Late-stage microscopic assay to identify inhibitors of NADPH oxidase 1 (NOX1): Inhibition of extracellular matrix degradation
Name: Late-stage microscopic assay to identify inhibitors of NADPH oxidase 1 (NOX1): Inhibition of extracellular matrix degradation. ..more
BioActive Compound: 1
Data Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Gary Bokoch, TSRI
Network: Molecular Libraries Probe Production Center Network (MLPCN)
Grant Proposal Number: 1 R03 MH083264-01A1
Grant Proposal PI: Gary Bokoch, TSRI
External Assay ID: NOX1_INH_FLUOR_MIC_2
Name: Late-stage microscopic assay to identify inhibitors of NADPH oxidase 1 (NOX1): Inhibition of extracellular matrix degradation.
Host defense mechanisms are diverse and include receptor-initiated signaling pathways, antibody and cytokine production, and the generation of reactive oxygen species (ROS) such as hydroxyl radical and hypochlorous acid to kill microorganisms (1). In activated phagocytic cells, the membrane integrated protein gp91phox serves as the catalytic cytochrome b subunit of the respiratory burst oxidase used to generate superoxide in an NADPH-dependent manner for host defense (2). Generation of ROS has also been identified in non-phagocytic cells (3). One important enzyme involved in ROS production in non-leukocyte tissues is NADPH oxidase 1 (NOX1), a homolog of gp91phox. NOX1 is highly expressed in colon epithelial cells where it can generate ROS to interact with normal and pathogenic bacteria (3-5). However, excess ROS production is associated with damage to the intestinal mucosa, particularly in mucosal lesions of inflammatory bowel disease (IBD) (4). Studies showing that NOX1 levels are increased in human prostate cancer (6) and that cells overexpressing NOX1 have a transformed appearance, exhibit anchorage-independent growth, and induce vascularized tumor formation in athymic mice (3, 7), suggest that NOX1 may also play a role in angiogenesis, cell growth, and tumor pathogenesis (8, 9). The identification of inhibitors of NOX1 may lead to potential candidates for excess cell proliferation, cancer, and IBD.
1. Takeya, R. and Sumimoto, H., Molecular mechanism for activation of superoxide-producing NADPH oxidases. Mol Cells, 2003. 16(3): p. 271-7.
2. Cheng, G., Cao, Z., Xu, X., van Meir, E.G., and Lambeth, J.D., Homologs of gp91phox: cloning and tissue expression of Nox3, Nox4, and Nox5. Gene, 2001. 269(1-2): p. 131-40.
3. Suh, Y.A., Arnold, R.S., Lassegue, B., Shi, J., Xu, X., Sorescu, D., Chung, A.B., Griendling, K.K., and Lambeth, J.D., Cell transformation by the superoxide-generating oxidase Mox1. Nature, 1999. 401(6748): p. 79-82.
4. Szanto, I., Rubbia-Brandt, L., Kiss, P., Steger, K., Banfi, B., Kovari, E., Herrmann, F., Hadengue, A., and Krause, K.H., Expression of NOX1, a superoxide-generating NADPH oxidase, in colon cancer and inflammatory bowel disease. J Pathol, 2005. 207(2): p. 164-76.
5. Rokutan, K., Kawahara, T., Kuwano, Y., Tominaga, K., Nishida, K., and Teshima-Kondo, S., Nox enzymes and oxidative stress in the immunopathology of the gastrointestinal tract. Semin Immunopathol, 2008. 30(3): p. 315-27.
6. Lim, S.D., Sun, C., Lambeth, J.D., Marshall, F., Amin, M., Chung, L., Petros, J.A., and Arnold, R.S., Increased Nox1 and hydrogen peroxide in prostate cancer. Prostate, 2005. 62(2): p. 200-7.
7. Arnold, R.S., Shi, J., Murad, E., Whalen, A.M., Sun, C.Q., Polavarapu, R., Parthasarathy, S., Petros, J.A., and Lambeth, J.D., Hydrogen peroxide mediates the cell growth and transformation caused by the mitogenic oxidase Nox1. Proc Natl Acad Sci U S A, 2001. 98(10): p. 5550-5.
8. Ushio-Fukai, M. and Nakamura, Y., Reactive oxygen species and angiogenesis: NADPH oxidase as target for cancer therapy. Cancer Lett, 2008. 266(1): p. 37-52.
9. Kobayashi, S., Nojima, Y., Shibuya, M., and Maru, Y., Nox1 regulates apoptosis and potentially stimulates branching morphogenesis in sinusoidal endothelial cells. Exp Cell Res, 2004. 300(2): p. 455-62.
10. Weaver, A.M., Invadopodia: specialized cell structures for cancer invasion. Clin Exp Metastasis. 2006. 23(2): p. 97-105.
11. Baldassarre, M., Pompeo, A., Beznoussenko, G., Castaldi, C., Cortellino, S., McNiven, M.A., Luini, A., and Buccione, R., Dynamin participates in focal extracellular matrix degradation by invasive cells. Mol Biol Cell. 2003. 14(3): p. 1074-1084.
NOX1, NADPH oxidase 1, cancer, inflammation, inhibitor, inhibition, late stage, dose response, invadopodia, DLD1 cells, actin, phalloidin, ECM, extracellular matrix, epifluorescence microscopy, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Center Network, MLPCN.
The purpose of this assay is to determine whether a NOX1 inhibitor compound that was shown to block invadopodia formation in DLD1 human colon cancer cells in a previous assay, "Late-stage microscopic assay to identify inhibitors of NADPH oxidase 1 (NOX1): Inhibition of invadopodia formation" (AID 434993), can block the ability of DLD1 cells to degrade extracellular matrix (ECM). The formation of functional invadopodia relates to the ability of cells to degrade the ECM (10, 11). This assay also tests whether NOX1 overexpression restores the capacity of the DLD1 cells to degrade ECM in the presence of NOX1 inhibitor compound. In this assay, cells are transfected with empty vector, tyrosine kinase c-Src (SrcYF), which is required for the formation of functional invadopodia, or SrcYF plus a NOX1 expression plasmid. After transfection, cells are plated on fluorescently labeled gelatin-coated coverslips. Cells are treated with test compound or control, then stained with invadopodia marker phalloidin (that stains actin), and then prepared for epifluorescence microscopy. As designed, a compound that inhibits NOX1 will inhibit the NOX1-mediated formation of functional invadopodia and reduce the ability of c-Src-transfected DLD1 cells to degrade the ECM.
Human DLD1 cells were maintained in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum, 2 mM glutamine, and antibiotics (100 units/ml penicillin and 100 g/ml streptomycin) at 37 C in 5% CO2. DLD1 cells were transfected with empty vector, vector with constitutive active Src (SrcYF), or SrcYF plus a NOX1 expression plasmid, in the presence of Lipofectamine 2000. 24 hrs after transfection, cells were trypsinized and plated on FITC-labeled gelatin-coated coverslips After 2 hours, cells were treated with compound SID 57287864, DPI (positive control), or DMSO (negative control) for 1.5 hours. 24 hours later, cells were fixed in 4% paraformaldehyde (PFA) at room temperature for 10 minutes. Successively, cells were permeabilized in 0.5% Triton for 10 minutes and blocked in 2% BSA in PBS for 45 minutes at room temperature. Cells were then immunolabeled with Alexa-Fluor 568-conjugated phalloidin and visualized by epifluorescence microscopy (60X). Cells were mounted on slides with Mowiol mounting medium (Calbiochem) according to the manufacturer's instructions. Epifluorescence images of fixed cells were acquired on an inverted microscope (Eclipse TE 2000-U, Nikon) equipped with an electronically controlled shutter, filter wheels, and a 14-bit cooled CCD camera (Cool SNAP HQ, Photometrics) controlled by MetaMorph software (Universal Imaging Corp.) by using a 60x/1.4 NA Plan Apo DIC or a 40x/1.4 NA Plan Apo Ph3 objective lens (Nikon).
Quantification from three independent biological experiments is reported for each condition. For each experiment, the total degradation area was obtained as sum of degradation areas calculated using Metamorph software from 25 random fields and reported as percentage (SrcYF + DMSO set as 100%).
PubChem Activity Outcome and Score:
A compound that exhibited a SEM of p < 0.05 compared to the DMSO control was declared active.
The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.
List of Reagents:
DLD1 cells (ATCC, CCL-221)
SrcYF (provided by Assay Provider)
NOX1 expression vector (provided by Assay Provider)
DMSO (Sigma 472301)
FITC (Molecular Probes F-1906)
Paraformaldehyde (Sigma P-6148)
Triton (Sigma T8787)
BSA (Sigma A9418)
Alexa-Fluor-568 phalloidin antibody (Molecular Probes, A12380)
Mowiol mounting medium (Calbiochem, 475904)
DMEM medium (GIBCO, part 25200)
Hank's Balanced Salt Solution (Invitrogen, part 14025-092)
100X Penicillin-Streptomycin mix (Invitrogen, part 15140)
Trypsin-EDTA solution (Invitrogen, part 25200-056)
Fetal Bovine Serum (Invitrogen, part 16140-071)
L-Glutamine (Invitrogen, 25030-081)
Lipofectamine (Invitrogen, part 52887)
DPI (Sigma, D2926)
150 mm tissue culture dishes (Corning, part 430599)
384-well plates (Corning, 3704)
This assay was performed in the laboratory of the Assay Provider with compounds ordered as powders. Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDs listed below.
Data Table (Concise)