bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5658: Development and optimization of CZ48-loaded microbubbles for controlled drug delivery .
Authors: Wang Yang , Li Siyang , Zheng Jian , Yan Xiufeng , Cao Zhisong , Vardeman Dana , Giovanella Beppino
Published: August 2014

CZ48, the 20-O-propionate ester of camptothecin, is a prodrug requiring activation by converting to parental CPT in vivo. To circumvent the problems of low water solubility, lack of absorption, and poor bioavailability, novel CZ48-loaded microbubbles were investigated in this study. CZ48-loaded microbubbles were prepared at particle sizes ranging from 0.5 μm to 6.7 μm using a double emulsion technique (W/O/W). The satisfactory encapsulation efficiency and drug-loading content were 85.73±2.41% and 26.07±0.76%, respectively. The successful encapsulation of the microbubbles was confirmed by confocal laser scanning microscope (CLSM) observations and X-ray diffraction (XRD) patterns. The comparison between the controlled drug release from CZ48-loaded microbubbles and the free drug release from plain CZ48 crystals was performed in vitro, showing that 55% of the CZ48 was steadily released by the CZ48-loaded microbubbles in the first 48 hrs while more than 90% of the CZ48 was rapidly released by CZ48 crystals within the first 5 hrs. In vivo pharmacokinetic studies indicated that the bioavailabilities of CZ48 and CPT from CZ48-loaded microbubbles raised 121% and 517%, respectively, after intravenous administration compared to the plain CZ48. Compared to the plain CZ48, CZ48-loaded microbubbles also showed a higher mean residence time (MRT) and a longer elimination half-life (T1/2), indicating that CZ48-loaded microbubbles were indeed able to release CZ48 drug in a finely controlled manner. These results demonstrated that such microbubbles could be a promising approach for the construction of an effectively controlled drug delivery system for CZ48 as well as for other drugs with lower water solubility.

Citation Format: Yang Wang, Siyang Li, Jian Zheng, Xiufeng Yan, Zhisong Cao, Dana Vardeman, Beppino Giovanella. Development and optimization of CZ48-loaded microbubbles for controlled drug delivery . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5658. doi:10.1158/1538-7445.AM2013-5658
Abstract 5665: New alternatives to photodynamic therapy based on light-enabled drug delivery.
Authors: Dcona M. Michael , Mitra Deboleena , Goehe Rachel W. , Gewirtz David A. , Lebman Deborah A. , Hartman Matthew C.T.
Published: August 2014

Conventional photodynamic therapy (PDT) aims to achieve tumor drug targeting by selectively killing cells upon illumination. PDT serves as a non-invasive treatment option for many types of non-metastatic, light-accessible cancers such as those involving the head and neck, lung, bladder, prostate, or esophagus. The various types of PDT all use the same basic strategy for killing cells; photosensitizing molecules are taken up by cancer cells, and, upon illumination, singlet oxygen is generated in the vicinity of the photosensitizer, killing the cell. A new alternative to PDT is photochemical internalization, in which singlet oxygen generated by a photosensitizer disrupts endosomes containing a toxin, releasing it into the cytoplasm and killing the cell. A drawback to both of these approaches is that moderate levels of oxygen are required, yet tumors are often hypoxic environments. Moreover, during PDT treatment, oxygen levels drop even further as the singlet oxygen is consumed; this can compromise treatment effectiveness. We have sought to address these challenges by developing new strategies for light-based treatment of cancer that are oxygen independent. In the first approach, which we call photocaged permeability, we have attached the anticancer drug doxorubicin to a cell impermeable small molecule via a light-cleavable linker. We show using flow cytometry and confocal microscopy that this conjugate is cell impermeable in the dark, yet upon illumination, doxorubicin is released and enters the cells. The IC50 for our conjugate in JH-EsoAd1 cells using non-cytotoxic doses of light is 1.6 μM, identical to that of doxorubicin alone. No cytotoxicity is observed at doses 30-fold higher in the dark. In a second approach, we have attached doxorubicin to folate receptor and PSMA ligands via a light cleavable linker in order to enable dual cancer cell targeting with both light and ligand specificity. The ability of the resulting conjugates to selectively kill cells expressing the requisite receptors with light will be described.

Citation Format: M. Michael Dcona, Deboleena Mitra, Rachel W. Goehe, David A. Gewirtz, Deborah A. Lebman, Matthew C.T. Hartman. New alternatives to photodynamic therapy based on light-enabled drug delivery. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5665. doi:10.1158/1538-7445.AM2013-5665
Abstract 5679: Automatic synthesis of efficient transcription inhibitors as anti-cancer agents designing sequence-specific DNA-binding molecules.
Authors: Nagase Hiroki , Koshikawa Nobuko , Watanabe Takayoshi
Published: August 2014

PURPOSE: Regulation of endogenous gene expression using synthetic chemicals, so called “Transcription therapy” is a unique and promising approach. Although we have reported in vitro and in vivo anti-tumor, anti-metastatic and various anti-disease efficacies of hairpin Pyrrole-Imidazole polyamide (hPIP),

PIP still needs to improve on selectivity of target DNA sequences and sensitivity to bind specific sequences. Hence, we synthesized PIPs targeting transcription-factor-binding-sites with several different structures and tested their efficacy, selectivity and sensitivity in vitro as well as in vivo model.

METHODS: We have developed an automatic system for

PIP synthesis by using peptide synthesizer of PSSM8 (Shimazu) and synthesized hPIPs or cyclic PIPs targeting either the MMP9 AP1 site or NFkB site to evaluate sensitivity and specificity. Each PIP, which has a different beta-alanine position, was tested for target DNA binding by surface plasmon resonance (SPR) using

BiaCore system. We also test cell invasion and migration in vitro and metastatic capability in an in vivo xenograft model.

RESULTS: Each hPIPs targeting AP1 or NFkB site showed significant reduction of MMP9 expression in MDA-MB-231 cells and HeLa cells and metastatic capacity in vivo. hPIP with crossed beta-alanine position showed the almost same dissociation constant as those with parallel beta-alanine position. Cyclic PIPs also showed similar dissociation constant as hPIPs and showed better sequence specificity with reasonable efficacy at least in vitro analysis.

CONCLUSION: The result suggested that various

PIPs with different target sites or architectures give distinct efficacy, selectivity and sensitivity in at least in vitro cancer cell culture system. Depending on pharmacokinetic properties of PIP the most suitable PIP can be selected for the future trial as seen in combinatorial chemistry technique.

Citation Format: Hiroki Nagase, Nobuko Koshikawa, Takayoshi Watanabe. Automatic synthesis of efficient transcription inhibitors as anti-cancer agents designing sequence-specific DNA-binding molecules. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5679. doi:10.1158/1538-7445.AM2013-5679
Abstract 5683: Blockade of glioma proliferation via allosteric inhibition of JAK2.
Authors: He Kunyan , Qi Qi , Chan Chi-Bun , Xiao Ge , Liu Xia , Tucker-Burden Carol , Wang Liya , Mao Hui , Lu Xiang , McDonald Frank E. , Luo Hongbo , Fan Qi-wen , Weiss William A. , Sun Shi-yong , Brat Daniel J. , Ye Keqiang
Published: August 2014

Gliomas are the most common primary brain tumors. Glioblastoma multiforme (GBM), WHO grade IV, is the most frequent and aggressive form, with a median survival of approximately 12 months following aggressive treatment with surgical resection, radiation and chemotherapy. Epidermal growth factor receptor (EGFR) is frequently amplified and mutated in human cancers including GBM. While the rationale for treating GBM patients with EGR inhibitors is strong, the efficacy of anti-EGFR small molecular inhibitors or monoclonal antibodies in treating gliomas with PTEN mutation or deletion has been modest. Here we show a novel approach to inhibiting EGFR that involves a small molecule (G5-7) that selectively blocks JAK2 phosphorylation of EGFR, inhibits cell proliferation and reduces VEGF secretion, and leads to glioma shrinkage via apoptosis and anti-angiogenesis. G5-7 inhibited EGFR and STAT3 tyrosine phosphorylation by JAK2 through binding to its N-terminal FERM domain. Interestingly, G5-7 selectively inhibited the proliferation of U87MG/EGFRvIII cells and human GBM explant neurosphere cultures but only weakly affected U87MG/PTEN cells or normal neural progenitor cultures. Moreover, oral administration of this compound markedly diminishes the brain tumor volumes in both subcutaneous and intracranial models, supporting that this compound itself and/or the metabolites might be able to penetrate the brain-blood barrier and exert the anti-proliferative actions through blocking the oncogenic pathways in vivo. Chronic treatment of mice with 50 mg/kg G5-7 exhibits no obvious toxicity in the animals. Our finding demonstrates that G5-7 is a novel allosteric JAK2 inhibitor with potent anti-tumor activity for GBM. Our report not onlyidentify a novel drug for treating the GBM, but also provide insight into the molecular mechanisms of how these small molecules impinge on the EGFR/PTEN signaling. Optimization of these novel compounds will lead to new treatments for the patients with these devastating tumors.

[K.H. and Q.Q. contributed equally to this work.]

Citation Format: Kunyan He, Qi Qi, Chi-Bun Chan, Ge Xiao, Xia Liu, Carol Tucker-Burden, Liya Wang, Hui Mao, Xiang Lu, Frank E. McDonald, Hongbo Luo, Qi-wen Fan, William A. Weiss, Shi-yong Sun, Daniel J. Brat, Keqiang Ye. Blockade of glioma proliferation via allosteric inhibition of JAK2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5683. doi:10.1158/1538-7445.AM2013-5683
Abstract 5684: Target tubulin polymerization with novel reverse ABI analogs interacting with the colchicine binding site.
Authors: Xiao Min , Ahn Sunjoo , Chen Jianjun , Wang Jin , Miller Duane D. , Dalton James T. , Li Wei
Published: August 2014

Cancer remains one of the leading causes of mortality worldwide. While current therapies are effective in treating early stage cancers, their efficacy against advanced cancers, especially multidrug-resistant cancers, is limited. Our lab recently reported a set of 2-aryl-4-benzoyl-imidazoles (ABI) derivatives with potent anti-proliferative activity against melanoma and prostate cancer cell lines and xenografts. Further optimization has led to the synthesis of a new series of 4-aryl-2-benzoyl-imidazoles with the aryl group and the benzoyl group reversed as compared to the previous ABI structure. The reverse ABI (RABI) compounds were evaluated for biological activity against 8 cancer cell lines including some which were multidrug-resistant. The in vitro results showed that several RABI compounds had excellent antiproliferative properties with IC50 values in the low namomolar range, comparable to existing tubulin-targeting agents such as paclitaxel, colchicine, or vinblastine, but with the ability to effectively circumvent Pgp-mediated drug resistance. The IC50 of the most active compound 12a was 9 nM. The mechanism of action (MOA) of the RABI compounds was investigated using cell cycle analysis, tubulin polymerization assays and molecular docking studies. These studies showed that their antitumor activity was achieved through inhibition of tubulin polymerization at the colchicine binding site. Our results suggest that the novel RABI compounds may be developed as a promising antitumor agent for advanced cancers.

Citation Format: Min Xiao, Sunjoo Ahn, Jianjun Chen, Jin Wang, Duane D. Miller, James T. Dalton, Wei Li. Target tubulin polymerization with novel reverse ABI analogs interacting with the colchicine binding site. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5684. doi:10.1158/1538-7445.AM2013-5684
Abstract 57: Validation of Next Generation Sequencing technologies in comparison to current diagnostic gold standards for BRAF, EGFR and KRAS mutational analysis.
Authors: McCourt Clare , Maxwell Perry , Catherwood Mark , Salto-Tellez Manuel
Published: August 2014

BACKGROUND: Next Generation Sequencing (NGS) has the potential of becoming an important tool in clinical diagnosis and therapeutic decision-making in oncology owing to its enhanced sensitivity in DNA mutation detection, fast-turnaround of samples in comparison to current gold standard methods and the potential to sequence a large number of cancer-driving genes at the one time. We aim to test the diagnostic accuracy of current NGS technology in the analysis of mutations that represent current standard-of-care, and its reliability to generate concomitant information on other key genes in human oncogenesis.

METHODS: Thirteen FFPE clinical samples (8 lung adenocarcinomas, 3 colon carcinomas and 2 malignant melanomas) already genotyped for EGFR, KRAS and BRAF mutations by current standard-of-care methods (Sanger Sequencing and q-PCR), were analyzed for detection of mutations in the same three genes using two NGS platforms and an additional 43 genes with one of these platforms. The results were analyzed using closed platform-specific proprietary bioinformatics software as well as open third party applications (including, among others, the ‘Open’ CLC Genomics Workbench).

RESULTS: Our results indicate that the existing format of the NGS technology performed well in detecting the clinically relevant mutations stated above, with apparent improved sensitivity, but may not be reliable for a broader unsupervised analysis of the wider genome in its current design. They also illustrate the relevance of different bioinformatics curation approaches, allowing different degrees of stringency, for the avoidance of potential false positives in the broader choice of genes selected, when compared with the expected mutation frequencies from sources such as the COSMIC database, for the tumor types analyzed. The systems are also able to detect other mutations outside the expected ones representing the current main "standard-of-care," the significance of which are uncertain at this point.

CONCLUSIONS: Our study represents a diagnostically lead validation of the major strengths and weaknesses of this technology in its current state of development before consideration for diagnostic use.

Citation Format: Clare McCourt, Perry Maxwell, Mark Catherwood, Manuel Salto-Tellez. Validation of Next Generation Sequencing technologies in comparison to current diagnostic gold standards for BRAF, EGFR and KRAS mutational analysis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 57. doi:10.1158/1538-7445.AM2013-57
Abstract 570: The integrated stress response kinase GCN2 regulates cell survival through p21/WAF1/CIP1 in response to nutrient deprivation.
Authors: Lehman Stacey L. , Ye Jiangbin , Koumenis Constantinos
Published: August 2014

The tumor microenvironment is often characterized by deficiencies in nutrients, such as oxygen, glucose, and amino acids. Activation of the nutrient-sensing kinase GCN2, which constitutes one arm of the Integrated

Stress Response (ISR), is one mechanism by which tumor cells cope with and adapt to nutrient stress. GCN2 phosphorylates the alpha subunit of the eukaryotic translation initiation factor eIF2, leading to global downregulation of translation to conserve amino acids and initiation of a transcriptional program through ATF4 to promote recovery from nutrient deprivation. Loss of GCN2 results in decreased tumor cell survival in vitro under conditions of amino acid deprivation and attenuated tumor growth in vivo in a variety of implanted animal tumor models. Our recent studies have shown that amino acid deprivation induces the cell cycle inhibitor p21 in a GCN2 dependent manner. GCN2 regulates p21 at the transcriptional and translational level. Induction of p21 mRNA occurs in both eIF2α S51A and p53 -/- cells, indicating that phosphorylation of eIF2α and expression of p53 are not required for GCN2 dependent transcriptional regulation. At the translational level, GCN2 phosphorylates eIF2α to prevent translational suppression of p21 transcript variants containing a 5’ uORF overlapping the true translation start site. Expression of p21 promotes cell survival during amino acid deprivation, suggesting a mechanism by which GCN2 regulates p21 in order to coordinate cell division with nutrient availability. In agreement with a pro-survival role, we observe p21 upregulation concomitant with ISR activation in a genetically engineered mouse of soft tissue sarcoma. These findings point to a novel mode of regulation of p21/WAF1/CIP1 by nutrient stress in the tumor microenvironment that contributes to tumor progression.

Citation Format: Stacey L. Lehman, Jiangbin Ye, Constantinos Koumenis. The integrated stress response kinase GCN2 regulates cell survival through p21/WAF1/CIP1 in response to nutrient deprivation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 570. doi:10.1158/1538-7445.AM2013-570
Abstract 573: USP7 and Daxx regulate mitosis progression and taxane sensitivity by affecting stability of Aurora A kinase.
Authors: Giovinazzi Serena , Morozov Viacheslav M. , Summers Matthew K. , Reinhold William C. , Ishov Alexander M.
Published: August 2014

A large number of patients are resistant to taxane-based chemotherapy. Functional mitotic checkpoints are essential for taxane sensitivity. Thus, mitotic regulators are potential markers for therapy response and could be targeted for anticancer therapy. In this study we identified a novel function of Ubiquitin Specific processing Protease-7 (USP7) that interacts and cooperates with protein Daxx in regulation of mitosis and taxane resistance. Depletion of USP7 impairs mitotic progression, stabilizes cyclin B and reduces stability of the mitotic E3 ubiquitin ligase, Checkpoint with Forkhead and RING finger (CHFR). Consequently, cells with depleted of USP7 accumulate Aurora A kinase, a CHFR substrate, thus elevating multipolar mitoses. We further show that these effects are independent from the USP7 substrate p53. Thus, USP7 and Daxx are necessary to regulate proper execution of mitosis, partially via regulation of CHFR and Aurora A kinase stability.

Results from colony formation assay, in silico analysis across the NCI60 platform and in breast cancer patients suggest that USP7 levels inversely correlate with response to taxanes, pointing at the USP7 protein as a potential predictive factor for taxane response in cancer patients. In addition, we demonstrated that inhibition of Aurora A attenuates USP7-mediated taxane resistance, suggesting that combinatorial drug regimens of Taxol and Aurora A inhibitors may improve the outcome of chemotherapy response in cancer patients resistant to taxane treatment. Finally our study offers novel insights on USP7 inhibition as cancer therapy.

Citation Format: Serena Giovinazzi, Viacheslav M. Morozov, Matthew K. Summers, William C. Reinhold, Alexander M. Ishov. USP7 and Daxx regulate mitosis progression and taxane sensitivity by affecting stability of Aurora A kinase. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 573. doi:10.1158/1538-7445.AM2013-573
Abstract 574: Investigating the role of CDC25B in inhibition of cellular proliferation.
Authors: Lee Caleb C. , Manfredi James
Published: August 2014

CDC25B is a dual specificity phosphatase that activates cyclin-dependent kinases to trigger entry into mitosis. Overexpression of CDC25B has been associated with breast and head and neck cancers as well as higher histological tumor grade in non-small cell lung cancers. In a transgenic mouse model, overexpression of Cdc25B in the mammary glands lead to accelerated mammary epithelial proliferation resulting in hyperplasia while in vitro, co-transfection of CDC25B with Ras allows for transformation of MEF's. However, in vitro experiments using ectopic expression of CDC25B in transformed cell lines have shown an anti-proliferative effect. This may be related to inappropriate cyclin-dependent kinase activation and premature mitotic entry, leading to both p53-dependent and independent checkpoints. In addition, S phase cells overexpressing CDC25B have higher rates of apoptosis due to premature entry into mitosis. CDC25B has also been shown to cause an early progression to S phase that is associated with increased amounts of DNA damage. Our goal is to expand on these results to further clarify the anti-proliferative effects of CDC25B as well as detail the mechanism by which tumor cells bypass these effects in vitro and in vivo.

Citation Format: Caleb C. Lee, James Manfredi. Investigating the role of CDC25B in inhibition of cellular proliferation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 574. doi:10.1158/1538-7445.AM2013-574
Abstract 575: Cdk1 prevents DNA rereplication in G2/M by phosphorylating and facilitating the removal of Cdc7 from chromatin at the end of S phase.
Authors: Knockleby James , Kim Byung Ju , Lee Hoyun
Published: August 2014

Cancer is inextricably linked with aneuploidy. Aneuploidy can arise for a number of reasons, including the deregulation of DNA and centrosome replication. Mechanisms have evolved that are designed to ensure that DNA is replicated only once per cell cycle. The initiation of DNA replication is a key target of these mechanisms, which includes regulating Cdc6, Cdt1 and Cyclin dependent kinases (Cdks). Although the regulatory mechanisms of Cdc6 and Cdt1 are known, the role of Cdk1 in particular is less clear. One potential target for Cdk1 regulation is the Dbf4-Cdc7 kinase (DDK), which acts as a licensing factor for DNA replication. DDK performs an essential and critical step in initiating DNA replication. As such, it is a target for novel cancer therapeutic drugs currently in clinical trials. Even so, the regulatory mechanisms dictating DDK function in human cells remains poorly understood.

A site-directed mutagenesis approach was undertaken to determine the in vivo function of Cdk-dependent phosphorylation sites on the primary Cdc7 sequence. Using over-expressed Cdc7 in human cells (HEK293T), the functional significance of Cdc7 phosphorylation was determined; both the lack of phosphorylation and constitutive phosphorylation of Cdc7 was analyzed. We found that Cdc7, the catalytic subunit of the DDK, is a target of Cdk1. Cdc7 is phosphorylated in G2/M phase by Cdk1. Phosphorylation of Cdc7 removes the catalytic subunit of DDK from origins of replication and chromatin with the assistance of the nuclear export factor CRM1, which relocates Cdc7 to the microtubule cytoskeleton. Finally, the Cdk1-dependent removal of Cdc7 from chromatin prevents the cell from initiating DNA replication, indicating that Cdc7 is a target of Cdk1-dependent inhibition of DNA re-replication.

We conclude that (1) Cdk1 phosphorylates Cdc7 in vivo. (2) Cdk1 dependent phosphorylation of Cdc7 regulates chromatin binding and DNA replication and (3) pCdc7 is associated with the nuclear export factor CRM1 in G2/M phase, suggesting a mechanism that couples the down-regulation of Cdc7 function at the end of S phase through nuclear localization and chromatin association. Taken together, our data provides key insight into the functional understanding of DDK.

Citation Format: James Knockleby, Byung Ju Kim, Hoyun Lee. Cdk1 prevents DNA rereplication in G2/M by phosphorylating and facilitating the removal of Cdc7 from chromatin at the end of S phase. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 575. doi:10.1158/1538-7445.AM2013-575
Abstract 576: Transforming growth factor-beta (TGF-β) inhibits growth of human myeloid leukemia cells and downregulates the expression of CDK inhibitor, p18INK4C.
Authors: Guardia Talia , Badaloo Reshma , Serrano Valentina , Toro Alejandra , Hu Xiaotang
Published: August 2014

The cell cycle progression controlled by cyclin-dependent kinases (CDKs) is counterbalanced by CDK inhibitors (CKIs). The CKIs have two families: CIP/KIP and INK. The CIP/KIP members are composed of p21WAF1/CIP1, p27KIP1 and p57KIP2, which bind to and inhibit both cyclin D-CDK4/6 kinases and cyclin-E/A-CDK2. The INK members consist of p15INK4B, p16INK4A, p18INK4C and p19INK4D. The members in this family only bind to and inhibit G1CDKs (CDK4 and CDK6). We have previously reported that p27KIP1 plays both a positive and a negative role in human myeloid leukemia cells. In this study, we investigated whether p18INK4C plays any role in TGF-β-mediated cell cycle control of human myeloid leukemia cells. TGF-β significantly inhibited proliferation of MV4-11 and TF-1 cells, with upregulation of p27KIP1and downregulation of multiple CDKs and cyclins. Surprisingly, TGF-β significantly inhibited the expression of p18INK4C, a traditional G1 CDK inhibitor. The inhibitory effect of TGFβ on p18INK4C was time dependent. The time course showed that early inhibition of p18INK4C occurred at 3h after initiating TGFβ treatment in the cells with a maximal inhibition of 70% being observed at 72h incubation with TGF-β. The control cells treated with the solvent for TGFβ did not show any significant changes in the levels of p18INK4C. As a sample loading control, the cells treated with or without TGF-β expressed about the same levels of actin.TGFβ-induced p18INK4C inhibition is also dose dependent with a maximal inhibition being detected by Western blot when 30ng/ml of TGFβ was added to the culture. Low concentrations (5ng/ml or less) did not markedly affect the expression of p18INK4C. The association of p18INK4C with CDK4 was also decreased in response to TGF-β treatment detected by immunoprecipitation. Our data also showed that the two cell lines tested barely expresses p15INK4B, p16INK4A, p19INK4D, and p21WAF1/CIP1 and these inhibitors do not appear to play an important role, either positively or negatively, in the cell cycle control of the leukemia cells. Our data suggest that p18INK4C may be required for cell cycle progression in human myeloid leukemia cells tested. Whether such a positive role of p18INK4C is caused by a possible gene mutation(s) and thereby contributes to leoplasia of the leukemia cells is currently under investigation (supported by NIH-NIGMS MBRS RISE: R25 GM059244, Barry University).

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Citation Format: Talia Guardia, Reshma Badaloo, Valentina Serrano, Alejandra Toro, Xiaotang Hu. Transforming growth factor-beta (TGF-β) inhibits growth of human myeloid leukemia cells and downregulates the expression of CDK inhibitor, p18INK4C. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 576. doi:10.1158/1538-7445.AM2013-576
Abstract 578: Plk1 decreases DNA double-strand break (DSB) formation during replication-stalling genotoxic stress.
Authors: Gebreselassie Daniel , Chun-Kost Gina , Leo Elisabetta , Pommier Yves , Saito Erika , Patierno Steven , Ceryak Susan M.
Published: August 2014

Polo-like kinase 1 (Plk1) is a key regulator of mitosis and its over-expression/ deregulation is linked to cellular transformation. Recent studies highlight its role in resumption of cell cycle progression after DNA damage. Plk1 has also been shown to enhance DNA replication under stress of stalled replication forks. We reported Plk1 activation-induced G2/M checkpoint bypass in normal human lung cells (HLFs), in the presence of acute genotoxic stress. The present aim was to determine the effect of Plk1 on DNA double strand breaks (DSBs) during genotoxic stress. In the present study, we measured DNA DSB induction in HLFs by Cr(VI), etoposide, and H2O2 after Plk1 activation using the comet assay and γH2AX immunofluorescence staining. We used equitoxic concentrations of the different genotoxins and found that DNA DSBs increased > 2 fold after exposure to 3 μM Cr(VI), 12.5 μM etoposide, and 150 μM H2O2, respectively, and persisted for at least 4h. Transfection of a Plk1 constitutively active (c/a) mutant (T210D) abrogated both Cr(VI)- and etoposide-induced DNA DSBs as early as 30 min for the duration of treatment, in comparison to the vector-transfected cells. In sharp contrast, Plk1 had no effect on H2O2-induced DNA DSBs. Consistent with DNA DSB formation, Cr(VI) exposure was associated with G1 arrest, at least at 4h, as determined by BrdU incorporation. Moreover, Plk1 enhanced S phase progression during Cr(VI) treatment, which was accompanied by increased replication fork velocity and longer inter-origin distances as determined by using a single-molecule approach based on molecular combing. Expression of a Plk1 “kinase-dead” mutant (K82R) also abrogated DNA DSBs during Cr(VI) treatment, suggesting that this effect may be independent of Plk1 kinase activity. In summary, our data underline the ability of Plk1 to override DNA DSB-induced checkpoints, and to decrease DSBs after replication-stalling genotoxin exposure. We postulate these effects to be due to the replication-enhancing ability of Plk1, which may occur at the expense of genomic stability. This work was supported in part by NIH grants CA107972 and ES017334 (S.C.); ES05304 (S.R.P.); and a PhRMA Foundation predoctoral fellowship (G.C.K.).

Citation Format: Daniel Gebreselassie, Gina Chun-Kost, Elisabetta Leo, Yves Pommier, Erika Saito, Steven Patierno, Susan M. Ceryak. Plk1 decreases DNA double-strand break (DSB) formation during replication-stalling genotoxic stress. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 578. doi:10.1158/1538-7445.AM2013-578
Abstract 583: Ghrelingene expression avoiding the D-ribose caused HepG2 cell death.
Authors: Shiu Jia Huei , Huang Chin Hao , Lee Tai Lin , Lee Shih Chieh , Liu Shu Ying , Tsai Meng Feng
Published: August 2014

Liver cancer patients could suffer from high saccharide environment due to the malfunction of liver. To mimic this high saccharide environment, HepG2 cells were culture with 30 mM D-riboses.

We found that this high concentration of saccharide cause cell death. Cinnamon was report to reduce glucose in model rats. We hypothesized that the administration of cinnamon extracts may benefit liver cancer patients whose metabolism is defected. To test the hypothesis, three different chemical subtypes of cinnamon extracts were added into the culture medium. The adding of all three cinnamon extracts protected HepG2 cells from D-ribose administration and increased around 20% of surviving cells. These protections were even better than the addition of aminoguanidine hemisulfate salt, a chemical used in the treatment of diabetes. The expressions of ghrelin gene and two ghrelin-related genes was monitored by RT-PCR. Ghrelin gene expression was up-regulated in cinnamon treated cells. Also, the ghrelin modify gene, MBOAT4, and ghrelin process enzyme, furin, gene were all detected in HepG2 cells and cinnnamon treated cells.

These results demonstrated that if ghrelin expressed in HepG2 cell and maybe liver as well, it can be processed properly. Both ghrelin transcript variants 1 and 3 recombinant plasmid were transfected into HepG2 cells and both transcript variants protected HepG2 cells from the addition of D-ribose without the administration of cinnamon extracts. We conclude that the cinnamon avoids HepG2 cell damage from D-ribose induced oxidative stress by inducing ghrelin gene expression, and through the ghrelin signaling increases cell surviving. Since ghrelin level is abnormally regulated in patients with hepatocellular carcinoma and post-hepatitic liver cirrhosis, the understanding of how ghrelin been regulated in liver may benefit liver disease patients.

Citation Format: Jia Huei Shiu, Chin Hao Huang, Tai Lin Lee, Shih Chieh Lee, Shu Ying Liu, Meng Feng Tsai. Ghrelingene expression avoiding the D-ribose caused HepG2 cell death. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 583. doi:10.1158/1538-7445.AM2013-583
Abstract 585: Salmonella typhimurium A1-R targets quiescent cancer cells.
Authors: Yano Shuya , Zhang Yong , Uehara Fuminari , Hiroshima Yukihiko , Miwa Shinji , Hoffman Robert M. , Zhao Ming
Published: August 2014

Quiescent cancer cells are resistant to cytotoxic agents which target only proliferating cancer cells. The cell-cycle phase of each cancer cell in real time was imaged using a fluorescence ubiquitination cell cycle indicator (FUCCI). With FUCCI, quiescent cancer cells express mKusabira-Orange fluorescent protein (red) and proliferating cells express mAzami-Green fluorescent protein (green). FUCCI-labeled cancer cells in monolayer culture and tumor spheres were treated with Salmonella typhimurium A1-R. Time-lapse confocal imaging showed that A1-R encircled and invaded quiescent cancer cells in monolayer culture, thereby eliminating them. Cancer cells in tumor spheres in serum-free culture become and remain quiescent. A1-R infected and killed quiescent cancer cells in tumor spheres. In contrast, cytotoxic agents did not kill quiescent cancer cells in tumor spheres. A1-R infection of FUCCI-expressing subcutaneous tumors growing in nude mice resulted in killing quiescent cancer cells resistant to cytotoxic agents. This study demonstrates that A1-R can kill quiescent cancer cells and suggests a new therapeutic paradigm potentially more effective than current therapeutics which are ineffective against quiescent cancer cells.

Citation Format: Shuya Yano, Yong Zhang, Fuminari Uehara, Yukihiko Hiroshima, Shinji Miwa, Robert M. Hoffman, Ming Zhao. Salmonella typhimurium A1-R targets quiescent cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 585. doi:10.1158/1538-7445.AM2013-585
Abstract 589: NQO1-dependent anticancer action with combination β-lapachone and ionizing radiation in the head and neck cancer.
Authors: Li Long Shan , Reddy Srilakshmi , Gao Jinming , Boothman David A. , Sumer Baran , Yordy John S.
Published: August 2014

The number of new cases of oral cavity, pharynx and larynx cancers is estimated be 52,610 in 2012 with an expected 11,500 deaths. Despite progressive improvements in outcome, current treatment approaches still result in an overall survival rate of approximately 50% at five years for locally advanced head and neck cancer (HNC). Additional therapeutic approaches are needed to further enhance outcomes. NAD(P)H:quinone oxidoreductase 1 (NQO1) is a two-electron oxidoreductase that reduces quinones to hydroquinones and has been shown to be overexpressed in a number of solid tumors. For the first time, our studies in 45 HNC cell lines identified NQO1 overexpression in more than 70% of HNC cell lines. Furthermore over-expression of NQO1 has been shown to be a critical determinant for tumor-selective therapy using β-lapachone (β-lap), a substrate of NQO1 that undergoes partial reduction and spontaneous reversion, creating a futile cycle that produces very high levels of reactive oxygen species. HNC cell lines that overexpress NQO1 are significantly killed by treatment with β-lap. The anti-cancer action of β-lap in HNC cells is initiated and processed through five essential downstream mediators: (i) reactive oxygen species (ROS), particularly hydrogen peroxide (H2O2); (ii) DNA single strand breaks (SSBs) induced by ROS; (iii) hyperactivation of poly(ADP-ribose)polymerase-1 (PARP1) and accompanying poly(ADP-ribose)-PARP1 (PAR) formation; (iv) dramatic NAD+/ATP depletion; and (v) programmed necrosis (necroptosis). In combination with ionizing radiation (IR), β-Lap radiosensitized NQO1(+) HNC cells under conditions where nontoxic doses of either agent alone did not achieve threshold levels of DNA lesions and /or SSBs required for PARP-1 hyper-activation. Furthermore, combination therapy with β-lap and IR significantly enhanced γ-H2AX and 53BP1 foci formation and PARP-1 hyper-activation which was associated with ATP loss and induction of programmed cell death. Furthermore, the anticancer effects of combination β-Lap and IR were blocked by the NQO1 inhibitor dicoumarol. Our findings offer a strategy for the clinical utilization of β-lap as a radiosensitizer in HNC.

Citation Format: Long Shan Li, Srilakshmi Reddy, Jinming Gao, David A. Boothman, Baran Sumer, John S. Yordy. NQO1-dependent anticancer action with combination β-lapachone and ionizing radiation in the head and neck cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 589. doi:10.1158/1538-7445.AM2013-589
Abstract 590: Enhancing effect of resveratrol on the antileukemic effects of arsenic trioxide.
Authors: Wu Edward J. , Goussetis Dennis J. , Platanias Leonidas C.
Published: August 2014

Arsenic trioxide (As2O3) has been approved by the FDA for the treatment of APL, in which it has major clinical activity. However, the requirement of high doses to generate antileukemic effects has been a limitation for As2O3‘s potential use in other subtypes of AML. Given As2O3‘s potent antileukemic properties in vitro, approaches to enhance its effects are of clinical and translational relevance. Resveratrol (RSV), a phytoalexin found naturally in grapes and other plants, has wide-ranging health benefits, and in leukemias, has been shown to induce apoptosis and act synergistically with other agents. In studies to examine the influence of RSV on As2O3‘s antileukemic effects, we found that RSV enhances As2O3-induced cell death in KT1 cells as shown by flow cytometric and MTT analyses to assess apoptosis and cell viability. Our studies demonstrate that combined As2O3 and RSV treatment significantly reduces leukemic progenitor colony formation in a dose dependent manner by K562, KT1, and U937 cell lines. It also results in enhanced suppressessive effects on primary leukemic progenitors from patients with myeloid leukemias. Altogether, our results suggest that use of RSV may provide an approach to enhance the antileukemic effects of As2O3 treatment by potentiating the effects of lower As2O3 doses.

Citation Format: Edward J. Wu, Dennis J. Goussetis, Leonidas C. Platanias. Enhancing effect of resveratrol on the antileukemic effects of arsenic trioxide. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 590. doi:10.1158/1538-7445.AM2013-590
Abstract 596: Low-dose 1,25-dihydroxyvitamin D3 combined with arsenic trioxide synergistically inhibits proliferation of acute myeloid leukemia cells by promoting apoptosis.
Authors: Ji-Yeon Bae , Kim Ji-Won , Kim Inho
Published: August 2014

Arsenic trioxide (As2O3) has shown substantial efficacy in the treatment of patients with acute promyelocytic leukemia (APL). However, since not all patients can achieve remission after treatment, it is necessary to investigate a novel method to overcome the problem. We investigated the anti-leukemic effect of low-dose 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in combination with As2O3 on HL-60 and K562 cells. The cell viability was in reverse proportion to As2O3 or 1,25(OH)2D3 concentration. In both HL-60 and K562 cells, after the combination treatment with As2O3 and 1,25(OH)2D3 at a 10:1 ratio, the combination index values were less than 1 in all treatment groups. In the RT-PCR and Western blot analysis, the combination treatment decreased Bcl-2 expression and increased Bax and Caspase-3 expressions more prominently than the single treatment. In the flow cytometric analysis performed in HL-60 cells, the proportion of late apoptosis was 4.9% in control, 30.0% in 1.0 μM As2O3, 8.1% in 100nM 1,25(OH)2D3, and 64.3% in 1.0 μM As2O3 plus 100 nM 1,25(OH)2D3. In conclusion, low-dose 1,25(OH)2D3 combined with As2O3 synergistically inhibited proliferation of HL-60 and K562 cells. In addition, this combination activated the apoptosis pathway more prominently than the single-drug treatment.

Citation Format: Bae Ji-Yeon, Ji-Won Kim, Inho Kim. Low-dose 1,25-dihydroxyvitamin D3 combined with arsenic trioxide synergistically inhibits proliferation of acute myeloid leukemia cells by promoting apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 596. doi:10.1158/1538-7445.AM2013-596
Abstract 597: The combination of valproic acid and fludarabine treatment induces a synergistic apoptotic response in chronic lymphocytic leukemia (CLL) patients involving the lysosomal protease cathepsin B.
Authors: Yoon Ju Yoon , Szwajcer David , Ishdorj Ganchimeg , Benjaminson Pat , Kumar Rajat , Johnston James B. , Gibson Spencer B.
Published: August 2014

Chronic Lymphocytic Leukaemia (CLL) is the most common hematological malignancy in the western world. Fludarabine, a nucleoside analogue, is commonly used in combinational treatments for CLL. However, patients commonly develop resistance to fludarabine based therapies. Valproic Acid (VPA), an inhibitor of histone deacetylases (HDACs), showed synergistic apoptotic responses when combined with fludarabine in human leukemic cells and primary CLL cells. The mechanism for this synergistic apoptotic response is unknown. We found that fludarabine and VPA treatment increased activates caspases-8, -9, -3, and caspase-2 and a decreased in expression of anti-apoptotic proteins Mcl-1 and XIAP. Treatment with fludarabine alone or in combination with VPA led to the loss of lysosome integrity suggesting a leakage of the lysosomal content into the cytosol in response to these drugs. Chemical inhibition of a specific lysosomal protease, cathepsin B, using CA074-Me was sufficient to stabilize Mcl-1 and XIAP while reducing caspase activation and apoptosis. Addition of purified cathepsin B to leukemic cell lysates led to the reduction in protein levels of Mcl-1, XIAP and pro-caspase-2, thus suggesting that the re-localization of cathepsin B into the cytosol is sufficient to degrade these proteins. VPA treatment increased cathepsin B levels in both leukemic cell lines and primary CLL cells. VPA also increased cathepsin B activity. In addition, six relapsed CLL patients who had received at least one prior therapy with fludarabine were treated with VPA alone or in combination with fludarabine. No responses were seen after 28 days using VPA alone but in five patients who continued on VPA with fludarabine, three patients showed reduced lymphocyte count after receiving at most five cycles of the combination therapy. When the CLL cells from VPA-treated CLL patients were examined, VPA administration induced increased levels of histone-3 acetylation and cathepsin B expression in vivo. Thus, VPA and fludarabine synergistic apoptotic response is mediated by cathepsin B activation leading to a decrease in anti-apoptotic proteins in CLL cells and likely contributes to the clinical response observed in relapsed, fludarabine-refractory CLL patients.

Citation Format: Ju Yoon Yoon, David Szwajcer, Ganchimeg Ishdorj, Pat Benjaminson, Rajat Kumar, James B. Johnston, Spencer B. Gibson. The combination of valproic acid and fludarabine treatment induces a synergistic apoptotic response in chronic lymphocytic leukemia (CLL) patients involving the lysosomal protease cathepsin B. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 597. doi:10.1158/1538-7445.AM2013-597
Abstract 60: The MedBook network for cancer precision medical care.
Authors: Goldstein Theodore C. , Baertsch Robert , Febbo Phillip G. , Stuart Josh , Haussler David
Published: August 2014

Precision cancer care will connect clinical information to genomic information. To facilitate this connection, we are creating MedBook, a social networking system that links patients, physicians, and researchers in the battle against cancer. The Stand Up To Cancer West Coast Dream Team is first creating a bioinformatic platform that will run statistical and machine learning data analysis in a cloud-computing environment applied first to castration resistant prostate cancer and then share the data analysis results with oncologists and their patients. Patients, physicians, and researchers can also share sample data, high-throughput genomic discoveries, and their personal experiences in a safe structured environment covering research, disease course, and treatment. All data will be extracted from HIPAA repositories and anonymized and retain institutional review board controls. The social media model allows both the capture of unstructured information as well as collaborative discussions, annotation, and editing of structured information, to learn from each patient. Eventually, MedBook will collect knowledge about the experience of the disease from individual patients, from their treatments, and from their outcomes, and store them alongside relevant genomic molecular data. Our approach allows a variety of high-throughput genomic information to be generated and shared in a wiki-style collaborative network. This presentation will summarize the current state of MedBook and its utilization by a team of doctors and researchers across five medical centers and many university laboratories.

Citation Format: Theodore C. Goldstein, Robert Baertsch, Phillip G. Febbo, Josh Stuart, David Haussler. The MedBook network for cancer precision medical care. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 60. doi:10.1158/1538-7445.AM2013-60

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.