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Class I HDAC inhibitor mocetinostat induces apoptosis by activation of miR-31 expression and suppression of E2F6

View Article: PubMed Central - PubMed

ABSTRACT

The class I selective inhibitor of the histone deacetylases, mocetinostat, has promising antitumor activities in both preclinical studies and the clinical trials. To understand how mocetinostat induces apoptosis, we examined the effects of mocetinostat on miR-31, a proapoptotic microRNA that was previously found to be epigenetically silenced in prostate cancer. We found that miR-31 was significantly upregulated by mocetinostat in prostate cancer cells. Antiapoptotic protein E2F6, the target of miR-31, was decreased by mocetinostat treatment. When miR-31 was blocked with an inhibitor, the ability of mocetinostat to induce apoptosis was reduced. We further demonstrated that mocetinostat enhanced the activity of docetaxel in apoptosis induction. While siRNA knockdown of E2F6 sensitized cancer cells to mocetinostat-induced apoptosis, overexpression of E2F6 blocked mocetinostat-induced apoptosis. In an orthotopic xenograft model, we demonstrated that mocetinostat activated miR-31, decreased E2F6, induced apoptosis, and significantly reduced prostate cancer growth. Importantly, we found that mocetinostat also increased miR-31 expression, decreased E2F6, and induced apoptosis in the primary prostate cancer stem cells. Thus, activation of miR-31 and downregulation of E2F6 constitute an important mechanism in mocetinostat-induced apoptosis in prostate cancer.

No MeSH data available.


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Mocetinostat activates miR-31 expression in prostate cancer cells. (a) DU-145 cells and (c) PC-3 cells were treated with 5 μM mocetinostat for 24 h. Total RNA was isolated from untreated (control) and mocetinostat-treated cells and real-time PCR analysis was performed as described in Materials and Methods. (b) DU-145 and (d) PC-3 cells were treated with various doses of mocetinostat for 24 h. Western blotting was performed with anti-E2F6 and antitubulin antibodies. (e and f) DU-145 cells were transfected with negative control or HDAC1 siRNAs. Real-time PCR and western blotting were performed as described in (a–d). The experiments have been repeated three times; data shown are mean values +S.D.
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fig2: Mocetinostat activates miR-31 expression in prostate cancer cells. (a) DU-145 cells and (c) PC-3 cells were treated with 5 μM mocetinostat for 24 h. Total RNA was isolated from untreated (control) and mocetinostat-treated cells and real-time PCR analysis was performed as described in Materials and Methods. (b) DU-145 and (d) PC-3 cells were treated with various doses of mocetinostat for 24 h. Western blotting was performed with anti-E2F6 and antitubulin antibodies. (e and f) DU-145 cells were transfected with negative control or HDAC1 siRNAs. Real-time PCR and western blotting were performed as described in (a–d). The experiments have been repeated three times; data shown are mean values +S.D.

Mentions: We recently demonstrated that the downregulation of miR-31 contributes to apoptosis resistance in prostate cancer cells.22 Since miR-31 was shown to be repressed by epigenetic mechanisms in prostate cancer,23 we hypothesized that mocetinostat may activate miR-31 expression. The effects of mocetinostat on miR-31 expression were determined by real-time PCR. As shown in Figures 2a and c, mocetinostat significantly induced miR-31 expression in both DU-145 and PC-3 cells. We have previously shown that miR-31 targets E2F6,22 which is a potent antiapoptotic protein that can inhibit UV- and hypoxia-induced apoptosis.26,27 As a result of miR-31 induction, mocetinostat significantly decreased E2F6 protein in DU-145 and PC-3 cells (Figures 2b and d). To determine whether HDAC inhibition by mocetinostat is responsible for the activation of miR-31, we used siRNA to specifically knock down HDAC1. As shown in Figures 2e and f, siRNA knockdown of HDAC1 activated miR-31 expression and decreased E2F6 protein. To further understand the mechanism of mocetinostat-induced apoptosis, we determined the effects of mocetinostat on the expression of the proapoptotic members of the Bcl-2 family proteins.28 Interestingly, Bad was significantly increased by mocetinostat treatment while the expression levels of the other proteins were either reduced (Puma, Bid, and Bax) or unchanged (Bak) (Figure 3a). Since Bad is a key proapoptotic protein that triggers the intrinsic pathway of apoptosis,29,30 we examined the effects of mocetinostat on the caspases. As shown in Figure 3b, mocetinostat treatment significantly increased the levels of activated (cleaved) caspase-9 and caspase-3. The cleaved products of PARP, substrates of the caspases, were also increased by mocetinostat.


Class I HDAC inhibitor mocetinostat induces apoptosis by activation of miR-31 expression and suppression of E2F6
Mocetinostat activates miR-31 expression in prostate cancer cells. (a) DU-145 cells and (c) PC-3 cells were treated with 5 μM mocetinostat for 24 h. Total RNA was isolated from untreated (control) and mocetinostat-treated cells and real-time PCR analysis was performed as described in Materials and Methods. (b) DU-145 and (d) PC-3 cells were treated with various doses of mocetinostat for 24 h. Western blotting was performed with anti-E2F6 and antitubulin antibodies. (e and f) DU-145 cells were transfected with negative control or HDAC1 siRNAs. Real-time PCR and western blotting were performed as described in (a–d). The experiments have been repeated three times; data shown are mean values +S.D.
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Related In: Results  -  Collection

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fig2: Mocetinostat activates miR-31 expression in prostate cancer cells. (a) DU-145 cells and (c) PC-3 cells were treated with 5 μM mocetinostat for 24 h. Total RNA was isolated from untreated (control) and mocetinostat-treated cells and real-time PCR analysis was performed as described in Materials and Methods. (b) DU-145 and (d) PC-3 cells were treated with various doses of mocetinostat for 24 h. Western blotting was performed with anti-E2F6 and antitubulin antibodies. (e and f) DU-145 cells were transfected with negative control or HDAC1 siRNAs. Real-time PCR and western blotting were performed as described in (a–d). The experiments have been repeated three times; data shown are mean values +S.D.
Mentions: We recently demonstrated that the downregulation of miR-31 contributes to apoptosis resistance in prostate cancer cells.22 Since miR-31 was shown to be repressed by epigenetic mechanisms in prostate cancer,23 we hypothesized that mocetinostat may activate miR-31 expression. The effects of mocetinostat on miR-31 expression were determined by real-time PCR. As shown in Figures 2a and c, mocetinostat significantly induced miR-31 expression in both DU-145 and PC-3 cells. We have previously shown that miR-31 targets E2F6,22 which is a potent antiapoptotic protein that can inhibit UV- and hypoxia-induced apoptosis.26,27 As a result of miR-31 induction, mocetinostat significantly decreased E2F6 protein in DU-145 and PC-3 cells (Figures 2b and d). To determine whether HDAC inhibition by mocetinostat is responsible for the activation of miR-31, we used siRNA to specifically knock down HDAC1. As shown in Figures 2e and f, siRNA knockdown of HDAC1 activated miR-31 expression and decreased E2F6 protein. To further understand the mechanism of mocetinostat-induced apoptosis, we determined the effects of mocetinostat on the expression of the proapoptotic members of the Bcl-2 family proteins.28 Interestingly, Bad was significantly increased by mocetinostat treatment while the expression levels of the other proteins were either reduced (Puma, Bid, and Bax) or unchanged (Bak) (Figure 3a). Since Bad is a key proapoptotic protein that triggers the intrinsic pathway of apoptosis,29,30 we examined the effects of mocetinostat on the caspases. As shown in Figure 3b, mocetinostat treatment significantly increased the levels of activated (cleaved) caspase-9 and caspase-3. The cleaved products of PARP, substrates of the caspases, were also increased by mocetinostat.

View Article: PubMed Central - PubMed

ABSTRACT

The class I selective inhibitor of the histone deacetylases, mocetinostat, has promising antitumor activities in both preclinical studies and the clinical trials. To understand how mocetinostat induces apoptosis, we examined the effects of mocetinostat on miR-31, a proapoptotic microRNA that was previously found to be epigenetically silenced in prostate cancer. We found that miR-31 was significantly upregulated by mocetinostat in prostate cancer cells. Antiapoptotic protein E2F6, the target of miR-31, was decreased by mocetinostat treatment. When miR-31 was blocked with an inhibitor, the ability of mocetinostat to induce apoptosis was reduced. We further demonstrated that mocetinostat enhanced the activity of docetaxel in apoptosis induction. While siRNA knockdown of E2F6 sensitized cancer cells to mocetinostat-induced apoptosis, overexpression of E2F6 blocked mocetinostat-induced apoptosis. In an orthotopic xenograft model, we demonstrated that mocetinostat activated miR-31, decreased E2F6, induced apoptosis, and significantly reduced prostate cancer growth. Importantly, we found that mocetinostat also increased miR-31 expression, decreased E2F6, and induced apoptosis in the primary prostate cancer stem cells. Thus, activation of miR-31 and downregulation of E2F6 constitute an important mechanism in mocetinostat-induced apoptosis in prostate cancer.

No MeSH data available.


Related in: MedlinePlus