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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.


E2F6 regulates mocetinostat-induced apoptosis. (a) DU-145 cells were transfected with the negative control or E2F6-targeting siRNAs. At 48 h after siRNA transfection, cell lysates were analyzed by western blotting with the indicated antibodies. (b) DU-145 cells were transfected with the negative control or E2F6 siRNAs. At 24 h after siRNA transfection, cells were treated with 1 μM mocetinostat for additional 24 h. Apoptosis was measured by Cell Death Detection ElisaPLUS analysis as described in Materials and Methods. The experiments have been repeated three times; data shown are mean values+S.D. (c) DU-145 cells were transfected with the empty expression vector or pcDNA3-HA-E2F6. At 24 h after transfection, E2F6 overexpression was confirmed by western blot. (d) DU-145 cells were transfected with the empty expression vector or pcDNA3-HA-E2F6. At 24 h after transfection, cells were treated with 5 μM of mocetinostat for additional 24 h and apoptosis was measured by Cell Death Detection ElisaPLUS analysis as described in Materials and Methods. The experiments have been repeated three times; data shown are mean values +S.D.
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fig4: E2F6 regulates mocetinostat-induced apoptosis. (a) DU-145 cells were transfected with the negative control or E2F6-targeting siRNAs. At 48 h after siRNA transfection, cell lysates were analyzed by western blotting with the indicated antibodies. (b) DU-145 cells were transfected with the negative control or E2F6 siRNAs. At 24 h after siRNA transfection, cells were treated with 1 μM mocetinostat for additional 24 h. Apoptosis was measured by Cell Death Detection ElisaPLUS analysis as described in Materials and Methods. The experiments have been repeated three times; data shown are mean values+S.D. (c) DU-145 cells were transfected with the empty expression vector or pcDNA3-HA-E2F6. At 24 h after transfection, E2F6 overexpression was confirmed by western blot. (d) DU-145 cells were transfected with the empty expression vector or pcDNA3-HA-E2F6. At 24 h after transfection, cells were treated with 5 μM of mocetinostat for additional 24 h and apoptosis was measured by Cell Death Detection ElisaPLUS analysis as described in Materials and Methods. The experiments have been repeated three times; data shown are mean values +S.D.

Mentions: To determine the role of E2F6 in mocetinostat-induced apoptosis, we used siRNA to knock down E2F6 in DU-145 cells. As shown in Figure 4a, E2F6 protein expression was significantly reduced by siRNA treatment. Knockdown of E2F6 sensitized DU-145 cells to apoptosis induced by mocetinostat (Figure 4b). We expressed E2F6 exogenously to determine if E2F6 can contribute to apoptosis resistance in prostate cancer cells. Overexpression of E2F6 was confirmed by western blotting (Figure 4c). When treated with mocetinostat, DU-145 cells overexpressing E2F6 were significantly more resistant to drug-induced apoptosis, comparing with empty vector-transfected cells (Figure 4d).


Class I HDAC inhibitor mocetinostat induces apoptosis by activation of miR-31 expression and suppression of E2F6
E2F6 regulates mocetinostat-induced apoptosis. (a) DU-145 cells were transfected with the negative control or E2F6-targeting siRNAs. At 48 h after siRNA transfection, cell lysates were analyzed by western blotting with the indicated antibodies. (b) DU-145 cells were transfected with the negative control or E2F6 siRNAs. At 24 h after siRNA transfection, cells were treated with 1 μM mocetinostat for additional 24 h. Apoptosis was measured by Cell Death Detection ElisaPLUS analysis as described in Materials and Methods. The experiments have been repeated three times; data shown are mean values+S.D. (c) DU-145 cells were transfected with the empty expression vector or pcDNA3-HA-E2F6. At 24 h after transfection, E2F6 overexpression was confirmed by western blot. (d) DU-145 cells were transfected with the empty expression vector or pcDNA3-HA-E2F6. At 24 h after transfection, cells were treated with 5 μM of mocetinostat for additional 24 h and apoptosis was measured by Cell Death Detection ElisaPLUS analysis as described in Materials and Methods. The experiments have been repeated three times; data shown are mean values +S.D.
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fig4: E2F6 regulates mocetinostat-induced apoptosis. (a) DU-145 cells were transfected with the negative control or E2F6-targeting siRNAs. At 48 h after siRNA transfection, cell lysates were analyzed by western blotting with the indicated antibodies. (b) DU-145 cells were transfected with the negative control or E2F6 siRNAs. At 24 h after siRNA transfection, cells were treated with 1 μM mocetinostat for additional 24 h. Apoptosis was measured by Cell Death Detection ElisaPLUS analysis as described in Materials and Methods. The experiments have been repeated three times; data shown are mean values+S.D. (c) DU-145 cells were transfected with the empty expression vector or pcDNA3-HA-E2F6. At 24 h after transfection, E2F6 overexpression was confirmed by western blot. (d) DU-145 cells were transfected with the empty expression vector or pcDNA3-HA-E2F6. At 24 h after transfection, cells were treated with 5 μM of mocetinostat for additional 24 h and apoptosis was measured by Cell Death Detection ElisaPLUS analysis as described in Materials and Methods. The experiments have been repeated three times; data shown are mean values +S.D.
Mentions: To determine the role of E2F6 in mocetinostat-induced apoptosis, we used siRNA to knock down E2F6 in DU-145 cells. As shown in Figure 4a, E2F6 protein expression was significantly reduced by siRNA treatment. Knockdown of E2F6 sensitized DU-145 cells to apoptosis induced by mocetinostat (Figure 4b). We expressed E2F6 exogenously to determine if E2F6 can contribute to apoptosis resistance in prostate cancer cells. Overexpression of E2F6 was confirmed by western blotting (Figure 4c). When treated with mocetinostat, DU-145 cells overexpressing E2F6 were significantly more resistant to drug-induced apoptosis, comparing with empty vector-transfected cells (Figure 4d).

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.