Complete regression of human malignant mesothelioma xenografts following local injection of midkine promoter-driven oncolytic adenovirus.

J Gene Med. 2010 Jul 14;

Authors: Kubo S, Kawasaki Y, Yamaoka N, Tagawa M, Kasahara N, Terada N, Okamura H

BACKGROUND: Malignant mesothelioma is a highly aggressive tumor with poor prognosis. We hypothesized that the tumor-specific midkine (Mdk) promoter could confer transcriptional targeting to oncolytic adenoviruses for effective treatment of malignant mesothelioma. METHODS: We analysed Mdk expression by quantitative reverse transcription-polymerase chain reaction in six human mesothelioma cell lines, and tested Mdk promoter activity by luciferase reporter assay. On the basis of these data, we constructed a replication-selective oncolytic adenovirus designated AdMdk-E1-iresTK. This virus contains a Mdk promoter-driven adenoviral E1 gene and herpes simplex virus-thymidine kinase (TK) suicide gene and cytomagalovirus promoter-driven enhanced green fluorescent protein marker gene. Selectivity of viral replication and cytolysis were characterized in normal versus mesothelioma cells in vitro, and intratumoral spread and antitumor efficacy were investigated in vivo. RESULTS: Mdk promoter activity was restricted in normal cells, but highly activated in mesothelioma cell lines. AdMdk-E1-iresTK was seen to efficiently replicate, produce viral progeny and spread in multiple mesothelioma cell lines. Lytic spread of AdMdk-E1-iresTK mediated the efficient killing of these mesothelioma cells, and its in vitro cytocidal effect was significantly enhanced by treatment with the prodrug, ganciclovir. Intratumoral injection of AdMdk-E1-iresTK caused complete regression of MESO4 and MSTO human mesothelioma xenografts in athymic mice. In vivo fluorescence imaging demonstrated intratumoral spread of AdMdk-E1-iresTK-derived signals, which vanished after tumor eradication. CONCLUSIONS: These data indicate that transcriptional targeting of viral replication by the Mdk promoter represents a promising general strategy for oncolytic virotherapy of cancers with up-regulated Mdk expression, including malignant mesothelioma. Copyright (c) 2010 John Wiley & Sons, Ltd.

PMID: 20635326 [PubMed - as supplied by publisher]

Noninvasive Imaging of Lipid Nanoparticle-Mediated Systemic Delivery of Small-Interfering RNA to the Liver.

Mol Ther. 2010 Jul 13;

Authors: Tao W, Davide JP, Cai M, Zhang GJ, South VJ, Matter A, Ng B, Zhang Y, Sepp-Lorenzino L

Mouse models with liver-specific expression of firefly luciferase were developed that enable a noninvasive and longitudinal assessment of small-interfering RNA (siRNA)-mediated gene silencing in hepatocytes of live animals via bioluminescence imaging. Using these models, a set of lipid nanoparticles (LNPs) with different compositions of cationic lipids, polyethylene glycol (PEG), and cholesterol, were tested for their abilities in delivering a luciferase siRNA to the liver via systemic administration. A dose-dependent luciferase knockdown by LNP/siRNA assemblies was measured by in vivo bioluminescence imaging, which correlated well with the results from parallel ex vivo analyses of luciferase mRNA and protein levels in the liver. RNA interference (RNAi)-mediated target silencing was further confirmed by the detection of RNAi-specific target mRNA cleavage. A single dose of LNP02L at 3 mg/kg (siRNA) caused 90% reduction of luciferase expression and the target repression lasted for at least 10 days. With identical components, LNPs containing 2% PEG are more potent than those with 5.4% PEG. Our results demonstrate that these liver-luciferase mouse models provide a powerful tool for a high-throughput evaluation of hepatic delivery platforms by noninvasive imaging and that the molar ratio of PEG lipid can affect the efficacy of LNPs in silencing liver targets via systemic administration.

PMID: 20628357 [PubMed - as supplied by publisher]

Antrodia camphorata suppresses lipopolysaccharide-induced nuclear factor-kappaB activation in transgenic mice evaluated by bioluminescence imaging.

Food Chem Toxicol. 2010 Jun 1;

Authors: Hseu YC, Huang HC, Hsiang CY

In an earlier study, we found that Antrodia camphorata inhibited the production of lipopolysaccharide (LPS)-induced cytokines, inducible nitric oxide synthase, and cyclooxygenase-2 by blocking nuclear factor-kappaB (NF-kappaB) activation in cultured RAW 264.7 macrophages. This study was aimed at evaluating the inhibitory effects of the fermented culture broth of A. camphorata in terms of LPS-induced NF-kappaB activation in transgenic mice by using a non-invasive, real-time NF-kappaB bioluminescence imaging technique. Transgenic mice carrying the luciferase gene under the control of NF-kappaB were given A. camphorata (570mg/kg, p.o.) for three consecutive days and then injected with LPS (4mg/kg, i.p.). In vivo imaging showed that treatment with LPS increased the luminescent signal, whereas A. camphorata suppressed the LPS-induced inflammatory response significantly. Ex vivo imaging showed that A. camphorata suppressed LPS-induced NF-kappaB activity in the small intestine, mesenteric lymph nodes, liver, spleen, and kidney. Immunohistochemical staining revealed that A. camphorata suppressed production of the LPS-induced tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and NF-kappaB p65 subunit in these organs. Furthermore, A. camphorata attenuated the productions of LPS-induced TNF-alpha and IL-1beta in serum from transgenic mice. We report the first confirmation of the anti-inflammatory action in vivo of this potentially beneficial mushroom.

PMID: 20621584 [PubMed - as supplied by publisher]

Betulinic acid induces apoptosis and inhibits hedgehog signalling in rhabdomyosarcoma.

Br J Cancer. 2010 Jun 29;103(1):43-51

Authors: Eichenmüller M, Hemmerlein B, von Schweinitz D, Kappler R

BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in childhood with the ability to resist apoptosis by the activation of survival promoting and anti-apoptotic proteins. METHODS: Efficacy of the apoptosis-inducing agent betulinic acid (BA) was determined in RMS cell cultures and in vivo by measuring cell viability, survival, apoptosis, hedgehog signalling activity, and neovascularisation. RESULTS: Betulinic acid had a strong cytotoxic effect on RMS cells in a dose-dependent manner. The BA treatment caused a massive induction of apoptosis mediated by the intrinsic mitochondrial pathway, which could be inhibited by the broad-range caspase inhibitor zVAD.fmk. Exposure of hedgehog-activated RMS-13 cells to BA resulted in a strong decrease in GLI1, GLI2, PTCH1, and IGF2 expression as well as hedgehog-responsive luciferase activity. Intraperitoneal injection of 20 mg BA per kg per day significantly retarded growth of RMS-13 xenografts in association with markedly higher counts of apoptotic cells and down-regulation of GLI1 expression compared with control tumours, while leaving microvascular density, cell proliferation, and myogenic differentiation unaffected. CONCLUSION: Our data show that induction of apoptosis and inhibition of hedgehog signalling are important features of the anti-tumourigenic effect of BA in RMS and advices this compound for the use in a multimodal therapy of this highly aggressive paediatric tumour.

PMID: 20517313 [PubMed - indexed for MEDLINE]

Biodistribution of Neural Stem Cells After Intravascular Therapy for Hypoxic-Ischemia.

Stroke. 2010 Jul 8;

Authors: Pendharkar AV, Chua JY, Andres RH, Wang N, Gaeta X, Wang H, De A, Choi R, Chen S, Rutt BK, Gambhir SS, Guzman R

BACKGROUND AND PURPOSE: Intravascular transplantation of neural stem cells represents a minimally invasive therapeutic approach for the treatment of central nervous system diseases. The cellular biodistribution after intravascular injection needs to be analyzed to determine the ideal delivery modality. We studied the biodistribution and efficiency of targeted central nervous system delivery comparing intravenous and intra-arterial (IA) administration of neural stem cells after brain ischemia. METHODS: Mouse neural stem cells were transduced with a firefly luciferase reporter gene for bioluminescence imaging (BLI). Hypoxic-ischemia was induced in adult mice and reporter neural stem cells were transplanted IA or intravenous at 24 hours after brain ischemia. In vivo BLI was used to track transplanted cells up to 2 weeks after transplantation and ex vivo BLI was used to determine single organ biodistribution. RESULTS: Immediately after transplantation, BLI signal from the brain was 12 times higher in IA versus intravenous injected animals (P<0.0001). After IA injection, 69% of the total luciferase activity arose from the brain early after transplantation and 93% at 1 week. After intravenous injection, 94% of the BLI signal was detected in the lungs (P=0.004) followed by an overall 94% signal loss at 1 week, indicating lack of cell survival outside the brain. Ex vivo single organ analysis showed a significantly higher BLI signal in the brain than in the lungs, liver, and kidneys at 1 week (P<0.0001) and 2 weeks in IA (P=0.007). CONCLUSIONS: IA transplantation results in superior delivery and sustained presence of neural stem cells in the ischemic brain in comparison to intravenous infusion.

PMID: 20616329 [PubMed - as supplied by publisher]

TAT-mediated transduction of NF-Ya peptide induces the ex vivo proliferation and engraftment potential of human hematopoietic progenitor cells.

Blood. 2010 Jul 8;

Authors: Domashenko AD, Danet-Desnoyers G, Aron A, Carroll MP, Emerson SG

Retroviral overexpression of NF-Ya, the regulatory subunit of the transcription factor NF-Y, activates the transcription of multiple genes implicated in hematopoietic stem cells (HSC) self-renewal and differentiation and directs HSC toward self-renewal. We asked whether TAT-NF-Ya fusion protein could be employed to transduce human CD34+ cells as a safer, more regulated alternative approach to gene therapy. Here we show that externally added recombinant protein was able to enter the cell nucleus and activate HoxB4, a target gene of NF-Ya, using real-time PCR RNA and luciferase-based protein assays. Following TAT-NF-Ya transduction, the proliferation of human CD34+ cells in the presence of myeloid cytokines was increased 4-fold. Moreover, TAT-NF-Ya treated human primary bone marrow cells showed a 4-fold increase in the percentage of huCD45+ cells recovered from the bone marrow of sublethally irradiated, transplanted NOD-scid IL2Rgamma (null) mice. These data demonstrate that TAT-peptide therapies are an alternative approach to retroviral stem cell therapies, and suggest that NF-Ya peptide delivery should be further evaluated as a therapeutic tool for HSC/progenitors ex vivo expansion and therapy.

PMID: 20616221 [PubMed - as supplied by publisher]

Modulation of collagen-induced arthritis by adenovirus-mediated intra-articular expression of modified collagen type II.

Arthritis Res Ther. 2010 Jul 8;12(4):R136

Authors: Tang B, Cullins DL, Zhou J, Zawaski JA, Park H, Brand DD, Hasty KA, Gaber MW, Stuart JM, Kang AH, Myers LK

ABSTRACT: INTRODUCTION: Rheumatoid arthritis (RA) is a systemic disease manifested by chronic inflammation in multiple articular joints, including the knees and small joints of the hands and feet. We have developed a unique modification to a clinically accepted method for delivering therapies directly to the synovium. Our therapy is based on our previous discovery of an analog peptide (A9) with amino acid substitutions made at positions 260 (I to A), 261 (A to B), and 263 (F to N) that could profoundly suppress immunity to type II collagen (CII) and arthritis in the collagen-induced arthritis model (CIA). METHODS: We engineered an adenoviral vector to contain the CB11 portion of recombinant type II collagen and used PCR to introduce point mutations at three sites within (CII124-402, 260A, 261B, 263D), (rCB11-A9) so that the resulting molecule contained the A9 sequence at the exact site of the wild-type sequence. RESULTS: We used this construct to target intra-articular tissues of mice and utilized the collagen-induced arthritis model to show that this treatment strategy provided a sustained, local therapy for individual arthritic joints, effective whether given to prevent arthritis or as a treatment. We also developed a novel system for in vivo bioimaging, using the firefly luciferase reporter gene to allow serial bioluminescence imaging to show that luciferase can be detected as late as 18 days post injection into the joint. CONCLUSIONS: Our therapy is unique in that we target synovial cells to ultimately shut down T cell-mediated inflammation. Its effectiveness is based on its ability to transform potential inflammatory T cells and/or bystander T cells into therapeutic (regulatory-like) T cells which secrete interleukin (IL)-4. We believe this approach has potential to effectively suppress RA with minimal side effects.

PMID: 20615221 [PubMed - as supplied by publisher]

Systemic Delivery of Bioactive Glucagon-Like Peptide 1 after Adenoviral-Mediated Gene Transfer in the Murine Salivary Gland.

Endocrinology. 2010 Jul 7;

Authors: Voutetakis A, Cotrim AP, Rowzee A, Zheng C, Rathod T, Yanik T, Loh YP, Baum BJ, Cawley NX

An adenoviral (Ad) vector that expresses bioactive glucagon-like peptide 1 (GLP-1) was generated, and its effectiveness at modulating glucose homeostasis was evaluated after transduction of murine salivary glands. The construct was engineered with the signal sequence of mouse GH to direct the peptide into the secretory pathway, followed by a furin cleavage site and the GLP-1(7-37) sequence encoding an Ala to Gly substitution at position 8 to achieve resistance to degradation. When expressed in Neuro2A and COS7 cells, an active form of GLP-1 was specifically detected by RIA in the conditioned medium of transduced cells, showed resistance to degradation by dipeptidyl-peptidase IV, and induced the secretion of insulin from NIT1 pancreatic beta-cells in vitro. In vivo studies demonstrated that healthy mice transduced with Ad-GLP-1 in both submandibular glands had serum GLP-1 levels approximately 3 times higher than mice transduced with the control Ad-luciferase vector. In fasted animals, serum glucose levels were similar between Ad-GLP-1 and Ad-luciferase transduced mice in keeping with GLP-1's glucose-dependent action. However, when challenged with glucose, Ad-GLP-1 transduced mice cleared the glucose significantly faster than control mice. In an animal model of diabetes induced by alloxan, progression of hyperglycemia was significantly attenuated in mice given the Ad-GLP-1 vector compared with control mice. These studies demonstrate that the bioactive peptide hormone, GLP-1, normally secreted from endocrine cells in the gut through the regulated secretory pathway, can be engineered for secretion into the circulatory system from exocrine cells of the salivary gland to affect glucose homeostasis.

PMID: 20610567 [PubMed - as supplied by publisher]

A Type-II Kinase Inhibitor Capable of Inhibiting the T315I "Gatekeeper" Mutant of Bcr-Abl.

J Med Chem. 2010 Jul 6;

Authors: Choi HG, Ren P, Adrian F, Sun F, Lee HS, Wang X, Ding Q, Zhang G, Xie Y, Zhang J, Liu Y, Tuntland T, Warmuth M, Manley PW, Mestan J, Gray NS, Sim T

The second generation of Bcr-Abl inhibitors nilotinib, dasatinib, and bosutinib developed to override imatinib resistance are not active against the T315I "gatekeeper" mutation. Here we describe a type-II T315I inhibitor 2 (GNF-7), based upon a 3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one scaffold which is capable of potently inhibiting wild-type and T315I Bcr-Abl as well as other clinically relevant Bcr-Abl mutants such as G250E, Q252H, Y253H, E255K, E255V, F317L, and M351T in biochemical and cellular assays. In addition, compound 2 displayed significant in vivo efficacy against T315I-Bcr-Abl without appreciable toxicity in a bioluminescent xenograft mouse model using a transformed T315I-Bcr-Abl-Ba/F3 cell line that has a stable luciferase expression. Compound 2 is among the first type-II inhibitors capable of inhibiting T315I to be described and will serve as a valuable lead to design the third generation Bcr-Abl kinase inhibitors.

PMID: 20604564 [PubMed - as supplied by publisher]

Inhibition of the transcriptional function of p53 by EWS-Fli1 chimeric protein in Ewing Family Tumors.

Cancer Lett. 2010 Aug 1;294(1):57-65

Authors: Li Y, Tanaka K, Fan X, Nakatani F, Li X, Nakamura T, Takasaki M, Yamamoto S, Iwamoto Y

The chromosomal translocation t(11;22)(q24;q12) generates the EWS-Fli1 fusion gene, which contributes to the development of Ewing Family Tumors (EFTs). Although p53 mutations are found only in 5-20% of EFTs, the p53 pathway is thought to be abrogated in EFTs. The role of EWS-Fli1 in the p53 pathway in the tumor is still poorly understood. In this study, using immunoprecipitation and co-localization, we show that EWS-Fli1 interacts with p53 within the nucleus in vivo. The introduction of EWS-Fli1 resulted in significant reduction of promoter activities and mRNA levels of p21 and mdm2, meanwhile it canceled p53-dependent growth suppression. In contrast, knockdown of EWS-Fli1 expression mediated by small interfering RNAs (siRNA) also augmented the induction of p21 and mdm2 in response to DNA damage. Furthermore, using serial deletion constructs of the EWS-Fli1 fusion protein, we determined that EWS-Fli1 binding to p53 as well as inhibition of p21 and mdm2 promoter activities was mediated by its N-terminal domain (amino acid residues 65-109). These observations suggest that the N-terminal region of EWS-Fli1 might associate with p53 and impair its transcriptional activity, subsequently inhibiting the expression of its downstream genes. These results might provide new insight into the oncogenesis of EFTs by EWS-Fli1 via the inhibition of p53 function.

PMID: 20153576 [PubMed - indexed for MEDLINE]

Noninvasive molecular imaging of apoptosis in vivo using a modified firefly luciferase substrate, Z-DEVD-aminoluciferin.

Cell Death Differ. 2010 Jun;17(6):1003-10

Authors: Hickson J, Ackler S, Klaubert D, Bouska J, Ellis P, Foster K, Oleksijew A, Rodriguez L, Schlessinger S, Wang B, Frost D

Apoptosis is a highly regulated process of programmed cell death essential for normal physiology. Dysregulation of apoptosis contributes to the development and progression of various diseases, including cancer, neurodegenerative disorders, and chronic heart failure. Quantitative noninvasive imaging of apoptosis in preclinical models would allow for dynamic longitudinal screening of compounds and facilitates a more rapid determination of therapeutic efficacy. In this study, we report the in vivo characterization of Z-DEVD-aminoluciferin, a modified firefly luciferase substrate that in apoptotic cells is cleaved by caspase-3 to liberate aminoluciferin, which can be consumed by luciferase to generate a luminescent signal. In two oncology models, namely SKOV3-luc and MDA-MB-231-luc-LN, at 24, 48, and 72 h after treatment with docetaxel, animals were injected with Z-DEVD-aminoluciferin and bioluminescent images were acquired. Significantly more light was detected at 24 (P<0.05), 48 (P<0.01), and 72 h (P<0.01) in the docetaxel-treated group compared with the vehicle-treated group, with caspase-3 activation at these time points confirmed using immunohistochemistry. Importantly, whereas significant differences between groups were detected as early as 24 h after treatment by molecular imaging, caliper measurements were unable to detect a difference for 4-5 additional days. Taken together, these data show that in vivo imaging of apoptosis using Z-DEVD-aminoluciferin could provide a sensitive and rapid method for early detection of drug efficacy, which could potentially be used by numerous therapeutic programs.

PMID: 20057500 [PubMed - indexed for MEDLINE]

Development of cell-penetrating peptide-modified MPEG-PCL diblock copolymeric nanoparticles for systemic gene delivery.

Int J Pharm. 2010 Jun 25;

Authors: Tanaka K, Kanazawa T, Shibata Y, Suda Y, Fukuda T, Takashima Y, Okada H

To develop a safe and efficient systemic non-viral gene vector, methoxy poly(ethylene glycol) (MPEG)/poly(varepsilon-caprolactone) (PCL) diblock copolymers conjugated with a Tat analog through the ester or disulfide linkage were synthesized and their suitability as a systemic non-viral gene carrier evaluated. The physicochemical properties of the MPEG-PCL diblock copolymers were determined by GPC, (1)H-NMR and FT-IR spectroscopy. The particle sizes and in vitro (COS7 and S-180 cells) transfection efficiencies and cytotoxicity were evaluated. Furthermore, the luciferase activity was then determined in various tissues after intravenous injection of MPEG-PCL-SS-Tat/pCMV-Luc complex into mice bearing S-180 cells. The particle sizes of the MPEG-PCL-Tat copolymers with or without pDNA were about 40 and 60nm, respectively. The luciferase activity in COS7 cells transfected with pCMV-Luc with MPEG-PCL-ester-Tat or MPEG-PCL-SS-Tat was higher than that with pDNA only. MPEG-PCL-SS-Tat greatly increased the transfection efficiency compared to MPEG-PCL-ester-Tat in COS7 and S-180 cells. In an in vitro cytotoxicity test MPEG-PCL-SS-Tat did not induce any remarkable cytotoxicity. In an in vivo experiment, the synthesized MPEG-PCL-SS-Tat copolymers promoted the delivery and expression of pDNA into tumor tissue in tumor-bearing mice. In conclusion, this vector might be applicable as a tumor-targeting non-viral systemic gene carrier in the clinical setting.

PMID: 20600726 [PubMed - as supplied by publisher]

Increased Expression of iASPP, Regulated by Hepatitis B Virus X Protein-Mediated NF-kappaB Activation, in Hepatocellular Carcinoma.

Gastroenterology. 2010 Jun 20;

Authors: Lu B, Guo H, Zhao J, Wang C, Wu G, Pang M, Tong X, Bu F, Liang A, Hou S, Fan X, Dai J, Wang H, Guo Y

BACKGROUND & AIMS: iASPP is an inhibitory member of the ankyrin-repeat-, SH3-domain- and proline-rich-region-containing protein (ASPP) family; iASPP expression is up-regulated in different human tumor types. We explored the molecular mechanism increased expression of iASPP and its role in hepatocellular carcinoma. METHODS: iASPP expression levels in human liver samples and cell lines were determined by PCR, immunoblot, and immunohistochemical analyses. Luciferase reporter, chromatin immunoprecipitation, and electrophoretic mobility shift assays were used to measure transcriptional activation by nuclear factor-kappaB (NF-kappaB). Effects on tumor growth were characterized using MTS, soft agar colony formation, and flow cytometry analyses. Tumorigenicity of hepatocellular carcinoma (HCC) cells was studied in nude mice. RESULTS: Compared with normal liver cells or tissues, iASPP was expressed at significantly higher levels in HCC cell lines (9/14), and liver samples from patients with HCC, cirrhosis, or hepatitis B virus infection. Increased expression of iASPP was significantly associated with time to recurrence and survival time of patients with HCC. NF-kappaB activation increased the expression of iASPP through p65/p50 binding to a putative NF-kappaB binding site in the iASPP promoter; HBx might up-regulate expression of iASPP. Transgenic expression of iASPP promoted tumor cell proliferation and resistance to chemotherapeutic drugs in vitro and in vivo. CONCLUSIONS: iASPP is up-regulated in HCC; it is a direct transcription target of NF-kappaB. Increased iASPP expression contributes to tumor progression via proliferative and anti-apoptotic effects. iASPP might be developed as a HCC therapeutic target or to sensitize cancer cells to chemotherapeutic drugs; it might also be used as a prognostic factor.

PMID: 20600029 [PubMed - as supplied by publisher]

Gaussia luciferase reporter assay for assessment of gene delivery systems in vivo.

Chin Med Sci J. 2010 Jun;25(2):95-9

Authors: Chen F, Xu Z, Lu J, Lü X, Mu WL, Wang YJ, Liu DP, Liang CC

OBJECTIVE: To develop an alternative method for assessment of gene delivery systems in vivo. METHODS: Mouse primary spleen lymphocytes were genetically modified in vitro by a retroviral vector harboring a Gaussia luciferase (Gluc) expression cassette. After implantation of these cells into recipient mice, the expression of Gluc was detected in whole blood or plasma collected. RESULTS: As little as 10 muL whole blood drawn from the recipient mice could guarantee prompt reading of Gluc activity with a luminometer. And the reading was found in good correlation with the number of genetically modified spleen lymphocytes implanted to the mice. CONCLUSIONS: Gluc may be useful as an in vivo reporter for gene therapy researches, and Gluc blood assay could provide an alternative method for assessment of gene delivery systems in vivo.

PMID: 20598231 [PubMed - in process]

Thymoquinone and cisplatin as a therapeutic combination in lung cancer: In vitro and in vivo.

J Exp Clin Cancer Res. 2010 Jul 1;29(1):87

Authors: Jafri SH, Glass J, Shi R, Zhang S, Prince M, Kleiner-Hancock H

ABSTRACT: BACKGROUND: Thymoquinone (TQ) is a compound extracted from Black Caraway seeds of Nigella Sativa and is active against various cancers. Cisplatin (CDDP) is the most active chemotherapeutic agent in Lung Cancer. Here we report activity of TQ against non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cell lines alone and in combination with Cisplatin (CDDP). METHODS: For proliferation MTT assay, cell viability trypan blue assay and for apoptosis Annexin-V FITC assay were used in NCI-H460 and NCI-H146 cell lines. Inhibition of invasion by TQ was assessed using Matrigel assay and its affect on release of various cytokines was determined using RayBio Human Cytokine detection kit. Mouse xenograft model using NCI-H460 was used to determine in vivo activity of TQ and CDDP. Inhibition of LPS induced NF-Kappa B expression by TQ was determined using transgenic mice expressing a luciferase reporter. RESULTS: TQ was able to inhibit cell proliferation, reduce cell viability and induce apoptosis. TQ at 100 uM and CDDP at 5 uM inhibited cell proliferation by nearly 90% and the combination showed synergism. TQ was able to induced apoptosis in both NCI-H460 and NCI-H146 cell lines. TQ also appears to affect the extracellular environment inhibiting invasion and reducing the production of two cytokines ENA-78 and Gro-alpha which are involved in neo-angiogenesis. Using a mouse xenograft model we were able to demonstrate that combination of TQ and CDDP was well tolerated and significantly reduced tumor volume and tumor weight without additional toxicity to the mice. In the combination arms (TQ5mg/kg/Cis 2.5mg/kg) tumor volume was reduced by 59% and (TQ20mg/kg /Cis 2.5mg/kg) by 79% as compared to control which is consistent with in vitro data. TQ down regulated NF-Kappa-B expression which may explain its various cellular activities and this activity may prove useful in overcoming CDDP resistance from over expression of NF-Kappa-B. CONCLUSION: Thus TQ and CDDP appear to be an active therapeutic combination in lung cancer.

PMID: 20594324 [PubMed - as supplied by publisher]

Antitumor Activity of Decoy Oligodeoxynucleotides Targeted to NF-kB in vitro and in vivo.

Asian Pac J Cancer Prev. 2010;11(1):193-200

Authors: Wang T, Li QH, Hao GP, Zhai J

Background: Nuclear factor-KappaB (NF-kB), a transcription factor, is abundantly expressed in many tumors and regulates many tumor-relative genes such as c-myc and caspase-8, which NF-kB-mediated genes activation serves as an anti-tumor pathway by regulating expression of these tumor-relative genes. Given the important roles of these genes in tumor control, the present study was to test the hypothesis that NF-kB decoy ODNs transfected into tumor cells in vitro and in vivo might affect growth and apoptosis. Methods: First, NF-kB decoy oligodeoxynucleotides were designed according to the NF-kB elements in the promoter region of c-myc gene. Then, supression effects of decoy ODNs on proliferation of five carcinoma cell lines were assessed by MTT assay. Apoptosis of carcinoma cells was determined by chromosome DNA ladder and flow cytometric analysis (FCM). Thirdly, suppression effects of NF-kB decoy ODNs on proliferation of carcinoma in vivo were investigated in nude mice. To confirm mechanisms of action, a pGL3-C-MYC luciferase expression vector containing a fragment of the c-myc promoter was constructed and co-transfected with NF-kB decoy ODNs into SKOV-3 cells by lipofectamineTM2000. Expression levels of endogenous c-myc and caspase-8 genes were assessed by northern blotting. Lastly, nuclear extracts were prepared from SKOV-3 cells and DNA-protein interactions were examined by electrophoretic mobility shift assay (EMSA). Results: Treatment of cancer cell lines with NF-kB decoy ODNs resulted in strong suppression of proliferation, especial of the SKOV-3 ovarian cancer cell line in vitro and in vivo. Induction of apoptosis of SKOV-3 was observed in DNA gel electrophoresis and FCM. Activity of luciferase was significantly reduced in the NF-kB decoy-transfected cells, but not in cells transfected with a control decoy. Furthermore, we found that transcripts of endogenous c-myc gene were reduced, while caspase-8 transcripts were induced. EMSA demonstrated specific binding of the NF-kB decoy to NF-kB protein. Conclusions: These findings indicatd that NF-kB activation plays an important role in proliferation in many cancers, esspecially ovarian carcinomas. Inhibitors of NF-kB may thus offer promise as a therapeutic approach for the treatment of tumors via manipulating expression of desired target genes. NF-kB decoy ODNs may allow development of therapeutic and investigative tools for human malignancies.

PMID: 20593956 [PubMed - as supplied by publisher]

In vivo quantitative bioluminescence tomography using heterogeneous and homogeneous mouse models.

Opt Express. 2010 Jun 7;18(12):13102-13

Authors: Liu J, Wang Y, Qu X, Li X, Ma X, Han R, Hu Z, Chen X, Sun D, Zhang R, Chen D, Chen D, Chen X, Liang J, Cao F, Tian J

Bioluminescence tomography (BLT) is a new optical molecular imaging modality, which can monitor both physiological and pathological processes by using bioluminescent light-emitting probes in small living animal. Especially, this technology possesses great potential in drug development, early detection, and therapy monitoring in preclinical settings. In the present study, we developed a dual modality BLT prototype system with Micro-computed tomography (MicroCT) registration approach, and improved the quantitative reconstruction algorithm based on adaptive hp finite element method (hp-FEM). Detailed comparisons of source reconstruction between the heterogeneous and homogeneous mouse models were performed. The models include mice with implanted luminescence source and tumor-bearing mice with firefly luciferase report gene. Our data suggest that the reconstruction based on heterogeneous mouse model is more accurate in localization and quantification than the homogeneous mouse model with appropriate optical parameters and that BLT allows super-early tumor detection in vivo based on tomographic reconstruction of heterogeneous mouse model signal.

PMID: 20588440 [PubMed - in process]

Polyplex nanomicelle promotes hydrodynamic gene introduction to skeletal muscle.

J Control Release. 2010 Apr 2;143(1):112-9

Authors: Itaka K, Osada K, Morii K, Kim P, Yun SH, Kataoka K

Skeletal muscle is an interesting target for gene therapy. To achieve effective gene introduction in skeletal muscle, a hydrodynamic approach by intravenous injection of plasmid DNA (pDNA) with transient isolation of the limb has attracted attention. In this study, we demonstrated that polyplex nanomicelle, composed of poly(ethyleneglycol) (PEG)-block-polycation and pDNA, showed excellent capacity of gene introduction to skeletal muscle. The evaluation of luciferase expression in the muscle revealed that the nanomicelle provided higher and sustained profiles of transgene expression compared with naked pDNA. Real-time in vivo imaging using a video-rate confocal imaging system suggested that the nanomicelle showed tolerability in the intracellular environment, resulting in the slow but sustained transgene expression. The nanomicelle induced less TNFalpha induction in the muscle than naked pDNA, indicating the safety of nanomicelle-based gene delivery into the skeletal muscle. Moreover, the nanomicelle showed significant tumor growth suppression for almost a month by introducing a pDNA expressing a soluble form of vascular endothelial growth factor (VEGF) receptor-1 (sFlt-1) to skeletal muscle to obtain anti-angiogenic effect on tumor growth. This feature of sustained effect gives an important advantage of gene therapy, especially on the points of cost effectiveness and high compliance. These results suggest that the hydrodynamic gene introduction to skeletal muscle using polyplex nanomicelle system possesses the potential for effective gene therapy.

PMID: 20043959 [PubMed - indexed for MEDLINE]

MicroRNA-122 modulates the rhythmic expression profile of the circadian deadenylase Nocturnin in mouse liver.

PLoS One. 2010;5(6):e11264

Authors: Kojima S, Gatfield D, Esau CC, Green CB

Nocturnin is a circadian clock-regulated deadenylase thought to control mRNA expression post-transcriptionally through poly(A) tail removal. The expression of Nocturnin is robustly rhythmic in liver at both the mRNA and protein levels, and mice lacking Nocturnin are resistant to diet-induced obesity and hepatic steatosis. Here we report that Nocturnin expression is regulated by microRNA-122 (miR-122), a liver specific miRNA. We found that the 3'-untranslated region (3'-UTR) of Nocturnin mRNA harbors one putative recognition site for miR-122, and this site is conserved among mammals. Using a luciferase reporter construct with wild-type or mutant Nocturnin 3'-UTR sequence, we demonstrated that overexpression of miR-122 can down-regulate luciferase activity levels and that this effect is dependent on the presence of the putative miR-122 recognition site. Additionally, the use of an antisense oligonucleotide to knock down miR-122 in vivo resulted in significant up-regulation of both Nocturnin mRNA and protein expression in mouse liver during the night, resulting in Nocturnin rhythms with increased amplitude. Together, these data demonstrate that the normal rhythmic profile of Nocturnin expression in liver is shaped in part by miR-122. Previous studies have implicated Nocturnin and miR-122 as important post-transcriptional regulators of both lipid metabolism and circadian clock controlled gene expression in the liver. Therefore, the demonstration that miR-122 plays a role in regulating Nocturnin expression suggests that this may be an important intersection between hepatic metabolic and circadian control.

PMID: 20582318 [PubMed - in process]

Non-nuclear estrogen receptor alpha signaling promotes cardiovascular protection but not uterine or breast cancer growth in mice.

J Clin Invest. 2010 Jun 23;

Authors: Chambliss KL, Wu Q, Oltmann S, Konaniah ES, Umetani M, Korach KS, Thomas GD, Mineo C, Yuhanna IS, Kim SH, Madak-Erdogan Z, Maggi A, Dineen SP, Roland CL, Hui DY, Brekken RA, Katzenellenbogen JA, Katzenellenbogen BS, Shaul PW

Steroid hormone receptors function classically in the nucleus as transcription factors. However, recent data indicate that there are also non-nuclear subpopulations of steroid hormone receptors, including estrogen receptors (ERs), that mediate membrane-initiated signaling of unclear basis and significance. Here we have shown that an estrogen-dendrimer conjugate (EDC) that is excluded from the nucleus stimulates endothelial cell proliferation and migration via ERalpha, direct ERalpha-Galphai interaction, and endothelial NOS (eNOS) activation. Analysis of mice carrying an estrogen response element luciferase reporter, ER-regulated genes in the mouse uterus, and eNOS enzyme activation further indicated that EDC specifically targets non-nuclear processes in vivo. In mice, estradiol and EDC equally stimulated carotid artery reendothelialization in an ERalpha- and G protein-dependent manner, and both agents attenuated the development of neointimal hyperplasia following endothelial injury. In contrast, endometrial carcinoma cell growth in vitro and uterine enlargement and MCF-7 cell breast cancer xenograft growth in vivo were stimulated by estradiol but not EDC. Thus, EDC is a non-nuclear selective ER modulator (SERM) in vivo, and in mice, non-nuclear ER signaling promotes cardiovascular protection. These processes potentially could be harnessed to provide vascular benefit without increasing the risk of uterine or breast cancer.

PMID: 20577047 [PubMed - as supplied by publisher]