cytochrome-c-t and p-hydroxycinnamaldehyde

Rhizomes of Alpinia galanga (Linn.) or 'Kha' in Thai are used in food and as folk medicine in South and Southeast Asia. The aims of this study were to identify the mechanism of cell death of human leukemic HL-60 and U937 cells induced by 4'-hydroxycinnamaldehyde (4'-HCA) isolated from A. galanga. 4'-HCA was cytotoxic to both cell lines in a dose-dependent manner (p<0.05) as demonstrated by MTT assay. Apoptosis induced by 4'-HCA was demonstrated by a variety of methods: visualization of propidium iodide (PI)-stained cells under fluorescence microscope, detection of subdiploid cells by PI-staining and flow cytometry, and assay of active caspase-3 using a specific fluorogenic substrate. 4'-HCA-treated cells (10 and 50 μg/ml for 4 h) showed significant increase in reactive oxygen species production and decreased mitochondrial transmembrane potential as detected by dichlorohydrofluorescein diacetate and 3,3'-dihexyloxacarbocyanine iodide respectively, together with flow cytometry. The apoptotic death involved cytochrome c release, increase in Bax level and concomitant decreases in levels of Bcl-2 and Bcl-xL (using Western blotting), and elevation in cytosolic and mitochondrial Ca²⁺ contents (using compartment-specific fluorescent Ca2+ dyes). These results indicate that 4'-HCA induces apoptosis of human leukemic cell through a combination of mitochondrial and ER stress pathways.

Topics: Alpinia; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Blotting, Western; Caspases; Cell Proliferation; Cinnamates; Cytochromes c; Endoplasmic Reticulum; Humans; Leukemia; Membrane Potential, Mitochondrial; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Tumor Cells, Cultured

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.

Topics: ADP-ribosyl Cyclase; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Line, Tumor; Cinnamates; Cytochromes c; Cytosol; Electrophoresis, Gel, Two-Dimensional; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Gene Expression Profiling; Heme Oxygenase-1; Humans; Membrane Potential, Mitochondrial; Membrane Proteins; Mitochondrial Swelling; Molecular Structure; Neoplasms; Oligonucleotide Array Sequence Analysis; Phosphatidate Phosphatase; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Transport; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transcription Factor CHOP