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Journal of Molecular Medicine

, Volume 96, Issue 12, pp 1307–1318 | Cite as

Trichostatin A, a histone deacetylase inhibitor, induces synergistic cytotoxicity with chemotherapy via suppression of Raf/MEK/ERK pathway in urothelial carcinoma

  • Wei-Chou Lin
  • Fu-Shun Hsu
  • Kuan-Lin Kuo
  • Shing-Hwa Liu
  • Chia-Tung Shun
  • Chung-Sheng Shi
  • Hong-Chiang Chang
  • Yu-Chieh Tsai
  • Ming-Chieh Lin
  • June-Tai Wu
  • Yu Kuo
  • Po-Ming Chow
  • Shih-Ming Liao
  • Shao-Ping Yang
  • Jo-Yu Hong
  • Kuo-How Huang
Original Article
  • 291 Downloads

Abstract

In this study, we aimed to investigate the antitumor effects of trichostatin A (TSA), an antifungal antibiotic that inhibits histone deacetylase (HDAC) family of enzymes, alone or in combination with anyone of the three chemotherapeutic agents (cisplatin, gemcitabine, and doxorubicin) for the treatment of human urothelial carcinoma (UC). Two high-grade human UC cell lines (T24 and NTUB1) were used. Cytotoxicity and apoptosis were assessed by MTT assay and flow cytometry, respectively. The expression of phospho-c-Raf, phospho-MEK1/2, and phospho-ERK1/2 was measured by western blotting. ERK siRNA knockdown and the specific MEK inhibitor U0126 were used to examine the role of Raf/MEK/ERK signaling pathway in combined cytotoxicity of TSA and chemotherapy. TSA co-treatment with any one of the three chemotherapeutic agents induced synergistic cytotoxicity (combination index < 1) and concomitantly suppressed chemotherapeutic drug-induced activation of Raf-MEK-ERK pathway. Combination of ERK siRNA knockdown and treatment with the specific MEK inhibitor (U0126) enhanced the cytotoxic effects of the chemotherapy on UC cells. These observations were confirmed in a xenograft nude mouse model. Moreover, activated Raf/MEK/ERK pathway was observed in human bladder UC specimens from patients with chemoresistant status. In conclusion, TSA elicits a synergistic cytotoxic response in combination with chemotherapy via targeting the Raf/MEK/ERK pathway. TSA elicits synergistic cytotoxic response in combination with three DNA-damaging drugs (cisplatin, gemcitabine, and doxorubicin). Activated Raf/MEK/ERK pathway is involved in chemoresistant mechanism of UC. Combining chemotherapeutic agents with HDAC inhibitor (TSA) or with targeting Raf/MEK/ERK pathway is promising to circumvent chemoresistance in UCs.

Keywords

Urothelial carcinoma Trichostatin A Histone deacetylase inhibitor Chemotherapy Drug resistance 

Abbreviations

ERK

extracellular signal-regulated kinase 1 and 2 (ERK1/2)

UC

urothelial carcinoma

HDAC

histone deacetylase

TSA

trichostatin A

Notes

Acknowledgements

This work was supported by grants from the Ministry of Science and Technology of Taiwan (104-2314-B-002-164-MY3 and 103-2314-B-002-161-MY3), National Taiwan University Hospital (103-S2349, 104-M2868, 105-S2978,105-28, 106-3415, and 107-S3784), and New Taipei City Hospital.

Author contributions

Wei-Chou Lin, Kuan-Lin Kuo, and Kuo-How Huang conceived of the presented idea and study design.

Kuan-Lin Kuo and Kuo-How Huang wrote the manuscript with support from Fu-Shun Hsu and Wei-Chou Lin.

Kuan-Lin Kuo, Shih-Ming Liao, Jo-Yu Hong, and Shao-Ping Yang carried out the experiment and performed the computations.

Kuo-How Huang, Shing-Hwa Liu, Chung-Sheng Shi, Hong-Chiang Chang, Fu-Shun Hsu, Wei-Chou Lin, Yu-Chieh Tsai, and June-Tai Wu verified the analytical methods and helped supervise the project.

Wei-Chou Lin and Chia-Dong Shun interpreted the results of immunostaining.

All authors discussed the results and contributed to the final manuscript.

Funding information

We also thank the personnel of the Second, Third, and Sixth Core Laboratories of National Taiwan University Hospital.

Compliance with ethical standards

The study that involve human participants and animal experiments have been approved by the institutional research ethics committee (no. 201112136RIC) and National Taiwan University College of Medicine and College of Public Health Institutional Animal Care and Use Committee (IACUC) (No. 20160117).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

109_2018_1697_MOESM1_ESM.jpg (45 kb)
Fig. S1 a, b NTUB1 (a) and T24 (b) cells were treated with 0.5 μM TSA alone or in combination with either cisplatin 10 μM, gemcitabine 2.5 μM, or doxorubicin 0.25 μM. Cell lysates were harvested and analyzed by western blotting with specific antibodies against phospho-Bcl2. (JPG 44 kb)
109_2018_1697_MOESM2_ESM.jpg (36 kb)
Fig. S2 a Western blot analysis by phospho-ERK1/2 and phospho-c-Raf antibodies on proteins extracted from UC tumors of two patients in chemo-sensitive and two patients in chemo-resistant status. b Comparative results of IHC scores in UC tumors from 5 patients in chemo-sensitive and 5 patients in chemo-resistant status.*p < 0.05 represents a significant difference between chemo-resistant and chemo-sensitive groups. (JPG 35 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Wei-Chou Lin
    • 1
  • Fu-Shun Hsu
    • 2
    • 3
  • Kuan-Lin Kuo
    • 4
    • 5
  • Shing-Hwa Liu
    • 4
  • Chia-Tung Shun
    • 1
  • Chung-Sheng Shi
    • 6
  • Hong-Chiang Chang
    • 5
  • Yu-Chieh Tsai
    • 7
  • Ming-Chieh Lin
    • 8
  • June-Tai Wu
    • 8
  • Yu Kuo
    • 8
    • 9
  • Po-Ming Chow
    • 5
  • Shih-Ming Liao
    • 5
  • Shao-Ping Yang
    • 5
  • Jo-Yu Hong
    • 5
  • Kuo-How Huang
    • 5
    • 10
  1. 1.Department of Pathology, College of MedicineNational Taiwan University, National Taiwan University HospitalTaipeiTaiwan
  2. 2.Graduate Institute of Clinical Medicine, College of MedicineNational Taiwan UniversityTaipeiTaiwan
  3. 3.Department of UrologyNew Taipei City HospitalNew Taipei CityTaiwan
  4. 4.Graduate Institute of Toxicology, College of MedicineNational Taiwan UniversityTaipeiTaiwan
  5. 5.Department of Urology, College of MedicineNational Taiwan University, National Taiwan University HospitalTaipeiTaiwan
  6. 6.Graduate Institute of Clinical Medical Sciences, College of MedicineChang Gung UniversityTaoyuanTaiwan
  7. 7.Department of Oncology, College of MedicineNational Taiwan University, National Taiwan University HospitalTaipeiTaiwan
  8. 8.Graduate Institute of Molecular Medicine, College of MedicineNational Taiwan UniversityTaipeiTaiwan
  9. 9.Department of RadiologyTaipei Veterans General HospitalTaipeiTaiwan
  10. 10.Department of Urology, College of MedicineNational Taiwan UniversityTaipeiTaiwan

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