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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

An epigenetic perspective on pharmacologic ascorbate in colon cancer

Aim: There is growing interest in pharmacologic ascorbate (Asc) and its therapeutic properties (Levine et al. Adv Nutr 2011;2:78). W e examined cell viability, histone deacetylase (HDAC) expression, and related protein modifications in cancer versus non- cancer colon epithelial cells following exposure to Asc. Methods: MTT assays were conducted in HCT116 colon cancer and CCD841 non-transformed colonic epithelial cells treated with 0.25 to 16 mM Asc or ascorbate-2-phosphate (AAp), in the presence and absence of catalase (CAT, 280 U/mg), or with 5 to 160 μM H₂O₂. Cell lysates obtained 6 h and 24 h post-treatment were immunoblotted as reported by Rajendran et al. Mol Cancer 2011;10:68. Results: In MTT assays, IC₅₀ data were as follows: 8 mM Asc (CCD841); 3 mM Asc (HCT116), >50mM AAp (HCT116), and 65 μM H₂O₂ (HCT116). CAT protected against both Asc- and H₂O₂- induced cytotoxicity. At 6 h, Asc and H₂O₂ altered the expression of HDACs (HDAC4, HDAC6, SIRT3) and enhanced the acetylation of histone (H3, H4) and non-histone proteins (tubulin, p53). Conclusions: Asc was more cytotoxic to colon cancer cells than non-cancer cells. Findings with the non-H₂O₂ producing compound AAp, and with CAT, implicated H₂O₂ in Asc-induced cytotoxicity. Asc was shown, for the first time, to alter epigenetic end-points related to HDAC changes in colon cancer cells

Kaiser_Matthew Project Presentation.pptx

Aim: There is growing interest in pharmacologic ascorbate (Asc) and its therapeutic properties (Levine et al. Adv Nutr 2011;2:78). W e examined cell viability, histone deacetylase (HDAC) expression, and related protein modifications in cancer versus non- cancer colon epithelial cells following exposure to Asc. Methods: MTT assays were conducted in HCT116 colon cancer and CCD841 non-transformed colonic epithelial cells treated with 0.25 to 16 mM Asc or ascorbate-2-phosphate (AAp), in the presence and absence of catalase (CAT, 280 U/mg), or with 5 to 160 μM H₂O₂. Cell lysates obtained 6 h and 24 h post-treatment were immunoblotted as reported by Rajendran et al. Mol Cancer 2011;10:68. Results: In MTT assays, IC₅₀ data were as follows: 8 mM Asc (CCD841); 3 mM Asc (HCT116), >50mM AAp (HCT116), and 65 μM H₂O₂ (HCT116). CAT protected against both Asc- and H₂O₂- induced cytotoxicity. At 6 h, Asc and H₂O₂ altered the expression of HDACs (HDAC4, HDAC6, SIRT3) and enhanced the acetylation of histone (H3, H4) and non-histone proteins (tubulin, p53). Conclusions: Asc was more cytotoxic to colon cancer cells than non-cancer cells. Findings with the non-H₂O₂ producing compound AAp, and with CAT, implicated H₂O₂ in Asc-induced cytotoxicity. Asc was shown, for the first time, to alter epigenetic end-points related to HDAC changes in colon cancer cells.Keywords: Linus Pauling Institute, Cancer, HDAC, Epigenetics, Ascorbate, Vitamin C, KaiserKeywords: Linus Pauling Institute, Cancer, HDAC, Epigenetics, Ascorbate, Vitamin C, Kaise

Matthew Kaiser at HHMI 2012.mp4

Aim: There is growing interest in pharmacologic ascorbate (Asc) and its therapeutic properties (Levine et al. Adv Nutr 2011;2:78). W e examined cell viability, histone deacetylase (HDAC) expression, and related protein modifications in cancer versus non- cancer colon epithelial cells following exposure to Asc. Methods: MTT assays were conducted in HCT116 colon cancer and CCD841 non-transformed colonic epithelial cells treated with 0.25 to 16 mM Asc or ascorbate-2-phosphate (AAp), in the presence and absence of catalase (CAT, 280 U/mg), or with 5 to 160 μM H₂O₂. Cell lysates obtained 6 h and 24 h post-treatment were immunoblotted as reported by Rajendran et al. Mol Cancer 2011;10:68. Results: In MTT assays, IC₅₀ data were as follows: 8 mM Asc (CCD841); 3 mM Asc (HCT116), >50mM AAp (HCT116), and 65 μM H₂O₂ (HCT116). CAT protected against both Asc- and H₂O₂- induced cytotoxicity. At 6 h, Asc and H₂O₂ altered the expression of HDACs (HDAC4, HDAC6, SIRT3) and enhanced the acetylation of histone (H3, H4) and non-histone proteins (tubulin, p53). Conclusions: Asc was more cytotoxic to colon cancer cells than non-cancer cells. Findings with the non-H₂O₂ producing compound AAp, and with CAT, implicated H₂O₂ in Asc-induced cytotoxicity. Asc was shown, for the first time, to alter epigenetic end-points related to HDAC changes in colon cancer cells.Keywords: Epigenetics, Vitamin C, Linus Pauling Institute, HDAC, Cancer, Ascorbate, Kaise

Kaiser_Matthew Project Presentation vPDF.pdf

Aim: There is growing interest in pharmacologic ascorbate (Asc) and its therapeutic properties (Levine et al. Adv Nutr 2011;2:78). W e examined cell viability, histone deacetylase (HDAC) expression, and related protein modifications in cancer versus non- cancer colon epithelial cells following exposure to Asc. Methods: MTT assays were conducted in HCT116 colon cancer and CCD841 non-transformed colonic epithelial cells treated with 0.25 to 16 mM Asc or ascorbate-2-phosphate (AAp), in the presence and absence of catalase (CAT, 280 U/mg), or with 5 to 160 μM H₂O₂. Cell lysates obtained 6 h and 24 h post-treatment were immunoblotted as reported by Rajendran et al. Mol Cancer 2011;10:68. Results: In MTT assays, IC₅₀ data were as follows: 8 mM Asc (CCD841); 3 mM Asc (HCT116), >50mM AAp (HCT116), and 65 μM H₂O₂ (HCT116). CAT protected against both Asc- and H₂O₂- induced cytotoxicity. At 6 h, Asc and H₂O₂ altered the expression of HDACs (HDAC4, HDAC6, SIRT3) and enhanced the acetylation of histone (H3, H4) and non-histone proteins (tubulin, p53). Conclusions: Asc was more cytotoxic to colon cancer cells than non-cancer cells. Findings with the non-H₂O₂ producing compound AAp, and with CAT, implicated H₂O₂ in Asc-induced cytotoxicity. Asc was shown, for the first time, to alter epigenetic end-points related to HDAC changes in colon cancer cells.Keywords: Cancer, Vitamin C, Linus Pauling Institute, Ascorbate, Kaiser, Epigenetics, HDA

EB Abstract Published.pdf

Aim: There is growing interest in pharmacologic ascorbate (Asc) and its therapeutic properties (Levine et al. Adv Nutr 2011;2:78). W e examined cell viability, histone deacetylase (HDAC) expression, and related protein modifications in cancer versus non- cancer colon epithelial cells following exposure to Asc. Methods: MTT assays were conducted in HCT116 colon cancer and CCD841 non-transformed colonic epithelial cells treated with 0.25 to 16 mM Asc or ascorbate-2-phosphate (AAp), in the presence and absence of catalase (CAT, 280 U/mg), or with 5 to 160 μM H₂O₂. Cell lysates obtained 6 h and 24 h post-treatment were immunoblotted as reported by Rajendran et al. Mol Cancer 2011;10:68. Results: In MTT assays, IC₅₀ data were as follows: 8 mM Asc (CCD841); 3 mM Asc (HCT116), >50mM AAp (HCT116), and 65 μM H₂O₂ (HCT116). CAT protected against both Asc- and H₂O₂- induced cytotoxicity. At 6 h, Asc and H₂O₂ altered the expression of HDACs (HDAC4, HDAC6, SIRT3) and enhanced the acetylation of histone (H3, H4) and non-histone proteins (tubulin, p53). Conclusions: Asc was more cytotoxic to colon cancer cells than non-cancer cells. Findings with the non-H₂O₂ producing compound AAp, and with CAT, implicated H₂O₂ in Asc-induced cytotoxicity. Asc was shown, for the first time, to alter epigenetic end-points related to HDAC changes in colon cancer cells.Keywords: Epigenetics, Vitamin C, Kaiser, Linus Pauling Institute, Ascorbate, Cancer, HDA