Activation of caspases and cleavage of Bid are required for tyrosine and phenylalanine deficiency-induced apoptosis of human A375 melanoma cells.
Deprivation of tyrosine (Tyr) and phenylalanine (Phe) inhibits growth and induces programmed cell death (apoptosis) of human A375 melanoma cells. Herein, we found that activation of caspases and release of mitochondrial cytochrome c are required for this process. Culturing A375 cells in Tyr/Phe-free medium, containing 10% dialyzed fetal bovine serum, results in activation of caspase-3-like activity. This is accompanied by decreased cell viability and increased apoptosis. Tyr/Phe deprivation also stimulates proteolytic cleavage of the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP). Western blot analysis showed that caspases 3, 7, 8, and 9 are activated by deprivation of Tyr/Phe. Tyr/Phe deprivation decreases mitochondrial membrane potential, induces cleavage of Bid, increases translocation of Bax from the cytosol to mitochondria, and results in release of cytochrome c from the mitochondria to the cytosol. Apoptosis due to Tyr/Phe deprivation is almost completely inhibited by the broad-spectrum cell-permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z.VAD.fmk). This inhibitor suppresses the cleavage of Bid, the release of cytochrome c from the mitochondria to the cytosol, and the cleavage of PARP. Decylubiquinone, a mitochondrial permeability transition pore inhibitor, does not suppress the activation of caspase 8 but suppresses release of cytochrome c, activation of caspase 9, and induction of apoptosis. These results indicate that activation of caspases, cleavage of Bid, and mitochondrial release of cytochrome c are required for apoptosis induced by Tyr/Phe deprivation.
Ge
Xiaokang
X
Cancer Prevention and Research Center, Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Pullman, WA 99164-6510, USA.
Fu
Ya-Min
YM
Li
Yi-Qi
YQ
Meadows
Gary G
GG
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D024322:Amino Acids, Aromatic / Q000172:deficiency*; D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D053222:Fas Ligand Protein; D006801:Humans; D008024:Ligands; D008545:Melanoma / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D008562:Membrane Glycoproteins / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism; D008954:Models, Biological; D010649:Phenylalanine / Q000172:deficiency / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000172:deficiency; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000494:pharmacology; D019014:fas Receptor / Q000378:metabolism
D000590:Amino Acid Chloromethyl Ketones; D024322:Amino Acids, Aromatic; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D002352:Carrier Proteins; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D003574:Cytochrome c Group; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008024:Ligands; D008562:Membrane Glycoproteins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D019014:fas Receptor; D014451:Ubiquinone; D014443:Tyrosine; D010649:Phenylalanine; C060262:2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone; D020169:Caspases