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. 2018 Oct 12;19(10):3127.
doi: 10.3390/ijms19103127.

Emodin Sensitizes Hepatocellular Carcinoma Cells to the Anti-Cancer Effect of Sorafenib through Suppression of Cholesterol Metabolism

Young-Seon Kim  1 , Yoon-Mi Lee  2 , Taek-In Oh  3 , Dong Hoon Shin  4 , Geon-Hee Kim  5 , Sang-Yeon Kan  6 , Hyeji Kang  7 , Ji Hyung Kim  8 , Byeong Mo Kim  9 , Woo Jong Yim  10 , Ji-Hong Lim  11   12
Affiliations

Emodin Sensitizes Hepatocellular Carcinoma Cells to the Anti-Cancer Effect of Sorafenib through Suppression of Cholesterol Metabolism

Young-Seon Kim et al. Int J Mol Sci. .

Abstract

Reduced therapeutic efficacy of sorafenib, a first-generation multikinase inhibitor, is often observed during the treatment of advanced hepatocellular carcinoma (HCC). Emodin is an active component of Chinese herbs, and is effective against leukemia, lung cancer, colon cancer, pancreatic cancer, and HCC; however, the sensitizing effect of emodin on sorafenib-based HCC therapy has not been evaluated. Here, we demonstrate that emodin significantly improved the anti-cancer effect of sorafenib in HCC cells, such as HepG2, Hep3B, Huh7, SK-HEP-1, and PLC/PRF5. Mechanistically, emodin inhibits sterol regulatory element-binding protein-2 (SREBP-2) transcriptional activity, which suppresses cholesterol biosynthesis and oncogenic protein kinase B (AKT) signaling. Additionally, attenuated cholesterol synthesis and oncogenic AKT signaling inactivated signal transducer and activator of transcription 3 (STAT3), an oncogenic transcription factor. Furthermore, emodin synergistically increased cell cycle arrest in the G1 phase and apoptotic cells in the presence of sorafenib. Animal models xenografted with HepG2 or SK-HEP-1 cells also showed that the combination of emodin and sorafenib was sufficient to inhibit tumor growth. Overall, these results suggested that the combination of emodin and sorafenib may offer a potential therapy for patients with advanced HCC.

Keywords: cholesterol; combination; emodin; hepatocellular carcinoma; sorafenib.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Emodin sensitizes hepatocellular carcinoma (HCC) cells to the anti-cancer effect of sorafenib. (A) Cell viability analysis after emodin, sorafenib, or the combination treatment of both drugs in five HCC cell lines. The cells were incubated with emodin (20 μM), sorafenib (2 μM), or the combination (20 μM emodin and 2 μM sorafenib) for 24, 48, or 72 h, as indicated. The values represent the mean ± SEM of three independent experiments performed in triplicate; * p < 0.05, ** p < 0.01, and *** p < 0.001. (B) Images of crystal violet staining. (C) The cell viability after different concentrations of sorafenib (0.5, 1, 2, 5, and 10 μM) with 20 μM emodin in Hep3B, HepG2, and Huh7 cells. The values represent the mean ± SEM of three independent experiments performed in triplicate; * p < 0.05 and ** p < 0.01.
Figure 2
Figure 2
Emodin did not sensitize HCC to the anti-cancer effect of doxorubicin or 5-fluorouracil. (A) The viability of three HCC cell lines after treatment with of emodin and doxorubicin. (B) The combinatory anti-cancer effect of emodin and 5-fluorouracil in the HCC cell lines. Cells were treated with the indicated drugs (1 μM doxorubicin or 10 μM 5-fluorouracil) for 48 h in the absence or presence of 20 μM emodin. The values are presented as the mean ± SEM of three independent experiments performed in duplicate; * p < 0.05 and ** p < 0.01.
Figure 3
Figure 3
The combination therapy of emodin and sorafenib causes cell cycle arrest and apoptosis in HCC cells. (A) The cell cycle analysis after treatment with the combination of emodin and sorafenib in HepG2 cells. (B) The cell cycle analysis after treatment with the combination of emodin and sorafenib in Hep3B, Huh7, SK-HEP-1, and PLC/PRF5 cells. The cells were incubated with emodin (20 μM), sorafenib (2 μM), or the combination (20 μM emodin and 2 μM sorafenib) for 16 h. The values represent the mean ± SEM of three independent experiments performed in duplicate. (C) Cell proliferation after treatment with the combination of emodin and sorafenib in five HCC cell lines. The cells were treated with the indicated drugs for 24 h prior to analysis. The values represent the mean ± SEM of three independent experiments performed in duplicate. (D) The induction of apoptosis after treatment with the combination of emodin and sorafenib in HepG2 cells. The cells were incubated with emodin and sorafenib for 72 h. The values represent the mean ± SEM of three independent experiments performed in duplicate; * p < 0.05 and ** p < 0.01.
Figure 4
Figure 4
Emodin suppresses SREBP-2 transcriptional activity and decreases intracellular cholesterol. (A) The steroid-responsive element (SRE) containing luciferase vectors (SRE–Luc–WT or SRE–Luc–Mut) were transiently transfected into SK-HEP-1 cells. Transfected cells were incubated with 10 or 20 μM emodin in the absence or presence of 10 μM simvastatin for 24 h. The values represent the mean ± SD of three independent experiments performed in duplicate; * p < 0.05 and ** p < 0.01. (B) The suppressive effect of emodin on simvastatin-induced cholesterogenic gene expression. SK-HEP-1 cells were incubated with 20 μM emodin in the absence or presence of 10 μM simvastatin for 24 h. The gene expression was measured by quantitative real-time PCR, and relative mRNA expression was normalized to the expression of 36B4. The values represent the mean ± SEM of three independent experiments performed in triplicate; * p < 0.05 and ** p < 0.01. (C) The suppressive effect of emodin on simvastatin-induced cholesterogenic gene expression in HepG2 cells. (D) The inhibitory effect of emodin on cholesterogenic enzyme expression in SK-HEP-1 cells. The cells were incubated with 5, 10, 20, or 40 μM emodin for 24 h, and protein expression was analyzed by Western blotting, as described in the Materials and Methods. Relative protein expression levels are shown. The values are presented as the mean ± SD of three independent experiments performed; * p < 0.05 and ** p < 0.01. (E) Intracellular cholesterol level after emodin treatment. Three HCC cell lines were cultured for 48 h in the absence or presence of 20 μM emodin or 20 μM simvastatin. The values are presented as the mean ± SD of three independent experiments performed in duplicate; * p < 0.05 and ** p < 0.01.
Figure 5
Figure 5
Suppression of cholesterol metabolism is required to sensitize HCC cells to the anti-cancer effect of sorafenib. (A) Cholesterogenic gene expression after the combination treatment of emodin and sorafenib in HepG2 and SK-HEP-1 cell lines. The cells were incubated with emodin (20 μM), sorafenib (2 μM), or their combination (20 μM emodin and 2 μM sorafenib) for 24 h. The values represent the mean ± SEM of three independent experiments performed in duplicate; * p < 0.05, ** p < 0.01, # p < 0.05, and ## p < 0.01. (B) The cell viability after the combination treatment of emodin and sorafenib in the absence or presence of 10% fetal bovine serum (FBS). Three cell lines were incubated with 10% FBS or FBS-free serum for 1 h prior to the combination treatment of emodin and sorafenib. Subsequently, the cells were incubated for 24 h with the indicated treatments. The values are presented as the mean ± SEM of three independent experiments performed in triplicate; * p < 0.05, ** p < 0.01, and # p < 0.05. (C) The cell viability after the combination treatment of emodin and sorafenib in the absence or presence of water-soluble cholesterol (0.5 mM). Three cell lines were incubated with or without 0.5 mM water-soluble cholesterol for 1 h prior to the combination treatment with emodin and sorafenib. Subsequently, the cells were incubated for a further 48 h. The values represent the mean ± SEM of three independent experiments performed in triplicate; * p < 0.05, ** p < 0.01, # p < 0.05, and ## p < 0.01.
Figure 6
Figure 6
Decreased intracellular cholesterol level by emodin causes the suppression of the oncogenic protein kinase B (AKT) signaling pathway. (A) Simvastatin suppressed the AKT signaling pathways. SK-HEP-1 cells were incubated with different concentrations of simvastatin (2, 5, 10, and 20 μM) for 12 h. (B) Emodin suppressed the phosphorylation of AKT and its target substrates. SK-HEP-1 cells were incubated with emodin (2, 5, 10, and 20 μM) for 12 h. (C) The supplementation of water-soluble cholesterol rescued the decreased phosphorylation of AKT caused by emodin. SK-HEP-1 and HepG2 cells were incubated in the presence or absence of 0.5 mM cholesterol for 1 h prior to emodin treatment. Subsequently, the cells were incubated for a further 12 h, and protein expression was measured by Western blotting.
Figure 7
Figure 7
The combination treatment of emodin and sorafenib synergistically suppressed STAT3 and the associated expression of cell cycle-regulating target genes. (A) Emodin inhibits the phosphorylation of STAT3. Three HCC cell lines were incubated with emodin (10 and 20 μM) for 24 h. (B) The synergistic effect of the emodin and sorafenib combination on STAT3 phosphorylation. The cells were incubated with 20 μM of emodin, 2 μM of sorafenib, or their combination (20 μM emodin and 2 μM sorafenib) for 24 h. (C) Simvastatin increased the suppressive effect of sorafenib on STAT3 phosphorylation. HepG2 and PLC/PRF5 cells were incubated with 2 μM of sorafenib, 10 μM of simvastatin, or the combination of sorafenib and simvastatin, as indicated, for 24 h. (D) The supplementation of cholesterol blocked the downregulation of STAT3 phosphorylation after the combination treatment of emodin and sorafenib. HepG2 cells were treated with water-soluble cholesterol (0.5 mM) for 1 h prior to the combination treatment of emodin and sorafenib, and the cells were incubated for a further 24 h. Protein expression was measured by Western blotting. (E) The combination of emodin and sorafenib decreased the expression of cell cycle-promoting STAT3 target genes. HepG2 cells were incubated for 24 h with the combination (20 μM emodin and 2 μM sorafenib). The values represent the mean ± SD of three independent experiments performed in duplicate; * p < 0.05 and ** p < 0.01.
Figure 8
Figure 8
Emodin synergistically increased the anti-cancer efficacy of sorafenib in vivo. (A) HepG2 and (B) SK-HEP-1 cells were injected into the flanks of nude mice, and the mice were then divided into four groups. When the tumor volumes reached 250 mm3, the mice were treated with DMSO, emodin (10 mg/kg/day), sorafenib (5 mg/kg/day), or the combination (10 mg/kg/day emodin and 5 mg/kg/day sorafenib) for three weeks; with the tumor volumes measured daily. Tumor growth curves are plotted as the mean ± SEM (n = 7); * p < 0.05 and ** p < 0.01. (C) The combination of emodin and sorafenib decreased the phosphorylation of STAT3 and increased cleaved-caspase-3. The protein expression in xenografted tumor tissues was measured by immunohistochemistry using the indicated antibodies.
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