Enhancement of diethylstilbestrol induced cytotoxicity by bcl-2 antisense oligodeoxynucleotides and a glutathione depletor for prostate cancer.
OBJECTIVE
Bcl-2 has been shown to prolong cancer cell survival by blocking apoptosis and have the function of scavenging reactive oxygen species. We assessed the efficacy of a novel strategy that relies on antisense bcl-2 oligodeoxynucleotides as well as a glutathione depletor combined with diethylstilbestrol (DES) for hormone independent prostate cancer.


METHODS
The cytotoxic effects of antisense bcl-2 oligodeoxynucleotides and DES on PC-3 cells were determined using spectrophotometry measurement. The expression of Bcl-2 protein was detected by Western blot analysis. Intracellular reactive oxygen species generation was estimated from the amount of intracellular dichlorofluorescein. Apoptosis was determined by the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate-biotin nick end labeling assay.


RESULTS
Antisense bcl-2 oligodeoxynucleotides decreased Bcl-2 protein levels and significantly inhibited PC-3 cell growth in a dose dependent manner. Antisense bcl-2 oligodeoxynucleotides significantly enhanced DES induced cytotoxicity. A significant increase in apoptotic cells was induced by the combination of antisense bcl-2 oligodeoxynucleotides and DES compared with the oligodeoxynucleotides alone. The glutathione depletor buthionine sulfoximine significantly decreased the intracellular concentration of glutathione and augmented DES induced cytotoxicity and reactive oxygen species generation in PC-3 cells. Neither the generation of reactive oxygen species nor the intracellular concentration of glutathione was influenced by antisense bcl-2 oligodeoxynucleotide treatment.


CONCLUSIONS
Antisense bcl-2 oligodeoxynucleotides significantly enhanced DES induced cytotoxicity in hormone independent prostate cancer cells through the apoptotic pathway independent of augmented reactive oxygen species generation, whereas the glutathione depletor augmented cytotoxicity and reactive oxygen species generation.
Kikuchi
Eiji
E
Department of Urology, Keio University School of Medicine, Tokyo, Japan.
Nakashima
Jun
J
Horiguchi
Yutaka
Y
Oya
Motosugu
M
Ohigashi
Takashi
T
Murai
Masaru
M
D017209:Apoptosis / Q000187:drug effects; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D004054:Diethylstilbestrol / Q000627:therapeutic use*; D005978:Glutathione / Q000037:antagonists & inhibitors*; D006801:Humans; D008297:Male; D020319:Oligodeoxyribonucleotides, Antisense / Q000627:therapeutic use*; D011471:Prostatic Neoplasms / Q000188:drug therapy*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000187:drug effects; D017382:Reactive Oxygen Species; D014407:Tumor Cells, Cultured
D020319:Oligodeoxyribonucleotides, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D019328:Buthionine Sulfoximine; D004054:Diethylstilbestrol; D005978:Glutathione