A hydrogen peroxide-generating agent, 6-formylpterin, enhances heat-induced apoptosis.
The enhancement of heat-induced apoptosis by 6-formylpterin, an intra-cellular generator of hydrogen peroxide (H2O2), was examined in human myelomonocytic lymphoma U937 cells. The cells were treated with either 6-formylpterin alone at a nontoxic concentration of 300 microM (37 degrees C), heat shock (44 degrees C per 20 min) alone or a combination of the two, then incubated at 37 degrees C for 6 h. Assessments of apoptosis, mitochondrial membrane potential and caspase-3 activation were performed by flow cytometry. Moreover, caspase-8 activation and changes in the intra-cellular Ca2+ concentration ([Ca2+]i) were examined. Bax, Bcl-2, Bcl-XL, Bid, cytochrome c and PKCd were detected by Western blotting. The induction of heat-induced apoptosis evaluated by morphological observation and DNA fragmentation were promoted by the addition of 6-formylpterin. Mitochondrial membrane potential was decreased and the activation of caspase-3 and -8 was enhanced in the cells treated with the combination. A decreased-expression of Bid was noted, although no significant changes in Bax, Bcl-2 and Bcl-XL expression were observed after the combined treatment. Furthermore, both the release of cytochrome c from mitochondria to cytosol and the translocation of PKCd from cytosol to mitochondria, which were induced by heat shock, were enhanced by the addition of 6-formylpterin. The number of cells with a higher [Ca2+]i was also increased by the addition of 6-formylpterin. These findings suggest that the increase in [Ca2+]i, the activation of the mitochondria-caspase dependent pathway and the translocation of PKCd to mitochondria play principal roles in the enhancement of heat-induced apoptosis by 6-FP.
Wada
S
S
Department of Oral and Maxillofacial Surgery, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan.
Cui
Z-G
ZG
Kondo
T
T
Zhao
Q-L
QL
Ogawa
R
R
Shoji
M
M
Arai
T
T
Makino
K
K
Furuta
I
I
D017209:Apoptosis; D002118:Calcium / Q000378:metabolism; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation; D005434:Flow Cytometry; D006358:Hot Temperature; D006801:Humans; D006861:Hydrogen Peroxide; D006979:Hyperthermia, Induced; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000502:physiology; D011493:Protein Kinase C / Q000096:biosynthesis; D051745:Protein Kinase C-delta; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011622:Pterins / Q000494:pharmacology*
D019253:Proto-Oncogene Proteins c-bcl-2; D011622:Pterins; C005926:2-amino-4-hydroxy-6-formylpteridine; D045304:Cytochromes c; D006861:Hydrogen Peroxide; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; C505407:CASP3 protein, human; C505530:CASP8 protein, human; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases; D002118:Calcium