A high-throughput image-based screen for the identification of Bax/Bak-independent caspase activators against drug-resistant cancer cells.
Despite the use of new generation target specific drugs or combination treatments, drug-resistance caused by defective apoptosis signaling remains a major challenge in cancer treatment. A common apoptotic defect in drug-resistant tumor is the failure of cancer cells to undergo Bax/Bak-dependent mitochondrial permeabilization due to impaired signaling of Bcl-2 family proteins. Therefore, Bax and Bak-independent caspase-activating compounds appear to be effective in killing such tumor cells. An image-based cellular platform of caspase sensors in Bax and Bak deficient background allowed us to identify several potential Bax/Bak-independent caspase-activating compounds from a limited high-throughput compound screening. FRET-based caspase sensor probe targeted at the nucleus enabled accurate and automated segmentation, yielding a Z-value of 0.72. Some of the positive hits showed promising activity against drug-resistant human cancer cells expressing high levels of Bcl-2 or Bcl-xL. Using this approach, we describe thiolutin, CD437 and TPEN as the most potentially valuable drug candidates for addressing drug-resistance caused by aberrant expression of Bcl-2 family proteins in tumor cells. The screen also enables the quantification of multiparameter apoptotic events along with caspase activation in HTS manner in live mode, allowing characterization of non-classical apoptosis signaling.
Cancer Research Program-1, Rajiv Gandhi Centre for Biotechnology, Poojappura, Thycaud P.O, Thiruvananthapuram, 695 014, Kerala, India.
Mahendra
M
Seervi
Praveen K
PK
Sobhan
Krupa Ann
KA
Mathew
Jeena
J
Joseph
Prakash Rajappan
PR
Pillai
T R
TR
Santhoshkumar
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D020169:Caspases / Q000378:metabolism*; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D004789:Enzyme Activation / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000235:genetics / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism*
D000970:Antineoplastic Agents; D019253:Proto-Oncogene Proteins c-bcl-2; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D020169:Caspases