Resveratrol induced serine phosphorylation of p53 causes apoptosis in a mutant p53 prostate cancer cell line.
OBJECTIVE
Resveratrol (Calbiochem, La Jolla, California) is a naturally occurring stilbene reported to cause apoptosis in various cultured cancer cells. In the current study the effect of resveratrol was determined in the androgen insensitive DU 145 prostate cancer cell line. Induction of apoptosis and activation of apoptosis related signal transduction pathways were measured.


METHODS
DU 145 cells were treated with resveratrol and apoptosis was measured by determining nucleosome content. Activation of mitogen activated protein kinase (MAPK) (extracellular signal-regulated kinase 1/2), p53 content and serine-15 phosphorylation of p53 were measured by immunoblot. Electrophoretic mobility shift assay of p53 binding to DNA, and measurement of p21 and glyceraldehyde-3-phosphate dehydrogenase messenger RNA were also done.


RESULTS
Resveratrol induced apoptosis in DU 145 cells. The stilbene activated MAPK and caused increased abundance of p53 and serine-15 phosphorylated p53. Resveratrol induced serine-15 phosphorylation of p53 was blocked by PD 98059 (Calbiochem), a MAPK kinase inhibitor, implicating MAPK activation in the phosphorylation of p53. PD 98059 also inhibited resveratrol induced apoptosis. These results suggest that apoptosis induction by resveratrol in DU 145 cells requires serine-15 phosphorylation of p53 by MAPK. Inhibition of MAPK dependent serine-15 phosphorylation resulted in reduced p53 binding to a p53 specific oligonucleotide on electrophoretic mobility shift assay. Pifithrin-alpha (Calbiochem), a p53 inhibitor, blocked resveratrol induced serine-15 phosphorylation of p53 and p53 binding to DNA. Resveratrol caused a p53 stimulated increase in p21 messenger RNA. Transfection of additional wild-type p53 into DU 145 cells induced apoptosis, which was further enhanced by resveratrol treatment.


CONCLUSIONS
Resveratrol causes apoptosis in DU 145 prostate cancer cells. This action depends on the activation of MAPK, increase in cellular p53 content, serine-15 phosphorylation of p53 and increased p53 binding to DNA.
Medical Research Service, Stratton Veterans Affairs Medical Center, Department of Medicine, Albany Medical College and Wadsworth Center, New York State Department of Health, Albany, New York, USA.
Hung-Yun
HY
Lin
Ai
A
Shih
Faith B
FB
Davis
Heng-Yuan
HY
Tang
Leon J
LJ
Martino
James A
JA
Bennett
Paul J
PJ
Davis
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D004252:DNA Mutational Analysis; D004789:Enzyme Activation / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008297:Male; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D000077185:Resveratrol; D012694:Serine / Q000378:metabolism*; D013267:Stilbenes / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D000972:Antineoplastic Agents, Phytogenic; D012333:RNA, Messenger; D013267:Stilbenes; D016159:Tumor Suppressor Protein p53; D010768:Phosphoserine; D012694:Serine; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D000077185:Resveratrol