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BioAssay: AID 1624

Confirmation screening of compounds that potentiate TRAIL-induced apoptosis of cancer cells.

Cytotoxic chemotherapy induces apoptosis via a pathway involving mitochondria, sometimes referred to as the "intrinsic pathway." An acquired resistance to anticancer drugs commonly results from the accumulation of defects in components of the mitochondrial pathway for apoptosis. Discovering and identifying alternative pathways for triggering tumor cells apoptosis offer hope for more effective more ..
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 Tested Compounds
 Tested Compounds
All(3)
 
 
Active(3)
 
 
 Tested Substances
 Tested Substances
All(3)
 
 
Active(3)
 
 
AID: 1624
Data Source: Burnham Center for Chemical Genomics (BCCG-A165-TRAIL-PPC-1-Assay)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2009-03-26
Modify Date: 2011-01-05

Data Table ( Complete ):           Active    All
Target
Sequence: tumor necrosis factor (ligand) superfamily, member 10 [Homo sapiens]
Description ..   
Protein Family: TNF

Gene:TNFSF10     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 3
Depositor Specified Assays
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AIDNameTypeProbeComment
1443uHTS for the identification of compounds that potentiate TRAIL-induced apoptosis of cancer cellsscreening
1447Identification of compounds which are cytotoxic to PPC-1 cells.screening
1773Comparison of the effects of compounds on TRAIL-mediated extrinsic apoptotic pathway in TRAIL-resistant MDA-MB-435 cells.other
1774Comparison of the effects of compounds on apoptotic pathways (intrinsic and ER stress pathway) in TRAIL-resistant MDA-MB-435 cells.other
2619SAR analysis of compounds that potentiate TRAIL-induced apoptosis in TRAIL-resistant cancer cells, MDA-MB-435.other
1745Confirmation screening of compounds that potentiate TRAIL-induced apoptosis in normal 184B5 cellsother
1755SAR analysis of compounds that potentiate TRAIL-induced apoptosis in MDA-MB-435 cells.confirmatory
1756SAR analysis of compounds cytotoxic to MDA-MB-435 cells.confirmatory
1787Isobologram analysis of chemical compounds and TRAIL to sensitize TRAIL-resistant tumor cells to TRAILother
1748Confirmation screening of compounds that potentiate TRAIL-induced apoptosis in TRAIL-resistant cancer cells, MCF7other
1640Summary of compounds that potentiate TRAIL-induced apoptosis of cancer cells.summary2
1746Confirmation screening of compounds that potentiate TRAIL-induced apoptosis in primary human hepatocytes.other
1747Confirmation screening of compounds that potentiate TRAIL-induced apoptosis in TRAIL-resistant cancer cells, MDA-MB-435other
2647SAR analysis of compounds that potentiate TRAIL-induced apoptosis in TRAIL-resistant cancer cells, MDA-MB-435/MDR.other
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Production Centers Network (MLPCN)
Grant Number: X01 MH083230-01
Assay Provider: Dr. Dmitri Rozanov, Sanford-Burnham Medical Research Institute, San Diego CA

Cytotoxic chemotherapy induces apoptosis via a pathway involving mitochondria, sometimes referred to as the "intrinsic pathway." An acquired resistance to anticancer drugs commonly results from the accumulation of defects in components of the mitochondrial pathway for apoptosis. Discovering and identifying alternative pathways for triggering tumor cells apoptosis offer hope for more effective outcomes. Members of the Tumor Necrosis Factor (TNF) family of "death receptors" induce apoptosis via a direct mechanism that proceeds without involving mitochondria - referred to as the "extrinsic pathway." These cytokine receptors are frequently employed by immune cells to attack tumors. The PI believes that a successful strategy can be implemented by identifying specific chemicals that will selectively potentiate the therapeutic effects of the Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL). Unlike other TNF-family members, TRAIL is a powerful and safe cancer therapeutic because it can induce broad spectrum apoptosis of different cancer cells but not normal cells. Unfortunately many cancer cells have proven to be resistant to TRAIL alone.

The goal of this project is to screen for chemical compounds that selectively sensitize tumor cells to the extrinsic apoptosis pathway activated by TRAIL, without affecting other cell death pathways and normal cells. These compounds would provide useful research tools for interrogating mechanisms of TRAIL-resistance, and they also might serve as the basis for future drug development programs to create a new generation of non-toxic anticancer drugs that restore sensitivity to endogenous pathways used by the immune system for eradicating tumors.

This assay was developed and performed to confirm hits originally identified in "uHTS for the identification of compounds that potentiate TRAIL-induced apoptosis of cancer cells" (AID 1443) and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally.

References:
Bodmer JL, Meier P, Tschopp J, Schneider P. Cysteine 230 is essential for the structure and activity of the cytotoxic ligand TRAIL. J Biol Chem 2000, 275:20632-7.

Lawrence D, Shahrokh Z, Marsters S, Achilles K, Shih D, Mounho B, Hillan K, Totpal K, DeForge L, Schow P, Hooley J, Sherwood S, Pai R, Leung S, Khan L, Gliniak B, Bussiere J, Smith CA, Strom SS, Kelley S, Fox JA, Thomas D, Ashkenazi A. Differential hepatocyte toxicity of recombinant Apo2L/TRAIL versions. Nat Med 2001, 7:383-5.

Singh TR, Shankar S, Chen X, Asim M, Srivastava RK. Synergistic interactions of chemotherapeutic drugs and tumor necrosis factor-related apoptosis-inducing ligand/Apo-2 ligand on apoptosis and on regression of breast carcinoma in vivo. Cancer Res 2003, 63:5390-400

Greil R, Anether G, Johrer K, Tinhofer I. Tracking death dealing by Fas and TRAIL in lymphatic neoplastic disorders: pathways, targets, and therapeutic tools. J Leukoc Biol 2003, 74:311-30.

Smyth MJ, Takeda K, Hayakawa Y, Peschon JJ, van den Brink MR, Yagita H. Nature's TRAIL-on a path to cancer immunotherapy. Immunity 2003, 18:1-6.
Protocol
Assay materials:

1) PPC-1 cells and TRAIL were provided by the assay provider
2) ATPLite (Perkin Elmer)

Cytotoxicity assay protocol:

Day 1

1) PPC-1 cells are grown in DMEM, 10% FBS, 4.5 g/l glucose, 2mM L-glutamine, 1mM Na-pyruvate, 15ug/ml gentamycin and are harvested at 80-90% confluency.
2) Cells are then seeded into a 96 well plate (Corning #3595) at a concentration of 2000-3000 cells/well in 80ul and incubated for 24 hours at 37 oC in a CO2 incubator to obtain 30-40% of confluence.

Day 2

1) Add 10 ul of compound or DMSO to wells to obtain 1.25-5 uM (final concentration which refers to 100 ul of solution per well).
2) Incubate cells 4 hours at 37 oC in a CO2 incubator.
3) Add 10 ul of TRAIL in PBS without Mg2+ and Ca2+ (total volume of solution per well is 100 ul) or 1% NaN3 (100% cytotoxicity). The measured value of 100% cytotoxicity is the background for measurement and is subtracted from all values.
4) Incubate cells for an additional 24 hours at 37 oC in a CO2 incubator.

Day 3

1) Add 12 ul of ATPlite reagent to each well and shake plate for 30 min at room temperature.
2) Read luminescence using a SpectraFluor Plus (Tecan) (integration time= maximum, gain=150).
Comment
Compounds in this assay were considered to be active if the cytotoxicity of TRAIL in the presence of the compound is greater than the sum of the cytotoxicity of the compound and the cytotoxicity of TRAIL separately at all three concentrations of compound(1.25, 2.5 and 5.0 uM).

Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the PMI assay is as follows:

1) First tier (0-40 range) is reserved for primary screening data and is not relevant in this assay.

2)Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable to this assay

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues

Compounds that are inactive in this assay are assigned a score of 81
Compounds that are active in this assay are assigned a score of 90
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Trail_StdThis qualifier is to be used with the next TID, IC50. If qualifier is "=", IC50 result equals to the value in that column;if qualifier is ">", IC50 result is greater than that value. if qualifier is "<", IC50 result is smaller than that value.Float%
21.25_uM_cmpd (1.25μM**)The cytotoxicity of 1.25 uM compoundFloat%
31.25_uM_plus_Trail (1.25μM**)The cytotoxicity of 1.25 uM compound plus 0.1 ng/ml TRAILFloat%
42.5_uM_cmpd (2.5μM**)The cytotoxicity of 2.5 uM compoundFloat%
52.5_uM_plus_Trail (2.5μM**)The cytotoxicity of 2.5 uM compound plus 0.1 ng/ml TRAILFloat%
65.0_uM_cmpd (5μM**)The cytotoxicity of 5.0 uM compoundFloat%
75.0_uM_plus_Trail (5μM**)The cytotoxicity of 5.0 uM compound plus 0.1 ng/ml TRAILFloat%

** Test Concentration.
Additional Information
Grant Number: X01 MH083230-01

Data Table (Concise)
Classification
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