Research ArticleExperimental Studies

MG132, A Proteasome Inhibitor, Induced Death of Calf Pulmonary Artery Endothelial Cells via Caspase-dependent Apoptosis and GSH Depletion

YONG HWAN HAN, HWA JIN MOON, BO RA YOU, YEON MI YANG, SUNG ZOO KIM, SUHN HEE KIM and WOO HYUN PARK
Anticancer Research March 2010, 30 (3) 879-885;

Abstract

MG132, a proteasome inhibitor, has been shown to induce apoptotic cell death through formation of reactive oxygen species (ROS). Here, we evaluated the effects of MG132 on the growth of endothelial cells, especially calf pulmonary artery endothelial cells (CPAECs). MG132 dose-dependently inhibited the growth of CPAECSs and human umbilical vein endothelial cells (HUVECs) at 24 hours. MG132 also induced apoptosis in both cell lines, which was accompanied by the loss of mitochondrial membrane potential. All the tested caspase inhibitors (pan-caspase, caspase-3, -8 and -9 inhibitor) significantly rescued CPAECs from MG132-induced cell death. MG132 increased ROS level and GSH depleted cell numbers of CPAECs. None of the caspase inhibitors reduced ROS level in MG132-treated CPAECs but did reduce apoptosis in these cells. In conclusion, MG132 inhibited the growth of endothelial cells, especially CPAECs via caspase-dependent apoptosis. MG132-induced CPAEC death was related to GSH depletion rather than a change in ROS level.

The ubiquitin–proteasomal system represents the major nonlysosomal pathway through which intracellular proteins involved in cell cycling, proliferation, differentiation and apoptosis are degraded in eukaryotic cells (1, 2). The inhibition of proteasome function has emerged as a useful strategy to maneuver apoptosis. MG132 (carbobenzoxy-Leu-Leu-leucinal) effectively blocks the proteolytic activity of proteasome complex (3). Proteasome inhibitors, including MG132, have been shown to induce apoptotic cell death through formation of reactive oxygen species (ROS) (4, 5). ROS formation and glutathione (GSH) depletion due to proteasome inhibitors may cause mitochondrial dysfunction and subsequent cytochrome c release, which leads to a loss of cell viability (6, 7). The mechanism underlying ROS generation after inhibition of proteasome is still unclear.

Vascular endothelium is involved in various regulatory responsibilities such as blood pressure, inflammation and angiogenesis (8). Fundamental to the transition of tumors from a latent to malignant state, angiogenesis involving formation of new blood vessels from pre-existing vasculature is a crucial part. The proliferation of endothelium cells (ECs; sprouting) is an early step of angiogenesis. Despite critical roles for vascular ECs in tumor biogenesis and progression, the effects of proteasome inhibitor on ECs remain relatively poorly understood.

In the present study, we evaluated the effects of MG132 on endothelial cells, especially calf pulmonary artery endothelial cells (CPAECs) in relation to cell death, ROS and GSH.

Materials and Methods

Cell culture. CPAECs from Korean Cell Line Bank (KCLB, Seoul, Korea) and primary human umbilical vein endothelial cells (HUVECs) from PromoCell GmbH (Heidelberg, Germany) were maintained in a humidified incubator containing 5% CO2 at 37°C. CPAECs were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (GIBCO BRL, Grand Island, NY, USA). HUVECs were cultured in complete endothelial cell growth medium (ECGM; PromoCell) with 2% FBS. HUVECs were washed and detached with Hepes balanced salt sodium (BSS) (30 mM Hepes), trypsin-EDTA and trypsin neutralization solution (Promocell). HUVECs were used between passages four and six.

Reagents. MG132 purchased from Calbiochem (San Diego, CA, USA) was dissolved in dimetylsufoxide (DMSO, Sigma, St. Louis, MO, USA) at 10 mM as a stock solution. Pan-caspase inhibitor (Z-VAD-FMK; benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone), and inhibitor of caspase-3 (Z-DEVD-FMK; benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone), caspase-8 (Z-IETD-FMK; benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone) and caspase-9 (Z-LEHD-FMK; benzyloxycarbonyl-Leu-Glu-His-Asp-fluoromethylketone) were obtained from R&D Systems, Inc. (Minneapolis, MN, USA) and were dissolved in DMSO at 10 mM as a stock solution. Cells were pretreated with each caspase inhibitor for 1 hour prior to treatment with MG132. DMSO (0.3%) was used as a control vehicle. All stock solutions were wrapped in foil and kept at −20°C.

Cell growth assay. The effect of MG132 on endothelial cell numbers and growth was determined by trypan blue cell counting or measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye absorbance of living cells as previously described (9). In brief, 1×105 cells per well were seeded in 24-well plates (Nunc, Roskilde, Denmark) for cell counting, and cells 3×104 cells per well were seeded in 96-well microtiter plates for MTT assay. After exposures to 0.1-10 μM MG132, with or without 15 μM each caspase inhibitor, for 24 hours, cells were collected with trypsin digestion for trypan blue exclusion cell counting or for MTT assay. Twenty microliters of MTT (Sigma) solution (2 mg/ml in PBS) were added to each well of 96-well plates. The plates were incubated for 4 additional hours at 37°C. MTT solution in the medium was aspirated off and 200 μl of DMSO were added to each well to solubilize the formazan crystals formed in viable cells. Optical density was measured at 570 nm using a microplate reader (Spectra MAX 340, Molecular Devices Co, Sunnyvale, CA, USA).

Annexin V staining. Apoptosis was determined by staining cells with annexin V-fluorescein isothiocyanate (FITC; PharMingen, San Diego, CA, USA) (Ex/Em=488 nm/519 nm) as previously described (10). In brief, 1×106 cells were incubated with the 10 μM MG132, with or without 15 μM each caspase inhibitor, for 24 hours. Cells were washed twice with cold PBS and then resuspended in 500 μl of binding buffer (10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2) at a density of 1×106 cells/ml. Five microliters of annexin V-FITC were then added to these cells, which were analyzed with a FACStar flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Measurement of mitochondrial membrane potential (MMP, ΔΨm). MMP (ΔΨm) levels were measured using the rhodamine 123 fluorescent dye (Ex/Em=485 nm/535 nm), as previously described (11). In brief, 1×106 cells were incubated with 10 μM MG132, with or without 15 μM each caspase inhibitor, for 24 hours. Cells were washed twice with PBS and incubated with rhodamine 123 (0.1 μg/ml; Sigma) at 37°C for 30 min. Rhodamine 123 staining intensity was determined by flow cytometry. Rhodamine 123-negative cells indicate the loss of MMP (ΔΨm) in CPAECs. MMP (ΔΨm) levels in cells except MMP (ΔΨm) loss cells were expressed as mean fluorescence intensity (MFI), which was calculated by CellQuest software (Becton and Dickinson).

Detection of intracellular ROS and O2•− levels. Intracellular ROS such as H2O2, •OH, and ONOO• were detected by means of an oxidation-sensitive fluorescent probe dye, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, Invitrogen Molecular Probes, OR, USA; Ex/Em=495 nm/529 nm) (12). As H2DCFDA is poorly selective for O2•−, dihydroethidium (DHE, Invitrogen Molecular Probes; Ex/Em=518 nm/605 nm), which is highly selective for O2•−, was used for its detection. In brief, 1×106 cells were incubated with 10 μM MG132, with or without 15 μM each caspase inhibitor, for 24 hours. Cells were then washed in PBS and incubated with 20 μM DCF or DHE at 37°C for 30 min according to the instructions of the manufacturer. Fluorescence was detected using a FACStar flow cytometer (Becton Dickinson). ROS and O2•− levels were expressed as mean fluorescence intensity (MFI), which was calculated by CellQuest software.

Detection of the intracellular GSH. Cellular GSH levels were analyzed using 5-chloromethylfluorescein diacetate (CMFDA, Invitrogen Molecular Probes; Ex/Em=522 nm/595 nm) as previously described (12). In brief, 1×106 cells were incubated with 10 μM MG132, with or without 15 μM each caspase inhibitor, for 24 hours. Cells were then washed with PBS and incubated with 5 μM CMFDA at 37°C for 30 min. Cytoplasmic esterases convert nonfluorescent CMFDA to fluorescent 5-chloromethylfluorescein (CMF), which can then react with GSH. CMF fluorescence intensity was determined using a FACStar flow cytometer (Becton Dickinson). A lack of CMF staining indicates GSH depletion in cells. Data were then expressed as the percentage of CMF-cells. CMF levels in CMF+ cells except GSH-depleted cells were expressed as MFI, which was calculated by CellQuest software (Becton Dickinson).

Statistical analysis. The results represent the mean of at least three independent experiments (mean±SD). The data were analyzed using Instat software (GraphPad Prism4, San Diego, CA, USA). The Student's t-test or one-way analysis of variance (ANOVA) with post hoc analysis using Tukey's multiple comparison test was used for parametric data. Statistical significance was defined as p<0.05.

Results

Effects of MG132 on the growth of CPAECs and HUVECs. We examined the effect of MG132 on the growth of CPAECs and HUVECs by trypan blue cell counting. Treatment with 0.1-10 μM MG132 reduced the population of viable (trypan blue-negative) CPAECs and HUVECs at 24 hours in a dose-dependent manner (Figure 1A and B). At the dose of 0.1 μM MG132, HUVECs were more strongly decreased than CPAECs were (Figure 1A and B). Collectively, the ratio of dead cells to viable cells was increased by MG132 treatment in both cell types. When the growth of cells after treatment with MG132 was also assessed by an MTT assay, dose-dependent reduction of cell growth was observed for both cell types, and the 50% inhibitory concentration of MG132 was almost the same, approximately 0.5 μM at 24 hours (Figure 1C and D). However, at the lower dose of 0.1 μM MG132, CPAECs were more resistant to this agent than were HUVECs (Figure 1C and D).

Effects of MG132 on apoptosis and MMP (ΔΨm) in CPAECs and HUVECs. Next, we determined whether MG132 induces apoptosis in endothelial cell. As shown in Figure 2A and B, the numbers of annexin V-staining cells dose-dependently increased in both cell types. Because apoptosis is closely related to the collapse of MMP (ΔΨm) (13), we elucidated the effect of MG132 on MMP (ΔΨm) using rhodamine 123. Treatment with MG132 induced the loss of MMP (ΔΨm) in both cells (Figure 2C and D). While 0.1 μM MG132 did not trigger MMP (ΔΨm) loss in CPAECs, this dose significantly did so in HUVECs (Figure 2C and D). In relation to MMP (ΔΨm) levels in rhodamine 123-positive cells, MG132 reduced MMP (ΔΨm) level in both cell types (Figure 2E and F). MMP (ΔΨm) was more down-regulated in CPAECs (Figure 2E and F).

Figure 1.
Figure 1.

Effects of MG132 on the growth of CPAECs and HUVECs in vitro. Exponentially growing cells were treated with the indicated concentrations of MG132 for 24 hours. Cell number (A and B) and cell growth (C and D) were assessed by trypan blue cell counting and an MTT assay, respectively. *P<0.05 compared with the MG132-untreated control cell group.

Effects of caspase inhibitors on cell growth and MG132-treated CPAECs. To determine which caspase was involved in the growth inhibition and apoptosis of CPAECs by MG132, cells were pre-treated with pan-caspase inhibitor, and inhibitors of caspase-3, caspase-8 and caspase-9 at a concentration of 15 μM before 10 μM MG132 treatment. Only pan-caspase inhibitor among the caspase inhibitors significantly reduced the growth inhibition by MG132 as measured by an MTT assay (Figure 3A). In addition, all the caspase inhibitors reduced annexin V-FITC-positive cell numbers in MG132-treated CPAECs (Figure 3B). All the inhibitors seemed to reduce basal annexin V staining cells in control CPAECs (Figure 3B). Pan-caspase inhibitor also reduced dead cell numbers in MG132-treated CPAECs (Figure 3C).

In relation to MMP (ΔΨm) in CPAECs, pan-caspase inhibitor reduced the loss of MMP (ΔΨm) induced by MG132 (Figure 3D). All the inhibitors reduced basal MMP (ΔΨm) loss in control CPAECs (Figure 3D). All the caspase inhibitors slightly increased MMP (ΔΨm) levels in MG132-treated CPAECs, but only the effects of caspase-3 inhibitor was significant (Figure 3E).

Effects of caspase inhibitors on ROS and GSH levels in MG132-treated CPAECs. We assessed the changes of the intracellular ROS levels in MG132 and caspase inhibitor-treated CPAECs. As shown in Figure 4A, ROS levels as shown by DCF were significantly increased in CPAECS treated with MG132 at 24 hours. Treatment with pan-caspase and caspase-8 inhibitors significantly increased ROS level in MG132-treated CPAECs (Figure 4A). All the caspase inhibitors except caspase-3 inhibitor seemed to increase the ROS level in control CPAECs (Figure 4A). The level of red fluorescence derived from DHE, which reflected O2•− accumulation, was increased in MG132-treated CPAECs (Figure 4B). Treatment with pan-caspase and caspase-8 inhibitors increased O2•− level in MG132-treated CPAECs but this was not significant (Figure 4B). Treatment with pan-caspase and caspase-9 inhibitors reduced O2•− level in control CPAECs (Figure 4B).

Figure 2.
Figure 2.

Effects of MG132 on apoptosis and MMP (ΔΨm) in CPAECs and HUVECs. Exponentially growing cells were treated with the indicated concentrations of MG132 for 24 hours. A and B: Percentage of annexin V positively stained cells. C and D: Percentage of rhodamine 123-negative cells indicating MMP (ΔΨm) loss. E and F: MMP (ΔΨm) in MG132-treated cells compared with control cells. *P<0.05 compared with the MG132-untreated control group.

When we analyzed the changes of GSH levels in CPAECs in the presence of MG132 and each caspase inhibitor, MG132 increased the number of GSH-depleted cells by about 33% compared with untreated control cells (Figure 4C). All the caspase inhibitors reduced the GSH-depleted cell numbers in MG132-treated CPAECs but only significantly so with pan-caspase inhibitor (Figure 4C). Furthermore, when only CMF-positive CPAECs were assessed, the MFI was not altered in MG132-treated CPAECs (Figure 4D). The caspase inhibitors significantly reduced the MFI in MG132-treated CPAECs (Figure 4D). Except for that of caspase-3 and -9 inhibitors significantly increased basal MFI in control CPAECs (Figure 4D).

Discussion

MG132 alone reduced the population of CPAECs and HUVECs. The ratio of dead cells to viable cells was increased after treatment with MG132 compared with MG132-untreated cells. MG132 also inhibited the growth of CPAECs and HUVECs in view of the MTT assay. MG132 induced apoptosis in both types of cell as evidenced by annexin V staining. Apoptosis is closely related to the collapse of MMP (ΔΨm) (13). Correspondingly, MG132 induced the loss of MMP (ΔΨm) and reduced MMP (ΔΨm) level in both types of cell. At the lower dose of 0.1 μM MG132, CPAECs were more resistant to the effect of this agent than were HUVECs. However, above 1 μM MG132, CPAECs seemed to be more sensitive to MG132 than HUVECs. The difference of susceptibility to MG132 between these cells is probably due to the different basal activities of mitochondria and antioxidant enzymes depending on cell type, tissue origin and species (14, 15). Our results demonstrate that MG132 inhibits the growth of CPAECs and HUVECs via apoptosis and MMP (ΔΨm) loss. In addition, we observed that MG132 increased the level of anonymous ubiquitinated proteins in CPAECs compared with untreated CPAEC control cells (data not shown). Taken together, these data suggest that the inhibition of the ubiquitin-proteasomal system is involved in growth inhibition and apoptosis in these cells.

Figure 3.
Figure 3.

Effects of caspase inhibitors on the cell growth, apoptosis and MMP (ΔΨm) in MG132-treated CPAECs. Exponentially growing cells were treated with MG132 and/or each caspase inhibitor (15 μM) for 24 hours. A: Cell growth, as assessed by an MTT assay. B: Percentage of annexin V-positive staining cells. C: Dead cell numbers. D and E: Percentage of rhodamine 123-negative cells (D) and MMP (ΔΨm) levels (E) in CPAECs, as measured with a FACStar flow cytometer. *P<0.05 compared with the MG132-untreated control group; #P<0.05 compared with cells treated with MG132 only.

To determine which caspases are required for the induction of apoptosis, MG132-treated CPAECs were incubated with different caspase inhibitors. Pan-caspase inhibitor significantly reduced the growth inhibition and dead cell number by MG132. The entire caspase inhibitors also decreased annexin V-FITC-positive cell numbers in MG132-treated CPAECs. In addition, pan-caspase inhibitor significantly prevented MMP (ΔΨm) loss by MG132. These data suggest that CPAEC death induced by MG132 occurs via caspase-dependent apoptosis. In particular, inhibitor of caspase-8, which is related to cell death receptor pathway in apoptosis (16), prevented apoptosis by MG132. This result suggests that MG132-induced CPAEC death is in part controlled by cell death receptor pathway.

Proteasome inhibitors including MG132 have been shown to induce apoptotic cell death through formation of ROS (4-6). Increasing evidence suggests that oxidative stress regulates apoptosis of endothelial cells. In fact, H2O2, tumor necrosis factor-α (TNF-α) and angiotensin II induce their apoptosis and stimulate the generation of ROS (17, 18). According to our results, ROS levels were significantly increased in CPAECs treated with MG132. However, pan-caspase and caspase-8 inhibitors showing antiapoptotic effect on MG132-treated CPAECs increased ROS levels, including these of O2•−. In addition, caspase inhibitors differently affected ROS levels in control CPAECs regardless of cell death. Therefore, these data suggest that the changes of ROS levels by MG132 and caspase inhibitor are not tightly correlated to CPAEC death. The exact role of ROS in MG132-induced cell death needs to be defined further.

GSH is a major non-protein antioxidant in cells. It is able to eliminate O2•− and provide electrons for enzymes such as GSH peroxidase, which reduces H2O2 to H2O. The redox state of cellular GSH is an important modulatory element in the protein ubiquitination pathways (19). It is reported that GSH depletion due to proteasome inhibitors leads to cell death (6, 7). MG132 increased the number of GSH-depleted cells in CPAECs. All the caspase inhibitors reduced the GSH-depleted cell numbers and pan-caspase inhibitor was significant. These results seem to be correlated to the annexin V-FITC results from CPAECs treated with MG132 and/or caspase inhibitors. Interestingly, all the caspase inhibitors reduced the GSH level in MG132-treated CPAECs. This phenomenon might reduce the increasing ROS by MG132 and caspase inhibitors. In addition, caspase-3 and -9 inhibitors increased basal GSH level in control CPAECs, implying that each caspase inhibitor differently regulates intracellular GSH levels.

Figure 4.
Figure 4.

Effects of caspase inhibitors on reactive oxygen species (ROS) and glutathione (GSH) levels in MG132-treated CPAECs. Exponentially growing cells were treated with MG132 and/or each caspase inhibitor (15 μM) for 24 hours. ROS and GSH levels in CPAECs were measured using a FACStar flow cytometer. A and B: Graphs indicate DCF (ROS) and DHE (O2•−) levels (%) compared with control CPAECs, respectively. C and D: Graphs show the percentage of (–) CMF (GSH-depleted) cells (C) and mean CMF (GSH) levels compared with control cells (D). *P<0.05 compared with the MG132-untreated control group. #P<0.05 compared with cells treated with MG132 only.

In conclusion, MG132 inhibited the growth of endothelial cells, especially CPAECs, apparently via caspase-dependent apoptosis. MG132-induced CPAEC death was related to GSH depletion rather than to changes in ROS level.

Acknowledgements

This research was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs and Republic of Korea (A084194) and the grant of the Korean Ministry of Education, Science and Technology (The regional Core Research Program/ Centers for Healthcare Technology Development).

Footnotes

    • Received September 26, 2009.
    • Revision received January 21, 2010.
    • Accepted January 21, 2010.

References

  1. ↵
    1. Orlowski RZ
    : The role of the ubiquitin-proteasome pathway in apoptosis. Cell Death Differ 6(4): 303-313, 1999.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Voges D,
    2. Zwickl P,
    3. Baumeister W
    : The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu Rev Biochem 68: 1015-1068, 1999.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Lee DH,
    2. Goldberg AL
    : Proteasome inhibitors: valuable new tools for cell biologists. Trends Cell Biol 8(10): 397-403, 1998.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Wu HM,
    2. Chi KH,
    3. Lin WW
    : Proteasome inhibitors stimulate activator protein-1 pathway via reactive oxygen species production. FEBS Lett 526(1-3): 101-105, 2002.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Perez-Galan P,
    2. Roue G,
    3. Villamor N,
    4. Montserrat E,
    5. Campo E,
    6. Colomer D
    : The proteasome inhibitor bortezomib induces apoptosis in mantle-cell lymphoma through generation of ROS and Noxa activation independent of p53 status. Blood 107(1): 257-264, 2006.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Ling YH,
    2. Liebes L,
    3. Zou Y,
    4. Perez-Soler R
    : Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic response to Bortezomib, a novel proteasome inhibitor, in human H460 non-small cell lung cancer cells. J Biol Chem 278(36): 33714-33723, 2003.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Qiu JH,
    2. Asai A,
    3. Chi S,
    4. Saito N,
    5. Hamada H,
    6. Kirino T
    : Proteasome inhibitors induce cytochrome c-caspase-3-like protease-mediated apoptosis in cultured cortical neurons. J Neurosci 20(1): 259-265, 2000.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Bassenge E
    : Endothelial function in different organs. Prog Cardiovasc Dis 39(3): 209-228, 1996.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Park WH,
    2. Seol JG,
    3. Kim ES,
    4. Hyun JM,
    5. Jung CW,
    6. Lee CC,
    7. Kim BK,
    8. Lee YY
    : Arsenic trioxide-mediated growth inhibition in MC/CAR myeloma cells via cell cycle arrest in association with induction of cyclin-dependent kinase inhibitor, p21, and apoptosis. Cancer Res 60(11): 3065-3071, 2000.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Han YH,
    2. Kim SZ,
    3. Kim SH,
    4. Park WH
    : Apoptosis in pyrogallol-treated Calu-6 cells is correlated with the changes of intracellular GSH levels rather than ROS levels. Lung Cancer 59(3): 301-314, 2008.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Han YH,
    2. Kim SZ,
    3. Kim SH,
    4. Park WH
    : Arsenic trioxide inhibits growth of As4.1 juxtaglomerular cells via cell cycle arrest and caspase-independent apoptosis. Am J Phys 293(2): F511-520, 2007.
    OpenUrl
  12. ↵
    1. Han YH,
    2. Kim SH,
    3. Kim SZ,
    4. Park WH
    : Caspase inhibitor decreases apoptosis in pyrogallol-treated lung cancer Calu-6 cells via the prevention of GSH depletion. Int J Oncol 33(5): 1099-1105, 2008.
    OpenUrlPubMed
  13. ↵
    1. Yang J,
    2. Liu X,
    3. Bhalla K,
    4. Kim CN,
    5. Ibrado AM,
    6. Cai J,
    7. Peng TI,
    8. Jones DP,
    9. Wang X
    : Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275(5303): 1129-1132, 1997.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Oberley LW,
    2. Oberley TD
    : Role of antioxidant enzymes in cell immortalization and transformation. Mol Cell Biochem 84(2): 147-153, 1988.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Yan T,
    2. Li S,
    3. Jiang X,
    4. Oberley LW
    : Altered levels of primary antioxidant enzymes in progeria skin fibroblasts. Biochem Biophys Res Comm 257(1): 163-167, 1999.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Ashkenazi A,
    2. Dixit VM
    : Death receptors: signaling and modulation. Science 281(5381): 1305-1308, 1998.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Hermann C,
    2. Zeiher AM,
    3. Dimmeler S
    : Shear stress inhibits H2O2-induced apoptosis of human endothelial cells by modulation of the glutathione redox cycle and nitric oxide synthase. Arterioscler Thromb Vasc Biol 17(12): 3588-3592, 1997.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Dimmeler S,
    2. Zeiher AM
    : Reactive oxygen species and vascular cell apoptosis in response to angiotensin II and pro-atherosclerotic factors. Regul Pept 90(1-3): 19-25, 2000.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Jahngen-Hodge J,
    2. Obin MS,
    3. Gong X,
    4. Shang F,
    5. Nowell TR Jr,
    6. Gong J,
    7. Abasi H,
    8. Blumberg J,
    9. Taylor A
    : Regulation of ubiquitin-conjugating enzymes by glutathione following oxidative stress. J Biol Chem 272(45): 28218-28226, 1997.
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Reprints and Permissions
MG132, A Proteasome Inhibitor, Induced Death of Calf Pulmonary Artery Endothelial Cells via Caspase-dependent Apoptosis and GSH Depletion
YONG HWAN HAN, HWA JIN MOON, BO RA YOU, YEON MI YANG, SUNG ZOO KIM, SUHN HEE KIM, WOO HYUN PARK
Anticancer Research Mar 2010, 30 (3) 879-885;
Twitter logo Facebook logo Mendeley logo

Jump to section