ASIC1α up-regulates MMP-2/9 expression to enhance mobility and proliferation of liver cancer cells via the PI3K/AKT/mTOR pathway

Abstract

A major challenge in the treatment of liver cancer is that a large proportion of patients fail to achieve long-term disease control, with death from liver cancer cell migration and invasion. Acid-sensitive ion channel 1α (ASIC1α) is involved in the migration, invasion, and proliferation of liver cancer cells. Therefore, we explored the mechanism of ASIC1α-mediated liver cancer cell migration and invasion. We determined the levels of ASIC1α by western blotting and immunofluorescence in HepG2 and SK-Hep1 cells cultured in various acidic conditions. In addition, wound healing assay, transwell invasion assay, and MTT assay were conducted to assess the migration, invasion, and proliferation abilities of liver cancer cells. Western blotting was conducted to determine the levels of MMP2, MMP9, ASIC1α, p-PI3Kp85, t-PI3Kp85, p-AKT(Ser473), t-AKT, p-mTOR (Ser2448), t-mTOR. We first found that the levels of ASIC1α in the HepG2 and SK-Hep1 cells in acidic conditions (pH 6.5) were significantly increased. Inhibition and knockdown of ASIC1α down-regulated MMP-2/9 expression and inhibited the migration, invasion, and proliferation of HepG2 and SK-Hep1 cells; overexpression of ASIC1α had the opposite effect. We further demonstrated that ASIC1α up-regulates MMP-2/9 via activation of the PI3K/AKT/mTOR pathway, thereby promoting migration, invasion, and proliferation of liver cancer cells. Overexpression of MMP-2/9 and activation of AKT reversed these effects on liver cancer cells caused by inhibition of ASIC1α. We conclude that ASIC1α can regulate migration, invasion, and proliferation of liver cancer cells through the MMP-2/9/PI3K/AKT/mTOR pathway. These observations may provide a new reference for liver cancer chemotherapy.

Keywords: Acid-sensitive ion channel 1α; HepG2 cells; Liver cancer; MMP-2/9; PI3K/AKT/mTOR; SK-Hep1cells.

Fig. 1

High expression of ASIC1α in HepG2 and SK-Hep1 cells which cultured in pH6.5. A1 Expression levels of ASIC1α in human liver cancer cell lines and a normal L-02 hepatic cell line were detected by western blotting. A2 and A3 Histogram showing the semiquantitative analyses of the gels from western blotting. B Expression levels and the cell distribution of ASIC1α in human liver cancer cell lines and a normal L-02 hepatic cell line were measured by immunofluorescence. Representative images were taken at × 400 magnification. Data were expressed as the mean ± SD, n = 3. *P < 0.05 and **P < 0.01 all versus L-02 group

Fig. 2

Inhibiting the activity of ASIC1α reduces the mobility and proliferation of HepG2 and SK-Hep1 Cells. A1 and A2 Expression levels of ASIC1α in different group cells were detected by western blotting. The concentrations of PcTx1 were 10, 20, 40 nM at 24 h. B1, B2, C1 and C2 Migration abilities of different group cells were examined by wound healing assays. Representative images were taken at × 100 magnification. The cells were treated with PcTx1 for 6 h before performing wound healing assays. D1 and D2 Invasion abilities of different group cells were determined by transwell invasion assays. Representative images were taken at × 200 magnification. Invasion assays were conducted at 24 h. The cells were treated with PcTx1 for 6 h before performing transwell invasion assays. E1, E2, E3 and E4 proliferation abilities of different group cells were determined by MTT assay. Data were expressed as the mean ± SD, n = 3. n.s: no significance, *P < 0.05, **P < 0.01 and ***P < 0.001 all versus pH6.5 group or PcTx1(0 nM) group

Fig. 3

Silencing of the ASIC1α gene inhibits the mobility and proliferation of HepG2 and SK-Hep1 cells. A1 and A2 Expression levels of ASIC1α in different group cells were detected by western blotting. B1, B2, C1 and C2 Migration abilities of different group cells were examined by wound healing assays. Representative images were taken at × 100 magnification. D1 and D2 Invasion abilities of different group cells were determined by transwell invasion assays. Representative images were taken at × 200 magnification. Invasion assays were conducted at 24 h. E proliferation abilities of different group cells were determined by MTT assay. Data were expressed as the mean ± SD, n = 3. n.s: no significance, *P < 0.05, **P < 0.01 and ***P < 0.001 all versus pH6.5 group or L-02 group

Fig. 4

Overexpression of the ASIC1α gene enhances the mobility and proliferation of HepG2 and SK-Hep1 cells. A1 and A2 Expression levels of ASIC1α in different group cells were detected by western blotting. B1, B2, C1 and C2 Migration abilities of different group cells were examined by wound healing assays. Representative images were taken at × 100 magnification. D1 and D2 Invasion abilities of different group cells were determined by transwell invasion assays. Representative images were taken at × 200 magnification. Invasion assays were conducted at 24 h. E proliferation abilities of different group cells were determined by MTT assay. Data were expressed as the mean ± SD, n = 3. n.s: no significance, *P < 0.05, **P < 0.01 and ***P < 0.001 all versus pH6.5 group or L-02 group

Fig. 5

Inhibiting the activity of ASIC1α reduces the mobility and proliferation of HepG2 and SK-Hep1 Cells by down-regulating the expression of MMP-2 and MMP-9. A1, A2 and A3 Expression levels of MMP-2 and MMP-9 in different group cells were detected by western blotting. The concentration of PcTx1 was 20 nM, and the concentration of cis-ACCP was 20 µM at 24 h. B1, B2 and B3 Migration abilities of different group cells were examined by wound healing assays. Representative images were taken at × 100 magnification. The cells were treated with PcTx1 or cis-ACCP for 6 h before performing wound healing assays. C1 and C2 Invasion abilities of different group cells were determined by transwell invasion assays. Invasion assays were conducted at 24 h. The cells were treated with PcTx1 or cis-ACCP for 6 h before performing transwell invasion assays. Representative images were taken at × 200 magnification. D proliferation abilities of different group cells were determined by MTT assay. MTT assays were conducted at 24 h. Data were expressed as the mean ± SD, n = 3. n.s: no significance, *P < 0.05 and ***P < 0.001 all versus pH6.5 group

Fig. 6

ASIC1a can regulate aberrant activation of the PI3K/AKT/MTOR pathway. A1, A2, B1, B2, C1 and C2 Expression levels of p-PI3Kp85, p-mTOR(Ser2448) and p-AKT(Ser473) in different group cells were detected by western blotting. The concentration of PcTx1 was 20 nM, the concentration of LY294002 was 1 µM, the concentration of Rapamycin was 0.5 nM, the concentration of MK-2206 was 0.1 µM, and all conducted at 24 h. Data were expressed as the mean ± SD, n = 3. n.s: no significance, *P < 0.05, **P < 0.01 and ***P < 0.001 all versus pH6.5 group

Fig. 7

ASIC1α regulates the expression of MMP-2 and MMP-9 via the PI3K/AKT/mTOR pathway in HepG2 and SK-Hep1 cells. A1, A2 and A3 Expression levels of MMP-2 and MMP-9 in different group cells were detected by western blotting. The concentration of NVP-BEZ235 was 4 nM and the concentration of MK-2206 was 0.1 µM at 24 h. B1, B2 and B3 Migration abilities of different group cells were examined by wound healing assays. Representative images were taken at × 100 magnification. C1 and C2 Invasion abilities of different group cells were determined by transwell invasion assays. Representative images were taken at × 200 magnification. The concentration of NVP-BEZ235 was 4 nM and the concentration of MK-2206 was 0.1 µM at 24 h. D proliferation abilities of different group cells were determined by MTT assay. The concentration of NVP-BEZ235 was 4 nM and the concentration of MK-2206 was 0.1 µM at 24 h. Data were expressed as the mean ± SD, n = 3. n.s: no significance, *P < 0.05, **P < 0.01 and ***P < 0.001 all versus pH6.5 + NVP-BEZ235 group

Fig. 8

Overexpression of MMP-2/9 and activation of AKT can rescue the effect of reduced HepG2 and SK-Hep1 cells migration, invasion and proliferation caused by inhibiting ASIC1α. A1, A2, B1, B2, C1 and C2 Expression levels of MMP-2, MMP-9 and p-AKT(Ser473) in different group cells were detected by western blotting. The concentration of PcTx1 was 20 nM and the concentration of SC79 was 8, 16 µM at 24 h. D1, D2 and D3 Migration abilities of different group cells were examined by wound healing assays. Representative images were taken at × 100 magnification. The cells were treated with PcTx1 or SC79 for 6 h before performing wound healing assays. E1 and E2 Invasion abilities of different group cells were determined by transwell invasion assays. Representative images were taken at × 200 magnification. Invasion assays were conducted at 24 h. The cells were treated with PcTx1 or SC79 for 6 h before performing transwell invasion assays. F proliferation abilities of different group cells were determined by MTT assay. The concentration of PcTx1 was 20 nM and the concentration of SC79 was 8 µM at 24 h. Data were expressed as the mean ± SD, n = 3. n.s: no significance, *P < 0.05, **P < 0.01 and ***P < 0.001 all versus Control group