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Histone deacetylase turnover and recovery in sulforaphane-treated colon cancer cells: competing actions of 14-3-3 and Pin1 in HDAC3/SMRT corepressor complex dissociation/reassembly

Rajendran P, Delage B, Dashwood WM, Yu TW, Wuth B, Williams DE, Ho E, Dashwood RH - Mol. Cancer (2011)

Bottom Line: Mechanism studies revealed no apparent effect of calpain, proteasome, protease or caspase inhibitors, but HDAC3 was rescued by cycloheximide or actinomycin D treatment.The current investigation supports a model in which protein kinase CK2 phosphorylates SMRT and HDAC3 in the nucleus, resulting in dissociation of the corepressor complex and enhanced binding of HDAC3 to 14-3-3 or Pin1.The latter pathway predominates in colon cancer cells exposed continuously to SFN, whereas the former pathway is likely to be favored when SFN has been removed within 24 h, allowing recovery from cell cycle arrest.

Affiliation: Linus Pauling Institute, Oregon State University, Corvallis, OR 97331-6512, USA.

ABSTRACT

Background: Histone deacetylase (HDAC) inhibitors are currently undergoing clinical evaluation as anti-cancer agents. Dietary constituents share certain properties of HDAC inhibitor drugs, including the ability to induce global histone acetylation, turn-on epigenetically-silenced genes, and trigger cell cycle arrest, apoptosis, or differentiation in cancer cells. One such example is sulforaphane (SFN), an isothiocyanate derived from the glucosinolate precursor glucoraphanin, which is abundant in broccoli. Here, we examined the time-course and reversibility of SFN-induced HDAC changes in human colon cancer cells.

Results: Cells underwent progressive G2/M arrest over the period 6-72 h after SFN treatment, during which time HDAC activity increased in the vehicle-treated controls but not in SFN-treated cells. There was a time-dependent loss of class I and selected class II HDAC proteins, with HDAC3 depletion detected ahead of other HDACs. Mechanism studies revealed no apparent effect of calpain, proteasome, protease or caspase inhibitors, but HDAC3 was rescued by cycloheximide or actinomycin D treatment. Among the protein partners implicated in the HDAC3 turnover mechanism, silencing mediator for retinoid and thyroid hormone receptors (SMRT) was phosphorylated in the nucleus within 6 h of SFN treatment, as was HDAC3 itself. Co-immunoprecipitation assays revealed SFN-induced dissociation of HDAC3/SMRT complexes coinciding with increased binding of HDAC3 to 14-3-3 and peptidyl-prolyl cis/trans isomerase 1 (Pin1). Pin1 knockdown blocked the SFN-induced loss of HDAC3. Finally, SFN treatment for 6 or 24 h followed by SFN removal from the culture media led to complete recovery of HDAC activity and HDAC protein expression, during which time cells were released from G2/M arrest.

Conclusion: The current investigation supports a model in which protein kinase CK2 phosphorylates SMRT and HDAC3 in the nucleus, resulting in dissociation of the corepressor complex and enhanced binding of HDAC3 to 14-3-3 or Pin1. In the cytoplasm, release of HDAC3 from 14-3-3 followed by nuclear import is postulated to compete with a Pin1 pathway that directs HDAC3 for degradation. The latter pathway predominates in colon cancer cells exposed continuously to SFN, whereas the former pathway is likely to be favored when SFN has been removed within 24 h, allowing recovery from cell cycle arrest.

Probing the pathways of protein turnover and stability in SFN-treated colon cancer cells. (A) HCT116 cells were treated with MG132, N-acetyl-Leu-Leu-norleucinal (ALLN), leupeptin, or PYR-41 [47], in the presence and absence of SFN (15 μM). Whole cell lysates obtained at 24 h were immunoblotted for HDAC3. For the concentrations of each inhibitor, see Methods. (B) HDAC activity in the whole cell lysates obtained at 24 h from HCT116 cells treated with DMSO, SFN, PYR-41 (PYR), or SFN+PYR. Data (mean ± SE, n = 3); **P < 0.01 versus the DMSO control. (C) HCT116 cells were treated with inhibitors, as shown, and the whole cell lysates were immunoblotted at 24 h for total cellular ubiquitin. (D) HCT116 cells were treated with cycloheximide (CHX), actinomycin D (Act D), SFN, SFN+CHX, or SFN+Act D. Whole cell lysates were immunoblotted at 6 h for HDAC3, HDAC4, SMRT, N-Cor and p21WAF1. Data are representative of findings from two or more separate experiments.
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Figure 6: Probing the pathways of protein turnover and stability in SFN-treated colon cancer cells. (A) HCT116 cells were treated with MG132, N-acetyl-Leu-Leu-norleucinal (ALLN), leupeptin, or PYR-41 [47], in the presence and absence of SFN (15 μM). Whole cell lysates obtained at 24 h were immunoblotted for HDAC3. For the concentrations of each inhibitor, see Methods. (B) HDAC activity in the whole cell lysates obtained at 24 h from HCT116 cells treated with DMSO, SFN, PYR-41 (PYR), or SFN+PYR. Data (mean ± SE, n = 3); **P < 0.01 versus the DMSO control. (C) HCT116 cells were treated with inhibitors, as shown, and the whole cell lysates were immunoblotted at 24 h for total cellular ubiquitin. (D) HCT116 cells were treated with cycloheximide (CHX), actinomycin D (Act D), SFN, SFN+CHX, or SFN+Act D. Whole cell lysates were immunoblotted at 6 h for HDAC3, HDAC4, SMRT, N-Cor and p21WAF1. Data are representative of findings from two or more separate experiments.

Mentions: Following the caspase studies, subsequent experiments assessed mRNA transcript levels via quantitative real-time PCR, for class I and class II HDACs. No concordance was seen with respect to SFN-induced changes in HDAC protein expression (data not presented). Next, selected inhibitors were used to probe different pathways of protein turnover and stability. Proteasome inhibitor MG132, calpain inhibitor N-acetyl-Leu-Leu-norleucinal (ALLN), and protease inhibitor leupeptin did not block the SFN-induced loss of HDAC3 protein expression (Figure 6A). On the contrary, loss of HDAC3 was enhanced when SFN was combined with these inhibitors. Prior reports described the synergistic interactions between HDAC inhibitors and proteasome inhibitors [43-46]. PYR-41, a purported inhibitor of the E1 ubiquitin-activating enzyme [47], blocked the SFN-induced loss of HDAC3 protein expression (Figure 6A, lanes 9 and 10). HDAC activities in the corresponding PYR and PYR+SFN whole cell lysates were identical to the vehicle control (Figure 6B).

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Histone deacetylase turnover and recovery in sulforaphane-treated colon cancer cells: competing actions of 14-3-3 and Pin1 in HDAC3/SMRT corepressor complex dissociation/reassembly

Rajendran P, Delage B, Dashwood WM, Yu TW, Wuth B, Williams DE, Ho E, Dashwood RH - Mol. Cancer (2011)

Probing the pathways of protein turnover and stability in SFN-treated colon cancer cells. (A) HCT116 cells were treated with MG132, N-acetyl-Leu-Leu-norleucinal (ALLN), leupeptin, or PYR-41 [47], in the presence and absence of SFN (15 μM). Whole cell lysates obtained at 24 h were immunoblotted for HDAC3. For the concentrations of each inhibitor, see Methods. (B) HDAC activity in the whole cell lysates obtained at 24 h from HCT116 cells treated with DMSO, SFN, PYR-41 (PYR), or SFN+PYR. Data (mean ± SE, n = 3); **P < 0.01 versus the DMSO control. (C) HCT116 cells were treated with inhibitors, as shown, and the whole cell lysates were immunoblotted at 24 h for total cellular ubiquitin. (D) HCT116 cells were treated with cycloheximide (CHX), actinomycin D (Act D), SFN, SFN+CHX, or SFN+Act D. Whole cell lysates were immunoblotted at 6 h for HDAC3, HDAC4, SMRT, N-Cor and p21WAF1. Data are representative of findings from two or more separate experiments.
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Figure 6: Probing the pathways of protein turnover and stability in SFN-treated colon cancer cells. (A) HCT116 cells were treated with MG132, N-acetyl-Leu-Leu-norleucinal (ALLN), leupeptin, or PYR-41 [47], in the presence and absence of SFN (15 μM). Whole cell lysates obtained at 24 h were immunoblotted for HDAC3. For the concentrations of each inhibitor, see Methods. (B) HDAC activity in the whole cell lysates obtained at 24 h from HCT116 cells treated with DMSO, SFN, PYR-41 (PYR), or SFN+PYR. Data (mean ± SE, n = 3); **P < 0.01 versus the DMSO control. (C) HCT116 cells were treated with inhibitors, as shown, and the whole cell lysates were immunoblotted at 24 h for total cellular ubiquitin. (D) HCT116 cells were treated with cycloheximide (CHX), actinomycin D (Act D), SFN, SFN+CHX, or SFN+Act D. Whole cell lysates were immunoblotted at 6 h for HDAC3, HDAC4, SMRT, N-Cor and p21WAF1. Data are representative of findings from two or more separate experiments.
Mentions: Following the caspase studies, subsequent experiments assessed mRNA transcript levels via quantitative real-time PCR, for class I and class II HDACs. No concordance was seen with respect to SFN-induced changes in HDAC protein expression (data not presented). Next, selected inhibitors were used to probe different pathways of protein turnover and stability. Proteasome inhibitor MG132, calpain inhibitor N-acetyl-Leu-Leu-norleucinal (ALLN), and protease inhibitor leupeptin did not block the SFN-induced loss of HDAC3 protein expression (Figure 6A). On the contrary, loss of HDAC3 was enhanced when SFN was combined with these inhibitors. Prior reports described the synergistic interactions between HDAC inhibitors and proteasome inhibitors [43-46]. PYR-41, a purported inhibitor of the E1 ubiquitin-activating enzyme [47], blocked the SFN-induced loss of HDAC3 protein expression (Figure 6A, lanes 9 and 10). HDAC activities in the corresponding PYR and PYR+SFN whole cell lysates were identical to the vehicle control (Figure 6B).

Bottom Line: Mechanism studies revealed no apparent effect of calpain, proteasome, protease or caspase inhibitors, but HDAC3 was rescued by cycloheximide or actinomycin D treatment.The current investigation supports a model in which protein kinase CK2 phosphorylates SMRT and HDAC3 in the nucleus, resulting in dissociation of the corepressor complex and enhanced binding of HDAC3 to 14-3-3 or Pin1.The latter pathway predominates in colon cancer cells exposed continuously to SFN, whereas the former pathway is likely to be favored when SFN has been removed within 24 h, allowing recovery from cell cycle arrest.

Affiliation: Linus Pauling Institute, Oregon State University, Corvallis, OR 97331-6512, USA.

ABSTRACT

Background:

Background: Histone deacetylase (HDAC) inhibitors are currently undergoing clinical evaluation as anti-cancer agents. Dietary constituents share certain properties of HDAC inhibitor drugs, including the ability to induce global histone acetylation, turn-on epigenetically-silenced genes, and trigger cell cycle arrest, apoptosis, or differentiation in cancer cells. One such example is sulforaphane (SFN), an isothiocyanate derived from the glucosinolate precursor glucoraphanin, which is abundant in broccoli. Here, we examined the time-course and reversibility of SFN-induced HDAC changes in human colon cancer cells.

Results: Cells underwent progressive G2/M arrest over the period 6-72 h after SFN treatment, during which time HDAC activity increased in the vehicle-treated controls but not in SFN-treated cells. There was a time-dependent loss of class I and selected class II HDAC proteins, with HDAC3 depletion detected ahead of other HDACs. Mechanism studies revealed no apparent effect of calpain, proteasome, protease or caspase inhibitors, but HDAC3 was rescued by cycloheximide or actinomycin D treatment. Among the protein partners implicated in the HDAC3 turnover mechanism, silencing mediator for retinoid and thyroid hormone receptors (SMRT) was phosphorylated in the nucleus within 6 h of SFN treatment, as was HDAC3 itself. Co-immunoprecipitation assays revealed SFN-induced dissociation of HDAC3/SMRT complexes coinciding with increased binding of HDAC3 to 14-3-3 and peptidyl-prolyl cis/trans isomerase 1 (Pin1). Pin1 knockdown blocked the SFN-induced loss of HDAC3. Finally, SFN treatment for 6 or 24 h followed by SFN removal from the culture media led to complete recovery of HDAC activity and HDAC protein expression, during which time cells were released from G2/M arrest.

Conclusion: The current investigation supports a model in which protein kinase CK2 phosphorylates SMRT and HDAC3 in the nucleus, resulting in dissociation of the corepressor complex and enhanced binding of HDAC3 to 14-3-3 or Pin1. In the cytoplasm, release of HDAC3 from 14-3-3 followed by nuclear import is postulated to compete with a Pin1 pathway that directs HDAC3 for degradation. The latter pathway predominates in colon cancer cells exposed continuously to SFN, whereas the former pathway is likely to be favored when SFN has been removed within 24 h, allowing recovery from cell cycle arrest.

View Similar Images In: Results  - Collection
View Article: Medline Plus - Pubmed Central - HTML -  PubMed
Show All Figures - Show MeSH
getmorefigures.php?pmc=3127849&rFormat=json&query=null&req=5