bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1611: Identification of target molecules and anti-leukemic effects of novel phospha sugar derivative, TMPP
Authors: Nakamura Satoki , Takemura Tomonari , Yokota Daisuke , HIrano Isao , Ono Takaaki , Shigeno Kazuyuki , Ohnishi Kazunori
Published: February 2011

[Background and Aims] We synthesized two phospha sugar derivatives, 2,3,4-tribromo-3-methyl-1-phenylphospholane 1-oxide (TMPP) by reacting 3-methyl-1-phenyl-2-phospholene 1-oxide with bromine, and we has reported their potential as an antileukemic agent in cell lines. In this study, we have investigated that TMPP have antileukemic effects as shown in various leukemia cell lines as well as primary AML patient specimens. Moreover, we have identified the target molecules of TMPP by using docking simulation. [Methods] In human leukemia cell lines (HL60, NB4, U937, NOMO-1, CEM, MOLT4, SUP-B15, and YRK2 cells), the anti-leukemic effects were elevated by cell proliferation assay, Cell cycle analysis, Western blotting. In AML cells derived from AML patients and normal hematopoietic progenitor cells derived from healthy volunteers, the changes of viability by treatment with TMPP were evaluated. By using docking simulation, the target molecules were identified in leukemia cells treated with TMPP. [Results] TMPP showed inhibitory effects on leukemia cell proliferation, with mean IC50 values of 10.2 nmol/L for TMPP, indicating that inhibition appeared to be dependent on the number of bromine atoms in the structure. Further, TMPP at 8.4 nmol/L induced G2/M cell cycle block in leukemia cells, and TMPP at 17.3 nmol/L induced apoptosis in these cells. TMPP treatment reduced cell cycle progression signals (FoxM1, KIS, Cdc25B, Cyclin D1, Cyclin A, and Aurora-B) and tumor cell survival (Bcl-2, p27Kip1 and p21Cip1). Further, treatment with TMPP significantly reduced the viability of AML specimens derived from AML patients, but only slightly reduced the viability of normal ALDHhi progenitor cells. We also found that TMPP showed the strong activity against Aurora kinase B and Bcl-2 by using docking simulation. [Conclusion] We demonstrate that TMPP inhibits Aurora kinase B and Bcl-2 and shows a dominant Aurora B kinase inhibition-related cell cycle arrest and the induction of mitochondrial potential catastrophe-related apoptosis in acute leukemia cells. Moreover, we identified Aurora kinase B and Bcl-2 as the target molecules of TMPP. We conclude that TMPP has great therapeutic potential in anticancer therapy in a wide range of cancers.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

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 1611.
Abstract 1620: Deregulation of cohesion establishment factor DCC1 promotes colorectal cancer cell growth
Authors: Yamaguchi Kiyoshi , Nakamura Yusuke , Furukawa Yoichi
Published: February 2011

Defective in sister chromatid cohesion 1 homolog (dcc1) was identified as a member of alternative replication factor C complex in yeast. Dcc1 has been known to play a crucial role in chromatid cohesion together with other subunits (ctf18 and ctf8), and that it is essential for DNA replication in yeast. We previously carried out genome-wide expression profile analysis of human colorectal tumors using cDNA microarray, and identified a set of genes whose expression was deregulated in tumor cells. In this study, we focused on human DCC1, because its expression was frequently up-regulated in the majority of colorectal cancer tissues. Quantitative PCR analysis corroborated its elevated expression in 12 of additional 20 colorectal tumors compared to their matched non-cancerous colonic mucosa. To examine whether DCC1 is involved in proliferation of cancer cells, we generated small interfering RNAs (siRNAs) targeting human DCC1. Notably, treatment of colorectal cancer cells with DCC1-specific siRNAs significantly suppressed their proliferation, suggesting that its elevated expression is essential for the growth of colorectal cancer cells. To clarify the mechanism of its overexpression, we carried out a reporter assay using 1-kb of its 5’-flanking region and 5’-UTR. As a result, we found that a region between −10 and +70 of transcription starting site was responsible for the activity, and identified three putative E2F1-binding sequences within this region. Further analysis revealed that the three motifs regulate the promoter activity. These data suggests that deregulated E2F1-signaling may play a role in the proliferation of cancer cells in part through the increased DCC1 expression, and that DCC1 may serve a target molecule for the development of new anti-cancer drugs.

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 1620.
Abstract 1621: PRAF2 knockdown inhibits cell proliferation and migration of neuroblastoma cells
Authors: Yco Lisette P. , Bachmann Andre S.
Published: February 2011

Prenylated Rab acceptor 1 domain family, member 2 (PRAF2) is a novel 19-kDa protein with four transmembrane-spanning domains that belongs the PRAF protein family that plays a role in vesicle trafficking and cargo transport. PRAF2 is strongly expressed in several cultured neuroblastoma (NB) cells (for example, SK-N-SH, SH-SY5Y, LAN-1). NB is a tumor that derived from the precursor cells of sympathetic nervous system and the most common extracranial solid tumor of childhood. Recently, our lab identified PRAF2 as a candidate prognostic marker of NB. In the present study, we examined the function of PRAF2 in cell cycle progression of human NB cells. In order to analyze the role of PRAF2, we generated a stable RNA interference (RNAi) short hairpin (sh) PRAF2 knockdown NB cell line (SK-N-SH shPRAF2). We observed that PRAF2 downregulation suppressed the cell viability of SK-N-SH cells. Moreover, PRAF2 knockdown also inhibited migration of SK-N-SH cells. Notably, PRAF2 downregulation also elevated the protein levels of cyclin-dependent kinase (Cdk) inhibitor p27Kip1 in these cells, therefore suggesting a role for PRAF2 in the regulation of NB cell cycle regulation and cell proliferation. In summary, our results suggest that reduction of PRAF2 protein level inhibits cell proliferation and migration in SK-N-SH cells that renders PRAF2 a promising target for therapeutic intervention.

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 1621.
Abstract 163: DTMETH: A novel method for increased length quantitative CpG methylation analysis
Authors: Liloglou Triantafillos , Filia Anastasia , Daskalos Alexandros , Nikolaidis Georgios , Bottomley Helen , Ma Peter N. , Finkelnburg Britta , Field John K.
Published: February 2011

DNA methylation analysis has been blooming in the last decade as there is increasing evidence for epigenetic control of new pathways involved in normal cell function as well as the pathogenesis of human disease. A large number of methods have been devised to address the needs of DNA methylation detection in clinical samples. A DNA methylation detection method of preference should be able to interrogate multiple CpGs over long stretches of DNA, quantify DNA methylation, include multiple bisulphite conversion controls, provide increased sensitivity and low background. Here we present DTMETH, a novel method for DNA methylation analysis that bears the above characteristics. In principle, the desired locus is amplified from bisulphite treated-DNA using primers at CpG-free areas. Primers bear short 5’ tails which contain two cytosines (forward) or guanines (reverse) that will serve as normalisation tools. After a spin-column cleanup process, the PCR product is subjected to DTMETH. This is a thermostable reaction utilising FS Taq and labelled ddC (Applied Biosystems). The latter will be only incorporated at methylated cytosines, as post-bisulphite unmethylated cytosines are converted to uracils. The DTMETH products are cleaned by ethanol precipitation and subjected to analysis on a capillary sequencer. ddC peak areas corresponding to methylated Cs are normalised by the peak areas of the control Cs on the primer tail. As proof of principle we present DNA methylation analysis for the p16 gene. We amplified a 269 bp region of the gene's 5’UTR containing 28 CpGs. The assays provide excellent linearity and reproducibility. We used it to screen 90 non-small cell lung carcinomas for p16 methylation in comparison to an established pyrosequencing assay. p16 hypermethylation was detected in 23 samples with both methods, 3 samples with DTMETH only, while 64 samples were negative. DTMETH combines the read lengths of dideoxy terminator sequencing, and enables quantitation without the need of cloning. It is highly versatile, offering easy assay design and due to its thermostable nature can easily overcome secondary structure issues arising from the low complexity bisulphite DNA sequence composition.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

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 163.
Abstract 1630: Critical role of Shp2 protein tyrosine phosphatase in tumorigenesis of carcinoma
Authors: Ren Yuan , Chen Zhengming , Chen Liwei , WinPiazza Hla , Enkemann Steven , Haura Eric , Wu Jie
Published: February 2011

Increasing evidence suggests that tumorigenic function requires coordinated action of both protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Certain PTPs, similar to PTKs, may play positive roles in the initiation and maintenance of human cancer. Shp2 is a non-receptor PTP encoded by the human PTPN11 gene. Gain-of-function Shp2 mutants have been linked to hematologic malignancies. While the role of Shp2 in carcinoma is less clear, Shp2 is a positive regulator of signaling by receptor tyrosine kinases such as EGFR, MET, and ALK that are involved in pathogenesis of carcinomas. To determine if Shp2 is required for tumor growth of carcinoma, we suppressed Shp2 expression and PTP activity using shRNAs and a PTP-inactive mutant in breast, prostate, and lung carcinoma cells. Shp2 knockdown and inactivation prevented tumor xenograft growth of these carcinoma cells. Biochemical analyses indicated that Shp2 inhibition suppressed Src, Erk1/2, and c-Myc activities, whereas it elevated p27 (CDKN1B) both in cell cultures and in tumors. These results suggest that Shp2 is a non-oncogene addiction gene critically involved in malignant growth of carcinoma and a potential target for development of a novel PTP-targeted therapy.

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 1630.
Abstract 1634: Reduced NIMA-related kinase 2 (Nek2) expression levels enhance paclitaxel sensitivity in breast cancer cells
Authors: Lee Jaehyung
Published: February 2011

While many clinical trials have shown that the taxane, paclitaxel (Taxol™), is a very powerful anticancer drug, paclitaxel is still controversial in its applicability for cancer treatment because of the resistance of tumor cells to its effects. Therefore, the purpose of this study is to determine whether the sensitivity of paclitaxel is improved with the addition of antisense oligonucleotides (ASO) against NIMA related kinase 2 (Nek2) in the breast cancer cells. Nek2, a serine/threonine kinase is a gene emerging as important to cancer development because of its regulatory role in mammalian cell mitosis. Moreover, abnormally increased Nek2 expression and activity levels are the main trait of various cancer cells. For these reasons, regulation of the Nek2 expression levels may prove important as a target for cancer treatment. Studies suggest that depletion of Nek2 results in mitotic arrest and ultimately leads to apoptosis in cancer cells. Because many cancer cells have demonstrated resistance to anticancer drugs, more effective therapeutic treatments for cancer patients are necessary. To overcome this treatment resistance, gene silencing through ASO has emerged as an attractive alternative. ASO are able to inhibit cancer progression because they bind to and depress, the expression of genes targeted for their involvement in tumor development. There are currently clinical trials using ASO for cancer patients suffering from a variety of cancers such as breast, colon, and prostate.

In this study, we investigated drug sensitivity by using plus ASOs targeted against Nek2 to treat breast cancer cells. MDA-MB-468 breast cancer cells transfected with Nek2 ASO were treated with paclitaxel to evaluate cell proliferation, cell cycle distribution, and apoptosis. We detected that MDA-MB-468 cells treated together with Nek2 ASO and paclitaxel showed more synergistic results compared with either agent alone. Nek2 ASO worked with paclitaxel in the arrest of the cell cycle at the G2-M phase and lead to apoptosis as well. This study suggests that Nek2 ASO and paclitaxel combination treatment of cancer cells may strongly improve the sensitivity of cancer cells. Thus, these results possibly provide a new strategy for the treatment of breast 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 1634.
Abstract 1638: Preclinical evaluation of the PI3K inhibitor BEZ235 in nasopharyngeal carcinoma cell lines
Authors: Ma Brigette B. , Lui Vivian WY , Ho Kakiu , Lau Cecilia PY , Ng Margaret HL , Cheng Suk-Hang , Tsang Chi-Man , Tsao Sai-Wah , Shi Michael , Hui Edwin P. , Chan Anthony TC
Published: February 2011

Undifferentiated nasopharyngeal carcinoma (NPC) is endemic to parts of Asia and ubiquitously associated with Epstein-Barr virus (EBV) infection. AKT is frequently activated in NPC and PIK3CA amplification can be found in 40% of NPC tissues. BEZ235 is a dual inhibitor of PI3K and mTOR, which results in inhibition of downstream effectors AKT, S6 ribosomal protein and 4EBP1. The preclinical activity of BEZ235 was evaluated in 6 NPC cell lines (Table 1). Western Blot analysis showed all 6 NPC cell lines showed basal activation of AKT and mTOR. The effect of BEZ235 on cell growth was evaluated by exposing NPC cell lines to increasing concentrations of BEZ235 (0.1nM, 0.5nM, 1nM, 10nM, 100nM, 1µM& 10µM) for up to 72 hrs followed by MTT assay. Over 80% of growth inhibition was achieved in all NPC cell lines except C666-1 (∼70% inhibition), with the respective IC50 concentrations in the nanomolar range (Table 1). Two representative cell lines (HONE1-LMP-1 and CNE-2 cells) were selected for assessing the effect of BEZ235 on AKT-mTOR signaling, apoptosis, cell cycle and synergism with chemotherapy. Treatment of HONE1-LMP-1 and CNE-2 cells with BEZ235 at 5nM and 50nM for 24-48 hours, resulted in cell cycle arrest at G1 phase, apoptosis (indicated by the induction of PARP cleavage), and moderate reduction of cyclin D1 expression. After BEZ235 treatment, the expression level of phosphorylated (p-)AKT, pS6K, p4EBP-1 and p-mTOR were reduced, but the level of p-p44/42 MAPK was increased in both HONE1-LMP-1 and CNE-2 cells. No synergistic effect on growth inhibition was observed when HONE1-LMP-1 and CNE-2 cells were treated with concomitant BEZ235 and chemotherapy (cisplatin, or paclitaxel). Our preliminary result suggests that BEZ235 has promising activity in most NPC cells, while the increase in p-p44/42 MAPK observed in some cell lines suggest the presence of compensatory MAPK activation that have been previously described with mTORC1 inhibitors in other cancer types. Further investigations are warranted.Table 1: IC50 (growth)% growth inhibition by BEZ235% cells at G1 arrest after BEZ235 (5nM) treatment for 24hrsEBV-associated NPC cell linesHK1-LMP14.1nM∼85%-HONE-1-LMP14.9nM∼75%62%C666-19.5nM∼70%-Poorly-differentiated NPC cell linesHONE-16.4nM∼85%-CNE-25.7nM∼90%58%Well-differentiated NPC cell lineHK14.7nM∼90%-

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 1638.
Abstract 1639: The novel c-MET inhibitor, ARQ 197, shows additive growrh-inhibitory effect with erlotinib through enhanced degradation of c-MET protein via ubiquitin/proteasome pathway
Authors: Kanome Tomoko , Kadofuku Tsuyoki , Yamaoka Toshimitsu , Hirose Takashi , Akinaga Shiro , Adachi Mitsuru , Saijo Nagahiro , Ohmori Tohru
Published: February 2011

Background

c-MET, a receptor of hepatocyte growth factor (HGF), is known to be overexpressed in a variety of human cancers. Recently several c-MET inhibitors are being developed as cancer chemotherapy against this compelling molecular target. ARQ 197 is a new small molecule c-MET inhibitor. Since ARQ 197 shows efficient anti-cancer activity without many severe adverse events in Phase I clinical trials, it is expected to be a useful anticancer agent for clinical treatment. This drug is known to bind to c-MET at neighbor area of its ATP binding site and it does not work as an ATP mimic. In this study, we investigated the molecular mechanism of inhibitory effect on c-MET by ARQ 197 in a non-small cell lung cancer cell (NSCLC) line.

Materials and Method

We established a c-MET-overexpressed NSCLC line, PC-9/MET, by long term exposure of PC-9 cells to EGFR-tyrosine kinase inhibitor. The cytotoxicity of ARQ 197 was measured by MTT assay. The expression of c-MET protein and mRNA were detected by Western blotting analysis and real time RT-PCR method, respectively. In a c-MET degradation analysis, bortezomib was used for inhibition of proteasome.

Results

ARQ 197 is equally cytotoxic to parental PC-9 and EGFRTKI-resistant PC-9/MET cell line with GI50 values around 200 nM (?). ARQ 197 showed an additive growth- inhibitory effect with erlotinib in PC-9/MET cells as assessed by an Isobologram analysis. In ARQ 197-treated PC-9/MET cells, a clear decrease of phospho c-MET protein as well as phospho AKT and ERK proteins was observed, however, ARQ 197 also induced a down-regulation of c-MET protein in the cells without any effect on c-MET mRNA level. This ARQ 197 induced degradation was reversed by a concomitant treatment with a proteasome inhibitor, bortezomib in the cells. However, bortezomib did not affect cytotoxicity of ARQ 197 in the same cells.

Conclusion

These data suggest that ARQ 197 may inhibit c-MET through downregulation of this protein in addition to inhibition of its kinase activity. Furthermore, it may enhance c-MET degradation by the ubiquitin/proteasome pathway. These c-MET inhibitory activities by ARQ 197 should be important for the additive combined effect with erlotinib in PC-9/MET 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 1639.
Abstract 1665: Selection of potential responders to FOLFOX therapy in advanced stage colorectal cancer
Authors: Midorikawa Yutaka , Tsuji Shingo , Takahashi Takao , Yagi Koichi , Mamiya Takao , Takayama Tadatoshi , Yoshida Kazuhiro , Sugiyama Yasuyuki , Aburatani Hiroyuki
Published: February 2011

Combinations of oxaliplatin combined with various schedules of antimetabolite 5- fluorouracil and leucovorin (FOLFOX) have been the first-line treatment for unresectable colorectal cancer (CRC) and survival benefits for the patients undergoing FOLFOX therapy were demonstrated. That is, the median duration of survival for the group treated with FOLFOX has been reported to be about 20 months, compared with 8 months for the best supportive care group. However, the rate of effect is not so high and has been reported to be approximately 50%. Molecular characterization gene expression profiling will undoubtedly improve the prediction of treatment responses and ultimately the clinical outcome of cancer patients, which has never been accomplished by conventional clinicopathological classification. To identify responders for FOLFOX therapy, we performed random forest analysis based on gene expression data about 49 unresectable colorectal cancer (CRC) patients. Random forest is a prediction model with the classification accuracy for each sample, outlier measure without additional test set analysis, and contribution for class division of each gene can be created. Through this analysis, predictor genes are selected by decision tree analysis using out-of-bag error estimation, which is repeated from 10,000 to 1,000,000 times, and rate of effect of therapy can be determined individually. Computed tomography was performed for all the patients after 4 course of FOLFOX therapy, and 11 patients were diagnosed as progressive disease (PD), 10 stable disease (SD), and 28 partial response (PR), respectively, i.e., the rate of effect of therapy was 57.1%. Using random forest, we could identify 8 predictor genes of response to FOLFOX therapy, SLC25A30, RPS3A, FZD7, MERTK, HABP4, PFKFB2, KIAA0329, and ZBTB41. Consensus clustering based on these predictor genes could classify tumor samples according to drug sensitivity. Furthermore, in cross validation with out-of-bags method, such predictor genes were available to predict PD, SD, and PR patients with an overall accuracy of 77.7% on average (30.7 to 99.8 %). Thus, we could obtain the individual information about efficacy of chemotherapy by about 20% improvement before therapy. Taken together, random forest analysis on gene expression data for unresectable CRC patients could predict responders for FOLFOX therapy with high accuracy rate, and this prediction can be one of factors of decision making of treatment for the patients with colorectal cancer, which will lead us to personalized therapy.

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 1665.
Abstract 1669: A quantitative real-time RT-PCR gene signature to detect circulating CD34+ cells as a pharmacodynamic biomarker following anti-EGFL7 treatment in preclinical models of efficacy
Authors: Kadel Edward E. , Huseni Mahrukh , Kan David , Desai Rupal , Yu Shang-Fan , Raja Rajiv , Ye Weilan , Hegde Priti
Published: February 2011

EGFL7 is an extracellular matrix protein that is highly expressed in endothelial cells of angiogenic vessels, including embryonic, tumor and injured vasculatures. Inhibition of EGFL7 activity by anti-EGFL7 monoclonal antibodies results in a reduction of tumor microvascular density (MVD) and tumor growth in genetically engineered models of cancer such as RIP-TβAg. Anti-EGFL7 therapy also leads to a decrease of circulating CD34+ cells in tumor-bearing mice. Utilizing in-silico and murine in-vivo analyses, we have identified a gene signature that can specifically monitor circulating CD34+ cell populations by quantitative real-time PCR using volumes as low as 50μl of mouse whole blood. This PCR-based assay shows a limit of quantification of 0.1 cells/μl of murine whole blood. Experiments comparing CD34+ progenitor cell quantification by flow cytometry to qPCR have shown equivalent performance and good correlation. We propose that using this qPCR based approach to evaluate changes in CD34+ cells in mouse blood in response to anti-angiogenesis therapies, including anti-EGFL7, may be an easy and viable alternative to the current flow cytometry-based assays.

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 1669.
Abstract 1671: A simple HPLC method to measure plasma mitotane and its two main metabolites in adrenocortical cancer patients
Authors: Garg Madhu B. , Sakoff Jennette A. , Ackland Stephen P.
Published: February 2011

Adrenocortical carcinoma (ACC) is a rare, aggressive tumour which is often detected in advanced stage. Mitotane (o,p’-DDD) is the drug of choice for unresectable, recurrent and metastatic ACC. Intraadrenal metabolic transformation is essential for its therapeutic effects but the metabolite responsible is unknown (o,p’-DDE or o,p’-DDA). Benefit depends on maintenance of adequate plasma drug levels >14μg/ml and toxicity occurs at >20μg/ml. The relationship between dose administered and steady state plasma level is variable, with up to 5-10 fold inter-patient variation. Plasma level monitoring during treatment allows more rapid and consistent attainment of levels between 14-20μg/ml. Measurement of o,p’-DDD and metabolites o,p'-DDE and o,p’-DDA could provide a better understanding of mitotane pharmacokinetics and pharmacodynamics.

We have developed and validated a simple HPLC method to measure plasma mitotane and its two principal metabolites using an internal standard (p,p’-DDD), which involves an isocratic elution and separation through a reversed phase phenyl HPLC column with UV detection (λ 226nm). All these compounds are well separated from each other and from other interfering peaks with a total run time of 21min. The method has been evaluated using 63 plasma samples from 11 patients on mitotane therapy, median age 53 years (range13-73) with start dose of 1-4.5 gm/day. Trough o,p’-DDD levels ranged between 0.9-37.7μg/ml, o,p'-DDE levels 0-4.4μg/ml and o,p’-DDA levels 8.5-90.8μg/ml indicating o,p’-DDA levels in patient plasma are about 1.2-18.1 times higher than the parent compound. 37 samples with o,p’-DDD levels <14μg/ml were recommended to steadily increase dose up to 12 gm/day to achieve target level and then continue at that dose. 10 samples with o,p’-DDD levels >20μg/ml were recommended to decrease dose. Correlation with clinical data would allow us to confirm a relationship between plasma level of o,p’-DDD, o,p'-DDE and o,p’-DDA and clinical outcome, including tumour response or stablisation and toxicity. We aim to establish a therapeutic drug monitoring facility for assessment of plasma mitotane and metabolite levels in patients in the Asia Pacific region. A result and dosing recommendation could be available within 7 days of receipt of sample.

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 1671.
Abstract 1675: Lung cancer chemotherapy: genetic variations in the transforming growth factor beta pathway as predictors of survival
Authors: Lin Moubin , Hildebrandt Michelle , Stewart David , Lippman Scott M. , Lu Charles , Spitz Margaret R. , Wu Xifeng
Published: February 2011

Treatments based on molecular determinants are likely to maximize therapeutic index for chemotherapy in non-small-cell lung cancer (NSCLC). The purpose of this study is to take a pathway-based approach to systematically evaluate the pharmacogenetic effects of transforming growth factor (TGF-β) pathway on lung cancer and identify potential genetic predictors of platinum-based chemotherapy. We genotyped 227 SNPS in 23 TGF-β pathways genes to evaluate their associations with overall survival in 598 late stage NSCLC patients receiving first-line platinum-based chemotherapy with or without radiotherapy. In individual analysis, 22 SNPs were significantly associated with overall survival, of which the strongest associations were found for BMP2:rs235756 (HR=1.45; 95%CI, 1.11-1.90; P=0.006) and SMAD3:rs4776342 (HR=1.25; 95%CI, 1.06-1.47; P=0.009). Cumulative effect analysis showed that individuals with multiple risk genotypes had a significant dose-dependent adverse affect on overall survival (Ptrend=2.44×10−15). Analysis stratified by treatment modality showed that SMAD3 haplotypes exhibited significant associations with outcomes of chemotherapy. Survival tree analysis categorized subgroups of patients with dramatically different survival pattern. The low-risk genetic profile group identified from patients treated with chemoradiation had a median survival time (MST) of 30.4 months. In comparison, the high-risk genetic profile group had a more than four-fold increased risk of death with MST of 6.8 months. The low-risk genetic profile group identified from patients treated with primary chemotherapy without definitive radiotherapy had a MST of 18.03 months, while the high-risk genetic profile group had a MST of 5.9 months and a four-fold increased risk of death. These results suggest that genetic variations in TGF-β pathway are potential predictors for clinical outcome in advanced NSCLC treated with platinum-based chemotherapy. We also found that genetic variations within the TGF-β signaling pathway may be able to identify potential subgroups of patients who will most benefit from platinum-based chemotherapy with or without radiotherapy.

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 1675.
Abstract 1687: Carcinogen induced inflammation and pancreatic cancer
Authors: Kadekar Sandeep , Silins Ilona , Hogberg Johan , Dreij Kristian , Stenius Ulla
Published: February 2011

National Toxicology Program (NTP) performs two-year cancer bioassays. We have analyzed the NTP rat data base and searched for gender differences in susceptibility to carcinogens. We identified 10 chemicals that induced exocrine pancreatic cancer in male rats only, but found no chemical that induced pancreatic cancer in female rats only. Furthermore, males were most affected by three chemicals that induced pancreatic cancer in both sexes. In an effort to understand this sex difference in susceptibility, we used a tool which builds on the technology published in BMC Bioinformatics 2009, 10, 303. This tool was built to assist cancer risk assessors in the analysis of literature data on modes of action (MOA). We used this tool in the analysis of 2041 PubMed abstracts on the 10 male-specific carcinogens. The tool automatically selected 634 abstracts as relevant for cancer and sorted them in taxonomies. The sorting suggested that induction of oxidative stress and inflammation was a common feature among the 10 carcinogens. As chronic pancreatitis is a risk factor for human exocrine pancreatic cancer, we investigated the effects of the 10 carcinogens on Ca2+ signaling and on inflammatory markers in pancreatic cancer cell line Panc-1. It was found that many the 10 male specific carcinogens rapidly increased intracellular [Ca2+] and interfered with extracellular ATP-induced Ca2+ release in Panc-1 cells. The effect on [Ca2+] was followed by activation of calcineurin and phosphorylation of Erk. Some of these effects were blocked by inhibitors of the purinergic P2X7 receptor, a receptor which may rapidly increase intracellular [Ca2+]. We further studied the induction of mRNA levels of inflammatory mediators, and found that IL8 was significantly up-regulated by most of the 10 chemicals. Our data suggest that inflammation is important for chemically induced exocrine pancreatic cancer in the rat and that male rats are more susceptible. Our data may have a bearing on human pancreatic cancer as men, and in particular young men, have a higher incidence of pancreatic cancer than women. Increased levels of the P2X7 receptor has also been detected in chronic pancreatitis in humans.

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 1687.
Abstract 1692: TRAF2 phosphorylation plays a critical role in cell adaptation to chronic oxidative stress
Authors: Habelhah Hasem , Blackwell Ken , Altaeva Aliya , Zhang Laiqun , Shi Zhaohui
Published: February 2011

The tumor microenvironment is characterized by hypoxia, low glucose and oxidative stress. Cancer-cell adaptation to such chronic stress has profound consequences for malignant progression and the response to therapy. Numerous studies have demonstrated that the NF-κB signaling pathway promotes tumor progression and metastasis, whereas its inhibition sensitizes cancer cells to stress-induced apoptosis. TNF receptor associated factor 2 (TRAF2) is an adaptor protein that regulates the activation of the JNK and NF-κB signaling cascades in response to TNFα stimulation. We mapped two phosphorylation sites (Ser-11 and Ser-55) on the N-terminal RING domain of TRAF2, and reported that phosphorylation at these sites increases basal and TNFα-induced NF-κB activation. Recently we found that oxidative stress induces TRAF2 phosphorylation at both Ser-11 and Ser-55, and that this phosphorylation plays a critical role in cell survival in the context of chronic oxidative stress: cells that expressed a TRAF2 mutant in which both Ser-11 and Ser-55 are replaced by Ala (TRAF2-S11/55A) underwent apoptosis even when the cells were subjected to sublethal oxidative stress. Analysis of both JNK and IKK pathways revealed that oxidative stress induces strong and prolonged JNK, but not IKK, activation in cells expressing TRAF2-S11/55A, suggesting that TRAF2 phosphorylation promotes cell survival by inhibiting the prolonged phase of JNK activation. Importantly, TRAF2 was found to be constitutively phosphorylated in some malignant cancer cell lines, as well as in some human tumor tissues. In addition, basal TRAF2 phosphorylation is elevated in H-Ras-V12-transformed cells, and its inhibition significantly sensitized the cells to oxidative stress-induced apoptosis. These results unveil a new layer of mechanism for cancer cell adaptation to oxidative stress modulated by TRAF2 phosphorylation, and suggest that inhibiting TRAF2 phosphorylation could be a new target for anti-cancer drug development.

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 1692.
Abstract 1699: The effects of hypoxia and oxidative stress on transcriptional regulation of carcinogen metabolizing enzymes
Authors: Richardson Amira , Williams Shardae , Kioko Stephen , Kirlin Ward
Published: February 2011

Enzymes are essential to every biochemical process; it is through the action of enzymes that the many metabolic pathways of the body can synchronize with one another to coordinate the processes needed to maintain life. Enzymes are also involved in the metabolism of xenobiotics including dietary chemicals, drugs and environmental chemicals. In this capacity they are very involved in chemical carcinogenesis. Cytochrome P450 1A1 (CYP1A1) and NAD(P)H: quinone oxidoreductase (NQO1) are two enzymes that play a vital role in the process of chemical carcinogenesis. The aryl hydrocarbon receptor (AhR), located in the cytoplasm, mediates various biological responses to environmental pollutants via the adaptive pathway. The binding of a xenobiotic to the AhR triggers transcriptional activation of xenobiotic metabolizing enzymes such as CYP1A1 and NQO1. Our current research investigates the contrast between oxidative stress and hypoxia on the transcriptional activation of CYP1A1 and NQO1 enzyme expression. Hypoxia is a frequent characteristic of tumors as a result of low vascualrization. The transcription factor HIF1alpha can serve as a marker of hypoxia, and aids as a key regulator of the hypoxic response. Previous research in our lab has shown that oxidative stress results in an increase in CYP1A1 and NQO1. We hypothesize that under hypoxic conditions the expression of NQO1 and CYP1A1 are altered which could indicate that hypoxia may possibly play a role in carcinogen metabolism. Two human cancerous cell lines, HEPG2 and HT29, were used to test this hypothesis. Both cells lines were treated with 500 µM of the hypoxic mimetic agent cobalt chloride and harvested at one, two and four hour increments. Hypoxic conditions were then confirmed via HIF1 alpha staining in immunocytochemistry. We then observed mRNA, protein and enzyme expression via RTPCR, Western Blots and enzyme assays, respectively. Altered levels of NQO1 and CYP1A1 suggest that these enzymes respond to hypoxia in a similar manner as they do to oxidative stress. This may provide preliminary evidence into one of the potential mechanisms by which hypoxia is chemopreventive. (Support: NIH MBRS/GM028248; RCMI RR033032).

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 1699.
Abstract 171: Cell memory of a genotoxic insult is mediated by methyltransferases
Authors: Rugo Rebecca E. , Mutamba James T. , Mohan K. Naga , Yee Tiffany , Chaillet J. Richard , Greenberger Joel S. , Engelward Bevin P.
Published: February 2011

Characterization of the direct effects of DNA damaging agents shows how DNA lesions lead to specific mutations. Yet, serum from Hiroshima survivors, Chernobyl liquidators, and radiotherapy patients can induce a clastogenic effect on naive cells, showing indirect induction of genomic instability that persists years after exposure. Such indirect effects are not restricted to ionizing radiation, as a chemical genotoxin induces a heritable and transmissible genomic instability phenotype. While such indirect induction of genomic instability is well described, the underlying mechanism has remained enigmatic. Here, we show that mouse embryonic stem (ES) cells exposed to γ-radiation remember the insult for weeks. Specifically, conditioned media from progeny of exposed cells can induce DNA damage and homologous recombination in naive cells. Notably, cells exposed to conditioned media also elicit a genome destabilizing effect on their neighbours, thus demonstrating transmission of genomic instability. Moreover, we show that the underlying basis for the memory of an insult is completely dependent on two of the major DNA cytosine methyltransferases (MTases), Dnmt1 and Dnmt3a. Targeted disruption of these genes completely eliminates transmission of genomic instability. Furthermore, transient inactivation of Dnmt1, using a tet-suppressible allele, clears the memory of the insult, thus protecting neighbouring cells from indirect induction of genomic instability. We have therefore shown that a single exposure can lead to long-term genome destabilizing effects that spread from cell to cell, and we provide a specific molecular mechanism for these persistent bystander effects. Collectively, our results impact current understanding of risks from toxin exposures and suggest specific mechanisms for suppressing genomic instability in people exposed to carcinogenic genotoxins.

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 171.
Abstract 1715: Leptin regulates the IL-1 system in breast cancer
Authors: Zhou Weiqiang , Gonzalez-Perez Ruben R.
Published: February 2011

Leptin and IL-1 are inflammatory cytokines promoting breast cancer angiogenesis that are often linked to worse prognosis. We hypothesize the leptin angiogenic effects in mammary tumor (MT) may be linked to, or regulated, in part by IL-1 signaling, therefore, leptin and IL-1 may have synergistic functions in MT progression. To test this hypothesis leptin-dose (0-6.2 nM) and time-course (0-48h) effects on levels of IL-1 system components (IL-1α, IL-1β, IL-1Ra and IL-1R tI) were investigated in mouse MT-4T1 cells. To determine the contribution of specific leptin-activated signaling pathways on IL-1 regulation several kinase inhibitors AG490 (JAK2/STAT3), PD98059 (MEK/MAPK/ERK1/2), Wortmannin (PI-3K/AKT1), Rapamycin (mToR), Go6976 (PKC-Ca dependent), SB203580 (p38 MAP kinase) and SP600125 (JNK) were used and IL-1 system component levels were determined by ELISA and Western blot. To determine if leptin could regulate its own receptor (long isoform, OB-Rb and all isoforms) flow cytometry was used. Our results show that basal levels of IL-1α were 14 fold higher than IL-1β. Remarkably, leptin up-regulates all IL-1 system components and all OB-R isoforms showing the highest effects at 1.2nM/24h. JAK2/STAT3 and MAPK/ERK 1/2 were essential for leptin upregulation of IL-1 system. In contrast, PI-3K/AKT1, PKC and p38 kinases were mainly linked to leptin regulation of IL-1 receptor-related proteins (IL-1R tI and IL-1Ra). JNK and mTOR were related to leptin-mediated induction of IL-1R tI but were not involved on IL-1α or IL-1Ra regulation. Interestingly, leptin-mediated activation of mTOR appears to be a negative regulator of IL-1α but acts as an inductor of IL-1Ra. In conclusion, leptin is an important regulator of IL-1 system in MT. In turn, IL-1 signaling can promote VEGF expression and tumor macrophage recruitment that could indirectly enhance leptin-mediated angiogenic effects and MT growth. This work was supported in part by NIH/NCI 1SC1CA138658-02; NIH/UAB Breast SPORE Career Development Award; BC 504370 Susan G. Komen Foundation for the Cure and the Georgia Cancer Coalition Distinguished Cancer Scholar Award (to RRGP).

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 1715.
Abstract 1720: Activating peroxisome proliferator-activated receptor γ mutant promotes tumor growth in vivo by enhancing angiogenesis
Authors: Zhou Jie , Tian Lifeng , Casimiro Mathew C. , Pestell Richard G. , Wang Chenguang
Published: February 2011

PPARγ, originally cloned as a transcription factor involved in fat cell differentiation, is activated by endogenous ligands, such as fatty acids produced by sterol response element binding protein 1 (SREBP1) and fatty acids synthase (FASN) pathways and synthetic ligands including the thiazolidinediones (TZD). PPARγ is expressed in a variety of cancer cells. Independent lines of evidence support a role for PPARγ to either inhibit or promote tumorigenesis. Human breast cancer is associated with ErbB2 amplification or overexpression in >30% of patients. Although PPARγ is reduced in ErbB2 induced tumor, the role of PPARγ in growth control of ErbB2-induced tumorigenesis is poorly understood. The addition of ligand activates the receptor by inducing a conformational change in the receptor, which can be recapitulated by mutation. To investigate the role of activated PPARγ signaling in breast cancer, we compared the function of a constitutively active PPARγ (PγCA) mutant with the wild-type PPARγ in ErbB2-induced mammary tumorigenesis in vivo. Tumor cells transduced with either PPARγ or PγCA were implanted into immune competent FVB mice. Enhanced tumor growth was observed in PγCA-transduced cells, which was associated with increased angiogenesis and endothelial stem cells as evidenced by increased number of cells stained with von Willebrand factor (vWF), c-Kit, CD133 and CD31. Genome-wide expression profiling identified a group of genes within the angiogenesis pathway, including Angptl4, as targets of activated PPARγ. PγCA also induced Angptl4 protein secretion in ErbB2-transformed mammary epithelial cells. Angptl4 promoted vascular endothelial cell migration and conversely immuno-depletion of Angptl4 reduced PγCA-mediated cellular migration. Collectively, these studies suggest that activated PPARγ induces Angptl4 to promote tumor growth through enhanced angiogenesis in vivo.

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 1720.
Abstract 1727: Interleukin-6 and intracrine androgens in prostate cancer cells
Authors: Chun Yae Yeon , Nadiminty Nagalakshmi , Lou Wei , Yang Joy , Evans Christopher P. , Kung Hsing-Jien , Gao Allen C.
Published: February 2011

INTRODUCTION AND OBJECTIVES: The standard systemic treatment for prostate cancer patients is androgen deprivation therapy. Although serum testosterone concentrations were significantly reduced after androgen deprivation therapy, levels of intraprostatic androgens are reproducibly measured at concentrations sufficient to activate androgen receptor (AR) and stimulate tumor growth, suggesting that prostate cancer cells may survive androgen deprivation therapies by increasing intracrine androgen synthesis within the prostate. However, factors that regulate de novo intracrine androgen synthesis have not been identified. Interleukin-6 (IL-6) has been implicated in the modulation of AR activation and growth and differentiation in prostate cancer. In this study, we investigate whether IL-6 regulates intraprostatic androgen synthesis in prostate cancer cells.

METHODS: Quantitative reverse transcription-PCR and western blot were performed to detect expression levels of steroidogenic enzymes. AKR1C3 promoter reporter was constructed and analyzed for IL-6-mediated AKR1C3 transcriptional activity. IL-6-mediated signaling was knocked down using siRNAs specific to IL-6R and gp130 and the effect on AKR1C3 expression was examined. To measure intraprostatic androgen levels, IL-6 overexpressing LNCaP-IL6+ cells were inoculated orthotopically into the prostate of castrated male nude mice and generated tumors. Intraprostatic androgen levels were measured by an enzyme immunoassay (Testosterone EIA kit) based on the competition between testosterone and a testosterone-acetylcholinesterase (AChE) conjugate (testosterone tracer) for a limited amount of testosterone in the sample.

RESULTS: We found that IL-6 increases the expression of genes encoding many steroidogenic enzymes including HSD3B2 and AKR1C3 involved in androgen biosynthesis. Down regulation of IL-6 receptor and gp130 expression using specific siRNA abolished IL-6 mediated AKR1C3 expression, suggesting that IL-6 signaling is responsible for AKR1C3 expression. IL-6 increases AKR1C3 promoter activity, indicating that the increase in IL-6-mediated AKR1C3 expression is in part at the transcriptional level. Treatment of IL-6 increased testosterone level in LNCaP cells. The tumor testosterone levels were detected at 378 pg/g in tumors generated from IL-6 overexpressing LNCaP-IL6+ cells inoculated orthotopically into the prostates of castrated male nude mice.

CONCLUSIONS: These results suggest that IL-6 increases levels of intracrine androgens through enhanced expression of genes mediating androgen metabolism in prostate cancer cells during androgen deprivation therapy.

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 1727.