Quinoline-based clioquinol and nitroxoline exhibit anticancer activity inducing FoxM1 inhibition in cholangiocarcinoma cells

Abstract

Purpose: Fork head box M1 (FoxM1) is an oncogenic transcription factor frequently elevated in numerous cancers, including cholangiocarcinoma (CCA). A growing body of evidence documents its diverse functions contributing to tumorigenesis and cancer progression. As such, discovery of agents that can target FoxM1 would be valuable for the treatment of CCA. The quinoline-based compounds, namely clioquinol (CQ) and nitroxoline (NQ), represent a new class of anticancer drug. However, their efficacy and underlying mechanisms have not been elucidated in CCA. In this study, anticancer activities and inhibitory effects of CQ and NQ on FoxM1 signaling were explored using CCA cells.

Methods: The effects of CQ and NQ on cell viability and proliferation were evaluated using the colorimetric 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-(4-sulfophenyl)-2H-tetrazolium (MTS assay). Colony formation and cell migration affected by CQ and NQ were investigated using a clonogenic and a wound healing assay, respectively. To demonstrate the agents' effects on FoxM1 signaling, expression levels of the target genes were quantitatively determined using real-time polymerase chain reaction.

Results: CQ and NQ significantly inhibited cell survival of HuCCT1 and Huh28 in a dose- and a time-dependent fashion. Further investigations using the rapidly proliferating HuCCT1 cells revealed significant suppression of cell proliferation and colony formation induced by low doses of the compounds. Treatment of CQ and NQ repressed expression of cyclin D1 but enhanced expression of p21. Most importantly, upon CQ and NQ treatment, expression of oncogenic FoxM1 was markedly decreased concomitant with downregulation of various FoxM1's downstream targets including cdc25b, CENP-B, and survivin. In addition, the compounds distinctly impaired HuCCT1 migration as well as inhibited expression of matrix metalloproteinase (MMP)-2 and MMP-9.

Conclusion: Collectively, this study reports for the first time the anticancer effects of CQ and NQ against CCA cells, and highlights new insights into the mechanism of actions of the quinoline-based compounds to disrupt FoxM1 signaling.

Keywords: 8-hydroxyquinoline derivatives; FoxM1; cholangiocarcinoma; clioquinol; migration; nitroxoline.

Figure 1

Chemical structures of CQ and NQ. Abbreviations: CQ, clioquinol; NQ, nitroxoline.

Figure 2

Cytotoxic effects of CQ and NQ on CCA cells. Notes: HuCCT1 and Huh28 cells were treated with (A) CQ (20, 25, and 30 μM) or (B) NQ (10, 20, and 40 μM) for 24, 48, and 72 hours (h). Viability of the compound-treated cells was compared with that of control treated with DMSO and expressed as a percentage of control. Results represent mean ± SD of three independent experiments. *P<0.05; **P<0.01. Abbreviations: CQ, clioquinol; NQ, nitroxoline; DMSO, dimethyl sulfoxide; SD, standard deviation; CCA, cholangiocarcinoma.

Figure 3

CQ and NQ inhibit proliferation and colony formation of CCA cells. Notes: (A) Proliferation rate of HuCCT1 was retarded by 5 and 10 μM CQ (top), or 2 and 5 μM NQ (bottom) compared with control treated with DMSO. Proliferation rate was monitored at 24, 48, and 72 hours (h) and normalized with time 0. (B) HuCCT1 cells were grown in 6-well plates (1×10³ cells/well) and cultured in the presence of CQ (5 and 10 μM), NQ (2 and 5 μM), or DMSO (top panel). Number of colony is expressed as a percentage of control (bottom panel). Bars represent means ± SD of three independent experiments. *P<0.05; **P<0.01. Abbreviations: CQ, clioquinol; NQ, nitroxoline; DMSO, dimethyl sulfoxide; SD, standard deviation; CCA, cholangiocarcinoma.

Figure 4

Changes in expression levels of cyclin D1 and p21 induced by CQ and NQ. Notes: CQ (25 and 50 μM for 8 hours) and NQ (10 and 20 μM for 8 hours) downregulated cyclin D1 mRNA (A), whereas upregulated p21 mRNA (B) in HuCCT1 and Huh28 cells. The mRNA levels were normalized to GAPDH reference gene. Results are presented as fold-change relative to control (cells treated with DMSO). Data are expressed as mean ± SD of three independent experiments. *P<0.05; **P<0.01. Abbreviations: CQ, clioquinol; NQ, nitroxoline; DMSO, dimethyl sulfoxide; SD, standard deviation.

Figure 5

Downregulation of FoxM1 expression mediated by CQ and NQ treatment in HuCCT1. Notes: (A) A dose-dependence inhibition of FoxM1 mRNA after exposure to 20, 25, and 30 μM CQ for 8 hours and (B) a time-dependence reduction in FoxM1 mRNA induced by 25 μM CQ for 4, 6, and 8 hours. Decrease in expression of FoxM1 mRNA induced by (C) 20, 30, and 40 μM NQ for 8 hours or (D) 30 μM NQ for 4, 6, and 8 hours. Results are presented as fold-change relative to control cells treated with DMSO. Data are expressed as mean ± SD of three independent experiments. *P<0.05; **P<0.01. (E) Inhibition of FoxM1 protein in HuCCT1 cells exposed to CQ (20, 30 μM) and NQ (10, 20 μM) for 24 hours. β-actin was used as a sample loading control. Abbreviations: CQ, clioquinol; NQ, nitroxoline; SD, standard deviation; DMSO, dimethyl sulfoxide.

Figure 6

A dose-dependent inhibition of CENP-B, cdc25b, and survivin by CQ and NQ in HuCCT1. Notes: Levels of CENP-B, cdc25b, and survivin mRNA were inhibited by increasing concentration of (A) CQ (20, 25, and 30 μM for 8 hours) and (B) NQ (20, 30, and 40 μM for 8 hours). Results are presented as fold-change relative to DMSO-treated control. Data are expressed as mean ± SD of three independent experiments. *P<0.05; **P<0.01. Abbreviations: CQ, clioquinol; NQ, nitroxoline; DMSO, dimethyl sulfoxide; SD, standard deviation.

Figure 7

Thiostrepton inhibits expression of FoxM1, CENP-B, cdc25b, and survivin in HuCCT1. Notes: The mRNA levels of (A) FoxM1 and (B) FoxM1’s targets including CENP-B, cdc25b, and survivin were determined in HuCCT1 treated with 10 μM thiostrepton for 12 hours. Results are presented as fold-change relative to DMSO-treated control. Data are expressed as mean ± SD of three independent experiments. **P<0.01. (C) Western blot analysis of FoxM1 protein after treatment of HuCCT1 cells with 10 μM thiostrepton for 24 hours. β-actin was used as a sample loading control. Abbreviations: SD, standard deviation; DMSO, dimethyl sulfoxide.

Figure 8

CQ, NQ, and thiostrepton inhibit HuCCT1 cell migration in a wound healing assay. Notes: HuCC1 cells treated with (A) 20 μM CQ, (B) 10 μM NQ, (C) 2.5 μM thiostrepton or DMSO were wounded. Phase-contrast pictures of the wounds were taken at 0 and 18 hours (h), and the open areas of the wound were quantified by ImageJ. Significant decrease in the wound closure rate of HuCCT1 cells treated with (D) CQ, (E) NQ, and (F) thiostrepton was observed at 18 hours compared with DMSO-treated control. *P<0.05; **P<0.01 vs DMSO-treated control. Abbreviations: CQ, clioquinol; NQ, nitroxoline; DMSO, dimethyl sulfoxide.

Figure 9

Expression and molecular docking of MMP-2 and MMP-9. Notes: Expression levels of MMP-2 and MMP-9 mRNA in HuCCT1 cells were reduced in response to treatment of (A) CQ, (B) NQ, and (C) thiostrepton. Data are expressed as mean ± SD of three independent experiments. *P<0.05. Interactions of NQ and CQ with MMP-2 and MMP-9 catalytic domains. NQ and CQ were docked into the active sites of MMP-2 (D and F) and MMP-9 (E and G). The Cα atom of NQ and CQ is shown in green and magenta sticks, respectively, while oxygen and nitrogen atoms are in red and blue colors, respectively. Zinc ions and amino acid residues at the active sites of the enzymes are represented by yellow spheres and light blue lines with a single-code indication, respectively. Abbreviations: CQ, clioquinol; NQ, nitroxoline; SD, standard deviation.