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A high-throughput image-based screen for the identification of Bax/Bak-independent caspase activators against drug-resistant cancer cells

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Abstract

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.

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Abbreviations

HTS:

High-throughput screening

DKO:

Double knockout

FRET:

Fluorescence resonance energy transfer

ECFP:

Enhanced cyan fluorescent protein

cyt c :

Cytochrome c

ER:

Endoplasmic reticulum

MMP:

Mitochondrial membrane permeabilization

TMRM:

Tetramethyl rhodamine methyl ester

UPR:

Unfolded protein response

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Acknowledgments

Immoratalized MEF wild type and Bax/Bak DKO cells were kindly provided by Dr. Stanely J. Korsemeyer (Harvard Medical School, Boston, MA , USA). Caspase-3 specific FRET probe, pcDNA3 DEVD (ECFP-DEVD-EYFP) and nuclear targeted SCAT3-NLS (ECFP-DEVD-Venus) plasmids were kindly provided by Dr. Gavin Welsh (University of Bristol, UK) and Dr. Masayuki Miura (University of Tokyo, Japan) respectively. Expression vectors pCDNA3 Bcl-2-EGFP, Bcl-xL-EGFP and ER localizing pcDNA3 Bcl-2Cb5-EGFP were provided by Dr. Clark Distelhorst (Case Western Reserve University, USA). This study was supported by grant from the Innovative Young Biotechnologist Award, Department of Biotechnology, Government of India and research fellowships from University Grants Commission (UGC) to MS, Council of Scientific and Industrial Research (CSIR) to JJ and Indian Council of Medical Research (ICMR) to PKS and KAM. We sincerely thank Prof. M. Radhakrishna Pillai for helping throughout, Mrs. Indu Ramachandran and Ms. Aparna Asok for FACS analysis.

Conflict of interest

The authors disclose no potential conflicts of interest.

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Authors and Affiliations

  1. Cancer Research Program-1, Rajiv Gandhi Centre for Biotechnology, Poojappura, Thycaud P.O, Thiruvananthapuram, 695 014, Kerala, India

    Mahendra Seervi, Praveen K. Sobhan, Krupa Ann Mathew, Jeena Joseph, Prakash Rajappan Pillai & T. R. Santhoshkumar

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  1. Mahendra Seervi
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  2. Praveen K. Sobhan
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  3. Krupa Ann Mathew
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  4. Jeena Joseph
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  5. Prakash Rajappan Pillai
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  6. T. R. Santhoshkumar
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Corresponding author

Correspondence to T. R. Santhoshkumar.

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Seervi, M., Sobhan, P.K., Mathew, K.A. et al. A high-throughput image-based screen for the identification of Bax/Bak-independent caspase activators against drug-resistant cancer cells. Apoptosis 19, 269–284 (2014). https://doi.org/10.1007/s10495-013-0921-8

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