AIF Apoptotic Pathway Mediates Cisplatin-induced Nephrotoxicity

IntroductionCisplatin (cis-diamminedichloroplatinum) is one of the most effective anticancer chemotherapeutic drugs. It has been used over 30 years in the clinic and has a broad spectrum of anticancer efficacy. Cisplatin has been widely used for the treatment of testicular, ovarian, bladder, head and neck, cervical, nonsmall and small cell lung carcinoma, etc. However, because of its accumulative toxicity to the kidney—there are approximate 20% cisplatin treated cases would complicate acute renal failure—its clinical application was limited. The mechanism of nephrotoxicity induced by cisplatin is complicated and varied. Cellular apoptosis is always an important one and researches on it mostly focused on caspases dependent pathways.Apoptosis-inducing factor (AIF) is a flavoprotein located on inner membrane of the cellular mitochondria. Reacting to some apoptotic stimuli, AIF is released from the inner membrane by a cleavage process and shifts to cytosol throuogh mitochondrial permeability transition pore (MPT), it then translocates to the nucleus. AIF could result in condensation of chromatin and break into large segments, which do not need capases cascade, so AIF is an important protein in caspase independent apoptotic pathway. In recent years, the phenomenon that CP induced apoptosis may mediated by AIF was paid more attention. However, the process and associated mechanisms are still obsure and largely in contradiction. Some research revealed that an important upstream regulatory factor of AIF is poly (ADP-ribose) polymerase (PARP). PARP is a nuclear protein which has dual function of facilitating cellular injury repair and promoting cellular apoptosis. Severe injury to cell would cause extensive activation of PARP and AIF nuclear translocation, which result in cellular apoptosis.3-aminobenzamide (3-AB), an inhibitor of PARP could eliminate the nuclear translocation of AIF. However, how this mechanism is functioned in CP induced nephrotoxicity, especially the research in vivo is rarely reported.In this study, experiments in vitro (treating HK-2 cell with CP) and in vivo (inducing nephrotoxicity by CP in rat) were performed. We investigated CP induced cellular necrosis and apoptosis and evaluated its effect on AIF mRNA, cytosol and nuclear AIF protein. Amifostine, Z-VAD-FMK, AIF siRNA and 3-AB were used as inhibitor to examine their effect on morphology change of nephrotoxicity and cellular apoptotic percentage. We also explore some regulatory factor of AIF dependent apoptotic pathway and their possible mechanisms to provide new academic and pratical base for further prevention and treatment of CP nephrotoxicity.PartⅠThe toxic effect of CP on HK-2 cellObjectives To investigate the toxicity of cisplatin (CP) on HK-2 cellMethods HK-2 cells were exposed to various concentrations (0-200μM) of CP for different times (0-24hr). Trypan blue staining was used to detect cell viability. Flow cytometer was applied to evaluate the necrosis and apoptosis percentage. Activated caspases expression after CP treatment with different times was measured by western blot. The protective effect of amifostine (AF) on CP treatment to HK-2 cells was assessed qualitatively and quantitatively by microscopic observation and DNA ladder.Results CP induced decreases of cell viability were significantly correlated with treating dose and time. Apoptotic percentage presented dose (0～100μM) and time (0～24hr) dependent manner, which was peaked at 100μM (CP treated for 24hr) and 24hr (CP treated with 50μM), and were 5.2 fold and 5.0 fold of control group. Exposure to CP treatment activated caspase-3,8,9. Caspase-8 and-9 was activated from 6hr and reached 8.1 and 3.0 folds, respectively, of 12hr group for 18hr. Caspase-3 was activated from 12hr and reached 10.5 and 93.7 folds of 12hr group for 18hr and 24hr respectively. AF was evidenced to be protective to necrosis and apoptosis of CP treated HK-2 cells.Conclusion The toxicity of CP to HK-2 cell presented to be dose and time-dependent. High dose CP mainly resulted in direct cellular necrosis and low dose mainly resulted in apoptosis. Caspase-8,-9 and-3 were activated in order with CP treatment. AF alleviated cellular injury and was protective against apoptosis.PartⅡAIF mediated HK-2 cell apoptosis induced by CPObjectives To investigate whether CP induced HK-2 cell death was mediated by AIF.Methods Cultured HK-2 cells were exposed to various concentration (0～200μM) for different times (0～12hr). Western blot and real time RT-PCR were applied to detect AIF protein and mRNA expression. Immunofluorescence was used to examine AIF distribution in HK-2 cells. The suppressive effect of pan-caspases inhibitor (Z-VAD-FMK) and AIF-siRNA on apoptosis of HK-2 cells was assessed qualitatively and quantitatively by TUNEL staining and flow cytometer.Results HK-2 cells exposed to various concentrations (0～200μM) of CP for different times (0-12hr) show increases of cytosolic AIF (cAIF), nuclear AIF (nAIF) and its mRNA level. As for cAIF, a 2.3-fold increase (P<0.05 vs control of non-treatment) was revealed at 25μM for 12hr and 1.7-fold (P<0.01 vs control) at 50μM for 3hr. Increase of nAIF expression showed a concentration and time-dependent manner, reached peaks at 150μM CP treatment for 12hr and 50μM CP treatment for 9hr, which were of 4.3 folds (P<0.005 vs 25μM group) and 3.7 folds (P<0.05 vs 3hr group). The expression of nAIF was consistent with cleaved-PARP expressive pattern. Real-time RT-PCR revealed that the AIF mRNA expression was increased when cells were treated with 50μM CP and its effect reached a peak at 9hr. Immunofluorescence demonstrated that AIF shift from cytosol to nuclei in some cultured HK-2 cells treated with CP. Adding Z-VAD-FMK and AIF-siRNA to CP treated HK-2 cells, the apoptotic percentages were reduced by 60.1% and 39.2% respectively. The inhibitory effect was even more significant with their combinative application.Conclusion CP up-regulated the expression level of AIF protein and transcription. The expression of nAIF and cleaved-PARP showed dose dependent manners and the expression of nAIF was also time dependent. CP induced AIF to shift from cytosolic mitochondria to nulei. Both AIF-siRNA and Z-VAD-FMK partially inhibited apoptosis of HK-2 and the effect was enhanced with combinative application.PartⅢThe protective effect of 3-aminobenzamide (3-AB) on nephrotoxicity induced by CPObjectives To investigate whether 3-aminobenzamide (3-AB) has protective effect on CP induced nephrotoxicity and the possible mechanism.Methods Cultured HK-2 cells were divided into four groups:control group, CP group, CP+3-AB group,3-AB group. Western Blot was used to detect PAR [poly (ADP-ribose)],μ-calpain and AIF protein expression. Hoechst 33258 staining and flow cytometer was performed to evaluate apoptosis. Western Blot was also used to detect phospho-Akt (p-Akt). SD rats were divided into four groups:control group, CP treated group,3-AB treated group and AF treated group. HE staining was applied to show the effect of 3-AB and AF. Immunohistochemistry was performed to detect AIF expression in renal tubules. Western Blot was used to detect PAR,μ-calpain and AIF protein expression in renal cortico-medullary junction.Results In vitro The expression of PAR,62KD AIF and 57KD AIF were all increased andμ-calpain was decreased (indicating activation) in CP group compared with control group. 3-AB reduced PAR and 57KD AIF expression and inhibitedμ-calpain activation.3-AB significantly reduced apoptosis induced by CP and show protective effect to HK-2 cells. 3-AB further enhanced p-Akt expression induced by CP. In Vivo Both 3-AB and AF alleviated CP induced renal injury.3-AB reduced AIF expression in proximal tubular cells. Compared with control group, the expression of PAR,62KD AIF and 57KD AIF were all increased andμ-calpain was decreased in CP treated group.3-AB treatment could reduce PAR and 57KD AIF expression and inhibiteμ-calpain activation.Conclusion PARP inhibitor 3-AB inhibited CP induced PAR formation, activation ofμ-calpain, and the expression of truncated AIF.3-AB had inhibitory effect on apoptosis of HK-2 cell induced by CP. Both 3-AB and AF alleviated nephrotoxicity induced by CP. 3-AB further activated PI3K/Akt signalling pathway induced by CP.Conclusions1. High dose CP mainly results in direct cellular necrosis and low dose mainly results in apoptosis. Both death receptor pathway and mitochondrial pathway are involved in HK-2 cell apoptosis induced by CP.2. AIF mediates HK-2 cell apoptosis induced by CP.3.3-aminobenzamide (3-AB) has protective effect on CP induced nephrotoxicity, which may be associated with its effect of reduction of PAR, inhibition ofμ-calpain, reduction of truncated AIF and activation of PI3K/Akt pathway.