Apoptosis induction of 2'-hydroxycinnamaldehyde as a proteasome inhibitor is associated with ER stress and mitochondrial perturbation in cancer cells.
2'-Hydroxycinnamaldehyde (HCA), isolated from the stem bark of Cinnamomum cassia, and 2'-benzoyloxycinnamaldehyde (BCA), one of HCA derivatives, have antiproliferative activities on several human cancer cell lines. Our previous study suggested that reactive oxygen species (ROS) and caspase-3 are the major regulators of HCA-induced apoptosis. In the present study, we demonstrated a novel molecular target using in vitro pull-down assay by biotin-labeled HCA (biotin-HCA) in SW620 cells. We analyzed 11 differential spots of 2-dimensional gel prepared with pull-downed proteins by biotin-HCA. Among them, five spots were identified as proteasome subunits. An in vitro 26S proteasome function assay using specific fluorogenic substrates showed that HCA potently inhibits L3-like activity of the proteasome. In addition, HCA showed inhibitory action against chymotrypsin-like, trypsin-like, and PGPH-like activities. DNA microarray showed that HCA induced heat shock family and ER stress-responsive genes, which reflects the accumulation of misfolded proteins by proteasome inhibition. On western blot analysis, it was confirmed that HCA induces glucose-regulated protein, 78 kDa (GRP78) and some representative endoplasmic reticulum (ER) stress-responsive proteins. Furthermore, HCA treatment decreased mitochondrial membrane potential. The effect of HCA on cytochrome c and Bax translocation between cytosol and mitochondrial membrane was clarified using western blot analysis. These results suggest that HCA-induced apoptosis is associated with the inhibition of the proteasome activity that leads in turn to the increase of ER stress and mitochondrial perturbation.
Hong
Su Hyung
SH
Department of Dental Microbiology, School of Dentistry, Kyungpook National University, Daegu 700-412, Republic of Korea.
Kim
Jina
J
Kim
Jung-Min
JM
Lee
So-Young
SY
Shin
Dae-Seop
DS
Son
Kwang-Hee
KH
Han
Dong Cho
DC
Sung
Young Kwan
YK
Kwon
Byoung-Mog
BM
D036541:ADP-ribosyl Cyclase / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002934:Cinnamates / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology*; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D020869:Gene Expression Profiling; D051547:Heme Oxygenase-1 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008933:Mitochondrial Swelling / Q000187:drug effects*; D015394:Molecular Structure; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D010711:Phosphatidate Phosphatase / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D021381:Protein Transport / Q000187:drug effects; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D051742:Transcription Factor CHOP / Q000235:genetics / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D002934:Cinnamates; D000091342:Endoplasmic Reticulum Chaperone BiP; C485932:HERPUD1 protein, human; C000717247:HSPA5 protein, human; D008565:Membrane Proteins; D061988:Proteasome Inhibitors; D051028:bcl-2-Associated X Protein; C117045:p-hydroxycinnamaldehyde; D051742:Transcription Factor CHOP; D045304:Cytochromes c; C496541:HMOX1 protein, human; D051547:Heme Oxygenase-1; C112948:lipid phosphate phosphatase; D010711:Phosphatidate Phosphatase; D036541:ADP-ribosyl Cyclase; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease