Reconstitution of galectin-3 alters glutathione content and potentiates TRAIL-induced cytotoxicity by dephosphorylation of Akt.
We investigated the role of galectin-3 in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptotic death in human breast carcinoma BT549 cells. We observed that parental galectin-3 null BT549 cells (BT549(par)) as well as control vector transfected (BT549(neo)) cells were resistant to TRAIL, while galectin-3 cDNA-transfected BT549 cells (BT549(gal-3)) were sensitive to TRAIL. Data from flow cytometry and immunoblotting analyses reveal that reconstitution of galectin-3 promoted cell death and PARP cleavage as well as caspase (-8, -9, and -3) activation during TRAIL treatment. However, unlike TRAIL treatment, galectin-3 transfectants were resistant to UV-B-induced PARP cleavage. Data from cDNA array analysis show that galectin-3 did not significantly enhance or reduce any apoptosis-related gene expression. Moreover, although galectin-3 restored pre-mRNA splicing activity and resulted in elevation of FLIPs protein, experiments with FLIPs cDNA-transfected cells show that overexpression of FLIPs did not sensitize cells to TRAIL. Interestingly, BT549(gal-3) cells demonstrated a approximately 2-fold increase in total glutathione content as well as a approximately 5-fold increase in GSSG content in comparison to BT549(par) and BT549(neo) cells, suggesting that galectin-3 overexpression may alter intraceullular oxidation/reduction reactions affecting the metabolism of glutathione and other thiols. In addition, galectin-3 overexpression inactivated Akt by dephosphorylation. Finally, overexpression of constitutively activated Akt protected BT549(gal-3) cells from TRAIL-induced cytotoxicity. Taken together, our data suggest that galectin-3-enhanced TRAIL-induced cytotoxicity is mediated through dephosphorylation of Akt, possibly through a redox-dependent process.
Lee
Yong J
YJ
Department of Surgery, University of Pittsburgh, The Hillman Cancer Center, 5117 Centre Avenue, Room G.5a, Pittsburgh, PA 15213, USA. leeyj@msx.upmc.edu
Song
Young K
YK
Song
Jae J
JJ
Siervo-Sassi
R Rita
RR
Kim
Hyeong-Reh C
HR
Li
Ling
L
Spitz
Douglas R
DR
Lokshin
Anna
A
Kim
Jin H
JH
D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins; D020169:Caspases / Q000378:metabolism; D037502:Galectin 3 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D005786:Gene Expression Regulation; D005978:Glutathione / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins / Q000494:pharmacology*; D010766:Phosphorylation; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D053221:TNF-Related Apoptosis-Inducing Ligand; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D051017:Apoptosis Regulatory Proteins; D037502:Galectin 3; D008562:Membrane Glycoproteins; D011518:Proto-Oncogene Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D011065:Poly(ADP-ribose) Polymerases; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020169:Caspases; D005978:Glutathione