Mechanism of action of compounds that potentiate TRAIL-induced apoptosis in PPC-1 cells
This dose response assay was developed and performed to explore the mechanism of action for 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. ..more
BioActive Compounds: 2
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
This dose response assay was developed and performed to explore the mechanism of action for 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.
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.
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) PPC-1 cells and TRAIL were provided by the assay provider
2) ATPLite (Perkin Elmer)
Cytotoxicity assay protocol:
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 384 well plate (Greiner # 781098 white, clear-bottom, 384, TC-treated plate) at a concentration of 500 cells/well in 45ul and incubated for 24 hours at 37 oC in a CO2 incubator to obtain 10-20% of confluence.
1) Add 5 ul of 20X compound in 10% DMSO to wells to obtain 10 uM (final assay concentration of DMSO is 0.5% in 100ul final assay volume per well).
*Drugs are tested in 10-point dilution series with dilution factor 2 starting from 10uM final assay concentration.
2) Add 45uL of Assay Media to each well
3) Incubate cells 4 hours at 37 oC in a CO2 incubator.
4) For 0.156 ng/ml TRAIL: Add 5 ul of 20X TRAIL in PBS without Mg2+ and Ca2+
For 0.000 ng/ml TRAIL: Add 5 ul of PBS without Mg2+ and Ca2+
*0.156 ng/mL TRAIL plus 10uM Doxirubicin is used as the 100% cytotoxicity positive control.
5) Incubate cells for an additional 24 hours at 37 oC in a CO2 incubator.
1) Add 10 ul of ATPlite reagent to each well and shake plate for 20 min at room temperature.
2) Read luminescence using Envision 384 US Lum (integration time= 0.1s, 0mm from top of plate.)
Compounds having a Maximal Effect with 0.156 ng/ml TRAIL that is greater than that without TRAIL are considered active.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the Bir1/2 assay is described below.
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 this assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data-and is not applicable in this assay.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
a. Inactive compounds of the confirmatory stage are assigned a score value equal 81.
b. For active compounds:
Score = 82 + 18*(maximal effect)/100
Active compounds will have a score >= 82.
Categorized Comment - additional comments and annotations
Data Table (Concise)