Inhibition of Wnt/β-catenin signaling downregulates P-glycoprotein and reverses multi-drug resistance of cholangiocarcinoma

Abstract

The development of multi-drug resistance (MDR) represents a major obstacle in the successful treatment of cancers. However, the factors and mechanisms that lead to MDR in cholangiocarcinoma (CCA), a chemoresistant bile duct carcinoma with a poor prognosis, remain unclear. In this study, we established a human MDR CCA cell line QBC939/5-FU. Compared with QBC939 cells, a rounder shape, a higher nuclear-cytoplasmic ratio, a shorter cell cycle, faster growth and resistance to chemotherapeutics are major characteristics of QBC939/5-FU cells. P-glycoprotein (P-gp) and β-catenin were upregulated in QBC939/5-FU cells. Furthermore, the drug susceptibility of QBC939 cells to common chemotherapeutics was significantly decreased after Wnt3a treatment, whereas inhibition of Wnt/β-catenin pathway by β-catenin siRNA reversed the MDR of QBC939/5-FU cells to chemotherapeutics. Molecular study revealed that activation of Wnt/β-catenin pathway resulted in upregulation of P-gp and contributed to MDR of QBC939/5-FU cells. Extraction of Siamese Crocodile 3 (ESC-3) bile enhanced the drug sensitivity of QBC939/5-FU cells to 5-FU, paralleled with downregulation of β-catenin and P-gp. The association of Wnt/β-catenin pathway and P-gp was further confirmed by the clinical data for CCA tissues. Our study represents the first implication of Wnt/β-catenin activation in the MDR of CCA, which may be a beneficial target for the clinical treatment of CCA.

Figure 1

Biological characteristics of QBC939/5‐FU cells. (a) Cellular morphology dyed with 0.005% crystal violet for 30 min. Zoom: 200×. (b) Growth curve assayed by MTT. (c) Cell cycle distribution determined by flow cytometry. (d) Effects of four common chemotherapeutics (60 μM, 24 h) on the proliferation of QBC939 and QBC939/5‐FU cells assayed by MTT. *P < 0.05, **P < 0.01. 5‐FU, 5‐fluorouracil; CDDP, cis‐diammineplatinum(II) dichloride; VCR, vincristine sulfate; MMC, mitogen enzyme C.

Figure 2

Upregulation of P‐gp and β‐catenin expression in QBC939/5‐FU cells. (a) Expression of MDR‐associated genes detected by real‐time RT‐PCR. (b) Protein expression of p‐IκB/IκB, p‐AKT/AKT, β‐catenin and P‐gp measured by western blot in QBC939 and QBC939/5‐FU cells. (c) Expression of β‐catenin and P‐gp detected by immunofluorescence staining. Minimal staining (membranous expression of β‐catenin and P‐gp) was observed in QBC939 cells, whereas QBC‐939/5‐FU cells showed high levels of fluorescent staining (nuclear and perinuclear expression of β‐catenin, and membranous expression of P‐gp), readily distinguished from background. Zoom: 200×. *P < 0.05, **P < 0.01. 5‐FU, 5‐fluorouracil; MDR, multi‐drug resistance; P‐gp, P‐glycoprotein.

Figure 3

Wnt/β‐catenin pathway alters MDR of QBC‐939/5‐FU cells. (a) The drug susceptibility to chemotherapeutics (60 μM) of QBC939 and QBC939/5‐FU cells transfected with siβ‐catenin, or (b) after Wnt3a (50 nM) treatment for 6 h, compared to respective control. Equivalent amount of water served as vehicle for Wnt3a. (c) CyclinD mRNA expression in QBC939 and QBC939/5‐FU cells transfected with siCtrl or siβ‐catenin, with/without Wnt3a treatment. (d) Protein expression of β‐catenin and P‐gp in QBC939 cells after Wnt3a (50 nM) treatment for 6 h assayed by western blot. (e) Effect of ESC‐3 (10 μg/mL) on the drug susceptibility of QBC939/5‐FU cells to 5‐FU detected by MTT assays. Methanol‐0.01 M NaH₂PO₄ (70:30, v/v) served as vehicle. (f) Protein expression of β‐catenin and P‐gp in QBC‐939/5‐FU cells with/without ESC‐3 treatment in the absence/presence of Wnt3a (50 nM) for 24 h. *P < 0.05, **P < 0.01, ***P < 0.001. 5‐FU, 5‐fluorouracil; CDDP, cis‐diammineplatinum(II) dichloride; VCR, vincristine sulfate; MDR, multi‐drug resistance; MMC, mitogen enzyme C; P‐gp, P‐glycoprotein.

Figure 4

Immunohistochemistry of β‐catenin and P‐gp in cholangiocarcinoma (CCA) tissues. The staining intension of β‐catenin and P‐gp in CCA tissues were classified into four grades, ±, +, ++ and +++, according to scoring criteria. Each grade of β‐catenin and P‐gp were from the same sample. Expression levels of β‐catenin and P‐gp were categorized as low (±, +) or high (++, +++). Zoom: 200×; P‐gp, P‐glycoprotein.