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. 2016 Jun 10;15(1):46.
doi: 10.1186/s12943-016-0531-5.

Combination of the novel histone deacetylase inhibitor YCW1 and radiation induces autophagic cell death through the downregulation of BNIP3 in triple-negative breast cancer cells in vitro and in an orthotopic mouse model

Hui-Wen Chiu  1   2 , Ya-Ling Yeh  3 , Yi-Ching Wang  4   5 , Wei-Jan Huang  6 , Sheng-Yow Ho  7   8 , Pinpin Lin  9 , Ying-Jan Wang  10   11   12
Affiliations

Combination of the novel histone deacetylase inhibitor YCW1 and radiation induces autophagic cell death through the downregulation of BNIP3 in triple-negative breast cancer cells in vitro and in an orthotopic mouse model

Hui-Wen Chiu et al. Mol Cancer. .

Abstract

Background: Triple-negative breast cancer (TNBC) is the most aggressive and invasive of the breast cancer subtypes. TNBC is a challenging disease that lacks targets for treatment. Histone deacetylase inhibitors (HDACi) are a group of targeted anticancer agents that enhance radiosensitivity. Bcl-2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) is a member of the Bcl-2 subfamily. BNIP3 is not found in normal breast tissue but is up-regulated in breast cancer. In the present study, we investigated the anti-cancer effects of a newly developed HDACi, YCW1, combined with ionizing radiation (IR) in TNBC in vitro and in an orthotopic mouse model. Furthermore, we examined the relationship between autophagy and BNIP3.

Methods: Trypan blue exclusion was used to investigate the viability of 4 T1 (a mouse TNBC cell line) and MDA-MB-231 cells (a human TNBC cell line) following combined YCW1 and IR treatment. Flow cytometry was used to determine apoptosis and autophagy. The expression levels of BNIP3, endoplasmic reticulum (ER) stress- and autophagic-related proteins were measured using western blot analysis. An orthotopic mouse model was used to investigate the in vivo effects of YCW1 and IR alone and in combination. Tumor volumes were monitored using a bioluminescence-based IVIS Imaging System 200.

Results: We found that YCW1 significantly enhanced toxicity in 4 T1 cells compared with suberoylanilide hydroxamic acid (SAHA), which was the first HDACi approved by the Food and Drug Administration for clinical use in cancer patients. The combined treatment of YCW1 and IR enhanced cytotoxicity by inducing ER stress and increasing autophagy induction. Additionally, the combined treatment caused autophagic flux and autophagic cell death. Furthermore, the expression level of BNIP3 was significantly decreased in cells following combined treatment. The downregulation of BNIP3 led to a significant increase in autophagy and cytotoxicity. The combined anti-tumor effects of YCW1 and IR were also observed in an orthotopic mouse model; combination therapy resulted in a significant increase in autophagy and decreased tumor tissue expression of BNIP3 in the tumor tissue.

Conclusions: These data support the possibility of using a combination of HDACi and IR in the treatment of TNBC. Moreover, BNIP3 may be a potential target protein for TNBC treatment.

Keywords: Autophagy; Histone deacetylase inhibitor; Radiation; Triple-negative breast cancer.

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Figures

Fig. 1
Fig. 1
Cytotoxic effects resulting from SAHA and YCW1 treatment in 4 T1 cells. a Dose-dependent effects of SAHA and YCW1 treatment on the viability of 4 T1 cells for 24 h. *, p < 0.05, SAHA versus YCW1. Data are presented as the mean ± standard deviation of three independent experiments. b Concentration-dependent effects of YCW1 on histone and nonhistone proteins for 24 h in 4 T1 cells
Fig. 2
Fig. 2
Cytotoxic effects resulting from YCW1 and/or IR in 4 T1 and MBA-MD-231 cells. a Concentration-dependent effects of YCW1 on the viability of 4 T1 and MBA-MD-231 cells. Cells were treated with 0.5, 1, 1.5 or 2 μM of YCW1 for 48 h. b Dose-dependent effects of IR on the viability of 4 T1 and MBA-MD-231 cells. Cells were treated with 2, 4, 6 or 8 Gy of IR for 48 h. *, p < 0.05, YCW1 or IR versus control group. c Cytotoxicity observed in cells treated with YCW1 (1 μM) and IR (4 Gy) for 48 h in 4 T1 and MBA-MD-231 cells. #, p < 0.05, IR versus combined treatment. *, p < 0.05, YCW1 versus combined treatment. Data are presented as the mean ± standard deviation of three independent experiments
Fig. 3
Fig. 3
Measurement of cell death and ER stress in 4 T1 cells. a The expression levels of ER stress-related proteins were measured by western blot analysis following treatment with YCW1 and IR alone or in combination. Cells were treated with YCW1 (1 μM) and IR (4 Gy) for 12 h. b Early apoptosis, detected using an Annexin V apoptosis detection kit, was measured using flow cytometry. Cells were treated with YCW1 (1 μM) and IR (4 Gy) for 48 h. c Immunofluorescence staining of the LC3 protein in 4 T1 cells treated with YCW1 (1 μM) and IR (4 Gy) for 48 h. Representative cell images showing punctate LC3 distribution using a confocal microscope. Quantitative data calculating the number of LC3 dots per cell. #, p < 0.05, IR versus combined treatment. *, p < 0.05, YCW1 versus combined treatment. Data are presented as the mean ± standard deviation of three independent experiments. d The expression levels of autophagic-related proteins were measured by western blot analysis following treatment with YCW1 and IR alone or in combination. The densities of the bands were quantified with a computer densitometer (AlphaImager™ 2200 System Alpha Innotech Corporation, CA, USA). Cells were treated with YCW1 (1 μM) and IR (4 Gy) for 48 h. #, p < 0.05, IR versus combined treatment. *, p < 0.05, YCW1 versus combined treatment
Fig. 4
Fig. 4
The ultrastructures of 4 T1 cells treated with YCW1 (1 μM) and IR (4 Gy) for 48 h were analyzed by TEM. N, nucleus. Black arrows, autophagosomes. Black arrowheads, autolysosome. White arrows, endoplasmic reticulum
Fig. 5
Fig. 5
Measurement of autophagic and cytotoxic effects in 4 T1 cells pretreated with Atg shRNA. a Western blotting for Atg5. The cells were transfected with Atg5 shRNA for 24 h. b Quantitative data calculating the number of LC3 dots per cell in the absence or presence of Atg5 shRNA. c Cytotoxic effects in the absence or presence of Atg5 shRNA. Cells were transfected with Atg5 shRNA for 24 h and were then incubated with YCW1 (1 μM) and IR (4 Gy) for 48 h. *, p < 0.05, YCW1 + IR versus Atg5 shRNA + YCW1 + IR. Data are presented as the mean ± standard deviation of three independent experiments. d Western blot analysis of LC3-I and LC3-II expression in 4 T1 cells. Cells were pretreated with BAF for 1 h prior to IR treatment and then treated with YCW1 (1 μM) and IR (4 Gy) for 48 h
Fig. 6
Fig. 6
BNIP3 knockdown by shRNA abrogates the growth of 4 T1 cells. a The expression levels of BNIP3 in 4 T1 cells were analyzed by western blot analysis following YCW and/or IR treatment. Cells were treated with YCW1 (1 μM) and IR (4 Gy) for 48 h. b Transfection efficacy was verified by western blot analysis. BNIP3 protein expression in 4 T1 cells transfected with scramble or BNIP3 shRNA for 24 h. c Quantitative data calculating the number of LC3 dots per cell transfected with scramble or BNIP3 shRNA. *, p < 0.05. d Cytotoxicity observed in cells transfected with scramble or BNIP3 shRNA using flow cytometry. *, p < 0.05. e Western blot analysis of BNIP3 protein expression in cells overexpressing BNIP3. f Quantitative data calculating the number of LC3 dots in the overexpression of BNIP3. Cells were transfected with BNIP3 for 24 h and were then incubated with YCW1 (1 μM) and IR (4 Gy) for 48 h. *, p < 0.05, YCW1 + IR versus BNIP3 + YCW1 + IR. Data are presented as the mean ± standard deviation of three independent experiments
Fig. 7
Fig. 7
Combined treatment with YCW1 and IR enhances the anti-tumor effects in an orthotopic breast cancer model. a 4 T1-luc cells were injected into the mammary fat pads of Balb/c mice, observed for luciferase signals and photographed using an IVIS 200. b Measurement of body weight in Balb/c mice taken once per week. c Quantification of the luciferase signals. *, p < 0.05, versus control. d IHC staining of orthotopic tumor tissues from the mice. IHC was used to determine the expression levels of LC3 and BNIP3 (×100 objective magnification). The percentage of LC3 and BNIP3-positive cells was determined using HistoQuest software (TissueGnostics)
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