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Inhibition of HDAC3- and HDAC6-Promoted Survivin Expression Plays an Important Role in SAHA-Induced Autophagy and Viability Reduction in Breast Cancer Cells.

Lee JY, Kuo CW, Tsai SL, Cheng SM, Chen SH, Chan HH, Lin CH, Lin KY, Li CF, Kanwar JR, Leung EY, Cheung CC, Huang WJ, Wang YC, Cheung CH - Front Pharmacol (2016)

Bottom Line: In this study, we found that SAHA is equally effective in targeting cells of different breast cancer subtypes and tamoxifen sensitivity.It also reduced survivin and XIAP protein stability in part through modulating the expression and activation of the 26S proteasome and heat-shock protein 90.Our findings emphasize the complexity of the regulatory roles in different HDAC isoforms and potentially assist in predicting the mechanism of novel HDAC inhibitors in targeted or combinational therapies in the future.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Medicine, National Cheng Kung University Tainan, Taiwan.

ABSTRACT
SAHA is a class I HDAC/HDAC6 co-inhibitor and an autophagy inducer currently undergoing clinical investigations in breast cancer patients. However, the molecular mechanism of action of SAHA in breast cancer cells remains unclear. In this study, we found that SAHA is equally effective in targeting cells of different breast cancer subtypes and tamoxifen sensitivity. Importantly, we found that down-regulation of survivin plays an important role in SAHA-induced autophagy and cell viability reduction in human breast cancer cells. SAHA decreased survivin and XIAP gene transcription, induced survivin protein acetylation and early nuclear translocation in MCF7 and MDA-MB-231 breast cancer cells. It also reduced survivin and XIAP protein stability in part through modulating the expression and activation of the 26S proteasome and heat-shock protein 90. Interestingly, targeting HDAC3 and HDAC6, but not other HDAC isoforms, by siRNA/pharmacological inhibitors mimicked the effects of SAHA in modulating the acetylation, expression, and nuclear translocation of survivin and induced autophagy in MCF7 and MDA-MB-231 cancer cells. Targeting HDAC3 also mimicked the effect of SAHA in up-regulating the expression and activity of proteasome, which might lead to the reduced protein stability of survivin in breast cancer cells. In conclusion, this study provides new insights into SAHA's molecular mechanism of actions in breast cancer cells. Our findings emphasize the complexity of the regulatory roles in different HDAC isoforms and potentially assist in predicting the mechanism of novel HDAC inhibitors in targeted or combinational therapies in the future.

No MeSH data available.


Related in: MedlinePlus

SAHA decreases the protein stability of survivin and XIAP in breast cancer cells. (A) Breast cancer cells were treated with 1x IC50 SAHA for 72 h. Cycloheximide (CHX) was added 72 h post-SAHA treatment to the cells to inhibit de novo protein synthesis. Cells were then harvested at the time points indicated and expression of survivin and XIAP was analyzed by Western blotting. Experiments were repeated three times and representative blots were shown. Signals in the blots (of all repeats) were quantitated and a graph was generated to compare the degradation rates. A statistically significant difference in the mean of the relative band intensity (of all repeats) of survivin and XIAP in cells treated with SAHA vs. without SAHA (control) at the same time point is denoted by “*” (p < 0.05), “**” (p < 0.01), or “***” (p < 0.001). (B) MCF7 cells were treated with cycloheximide to inhibit the de novo protein synthesis process. Nucleic proteins and cytoplasmic proteins were isolated using cells fractionation assay. Expression of survivin was analyzed by Western blotting. Equal protein loading was verified by either lamin A/C or GAPDH. The numbers under each blot are intensity of the blot relative to that of the control (0 h). Signals in the blots (of all repeats) were quantitated and a graph was generated to compare the degradation rates. A statistically significant difference in the mean of the relative band intensity (of all repeats) of nuclear survivin and cytosolic survivin is denoted by “*” (p < 0.05). (C) MCF7 cells were treated with SAHA and expression of the acetylated survivin was analyzed by Western blotting. (D) Breast cancer cells were treated with 2x IC50 SAHA for 24 h. Intracellular distribution of survivin was analyzed using immunofluorescence confocal microscopy. Nucleus was stained blue with DAPI. Survivin was labeled red in the photo. Relative expression of nucleic and cytoplasmic survivin in cells treated with/without SAHA was quantified. Experiment was repeated three times. A statistically significant difference in the nucleus/cytoplasm ratio of red fluorescence (survivin) intensity in cells with SAHA vs. without SAHA (control) is denoted by either “**” (p < 0.01) or “***” (p < 0.001). Percentage of cells with red fluorescence signal higher in the nucleus than in the cytosol in cells treated with or without SAHA was also quantified. (E) MCF7 cells were transfected with HDAC1, 2, 3, or 6 siRNA for 36 h. Expression of acetylated survivin was determined by Western blotting. Signals in the acetylated survivin blots (of all repeats) were quantitated and a graph was generated to show the effect of different HDAC isoforms on the expression of acetylated survivin. A statistically significant difference in the expression of acetylated survivin in cells treated with HDAC1, 2, 3, or 6 siRNA vs. scramble siRNA is denoted by “*” (p < 0.05). (F) MCF7 cells were treated with or without BML281 and RGFP966 for 48 h. Expression of acetylated survivin was determined Western blotting. (G) MCF7 cells were transfected with scramble, HDAC3 or HDAC6 siRNA for 36 h. Intracellular distribution of survivin was analyzed using immunofluorescence confocal microscopy. Nucleus was stained blue with DAPI. Survivin was labeled red in the photo. Relative expression of nucleic and cytoplasmic survivin in cells treated with scramble, HDAC3 or HDAC6 siRNA was quantified. Experiment was repeated three times. A statistically significant difference in the nucleus/cytoplasm ratio of red fluorescence (survivin) intensity in cells treated with HDAC3 or 6 siRNA vs. scramble siRNA is denoted by “**” (p < 0.01). Percentage of cells with red fluorescence signal higher in the nucleus than in the cytosol in cells treated with or without HDAC3 and HDAC6 siRNA was also quantified.
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Figure 4: SAHA decreases the protein stability of survivin and XIAP in breast cancer cells. (A) Breast cancer cells were treated with 1x IC50 SAHA for 72 h. Cycloheximide (CHX) was added 72 h post-SAHA treatment to the cells to inhibit de novo protein synthesis. Cells were then harvested at the time points indicated and expression of survivin and XIAP was analyzed by Western blotting. Experiments were repeated three times and representative blots were shown. Signals in the blots (of all repeats) were quantitated and a graph was generated to compare the degradation rates. A statistically significant difference in the mean of the relative band intensity (of all repeats) of survivin and XIAP in cells treated with SAHA vs. without SAHA (control) at the same time point is denoted by “*” (p < 0.05), “**” (p < 0.01), or “***” (p < 0.001). (B) MCF7 cells were treated with cycloheximide to inhibit the de novo protein synthesis process. Nucleic proteins and cytoplasmic proteins were isolated using cells fractionation assay. Expression of survivin was analyzed by Western blotting. Equal protein loading was verified by either lamin A/C or GAPDH. The numbers under each blot are intensity of the blot relative to that of the control (0 h). Signals in the blots (of all repeats) were quantitated and a graph was generated to compare the degradation rates. A statistically significant difference in the mean of the relative band intensity (of all repeats) of nuclear survivin and cytosolic survivin is denoted by “*” (p < 0.05). (C) MCF7 cells were treated with SAHA and expression of the acetylated survivin was analyzed by Western blotting. (D) Breast cancer cells were treated with 2x IC50 SAHA for 24 h. Intracellular distribution of survivin was analyzed using immunofluorescence confocal microscopy. Nucleus was stained blue with DAPI. Survivin was labeled red in the photo. Relative expression of nucleic and cytoplasmic survivin in cells treated with/without SAHA was quantified. Experiment was repeated three times. A statistically significant difference in the nucleus/cytoplasm ratio of red fluorescence (survivin) intensity in cells with SAHA vs. without SAHA (control) is denoted by either “**” (p < 0.01) or “***” (p < 0.001). Percentage of cells with red fluorescence signal higher in the nucleus than in the cytosol in cells treated with or without SAHA was also quantified. (E) MCF7 cells were transfected with HDAC1, 2, 3, or 6 siRNA for 36 h. Expression of acetylated survivin was determined by Western blotting. Signals in the acetylated survivin blots (of all repeats) were quantitated and a graph was generated to show the effect of different HDAC isoforms on the expression of acetylated survivin. A statistically significant difference in the expression of acetylated survivin in cells treated with HDAC1, 2, 3, or 6 siRNA vs. scramble siRNA is denoted by “*” (p < 0.05). (F) MCF7 cells were treated with or without BML281 and RGFP966 for 48 h. Expression of acetylated survivin was determined Western blotting. (G) MCF7 cells were transfected with scramble, HDAC3 or HDAC6 siRNA for 36 h. Intracellular distribution of survivin was analyzed using immunofluorescence confocal microscopy. Nucleus was stained blue with DAPI. Survivin was labeled red in the photo. Relative expression of nucleic and cytoplasmic survivin in cells treated with scramble, HDAC3 or HDAC6 siRNA was quantified. Experiment was repeated three times. A statistically significant difference in the nucleus/cytoplasm ratio of red fluorescence (survivin) intensity in cells treated with HDAC3 or 6 siRNA vs. scramble siRNA is denoted by “**” (p < 0.01). Percentage of cells with red fluorescence signal higher in the nucleus than in the cytosol in cells treated with or without HDAC3 and HDAC6 siRNA was also quantified.

Mentions: To determine whether SAHA also affects the expression of survivin and XIAP through other mechanisms, the protein stability of both survivin and XIAP was evaluated. Western blot analysis revealed that the protein stability of survivin was significantly decreased in both MCF7 and MDA-MB-231 cells treated with SAHA (at IC50 conc.) relative to the control (Figure 4A). The protein stability of XIAP was also significantly decreased in SAHA-treated MDA-MB-231 cells and slightly decreased in SAHA-treated MCF7 cells as compared to that of the untreated cells (Figure 4A).


Inhibition of HDAC3- and HDAC6-Promoted Survivin Expression Plays an Important Role in SAHA-Induced Autophagy and Viability Reduction in Breast Cancer Cells.

Lee JY, Kuo CW, Tsai SL, Cheng SM, Chen SH, Chan HH, Lin CH, Lin KY, Li CF, Kanwar JR, Leung EY, Cheung CC, Huang WJ, Wang YC, Cheung CH - Front Pharmacol (2016)

SAHA decreases the protein stability of survivin and XIAP in breast cancer cells. (A) Breast cancer cells were treated with 1x IC50 SAHA for 72 h. Cycloheximide (CHX) was added 72 h post-SAHA treatment to the cells to inhibit de novo protein synthesis. Cells were then harvested at the time points indicated and expression of survivin and XIAP was analyzed by Western blotting. Experiments were repeated three times and representative blots were shown. Signals in the blots (of all repeats) were quantitated and a graph was generated to compare the degradation rates. A statistically significant difference in the mean of the relative band intensity (of all repeats) of survivin and XIAP in cells treated with SAHA vs. without SAHA (control) at the same time point is denoted by “*” (p < 0.05), “**” (p < 0.01), or “***” (p < 0.001). (B) MCF7 cells were treated with cycloheximide to inhibit the de novo protein synthesis process. Nucleic proteins and cytoplasmic proteins were isolated using cells fractionation assay. Expression of survivin was analyzed by Western blotting. Equal protein loading was verified by either lamin A/C or GAPDH. The numbers under each blot are intensity of the blot relative to that of the control (0 h). Signals in the blots (of all repeats) were quantitated and a graph was generated to compare the degradation rates. A statistically significant difference in the mean of the relative band intensity (of all repeats) of nuclear survivin and cytosolic survivin is denoted by “*” (p < 0.05). (C) MCF7 cells were treated with SAHA and expression of the acetylated survivin was analyzed by Western blotting. (D) Breast cancer cells were treated with 2x IC50 SAHA for 24 h. Intracellular distribution of survivin was analyzed using immunofluorescence confocal microscopy. Nucleus was stained blue with DAPI. Survivin was labeled red in the photo. Relative expression of nucleic and cytoplasmic survivin in cells treated with/without SAHA was quantified. Experiment was repeated three times. A statistically significant difference in the nucleus/cytoplasm ratio of red fluorescence (survivin) intensity in cells with SAHA vs. without SAHA (control) is denoted by either “**” (p < 0.01) or “***” (p < 0.001). Percentage of cells with red fluorescence signal higher in the nucleus than in the cytosol in cells treated with or without SAHA was also quantified. (E) MCF7 cells were transfected with HDAC1, 2, 3, or 6 siRNA for 36 h. Expression of acetylated survivin was determined by Western blotting. Signals in the acetylated survivin blots (of all repeats) were quantitated and a graph was generated to show the effect of different HDAC isoforms on the expression of acetylated survivin. A statistically significant difference in the expression of acetylated survivin in cells treated with HDAC1, 2, 3, or 6 siRNA vs. scramble siRNA is denoted by “*” (p < 0.05). (F) MCF7 cells were treated with or without BML281 and RGFP966 for 48 h. Expression of acetylated survivin was determined Western blotting. (G) MCF7 cells were transfected with scramble, HDAC3 or HDAC6 siRNA for 36 h. Intracellular distribution of survivin was analyzed using immunofluorescence confocal microscopy. Nucleus was stained blue with DAPI. Survivin was labeled red in the photo. Relative expression of nucleic and cytoplasmic survivin in cells treated with scramble, HDAC3 or HDAC6 siRNA was quantified. Experiment was repeated three times. A statistically significant difference in the nucleus/cytoplasm ratio of red fluorescence (survivin) intensity in cells treated with HDAC3 or 6 siRNA vs. scramble siRNA is denoted by “**” (p < 0.01). Percentage of cells with red fluorescence signal higher in the nucleus than in the cytosol in cells treated with or without HDAC3 and HDAC6 siRNA was also quantified.
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Figure 4: SAHA decreases the protein stability of survivin and XIAP in breast cancer cells. (A) Breast cancer cells were treated with 1x IC50 SAHA for 72 h. Cycloheximide (CHX) was added 72 h post-SAHA treatment to the cells to inhibit de novo protein synthesis. Cells were then harvested at the time points indicated and expression of survivin and XIAP was analyzed by Western blotting. Experiments were repeated three times and representative blots were shown. Signals in the blots (of all repeats) were quantitated and a graph was generated to compare the degradation rates. A statistically significant difference in the mean of the relative band intensity (of all repeats) of survivin and XIAP in cells treated with SAHA vs. without SAHA (control) at the same time point is denoted by “*” (p < 0.05), “**” (p < 0.01), or “***” (p < 0.001). (B) MCF7 cells were treated with cycloheximide to inhibit the de novo protein synthesis process. Nucleic proteins and cytoplasmic proteins were isolated using cells fractionation assay. Expression of survivin was analyzed by Western blotting. Equal protein loading was verified by either lamin A/C or GAPDH. The numbers under each blot are intensity of the blot relative to that of the control (0 h). Signals in the blots (of all repeats) were quantitated and a graph was generated to compare the degradation rates. A statistically significant difference in the mean of the relative band intensity (of all repeats) of nuclear survivin and cytosolic survivin is denoted by “*” (p < 0.05). (C) MCF7 cells were treated with SAHA and expression of the acetylated survivin was analyzed by Western blotting. (D) Breast cancer cells were treated with 2x IC50 SAHA for 24 h. Intracellular distribution of survivin was analyzed using immunofluorescence confocal microscopy. Nucleus was stained blue with DAPI. Survivin was labeled red in the photo. Relative expression of nucleic and cytoplasmic survivin in cells treated with/without SAHA was quantified. Experiment was repeated three times. A statistically significant difference in the nucleus/cytoplasm ratio of red fluorescence (survivin) intensity in cells with SAHA vs. without SAHA (control) is denoted by either “**” (p < 0.01) or “***” (p < 0.001). Percentage of cells with red fluorescence signal higher in the nucleus than in the cytosol in cells treated with or without SAHA was also quantified. (E) MCF7 cells were transfected with HDAC1, 2, 3, or 6 siRNA for 36 h. Expression of acetylated survivin was determined by Western blotting. Signals in the acetylated survivin blots (of all repeats) were quantitated and a graph was generated to show the effect of different HDAC isoforms on the expression of acetylated survivin. A statistically significant difference in the expression of acetylated survivin in cells treated with HDAC1, 2, 3, or 6 siRNA vs. scramble siRNA is denoted by “*” (p < 0.05). (F) MCF7 cells were treated with or without BML281 and RGFP966 for 48 h. Expression of acetylated survivin was determined Western blotting. (G) MCF7 cells were transfected with scramble, HDAC3 or HDAC6 siRNA for 36 h. Intracellular distribution of survivin was analyzed using immunofluorescence confocal microscopy. Nucleus was stained blue with DAPI. Survivin was labeled red in the photo. Relative expression of nucleic and cytoplasmic survivin in cells treated with scramble, HDAC3 or HDAC6 siRNA was quantified. Experiment was repeated three times. A statistically significant difference in the nucleus/cytoplasm ratio of red fluorescence (survivin) intensity in cells treated with HDAC3 or 6 siRNA vs. scramble siRNA is denoted by “**” (p < 0.01). Percentage of cells with red fluorescence signal higher in the nucleus than in the cytosol in cells treated with or without HDAC3 and HDAC6 siRNA was also quantified.
Mentions: To determine whether SAHA also affects the expression of survivin and XIAP through other mechanisms, the protein stability of both survivin and XIAP was evaluated. Western blot analysis revealed that the protein stability of survivin was significantly decreased in both MCF7 and MDA-MB-231 cells treated with SAHA (at IC50 conc.) relative to the control (Figure 4A). The protein stability of XIAP was also significantly decreased in SAHA-treated MDA-MB-231 cells and slightly decreased in SAHA-treated MCF7 cells as compared to that of the untreated cells (Figure 4A).

Bottom Line: In this study, we found that SAHA is equally effective in targeting cells of different breast cancer subtypes and tamoxifen sensitivity.It also reduced survivin and XIAP protein stability in part through modulating the expression and activation of the 26S proteasome and heat-shock protein 90.Our findings emphasize the complexity of the regulatory roles in different HDAC isoforms and potentially assist in predicting the mechanism of novel HDAC inhibitors in targeted or combinational therapies in the future.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Medicine, National Cheng Kung University Tainan, Taiwan.

ABSTRACT
SAHA is a class I HDAC/HDAC6 co-inhibitor and an autophagy inducer currently undergoing clinical investigations in breast cancer patients. However, the molecular mechanism of action of SAHA in breast cancer cells remains unclear. In this study, we found that SAHA is equally effective in targeting cells of different breast cancer subtypes and tamoxifen sensitivity. Importantly, we found that down-regulation of survivin plays an important role in SAHA-induced autophagy and cell viability reduction in human breast cancer cells. SAHA decreased survivin and XIAP gene transcription, induced survivin protein acetylation and early nuclear translocation in MCF7 and MDA-MB-231 breast cancer cells. It also reduced survivin and XIAP protein stability in part through modulating the expression and activation of the 26S proteasome and heat-shock protein 90. Interestingly, targeting HDAC3 and HDAC6, but not other HDAC isoforms, by siRNA/pharmacological inhibitors mimicked the effects of SAHA in modulating the acetylation, expression, and nuclear translocation of survivin and induced autophagy in MCF7 and MDA-MB-231 cancer cells. Targeting HDAC3 also mimicked the effect of SAHA in up-regulating the expression and activity of proteasome, which might lead to the reduced protein stability of survivin in breast cancer cells. In conclusion, this study provides new insights into SAHA's molecular mechanism of actions in breast cancer cells. Our findings emphasize the complexity of the regulatory roles in different HDAC isoforms and potentially assist in predicting the mechanism of novel HDAC inhibitors in targeted or combinational therapies in the future.

No MeSH data available.


Related in: MedlinePlus