Development of human prostate cancer stem cells involves epigenomic alteration and PI3K/AKT pathway activation

Abstract

Background: Human prostate cancer spheres endowed with stem cell properties have been obtained from androgen-dependent cell line LNCaP after exposure to an epigenomic modulator phenethyl isothiocynate (PEITC). Sphere cells can self-renew and grow with androgen, and also without androgen. Little is known about the signaling pathway and mechanism in the development of the stem cells in the spheres.

Methods: Expression of phosphoinositol-3 kinase (PI3K) pathway members and histone acetylation were quantified in the tumor spheres and LNCaP cells by western immunoblotting.

Results: The level of phosphorylated AKT was significantly increased in the sphere stem cells than the LNCaP cells at an average of 7.4 folds (range 5.8-10.7 folds), whereas the P27 level was elevated 5.4 folds (range 4.8-6.3 folds) (P < 0.05). The acetylation level on histone H3 lysine 9 was decreased.

Conclusions: PEITC appears to regulate the epigenome through histone acetylation and activate the PI3K/AKT pathway in the LNCaP cells. This mechanism may be responsible in part for the development of the prostate cancer stem cells.

Keywords: AKT; Cancer stem cells; Histone acetylation; PI3K; Prostate cancer; Sphere.

Fig. 1

Cell cycle analysis of prostate tumor spheres and LNCaP cells. Cell cycle phase distribution in G1, S, G2M, and S + G2M was analyzed by flow cytometry study of LNCaP cells (grey color column) and sphere cells (solid bar). Columns and vertical bars are means and ranges from independent experiments

Fig. 2

Histone H3 acetylation and HDAC1 expression in the sphere and LNCaP cells. Capillary electrophoresis-western blot analysis was used to assess the level of acetylated H3K9 and HDAC1 expression. The electropherogram of acetylated H3K9, HDAC1, and β-actin proteins from the lysates of the LNCaP cells (LN) and spheres (SpH) is shown in the left panel, where Y axis represents the chemiluminescence and the X axis molecular weight (kDa). The right panel of the figure shows the Western blot images of H3K9, HDAC1 and β-actin. The β-actin was used as a loading control

Fig. 3

Analysis of phosphoinositol-3 kinase pathway members. Upper left panel: representative blots of p-AKT and AKT expression in the LNCaP cells and the spheres. β-actin was used as a loading control. Upper right panel: representative blots from capillary electrophoresis-western blot of protein p27 and mTOR from the spheres and the LNCaP cells. β-actin was used as a loading control. The bar graph in the lower panel depicts quantity of the protein expression in chemiluminescence units (area under peak) for each protein assayed by the capillary electrophoresis-western blot analysis. Solid bars indicate the values of the spheres, and open bars the values of LNCaP cells. Columns and vertical bars are means and ranges from independent experiments. The levels of p-AKT and P27 in the spheres and LNCaP cells were statistically significant (P < 0.05)