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Nitric oxide sensitizes prostate carcinoma cell lines to TRAIL-mediated apoptosis via inactivation of NF-κB and inhibition of Bcl-xL expression

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to be selective in the induction of apoptosis in cancer cells with minimal toxicity to normal tissues and this prompted its potential therapeutic application in cancer. However, not all cancers are sensitive to TRAIL-mediated apoptosis and, therefore, TRAIL-resistant cancer cells must be sensitized first to become sensitive to TRAIL. Treatment of prostate cancer (CaP) cell lines (DU145, PC-3, CL-1, and LNCaP) with nitric oxide donors (e.g. (Z)-1-[2-(2-aminoethyl)-N-(2-ammonio-ethyl)amino]diazen-1-ium-1, 2-diolate (DETANONOate)) sensitized CaP cells to TRAIL-induced apoptosis and synergy was achieved. The mechanism by which DETANONOate mediated the sensitization was examined. DETANONOate inhibited the constitutive NF-κB activity as assessed by EMSA. Also, p50 was S-nitrosylated by DETANONOate resulting in inhibition of NF-κB. Inhibition of NF-κB activity by the chemical inhibitor Bay 11-7085, like DETANONOate, sensitized CaP to TRAIL apoptosis. In addition, DETANONOate downregulated the expression of Bcl-2 related gene (Bcl-xL) which is under the transcriptional regulation of NF-κB. The regulation of NF-κB and Bcl-xL by DETANONOate was corroborated by the use of Bcl-xL and Bcl-x κB reporter systems. DETANONOate inhibited luciferase activity in the wild type and had no effect on the mutant cells. Inhibition of NF-κB resulted in downregulation of Bcl-xL expression and sensitized CaP to TRAIL-induced apoptosis. The role of Bcl-xL in the regulation of TRAIL apoptosis was corroborated by inhibiting Bcl-xL function by the chemical inhibitor 2-methoxyantimycin A3 and this resulted in sensitization of the cells to TRAIL apoptosis. Signaling by DETANONOate and TRAIL for apoptosis was examined. DETANONOate altered the mitochondria by inducing membrane depolarization and releasing modest amounts of cytochrome c and Smac/DIABLO in the absence of downstream activation of caspases 9 and 3. However, the combination of DETANONOate and TRAIL resulted in activation of the mitochondrial pathway and activation of caspases 9 and 3, and induction of apoptosis. These findings demonstrate that DETANONOate-mediated sensitization of CaP to TRAIL-induced apoptosis is via inhibition of constitutive NF-κB activity and Bcl-xL expression.

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Abbreviations

Bcl-xL:

Bcl-2 related gene

CaP:

prostate cancer

DETANONOate:

(Z)-1-[2-(2-aminoethyl)-N-(2-ammonio-ethyl)amino]diazen-1-ium-1, 2-diolate

DHT:

5-α dihydrotestosterone

DR:

death receptor

DTT:

1,4-dithiothreitol

EDTA:

ethylenediaminetetraacetic acid

FBS:

fetal bovine serum

FITC:

fluorescein isothiocyanate

IAP:

inhibitor of apoptosis protein

IKK:

IκB kinase complex

JNK:

c-Jun N-terminal kinase

2MAM-A3:

2-methoxyantimycin A3

NF-κB:

nuclear factor κB

NO:

nitric oxide

PAGE:

polyacrylamide gel electrophoresis

PBS:

phosphate-buffered saline

PI:

propidium iodide

RIPA:

radioimmunoprecipitation assay (buffer)

SDS:

sodium dodecyl sulfate

Smac/DIABLO:

second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low PI

TNF-α:

tumor necrosis factor alpha

TPA:

12-O-tetradecanoylphorbolacetate

TRAIL:

tumor necrosis factor-related apoptosis-inducing ligand

XIAP:

X-linked inhibitor of apoptosis

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Acknowledgements

This study was supported by the UCLA SPORE in Prostate Cancer (P50 CA92131-01A1), a grant from the Department of Defense (DOD/US Army DAMD 17-02-1-0023), by Fogarty Fellowships (D43 TW00013-14) (SH-Y, MV), and UC MEXUS-CONACYT (SH-Y). We acknowledge the assistance of Kate Dinh in the preparation of the manuscript.

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Correspondence to Benjamin Bonavida.

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Huerta-Yepez, S., Vega, M., Jazirehi, A. et al. Nitric oxide sensitizes prostate carcinoma cell lines to TRAIL-mediated apoptosis via inactivation of NF-κB and inhibition of Bcl-xL expression. Oncogene 23, 4993–5003 (2004). https://doi.org/10.1038/sj.onc.1207655

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