YC-1 induces apoptosis of human renal carcinoma A498 cells in vitro and in vivo through activation of the JNK pathway.
OBJECTIVE
The aim of this study was to elucidate the mechanism of YC-1{3-(5'-hydroxy methyl-2'-furyl)-1-benzylindazole}-induced human renal carcinoma cells apoptosis and to evaluate the potency of YC-1 in models of tumour growth in mice.


METHODS
YC-1-mediated apoptosis was assessed by analysis of MTT, SRB, DAPI staining and flow cytometry analysis. Knockdown of JNK protein was achieved by transient transfection using siRNA. The mechanisms of action of YC-1 on different signalling pathways involved were studied using western blot. Fas clustering was analysed by confocal microscopy and in vivo efficacy was examined in a A498 xenograft model.


RESULTS
YC-1 displayed cytotoxicity in renal carcinoma cells at 10(-7)-10(-8) M. Increased condensation of chromatin was observed and an increase in the cell population in subG1 phase. Moreover, YC-1 triggered mitochondria-mediated and caspase-dependent pathways. YC-1 significantly induced Fas ligand expression, but did not modify either the protein levels of death receptors or ligands. In addition, Fas clustering in cells responsive to YC-1 was observed, suggesting involvement of a Fas-mediated pathway. Furthermore, YC-1 markedly induced phosphorylation of JNK and a JNK inhibitor, SP600125, and siRNA JNK1/2 significantly reversed YC-1-induced cytotoxicity and protein expression. We suggest that YC-1 induced JNK phosphorylation, the upregulation of FasL and Fas receptor clustering to promote the activation of caspases 8 and 3, resulting in apoptosis. Finally, we demonstrated the antitumour effect of YC-1 in vivo.


CONCLUSIONS
These data suggest that YC-1 is a good candidate for development as an anticancer drug.
Wu
S Y
SY
Department of Pharmacology, College of Medicine, Pharmacological Institute, National Taiwan University, Taipei, Taiwan.
Pan
S L
SL
Chen
T H
TH
Liao
C H
CH
Huang
D Y
DY
Guh
J H
JH
Chang
Y L
YL
Kuo
S C
SC
Lee
F Y
FY
Teng
C M
CM
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002292:Carcinoma, Renal Cell / Q000188:drug therapy* / Q000473:pathology; D053148:Caspase 3 / Q000187:drug effects / Q000378:metabolism; D053181:Caspase 8 / Q000187:drug effects / Q000378:metabolism; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D020536:Enzyme Activators / Q000008:administration & dosage / Q000494:pharmacology*; D053222:Fas Ligand Protein / Q000187:drug effects / Q000378:metabolism; D016193:G1 Phase / Q000187:drug effects; D006801:Humans; D007191:Indazoles / Q000008:administration & dosage / Q000494:pharmacology*; D048031:JNK Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D010766:Phosphorylation / Q000187:drug effects; D019014:fas Receptor / Q000187:drug effects / Q000378:metabolism
D002843:Chromatin; D020536:Enzyme Activators; D053222:Fas Ligand Protein; D007191:Indazoles; D019014:fas Receptor; C090937:3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole; D048031:JNK Mitogen-Activated Protein Kinases; D053148:Caspase 3; D053181:Caspase 8