Korean J Androl.  2011 Apr;29(1):43-52. 10.5534/kja.2011.29.1.43.

Role of Clusterin and Tumor Necrosis Factor Receptors on the Apoptosis of Prostate Cancer Cells

Affiliations
  • 1Department of Urology, Sungkyunkwan University School of Medicine, Seoul, Korea. urojoo@dreamwiz.com

Abstract

PURPOSE
In prostate cancer, the anti-apoptotic mechanism of sulfated glycoprotein-2 (clusterin) against tumor necrosis factor-alpha (TNF-alpha) receptors and the action of type 2 TNF-alpha receptor (TNFR2) were investigated.
MATERIALS AND METHODS
TNF-alpha, agonistic-TNF type 1 receptor (TNFR1) antibody, agonistic-TNF-R2 antibody and their combination were treated in PC3 cell line with or without anti-clusterin. Cytotoxicity was assessed by trypan blue dye exclusion assay. By using flowcytometric analysis, the exact amount of apoptosis and their changes were assessed.
RESULTS
Apoptosis was significantly increased in both agonistic-TNFR1 antibody and TNF-alpha treated cases after blocking the activity of clusterin. The more the anti-clusterin antibody added, the more the apoptosis occurred. The increase of total apoptosis was greater in TNF-alpha treated cells than in agonistic-TNFR1 antibody treated ones. However, there was no increase of apoptosis in agonistic-TNFR2 antibody and TNF-alpha with agonistic-TNFR2 antibody treated cases, respectively.
CONCLUSIONS
Clusterin prevents TNF-alpha induced apoptosis by affecting TNFR1. The difference in degree of apoptosis between agonistic-TNFR1 antibody treated cells and TNF-alpha treated ones suggests the possibility of the action of TNFR2. It may be associated with affinity of TNF-alpha to the tumor cell surface.

Keyword

Prostate neoplasm; Clusterin; Tumor necrosis factor-alpha; Receptors; Tumor necrosis factor; Type II

MeSH Terms

Apoptosis
Cell Line
Clusterin
Diminazene
Prostate
Prostatic Neoplasms
Receptors, Tumor Necrosis Factor
Receptors, Tumor Necrosis Factor, Type I
Receptors, Tumor Necrosis Factor, Type II
Trypan Blue
Tumor Necrosis Factor-alpha
Clusterin
Diminazene
Receptors, Tumor Necrosis Factor
Receptors, Tumor Necrosis Factor, Type I
Receptors, Tumor Necrosis Factor, Type II
Trypan Blue
Tumor Necrosis Factor-alpha

Figure

  • Fig. 1. Assessment of the response of PC3 cell to the treatment of TNF-α with varying amount of anti-clusterin antibody. (A) Cell number was counted with Coulter hemocytometer. (B) Cell viability was evaluated by trypan-blue dye extraction assay. Each column represents mean value (bars, SD) of triple replicates experiments. Anti-SGP-2: anti-clusterin antibody. ∗p<0.05.

  • Fig. 2. Assessment of the response of PC3 cell to the treatment of agonistic-TNFR1 antibody with varying amount of anti-clusterin antibody. (A) Cell number was counted with Coulter hemocytometer. (B) Cell viability was evaluated by trypan-blue dye extraction assay. Each column represents mean value (bars, SD) of triple replicates experiments. Anti-SGP-2: anti-clusterin antibody. ∗p<0.05.

  • Fig. 3. Effects of anti-clusterin antibody to early and total apoptosis. (A) Cases of TNF-α treatment. (B) Cases of agonistic-TNFR1 antibody treatment. Anti-SGP-2: anti-clusterin antibody. ∗p<0.05.

  • Fig. 4. Effects of anti-clusterin antibody to early and total apoptosis. (A) Cases of TNF-α with agonistic-TNFR2 antibody treatment. (B) Cases of agonistic-TNFR1 antibody with agonistic-TNFR2 antibody treatment. Anti-SGP-2: anti-clusterin antibody. ∗p<0.05.

  • Fig. 5. Flowcytometric analysis of the apoptosis. (A) Cells treated with TNF-α plus agonistic-TNFR2 antibody in the presence of anti-clusterin antibody for 24 hours. Early and late apoptosis were not increased after 24 hours. (B) Cells treated with agonistic-TNFR1 antibody plus agonistic-TNFR2 antibody in the presence of anti-clusterin antibody for 24 hours. Early apoptosis was increased. (C) and (D) Cells treated with TNF-α plus agonistic-TNFR2 antibody and agonistic-TNFR1 antibody plus agonistic-TNFR2 antibody in the presence of anti-clusterin antibody for 36 hours respectively. The vertical scale denotes propidium iodide and the horizontal scale indicates fluorescein isothiocyanate labelled annexin V (lower left: normal cells, lower right: early apoptotic cells, upper right: late apoptotic cells, upper left: necrotic cells).


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