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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 concurrently induces autophagy and down-regulates the expression of survivin and XIAP in human breast cancer cells. (A,B) MCF7 and MDA-MB-231 cells were treated with SAHA and expression of different proteins was analyzed by Western blotting. Equal protein loading was verified by actin. Experiments were repeated three times. The numbers under each blot are intensity of the blot relative to that of the untreated control (either “–ve control” or “0 h”). Signals in the survivin and XIAP blots (of all repeats) were quantitated and a graph was generated to show the effect of SAHA on the expression of survivin and XIAP. A statistically significant difference in the expression of survivin and XIAP in cells treated with SAHA vs. without SAHA (either “–ve control” or “0 h”) is denoted by “*” (p < 0.05) and “**” (p < 0.01). (C) MCF7 and MDA-B-231 breast cancer cells were treated with various concentrations of SAHA for 72 h and subsequently stained with MDC. AVOs in cells were observed under a fluorescence microscope. Red arrows indicate puncta formation.
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Figure 1: SAHA concurrently induces autophagy and down-regulates the expression of survivin and XIAP in human breast cancer cells. (A,B) MCF7 and MDA-MB-231 cells were treated with SAHA and expression of different proteins was analyzed by Western blotting. Equal protein loading was verified by actin. Experiments were repeated three times. The numbers under each blot are intensity of the blot relative to that of the untreated control (either “–ve control” or “0 h”). Signals in the survivin and XIAP blots (of all repeats) were quantitated and a graph was generated to show the effect of SAHA on the expression of survivin and XIAP. A statistically significant difference in the expression of survivin and XIAP in cells treated with SAHA vs. without SAHA (either “–ve control” or “0 h”) is denoted by “*” (p < 0.05) and “**” (p < 0.01). (C) MCF7 and MDA-B-231 breast cancer cells were treated with various concentrations of SAHA for 72 h and subsequently stained with MDC. AVOs in cells were observed under a fluorescence microscope. Red arrows indicate puncta formation.

Mentions: To determine the effectiveness of SAHA in targeting various types of breast cancer in vitro, IC50-values for SAHA were determined. In this study, the ER+/caspase-3-deficient/p53-expressing MCF7 and its tamoxifen-resistant sub-lines (MCF7- TamR8 and MCF7-TamC3) were used. As shown in Table 1, MTT cell viability assay revealed that the IC50-values of SAHA in MCF7-TamC3 (0.9 ± 0.1 μM) and MCF7-TamR8 (1.2 ± 0.3 μM) were similar to that in the parental tamoxifen-sensitive MCF7 (0.7 ± 0.1 μM) cells. SAHA was also effective in targeting SK- BR-3 (ER−/HER2+, caspase-3, and p53 mutant-expressing) and the triple-negative MDA-MB-231 (ER−/HER2−/PR−, caspase-3, and p53 mutant-expressing) breast cancer cells at low micromolar concentrations (Table 1). Taken together, our results revealed that SAHA is effective in reducing cell viability of various breast cancer subtypes regardless of the expression and status of ER, HER2, caspase-3, and p53. Western blot analysis was used to investigate the molecular effects of SAHA in breast cancer cells. Two different breast cancer cell lines, MCF7 (ER+/tamoxifen-sensitive) and MDA-MB-231 (ER−/HER2−/PR−/tamoxifen-resistant), were selected for the following molecular investigations. As shown in Figure 1A, SAHA increased the expression of acetylated α-tubulin, which is a function indicator of the drug, in both MCF7 and MDA-MB-231 cells in a concentration-dependent manner. SAHA also increased the conversion of LC3B-II and expression of beclin-1, and decreased the expression of p62/SQSTM1, which are molecular markers for autophagy (Figures 1A,B). SAHA-treated cells were stained with monodansylcadaverine (MDC) to further determine the formation of acidic vesicular organelles (AVOs). MDC is a fluorescent compound commonly used for the detection of AVOs including lysosome and autolysosome (Niemann et al., 2000; Munafo and Colombo, 2001). Similar to the results of cancer cells treated with the known autophagy inducer, resveratrol, SAHA treatment also increased the formation of green fluorescent puncta, indicating the increased formation of AVOs (Supplementary Figure 1 and Figure 1C). Taken together, these results indicate that SAHA, at the tested concentrations, did function normally at the molecular level and induced autophagy in both MCF7 and MDA-MB-231 cells. Surprisingly, cleavage of caspase-3 and PARP, which are molecular markers for caspase-3 activation, was only observed in the pro-caspase-3 expressing MDA-MB-231 cells treated with high concentration (2x IC50) of SAHA (Figure 1A), suggesting that caspase-3 activation might only play a role in the cell viability reduction induced by SAHA at high concentrations but not in moderate-to-low concentrations.


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 concurrently induces autophagy and down-regulates the expression of survivin and XIAP in human breast cancer cells. (A,B) MCF7 and MDA-MB-231 cells were treated with SAHA and expression of different proteins was analyzed by Western blotting. Equal protein loading was verified by actin. Experiments were repeated three times. The numbers under each blot are intensity of the blot relative to that of the untreated control (either “–ve control” or “0 h”). Signals in the survivin and XIAP blots (of all repeats) were quantitated and a graph was generated to show the effect of SAHA on the expression of survivin and XIAP. A statistically significant difference in the expression of survivin and XIAP in cells treated with SAHA vs. without SAHA (either “–ve control” or “0 h”) is denoted by “*” (p < 0.05) and “**” (p < 0.01). (C) MCF7 and MDA-B-231 breast cancer cells were treated with various concentrations of SAHA for 72 h and subsequently stained with MDC. AVOs in cells were observed under a fluorescence microscope. Red arrows indicate puncta formation.
© Copyright Policy
Related In: Results  -  Collection

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Figure 1: SAHA concurrently induces autophagy and down-regulates the expression of survivin and XIAP in human breast cancer cells. (A,B) MCF7 and MDA-MB-231 cells were treated with SAHA and expression of different proteins was analyzed by Western blotting. Equal protein loading was verified by actin. Experiments were repeated three times. The numbers under each blot are intensity of the blot relative to that of the untreated control (either “–ve control” or “0 h”). Signals in the survivin and XIAP blots (of all repeats) were quantitated and a graph was generated to show the effect of SAHA on the expression of survivin and XIAP. A statistically significant difference in the expression of survivin and XIAP in cells treated with SAHA vs. without SAHA (either “–ve control” or “0 h”) is denoted by “*” (p < 0.05) and “**” (p < 0.01). (C) MCF7 and MDA-B-231 breast cancer cells were treated with various concentrations of SAHA for 72 h and subsequently stained with MDC. AVOs in cells were observed under a fluorescence microscope. Red arrows indicate puncta formation.
Mentions: To determine the effectiveness of SAHA in targeting various types of breast cancer in vitro, IC50-values for SAHA were determined. In this study, the ER+/caspase-3-deficient/p53-expressing MCF7 and its tamoxifen-resistant sub-lines (MCF7- TamR8 and MCF7-TamC3) were used. As shown in Table 1, MTT cell viability assay revealed that the IC50-values of SAHA in MCF7-TamC3 (0.9 ± 0.1 μM) and MCF7-TamR8 (1.2 ± 0.3 μM) were similar to that in the parental tamoxifen-sensitive MCF7 (0.7 ± 0.1 μM) cells. SAHA was also effective in targeting SK- BR-3 (ER−/HER2+, caspase-3, and p53 mutant-expressing) and the triple-negative MDA-MB-231 (ER−/HER2−/PR−, caspase-3, and p53 mutant-expressing) breast cancer cells at low micromolar concentrations (Table 1). Taken together, our results revealed that SAHA is effective in reducing cell viability of various breast cancer subtypes regardless of the expression and status of ER, HER2, caspase-3, and p53. Western blot analysis was used to investigate the molecular effects of SAHA in breast cancer cells. Two different breast cancer cell lines, MCF7 (ER+/tamoxifen-sensitive) and MDA-MB-231 (ER−/HER2−/PR−/tamoxifen-resistant), were selected for the following molecular investigations. As shown in Figure 1A, SAHA increased the expression of acetylated α-tubulin, which is a function indicator of the drug, in both MCF7 and MDA-MB-231 cells in a concentration-dependent manner. SAHA also increased the conversion of LC3B-II and expression of beclin-1, and decreased the expression of p62/SQSTM1, which are molecular markers for autophagy (Figures 1A,B). SAHA-treated cells were stained with monodansylcadaverine (MDC) to further determine the formation of acidic vesicular organelles (AVOs). MDC is a fluorescent compound commonly used for the detection of AVOs including lysosome and autolysosome (Niemann et al., 2000; Munafo and Colombo, 2001). Similar to the results of cancer cells treated with the known autophagy inducer, resveratrol, SAHA treatment also increased the formation of green fluorescent puncta, indicating the increased formation of AVOs (Supplementary Figure 1 and Figure 1C). Taken together, these results indicate that SAHA, at the tested concentrations, did function normally at the molecular level and induced autophagy in both MCF7 and MDA-MB-231 cells. Surprisingly, cleavage of caspase-3 and PARP, which are molecular markers for caspase-3 activation, was only observed in the pro-caspase-3 expressing MDA-MB-231 cells treated with high concentration (2x IC50) of SAHA (Figure 1A), suggesting that caspase-3 activation might only play a role in the cell viability reduction induced by SAHA at high concentrations but not in moderate-to-low concentrations.

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