hRFI overexpressed in HCT116 cells modulates Bcl-2 family proteins when treated with 5-fluorouracil.
Exogenous overexpression of hRFI, originally isolated in our laboratory, inhibits not only death receptor-mediated apoptosis but also the mitochondrial apoptosis induced by several chemotherapeutic agents including 5-fluorouracil (5-FU). Recently, it has become clear that hRFI targets and degradates caspase-8 and -10 in death receptor-mediated apoptosis by E3 ubiquitin activity in a ring finger domain homologous to that of X-chromosome-linked inhibitor of apoptosis protein (XIAP). However, the cellular mechanism of the inhibition of mitochondrial apoptosis by hRFI has not been fully elucidated. We prepared HCT116 overexpressing hRFI (HCT116/hRFI) cells and comprehensively analyzed the expression changes of 51 apoptosis-related genes with or without 5-FU treatment between HCT116/hRFI and mock cells using microfluidic low-density arrays. As a result, we identified four genes (Bcl-2, Bcl-XL, cIAP2, and CFLAR) whose expression was four or more times higher in HCT116/ hRFI cells than in HCT116/LacZ cells, and found that Bcl-2 and the ratio of Bcl-2/Bax or Bcl-2/Bak were upregulated when HCT116/hRFI cells were treated with 5-FU. Furthermore, we also validated the up-regulation of Bcl-2 and Bcl-XL in HCT116/hRFI cells treated with 5-FU by Western blot analysis. Such evidence suggests that the modulation of Bcl-2 family proteins seen in 5-FU treatment plays an important role in the anti-apoptotic function of HCT116/hRFI cells.
Sasaki
Shin
S
Department of Surgical Oncology, The University of Tokyo, Hongo, Tokyo 113-8655, Japan. sasaki-1su@h.u-tokyo.ac.jp
Watanabe
Toshiaki
T
Kobunai
Takashi
T
Konishi
Tsuyoshi
T
Nagase
Hideki
H
Sugimoto
Yoshikazu
Y
Oka
Toshinori
T
Nagawa
Hirokazu
H
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002352:Carrier Proteins / Q000235:genetics*; D005472:Fluorouracil / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D045325:HCT116 Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D046228:Microarray Analysis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D014162:Transfection; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000235:genetics / Q000378:metabolism; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000235:genetics / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
D000964:Antimetabolites, Antineoplastic; D002352:Carrier Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; C461219:RNF34 protein, human; D051636:X-Linked Inhibitor of Apoptosis Protein; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D005472:Fluorouracil