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1.  RhoGDIα suppresses growth and survival of prostate cancer cells 
The Prostate  2011;72(4):392-398.
Treatment for primary prostate cancer (CaP) is the withdrawal of androgens. However, CaP eventually progresses to grow in a castration-resistant state. The mechanisms involved in the development and progression of castration-resistant prostate cancer (CRPC) remain unknown. We have previously generated LNCaP-IL6+ cells by treating LNCaP cells chronically with interleukin-6 (IL-6), which have acquired the ability to grow in androgen-deprived conditions.
We compared the protein expression profile of LNCaP and LNCaP-IL6+ cells using two-dimensional gel electrophoresis. The gels were then silver stained in order to visualize proteins and the differentially expressed spots were identified and characterized by micro sequencing using MALDI-PMF mass spectrometry.
In this study, we have identified RhoGDIα (GDIα) as a suppressor of prostate cancer growth. Expression of GDIα was reduced in LNCaP-IL6+ cells and was downregulated in more aggressive prostate cancer cells compared to LNCaP cells. Over expression of GDIα inhibited the growth of prostate cancer cells and caused LNCaP-IL6+ cells reversal to androgen-sensitive state, while down regulation of GDIα enhanced growth of androgen-sensitive LNCaP prostate cancer cells in androgen-deprived conditions. In addition, GDIα suppressed the tumorigenic ability of prostate tumor xenografts in vivo.
These results demonstrate that loss of GDIα expression promotes the development and progression of prostate cancer.
PMCID: PMC3961824  PMID: 21681778
Prostate cancer; RhoGDIα; IL-6
2.  NF-κB2/p52 Induces Resistance to Enzalutamide in Prostate Cancer: Role of Androgen Receptor and Its Variants 
Molecular cancer therapeutics  2013;12(8):1629-1637.
Resistance of prostate cancer cells to the next-generation antiandrogen enzalutamide may be mediated by a multitude of survival signaling pathways. In this study, we tested whether increased expression of NF-κB2/p52 induces prostate cancer cell resistance to enzalutamide and whether this response is mediated by aberrant androgen receptor (AR) activation and AR splice variant production. LNCaP cells stably expressing NF-κB2/p52 exhibited higher survival rates than controls when treated with enzalutamide. C4-2B and CWR22Rv1 cells chronically treated with enzalutamide were found to express higher levels of NF-κB2/p52. Downregulation of NF-κB2/p52 in CWR22Rv1 cells chronically treated with enzalutamide rendered them more sensitive to cell growth inhibition by enzalutamide. Analysis of the expression levels of AR splice variants by quantitative reverse transcription PCR and Western blotting revealed that LNCaP cells expressing p52 exhibit higher expression of AR splice variants. Downregulation of expression of NF-κB2/p52 in VCaP and CWR22Rv1 cells by short hairpin RNA abolished expression of splice variants. Downregulation of expression of either full-length AR or the splice variant AR-V7 led to an increase in sensitivity of prostate cancer cells to enzalutamide. These results collectively demonstrate that resistance to enzalutamide may be mediated by NF-κB2/p52 via activation of AR and its splice variants.
PMCID: PMC3941973  PMID: 23699654
3.  Inhibition of Stat3 Activation by Sanguinarine Suppresses Prostate Cancer Cell Growth and Invasion 
The Prostate  2011;72(1):82-89.
Signal transducer and activator of transcription 3 (Stat3) is an oncogenic transcriptional factor that plays a critical role in carcinogenesis and cancer progression and is a potential therapeutic target. Sanguinarine, a benzophenanthridine alkaloid derived primarily from the bloodroot plant, was identified previously as a novel inhibitor of survivin that selectively kills prostate cancer cells over “normal” prostate epithelial cells.
DU145, C4-2B, and LNCaP cells were treated with sanguinarine. The phosphorylation status of Stat3 and related proteins were measured with Western blots. Activation of transcription by Stat3 was measured with luciferase reporter assay. The effect of sanguinarine on anchorage-independent growth was examined with soft agar assay, and on cell migration and invasion of DU145 cells were measured with scratch assay and invasion assay, respectively.
In this study, we identified sanguinarine as a potent inhibitor of Stat3 activation which was able to suppress prostate cancer growth, migration, and invasion. Sanguinarine inhibits constitutive as well as IL6-induced phosphorylation of Stat3 at both Tyr705 and Ser727 in prostate cancer cells. The inhibition of Stat3 phosphorylation by sanguinarine correlates with reduction of Janus-activated Kinase 2 (Jak2) and Src phosphorylation. Sanguinarine downregulates the expression of Stat3-mediated genes such as c-myc and survivin and inhibits the Stat3 responsive element luciferase reporter activity. Sanguinarine inhibits the anchorage-independent growth of DU145 and LN-S17 cells expressing constitutively activated Stat3. Migration and invasion abilities of DU145 cells were also inhibited by sanguinarine in a manner similar to the dominant negative form of Stat3.
These data demonstrate that sanguinarine is a potent Stat3 inhibitor and it could be developed as a therapeutic agent for prostate cancer with constitutive activation of Stat3.
PMCID: PMC3938016  PMID: 21538419
prostate cancer; Stat3; sanguinarine; invasion
4.  Effects of Dexamethasone on Mesenchymal Stromal Cell Chondrogenesis and Aggrecanase Activity: Comparison of Agarose and Self-Assembling Peptide Scaffolds 
Cartilage  2012;4(1):63-74.
Dexamethasone (Dex) is a synthetic glucocorticoid that has pro-anabolic and anti-catabolic effects in cartilage tissue engineering systems, though the mechanisms by which these effects are mediated are not well understood. We tested the hypothesis that the addition of Dex to chondrogenic medium would affect matrix production and aggrecanase activity of human and bovine bone marrow stromal cells (BMSCs) cultured in self-assembling peptide and agarose hydrogels.
We cultured young bovine and adult human BMSCs in (RADA)4 self-assembling peptide and agarose hydrogels in medium containing TGF-β1±Dex and analyzed extracellular matrix composition, aggrecan cleavage products, and the effects of the glucocorticoid receptor antagonist RU-486 on proteoglycan content, synthesis, and catabolic processing.
Dex improved proteoglycan synthesis and retention in agarose hydrogels seeded with young bovine cells, but decreased proteoglycan accumulation in peptide scaffolds. These effects were mediated by the glucocorticoid receptor. Adult human BMSCs showed minimal matrix accumulation in agarose, but accumulated ~50% as much proteoglycan and collagen as young bovine BMSCs in peptide hydrogels. Dex reduced aggrecanase activity in (RADA)4 and agarose hydrogels, as measured by anti-NITEGE Western blotting, for both bovine and human BMSC-seeded gels.
The effects of Dex on matrix production are dependent on cell source and hydrogel identity. This is the first report of Dex reducing aggrecanase activity in a tissue engineering culture system.
PMCID: PMC3922645  PMID: 24533173
Chondrogenesis; mesenchymal stem cells; degradative enzymes; articular cartilage; extracellular matrix
5.  Select G-protein coupled receptors modulate agonist-induced signaling via a ROCK, LIMK and β-arrestin 1 pathway 
Cell reports  2013;5(4):10.1016/j.celrep.2013.10.015.
G-protein coupled receptors (GPCRs) are typically present in a basal, inactive state, but when bound to agonist they activate downstream signaling cascades. In studying arrestin regulation of opioid receptors in dorsal root ganglia (DRG) neurons, we find that agonists of delta opioid receptors (δORs) activate cofilin through Rho-associated coiled-coiled containing protein kinase (ROCK), LIM domain kinase (LIMK) and β- arrestin 1 (β-arr1), to regulate actin polymerization. This controls receptor function, as assessed by agonist-induced inhibition of voltage-dependent Ca2+ channels in DRGs. Agonists of opioid-receptor like receptors (ORL1) similarly influence the function of this receptor through ROCK, LIMK and β-arr1. Functional evidence of this cascade was demonstrated in vivo where the behavioral effects of δOR or ORL1 agonists were enhanced in the absence of β-arr1 or prevented by inhibiting ROCK. This pathway allows δOR and ORL1 agonists to rapidly regulate receptor function.
PMCID: PMC3870884  PMID: 24239352
6.  Sequestration of the δ Opioid Receptor 
The Journal of biological chemistry  1996;271(46):29279-29285.
The primary structure of the opioid receptors have revealed that many of the structural features that are conserved in other G protein-coupled receptors are also conserved in the opioid receptors. Upon exposure to agonists, some G protein-coupled receptors internalize rapidly, whereas other structurally homologous G protein-coupled receptors do not. It is not known whether opioid receptors are regulated by rapid endocytosis. In transfected Chinese hamster ovary cells expressing the epitope-tagged wild type δ opioid receptor, exposure to 100 nM [D-Ala2,D-Leu5]enkephalin causes internalization of the receptor within 30 min as determined by confocal microscopy. The rate of internalization of the wild type receptor is rapid with a half-maximal reduction by about 10 min, as determined by the reduction in mean surface receptor fluorescence intensity measured using flow cytometry. In contrast, the cells expressing receptors lacking the C-terminal 15 or 37 amino acids exhibit a substantially slower rate of internalization. Furthermore, the cells expressing receptors with point mutations of any of the Ser/Thr between Ser344 and Ser363 in the C-terminal tail exhibit a significant reduction in the rate of receptor internalization. These results suggest that a portion of the C-terminal tail is involved in receptor internalization. Agents that block the formation of clathrin-coated pits considerably reduce the extent of agonist-mediated internalization of the wild type receptor. Taken together, these results suggest that the wild type opioid receptor undergoes rapid agonist-mediated internalization via a classic endocytic pathway and that a portion of the C-terminal tail plays an important role in this internalization process.
PMCID: PMC3856721  PMID: 8910588
7.  Stable-isotope GC-MS/MS determination of aminoethylcysteine ketimine decarboxylated dimer in biological samples 
Analytical biochemistry  2012;430(1):4-15.
Aminoethylcysteine ketimine decarboxylated dimer [AECK-DD; systematic name: 1,2–3,4–5,6–7,8-octahydro-1,8a-diaza-4,6-dithiafluoren-9(8aH)-one] is a previously described metabolite of cysteamine that has been reported to be present in mammalian brain, urine, plasma, cells in culture and vegetables, and to possess potent anti-oxidative properties. Here, we describe a stable-isotope GC-MS/MS method for specific and sensitive determination of AECK-DD in biological samples. 13C2-AECK-DD was synthesized and used as the internal standard. Derivatization was carried out by N-pentafluorobenzylation with pentafluorobenzyl bromide in acetonitrile. Quantification was performed by selected-reaction monitoring of the mass transitions m/z 328 to m/z 268 for AECK-DD and m/z 330 to m/z 270 for 13C2-AECK-DD in the electron-capture negative-ion chemical ionization mode. The procedure was systematically validated for human plasma and urine samples. AECK-DD was not detectable in human plasma above ~ 4 nM, but was present in urine samples of healthy humans at a maximal concentration of 46 nM. AECK-DD was detectable in rat brain at very low levels of about 8 pmol/g wet weight. Higher levels of AECK-DD were detected in mouse brain (~1 nmol/g wet weight). Among nine dietary vegetables evaluated, only shallots were found to contain trace amounts of AECK-DD (~ 6.8 pmol/g fresh tissue).
PMCID: PMC3462305  PMID: 22858756
AECK-DD; Brain; Cysteamine; GC-MS/MS; Organic cation transporters; Validation; Vegetables
9.  Drug-primed reinstatement of cocaine seeking in mice: increased excitability of medium-sized spiny neurons in the nucleus accumbens 
ASN NEURO  2013;5(4):e00122.
To examine the mechanisms of drug relapse, we first established a model for cocaine IVSA (intravenous self-administration) in mice, and subsequently examined electrophysiological alterations of MSNs (medium-sized spiny neurons) in the NAc (nucleus accumbens) before and after acute application of cocaine in slices. Three groups were included: master mice trained by AL (active lever) pressings followed by IV (intravenous) cocaine delivery, yoked mice that received passive IV cocaine administration initiated by paired master mice, and saline controls. MSNs recorded in the NAc shell in master mice exhibited higher membrane input resistances but lower frequencies and smaller amplitudes of sEPSCs (spontaneous excitatory postsynaptic currents) compared with neurons recorded from saline control mice, whereas cells in the NAc core had higher sEPSCs frequencies and larger amplitudes. Furthermore, sEPSCs in MSNs of the shell compartment displayed longer decay times, suggesting that both pre- and postsynaptic mechanisms were involved. After acute re-exposure to a low-dose of cocaine in vitro, an AP (action potential)-dependent, persistent increase in sEPSC frequency was observed in both NAc shell and core MSNs from master, but not yoked or saline control mice. Furthermore, re-exposure to cocaine induced membrane hyperpolarization, but concomitantly increased excitability of MSNs from master mice, as evidenced by increased membrane input resistance, decreased depolarizing current to generate APs, and a more negative Thr (threshold) for firing. These data demonstrate functional differences in NAc MSNs after chronic contingent versus non-contingent IV cocaine administration in mice, as well as synaptic adaptations of MSNs before and after acute re-exposure to cocaine. Reversing these functional alterations in NAc could represent a rational target for the treatment of some reward-related behaviors, including drug addiction.
PMCID: PMC3789142  PMID: 24000958
intravenous self-administration; nucleus accumbens; relapse; synaptic transmission; AHP, afterhyperpolarization; AL, active lever; BIC, bicuculline; Coc, cocaine; CsMeth, Cs-Methanesulfonate; FR, fixed ratio; GABA, γ-aminobutyric acid; IAL, inactive lever; IC, current clamp; IVSA, intravenous self-administration; KGluc, K-gluconate internal solution; mEPSCs, miniature excitatory postsynaptic currents; MSNs, medium-sized spiny neurons; NAc, nucleus accumbens; Rin, resistance; rm, repeated measure; RMP, resting membrane potential; sEPSCs, spontaneous excitatory postsynaptic currents; sIPSCs, spontaneous inhibitory postsynaptic currents; Thr, threshold; TTX, tetrodotoxin; VC, voltage clamp
10.  EWS-FLI-1-targeted cytotoxic T-cell killing of multiple tumor types belonging to the Ewing Sarcoma Family of Tumors* 
The Ewing Sarcoma Family of Tumors (ESFTs) comprises a group of aggressive, malignant bone and soft tissue tumors that predominantly affect children and young adults. These tumors frequently share expression of the EWS-FLI-1 translocation, which is central to tumor survival but not present in healthy cells. In this study, we examined EWS-FLI-1 antigens for their capacity to induce immunity against a range of ESFT types.
Computer prediction analysis of peptide binding, HLA-A2.1 stabilization assays, and induction of Cytotoxic T-Lymphocytes (CTL) in immunized HLA-A2.1 transgenic mice were used to assess the immunogenicity of native and modified peptides derived from the fusion region of EWS-FLI-1 type 1. CTL-killing of multiple ESFT family members in vitro, and control of established xenografts in vivo, was assessed. We also examined whether these peptides could induce human CTLs in vitro.
EWS-FLI-1 type 1 peptides were unable to stabilize cell surface HLA-A2.1 and induced weak CTL activity against Ewing Sarcoma cells. In contrast, peptides with modified anchor residues induced potent CTL killing of Ewing Sarcoma cells presenting endogenous (native) peptides. The adoptive transfer of CTL specific for the modified peptide YLNPSVDSV resulted in enhanced survival of mice with established Ewing Sarcoma xenografts. YLNPSVDSV-specific CTL displayed potent killing of multiple ESFT types in vitro: Ewing Sarcoma, pPNET, Askin’s Tumor, and Biphenotypic Sarcoma. Stimulation of human Peripheral Blood Mononuclear Cells with YLNPSVDSV peptide resulted in potent CTL-killing.
These data show that YLNPSVDSV peptide is a promising antigen for ESFT immunotherapy and warrants further clinical development.
PMCID: PMC3463738  PMID: 22879388
Ewing Sarcoma Family of Tumors; Ewing Sarcoma; pPNET; Askin’s Tumor; Biphenotypic sarcoma; EWS-FLI-1; Immunotherapy; vaccine; cancer; HLA-A2.1; HLA-A*0201
11.  Pharmacological Tuning of Heat Shock Protein 70 Modulates Polyglutamine Toxicity and Aggregation 
ACS chemical biology  2012;7(9):1556-1564.
Nine neurodegenerative disorders are caused by the abnormal expansion of polyglutamine (polyQ) regions within distinct proteins. Genetic and biochemical evidence has documented that the molecular chaperone, heat shock protein 70 (Hsp70), modulates polyQ toxicity and aggregation, yet it remains unclear how Hsp70 might be used as a potential target in polyQ-related diseases. We have utilized a pair of membrane-permeable compounds that tune the activity of Hsp70 by either stimulating or by inhibiting its ATPase functions. Using these two pharmacological agents in both yeast and PC12 cell models of polyQ aggregation and toxicity, we were surprised to find that stimulating Hsp70 solubilized polyQ conformers and simultaneously exacerbated polyQ-mediated toxicity. By contrast, inhibiting Hsp70’s ATPase activity protected against polyQ toxicity and promoted aggregation. These findings clarify Hsp70’s role as a possible drug target in polyQ disorders and suggest that Hsp70 uses ATP hydrolysis to help partition polyQ proteins into structures with varying levels of proteotoxicity. Our results thus support an emerging concept in which certain kinds of polyQ aggregates may be protective, while more soluble polyQ species are toxic.
PMCID: PMC3448832  PMID: 22709427
polyQ; protein misfolding; molecular chaperones; heat shock protein 70 (Hsp70); proteostasis; chemical genetics; chemical probes
12.  Assessment of Intracellular Mucin Content In Vivo 
Airway mucus presents a first line of defense against inhaled materials. It also, however, is a significant pathological contributor to chronic lung diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease. Thus, gaining a better understanding of the mechanisms of mucus production and secretion is an important goal for improving respiratory health. Mucins, the chief glycoprotein components of airway mucus, are very large polymeric glycoproteins, and measuring their production and secretion in experimental animals present significant technical challenges. Over the past several years, we have developed assays for accurately quantifying mucin production and secretion using histological and biochemical assays. These methods are described here.
PMCID: PMC3751172  PMID: 22259143
airways; asthma; cystic fibrosis; chronic obstructive pulmonary disease; goblet cell; lungs; mouse; mucin; mucous; mucus
13.  CTA095, a Novel Etk and Src Dual Inhibitor, Induces Apoptosis in Prostate Cancer Cells and Overcomes Resistance to Src Inhibitors 
PLoS ONE  2013;8(8):e70910.
Etk is a non-receptor tyrosine kinase, which provides a strong survival signal in human prostate cancer cells. Src, another tyrosine kinase that cross-activates with Etk, has been shown to play an important role in prostate cancer metastasis. Herein, we discovered a new class of Etk inhibitors. Within those inhibitors, CTA095 was identified as a potent Etk and Src dual inhibitor. CTA095 was found to induce autophagy as well as apoptosis in human prostate cancer cells. In addition, CTA095 inhibited HUVEC cell tube formation and “wound healing” of human prostate cancer cells, implying its role in inhibition of angiogenesis and metastasis of human prostate cancer. More interestingly, CTA095 could overcome Src inhibitor resistance in prostate cancer cells. It induces apoptosis in Src inhibitor resistant prostate cancer cells, likely through a mechanism of down regulation of Myc and BCL2. This finding indicates that simultaneously targeting Etk and Src could be a promising approach to overcome drug resistance in prostate cancer.
PMCID: PMC3744530  PMID: 23967135
14.  Histopathology in Surgically Treated Renal Cell Carcinoma: Survival Differences by Subtype and Stage 
The Journal of urology  2012;188(2):391-397.
Previous studies of the impact of renal cell carcinoma histopathology on survival are conflicting and generally limited to institutional analyses. Thus, we determined the role of renal cell carcinoma histopathology on the stage specific survival rate in a large population based cohort.
Materials and Methods
We used the 2000 to 2005 National Cancer Institute SEER (Surveillance, Epidemiology and End Results) database to identify 17,605 patients who underwent surgery for renal cell carcinoma and met study inclusion criteria. Patients were stratified by histological subtype (clear cell, papillary, chromophobe, collecting duct and sarcomatoid differentiation) and pathological stage. We performed Cox proportional hazard modeling and Kaplan-Meier survival analysis to determine overall and cancer specific survival.
Patients with papillary and chromophobe pathology were less likely to present with T3 or greater disease (17.6% and 16.9% ,respectively) while patients with collecting duct and sarcomatoid variants were more likely to present with T3 or greater disease (55.7% and 82.8%, respectively) compared to those with clear cell histology (p <0.001). On multivariate analysis histology was significantly associated with overall and cancer specific survival. Patients with chromophobe pathology had improved survival (HR 0.56, 95% CI 0.40–0.78) while those with collecting duct and sarcomatoid variants had worse survival (HR 2.07, 95% CI 1.44–2.97 and 2.26, 95% CI 1.93–2.64, respectively).
Renal cell carcinoma histological subtype predicts overall and cancer specific survival. Patients with collecting duct and sarcomatoid variants of renal cell carcinoma have poor survival, even those who present with low stage disease. These data suggest inherent differences in renal cell carcinoma biology and may ultimately form the basis of future histologically targeted therapies.
PMCID: PMC3714400  PMID: 22698625
kidney; carcinoma; renal cell; SEER program; survival analysis; histology
15.  Kidney tumor biomarkers revealed by simultaneous multiple matrix metabolomics analysis 
Cancer Research  2012;72(14):3471-3479.
Metabolomics is increasingly being utilized in cancer biology for biomarker discovery and identification of potential novel therapeutic targets. However, a systematic metabolomics study of multiple biofluids to determine their interrelationships and to describe their utility as tumor proxies is lacking. Using a mouse xenograft model of kidney cancer, characterized by sub-capsular implantation of Caki-1 clear cell human kidney cancer cells, we examined tissue, serum, and urine all obtained simultaneously at baseline (urine) and at, or close to, animal sacrifice (tissue and plasma). Uniform metabolomics analysis of all three “matrices” was accomplished using GC- and LC-MS. Of all the metabolites identified (267 in tissue, 246 in serum, 267 in urine), 89 were detected in all 3 matrices, and the majority were altered in the same direction. Heat maps of individual metabolites showed that alterations in serum were more closely related to tissue than was urine. Two metabolites, cinnamoylglycine and nicotinamide, were concordantly and significantly (when corrected for multiple testing) altered in tissue and serum, and cysteine-glutathione disulfide showed the highest change (232.4-fold in tissue) of any metabolite. Based on these and other considerations, three pathways were chosen for biological validation of the metabolomic data, resulting in potential therapeutic target identification. These data show that serum metabolomics analysis is a more accurate proxy for tissue changes than urine, that tryptophan degradation (yielding anti-inflammatory metabolites) is highly represented in renal cell carcinoma, and support the concept that PPAR-alpha antagonism may be a potential therapeutic approach for this disease.
PMCID: PMC3399039  PMID: 22628425
16.  Active Surveillance for Prostate Cancer Compared With Immediate Treatment 
Cancer  2011;118(14):3512-3518.
The costs associated with a contemporary active surveillance strategy compared with immediate treatment for prostate cancer are not well characterized. The purpose of this study is to elucidate the health care costs of an active surveillance paradigm for prostate cancer.
A theoretical cohort of 120,000 men selecting active surveillance for prostate cancer was created. The number of men remaining on active surveillance and those exiting to each of 5 treatments over 5 years were simulated in a Markov model. Estimated total costs after 5 years of active surveillance with subsequent delayed treatment were compared with immediate treatment. Sensitivity analyses were performed to test the effect of various surveillance strategies and attrition rates. Additional analyses to include 10 years of follow-up were performed.
The average simulated cost of treatment for 120,000 men initiating active surveillance with 5 years of follow-up and subsequent delayed treatment resulted in per patient cost savings of $16,042 (95% confidence interval [CI], $16,039-$16,046) relative to initial curative treatment. This represents a $1.9 billion dollar savings to the cohort. The strict costs of active surveillance exceeded those of brachytherapy in the ninth year of follow-up. A yearly biopsy within the active surveillance cohort increased costs by 22%, compared with every other year biopsy. At 10 years of follow-up, active surveillance still resulted in a cost benefit; however, the savings were reduced by 38% to $9944 (95% CI, $9941-$9948) per patient relative to initial treatment.
These data demonstrate that active surveillance represents a considerable cost savings over immediate treatment for prostate cancer in a theoretical cohort after 5 and 10 years of follow-up.
PMCID: PMC3698480  PMID: 22180322
prostate cancer; active surveillance; economics; Markov model; cost
17.  Evaluation of BMP-2 gene-activated muscle grafts for cranial defect repair 
Journal of Orthopaedic Research  2011;30(7):1095-1102.
Large, osseous, segmental defects heal poorly. Muscle has a propensity to form bone when exposed to an osteogenic stimulus such as that provided by transfer and expression of cDNA encoding bone morphogenetic protein-2 (BMP-2). The present study evaluated the ability of genetically modified, autologous muscle to heal large cranial defects in rats. Autologous grafts (8mm × 2mm) were punched from the biceps femoris muscle and transduced intraoperatively with recombinant adenovirus vector containing human BMP-2 or green fluorescent protein cDNA. While the muscle biopsies were incubating with the vector, a central parietal 8mm defect was surgically created in the calvarium of the same animal. The gene-activated muscle graft was then implanted into the cranial defect. After 8 weeks, crania were examined radiographically, histologically, and by micro-computed tomography and dual energy X-ray absorptiometry. Although none of the defects were completely healed in this time, muscle grafts expressing BMP-2 deposited more than twice as much new bone as controls. Histology confirmed the anatomical integrity of the newly formed bone, which was comparable in thickness and mineral density to the original cranial bone. This study confirms the in vivo osteogenic properties of genetically modified muscle and suggests novel strategies for healing bone.
PMCID: PMC3349003  PMID: 22213093
Gene Therapy; Bone Healing; Muscle; BMP-2; Osteogenesis
18.  Evidence that Behavioral Phenotypes of Morphine in β-arr2−/− Mice Are Due to the Unmasking of JNK Signaling 
Neuropsychopharmacology  2012;37(8):1953-1962.
The altered behavioral effects of morphine, but not most other mu agonists, in mice lacking β-arrestin 2, suggest that this scaffolding protein regulates the signaling cascade of this commonly used analgesic. One of the cascades that could be regulated by β-arrestin 2 is cJun-N-terminal kinase (JNK), which binds with β-arrestin 2 and modulates the analgesic effects of morphine. Using neurons lacking β-arrestin 2 (β-arr2−/−) to examine this interaction, we found that β-arr2−/− neurons show altered intracellular distribution of JNK and cJun, and that morphine, but not fentanyl, increased the nuclear localization of the phosphorylated, therefore activated, form of cJun, a JNK target in dorsal root ganglia neurons. This suggests that deleting β-arrestin 2 affects the JNK cascade. We therefore examined whether some of the behavioral phenotypes of mice lacking β-arrestin 2 could be a result of altered JNK signaling. Indeed, two different JNK inhibitors reversed the enhanced analgesic effect of morphine, a known phenotype of β-arr2−/− mice, to +/+ levels. Both the reduced locomotor effect of morphine and the psychomotor sensitization to repeated morphine administration in β-arr2−/− mice were also returned to +/+ levels by inhibiting JNK. In contrast, the behavioral effects of fentanyl were neither genotype-dependent nor affected by JNK inhibition. Furthermore, a PKC inhibitor had a similar effect as inhibiting JNK in reducing the enhanced analgesic effect of morphine in β-arr2−/− mice to +/+ levels. In summary, removing β-arrestin 2 reveals mu receptor activation of the JNK cascade in a ligand-specific manner explaining several behavioral phenotypes of β-arr2−/− mice.
PMCID: PMC3376327  PMID: 22491351
mu opioid receptor; JNK; β-arrestin 2; morphine; analgesia; locomotion; addiction & substance abuse; analgesia; animal models; beta-arrestin 2; JNK; morphine; mu opioid receptor; opioids; pain/analgesics
19.  Urinary acylcarnitines are altered in human kidney cancer 
Kidney cancer often diagnosed at late stages when treatment options are severely limited. Thus, greater understanding of tumor metabolism leading ultimately to novel approaches to diagnosis are needed. Our laboratory has been utilizing metabolomics to evaluate compounds appearing in kidney cancer patients’ biofluids at concentrations different from control patients. Here, we collected urine samples from kidney cancer patients and analyzed them by chromatography coupled to mass spectrometry. Once normalized to control for urinary concentration, samples were analyzed by two independent laboratories. After technical validation, we now show differential urinary concentrations of several acylcarnitines as a function of both cancer status and kidney cancer grade, with most acylcarnitines being increased in the urine of cancer patients and in those patients with high cancer grades. This finding was validated in a mouse xenograft model of human kidney cancer. Biological validation shows carbon chain length-dependent effects of the acylcarnitines on cytotoxicity in vitro, and higher chain length acylcarnitines demonstrated inhibitory effects on NF-κB activation, suggesting an immune modulatory effect of these compounds. Thus, acylcarnitines in the kidney cancer urine may reflect alterations in metabolism, cell component synthesis, and/or immune surveillance, and may help explain the profound chemotherapy resistance seen with this cancer. This study shows for the first time the value of a novel class of metabolites which may lead to new therapeutic approaches for cancer and may prove useful in cancer biomarker studies. Furthermore, these findings open up a new area of investigation into the metabolic basis of kidney cancer.
PMCID: PMC3258465  PMID: 21732340
20.  Synovial fibroblasts spread rheumatoid arthritis to unaffected joints 
Nature medicine  2009;15(12):1414-1420.
Active rheumatoid arthritis is characterized by originating from few but affecting subsequently the majority of joints. Thus far, the pathways of the progression of the disease are largely unknown. As rheumatoid arthritis synovial fibroblasts (RASFs) are key players in joint destruction and migrate in vitro, the current study evaluated the potential of RASFs to spread the disease in vivo. To simulate the primary joint of origin, healthy human cartilage was co-implanted subcutaneously into SCID mice together with RASFs. At the contralateral flank, healthy cartilage was implanted without cells. RASFs showed an active movement to the naïve cartilage via the vasculature independent of the site of application of RASFs into the SCID mouse, leading to a strong destruction of the target cartilage. These findings support the hypothesis that the characteristic clinical phenomenon of destructive arthritis spreading between joints is mediated, at least in part, by the transmigration of activated RASFs.
PMCID: PMC3678354  PMID: 19898488
21.  Development of an in vitro bioassay for measuring susceptibility to macrocyclic lactone anthelmintics in Dirofilaria immitis☆ 
Graphical abstract
•We optimized a larval migration inhibition assay for use with Dirofilaria immitis.•Consistent and reproducible dose–response data were generated.•High control migration rates (>90%) were observed.•The IC50 is the preferred parameter for this assay.•IC50 and IC95 values were better defined with eprinomectin than ivermectin.
For more than 20 years, anthelmintics of the macrocyclic lactone (ML) drug class have been widely and effectively used as preventives against the canine heartworm, Dirofilaria immitis. However, in recent years an increased number of lack of efficacy (LOE) cases are being reported, in which dogs develop mature heartworm infections despite receiving monthly prophylactic doses of ML drugs. While this situation is raising concerns that heartworms may be developing resistance to MLs, compelling evidence for this is still lacking. Resolution of this dilemma requires validated biological or molecular diagnostic assays, but, unfortunately, no such tests currently exist. To address this need, we developed and optimized a larval migration inhibition assay (LMIA) for use with D. immitis third-stage larvae. The LMIA was used to measure the in vitro dose–response of two ML drugs (ivermectin and eprinomectin) on a known ML-susceptible laboratory strain of D. immitis. A nonlinear regression model was fit to the dose–response data, from which IC50 values were calculated; the mean IC50 and 95% confidence interval for IVM was 4.56 μM (1.26–16.4 μM), greater than that for EPR at 2.02 μM (1.68–2.42 μM), and this difference was significant (p = 0.0428). The R2 value for EPR assays (0.90) was also greater than that for IVM treatment (0.71). The consistency and reproducibility of the dose–response data obtained with this assay suggests that it may be a useful technique for investigating the relative susceptibilities to ML drugs in other D. immitis populations.
PMCID: PMC3862408  PMID: 24533299
Dirofilaria immitis; In vitro; Macrocyclic lactone; Third-stage larva
22.  Investigation of the impact of urine handling procedures on interpretation of urinalysis findings and product safety in subjects treated with ezogabine 
Ezogabine (also known by the international nonproprietary name of retigabine) is an antiepileptic drug codeveloped and comarketed by Valeant Pharmaceuticals North America and GlaxoSmithKline, which reduces neuronal excitability by enhancing the activity of potassium channels and has the potential to have effects on the urinary system through a pharmacologic action on bladder smooth muscle. In a single post-herpetic neuralgia trial, but not in an extensive epilepsy development program, proteinuria was unexpectedly reported in patients receiving ezogabine. Consequently, investigations were conducted to determine whether the reported proteinuria represented a true or false-positive finding.
Patients receiving ezogabine 900–1200 mg/day in an open-label extension (Study 303) of a Phase III epilepsy trial voluntarily provided urine samples. Fresh samples were analyzed immediately at the study site, and stabilized aliquots were analyzed 1–3 days after collection at two central laboratories. In an open-label study in healthy volunteers receiving ezogabine 600–900 mg/day (Study RTG114137), urine samples were analyzed fresh (<2 hours after collection) and, using two different stabilizers and storage at room temperature, after 24 and 72 hours. Fluid intake was restricted prior to one sample point. Albumin:creatinine ratios were assessed in both studies.
In Study 303, there was notable variation in clarity, color, and proteinuria between fresh and stored urine samples, and between samples analyzed at different laboratories. In RTG114137, reporting rates of proteinuria were elevated following storage using one stabilizer, and the frequency of color change from fresh to stored samples differed between the stabilizers and between 24 and 72 hours with one stabilizer. Following fluid restriction, proteinuria rates were elevated with both stabilizers. Poor tolerability of ezogabine 750–900 mg/day resulted in limited titration beyond 750 mg/day and early termination of RTG114137.
Hydration status, interval between urine collection and analysis, and the type of stabilizer used are all factors that may lead to false-positive proteinuria findings in patients receiving ezogabine and should be borne in mind if abnormalities are reported.
PMCID: PMC3650885  PMID: 23671389
antiepileptic drugs; ezogabine; retigabine; urinary safety; urinalysis
23.  Chronic inflammatory injury results in increased coupling of delta opioid receptors to voltage-gated Ca2+ channels 
Molecular Pain  2013;9:8.
Opioid receptors regulate a diverse array of physiological functions. Mu opioid receptor agonists are well-known analgesics for treating acute pain. In contrast, animal models suggest that chronic pain is more effectively relieved by delta opioid receptor agonists. A number of studies have shown that chronic pain results in increased function of delta opioid receptors. This is proposed to result from enhanced trafficking of the delta opioid receptor to the cell membrane induced by persistent tissue injury. However, recent studies have questioned this mechanism, which has resulted in some uncertainty as to whether delta opioid receptors are indeed upregulated in chronic pain states. To clarify this question, we have examined the effect of chronic inflammatory pain over time using both an ex vivo measure of delta function: receptor-Ca2+ channel coupling, and an in vivo measure; the relief of chronic pain by a delta opioid receptor agonist. In addition, as beta-arrestin 2 can regulate delta opioid receptor trafficking and signaling, we have further examined whether deleting this scaffolding and signal transduction molecule alters delta opioid receptor function.
We used the Complete Freund’s Adjuvant model of inflammatory pain, and examined the effectiveness of the delta agonist, SNC80, to both inhibit Ca2+ channels in primary afferent neurons and to attenuate mechanical allodynia. In naïve beta-arrestin 2 wildtype and knockout mice, SNC80 neither significantly inhibited voltage-dependent Ca2+ currents nor produced antinociception. However, following inflammatory pain, both measures showed a significant and long-lasting enhancement of delta opioid receptor function that persisted for up to 14 days post-injury regardless of genotype. Furthermore, although this pain model did not alter Ca2+ current density, the contribution of N-type Ca2+ channels to the total current appeared to be regulated by the presence of beta-arrestin 2.
Our results indicate that there is an upregulation of delta opioid receptor function following chronic pain. This gain of function is reflected in the increased efficacy of a delta agonist in both behavioral and electrophysiological measures. Overall, this work confirms that delta opioid receptors can be enhanced following tissue injury associated with chronic pain.
PMCID: PMC3621800  PMID: 23497324
Primary afferent; SNC80; Delta opioid receptor; Chronic pain; Dorsal root ganglia; Ca2+ channel
24.  BMP12 and BMP13 gene transfer induce ligamentogenic differentiation in mesenchymal progenitor and anterior cruciate ligament cells 
Cytotherapy  2010;12(4):505-513.
Background aims
To date there are only very few data available on the ligamentogenic differentiation capacity of mesenchymal stromal/progenitor cells (MSC) and anterior cruciate ligament (ACL) fibroblasts.
We describe the in vitro potential of MSC and ACL cells to undergo ligamentogenic differentiation upon transduction with adenoviral vectors encoding the human cDNA for bone morphogenetic protein (BMP) 12 and BMP13, also known as growth and differentiation factors (GDF) 6 and 7, respectively.
Transgene expression for at least 14 days was confirmed by Western blot analyzes. After 21 days of cell culture within collagen type I hydrogels, histochemical (hematoxylin/eosin (H&E), Azan and van Gieson), immunohistochemical and polymerase chain reaction (PCR) analyzes of the genetically modified constructs of both cell types revealed elongated, viable fibroblast-like cells embedded in a ligament-like matrix rich in collagens, vimentin, fibronectin, decorin, elastin, scleraxis, tenascin, and tenomodulin.
It appears that both MSC and ACL fibroblasts are capable of ligamentogenic differentiation with these factors. This information may aid in the development of biologic approaches to repair and restore ACL after injury.
PMCID: PMC3580941  PMID: 20334610
adenovirus; anterior cruciate ligament; BMP12; BMP13; collagen hydrogel; fibroblasts; gene transfer; mesenchymal stromal cells
25.  Arginase Treatment Prevents the Recovery of Canine Lymphoma and Osteosarcoma Cells Resistant to the Toxic Effects of Prolonged Arginine Deprivation 
PLoS ONE  2013;8(1):e54464.
Rapidly growing tumor cells require a nutrient-rich environment in order to thrive, therefore, restricting access to certain key amino acids, such as arginine, often results in the death of malignant cells, which frequently display defective cell cycle check-point control. Healthy cells, by contrast, become quiescent and remain viable under arginine restriction, displaying full recovery upon return to arginine-rich conditions. The use of arginase therapy to restrict available arginine for selectively targeting malignant cells is currently under investigation in human clinical trials. However, the suitability of this approach for veterinary uses is unexplored. As a prelude to in vivo studies in canine malignancies, we examined the in vitro effects of arginine-deprivation on canine lymphoid and osteosarcoma cell lines. Two lymphoid and 2 osteosarcoma cell lines were unable to recover following 6 days of arginine deprivation, but all remaining cell lines displayed full recovery upon return to arginine-rich culture conditions. These remaining cell lines all proved susceptible to cell death following the addition of arginase to the cultures. The lymphoid lines were particularly sensitive to arginase, becoming unrecoverable after just 3 days of treatment. Two of the osteosarcoma lines were also susceptible over this time-frame; however the other 3 lines required 6–8 days of arginase treatment to prevent recovery. In contrast, adult progenitor cells from the bone marrow of a healthy dog were able to recover fully following 9 days of culture in arginase. Over 3 days in culture, arginase was more effective than asparaginase in inducing the death of lymphoid lines. These results strongly suggest that short-term arginase treatment warrants further investigation as a therapy for lymphoid malignancies and osteosarcomas in dogs.
PMCID: PMC3554772  PMID: 23365669

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