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
- Received February 24, 2009.
- Accepted June 26, 2009.
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.
- The American Society for Pharmacology and Experimental Therapeutics
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