Tocotrienols Induce Apoptosis and Autophagy in Rat Pancreatic Stellate Cells Through the Mitochondrial Death Pathway  Mariana Rickmann, Eva C. Vaquero, Juan Ramón Malagelada, Xavier Molero  Gastroenterology  Volume 132, Issue 7, Pages 2518-2532 (June 2007) DOI: 10.1053/j.gastro.2007.03.107 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Tocotrienols (TRF) reduce viability of activated PSCs. (A) Total cell number as measured with the CyQUANT kit following 24-hour treatment with TRF and α-tocopherol at the stated concentrations. Data are presented as percentage of control cells. Horizontal dotted line indicates 100% of control cells. Overall, data are expressed as mean ± SEM. *P < .05 versus control; n = 3. (B) Cell PI uptake as measured by flow cytometry following 24-hour treatment with TRF or α-tocopherol (α-TCF) at the stated concentrations. *P < .05 versus control; n = 3. (C) Cell PI uptake as measured by flow cytometry in cells treated with TRF (20 μmol/L) or α-tocopherol (200 μmol/L) for the indicated times. *P < .001 versus control; n ≥ 4. (D) LDH release in cells treated with TRF (20 μmol/L) and α-tocopherol (200 μmol/L) for the indicated times. *P < .001 versus control; n ≥ 4. Gastroenterology 2007 132, 2518-2532DOI: (10.1053/j.gastro.2007.03.107) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Lack of effect of tocotrienols (TRF) on cell cycle distribution in activated PSCs. Cells were treated with TRF (20 μmol/L), α-tocopherol (200 μmol/L), or vehicle. After 24 hours, DNA content was analyzed by flow cytometry as described in the Materials and Methods section. Percentage of cells in G0-G1, S, and G2-M phases shown on the histograms was calculated using the Cell Quest software. Representative histograms from 3 different experiments are shown. Gastroenterology 2007 132, 2518-2532DOI: (10.1053/j.gastro.2007.03.107) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Tocotrienols (TRF) induce apoptosis in activated PSCs. Cells were treated with TRF (20 μmol/L), α-tocopherol (200 μmol/L), or vehicle (control) for the indicated times. (A) DNA fragmentation. Data are expressed as mean ± SEM and presented relative to untreated cells. Horizontal dotted line indicates arbitrary level of 1 assigned to control cells. *P < .001 versus control; n ≥ 4. (B) Caspase-3, (C) caspase-8, and (D) caspase-9 activities measured by fluorogenic assays in cell lysates using specific substrates as described in the Materials and Methods section. Data are expressed as mean ± SEM. *P < .05 versus control; n ≥ 5. Gastroenterology 2007 132, 2518-2532DOI: (10.1053/j.gastro.2007.03.107) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Tocotrienols (TRF) induce autophagy in activated PSCs. Cells were treated with TRF (20 μmol/L), α-tocopherol (200 μmol/L), or vehicle (control) for 24 hours. Cells incubated in amino acid free medium (−aa) for 2 hours were included as a positive control for autophagy. (A) In vivo identification of MDC-labeled autophagolysosome vacuoles by fluorescence microscopy. Prominent cytoplasmic MDC-labeled vacuoles in TRF-treated cells are indicated by arrows. Punctate cytoplasmic vacuoles are observed in amino acid starved cells, whereas a diffuse background staining without cytoplasmic vacuoles is observed in control and α-tocopherol treated cells (original magnification, ×20). (B). Representative immunoblot analysis of LC3-I (cytoplasmic form) and LC3-II (membrane-bound form) expression in cell lysates. β-actin in stripped membranes is shown to demonstrate equal protein loading. (C) Graphic representation of LC3-II expression normalized with β-actin as determined by densitometry analysis of immunoblot shown in panel B. Data are expressed as mean ± SEM of 3 independent experiments. *P < .05 versus control. Gastroenterology 2007 132, 2518-2532DOI: (10.1053/j.gastro.2007.03.107) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Tocotrienols (TRF) trigger the collapse of Δψm and release of cytochrome c by targeting mPTP in activated PSCs. (Panels A–C) Changes in Δψm as measured by flow cytometry in double-stained cells with DiOC6(3) and PI. To exclude mitochondrial depolarization associated with cell death, Δψm was monitored in PI excluding (living) cells, as illustrated in panel A inset. (A) Dose-dependent effects of TRF on Δψm following 24-hour treatment. Cells treated with the protonophore CCCP (50 μmol/L) for 1 hour were included as a reference for complete mitochondrial depolarization. Data are expressed as mean ± SEM; *P < .05 and **P < .0001 versus control; n ≥ 3. (B) Kinetics of Δψm in cells treated with TRF (20 μmol/L), α-tocopherol (200 μmol/L), or vehicle (control). Data are expressed as mean ± SEM and presented as percentage of control cells. *P < .01 versus control and #P < .01 versus α-tocopherol; n ≥ 5. (C). Representative flow cytometric analysis of Δψm showing the effects of CsA (10 μmol/L) in cells treated with TRF (20 μmol/L) for 24 hours (left panel) and with α-tocopherol (200 μmol/L) for 3 hours (right panel). Numbers indicate the percentage of Δψm compared with control cells. Data are expressed as mean ± SEM of 3 independent experiments. (D) Representative immunoblot detection of cytochrome c in cytosolic fractions (CytF) following 24-hour TRF (20 μmol/L) or α-tocopherol (200 μmol/L) treatment with or without CsA. In the cytosol, cytochrome c is detected in polymeric form, appearing as a 58-kilodalton band. Detection of succinate-ubiquinone oxidoreductase (SOX) exclusively in mitochondria-enriched heavy membrane fractions (MitF), and not in cytosolic fractions, excludes mitochondrial contamination. β-actin in stripped membranes is shown to demonstrate equal protein loading. (E) Graphic representation of cytochrome c expression normalized with β-actin in cytosolic fractions as determined by densitometry analysis of Western blots shown in panel D. Data are expressed as mean ± SEM of 3 independent experiments. Gastroenterology 2007 132, 2518-2532DOI: (10.1053/j.gastro.2007.03.107) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Blockade of mPTP opening, but not caspase inhibition, prevents activated PSC death induced by tocotrienols (TRF). Effects of zVAD-fmk (100 μmol/L) or CsA (10 μmol/L) on (A) DNA fragmentation, (B) LDH release, and (C) PI uptake at 24 hours after treatment with TRF (20 μmol/L) or vehicle (control). Throughout, data are expressed as mean ± SEM; n ≥ 3. (D) Phase-contrast microscopy showing morphologic changes following 24-hour TRF (20 μmol/L) treatment with or without inhibitors (original magnification, ×20). (E) Representative immunoblot analysis showing the effect of zVAD-fmk and CsA in LC3-I (cytoplasmic form) and LC3-II (membrane-bound form) expression in cells treated with TRF for 24 hours. β-actin in stripped membranes is shown to demonstrate equal protein loading. (F) Graphic representation of LC3-II expression normalized with β-actin as determined by densitometry analysis of Western blots shown in panel E. Data are expressed as mean ± SEM of 3 independent experiments; *P < .05 versus control and #P < .05 versus TRF without inhibitors. Gastroenterology 2007 132, 2518-2532DOI: (10.1053/j.gastro.2007.03.107) Copyright © 2007 AGA Institute Terms and Conditions

Figure 7 Tocotrienols (TRF) do not trigger death in quiescent PSCs or in acinar cells. (A) Inmunofluorescence expression of α-SMA and GFAP after replating activated PSCs on uncoated or on Matrigel-coated glass slides for the indicated times. Nuclei were counterstained with DAPI (original magnification, ×40). (B) Effect of TRF on DNA fragmentation in Matrigel inactivated PSCs. Twenty-four hours after replating cells on Matrigel, TRF (20 μmol/L) was added for another 24 hours before DNA fragmentation analysis. Data are expressed as mean ± SEM; n = 3. (C) DNA fragmentation and (D) caspase-3 activity in pancreatic acinar cells incubated with TRF (20 μmol/L), α-tocopherol (200 μmol/L), or vehicle (control) for 24 hours. TNF-α (20 ng/mL) + MG-132 (5 μmol/L) was used as a positive control to induce apoptosis. Data are expressed as mean ± SEM. *P < .001 versus control; n = 3. Gastroenterology 2007 132, 2518-2532DOI: (10.1053/j.gastro.2007.03.107) Copyright © 2007 AGA Institute Terms and Conditions

Figure 8 Individual tocotrienols cause ΔΨm loss, reduction of cell viability, and apoptosis in activated PSCs. (A–D) Comparative effects of α-, β-, γ-, and δ-tocotrienol isomers on ΔΨm and cell viability as measured by flow cytometry in cells double stained with DiOC6(3) and PI. Cells were treated for 24 hours with increasing concentrations of tocotrienols. Throughout, DiOC6(3) values are presented as percentage of control cells (considered as 100%) and analyzed on PI excluding cells. Bar graphs presented for each isomer on the right side show the effect of CsA (10 μmol/L) in preventing ΔΨm loss in cells exposed to 15 μmol/L of α-, β-, γ-, and δ-tocotrienol. (E) Cell PI uptake and (F) DNA fragmentation following 24-hour treatment with α-, β-, γ-, and δ-tocotrienol (15 μmol/L) with or without CsA. Data are expressed as mean ± SEM. Horizontal dotted line indicates arbitrary level of 1 assigned to control cells. *P < .05 versus control and #P < .05 versus the corresponding tocotrienol isomer without CsA; n ≥ 3. Gastroenterology 2007 132, 2518-2532DOI: (10.1053/j.gastro.2007.03.107) Copyright © 2007 AGA Institute Terms and Conditions