FSCN‑1 increases doxorubicin resistance in hepatocellular carcinoma through promotion of epithelial-mesenchymal transition

Abstract

Resistance to chemotherapy drugs remains a significant problem for the treatment of many types of cancer. Fascin‑1 (FSCN‑1) is an actin-bundling protein involved in the invasion and metastasis of a variety of tumors. However, its involvement in drug resistance in hepatocellular carcinoma (HCC) remains unclear. The present study aimed to investigate the function of FSCN‑1 in HCC resistance to doxorubicin (DOX). FSCN‑1 expression was increased in DOX-resistant HCC cell lines (SNU449 and SNU387) compared with DOX-sensitive cell lines (Huh7 and Hep3B). The resistance of HCC cells to DOX was decreased following FSCN‑1 knockdown with small interfering RNA. FSCN‑1 knockdown also significantly altered the expression of key markers of epithelial-mesenchymal transition (EMT). Notably, vimentin expression was reduced and epithelial‑cadherin expression was increased. Furthermore, when EMT was suppressed through knockdown of Twist, an essential pathway of DOX-induced EMT, the viability of HCC cells following treatment with DOX was not affected by FSCN‑1 expression. Furthermore, FSCN‑1 knockdown eliminated hypoxia-induced doxorubicin resistance and EMT. The results of the present study indicated that FSCN‑1 expression increased DOX resistance in HCC cells via the promotion of EMT, and this phenomenon was maintained in a hypoxic environment. FSCN‑1 potentially represents a novel target to overcome resistance to DOX in HCC.

Figure 1

Expression of FSCN-1 is associated with DOX resistance in hepatocellular carcinoma cell lines. (A) Cell viability of SNU449, SNU387, Hep3B and Huh7 cells in the presence of different concentrations of DOX was assessed using Cell Counting Kit-8. (B) Basal level of FSCN-1 expression in SNU449, SNU387, Hep3B and Huh7 cells were assessed using western blotting. FSCN-1, Fascin-1; DOX, doxorubicin.

Figure 2

FSCN-1 expression is associated with DOX resistance in HCC cell lines. (A) Western blot analysis of FSCN-1 expression in HCC cell lines following siRNA transfection. Cell viability following FSCN-1 knockdown or transfection with negative siRNA in (B) Hep3B, (C) Huh7, (D) SNU387 and (E) SNU449 cells in the presence of different concentrations of DOX was assessed using Cell Counting Kit-8. EDU staining assays (DNA copy number) following DOX treatment and FSCN-1 knockdown in (F) Hep3B, (G) Huh7, (H) SNU387 and (I) SNU449 cells compared with the control (magnification, ×200). DOX concentrations (μg/ml) were as follows: Hep3B, 0.6751; Huh7, 0.4620; SNU387, 1.154; SNU449, 2.402. ^*P<0.05 and ^**P<0.01 vs. DOX. FSCN-1, Fascin-1; DOX, doxorubicin; HCC, hepatocellular carcinoma; siRNA, small interfering RNA.

Figure 3

EMT detection with or without FSCN-1 knockdown. (A) Western blot showing the expression of EMT markers (vimentin and E-cadherin) in the hepatocellular carcinoma cell lines with or without FSCN-1 siRNA transfection. (B) Confocal immunofluorescence images revealing the expression of E-cadherin and vimentin (magnification, ×200). EMT, epithelial-mesenchymal transition; FSCN-1, Fascin-1; siRNA, small interfering RNA; E-cadherin, epithelial-cadherin.

Figure 4

Knockdown of FSCN-1-reverses doxorubicin-induced EMT in different HCC cell lines. (A) Western blot analysis of the expression of EMT markers (vimentin and E-cadherin) in the various HCC cell lines, with or without FSCN-1 knockdown, and in the presence or absence of DOX. (B) Confocal immunofluorescence images showing the expression of EMT markers (magnification, ×200). DOX concentrations (μg/ml) were as follows: Hep3B, 0.6751; Huh7, 0.4620; SNU387, 1.154; SNU449, 2.402. FSCN-1, Fascin-1; EMT, epithelial-mesenchymal transition; HCC, hepatocellular carcinoma; E-cadherin, epithelial-cadherin; DOX, doxorubicin; siRNA, small interfering RNA.

Figure 5

FSCN-1 increases the resistance of HCC to DOX by promoting epithelial-mesenchymal transition. Western blot analysis shows the effective knockdown of (A) Twist and (B) FSCN-1 in HCC cells. Cell viability following DOX treatment was assessed in (C) Huh7, (D) Hep3B, (E) SNU387 and (F) SNU449 cells following Twist knockdown or Twist + FSCN-1 knockdown with Cell Counting Kit-8. FSCN-1, Fascin-1; HCC, hepatocellular carcinoma; DOX doxorubicin; siRNA, small interfering RNA.

Figure 6

FSCN-1 knockdown reverses hypoxia-induced drug resistance and EMT. Cell viability was assessed following DOX treatment in (A) Huh7 and (B) Hep3B cells with or without FSCN-1 knockdown, in a hypoxic environment, using Cell Counting Kit-8. (C) Western blot analysis of vimentin and E-cadherin expression in HCC cells following hypoxia treatment. (D) Confocal immunofluorescence images showing the expression of EMT markers (magnification, ×200). FSCN-1, Fascin-1; EMT, epithelial-mesenchymal transition; DOX, doxorubicin; siRNA, small interfering RNA.