bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3417: Ezh2 is up-regulated and correlates with Ki67 and CD31 expression in human pediatric rhabdomyosarcoma
Authors: Ciarapica Roberta , Pezzullo Marco , Verginelli Federica , Boldrini Renata , Sio Luigi De , Stifani Stefano , Giordano Antonio , Rota Rossella
Published: February 2011

Rhabdomyosarcoma (RMS) is a pediatric tumor believed to originate from perturbations of the normal development of the muscle lineage. The Polycomb Group (PcG) protein Ezh2, which was originally implicated in inhibiting homeobox gene expression during embryonic development, was recently shown to act as a negative regulator of muscle differentiation. Moreover, Ezh2 is a histone methyltransferase that is aberrantly overexpressed in most human cancers. Comparing the level of the expression of Ezh2 in RMS cell lines, a small group of tumor samples with normal skeletal muscle myoblasts (SkMC), and normal muscle tissues, we have previously observed that Ezh2 is highly upregulated in rhabdomyosarcoma tumors (Ciarapica et al., 2009). The aim of this study was to characterize the relevance of Ezh2 expression in RMS. Ezh2 expression was analyzed by immunohistochemistry in a cohort of 32 RMS primary tumors and correlated with the expression of the cell proliferation marker, Ki67, and the angiogenic marker, CD31. Results of these expression studies were then correlated with the clinicopathological features of the tumor samples. No significant difference in the clinicopathological variables was observed between the two major histophatological groups of RMS, the most aggressive alveolar RMS (ARMS) and the embryonal RMS (ERMS). Ezh2 expression was not correlated with the clinical parameters of the cohorts, but strikingly, a significant positive correlation was observed between Ezh2 and Ki67 expression in the whole RMS sample (r0 = 0,560; p = 0,013). The number of the large vessels was correlated with the total number of vessels (r0= 0,730; p<0,0001) and with the expression of Ki67 (r0= 0,421; p=0,02). In ARMS samples, the number of large vessels was significantly correlated with the expression of Ezh2 (r0= 0,559; p=0,047), the total number of vessels (r0= 0,781; p=0,02), and the expression of Ki67 (r0= 0,738; p=0,004). This study confirmed our previous observation regarding the aberrantly high expression of Ezh2 in rhabdomyosarcoma tumors on a larger cohort of samples. Moreover, the present data established a positive correlation between Ezh2 expression and the proliferative and angiogenic potential of rhabdomyosarcoma neoplasia.

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 3417.
Abstract 3418: Characterization of a survival pathway controlled by FGFR4 in alveolar Rhabdomyosarcoma
Authors: Wachtel Marco , Schäfer Beat
Published: February 2011

The inefficiency of the currently used conventional treatment modalities for alveolar Rhabdomyosarcoma (aRMS) stimulate the search for alternative treatment approaches such as targeted therapies exploiting addiction of tumors cells to specific signalling pathways. Indeed, inhibition of specific nodal points in such pathways such as growth factor receptors (e.g. IGF1R) was effective in preclinical model systems of aRMS and might advance to early clinical studies. However, experience from clinical studies of adult tumors suggests that tumor cells can escape such therapies targeting solely one signalling molecule via (re)activation of alternative pathways. Therefore, the evolution of tumor resistance needs to be addressed already in preclinical studies and might then be targeted upfront by employing an evidence-based combination approach.

Here, we describe a FGFR regulated survival pathway which is able to rescue aRMS cells from apoptosis induced by blocking other growth factor pathways. The relevant receptor is FGFR4, a direct target of the oncogenic PAX3/FKHR fusion protein, whose activation efficiently blocks apoptosis induced by an IGF1R inhibitor in different aRMS cell lines. On the other hand, combined inhibition of both FGFR4 and IGF1R pathways synergistically reduces aRMS cell viability, involving upregulation of Bcl-xl. Interestingly, the survival pathway downstream of FGFR4 does neither involve MEK/ERK nor Akt. Experiments defining the components of this pathway using genetic siRNA screens are currently underway.

Taken together, our data hint to a potential escape mechanism of tumor cells from therapies targeting the IGF1R/AKT axis via FGFR4 and suggests that combined inhibition of the FGFR4 and IGF1R pathways might be superior to single treatments.

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 3418.
Abstract 3425: Expression and functional role of erbB2 in human hepatoblastoma
Authors: Steiger Barbara , Leuschner Ivo , Denkhaus Dorota , von Schweinitz Dietrich , Pietsch Torsten
Published: February 2011

Background: The receptor tyrosine kinase erbB2 is known to play a role in the development of hepatic progenitor cells and is often found to be expressed and mutated in different solid tumors. Activated erbB2 is a marker of poor prognosis in several malignancies and to date, successful strategies have been developed to target erbb2 receptor. The aim of this study was to determine the expression of erbb2 in human hepatoblastoma, the most common primary hepatic malignancy in early childhood, and its functional role in hepatoblastoma cell biology.

Methods: Expression of erbB2 on the protein level was analyzed by Western Blot, immunofluorescence and RT-PCR of lysates of primary tumors and cell lines HuH6, HepT1, HepT3 and HepG2. Immunohistochemical staining was performed to determine phosphorylated erbB2 and subsequent kinases. Single strand conformation polymorphism (SSCP) analysis was used to scan known hotspots for mutations (exons 6, 8, 18-23 of erbB2) in a panel of 56 tumor samples. Cells were stimulated with rhHRG1-β. Activation of subsequent kinases was determined via Western Blot and MTT assay was used to determine proliferation after stimulation and overexpression of erbb2 via transient transfection. The influence of rhHRG1-β on the activity of the Wnt/ß-catenin pathway was measured by transfection with the TOP-/FOP-Flash reporter assay. The effects of erbB2 kinase knockdown by transient transfection with siRNA against erbB2 on apoptosis and proliferation were also evaluated.

Results: Expression of erbB2 was detected in all four cell lines and most of the primary tumors. Immunohistochemistry indicated expression of phosphorylated (activated) erbb2 proteins in 90% and activation of subsequent kinases, which are downstream targets of erbb2. Mutations were not present in the erbB2 gene. Stimulation with HRG1-β induced phosphorylation of Akt, Erk1/2 and p70S6K and resulted in increased cell proliferation. This effect was not mediated by an increase in activity of the Wnt/ß-catenin signaling pathway because TOP-Flash reporter activity was not further increased in the presence of rhHRG1-β. Overexpression of erbB2 resulted in enhanced proliferation after stimulation with HRG1-β in all four cell lines, whereas knockdown of erbB2 decreased the proliferative effect of HRG1-β on hepatoblastoma cell lines. Knockdown of erbB2 did not induce apoptosis in hepatoblastoma cell lines.

Conclusion: These results indicate that erbB2 might play an important functional role in hepatoblastoma cell biology. Further experiments are needed to evaluate the clinical significance.

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 3425.
Abstract 3426: The lower incidence of loss of IGF2 imprinting, but not that of WT1, WTX or CTNNB1 abnormality may cause different incidence rates of Wilms tumor between Japanese and Caucasian populations
Authors: Haruta Masayuki , Arai Yasuhito , Honda Shohei , Ohshima Junjiro , Kasai Fumio , Soejima Hidenobu , Okita Hajime , Hata Jun-ichi , Fukuzawa Masahiro , Kaneko Yasuhiko
Published: February 2011

Incidence rates of Wilms tumor (WT) markedly differ among ethnic groups; the epidemiological studies showed that the incidence of WT in Japanese children is about half of that of WT in Caucasian children. In Hawaii and Britain, the rates of WT in Asian descent were less than two-third or a half of those in Caucasian children, and these findings suggest that the different incidences of WT may be caused by genetic factor(s), but not by environmental factor(s). Development of WT is associated with mutations of certain genes, including WT1, WTX and CTNNB1, and abnormal expression of IGF2, namely, loss of imprinting (LOI). The frequency of these abnormalities occurs in 10-15% (WT1), 7-15% (WTX), 5-13% (CTNNB1) and 30-60% (IGF2 LOI) of sporadic WTs based on the studies in North-American or European children. To demonstrate the molecular characteristic in Japanese WT, we examined mutations/deletions of WT1, WTX and CTNNB1 using Southern blot, SNP array, sequencing and/or real-time PCR, and expression pattern of IGF2 using the ApaI polymorphism site in exon 9 and the methylation status of the CTCF binding site 6 at H19-differential methylated region (DMR) in 101 sporadic and 13 syndromic WTs in Japanese children. Abnormalities of WT1, WTX or CTNNB1 were detected in 24 (23.8%), 24 (23.8%), and 21 (20.8%)of 101 sporadic WTs, respectively, and in 13 (100%), 0, and 10 (76.9%) of 13 syndromic WTs, respectively. LOI of IGF2 was found in 24 (23.8%) of sporadic WTs and in 1 (7.7%) of syndromic WTs. Thus, the incidence of WT1, WTX or CTNNB1 abnormality was higher, but that of LOI of IGF2 was lower in Japanese children than in Caucasian counterparts. Because the incidence of WT in Japanese children is about half of that of WT in Caucasian ones, the population-based incidences of WT1, WTX or CTNNB1 abnormality may be similar between the two ethnic groups. In contrast, the incidence rate of IGF2-LOI in Japanese children was lower than those of IGF2-LOI in Caucasian children, and the difference in the population-based incidences is much larger. A recent study of 47 Japanese patients with Beckwith-Wiedemann syndrome revealed a significantly lower frequency of H19-DMR hypermethylation in Japanese patients than in North American and European patients (Sasaki et al, Eur J Hum Genet 2007). This and the present findings suggest that susceptibility to epigenetic alterations differs between Japanese and Caucasian populations.

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 3426.
Abstract 3441: Circulating perivascular progenitors, a target of PDGFRbeta inhibitors
Authors: Mancuso Patrizia , Calleri Angelica , Marighetti Paola , Quarna Jessica , Curigliano Giuseppe , Martin-Padura Ines , Bertolini Francesco
Published: February 2011

Cancer blood vessels consist of two interacting types of cells: inner lining endothelial cells and surrounding perivascular cells (pericytes, vascular smooth muscle cells or mural cells). Marrow-derived PDGFRbeta(CD140b)+ progenitor perivascular cells (PPC) can differentiate into pericytes and regulate vessel stability and vascular survival in tumours (Song et al, Nature Cell Biol 2005). Similarly to what we have done with circulating endothelial cells and progenitors (Mancuso et al, Clin Cancer Res 2009), we have developed and validated a flow cytometry procedure for the enumeration of circulating PPC and the study of their viability in murine models of cancer and in cancer patients. DNA+CD45-CD31-CD140b+ cells were enumerated by six-colour flow cytometry, their perivascular nature was confirmed by electron microscopy and RT-PCR expression of CD140b, and their viability was assessed by 7AAD. PPC were found to circulate in cancer-bearing mice, and to be increased (compared to age-matched healthy controls) in patients affected by some types of cancer such as metastatic breast and kidney cancer. In murine models of cancer and in cancer patients, at variance with the kinetcs of circulating endothelial progenitors (Bertolini et al, Cancer Res 2003; Shaked et al, Cancer Cell 2008), cyclophosphamide-based chemotherapy reduced the number of viable PPC, most likely because of an increase of PPC recruitment towards vessels damaged by cyclophosphamide administration (Bertolini et al, Lancet Oncology 2001). Conversely, the administration of the tyrosine kinase inhibitor (TKI) sunitinib, known to inhibit PDGFRbeta, was associated in murine models and human cancer patients with an increase of apoptotic circulating PPC, suggesting a direct targeting of these cells. Studies are ongoing to validate PPC enumeration and viability assessment as a surrogate marker of clinical efficacy in preclinical models and in cancer patients receiving multiple TKI inhibitors including an anti-PDGFRbeta activity.

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 3441.
Abstract 3448: Synergistic effect of benzo(a)pyrene (BaP) and dietary fat on biotransformation enzymes, DNA adducts and colon tumors in ApcMin mice
Authors: Harris Deacqunita L. , Washington Mary K. , Hood Darryl B. , Roberts Lyman J. , Ramesh Aramandla
Published: February 2011

Studies in our laboratory thus far have shown formation of colon tumors in ApcMin mice subsequent to ingestion of fat containing BaP, an environmental carcinogen. These findings have human health relevance in that around 60,000 lives/year are lost to colon cancer in US alone. Diet and environment have been implicated in the development of sporadic colon tumors. Since biotransformation of toxicants is one of the factors that trigger carcinogenesis, the objective of this study was to determine how dietary fat potentiates the development of colon tumors through altered BaP biotransformation, using the ApcMin mouse model. Benzo(a)pyrene was administered to ApcMin mice in unsaturated- (peanut oil) and saturated- (coconut oil) fats at doses of 50 and 100 μg/kg via oral gavage over a 60-day period. Blood, colon and liver were collected at the end of exposure period. The expression of BaP biotransformation enzymes (CYP1A1, CYP1B1 and GST) in liver and colon were assayed at the level of mRNA and activities. Tissue samples were analyzed by reverse phase-HPLC for BaP metabolites, and 32P-postlabeling method for BaP- DNA adducts. BaP exposure through dietary fat altered its biotransformation in a dose-dependent manner, with 100 μg/kg dose group registering an elevated expression of BaP biotransformation enzymes, greater concentration of BaP metabolites, BaP-DNA adducts and more adenomas compared to the 50 μg/kg dose group (p < 0.05). This effect was more pronounced for saturated fat group compared to unsaturated fat group (p < 0.05). These findings establish that saturated fat contributes to sustained induction of BaP biotransformation enzymes and extensive metabolism of this carcinogen. As a consequence, the reactive metabolites such as epoxides and quinones generated in colon bind with DNA, and form adducts resulting in colon tumors in a long term exposure situation (funded by NIH grants S11ES014156, 1F31ES017391-01, 5T32 HL007735-14 and RO3CA130112-01).

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 3448.
Abstract 3453: Phylogenetic fingerprinting of the fecal microbiota in colorectal cancer patients
Authors: Brim Hassan , Zoetendal Erwin , Lee Edward , Nelson Karen , Torralba Manolito , Smoot Duane , Sears cynthia , Ashktorab Hassan
Published: February 2011

Colonic adenomatous polyps are common tumors occurring in ∼50% of Western populations with ∼10% risk of malignant progression. To date, the environmental exposures proposed to promote the development of colorectal cancer (CRC) have been primarily dietary agents. However, the local environment to which the colonic mucosa is exposed is created by the bacterial microbiota of the colon and their metabolic products that include toxins. A member of the Bacteroidetes division, Bacteroides fragilis is a common human commensal, occupying a mucosal niche in the colon. One molecular class of B. fragilis, enterotoxigenic B. fragilis (ETBF) induces polyp formation in Min (multiple intestinal neopplasia) mice through a STAT3 pathway.

Here we analyzed fecal DNA for gut flora composition in African Americans (n=12) with colon adenoma. A 16S rDNA cloning/sequencing experiment revealed that patients with colon adenomas have a higher rate of Bacteroides bacteria than healthy ones. These data was further confirmed using a HITChip (Human Intestinal Tract Chip) where samples from adenoma patients tended to cluster together pointing to common bacterial profiles.

STAT3 induced in adenoma and tumor by IHC, indicating, signal activation perhaps through ETBF like bacteria.

In conclusion, High throughput and sequencing approaches may help to better detect gut flora components including Bacteroides fragilis that induces colorectal carcinogenesis. In addition, the STAT3 expression results would confirm possible ETBF involvement on colon cancer risk via activating STAT3 downstream factor and molecules that are known to play a key role in the regulation of epithelial proliferation

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 3453.
Abstract 3455: Arsenic trioxide modulates DNA synthesis and apoptosis in lung carcinoma cells
Authors: Walker Alice M. , Stevens Jacqueline J. , Tchounwou Paul B.
Published: February 2011

Arsenic trioxide, the trade name Trisenox, is a drug used to treat acute promyleocytic leukemia (APL). Studies have demonstrated that arsenic trioxide slows the cancer cells growth. Although it has been reported that arsenic acts on cells by influencing numerous signal transduction pathways, cell cycle progression, and/or apoptosis, its apoptotic mechanisms are very complex. The primary objective of this research is to evaluate the effects of arsenic trioxide on DNA synthesis and to determine whether arsenic induced-apoptosis is mediated via caspase activation, p38 mitogen-activated protein kinase (MAPK), and cell cycle arrest. To achieve this goal, the lung cancer (A549) cells were cultured following standard protocol, and exposed to various doses (0, 2, 4, and 6 µg/ml) of arsenic trioxide for 48 h. The effect of arsenic trioxide on DNA synthesis was determined by [3H]thymidine incorporation assay. Apoptosis was determined by caspase-3 fluorescein isothiocyanate (FITC) assay, and immunoblot analysis (p38 MAP kinase activity). The cell cycle analysis was evaluated propidium iodide assay. [3H]Thymidine incorporation assay revealed dose-related cytotoxic response at higher levels of exposure. Our findings revealed that arsenic trioxide modulated caspase 3 activity and induced p38 map kinase activation in lung carcinoma (A549) cells. The data revealed no cell cycle arrest at the sub-G0 in A549 cells when treated with arsenic trioxide.

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 3455.
Abstract 3456: Characterization of dibenzo[a,l]pyrene-trans-11,12-diol glucuronidation by UDP-glucuronosyltransferases
Authors: Olson Kristine C. , Sun Dongxiao , Chen Gang , Sharma Arun K. , Amin Shantu , Ropson Ira J. , Spratt Thomas E. , Lazarus Philip
Published: February 2011

A procarcinogen found most notably in tobacco smoke is dibenzo[a,l]pyrene (DB[a,l]P), which can be further metabolized into its epoxide, diol, and carcinogenic diol-epoxide forms. DB[a,l]P metabolites are some of the most potent carcinogens of the polycyclic aromatic hydrocarbon (PAH) family of compounds due to a structural feature called the fjord region. The activated metabolites of DB[a,l]P bind mostly to deoxyadenosine in DNA to form stable adducts, which can potentially lead to DNA damage. UDP-glucuronosyltransferase (UGT) enzymes mediate the attachment of a UDP-glucuronic acid to a wide variety of endogenous and exogenous substrates including several carcinogens, resulting in increases in water solubility and elimination of the carcinogen from the body. The focus of the current study was to examine the detoxification of the carcinogenic diol form (DB[a,l]P-trans-11,12-diol) by glucuronidation. In order to understand the metabolism of this potent PAH by human UGT enzymes, HEK293 cell lines over-expressing individual UGTs were screened for glucuronidation activity toward DB[a,l]P-11,12-diol. Glucuronide products were quantified using an ultra-performance liquid chromatography (UPLC) method. Six UGTs (UGTs 1A1, 1A7, 1A8, 1A9, 1A10 and UGT2B7) were found to be highly active. Three glucuronide conjugates were observed in activity assays with UGT1A1 and 1A10, while only two glucuronides were formed with UGTs 1A7, 1A8, 1A9, and 2B7. Enzyme kinetic analysis indicated that UGT1A9 is the most active isoform among all the UGTs, exhibiting the highest Vmax/Km for the two glucuronide products it formed. These results suggest that multiple UGTs are involved in stereospecific glucuronidation of DB[a,l]P metabolites in humans. In addition to the further characterization of (DB[a,l]P-trans-11,12-diol) glucuronide conjugates, studies examining the role of polymorphisms in active UGTs on cancer risk are currently on-going.

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 3456.
Abstract 3472: Inhaled carcinogen induced lung cancer in mice
Authors: Zou Yiyu , Qiu Xuan , Perez-Soler Roman
Published: February 2011

Inhaled carcinogen-induced lung cancer originates at the bronchial epithelium. Traditional animal models with tumor grafting or intraperitoneal/intravenous injection of tobacco carcinogens either lack the natural stages of tumor initiation and progression, or lack the advantage of modeling the disease through direct exposure of the airway epithelium to the causative agents. To overcome these problems, we used intratracheal inoculation of the major tobacco carcinogens benzo[a]pyrene (BP) and nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to induce lung premalignancy and lung cancer in two different strains of mice. This mouse model of inhaled carcinogen-induced lung cancer consistently mimics the carcinogenesis process in tobacco-induced human lung cancer that progresses through a series of identifiable histopathological stages. In our model the lung carcinogenesis clearly progresses through three different stages: 1. Early transitional stage with no pathological lesions (0-15 weeks after first dose of the carcinogens); 2. Premalignant stage with dysplasia in the lungs in 100% of both A/J and ICR mice at 16 weeks after the first dose of the carcinogens; 3. Malignant stage with tumor nodules developed in the lungs in 100% of A/J and > 60% of ICR mice at 20-24 weeks. We have been using this model in trying to identify key molecular players in each stage that could possibly serve as therapeutic targets for lung cancer treatment. Currently it is one of best available models for studying environmental carcinogen-induced lung cancer and warrants further characterization. Future studies using this model are expected to enhance our understanding of the disease and facilitate our efforts of developing early therapeutic interventions. Supported by NIH grants CA96515 and CA104297.

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 3472.
Abstract 3474: Formation of myosmine under oxidation of nicotine
Authors: Zwickenpflug Wolfgang , Hoegg Christof
Published: February 2011

Nicotine and myosmine are alkaloids in tobacco products at which nicotine occurs in 200fold higher concentrations than myosmine. Nicotine is restricted on tobacco mainly, but myosmine is widespread in the biosphere and food materials additionally. Activation of nicotine showed amongst others N-nitrosation conversion to a compound class, the tobacco specific N-nitrosamines (TSNA). N’-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) represent nicotine derived substances. These are assumed to be responsible for tumorigenesis and cancer disease, especially from the lung after activation to highly reactive intermediate species pyridyloxobutanone (POB). POB is known in the context of CYP 450 dependent activation of TSNA and was detected in DNA of tissues from laboratory animals, after TSNA application. The POB-DNA adducts release under hydrolysis 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) which was observed also during myosmine metabolism in rats, under N-nitrosation and under peroxidation procedures of this compound. Partially, nicotine and myosmine seem to be metabolized on similar way and support assumption from employment of identical CYP450 isozymes. Oxidation processes of nicotine and myosmine employing non CYP450 dependent systems undergoe different pathways involving N or N’ ring positions of their pyridine and pyrrole structures. Peroxidation of nicotine is reported under formation of nicotine N-oxides mainly. First results of finding higher myosmine contents in cigarette filter tips from smoked in relation to unburned cigarettes gave rise to deliberation of possible conversion of nicotine to myosmine. Following, (−)-nicotine in concentrations equivalent to amount in cigarettes 4.9-5.5µmol was incubated with hydrogen peroxide at 37°C and analyzed. To simulate the assumed reaction on biological systems, (−)-nicotine was incubated with horse radish peroxidase (HRP) and occurence of the relevant compounds analyzed. The assays were directly measured with high pressure liquid chromatography (HPLC) instrumentation and confirmed by cochromatography with respective standard compounds. Our results showed formation of myosmine under peroxidation from (−)-nicotine. This would mean conversion of nicotine to myosmine releases activated methyl-species, which could undergo protein or DNA methylation. This possible effect would indicate ability of nicotine to become activated not only as nitrosated but also as peroxidated species. Additional experiments are demanded to elucidate possible carcinogenic risk of nicotine conversion to myosmine. Acknowledgement: This work was supported by DFG grant 59/2-2

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 3474.
Abstract 3486: Interaction of flaxseed oil with trastuzumab in HER2 overexpressing human breast tumors (BT-474)
Authors: Mason Julie K. , Chen Jianmin , Thompson Lilian U.
Published: February 2011

BACKGROUND: Trastuzumab (TRAS) is a first line therapy for breast tumors that overexpress the human epidermal growth factor receptor 2 (HER2) but tumors develop resistance within one year of treatment. Flaxseed oil (FO) rich in n-3 fatty acid alpha linolenic acid (ALA) has been shown to reduce HER2 expression and signalling in estrogen receptor positive human breast tumors. Our objective was to determine whether combining dietary FO with TRAS treatment will enhance TRAS effectiveness in reducing tumor growth.

METHODS: Ovariectomized mice with established HER2 overxpressing tumors (BT-474) and estrogen pellet implant were fed the basal diet (BD) (control) or treated with TRAS (1 or 2.5mg/kg body weight) and fed either the BD or BD supplemented with 4% FO for 4 weeks. Palpable tumor area was measured weekly. After animal sacrifice, excised tumors were analyzed using immunohistochemistry for cell proliferation (Ki-67) and apoptosis and using Western blot analysis for HER2, MAPK and Akt.

RESULTS: Tumors in the control, TRAS1, TRAS2.5, and TRAS1+FO, had palpable tumor areas that significantly increased (p<0.05) by 164%, 104%, 60% and 107%, respectively at the end of the study compared to week 0 while tumors in the FO+TRAS2.5 group did not significantly increase in size. FO+TRAS2.5 treated tumors also had significantly lower (p<0.05) palpable tumor area compared to the TRAS2.5 and other treated tumors at week 4. There was no difference in palpable tumor area between TRAS1 and FO+TRAS1 treated tumors. FO+TRAS2.5 caused a 39% greater increase in apoptosis (p<0.05) and a 61% greater reduction in cell proliferation (p<0.05) compared to TRAS2.5 treatment alone. Western blots support these results and indicate enhanced reductions in HER2, MAPK and Akt expression when TRAS2.5 treatment was combined with dietary FO.

CONCLUSIONS: ALA-rich FO enhanced the effectiveness of TRAS at 2.5mg/kg in reducing tumor growth and the mechanisms may involve reduced cell proliferation and increased apoptosis through modulation of HER2 signalling pathways. This is significant as it could lead to a simple, inexpensive complementary treatment for HER2+ patients being treated with TRAS.

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 3486.
Abstract 349: PI3K inhibitor GDC-0941 synergizes with mTOR inhibitors by blocking negative feedback and causes distinct effects on signaling and apoptosis compared to a dual PI3K/mTOR inhibitor
Authors: Vijapurkar Ulka , Robillard Liliane , Friedman Lori S. , Belvin Marcia P.
Published: February 2011

Background: The PI3K/Akt/mTOR pathway is deregulated in many cancers. and activation of Akt leads to the stimulation of cell survival and proliferation pathways. Akt activation also regulates mTOR to control cell growth in response to growth factors and nutrients. The PI3K pathway is therefore an attractive target for cancer therapy. Multiple targeted inhibitors of PI3K, mTOR and PI3K/mTOR are currently in clinical trials, whileTORC1 inhibitors including rapamycin and analogs are approved. In this study we assessed the consequence of the combination of a PI3K inhibitor, GDC-0941, plus a TORC1 or TORC1/2 inhibitor compared to dual PI3K/mTOR inhibitor, in cancer cell lines with different genetic backgrounds. Method: We analyzed the effects of PI3K, TORC1 or TORC1/2 inhibition alone or in combination on the viability, cell cycle profile and apoptosis of cancer cell lines with varying PI3K, PTEN, K-ras, and LKB status. Effects on signaling were analyzed by measuring the phosphorylation of signaling proteins by reverse phase protein array and other techniques. Effects on viability and apoptosis were assessed, along with a calculation of synergy for combinations. Results: Inhibition of PI3K with GDC-0941 or a dual PI3K/mTOR inhibitor inhibited proliferation of cancer cell lines and in certain tumor lines induced apoptosis. Treatment also resulted in the inactivation of downstream markers including pAKT, pPRAS40 and pS6. TORC1/2 inhibition resulted in a potent inhibition of pS6, however it had differential effects on phospho-AKT and survival proteins pBad and pGSK3b, likely due to the release of the negative feedback regulation. The signaling effects demonstrated by TORC1/2 inhibition also differed from TORC1 inhibition by rapamycin. Inhibition of mTOR resulted in a cytostatic response in all lines tested. Combination of PI3K and mTOR inhibitors (either TORC1 or TORC1/2) caused a significant decrease in cell viability, enhanced apoptosis and caused a greater, sustained knockdown of the signaling pathway. However, treatment with a dual PI3K/mTOR inhibitor led to slight but consistently lower levels of apoptosis than the combination of the separate agents. There were also differences in phosphoprotein status of some of the pathway components. Conclusion: Inhibiting multiple nodes within the same signaling pathway is synergistic and can be an effective strategy to enhance potency in cancer cells. However, combinations of selective targeted agents do not necessarily cause identical effects as a single molecule that inhibit multiple targets simultaneously.

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 349.
Abstract 3490: Selective estrogen receptor modulators enhance efficacy of small molecule CDK inhibitors on human breast cancer cells differing in the expression of ER
Authors: Wesierska-Gadek Jozefa , Zulehner Nora , Komina Oxana , Maurer Margarita
Published: February 2011

Inactivation of cyclin-dependent kinases (CDKs) by pharmacological inhibitors is a very effective weapon against malignant cells. Roscovitine (ROSC), a selective CDK inhibitor primarily affecting CDK2, CDK1, and CDK7, reduces the number of human cancer cells in a concentration-dependent manner. At lower doses ROSC arrests cell cycle progression and at higher doses it induces apoptosis. ROSC inhibits efficiently proliferation of human ER+-ve MCF-7 breast cancer cells by induction of cell cycle arrest at the G2/M transition [1] and concomitantly initiates apoptosis by a p53-dependent pathway [2]. However, the effect of ROSC was much weaker in MCF-7 cells maintained in the presence of estrogen-mimicking compounds [3]. Therefore, we decided to examine the action of selective CDK inhibitors on other breast cancer cell lines differing in the status of p53 and ER and to prove the impact of selective estrogen receptor modulators (SERMs) on the efficacy of ROSC and olomoucine II (OLO II), a new pharmacological CDK inhibitor. ROSC and OLO II were effective on all tested breast cancer cell lines. They arrested MCF-7 and BT-20 cells at G2/M transition and SKBR3 cells in G1 phase and additionally induced apoptosis. The effect of the inhibition of CDKs on distinct cell cycle regulators and pro-survival factors was studied. Interestingly, SERM strongly affected all tested cell lines irrespective of their ER status. Its combination with CDK inhibitors had a different impact. It enhanced G1 or G2 arrest. Our results provide evidence that both CDK inhibitors exert a strong anti-proliferative and pro-apoptotic effect and that their mode of action depends on the cellular context. Moreover, our findings demonstrate that pharmacological CDK inhibitors can be combined with anti-estrogen therapy. The strong effect of anti-estrogens on ER-negative cancer cells indicates that SERMs crosstalk with other steroid hormone receptors.

[1] Wojciechowski J. et al. 2003. Rapid onset of nucleolar disintegration preceding cell cycle arrest in roscovitine-induced apoptosis of human MCF-7 breast cancer cells. Int J Cancer 106: 486-95.

[2] Wesierska-Gadek J. et al. 2005. Roscovitine-induced up-regulation of p53AIP1 protein precedes the onset of apoptosis in human breast cancer cells. Mol Cancer Ther 4: 113-124.

[3] Wesierska-Gadek J. et al. 2006. Phenol red reduces ROSC mediated cell cycle arrest and apoptosis in human MCF-7 cells. J Cell Biochem. 98: 1369-1379.

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 3490.
Abstract 351: Mutations of genes in the c-Kit receptor tyrosine kinase signaling pathway are inactive in adenoid cystic carcinoma of the salivary glands: Implications for c-Kit targeted therapy
Authors: Tetsu Osamu , Phuchareon Janyaporn , Chou Annie , Cox Darren P. , Eisele David W. , Jordan Richard CK
Published: February 2011

Adenoid cystic carcinoma (ACC) is a malignant salivary gland tumor well-known for spreading in wide local area and invasion of vital structures of the head and neck. Unfortunately, conventional therapies have not improved longer-term survival, resulting in significant morbidity and a very low overall survival rate. Improved systemic therapies are clearly needed for this population. ACC tumors overexpress the receptor tyrosine kinase c-Kit, which is thought to be a therapeutic target. However, the role of c-Kit in the pathogenesis of ACC is not currently understood. We addressed this question through a functional genomic study with 17 sporadic ACC tumor specimens. We found heterogeneous missense mutations in three genes in the c-Kit signaling pathway, although mutations were identified at a low frequency. Two ACC tumors had distinct missense mutations in the gene KIT, resulting in G664R and R796G amino acid substitutions in the c-Kit kinase domain. Furthermore, two ACC tumors without KIT mutations had missense mutations in either KRAS or BRAF, causing S17N K-Ras and V590I B-Raf mutants. KRAS and BRAF are downstream effectors of c-Kit. We explored the functional consequences of these amino acid substitutions and found that all of c-Kit, K-Ras and B-Raf mutations in our ACC samples were inactive. These observations suggest that the c-Kit signaling pathway must be dispensable for maintaining established ACC. As a result, we believe that selective c-Kit inhibition is not a suitable treatment for the 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 351.
Abstract 3514: LT-00673, a highly potent PARP inhibitor, shows antitumor activity against tumor cells with DNA repair deficiencies
Authors: Shen Yuqiao Jerry , Feng Ying , Wang Bing , Chu Daniel , Myers Peter , Post Leonard
Published: February 2011

Poly (ADP-ribose) polymerases (PARP) −1 and −2 are key nuclear enzymes that are activated by DNA damage and play a critical role in the base excision DNA repair (BER) pathway. Previous studies have indicated that inhibitors of PARP-1 and −2 could enhance the tumor cytotoxicity of DNA damaging agents by preventing cancer cells from repairing DNA damage. In addition, PARP inhibitors have been shown to selectively kill cancer cells that are deficient in BRCA-1 or BRCA-2 function. More recently, it was further suggested that other genetic deficiencies, such as PTEN gene mutation and malfunction of DNA mismatch repair, may also sensitize cells to cytotoxicity of PARP inhibitors. We have previously reported a novel PARP inhibitor, called LT-00673, that inhibits PARP-1 with IC50 of 0.58 nM. LT-00673 is also a potent inhibitor of PARP-2, but does not inhibit poly (ADP-ribose) glycohydrolase (PARG). In cell culture studies, LT-00673 potentiates chemotherapeutic agents such as temozolomide and SN-38, and displays potent single-agent cytotoxicity in BRCA-1 and BRCA-2 mutant cells. In xenograft tumor model studies, daily oral dosing of LT-00673 significantly enhances the antitumor effects of cytotoxic therapies in a dose-dependent manner. Furthermore, once daily oral dosing of LT-00673 at 0.33 mg/kg/day results in tumor regression in the MX-1 model (BRCA-1 deficient). In the current study, we further investigated the pharmacokinetic properties of LT-00673 in dogs and rats, and compared antitumor efficacies of different dosing schedules of LT-00673 using xenograft tumor models. We also demonstrated that several PTEN-deficient human cancer cell lines are highly sensitive to LT-00673 as a single 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 3514.
Abstract 3523: C7, C9-dimethylifosfamide analogs evaluation for antitumor activity and toxicity: Detoxification pathways might explain nephrotoxicity
Authors: GIRAUD Bérénice , HEBERT Guillaume , DEROUSSENT Alain , DAUDIGEOS Estelle , MARTELLI Sonia , VASSAL Gilles , PACI Angelo
Published: February 2011

Ifosfamide (IFO) is an anticancer drug, widely used against various types of human solid tumors. This prodrug produced active alkylating isophosphoramide mustard. But its side-chain oxidative metabolism is associated with toxicities in high-dose chemotherapy. As acrolein urotoxicity is limited by coadministration of mesna, chloroacetaldehyde (CAA) is responsible for neurotoxicity and nephrotoxicity. C7, C9 dimethyl-ifosfamide analogs have been synthesized to prevent these side effects. In vitro metabolism and anticancer activity investigations demonstrated that the generated dimethylated mustards were faster alkylating agents (28 fold faster) than isophosphoramide mustard and IC50 values on 9L glioblastoma cells showed higher cytotoxicity of analogs, especially the 7S,9S enantiomer (1). This later was selected for in vivo investigations. As these analogs were still metabolized through the same N-deschloroalkylation pathway, monoochloroacetone (MCA) is produced instead of CAA. In a first step, in vitro/ex vivo experiments were conducted to investigate its toxicity. The toxicity of both metabolites was compared by evaluation of cell viability using canine kidney cell lines (MDCK) and rabbit renal proximal tubules (RRPT). Cell viability experiments showed that similar IC50 values were obtained for CAA and MCA (21 and 23 µM) on MDCK. To determine detoxification pathways, several experiments were conducted on RRPT. Whereas these compounds could react directly with glutathione (GSH), identification of GSH and its conjugates (GSR) was performed by LC/MS after incubation of MDCK and RRPT with 200 µM CAA and MCA. Both enzymatic reduction and GSH conjugation pathways occurred. Conjugates were then quantified by LC-MS/MS over 1-650 µM for GSH and over 1-200 µM for GSR from 20 to 120 min. The results demonstrated a rapid depletion of GSH and few differences in detoxification's kinetics for CAA and MCA. As MCA in vivo generated levels are still unknown, 7S,9S dimethyl-IFO analog is currently under investigation on human tumor cells xenografted in mice in order to confirm its antitumor activity and to evaluate its toxicity.

(1) Storme T, Deroussent A, Mercier L, Prost E, Re M, Munier F, Martens T, Bourget P, Vassal G, Royer J and Paci A. J Pharmacol Exp Ther. 2009, 328(2):598-609.

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 3523.
Abstract 3524: Plasma pharmacokinetics and metabolism of benzaldehyde dimethane sulfonate (NSC281612, DMS612, BEN) in mice
Authors: Parise Robert A. , Eiseman Julie L. , Egorin Merrill J. , Clausen Dana M. , Covey Joseph M. , Beumer Jan H.
Published: February 2011

Introduction: BEN is an alkylating agent in early clinical trials based on its in vitro and in vivo activity against renal cell carcinoma. We have previously reported that BEN is rapidly metabolized into its carboxylic acid (BA) and that BEN and BA undergo chemical conversion in vitro. In support of current clinical trials, we characterized the pharmacokinetics of BEN, BA, and their products in mice.

Materials & Methods: Female CD2F1 mice were dosed with 20 mg/kg BEN i.v. Twelve blood samples were obtained between 0 and 24 hrs and urine was collected at 0-6 hrs and 6-24 hrs. Formic acid was added to all samples (5%, v/v) and the sample processing was performed on ice to stop ex vivo chemical conversions. Plasma and urine concentrations of BEN, and its metabolites and conversion products were quantitated with LC-MS/MS. Plasma and urine pharmacokinetic parameters were calculated non-compartmentally.

Results: BEN was rapidly converted to at least 13 different metabolites or products. The half-life (T1/2) of BEN was approximately 60 min. Half-lives of metabolites were all >2 h. The analytes that could be quantitated in urine represented approximately 30% of the dose administered.AnalyteCmax (µg/ml)Tmax (min)T½ (min)AUC0-inf (mg/mL*min)Vd/F (L/kg)Cl/F (mL/min/kg)% Dose 0-24 h urineBEN4.355922.974.50.96.6BA26.1531315.72.80.18.5BA-(OH)20.71107323.988.10.815.5BEN-(Cl)20.001151080.2471264481.1NDBA-(Cl)20.027580.58938534NDBEN-(OH)20.6565416.811732.9NDBA-Cl-OH 513    BA-Cl 528    BA-OH 529    BA-Gluc 1031    BA-(OH)2-Gluc 1531    BA-Cl-OH-Gluc 3049    BA-Cl-Gluc 15107    BA-OH-Gluc 1036    

ND, not detectable; NQ, not quantifiable.

Conclusions: BEN is rapidly metabolized to BA and chemically converted to many subsequent products, some of which are glucuronidated. Several metabolites and conversion products contain chloroethyl or methanesulphonate moieties, which would allow them to alkylate cellular targets and exhibit anti-neoplastic properties or cause toxicity. The enzymes responsible for the metabolism of BEN and BA need to be identified.

Support: N01-CM07106, N01-CM52202, P30-CA47904 from the NCI.

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 3524.