Late stage assay provider counterscreen for activators of Aryl hydrocarbon receptor (AHR): Absorbance-based cell-based assay to identify compounds that modulate proliferation of ER-positive breast cancer cells (MCF7), Set 2
Name: Late stage assay provider counterscreen for activators of Aryl hydrocarbon receptor (AHR): Absorbance-based cell-based assay to identify compounds that modulate proliferation of ER-positive breast cancer cells (MCF7), Set 2. ..more
BioActive Compounds: 3
Depositor Specified Assays
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Michael Denison, University of California, Davis
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1-X01-DA026558-01
Grant Proposal PI: Michael Denison
External Assay ID: MCF7_GROWTH_INH_ABS_0096_1X%INH MCSRUN
Name: Late stage assay provider counterscreen for activators of Aryl hydrocarbon receptor (AHR): Absorbance-based cell-based assay to identify compounds that modulate proliferation of ER-positive breast cancer cells (MCF7), Set 2.
Transcription factors are critical regulators of gene expression (1). Under conditions such as environmental stress and exposure to endogenous toxins, transcription factors can rapidly modulate the transcription of genes whose products regulate cell proliferation and metabolism. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor of the basic helix-loop-helix protein superfamily involved in the biological response to aromatic hydrocarbons, and regulates the expression of xenobiotic-metabolizing enzymes such as cytochrome P450, aldehyde dehydrogenase, quinone reductase, and other phase I and phase II detoxification genes (2, 3). In response to various compounds, including the environmental pollutants dioxins, benzo(a)pyrene, dietary contaminants, grapefruit juice, endogenous toxins, and plant products such as carotinoids, nicotine and caffeine (2, 4-6), cytosolic AHR complexes with chaperones hsp90, p23, and XAP2, translocates to the nucleus where it dimerizes with the AHR nuclear translocator (ARNT) to influence target gene transcription (7, 8). Gain-of-function studies in mice reveal the oncogenic potential of AHR (9), while other reports show roles for AHR in diverse biologic events such as organ development (10, 11), immune function and allergy (12), and estrogen responsiveness (13). The identification of agonists of AHR will provide useful tools to elucidate the roles of this receptor in cell metabolism, transcriptional control, and tumor formation (14-16).
1. Ptashne, M., Regulation of transcription: from lambda to eukaryotes. Trends Biochem Sci, 2005. 30(6): p. 275-9.
2. McMillan, B.J. and Bradfield, C.A., The aryl hydrocarbon receptor sans xenobiotics: endogenous function in genetic model systems. Mol Pharmacol, 2007. 72(3): p. 487-98.
3. Puga, A., Tomlinson, C.R., and Xia, Y., Ah receptor signals cross-talk with multiple developmental pathways. Biochem Pharmacol, 2005. 69(2): p. 199-207.
4. Bock, K.W. and Kohle, C., Ah receptor: dioxin-mediated toxic responses as hints to deregulated physiologic functions. Biochem Pharmacol, 2006. 72(4): p. 393-404.
5. Denison, M.S. and Nagy, S.R., Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals. Annu Rev Pharmacol Toxicol, 2003. 43: p. 309-34.
6. de Waard, P.W., Peijnenburg, A.A., Baykus, H., Aarts, J.M., Hoogenboom, R.L., van Schooten, F.J., and de Kok, T.M., A human intervention study with foods containing natural Ah-receptor agonists does not significantly show AhR-mediated effects as measured in blood cells and urine. Chem Biol Interact, 2008.
7. Hankinson, O., The aryl hydrocarbon receptor complex. Annu Rev Pharmacol Toxicol, 1995. 35: p. 307-40.
8. Petrulis, J.R. and Perdew, G.H., The role of chaperone proteins in the aryl hydrocarbon receptor core complex. Chem Biol Interact, 2002. 141(1-2): p. 25-40.
9. Andersson, P., McGuire, J., Rubio, C., Gradin, K., Whitelaw, M.L., Pettersson, S., Hanberg, A., and Poellinger, L., A constitutively active dioxin/aryl hydrocarbon receptor induces stomach tumors. Proc Natl Acad Sci U S A, 2002. 99(15): p. 9990-5.
10. Ramos, K.S., Transcriptional profiling and functional genomics reveal a role for AHR transcription factor in nephrogenesis. Ann N Y Acad Sci, 2006. 1076: p. 728-35.
11. Walisser, J.A., Glover, E., Pande, K., Liss, A.L., and Bradfield, C.A., Aryl hydrocarbon receptor-dependent liver development and hepatotoxicity are mediated by different cell types. Proc Natl Acad Sci U S A, 2005. 102(49): p. 17858-63.
12. Lawrence, B.P., Denison, M.S., Novak, H., Vorderstrasse, B.A., Harrer, N., Neruda, W., Reichel, C., and Woisetschlager, M., Activation of the aryl hydrocarbon receptor is essential for mediating the anti-inflammatory effects of a novel low-molecular-weight compound. Blood, 2008. 112(4): p. 1158-65.
13. Ohtake, F., Takeyama, K., Matsumoto, T., Kitagawa, H., Yamamoto, Y., Nohara, K., Tohyama, C., Krust, A., Mimura, J., Chambon, P., Yanagisawa, J., Fujii-Kuriyama, Y., and Kato, S., Modulation of oestrogen receptor signalling by association with the activated dioxin receptor. Nature, 2003. 423(6939): p. 545-50.
14. Zhao, B., Baston, D.S., Hammock, B., and Denison, M.S., Interaction of diuron and related substituted phenylureas with the Ah receptor pathway. J Biochem Mol Toxicol, 2006. 20(3): p. 103-13.
15. Garrison, P.M., Tullis, K., Aarts, J.M., Brouwer, A., Giesy, J.P., and Denison, M.S., Species-specific recombinant cell lines as bioassay systems for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin-like chemicals. Fundam Appl Toxicol, 1996. 30(2): p. 194-203.
16. Han, D., Nagy, S.R., and Denison, M.S., Comparison of recombinant cell bioassays for the detection of Ah receptor agonists. Biofactors, 2004. 20(1): p. 11-22.
17. Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren, J.T., Bokesch, H., Kenney, S. and Boyd, M.R. (1990) New colorimetric cytotoxicity assay for anticancer-drug screening, J Natl Cancer Inst 82, 1107-1112.
18. Vichai, V. and Kirtikara, K. (2006) Sulforhodamine B colorimetric assay for cytotoxicity screening, Nat Protocol 1, 1112-1116.
19. Papazisis, K.T., Geromichalos, G.D., Dimitriadis, K.A. and Kortsaris, A.H. (1997) Optimization of the sulforhodamine B colorimetric assay, J Immunol Meth 208, 151-158.
late stage, powders, purchased, synthesized, AHR, aryl hydrocarbon receptor, receptor, transcription factor, triplicate, dose response, counterscreen, assay provider, cell, liver, proliferation, viability, growth, MCF7, breast, sulforhodamine B, activator, agonist, activation, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to determine the ability of test compounds to inhibit estrogen-dependent and/or -independent cell proliferation of human breast (T47D), ovarian (BG1) and liver (HepG2) cells in an AHR-dependent manner in vitro. Estrogen-responsive cells (T47D and BG1) are preincubated in 96-well culture plates with phenol red-free media containing charcoal-stripped serum for 5 days (to reduce endogenous estrogen levels) before use. Cells in 96-well culture plates grown in the presence (T47D and BG1) and absence (HepG2) of estradiol 17b and in the absence or presence of test compound for 7 - 10 days (TCDD is used as a positive control). Media is removed, the cells fixed and total cell biomass stained with sulforhodamine B, washed and absorbance at 490 nm determined. This assay provides a measure of total cell numbers over a wide range of cell numbers (33). Since proliferation of cancer cell growth can be regulated by multiple mechanisms (both hormone-dependent and independent) the assays here are designed to identify those AHR agonists that will also inhibit estrogen-dependent and -independent proliferation of these cancer cell lines, with the extent of inhibition directly related to the level of AHR activation. Comparison of the inhibition of proliferation by test compounds of T47D and BG1 cells grown in the absence or presence of estrogen will allow specific determination of AHR-dependent inhibition of proliferation that is estrogen-/estrogen receptor-dependent. Determination of the inhibition of proliferation of estrogen receptor-deficient HepG2 cells and of T47D and BG1 cells grown in the absence of estrogen, will allow specific determination of AHR-dependent inhibition of proliferation that is estrogen-/estrogen receptor-independent. Thus, multiple mechanisms of inhibition of cell proliferation will be assessed. Details of this assay protocol can be found in (Skehan et al., 1990; Vichai and Kirtikara, 2006; Papazisis et al., 1997) (17-19). Compounds were tested using a dose response series up to a maximum nominal concentration of 10 uM.
1. Cells were harvested at 90% confluent by addition of 2 ml trypsin, counted and plated in 24-wells plates at 2,000 cells/well in a-MEM media containing 10% fetal bovine serum. For these studies, ER-positive (human breast cancer (MCF7)) and ER-negative (human hepatoma (HepG2)) cells were examined.
2. The plates were incubated at 37 C for 24 hour.
3. After 24 hours, the medium was changed and cells were treated with DMSO (1 ul/ml), TCDD (10 nM) or 10 uM of the test compounds and incubated for 8-10 days at 37 C. For concentration response analysis a range of 10 nM to 10 uM of the test compounds were used. The effect of all chemicals (at all concentrations) were examined in at least triplicate incubations. During the incubation period, medium was changed and cells were re-dosed with chemical every 48 hours.
4. Following removal of media by aspiration, 250 ml of 10% cold (4 C) TCA were gently added to the wells.
5. Plates were incubated for 30 min at 4 C, then washed 5 times with deionized water and left to dry at room temperature for at least 1 hour.
6. Cells were stained by the addition and incubation (20 min) with 250 ml of SRB dissolved in 1% acetic acid.
7. Unbound dye was removed by washing with 1% acetic acid five times.
8. Bound dye was solubilized with 150 ml of unbuffered Tris base (10 mM, pH 10.5) for 10 min on a shaker.
9. Finally, an aliquot (130 ml) of the Tris buffer containing SRB dye was transferred to a 96-well plate. The total absorbance at 490 nm was determined, and specific absorbance determined after subtraction of background absorbance (i.e., absorbance at 650 nm). Results are presented as a percent of proliferation (absorbance) of cells exposed to DMSO, with negative values representing inhibition and positive values representing enhancement of proliferation.
IC50 values from sigmoidal concentration-response curves were determined using the four-parameter Hill equation (SigmaPlot (Systat)). The compounds exhibited an estimated IC50 of 3 uM, but the dose response curve was steep, so the value is approximate.
PubChem Activity Outcome and Score:
Compounds that inhibit proliferation by less than 20% were considered inactive. Compounds that inhibit proliferation equal to or greater than 20% were considered active.
The reported PubChem Activity Score has been normalized to 100% observed max % inhibition. Negative max % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-96. There are no inactive compounds.
This assay was run by the assay provider. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the plate, compounds that modulate cell growth and/or well absorbance. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)