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Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model
Published on: April 16, 2014
Effect of a Smac Mimetic (TL32711, Birinapant) on the Apoptotic Program and Apoptosis Biomarkers Examined with
Apurva K Srivastava1, Soumya Jaganathan2, Laurie Stephen3
1Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland. srivastavaa4@mail.nih.gov.
Purpose:
To support clinical pharmacodynamic evaluation of the Smac mimetic TL32711 (birinapant) and other apoptosis-targeting drugs, we describe the development, validation, and application of novel immunoassays for 15 cytosolic and membrane-associated proteins indicative of the induction, onset, and commitment to apoptosis in human tumors.
Experimental Design:
The multiplex immunoassays were constructed on the Luminex platform with apoptosis biomarkers grouped into three panels. Panel 1 contains Bak, Bax, total caspase-3, total lamin-B (intact and 45 kDa fragment), and Smac; panel 2 contains Bad, Bax-Bcl-2 heterodimer, Bcl-xL, Bim, and Mcl1; and panel 3 contains active (cleaved) caspase-3, Bcl-xL-Bak heterodimer, Mcl1-Bak heterodimer, pS99-Bad, and survivin. Antibody specificity was confirmed by immunoprecipitation and Western blot analysis.
Results:
Two laboratories analytically validated the multiplex immunoassays for application with core-needle biopsy samples processed to control preanalytical variables; the biologic variability for each biomarker was estimated from xenograft measurements. Studies of TL32711 in xenograft models confirmed a dose-dependent increase in activated caspase-3 6 hours after dosing and provided assay fit-for-purpose confirmation. Coincident changes in cytosolic lamin-B and subsequent changes in Bcl-xL provided correlative evidence of caspase-3 activation. The validated assay is suitable for use with clinical specimens; 14 of 15 biomarkers were quantifiable in patient core-needle biopsies.
Conclusions:
The validated multiplex immunoassays developed for this study provided proof of mechanism data for TL32711 and are suitable for quantifying apoptotic biomarkers in clinical trials.
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