Hepatic stellate cells promote upregulation of epithelial cell adhesion molecule and epithelial-mesenchymal transition in hepatic cancer cells

Nagahara,Teruya; Shiraha,Hidenori; Sawahara,Hiroaki; Uchida,Daisuke; Takeuchi,Yasuto; Iwamuro,Masaya; Kataoka,Junro; Horiguchi,Shigeru; Kuwaki,Takeshi; Onishi,Hideki; Nakamura,Shinichiro; Takaki,Akinobu; Nouso,Kazuhiro; Yamamoto,Kazuhide

doi:10.3892/or.2015.4126

Hepatic stellate cells promote upregulation of epithelial cell adhesion molecule and epithelial-mesenchymal transition in hepatic cancer cells

Authors:
- Teruya Nagahara
- Hidenori Shiraha
- Hiroaki Sawahara
- Daisuke Uchida
- Yasuto Takeuchi
- Masaya Iwamuro
- Junro Kataoka
- Shigeru Horiguchi
- Takeshi Kuwaki
- Hideki Onishi
- Shinichiro Nakamura
- Akinobu Takaki
- Kazuhiro Nouso
- Kazuhide Yamamoto
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Affiliations: Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Shikata-cho, Kita-ku, Okayama 700-8558, Japan
Published online on: July 13, 2015 https://doi.org/10.3892/or.2015.4126
Pages: 1169-1177
Copyright: © Nagahara et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].

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Abstract

Microenvironment plays an important role in epithelial-mesenchymal transition (EMT) and stemness of cells in hepatocellular carcinoma (HCC). Epithelial cell adhesion molecule (EpCAM) is known as a tumor stemness marker of HCC. To investigate the relationship between microenvironment and stemness, we performed an in vitro co-culture assay. Four HCC cell lines (HepG2, Hep3B, HuH-7 and PLC/PRF/5) were co-cultured with the TWNT-1 immortalized hepatic stellate cells (HSCs), which create a microenvironment with HCC. Cell proliferation ability was analyzed by flow cytometry (FCM) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while migration ability was assessed by a wound healing assay. Expression of EpCAM was analyzed by immunoblotting and FCM. HCC cell lines were co-cultured with TWNT-1 treated with small interfering RNA (siRNA) for TGF-β and HB-EGF; we then analyzed proliferation, migration ability and protein expression using the methods described above. Proliferation ability was unchanged in HCC cell lines co-cultured with TWNT-1. Migration ability was increased in HCC cell lines (HepG2, Hep3B, HuH-7 and PLC/PRF/5) directly (216.2±67.0, 61.0±22.0, 124.0±66.2 and 51.5±40.3%) and indirectly (102.5±22.0, 84.6±30.9, 86.1±25.7 and 73.9±29.7%) co-cultured with TWNT-1 compared with the HCC uni-culture. Immunoblot analysis revealed increased EpCAM expression in the HCC cell lines co-cultured with TWNT-1. Flow cytometry revealed that the population of E-cadherin-/N-cadherin+ and EpCAM-positive cells increased and accordingly, EMT and stemness in the HCC cell line were activated. These results were similar in the directly and indirectly co-cultured samples, indicating that humoral factors were at play. Conversely, HCC cell lines co-cultured with siRNA‑treated TWNT-1 showed decreased migration ability, a decreased population of EpCAM-positive and E-cadherin-/N-cadherin+ cells. Taken together, humoral factors secreted from TWNT-1 promote upregulation of EpCAM and EMT in hepatic cancer cells.

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