Valproic Acid Addresses Neuroendocrine Differentiation of LNCaP Cells and Maintains Cell Survival

Abstract

Purpose: Neuroendocrine differentiation of prostate cancer, induced by androgen deprivation therapy, is mainly related to advanced disease and poor clinical outcome. Genetic and epigenetic alterations are the key elements of the prostate carcinogenesis. A group of compounds able to induce changes in this sense is inhibitors of histone deacetylase, to which it belongs valproic acid (VPA). In the present paper, we evaluated the role of this molecule on the neuroendocrine differentiation of LNCaP cells together with the effect on proliferation and survival signals.

Methods: Cell growth was analyzed by MTT and flow cytometry, while expression of proteins through Western blot analysis.

Results: Our results have documented that VPA in LNCaP cells reduces cell proliferation, decreases the S phase and Cyclin A, and up-regulates the cyclin-dependent kinase inhibitors p21waf and p27. The acquisition of androgen-independent condition is consistent with an induction of β-III Tubulin and gamma Enolase, both markers of neuroendocrine phenotype. However, all these features cease with the removal of valproate from the culture medium, demonstrating the transitory nature of the epigenetic event. The VPA treatment does not compromise the survival phosphorylated signals of Akt, ERK1/2 and mTOR/p70S6K that remain up-regulated. Consistently, there is an increase of phospho-FOXO3a, to which corresponds the decreased expression of the corresponding oncosuppressor protein.

Conclusion: Overall, our findings indicate that VPA in LNCaP prostate tumor cells, although it reduces cell proliferation, is able to drive neuroendocrine phenotype and to maintain the survival of these cells. Keeping in mind that neuroendocrine differentiation of prostate cancer appears to be associated with a poor prognosis, it is necessary to develop new treatments that do not induce neurodifferentiation but able to counteract cell survival.

Keywords: cell cycle; cell proliferation; neuroendocrine tumor; prostate cancer.

Figure 1

Effects of valproic acid on human prostate LNCaP cell growth. MTT growth assays in LNCaP cells treated for 24, 48, 72 and 96 hrs with vehicle (C) or 1mM of Valproic acid (VPA). The histograms represent the mean ± SD of three separate experiments, performed in triplicate. *p<0.05, **p<0.01 vs C.

Figure 2

Effects of valproic acid on cell cycle distribution in prostate cancer cells. Cell cycle profile of LNCaP cells treated for 48, 72 and 96 hrs with vehicle (C) or 1mM of VPA (A). The latter condition at 96 hrs was reproduced in two other plates, drug-withdrawal and incubated for 72 and 96 hrs with refreshed medium (RM) (C), as described in the 'Material and Methods section. Cells were stained with propidium iodide and analyzed on a FACScan flow cytometer. Quantitative analysis of percentage gated cells at G0/G1, S and G2/M phases in the above reported experimental conditions (B and D respectively). The results are representative of three independent experiments, with similar results.

Figure 3

Influence of treatment on cell cycle proteins in LNCaP cells. LNCaP cells were treated with vehicle (C) or 1mM of VPA for 48 and 72 h. The latter condition was reproduced in triplicate, two of these plates were replaced by drug-free media (-VPA) and harvested for lysis respectively after 72 and 96 hrs. Replacement of media was also done for the control. (A). Equal amounts of total cellular extracts were analyzed for Cyclin D1, Cyclin A, p21Cip1/WAF1 and p27 protein levels by immunoblotting analysis. GAPDH was used as loading control. The Western blot is representative of three experiments, with similar results. The histograms represent the mean ± SD of three separate experiments in which band intensities were evaluated in terms of optical density arbitrary units and expressed as percentage versus vehicle-treated samples, respectively (B). *p<0.05, **p<0.01 vs C.

Figure 4

Changes of AR, PSA, β-III Tubulin and γ-Enolase protein expressions together with morphological modifications of human prostate cancer cells under valproic acid. LNCaP cells were treated with vehicle (C) or 1mM of VPA for 48 and 72 hrs. The latter condition was reproduced in triplicate, two of these plates were replaced by drug-free media (-VPA) and harvested for lysis respectively after 72 and 96 hrs. Replacement of media was also done for the corresponding control (A). Equal amounts of total cellular extracts were analyzed for androgen receptor (AR), prostatic-specific antigen (PSA), β-III Tubulin and γ-Enolase protein levels by immunoblotting analysis. GAPDH was used as loading control. Optical microscope imaging of untreated (left) and treated (right) LNCaP cells with VPA (1mM for 72 hrs) (C). Magnification 200×. The arrows in panel C indicate the cytoplasmic extensions similar to axonal figures. The W.B. is representative of three experiments, with similar results. The histograms represent the mean ± SD of three separate experiments in which band intensities were evaluated in terms of optical density arbitrary units and expressed as percentage versus vehicle-treated samples, respectively (B). *p<0.05, **p<0.01 vs C.

Figure 5

Valproic acid sustains phosphorylative transductional signals in LNCaP cells. LNCaP cells were treated with 1mM of VPA at different times (30ʹ, 3, 6, 12, 48 and 72 hrs) before lysis (A, C). Equal amounts of total cellular extracts were analyzed for the phosphorylated and total forms of ERK1/2, Akt, mTOR, p70 and FOXO 3 proteins by Western blot. GADPH was probed as a loading control. The W.B. is representative of three experiments, with similar results. The histograms represent the mean ± SD of three separate experiments in which band intensities were evaluated in terms of optical density arbitrary units and expressed as fold change versus vehicle-treated samples (B, D). *p<0.05, **p<0.01 vs C.