Spleen tyrosine kinase (Syk) is a potent target for GvHD prevention at different cellular levels.

Leukemia. 2012 Jan 17;

Authors: Leonhardt F, Zirlik K, Buchner M, Prinz G, Hechinger AK, Gerlach UV, Fisch P, Schmitt-Gräff A, Reichardt W, Zeiser R

Abstract
Acute graft-versus-host disease (GvHD) limits the applicability of allogeneic hematopoietic cell transplantation for the treatment of leukemia. GvHD occurs as a consequence of multiple activating events in antigen-presenting cells (APCs) and T cells (Tcs). Spleen tyrosine kinase (Syk) is an intracellular non-receptor tyrosine kinase involved in multiple signaling events of immune cells. Therefore, we hypothesized that Syk may be a promising target to inhibit GvHD, which involves activation of different immune cell populations. In vivo expansion of luciferase(+) donor Tcs in mice developing GvHD was reduced by treatment with the Syk inhibitor Fostamatinib, which led to increased survival and reduced histologically confirmed GvHD severity. Importantly, in vivo and in vitro cytotoxicity against leukemia target cells and anti-murine cytomegalovirus immune responses were not impacted by Fostamatinib. In APCs Syk inhibition reduced the expression of costimulatory molecules and disrupted cytoskeletal organization with consecutive APC migratory defects in vitro and in vivo while phagocytic activity remained intact. On the basis of these immunomodulatory effects on different cell populations, we conclude that Syk targeting in alloantigen-activated Tcs and APCs with pharmacologic inhibitors, already applied successfully in anti-lymphoma therapy, has clinical potential to reduce GvHD, especially as anti-leukemia and anti-viral immunity were preserved.Leukemia advance online publication, 3 February 2012; doi:10.1038/leu.2012.10.

PMID: 22301676 [PubMed - as supplied by publisher]

Transgene expression and local tissue distribution of naked and polymer-condensed plasmid DNA after intradermal administration in mice.

J Control Release. 2012 Jan 24;

Authors: Palumbo RN, Zhong X, Panus D, Han W, Ji W, Wang C

Abstract
DNA vaccination using cationic polymers as carriers has the potential to be a very powerful method of immunotherapy, but typical immune responses generated have been less than robust. To better understand the details of DNA vaccine delivery in vivo, we prepared polymer/DNA complexes using three structurally distinct cationic polymers and fluorescently labeled plasmid DNA and injected them intradermally into mice. We analyzed transgene expression (luciferase) and the local tissue distribution of the labeled plasmid at the injection site at various time points (from hours to days). Comparable numbers of luciferase expressing cells were observed in the skin of mice receiving naked plasmid or polyplexes one day after transfection. At day 4, however, the polyplexes appeared to result in more transfected skin cells than naked plasmid. Live animal imaging revealed that naked plasmid dispersed quickly in the skin of mice after injection and had a wider distribution than any of the three types of polyplexes. However, naked plasmid level dropped to below detection limit after 24h, whereas polyplexes persisted for up to 2weeks. The PEGylated polyplexes had a significantly wider distribution in the tissue than the nonPEGylated polyplexes. PEGylated polyplexes also distributed more broadly among dermal fibroblasts and allowed greater interaction with antigen-presenting cells (APCs) (dendritic cells and macrophages) starting at around 24h post-injection. By day 4, co-localization of polyplexes with APCs was observed at the injection site regardless of polymer structure, whereas small amounts of polyplexes were found in the draining lymph nodes. These in vivo findings demonstrate the superior stability of PEGylated polyplexes in physiological milieu and provide important insight on how cationic polymers could be optimized for DNA vaccine delivery.

PMID: 22300619 [PubMed - as supplied by publisher]

Significant biological role of sp1 transactivation in multiple myeloma.

Clin Cancer Res. 2011 Oct 15;17(20):6500-9

Authors: Fulciniti M, Amin S, Nanjappa P, Rodig S, Prabhala R, Li C, Minvielle S, Tai YT, Tassone P, Avet-Loiseau H, Hideshima T, Anderson KC, Munshi NC

Abstract
PURPOSE: The transcription factor specificity protein 1 (Sp1) controls number of cellular processes by regulating the expression of critical cell cycle, differentiation, and apoptosis-related genes containing proximal GC/GT-rich promoter elements. We here provide experimental and clinical evidence that Sp1 plays an important regulatory role in multiple myeloma (MM) cell growth and survival.
EXPERIMENTAL DESIGN: We have investigated the functional Sp1 activity in MM cells using a plasmid with Firefly luciferase reporter gene driven by Sp1-responsive promoter. We have also used both siRNA- and short hairpin RNA-mediated Sp1 knockdown to investigate the growth and survival effects of Sp1 on MM cells and further investigated the anti-MM activity of terameprocol (TMP), a small molecule that specifically competes with Sp1-DNA binding in vitro and in vivo.
RESULTS: We have confirmed high Sp1 activity in MM cells that is further induced by adhesion to bone marrow stromal cells (BMSC). Sp1 knockdown decreases MM cell proliferation and induces apoptosis. Sp1-DNA binding inhibition by TMP inhibits MM cell growth both in vitro and in vivo, inducing caspase-9-dependent apoptosis and overcoming the protective effects of BMSCs.
CONCLUSIONS: Our results show Sp1 as an important transcription factor in myeloma that can be therapeutically targeted for clinical application by TMP.

PMID: 21856768 [PubMed - indexed for MEDLINE]

A new model of rectal cancer with regional lymph node metastasis allowing in vivo evaluation by imaging biomarkers.

Biomed Pharmacother. 2011 Sep;65(6):401-6

Authors: Minicozzi AM, Conti G, Merigo G, Marzola P, Boschi F, Calderan L, Pacca R, Sbarbati A, Cordiano C

Abstract
OBJECT: The work is aimed to develop a murine model of rectal cancer, which could be used to monitor lymph node metastasis development by magnetic resonance imaging (MRI) and optical imaging (OI) techniques.
SUBJECTS AND METHODS: Ht-29 cancer cells were directly injected into the submucosal layer of the rectum of athymic nude mice using trans-anal rectal cancer cell injection (TARCI). Thirty-six mice were inoculated with 10×10(5) cells and five mice were treated with sterile phosphate buffer solution. One to 4 weeks after cell injection, tumor growth was evaluated in vivo using T2-weighted MRI at 4.7T. A further group of animal (n=6) treated with ht-29_luc cells, with the same protocol, was monitored by optical imaging. In both groups, the presence of the primary tumor and of lymph nodes metastasis was confirmed by histology.
RESULTS: In all animals, primary tumors were detectable by MRI, 1 week from TARCI. After 4 weeks primary tumors showed a mean longitudinal diameter of about 2cm. All animals developed regional lymph node metastases. Others organs (e.g. lung or liver) were not affected. In fat-suppressed, T2-weighted MRI, lymph nodes appeared as small areas characterized by hyper-intense signal compared to muscle. OI permitted evaluation of the primary tumor growth in perineal region.
CONCLUSIONS: TARCI of ht-29 cells into the rectum of nude mice is a feasible way to obtain a easily reproducible model of regional lymph node metastases could be monitored by magnetic resonance and optical imaging techniques.

PMID: 21719244 [PubMed - indexed for MEDLINE]

Regulatory Role of mir-203 in Prostate Cancer Progression and Metastasis.

Clin Cancer Res. 2011 Aug 15;17(16):5287-98

Authors: Saini S, Majid S, Yamamura S, Tabatabai L, Suh SO, Shahryari V, Chen Y, Deng G, Tanaka Y, Dahiya R

Abstract
PURPOSE: Advanced metastatic prostate cancer (PCa) is a fatal disease, with only palliative therapeutic options. Though almost 80% of cases of metastatic PCa present bone metastasis, our current understanding of the molecular mechanisms that govern this metastatic dissemination remains fragmentary. The main objective of the present study was to identify microRNA (miRNA) genes that regulate metastatic PCa.
EXPERIMENTAL DESIGN: miRNA expression profiling was done in human prostate cell lines to identify dysregulated miRNA components of advanced PCa. miR-203 expression was assessed in prostate carcinoma cell lines and clinical specimens by real-time PCR and in situ hybridization. To assess the biological significance of miR-203, miR-203 was reexpressed in bone metastatic PCa cell lines followed by in vitro and in vivo functional assays.
RESULTS: miR-203 expression is specifically attenuated in bone metastatic PCa suggesting a fundamental antimetastatic role for this miRNA. Reintroduction of miR-203 in bone metastatic PCa cell lines suppresses metastasis via inhibition of several critical steps of the metastatic cascade including epithelial-mesenchymal transition, invasion, and motility. Ectopic miR-203 significantly attenuated the development of metastasis in a bone metastatic model of PCa. Importantly, miR-203 regulates a cohort of pro-metastatic genes including ZEB2, Bmi, survivin, and bone-specific effectors including Runx2, a master regulator of bone metastasis.
CONCLUSIONS: miR-203 is an "antimetastatic" miRNA in PCa that acts at multiple steps of the PCa metastatic cascade via repression of a cohort of prometastatic targets. miR-203 may be an attractive target for therapeutic intervention in advanced PCa.

PMID: 21159887 [PubMed - indexed for MEDLINE]

Pressure-Overload Dependent Membrane-Type 1 Matrix Metalloproteinase Induction: Relationship to LV Remodeling and Fibrosis.

Am J Physiol Heart Circ Physiol. 2012 Jan 27;

Authors: Zile MR, Baicu CF, Stroud RE, Van Laer AO, Arroyo J, Mukherjee R, Jones JA, Spinale FG

Abstract
Background: Increased myocardial extracellular matrix collagen represents an important structural milestone during the development of left ventricular (LV) pressure overload (PO); however, the proteolytic pathways that contribute to this process are not fully understood. This study tested the hypothesis that membrane type-1 matrix metalloproteinase (MT1-MMP) is directly induced at the transcriptional level in-vivo during PO and is related to changes in LV collagen content. Methods/Results: PO was induced in vivo by transverse aortic constriction in transgenic mice containing the full length human MT1-MMP promoter region ligated to luciferase (MT1-MMP prom mice). MT1-MMP promoter activation (luciferase expression), expression, and activity, collagen volume fraction (CVF), and left atrial dimension were measured at 1 (n= 8), 2 (n= 12) and 4 (n= 17) weeks following PO. Non-PO mice (n= 10) served as controls. Luciferase expression increased by 5-fold at 1 week, fell at 2 weeks and increased again by 9-fold at 4 weeks of PO (p<0.05). MT1-MMP expression and activity increased at 1 week fell at 2 weeks and increased again at 4 weeks after PO. CVF increased at 1 week, remained unchanged at 2 weeks and increased by 3 fold at 4 weeks of PO (p<0.05). There was a strong positive correlation between CVF and MT1-MMP activity (r=0.80, p<0.05). Left atrial dimension remained unchanged at 1 and 2 weeks but increased by 25% at 4 weeks of PO. When a mechanical load was applied in vitro to LV papillary muscles isolated from MT1-MMP prom mice, increased load caused MT1-MMP promoter activation to increase by 2 fold and MT1-MMP expression to increase by 5 fold (p< 0.05). Conclusions: These findings challenge the canonical belief that PO suppresses overall matrix proteolytic activity, but rather supports the concept that certain proteases, such as MT1-MMP, play a pivotal role in pressure-overload induced matrix remodeling and fibrosis.

PMID: 22287584 [PubMed - as supplied by publisher]

The mouse gene encoding the carnitine biosynthetic enzyme 4-N-trimethylaminobutyraldehyde dehydrogenase is regulated by peroxisome proliferator-activated receptor α

Biochim Biophys Acta. 2012 Jan 21;

Authors: Wen G, Ringseis R, Rauer C, Eder K

Abstract
Genes involved in carnitine uptake and synthesis, such as organic cation transporter-2 (OCTN2) and γ-butyrobetaine dioxygenase (BBD), have been shown to be regulated by peroxisome proliferator-activated receptor (PPAR)α directly. Whether other genes encoding enzymes involved in the carnitine synthesis pathway, such as 4-N-trimethylaminobutyraldehyde dehydrogenase (TMABA-DH) and trimethyllysine dioxygenase (TMLD), are also direct PPARα target genes is less clear. In silico-analysis of the mouse TMLD promoter and first intron and the TMABA-DH promoter revealed several putative peroxisome proliferator response elements (PPRE) with high similarity to the consensus PPRE. Luciferase reporter gene assays using either a 2kb TMLD promoter or a 4kb TMLD first intron reporter constructs revealed no functional PPRE. In contrast, reporter gene assays using wild-type and mutated 5´-truncation TMABA-DH promoter reporter constructs showed that one PPRE located at position -132 in the proximal promoter is probably functional. Using gel shift assays we observed in vitro-binding of PPARα to this PPRE. Moreover, using chromatin immunoprecipitation assays we found that PPARα also binds in vivo to a nucleotide sequence spanning the PPRE at -132, which confirms that this PPRE is functional. In conclusion, the present study shows that the mouse TMABA-DH gene is a direct PPARα target gene. Together with the recent identification of the mouse BBD and the mouse OCTN2 genes as PPARα target genes this finding confirm that PPARα plays a key role in the regulation of carnitine homeostasis by controlling genes involved in carnitine synthesis and carnitine uptake.

PMID: 22285688 [PubMed - as supplied by publisher]

Snail1 induces epithelial-to-mesenchymal transition and tumor initiating stem cell characteristics.

BMC Cancer. 2011;11:396

Authors: Dang H, Ding W, Emerson D, Rountree CB

Abstract
BACKGROUND: Tumor initiating stem-like cells (TISCs) are a subset of neoplastic cells that possess distinct survival mechanisms and self-renewal characteristics crucial for tumor maintenance and propagation. The induction of epithelial-mesenchymal-transition (EMT) by TGFβ has been recently linked to the acquisition of TISC characteristics in breast cancer. In HCC, a TISC and EMT phenotype correlates with a worse prognosis. In this work, our aim is to elucidate the underlying mechanism by which cells acquire tumor initiating characteristics after EMT.
METHODS: Gene and protein expression assays and Nanog-promoter luciferase reporter were utilized in epithelial and mesenchymal phenotype liver cancer cell lines. EMT was analyzed with migration/invasion assays. TISC characteristics were analyzed with tumor-sphere self-renewal and chemotherapy resistance assays. In vivo tumor assay was performed to investigate the role of Snail1 in tumor initiation.
CONCLUSION: TGFβ induced EMT in epithelial cells through the up-regulation of Snail1 in Smad-dependent signaling. Mesenchymal liver cancer post-EMT demonstrates TISC characteristics such as tumor-sphere formation but are not resistant to cytotoxic therapy. The inhibition of Snail1 in mesenchymal cells results in decreased Nanog promoter luciferase activity and loss of self-renewal characteristics in vitro. These changes confirm the direct role of Snail1 in some TISC traits. In vivo, the down-regulation of Snail1 reduced tumor growth but was not sufficient to eliminate tumor initiation. In summary, TGFβ induces EMT and TISC characteristics through Snail1 and Nanog up-regulation. In mesenchymal cells post-EMT, Snail1 directly regulates Nanog expression, and loss of Snail1 regulates tumor growth without affecting tumor initiation.

PMID: 21929801 [PubMed - indexed for MEDLINE]

HIF-1-mediated up-regulation of cardiotrophin-1 is involved in the survival response of cardiomyocytes to hypoxia.

Cardiovasc Res. 2011 Nov 1;92(2):247-55

Authors: Robador PA, San José G, Rodríguez C, Guadall A, Moreno MU, Beaumont J, Fortuño A, Díez J, Martínez-González J, Zalba G

Abstract
AIMS: Cardiotrophin-1 (CT-1) is a cytokine of the interleukin-6 superfamily which is up-regulated in cardiac diseases, in part via hypoxia-dependent mechanisms. However, no evidence for a direct regulation of CT-1 gene (CTF1) promoter by hypoxia inducible factor-1 (HIF-1) has been provided.
METHODS AND RESULTS: Hypoxia increased CT-1 mRNA levels in the murine adult cardiomyocyte cell line HL-1 in a time-dependent manner. Interestingly, in a murine model (C57BL/6), we show that systemic hypoxia also significantly up-regulated CT-1 in myocardial tissue. The effect of hypoxia on CT-1 expression was mediated through a transcriptional mechanism, since hypoxia increased luciferase activity of constructs containing CTF1 promoter sequences. The increase in CT-1 levels was significantly reduced by drugs that prevent calcium mobilization, such as lercanidipine, or that inhibit the activation of the PI3K/Akt pathway (wortmannin) or mammalian target of rapamycin (rapamycin). The CT-1 elevation was similarly induced by HIF-1α over-expression in co-transfection experiments and prevented by HIF-1α silencing. The direct interaction of HIF-1α with the CTF1 promoter was confirmed through site-directed mutagenesis of hypoxia response elements, electrophoreric mobility shift, and ChIP assays. Hypoxia induced HL-1 apoptosis (measured as annexin-V binding or caspase 3/7 activity) which was increased when CT-1 was silenced in knocked-down cells by lentiviral vectors.
CONCLUSION: Hypoxia increased CT-1 levels in cardiac cells (in vitro and in vivo) through a direct regulation of CTF1 promoter by HIF-1α. This CT-1 activation by hypoxia may protect cells from apoptosis, thus supporting a protective role for CT-1 as a survival factor for cardiomyocytes.

PMID: 21771897 [PubMed - indexed for MEDLINE]

Eμ/miR-125b transgenic mice develop lethal B-cell malignancies.

Leukemia. 2011 Dec;25(12):1849-56

Authors: Enomoto Y, Kitaura J, Hatakeyama K, Watanuki J, Akasaka T, Kato N, Shimanuki M, Nishimura K, Takahashi M, Taniwaki M, Haferlach C, Siebert R, Dyer MJ, Asou N, Aburatani H, Nakakuma H, Kitamura T, Sonoki T

Abstract
MicroRNA-125b-1 (miR-125b-1) is a target of a chromosomal translocation t(11;14)(q24;q32) recurrently found in human B-cell precursor acute lymphoblastic leukemia (BCP-ALL). This translocation results in overexpression of miR-125b controlled by immunoglobulin heavy chain gene (IGH) regulatory elements. In addition, we found that six out of twenty-one BCP-ALL patients without t(11;14)(q24;q32) showed overexpression of miR-125b. Interestingly, four out of nine patients with BCR/ABL-positive BCP-ALL and one patient with B-cell lymphoid crisis that had progressed from chronic myelogenous leukemia overexpressed miR-125b. To examine the role of the deregulated expression of miR-125b in the development of B-cell tumor in vivo, we generated transgenic mice mimicking the t(11;14)(q24;q32) (Eμ/miR-125b-TG mice). Eμ/miR-125b-TG mice overexpressed miR-125b driven by IGH enhancer and promoter and developed IgM-negative or IgM-positive lethal B-cell malignancies with clonal proliferation. B cells obtained from the Eμ/miR-125b-TG mice were resistant to apoptosis induced by serum starvation. We identified Trp53inp1, a pro-apoptotic gene induced by cell stress, as a novel target gene of miR-125b in hematopoietic cells in vitro and in vivo. Our results provide direct evidence that miR-125b has important roles in the tumorigenesis of precursor B cells.

PMID: 21738213 [PubMed - indexed for MEDLINE]

RNAi-mediated gene silencing in tumour tissue using replication-competent retroviral vectors.

Gene Ther. 2011 Oct;18(10):953-60

Authors: Schaser T, Wrede C, Duerner L, Sliva K, Cichutek K, Schnierle B, Buchholz CJ

Abstract
RNAi represents a powerful technology to specifically downregulate the expression of target genes. For cancer research and therapy, an efficient in vivo delivery system is supposed to distribute RNAi to all tumour cells upon systemic administration. We present replication-competent murine leukaemia virus (MLV) vectors, which deliver RNAi to tumour tissue upon tail vein injection. In HT1080 cells stably expressing GFP or luciferase, GFP expression was suppressed by more than 80% and luciferase (luc) activity by more than 90%, even when only 0.1% of the cells were initially infected with reporter gene specific vectors. To demonstrate its potential, PLK1- and MMP14-specific small hairpin RNA expression cassettes were applied in the system. Upon infection, PLK1 and MMP14 levels were reduced on mRNA and protein level. MLV-shPLK1-infected cells were arrested in the G2-phase and underwent apoptosis. MLV-shMMP14-infected cells showed reduced MMP2 activity, as well as substantially reduced invasion and tumour growth. In vivo, MLV-shLuc silenced luc expression in HT1080-luc tumour tissue by more than 80% and MLV-shPLK1 reduced tumour growth substantially, demonstrating the therapeutic relevance of this system. This RNAi vector system allows long-term downregulation of target gene expression as well as efficient delivery to and distribution throughout tumour tissue in vivo.

PMID: 21472010 [PubMed - indexed for MEDLINE]

Correlation between epidermal growth factor receptor-specific nanobody uptake and tumor burden: a tool for noninvasive monitoring of tumor response to therapy.

Mol Imaging Biol. 2011 Oct;13(5):940-8

Authors: Gainkam LO, Keyaerts M, Caveliers V, Devoogdt N, Vanhove C, Van Grunsven L, Muyldermans S, Lahoutte T

Abstract
PURPOSE: Nanobodies represent an interesting class of probes for the generic development of molecular imaging agents. We studied the relationship between tumor uptake of the epidermal growth factor receptor (EGFR)-specific nanobody (99m)Tc-7C12 and tumor burden and evaluated the possibility of using this probe to monitor tumor response to erlotinib.
PROCEDURES: The specificity and affinity of (99m)Tc-7C12 was determined on A431 cells. Cells expressing firefly luciferase were used to evaluate tumor burden using bioluminescence imaging. We evaluated the effect of erlotinib on tumor burden and (99m)Tc-7C12 uptake in vitro as well as in vivo. In vivo bioluminescence imaging was performed followed by pinhole single-photon emission computed tomography/micro-computed tomography.
RESULTS: (99m)Tc-7C12 binds specifically to the receptor with high affinity (3.67 ± 0.59 nM). Erlotinib reduced tumor uptake and cell viability in a concentration-dependent manner. Tumor uptake of (99m)Tc-7C12 showed good correlation with tumor burden. Erlotinib treatment resulted in a progressive reduction of tumor burden and tumor uptake of (99m)Tc-7C12.
CONCLUSION: (99m)Tc-7C12 binds to EGFR with high affinity and specificity. Tumor uptake is correlated with tumor burden. Quantification of (99m)Tc-7C12 uptake is promising for monitoring therapy response of EGFR-expressing tumors.

PMID: 20865332 [PubMed - indexed for MEDLINE]

Topical androgen antagonism promotes cutaneous wound healing without systemic androgen deprivation by blocking β-catenin nuclear translocation and cross-talk with TGF-β signaling in keratinocytes.

Wound Repair Regen. 2012 Jan;20(1):61-73

Authors: Toraldo G, Bhasin S, Bakhit M, Guo W, Serra C, Safer JD, Bhawan J, Jasuja R

Abstract
Orchidectomy in rodents and lower testosterone levels in men are associated with improved cutaneous wound healing. However, due to the adverse effects on skeletal and sexual tissues, systemic androgen blockade is not a viable therapeutic intervention. Accordingly, we tested the hypothesis that topical application of an androgen antagonist would elicit accelerated wound healing without systemic androgen antagonism. Full-thickness cutaneous wounds were created on adult C57BL6/J mice. Daily topical application of androgen receptor antagonist, flutamide, resulted in improved gap closure similar to orchiectomized controls and faster than orchidectomized mice treated with topical testosterone. In vivo data showed that the effects of androgen antagonism on wound closure primarily accelerate keratinocytes migration without effecting wound contraction. Consequently, mechanisms of testosterone action on reepithelialization were investigated in vitro by scratch wounding assays in confluent keratinocytes. Testosterone inhibited keratinocyte migration and this effect was in part mediated through promotion of nuclear translocation of β-catenin and by attenuating transforming growth factor-β (TGF-β) signaling through β-catenin. The link between Wnt and TGF beta signaling was confirmed by blocking β-catenin and by following TGF-β-induced transcription of a luciferase reporter gene. Together, these data show that blockade of β-catenin can, as a potential target for novel therapeutic interventions, accelerate cutaneous wound healing.

PMID: 22276587 [PubMed - in process]

The HIV-1 protease inhibitor nelfinavir impairs proteasome activity and inhibits the multiple myeloma cells proliferation in vitro and in vivo.

Haematologica. 2012 Jan 22;

Authors: Bono C, Karlin L, Harel S, Mouly E, Labaume S, Galicier L, Apcher S, Sauvageon H, Fermand JP, Bories JC, Arnulf B

Abstract
Background. Multiple Myeloma is characterized by the accumulation of tumor plasma cells in the bone marrow. Despite improvement brought by proteasome inhibitors such as bortezomib, myeloma remains an incurable disease. In a variety of human cancers, human immunodeficiency virus protease inhibitors (nelfinavir) effectively inhibit tumor progression, but their impact on myeloma is unknown. We assessed in vitro and in vivo effects of the nelfinavir, on myeloma. Design and Methods. The effect of nelfinavir (1-10microM) on proteasome activity, proliferation and viability of myeloma cell line and plasma cells from patients was assessed by measuring PERK, AKT, STAT3 and ERK1/2 phosphorylation and CHOP expression with immunoblotting or flow cytometry. The in vivo effect was assessed in NOD/SCID mice injected with luciferase expressing human myeloma cell lines and treated with nelfinavir at 75mg/kg/day. Tumor progression was evaluated using a bioluminescent system. Results. Nelfinavir inhibited the 26S chymotrypsin-like proteasome activity, impaired proliferation and triggered apoptosis of the myeloma cell lines and fresh plasma cells. It activated the proapoptotic unfolded protein response pathway by inducing PERK phosphorylation and CHOP expression. Cell death triggered by nelfinavir treatment correlated with decreased phosphorylation of AKT, STAT3 and ERK1/2. Nelfinavir enhanced the anti-proliferative activity of bortezomib, dexamethasone and histone deacetylase inhibitors and delayed tumor growth in a myeloma mouse model.Conclusions. These results suggest that nelfinavir used at pharmacological dosage, alone or in combination, may be useful in the treatment of myeloma. Our data provide a preclinical basis for clinical trials using nelfinavir in patients with myeloma.

PMID: 22271897 [PubMed - as supplied by publisher]

Natural killer cell lines preferentially kill clonogenic multiple myelomacells and decrease myeloma engraftment in a bioluminescentxenograft mouse model.

Haematologica. 2012 Jan 22;

Authors: Swift BE, Williams BA, Kosaka Y, Wang XH, Medin JA, Viswanathan S, Martinez-Lopez J, Keating A

Abstract
Background: Novel therapies capable of targeting drug resistant clonogenic MM cells are required for more effective treatment of multiple myeloma. This study investigates the cytotoxicity of natural killer cell lines against bulk and clonogenic multiple myeloma and evaluates the tumor burden after NK cell therapy in a bioluminescent xenograft mouse model.Design and Methods: The cytotoxicity of natural killer cell lines was evaluated against bulk multiple myeloma cell lines using chromium release and flow cytometry cytotoxicity assays. Selected activating receptors on natural killer cells were blocked to determine their role in multiple myeloma recognition. Growth inhibition of clonogenic multiple myeloma cells was assessed in a methylcellulose clonogenic assay in combination with secondary replating to evaluate the self-renewal of residual progenitors after natural killer cell treatment. A bioluminescent mouse model was developed using the human U266 cell line transduced to express green fluorescent protein and luciferase (U266eGFPluc) to monitor disease progression in vivo and assess bone marrow engraftment after intravenous NK-92 cell therapy. Results: Three multiple myeloma cell lines were sensitive to NK-92 and KHYG-1 cytotoxicity mediated by NKp30, NKp46, NKG2D and DNAM-1 activating receptors. NK-92 and KHYG-1 demonstrated 2-3 fold greater inhibition of clonogenic multiple myeloma growth, compared with killing of the bulk tumor population. In addition, the residual colonies after treatment formed significantly fewer colonies compared to the control in a secondary replating for a cumulative clonogenic inhibition of 89-99% at the 20:1 effector to target ratio. Multiple myeloma tumor burden was reduced by NK-92 in a xenograft mouse model as measured by bioluminescence imaging and reduction in bone marrow engraftment of U266eGFPluc cells by flow cytometry. Conclusions: This study demonstrates that NK-92 and KHYG-1 are capable of killing clonogenic and bulk multiple myeloma cells. In addition, multiple myeloma tumor burden in a xenograft mouse model was reduced by intravenous NK-92 cell therapy. Since multiple myeloma colony frequency correlates with survival, our observations have important clinical implications and suggest that clinical studies of NK cell lines to treat MM are warranted.

PMID: 22271890 [PubMed - as supplied by publisher]

Scavenging of CXCL12 by CXCR7 promotes tumor growth and metastasis of CXCR4-positive breast cancer cells.

Oncogene. 2012 Jan 23;

Authors: Luker KE, Lewin SA, Mihalko LA, Schmidt BT, Winkler JS, Coggins NL, Thomas DG, Luker GD

Abstract
Chemokine CXCL12 and receptor CXCR4 control multiple steps in primary tumor growth and metastasis in breast cancer and more than 20 other human malignancies. Mechanisms that regulate availability of CXCL12 in tumor microenvironments will substantially impact cancer progression and ongoing efforts to target the CXCL12-CXCR4 pathway for cancer chemotherapy. We used dual luciferase imaging to investigate CXCR7-dependent scavenging of CXCL12 in breast tumors in vivo and quantify effects of CXCR7 on tumor growth and metastasis of a separate population of CXCR4+ breast cancer cells. In a mouse xenograft model of human breast cancer, in vivo imaging showed that malignant cells expressing CXCR7 reduced bioluminescent CXCL12 secreted in the primary tumor microenvironment. Capitalizing on sensitive detection of bioluminescent CXCL12, we also demonstrated that CXCR7+ cells reduced amounts of chemokine released from orthotopic tumors into the circulation. Immunofluorescence staining of human primary breast cancers showed expression of CXCR4 and CXCR7 on malignant cells in ≈30% of cases. In most cases, CXCR4 and CXCR7 predominantly were expressed on separate populations of malignant cells in a tumor. We modeled these cases of human breast cancer by co-implanting tumor xenografts with CXCR4+ breast cancer cells, human mammary fibroblasts secreting CXCL12, and CXCR7+ or control breast cancer cells. Bioluminescence imaging showed that CXCR7+ breast cancer cells enhanced proliferation of CXCR4+ breast cancer cells in orthotopic tumors and spontaneous metastases. Treatment with a small-molecule inhibitor of CXCR7 chemokine limited the growth of CXCR4+ breast cancer cells in tumors that also contained malignant CXCR7+ cells. These studies establish a new in vivo imaging method to quantify chemokine scavenging by CXCR7 in the tumor microenvironment and identify that CXCR7+ cells promote growth and metastasis of CXCR4+ breast cancer cells.Oncogene advance online publication, 23 January 2012; doi:10.1038/onc.2011.633.

PMID: 22266857 [PubMed - as supplied by publisher]

N-Alkyl-PEI-functionalized iron oxide nanoclusters for efficient siRNA delivery.

Small. 2011 Oct 4;7(19):2742-9

Authors: Liu G, Xie J, Zhang F, Wang Z, Luo K, Zhu L, Quan Q, Niu G, Lee S, Ai H, Chen X

Abstract
Small-interfering RNA (siRNA) is an emerging class of therapeutics, which works by regulating the expression of a specific gene involved in disease progression. Despite the promises, effective transport of siRNA with minimal side effects remains a challenge. In this study, a nonviral nanoparticle gene carrier is developed and its efficiency for siRNA delivery and transfection is validated at both in vitro and in vivo levels. Such a nanocarrier, abbreviated as Alkyl-PEI2k-IO, was constructed with a core of iron oxide nanoparticles (IOs) and a shell of alkylated polyethyleneimine of 2000 kDa molecualr weight (Alkyl-PEI2k). It is found to be able to bind with siRNA, resulting in well-dispersed nanoparticles with a controlled clustering structure and narrow size distribution. Electrophoresis studies show that the Alkyl-PEI2k-IOs could retard siRNA completely at N:P ratios (i.e., PEI nitrogen to nucleic acid phosphate) above 10, protect siRNA from enzymatic degradation in serum, and release complexed siRNA efficiently in the presence of polyanionic heparin. The knockdown efficiency of the siRNA-loaded nanocarriers is assessed with 4T1 cells stably expressing luciferase (fluc-4T1) and further, with a fluc-4T1 xenograft model. Significant down-regulation of luciferase is observed, and unlike high-molecular-weight analogues, the Alkyl-PEI2k-coated IOs show good biocompatibility. In conclusion, Alkyl-PEI2k-IOs demonstrate highly efficient delivery of siRNA and an innocuous toxic profile, making it a potential carrier for gene therapy.

PMID: 21861295 [PubMed - indexed for MEDLINE]

Preclinical activity, pharmacodynamic and pharmacokinetic properties of a selective HDAC6 inhibitor, ACY-1215, in combination with bortezomib in multiple myeloma.

Blood. 2012 Jan 19;

Authors: Santo L, Hideshima T, Kung AL, Tseng JC, Tamang D, Yang M, Jarpe M, van Duzer JH, Mazitschek R, Ogier WC, Cirstea D, Rodig S, Eda H, Scullen T, Canavese M, Bradner J, Anderson KC, Jones SS, Raje N

Abstract
Histone deacetylase (HDAC) enzymatic activity has been linked to the transcription of DNA in cancers including multiple myeloma (MM). Consequently, HDAC inhibitors, alone and in combination, are being actively studied as novel therapies in MM. Here we investigated the preclinical activity of an HDAC6 selective inhibitor ACY-1215 in MM, alone and in combination with bortezomib. Low doses of ACY-1215 combined with bortezomib triggered synergistic anti-MM activity, resulting in protracted ER stress and apoptosis via activation of caspase-3,-8,-9 and poly (ADP) ribosome polymerase. In vivo anti-MM activity of ACY-1215 in combination with bortezomib was confirmed using two different xenograft SCID mouse models: human MM injected subcutaneously (plasmacytoma model); and luciferase-expressing human MM injected intravenously (disseminated MM model). Tumor growth was significantly delayed and overall survival significantly prolonged in animals treated with combined therapy. Importantly, pharmacokinetic data showed peak plasma levels of ACY-1215 at 4 h following treatment coincident with an increase in acetylated-α tubulin, a marker of HDAC6 inhibition, by immunohistochemistry and Western blotting analysis. These studies provide preclinical rationale for acetylated-α tubulin use as a pharmacodynamic biomarker in future clinical trials.

PMID: 22262760 [PubMed - as supplied by publisher]

The human lactase persistence-associated SNP -13910*T enables in vivo functional persistence of lactase promoter-reporter transgene expression.

Hum Genet. 2012 Jan 19;

Authors: Fang L, Ahn JK, Wodziak D, Sibley E

Abstract
Lactase is the intestinal enzyme responsible for digestion of the milk sugar lactose. Lactase gene expression declines dramatically upon weaning in mammals and during early childhood in humans (lactase nonpersistence). In various ethnic groups, however, lactase persists in high levels throughout adulthood (lactase persistence). Genetic association studies have identified that lactase persistence in northern Europeans is strongly associated with a single nucleotide polymorphism (SNP) located 14 kb upstream of the lactase gene: -13910*C/T. To determine whether the -13910*T SNP can function in vivo to mediate lactase persistence, we generated transgenic mice harboring human DNA fragments with the -13910*T SNP or the ancestral -13910*C SNP cloned upstream of a 2-kb rat lactase gene promoter in a luciferase reporter construct. We previously reported that the 2-kb rat lactase promoter directs a post-weaning decline of luciferase transgene expression similar to that of the endogenous lactase gene. In the present study, the post-weaning decline directed by the rat lactase promoter is impeded by addition of the -13910*T SNP human DNA fragment, but not by addition of the -13910*C ancestral SNP fragment. Persistence of transgene expression associated with the -13910*T SNP represents the first in vivo data in support of a functional role for the -13910*T SNP in mediating the human lactase persistence phenotype.

PMID: 22258180 [PubMed - as supplied by publisher]

A Deimmunized Bispecific Ligand-Directed Toxin That Shows an Impressive Anti-Pancreatic Cancer Effect in a Systemic Nude Mouse Orthotopic Model.

Pancreas. 2012 Jan 17;

Authors: Oh S, Todhunter DA, Panoskaltsis-Mortari A, Buchsbaum DJ, Toma S, Vallera DA

Abstract
OBJECTIVE: The objective was to test a bispecific ligand-directed toxin (BLT), with reduced immunogenicity for enhanced efficacy in targeting orthotopic pancreatic cancer in vivo. METHOD: A new BLT was created in which both human epidermal growth factor (EGF) and interleukin 4 cytokines were cloned onto the same single chain molecule with deimmunized pseudomonas exotoxin (dEGF4KDEL). Key amino acids dictating B-cell generation of neutralizing antitoxin antibodies were mutated. Bioassays were used to determine whether mutation reduced potency, and enzyme-linked immunosorbent assay studies were performed to determine whether antitoxin antibodies were reduced. A genetically altered luciferase MIA PaCa-2 xenograft model was used to image in real time and determine effects on systemic malignant human cancer. Bispecific ligand-directed toxins targeting B cells were used as specificity controls. RESULTS: Deimmunized EGF4KDEL was significantly effective after systemic injection against established orthotopic MIA PaCa-2 pancreatic cancer and selectively prevented metastasis. Mutagenesis significantly reduced antitoxin levels in vivo with no apparent activity loss in vitro. The drug was effective against 3 human pancreatic cancer lines in vitro, MIA PaCa-2, SW1990, and S2VP10. CONCLUSIONS: Despite the metastatic nature of the MIA PaCa-2 orthotopic tumor xenografted in nude mice, high percentages of tumors responded to extended dEGFKDEL treatment resulting in significant anticancer effects and disease-free survivors.

PMID: 22258068 [PubMed - as supplied by publisher]