Cellular prostatic acid phosphatase (cPAcP) serves as a useful biomarker of histone deacetylase (HDAC) inhibitors in prostate cancer cell growth suppression

Abstract

Background: Prostate cancer (PCa) is the most commonly diagnosed solid tumor and the second leading cancer death in the United States, and also one of the major cancer-related deaths in Chinese. Androgen deprivation therapy (ADT) is the first line treatment for metastatic PCa. PCa ultimately relapses with subsequent ADT treatment failure and becomes castrate-resistant (CR). It is important to develop effective therapies with a surrogate marker towards CR PCa.

Method: Histone deacetylase (HDAC) inhibitors were examined to determine their effects in androgen receptor (AR)/cellular prostatic acid phosphatase (cPAcP)-positive PCa cells, including LNCaP C-33, C-81, C4-2 and C4-2B and MDA PCa2b androgen-sensitive and androgen-independent cells, and AR/cPAcP-negative PCa cells, including PC-3 and DU 145 cells. Cell growth was determined by cell number counting. Western blot analyses were carried out to determine AR, cPAcP and PSA protein levels.

Results: cPAcP protein level was increased by HDAC inhibitor treatment. Valproic acid, a HDAC inhibitor, suppressed the growth of AR/cPAcP-positive PCa cells by over 50% in steroid-reduced conditions, higher than on AR/cPAcP-negative PCa cells. Further, HDAC inhibitor pretreatments increased androgen responsiveness as demonstrated by PSA protein level quantitation.

Conclusion: Our results clearly demonstrate that HDAC inhibitors can induce cPAcP protein level, increase androgen responsiveness, and exhibit higher inhibitory activities on AR/cPAcP-positive PCa cells than on AR/cPAcP-negative PCa cells. Upon HDAC inhibitor pretreatment, PSA level was greatly elevated by androgens. This data indicates the potential clinical importance of cPAcP serving as a useful biomarker in the identification of PCa patient sub-population suitable for HDAC inhibitor treatment.

Keywords: Biomarker; Cellular prostatic acid phosphatase; Histone deacetylase inhibitor; Prostate cancer.

Figure 1

The basal expression levels of AR, cPAcP and PSA were determined in different PCa cell lines. LNCaP C-33/C-81, LNCaP C4-2/C4-2B, MDA PCa2B AS/AI, PC-3 and DU 145 cells that were plated in regular medium for 3 days and then changed with fresh medium for 1 day. The cells were harvested and the total protein was subjected to western blot analyses of functional proteins expression. β-Actin was analyzed and used as a loading control.

Figure 2

Effect of VPA on androgen responsiveness of PCa cell lines. a LNCaP C-33; b LNCaP C4-2B; c MDA PCa2B AS cells were seeded in 6-wells plate and then treated with 1 mM VPA or solvent for 48 h. Cell were then maintained in a steroid-reduced medium with or without 10 nM DHT for 2 days. Total cell number was counted. The ratio of cell growth was calculated by normalizing the cell number to that of control cells (column #1, left panel, n = 3×2). Total cell lysate proteins from 3-day DHT treatment were analyzed for cPAcP, PSA, AR protein. β-Actin was analyzed and used as a loading control (right panel).

Figure 3

Effects of different HDAC inhibitors on androgen responsiveness of PCa cells. LNCaP C-81 cells were plated in 6-wells plate and then treated with a 1 mM NaB; b 2.5 µM SAHA; c 2.5 µM PxD101; d 1.0 µM MS-275; e 0.5 µM AR42 or solvent alone for 48 h. Cell were then maintained in a steroid-reduced medium with or without 1 and 10 nM DHT for 3 days. Total cell number was counted. The ratio of cell growth was calculated by normalizing the cell number to that of control cells (column #1, left panel, n = 3×2). Total cell lysate proteins were analyzed for cPAcP, PSA and AR protein. β-Actin was analyzed and used as a loading control (right panel).

Figure 4

The growth inhibition of VPA treatment on various PCa cells. LNCaP C-33/C-81, LNCaP C4-2/C4-2B, MDA PCa2B AS/AI, NE 1-3/1-8, PC-3 and DU 145 PCa cell lines were plated in 6-wells plates and then treated with 1 mM VPA or solvent for 48 h. Cells were then maintained in a steroid-reduced medium for 3 days. Total cell number was counted. The ratio of cell growth was calculated by normalizing the cell number to that of control cells (n = 3×2).