Introduction: Autophagic cell death, otherwise known as one of non-apoptotic cell death, is characterized by the appearance of large autophagic vacuoles in the cytoplasm. We have shown that histone deacetylase (HDAC) inhibitor depsipeptide (FK) induce apoptosis on osteosarcoma cells in vitro and in vivo through Fas/Fas ligand system (Oncogene 22; 9231, 2003). Here we show that FK induced autophagic cell death on Malignant Rhabdoid Tumor (MRT) cells in vitro and in vivo through AIF translocation. Methods: We used three human MRT cell lines (KP-MRT-NS, STM91-01, and TTC549). The cytotoxic effects of FK were determined in WST-1 assays, sub-G1 populations and cell viability by PI staining with flow cytometric analysis. Analysis of AIF translocation was performed by immunofluorescence and immunoblotting. Autophagic cell death was detected by electron microscopy and by the conversion of LC3-I to LC3-II with Western blot. Antitumor activity in vivo was studied using human MRT xenografts in male nude mice. When implanted tumors became evident, we started to treat mice with FK by i.v. Immunoelectron microscopy was used to study the subcellular localisation of AIF in tumor tissue. AIF was detected using a polyclonal antibody and secondary anti-rabbit antibody conjugated to colloidal gold particles. Results: FK induced apoptosis in all MRT cells with IC50 for 72 hours less than 5nM. The amount in sub-G1 fractions was from 44.7% (KP-MRT-NS) to 73.3% (TTC549) for 72hr. To our surprise, preincubation of MRT cells with the pan-caspase inhibitor z-VAD-fmk could not completely rescue cell death induced by FK though z-VAD-fmk could inhibit the above sub-G1 populations. The ultrastructural morphology of FK-treated cells revealed that these non-apoptotic cells included autophagic cell death. Western blotting using anti-LC3 antibody also showed that FK-treatment induced autophagic cell death because of increased level of LC3-II. We detected AIF translocation into the nucleus after treatment with FK both by immunofluorescence and immunoblotting and this translocation is related with autophagic cell death. In vivo assay showed dose of 1.2, 2.5, and 5.0mg/kg caused a dose-dependent mean tumor size reduction of 59%, 73%, and 87%, compared to the mean tumor volume of control mice. Mice exhibited regression of tumor burden upon treatment with 5.0mg/kg (-13%). Subcellular translocation of AIF has been observed in tumor tissue after treatment with FK. Electron microscopy showed FK induced autophagic cell death in addition to apoptosis or necrosis in vivo. Using immunoelectron microscopy we identified colloidal gold particles detecting AIF presented outside of mitochondria and in the nucleus of autophagic cells. Conclusion: We could detect new type of cell death by HDAC inhibitor both in vivo and in vitro. These are autophagic cell death through AIF translocation into the nucleus.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]