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Blocking autophagy enhances the apoptosis effect of bufalin on human hepatocellular carcinoma cells through endoplasmic reticulum stress and JNK activation

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Abstract

Bufalin extracts are a part of traditional Chinese medicine, Chansu. In the current study, we investigated the effect of bufalin on the proliferation of the human hepatocellular carcinoma (HCC) cell lines, Huh-7 and HepG-2, and explored the therapeutic potential of the drug. Our results demonstrated that bufalin markedly inhibited cell proliferation and promoted apoptosis in the Huh-7 and HepG-2 cells in vitro. The underlying mechanism of the bufalin-induced apoptosis was the induction of endoplasmic reticulum (ER) stress via the IRE1–JNK pathway. In addition, during the ER stress response, the autophagy pathway, characterized by the conversion of LC3-I to LC3-II, was activated, resulting in increased Beclin-1 protein levels, decreased p62 expression and stimulation of autophagic flux. Our data supported the pro-survival role of bufalin-induced autophagy when the autophagy pathway was blocked with specific chemical inhibitors; the involvement of the IRE1 pathway in the ER stress-induced autophagy was also demonstrated when the expression of IRE1 and CHOP was silenced using siRNA. These data indicate that combining bufalin with a specific autophagy inhibitor could be a promising therapeutic approach for the treatment of HCC.

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Abbreviations

HCC:

Hepatocellular carcinoma

DMEM:

Dulbecco’s modified eagle medium

DMSO:

Dimethyl sulfoxide

PI:

Propidium iodide

CCK-8:

Cell Counting Kit-8

SD:

Standard deviation

CHOP:

Enhancer binding protein-homologous protein

PARP:

Poly(ADP-Ribose) polymerase

ANOVA:

Analysis of variance

IRE1:

Inositol-requiring enzyme 1

ATF6:

Activating transcription factor 6

PERK:

PKR-like ER kinase

UPR:

Unfolded protein response

ASK1:

Apoptosis-signal-regulating kinase

TRAF2:

TNF-receptor-associated factor 2

ER:

Endoplasmic reticulum

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Authors and Affiliations

  1. Department of Gastroenterology, The Fourth Affiliated Hospital of Harbin Medical University, Harbin, 150001, China

    Fengli Hu, Jiwu Han, Liwei Zhuang, Yong Liu & Tiefu Liu

  2. Department of General Surgery, The Fourth Affiliated Hospital of Harbin Medical University, Harbin, China

    Bo Zhai & Xiaodong Ming

  3. Department of General Surgery, The Hepatosplenic Surgery Center, The First Affiliated Hospital of Harbin Medical University, Harbin, 150001, China

    Shangha Pan

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  1. Fengli Hu
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Correspondence to Tiefu Liu.

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Hu, F., Han, J., Zhai, B. et al. Blocking autophagy enhances the apoptosis effect of bufalin on human hepatocellular carcinoma cells through endoplasmic reticulum stress and JNK activation. Apoptosis 19, 210–223 (2014). https://doi.org/10.1007/s10495-013-0914-7

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