XAF1 mediates apoptosis through an extracellular signal-regulated kinase pathway in colon cancer.
BACKGROUND
XIAP-associated factor 1 (XAF1) negatively regulates the function of the X-linked inhibitor of apoptosis protein (XIAP), a member of the IAP family that exerts antiapoptotic effects. The extracellular signal-regulated kinase (ERK) pathway is thought to increase cell proliferation and to protect cells from apoptosis. The aim of the study was to investigate the correlation between the ERK1/2 signaling pathway and XAF1 in colon cancer.


METHODS
Four human colon cancer cell lines, HCT1116 and Lovo (wildtype p53), DLD1 and SW1116 (mutant p53), were used. Lovo stable transfectants with XAF1 sense and antisense were established. The effects of dominant-negative MEK1 (DN-MEK1) and MEK-specific inhibitor U0126 on the ERK signaling pathway and expression of XAF1 and XIAP proteins were determined. The transcription activity of core XAF1 promoter was assessed by dual luciferase reporter assay. Cell proliferation was measured by MTT assay. Apoptosis was determined by Hoechst 33258 staining.


RESULTS
U0126 increased the expression of XAF1 in a time- and dose-dependent manner. A similar result was obtained in cells transfected with DN-MEK1 treatment. Conversely, the expression of XIAP was down-regulated. Activity of the putative promoter of the XAF1 gene was significantly increased by U0126 treatment and DN-MEK1 transient transfection. rhEGF-stimulated phosphorylation of ERK appeared to have little or no effect on XAF1 expression. Overexpression of XAF1 was more sensitive to U0126-induced apoptosis, whereas down-regulation of XAF1 by antisense reversed U0126-induced inhibition of cell proliferation.


CONCLUSIONS
XAF1 expression was up-regulated by inhibition of the ERK1/2 pathway through transcriptional regulation, which required de novo protein synthesis. The results suggest that XAF1 mediates apoptosis induced by the ERK1/2 pathway in colon cancer.
Yu
Li Fen
LF
Department of Gastroenterology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Wang
Jide
J
Zou
Bing
B
Lin
Marie C M
MC
Wu
Yun Lin
YL
Xia
Harry H X
HH
Sun
Yun Wei
YW
Gu
Qing
Q
He
Hua
H
Lam
S K
SK
Kung
Hsiang Fu
HF
Wong
Benjamin C Y
BC
D048868:Adaptor Proteins, Signal Transducing; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D002070:Butadienes / Q000494:pharmacology; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1; D009363:Neoplasm Proteins / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D015854:Up-Regulation; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D002070:Butadienes; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; D009570:Nitriles; C113580:U 0126; D051636:X-Linked Inhibitor of Apoptosis Protein; C417551:XAF1 protein, human; D004815:Epidermal Growth Factor; D048049:Extracellular Signal-Regulated MAP Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048369:MAP Kinase Kinase 1