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1.  Potentiation of carboplatin-mediated DNA damage by the Mdm2 modulator Nutlin-3a in a humanized orthotopic breast-to-lung metastatic model 
Molecular cancer therapeutics  2015;14(12):2850-2863.
Triple-negative breast cancers (TNBCs) are typically resistant to treatment, and strategies that build upon frontline therapy are needed. Targeting the murine double minute 2 (Mdm2) protein is an attractive approach as Mdm2 levels are elevated in many therapy-refractive breast cancers. The Mdm2 protein-protein interaction inhibitor Nutlin-3a blocks the binding of Mdm2 to key signaling molecules such as p53 and p73α, and can result in activation of cell-death signaling pathways. In the present study, the therapeutic potential of carboplatin and Nutlin-3a to treat TNBC was investigated, as carboplatin is under evaluation in clinical trials for TNBC. In mutant p53 TMD231 TNBC cells, carboplatin and Nutlin-3a led to increased Mdm2 and was strongly synergistic in promoting cell death in vitro. Furthermore, sensitivity of TNBC cells to combination treatment was dependent on p73α. Following combination treatment, γH2AX increased and Mdm2 localized to a larger degree to chromatin compared to single-agent treatment, consistent with previous observations that Mdm2 binds to the Mre11/Rad50/Nbs1 complex associated with DNA and inhibits the DNA-damage response. In vivo efficacy studies were conducted in the TMD231 orthotopic mammary fat pad model in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Using an intermittent dosing schedule of combined carboplatin and Nutlin-3a, there was a significant reduction in primary tumor growth and lung metastases compared to vehicle and single-agent treatments. Additionally, there was minimal toxicity to the bone marrow and normal tissues. These studies demonstrate that Mdm2 holds promise as a therapeutic target in combination with conventional therapy and may lead to new clinical therapies for TNBC.
PMCID: PMC4674357  PMID: 26494859
Breast cancer; Animal models of cancer; Noninvasive imaging in animal models; Other tumor suppressor genes; Combination chemotherapy; Xenograft models; Cellular responses to anticancer drugs; Modulation of DNA repair; Reversal of drug resistance; Preclinical toxicology; Mdm2; carboplatin; p73
Health physics  2015;109(5):511-521.
The threat of radiation exposure from warfare or radiation accidents raises the need for appropriate animal models to study the acute and chronic effects of high dose rate radiation exposure. The goal of this study was to assess the late development of fibrosis in multiple organs (kidney, heart, and lung) in survivors of the C57BL/6 mouse model of the hematopoietic-acute radiation syndrome (H-ARS). Separate groups of mice for histological and functional studies were exposed to a single uniform total body dose between 8.53 and 8.72 Gy of gamma radiation from a 137Cs radiation source and studied 1–21 months later. Blood urea nitrogen levels were elevated significantly in the irradiated mice at 9 and 21 mo (from ~22 to 34 ± 3.8 and 69±6.0 mg/dl, p<0.01 vs non-irradiated controls) and correlated with glomerosclerosis (29±1.8% vs 64±9.7% of total glomeruli, p<0.01 vs non-irradiated controls). Glomerular tubularization and hypertrophy and tubular atrophy were also observed at 21 mo post-total body irradiation (TBI). An increase in interstitial, perivascular, pericardial and peri-bronchial fibrosis/collagen deposition was observed from ~9–21 mo post-TBI in kidney, heart and lung of irradiated mice relative to age-matched controls. Echocardiography suggested decreased ventricular volumes with a compensatory increase in left ventricular ejection fraction. The results indicate that significant delayed effects of acute radiation exposure occur in kidney, heart, and lung in survivors of the murine H-ARS TBI model which mirrors pathology detected in larger species and humans at higher radiation doses focused on specific organs.
PMCID: PMC4593322  PMID: 26425910
Health effects; mice; radiation dose; radiation damage
3.  Novel Use of Folate-Targeted Intraoperative Fluorescence, OTL38, in Robot-Assisted Laparoscopic Partial Nephrectomy: Report of the First Three Cases 
Partial nephrectomy is now the preferred surgical option for small renal tumors because it allows nephron preservation without compromising oncologic clearance. Its outcomes depend on the surgeon's ability to continuously identify the edges of the tumor during resection, thus leaving an adequate margin around the tumor without excessive removal of normal parenchyma, as well as keeping a short ischemic time. Folate receptors are highly abundant in the normal kidney, and there is a difference in folate receptor expression between malignant and normal renal tissues. Thus, the use of fluorescent agents that target folate receptors should result in differential fluorescence between the tumor and surrounding parenchyma during partial nephrectomy, which, in turn, helps tumor demarcation for identification and resection. A phase 2 study on the novel use of OTL38 in robot-assisted laparoscopic partial nephrectomy is currently in progress in our institution. The outcomes of the first three cases have shown the possible advantages of OTL38 in intraoperative tumor identification before resection and recognition of residual disease in the surrounding parenchyma after resection. The tumors typically appeared dark while the surrounding parenchyma showed brighter fluorescence. Immediately after tumor resection, the margins of all the specimens appeared to have a uniformly bright fluorescence, suggestive of an intact margin of normal renal parenchyma along the plane of excision. The pattern of intraoperative fluorescence correlates well with immunohistochemistry. No OTL38-related adverse effects have been seen among these three patients. We present the outcomes of these three cases, illustrated with intraoperative and immunohistochemistry images.
PMCID: PMC5107661  PMID: 27868096
OTL38; folate-targeted intraoperative fluorescence; partial nephrectomy
4.  Outer Surface Protein OspC Is an Antiphagocytic Factor That Protects Borrelia burgdorferi from Phagocytosis by Macrophages 
Infection and Immunity  2015;83(12):4848-4860.
Outer surface protein C (OspC) is one of the major lipoproteins expressed on the surface of Borrelia burgdorferi during tick feeding and the early phase of mammalian infection. OspC is required for B. burgdorferi to establish infection in both immunocompetent and SCID mice and has been proposed to facilitate evasion of innate immune defenses. However, the exact biological function of OspC remains elusive. In this study, we showed that the ospC-deficient spirochete could not establish infection in NOD-scid IL2rγnull mice that lack B cells, T cells, NK cells, and lytic complement. The ospC mutant also could not establish infection in anti-Ly6G-treated SCID and C3H/HeN mice (depletion of neutrophils). However, depletion of mononuclear phagocytes at the skin site of inoculation in SCID and C3H/HeN mice allowed the ospC mutant to establish infection in vivo. In phagocyte-depleted mice, the ospC mutant was able to colonize the joints and triggered neutrophilia during dissemination. Furthermore, we found that phagocytosis of green fluorescent protein (GFP)-expressing ospC mutant spirochetes by murine peritoneal macrophages and human THP-1 macrophage-like cells, but not in PMN-HL60, was significantly higher than parental wild-type B. burgdorferi strains, suggesting that OspC has an antiphagocytic property. In addition, overproduction of OspC in spirochetes also decreased the uptake of spirochetes by murine peritoneal macrophages. Together, our findings provide evidence that mononuclear phagocytes play a key role in clearance of the ospC mutant and that OspC promotes spirochetes' evasion of macrophages during early Lyme borreliosis.
PMCID: PMC4645385  PMID: 26438793
5.  FGF23 is elevated in multiple myeloma and increases heparanase expression by tumor cells 
Oncotarget  2015;6(23):19647-19660.
Multiply myeloma (MM) grows in and destroys bone, where osteocytes secrete FGF23, a hormone which affects phosphate homeostasis and aging. We report that multiple myeloma (MM) cells express receptors for and respond to FGF23. FGF23 increased mRNA for EGR1 and its target heparanase, a pro-osteolytic factor in MM. FGF23 signals through a complex of klotho and a classical FGF receptor (FGFR); both were expressed by MM cell lines and patient samples. Bone marrow plasma cells from 42 MM patients stained positively for klotho, while plasma cells from 8 patients with monoclonal gammopathy of undetermined significance (MGUS) and 6 controls were negative. Intact, active FGF23 was increased 2.9X in sera of MM patients compared to controls. FGF23 was not expressed by human MM cells, but co-culture with mouse bone increased its mRNA. The FGFR inhibitor NVP-BGJ398 blocked the heparanase response to FGF23. NVP-BGJ398 did not inhibit 8226 growth in vitro but significantly suppressed growth in bone and induction of the osteoclast regulator RANK ligand, while decreasing heparanase mRNA. The bone microenvironment provides resistance to some anti-tumor drugs but increased the activity of NVP-BGJ398 against 8226 cells. The FGF23/klotho/heparanase signaling axis may offer targets for treatment of MM in bone.
PMCID: PMC4637311  PMID: 25944690
multiple myeloma; osteocytes; FGF23; FGF receptor; klotho
6.  The mushroom Ganoderma lucidum suppresses breast-to-lung cancer metastasis through the inhibition of pro-invasive genes 
International Journal of Oncology  2014;44(6):2009-2015.
Breast cancer metastasis is one of the major reasons for the high morbidity and mortality of breast cancer patients. In spite of surgical interventions, chemotherapy, radiation therapy and targeted therapy, some patients are considering alternative therapies with herbal/natural products. In the present study, we evaluated a well-characterized extract from the medicinal mushroom Ganoderma lucidum (GLE) for its affects on tumor growth and breast-to-lung cancer metastasis. MDA-MB-231 human breast cancer cells were implanted into the mammary fat pads of nude mice. GLE (100 mg/kg/every other day) was administered to the mice by an oral gavage for 4 weeks, and tumor size was measured using microcalipers. Lung metastases were evaluated by hematoxylin and eosin (H&E) staining. Gene expression in MDA-MB-231 cells was determined by DNA microarray analysis and confirmed by quantitative PCR. Identified genes were silenced by siRNA, and cell migration was determined in Boyden chambers and by wound-healing assay. Although an oral administration of GLE only slightly suppressed the growth of large tumors, the same treatment significantly inhibited the number of breast-to-lung cancer metastases. GLE also downregulated the expression of genes associated with invasive behavior (HRAS, VIL2, S100A4, MCAM, I2PP2A and FN1) in MDA-MB-231 cells. Gene silencing of HRAS, VIL2, S100A4, I2PP2A and FN1 by siRNA suppressed migration of MDA-MB-231 cells. Our study suggests that an oral administration of GLE can inhibit breast-to-lung cancer metastases through the downregulation of genes responsible for cell invasiveness. The anti-metastatic benefits of GLE warrant further clinical studies.
PMCID: PMC4735696  PMID: 24718855
mushroom; Ganoderma lucidum; polysaccharides; triterpenes; breast-to-lung cancer metastases; gene expression; cell migration
7.  Pachymic Acid Inhibits Growth and Induces Apoptosis of Pancreatic Cancer In Vitro and In Vivo by Targeting ER Stress 
PLoS ONE  2015;10(4):e0122270.
Pachymic acid (PA) is a purified triterpene extracted from medicinal fungus Poria cocos. In this paper, we investigated the anticancer effect of PA on human chemotherapy resistant pancreatic cancer. PA triggered apoptosis in gemcitabine-resistant pancreatic cancer cells PANC-1 and MIA PaCa-2. Comparative gene expression array analysis demonstrated that endoplasmic reticulum (ER) stress was induced by PA through activation of heat shock response and unfolded protein response related genes. Induced ER stress was confirmed by increasing expression of XBP-1s, ATF4, Hsp70, CHOP and phospho-eIF2α. Moreover, ER stress inhibitor tauroursodeoxycholic acid (TUDCA) blocked PA induced apoptosis. In addition, 25 mg kg-1 of PA significantly suppressed MIA PaCa-2 tumor growth in vivo without toxicity, which correlated with induction of apoptosis and expression of ER stress related proteins in tumor tissues. Taken together, growth inhibition and induction of apoptosis by PA in gemcitabine-resistant pancreatic cancer cells were associated with ER stress activation both in vitro and in vivo. PA may be potentially exploited for the use in treatment of chemotherapy resistant pancreatic cancer.
PMCID: PMC4411097  PMID: 25915041
8.  Role of Complement Activation in Obliterative Bronchiolitis Post Lung Transplantation 
Journal of immunology (Baltimore, Md. : 1950)  2013;191(8):10.4049/jimmunol.1202242.
Obliterative bronchiolitis (OB) post lung transplantation involves IL-17 regulated autoimmunity to type V collagen and alloimmunity, which could be enhanced by complement activation. However, the specific role of complement activation in lung allograft pathology, IL-17 production, and OB are unknown. The current study examines the role of complement activation in OB. Complement regulatory protein (CRP) (CD55, CD46, Crry/CD46) expression was down regulated in human and murine OB; and C3a, a marker of complement activation, was up regulated locally. IL-17 differentially suppressed Crry expression in airway epithelial cells in vitro. Neutralizing IL-17 recovered CRP expression in murine lung allografts and decreased local C3a production. Exogenous C3a enhanced IL-17 production from alloantigen or autoantigen (type V collagen) reactive lymphocytes. Systemically neutralizing C5 abrogated the development of OB, reduced acute rejection severity, lowered systemic and local levels of C3a and C5a, recovered CRP expression, and diminished systemic IL-17 and IL-6 levels. These data indicated that OB induction is in part complement dependent due to IL-17 mediated down regulation of CRPs on airway epithelium. C3a and IL-17 are part of a feed forward loop that may enhance CRP down regulation, suggesting that complement blockade could be a therapeutic strategy for OB.
PMCID: PMC3873138  PMID: 24043901
9.  Peptide-Mediated Inhibition of Mitogen-Activated Protein Kinase–Activated Protein Kinase–2 Ameliorates Bleomycin-Induced Pulmonary Fibrosis 
Mitogen-activated protein kinase–activated protein kinase–2 (MAPKAPK2, or MK2), a serine/threonine kinase downstream of p38 mitogen–activated protein kinase, has been implicated in inflammation and fibrosis. Compared with pathologically normal lung tissue, significantly higher concentrations of activated MK2 are evident in lung biopsies of patients with idiopathic pulmonary fibrosis (IPF). Expression is localized to fibroblasts and epithelial cells. In the murine bleomycin model of pulmonary fibrosis, we observed robust, activated MK2 expression on Day 7 (prefibrotic stage) and Day 14 (postfibrotic stage). To determine the effects of MK2 inhibition during the postinflammatory/prefibrotic and postfibrotic stages, C57BL/6 mice received intratracheal bleomycin instillation (0.025 U; Day 0), followed by PBS or the MK2 inhibitor (MK2i; 37.5 μg/kg), administered via either local (nebulized) or systemic (intraperitoneal) routes. MK2i or PBS was dosed daily for 14 days subsequent to bleomycin injury, beginning on either Day 7 or Day 14. Regardless of mode of administration or stage of intervention, MK2i significantly abrogated collagen deposition, myofibroblast differentiation and activated MK2 expression. MK2i also decreased circulating TNF-α and IL-6 concentrations, and modulated the local mRNA expression of profibrotic cytokine il-1β, matrix-related genes col1a2, col3a1, and lox, and transforming growth factor–β family members, including smad3, serpine1 (pai1), and smad6/7. In vitro, MK2i dose-dependently attenuated total MK2, myofibroblast differentiation, the secretion of collagen Type I, fibronectin, and the activation of focal adhesion kinase, whereas activated MK2 was attenuated at optimal doses. The peptide-mediated inhibition of MK2 affects both inflammatory and fibrotic responses, and thus may offer a promising therapeutic target for IPF.
PMCID: PMC3727887  PMID: 23470623
MK2; IPF; established fibrosis; transforming growth factor–β; SMAD
10.  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
11.  The Protein Tyrosine Phosphatase, Shp2, Positively Contributes to FLT3-ITD-Induced Hematopoietic Progenitor Hyperproliferation and Malignant Disease In Vivo 
Leukemia  2012;27(2):398-408.
Internal tandem duplications in the fms-like tyrosine kinase receptor (FLT3-ITDs) confer a poor prognosis in acute myeloid leukemia. We hypothesized that increased recruitment of the protein tyrosine phosphatase, Shp2, to FLT3-ITDs contributes to FLT3 ligand (FL)-independent hyperproliferation and STAT5 activation. Co-immunoprecipitation demonstrated constitutive association of Shp2 with the FLT3-ITD, N51-FLT3, as well as with STAT5. Knock-down of Shp2 in Baf3/N51-FLT3 cells significantly reduced proliferation while having little effect on WT-FLT3-expressing cells. Consistently, mutation of N51-FLT3 tyrosine 599 to phenylalanine or genetic disruption of Shp2 in N51-FLT3-expressing bone marrow low density mononuclear cells reduced proliferation and STAT5 activation. In transplants, genetic disruption of Shp2 in vivo yielded increased latency to and reduced severity of FLT3-ITD-induced malignancy. Mechanistically, Shp2 co-localizes with nuclear phospho-STAT5, is present at functional interferon-γ activation sites (GAS) within the BCL2L1 promoter, and positively activates the human BCL2L1 promoter, suggesting that Shp2 works with STAT5 to promote pro-leukemogenic gene expression. Further, using a small molecule Shp2 inhibitor, the proliferation of N51-FLT3-expressing bone marrow progenitors and primary AML samples was reduced in a dose-dependent manner. These findings demonstrate that Shp2 positively contributes to FLT3-ITD-induced leukemia and suggest that Shp2 inhibition may provide a novel therapeutic approach to acute myeloid leukemia.
PMCID: PMC3916934  PMID: 23103841
Acute Myeloid Leukemia; FLT3-ITD; Shp2; STAT5
12.  Type V Collagen Induced Tolerance Suppresses Collagen Deposition, TGF-β and Associated Transcripts in Pulmonary Fibrosis 
PLoS ONE  2013;8(10):e76451.
Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease characterized by progressive scarring and matrix deposition. Recent reports highlight an autoimmune component in IPF pathogenesis. We have reported anti-col(V) immunity in IPF patients. The objective of our study was to determine the specificity of col(V) expression profile and anti-col(V) immunity relative to col(I) in clinical IPF and the efficacy of nebulized col(V) in pre-clinical IPF models.
Col(V) and col(I) expression profile was analyzed in normal human and IPF tissues. C57-BL6 mice were intratracheally instilled with bleomycin (0.025 U) followed by col(V) nebulization at pre-/post-fibrotic stage and analyzed for systemic and local responses.
Compared to normal lungs, IPF lungs had higher protein and transcript expression of the alpha 1 chain of col(V) and col(I). Systemic anti-col(V) antibody concentrations, but not of anti-col(I), were higher in IPF patients. Nebulized col(V), but not col(I), prevented bleomycin-induced fibrosis, collagen deposition, and myofibroblast differentiation. Col(V) treatment suppressed systemic levels of anti-col(V) antibodies, IL-6 and TNF-α; and local Il-17a transcripts. Compared to controls, nebulized col(V)-induced tolerance abrogated antigen-specific proliferation in mediastinal lymphocytes and production of IL-17A, IL-6, TNF-α and IFN-γ. In a clinically relevant established fibrosis model, nebulized col(V) decreased collagen deposition. mRNA array revealed downregulation of genes specific to fibrosis (Tgf-β, Il-1β, Pdgfb), matrix (Acta2, Col1a2, Col3a1, Lox, Itgb1/6, Itga2/3) and members of the TGF-β superfamily (Tgfbr1/2, Smad2/3, Ltbp1, Serpine1, Nfkb/Sp1/Cebpb).
Anti-col(V) immunity is pathogenic in IPF, and col(V)-induced tolerance abrogates bleomycin-induced fibrogenesis and down regulates TGF- β-related signaling pathways.
PMCID: PMC3804565  PMID: 24204629
13.  Preclinical Models of Wound Healing: Is Man the Model? Proceedings of the Wound Healing Society Symposium 
Advances in Wound Care  2013;2(1):1-4.
A review of therapeutic effects in preclinical and clinical studies suggests that concordance between large animal (pig=78%), small laboratory animal (53%) and in vitro (57%) results with those observed in humans is only partial. Pig models of wound healing provide major advantages over other animal models. Since the vast majority of wound-healing research is done in rodents and in vitro, the low concordance rate is a significant impediment to research that will have any clinical impact.
Critical Issues
To generate clinically relevant experimental data, hypothesis generation should begin, or at least involve human wound tissue samples. Such tissue could be used to test a predetermined hypothesis generated based on, say, murine data. Alternatively, such tissue could be analyzed using high-throughput cell biology techniques (e.g., genomics, proteomics, or metabolomics) to identify novel mechanisms involved in human wounds. Once the hypothesis has been formulated and confirmed using human samples, identification of these same mechanisms in animals represents a valid approach that could be used for more in-depth investigations and experimental manipulations not feasible with humans.
Future Directions
This consensus statement issued by the Wound Healing Society symposium strongly encourages all wound researchers to involve human wound tissue validation studies to make their animal and cell biology studies more translationally and clinically significant.
PMCID: PMC3840478  PMID: 24527316
14.  Allergic Airway Disease in Mice Alters T and B Cell Responses during an Acute Respiratory Poxvirus Infection 
PLoS ONE  2013;8(4):e62222.
Pulmonary viral infections can exacerbate or trigger the development of allergic airway diseases via multiple mechanisms depending upon the infectious agent. Respiratory vaccinia virus transmission is well established, yet the effects of allergic airway disease on the host response to intra-pulmonary vaccinia virus infection remain poorly defined. As shown here BALB/c mice with preexisting airway disease infected with vaccinia virus developed more severe pulmonary inflammation, higher lung virus titers and greater weight loss compared with mice inoculated with virus alone. This enhanced viremia was observed despite increased pulmonary recruitment of CD8+ T effectors, greater IFNγ production in the lung, and high serum levels of anti-viral antibodies. Notably, flow cytometric analyses of lung CD8+ T cells revealed a shift in the hierarchy of immunodominant viral epitopes in virus inoculated mice with allergic airway disease compared to mice treated with virus only. Pulmonary IL-10 production by T cells and antigen presenting cells was detected following virus inoculation of animals and increased dramatically in allergic mice exposed to virus. IL-10 modulation of host responses to this respiratory virus infection was greatly influenced by the localized pulmonary microenvironment. Thus, blocking IL-10 signaling in virus-infected mice with allergic airway disease enhanced pulmonary CD4+ T cell production of IFNγ and increased serum anti-viral IgG1 levels. In contrast, pulmonary IFNγ and virus-specific IgG1 levels were reduced in vaccinia virus-treated mice with IL-10 receptor blockade. These observations demonstrate that pre-existing allergic lung disease alters the quality and magnitude of immune responses to respiratory poxviruses through an IL-10-dependent mechanism.
PMCID: PMC3631162  PMID: 23620814
15.  Mushroom Ganoderma lucidum Prevents Colitis-Associated Carcinogenesis in Mice 
PLoS ONE  2012;7(10):e47873.
Epidemiological studies suggest that mushroom intake is inversely correlated with gastric, gastrointestinal and breast cancers. We have recently demonstrated anticancer and anti-inflammatory activity of triterpene extract isolated from mushroom Ganoderma lucidum (GLT). The aim of the present study was to evaluate whether GLT prevents colitis-associated carcinogenesis in mice.
Methods/Principal Findings
Colon carcinogenesis was induced by the food-borne carcinogen (2-Amino-1-methyl-6-phenylimidazol[4,5-b]pyridine [PhIP]) and inflammation (dextran sodium sulfate [DSS]) in mice. Mice were treated with 0, 100, 300 and 500 mg GLT/kg of body weight 3 times per week for 4 months. Cell proliferation, expression of cyclin D1 and COX-2 and macrophage infiltration was assessed by immunohistochemistry. The effect of GLT on XRE/AhR, PXR and rPXR was evaluated by the reporter gene assays. Expression of metabolizing enzymes CYP1A2, CYP3A1 and CYP3A4 in colon tissue was determined by immunohistochemistry. GLT treatment significantly suppressed focal hyperplasia, aberrant crypt foci (ACF) formation and tumor formation in mice exposed to PhIP/DSS. The anti-proliferative effects of GLT were further confirmed by the decreased staining with Ki-67 in colon tissues. PhIP/DSS-induced colon inflammation was demonstrated by the significant shortening of the large intestine and macrophage infiltrations, whereas GLT treatment prevented the shortening of colon lengths, and reduced infiltration of macrophages in colon tissue. GLT treatment also significantly down-regulated PhIP/DSS-dependent expression of cyclin D1, COX-2, CYP1A2 and CYP3A4 in colon tissue.
Our data suggest that GLT could be considered as an alternative dietary approach for the prevention of colitis-associated cancer.
PMCID: PMC3484149  PMID: 23118901
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.  BreastDefend™ prevents breast-to-lung cancer metastases in an orthotopic animal model of triple-negative human breast cancer 
Oncology Reports  2012;28(4):1139-1145.
We have recently demonstrated that a natural dietary supplement BreastDefend (BD), which contains extracts from medicinal mushrooms (Coriolus versicolor, Ganoderma lucidum, Phellinus linteus), medicinal herbs (Scutellaria barbata, Astragalus membranaceus, Curcuma longa), and purified biologically active nutritional compounds (diindolylmethane and quercetin), inhibits proliferation and metastatic behavior of MDA-MB-231 invasive human breast cancer cells in vitro. In the present study, we evaluated whether BD suppresses growth and breast-to lung cancer metastasis in an orthotopic model of human breast cancer cells implanted in mice. Oral application of BD (100 mg/kg of body weight for 4 weeks) by intragastric gavage did not affect body weight or activity of liver enzymes and did not show any sign of toxicity in liver, spleen, kidney, lung and heart tissues in mice. Moreover, BD significantly decreased the change in tumor volume over time compared to the control group (p=0.002). BD treatment also markedly decreased the incidence of breast-to-lung cancer metastasis from 67% (control) to 20% (BD) (p<0.05) and the number of metastases from 2.8 (0.0, 48.0) in the control group to 0.0 (0.0, 14.2) in the BD treatment group (p<0.05). Finally, anti-metastatic activity of BD in vivo was further confirmed by the downregulation of expression of PLAU (urokinase plasminogen activator, uPA) and CXCR4 (C-X-C chemokine receptor-4) genes in breast tumors. In conclusion, BD may be considered as a biological therapeutic agent against invasive breast cancers.
PMCID: PMC3583511  PMID: 22842551
triple negative breast cancer cells; lung cancer metastasis; dietary supplement; urokinase plasminogen activator; C-X-C chemokine receptor-4
The Prostate  2010;71(7):748-754.
Proliferating-cell nuclear antigen (PCNA) plays an important role in DNA replication and repair. The expression and potential utility of this marker in prostatic neoplasia is uncertain. With the development of this new caPCNA selective antibody, we explored the potential utility of this marker in prostate cancer.
Using a traditional primary Fab2′ rabbit anti-caPCNA antibody-HRP conjugated secondary anti-Fab2′ antibody format, the expression of the caPCNA was analyzed in prostate tissue from 89 radical prostatectomy specimens. The caPCNA expression was correlated with clinicopathologic characteristics.
The fraction of cells staining positively with caPCNA antibody in prostatic adenocarcinoma (mean, 23%) was significantly higher than that in benign prostatic epithelium (mean, 2%; p < 0.001) or high-grade prostatic intraepithelial neoplasia (PIN) (mean, 6%; p < 0.05). Moreover, the intensity of caPCNA expression in prostatic adenocarcinoma (mean, 2.9) was significantly higher than that in benign prostatic tissue (mean, 0.7; p < 0.001) or high-grade PIN (mean, 2.0; p < 0.001). Benign prostatic epithelium showed only minimal or negative reactivity. There was significant correlation between the percentage of caPCNA expression and primary Gleason grade (p = 0.01), and with Gleason score (p = 0.02). Adenocarcinomas with positive vascular invasion had a significantly higher percentage of cells staining with caPCNA antibody (p < 0.0001) and a higher intensity of caPCNA expression (p = 0.04).
Our data indicate that increased expression of the cancer-associated isoform of PCNA is common in prostatic adenocarcinoma and its precursor and may be a useful biomarker.
PMCID: PMC3116049  PMID: 21031434
Prostate; biomarkers; proliferating-cell nuclear antigen (PCNA); Gleason grading; neoplasia; high-grade prostatic intraepithelial neoplasia (PIN); targeted therapy; progression; carcinogenesis
19.  High quality DNA obtained with an automated DNA extraction method with 70+ year old formalin-fixed celloidin-embedded (FFCE) blocks from the indiana medical history museum 
DNA and RNA have been used as markers of tissue quality and integrity throughout the last few decades. In this research study, genomic quality DNA of kidney, liver, heart, lung, spleen, and brain were analyzed in tissues from post-mortem patients and surgical cancer cases spanning the past century. DNA extraction was performed on over 180 samples from: 70+ year old formalin-fixed celloidin-embedded (FFCE) tissues, formalin-fixed paraffin-embedded (FFPE) tissue samples from surgical cases and post-mortem cases from the 1970’s, 1980’s, 1990’s, and 2000’s, tissues fixed in 10% neutral buffered formalin/stored in 70% ethanol from the 1990’s, 70+ year old tissues fixed in unbuffered formalin of various concentrations, and fresh tissue as a control. To extract DNA from FFCE samples and ethanol-soaked samples, a modified standard operating procedure was used in which all tissues were homogenized, digested with a proteinase K solution for a long period of time (24-48 hours), and DNA was extracted using the Autogen Flexstar automated extraction machine. To extract DNA from FFPE, all tissues were soaked in xylene to remove the paraffin from the tissue prior to digestion, and FFPE tissues were not homogenized. The results were as follows: celloidin-embedded and paraffin-embedded tissues yielded the highest DNA concentration and greatest DNA quality, while the formalin in various concentrations, and long term formalin/ethanol-stored tissue yielded both the lowest DNA concentration and quality of the tissues tested. The average DNA yield for the various fixatives was: 367.77 μg/ mL FFCE, 590.7 μg/mL FFPE, 53.74 μg/mL formalin-fixed/70% ethanol-stored and 33.2 μg/mL unbuffered formalin tissues. The average OD readings for FFCE, FFPE, formalin-fixed/70% ethanol-stored tissues, and tissues fixed in unbuffered formalin were 1.86, 1.87, 1.43, and 1.48 respectively. The results show that usable DNA can be extracted from tissue fixed in formalin and embedded in celloidin or paraffin from the early 1900’s to present, and may be amplified through PCR and used for clinical and experimental studies.
PMCID: PMC3353536  PMID: 22611472
DNA; celloidin; museum specimens
20.  NAHA, a Novel Hydroxamic Acid-Derivative, Inhibits Growth and Angiogenesis of Breast Cancer In Vitro and In Vivo 
PLoS ONE  2012;7(3):e34283.
We have recently synthesized novel N-alkylated amino acid-derived hydroxamate, 2-[Benzyl-(2-nitro-benzenesulfonyl)-amino]-N-hydroxy-3-methyl-N-propyl-butyramide (NAHA). Here, we evaluate the anticancer activity of NAHA against highly invasive human breast cancer cells MDA-MB-231 in vitro and in vivo.
Methodology/Principal Findings
Cell growth was evaluated by MTT and soft agar assays. Protein expression was determined by DNA microarray and Western blot analysis. Metastatic potential was evaluated by cell adhesion, migration, invasion, capillary morphogenesis, and ELISA assays. The anticancer activity in vivo was evaluated in mouse xenograft model. NAHA inhibited proliferation and colony formation of MDA-MB-231 cells together with the down-regulation of expression of Cdk2 and CDC20 proteins. NAHA inhibited cell adhesion, migration, and invasion through the suppression of secretion of uPA. NAHA suppressed secretion of VEGF from MDA-MB-231 cells and inhibited capillary morphogenesis of human aortic endothelial cells (HAECs). Finally, NAHA at 50 mg/kg was not toxic and decreased tumor volume and tumor weight in vivo. This suppression of tumor growth was associated with the inhibition of mitotic figures and induction of apoptosis, and the reduction of CD31 and VEGF positive cells in tumors.
NAHA could be a novel promising compound for the development of new drugs for the therapy of invasive breast cancers.
PMCID: PMC3315582  PMID: 22479587
21.  Tissue-specific alterations of PRL-1 and PRL-2 expression in cancer 
The PRL-1 and PRL-2 phosphatases have been implicated as oncogenic, however the involvement of these molecules in human neoplasms is not well understood. To increase understanding of the role PRL-1 and PRL-2 play in the neoplastic process, in situ hybridization was used to examine PRL-1 and PRL-2 mRNA expression in 285 normal, benign, and malignant human tissues of diverse origin. Immunohistochemical analysis was performed on a subset of these. PRL-1 and PRL-2 mRNA expression was also assessed in a small set of samples from a variety of diseases other than cancer. Where possible, associations with clinicopathological characteristics were evaluated. Alterations in PRL-1 or -2 expression were a frequent event, but the nature of those alterations was highly tumor type specific. PRL-1 was significantly overexpressed in 100% of hepatocellular and gastric carcinomas, but significantly under-expressed in 100% of ovarian, 80% of breast, and 75% of lung tumors. PRL-2 expression was significantly increased in 100% of hepatocellular carcinomas, yet significantly downregulated in 54% of kidney carcinomas. PRL-1 expression was correlated to patient gender in the bladder and to patient age in the brain and skeletal muscle. PRL-1 expression was also associated with tumor grade in the prostate, ovary, and uterus. These results suggest a pleiotropic role for PRL-1 and PRL-2 in the neoplastic process. These molecules may associate with tumor progression and serve as clinical markers of tumor aggressiveness in some tissues, but be involved in inhibition of tumor formation or growth in others.
PMCID: PMC3276379  PMID: 22347524
Phosphatase of regenerating liver; PRL-1; PRL-2; in situ hybridization; cancer
23.  Reduced Expression of DNA Repair and Redox Signaling Protein APE1/Ref-1 Impairs Human Pancreatic Cancer Cell Survival, Proliferation, and Cell Cycle Progression 
Cancer investigation  2010;28(9):885-895.
Pancreatic cancer is a deadly disease that is virtually never cured. Understanding the chemoresistance intrinsic to this cancer will aid in developing new regimens. High expression of APE1/Ref-1, a DNA repair and redox signaling protein, is associated with resistance, poor outcome, and angiogenesis; little is known in pancreatic cancer. Immunostaining of adenocarcinoma shows greater APE1/Ref-1 expression than in normal pancreas tissue. A decrease in APE1/Ref-1 protein levels results in pancreatic cancer cell growth inhibition, increased apoptosis, and altered cell cycle progression. Endogenous cell cycle inhibitors increase when APE1/Ref-1 is reduced, demonstrating its importance to proliferation and growth of pancreatic cancer.
PMCID: PMC2966714  PMID: 20919954
Pancreatic cancer; APE1/Ref-1; siRNA; cell cycle
24.  New insights into the protein C pathway: potential implications for the biological activities of drotrecogin alfa (activated) 
Critical Care  2005;9(Suppl 4):S38-S45.
It has been hypothesized that the protein C pathway is a pivotal link between the inflammation and coagulation cascades. The demonstration that a survival benefit is associated with administration of drotrecogin alfa (activated) (recombinant human activated protein C [APC]) in severe sepsis patients has provided new insights into the protein C pathway. APC was originally identified based on its antithrombotic properties, which result from the inhibition of activated Factors V and VIII. In the early 1990s, any potential anti-inflammatory properties of APC were thought to relate primarily to its inhibition of thrombin generation. However, the mid-1990s saw the identification of the endothelial protein C receptor (EPCR), which has subsequently been shown to be neither endothelial specific nor protein C specific, but has a primary function as a cofactor for enhancing the generation of APC or behaving as an APC receptor. Thus, the potential biologic activities of APC can be classed into two categories related either to the limiting of thrombin generation or to cellular effects initiated by binding to the EPCR. Intracellular signaling initiated by binding of APC to its receptor appears to be mediated by interaction with an adjacent protease-activated receptor (PAR), or by indirect activation of the sphingosine 1-phosphate pathway. Based mostly on in vitro studies, binding of APC to its receptor on endothelial cells leads to a decrease in thrombin-induced endothelial permeability injury, while such binding on blood cells, epithelial cells, and neurons has been shown to inhibit chemotaxis, be anti-apoptotic, and be neuroprotective, respectively. In the Recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis (PROWESS) study, drotrecogin alfa (activated) was associated with improved cardiovascular function, respiratory function, and a prevention of hematologic dysfunction. This article discusses the way in which the interactions of APC may alter the microcirculation.
PMCID: PMC3226161  PMID: 16168074
25.  FGF-21 as a novel metabolic regulator 
Journal of Clinical Investigation  2005;115(6):1627-1635.
Diabetes mellitus is a major health concern, affecting more than 5% of the population. Here we describe a potential novel therapeutic agent for this disease, FGF-21, which was discovered to be a potent regulator of glucose uptake in mouse 3T3-L1 and primary human adipocytes. FGF-21–transgenic mice were viable and resistant to diet-induced obesity. Therapeutic administration of FGF-21 reduced plasma glucose and triglycerides to near normal levels in both ob/ob and db/db mice. These effects persisted for at least 24 hours following the cessation of FGF-21 administration. Importantly, FGF-21 did not induce mitogenicity, hypoglycemia, or weight gain at any dose tested in diabetic or healthy animals or when overexpressed in transgenic mice. Thus, we conclude that FGF-21, which we have identified as a novel metabolic factor, exhibits the therapeutic characteristics necessary for an effective treatment of diabetes.
PMCID: PMC1088017  PMID: 15902306

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