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. 2015 May 4;20(5):8000-19.
doi: 10.3390/molecules20058000.

NBM-T-BBX-OS01, Semisynthesized from Osthole, Induced G1 Growth Arrest through HDAC6 Inhibition in Lung Cancer Cells

Jih-Tung Pai  1 , Chia-Yun Hsu  2 , Kuo-Tai Hua  3 , Sheng-Yung Yu  4 , Chung-Yang Huang  5 , Chia-Nan Chen  6 , Chiung-Ho Liao  7 , Meng-Shih Weng  8
Affiliations

NBM-T-BBX-OS01, Semisynthesized from Osthole, Induced G1 Growth Arrest through HDAC6 Inhibition in Lung Cancer Cells

Jih-Tung Pai et al. Molecules. .

Abstract

Disrupting lung tumor growth via histone deacetylases (HDACs) inhibition is a strategy for cancer therapy or prevention. Targeting HDAC6 may disturb the maturation of heat shock protein 90 (Hsp90) mediated cell cycle regulation. In this study, we demonstrated the effects of semisynthesized NBM-T-BBX-OS01 (TBBX) from osthole on HDAC6-mediated growth arrest in lung cancer cells. The results exhibited that the anti-proliferative activity of TBBX in numerous lung cancer cells was more potent than suberoylanilide hydroxamic acid (SAHA), a clinically approved pan-HDAC inhibitor, and the growth inhibitory effect has been mediated through G1 growth arrest. Furthermore, the protein levels of cyclin D1, CDK2 and CDK4 were reduced while cyclin E and CDK inhibitor, p21Waf1/Cip1, were up-regulated in TBBX-treated H1299 cells. The results also displayed that TBBX inhibited HDAC6 activity via down-regulation HDAC6 protein expression. TBBX induced Hsp90 hyper-acetylation and led to the disruption of cyclin D1/Hsp90 and CDK4/Hsp90 association following the degradation of cyclin D1 and CDK4 proteins through proteasome. Ectopic expression of HDAC6 rescued TBBX-induced G1 arrest in H1299 cells. Conclusively, the data suggested that TBBX induced G1 growth arrest may mediate HDAC6-caused Hsp90 hyper-acetylation and consequently increased the degradation of cyclin D1 and CDK4.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Structure of NBM-T-BBX-OS01 (TBBX).
Figure 2
Figure 2
The growth inhibition effect of TBBX in H1299 lung cancer cells. (A) A549, H1299, H460 and H1155 cells were incubated with a serial dosage (0, 2.5, 5, 7.5, 10 and 20 μM) of TBBX or suberoylanilide hydroxamic acid (SAHA) for 24 h. Afterward, MTT assay was performed for cell viability as described in Materials and Methods. Data shown are representative of at least three independent experiments. (B) H1299 cells were seeded in a 10 cm petri dish for 24 h. Cells were then treated with various doses of TBBX for 24 h. After cells were harvested, flow cytomertic analysis was performed for cell cycle distribution. Data were the mean ± S.D. of triplicate samples. Significant difference was observed from the control group (* p < 0.05).
Figure 2
Figure 2
The growth inhibition effect of TBBX in H1299 lung cancer cells. (A) A549, H1299, H460 and H1155 cells were incubated with a serial dosage (0, 2.5, 5, 7.5, 10 and 20 μM) of TBBX or suberoylanilide hydroxamic acid (SAHA) for 24 h. Afterward, MTT assay was performed for cell viability as described in Materials and Methods. Data shown are representative of at least three independent experiments. (B) H1299 cells were seeded in a 10 cm petri dish for 24 h. Cells were then treated with various doses of TBBX for 24 h. After cells were harvested, flow cytomertic analysis was performed for cell cycle distribution. Data were the mean ± S.D. of triplicate samples. Significant difference was observed from the control group (* p < 0.05).
Figure 3
Figure 3
Effects of TBBX on the expressions of cyclins, and CDKs in H1299 lung cancer cells. H1299 lung cancer cells were initially synchronized by serum-free medium and then serum-supplemented medium containing various doses of TBBX (0, 2.5, 5, 7.5, and 10 μM). After the cells were harvested, Western blot analyses were performed with anti-cyclin D1, E, CDK2, CDK4, and β-actin antibodies. Data shown are representative of at least three independent experiments. Significant difference was observed from the control group (* p < 0.05).
Figure 4
Figure 4
Effects of TBBX on the expression of CDK inhibitors, p21Waf1/Cip1 and p27Kip1, in lung carcinoma H1299 cells. H1299 lung cancer cells were initially synchronized by serum-free medium and then serum-supplemented medium containing various doses of TBBX (0, 2.5, 5, 7.5, and 10 μM) for 24 h. After the cells were harvested, (A) Western blot analyses were performed with anti-p21Waf1/Cip1, p27Kip1 and anti-β-actin antibodies. (B) H1299 cells were treated with TBBX for 12 h and total mRNAs were extracted afterward. After the extraction of total mRNAs, p21Waf1/Cip1 and GAPDH RT-PCR were performed as described in Materials and Methods. Data shown are representative of at least three independent experiments. Significant difference was observed from the control group (* p < 0.05).
Figure 5
Figure 5
Effects of TBBX on the class I HDAC activity and protein expression in H1299 lung cancer cells. (A) Direct inhibition class I HDAC activity assay by TBBX was performed as described in Materials and Methods. (B) H1299 cells were treated with various dosage of TBBX (0, 2.5, 5, 7.5, and 10 μM) for 24 h. After treatment, cells were harvested and Western blot was done by anti-HDAC1, HDAC2, HDAC3 and anti-β-actin antibodies. Data shown are representative of at least three independent experiments.
Figure 6
Figure 6
TBBX induced Hsp90 hyper-acetylation and disrupted cyclin D1/Hsp90 and CDK4/Hsp90 interaction and leading to cyclin D1 and CDK4 degradation in H1299 lung cancer cells. (A) H1299 cells were incubated with or without 10 μM MG132 for 30 min before 10 μM TBBX stimulation for 24 h. Cells were harvested to detect cyclin D1 and CDK4 expression by Western blotting analyses. (B) H1299 cells were incubated with 10 μM TBBX for 24 h. Cells were then harvested and immuno-precipitation analysis was down with anti-Hsp90 antibody. The immuno-precipitates were then detected by Western blotting with anti-cyclin D1, anti-CDK4 and anti-acetyl-lysine antibodies as described in Materials and Methods. Data shown are representative of at least three independent experiments. Significant difference was observed from the control group (* p < 0.05).
Figure 7
Figure 7
Effect of TBBX on class IIb HDAC6 activity and protein expression in H1299 lung cancer cells. (A) Nuclear extracts containing HDAC6 enzymes were incubated with various concentrations of TBBX (0, 2.5, 5, 7.5 and 10 μM) and HDAC6 activity assay was determined as described in Materials and Methods. The trichostatin A (TSA, 2 μM) were examined as positive control. (B) H1299 cells were incubated with various dosage of TBBX (0, 2.5, 5, 7.5 and 10 μM) for 24 h. Cells were harvested and HDAC6 activity was determined as described in Material and Methods. (C) H1299 cells were treated with various dosage of TBBX (0, 2.5, 5, 7.5, and 10 μM) for 24 h. After treatment, cells were harvested and Western blot was done by anti-HDAC6, acetyl-tubulin and anti-β-actin antibodies. Data were the mean ± S.D. of triplicate samples. Significant difference was observed from the control group (* p < 0.05).
Figure 8
Figure 8
Ectopic expression of HDAC6 rescued TBBX-induced G1 growth arrest in H1299 lung cancer cell. H1299 cells were transient transfected HDAC6-overexpressed plasmid or vector only as described in Materials and Methods. Afterward, cells were treated with 10 μM TBBX for 24 h. (A) Cells were then harvested and Western blotting analyses performed by anti-HDAC6, acetyl-α-tubulin, flag-tag and anti-α-tubulin. Data shown are representative of at least three independent experiments. (B) After TBBX treatment, cell cycle analyses were done as described in Materials and Methods. Data were the mean ± S.D. of triplicate samples. Significant difference was observed from the control group (* p < 0.05).
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