Confirmation screening of compounds that potentiate TRAIL-induced apoptosis in primary human hepatocytes.
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 ..
BioActive Compounds: 3
Depositor Specified Assays
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 confirm the results of the primary screen for chemical compounds that selectively sensitize TRAIL-resistant 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.
In this assay we are testing normal cells because we would like to determine if the combined treatment of TRAIL with the identified compounds is safe to normal cells especially human primary hepatocytes. Hepatocytes are the most sensitive cells to the known compounds which potentiate TRAIL-mediated cytotoxicity.
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.
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.
1) Primary Human Hepatocytes and TRAIL were provided by the assay provider
2) ATPLite (Perkin Elmer)
Cytotoxicity assay protocol:
1) Hepatocytes are grown in DMEM, 10% FBS, 4.5 g/l glucose, 2mM L-glutamine, 1mM Na-pyruvate, 15ug/ml gentamycin (DMEM/10%FBS). Hepatocytes are harvested at 80-90% confluency.
2) Hepatocytes are then seeded into a 96 well plate (Corning #3595) at a concentration of 2-3 x 10^3 cells/well in 80ul and incubated for 24 hours at 37 oC in a CO2 incubator to obtain 30-40% of confluence.
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 hepatocytes 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 final concentration of TRAIL is 1000 ng/ml. 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.
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).
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
** Test Concentration.
Data Table (Concise)