Interstitial gene delivery in human xenograft prostate tumors using titanium metal seeds.

Abstract:

:Gene therapy is a promising approach for the treatment of cancers. Strategies for gene vector delivery include systemic and local-regional approaches. Intratumoral delivery of vectors has generally employed direct injections into single or multiple locations throughout the tumor volume. However, this approach leads to nonuniform distributions of reagents within tumors and becomes cumbersome as the required number of injections is increased. We have investigated the effectiveness of an interstitial plasmid gene delivery based on using tiny metallic seeds (GeneSeeds) analogous to technology used for brachytherapy. Feasibility for interstitial use of GeneSeeds was demonstrated expressing reporter plasmids (green fluorescence protein or beta-galactosidase) in human xenograft prostate tumors. Immunohistochemical analysis confirmed effective interstitial delivery, vector expression, and distributions of reporter genes within tumors. Applicability of GeneSeeds for delivery of radiosensitizing cytokines was examined by generating a cytokine [tumor necrosis factor-alpha (TNF-alpha)] expressing vector under the cytomegaloviral promoter and interstitially implanting GeneSeeds with this vector into prostate cancer tumors. TNF-alpha protein expression was observed around the ends of seeds and decreasing in an exponential gradient as a function of distance. The expression of TNF-alpha resulted in tumor growth delay of a human prostate cancer xenograft. These results demonstrate the feasibility of applying interstitial delivery of gene expressing vectors for the treatment of human cancers.

journal_name

Mol Cancer Ther

authors

Jung M,Zhang Y,Dimtchev A,Subramanian MR,Suthanthiran K,Dritschilo A

keywords:

subject

Has Abstract

pub_date

2004-06-01 00:00:00

pages

655-9

issue

6

eissn

1535-7163

issn

1538-8514

pii

3/6/655

journal_volume

3

pub_type

杂志文章
  • Therapeutic potential of antisense oligodeoxynucleotides to down-regulate thymidylate synthase in mesothelioma.

    abstract::Malignant mesothelioma is an aggressive tumor of the serosal surfaces of the lungs, heart, and abdomen. Survival rates are poor and effective treatments are not available. However, recent therapeutic regimens targeting thymidylate synthase (TS) in malignant mesothelioma patients have shown promise. We have reported th...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-06-0073

    authors: Flynn J,Berg RW,Wong T,van Aken M,Vincent MD,Fukushima M,Koropatnick J

    更新日期:2006-06-01 00:00:00

  • A New Class of Bifunctional Major Histocompatibility Class I Antibody Fusion Molecules to Redirect CD8 T Cells.

    abstract::Bifunctional antibody fusion proteins engaging effector T cells for targeted elimination of tumor cells via CD3 binding have shown efficacy in both preclinical and clinical studies. Different from such a polyclonal T-cell recruitment, an alternative concept is to engage only antigen-specific T-cell subsets. Recruitmen...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-16-0207

    authors: Schmittnaegel M,Hoffmann E,Imhof-Jung S,Fischer C,Drabner G,Georges G,Klein C,Knoetgen H

    更新日期:2016-09-01 00:00:00

  • ORAI1-mediated calcium influx in lactation and in breast cancer.

    abstract::The entry of calcium into the mammary epithelial cell from the maternal plasma (i.e., calcium influx mechanisms) during lactation is poorly understood. As alterations in calcium channels and pumps are a key feature of some cancers, including breast cancer, understanding these calcium influx pathways may have significa...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-10-0923

    authors: McAndrew D,Grice DM,Peters AA,Davis FM,Stewart T,Rice M,Smart CE,Brown MA,Kenny PA,Roberts-Thomson SJ,Monteith GR

    更新日期:2011-03-01 00:00:00

  • Janus kinase inhibitor INCB20 has antiproliferative and apoptotic effects on human myeloma cells in vitro and in vivo.

    abstract::Protein tyrosine kinases of the Janus kinase (JAK) family are associated with many cytokine receptors, which, on ligand binding, regulate important cellular functions such as proliferation, survival, and differentiation. In multiple myeloma, JAKs may be persistently activated due to a constant stimulation by interleuk...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-08-0149

    authors: Burger R,Le Gouill S,Tai YT,Shringarpure R,Tassone P,Neri P,Podar K,Catley L,Hideshima T,Chauhan D,Caulder E,Neilan CL,Vaddi K,Li J,Gramatzki M,Fridman JS,Anderson KC

    更新日期:2009-01-01 00:00:00

  • Targeting the Akt/mammalian target of rapamycin pathway for radiosensitization of breast cancer.

    abstract::The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is known to be activated by radiation. The mammalian target of rapamycin (mTOR) is downstream of Akt, and we investigated the effects of radiation on Akt/mTOR signaling in breast cancer cell models. RAD001 (everolimus), a potent derivative of the mTOR inhibitor rapa...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-05-0400

    authors: Albert JM,Kim KW,Cao C,Lu B

    更新日期:2006-05-01 00:00:00

  • Camptothecin- and etoposide-induced apoptosis in human leukemia cells is independent of cell death receptor-3 and -4 aggregation but accelerates tumor necrosis factor-related apoptosis-inducing ligand-mediated cell death.

    abstract::During camptothecin- and etoposide (VP-16)-induced apoptosis in HL-60 cells, the expression level of cell death receptor-3 (DR3), cell death receptor-4 (DR4), and FAS remained mostly unchanged, whereas the expression of silencers of death domain (SODD) and FLICE inhibitory proteins, inhibitors of the cell death recept...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:

    authors: Bergeron S,Beauchemin M,Bertrand R

    更新日期:2004-12-01 00:00:00

  • Knock-down of Bcl-2 by antisense oligodeoxynucleotides induces radiosensitization and inhibition of angiogenesis in human PC-3 prostate tumor xenografts.

    abstract::Expression of the proto-oncogene Bcl-2 is associated with tumor progression. Bcl-2's broad expression in tumors, coupled with its role in resistance to chemotherapy and radiation therapy-induced apoptosis, makes it a rational target for anticancer therapy. Antisense Bcl-2 oligodeoxynucleotide (ODN) reagents have been ...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-06-0367

    authors: Anai S,Goodison S,Shiverick K,Hirao Y,Brown BD,Rosser CJ

    更新日期:2007-01-01 00:00:00

  • 3-Dimensional Patient-Derived Lung Cancer Assays Reveal Resistance to Standards-of-Care Promoted by Stromal Cells but Sensitivity to Histone Deacetylase Inhibitors.

    abstract::There is a growing recognition that current preclinical models do not reflect the tumor microenvironment in cellular, biological, and biophysical content and this may have a profound effect on drug efficacy testing, especially in the era of molecular-targeted agents. Here, we describe a method to directly embed low-pa...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-15-0598

    authors: Onion D,Argent RH,Reece-Smith AM,Craze ML,Pineda RG,Clarke PA,Ratan HL,Parsons SL,Lobo DN,Duffy JP,Atherton JC,McKenzie AJ,Kumari R,King P,Hall BM,Grabowska AM

    更新日期:2016-04-01 00:00:00

  • Fasudil inhibits vascular endothelial growth factor-induced angiogenesis in vitro and in vivo.

    abstract::Vascular endothelial growth factor (VEGF)-induced endothelial cell migration is an important component of tumor angiogenesis. Rho and Rho-associated kinase (ROCK) are key regulators of focal adhesion, stress fiber formation, and thus cell motility. Inhibitors of this pathway have been shown to inhibit endothelial cell...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-06-0689

    authors: Yin L,Morishige K,Takahashi T,Hashimoto K,Ogata S,Tsutsumi S,Takata K,Ohta T,Kawagoe J,Takahashi K,Kurachi H

    更新日期:2007-05-01 00:00:00

  • Long Noncoding RNA MALAT1 Contributes to Sorafenib Resistance by Targeting miR-140-5p/Aurora-A Signaling in Hepatocellular Carcinoma.

    abstract::Long noncoding RNAs (lncRNA) have been found to play critical roles in tumorigenesis and the development of various cancers, including hepatocellular carcinoma (HCC). Metastasis associated with lung adenocarcinoma transcript-1 (MALAT1) has been identified as an oncogene and prognostic biomarker in HCC. Here, we demons...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-19-0203

    authors: Fan L,Huang X,Chen J,Zhang K,Gu YH,Sun J,Cui SY

    更新日期:2020-05-01 00:00:00

  • In vivo optical imaging of human lymphoma xenograft using a library-derived peptidomimetic against alpha4beta1 integrin.

    abstract::Increasing literature suggests that cell adhesion molecule alpha4beta1 integrin plays a pivotal role in autoimmune diseases and cancer development. Noninvasive visualization of alpha4beta1 integrin in vivo will facilitate the understanding of its involvement in disease progression and development of targeted therapies...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-07-0575

    authors: Peng L,Liu R,Andrei M,Xiao W,Lam KS

    更新日期:2008-02-01 00:00:00

  • DZNep is a global histone methylation inhibitor that reactivates developmental genes not silenced by DNA methylation.

    abstract::DNA methylation, histone modifications, and nucleosomal occupancy collaborate to cause silencing of tumor-related genes in cancer. The development of drugs that target these processes is therefore important for cancer therapy. Inhibitors of DNA methylation and histone deacetylation have been approved by the Food and D...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-09-0013

    authors: Miranda TB,Cortez CC,Yoo CB,Liang G,Abe M,Kelly TK,Marquez VE,Jones PA

    更新日期:2009-06-01 00:00:00

  • Inhibition of cell growth by NB1011 requires high thymidylate synthase levels and correlates with p53, p21, bax, and GADD45 induction.

    abstract::NB1011, a phosphoramidate derivative of (E)-5-(2-bromovinyl)-2'-deoxyuridine, is a novel small molecule anticancer agent. NB1011 is selectively active against tumor cells expressing high levels of thymidylate synthase (TS), a critical enzyme in DNA biosynthesis. NB1011 is different from the current TS-targeted drugs, ...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:

    authors: Neuteboom ST,Karjian PL,Boyer CR,Beryt M,Pegram M,Wahl GM,Shepard HM

    更新日期:2002-04-01 00:00:00

  • Inhibition of p38 MAPK-dependent excision repair cross-complementing 1 expression decreases the DNA repair capacity to sensitize lung cancer cells to etoposide.

    abstract::Etoposide (VP-16), a topoisomerase II inhibitor, is an effective anticancer drug currently used for the treatment of a wide range of cancers. Excision repair cross-complementary 1 (ERCC1) is a key protein involved in the process of nucleotide excision repair. High level of ERCC1 expression in cancers is associated wit...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-11-0684

    authors: Tsai MS,Weng SH,Chen HJ,Chiu YF,Huang YC,Tseng SC,Kuo YH,Lin YW

    更新日期:2012-03-01 00:00:00

  • Dual PI3K/mTOR Inhibitors Induce Rapid Overactivation of the MEK/ERK Pathway in Human Pancreatic Cancer Cells through Suppression of mTORC2.

    abstract::The PI3K/AKT/mTOR pathway, which is aberrantly stimulated in many cancer cells, has emerged as a target for therapy. However, mTORC1/S6K also mediates negative feedback loops that attenuate upstream signaling. Suppression of these feedback loops opposes the growth-suppressive effects of mTOR inhibitors and leads to dr...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-14-0669

    authors: Soares HP,Ming M,Mellon M,Young SH,Han L,Sinnet-Smith J,Rozengurt E

    更新日期:2015-04-01 00:00:00

  • Therapeutic efficacy of CEP-33779, a novel selective JAK2 inhibitor, in a mouse model of colitis-induced colorectal cancer.

    abstract::Constitutively activated STAT3 and STAT5 are expressed in a wide variety of human malignancies including solid and hematopoietic cancers and often correlate with a poor prognosis and resistance to multiple therapies. Given the well established role of STAT3 in tumorigenesis, inhibition of Janus-activated kinase 2 (JAK...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-11-0951

    authors: Seavey MM,Lu LD,Stump KL,Wallace NH,Hockeimer W,O'Kane TM,Ruggeri BA,Dobrzanski P

    更新日期:2012-04-01 00:00:00

  • Enterolactone inhibits the growth of 7,12-dimethylbenz(a)anthracene-induced mammary carcinomas in the rat.

    abstract::The inverse association between a high enterolactone (ENL) concentration in both urine and serum, and the risk of breast cancer found in epidemiological studies suggests a chemopreventive action for ENL. However, no causal relationship has been established in clinical studies or in experimental models for breast cance...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:

    authors: Saarinen NM,Huovinen R,Wärri A,Mäkelä SI,Valentín-Blasini L,Sjöholm R,Ammälä J,Lehtilä R,Eckerman C,Collan YU,Santti RS

    更新日期:2002-08-01 00:00:00

  • Targeting CXCR2 enhances chemotherapeutic response, inhibits mammary tumor growth, angiogenesis, and lung metastasis.

    abstract::Breast cancer is one of the leading causes of cancer deaths among females. Many challenges exist in the current management of advanced stage breast cancer as there are fewer recognized therapeutic strategies, often because of therapy resistance. How breast cancer cells evade chemotherapy and the underlying mechanism r...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-12-0529

    authors: Sharma B,Nawandar DM,Nannuru KC,Varney ML,Singh RK

    更新日期:2013-05-01 00:00:00

  • Radiosensitization In Vivo by Histone Deacetylase Inhibition with No Increase in Early Normal Tissue Radiation Toxicity.

    abstract::As the population ages, more elderly patients require radiotherapy-based treatment for their pelvic malignancies, including muscle-invasive bladder cancer, as they are unfit for major surgery. Therefore, there is an urgent need to find radiosensitizing agents minimally toxic to normal tissues, including bowel and blad...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-17-0011

    authors: Groselj B,Ruan JL,Scott H,Gorrill J,Nicholson J,Kelly J,Anbalagan S,Thompson J,Stratford MRL,Jevons SJ,Hammond EM,Scudamore CL,Kerr M,Kiltie AE

    更新日期:2018-02-01 00:00:00

  • Induction of Thioredoxin-Interacting Protein by a Histone Deacetylase Inhibitor, Entinostat, Is Associated with DNA Damage and Apoptosis in Esophageal Adenocarcinoma.

    abstract::In 2017, an estimated 17,000 individuals were diagnosed with esophageal adenocarcinoma (EAC), and less than 20% will survive 5 years. Positron emission tomography avidity is indicative of high glucose utilization and is nearly universal in EAC. TXNIP blocks glucose uptake and exhibits proapoptotic functions. Higher ex...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-17-1240

    authors: Feingold PL,Surman DR,Brown K,Xu Y,McDuffie LA,Shukla V,Reardon ES,Crooks DR,Trepel JB,Lee S,Lee MJ,Gao S,Xi S,McLoughlin KC,Diggs LP,Beer DG,Nancarrow DJ,Neckers LM,Davis JL,Hoang CD,Hernandez JM,Schrump DS,R

    更新日期:2018-09-01 00:00:00

  • 10D1F, an Anti-HER3 Antibody that Uniquely Blocks the Receptor Heterodimerization Interface, Potently Inhibits Tumor Growth Across a Broad Panel of Tumor Models.

    abstract::In recent years, HER3 has increasingly been implicated in the progression of a variety of tumor types and in acquired resistance to EGFR and HER2 therapies. Whereas EGFR and HER2 primarily signal through the MAPK pathway, HER3, as a heterodimer with EGFR or HER2, potently activates the PI3K pathway. Despite its critic...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-19-0515

    authors: Thakkar D,Sancenon V,Taguiam MM,Guan S,Wu Z,Ng E,Paszkiewicz KH,Ingram PJ,Boyd-Kirkup JD

    更新日期:2020-02-01 00:00:00

  • Determinants of mitotic catastrophe on abrogation of the G2 DNA damage checkpoint by UCN-01.

    abstract::Genotoxic stress such as ionizing radiation halts entry into mitosis by activation of the G(2) DNA damage checkpoint. The CHK1 inhibitor 7-hydroxystaurosporine (UCN-01) can bypass the checkpoint and induce unscheduled mitosis in irradiated cells. Precisely, how cells behave following checkpoint abrogation remains to b...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-10-0809

    authors: On KF,Chen Y,Ma HT,Chow JP,Poon RY

    更新日期:2011-05-01 00:00:00

  • Combined Inhibition of NEDD8-Activating Enzyme and mTOR Suppresses NF2 Loss-Driven Tumorigenesis.

    abstract::Inactivation of NF2/Merlin causes the autosomal-dominant cancer predisposition syndrome familial neurofibromatosis type 2 (NF2) and contributes to the development of malignant pleural mesothelioma (MPM). To develop a targeted therapy for NF2-mutant tumors, we have exploited the recent realization that Merlin loss driv...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-16-0821

    authors: Cooper J,Xu Q,Zhou L,Pavlovic M,Ojeda V,Moulick K,de Stanchina E,Poirier JT,Zauderer M,Rudin CM,Karajannis MA,Hanemann CO,Giancotti FG

    更新日期:2017-08-01 00:00:00

  • Novel semisynthetic analogues of betulinic acid with diverse cytoprotective, antiproliferative, and proapoptotic activities.

    abstract::Betulinic acid (BA), a pentacyclic triterpene isolated from birch bark and other plants, selectively inhibits the growth of human cancer cell lines. However, the poor potency of BA hinders its clinical development, despite a lack of toxicity in animal studies even at high concentrations. Here, we describe six BA deriv...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-07-0180

    authors: Liby K,Honda T,Williams CR,Risingsong R,Royce DB,Suh N,Dinkova-Kostova AT,Stephenson KK,Talalay P,Sundararajan C,Gribble GW,Sporn MB

    更新日期:2007-07-01 00:00:00

  • Nuclear Export of Ubiquitinated Proteins Determines the Sensitivity of Colorectal Cancer to Proteasome Inhibitor.

    abstract::Although proteasome inhibitors such as bortezomib had significant therapeutic effects in multiple myeloma and mantel cell lymphoma, they exhibited minimal clinical activity as a monotherapy for solid tumors, including colorectal cancer. We found in this study that proteasome inhibition induced a remarkable nuclear exp...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-16-0553

    authors: Wu T,Chen W,Zhong Y,Hou X,Fang S,Liu CY,Wang G,Yu T,Huang YY,Ouyang X,Li HQ,Cui L,Yang Y

    更新日期:2017-04-01 00:00:00

  • Targeting BRAFV600E in thyroid carcinoma: therapeutic implications.

    abstract::B-Raf is an important mediator of cell proliferation and survival signals transduced via the Ras-Raf-MEK-ERK cascade. BRAF mutations have been detected in several tumors, including papillary thyroid carcinoma, but the precise role of B-Raf as a therapeutic target for thyroid carcinoma is still under investigation. We ...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-06-0449

    authors: Mitsiades CS,Negri J,McMullan C,McMillin DW,Sozopoulos E,Fanourakis G,Voutsinas G,Tseleni-Balafouta S,Poulaki V,Batt D,Mitsiades N

    更新日期:2007-03-01 00:00:00

  • Fatty acid synthase inhibition results in a magnetic resonance-detectable drop in phosphocholine.

    abstract::Expression of fatty acid synthase (FASN), the key enzyme in de novo synthesis of long-chain fatty acids, is normally low but increases in cancer. Consequently, FASN is a novel target for cancer therapy. However, because FASN inhibitors can lead to tumor stasis rather than shrinkage, noninvasive methods for assessing F...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-08-0015

    authors: Ross J,Najjar AM,Sankaranarayanapillai M,Tong WP,Kaluarachchi K,Ronen SM

    更新日期:2008-08-01 00:00:00

  • Suppression of Prostate Cancer Pathogenesis Using an MDA-9/Syntenin (SDCBP) PDZ1 Small-Molecule Inhibitor.

    abstract::Metastasis is the primary determinant of death in patients with diverse solid tumors and MDA-9/Syntenin (SDCBP), a pro-metastatic and pro-angiogenic gene, contributes to this process. Recently, we documented that by physically interacting with IGF-1R, MDA-9/Syntenin activates STAT3 and regulates prostate cancer pathog...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-18-1019

    authors: Das SK,Kegelman TP,Pradhan AK,Shen XN,Bhoopathi P,Talukdar S,Maji S,Sarkar D,Emdad L,Fisher PB

    更新日期:2019-11-01 00:00:00

  • Caspase-3-dependent mitotic checkpoint inactivation by the small-molecule inducers of mitotic slippage SU6656 and geraldol.

    abstract::Microtubule-targeting cancer drugs such as paclitaxel block cell-cycle progression at mitosis by prolonged activation of the mitotic checkpoint. Cells can spontaneously escape mitotic arrest and enter interphase without chromosome segregation by a process termed mitotic slippage that involves the degradation of cyclin...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-10-0909

    authors: Riffell JL,Jänicke RU,Roberge M

    更新日期:2011-05-01 00:00:00

  • Comparison of antitumor effects of multitargeted tyrosine kinase inhibitors in acute myelogenous leukemia.

    abstract::We compared the antitumor activities of the multitargeted tyrosine kinase inhibitors imatinib, sorafenib, and sunitinib to determine which inhibitor is best suited to be used for the treatment of acute myelogenous leukemia (AML). In nine human AML cell lines, sorafenib and sunitinib were more potent inhibitors of cell...

    journal_title:Molecular cancer therapeutics

    pub_type: 杂志文章

    doi:10.1158/1535-7163.MCT-07-2218

    authors: Hu S,Niu H,Minkin P,Orwick S,Shimada A,Inaba H,Dahl GV,Rubnitz J,Baker SD

    更新日期:2008-05-01 00:00:00