bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5413: Is a non-conjugated triple-helix forming oligonucleotide targeting the genomic HPRT locus capable of sequence specific mutagenesis
Authors: Reshat Reshat , Priestley Catherine , Fellows Mick , O'Donovan Mike , Gooderham Nigel J.
Published: June 2012

Oligonucleotide strands have been shown to be capable of binding to duplex DNA in a sequence specific manner to form a triple-helix or triplex structure. Such phenomenons have been shown to induce mutation, as demonstrated using plasmid-based reporter constructs (Wang et al 1996, Science, 271, p802). These studies have engendered concern from the European Medicines Agency (EMEA) in regard to biotechnology derived pharmaceuticals such that antisense oligonucleotides may also form triplex structures in a sequence specific manner at genomic DNA with potentially mutagenic effects. In this study, we have examined the mutagenic potential of a non-conjugated triplex forming oligonucleotide (TFO27) targeting the genomic hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus. The mutagenicity of TFO27 was assessed using the human lymphoblastoid TK6 cell line through 6-thioguanine resistance (6TGr).

Electrophoretic mobility shift assays were used to demonstrate triplex formation by TFO27 at the target motif at sub-nanomolar concentrations. As expected, a control oligonucleotide, SCR27, failed to form a triplex at the target motif. Subsequently, a range of transfection facilitators were evaluated for optimal delivery of a fluorescently labelled oligonucleotide probe.

In preliminary experiments treating 3×106 cells, TFO27 appeared to produce a dose dependent increase in 6TGr mutants. Furthermore, TFO27 failed to induce mutation at the non-targeted thymidine kinase (TK) locus suggesting locus specificity for its mode of action. Moreover, SCR27 failed to induce mutation at both loci. However, TFO27 failed to induce mutation at the HPRT locus in experiments treating 10×106 cells. Increasing the oligonucleotide concentration had no effect on the number of 6TGr recovered.

These contradictory findings are difficult to explain, but emphasise the importance of using appropriate target cell numbers in mutation assays. Overall, our data would suggest that triplex formation could occur in cells but chromatin structure, intranuclear pH, ion concentrations and nucleases are all potential barriers that probably influence the dynamics of triplex formation and stability. In view of these factors, we suggest that studies of the mutagenic potential of triplex formation are better performed at genomic loci rather than extra-chromosomal test systems.

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 5413. doi:10.1158/1538-7445.AM2011-5413
Abstract 5414: Novel tissue-targeted adenoviral vector for squamous cell carcinoma therapy expressing cell cycle regulator cdk2ap1 and molecular imaging genes
Authors: Zolochevska Olga , Figueiredo Marxa L.
Published: June 2012

Oral squamous cell carcinoma (OSCC) affects about 30,000 Americans each year, and although the overall prognosis has improved in recent years for localized carcinomas, metastases can decrease survival rates by half. For tongue and palate lesions, surgery is usually the initial treatment, but metastases aggressively develop at sites where conventional therapies are ineffective. Therefore, our goal has been to develop more effective therapeutic modalities for the disease utilizing expression of cell cycle arrest and apoptosis-modulating genes. Our efforts have focused on gaining understanding of the biology and therapeutic potential of p12cdk2ap1 (p12), an S-phase growth suppressor and proapoptotic gene commonly downregulated in OSCC. In vivo, we have showed that expressing p12 using a nonviral vector can result in effective reduction in tumor size and a dramatically altered tumor phenotype. Moreover, we have showed that p12 can have cell growth inhibitory effects in a cell-autonomous as well as non-cell autonomous manner, rendering expression of this cell cycle regulator a very promising new gene therapy strategy. Therefore, we hypothesized that this growth regulator molecule will comprise a highly efficient antiumor gene therapy for OSCC. We have developed a novel viral delivery vector system with high activity and high specificity expression in oral tumor cells. This Adenovirus (Ad) utilizes a two-step transcriptional amplification system driven by the SCCA2 promoter and a bidirectional target cassette containing the sequences for the p12 and ether firefly luciferase (Luc) or thymidine kinase (TK) imaging genes. We constructed and are characterizing novel configurations of these dE1dE3 Ad-SCCA2-p12/Luc or p12/TK bidirectional vectors for gene therapy and molecular imaging applications. We are currently testing the specificity of these vectors and their effectiveness in tumor growth inhibition at either floor of mouth, orthotopic, or distant sites following intratumoral or systemic administration. We propose this new vector design may hold promise not only as a novel SCC-specific gene therapy but perhaps as a molecular imaging detection agent for tumor cells that have metastasized to distant and thus difficult to treat sites.

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 5414. doi:10.1158/1538-7445.AM2011-5414
Abstract 5419: The plant alkaloid ellipticine is detoxified by cytochrome P450 1A1/2 (CYP 1A1/2) but activated and forms DNA adducts if CYP1A1 or 1A2 is combined with cytochrome b5: an explanation for an in vivo/in vitro discrepancy
Authors: Frei Eva , Kotrbova Vera , Martinek Vaclav , Breuer Andrea , Stiborova Marie
Published: June 2012

Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b] carbazole) is an alkaloid with anti-cancer activity elicited by interfering with many cellular functions. One of these is the formation of DNA adducts upon activation by CYP or peroxidases. The metabolites responsible for detoxication or DNA adduct formation have been characterised and CYP3A1 and the othologous human enzyme CYP3A4 are the most efficient activating enzymes in microsomes from either species (Stiborová M et al. Cancer Res 64 (2004) 8374-8380). In vivo also CYP1A effectively activate ellipticine to DNA adducts, while isolated CYP1A1/2 detoxify ellipticine mainly to its 9-hydroxy derivative. Cytochrome b5 has been shown to modulate CYP catalysis by either transferring the second electron to CYPs, or by allosteric interaction thereby leading to higher CYP enzyme activity (Schenkman, J.B., I. Jansson Pharmacol. Ther. 97(2003) 139-52). Isolated cytochrome b5 was added to isolated CYP1A1 or 1A2 reconstituted with NADPH-CYP reductase in liposomes at different ratios of the three enzymes. Apo-cytochrome b5 (containing no heme) was generated in transfected E.coli (Kotrbová V. et al. Protein Expr Purif., 66(2009) 203-209) and used as such or reconstituted with heme or Mn-protoporphyrin IX. Metabolites were analysed by HPLC and UV detection and quantified with standards. DNA adducts were determined by the nuclease P1 version of the 32P-postlabeling assay. With both CYP1A1 and 1A2 the pattern of ellipticine metabolites in incubations containing cytochrome b5 shifted from detoxication to activation. This shift depended on the ratio of CYP1A to cytochrome b5 with optimal yield of 12-hydroxy- and 13-hydroxy ellipticine at a ratio of 1:5, CYP1A to cytochrome b5. The shift was more pronounced for CYP1A1 than for 1A2 leading to 6.5 fold higher DNA adduct levels than without cytochrome b5 correlating with 6 fold higher 13-hydroxy-ellipticine amounts, the metabolite responsible for the major deoxyguanosin adduct. The cytochrome b5 holo-enzyme is necessary for this effect, since neither the apo-enzyme nor the apo-enzyme reconstituted with Mn-protoporphyrin IX had the same effect. Other heme containing proteins, like myoglobin or cytochrome c had no effect. Ellipticine is, to our knowledge, the first xenobiotic compound where cytochrome b5 shifts the metabolism by CYP from detoxication to activation and DNA adduct formation. This is important also when analysing the pharmacological effects in vivo, since ellipticine induces both CYP1A and cytochrome b5.

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 5419. doi:10.1158/1538-7445.AM2011-5419
Abstract 542: TGF-β inhibitors enhance chemotherapeutic efficacy via abrogation of autocrine and paracrine signaling in a triple negative breast cancer model
Authors: Biswas Tanuka , Sun LuZhe
Published: June 2012

Triple negative breast cancer (TNBC) accounts for approximately 15-20% of total invasive breast cancers. This subtype of cancer is often correlated with poor prognosis, visceral metastases and relapse. Molecular characterization reveals that these tumors are negative for hormone receptors and they often develop chemo resistance. Tumor microenvironment, among other factors, has emerged as a definite player in the progression of breast cancer and may also confer chemo resistance. In the current study, we demonstrate that MDA-MB-231 cells show greater resistance to paclitaxel in heterotypic culture with RMF/EG-hTERT (reduction mammary fibroblast with enhanced GFP) as compared to homotypic culture. Further, we show that RMF/EG-hTERT cells can enhance the motility of MDA-MB-231 cells in trans well migration assays, which could be attenuated by the TGF-β inhibitor sRIII (soluble receptor type III). TGF-β signaling was shown to activate pro-survival pathways like PI3K/Akt and MAPK/ERK pathways in mammary fibroblasts suggesting that they may mediate the paracrine tumor-promoting activity of TGF-β.

Autocrine TGF-β signaling has been shown to promote EMT and cancer stem cell-like features in TNBC cells and appears to be a potential therapeutic target. We show that paclitaxel in combination with sRIII was more effective in inhibiting the growth of MDA-MB-231 cells than either agent alone. The combination of a TGF-β inhibitor with paclitaxel was also more effective in inhibiting tumor sphere formation than either agent alone.

Our future research will be focused on the mechanisms of autocrine and paracrine TGF-β signaling in mediating chemo resistance and the tumor-promoting activity of mammary stromal cells. Our research will reveal the potential use of TGF-β inhibitors as novel therapeutics in combination with chemotherapy.

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 542. doi:10.1158/1538-7445.AM2011-542
Abstract 5427: Andrographolide is a novel inhibitor of interleukin-6 and suppresses prostate tumor growth
Authors: Chun Jae Yeon , Tummala Ramakumar , Nadiminty Nagalakshmi , Lou Wei , Liu Chengfei , Yang Joy , Evans Christopher P. , Gao Allen C.
Published: June 2012

INTRODUCTION AND OBJECTIVES: Elevated interleukin-6 (IL-6), a major mediator of the inflammatory response, has been implicated in androgen receptor (AR) activation, cellular growth and differentiation, plays important roles in the development and progression of prostate cancer, and is a potential target in cancer therapy.

METHODS: In an attempt to identify potential inhibitors of IL-6, DU145 human prostate cancer cells that express IL-6 autocrine loop were treated with compounds from the Natural compound library (TimTec LLC, Newark, DE) for 24 hours. The Natural compound Library consisted of 440 natural compounds with minimal purity of 95%. The media were collected and used for measurement of IL-6 protein by ELISA. Compounds that decreased the levels of IL-6 protein by 50% were selected for further characterization.

RESULTS: We found that andrographolide, a diterpenoid lactone isolated from a traditional Chinese and Indian medicinal plant Andrographis paniculata, could inhibit IL-6 expression and suppress IL-6-mediated signals. Andrographolide inhibits IL-6 expression at both mRNA and protein levels in a dose-dependent manner. Andrographolide suppresses both IL-6 autocrine- and paracrine-loop induced cell signaling including Stat3 and Erk phosphorylation. Furthermore, andrographolide inhibits cell viability and induces apoptotic cell death in both androgen-stimulated and castration-resistant human prostate cancer cells without causing significant toxicity to normal immortalized prostate epithelial cells. Moreover, treatment of andrographolide in mice bearing castration-resistant DU145 human prostate tumors that express constitutive IL-6 autocrine loop significantly suppresses tumor growth.

CONCLUSIONS: Taken together, these results demonstrate that andrographolide could be developed as a therapeutic agent to treat both androgen-stimulated and castration-resistant prostate cancer possibly by suppressing IL-6 expression and IL-6-induced signaling.

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 5427. doi:10.1158/1538-7445.AM2011-5427
Abstract 5435: The potential of ginseng for cancer chemoprevention
Authors: Wang Chong-Zhi , Calway Tyler D. , Yuan Chun-Su
Published: June 2012

The clinical management of cancer invariably involves diverse conventional modalities, including surgery, radiation, and chemotherapy. However, the complexity of human cancer requires some alternative management to improve the therapeutic efficacy of conventional treatment and/or the quality of life of cancer patients. Medicinal botanicals have recently gained more attention for cancer management. Numerous effective anticancer drugs have been developed from botanicals, and identifying new herbal sources to develop ideal chemoprevention remains an essential step in advancing the treatment of colorectal cancer. In this study, potential roles of ginseng herbs, especially American ginseng and notoginseng, in cancer chemoprevention are evaluated. The major pharmacologically active constituents of ginsengs are ginsenosides, which can be mainly classified into protopanaxadiol and protopanaxatriol groups. The recognized active anticancer compounds from American ginseng and notoginseng are ginsenosides Rg3, Rh2, and protopanaxadiol. The structure-activity relationship between their chemical structures and pharmacological activities is discussed. Sugar molecules within a ginsenoside have a high impact on cancer cells. Anticancer activities increase with the decrease of sugar number. In addition, we observed that various steaming temperatures and time treatments of the ginseng herbs can change their ginsenoside profiles and enhance their anticancer activities. This heat treatment process may increase the efficacy of ginseng in cancer chemoprevention. (This work was supported in part by the NIH/NCCAM grants AT003255, AT004418 and AT005362).

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 5435. doi:10.1158/1538-7445.AM2011-5435
Abstract 5438: The ex vivo approach of testing drug activity with clinical leukemia specimens
Authors: Lu Jiwei , Zhang Dongqing , Chen Li , Wang Licui , Chen Shi , Shu Lili , Chen Taiping
Published: June 2012

There is an urgent need to establish better preclinical evaluation platforms for anticancer drug development. To understand the therapeutic potential for leukemia patients, we establish an ex vivo platform for anti-leukemia compounds on freshly isolated leukemia blast cells. The primary leukemia cells were isolated from patients’ fresh bone marrow samples with Human Mononuclear Isolation Medium HISTOPAQUE-1077(Sigma-Aldrich) and subjected to different concentrations of testing anti-leukemia compounds. Then, the treated specimens were detected with annexin V/7-AAD and PI/FACS assays by flow cytometry to measure apoptosis and cell cycle. We have examined 54 cases (7 of ALL, 26 of AML, 21 of undetermined) of leukemia patients’ fresh bone marrow samples with 5 compounds. The leukemia blast cells displayed a dose-dependent drug reactivity which could be estimated through IC50. Combined with the patients’ clinical information, we found that patients showed distinct sensitivity to anti-leukemia compounds, which may guide further research on drug discovery. Therefore, these data provide a foundation for the preclinical investigation of anti-leukemia compounds. The ex vivo system constitutes a more sensitive and quantitative platform for the therapeutic potential of anti-leukemia compounds.

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 5438. doi:10.1158/1538-7445.AM2011-5438
Abstract 5444: Real-time RT-PCR of terminal deoxynucleotidyl transferase (TdT) is a useful tool for identification of minimal leukemia in cerebrospinal fluid
Authors: Belgaumi Asim F. , Bu Rong , Hussain Azhar R. , Al-Obaisi Khadija A S , Al-Mahr Mohammed , Uddin Shahab , Al-Kuraya Khawla S.
Published: June 2012

Accurate detection of central nervous system (CNS) involvement in children with newly diagnosed acute lymphoblastic leukemia (ALL) could have profound prognostic and therapeutic implications. The detection of cerebrospinal fluid (CSF) infiltration by leukemic cells as performed by morphological assessment is subjective and unable to detect low levels of involvement. Therefore, newer and more accurate techniques are required to more efficiently detect minimal leukemic cell involvement. For this reason, a quantitative, Real-time RT-PCR targeting the TdT molecule was developed and tested to allow an objective and sensitive methodology for detecting CNS leukemia. 35 diagnostic CSF samples were subjected to the qRT-PCR assay for TdT. Only 4/35 samples were positive by cytomorphological detection. Of these 4 samples, one sample was diagnosed with CNS disease (CNS 3) and three samples had lower numbers of leukemic blasts (CNS 2). When compared to morphology results, as the gold standard, we found 24 out of the 35 samples to be positive by qRT-PCR. 20 CSF samples that were negative by morphology had detectable TdT by RT-PCR, possibly indicating leukemic infiltration below the level of cytomorphological detection. Serial samples for the 4 CNS-positive patients were tested by qRT-PCR and showed persistent positivity with gradual reduction in quantity of TdT with chemotherapy administration. 11 samples had no detectable TdT by RT-PCR and were found to be negative by cytomorphological assessment as well. In patients with no cytomorphologically detected blasts, TdT positivity was not associated with the level of peripheral blood WBC count. This methodology provides an objective, quantitative and sensitive tool for the detection of leukemic blasts in the CSF. This allows us to detect molecular CNS involvement in CNS negative patients. Although the outcome for all CNS negative patients is good, this has been achieved with fairly intensive prophylactic CNS-directed therapy, reduction in CNS-directed therapy may be possible in molecularly negative patients.

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 5444. doi:10.1158/1538-7445.AM2011-5444
Abstract 5450: CYP2A6 and ERCC1 polymorphisms correlate with efficacy of S-1 plus cisplatin in metastatic gastric cancer patients
Authors: Park Sook Ryun , Kong Sun-Young , Nam Byung-Ho , Choi Il Ju , Kim Chan Gyoo , Lee Jong Yeul , Cho Soo-Jeong , Kim Young-Woo , Ryu Keun Won , Lee Jun Ho , Rhee Jiyoung , Park Young-Iee , Kim Noe Kyeong
Published: June 2012

Introduction: S-1 contains tegafur, which is converted to 5-fluorouracil by CYP2A6. We evaluated the association between polymorphisms of CYP2A6, ERCC1 and XRCC1 and treatment outcomes of metastatic gastric cancer (MGC) patients treated with S-1/cisplatin.

Methods: Among chemonaive MGC patients (n=108) who received S-1 40 mg/m2 b.i.d. on days 1-14 and cisplatin 60 mg/m2 on day 1 of a 3-week cycle, we analyzed the wild-type allele [W] (CYP2A6*1), four CYP2A6 variants [V] that abolish (CYP2A6*4) or reduce (CYP2A6*7, *9, *10) enzyme activity, and the polymorphisms of ERCC1 [8525C>T (rs11615), 19442C>A (rs3212986)] and XRCC1 [1196G>A (rs25487)].

Results: Patients having fewer CYP2A6 variants had significantly better response rates (W/W vs W/V other than *1/*4 vs V/V or *1/*4 = 66.7% vs 58.3% vs 32.3%; P=0.008), time to progression (TTP) (median, 7.2 vs 6.1 vs 3.5 months, P=0.021), and overall survival (median, 23.2 vs 15.4 vs 12.0 months, P=0.004). ERCC1 19442C>A was also associated with response rate (C/C, 46.7% vs C/A, 55.3% vs A/A, 87.5%) (P=0.048) and TTP (median, 4.4 vs 7.6 vs 7.9 months) (P=0.012). When we classified patients according to the number of risk genotypes of CYP2A6 (V/V or 1/*4) and ERCC1 19442C>A (C/C), patients carrying two vs those carrying none showed an adjusted odds ratio of 0.113 (95% CI, 0.025-0.509) (P=0.004) for response and adjusted hazard ratios of 3.748 (95% CI, 1.900-7.393) (P=0.0001) for TTP and 2.961 (95% CI, 1.371-6.393) (P=0.006) for death.Conclusion: CYP2A6 and ERCC1 19442C>A polymorphisms are important predictors for the efficacy of S-1/cisplatin in MGC patients.

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 5450. doi:10.1158/1538-7445.AM2011-5450
Abstract 5454: Drug effect analysis of methoxyamine through quantifying the drug on its therapeutic target
Authors: Li Lan , Xu Yan , Liu Lili , Gerson Stanton L.
Published: June 2012

Methoxyamine (MX) is the first DNA base-excision-repair (BER) inhibitor evaluated in humans. MX potentiates the cytotoxicity of alkylating and antimetabolic agents by condensing with DNA apurinic/pymidinic (AP) sites which are produced through the removal of alkylated or abnormal nucleobases from DNA backbone by DNA glycosylases. The formation of AP-MX complex blocks the BER pathway, and hence reverses tumor resistance to the chemotherapeutic agents. The further repair of MX-bound DNA by topoisomerase II results in ds-DNA breaks, and induces tumor cell death.

In this work, we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative analysis of AP-MX complex released from MX-bound DNA by a multi-enzyme system. The enzyme-released AP-MX (i.e., deoxyribose-MX, or dR-MX) was first separated from the enzyme digestion matrix on a Hypercarb® column, and then quantitated by a triple quadrupole mass analyzer using multiple-reaction-monitoring (MRM) mode with a mass transition of m/z 164 > 117. The calibration curve which corresponds to 3.55 to 227 AP-MX complexes/107 bases was established using AP-MX calibrators obtained by serial dilution of DNA oligomer containing known amount of AP-MX complex in blank calf thymus DNA. The method developed has been applied to AP-MX analysis of DNA extracts from T98G cells treated with temozolomide with MX, and blood samples from an ongoing Phase I study of methoxyamine and temozolomide in patient with advanced solid tumors. The dose, AP-MX complex, and time profiles have been obtained. This method provides a quantitative measure of methoxyamine at its proposed target sites.

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 5454. doi:10.1158/1538-7445.AM2011-5454
Abstract 5460: Dual efficacy of Lazaroid U-74389G liposomes in glioblastoma mouse model
Authors: Ibrahim Fady Makram , Cui Kemi , New Pamela , Wang Lulu , Zhao Hong , Wong Stephen , Chow Diana S-L
Published: June 2012

Purpose: Lazaroid U-74389G (LAZ) is a 21-aminosteroid that has radioprotective effect against radiation-induced lipid peroxidation. In-vitro antiproliferative effect was reported against Glioblastoma primary cell lines. The aim of this study was to use a liposomal formulation in a proof-of-concept efficacy study to evaluate LAZ radioprotective and antiproliferative effects in Glioblastoma mouse model.

Methods: LAZ PEGylated liposomes (Lipo G) were developed in our laboratory. Lipo G size was 80-90 nm with Zeta potential of -22mv. Glioblastoma cell line U87-Lexpressing firefly luciferase reporter gene (100,000 cells in 2 μL) was injected intracranially in each male SCID hairless outbred mouse. The mice were randomized among 4 treatment groups (n=8-9, each): brain model (M) without treatment (control), radiation of 2Gy once a week (M+R), Lipo G at 5 mg/kg dose IP twice a week (M+L) and radiation with Lipo G (M+R+L). The treatment lasted three weeks. The tumor size was monitored using non-invasive bioluminescence imaging (BLI), in each mouse, expressed as the relative BLI photon intensity on the second and third week to that of the first measurement. The mice were sacrificed at the end of the third week and the whole brain was harvested for immunohistochemistry examinations. Lipid peroxidation of brain tissues was quantified by measuring malondialdehyde (MDA) as a surrogate biomarker. The survival was evaluated using Kaplan Meier analysis at P = 0.05.

Results: The relative BLI intensity was 4002.03±1737.67, 2034±737.72, 1387.36±684.53 and 2498.89±2521.32 % for M, M+R, M+L and M+R+L, respectively. The tumor size of the M+L group was reduced by 65% compared to the control group. For the radiation treated groups (M+R and M+R+L), there was no significant difference in the tumor size suppression compared to the control group. MDA brain concentration in M+L and M+R+L groups was significantly less than that in M+R group (8.27±0.78 and 10.37±3.30 µM/gm vs. 23.09± 3.79 µM/gm). The survival mean was 22.67, 25.33, 25.22 and 27.13 days for M, M+R, M+R+L and M+L groups, respectively. The mean survival duration of LAZ treated groups (M+L and M+R+L) was significantly longer than that of the control group (27.13 and 25.22 day vs. 22.67 day, respectively).

Conclusion: LAZ liposomal formulations administered at 5 mg/kg dose delivered a therapeutic concentration of LAZ to the brain for the efficacy of suppressing Glioblastoma tumor growth (65% reduction). The liposomal LAZ also protected the brain tissue from radiation-induced lipid peroxidation by reducing MDA concentration (lipid peroxidation surrogate) to 50%.

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 5460. doi:10.1158/1538-7445.AM2011-5460
Abstract 5476: Small molecule activation of BAX-mediated apoptosis through direct engagement of the BAX trigger site
Authors: Gavathiotis Evripidis , Reyna Denis , Walensky Loren
Published: June 2012

BCL-2 family proteins are key regulators of the mitochondrial apoptotic pathway in health and disease. Anti-apoptotic members such as BCL-2 and BCL-XL have been implicated in the initiation, progression, and chemoresistance of human cancer. The anti-apoptotic BCL-2/BCL-XL groove that binds and sequesters pro-apoptotic BH3 death helices has been successfully targeted by small molecules and peptides. Such compounds induce tumor cell apoptosis and are being advanced in clinical trials as promising next-generation cancer therapeutics. We recently localized a distinct BH3 binding groove on pro-apoptotic BAX and demonstrated that engagement of the novel site by a triggering BIM BH3 helix directly activates BAX (Gavathiotis et al. Nature, 2008), unleashing a succession of key conformational changes that culminates in the formation of a toxic mitochondrial pore (Gavathiotis et al. Mol Cell, 2010). Here we applied these new structural insights in a computational screen to identify small molecules capable of directly engaging the BAX trigger site for therapeutic induction of BAX-mediated apoptosis in human cancer. We confirmed by NMR analysis that a series of identified small molecules indeed bound to the BAX trigger site and in fluorescence polarization assays were strikingly selective for the BH3-binding groove of BAX as compared to that of anti-apoptotic targets. We demonstrate that molecular binding triggers dose-responsive BAX activation, as assessed by in vitro oligomerization, membrane permeabilization, and cell death assays. Importantly, the small molecule BAX activators induce apoptosis of cancer cells in a BAX-dependent manner, highlighting both the selectivity and pharmacologic potential of direct BAX activation as a novel strategy for cancer therapy.

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 5476. doi:10.1158/1538-7445.AM2011-5476
Abstract 5479: Mcl-1 is differentially regulated in acute myeloid leukemia cells treated with all trans retinoic acid and arsenic trioxide
Authors: Wang Rui , Waxman Samuel , Jing Yongkui
Published: June 2012

Both all trans retinoic acid (ATRA) and arsenic trioxide (ATO) induce complete remission in acute promyelocytic leukemia (APL) patients. Using the APL derived NB4 cell line containing the PML-RARα fusion protein it has been found that ATRA induced differentiation while ATO mainly induced apoptosis. Previously it has been reported that the antiapoptotic proteins Bcl-2 and Bfl-1/A1 were regulated during ATRA-induced differentiation process in APL cells. However, the regulation of Mcl-1, a major antiapoptotic protein required during myeloid granulocyte differentiation, has not been determined. Recently it has been shown that ERK phosphorylates Mcl-1 and regulates its stability. The regulation of Mcl-1 and p-ERK by ATRA and ATO were investigated in NB4 and its subclone R4 cells, HL-60 and its subclone HL-60/Res cells. Both NB4 and R4 cells contain PML-RARα while HL-60 and HL-60/Res cells contain RARα. However, the ATRA binding domain in PML-RARα and RARα is mutant in R4 and HL-60/Res cells, respectively. The levels of Mcl-1 were induced by ATRA in both NB4 and HL-60 cells, but not in R4 cells nor HL-60/Res cells. The induction of Mcl-1 in NB4 and HL-60 cells correlated with the induction of differentiation and the increased level of p-ERK. Unlike ATRA treatment, ATO treatment decreased the levels of Mcl-1 in both NB4 and R4 cells, but not in HL-60 nor in HL-60/Res cells, which correlated with the induction of apoptosis and down-regulation of p-ERK. Both NB4 and R4 cells contain PML-RARα which is degraded by ATO, suggesting that PML-RARα plays a role in ATO-mediated down-regulation of Mcl-1. To test the effect of PML-RARα on the regulation of Mcl-1 after ATO treatment, U937/PR9 cells with the inducible expression of PML-RARα were used. Expression of PML-RARα increased the levels of Mcl-1 and p-ERK. ATO treatment decreased the levels of both Mcl-1 and p-ERK. These data suggest that ATRA increases the level of Mcl-1 through activating ERK due to overcoming PML-RARα transcriptional repression and activating RARα while ATO decreases the level of Mcl-1 through inactivation of ERK due to inducing degradation of PML-RARα. The increased level of Mcl-1 after ATRA treatment protects against cell death and promotes differentiation while the decreased level of Mcl-1 after ATO-treatment sensitizes apoptosis induction.

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 5479. doi:10.1158/1538-7445.AM2011-5479
Abstract 5481: Antitumor activity of a novel JAK/Stat3 inhibitor, NSC-743380
Authors: Guo Wei , Wu Shuhong , Fang Bingliang , Wang Li , Liu Xiaoying
Published: June 2012

We recently developed two compounds, NSC-741909 and NSC-743380, which were highly active against a subset of NCI-60 cancer cell lines. This study investigated the in vivo activities of NSC-743380 and NSC-741909 in human tumor xenografts in nude mice and the mechanism of NSC-743380 antitumor activity. In vitro antitumor activity was determined by sulforhodamine assay of cell viability. In vivo antitumor activity was determined in xenograft tumors established from the human renal cancer cell line A498. NSC-743380-induced apoptosis and changes in gene expression levels and signaling pathway activities were identified by Western blot analyses, flow cytometric analyses, or both. The role of Stat3 in NSC-743380-induced antitumor activity was determined by enforced expression of a constitutively active Stat3 construct in sensitive cancer cells. NSC-743380 is highly active against a subset of cancer cell lines derived from lung, colon, ovary, kidney, and breast cancers and has an antitumor spectrum similar to that of NSC-741909 in the NCI-60 cancer cell line panel. The in vivo study revealed that NSC-743380 has an improved toxicity profile and greater antitumor activity than NSC-741909. Treatment with NSC-743380 caused tumor regression at all three dose levels, and a portion of animals at all three doses were tumor free. Mechanistic studies revealed that treatment with NSC-743380 inhibited JAK/Stat3 phosphorylation and this inhibition was dependent on both dose and time. Our results demonstrate that NSC-743380 is a novel JAK/Stat3 inhibitor that has selective antitumor activity against a subset of cancer cell lines and has favorable in vivo antitumor activities.

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 5481. doi:10.1158/1538-7445.AM2011-5481
Abstract 5485: mTOR inhibition promotes autophagy that reduces chemosensitivity and is partially restored by chloroquine
Authors: Huang Shengbing , Yu Chunrong , Sinicrope Frank A.
Published: June 2012

BACKGROUND. The mammalian target of rapamycin (mTOR) kinase forms two multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, cell survival, and autophagy. We determined whether mTOR inhibition can induce cytoprotective autophagy resulting in anti-cancer drug resistance, and whether inhibition of autophagy using the lysomotrophic agent, chloroquine, can restore chemosensitivity.

EXPERIMENTAL METHODS. Inhibition of mTOR was accomplished in human colon cancer cells (HT-29, DLD1) using lentiviral shRNA or by AZD8055, a TORC1 and TORC2 kinase inhibitor. These cells were then incubated with 5-FU (0-100 µM) or CPT-11 (0-20 µM) alone and in combination with chloroquine. LC3I/II expression, phospho-histone H2A.X (a DNA damage marker), and caspase cleavage were analyzed by Western blotting using highly specific antibodies, and clonogenic survival assays were also performed.

RESULTS. AZD8055 was shown to attenuate caspase-3 and -9 cleavage and to suppress the expression of phospho-histone H2A.X induced by 5-FU or CPT-11 in colon cancer cells. AZD8055 was also found to antagonize the ability of 5-FU to inhibit clonogenic survival. Similarly, shRNA knockdown of mTOR attenuated 5-FU-induced caspase cleavage and phospho-histone H2A.X expression. Inhibition of mTOR by AZD8055 or shRNA enhanced the conversion of the autophagosome-associated protein light chain 3 (LC3) from a cytosolic (LC3I) to a membrane-bound (LC3II) form, consistent with induction of autophagy. Inhibition of autophagy by chloroquine was shown to attenuate the ability of AZD8055 to suppress 5-FU-induced caspase cleavage. Consistent with this result, chloroquine sensitized DLD1 cells to AZD8055 and 5-FU in a clonogenic survival assay.

CONCLUSION. Inhibition of mTOR can protect colon cancer cells from DNA damage induced by cytotoxic chemotherapy. This cytoprotection was due, in part, to induction of pro-survival autophagy since chloroquine was able to partially restore chemosensitivity.

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 5485. doi:10.1158/1538-7445.AM2011-5485
Abstract 5488: Novel death pathway in a p53 null human leukemia cell line treated with PNC-27
Authors: Fenelus Maly , Mathew Sheena M. , Zolkind Paul A. , Adler Victor , Yazdi Ehsan S. , Michl Josef , Pincus Matthew R. , Bowne Wilbur B.
Published: June 2012

Background: PNC-27 is a p53-derived peptide from the human double minute binding domain (HDM-2). This peptide has been shown to induce rapid tumor cell necrosis of selected cancer cells, while sparing their normal cellular counterparts (PNAS 2010; 107: 1918-1923). PNC-27 binds to HDM-2 in the cancer cell membrane, causing pore-formation and permeabilization of the membrane. We now propose an additional target for PNC-27 involving mitochondrial membrane-bound B-cell lymphoma-2 (Bcl-2), which contains a BH3 protein-protein interaction domain. BH3 serves as a potent mediator of cell death in pro-apoptotic proteins (such as Bax and Bad), as well as anti-apoptotic proteins, Bcl-2 and Bcl-XL. These Bcl-2 proteins have been proposed to control function and stability of the outer mitochondrial membrane.

Methods: The anti-cancer activity and mechanism of PNC-27 (p53 aa12-26-penetratin) was studied against p53 null K562, a human leukemia cancer cell line. Necrosis was determined by measuring lactate dehydrogenase (LDH), while apoptosis was determined by measuring early markers of apoptosis (caspases 3, 7). Co-immunoprecipitation (IP) was performed between PNC-27 and subcellular components to identify potential targets for PNC-27 in K562 cells. HDM-2, Bcl-2, Cytochrome C, and ATP synthesis in PNC-27-treated cells were studied.

Results: We found that PNC-27, but not control peptide PNC-29, is cytotoxic to K562 cells, inducing nearly 90% inhibition of proliferation with LDH release. PNC-27 peptide induced Cytochrome C release as well as downregulation of ATP synthesis. Furthermore, endoge$$nous HDM-2 and Bcl-2 was found to IP with exogenous PNC-27 in K562 cells, inducing downregulation of HDM-2 and Bcl-2.

Conclusions: P53 N-terminal derived peptide (PNC-27) potentially alters the mitochondrial membrane of K562 cells via induction of outer membrane permeabilization; with resultant down-regulation of Bcl-2, induction of Cytochrome C release, and decreased ATP synthesis. Our results suggest a second target and mechanism of cell death using PNC-27 in a p53 null K562 cell line; a p53 derived peptide that also targets Bcl-2 leading to rapid tumor cell necrosis.

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 5488. doi:10.1158/1538-7445.AM2011-5488
Abstract 5493: Lethal mutagenesis of cancer cells by nucleoside analogs
Authors: Fox Edward J. , Shen Jiang-Cheng , Loeb Lawrence A.
Published: June 2012

The evolution of cancer and RNA viruses is characterized by mutation accumulation facilitating phenotypic plasticity and environmental adaptation. We have demonstrated that serial passage of virus from HIV-infected human lymphoblastoid cells in the presence of mutagenic nucleosides results in mutation accumulation, leading to error catastrophe, and viral extinction. In limited phase II clinical studies, administration of a cytidine analog, KP-1461, to individuals infected with HIV results in increased viral mutations. We hypothesize that cancer cells treated with mutagenic nucleoside analogs will progressively accumulate mutations until the error threshold for viability is exceeded and the tumor ablated. We have serially passaged mismatch repair proficient and deficient colon cancer cells in the presence of 36 different mutagenic deoxynucleoside analogs and established a new protocol for measuring both mis-incorporation and repair during DNA synthesis in cells. DNA templates, purified from phage, are blocked with terminal non-hydrolysable nucleotides to prevent degradation by cellular nucleases. After transfection of double-stranded DNA into human cells, we can recover >50,000 molecules/cell. After transfection of single-stranded DNA molecules hybridized to a 5′-biotin labeled primer, extended primers are recovered on streptavidin beads; the frequency of misincorporation into the newly synthesized DNA varies from 10-3 to 10-6, depending on the genetic background of the tranfected cells. The versatility of this assay will facilitate studies on the efficiency of DNA repair, the role of different human DNA polymerases in DNA synthesis and bypass of DNA damage, and the stratification of individuals with respect to DNA repair capacity and fidelity. Enhancing the mutation frequency of cancer cells represents a new avenue for treatment, particularly for patients with tumors that have accumulated large numbers of mutations and/or who failed extensive chemotherapy.

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 5493. doi:10.1158/1538-7445.AM2011-5493
Abstract 5497: An inhibitor of phosphatase PP2A enhances in vivo anticancer activity of a phospho (ADP-ribosyl) polymerase inhibitor (PARPi) combined with temozolomide (TMZ)
Authors: Lu Jie , Ikejiri Barbara , Yang Chunzhang , Thompson Eli , Mushlin Harry , Kovach John S. , Lonser Russell , Zhuang Zhengping
Published: June 2012

We previously showed that a novel inhibitor of PP2A enhances the in vivo activity of TMZ against human glioblastoma xenografts (Lu et al, PNAS 2009;104:11697-11702). Several mechanisms underlie this effect of PP2A inhibition and include an increased rate of cancer cell entry into DNA synthesis, inhibition of cell cycle arrest at G1 and G2/M, and a marked reduction in p53. Because inhibitors of PARP1 have been shown to enhance the activity of cytotoxic cancer drugs by inhibiting repair of single-strand breaks in cells with impaired mechanisms for double-stranded DNA repair (BrCa mutants, triple negative breast cancer cells), we reasoned that blocking other types of DNA-damage defense mechanisms by PP2A inhibition might further enhance the activity of a PARPi combined with TMZ in cancers, not necessarily already deficient in DNA repair. We treated mice bearing xenografts of human melanoma, line A2058, with single agent LB-100, a novel water soluble PP2A inhibitor (Lixte Biotechnology Holdings, Inc., East Setauket, NY; Lu et al, J Neurosurg 2010; 113: 225-233) at 1.5mg/kg by continuous intraperitoneal (i.p.) infusion for 21 days and TMZ at 80mg/kg i.p. days 3,6,9,12,15,18. LB-100 and TMZ were given alone and in combination with each other and in 2-drug combinations with the PARPi, ABT-888 (Sigma), at 6.25 mg/kg i.p. on days 3,6,9,12,15,18, and all three drugs were given together at the same doses and schedules as when used alone or in 2-drug combinations. (ABT-888 was inactive as a single agent and was not used alone). The combination of LB-100 + TMZ was more inhibitory to the xenografts at 21 days than ABT-888 + TMZ, but the 3-drug regimen was most effective and no more toxic than any of the 2-drug regimens. Histologic analysis of xenografts 24 hours after treatment of animals with each of the single agents, each 2-drug regimen, and the 3-drug regimen showed increased apoptosis and necrosis by the 3-drug combination compared to the two-drug regimens, which in turn were more effective that the single agents. Similar results were found in vitro by flow cytometric analysis of A2058 cells 24 hours after exposure to the single agent and 2- and 3-drug regimens. Inclusion of a PP2A inhibitor may increase and extend the effectiveness of cytotoxic regimens containing a PARPi to cancers without acquired mutations in DNA repair.

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 5497. doi:10.1158/1538-7445.AM2011-5497
Abstract 5502: Absence of ERCC1 expression is predictive of complete pathologic response to platinum-based therapy for women with triple negative breast cancer
Authors: Cioffi-Lavina Maureen , Hurley Jude , Jorda Merce , Walker Gail , Gomez-Fernandez Carmen
Published: June 2012

Background: Platinum-based chemotherapy exerts its cytotoxic effects by forming intra-strand DNA adducts that inhibit replication. Excision Repair Cross Complementing group 1 protein (ERCC1) plays a critical, yet undesirable, role in repair of DNA damage induced by platinum. Absence of immunohistochemical expression of ERCC1 has been shown to be associated with response to platinum-based chemotherapy regimens in non small cell lung carcinomas. Experimental data suggests that triple negative breast cancers may have increased sensitivity to platinum-based chemotherapy, particularly in BRCA1 mutation carriers, whose cancers cluster among the basal-like subtype, as defined by gene expression profiling. We investigated the immunohistochemical expression of ERCC1 in relation to response to neoadjuvant platinum-based chemotherapy in a cohort of patients with triple negative breast cancers.

Materials and Methods: We reviewed the diagnostic core biopsies of 32 triple-negative breast cancer patients treated with neoadjuvant docetaxel plus platinum-based salts at our institution between 1999 and 2007. Fifteen (46.9%) study patients had pathologic complete response (pCR), defined by the absence of invasive carcinoma in the breast and axilla. Immunohistochemistry for ERRC1 (ABCAM) was performed in all cases using the LSAB method. A nuclear immunohistochemical reaction in >1% of the tumor cells was scored as positive for ERCC1. Fisher's exact test was used to compare pCR rates by ERCC1 status.

Results: Sixteen of 32 (50%) triple negative breast carcinomas were positive for ERCC1 by immunohistochemistry. Reactivity was present in 10 to 50% of the tumor cell nuclei. The remaining 16/32 (50%) carcinomas were definitively negative for ERCC1. Significantly higher pCR rates were found in patients with ERCC1(−) tumors, 11/16 (68.8%), compared to 4/16 (25%) patients with ERCC1(+) tumors, p=0.032. Findings were similar for pCR confined to the breast (75.0 v 31.3%, p=0.032), and for pCR in the axilla (75.0 v 43.8%), although the latter result lost statistical significance, p=−0.149.

Conclusion: Absence of immunohistochemical expression for ERCC1 is predictive of pathologic complete response to platinum-based neoadjuvant chemotherapy for patients with triple negative breast cancers.

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 5502. doi:10.1158/1538-7445.AM2011-5502