J Pathol Transl Med.  2019 Mar;53(2):94-103. 10.4132/jptm.2019.01.14.

Guanabenz Acetate Induces Endoplasmic Reticulum Stress–Related Cell Death in Hepatocellular Carcinoma Cells

• Kang HJ
• Seol HS
• Lee SE
• Suh YA
• Kim J
• Jang SJ
• Yu E

• KMID: 2465448
• DOI: http://doi.org/10.4132/jptm.2019.01.14

Abstract

BACKGROUND
Development of chemotherapeutics for the treatment of advanced hepatocellular carcinoma (HCC) has been lagging. Screening of candidate therapeutic agents by using patient-derived preclinical models may facilitate drug discovery for HCC patients.
METHODS
Four primary cultured HCC cells from surgically resected tumor tissues and six HCC cell lines were used for high-throughput screening of 252 drugs from the Prestwick Chemical Library. The efficacy and mechanisms of action of the candidate anti-cancer drug were analyzed via cell viability, cell cycle assays, and western blotting.
RESULTS
Guanabenz acetate, which has been used as an antihypertensive drug, was screened as a candidate anti-cancer agent for HCC through a drug sensitivity assay by using the primary cultured HCC cells and HCC cell lines. Guanabenz acetate reduced HCC cell viability through apoptosis and autophagy. This occurred via inhibition of growth arrest and DNA damage-inducible protein 34, increased phosphorylation of eukaryotic initiation factor 2α, increased activating transcription factor 4, and cell cycle arrest.
CONCLUSIONS
Guanabenz acetate induces endoplasmic reticulum stress-related cell death in HCC and may be repositioned as an anti-cancer therapeutic agent for HCC patients.

Keyword

Hepatocellular carcinoma; Primary cell culture; Drug sensitivity; Drug repositioning; Guanabenz acetate

MeSH Terms

Activating Transcription Factor 4
Apoptosis
Autophagy
Blotting, Western
Carcinoma, Hepatocellular*
Cell Cycle
Cell Cycle Checkpoints
Cell Death*
Cell Line
Cell Survival
DNA
Drug Discovery
Drug Repositioning
Endoplasmic Reticulum*
Guanabenz*
Humans
Mass Screening
Peptide Initiation Factors
Phosphorylation
Primary Cell Culture
Activating Transcription Factor 4
DNA
Guanabenz
Peptide Initiation Factors

Figure

• 

  Fig. 1. (A) Histological features of surgically resected hepatocellular carcinomas, cases 1–4, respectively. (B) Cytological characteristics of primary cultured hepatocellular carcinoma cells with 3 passages, cases 1–4, respectively.

• 

  Fig. 2. (A) Drug sensitivity analysis using MTT assay of four primary cultured hepatocellular carcinoma (HCC) cells, revealing a candidate therapeutic agent for HCC. Plots show analyses of HCC cells from cases 1–4, respectively. The 41st of 252 drugs is guanabenz acetate, which shows a significant reduction of cell proliferation (IC50=20 μM) 3 days after drug treatment in cases 1, 2, and 4 (highlighted in red). (B) MTT assay to evaluate the inhibitory effect of guanabenz acetate and sorafenib on viability of stable HCC cell lines. Guanabenz acetate reduced the viability by more than 50% at 30 μM in Hep3B cells, and at 50 μM in Huh7 cells. Guanabenz acetate reduced the viability by more than 50% at 100 μM in SNU423, SNU449, and SNU475 cells. Cell viability of SNU398 cells was not reduced below 50%, even at 100 μM of guanabenz acetate. Dark bars, sorafenib treatment; light bars, guanabenz acetate treatment.

• 

  Fig. 3. (A) Western blotting showing that the effect of guanabenz acetate (GA) is associated with the protein kinase RNA-like endoplasmic reticulum kinase signaling pathway in hepatocellular carcinoma (HCC). Similar patterns of eIF2α phosphorylation (p-eIF2α) and activating transcription factor 4 (ATF4) protein expression are observed in HCC cells treated with GA and HCC cells transfected with growth arrest and DNA damage-inducible protein 34 (GADD34) siRNA. (B–D) Western blotting showing apoptosis- and autophagy-related protein levels after GA treatment of HCC cells. (B) The protein expression levels of BAX and Bcl-xL are increased and decreased, respectively, by GA in both Hep3B and Huh7 cells. (C) The protein expression level of procaspase-9 is decreased and that of cleaved caspase-9 is increased by GA in Hep3B and Huh7 cells. (D) The levels of autophagy-related proteins, LC3-I and LC3-II, are upregulated in GA-treated Hep3B cells, and LC3-II is upregulated in GA-treated Huh 7 cells.

• 

  Fig. 4. Flow cytometry and western blotting showing that the type of cell cycle arrest induced by guanabenz acetate (GA) varies between hepatocellular carcinoma cell lines. (A) Flow cytometry shows increased percentage of G1 phase cells in GA-treated Hep3B cells. (B) Flow cytometry shows increased percentage of G2 phase cells in GA-treated Huh7 cells. (C) Western blotting shows the effect of GA treatment on expression levels of checkpoint proteins in the G1/S and G2/M transition and cell cycle regulating factors in Hep3B and Huh7 cells.

• 

  Fig. 5. Mechanisms of guanabenz acetate stimulation of the protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK) signaling pathway. Guanabenz acetate selectively inhibits growth arrest and DNA damage-inducible protein 34 (GADD34) and increases phosphorylation of eukaryotic initiation factor 2α (eIF2α). Phosphorylation of eIF2α leads to increased activating transcription factor 4 (ATF4), which triggers cell apoptosis and autophagy.

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