bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3588: Common and different effects induced in primary human mesothelial cells and mice exposed to chrysotile or crocidolite asbestos.
Authors: Qi Fang , Okimoto Gordon , Jube Sandro , Pass Harvey I. , Laczko Rozalia , DeMay Richard M. , Khan Ghazal , Tiirikainen Maarit , Yang Haining , Gaudino Giovanni , Carbone Michele
Published: August 2014

Crocidolite is considered the most oncogenic asbestos type. Chrysotile accounts for >90% of asbestos used worldwide, however its capacity to induce MM is still debated. We linked crocidolite carcinogenesis to the release of High-Mobility Group Box-1 protein (HMGB1) and activation of downstream genes (TNF-α and NF-κB). We compared the carcinogenicity of crocidolite and chrysotile in primary human mesothelial cells (HM). Microarray analyses of HM exposed to crocidolite or chrysotile identified 57 genes that were significantly induced or repressed. Forty-eight hours after exposure we observed induction of HMGB1 and NF-κB targeted genes and E-cadherin and SFRP4 down-regulation. In vitro, both crocidolite and chrysotile induced HMGB1 and TNF-α secretion, induced β-catenin nuclear translocation and caused HM death and epithelial-mesenchymal transition in surviving cells. Five weeks from exposure these effects were sustained in HM exposed to crocidolite; the expression of these genes had returned to normal in HM exposed to chrysotile. Soon after injection, serum HMGB1 levels were elevated in mice injected with either crocidolite or chrysotile, and remained elevated 10 weeks post-injection only in mice injected with crocidolite. Our results show that chrysotile induces the same intracellular signaling elicited by crocidolite, however these effects are transient. Accordingly, HM exposure to chrysotile in the presence of TNF-α or in co-culture with macrophages, induced foci formation with a lower transforming efficiency compared to crocidolite.

Citation Format: Fang Qi, Gordon Okimoto, Sandro Jube, Harvey I. Pass, Rozalia Laczko, Richard M. DeMay, Ghazal Khan, Maarit Tiirikainen, Haining Yang, Giovanni Gaudino, Michele Carbone. Common and different effects induced in primary human mesothelial cells and mice exposed to chrysotile or crocidolite asbestos. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3588. doi:10.1158/1538-7445.AM2013-3588
Abstract 3589: Microbiomic analysis of colon cancer tumors and their matched normal reveals distinct gut floras.
Authors: Brim Hassan , Yooseph Shibu , Torralba Manolito , Lee Edward , Nelson Karen , Ashktorab Hassan
Published: August 2014

Background: The gut flora has long been suspected to play a role in colon carcinogenesis either through their secreted toxins or through their processing of ingested nutrients leading to the production of carcinogenic products. We previously have shown that stool samples flora from patients with pre-neoplastic colon lesions differ from those from healthy individuals. However, bacteria in stool samples are not representative of the colon adherent bacteria that are more prone to affect colon mucosa homeostasis. Here we analyzed the microbiota composition in 10 colon tumors and their matched normal tissue from 10 African American patients.

Materials & Methods: DNA extracts from 10 pairs of colon tumors and matched normal tissue, was used to amplify the V1-V3 regions of the 16S rRNA gene. The PCR primers included the A and B adaptor sequences for 454 pyrosequencing as well as a unique 12 bp barcode incorporated onto the reverse primer such that each sample receives its own unique barcode. The amplicons were purified, quantified, normalized, and then pooled in preparation for emulsion PCR followed by 454 sequencing using Titanium chemistry. After deconvolution and trimming, the sequences were put through chimera checking (using chimera slayer). Those sequences of length >=100 bp that were not labeled as chimeras were then processed using RDP's classifier.

Results: Overall, each tumor sample microbiota was the closest to its matched normal based on the general bacterial profile. Bacteroidetes and Firmicutes were the dominant bacterial groups in all analyzed samples. Using a Principal Component Analysis at the genus level with bacterial genera that are represented at least 5% in one sample revealed two distinctly separated healthy tissue groups, one defined primarily by Bacteroides and the second defined by Prevotella bacteria. No tumor sample was within these two groups. It is noteworthy that Fusobacteria, that was shown by other similar studies as primarily abundant in tumors, was not defining any tumor group in our study.

Conclusion: Our study reveals the presence of significant microbiota changes between tumor samples and matched normal tissue even though our analysis were done only at the genus level. Further analysis at the Operational Taxonomic Units (sub-genus level) for each pair of samples are underway and will further dissect the observed differences.

Citation Format: Hassan Brim, Shibu Yooseph, Manolito Torralba, Edward Lee, Karen Nelson, Hassan Ashktorab. Microbiomic analysis of colon cancer tumors and their matched normal reveals distinct gut floras. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3589. doi:10.1158/1538-7445.AM2013-3589
Abstract 359: Systematic manipulation of brain tumor diversity using piggybac transposon mediated transgenesis approach in rat.
Authors: Chen Fuyi , Becker Albert , LoTurco Joseph
Published: August 2014

Animal models are important for interrogating the native biology of tumor formation, invasiveness, and may lead to novel therapeutic approaches. Here we describe the development of a rat glioma model which provides a fast and convenient approach to reproducibly induce brain tumors of different type in rat. We utilize a piggybac transposon system delivered by in utero electroporation (PB-IUE) to express oncogenic HRasV12 in different cell types in CNS. When expression of HRasV12 was driven by ubiquitous CAG promoter in radial glia cells, glioblastoma multiforme (GBM) was induced. When we specifically express HRasV12 in astrocytes using a mouse GFAP promoter, malignant astroglioma resembling GBM was induced in rat brains. In contrast, anaplastic oligoastrocytoma was induced when HRasV12 were expressed in oligodendrocytes under the control a rat MBP promoter. Surprisingly when co-expressing a basic helix loop helix protein Neurogenin2 (Ngn2) with HRasV12 both driven by CAG promoter, primitive neuroectodermal tumor (PNET) like tumors were generated. Molecular classification of tumors induced by CAG and GFAP HRasV12 matched mesenchymal GBM subtypes. Our data demonstrate the induction of different tumor types using a non-viral piggyBac transposon mediated transgenesis approach. The binary plasmid system we developed provides a method to direct different oncogenetic signals to different cell types. The models generated with this system should facilitate our understanding of the sources of tumor diversity.

Citation Format: Fuyi Chen, Albert Becker, Joseph LoTurco. Systematic manipulation of brain tumor diversity using piggybac transposon mediated transgenesis approach in rat. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 359. doi:10.1158/1538-7445.AM2013-359
Abstract 360: Erlotinib inhibits growth of a patient derived chordoma tumor xenograft.
Authors: Siu I-Mei , Burger Peter C. , Zhao Qi , Ruzevick Jacob , Connis Nick , Hann Christine L. , Gallia Gary L.
Published: August 2014

Chordomas are rare primary bone tumors that occur along the neuraxis. Current treatment includes surgical resection and often postoperative radiotherapy. Despite treatment, local recurrence occurs in the majority of patients and metastasis is frequent. Treatment options for patients with recurrence is limited and, notably, there are no FDA approved chemotherapeutic agents. Median survival is approximately 7 years. The lack of treatment options is in part due to the paucity of preclinical model systems for this tumor. We have previously established a serially transplantable animal model directly from human chordoma tissue. Further analysis of this patient derived xenograft demonstrates that subsequent passages continue to resemble the original patient tumor histologically and immunohistochemically and maintain nuclear expression of brachyury, a marker for chordoma. Genome wide variation between the patient's tumor and xenografts continues to be more than 99% concordant. To further characterize this model, a receptor tyrosine kinase screen of more than 70 kinases was evaluated and epidermal growth factor receptor (EGFR) was found to be the most activated kinase. In vitro studies demonstrate that EGFR inhibitors, such as erlotinib and gefitinib, inhibit growth of a validated human chordoma cell line, U-CH1. In vivo studies demonstrate that erlotinib significantly inhibits growth of this patient derived chordoma xenograft. Evaluation of tumors post-treatment reveals that erlotinib reduces tumor proliferation and phosphorylation of EGFR. Taken together, these findings demonstrate targeting EGFR can inhibit chordoma growth in vivo and support further clinical evaluation of EGFR inhibitors in the treatment of this disease.

Citation Format: I-Mei Siu, Peter C. Burger, Qi Zhao, Jacob Ruzevick, Nick Connis, Christine L. Hann, Gary L. Gallia. Erlotinib inhibits growth of a patient derived chordoma tumor xenograft. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 360. doi:10.1158/1538-7445.AM2013-360
Abstract 3602: Effect of alcohol on DB[a,l]P, an environmental pollutant and a tobacco smoke constituent, on DNA adduct formation in mouse oral tissues.
Authors: Zhang Shang-Min , Chen Kun-Ming , Richie John R. , Calcagnotto Ana , Aliaga Cesar , Berg Arthur , Sun Yuan-Wan , Sharma Arun K. , Amin Shantu , El-Bayoumy Karam
Published: August 2014

Alcohol acts synergistically with tobacco smoke to greatly increase the risk of oral squamous cell carcinoma (OSCC) up to 20-fold; the mechanisms of this synergism remain unknown. Research in this area has been hampered by the lack of appropriate animal models that would mimic chronic simultaneous human exposure to alcohol and representative carcinogens in tobacco or tobacco smoke as a whole. Recently, we developed a more relevant animal model (Int. J. Cancer, 2012, 2783-90) and showed that dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and a tobacco smoke component, induces SCC in oral tissues of mice. We also showed that its diol epoxide metabolite, (±)-anti-DB[a,l]PDE, is a potent and specific carcinogen in tongue and other oral tissues in mice. We hypothesize that chronic alcohol consumption will increase the carcinogenic potency of DB[a,l]P in the mouse oral cavity by multiple mechanisms including the depletion of glutathione (GSH) and increasing levels of covalent DB[a,l]PDE-DNA adducts.

As an initial study to test our hypothesis, mice were fed Isocaloric Lieber-DeCarli-type liquid diets formulated by BioServ (Frenchtown, N.J.) with or without ethanol (35% of total calories, increased gradually from 0, 11, 22 to 35%). After 3 weeks, mice were treated with 2 daily doses of DB[a,l]P (240 nmol each) and/or tobacco smoke particulates (TSP, 2R4F, 2.5 mg/mouse). Mice were sacrificed 24 hrs later and DB[a,l]PDE-DNA adduct levels were quantified by our recently developed LC-MS/MS method (Chem. Res. Toxicol. 2011, 1297-303). We showed that alcohol consumption significantly increased the levels of (-)-anti-trans-DB[a,l]P-dA in the absence of TSP (25%) or in the presence of TSP (75%). We also demonstrated that GSH levels in buccal mucosa were decreased (38%) in mice fed with liquid diet (35% calories from ethanol) compared with mice fed with control diet (0.89±0.13 μmol/g vs 1.23±0.17 μmol/g). Collectively, our preliminary results support our hypothesis that alcohol consumption will increase the carcinogenic potency of DB[a,l]P by altering the metabolic detoxification via depleting GSH and increasing DNA damage in oral tissues of mice.

Citation Format: Shang-Min Zhang, Kun-Ming Chen, John R. Richie, Ana Calcagnotto, Cesar Aliaga, Arthur Berg, Yuan-Wan Sun, Arun K. Sharma, Shantu Amin, Karam El-Bayoumy. Effect of alcohol on DB[a,l]P, an environmental pollutant and a tobacco smoke constituent, on DNA adduct formation in mouse oral tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3602. doi:10.1158/1538-7445.AM2013-3602
Abstract 3603: Zidovudine (AZT)-induced aneuploidy, mediated by Stathmin 1 (STMN1), involves a loss of polymerized β-tubulin.
Authors: Rivera Andrea V. , Sanchez Vanesa C. , Poirier Miriam C. , Olivero Ofelia A.
Published: August 2014

The nucleoside reverse transcriptase inhibitor AZT induces multiple manifestations of genotoxicity, among which aneuploidy, observed as micronuclei containing whole chromosomes, has been reported (Borojerdi et al., Mut Res, 2009). We have seen a loss of β-tubulin polymerization in about 23% of normal human mammary epithelial cells (NHMECs) exposed for 24 hr to 200 μM AZT, and demonstrated down-regulation of hsa-miR-770-5p, a microRNA known to target STMN1, in the same AZT-exposed cells. STMN1 prevents microtubule polymerization, resulting in destabilization of the mitotic spindle and chromosome misalignment. We therefore hypothesized that, if AZT exposure downregulates hsa-miR-770-5p, there should be increased expression of STMN1, which would result in loss of microtubule polymerization leading to aneuploidy. In this study we exposed cells of the human breast line MCF-10A to 100 μM AZT continuously for 1 to 3 passages (P). By Western Blot we showed that total STMN1 was 3.5-fold increased at P1, in AZT-exposed cells, compared to the unexposed controls. Using the same STMN1 antiserum, by immunohistochemistry (IHC), we observed that some cells stained heavily for STMN1, while others had virtually no staining, and that the overall STMN1 fluorescence was increased in AZT-exposed cells, compared to unexposed controls. When tubulin polymerization was evaluated by IHC, using β-tubulin antiserum, 10.1% of AZT-exposed cells showed a loss of tubulin polymerization, while only 0.7% of unexposed cells showed the same effect (p=0.004). The data suggest that AZT induced aneuploidy may be mediated by inhibition of hsa-miR-770-5p, upregulation of STMN1, impaired polymerization of β-tubulin, aberrant mitotic spindle formation and consequent chromosome misalignment resulting in aneuploidy. If confirmed, this will constitute a novel mechanism of AZT-induced genotoxicity that may or may not be related to AZT-DNA incorporation. Ongoing experiments will explore these relationships further by localizing STMN1 and β-tubulin in specific cells, and examining the same cultures for spindle aberrations at mitosis.

Citation Format: Andrea V. Rivera, Vanesa C. Sanchez, Miriam C. Poirier, Ofelia A. Olivero. Zidovudine (AZT)-induced aneuploidy, mediated by Stathmin 1 (STMN1), involves a loss of polymerized β-tubulin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3603. doi:10.1158/1538-7445.AM2013-3603
Abstract 3613: Toenail selenium is associated with a decreased risk of advanced prostate cancer.
Authors: Geybels Milan S. , Verhage Bas A.J. , van Schooten Frederik J. , Goldbohm Alexandra , van den Brandt Piet A.
Published: August 2014

Introduction: Selenium status has been associated with a reduced risk of total prostate cancer (PCa) and there is evidence that the association is more pronounced for advanced, clinically relevant PCa. This association, however, has been studied over a relatively narrow range of selenium status and data from low-selenium populations are missing. Most prior studies of selenium status and PCa have used plasma/serum selenium, which reflects recent selenium intake, and few studies have used toenail selenium, which reflects longer exposure time windows. Methods: We studied the association of toenail selenium and advanced PCa risk in the Netherlands Cohort study, which includes 58,279 men aged 55 to 69 years. The study has a case-cohort design; a random subcohort was sampled at baseline in 1986 and incident advanced (stage III/IV) PCa cases were identified during 17.3 years of follow-up. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated using Cox proportional hazards regression models. Results: The study population included 898 advanced PCa cases and 1,203 subcohort members. The average toenail selenium concentration among subcohort members was 0.549 μg/g (standard deviation: 0.128). Toenail selenium was associated with a reduced risk of advanced PCa and adjusted HRs for increasing quintiles of toenail selenium were 1.00 (reference), 0.69 (95% CI: 0.52, 0.90), 0.45 (95% CI: 0.34, 0.60), 0.32 (95% CI: 0.24, 0.44), and 0.24 (95% CI: 0.17, 0.33) (P for trend <0.01). The association was more pronounced for men diagnosed during later follow-up, and adjusted HRs (0.045 μg/g increment; size central quintile) for men diagnosed during ≤6 years, >6 to 12 years, and >12 years of follow-up were 0.91 (95% CI: 0.85, 0.98), 0.85 (95% CI: 0.75, 0.96), and 0.77 (95% CI: 0.71, 0.84), respectively. Conclusion: Toenail selenium was associated with a substantial decrease in risk of advanced PCa, particularly during later follow-up. If our results can be confirmed, a prevention trial of selenium and PCa in a low-selenium population may be justified. Selenium exerts important biological functions through its presence in selenoproteins and genetic variation in the major selenoproteins glutathione peroxidase 1 (GPX1) and selenoprotein P (SEPP1) has been associated with the risk of PCa. In a next analysis, in the same population, we will study the association of common variation in GPX1 and SEPP1 with advanced PCa risk, and we will evaluate SNP-selenium interactions.

Citation Format: Milan S. Geybels, Bas A.J. Verhage, Frederik J. van Schooten, Alexandra Goldbohm, Piet A. van den Brandt. Toenail selenium is associated with a decreased risk of advanced prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3613. doi:10.1158/1538-7445.AM2013-3613
Abstract 3627: Occupational asbestos exposure is associated with head and neck cancer.
Authors: Langevin Scott M. , O'Sullivan Mattie H. , Valerio Jennifer L. , Applebaum Kate M. , Eliot Melissa , McClean Michael D. , Kelsey Karl T.
Published: August 2014

Asbestos describes a group of naturally occurring silicate mineral fibers that were frequently used in industry during the 20th century due to their desirable flame retardant and tensile properties. Although use has fallen over the last three decades, exposure continues and the burden of disease is considerable. The primary routes of exposure to the fibers include inhalation and ingestion. Asbestos is a known risk factor for several malignant respiratory diseases, including lung cancer and mesothelioma, and has more recently been implicated in pharyngeal and laryngeal cancer. However, studies of asbestos and cancers of the larynx or pharynx with adequate sample-size and control for other risk factors for head and neck squamous cell carcinoma (HNSCC) such as smoking and drinking habit remain somewhat sparse. We report findings from a case-control study consisting of 934 incident HNSCC cases from the greater Boston region and 1180 population-based controls, frequency matched on age (+/- 3 years), sex, and town or neighborhood of residence. Study subjects completed a self-administered questionnaire that provided detailed data on history of asbestos exposure, sociodemographics and personal characteristics, alcohol and tobacco use, personal and family cancer history, and other relevant dietary, occupational, residential and medical exposures. Occupational asbestos exposure was dichotomized as exposed or never exposed based on self reported occupation data. Multivariate unconditional logistic regression was used to assess the association between asbestos exposure and HNSCC, overall and by primary tumor site and smoking history. A total of 217 cases (23.2%) and 226 controls (19.2%) reported an occupational exposure to asbestos. We observed a borderline association of asbestos exposure and laryngopharyngeal cancer (OR = 1.23, 95% CI: 0.95-1.59), adjusted for age, sex, race, smoking, alcohol consumption, and education. After stratifying by smoking history, a significant association was observed between asbestos exposure and laryngopharyngeal cancer among ever-smokers (OR = 1.36, 95% CI: 1.00-1.84). These observations are consistent with the mounting evidence that asbestos is a risk factor for pharyngeal and laryngeal cancer.

Citation Format: Scott M. Langevin, Mattie H. O'Sullivan, Jennifer L. Valerio, Kate M. Applebaum, Melissa Eliot, Michael D. McClean, Karl T. Kelsey. Occupational asbestos exposure is associated with head and neck cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3627. doi:10.1158/1538-7445.AM2013-3627
Abstract 3630: Fat intake interacts with cigarette smoking to alter lung cancer risk.
Authors: Zhang Jianjun , Tanaka Hideo , Ito Hidemi , Matsuo Keitaro
Published: August 2014

Although cigarette smoking is the primary cause of lung cancer, it alone cannot explain large difference in risk of this malignancy worldwide. During the last three decades, age-adjusted male lung cancer mortality rates have been remarkably higher in Hungary than in Japan, but per capita cigarette consumption has remained lower in Hungary than in Japan. A similar pattern of difference has been observed between other Asian and western countries, (e.g., China, South Korea, Poland, and the US). It remains unclear why this lung cancer paradox occurs. Lung cancer incidence is generally high in countries with high intake of saturated fat. In addition, animal studies revealed a synergistic effect of fat and tobacco carcinogens on the development of lung tumors. Therefore, it is possible that different types of fat interact with cigarette smoking to modulate lung cancer risk. We sought to investigate this hypothesis in a hospital-based case-control study conducted from 2000 to 2005 in Nagoya, Japan. Cases (n=500), ages 20-79, were patients pathologically diagnosed with lung cancer at the Aichi Cancer Center. Controls (n=500) were recruited from visitors to the outpatient department of the same institution, and matched to cases by age (within 5 years) and sex. Fat intake was assessed with a validated food frequency questionnaire, and cigarette smoking was evaluated with a pre-tested risk factor questionnaire. Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated using unconditional logistic regression. After adjustment for age, sex, BMI, energy intake, and alcohol drinking, lung cancer risk among heavy smokers (≥40 pack-years) decreased with increasing intake of monounsaturated fat (MUFA). Compared with never or light smokers (≤20 pack-years), ORs (95% CI) were 8.44 (4.53, 15.7), 4.34 (2.32, 8.13), and 3.63 (1.84, 7.04) for heavy smokers who were in the 1st(lowest), 2nd, and 3rd tertiles of MUFA intake, respectively. A similar pattern of interaction was present for intake of polyunsaturated fat (PUFA) and for moderate smokers (20-39 pack-years). To our knowledge, the present study is among the first to show that intake of unsaturated fat modifies the association between cigarette smoking and lung cancer risk, and thereby sheds new light on the etiology and prevention of this malignancy.

Citation Format: Jianjun Zhang, Hideo Tanaka, Hidemi Ito, Keitaro Matsuo. Fat intake interacts with cigarette smoking to alter lung cancer risk. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3630. doi:10.1158/1538-7445.AM2013-3630
Abstract 3656: Methodology design for identifications of protein targets of chemopreventive isothiocyanates using click chemistry.
Authors: Fu Ying , Mi Lixin , Aggarwal Monika , Appella Daniel M. , Chung Fung-Lung
Published: August 2014

Isothiocyanates (ITCs) derived from cruciferous vegetables are inhibitors of carcinogenesis in animal models and can induce cell cycle arrest and apoptosis in a variety of cancer cell lines. It is known that ITCs can conjugate with glutathione (GSH) and induce cellular oxidative stress; ITCs can also bind to intracellular proteins (e.g. tubulin) which may constitute a mechanism underlying their cancer preventive effects. To fully understand the cellular responses to ITCs, it is crucial to develop an unbiased method that can rapidly identify their protein targets. In this study, the direct protein targets of phenethyl isothiocyanate (PEITC) were investigated by using click chemistry. An alkyne-tagged ITC was used as an analog of PEITC, a known cancer prevention agent. The tagged ITC (N-(2-(4-methoxyalkyne)phenethyl)isothiocyanate (NPPEITC) was designed and synthesized with a moiety for copper-catalyzed Huisgen 1,3 dipolar cycloaddition reaction (click chemistry), the tagged ITC, NPPEITC, was also found to show similar biological activities to PEITC; The click-chemistry reaction conditions of copper-catalyzed azide-alkyne cycloaddtion (CuAAC) for NPPEITC and N-(2-(4-methoxyalkyne)phenylethyl)acetamide (NPA, a negative control) with biotin azide were optimized and the reaction product was isolated and analyzed by mass spectrometry. A model click reaction between NPPEITC and tubulin was employed to validate this method. The western-blot showed that the tubulin protein is modified by biotin after NPPEITC treatment then followed by click reaction with biotin azide. In contrast, no biotin was found on NPA treated tubulin. Secondly, cancer cells exposed to the tagged ITC (NPPEITC or NPA) was lysed and exposed to click chemistry reagents to conjugate ITC-adducted proteins with biotin for subsequent affinity purification; the ITC conjugated proteins were identified and quantified using proteomic analysis by LC/MS/MS.(Supported by NCI grant CA100853)

Citation Format: Ying Fu, Lixin Mi, Monika Aggarwal, Daniel M. Appella, Fung-Lung Chung. Methodology design for identifications of protein targets of chemopreventive isothiocyanates using click chemistry. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3656. doi:10.1158/1538-7445.AM2013-3656
Abstract 3659: Next generation sulindac.
Authors: Li Nan , Xi Yaguang , Tinsley Heather N. , Gurpinar Evrim , Gary Bernard D. , Zhu Bing , Li Yonghe , Keeton Adam B. , Grizzle William E. , Piazza Gary A.
Published: August 2014

Sulindac displays promising antineoplastic activity, but toxicity from cyclooxygenase (COX) inhibition limits its long-term use for chemoprevention. Previous studies have concluded that the tumor cell growth inhibitory activity of sulindac is COX-independent and suggest that safer and more efficacious drugs can be developed by targeting the underlying mechanism. Here we report that sulindac sulfide (SS) inhibits cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) activity, increases intracellular cGMP levels and activates protein kinase G at concentrations that suppress proliferation and induce apoptosis of colon tumor cells. Normal colonocytes were refractory to the anti-proliferative and pro-apoptotic effects of SS as well as its ability to induce cGMP signaling. PDE5-specific inhibitors such as tadalafil also inhibited the growth of colon tumor cells that expressed high levels of the cGMP-specific PDE5 isozyme compared with colonocytes. RNAi knockdown of PDE5 selectively inhibited proliferation and induced apoptosis of colon tumor cells as did SS. SS, 8-bromo-cGMP, and PDE5 siRNA reduced β-catenin levels, as well as the expression of cyclin D and survivin that are regulated by β-catenin-dependent TCF transcriptional activity by a mechanism involving the transcriptional repression of β-catenin. The significance of these findings are highlighted by studies in the FCCC Min mouse model of intestinal tumorigenesis showing that a novel non-COX inhibitory derivative of SS can strongly inhibit colon tumor formation by a mechanism that appears to involve cGMP PDE inhibition. These observations suggest that safer and more efficacious drugs can be developed for colorectal cancer chemoprevention by targeting PDE5 and possibly other cGMP degrading isozymes.

Citation Format: Nan Li, Yaguang Xi, Heather N. Tinsley, Evrim Gurpinar, Bernard D. Gary, Bing Zhu, Yonghe Li, Adam B. Keeton, William E. Grizzle, Gary A. Piazza. Next generation sulindac. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3659. doi:10.1158/1538-7445.AM2013-3659
Abstract 3670: The role of reactive oxygen species in anticancer effects of diallyl trisulfide, a bioactive component of garlic.
Authors: Arlotti Julie A. , Chandra-Kuntal Kumar , Singh Shivendra V.
Published: August 2014

Diallyl trisulfide (DATS) is a bioactive component of processed garlic with in vivo activity against chemically-induced as well as oncogene-driven cancer in rodent models. However, the mechanisms underlying anticancer effects of DATS (eg, growth inhibition, apoptosis induction, and suppression of migration in cancer cells) are not fully understood. The present study reveals a critical role for reactive oxygen species (ROS) in cellular anticancer responses to DATS. Exposure of human breast cancer cells (MDA-MB-231 and MCF-7) as well as a cell line derived from spontaneously developing mammary tumor of a MMTV-neu transgenic mouse (BRI-JM04) to DATS resulted in dose-dependent inhibition of cell viability and apoptosis induction. Interestingly, a non-tumorigenic normal human mammary cell line (MCF-10A) was resistant to growth inhibition and apoptosis induction by DATS. The DATS-induced apoptosis in MDA-MB-231, MCF-7, and BRI-JM04 cells was associated with ROS production as evidenced by fluorescence microscopy and flow cytometry using a chemical probe (MitoSOX Red). Ectopic expression of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) as well as Mn-SOD conferred significant protection against DATS-induced ROS production and apoptotic cell death. The DATS treatment caused activation of Bax and Bak, and activation of Bak was nearly fully abolished by overexpression of Mn-SOD. Likewise, inhibition of MDA-MB-231 and MCF-7 cell migration resulting from DATS treatment was partially but statistically significantly attenuated by stable overexpression of Cu,Zn-SOD and Mn-SOD. This protection against inhibition of cell migration was attributable to abrogation of DATS-mediated induction of E-cadherin and suppression of vimentin. These results indicate that ROS play an important role in anticancer effects of DATS at least in human breast cancer cells. This study was supported by grant CA113363-07 awarded by the National Cancer Institute.

Citation Format: Julie A. Arlotti, Kumar Chandra-Kuntal, Shivendra V. Singh. The role of reactive oxygen species in anticancer effects of diallyl trisulfide, a bioactive component of garlic. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3670. doi:10.1158/1538-7445.AM2013-3670
Abstract 3671: The role of FOXQ1 in benzyl isothiocyanate-mediated inhibition of epithelial-mesenchymal transition in human breast cancer cells.
Authors: Sehrawat Anuradha , Kim Su-Hyeong , Vogt Andreas , Singh Shivendra V.
Published: August 2014

We have shown previously that breast cancer chemoprevention by benzyl isothiocyanate (BITC) in mouse mammary tumor virus-neu transgenic mice is associated with induction of E-cadherin protein expression. Loss of E-cadherin expression concomitant with induction of mesenchymal markers (eg, vimentin and fibronectin) is a biochemical hallmark of epithelial-to-mesenchymal transition (EMT). EMT is a developmental process but implicated in progression of cancer to aggressive state. We have also shown previously that BITC treatment inhibits EMT in cultured MDA-MB-231 human breast cancer cells. The present study extends these observations and investigates the mechanism by which BITC inhibits EMT. Initially, we designed experiments using another triple-negative human breast cancer cell line (SUM159) to determine generality of the BITC-mediated inhibition of EMT. Exposure of SUM159 cells to pharmacologically-relevant concentrations of BITC (2.5 and 5 μM) resulted in induction of E-cadherin protein expression, which was accompanied by suppression of vimentin protein level especially at the 5 μM dose. Protein levels of slug and snail, which are transcriptional repressors of E-cadherin, were also decreased markedly after treatment of SUM159 cells with BITC, but this effect was relatively more pronounced on slug protein level compared with snail. Similar to MDA-MB-231 cells, the BITC-mediated induction of E-cadherin protein expression in SUM159 cells was accompanied by its transcriptional upregulation as revealed by E-cadherin luciferase reporter assay. Immunofluorescence microscopy confirmed induction of E-cadherin protein and suppression of vimentin protein expression in BITC-treated SUM159 cells. BITC treatment resulted in downregulation of Forkhead Box Q1 transcription factor (FOXQ1) protein and FOXQ1 mRNA in MDA-MB-231, SUM159, and MDA-MB-468 cells. The BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with suppression of FOXQ1 protein level. Ectopic expression of FOXQ1 in a mammary epithelial cell line conferred marked protection against BITC-mediated inhibition of EMT as well as cell migration. In conclusion, the present study implicates FOXQ1 suppression in BITC-mediated inhibition of EMT in human breast cancer cells. This study was supported by the grant CA129347-06 awarded by the National Cancer Institute.

Citation Format: Anuradha Sehrawat, Su-Hyeong Kim, Andreas Vogt, Shivendra V. Singh. The role of FOXQ1 in benzyl isothiocyanate-mediated inhibition of epithelial-mesenchymal transition in human breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3671. doi:10.1158/1538-7445.AM2013-3671
Abstract 3673: AMPK-SIRT1 axis: a potential therapeutic target for prostate cancer management.
Authors: Li Guiming , Rivas Paul , Bedolla Roble , Reddick Robert L. , Ghosh Rita , Kumar Addanki P.
Published: August 2014

Recently we have shown that resveratrol (RES) intervention prevents development of high-grade prostatic intraepithelial neoplastic (HGPIN) lesions in prostate-specific PTEN knockout mouse model targeting SIRT1/mTORC1 axis. It is known that insufficient nutrient supply combined with high proliferation keeps solid tumors including prostate under hypoxic and metabolic stress. Tumor cells adapt to survive under such conditions through activation of AMP-activated kinase (AMPK). AMPK has been reported to be activated in prostate tumors. Therefore targeting AMPK and associated signaling pathways will be a promising approach for prostate cancer management. Accordingly we investigated the role of AMPK in RES-induced growth inhibitory effects using multiple human prostate cancer cell lines and preclinical animal model. These data show that RES treatment (50 μM, 24 h) results in activation of SIRT1, significant inhibition of AMPK phosphorylation and cell survival in human prostate cancer cells (RWPE-1, LNCaP, C42B and DU145). The observed molecular changes were associated with induction of apoptosis and autophagy following treatment with RES. Further dietary administration of RES (0.1 and 2%) to 4-5 week old prostate-specific PTEN knockout mice for 11 and 14 weeks showed prevention of HGPIN development. Interestingly intervention for 7 weeks showed prevention of HGPIN development at lower dose but not at high dose. On the other hand, 28-week intervention had no significant effect on the development of HGPIN lesions. Immunohistochemical evaluation showed modulation of AMPK, pS6K and SIRT1 in the prostate. Overall these data provide novel insights into RES-induced prevention of HGPIN development via AMPK/SIRT1/mTORC1 axis. Supported in part by NIH (CA 137518 and 135451 APK).

Citation Format: Guiming Li, Paul Rivas, Roble Bedolla, Robert L. Reddick, Rita Ghosh, Addanki P. Kumar. AMPK-SIRT1 axis: a potential therapeutic target for prostate cancer management. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3673. doi:10.1158/1538-7445.AM2013-3673
Abstract 3674: Chemoprevention of endometrial cancer with urolithin A, a metabolite of berry ellagitannins.
Authors: Huang Yi-Wen , Kuo Chieh-Ti , Siddiqui Jibran , Chen Jo-Hsin , Shiau Chung-Wai , Stoner Gary D.
Published: August 2014

Berries have been reported to elicit chemopreventive effects in cell culture, in animal model systems and in humans. Bio-fractionation studies indicate that the chemopreventive effects of berries can be ascribed to their component anthocyanins, ellagitannins and compounds in the non-alcohol soluble (fiber) fraction. In the present study, 17 berry-related compounds were evaluated at concentrations ranging from 1- 50 μM for their anti-proliferative effects on cultured human endometrial cancer cell lines (ECC-1, Ishikawa and HEC1A). These compounds include the anthocyanins; cyandin-3-glucoside and cyanidin-3-rutinoside and their metabolite, protocatechuic acid; ellagic acid and its metabolites, urolithins A and B; and three oligosaccharides, four short-chain carboxylic acids and four lignans present in the fiber fraction. Cell proliferation assays indicated that urolithin A was the most effective inhibitor of the growth of all three endometrial cancer cell lines. Cell cycle analysis by flow cytometry revealed that urolithin A induced cell cycle arrest at the G2/M phase. This was associated with up-regulation of the protein expression of cyclins B1 and E2, p21, phospho-cdc2 and myt1 as determined by western blot analysis. Endometrial cells depend on progesterone to limit cellular growth because it is antagonistic to estrogen-mediated cell proliferation. It is of importance therefore, that urolithin A was found to reduce estrogen receptor alpha and induce progesterone receptor expression in all three cell lines. In summary, our findings suggest that urolithin A may be chemopreventive for endometrial cancer through modulating genes that control the G2/M phase of the cell cycle and that regulate steroid receptor expression. This research was supported, in part, by MCW Startup Funds to Y-W Huang.

Citation Format: Yi-Wen Huang, Chieh-Ti Kuo, Jibran Siddiqui, Jo-Hsin Chen, Chung-Wai Shiau, Gary D. Stoner. Chemoprevention of endometrial cancer with urolithin A, a metabolite of berry ellagitannins. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3674. doi:10.1158/1538-7445.AM2013-3674
Abstract 3680: Targeting IGF-1R for inhibition of pancreatic cancer metastasis by pomegranate fraction.
Authors: Vadhanam Manicka V. , Gupta Ramesh C. , McNally Lacey R.
Published: August 2014

Patients with pancreatic adenocarcinoma have the lowest 5-year survival rate with more than 95% of the patients dying within 5 years and median survival of <6 months. The incidence of pancreatic cancer is also increasing by 1.5% per year since 2004. At present, there are limited treatment options and no early detection strategies for the disease. Although ∼ 20% of patients are eligible for surgical and chemotherapy intervention, these patients typically die of metastasis. Therefore, the prevention of these metastases would greatly improve patient care. Insulin-like growth factor receptor 1 (IGF-1R) is commonly over-expressed in metastases as compared to primary tumor. The aggressive forms of tumors overexpress the receptor when compared to less aggressive, non-metastatic tumors. In order to test this concept, we tested two metastatic pancreatic cell lines, S2VP10 and S2CP9 and three non-metastatic cell lines, Panc-1, AsPc-1 and MiaPaCa-2. Immunoblotting revealed the overexpression of IGF-1R specifically in the metastatic pancreatic cell lines. Pomegranate has recently gained much attention for its chemopreventive efficacy. We had previously demonstrated that the less-polar pomegranate component of the peel target IGF-1R signaling pathway in prostate cells. To test the efficacy of the pomegranate component against pancreatic cancer cells, we treated the highly aggressive S2VP10 cells with fractions derived from different parts of pomegranate. The water-insoluble fraction of pomegranate peel was most efficacious with an IC50 of 62 μg/ml. Molecules related to IGF-1R signaling were analyzed by immunoblotting. IRS-1, a docking protein for the receptor decrease significantly in a dose-dependent manner. A dose-dependent decrease of phosphorylation of the IGF-1R receptor was also observed. Dose-dependent decreased protein levels of BCL2 and increased levels of caspase 9 confirmed the effect of pomegranate fraction on apoptosis; and increased levels of p21 protein and decreased levels of cyclin D1 confirmed the effect on cell proliferation. Thus, we hypothesize that targeting IGF-1R signaling axis with pomegranate fraction will decrease survival of pancreatic tumor cells and decrease metastasis. Studies are currently underway to assess the anti-metastatic effect in vivo.

Citation Format: Manicka V. Vadhanam, Ramesh C. Gupta, Lacey R. McNally. Targeting IGF-1R for inhibition of pancreatic cancer metastasis by pomegranate fraction. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3680. doi:10.1158/1538-7445.AM2013-3680
Abstract 3682: Differential effect of withaferin A treatment on activation of Notch1 and Notch2 in human breast cancer cells.
Authors: Lee Joomin , Sehrawat Anuradha , Singh Shivendra V.
Published: August 2014

Withaferin A (WA), a constituent of the Ayurvedic medicine plant Withania somnifera, exhibits in vivo efficacy against human breast cancer xenografts in athymic mice as well as spontaneous mammary cancer development in MMTV-neu transgenic mice. However, the mechanism underlying anticancer effect of WA is not fully understood. The present study was undertaken to determine the role of Notch family of transcription factors in anticancer effects of WA using human breast cancer cells as a model. Notch activation is linked to mammary carcinogenesis as evidenced by the following observations: (a) overexpression of activated Notch1 and Notch3 in transgenic mice blocks mammary gland development but induces tumorigenesis of the breast; (b) high level expression of Notch1 and its ligands is associated with poor outcome in breast cancer; (c) expression of Notch ligand Jagged1 is associated with recurrence in lymph node-negative breast cancer; and (d) Notch1 is involved in migration and invasion of human breast cancer cells. The present study reveals differential effect of WA treatment on activation of Notch1 versus Notch2 in human breast cancer cells. Exposure of MDA-MB-231 (an estrogen-independent cell line with mutant p53) and MCF-7 (an estrogen-responsive cell line with wild-type p53) human breast cancer cells to pharmacological concentrations of WA (2 and 4 μM) resulted in cleavage (suggesting activation) of Notch2, which was accompanied by transcriptional activation of Notch as evidenced by RBP-Jk, HES-1A/B, and HEY-1 luciferase reporter assays. On the other hand, WA treatment caused a decrease in levels of both transmembrane and cleaved (active) Notch1. The WA-mediated activation of Notch2 was associated with induction of γ-secretase complex components presenilin1 and/or nicastrin. Inhibition of MDA-MB-231 and MDA-MB-468 cell migration resulting from WA exposure was statistically significantly augmented by knockdown of Notch2 using siRNA and shRNA. Activation of Notch2 was not observed in cells treated with withanone or withanolide A, which are structural analogs of WA. Moreover, withanone and withanolide A were practically inactive against MDA-MB-231 cell migration. The results of the present study suggest that anti-metastatic effect of WA may be augmented in the presence of Notch2 inhibitors. This study was supported by the grant CA142604 awarded by the National Cancer Institute.

Citation Format: Joomin Lee, Anuradha Sehrawat, Shivendra V. Singh. Differential effect of withaferin A treatment on activation of Notch1 and Notch2 in human breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3682. doi:10.1158/1538-7445.AM2013-3682