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. 2019 Mar 22;19(1):262.
doi: 10.1186/s12885-019-5482-y.

Short-term stimulation with histone deacetylase inhibitor trichostatin a induces epithelial-mesenchymal transition in nasopharyngeal carcinoma cells without increasing cell invasion ability

Zhihua Shen  1 , Xiaomin Liao  2 , Zhongming Shao  2 , Muyin Feng  2 , Jianling Yuan  1 , Sisi Wang  2 , Siyuan Gan  2 , Yanping Ha  2 , Zhiwei He  1 , Wei Jie  3
Affiliations

Short-term stimulation with histone deacetylase inhibitor trichostatin a induces epithelial-mesenchymal transition in nasopharyngeal carcinoma cells without increasing cell invasion ability

Zhihua Shen et al. BMC Cancer. .

Abstract

Background: Epithelial-mesenchymal transition (EMT) may be one of the reasons for the failure in some clinical trials regarding histone deacetylase inhibitors (HDACIs)-treated solid tumors. We investigated the effects of a pan-HDACI trichostatin A (TSA) on the proliferation and EMT of nasopharyngeal carcinoma (NPC) cells.

Methods: Poorly-differentiated NPC cell line CNE2 and undifferentiated C666-1 were treated with various concentrations of TSA, the cell viability was assessed by CCK-8 assay, the morphology was photographed, and the mRNA level of HDACs was assessed by semiquantitative PCR. After determination the cell cycle distributions, cells were subjected to western blotting analysis of cell cycle and EMT-associated genes expression. And the changes in migration ability were assessed by transwell migration assay and scratch wound healing assay. Finally, histone deacetylases activator ITSA-1 was used to assess the reverse of TSA-induced changes in NPC cells.

Results: TSA inhibited the proliferation of CNE2 and C666-1 cells in a concentration-dependent manner and arrested the cell cycle at G1 phases. TSA reduced PCNA, cyclin D1, cyclin E1, CDK2, p16 and p21 expressions and stimulated CDK6 levels. TSA stimulation for 48 h could effectively induce the EMT in CNE2 and C666-1 cells, which showed an increase of spindle-like cells and promoted expression of Vimentin and Snail1 expression in a concentration-dependent manner. Surprisingly, this short period of TSA treatment that induced EMT also impeded the migration ability of CNE2 and C666-1 cells. Interestingly, ITSA-1 rescued TSA-impeded CNE2 and C666-1 cells' proliferation, migration and HDACs expression, also re-induced the cells to turn into epithelial cell phenotypes.

Conclusions: These results indicate that short-term stimulation of TSA effectively inhibits cell proliferation and induce EMT-like changes in NPC cells but not increase its invasion ability.

Keywords: Epithelial mesenchymal transition; Histone deacetylase inhibitor; ITSA-1; Migration; Nasopharyngeal carcinoma; Proliferation; Trichostatin a.

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Conflict of interest statement

Ethics approval and consent to participate

All usage of cell lines did not require Ethic approval.

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Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig 1
Fig 1
TSA inhibits NPC cells proliferation. a CNE2 and C666–1 cells plated in 96-well plate were treated with the indicated various concentrations of TSA for 0 h, 24 h and 48 h, and CCK-8 assay was performed. N.S., no significance; **P < 0.01 vs. 0 h; ***P < 0.001 vs. 0 h; #P < 0.05 vs. TSA 0 ng/ml; ##P < 0.01 vs. TSA 0 ng/ml; ###P < 0.001 vs. TSA 0 ng/ml. b Western blot of PCNA protein expression in CNE2 and C666–1 cells treated with various concentrations of TSA for 48 h
Fig 2
Fig 2
Effects of TSA on the cell cycle distribution of NPC cells. a Flow cytometric analysis was performed to analyze the cell cycle distribution of CEN2 and C666–1 cells treated with 0 and 200 ng/ml TSA for 48 h. *P < 0.05; #P < 0.05. b Western blot analysis was performed for the indicated proteins in CNE2 and C666–1 cells treated with 0 and 200 ng/ml TSA for 48 h
Fig 3
Fig 3
Morphology of NPC cells treated with TSA. CNE2 and C666–1 cells were treated with 0 and 200 ng/ml TSA for 0, 24 and 48 h, and then cells were photographed under inverted microscope. Scale bars = 200 μm or 100 μm
Fig 4
Fig 4
TSA induces EMT-associated marker expression in NPC cells in a concentration-dependent manner. CNE2 and C666–1 cells were treated with various concentrations of TSA for short period within 48 h, and then cells were harvested and subjected to real-time PCR a and western blot b analysis of EMT-associated E-cadherin, Vimentin, Snail1 and Twist1 gene and protein expressions. *P < 0.05 vs. TSA 0 ng/ml; **P < 0.01 vs. TSA 0 ng/ml; ***P < 0.001 vs. TSA 0 ng/ml; #P < 0.05 vs. TSA 0 ng/ml; ##P < 0.01 vs. TSA 0 ng/ml
Fig 5
Fig 5
TSA attenuates NPC cells motility within short periods of treatment. CNE2 and C666–1 cell were treated with 0 and 200 ng/ml TSA for 48 h and scratch wound healing assay a and transwell migration assay b were performed. In a **P < 0.01 vs. 24 h, #P < 0.05; ##P < 0.01; in b ***P < 0.001
Fig. 6
Fig. 6
ITSA-1 reverses TSA-impacted changes in NPC cells. a CCK-8 assay was used to detect the cell viability of TSA (200 ng/ml) and TSA + ITSA-1(10 μmol/l) - treated CNE2 and C666–1 cells. N.S., no significance; ***P < 0.001. b Western blot was used to examine the protein levels of cell proliferation (PCNA), cell cycle (cyclinD1, CDK2 and p16) and EMT-associated genes (E-cadherin and Vimentin). c Morphology of TSA and ITSA-impacted NPC cells. Scale bars = 50 μm. d & e Scratch wound healing assay was performed to test the migration ability of CNE2 and C666–1 cell treated with TSA or TSA + ITSA-1 for 48 h. Scale bars = 200 μm. *P < 0.05; **P < 0.01
Fig 7
Fig 7
Expression and functional role of HDACs in TSA and ITSA-1-treated NPC cells. a Semiquantitative PCR was used to detect the mRNA level of HDAC1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 in TSA and TSA + ITSA-1 treated CNE2 and C666–1 cells. M, DNA marker. b Schematic diagram of the functional mechanisms of TSA and ITSA-1-impacted HDACs on NPC cells
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