Korean J Physiol Pharmacol.  2024 Jan;28(1):83-91. 10.4196/kjpp.2024.28.1.83.

ACY-241, a histone deacetylase 6 inhibitor, suppresses the epithelial–mesenchymal transition in lung cancer cells by downregulating hypoxia-inducible factor-1 alpha

• Park SJ
• Lee N
• Jeong CH

• KMID: 2550291
• DOI: http://doi.org/10.4196/kjpp.2024.28.1.83

Abstract

Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor activated under hypoxic conditions, and it plays a crucial role in cellular stress regulation. While HIF-1α activity is essential in normal tissues, its presence in the tumor microenvironment represents a significant risk factor as it can induce angiogenesis and confer resistance to anti-cancer drugs, thereby contributing to poor prognoses. Typically, HIF-1α undergoes rapid degradation in normoxic conditions via oxygen-dependent degradation mechanisms. However, certain cancer cells can express HIF-1α even under normoxia. In this study, we observed an inclination toward increased normoxic HIF-1α expression in cancer cell lines exhibiting increased HDAC6 expression, which prompted the hypothesis that HDAC6 may modulate HIF-1α stability in normoxic conditions. To prove this hypothesis, several cancer cells with relatively higher HIF-1α levels under normoxic conditions were treated with ACY-241, a selective HDAC6 inhibitor, and small interfering RNAs for HDAC6 knockdown. Our data revealed a significant reduction in HIF-1α expression upon HDAC6 inhibition. Moreover, the downregulation of HIF-1α under normoxic conditions decreased zinc finger E-box-binding homeobox 1 expression and increased E-cadherin levels in lung cancer H1975 cells, consequently suppressing cell invasion and migration. ACY-241 treatment also demonstrated an inhibitory effect on cell invasion and migration by reducing HIF-1α level. This study confirms that HDAC6 knockdown and ACY-241 treatment effectively decrease HIF-1α expression under normoxia, thereby suppressing the epithelial– mesenchymal transition. These findings highlight the potential of selective HDAC6 inhibition as an innovative therapeutic strategy for lung cancer.

Keyword

ACY-241 (Citarinostat); Epithelial-mesenchymal transition; Histone deacetylase 6; Hypoxia-inducible factor 1, alpha subunit

Figure

• 

  Fig. 1 HDAC6 knockdown reduces the protein levels of HIF-1α under normoxic conditions. (A) Western blot analysis of HDAC6 and HIF-1α levels under normoxic conditions in breast cancer and lung cancer cells. (B) Cells were transfected with siHDAC6 for 48 h. HDAC6, HIF-1α, Ac-α-tubulin, and α-tubulin expression levels were measured using western blot analysis. GAPDH was used as the loading control. (C) Cells were transfected with siHDAC6 for 48 h. HIF-1α expression was measured using qRT-PCR. The expression value was normalized to the 18s rRNA level. Statistical analysis was performed using Student’s t-test. Error bars present the mean ± standard deviation (n = 3). NS > 0.05 vs. siCON. HDAC6, histone deacetylase 6; HIF-1α, hypoxia-inducible factor 1 alpha; siHDAC6, small interfering RNA against HDAC6; Ac-α-tubulin, acetylated alpha tubulin; GAPDH, glyceraldehyde 3-phosphate; siCON, negative control siRNA; NS, non-significant.

• 

  Fig. 2 ACY-241 decreases the HIF-1α protein level. (A) After treatment with ACY-241 for 48 h, the cell viability was measured using the CCK-8 assay. Statistical analysis was performed using one‐way ANOVA (Tukey's multiple comparisons test). Error bars present the mean ± standard deviation (n = 6). ***p < 0.001 vs. CON. (B) Cells were treated with ACY-241 for 48 h, after which the protein levels of HDAC6, HIF-1α, Ac-α-tubulin, and α-tubulin were measured via western blot analysis. GAPDH was used as the loading control. (C) Cells were treated with ACY-241 for 48 h and with MG-132 4 h before sampling, after which the protein levels of HIF-1α were measured via Western blot analysis. GAPDH was used as the loading control. HDAC6, histone deacetylase 6; HIF-1α, hypoxia-inducible factor 1 alpha; Ac-α-tubulin, acetylated alpha tubulin; GAPDH, glyceraldehyde 3-phosphate; CON, control.

• 

  Fig. 3 Knockdown of HIF-1α inhibits invasion and migration of H1975 cells. (A) Cells were transfected with siHIF-1α for 48 h. Cell viability was measured using the CCK-8 assay. Statistical analysis was performed using Student’s t-test. Error bars present the mean ± standard deviation (n = 6). ***p < 0.001 vs. siCON. (B) Transwell assays were performed by incubating H1975 cells for 36 h under normoxic conditions after siHIF-1α treatment. Representative images of cell migration and invasion captured under a microscope. Statistical analysis was performed using one‐way ANOVA. Error bars present the mean ± standard deviation (n = 3). ***p < 0.001 vs. siCON. (C) Cells were transfected with siHIF-1α for 48 h. The HIF-1α, E-cadherin, and ZEB1 expression levels were measured using western blotting. GAPDH was used as a loading control. Quantitation of protein intensity was performed using the ImageJ software. Statistical analysis was performed using Student’s t-test. Error bars present the mean ± standard deviation (n = 3). ***p < 0.001 vs. siCON. (D) qRT-PCR analysis of ZEB1. Cells were transfected with siHIF-1α for 48 h. The expression value was normalized to the 18s rRNA level. Statistical analyses were performed using the Student’s t-test. Error bars present the mean ± standard deviation (n = 3). ***p < 0.001 vs. siCON. HDAC6, histone deacetylase 6; HIF-1α, hypoxia-inducible factor 1 alpha; ZEB1, zinc finger E-box-binding homeobox 1; siHIF-1α, small interfering RNA against HIF-1α; GAPDH, glyceraldehyde 3-phosphate; siCON, negative control siRNA.

• 

  Fig. 4 ACY-241 inhibits migration and invasion in H1975 cell lines. (A) Wound healing assay of H1975 cells treated with different concentrations of ACY-241. Microphotographs of the wounded area were taken at 0, 24, and 36 h to observe cell migration into the wounded area. Statistical analysis was performed using one‐way ANOVA (Tukey's multiple comparisons test). Error bars present the mean ± standard deviation (n = 6). ***p < 0.001 vs. CON. (B) Transwell assays were performed using different concentrations of ACY-241 for 36 h. Representative images of cell migration and invasion were captured under a microscope. Statistical analysis was performed using one‐way ANOVA (Tukey's multiple comparisons test). Error bars present the mean ± standard deviation (n = 3). ***p < 0.001 vs. CON. (C) Cells were treated with ACY-241 for 48 h, after which the protein levels of HDAC6, HIF-1α, E-cadherin, and ZEB1 were monitored via Western blot analysis. GAPDH was used as a loading control. Quantitation of protein intensity was performed using the ImageJ software. Statistical analysis was performed using one‐way ANOVA (Tukey's multiple comparisons test). Error bars present the mean ± standard deviation (n = 3). **p < 0.01; ***p < 0.001 vs. CON. (D) qRT-PCR analysis of ZEB1. Cells were treated with ACY-241 for 48 h. The expression value was normalized to the 18s rRNA level. Statistical analyses were performed using the one‐way ANOVA (Tukey's multiple comparisons test). Error bars present the mean ± standard deviation (n = 3). **p < 0.01; ***p < 0.001 vs. CON. (E) Under normoxic conditions, cancer cells with higher expression of HDAC6 have been shown to increase the stability of HIF-1α protein. The stabilized HIF-1α induces the expression of ZEB1, which decreases the expression of E-cadherin, leading to increased invasion and migration of cancer cells (left). Conversely, reducing HDAC6 through HDAC6 knockdown, or inhibiting HDAC6 activity with ACY-241, decreases the protein level of HIF-1α, which in turn leads to a reduction in epithelial-mesenchymal transition (right). HDAC6, histone deacetylase 6; HIF-1α, hypoxia-inducible factor 1 alpha; ZEB1, zinc finger E-box-binding homeobox 1; GAPDH, glyceraldehyde 3-phosphate ; CON, control.

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