[Apoptosis inducing factor mediates cisplatin-induced apoptosis of renal tubular epithelial cells].
OBJECTIVE
To investigate the involvement of apoptosis inducing factor (AIF) in caspase-independent pathway mediating apoptosis of cultured renal tubular epithelial cells induced by cisplatin (CP).


METHODS
Western Blot analysis and real-time PCR were performed to detect cytosol AIF (cAIF), nuclear AIF (nAIF) and AIF mRNA expression in cultured renal epithelial cells (HK-2) treated with cisplatin (CP) at various concentrations (0 - 200 micromol/L) and time courses (0 - 12 h). Immunofluorescence analysis was used to detect the AIF protein distribution in HK-2 cells. Pan-caspase inhibitor (Z-VAD-FMK) and AIF-siRNA treatment, TUNEL and flow cytometer were used to measure the suppression of apoptosis induced by CP in HK-2 cells.


RESULTS
The expressions of cAIF, nAIF protein and AIF mRNA were all increased to some extent in HK-2 cells treated with CP at various concentrations and time points. cAIF expression was 2.3-fold (P < 0.05) increased after 25 micromol/L CP treatment for 12 h and 1.7-fold (P < 0.01) increased after 50 micromol/L CP treatment for 3 h, compared with that of control groups, and showed a concentration- and time-dependent increment. The nAIF expression reached a peak (4.3-fold increase) (P < 0.005) after 150 micromol/L CP treatment for 12 h and 3.7-fold incease (P < 0.05) after 50 micromol/L CP treatment for 9 h, compared with that of the 25 micromol/L group and 3 h group, respectively. The expression of nAIF was approximately consistent with cleaved-PARP expressive pattern. Real-time PCR showed that AIF mRNA increased gradually with prolonged treatment with 50 micromol/L CP and reached a peak at 9 h. Immunofluorescence assay showed AIF translocation from cytosol to nuclei in some cultured HK-2 cells treated with CP. Applying pan-caspase inhibitor (Z-VAD-FMK) and AIF-siRNA to CP-treated HK-2 cells, the apoptotic rates were decreased by 60.1% and 39.2%, respectively. The inhibitory effect on HK-2 cell apoptosis was even more significant with combination of both Z-VAD-FMK and AIF-siRNA.


CONCLUSIONS
The AIF activation and translocation to nuclei with the increment of its mRNA expression mediates CP-induced apoptosis of renal tubular epithelial cells in vitro. It may provide a new therapeutic target for protecting from nephrotoxciity of cisplatin.
Liu
Ye
Y
Department of Pathology, Shanghai Medical College, Fudan University, Shanghai 200032, China.
Guo
Ye
Y
Wu
Hui-juan
HJ
Zhang
Zhi-gang
ZG
Guo
Mu-yi
MY
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D051033:Apoptosis Inducing Factor / Q000235:genetics / Q000378:metabolism*; D061945:Caspase Inhibitors; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002945:Cisplatin / Q000008:administration & dosage / Q000494:pharmacology*; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D007684:Kidney Tubules / Q000166:cytology*; D021381:Protein Transport; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics
C494829:AIFM1 protein, human; D000590:Amino Acid Chloromethyl Ketones; D000970:Antineoplastic Agents; D051033:Apoptosis Inducing Factor; D061945:Caspase Inhibitors; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D002945:Cisplatin