Niclosamide suppresses migration of hepatocellular carcinoma cells and downregulates matrix metalloproteinase-9 expression

Abstract

Metastasis negatively affects the prognosis of hepatocellular carcinoma (HCC). In the present study, niclosamide, which is known to suppress the proliferation of HCC cells, was investigated for possible suppressant effects on the migration of HCC cells. HLF and PLC/PRF/5 HCC cells were cultured in the presence of niclosamide. Cell proliferation was analyzed using the MTS assay. Cell migration was measured by performing a scratch assay. Expression levels of cyclin D1 and matrix metalloproteinase 9 (MMP9) were analyzed by performing revers transcription-quantitative polymerase chain reaction. Compared with the control treatment, treatment with 10 µm niclosamide suppressed the proliferation of the HLF and PRL/PRF/5 cells to 49.9±3.7 and 17.9±11.5% (P<0.05), respectively. Furthermore, compared with the control treatment, treatment with 1.0 µM niclosamide downregulated the expression of cyclin D1 to 52.4±4.4 and 23.9±5.4% (P<0.05) in the HLF and PRL/PRF/5 cells, respectively. In the scratch assay, treatment of the HLF cells with niclosamide (1.0 µm) decreased the distance of the scratched line from the growing edge to 4.6±1.0 mm compared with the 9.2±1.4 mm observed with the control treatment (P<0.05). Similarly, treatment of the PRL/PRF/5 cells with niclosamide (1.0 µm) also decreased the distance of the scratched line from the growing edge to 3.0±0.8 mm compared with the 5.5±0.9 mm observed with the control treatment (P<0.05). Further, MMP9 expression levels in the HLF cells treated with 1.0 µm niclosamide decreased to 22.4±1.76% (P<0.05) compared with those in the untreated control HLF cells. Similarly, expression level of MMP9 in the PRL/PRF/5 cells treated with 1.0 µm niclosamide deceased to 18.7±10.7% (P<0.05) compared with those in the untreated control PRL/PRF/5 cells. Overall, niclosamide downregulated the expression of MMP9 in and suppressed the migration of HCC cells.

Keywords: MTS assay; quantitative polymerase chain reaction; scratch assay.

Figure 1.

Cell proliferation assay. (A) HLF cells or (B) PLC/PRL/5 cells were cultured with niclosamide and subjected to MTS assays. The error bars represent the standard deviation (n=3). *P<0.05 vs. 0 µM.

Figure 2.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) of cyclin D1. (A) HLF cells or (B) PLC/PRL/5 cells were cultured with niclosamide, and subjected to qRT-PCR for detection of cyclin D1 expression. The error bars represent the standard deviation (n=3). *P<0.05 vs. 0 µM.

Figure 3.

Scratch assay. (A and B) HLF or (C and D) PLC/PRL/5 cells were cultured on chamber slides. Upon attaining confluence, the cells were scratched with the tip of a 200-µl pipette tip and treated (B and D) with 1.0 µM niclosamide, or (A and C) without niclosamide. The white line represents the location of the original leading edge of the scratch. The distance from the line to the new growing edge of the cells was measured in the (E) HLF and (F) PLC/PRL/5 cells. Original magnification, ×100; scale bar, 200 µm; error bar, standard deviation; n=3; *P<0.05 vs. 0 µM.

Figure 4.

Real-time quantitation polymerase chain reaction (qRT-PCR) of matrix metalloproteinase 9. HLF cells (A) and PLC/PRL/5 cells (B) were cultured with niclosamide and subjected to qRT-PCR with matrix metalloproteinase 9. Error bar: standard deviation, *P<0.05 vs. 0 µM, n=3.