Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul 110‑799, Republic of Korea.
Abstract
Sorafenib has been approved for the treatment of advanced stage hepatocellular carcinoma but has limited efficacy. Ursodeoxycholic acid exerts cytoprotective activities in hepatocytes and is believed to suppress tumorigenesis through cell cycle arrest and induction of apoptosis. The present study examined whether co‑treatment with ursodeoxycholic acid has a synergistic effect on the antitumor activity of sorafenib in hepatocellular carcinoma cells. Notably, co‑treatment with both agents more effectively inhibited cell proliferation than sorafenib or ursodeoxycholic acid alone. Furthermore, co‑treatment inhibited the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and activated extracellular signal‑regulated kinase (ERK), a mitogen‑activated protein kinase, accompanied by excessive intracellular reactive oxygen species generation in hepatocellular carcinoma cells. Thus, chemotherapy with sorafenib and ursodeoxycholic combination may be efficacious in hepatocellular carcinoma by inhibiting cell proliferation and inducing apoptosis through reactive oxygen species‑dependent activation of ERK and dephosphorylation of STAT3. The present findings may represent a promising therapeutic strategy for patients with advanced hepatocellular carcinoma.
Figures
Figure 1Sorafenib and UDCA inhibit hepatocellular carcinoma cell viability. (A) Huh-BAT and (B) HepG2 cells were treated for 48 h with different concentrations of sorafenib (1, 3, 5 and 10 μM), UDCA (250, 500, 750 and 1,000 μM), or co-treated with 5 μM sorafenib and 750 μM UDCA. Cell viability was measured using the MTT assay. **P<0.01, compared with control; #P<0.05 and ##P<0.01 compared with sorafenib alone. UDCA, ursodeoxycholic acid.Figure 2Sorafenib and UDCA induce caspase-dependent apoptosis in HCC cells. (A) Huh-BAT and HepG2 cells were treated with 5 μM sorafenib and 750 μM UDCA for 48 h. Cell morphology was observed by phase-contrast microscopy. Scale bar, 100 μm. (B) Representative plots and (C) quantification of flow cytometry analysis of cell apoptosis by Annexin V/PI staining. Cells were treated with 5 μM sorafenib, 750 μM UDCA, or both for 48 h. The % of apoptotic cells was evaluated from three independent experiments. Data are presented as means ± standard deviation. *P<0.05 and **P<0.01 compared with control untreated cells. (D) Expression levels of cleaved caspase-3 and caspase-9 were determined by western blotting following 48-h treatment with 5 μM sorafenib, 750 μM UDCA, or both. UDCA, ursodeoxycholic acid; FITC, fluorescein isothiocyanate.Figure 3Sorafenib and UDCA suppress activation of STAT3 in a time- and concentration-dependent manner. (A) Huh-BAT cells were treated with various doses of UDCA or sorafenib for 48 h. (B) Huh-BAT cells were treated with 750 μM UDCA or 5 μM sorafenib for various timepoints. (C) HepG2 cells were treated with various doses of UDCA or sorafenib for 48 h. (D) HepG2 cells were treated with 750 μM UDCA or 5 μM sorafenib for various timepoints. (E) Cells were treated with 5 μM sorafenib, 750 μM UDCA or both for 48 h. Protein expression levels of STAT3 and phospho-STAT3 were evaluated by western blotting. β-actin was used as a loading control. Representative blots and quantifications are shown for all panels. Data are presented as means ± standard deviation. **P<0.01 compared with control untreated cells. (F) Huh-BAT and HepG2 cells were treated with 5 μM sorafenib, 750 μM UDCA, or both for 48 h. Treatment with 50 ng/ml IL-6 for 30 min was used as a positive control for STAT3 activation. Following treatments, localization of phospho-STAT3 was evaluated using immunofluorescence. Magnification, ×400. UDCA, ursodeoxycholic acid; STAT3, signal transducer and activator of transcription 3; IL-6, interkleukin-6.Figure 4Sorafenib and UDCA trigger ROS production and activate ERK in a time- and concentration-dependent manner. (A) Huh-BAT and HepG2 cells were treated with 5 μM sorafenib, 750 μM UDCA, or both, or with 5 mM NAC for 48 h. ROS generation was measured by confocal fluorescence microscopy. Magnification, ×400. (B) Quantitative analysis of ROS generation. (C) Huh-BAT cells were treated with various doses of UDCA or sorafenib for 48 h. (D) Huh-BAT cells were treated with 750 μM UDCA or 5 μM sorafenib for various timepoints. (E) HepG2 cells were treated with various doses of UDCA or sorafenib for 48 h. (F) HepG2 cells were treated with 750 μM UDCA or 5 μM sorafenib for various timepoints. (G) Cells were treated with 5 μM sorafenib, 750 μM UDCA or both for 48 h. Protein expression levels of ERK and phospho-ERK were evaluated by western blotting. β-actin was used as a loading control. Representative blots and quantifications are shown. Data are presented as means ± standard deviation. *P<0.05 and **P<0.01 compared with control untreated cells. UDCA, ursodeoxycholic acid; ROS, reactive oxygen species; ERK, extracellular signal-regulated kinase; NAC, N-acetyl cysteine.Figure 5ROS production and ERK activation are associated with downregulation of STAT3 phosphorylation in Huh-BAT cells co-treated with sorafenib and UDCA. (A) Huh-BAT cells were co-treated with 5 μM sorafenib and 750 μM UDCA for 48 h, followed by 5 mM NAC. Following treatments, ROS intensity was measured using a fluorescence microplate reader. The relative ROS levels were normalized to the mean intensity of ROS in the control untreated cells (presented here as 1.0). (B) Huh-BAT cells were pretreated with 5 mM NAC, or (C) pretreated with 5 μM U0126, prior to 5 μM sorafenib and 750 μM UDCA treatments. Samples were immunoblotted with primary antibodies to STAT3, phospho-STAT3, ERK, and phospho-ERK. β-actin was used as a loading control. *P<0.05 and **P<0.01 compared with control untreated cells. ROS, reactive oxygen species; ERK, extracellular signal-regulated kinase; STAT3, signal transducer and activator of transcription 3; UDCA, ursodeoxycholic acid; NAC, N-acetyl cysteine.
그림 출처: PubMed Central 공개 논문 (PMC6192782)
그 밖의 정보
출판 형태
Journal Article
저널
International journal of molecular medicine · eISSN 1791-244X
전자 출판
2018-08-03
MeSH
Apoptosis / drug effects · Blotting, Western · Carcinoma, Hepatocellular / drug therapy / metabolism · Cell Survival / drug effects · Extracellular Signal-Regulated MAP Kinases / metabolism · Hep G2 Cells · Humans · Liver Neoplasms / drug therapy / metabolism · Phosphorylation / drug effects · Reactive Oxygen Species / metabolism · STAT3 Transcription Factor / metabolism · Signal Transduction / drug effects · Sorafenib / pharmacology / therapeutic use · Ursodeoxycholic Acid / pharmacology / therapeutic use
