J Korean Surg Soc.  2011 Oct;81(4):235-241. 10.4174/jkss.2011.81.4.235.

Mycophenolic acid mediated mitochondrial membrane potential transition change lead to T lymphocyte apoptosis

• Choi SJ
• Lee HK
• Kim NH
• Chung SY

• KMID: 1445743
• DOI: http://doi.org/10.4174/jkss.2011.81.4.235

Abstract

PURPOSE
This study demonstrated that apoptosis induced by mycophenolic acid (MPA) is mediated by mitochondrial membrane potential transition (MPT) changes in Jurkat cells.
METHODS
Cell viability and MPT changes were measured by flow cytometry. Western blotting was performed to evaluate the expression of Bcl-2 family proteins, Bid, truncated Bid (tBid), cytochrome c, voltage dependent anion channel (VDAC), poly ADP-ribose polymerase (PARP), and protein kinase C-delta (PKC-delta). The catalytic activity of caspase-9 and -3 was also measured.
RESULTS
Cell viability was decreased in time- and dose-dependent manners. Bcl-2 protein expression was decreased, but Bax protein expression was identified. A decreased Bcl-XL /Bcl-XS ratio was also noted. The expression of tBid protein also increased in a time-dependent manner in Jurkat cells treated with MPA. While normal MPT appeared as orange fluorescence, abnormal MPT corresponded to green fluorescence. Green fluorescence increased as orange decreased in the MPA-treated cells. Significantly increased concentrations of MPA induced the release of cytosolic cytochrome c. MPA also augmented the catalytic activity of caspase-9 and caspase-3 in Jurkat cells. Our findings demonstrated that MPA-induced apoptosis is mediated by MPT changes accompanied by decreased Bcl-XL expression and the appearance of tBid protein. The release of cytosolic cytochrome c from mitochondria and increased catalytic activity of caspase-9 and caspase-3 were observed in MPA-treated Jurkat cells.
CONCLUSION
These results suggest that mitochondrial dysfunction caused by MPA induces human T lymphocyte apoptosis.

Keyword

Mycophenolic acid; Mitochondrial membrane potential; Apoptosis

MeSH Terms

Adenosine Diphosphate Ribose
Apoptosis
bcl-2-Associated X Protein
BH3 Interacting Domain Death Agonist Protein
Blotting, Western
Caspase 3
Caspase 9
Cell Survival
Citrus sinensis
Cytochromes c
Cytosol
Flow Cytometry
Fluorescence
Humans
Jurkat Cells
Lymphocytes
Membrane Potential, Mitochondrial
Mitochondria
Mitochondrial Membranes
Mycophenolic Acid
Protein Kinase C-delta
Proteins
Adenosine Diphosphate Ribose
BH3 Interacting Domain Death Agonist Protein
Caspase 3
Caspase 9
Cytochromes c
Mycophenolic Acid
Protein Kinase C-delta
Proteins
bcl-2-Associated X Protein

Figure

• 

  Fig. 1 Bcl-XL/Bcl-XS ratio decreases in Jurkat cells treated with mycophenolic acid (mycophenolic acid [MPA]; 5 µM). Cells were treated with 5 µM MPA for various periods of time. Equal amounts of protein from cell lysate were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, and immunoblotted with anti-Bcl-XL and anti-Bcl-XS antibodies.

• 

  Fig. 2 Mycophenolic acid (MPA) induces the truncated Bid (tBid) protein in Jurkat cells. Cells were treated with 5 µM MPA for various periods and separated into cytosolic and mitochondrial fractions. The cytosolic fraction was separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotted for Bid and tBid. Antibody binding was visualized by enhanced chemilluminescence.

• 

  Fig. 3 Mycophenolic acid induces changes in mitochondrial membrane potential transition. Cells were stained with JC-1 and analyzed using flow cytometry. MPT, membrane potential transition.

• 

  Fig. 4 Mycophenolic acid (MPA) induces the cytosolic release of cytochrome c from mitochondria in Jurkat cells. Cells were treated with 5 µM MPA for various periods of time. Cells were separated into cytosolic and mitochondrial fractions. The two fractions were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotted for cytochrome c and voltage dependent anion channel (VDAC). Immunoreactive signals were visualized by enhanced chemilluminescence.

• 

  Fig. 5 Mycophenolic acid (MPA) increases the catalytic activity of caspase-9 and -3 of Jurkat cells in a time-dependent manner. Cells were treated with 5 µM MPA for various periods of time and then lysed to measure the activity of these proteases using fluorogenic biosubstrates. The lysate were incubated with Ac-LEHD-AFC to examine caspase-9 (A) or Ac-DEVD-AMC to monitor caspase-3 (B). Protein kinase C-δ (PKC-δ) cleavage was measured by Western blotting with an anti-PKC-δ antibody (C). a)P < 0.05 by student t-test, compared to control group.

Reference

1. Gross A, McDonnell JM, Korsmeyer SJ. BCL-2 family members and the mitochondria in apoptosis. Genes Dev. 1999. 13:1899–1911.

2. Hockenbery D, Nuñez G, Milliman C, Schreiber RD, Korsmeyer SJ. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature. 1990. 348:334–336.

3. Crompton M. Bax, Bid and the permeabilization of the mitochondrial outer membrane in apoptosis. Curr Opin Cell Biol. 2000. 12:414–419.

4. Lee WA, Gu L, Miksztal AR, Chu N, Leung K, Nelson PH. Bioavailability improvement of mycophenolic acid through amino ester derivatization. Pharm Res. 1990. 7:161–166.

5. Schulze-Osthoff K, Ferrari D, Los M, Wesselborg S, Peter ME. Apoptosis signaling by death receptors. Eur J Biochem. 1998. 254:439–459.

6. Cory S, Adams JM. The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer. 2002. 2:647–656.

7. Vander Heiden MG, Chandel NS, Williamson EK, Schumacker PT, Thompson CB. Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria. Cell. 1997. 91:627–637.

8. Annis MG, Soucie EL, Dlugosz PJ, Cruz-Aguado JA, Penn LZ, Leber B, et al. Bax forms multispanning monomers that oligomerize to permeabilize membranes during apoptosis. EMBO J. 2005. 24:2096–2103.

9. Huang DC, Strasser A. BH3-Only proteins-essential initiators of apoptotic cell death. Cell. 2000. 103:839–842.

10. Lee YC, Kim JJ, Park HR, Lee S, Woo YM, Cho MH, et al. A study for apoptosis and its mechanism of allogeneic activated T lymphocytes induced by mouse liver immature dendritic cells. J Korean Surg Soc. 2004. 66:1–4.

11. Li PF, Dietz R, von Harsdorf R. p53 regulates mitochondrial membrane potential through reactive oxygen species and induces cytochrome c-independent apoptosis blocked by Bcl-2. EMBO J. 1999. 18:6027–6036.

12. Desagher S, Martinou JC. Mitochondria as the central control point of apoptosis. Trends Cell Biol. 2000. 10:369–377.

13. Gogvadze V, Orrenius S, Zhivotovsky B. Multiple pathways of cytochrome c release from mitochondria in apoptosis. Biochim Biophys Acta. 2006. 1757:639–647.

14. Hirata H, Takahashi A, Kobayashi S, Yonehara S, Sawai H, Okazaki T, et al. Caspases are activated in a branched protease cascade and control distinct downstream processes in Fas-induced apoptosis. J Exp Med. 1998. 187:587–600.

15. Creagh EM, Martin SJ. Caspases: cellular demolition experts. Biochem Soc Trans. 2001. 29(Pt 6):696–702.

16. Budihardjo I, Oliver H, Lutter M, Luo X, Wang X. Biochemical pathways of caspase activation during apoptosis. Annu Rev Cell Dev Biol. 1999. 15:269–290.

17. Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM, et al. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature. 1999. 397:441–446.

18. Green DR, Reed JC. Mitochondria and apoptosis. Science. 1998. 281:1309–1312.

19. Ly JD, Grubb DR, Lawen A. The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update. Apoptosis. 2003. 8:115–128.