Dual mechanisms of sHA 14-1 in inducing cell death through endoplasmic reticulum and mitochondria.
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 Ca(2+)-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 (Delta psi m) 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 Delta psi m 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.
Hermanson
David
D
Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota, USA.
Addo
Sadiya N
SN
Bajer
Anna A
AA
Marchant
Jonathan S
JS
Das
Sonia Goutam Kumar
SG
Srinivasan
Balasubramanian
B
Al-Mousa
Fawaz
F
Michelangeli
Francesco
F
Thomas
David D
DD
Lebien
Tucker W
TW
Xing
Chengguo
C
D017209:Apoptosis; D001578:Benzopyrans / Q000737:chemistry / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism
D001578:Benzopyrans; D009570:Nitriles; D019253:Proto-Oncogene Proteins c-bcl-2; C543325:ethyl 2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate; C456800:ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases