment and the development of resistance are critical to improve the therapeutic options for patients. Here, we report that expression of the CCAAT/enhancer binding protein delta (CEBPD, C/EBPδ, NFIL-6β) is induced by cisplatin in the human bladder urothelial carcinoma NTUB1 cell line and is specifically elevated in a cisplatin resistant subline. Expression of CEBPD reduces cisplatin-induced reactive oxygen species (ROS) and apoptosis in NTUB1 cells by inducing the expression of Cu/Zn-superoxide dismutase (SOD1) via direct promoter trans-activation. Several reports have implicated CEBPD as a tumor suppressor gene. This study reveals a novel role for CEBPD in conferring drug resistance, suggesting that it can also be pro-oncogenic. Furthermore, our data suggest that SOD inhibitors, which are already used as antiangiogenic agents, may be suitable for combinatorial chemotherapy to prevent or treat cisplatin resistance in bladder and possibly other cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2558.