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Volume 10, Issue 7, Pages 609-618 (July 2003) Mitochondrial-Targeted Fatty Acid Analog Induces Apoptosis with Selective Loss of Mitochondrial Glutathione in Promyelocytic Leukemia Cells Karl Johan Tronstad, Bjørn Tore Gjertsen, Camilla Krakstad, Kjetil Berge, Odd Terje Brustugun, Stein Ove Døskeland, Rolf Kristian Berge Chemistry & Biology Volume 10, Issue 7, Pages 609-618 (July 2003) DOI: 10.1016/S1074-5521(03)00142-X

Figure 1 Induction of Apoptosis in Cells Treated with TTA and 8CPT-cAMP Wild-type and Bcl-2-overexpressing IPC-81 cells were treated with either 500 μM BSA-bound TTA (A) or 200 μM 8CPT-cAMP (cAMP) (B). Apoptosis was determined by microscopic examination of cell surface and nuclei after staining with Hoechst 33342. The data are presented as mean ± SD. *p < 0.05 compared to untreated control. Chemistry & Biology 2003 10, 609-618DOI: (10.1016/S1074-5521(03)00142-X)

Figure 2 Cytochrome c Release Mitochondrial and cytosolic fractions were isolated from wild-type IPC-81 (wt) cells, and IPC-81 cells overexpressing Bcl-2 or ICER. Cytochrome c (Cyt c) was determined as described in Experimental Procedures. Cells were treated with 500 μM BSA-bound TTA (A and B) or 200 μM 8CPT-cAMP (cAMP) (C and D). The inserted curve in (B) shows the increase of cytosolic cytochrome c at early time points (0.5–4 hr) in TTA-treated cells. (E and F) Cytochrome c was measured in cytosol and mitochondria from IPC-81 wild-type, IPC-81 Bcl-2, and IPC-81 ICER cells after 12 and 6 hr of exposure to 500 μM TTA and 200 μM 8CPT-cAMP, respectively. The data are given as mean ± SD. *p < 0.05 compared to untreated control. Chemistry & Biology 2003 10, 609-618DOI: (10.1016/S1074-5521(03)00142-X)

Figure 3 The Role of Caspases in 8CPT-cAMP- and TTA-Induced Cell Death IPC-81 cells were exposed to 500 μM BSA-bound TTA or 200 μM 8CPT-cAMP (cAMP). (A) Capase-3 activity was measured in cellular extracts. (B) The level of procaspase-3 and cleavage pattern of PARP was assessed by Western analysis. (C) Apoptosis was scored in cultures treated with 8CPT-cAMP (6 hr) or TTA (20 hr) in absence or presence of zVAD-fmk (zVAD). (D) Morphological changes were visualized by phase contrast (left) and Hoechst fluorescence (right) microscopy. The data are given as mean ± SD. ap < 0.05 compared to untreated control, bp < 0.05 compared to the same treatment (cAMP) in absence of zVAD-fmk. Chemistry & Biology 2003 10, 609-618DOI: (10.1016/S1074-5521(03)00142-X)

Figure 4 Modulation of Mitochondrial Glutathione Early in Apoptosis The content of total free glutathione (GSSG + GSH) and nonoxidized free glutathione (GSH) was determined by HPLC, and the respective GSH/(GSSG + GSH) ratios were calculated in the mitochondrial (A–D) and cytosolic (E–F) fractions after treatment with 500 μM TTA (A, B, E, and F) or 200 μM 8CPT-cAMP (cAMP) (C, D, G, and H). The results are presented as mean ± SD. *p < 0.05 compared to untreated control. Chemistry & Biology 2003 10, 609-618DOI: (10.1016/S1074-5521(03)00142-X)

Figure 5 Apoptotic Nuclear Morphology in Wild-Type and Bcl-2-Overexpressing IPC-81 Cells Cells were treated with 200 μM TTA bound to serum protein (A) or 200 μM 8CPT-cAMP (B). Cells were fixed in 4% paraformaldehyde with daunorubicin (5 μM), mounted on slides, and examined by laser scanning confocal microscopy Chemistry & Biology 2003 10, 609-618DOI: (10.1016/S1074-5521(03)00142-X)

Figure 6 Depolarization of Δψ after Treatment with TTA and 8CPT-cAMP Mitochondrial depolarization was determined by flow-cytometric measurements after incubation with the Δψ-sensitive dye JC-1. Dissipation of Δψ is indicated by a decrease in red fluorescence (590 nm). (A) Presents representative histograms from the flow-cytometric analysis. The level of red fluorescence relative to control is presented as mean ± SD for wild-type (B) and Bcl-2-overexpressing (C) IPC-81 cells treated with either 200 μM serum protein bound TTA or 200 μM 8CPT-cAMP (cAMP). *p < 0.05 compared to untreated control. Chemistry & Biology 2003 10, 609-618DOI: (10.1016/S1074-5521(03)00142-X)

Figure 7 Mitochondrial Localization and Glutathione Distribution Cells were labeled with MTR in combination with CMFDA as described in Experimental Procedures. The samples were studied by laser scanning confocal microscopy. Red and green fluorescence indicates mitochondria and glutathione, respectively. (A) IPC-81 wild-type, TTA treatment; (B) IPC-81 Bcl-2, TTA treatment; (C) IPC-81 wild-type, 8CPT-cAMP treatment; (D) IPC-81 Bcl-2, 8CPT-cAMP treatment. Chemistry & Biology 2003 10, 609-618DOI: (10.1016/S1074-5521(03)00142-X)