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, 6 (1), e03255
eCollection

Curcumin Enhances Chemotherapeutic Effects and Suppresses ANGPTL4 in Anoikis-Resistant Cholangiocarcinoma Cells

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Curcumin Enhances Chemotherapeutic Effects and Suppresses ANGPTL4 in Anoikis-Resistant Cholangiocarcinoma Cells

Tin Tin San et al. Heliyon.

Abstract

Anoikis resistance is a critical feature involved in tumor progression and chemoresistance. Finding approaches to improve the effect of chemotherapy on anoikis-resistant cancer cells is therefore critically important. In this study, we examined the effects of curcumin in anoikis-resistant cholangiocarcinoma (CCA) cells, including HuCCT1 and TFK-1 that were anchorage-independently cultured (AI-cells) using poly (2-hydroxyethyl methacrylate). The AI-CCA cells were treated with curcumin alone or in combination with anti-cancer agents and their responses to each treatment were determined by cell viability assay. Gene expression in AI-cells was determined by quantitative real-time PCR. The potential involvement of angiopoietin-like 4 (ANGPTL4) in anoikis resistance was examined by gene knockdown. It was found that AI-cells tended to resist anti-cancer agents tested, especially AI-HuCCT1, which significantly resisted gemcitabine and suberoylanilide hydroxamic acid (SAHA). Curcumin alone significantly inhibited viability and colony formation of AI-cells. Moreover, curcumin combination significantly enhanced the treatment effect of SAHA on AI-HuCCT1 and AI-TFK-1 cells. Gene expression analysis revealed that ANGPTL4 was markedly upregulated in AI-CCA cells and its knockdown tended to sensitize AI-cells to cell death and treatments. In addition, curcumin treatment decreased phosphorylated STAT3 and expression levels of Mcl-1, HDACs and ANGPTL4. Altogether, these findings reveal the beneficial property of curcumin to potentiate chemotherapeutic effects on anoikis-resistant CCA cells, which might suggest the potential use of curcumin for cancer treatment.

Keywords: Cancer research; Molecular biology; Natural product.

Figures

Figure 1
Figure 1
Anoikis induction of CCA cell lines and effect of curcumin on L929. (A) The percentage of viable HuCCT1 and TFK-1 cells cultured under anchorage-independent condition (AI-cells) for 24 h and 48 h. (B) Viability of non-malignant L929 and CCA cell lines after treatment with curcumin for 48 h. Percentage of cell viability was compared against control (0.1% DMSO). Data represent the mean ± SE of three independent experiments. *P < 0.05.
Figure 2
Figure 2
Effects of curcumin on viability and colony formation of AI-CCA cells. (A) MTS assay of adherent and AI-cells (HuCCT1 and TFK-1) treated with curcumin. Percentage of viable cells was calculated against 0.1% DMSO-treated controls. (B) Colony formation assay of AI-HuCCT1 and AI-TFK-1. Colony number of the cells treated with curcumin was analyzed versus 0.1% DMSO-treated cells. Data represent the mean ± SE of three independent experiments. *P < 0.05 and **P < 0.01 vs. control. (C) Western blot analysis showed the inhibition of p-STAT3 and Mcl-1 and the increase in cleaved PARP in AI-cells treated with curcumin for 48 h. GAPDH was detected as loading control for western blotting. Original images are shown in Sup Fig. 1.
Figure 3
Figure 3
Effect of curcumin combined with gemcitabine, cisplatin, or SAHA. (A) Response of AI-HuCCT1 and AI-TFK-1 cells to gemcitabine, cisplatin, and SAHA measured by MTS assay at 48 h after treatments. (B) Viability of AI-HuCCT1 and AI-TFK-1 cells treated with individual agents or in combination with curcumin for 48 h. Untreated cells were used as control for gemcitabine and 0.1% DMSO-treated cells was used as control for cisplatin and SAHA. Data represent the mean ± SE of three independent experiments. *P < 0.05 and **P < 0.01 vs. controls. Gem, gemcitabine; Cis, cisplatin; Cur, curcumin. (C) Expression of HDACs was detected by western blotting. Original images are shown in Sup Fig. 2. (D) Relative band intensity of HDAC1, 3, and 6 in AI-cells treated with 40 μM curcumin for 24 and 48 h.
Figure 4
Figure 4
Gene expression in anchorage-independently grown HuCCT1 and TFK-1 cells. (A) Schematic representation of adherent and AI culture of HuCCT1 and TFK-1 cells for gene expression analysis. The relative expression levels of genes involved in EMT (B), epigenetic (C), cell cycle (D), and hypoxia (E) in AI-cells was compared against that of adherent cells. The mRNA levels were normalized to GAPDH reference gene. Data represent the mean ± SE of three independent experiments. *P < 0.05 and **P < 0.01 vs. controls.
Figure 5
Figure 5
Inhibition of ANGPTL4 by siRNA and curcumin in AI-CCA cells. (A) ANGTPL4 expression in AI-HuCCT1 and AI-TFK-1 cells was decreased by siRNA. (B) Survival and (C) response to anti-cancer drugs of ANGPTL4-knockdown cells under suspension condition. Data represent the mean ± SE of three independent experiments. *P < 0.05 and **P < 0.01 vs. controls. Gem, gemcitabine; Cis, cisplatin. Inhibition of ANGPTL4 expression at mRNA (D) and protein (E) level after curcumin treatment. GAPDH was detected as loading control for western blotting. Original images are shown in Sup Fig. 3.
Supplementary figure1
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