Inhibition of hepatocellular carcinoma progression by artesunate via modulation of the TLR4/MyD88/NF-κB signaling pathway

Abstract

Background: Liver cancer remains a frequent cause of cancer-related death, and thus targeted drugs urgently need to be developed. Artesunate (ART) inhibits the progression of liver cancer; however, its mechanism of action remains unclear. The primary aim of this study is to clarify whether ART inhibits the progression of hepatocellular carcinoma (HCC) cells by suppressing the Toll-like receptor 4 (TLR4)/MyD88/nuclear factor (NF)-κB pathway.

Methods: In vitro studies demonstrated the effects on cell proliferation, invasion, and migration through a series of phenotypic experiments. Specifically, the CCK8 was used to assess the impact on cell proliferation, while the Transwell assay was employed to evaluate the effect on cell invasion. A xeno-inhibitory tumor model was established in vivo to verify the therapeutic effects of ART. Western blotting was used to detect changes in the TLR4/MyD88/NF-κB pathway.

Results: The study showed that ART inhibits HCC cell proliferation, invasion, and migration and induces apoptosis in a dose-dependent manner. In vivo studies indicated shown that ART treatment in xenograft tumor models could consistently reduce tumor growth. Moreover, ART inhibited the viability, colony formation, migration, and invasion ability of HCC cells while promoting their apoptosis in a dose-dependent manner. The treatment of xenograft models with ART consistently reduced tumor growth. Furthermore, Western blot analysis demonstrated that the levels of TLR4 and its known downstream effectors (TRAF6, MyD88, and NF-κB) were markedly downregulated after ART treatment in Huh-7 and liposaccharide-stimulated Huh-7 cells.

Conclusions: These results indicate that ART has a potent effect on the development of HCC cells, the underlying mechanisms of which may be associated with alterations in the TLR4/MyD88/NF-κB signaling pathway in HCC. Therefore, further development of ART as a therapeutic agent is warranted.

Keywords: Artesunate (ART); MyD88; Toll-like receptor (TLR); hepatocellular carcinoma (HCC); nuclear factor-κB (NF-κB).

Figure 1

Effects of ART on the viability and colony formation of HCC cells. (A) ART 2D and 3D molecular structure formulae. (B) The changes in cell viability of Huh-7, SNU-182, and HepG2 cells treated with ART for 48 h were detected using CCK8. (C,D) Observe the morphological changes of Huh-7 and SNU-182 HCC cells treated with ART (40 µg/mL) through a microscope. Then, calculate the changes in the number of viable cells by trypan blue staining. (E) The expression level of TLR4 mRNA in ART-treated THLE-2, HepG2, HLE, Huh-7, C3A, and SNU-182 cells was detected using qPCR after 48 h. (F) ART suppression of Huh-7 cell colony formation was demonstrated via a colony formation assay (stained with 0.5% crystal violet). 2D, two-dimensional; 3D, three-dimensional; ART, artesunate; CCK8, cell counting kit-8; HCC, hepatocellular carcinoma; qPCR, quantitative polymerase chain reaction; TLR4, Toll-like receptor 4.

Figure 2

ART could inhibit the invasion and migration abilities of hepatoma cells Huh-7 and SNU-182. (A) The cell migration ability of Huh-7 and SNU-182 cells was detected by cell scratch assay at 0 h, 24 h and 48 h under different concentrations of ART. (B) The invasion ability of Huh-7 and SNU-182 cells was detected by the Transwell method and stained with 0.5% crystal violet. ART, artesunate.

Figure 3

ART could inhibit the proliferation ability of hepatoma Huh-7 and SNU-182 cells and induce the apoptosis of Huh-7 and SNU-182 cells. (A) The proliferative ability of Huh-7 and SNU-182 cells was detected by the CCK8 method at 24, 48 and 72 h under different concentrations of ART. (B) The apoptosis of Huh-7 and SNU-182 cells was detected by flow cytometry. ART, artesunate; CCK8, cell counting kit-8.

Figure 4

Effects of ART on HCC cell growth in vivo. (A) Schematic diagrams of the tumor sizes of tumor-bearing mice in each group after treatment with different concentrations of ART (50, 100, 200 mg/kg). (B) After treating the tumor-bearing mice as required by different groups, measure the length and width of the tumor tissues weekly for 4 consecutive weeks. Calculate the tumor volume and plot the curve of the tumor volume changing with time. (C) Fluorescent images of mice bearing Huh-7 cell xenografts were obtained on the 10th and 20th days after inoculation to observe the tumor growth in the tumor-bearing mice. (D) The relative expression levels of MyD88, NF-κB, TLR4, and TRAF6 proteins in mouse tumor tissues were detected using western blot assay. ART, artesunate; HCC, hepatocellular carcinoma; TLR4, Toll-like receptor 4; MyD88, myeloid differentiation primary response 88; TRAF6, tumor necrosis factor receptor-associated factor 6.

Figure 5

Effects of ART on TLR4/MyD88/NF-κB signaling pathways. (A) The mRNA expression levels of MyD88, NF-κB, TLR4, and TRAF6 in Huh-7 cells treated with ART for 48 h were detected using qPCR. (B) The mRNA expression levels of MyD88, NF-κB, TLR4, and TRAF6 in SNU-182 cells treated with ART for 48 h were detected using qPCR. (C) The expressions of TLR4, TRAF6, MyD88, and NF-κB proteins were determined via Western blotting of Huh-7. (D) The expressions of TLR4, TRAF6, MyD88, and NF-κB proteins were determined via the Western blotting of SNU-182 cells. ART, artesunate; MyD88, myeloid differentiation primary response 88; NF, nuclear factor; qPCR, quantitative polymerase chain reaction; TLR4, Toll-like receptor 4; TRAF6, tumor necrosis factor receptor-associated factor 6.

Figure 6

Effects of ART on cell viability and TLR4/MyD88/NF-κB signaling pathways in LPS-stimulated Huh-7 cells. (A) The effect of CCK8 on Huh-7 and SNU-182 cell viability after LPS or ART treatment for 24, 48, and 72 h was detected. (B) A western blot assay detected the expression of TLR4, TRAF6, MyD88, and NF-κB proteins in Huh-7. (C) A Western blot assay detected the expression of TLR4, TRAF6, MyD88, and NF-κB proteins in SNU-182. ART, artesunate; LPS, lipopolysaccharide; MyD88, myeloid differentiation primary response 88; NF, nuclear factor; TLR4, Toll-like receptor 4; TRAF6, tumor necrosis factor receptor-associated factor 6.

Figure 7

Mechanism by which ART inhibits hepatocellular carcinoma cells. LPS could activate the TLR4/MyD88/NF-κB signaling pathway in hepatocellular carcinoma. ART could inhibit the TLR4/MyD88/NF-κB signaling pathway, thereby inhibiting proliferation, invasion, and migration and inducing apoptosis in hepatocellular carcinoma cells. ART, artesunate; LPS, lipopolysaccharide; MyD88, myeloid differentiation primary response 88; NF, nuclear factor; TLR4, Toll-like receptor 4; TRAF6, tumor necrosis factor receptor-associated factor 6.