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Valproic acid induces autophagy by suppressing the Akt/mTOR pathway in human prostate cancer cells

View Article: PubMed Central - PubMed

ABSTRACT

Previous studies have demonstrated that the chronic administration of valproic acid (VPA) suppresses angiogenesis in vivo; however, the mechanisms implicated in VPA-induced autophagy remain unclear. The current study aimed to assess VPA-induced autophagy in three prostate cancer cell lines (PC3, DU145 and LNCaP), in addition to analyzing the Akt/mammalian target of rapamycin (mTOR) signal pathway. Prostate cancer cell lines were cultured with various doses of VPA. Cell cycle was analyzed using flow cytometry, and autophagy markers [1A/1B-light chain 3 (LC3)-II and Beclin-1] were examined using transmission electron microscopy, fluorescent microscopy and western blotting. Activation of the Akt/mTOR signal pathway was also assessed by western blotting. The results demonstrated that VPA induced autophagosomes and suppressed the Akt/mTOR signal pathway. This was confirmed by detection of increased LC3-II and Beclin-1 in VPA-treated cells compared with untreated controls. Phosphorylated forms of Akt (PC3, P=0.048; DU145, P=0.045; LNCaP, P=0.039) and mTOR (PC3, P=0.012; DU145, P=0.41; LNCaP, P=0.35) were significantly reduced following VPA treatment. These results suggest that VPA may function as a histone deacetylase inhibitor, suppressing the growth of prostate cancer cells by modulating autophagy pathways, including inhibition of the Akt/mTOR pathway. Further experiments are required to determine the significance of all involved pathways regarding VPA-induced growth inhibition.

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Expression of specific autophagic proteins, LC3-II and Beclin-1, in prostate cancer cells. There was an overlap in the distribution and expression of (A) LC3 and (B) Beclin-1 protein, which merged with DAPI nuclear staining (blue). Cells untreated or treated with VPA (2.5 mmol/l for 48 h) were fixed and probed with polyclonal rabbit anti-LC3-II and anti-Beclin-1 antibodies, in addition to DyLight 549-conjugated (red) secondary antibodies. (C) Western blot analysis detected the induction of LC3-II and Beclin-1 protein expression in cells treated with VPA (1.2, 2.5 and 5.0 mmol/l) compared with the non-treated control. LC3-II and Beclin-1 expression increased in a dose-dependent manner in all cell lines after 48 h incubation with VPA. (D) Histograms representing the level of LC3-II and Beclin-1 expression in LNCaP and DU145 cells (normalized to β-actin). In LNCaP cells, LC3-II and Beclin-1 protein levels in the 2.5 mmol/l and 5.0 mmol/l VPA treatment groups were significantly increased compared with the control groups. In addition, 1.2 mmol/l VPA treatment did not significantly increase LC3-II and Beclin-1 protein levels. In DU145 cells, LC3-II protein levels in 2.5 mmol/l and 5.0 mmol/l VPA treatment groups were significantly increased compared with the control groups. Beclin-1 protein levels in all three VPA treatment groups were significantly increased compared with the control groups. *P<0.05. LC3, 1A/1B-light chain 3; VPA, valproic acid.
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f3-ol-0-0-4880: Expression of specific autophagic proteins, LC3-II and Beclin-1, in prostate cancer cells. There was an overlap in the distribution and expression of (A) LC3 and (B) Beclin-1 protein, which merged with DAPI nuclear staining (blue). Cells untreated or treated with VPA (2.5 mmol/l for 48 h) were fixed and probed with polyclonal rabbit anti-LC3-II and anti-Beclin-1 antibodies, in addition to DyLight 549-conjugated (red) secondary antibodies. (C) Western blot analysis detected the induction of LC3-II and Beclin-1 protein expression in cells treated with VPA (1.2, 2.5 and 5.0 mmol/l) compared with the non-treated control. LC3-II and Beclin-1 expression increased in a dose-dependent manner in all cell lines after 48 h incubation with VPA. (D) Histograms representing the level of LC3-II and Beclin-1 expression in LNCaP and DU145 cells (normalized to β-actin). In LNCaP cells, LC3-II and Beclin-1 protein levels in the 2.5 mmol/l and 5.0 mmol/l VPA treatment groups were significantly increased compared with the control groups. In addition, 1.2 mmol/l VPA treatment did not significantly increase LC3-II and Beclin-1 protein levels. In DU145 cells, LC3-II protein levels in 2.5 mmol/l and 5.0 mmol/l VPA treatment groups were significantly increased compared with the control groups. Beclin-1 protein levels in all three VPA treatment groups were significantly increased compared with the control groups. *P<0.05. LC3, 1A/1B-light chain 3; VPA, valproic acid.

Mentions: Accumulation of LC3-II is associated with the extent of autophagosome formation (13). Following DyLight-549 staining, the cells in the VPA treatment group exhibited red fluorescence in the cytoplasm, but not in the nucleolus, and the fluorescence was stronger than that of the non-treated control group, thus indicating the formation of autophagolysosomes (Fig. 3A). These autophagic changes were further confirmed by western blotting as presented in Fig. 3C and D (LNCaP, P=0.019; DU145, P=0.043). Furthermore, Beclin-1 and LC3-II exhibited strong immunofluorescence in the cytoplasm of the DU145 and LNCaP cells compared with the non-treated control group (Fig. 3A and B), which was also detected by western blot analysis (LNCaP, P=0.033; DU145, P=0.027) (Fig. 3C and 3D).


Valproic acid induces autophagy by suppressing the Akt/mTOR pathway in human prostate cancer cells
Expression of specific autophagic proteins, LC3-II and Beclin-1, in prostate cancer cells. There was an overlap in the distribution and expression of (A) LC3 and (B) Beclin-1 protein, which merged with DAPI nuclear staining (blue). Cells untreated or treated with VPA (2.5 mmol/l for 48 h) were fixed and probed with polyclonal rabbit anti-LC3-II and anti-Beclin-1 antibodies, in addition to DyLight 549-conjugated (red) secondary antibodies. (C) Western blot analysis detected the induction of LC3-II and Beclin-1 protein expression in cells treated with VPA (1.2, 2.5 and 5.0 mmol/l) compared with the non-treated control. LC3-II and Beclin-1 expression increased in a dose-dependent manner in all cell lines after 48 h incubation with VPA. (D) Histograms representing the level of LC3-II and Beclin-1 expression in LNCaP and DU145 cells (normalized to β-actin). In LNCaP cells, LC3-II and Beclin-1 protein levels in the 2.5 mmol/l and 5.0 mmol/l VPA treatment groups were significantly increased compared with the control groups. In addition, 1.2 mmol/l VPA treatment did not significantly increase LC3-II and Beclin-1 protein levels. In DU145 cells, LC3-II protein levels in 2.5 mmol/l and 5.0 mmol/l VPA treatment groups were significantly increased compared with the control groups. Beclin-1 protein levels in all three VPA treatment groups were significantly increased compared with the control groups. *P<0.05. LC3, 1A/1B-light chain 3; VPA, valproic acid.
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f3-ol-0-0-4880: Expression of specific autophagic proteins, LC3-II and Beclin-1, in prostate cancer cells. There was an overlap in the distribution and expression of (A) LC3 and (B) Beclin-1 protein, which merged with DAPI nuclear staining (blue). Cells untreated or treated with VPA (2.5 mmol/l for 48 h) were fixed and probed with polyclonal rabbit anti-LC3-II and anti-Beclin-1 antibodies, in addition to DyLight 549-conjugated (red) secondary antibodies. (C) Western blot analysis detected the induction of LC3-II and Beclin-1 protein expression in cells treated with VPA (1.2, 2.5 and 5.0 mmol/l) compared with the non-treated control. LC3-II and Beclin-1 expression increased in a dose-dependent manner in all cell lines after 48 h incubation with VPA. (D) Histograms representing the level of LC3-II and Beclin-1 expression in LNCaP and DU145 cells (normalized to β-actin). In LNCaP cells, LC3-II and Beclin-1 protein levels in the 2.5 mmol/l and 5.0 mmol/l VPA treatment groups were significantly increased compared with the control groups. In addition, 1.2 mmol/l VPA treatment did not significantly increase LC3-II and Beclin-1 protein levels. In DU145 cells, LC3-II protein levels in 2.5 mmol/l and 5.0 mmol/l VPA treatment groups were significantly increased compared with the control groups. Beclin-1 protein levels in all three VPA treatment groups were significantly increased compared with the control groups. *P<0.05. LC3, 1A/1B-light chain 3; VPA, valproic acid.
Mentions: Accumulation of LC3-II is associated with the extent of autophagosome formation (13). Following DyLight-549 staining, the cells in the VPA treatment group exhibited red fluorescence in the cytoplasm, but not in the nucleolus, and the fluorescence was stronger than that of the non-treated control group, thus indicating the formation of autophagolysosomes (Fig. 3A). These autophagic changes were further confirmed by western blotting as presented in Fig. 3C and D (LNCaP, P=0.019; DU145, P=0.043). Furthermore, Beclin-1 and LC3-II exhibited strong immunofluorescence in the cytoplasm of the DU145 and LNCaP cells compared with the non-treated control group (Fig. 3A and B), which was also detected by western blot analysis (LNCaP, P=0.033; DU145, P=0.027) (Fig. 3C and 3D).

View Article: PubMed Central - PubMed

ABSTRACT

Previous studies have demonstrated that the chronic administration of valproic acid (VPA) suppresses angiogenesis in vivo; however, the mechanisms implicated in VPA-induced autophagy remain unclear. The current study aimed to assess VPA-induced autophagy in three prostate cancer cell lines (PC3, DU145 and LNCaP), in addition to analyzing the Akt/mammalian target of rapamycin (mTOR) signal pathway. Prostate cancer cell lines were cultured with various doses of VPA. Cell cycle was analyzed using flow cytometry, and autophagy markers [1A/1B-light chain 3 (LC3)-II and Beclin-1] were examined using transmission electron microscopy, fluorescent microscopy and western blotting. Activation of the Akt/mTOR signal pathway was also assessed by western blotting. The results demonstrated that VPA induced autophagosomes and suppressed the Akt/mTOR signal pathway. This was confirmed by detection of increased LC3-II and Beclin-1 in VPA-treated cells compared with untreated controls. Phosphorylated forms of Akt (PC3, P=0.048; DU145, P=0.045; LNCaP, P=0.039) and mTOR (PC3, P=0.012; DU145, P=0.41; LNCaP, P=0.35) were significantly reduced following VPA treatment. These results suggest that VPA may function as a histone deacetylase inhibitor, suppressing the growth of prostate cancer cells by modulating autophagy pathways, including inhibition of the Akt/mTOR pathway. Further experiments are required to determine the significance of all involved pathways regarding VPA-induced growth inhibition.

No MeSH data available.


Related in: MedlinePlus