Phosphorothioate oligodeoxynucleotides and G3139 induce apoptosis in 518A2 melanoma cells.
In a previous study, we showed that G3139, an antisense phosphorothioate oligonucleotide that down-regulates the expression of Bcl-2 protein, did not cause chemosensitization of 518A2 melanoma cells. In this work, we show that G3139, and the 2-base mismatch, G4126, can initiate apoptosis in this and other melanoma cell lines as shown by increased cell surface Annexin V expression, typical nuclear phenotypic changes as assessed by 4',6-diamidino-2-phenylindole staining, activation of caspase-3 (but not caspase-8) and Bid, appearance of DEVDase (but not IETDase) activity, and cleavage of poly(ADP-ribose)-polymerase 1. Depolarization of the mitochondrial membrane occurs as a relatively late event. All of these processes seem to be substantially, but perhaps not totally, Bcl-2 independent as shown by experiments employing an anti-Bcl-2 small interfering RNA, which as shown previously down-regulated Bcl-2 protein expression but did not produce apoptosis or chemosensitization in melanoma cells. In fact, these G3139-induced molecular events were not dramatically altered in cells that forcibly overexpressed high levels of Bcl-2 protein. Addition of irreversible caspase inhibitors (e.g., the pan-caspase inhibitor zVAD-fmk) to G3139-treated cells almost completely blocked cytotoxicity. Examination of the time course of the appearance of caspase-3 and cleaved poly(ADP-ribose)-polymerase 1 showed that this could be correlated with the release of cytochrome c from the mitochondria, an event that begins only approximately 4 hours after the end of the oligonucleotide/LipofectAMINE 2000 5-hour transfection period. Thus, both G3139 and cytotoxic chemotherapy activate the intrinsic pathway of apoptosis in these cells, although Bcl-2 expression does not seem to contribute strongly to chemoresistance. These findings suggest that the attainment of G3139-induced chemosensitization in these cells will be difficult.
Lai
Johnathan C
JC
Department of Biomedical Engineering, Columbia University, New York, New York, USA.
Benimetskaya
Luba
L
Khvorova
Anastasia
A
Wu
Sijian
S
Hua
Emily
E
Miller
Paul
P
Stein
C A
CA
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein; D002352:Carrier Proteins / Q000378:metabolism; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000032:analysis / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics; D004789:Enzyme Activation; D006801:Humans; D008055:Lipids / Q000494:pharmacology; D008545:Melanoma / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology*; D014162:Transfection
D000970:Antineoplastic Agents; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D002352:Carrier Proteins; D061945:Caspase Inhibitors; D008055:Lipids; C086724:Lipofectamine; D020319:Oligodeoxyribonucleotides, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2; D013873:Thionucleotides; C408162:oblimersen; D045304:Cytochromes c; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases