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1.  Longitudinal Bioluminescence Imaging of Primary versus Abdominal Metastatic Tumor Growth in Orthotopic Pancreatic Tumor Models in NSG Mice 
Pancreas  2015;44(1):64-75.
The purpose of the present study was to develop and validate noninvasive bioluminescence imaging methods for differentially monitoring primary and abdominal metastatic tumor growth in mouse orthotopic models of pancreatic cancer.
A semiautomated maximum entropy segmentation method was implemented for the primary tumor region-of-interest, and a rule-based method for manually drawing a region-of-interest for the abdominal metastatic region was developed for monitoring tumor growth in orthotopic models of pancreatic cancer. The two region-of-interest methods were validated by having two observers independently segment Panc-1 tumors, and the results compared with the number of mesenteric lymph node nodules, and histopathological assessment of liver metastases. The findings were extended to orthotopic tumors of the more metastatic MIA PaCa-2 and AsPC-1 cells where separate groups of animals were implanted with different numbers of cells.
The results demonstrated that the segmentation methods were highly reliable, reproducible and robust, and allowed statistically significant discrimination in the growth rates of primary and abdominal metastatic tumors of different cell lines implanted with different numbers of cells.
The present results demonstrate that primary tumors and abdominal metastatic foci in orthotopic pancreatic cancer models can be reliably quantified separately and noninvasively over time with bioluminescence imaging.
PMCID: PMC4262664  PMID: 25406955
Bioluminescence Imaging; Molecular Imaging; Pancreatic cancer; Metastases; ROI Segmentation; NSG Mice
2.  Emerging targets for glioblastoma stem cell therapy 
Journal of Biomedical Research  2015;30(1):19-31.
Glioblastoma multiforme (GBM), designated as World Health Organization (WHO) grade IV astrocytoma, is a lethal and therapy-resistant brain cancer comprised of several tumor cell subpopulations, including GBM stem cells (GSCs) which are believed to contribute to tumor recurrence following initial response to therapies. Emerging evidence demonstrates that GBM tumors are initiated from GSCs. The development and use of novel therapies including small molecule inhibitors of specific proteins in signaling pathways that regulate stemness, proliferation and migration of GSCs, immunotherapy, and non-coding microRNAs may provide better means of treating GBM. Identification and characterization of GSC-specific signaling pathways would be necessary to identify specific therapeutic targets which may lead to the development of more efficient therapies selectively targeting GSCs. Several signaling pathways including mTOR, AKT, maternal embryonic leucine zipper kinase (MELK), NOTCH1 and Wnt/β-catenin as well as expression of cancer stem cell markers CD133, CD44, Oct4, Sox2, Nanog, and ALDH1A1 maintain GSC properties. Moreover, the data published in the Cancer Genome Atlas (TCGA) specifically demonstrated the activated PI3K/AKT/mTOR pathway in GBM tumorigenesis. Studying such pathways may help to understand GSC biology and lead to the development of potential therapeutic interventions to render them more sensitive to chemotherapy and radiation therapy. Furthemore, recent demonstration of dedifferentiation of GBM cell lines into CSC-like cells prove that any successful therapeutic agent or combination of drugs for GBM therapy must eliminate not only GSCs, but the differentiated GBM cells and the entire bulk of tumor cells.
PMCID: PMC4726830  PMID: 26616589
glioblastoma multiforme; stem cells; dedifferentiation; CD133; CD44; ALDH1A1; SOX2
3.  Temozolomide-mediated DNA methylation in human myeloid precursor cells:differential involvement of intrinsic and extrinsic apoptotic pathways 
An understanding of how hematopoietic cells respond to therapy that causes myelosuppression will help develop approaches to prevent this potentially life-threatening toxicity. The goal of this study was to determine how human myeloid precursor cells (MP) respond to temozolomide (TMZ)-induced DNA damage.
Experimental Design
We developed an ex vivo primary human MP cells model system to investigate the involvement of cell-death pathways using a known myelosuppressive regimen of O6-benzylguanine (6BG) and TMZ.
Exposure to 6BG/TMZ led to increases in p53, p21, γ-H2AX, and mitochondrial DNA damage. Increases in mitochondrial membrane depolarization correlated with increased caspase-9 and caspase-3 activities following 6BG/TMZ treatment. These events correlated with decreases in activated AKT, downregulation of the DNA repair protein O6methylguanine-DNA methyltransferase (MGMT), and increased cell death. During MP cell expansion, FAS/CD95/APO1(FAS) expression increased over time and was present on ~100% of the cells following exposure to 6BG/TMZ. While c-flipshort, an endogenous inhibitor of FAS-mediated signaling, was decreased in 6BG/TMZ-treated versus control, 6BG-, or TMZ alone-treated cells, there were no changes in caspase-8 activity. Additionally, there were no changes in the extent of cell death in MP cells exposed to 6BG/TMZ in the presence of neutralizing or agonistic anti-FAS antibodies, indicating that FAS-mediated signaling was not operative.
In human MP cells, 6BG/TMZ-initiated apoptosis occurred by intrinsic, mitochondrial-mediated and not extrinsic, FAS-mediated apoptosis. Human MP cells represent a clinically relevant model system for gaining insight into how hematopoietic cells respond to chemotherapeutics and offer an approach for selecting effective chemotherapeutic regimens with limited hematopoietic toxicity.
PMCID: PMC3711223  PMID: 23536437
4.  Glioblastoma stem cells (GSCs) epigenetic plasticity and interconversion between differentiated non-GSCs and GSCs 
Genes & Diseases  2015;2(2):152-163.
Cancer stem cells (CSCs) or cancer initiating cells (CICs) maintain self-renewal and multilineage differentiation properties of various tumors, as well as the cellular heterogeneity consisting of several subpopulations within tumors. CSCs display the malignant phenotype, self-renewal ability, altered genomic stability, specific epigenetic signature, and most of the time can be phenotyped by cell surface markers (e.g., CD133, CD24, and CD44). Numerous studies support the concept that non-stem cancer cells (non-CSCs) are sensitive to cancer therapy while CSCs are relatively resistant to treatment. In glioblastoma stem cells (GSCs), there is clonal heterogeneity at the genetic level with distinct tumorigenic potential, and defined GSC marker expression resulting from clonal evolution which is likely to influence disease progression and response to treatment. Another level of complexity in glioblastoma multiforme (GBM) tumors is the dynamic equilibrium between GSCs and differentiated non-GSCs, and the potential for non-GSCs to revert (dedifferentiate) to GSCs due to epigenetic alteration which confers phenotypic plasticity to the tumor cell population. Moreover, exposure of the differentiated GBM cells to therapeutic doses of temozolomide (TMZ) or ionizing radiation (IR) increases the GSC pool both in vitro and in vivo. This review describes various subtypes of GBM, discusses the evolution of CSC models and epigenetic plasticity, as well as interconversion between GSCs and differentiated non-GSCs, and offers strategies to potentially eliminate GSCs.
PMCID: PMC4484766  PMID: 26137500
Cancer stem cells; Epigenetic; GBM plasticity; Glioblastoma; Stemness; Dedifferentiation; GBM stem cells
5.  Humanized bone-marrow mouse model as a pre-clinical tool to assess therapy-mediated hematotoxicity 
Pre-clinical in vivo studies can help guide the selection of agents and regimens for clinical testing. However, one of the challenges in screening anti-cancer therapies is the assessment of off-target human toxicity. There is a need for in vivo models that can simulate efficacy and toxicities of promising therapeutic regimens. For example, hematopoietic cells of human origin are particularly sensitive to a variety of chemotherapeutic regimens but in vivo models to assess potential toxicities have not been developed. In this study, a xenograft model containing humanized bone marrow is utilized as an in vivo assay to monitor hematotoxicity.
Experimental Design
A proof-of-concept, temozolomide-based regimen was developed that inhibits tumor xenograft growth. This regimen was selected for testing since it has been previously shown to cause myelosuppression in mice and humans. The dose-intensive regimen was administered to NOD/SCID/γchainnull mice reconstituted with human hematopoietic cells and the impact of treatment on human hematopoiesis was evaluated.
The dose-intensive regimen resulted in significant decreases in growth of human-glioblastoma xenografts. When this regimen was administered to mice containing humanized bone marrow, flow cytometric analyses indicated that the human bone-marrow cells were significantly more sensitive to treatment than the murine bone-marrow cells, and that the regimen was highly toxic to human-derived hematopoietic cells of all lineages (progenitor, lymphoid, and myeloid).
The humanized bone-marrow xenograft model described has the potential to be used as a platform for monitoring the impact of anti-cancer therapies on human hematopoiesis and could lead to subsequent refinement of therapies prior to clinical evaluation.
PMCID: PMC3078977  PMID: 21487065
human xenograft model; myelosuppression; stem-cell
6.  Differential Secondary Reconstitution of In Vivo-Selected Human SCID-Repopulating Cells in NOD/SCID versus NOD/SCID/γ chainnull Mice 
Bone Marrow Research  2010;2011:252953.
Humanized bone-marrow xenograft models that can monitor the long-term impact of gene-therapy strategies will help facilitate evaluation of clinical utility. The ability of the murine bone-marrow microenvironment in NOD/SCID versus NOD/SCID/γ chainnull mice to support long-term engraftment of MGMTP140K-transduced human-hematopoietic cells following alkylator-mediated in vivo selection was investigated. Mice were transplanted with MGMTP140K-transduced CD34+ cells and transduced cells selected in vivo. At 4 months after transplantation, levels of human-cell engraftment, and MGMTP140K-transduced cells in the bone marrow of NOD/SCID versus NSG mice varied slightly in vehicle- and drug-treated mice. In secondary transplants, although equal numbers of MGMTP140K-transduced human cells were transplanted, engraftment was significantly higher in NOD/SCID/γ chainnull mice compared to NOD/SCID mice at 2 months after transplantation. These data indicate that reconstitution of NOD/SCID/γ chainnull mice with human-hematopoietic cells represents a more promising model in which to test for genotoxicity and efficacy of strategies that focus on manipulation of long-term repopulating cells of human origin.
PMCID: PMC3200073  PMID: 22046557
7.  A Heterobivalent Ligand Inhibits Mast Cell Degranulation via Selective Inhibition of Allergen-IgE Interactions In Vivo 
Current treatments for allergies include epinephrine and anti-histamines which treat the symptoms after an allergic response has taken place, steroids that result in local and systemic immune suppression and IgE-depleting therapies which can only be used for a narrow range of clinical IgE titers. The limitations of current treatments motivated the design of a heterobivalent inhibitor (HBI) of IgE-mediated allergic responses that selectively inhibits allergen-IgE interactions, thereby preventing IgE clustering and mast cell degranulation. The HBI was designed to simultaneously target the allergen binding site and the adjacent conserved nucleotide binding site (NBS) found on the Fab of IgE antibodies. The bivalent targeting was accomplished by linking a hapten to an NBS ligand with an ethylene glycol linker. The hapten moiety of HBI enables selective targeting of a specific IgE while the NBS ligand enhances the avidity for the IgE. Simultaneous bivalent binding to both sites provided HBI with 120 fold enhancement in avidity for the target IgE compared to the monovalent hapten. The increased avidity for IgE made HBI a potent inhibitor of mast cell degranulation in the rat basophilic leukemia (RBL) mast cell model, in the passive cutaneous anaphylaxis (PCA) mouse model of allergy, and in mice sensitized to the model allergen. Additionally, HBI did not have any observable systemic toxic effects even at elevated doses. Taken together, these results establish the HBI design as a broadly applicable platform with therapeutic potential for the targeted and selective inhibition of IgE-mediated allergic responses including food, environmental, and drug allergies.
PMCID: PMC3951166  PMID: 24489096
8.  In vivo selection of hematopoietic stem cells transduced at a low multiplicity-of-infection with a foamy viral MGMTP140K vector 
Experimental hematology  2008;36(3):283-292.
Using a clinically relevant transduction strategy, we investigated to what extent hematopoietic stem cells in lineage-negative bone marrow (Linneg BM) could be genetically modified with a FV vector that expresses the DNA repair protein, O6-methylguanine DNA methyltransferase (MGMTP140K) and selected in vivo with submyeloablative versus myeloablative alkylator therapy.
Linneg BM was transduced at a low multiplicity-of-infection (MOI), with the FV vector, MD9-P140K, that co-expresses MGMTP140K and the enhanced green fluorescent protein, transplanted into C57BL/6 mice, and mice treated with submyeloablative or myeloablative alkylator therapy. The BM was analyzed for the presence of in vivo selected, MD9-P140K-transduced cells at 6 months post-transplantation and subsequently transplanted into secondary recipient animals.
Following submyeloablative therapy, 55% of the mice expressed MGMTP140K in the BM. Proviral integration was observed in ∼50% of committed BM-derived progenitors and analysis of proviral insertion sites indicated up to 2 integrations per transduced progenitor colony. Transduced BM cells selected with submyeloablative therapy reconstituted secondary recipient mice for up to 6 months post-transplantation. In contrast, following delivery of myeloablative therapy to primary recipient mice, only 25% survived. Hematopoietic stem cells were transduced since BM cells from the surviving animals reconstituted secondary recipients with MGMTP140K positive cells for 5-6 months.
In vivo selection of MD9-P140K-transduced BM cells was more efficient following submyeloablative versus myeloablative therapy. These data indicate that a critical number of transduced-stem cells must be present to produce sufficient numbers of genetically modified progeny to protect against the acute toxicity associated with myeloablative therapy.
PMCID: PMC2699892  PMID: 18279716
gene therapy; hematopoietic stem cells; foamy virus vector; O6-methylguanine DNA methyltransferase (MGMT)
9.  In Vivo Measurements of Tumor Metabolism and Growth after Administration of Enzastaurin Using Small Animal FDG Positron Emission Tomography 
Journal of Oncology  2009;2009:596560.
Background. The use of 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) may help to establish the antitumor activity of enzastaurin, a novel protein kinase C-beta II (PKC-βII) inhibitor, in mouse xenografts. Methods. The hematologic cell line RAJI and the solid tumor cell line U87MG were each implanted in NOD/SCID mice. Standard tumor growth measurements and [18F]FDG PET imaging were performed weekly for up to three weeks after tumor implantation and growth. Results. Concomitant with caliper measurements, [18F]FDG PET imaging was performed to monitor glucose metabolism. Heterogeneity of glucose uptake in various areas of the tumors was observed after vehicle or enzastaurin treatment. This heterogeneity may limit the use of [18F]FDG PET imaging to measure enzastaurin-associated changes in xenograft tumors. Conclusion. [18F]FDG PET imaging technique does not correlate with standard caliper assessments in xenografts to assess the antitumor activity of enzastaurin. Future studies are needed to determine the use of [18F]FDG PET imaging in preclinical models.
PMCID: PMC2688651  PMID: 19503801
11.  Human Proangiogenic Circulating Hematopoietic Stem and Progenitor Cells Promote Tumor Growth in an Orthotopic Melanoma Xenograft Model 
Angiogenesis  2013;16(4):953-962.
We previously identified a distinct population of human circulating hematopoietic stem and progenitor cells (CHSPCs; CD14-glyA-CD34+AC133+/-CD45dimCD31+ cells) in the peripheral blood (PB) and bone marrow, and their frequency in the PB can correlate with disease state. The proangiogenic subset (pCHSPC) play a role in regulating tumor progression, for we previously demonstrated a statistically significant increase in C32 melanoma growth in NOD.Cg-Prkdcscid (NOD/SCID) injected with human pCHSPCs (p<0.001). We now provide further evidence that pCHSPCs possess proangiogenic properties. In vitro bio-plex cytokine analyses and tube forming assays indicate that pCHSPCs secrete a proangiogenic profile and promote vessel formation respectively. We also developed a humanized bone marrow-melanoma orthotopic model to explore in vivo the biological significance of the pCHSPC population. Growth of melanoma xenografts increased more rapidly at 3-4 weeks post-tumor implantation in mice previously transplanted with human CD34+ cells compared to control mice. Increases in pCHSPCs in PB correlated with increases in tumor growth. Additionally, to determine if we could prevent the appearance of pCHSPCs in the PB, mice with humanized bone marrow-melanoma xenografts were administered Interferon α-2b, which is used clinically for treatment of melanoma. The mobilization of the pCHSPCs was decreased in the mice with the humanized bone marrow-melanoma xenografts. Taken together, these data indicate that pCHSPCs play a functional role in tumor growth. The novel in vivo model described here can be utilized to further validate pCHSPCs as a biomarker of tumor progression. The model can also be used to screen and optimize anticancer/anti-angiogenic therapies in a humanized system.
PMCID: PMC4128550  PMID: 23877751
proangiogenic; circulating hematopoietic stem and progenitor cells; biomarker; tumor growth
12.  Multimodality Imaging Methods for Assessing Retinoblastoma Orthotopic Xenograft Growth and Development 
PLoS ONE  2014;9(6):e99036.
Genomic studies of the pediatric ocular tumor retinoblastoma are paving the way for development of targeted therapies. Robust model systems such as orthotopic xenografts are necessary for testing such therapeutics. One system involves bioluminescence imaging of luciferase-expressing human retinoblastoma cells injected into the vitreous of newborn rat eyes. Although used for several drug studies, the spatial and temporal development of tumors in this model has not been documented. Here, we present a new model to allow analysis of average luciferin flux () through the tumor, a more biologically relevant parameter than peak bioluminescence as traditionally measured. Moreover, we monitored the spatial development of xenografts in the living eye. We engineered Y79 retinoblastoma cells to express a lentivirally-delivered enhanced green fluorescent protein-luciferase fusion protein. In intravitreal xenografts, we assayed bioluminescence and computed , as well as documented tumor growth by intraocular optical coherence tomography (OCT), brightfield, and fluorescence imaging. In vivo bioluminescence, ex vivo tumor size, and ex vivo fluorescent signal were all highly correlated in orthotopic xenografts. By OCT, xenografts were dense and highly vascularized, with well-defined edges. Small tumors preferentially sat atop the optic nerve head; this morphology was confirmed on histological examination. In vivo, in xenografts showed a plateau effect as tumors became bounded by the dimensions of the eye. The combination of modeling and in vivo intraocular imaging allows both quantitative and high-resolution, non-invasive spatial analysis of this retinoblastoma model. This technique will be applied to other cell lines and experimental therapeutic trials in the future.
PMCID: PMC4047070  PMID: 24901248
13.  Peripheral blood biomarkers of solid tumor angiogenesis in dogs: A polychromatic flow cytometry pilot study 
A subset of peripheral blood hematopoietic stem and progenitor cells of bone marrow origin is elevated in humans with solid cancers before treatment and declines with therapy. This biomarker of angiogenesis is not specific to tumor type and has great potential in the objective assessment of treatment response in clinical trials. This pilot study was designed to develop a biomarker of neoangiogenesis in dogs for the diagnosis of cancer, the measurement of treatment response, and the provision of objective data in clinical trials. Polychromatic flow cytometry was used to quantify two subsets of circulating hematopoietic stem and progenitor cells in dogs with spontaneous solid tumors before (n = 8) and after (n = 3) treatment, and normal controls (n = 6).
Pro-angiogenic peripheral blood cells of bone marrow origin were detected in all eight cases and the six normal controls; however, there was no statistically significant difference between the two groups. Interestingly, an apparent decline in pro-angiogenic cells was observed after treatment. Bone marrow derived hematopoietic cells appear to contribute to tumor angiogenesis in dogs, as has been previously reported in humans. While the methodology for pro-angiogenic cell quantification in a small number of dogs in the current study did not result in a significant difference from normal controls, an optimized canine polychromatic flow cytometry protocol holds great promise in the development of a canine cancer model and for the objective measurements of treatment response in clinical trials.
PMCID: PMC3570660  PMID: 23063489
Dog; Cancer; Biomarker; Angiogenesis; Polychromatic flow cytometry
14.  Small-Molecule Inhibition of the uPAR·uPA Interaction: Synthesis, Biochemical, Cellular, in vivo Pharmacokinetics and Efficacy Studies in Breast Cancer Metastasis 
Bioorganic & medicinal chemistry  2013;21(7):2145-2155.
The uPAR·uPA protein-protein interaction (PPI) is involved in signaling and proteolytic events that promote tumor invasion and metastasis. A previous study had identified 4 (IPR-803) from computational screening of a commercial chemical library and shown that the compound inhibited uPAR·uPA PPI in competition biochemical assays and invasion cellular studies. Here, we synthesize 4 to evaluate in vivo pharmacokinetic (PK) and efficacy studies in a murine breast cancer metastasis model. First, we show, using fluorescence polarization and saturation transfer difference (STD) NMR, that 4 binds directly to uPAR with sub-micromolar affinity of 0.2 μM. We show that 4 blocks invasion of breast MDA-MB-231, and inhibits matrix metalloproteinase (MMP) breakdown of the extracellular matrix (ECM). Derivatives of 4 also inhibited MMP activity and blocked invasion in a concentration-dependent manner. 4 also impaired MDA-MB-231 cell adhesion and migration. Extensive in vivo PK studies in NOD-SCID mice revealed a half-life of nearly 5 hours and peak concentration of 5 μM. Similar levels of the inhibitor were detected in tumor tissue up to 10 hours. Female NSG mice inoculated with highly malignant TMD-MDA-MB-231 in their mammary fat pads showed that 4 impaired metastasis to the lungs with only four of the treated mice showing severe or marked metastasis compared to ten for the untreated mice. Compound 4 is a promising template for the development of compounds with enhanced PK parameters and greater efficacy.
PMCID: PMC3625246  PMID: 23411397
15.  Soypeptide lunasin in cytokine immunotherapy for lymphoma 
Cancer Immunology, Immunotherapy  2013;63(3):283-295.
Immunostimulatory cytokines can enhance anti-tumor immunity and are part of the therapeutic armamentarium for cancer treatment. We have previously reported that post-transplant lymphoma patients have an acquired deficiency of signal transducer and activator of transcription 4, which results in defective IFNγ production during clinical immunotherapy. With the goal of further improving cytokine-based immunotherapy, we examined the effects of a soybean peptide called lunasin that synergistically works with cytokines on natural killer (NK) cells. Peripheral blood mononuclear cells of healthy donors and post-transplant lymphoma patients were stimulated with or without lunasin in the presence of IL-12 or IL-2. NK activation was evaluated, and its tumoricidal activity was assessed using in vitro and in vivo tumor models. Chromatin immunoprecipitation assay was performed to evaluate the histone modification of gene loci that are regulated by lunasin and cytokine. Adding lunasin to IL-12- or IL-2-stimulated NK cells demonstrated synergistic effects in the induction of IFNG and GZMB involved in cytotoxicity. The combination of lunasin and cytokines (IL-12 plus IL-2) was capable of restoring IFNγ production by NK cells from post-transplant lymphoma patients. In addition, NK cells stimulated with lunasin plus cytokines displayed higher tumoricidal activity than those stimulated with cytokines alone using in vitro and in vivo tumor models. The underlying mechanism responsible for the effects of lunasin on NK cells is likely due to epigenetic modulation on target gene loci. Lunasin represents a different class of immune modulating agent that may augment the therapeutic responses mediated by cytokine-based immunotherapy.
PMCID: PMC3928510  PMID: 24363024
Lunasin; NK; Cytokine immunotherapy; Lymphoma
16.  Virtual Screening Targeting the Urokinase Receptor, Biochemical and Cell-Based Studies, Synthesis, Pharmacokinetic Characterization, and Effect on Breast Tumor Metastasis 
Journal of Medicinal Chemistry  2011;54(20):7193-7205.
Virtual screening targeting the urokinase receptor (uPAR) led to (3R)-4-cyclohexyl-3-(hexahydrobenzo[d][1,3]dioxol-5-yl)-N-((hexahydrobenzo[d][1,3]dioxol-5-yl)methyl)butan-1-aminium 1 (IPR-1) and 4-(4-((3,5-dimethylcyclohexyl)carbamoyl)-2-(4-isopropylcyclohexyl)pyrazolidin-3-yl)piperidin-1-ium 3 (IPR-69). Synthesis of an analog of 1, namely 2 (IPR-9), and 3 led to breast MDA-MB-231 invasion, migration and adhesion assays with IC50 near 30 μM. Both compounds blocked angiogenesis with IC50 of 3 μM. Compounds 2 and 3 inhibited cell growth with IC50 of 6 and 18 μM and induced apoptosis. Biochemical assays revealed lead-like properties for 3, but not 2. Compound 3 administered orally reached peak concentration of nearly 40 μM with a half-life of about 2 hours. In NOD-SCID mice inoculated with breast TMD-231 cells in their mammary fat pads, compound 3 showed a 20% reduction in tumor volumes and less extensive metastasis was observed for the treated mice. The suitable pharmacokinetic properties of 3 and the encouraging preliminary results in metastasis make it an ideal starting point for next generation compounds.
PMCID: PMC3280887  PMID: 21851064
17.  Impact of APE1/Ref-1 Redox Inhibition on Pancreatic Tumor Growth 
Molecular cancer therapeutics  2011;10(9):1698-1708.
Pancreatic cancer is an especially deadly form of cancer with a survival rate <2%. Pancreatic cancers respond poorly to existing chemotherapeutic agents and radiation, and progress for the treatment of pancreatic cancer remains elusive. To address this unmet medical need, a better understanding of critical pathways and molecular mechanisms involved in pancreatic tumor development, progression and resistance to traditional therapy is therefore critical. Reduction-oxidation (redox) signaling systems are emerging as important targets in pancreatic cancer. AP endonuclease1/ Redox effector factor 1 (APE1/Ref-1) is upregulated in human pancreatic cancer cells and modulation of its redox activity blocks the proliferation and migration of pancreatic cancer cells as well as pancreatic cancer-associated endothelial cells (PCECs) in vitro. Modulation of APE1/Ref-1 using a specific inhibitor of APE1/Ref-1’s redox function, E3330 leads to a decrease in transcription factor activity for NFκB, AP-1, and HIF1 in vitro. This study aims to further establish the redox signaling protein APE1/Ref-1 as a molecular target in pancreatic cancer. Here, we show that inhibition of APE1/Ref-1 via E3330 results in tumor growth inhibition in cell lines as well as pancreatic cancer xenograft models in mice. Pharmacokinetic (PK) studies also demonstrate that E3330 attains >10 μM blood concentrations and is detectable in tumor xenografts. Through inhibition of APE1/Ref-1, the activity of NFκB, AP-1, and HIF1α which are key transcriptional regulators involved in survival, invasion and metastasis is blocked. These data indicate that E3330, inhibitor of APE1/Ref-1, has potential in pancreatic cancer and clinical investigation of APE1/Ref-1 molecular target is warranted.
PMCID: PMC3170439  PMID: 21700832
Pancreatic cancer; animal models of cancer; new targets; xenograft models; cellular responses to anticancer drugs; cellular pharmacology; pharmacokinetics and pharmacodynamics; agents with other mechanisms of action
18.  Targeting the Anti-Apoptotic Protein c-FLIP for Cancer Therapy 
Cancers  2011;3(2):1639-1671.
Cellular FLICE (FADD-like IL-1beta-converting enzyme)-inhibitory protein (c-FLIP) is a major resistance factor and critical anti-apoptotic regulator that inhibits tumor necrosis factor-alpha (TNF-alpha), Fas-L, and TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis as well as chemotherapy-triggered apoptosis in malignant cells. c-FLIP is expressed as long (c-FLIPL), short (c-FLIPS), and c-FLIPR splice variants in human cells. c-FLIP binds to FADD and/or caspase-8 or -10 in a ligand-dependent and-independent fashion, which in turn prevents death-inducing signaling complex (DISC) formation and subsequent activation of the caspase cascade. Moreover, c-FLIPL and c-FLIPS are known to have multifunctional roles in various signaling pathways, as well as activating and/or upregulating several cytoprotective signaling molecules. Upregulation of c-FLIP has been found in various tumor types, and its downregulation has been shown to restore apoptosis triggered by cytokines and various chemotherapeutic agents. Hence, c-FLIP is an important target for cancer therapy. For example, small interfering RNAs (siRNAs) that specifically knockdown the expression of c-FLIPL in diverse human cancer cell lines augmented TRAIL-induced DISC recruitment and increased the efficacy of chemotherapeutic agents, thereby enhancing effector caspase stimulation and apoptosis. Moreover, small molecules causing degradation of c-FLIP as well as decreasing mRNA and protein levels of c-FLIPL and c-FLIPS splice variants have been found, and efforts are underway to develop other c-FLIP-targeted cancer therapies. This review focuses on (1) the functional role of c-FLIP splice variants in preventing apoptosis and inducing cytokine and drug resistance; (2) the molecular mechanisms that regulate c-FLIP expression; and (3) strategies to inhibit c-FLIP expression and function.
PMCID: PMC3281420  PMID: 22348197
c-FLIP; apoptosis; death receptors; cancer; chemotherapy
19.  Application of Polychromatic Flow Cytometry to Identify Novel Subsets of Circulating Cells with Angiogenic Potential 
Defining whether human circulating pro-angiogenic cells represent a subset of the hematopoietic system and express CD45 or are hematopoietic derivatives that do not express CD45 (and are called endothelial progenitor cells) remains controversial. We have previously developed a polychromatic flow cytometry (PFC) protocol to isolate subsets of hematopoietic cells and we now identify the circulating pool of CD34+CD45dim cells representing functional circulating hematopoietic stem and progenitor cells (CHSPCs) that can be separated on the basis of AC133 expression and report that the AC133+ subset of the CHSPCs enhances the growth of tumor blood vessels in vivo in immunodeficient mice. In addition, the ratio of AC133+ pro-angiogenic CHSPCs to AC133− non-angiogenic CHSPCs unambiguously correlates with the severity of the clinical state of patients with peripheral arterial disease. In sum, a PFC protocol validated via in vitro and in vivo analyses, can be used to interrogate the roles of human hematopoietic elements in the growth and maintenance of the vasculature.
PMCID: PMC2931367  PMID: 20803735
Polychromatic flow cytometry; circulating progenitor cells; endothelial progenitor cells; angiogenesis; peripheral arterial disease
20.  Targeting the Anti-Apoptotic Protein c-FLIP for Cancer Therapy 
Cancers  2011;3(2):1639-1671.
Cellular FLICE (FADD-like IL-1beta-converting enzyme)-inhibitory protein (c-FLIP) is a major resistance factor and critical anti-apoptotic regulator that inhibits tumor necrosis factor-alpha (TNF-alpha), Fas-L, and TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis as well as chemotherapy-triggered apoptosis in malignant cells. c-FLIP is expressed as long (c-FLIPL), short (c-FLIPS), and c-FLIPR splice variants in human cells. c-FLIP binds to FADD and/or caspase-8 or -10 in a ligand-dependent and-independent fashion, which in turn prevents death-inducing signaling complex (DISC) formation and subsequent activation of the caspase cascade. Moreover, c-FLIPL and c-FLIPS are known to have multifunctional roles in various signaling pathways, as well as activating and/or upregulating several cytoprotective signaling molecules. Upregulation of c-FLIP has been found in various tumor types, and its downregulation has been shown to restore apoptosis triggered by cytokines and various chemotherapeutic agents. Hence, c-FLIP is an important target for cancer therapy. For example, small interfering RNAs (siRNAs) that specifically knockdown the expression of c-FLIPL in diverse human cancer cell lines augmented TRAIL-induced DISC recruitment and increased the efficacy of chemotherapeutic agents, thereby enhancing effector caspase stimulation and apoptosis. Moreover, small molecules causing degradation of c-FLIP as well as decreasing mRNA and protein levels of c-FLIPL and c-FLIPS splice variants have been found, and efforts are underway to develop other c-FLIP-targeted cancer therapies. This review focuses on (1) the functional role of c-FLIP splice variants in preventing apoptosis and inducing cytokine and drug resistance; (2) the molecular mechanisms that regulate c-FLIP expression; and (3) strategies to inhibit c-FLIP expression and function.
PMCID: PMC3281420  PMID: 22348197
c-FLIP; apoptosis; death receptors; cancer; chemotherapy
21.  Enhanced homing and engraftment of fresh but not ex vivo cultured murine marrow cells in submyeloablated hosts following CD26 inhibition by Diprotin A 
Experimental hematology  2009;37(7):814-823.
We recently reported that murine marrow cultured ex vivo for gamma-retrovirus transduction engrafts ~10 fold less well than fresh marrow upon transplantation into submyeloablated hosts. Here, we evaluated homing efficiency as a potential mechanism for this engraftment disparity, and whether CD26 inhibition with the tripeptide Diprotin A (DipA) would enhance engraftment of ex vivo cultured cells in submyeloablated hosts.
Homing and engraftment of fresh and ex vivo cultured lineage-negative (lin-) marrow cells in submyeloablated congenic hosts with and without DipA treatment was evaluated. Expression of CXCR4 and CD26 on fresh and cultured lin- marrow cells was compared.
Homing of lin- cells cultured for gamma-retrovirus transduction was ≥3-fold less than that of fresh lin- cells 20 hours after transplantation into submyeloablated hosts. DipA treatment of fresh lin- cells resulted in ≥-fold increased homing and engraftment in submyeloablated hosts. DipA treatment, however, did not significantly improve homing or engraftment of cells undergoing a three-day culture protocol for gamma-retrovirus transduction in submyeloablated hosts. CXCR4 expression on lin- cells was significantly decreased following three days of culture; CXCR4 expression was not significantly altered following overnight culture.
Ex vivo culture of lin- cells for gamma-retroviral transduction downregulates CXCR4 expression and markedly impairs homing and engraftment of murine lin- marrow in submyeloablated hosts. While inhibition of CD26 activity with DipA increases homing and engraftment of fresh lin- cells, DipA treatment does not improve homing and engraftment of cultured lin- marrow cells in submyeloablated congenic hosts.
PMCID: PMC2700780  PMID: 19540435
22.  TGF-β1–induced expression of human Mdm2 correlates with late-stage metastatic breast cancer 
The E3 ubiquitin ligase human murine double minute (HDM2) is overexpressed in 40%–80% of late-stage metastatic cancers in the absence of gene amplification. Hdm2 regulates p53 stability via ubiquitination and has also been implicated in altering the sensitivity of cells to TGF-β1. Whether TGF-β1 signaling induces Hdm2 expression leading to HDM2-mediated destabilization of p53 has not been investigated. In this study, we report that TGF-β1–activated SMA- and MAD3 (Smad3/4) transcription factors specifically bound to the second promoter region of HDM2, leading to increased HDM2 protein expression and destabilization of p53 in human cancer cell lines. Additionally, TGF-β1 expression led to Smad3 activation and murine double minute 2 (Mdm2) expression in murine mammary epithelial cells during epithelial-to-mesenchymal transition (EMT). Furthermore, histological analyses of human breast cancer samples demonstrated that approximately 65% of late-stage carcinomas were positive for activated Smad3 and HDM2, indicating a strong correlation between TGF-β1–mediated induction of HDM2 and late-stage tumor progression. Identification of Hdm2 as a downstream target of TGF-β1 represents a critical prosurvival mechanism in cancer progression and provides another point for therapeutic intervention in late-stage cancer.
PMCID: PMC2798681  PMID: 19955655
23.  Involvement of Platelet-Activating Factor in Ultraviolet B-Induced Hyperalgesia 
Ultraviolet B (UVB) radiation causes cutaneous inflammation. One important clinical consequence of UVB-induced inflammation is increased pain or hyperalgesia, which is likely mediated by enhanced sensitivity of cutaneous sensory neurons. Previous studies have demonstrated that UVB radiation generates the lipid mediator, platelet-activating factor (PAF), as well as oxidized phospholipids that act as PAF-mimetics. These substances exert effects through the PAF receptor (PAF-R). This study was designed to assess whether PAF-R is involved in UVB-induced hyperalgesia. Intradermal injection of carbamoyl PAF (CPAF; 1-hexadecyl-2-N-methylcarbamoyl glycerophosphocholine) resulted in an enhanced response to mechanical stimuli in wild-type mice but not in PAF-R knockout (KO) mice. There was no significant change in paw withdrawal to noxious thermal stimuli in either genotype after intradermal injection of CPAF. Exposure of the hind paw to 1,500 J m−2 UVB radiation caused an increased sensitivity to both mechanical and thermal stimulation in wild-type mice but not in PAF-R KO mice. The thermal hyperalgesia caused by UVB irradiation was inhibited in mice that lacked PAF-R in bone marrow-derived cells. These data demonstrate that the PAF-R is important for UVB-induced hyperalgesia. Further investigation of the role of PAF-R signaling in UVB-induced hyperalgesia could provide better understanding of the pathological processes initiated by UVB-induced skin damage.
PMCID: PMC2717090  PMID: 18580961
24.  UVB Radiation-Mediated Inhibition of Contact Hypersensitivity Reactions Is Dependent on the Platelet-Activating Factor System 
Through its ability to both induce immunosuppression and act as a carcinogen, UVB radiation plays a major role in cutaneous malignancies. Recent studies have indicated that UVB-mediated inhibition of delayed-type hypersensitivity reactions is mediated, in part, by the lipid mediator platelet-activating factor (PAF). The objective of this study was to further define the mechanism by which UVB inhibits contact hypersensitivity (CHS) reactions. UVB irradiation resulted in an inhibition of subsequent CHS to the chemical DNFB in wild-type, but not in PAF-R-deficient mice. UVB-mediated inhibition of CHS was also blocked by a cyclooxygenase-2 (COX-2) inhibitor or a neutralizing antibody directed against IL-10. UVB irradiation upregulated IL-10 mRNA levels in lymph nodes and spleen only to significant levels in PAF-R-expressing mice. Bone marrow transplantation studies demonstrated that UVB-mediated immunomodulatory effects were dependent on PAF-R-positive bone marrow. These studies suggest that UVB irradiation results in epidermal production of PAF agonists, which then act on PAF-R-positive bone marrow-derived cells to upregulate IL-10 through COX-2-generated prostaglandins.
PMCID: PMC2643301  PMID: 18200048
25.  The Xpc gene markedly affects cell survival in mouse bone marrow 
Mutagenesis  2009;24(4):309-316.
The XPC protein (encoded by the xeroderma pigmentosum Xpc gene) is a key DNA damage recognition factor that is required for global genomic nucleotide excision repair (G-NER). In contrast to transcription-coupled nucleotide excision repair (TC-NER), XPC and G-NER have been reported to contribute only modestly to cell survival after DNA damage. Previous studies were conducted using fibroblasts of human or mouse origin. Since the advent of Xpc−/− mice, no study has focused on the bone marrow of these mice. We used carboplatin to induce DNA damage in Xpc−/− and strain-matched wild-type mice. Using several independent methods, Xpc−/− bone marrow was ∼10-fold more sensitive to carboplatin than the wild type. Importantly, 12/20 Xpc−/− mice died while 0/20 wild-type mice died. We conclude that G-NER, and XPC specifically, can contribute substantially to cell survival. The data are important in the context of cancer chemotherapy, where Xpc gene status and G-NER may be determinants of response to DNA-damaging agents including carboplatin. Additionally, altered cell cycles and altered DNA damage signalling may contribute to the cell survival end point.
PMCID: PMC2701989  PMID: 19372135

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