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1.
Int J Mol Sci ; 22(11)2021 May 21.
Article in English | MEDLINE | ID: mdl-34063776

ABSTRACT

Calcineurin inhibitors are highly efficacious immunosuppressive agents used in pediatric kidney transplantation. However, calcineurin inhibitor nephrotoxicity (CNIT) has been associated with the development of chronic renal allograft dysfunction and decreased graft survival. This study evaluated 37 formalin-fixed paraffin-embedded biopsies from pediatric kidney transplant recipients using gene expression profiling. Normal allograft samples (n = 12) served as negative controls and were compared to biopsies exhibiting CNIT (n = 11). The remaining samples served as positive controls to validate CNIT marker specificity and were characterized by other common causes of graft failure such as acute rejection (n = 7) and interstitial fibrosis/tubular atrophy (n = 7). MiRNA profiles served as the platform for data integration. Oxidative phosphorylation and mitochondrial dysfunction were the top molecular pathways associated with overexpressed genes in CNIT samples. Decreased ATP synthesis was identified as a significant biological function in CNIT, while key toxicology pathways included NRF2-mediated oxidative stress response and increased permeability transition of mitochondria. An integrative analysis demonstrated a panel of 13 significant miRNAs and their 33 CNIT-specific gene targets involved with mitochondrial activity and function. We also identified a candidate panel of miRNAs/genes, which may serve as future molecular markers for CNIT diagnosis as well as potential therapeutic targets.


Subject(s)
Biomarkers/metabolism , Calcineurin Inhibitors/toxicity , Graft Survival/genetics , Kidney Diseases/chemically induced , Kidney Diseases/genetics , Transcriptome/genetics , Biopsy/methods , Calcineurin Inhibitors/therapeutic use , Child , Computational Biology/methods , Gene Expression Profiling/methods , Graft Rejection/drug therapy , Graft Rejection/genetics , Humans , Immunosuppressive Agents/therapeutic use , Immunosuppressive Agents/toxicity , Kidney/drug effects , Kidney/metabolism , Kidney Transplantation/adverse effects , MicroRNAs/genetics , Oxidative Stress/drug effects , Oxidative Stress/genetics , Transplant Recipients , Transplantation, Homologous/methods
2.
Ear Nose Throat J ; 100(1): 55-59, 2021 Jan.
Article in English | MEDLINE | ID: mdl-33307794

ABSTRACT

Invasive fungal sinusitis is a morbid pathology that typically affects immunocompromised patients and may quickly progress to fulminant disease. The purpose of this study was to measure the sensitivity and specificity of touch preparation of nasal debridement specimens as a rapid diagnostic tool for invasive fungal sinusitis. A retrospective chart review was performed of 22 patients undergoing nasal debridement due to suspicion for invasive fungal sinusitis over a 10-year period. Thirteen patients had touch preparation of nasal specimens followed by routine histologic processing; two of these patients underwent 2, and 1 patient had 3 separate debridements, for a total of 17 touch preparations performed. The sensitivity and specificity of touch preparation were calculated by correlating the initial results with the presence of fungal invasion on final pathologic analysis. The sensitivity of touch preparation was 56% (95% confidence interval [CI]: 0.23-0.85), specificity was 100% (95% CI: 0.60-1.00), positive predictive value was 100% (95% CI: 0.46-1.00), and negative predictive value was 67% (95% CI: 0.35-0.89). This procedure may be a useful adjunct in situations requiring rapid diagnosis of invasive fungal sinusitis but should not be used as the sole criteria for determining the need for surgical intervention.


Subject(s)
Invasive Fungal Infections/diagnosis , Mycological Typing Techniques/statistics & numerical data , Sinusitis/diagnosis , Adolescent , Adult , Aged , Debridement , Female , Humans , Invasive Fungal Infections/classification , Invasive Fungal Infections/microbiology , Male , Middle Aged , Mycological Typing Techniques/methods , Nose/microbiology , Pilot Projects , Predictive Value of Tests , Retrospective Studies , Sensitivity and Specificity , Sinusitis/classification , Sinusitis/microbiology , Touch , Young Adult
3.
Ear Nose Throat J ; 97(1-2): E32-E36, 2018.
Article in English | MEDLINE | ID: mdl-29493729

ABSTRACT

Invasive fungal sinusitis is a morbid pathology that typically affects immunocompromised patients and may quickly progress to fulminant disease. The purpose of this study was to measure the sensitivity and specificity of touch preparation of nasal debridement specimens as a rapid diagnostic tool for invasive fungal sinusitis. A retrospective chart review was performed of 22 patients undergoing nasal debridement due to suspicion for invasive fungal sinusitis over a 10-year period. Thirteen patients had touch preparation of nasal specimens followed by routine histologic processing; 2 of these patients underwent two and 1 patient had three separate debridements, for a total of 17 touch preparations performed. The sensitivity and specificity of touch preparation were calculated by correlating the initial results with the presence of fungal invasion on final pathologic analysis. The sensitivity of touch preparation was 56% (95% confidence interval [CI]: 0.23 to 0.85), specificity was 100% (95% CI: 0.60 to 1.00), positive predictive value was 100% (95% CI: 0.46 to 1.00), and negative predictive value was 67% (95% CI: 0.35 to 0.89). This procedure may be a useful adjunct in situations requiring rapid diagnosis of invasive fungal sinusitis but should not be used as the sole criterion for determining the need for surgical intervention.


Subject(s)
Invasive Fungal Infections/diagnosis , Mycological Typing Techniques/statistics & numerical data , Sinusitis/diagnosis , Adolescent , Adult , Debridement , Female , Humans , Invasive Fungal Infections/classification , Invasive Fungal Infections/microbiology , Male , Middle Aged , Mycological Typing Techniques/methods , Nose , Pilot Projects , Predictive Value of Tests , Retrospective Studies , Sensitivity and Specificity , Sinusitis/classification , Sinusitis/microbiology , Touch , Young Adult
5.
Diagn Pathol ; 10: 169, 2015 Sep 17.
Article in English | MEDLINE | ID: mdl-26376646

ABSTRACT

BACKGROUND: Targeted next generation sequencing (NGS) technology to assess the mutational status of multiple genes on formalin-fixed, paraffin embedded (FFPE) tumors is rapidly being adopted in clinical settings, where quality control (QC) practices are required. Establishing reliable FFPE QC materials for NGS can be challenging and/or expensive. Here, we established a reliable and cost-effective FFPE QC material for routine utilization in the Ion AmpliSeq™ Cancer Hotspot Panel v2 (CHP2) assay. METHODS: The performance characteristics of the CHP2 assay were determined by sequencing various cell line mixtures and 55 different FFPE tumors on the Ion Torrent PGM platform. A FFPE QC material was prepared from a mixture of cell lines derived from different cancers, comprising single nucleotide variants and small deletions on actionable genes at different allelic frequencies. RESULTS: The CHP2 assay performed with high precision and sensitivity when custom variant calling pipeline parameters where established. In addition, all expected somatic variants in the QC material were consistently called at variant frequencies ranging from 9.1 % (CV = 11.1 %) to 37.9 % (CV = 2.8 %). CONCLUSIONS: The availability of a reliable and cost-effective QC material is instrumental in assessing the performance of this or any targeted NGS assay that detects somatic variants in fixed solid tumor specimens.


Subject(s)
Biomarkers, Tumor/genetics , DNA Mutational Analysis/standards , Fixatives , Formaldehyde , High-Throughput Nucleotide Sequencing/standards , Mutation , Neoplasms/genetics , Tissue Fixation , Artifacts , Cell Line, Tumor , Cost-Benefit Analysis , DNA Mutational Analysis/economics , Gene Frequency , Genetic Predisposition to Disease , Health Care Costs , High-Throughput Nucleotide Sequencing/economics , Humans , Limit of Detection , Paraffin Embedding , Polymorphism, Single Nucleotide , Predictive Value of Tests , Quality Control , Reproducibility of Results
6.
Exp Mol Pathol ; 97(3): 515-24, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25446840

ABSTRACT

Periostin is a modular glycoprotein frequently observed to be a major constituent of the extracellular milieu of mass-forming intrahepatic cholangiocarcinoma and other desmoplastic malignant tumors. In intrahepatic cholangiocarcinoma, as well as in desmoplastic pancreatic ductal adenocarcinoma, periostin is overexpressed and hypersecreted in large part, if not exclusively, by cancer-associated fibroblasts within the tumor stroma. Through its interaction with specific components of the extracellular tumor matrix, particularly collagen type I and tenascin-C, and with cell surface receptors, notably integrins leading to activation of the Akt and FAK signaling pathways, this TGF-ß family-inducible matricellular protein appears to be functioning as a key extracellular matrix molecule regulating such critically important and diverse malignant tumor behaviors as tumor fibrogenesis and desmoplasia, invasive malignant cell growth, chemoresistance, and metastatic colonization. This review will discuss current evidence and basic molecular mechanisms implicating periostin as a mediator of intrahepatic cholangiocarcinoma invasive growth. In addition, its significance as a potential prognostic biomarker for intrahepatic cholangiocarcinoma patients, as well as future possibilities and challenges as a molecular target for cholangiocarcinoma therapy and/or prevention, will be critically evaluated.


Subject(s)
Bile Duct Neoplasms/metabolism , Bile Duct Neoplasms/pathology , Bile Ducts, Intrahepatic/metabolism , Bile Ducts, Intrahepatic/pathology , Cell Adhesion Molecules/metabolism , Cholangiocarcinoma/metabolism , Cholangiocarcinoma/pathology , Humans
7.
Blood ; 118(19): 5189-200, 2011 Nov 10.
Article in English | MEDLINE | ID: mdl-21911831

ABSTRACT

Effects of Chk1 and MEK1/2 inhibition were investigated in cytokinetically quiescent multiple myeloma (MM) and primary CD138(+) cells. Coexposure to the Chk1 and MEK1/2 inhibitors AZD7762 and selumetinib (AZD6244) robustly induced apoptosis in various MM cells and CD138(+) primary samples, but spared normal CD138(-) and CD34(+) cells. Furthermore, Chk1/MEK1/2 inhibitor treatment of asynchronized cells induced G(0)/G(1) arrest and increased apoptosis in all cell-cycle phases, including G(0)/G(1). To determine whether this regimen is active against quiescent G(0)/G(1) MM cells, cells were cultured in low-serum medium to enrich the G(0)/G(1) population. G(0)/G(1)-enriched cells exhibited diminished sensitivity to conventional agents (eg, Taxol and VP-16) but significantly increased susceptibility to Chk1 ± MEK1/2 inhibitors or Chk1 shRNA knock-down. These events were associated with increased γH2A.X expression/foci formation and Bim up-regulation, whereas Bim shRNA knock-down markedly attenuated lethality. Immunofluorescent analysis of G(0)/G(1)-enriched or primary MM cells demonstrated colocalization of activated caspase-3 and the quiescent (G(0)) marker statin, a nuclear envelope protein. Finally, Chk1/MEK1/2 inhibition increased cell death in the Hoechst-positive (Hst(+)), low pyronin Y (PY)-staining (2N Hst(+)/PY(-)) G(0) population and in sorted small side-population (SSP) MM cells. These findings provide evidence that cytokinetically quiescent MM cells are highly susceptible to simultaneous Chk1 and MEK1/2 inhibition.


Subject(s)
MAP Kinase Kinase 1/antagonists & inhibitors , MAP Kinase Kinase 2/antagonists & inhibitors , Multiple Myeloma/drug therapy , Multiple Myeloma/metabolism , Protein Kinase Inhibitors/therapeutic use , Protein Kinases/metabolism , Apoptosis/drug effects , Benzimidazoles/administration & dosage , Benzimidazoles/therapeutic use , Cell Line, Tumor , Checkpoint Kinase 1 , DNA Damage , G1 Phase , Humans , Interleukin-6/metabolism , Multiple Myeloma/pathology , Protein Kinase Inhibitors/administration & dosage , Resting Phase, Cell Cycle , Syndecan-1/metabolism , Thiophenes/administration & dosage , Thiophenes/therapeutic use , Urea/administration & dosage , Urea/analogs & derivatives , Urea/therapeutic use
8.
Blood ; 117(6): 1947-57, 2011 Feb 10.
Article in English | MEDLINE | ID: mdl-21148814

ABSTRACT

Ras/MEK/ERK pathway activation represents an important compensatory response of human multiple myeloma (MM) cells to checkpoint kinase 1 (Chk1) inhibitors. To investigate the functional roles of Src in this event and potential therapeutic significance, interactions between Src and Chk1 inhibitors (eg, UCN-01 or Chk1i) were examined in vitro and in vivo. The dual Src/Abl inhibitors BMS354825 and SKI-606 blocked Chk1-inhibitor-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation, markedly increasing apoptosis in association with BimEL up-regulation, p34(cdc2) activation, and DNA damage in MM cell lines and primary CD138(+) MM samples. Loss-of-function Src mutants (K297R, K296R/Y528F) or shRNA knock-down of Src prevented the ERK1/2 activation induced by Chk1 inhibitors and increased apoptosis. Conversely, constitutively active Ras or mitogen-activated protein kinase/ERK kinase 1 (MEK1) significantly diminished the ability of Src inhibitors to potentiate Chk1-inhibitor lethality. Moreover, Src/Chk1-inhibitor cotreatment attenuated MM-cell production of vascular endothelial growth factor and other angiogenic factors (eg, ANG [angiogenin], TIMP1/2 [tissue inhibitor of metalloproteinases 1/2], and RANTES [regulated on activation normal T-cell expressed and secreted]), and inhibited in vitro angiogenesis. Finally, coadministration of BMS354825 and UCN-01 suppressed human MM tumor growth in a murine xenograft model, increased apoptosis, and diminished angiogenesis. These findings suggest that Src kinase is required for Chk1-inhibitor-mediated Ras → ERK1/2 signaling activation, and that disruption of this event sharply potentiates the anti-MM activity of Chk1 inhi-bitors in vitro and in vivo.


Subject(s)
Apoptosis/physiology , Multiple Myeloma/pathology , Multiple Myeloma/physiopathology , Protein Kinases/physiology , src-Family Kinases/antagonists & inhibitors , Aniline Compounds/pharmacology , Animals , Apoptosis/drug effects , CDC2 Protein Kinase/metabolism , Cell Line, Tumor , Checkpoint Kinase 1 , DNA Damage , Dasatinib , Female , Gene Knockdown Techniques , Humans , In Vitro Techniques , MAP Kinase Signaling System/drug effects , Mice , Mice, Nude , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Mutation , Neovascularization, Pathologic/drug therapy , Nitriles/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Quinolines/pharmacology , RecQ Helicases/antagonists & inhibitors , Thiazoles/pharmacology , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/physiology , src-Family Kinases/genetics , src-Family Kinases/physiology
9.
Hepatology ; 52(3): 975-86, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20607690

ABSTRACT

UNLABELLED: Overexpression of epidermal growth factor receptor (ErbB1) and/or ErbB2 has been implicated in the pathogenesis of cholangiocarcinoma, suggesting that combined ErbB1/ErbB2 targeting might serve as a target-based therapeutic strategy for this highly lethal cancer. To test this strategy, we investigated targeting with the ErbB1 inhibitor tryphostin AG1517 and the ErbB2 inhibitor tryphostin AG879, in combination and alone, as well as with the dual ErbB1/ErbB2 inhibitor lapatinib, to assess the effectiveness of simultaneous targeting of ErbB1 and ErbB2 signaling over single inhibitor treatments in suppressing cholangiocarcinoma cell growth in vitro and the therapeutic efficacy of lapatinib in vivo. Our in vitro studies were carried out using rat (BDEneu and C611B) and human (HuCCT1 and TFK1) cholangiocarcinoma cell lines. The efficacy of lapatinib to significantly suppress liver tumor growth was tested in an orthotopic, syngeneic rat model of intrahepatic cholangiocarcinoma progression. Our results demonstrated that simultaneous targeting of ErbB1 and ErbB2 signaling was significantly more effective in suppressing the in vitro growth of both rat and human cholangiocarcinoma cells than individual receptor targeting. Lapatinib was an even more potent inhibitor of cholangiocarcinoma cell growth and inducer of apoptosis than either tryphostin when tested in vitro against these respective cholangiocarcinoma cell lines, regardless of differences in their levels of ErbB1 or ErbB2 protein expression and/or mechanism of activation. Lapatinib treatment also produced a significant suppression of intrahepatic cholangiocarcinoma growth when administered early to rats, but was without effect in inhibiting liver tumor growth in rats with more advanced tumors. CONCLUSION: Our findings suggest that simultaneous targeting of ErbB1 and ErbB2 could be a potentially selective strategy for cholangiocarcinoma therapy, but is likely to be ineffective by itself against advanced cancer.


Subject(s)
Bile Duct Neoplasms/drug therapy , Bile Ducts, Intrahepatic , Cholangiocarcinoma/drug therapy , ErbB Receptors/antagonists & inhibitors , Quinazolines/therapeutic use , Receptor, ErbB-2/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Bile Duct Neoplasms/metabolism , Bile Duct Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Cholangiocarcinoma/metabolism , Cholangiocarcinoma/pathology , Disease Models, Animal , ErbB Receptors/metabolism , Humans , Lapatinib , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Quinazolines/pharmacology , Rats , Receptor, ErbB-2/metabolism , Signal Transduction/drug effects , Tyrphostins/pharmacology , Tyrphostins/therapeutic use
10.
J Biol Chem ; 285(13): 10064-10077, 2010 Mar 26.
Article in English | MEDLINE | ID: mdl-20065354

ABSTRACT

Mechanisms underlying histone deacetylase inhibitor (HDACI)-mediated NF-kappaB activation were investigated in human leukemia cells. Exposure of U937 and other leukemia cells to LBH-589 induced reactive oxygen species (ROS) followed by single strand (XRCC1) and double strand (gamma-H2AX) DNA breaks. Notably, LBH-589 lethality was markedly attenuated by small interfering RNA (siRNA) knockdown of the DNA damage-linked histone, H1.2. LBH-589 triggered p65/RelA activation, NF-kappaB-dependent induction of Mn-SOD2, and ROS elimination. Interference with LBH-589-mediated NF-kappaB activation (e.g. in I kappaB alpha super-repressor transfected cells) diminished HDACI-mediated Mn-SOD2 induction and increased ROS accumulation, DNA damage, and apoptosis. The Mn-SOD2 mimetic TBAP (manganese(III)-tetrakis 4-benzoic acid porphyrin) prevented HDACI-induced ROS and NF-kappaB activation while dramatically attenuating DNA damage and cell death. In contrast, TRAF2 siRNA knockdown, targeting receptor-mediated NF-kappaB activation, blocked TNFalpha- but not HDACI-mediated NF-kappaB activation and lethality. Consistent with ROS-mediated DNA damage, LBH-589 exposure activated ATM (on serine 1981) and increased its association with NEMO. Significantly, siRNA NEMO or ATM knockdown blocked HDACI-mediated NF-kappaB activation, resulting in diminished MnSOD2 induction and enhanced oxidative DNA damage and cell death. In accord with the recently described DNA damage/ATM/NEMO pathway, SUMOylation site mutant NEMO (K277A or K309A) cells exposed to LBH-589 displayed diminished ATM/NEMO association, NEMO and p65/RelA nuclear localization/activation, and MnSOD2 up-regulation. These events were accompanied by increased ROS production, gamma-H2AX formation, and cell death. Together, these findings indicate that in human leukemia cells, HDACIs activate the cytoprotective NF-kappaB pathway through an ATM/NEMO/SUMOylation-dependent process involving the induction of ROS and DNA damage and suggest that blocking NF-kappaB activation via the atypical ATM/NEMO nuclear pathway can enhance HDACI antileukemic activity.


Subject(s)
Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Leukemic , Histone Deacetylase Inhibitors/metabolism , Leukemia/drug therapy , Leukemia/enzymology , NF-kappa B/metabolism , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/metabolism , Cell Line, Tumor , DNA-Binding Proteins/metabolism , Enzyme Activation , HL-60 Cells , Humans , I-kappa B Kinase/metabolism , Jurkat Cells , Mutation , Protein Serine-Threonine Kinases/metabolism , Reactive Oxygen Species , SUMO-1 Protein/metabolism , Tumor Suppressor Proteins/metabolism , U937 Cells
11.
Clin Gastroenterol Hepatol ; 7(11 Suppl): S68-78, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19896103

ABSTRACT

In this review, we will examine various molecular biomarkers for their potential to serve as independent prognostic factors for predicting survival outcome in postoperative patients with progressive intrahepatic cholangiocarcinoma. Specific rodent models of intrahepatic cholangiocarcinoma that mimic relevant cellular, molecular, and clinical features of the human disease are also described, not only in terms of their usefulness in identifying molecular pathways and mechanisms linked to cholangiocarcinoma development and progression, but also for their potential value as preclinical platforms for suggesting and testing novel molecular strategies for cholangiocarcinoma therapy. Last, recent studies aimed at addressing the role of desmoplastic stroma in promoting intrahepatic cholangiocarcinoma progression are highlighted in an effort to underline the potential value of targeting tumor stromal components together with that of cholangiocarcinoma cells as a novel therapeutic option for this devastating cancer.


Subject(s)
Cholangiocarcinoma/pathology , Liver Neoplasms/pathology , Animals , Biomarkers , Cholangiocarcinoma/diagnosis , Cholangiocarcinoma/therapy , Disease Models, Animal , Disease Progression , Humans , Liver Neoplasms/diagnosis , Liver Neoplasms/therapy , Prognosis , Rodentia
12.
Cancer Res ; 69(17): 6915-23, 2009 Sep 01.
Article in English | MEDLINE | ID: mdl-19723667

ABSTRACT

Sphingosine-1-phosphate is a potent sphingolipid mediator of diverse processes important for brain tumors, including cell growth, survival, migration, invasion, and angiogenesis. Sphingosine kinase 1 (SphK1), one of the two isoenzymes that produce sphingosine-1-phosphate, is up-regulated in glioblastoma and has been linked to poor prognosis in patients with glioblastoma multiforme (GBM). In the present study, we found that a potent isotype-specific SphK1 inhibitor, SK1-I, suppressed growth of LN229 and U373 glioblastoma cell lines and nonestablished human GBM6 cells. SK1-I also enhanced GBM cell death and inhibited their migration and invasion. SK1-I rapidly reduced phosphorylation of Akt but had no significant effect on activation of extracellular signal-regulated kinase 1/2, another important survival pathway for GBM. Inhibition of the concomitant activation of the c-Jun-NH(2)-kinase pathway induced by SK1-I attenuated death of GBM cells. Importantly, SK1-I markedly reduced the tumor growth rate of glioblastoma xenografts, inducing apoptosis and reducing tumor vascularization, and enhanced the survival of mice harboring LN229 intracranial tumors. Our results support the notion that SphK1 may be an important factor in GBM and suggest that an isozyme-specific inhibitor of SphK1 deserves consideration as a new therapeutic agent for this disease.


Subject(s)
Amino Alcohols/pharmacology , Central Nervous System Neoplasms/drug therapy , Central Nervous System Neoplasms/enzymology , Enzyme Inhibitors/pharmacology , Glioblastoma/drug therapy , Glioblastoma/enzymology , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Animals , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Central Nervous System Neoplasms/blood supply , Central Nervous System Neoplasms/pathology , Enzyme Inhibitors/therapeutic use , Female , Glioblastoma/blood supply , Glioblastoma/pathology , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Male , Mice , Neoplasm Transplantation , Neovascularization, Pathologic/drug therapy , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , Sphingosine/analogs & derivatives , Sphingosine/pharmacology
13.
Mol Cancer Ther ; 7(10): 3285-97, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18852132

ABSTRACT

The role of reactive oxygen species (ROS) production on DNA damage and potentiation of fludarabine lethality by the histone deacetylase inhibitor (HDACI) LAQ-824 was investigated in human leukemia cells. Preexposure (24 h) of U937, HL-60, Jurkat, or K562 cells to LAQ-824 (40 nmol/L) followed by fludarabine (0.4 micromol/L) dramatically potentiated apoptosis (>or=75%). LAQ-824 triggered an early ROS peak (30 min-3 h), which declined by 6 h, following LAQ-824-induced manganese superoxide dismutase 2 (Mn-SOD2) upregulation. LAQ-824/fludarabine lethality was significantly diminished by either ROS scavengers N-acetylcysteine or manganese (III) tetrakis (4-benzoic acid) porphyrin or ectopic Mn-SOD2 expression and conversely increased by Mn-SOD2 antisense knockdown. During this interval, LAQ-824 induced early (4-8 h) increases in gamma-H2AX, which persisted (48 h) secondary to LAQ-824-mediated inhibition of DNA repair (e.g., down-regulation of Ku86 and Rad50, increased Ku70 acetylation, diminished Ku70 and Ku86 DNA-binding activity, and down-regulated DNA repair genes BRCA1, CHEK1, and RAD51). Addition of fludarabine further potentiated DNA damage, which was incompatible with cell survival, and triggered multiple proapoptotic signals including activation of nuclear caspase-2 and release of histone H1.2 into the cytoplasm. The latter event induced activation of Bak and culminated in pronounced mitochondrial injury and apoptosis. These findings provide a mechanistic basis for understanding the role of early HDACI-induced ROS generation and modulation of DNA repair processes in potentiation of nucleoside analogue-mediated DNA damage and lethality in leukemia. Moreover, they show for the first time the link between HDACI-mediated ROS generation and the recently reported DNA damage observed in cells exposed to these agents.


Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Hydroxamic Acids/pharmacology , Leukemia/enzymology , Reactive Oxygen Species/metabolism , Vidarabine/analogs & derivatives , Acetylation/drug effects , Apoptosis/drug effects , Caspase 2/metabolism , Cell Line, Tumor , Cytosol/drug effects , Cytosol/metabolism , DNA Damage , DNA Repair/drug effects , Down-Regulation/drug effects , Drug Screening Assays, Antitumor , Drug Synergism , Enzyme Activation/drug effects , Histones/metabolism , Humans , Leukemia/pathology , Superoxide Dismutase/metabolism , Vidarabine/pharmacology , bcl-2 Homologous Antagonist-Killer Protein/metabolism
14.
Blood ; 112(6): 2439-49, 2008 Sep 15.
Article in English | MEDLINE | ID: mdl-18614762

ABSTRACT

The role of the Ras/MEK/ERK pathway was examined in relation to DNA damage in human multiple myeloma (MM) cells exposed to Chk1 inhibitors in vitro and in vivo. Exposure of various MM cells to marginally toxic concentrations of the Chk1 inhibitors UCN-01 or Chk1i modestly induced DNA damage, accompanied by Ras and ERK1/2 activation. Interruption of these events by pharmacologic (eg, the farnesyltransferase inhibitor R115777 or the MEK1/2 inhibitor PD184352) or genetic (eg, transfection with dominant-negative Ras or MEK1 shRNA) means induced pronounced DNA damage, reflected by increased gammaH2A.X expression/foci formation and by comet assay. Increased DNA damage preceded extensive apoptosis. Notably, similar phenomena were observed in primary CD138(+) MM cells. Enforced MEK1/2 activation by B-Raf transfection prevented R115777 but not PD184352 from inactivating ERK1/2 and promoting Chk1 inhibitor-induced gammaH2A.X expression. Finally, coadministration of R115777 diminished UCN-01-mediated ERK1/2 activation and markedly potentiated gammaH2A.X expression in a MM xenograft model, associated with a striking increase in tumor cell apoptosis and growth suppression. Such findings suggest that Ras/MEK/ERK activation opposes whereas its inhibition dramatically promotes Chk1 antagonist-mediated DNA damage. Together, these findings identify a novel mechanism by which agents targeting the Ras/MEK/ERK pathway potentiate Chk1 inhibitor lethality in MM.


Subject(s)
DNA Damage/drug effects , MAP Kinase Signaling System/physiology , Multiple Myeloma/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Kinases/drug effects , ras Proteins/physiology , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Apoptosis , Checkpoint Kinase 1 , Humans , Mice , Mice, SCID , Multiple Myeloma/pathology , Protein Kinase Inhibitors/therapeutic use , Quinolones/pharmacology , Quinolones/therapeutic use , Staurosporine/analogs & derivatives , Staurosporine/pharmacology , Staurosporine/therapeutic use , Xenograft Model Antitumor Assays
15.
Blood ; 112(4): 1382-91, 2008 Aug 15.
Article in English | MEDLINE | ID: mdl-18511810

ABSTRACT

The potent bioactive sphingolipid mediator, sphingosine-1-phosphate (S1P), is produced by 2 sphingosine kinase isoenzymes, SphK1 and SphK2. Expression of SphK1 is up-regulated in cancers, including leukemia, and associated with cancer progression. A screen of sphingosine analogs identified (2R,3S,4E)-N-methyl-5-(4'-pentylphenyl)-2-aminopent-4-ene-1,3-diol, designated SK1-I (BML-258), as a potent, water-soluble, isoenzyme-specific inhibitor of SphK1. In contrast to pan-SphK inhibitors, SK1-I did not inhibit SphK2, PKC, or numerous other protein kinases. SK1-I decreased growth and survival of human leukemia U937 and Jurkat cells, and enhanced apoptosis and cleavage of Bcl-2. Lethality of SK1-I was reversed by caspase inhibitors and by expression of Bcl-2. SK1-I not only decreased S1P levels but concomitantly increased levels of its proapoptotic precursor ceramide. Conversely, S1P protected against SK1-I-induced apoptosis. SK1-I also induced multiple perturbations in activation of signaling and survival-related proteins, including diminished phosphorylation of ERK1/2 and Akt. Expression of constitutively active Akt protected against SK1-I-induced apoptosis. Notably, SK1-I potently induced apoptosis in leukemic blasts isolated from patients with acute myelogenous leukemia but was relatively sparing of normal peripheral blood mononuclear leukocytes. Moreover, SK1-I markedly reduced growth of AML xenograft tumors. Our results suggest that specific inhibitors of SphK1 warrant attention as potential additions to the therapeutic armamentarium in leukemia.


Subject(s)
Leukemia/drug therapy , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Sphingosine/analogs & derivatives , Animals , Apoptosis/drug effects , Cell Proliferation/drug effects , Enzyme Inhibitors/therapeutic use , Humans , Mice , Mice, SCID , Proto-Oncogene Proteins c-bcl-2/metabolism , Sphingosine/therapeutic use , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
16.
Cancer Res ; 67(19): 9490-500, 2007 Oct 01.
Article in English | MEDLINE | ID: mdl-17909059

ABSTRACT

Interactions between the multikinase inhibitor sorafenib and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were examined in malignant hematopoietic cells. Pretreatment (24 h) of U937 leukemia cells with 7.5 micromol/L sorafenib dramatically increased apoptosis induced by sublethal concentrations of TRAIL/Apo2L (75 ng/mL). Similar interactions were observed in Raji, Jurkat, Karpas, K562, U266 cells, primary acute myelogenous leukemia blasts, but not in normal CD34+ bone marrow cells. Sorafenib/TRAIL-induced cell death was accompanied by mitochondrial injury and release of cytochrome c, Smac, and AIF into the cytosol and caspase-9, caspase-3, caspase-7, and caspase-8 activation. Sorafenib pretreatment down-regulated Bcl-xL and abrogated Mcl-1 expression, whereas addition of TRAIL sharply increased Bid activation, conformational change of Bak (ccBak) and Bax (ccBax), and Bax translocation. Ectopic Mcl-1 expression significantly attenuated sorafenib/TRAIL-mediated lethality and dramatically reduced ccBak while minimally affecting levels of ccBax. Similarly, inhibition of the receptor-mediated apoptotic cascade with a caspase-8 dominant-negative mutant significantly blocked sorafenib/TRAIL-induced lethality but not Mcl-1 down-regulation or Bak/Bax conformational change, indicating that TRAIL-mediated receptor pathway activation is required for maximal lethality. Sorafenib/TRAIL did not increase expression of DR4/DR5, or recruitment of procaspase-8 or FADD to the death-inducing signaling complex (DISC), but strikingly increased DISC-associated procaspase-8 activation. Sorafenib also down-regulated cFLIP(L), most likely through a translational mechanism, in association with diminished eIF4E phosphorylation, whereas ectopic expression of cFLIP(L) significantly reduced sorafenib/TRAIL lethality. Together, these results suggest that in human leukemia cells, sorafenib potentiates TRAIL-induced lethality by down-regulating Mcl-1 and cFLIP(L), events that cooperate to engage the intrinsic and extrinsic apoptotic cascades, culminating in pronounced mitochondrial injury and apoptosis.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Benzenesulfonates/pharmacology , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Leukemia/drug therapy , Neoplasm Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Pyridines/pharmacology , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Apoptosis/physiology , Benzenesulfonates/administration & dosage , CASP8 and FADD-Like Apoptosis Regulating Protein/biosynthesis , Caspase 8/metabolism , Death Domain Receptor Signaling Adaptor Proteins/metabolism , Down-Regulation/drug effects , Drug Synergism , Humans , Leukemia/metabolism , Leukemia/pathology , Myeloid Cell Leukemia Sequence 1 Protein , Neoplasm Proteins/biosynthesis , Niacinamide/analogs & derivatives , Phenylurea Compounds , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Pyridines/administration & dosage , Receptors, TNF-Related Apoptosis-Inducing Ligand/biosynthesis , Receptors, Tumor Necrosis Factor/biosynthesis , Sorafenib , TNF-Related Apoptosis-Inducing Ligand/administration & dosage , U937 Cells , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/metabolism
17.
Mol Cancer Ther ; 6(2): 692-702, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17308065

ABSTRACT

The mechanism and functional significance of XIAP and Mcl-1 down-regulation in human leukemia cells exposed to the histone deacetylase inhibitor vorinostat and the cyclin-dependent kinase inhibitor flavopiridol was investigated. Combined exposure of U937 leukemia cells to marginally toxic concentrations of vorinostat and flavopiridol resulted in a marked increase in mitochondrial damage and apoptosis accompanied by pronounced reductions in XIAP and Mcl-1 mRNA and protein. Down-regulation of Mcl-1 and XIAP expression by vorinostat/flavopiridol was associated with enhanced inhibition of phosphorylation of RNA polymerase II and was amplified by caspase-mediated protein degradation. Chromatin immunoprecipitation analysis revealed that XIAP and Mcl-1 down-regulation were also accompanied by both decreased association of nuclear factor-kappaB (XIAP) and increased E2F1 association (Mcl-1) with their promoter regions, respectively. Ectopic expression of Mcl-1 but not XIAP partially protected cells from flavopiridol/vorinostat-mediated mitochondrial injury at 48 h, but both did not significantly restored clonogenic potential. Flavopiridol/vorinostat-mediated transcriptional repression of XIAP, Mcl-1-enhanced apoptosis, and loss of clonogenic potential also occurred in primary acute myelogenous leukemia (AML) blasts. Together, these findings indicate that transcriptional repression of XIAP and Mcl-1 by flavopiridol/vorinostat contributes functionally to apoptosis induction at early exposure intervals and raise the possibility that expression levels may be a useful surrogate marker for activity in current trials.


Subject(s)
Antineoplastic Agents/pharmacology , Flavonoids/pharmacology , Hydroxamic Acids/pharmacology , Neoplasm Proteins/metabolism , Piperidines/pharmacology , Proto-Oncogene Proteins c-bcl-2/metabolism , X-Linked Inhibitor of Apoptosis Protein/metabolism , Apoptosis/drug effects , Apoptosis Inducing Factor/metabolism , Blast Crisis , Blotting, Western , Butyrates/pharmacology , Caspases/metabolism , Chromatin Immunoprecipitation , Cyclin-Dependent Kinases/antagonists & inhibitors , Cytochromes c/metabolism , Down-Regulation , Drug Interactions , Histone Deacetylase Inhibitors , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/metabolism , Membrane Potential, Mitochondrial/drug effects , Myeloid Cell Leukemia Sequence 1 Protein , Neoplasm Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic/drug effects , Tumor Stem Cell Assay , U937 Cells/drug effects , Vorinostat , X-Linked Inhibitor of Apoptosis Protein/antagonists & inhibitors , X-Linked Inhibitor of Apoptosis Protein/genetics
18.
Mol Pharmacol ; 69(1): 288-98, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16219908

ABSTRACT

Interactions between the cyclin-dependent kinase (CDK) inhibitor flavopiridol and histone deacetylase (HDAC) inhibitors (suberoylanilide hydroxamide and sodium butyrate) were examined in human leukemia cells (U937 and HL-60) ectopically expressing Bcl-2/Bcl-x(L) and in primary AML cells. Coadministration of flavopiridol with HDAC inhibitors synergistically potentiated mitochondrial damage (cytochrome c, second mitochondria-derived activator of caspases/direct IAP binding protein with low pI, and apoptosis-inducing factor release), caspase activation, poly(ADP-ribose) polymerase degradation, and cell death in both wild type and Bcl-2- or Bcl-x(L)-overexpressing cells and induced a pronounced loss of clonogenicity. In contrast, Bcl-2 and Bcl-x(L) largely blocked these events in cells exposed to the cytotoxic agent 1-beta-d-arabinofuranosylcytosine (ara-C). Enforced expression of dominant-negative Fas-associated death domain failed to protect cells from the flavopiridol/histone deacetylase inhibitor (HDACI) regimen, arguing against the involvement of the receptor pathway in lethality. Ectopic expression of a phosphorylation loop-deleted Bcl-2 or Bcl-2 lacking the serine(70) phosphorylation site, which dramatically protected cells from ara-C lethality, delayed but did not prevent flavopiridol/HDAC inhibitor-induced mitochondrial injury, cell death, or loss of clonogenicity. Ectopic expression of Bcl-2 or Bcl-x(L) was also unable to prevent the flavopiridol/HDACI regimen from inducing a conformational change in and mitochondrial translocation of Bax, and it did not attenuate Bax dimerization. As a whole, these findings indicate that in contrast to certain conventional cytotoxic agents such as ara-C, overexpression of Bcl-2 or Bcl-x(L) are largely ineffective in preventing perturbations in Bax, mitochondrial injury, and cell death in human leukemia cells subjected to simultaneous CDK and HDAC inhibition. They also raise the possibility that a strategy combining CDK and HDAC inhibitors may be effective against drug-resistant leukemia cells overexpressing Bcl-2 or Bcl-x(L).


Subject(s)
Cell Death/drug effects , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Histone Deacetylase Inhibitors , Leukemia/metabolism , Mitochondria/drug effects , Piperidines/pharmacology , Proto-Oncogene Proteins c-bcl-2/metabolism , Blotting, Western , Humans , Leukemia/pathology , U937 Cells
19.
Mol Pharmacol ; 69(1): 216-25, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16189296

ABSTRACT

Determinants of differentiation and apoptosis induction by the novel histone deacetylase inhibitor (HDACI) LAQ824 were examined in human leukemia cells (U937 and Jurkat). Exposure of U937 cells to a low concentration of LAQ824 (30 nM) resulted in a delayed (2 h) increase in reactive oxygen species (ROS), induction of p21(WAF1/CIP1), pRb dephosphorylation, growth arrest of cells in G(0)/G(1) phase, and differentiation. On the other hand, exposure of cells to a higher concentration of LAQ824 (75 nM) resulted in the early (30 min) generation of ROS, arrest of cells in G(2)/M phase, down-regulation of XIAP (at the transcriptional level) and Mcl-1 (through a caspase-mediated process), the acid sphingomyelinase-dependent generation of ceramide, and profound mitochondrial injury, caspase activation, and apoptosis. LAQ824-induced lethality in U937 cells did not involve the extrinsic apoptotic pathway, nor was it associated with death receptor up-regulation; instead, it was markedly inhibited by ectopic expression of Bcl-2, Bcl-x(L), XIAP, and Mcl-1. The free radical scavenger N-acetyl cysteine blocked LAQ824-mediated ROS generation, mitochondrial injury, Mcl-1 down-regulation, ceramide generation, and apoptosis, suggesting a primary role for oxidative injury in LAQ824 lethality. Together, these findings indicate that LAQ824-induced lethality represents a multifactorial process in which LAQ824-mediated ROS generation is necessary but not sufficient to induce apoptosis, and that the degree of XIAP and Mcl-1 down-regulation and ceramide generation determines whether this agent engages a maturation rather than an apoptotic program.


Subject(s)
Apoptosis/drug effects , Ceramides/biosynthesis , Down-Regulation , Enzyme Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Hydroxamic Acids/pharmacology , Leukemia/pathology , Oxidative Stress , Sphingomyelin Phosphodiesterase/metabolism , X-Linked Inhibitor of Apoptosis Protein/physiology , Humans , Jurkat Cells , Reverse Transcriptase Polymerase Chain Reaction , U937 Cells
20.
Mol Cancer Ther ; 4(11): 1772-85, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16275999

ABSTRACT

Interactions between the novel histone deacetylase inhibitor LAQ824 and the cyclin-dependent kinase inhibitor roscovitine were examined in human leukemia cells. Pretreatment (24 hours) with a subtoxic concentration of LAQ824 (30 nmol/L) followed by a minimally toxic concentration of roscovitine (10 micromol/L; 24 hours) resulted in greater than additive effects on apoptosis in U937, Jurkat, and HL-60 human leukemia cells and blasts from three patients with acute myelogenous leukemia. These events were associated with enhanced conformational changes in Bax; mitochondrial release of cytochrome c, Smac/DIABLO, and apoptosis-inducing factor; and a marked increase in caspase activation. LAQ824/roscovitine-treated cells displayed caspase-dependent down-regulation of p21(CIP1) and Mcl-1 and a pronounced caspase-independent reduction in X-linked inhibitor of apoptosis (XIAP) expression. The lethality of this regimen was significantly attenuated by ectopic expression of XIAP, a nuclear localization signal-defective p21(CIP1) mutant, Mcl-1, and Bcl-2. Combined exposure to LAQ824 and roscovitine resulted in a significant reduction in XIAP mRNA levels and diminished phosphorylation of the carboxyl-terminal domain of RNA polymerase II. Notably, roscovitine blocked LAQ824-mediated differentiation. Finally, LAQ824 and roscovitine individually and in combination triggered an increase in generation of reactive oxygen species; moreover, coadministration of the free radical scavenger N-acetylcysteine prevented LAQ824/roscovitine-mediated mitochondrial injury and apoptosis. Collectively, these findings suggest that combined treatment of human leukemia cells with LAQ824 and roscovitine disrupts maturation and synergistically induces apoptosis, lending further support for an antileukemic strategy combining novel histone deacetylase and cyclin-dependent kinase inhibitors.


Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Hydroxamic Acids/pharmacology , Leukemia/drug therapy , Leukemia/enzymology , Purines/pharmacology , Apoptosis , Apoptosis Inducing Factor/metabolism , Blotting, Western , Cell Cycle , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cytochromes c/metabolism , Cytosol/metabolism , Down-Regulation , Flow Cytometry , HL-60 Cells , Humans , Jurkat Cells , Membrane Potentials , Mitochondria/pathology , Myeloid Cell Leukemia Sequence 1 Protein , Neoplasm Proteins/metabolism , Protein Conformation , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-bcl-2/metabolism , Reactive Oxygen Species , Reverse Transcriptase Polymerase Chain Reaction , Roscovitine , Time Factors , U937 Cells , X-Linked Inhibitor of Apoptosis Protein/metabolism
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