bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2091: Nitrooleic acids induce localization of heat shock protein 70 in PAM212 keratinocyte caveolae
Authors: Zheng Ruijin , Black Adrienne T. , Heck Diane E. , Laskin Debra L. , Laskin Jeffrey D.
Published: July 2012

Keratinocytes can be stimulated to express inducible nitric oxide synthase and generate nitric oxide by proinflammatory mediators including γ-interferon and lipopolysaccharide. Once formed, nitric oxide and its oxidation products form signaling molecules via redox mediated nitration reactions. Important targets for nitration in mammalian cells are unsaturated fatty acids. This can result in the production of reactive electrophilic species including 9-nitrooleate (9-NO) and 10-nitrooleate (10-NO). Oxidative stress is known to upregulate expression of heat shock proteins which are important chaperones functioning in protein-protein interactions including folding and trafficking. In the present studies we characterized nitro-fatty acid-induced expression of heat shock protein 70 (hsp70) in PAM212 mouse keratinocytes. Western blot analysis showed low constitutive levels of hsp70. Treatment of keratinocytes with 5-25 μM 9-NO or 10-NO for 6 hr markedly increased hsp70 expression. 10-NO was more active than 9-NO. Constitutive hsp70 was found to be localized in cytosolic fractions of keratinocytes, 9- or 10-NO did not alter constitutive cytosolic hsp70. In contrast, following treatment of keratinocytes with the nitro-fatty acids, hsp70 was selectively upregulated in lipid raft membrane fractions. Both hsp70 and caveolin-1 were co-localized in the lipid raft fractions of the membranes demonstrating that the lipid rafts are caveolae. In mouse keratinocytes, nitro-fatty acids readily stimulate MAP kinase signaling including JNK and p38 MAP kinases. Inhibitors of JNK (SP600125, 20 μM) or p38 (SB203580, 10 μM) had no effect on induction of hsp70 or its localization in caveolae indicating that nitro-fatty acid-induced expression of this protein was MAP kinase-independent. Taken together these data demonstrate that nitro-fatty acids are important in the control of heat shock protein expression. Moreover, selective localization of hsp70 in caveolae suggests that it functions in protecting caveolar proteins from cellular stress. Supported by CA132624, ES004738, ES005022, GM034310 and AR055073.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2091. doi:10.1158/1538-7445.AM2011-2091
Abstract 2092: Pathway specific gene profiling analysis reveals potential target genes of the stress oncoprotein LEDGF/p75 in prostate cancer cells
Authors: Basu Anamika , Drame Awa , De Leon Marino , Casiano Carlos A.
Published: July 2012

Chronic inflammation in the prostate microenvironment, with its associated increase in oxidative stress, is critical for prostate carcinogenesis since it leads to DNA mutations and epigenetic changes, as well as deregulation of stress and antioxidant proteins that protect prostate cells against oxidative damage and cell death. The lens epithelium-derived growth factor p75 (LEDGF/p75), an oxidative stress-induced transcription coactivator, is overexpressed in prostate tumors and other tumor types. LEDGF/p75 is emerging as a stress oncoprotein that protects cancer cells against lysosomal cell death and promotes resistance to chemotherapeutic drugs. We hypothesize that the pro-survival function of LEDGF/p75 is associated with its ability to promote the transcriptional activation of protective stress genes. To date, only very few candidate target genes of LEDGF/p75 have been identified in cancer cells. Towards this goal we initiated a stress focused gene profiling analysis of PC3 prostate cancer cells overexpressing or lacking LEDGF/p75, using the Real Time PCR Array System (SABiosciences). We selected an array that contained 80 genes involved in the cellular stress response and antioxidant defense. siRNA oligos targeting the C-terminus of the LEDGF/p75 mRNA sequence were used to induce transient LEDGF/p75 knockdown in PC3 cell. LEDGF/p75 was also stably overexpressed in PC3 cells by transduction with a lentiliviral vector. Both transient siRNA-mediated knockdown and overexpression of LEDGF/p75 were associated with significant changes in the expression of several stress/antioxidant genes, using a cutoff fold change value of 1.5 (P<0.001). We selected for further analysis common genes that were significantly downregulated when LEDGF/p75 was knocked down and upregulated when this protein was overexpressed. These genes are phosphoinositide-binding protein E, cytoglobulin, superoxide dismutase 3, thyroid peroxidase, titin, glutathione transferase zeta1 and albumin. The expression of these genes in response to LEDGF/p75 expression was further validated using real-time RT-PCR. This study identifies candidate stress genes potentially regulated by LEDGF/p75 in PC3 cells, and provides a rationale for investigating the mechanisms by which this emerging stress oncoprotein promotes resistance to lysosomal cell death and chemotherapy in prostate tumor cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2092. doi:10.1158/1538-7445.AM2011-2092
Abstract 2104: Frequent inactivation of A20 and its prognostic significance in Middle Eastern colorectal carcinoma
Authors: Abubaker Jehad , Bavi Prashant , Uddin Shahab , Jehan Zeenath , Ahmed Maqbool , Al-Sanea Nasser , Abduljabbar Alaa , Ashari Luai H. , Alhomoud Samar , Al-Dayel Fouad , Sultana Mehar , Al-Haqawi Wael , Al-Rasheed Maha , Hassan Nurul , Qadri Zeeshan , George Thara , Prabhakaran Sarita E. , Balde Valorie , Siraj Abdul K. , Al-Kuraya Khawla S.
Published: July 2012

A20, also known as TNF alpha induced protein3 (TNFAIP3), is a tumor suppressor gene and a well-known negative regulator of the NF-kB pathway in hematolymphoid neoplasms. In colorectal carcinoma (CRC), aberrant NF-kB regulation has been associated with poor prognosis and resistance to therapy. However, role of A20 gene in CRC is unknown. Therefore, we sought to elucidate the biological role of A20, mechanism of its inactivation and its prognostic role in colorectal carcinoma. We investigated the genetic and epigenetic abnormalities of A20 gene in CRC. In a tissue microarray cohort of 434 CRC, we studied loss of A20 protein expression by immunohistochemistry and A20 deletions by FISH. We also screened 116 random CRC and 14 CRC cell lines for mutations in A20 gene and A20 promoter methylation. Of the 116 CRC samples, A20 deletions were seen in 15.3% and incidence of A20 mutation was 2.5%; most of the mutations (4/5) were missense mutations. A20 promoter hyper-methylation was seen in 50.8% and was correlated with loss of protein expression (p=0.0079). A20 inactivation, defined as loss or reduced expression of protein was observed in 63% of CRC and A20 inactivation was an independent prognostic marker for poor survival in all CRC. In addition A20 expression retained its prognostic value in the following subgroups: Stage III; Stage II and III; and Stage III and IV. A20 was inactivated in majority of the CRC due to promoter methylation and can be targeted to modulate NF-κb expression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2104. doi:10.1158/1538-7445.AM2011-2104
Abstract 2108: New analogues of benzyl-isothiourea showed Rb-Raf disruption and anti-cancer activity
Authors: Verma Monika , Johnson Jackie , Kovacs Michelle , Singh Sandeep , Wu Xin , Lawrence Nicholas , Sebti Said , Chellappan Srikumar
Published: July 2012

The retinoblastoma tumor suppressor protein Rb, is a vital regulator of the mammalian cell cycle which is inactivated by phosphorylation. We had shown that Raf-1 kinase binds and phosphorylates Rb early in the G1 phase facilitating its subsequent phosphorylation by cyclin dependent kinases. This phosphorlation released transcriptionally active E2Fs from Rb to facilitate S-phase entry. Disruption of Rb-Raf protein-protein interaction could prevent proliferation and angiogenesis, leading to inhibition of tumor growth in mouse models. We had earlier identified an orally active small molecule, Rb/Raf-1 disruptor 251 (RRD-251) that potently and selectively disrupts Rb/Raf-1 but not Rb/E2F, Rb/prohibitin, Rb/cyclin E or Rb/HDAC interactions. Here, we have made efforts to analyze additional analogues of RRD251; we focused on XW-19B and XW-35B. These compounds inhibited Rb-Raf interaction in vitro and in vivo as shown by ELISA and Immunoprecipitation-Western blot analysis. They also inhibited cell viability, proliferation, migration, invasion, angiogenesis and adherence-independent growth of human lung cancer cell lines A549. XW-19B inhibited tumor growth significantly in human A549 xenograft model in nude mice, compared to control while XW-35B showed certain amount of toxicity to animals at 50MPK. Overall, these results showed that Rb-Raf disruptors XW-19B and XW-35B have potential anticancer activity. Thus, selective targeting of Rb/Raf-1 interaction seems to be a promising approach for developing novel chemotherapeutic agents.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2108. doi:10.1158/1538-7445.AM2011-2108
Abstract 2116: The Cables1 protein complex couples survival signaling to cell death machinery
Authors: Shi Zhi , Park Hae Ryoun , Du Yuhong , Fu Haian , Khuri Fadlo R.
Published: July 2012

Cables1 is a candidate tumor suppressor and functions as a negative regulator of cell growth through inhibition of Cdks. Indeed, loss of Cables1 expression has been observed with high frequency in many cancers. However, little is known about how Cables1 itself is regulated. Here we report that Cables1 is regulated by a phosphorylation-dependent, 14-3-3-mediated mechanism. Further studies through mutational analysis have led to the identification of two residues of Cables1 that are critical for 14-3-3 binding. These residues of Cables1 serve as a substrate of serine/threonine kinase Akt. Akt could directly bind to and phosphorylate Cables1, recruiting 14-3-3 to the complex. Moreover, Cables1 overepxression induces cell apoptosis and inhibits cell growth in part through stabilizing p21 and decreasing Cdk2 kinase activity. Activated Akt is able to prevent cell apoptosis induced by Cables1. Importantly, the level of phosphorylated Cables1 has been correlated with that of pAkt in human lung cancer patient tissues, supporting a potentially significant event in lung cancer tumorigenesis. Together, our results suggest a dynamic regulatory system by which activated Akt phosphorylates Cables1 and recruits 14-3-3 to neutralize a potent tumor suppressor function.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2116. doi:10.1158/1538-7445.AM2011-2116
Abstract 2142: Signaling pathways involved in apoptosis-resistance against doxorubicin analogues in murine mammary cells; Role of protein kinase C delta (PKCΔ)
Authors: Bessone María Inés Díaz , Berardi Damian E. , Campodónico Paola B. , Lothstein Leonard , Todaro Laura B. , Bal de Kier Joffé Elisa D. , Urtreger Alejandro J.
Published: July 2012

In this work we have analyzed whether the overexpression of PKCΔ in murine mammary cells (NMuMG) is able to alter cell death susceptibility to two chemotherapeutic drugs derived from doxorubicin (AD198 and AD288), and evaluate the signaling pathways involved in this process. Similarly to doxorubicin, the mechanism of action of AD288 involves the generation of DNA damage whereas, it has been proposed that AD198 induces apoptosis by activating PKC isoforms, in particular PKCΔ.

By western blot we could determine that NMuMG-PKCΔ cells showed an increase in the activation and/or expression of different molecules and signaling pathways involved in cell survival such as Bcl-2, pBad, PI3K/Akt y NFκB. Therefore PKCΔ overexpression induced an increase in cell death resistance against both ADs (survival: AD198: 80±10% vs. 41±8% and AD288: 90±15% vs. 60±10% in NMuMG-PKCΔ and NMuMG-vector cells respectively).

In order to evaluate the role of PI3K/Akt pathway in cell death resistance induced by both ADs, cells were treated with LY294002, a PI3K inhibitor. The inhibition of this signaling pathway in NMuMG-PKCΔ cells increased 3-fold AD198 cytotoxic effect, whereas did not alter AD288 effect.

The role of NFκB signaling pathway in the resistance against cell death induced by each AD was determined by transient transfection with a variant of its repressor IκB. The inhibition of this signaling pathway significantly increased cell death susceptibility to both drugs (P<0.05 student's t test).

In this work we have demonstrated that the anti-apoptotic signals generated by PKCΔ in NMuMG mammary cells could be reverted by inhibiting PI3K/Akt and/or NFκB signaling pathways. In addition, it was shown that the pathway involved was dependent on the mechanism of action of the cytotoxic drug used.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2142. doi:10.1158/1538-7445.AM2011-2142
Abstract 3907: Gene expression profile of aging-related genes in human gastric cancer development
Authors: Kim Han Sang , Kim Sang Cheol , Park Chan Hee , Shin Sang Joon , Ahn Joong Bae , Jeung Hei-Cheul , Chung Hyun Cheol , Roh Jae Kyung , Rha Sun Young
Published: July 2012

Background: Aging process modifies gene expression pattern and might be related to certain disease like cancer. As clinical outcome worsens with age, different molecular behavior of cancer between aging should be elucidated to accomplish future personalized medicine, especially in geriatric cancer patients.

Methods: Primary gastric cancer tissues and corresponding normal mucosa were obtained from 81 patients. cDNA microarray analysis was performed using 14,125 probes. To select differentially expressed genes between aging, multiple regression model considering age, sex, stage, and pathology was applied. Functional study and pathway analysis was performed through database for annotation, visualization and integrated discovery (DAVID) and Ingenuity pathway analysis.

Results: In normal tissues, two hundred and ninety-six genes were determined as aging-related genes that were involved in aging-process relation with mitochondria and ubiquitination. In tumor tissues, sixty-two genes were identified that showed significantly different expression according to age. Twenty genes were up-regulated and forty-two genes were down-regulated. Apoptosis (6 genes), carbohydrate metabolism (4 genes), cell cycle (8 genes), cellular proliferation (7 genes), DNA repair (3 genes), and inflammation (5 genes) were related. In pathway analysis with tumor tissues, gene expression was down-regulated with age in proliferation pathways including growth hormone, PI3K/Akt, TGF-β, and TNFR signaling. Furthermore, genes involved in tumor suppressor pathways involving apoptosis and PTEN were down-regulated in older tumors.

Conclusions: As gastric cancer develops later, carcinogenesis is less dependent from proliferation, but also tumor suppressor pathways. Our results could be one explanation of less chemotherapy response and enhancing defense mechanism is needed in old-age gastric cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3907. doi:10.1158/1538-7445.AM2011-3907
Abstract 4077: Exposure to bile acids alters intracellular expression of MnSOD in Barrett's esophagus
Authors: Farmer Russell W. , Li Yan , Hill Charles R. St. , Schiffman Suzanne , Harper Nicholas L. , Kang Xinqin , Martin Robert C.
Published: July 2012

Introduction: Oxidative stress secondary to bile acid exposure has been associated with metaplastic degeneration of normal esophageal mucosa into Barrett's Esophagus cells and eventually Esophageal Adenocarcinoma. We have previously reported that the macromolecular response of BE cells to this stress is largely regulated by the expression of Manganese Superoxide Dismutase (MnSOD). As the mitochondrion plays a vital role in MnSOD activation, the purpose of this study was to determine the location and activity of MnSOD within the BE cell after exposure to oxidative stress.

Methods: BE cells were cultured to 80-90% confluence in 75ml flasks. The cells were then exposed for four hours to 0.4mM concentrations of taurocholic acid (TCA) or a 0.4mM 1:1 mixture of bile salts including sodium cholate (CA), sodium deoxycholate (DCA), sodium glycocholate (GCA), and sodium taurocholate (TCA) to mimic natural biliary refluxate. Proteins were extracted and separated into mitochondrial, nuclear, and cytoplasmic fragments followed by western blot analysis. Enzymatic Activity of MnSOD was determined by silencing of other SOD molecules with potassium cyanate. Lethality testing of biliary exposure was performed with MTT assays.

Results: Mitochondrial expression of MnSOD was significantly increased in the cells exposed to a mixture of bile acids vs. TCA and control. This was present on both qualitative and quantitative analyses of Western blots. Cytoplasmic expression of MnSOD was also present and conserved between treated and untreated cells. Control cells lines showed no significant change in baseline MnSOD expression. This expression was confirmed by increased MnSOD activity in the mitochondria of cells exposed to mixed bile. The lethality of mixed bile for Barrett's cells was confirmed by MTT assay, with isolated bile salts and controls having a significantly higher fraction of living cells.

Conclusion: This is the first study to evaluate microcellular MnSOD expression which is increased in BE cells in response to the oxidative stress of bile exposure, specifically in the mitochondrion. Mitochondrial MnSOD activity is increased within the BE cell after exposure to bile. Further investigation is required to determine the potential correlation between bile exposure and BE to adenocarcinoma progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4077. doi:10.1158/1538-7445.AM2011-4077
Abstract 4078: ANT-ligands small molecules induce apoptosis of cancer cells by inhibiting ATP/ADP translocation
Authors: Borgne-Sanchez Annie , Buron Nelly , Porceddu Mathieu , Jacotot Etienne , Brenner Catherine
Published: July 2012

Since fifteen years ago, the mitochondrion has been progressively recognized as an integrator-coordinator of apoptosis. In this context recent anti-cancer drugs in development are targeting mitochondrial apoptotic machinery to induce tumor cell death. By this strategy, drugs can interact with apoptosis-regulating proteins of Bcl-2 family to trigger Bax/Bak oligomerization and mitochondrial membrane permeabilization. This event conducts to the release of pro-apoptotic factors such as cytochrome c into the cytosol that are required for caspases activation and apoptosis process. In this study, we selected another strategy and investigated the effect of ANT-targeting small molecules to induce cancer cells apoptosis. The adenine nucleotide translocator (ANT) is a mitochondrial protein of the inner membrane involved in the exchange of ADP and ATP between the mitochondrial matrix and the cytosol. Medicinal chemistry approach coupled with in silico studies yield to several small organic compounds, which proved to be specific for ANT and fulfill druggability criteria (good cell penetration and biodisponibility). We identified a family of compounds that inhibited ATP translocation and induced apoptosis by a pathway involving Bax-dependent cytochrome c release and caspases activation. One of the hit compound was optimized to obtain molecules able to induce apoptosis of HT-29 and BxPC3 cells at 100 nM. We are currently investigating which ANT isoform(s) is (are) targeted in cells. In vivo tumor regression experiments will be performed to achieve the proof of concept of ANT-ligands small molecules as an interesting anti-cancer strategy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4078. doi:10.1158/1538-7445.AM2011-4078
Abstract 4081: Sensitivity of gastric cancer cell lines against 2-deoxy-D-glucose is independent of PI3K/mTOR inhibition
Authors: Eder Sandra , Strommer Sabine , Füreder Thorsten , Freitag Rene , Dorn Sebastian , Wacheck Volker
Published: July 2012

2-deoxy-D-glucose (2-DG) is currently studied in Phase I clinical trials as a novel therapeutic strategy for anti-cancer therapy. Several preclinical studies have suggested that the anti-proliferative activity of 2-DG might be linked, amongst others, to the inhibition of the PI3K/mTOR signalling pathway. Blocking the PI3K/mTOR pathway has become a major focus for molecular targeted drug development. In the current study we assessed the biological relevance of PI3K/mTOR signalling for the anti-proliferative activity of 2-DG in gastric cancer cells.

The gastric cancer cell lines (N87, MKN-28, MKN-45) were treated with 2-DG and evaluated for cell proliferation, apoptosis induction, cell cycle arrest, and PI3K/mTOR signalling. Everolimus, a specific mTORC1 inhibitor, was employed as positive control.

Treatment with 2-DG potently suppressed cell proliferation resulting in an up to 300% reduced IC50 in the gastric cancer cell lines. Cell cycle inhibition and apoptosis induction varied substantially among cell lines. However, the distinct 2-DG sensitivity of gastric cancer cell lines was not reflected by any alteration in the PI3K/mTOR signalling pathway. In all cancer cell lines, no major inhibition of pERK and pAKT was observed, whereas both, 2-DG as well as everolimus strongly inhibited phosphorylation of S6. In contrast to 2-DG everolimus induced also a clear inhibition of 4EBP1 phosphorylation.

In conclusion, inhibition of PI3K/mTOR signalling pathway by 2-DG differs from inhibition by everolimus and is not linked with 2-DG anti-proliferative sensitivity in gastric cancer cell lines.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4081. doi:10.1158/1538-7445.AM2011-4081
Abstract 4085: Inhibition of rodent mammary carcinogenesis by wheel running and/or dietary energy restriction
Authors: Jiang Weiqin , Zhu Zongjian , McGinley John N. , Zacher Jarrod , Neil Elizabeth , Thompson Henry J.
Published: July 2012

The prevention of breast cancer by the combined effects of physical activity (PA) and dietary energy restriction (DER) has received limited investigation. The objective of this experiment was to determine the inhibitory effects of PA and/or DER on mammary carcinogenesis and associated mechanisms. Female Sprague Dawley rats were injected with 1-methyl-1-nitrosourea (50 mg/kg) and 7 days thereafter randomized to: 1) sedentary control, 2) PA at 3000 revolutions (3.36 km) per day; or the energy balance equivalents of PA: 3) 20% DER or 4) PA at 1500 revolutions per day + 10% DER. In order to investigate the effects of physical activity at a constant intensity, rats were given free access to a motorized activity wheel (55 m/min) with reinforcement of running behavior using a food reward system. The duration of this experiment was 8 weeks. As intended, final body weights of the sedentary control were higher than PA, DER or PA&DER, but the intervention groups had similar final weights. Overall, PA and/or DER reduced mammary cancer incidence, cancer multiplicity, and tumor burden and prolonged cancer latency (p<0.05) in comparison to the sedentary control group. However, the differences in these cancer endpoints among the intervention groups were not statistically different. Experiments are in progress to determine the extent to which host systemic factors and cell autonomous mechanisms are differentially affect by PA, DER, or their combination in this context of energy balance equivalence. Supported by PHS grant CA100693 from the National Cancer Institute.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4085. doi:10.1158/1538-7445.AM2011-4085
Abstract 4087: Metabolic changes associated with acquired cisplatin resistance
Authors: Sullivan Elizabeth J. , Kurtoglu Metin , Wangpaichitr Medhi , Savaraj Niramol , Lampidis Theodore
Published: July 2012

Although cisplatin is the drug of choice in treating lung cancer patients, acquired resistance appears to be a common and serious drawback to its effectiveness in the clinic. Using two pairs of cisplatin sensitive and resistant lung cancer cell lines developed in vitro, we have found significantly lowered hexokinase II (HKII) levels in both resistant cell lines. As a consequence of lowered HKII, these cell lines, when grown under anaerobic conditions, are markedly more sensitive to the glycolytic inhibitor 2-deoxy-D-glucose (2DG) than their parental cell lines from which they were derived. This was our first indication that lowered HKII levels in these cisplatin resistant cells has metabolic consequences in different environmental conditions. Thus, it appears that a fundamental difference in glucose metabolism is associated with resistance to cisplatin in these cell lines. It is the aim of this study to explore whether reduced HKII is a common occurrence in cisplatin resistant cells and whether these differences uncover metabolic targets that can be exploited for therapeutic gain.

Much like the Atkins diet, when carbohydrates are lowered or glycolysis is inhibited, other sources of energy such as fatty acids and amino acids can be used to maintain cell viability. A trypan blue cytotoxicity assay was used to determine the sensitivity of our cisplatin resistant cell lines to a pharmacological inhibitor, etomoxir, toward fatty acid oxidation. Thus, we tested and found that one of these cisplatin resistant cell lines is sensitive to interference with fatty acid oxidation. Next, we placed our cell lines in glutamine free medium, and found that the second cisplatin resistant cell line is sensitive to glutamine deprivation. Moreover, when lactate production was measured in anaerobic conditions, cisplatin resistant cell lines produced less lactate when challenged with the glycolytic inhibitor, 2DG, than their cisplatin sensitive parental cell lines.

Overall, our results indicate that lowered HKII levels in two cisplatin resistant cell lines appear to be associated with a decreased reliance on glucose metabolism, with a shift to either fatty acid oxidation or glutaminolysis. Moreover, these cisplatin resistant cells are sensitive to glycolytic inhibition when placed in oxygen-deprived conditions. Thus, these new metabolic targets may be exploited for therapeutic gain in the clinic.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4087. doi:10.1158/1538-7445.AM2011-4087
Abstract 4090: The role of caspase-2 in apoptotic signaling in AML cells
Authors: Haag Petra , Lindberg Marita Lagergren , Kanter Lena , Stenke Leif , Lewensohn Rolf , Viktorsson Kristina
Published: July 2012

We are investigating signaling molecules involved in apoptotic cell death in acute myeloid leukemia (AML) cells after treatment with the targeted drug gemtuzumab ozogamicin (GO) and the conventional drug daunorubicin, with focus on caspase-2.

New effective therapeutic drugs are highly desirable in the treatment of AML. Although a majority of treated patients initially respond to conventional treatment, most patients will relapse within 1-2 years. Also, the treatment is associated with severe off-target effects. GO is a novel therapeutic approach, consisting of a monoclonal CD33 antibody coupled to calicheamicin, which cause DNA double strand breaks (DSB). We demonstrated that activation of Bak and Bax is critical for GO- and daunorubicin- induced apoptotic signaling in AML cells. The activation was followed by mitochondrial depolarization, caspase-3 activation and nuclear fragmentation.

We continued to examine potential proapoptotic signaling events upstream of mitochondria in HL60 AML cells in vitro. We identified caspase-2 and the pro-apoptotic BH3-only protein Bid as two candidates. By analyzing caspase-2 by western blotting after GO treatment we found that GO caused cleavage of caspase-2 after 24h, which was prior to mitochondria-mediated signaling. Also, full-length Bid was cleaved to its truncated, active form tBid. The importance of caspase-2 activation in the signaling cascade after GO treatment was demonstrated using z-VDVAD-fmk, a caspase-2 inhibitor. Preincubation with this inhibitor reduced caspase-3 activation with around 50%, suggesting that caspase-2 is an important molecule, located upstream of caspase-3, in the signaling between DNA double stranded breaks and apoptosis signaling in AML cells.

In conclusion, we demonstrate that GO and daunorubicin, at clinically applicable concentrations, induce pro-apoptotic signaling which involve activation of caspase-2, and activation of the BH3-only protein Bid. Blocking caspase-2 activity reduced caspase-3 activation with 50%. This suggests multiple signaling pathways capable of activating caspase-3. The caspase-2 pathway appears central for downstream caspase-3 activity, after GO treatment in AML. Our findings may highlight possible resistance mechanisms in AML, which might have profound therapeutic implications.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4090. doi:10.1158/1538-7445.AM2011-4090
Abstract 4107: p38-HSP27 as modulators from survival to apoptosis: Combined treatment with TRAIL and curcumin to prostate cancer cells
Authors: Yoon Hyo R. , Sun Bo K. , Nguyen Hoan N. , Kim So Y. , Oh Seeun , Choi Hye J. , Kim Joo-Hang , Song Jae J.
Published: July 2012

The effect of combination of TRAIL and curcumin enhanced apoptotic cell death in many other studies. However, the exact molecular mechanism of enhanced apoptotic cell death by TRAIL plus curcumin was not yet completely understood. In this study, we clearly observed the depression of phosphorylated Akt and increase of phosphorylated p38 during combined treatment with TRAIL and curcumin. Previously, we observed that TRAIL induced the catalytic activation of Akt in TRAIL-sensitive cells and here, the catalytic activation of Akt was reversely decreased by curcumin pretreatment followed by TRAIL. We also found that significant increase of p38 phosphorylation was deeply linked to the decrease of Akt catalytic activity by regulation of the level of phosphorylated HSP27, which leads to the change of interaction between TRAF6 and Src, and subsequent Src-Akt inactivation. For the examination of relationship of p38-HSP27 with Akt activity, in vitro Akt kinase assay, and siRNA technique were used to confirm the biological effect of p38-HSP27 as modulators from survival to apoptosis by combined treatment with TRAIL and curcumin to prostate cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4107. doi:10.1158/1538-7445.AM2011-4107
Abstract 4108: BH3 only Bcl-2 family member BNIP3 repressed expression of death receptor 5 (DR5) in glioblastoma cells: Implications for regulation of the tumor necrosis factor related apoptosis inducing ligand (TRAIL) cell death pathway
Authors: Henson Elizabeth S. , Burton Teralee R. , Azad Meghan B. , Eisenstat David D. , Gibson Spencer B.
Published: July 2012

Glioblastoma multiforme (GBM) is the most aggressive form of brain cancer. The median survival for patients with GBM is less than 15 months, even with treatment such as surgery, radiation and chemotherapy with temozolomide. Resistance to the standard treatment regimes represents a serious clinical challenge. GBM tumors contain extensive regions of necrosis, indicative of hypoxia. BNIP-3 (Bcl-2 nineteen kilodalton interacting protein) is a BH-3 only, pro-cell death member of the Bcl-2 family. BNIP-3 is up-regulated in hypoxia and leads to a non-classical programmed cell death through the mitochondria. In the majority of glioblastoma multiforme tumors, BNIP3 is localized to the nucleus and does not induce cell death. Our group has shown that when located in the nucleus, BNIP3 binds to DNA including the promoters of cell death genes such as AIF (apoptosis inducing factor). Herein, we found that BNIP3 bound to the promoter region of death receptor 5 (DR5). DR5 binds to the tumor necrosis factor related apoptosis inducing ligand (TRAIL) leading to induction of apoptosis. TRAIL is currently in phase 2 clinical trials for a wide variety of tumor types but glioblastoma tumors are resistant to TRAIL induced apoptosis. Our data showed that binding of BNIP3 to the promoter of DR5 prevented its expression and led to resistance to TRAIL induced cell death in glioma cells. Furthermore, when a BNIP3 construct was transiently over-expressed in the nucleus, DR5 mRNA and protein levels were reduced; also when BNip3 was knocked down using shRNA, DR5 message and protein levels increased. In BNIP3 knockout mice, we demonstrated that increased DR5 levels were increased in primary astrocytes compared to wild type controls as detected by western blot and immunofluorescence. We also showed similar results in brain sections from BNIP3 knockout mice. In paraffin embedded GBM tumors, we found that tumors that had BNIP3 in the nucleus had decreased levels of DR5, whereas tumors without BNIP3 in the nucleus had higher levels of DR5. Taken together, this represents a novel mechanism for regulation of DR5 expression, contributing to TRAIL resistance in GBM tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4108. doi:10.1158/1538-7445.AM2011-4108
Abstract 4200: EXO1 is required for normal SN1 methylator-induced DNA damage response
Authors: Izumchenko Eugene
Published: July 2012

The mismatch repair (MMR) system plays an important role in mutation avoidance and its inactivation, which is commonly seen in both hereditary and sporadic cancers, results in genomic instability and increased cancer risk.

SN1 DNA methylating agents are potent chemotherapeutics that can methylate the O6 position of guanine. This O6meG adduct subsequently mispairs with thymine during DNA replication, MMR recognizes these O6meG/T mispairs, and consequently activates DNA damage response (DDR). While the exact mechanism by which MMR activates DDR signaling remains unresolved, the DNA repair activity of MMR has been implicated as important in this process.

Exonuclease I (EXO1) plays a crucial role in eukaryotic MMR by removing the strand containing the mismatched base. Several studies describe direct association between EXO1 and several MMR proteins (ie, MSH2, MLH1) and that EXO1 is activated during MMR processing of O6meG lesions. However, whether EXO1 is required to activate MMR-dependent response to O6meG remains unknown.

Here we show that Lentiviral shRNA-mediated EXO1 knockdown in mouse embryonic fibroblasts (MEFs) results in decreased G2/M arrest in response to treatment with the SN1 methylator MNNG. Accordingly, we observed reduced phosphorylation of the checkpoint proteins Cdc2 and Chk1, consistent with reduced MMR-dependent signaling in response to MNNG. We also found that EXO1 knockdown resulted in a limited effect on cell proliferation and loss of viability while MEFs transduced with control virus displayed clear growth arrest and a heightened cytotoxic response following MNNG exposure. Taken together, these data support our interpretation that reduced EXO1 activity results in an alkylation-tolerant phenotype similar to documented defects stemming from loss of MMR. Moreover, these findings seemingly strengthen the hypothesis that MMR-associated DNA repair activity is required for response to O6meG lesions. However, several lines of evidence suggest that EXO1 may function in both a catalytic and a structural manner during MMR-mediated mutation avoidance. Therefore, we are currently testing if EXO1 is required for MMR complex assembly at sites of mismatches within duplex DNA, and if exonuclease activity is required for normal response to SN1 methylators.

Our long-term goal is to better understand the mechanisms that guide response to SN1 alkylators since deducing MMR-mediated DNA damage response will yield essential insights for designing drugs targeting these pathways, and predicting drug response in cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4200. doi:10.1158/1538-7445.AM2011-4200
Abstract 4273: Circulating human prostate cancer cells from an orthotopic mouse model rapidly captured by immunomagnetic beads and imaged by GFP expression
Authors: Kolostova Katarina , Pinterova Daniela , Hoffman Robert M. , Bobek Vladimir
Published: July 2012

Circulating tumor cells (CTC) are potential precursors of metastasis. They are also of use in diagnosing malignancy and for prognostic purposes. Our laboratory has previously isolated CTC from orthotopic nude mouse models of human prostate cancer cells where the PC-3 cancer cells express green fluorescent protein (GFP). It was found that only orthotopic tumors produced CTC and not subcutaneous tumors, which may explain why orthotopic tumors metastasize and subcutaneous tumors do not. However, in this previous study, CTC were observed only after culture. In the present study, using the GFP-expressing PC-3 orthotopic model and immunomagnetic beads coated with anti-EpCAM and anti-PSMA, GFP-expressing CTC were isolated within 15 minutes and were readily visualized by GFP fluorescence. The immunomagnetic-bead-captured GFP-expressing PC-3 CTC could be immediately placed in 3-dimensional sponge cell culture where they proliferated. The combination of GFP-expression and immunomagnetic beads is very powerful method to obtain CTC for either immediate analysis or for biological characterization in vivo or 3-dimensional culture.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4273. doi:10.1158/1538-7445.AM2011-4273
Abstract 429: The value of Tenascin-C expression in neoadjuvant breast cancer therapy
Authors: Bastelberger Stephan , Swierczynski Stefan , Pradel Leon , Beil Jane , Hinterseer Elisabeth , Rendl Gundula , Hauser-Kronberger Cornelia , Dietze Otto
Published: July 2012

Introduction: The extra cellular matrix molecule Tenascin-C (TNC) in its many isoforms is highly expressed during tissue repair and pathological conditions such as cancer and stress. Utilising the selective pressure of neoadjuvant chemotherapy on carcinoma cells we screened for a subgroup of breast cancer patients with elevated TNC expression and a bad therapy response. TNC expression was correlated with biomarkers, histopathological (stage) and clinical data. Breast cancer cell lines were tested on TNC levels and their expression behaviour under competitive conditions.

Methods: Paraffin sections from 68 preoperatively treated breast cancer patients were used for automated immunohistochemistry (IHC) with antibodies targeting TNC, AE1/AE3, Ki67, CD3 and p53. In addition F-18 FDG PET-CT was performed to evaluate therapy response. Protein- and gene expression experiments performed onto six breast cancer cell lines (MCF-7, MDA-MB231, MDA-MB453, SK-BR3, SUM-159, T47-D) showed TNC-expression in MDA-MB453 breast cancer cell line. Gene expression of TNC in hedgehog positive cells (MCF-7) was evaluated after co-cultivation with TNC positive cells (MDA-MB453) by qRT-PCR. The qRT-PCR results confirmed the protein expression results on in vitro stained cell-slides after cell-cell interactions and different growth times. Growth and migration behaviour was studied in 3D-matrigels.

Results: We demonstrate that TNC expression in breast cancer is immensely reduced after neoadjuvant chemotherapy (43% of patients lost TNC expression) and that expression profiles after treatment correlate with tumor grade, lymph node status and estrogen receptor status, indicating prognostic value, probably for endocrine treatment. Post-therapeutical correlation and co-localisation of the proliferation marker Ki67 in cancer cells and TNC in the surrounding tumor-stroma of patients having a less beneficial response to chemotherapy. The evaluation of expression levels revealed a linear correlation of TNC and Ki67. Submitting cell lines to selective environmental pressure on cell chamber slides, we observed differentiated staining patterns and cellular localization for TNC. After co-cultivation with MDA-MB453, the hedgehog positive cell line MCF-7 expressed highly elevated levels of TNC.

Conclusions: These findings concur with the role of TNC in tissue reorganisation, wound healing and it's growth promoting properties in cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 429. doi:10.1158/1538-7445.AM2011-429
Abstract 4307: Establishment of a patient-derived diffuse infiltrative pontine glioma primary cell line and animal model: Implications for future investigations and treatment
Authors: Hashizume Rintaro , Aoki Yasuyuki , Liu Sharon , Smirnov Ivan , Phillips Joanna J. , McKnight Tracy , James Charles D. , Gupta Nalin
Published: July 2012

Diffuse infiltrating brainstem glioma arises almost exclusively in children and despite advances in imaging, neurosurgery and radiotherapy, the majority of patients die within 2 years after initial diagnosis. Because of the infiltrative nature and anatomic location in an eloquent area of the brain, most brainstem gliomas are treated without a surgical biopsy. The corresponding lack of tissue samples has resulted in a limited understanding of the underlying genetic and biochemical abnormalities associated with these tumors, and is a substantial obstacle for the development of animal model designed specifically for the preclinical testing of specific molecular target of brainstem gliomas. We established a brainstem glioma cells originated from human biopsy sample of a low grade brainstem glioma. The brainstem glioma cells were subsequently modified with hTERT (human telomerase ribonucleoprotein reverse transcriptase) to immortalize the cells and analyzed genetic and biochemical abnomalities of the cells. The established human brainstem glioma cells were tumorgenic in athymic rodents and the developed brainstem tumor was recapitulated the infiltrative growth of diffuse infiltrating brainstem glioma in patients, and can be monitored for tumor growth noninvasively by bioluminescence imaging (BLI).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4307. doi:10.1158/1538-7445.AM2011-4307
Abstract 4314: A mouse model of cerebral cavernous malformations
Authors: Yang Guili , Wei Huijie , Han Zhenying , Zhang Zhisong , Duan Biyan , Zhang Jianning , Li Lu-yuan
Published: July 2012

Cerebral cavernous malformations (CCM) are prevalent cerebral vascular lesions involving aberrant angiogenesis that can lead to hemorrhage, epilepsy, and other neurological manifestations. CCM consists of brittle, proliferating caverns filled with blood and lined with endothelial cells. However, the underlying mechanism of CCM development is poorly understood. The cytokine vascular endothelial cell growth inhibitor (VEGI; TNFSF15; TL1A) is an endogenous negative regulator of angiogenesis produced largely by endothelial cells and exerts a specific inhibitory activity on the proliferation of endothelial cells. We found that the production of VEGI by the endothelium is nearly completely diminished in the CCM tissues. In sharp contrast, the expression of VEGI is readily detectable on the vasculature in nearby brain tissues. On the other hand, angiogenic factors such as vascular endothelial cell growth factor (VEGF) is known to be highly expressed in CCM lesions. We show here that an imbalance of angiogenesis promoters and inhibitors, namely, the down-regulation of VEGI and up-regulation of VEGF, are attributable to the development of CCM. We have constructed a mouse CCM model by implanting mouse endothelial cell line bEnd3 cells in the brain of a female BALB/C mouse. The cavernous blood vessels formed in the model closely resemble those observed in CCM and thus may be used to study the pathology and treatment options of CCM.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4314. doi:10.1158/1538-7445.AM2011-4314