Dual Mechanisms of sHA 14-1 in Inducing Cell Death through Endoplasmic Reticulum and Mitochondria

  1. David Hermanson,
  2. Sadiya N. Addo,
  3. Anna A. Bajer,
  4. Jonathan S. Marchant,
  5. Sonia Goutam Kumar Das,
  6. Balasubramanian Srinivasan,
  7. Fawaz Al-Mousa,
  8. Francesco Michelangeli,
  9. David D. Thomas,
  10. Tucker W. LeBien and
  11. Chengguo Xing
  1. Departments of Medicinal Chemistry (D.H., S.N.A., S.G.K.D., B.S., C.X.), Laboratory Medicine and Pathology (A.A.B., T.W.L.), Pharmacology (J.S.M.), Biochemistry, Molecular Biology, and Biophysics (D.D.T.), and the Masonic Cancer Center (T.W.L., C.X.), University of Minnesota, Minneapolis, Minnesota; and School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom (F.A., F.M.)
  1. Address correspondence to:
    Dr. Chengguo Xing, 308 Harvard St. SE, Minneapolis, MN 55455. E-mail: xingx009{at}umn.edu

Abstract

HA 14-1 is a small-molecule Bcl-2 antagonist that promotes apoptosis in malignant cells, but its mechanism of action is not well defined. We recently reported that HA 14-1 has a half-life of only 15 min in vitro, which led us to develop a stable analog of HA 14-1 (sHA 14-1). The current study characterizes its mode of action. Because of the antiapoptotic function of Bcl-2 family proteins on the endoplasmic reticulum (ER) and mitochondria, the effect of sHA 14-1 on both organelles was evaluated. sHA 14-1 induced ER calcium release in human leukemic cells within 1 min, followed by induction of the ER stress-inducible transcription factor ATF4. Similar kinetics and stronger intensity of ER calcium release were induced by the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin, accompanied by similar kinetics and intensity of ATF4 induction. sHA 14-1 directly inhibited SERCA enzymatic activity but had no effect on the inositol triphosphate receptor. Evaluation of the mitochondrial pathway showed that sHA 14-1 triggered a loss of mitochondrial transmembrane potential (Δψm) and weak caspase-9 activation, whereas thapsigargin had no effect. (R)-4-(3-Dimethylamino-1-phenylsulfanylmethyl-propylamino)-N-{4-[4-(4′-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl]-benzoyl}-3-nitrobenzenesulfonamide (ABT-737), a well established small-molecule Bcl-2 antagonist, rapidly induced loss of Δψm and caspase-9 activation but had no effect on the ER. The pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone had some protective effect on sHA 14-1-induced cell death. These collective results suggest a unique dual targeting mechanism of sHA 14-1 on the apoptotic resistance machinery of tumor cells that includes antiapoptotic Bcl-2 family proteins and SERCA proteins.

Footnotes

  • This work was supported by National Institutes of Health National Cancer Institute [Grant R01-CA114294]; the American Association of College of Pharmacy; and the Leukemia Research Fund, Masonic Cancer Center, University of Minnesota.

  • D.H. and S.N.A. contributed equally to this work and are cofirst authors. T.W.L. and C.X. contributed equally to this work and are cosenior authors.

  • ABBREVIATIONS: ER, endoplasmic reticulum; IP3R, inositol phosphate 3 receptor; SERCA, sarcoendoplasmic reticulum Ca2+-ATPase; HA 14-1, ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate; sHA 14-1, ethyl-2-amino-6-phenyl-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate; isHA 14-1, ethyl-2-amino-6-phenyl-4-(2-morpholino-2-oxoethyl)-4H-chromene-3-carboxylate; ABT-737, (R)-4-(3-dimethylamino-1-phenylsulfanylmethyl-propylamino)-N-{4-[4-(4′-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl]-benzoyl}-3-nitrobenzenesulfonamide; AM, acetoxymethyl ester; ATF4, activating transcription factor 4; FCCP, carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone; PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid); DMSO, dimethyl sulfoxide; SR, sarcoplasmic reticulum; PI, propidium iodide; Δψm, mitochondrial transmembrane potential; TMRE, tetramethylrhodamine; Z-VAD-fmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; TG, thapsigargin; ROS, reactive oxygen species.

    • Accepted June 26, 2009.
    • Received February 24, 2009.
« Previous | Next Article »Table of Contents

This Article

  1. Published online before print June 26, 2009, doi: 10.1124/mol.109.055830 Molecular Pharmacology September 2009 vol. 76 no. 3 667-678
  1. » Abstract
  2. Full Text
  3. Full Text (PDF)
  4. All Versions of this Article:
    1. mol.109.055830v1
    2. 76/3/667 most recent

Classifications

Responses

  1. Submit a response
  2. No responses published

Current Issue

  1. November 2009, 76 (5)
  1. Current Issue
  1. Alert me to new issues of Molecular Pharmacology