Excretory/secretory products of the carcinogenic liver fluke are endocytosed by human cholangiocytes and drive cell proliferation and IL6 production

Abstract

Liver fluke infection caused by Opisthorchis viverrini remains a major public health problem in many parts of Asia including Thailand, Lao PDR, Vietnam and Cambodia, where there is a strikingly high incidence of cholangiocarcinoma (CCA - hepatic cancer of the bile duct epithelium). Among other factors, uptake of O. viverrini excretory/secretory products (OvES) by biliary epithelial cells has been postulated to be responsible for chronic inflammation and proliferation of cholangiocytes, but the mechanisms by which cells internalise O. viverrini excretory/secretory products are still unknown. Herein we incubated normal human cholangiocytes (H69), human cholangiocarcinoma cells (KKU-100, KKU-M156) and human colon cancer (Caco-2) cells with O. viverrini excretory/secretory products and analysed the effects of different endocytic inhibitors to address the mechanism of cellular uptake of ES proteins. Opisthorchis viverrini excretory/secretory products was internalised preferentially by liver cell lines, and most efficiently/rapidly by H69 cells. There was no evidence for trafficking of ES proteins to cholangiocyte organelles, and most of the fluorescence was detected in the cytoplasm. Pretreatment with clathrin inhibitors significantly reduced the uptake of O. viverrini excretory/secretory products, particularly by H69 cells. Opisthorchis viverrini excretory/secretory products induced proliferation of liver cells (H69 and CCA lines) but not intestinal (Caco-2) cells, and proliferation was blocked using inhibitors of the classical endocytic pathways (clathrin and caveolae). Opisthorchis viverrini excretory/secretory products drove IL6 secretion by H69 cells but not Caco-2 cells, and cytokine secretion was significantly reduced by endocytosis inhibitors. This the first known study to address the endocytosis of helminth ES proteins by host epithelial cells and sheds light on the pathways by which this parasite causes one of the most devastating forms of cancer in south-eastern Asia.

Keywords: Carcinogenesis; Cholangiocyte; Endocytosis; Excretory/secretory products; IL6; Opisthorchis viverrini.

Fig. 1

Internalization of Opisthorchis viverrini excretory/secretory products (OvES) by biliary cells (H69, KKU-100, KKU-M156) and colon cancer cells (Caco-2) at 15, 30, 45 and 60 min post-incubation. Internalization of OvES (green fluorescence) by different cell types over time (A). Orthogonal views using Z-stack immunofluorescence microscopy showing three-dimensional internalization of OvES at 0 min (B), 15 min (C) and 120 min (D) in H69 cholangiocytes. Note that OvES is more readily internalized by biliary cells than by colonic epithelial cells. Nucleus was stained with propidium iodide (red fluorescence).

Fig. 2

Mean intracellular fluorescence depicting the internalization of Opisthorchis viverrini excretory/secretory products (OvES) by normal cholangiocytes (H69), cholangiocarcinoma (KKU-100, KKU-M156) and colon cancer (Caco-2) cell lines over 2 h. Fluorescence intensity was measured by flow cytometry (A). Histogram is the average intensity of OvES internalization from three independent experiments ± S.E.M. (B).

Fig. 3

Confocal fluorescence microscopy images showing internalization of Opisthorchis viverrini excretory/secretory products (OvES) in normal (H69) and cholangiocarcinoma (KKU-100 and KKU-M156) biliary cells with and without endocytosis inhibitors. Cells treated with an inhibitor of the clathrin pathway (chlorpromazine, CPZ) showed reduced internalization of OvES (green fluorescence) in all cell types tested. Similarly, sucrose-treated cells showed substantially reduced internalization of OvES in the cholangiocarcinoma lines KKU-100 and KKU-M156 and partially reduced internalization in H69 cholangiocytes.

Fig. 4

Endocytosis pathway inhibitors block the uptake of Opisthorchis viverrini excretory/secretory products (OvES) by normal (H69) and cholangiocarcinoma (KKU-100 and KKU-M156) biliary cell lines using flow cytometry. Cells were pretreated with the endocytosis inhibitors chlorpromazine (CPZ), sucrose (clathrin-mediated inhibitor) and filipin (caveolae-mediated inhibitor) before incubation with OvES for 2 h. Fluorescence intensity was measured by flow cytometry (A). The histogram depicts the average of three independent experiments ± S.E.M. (B).

Fig. 5

Co-staining of H69 cholangiocytes for internalized Opisthorchis viverrini excretory/secretory products (OvES) and cell organelles revelaed an absence of co-localization. OvES is observed as punctate green fluorescence. Cells were co-stained for endoplasmic reticulum (ER) with ER-Tracker-red (A), Golgi with Golgi-tracker-red (B), early endosomes with Anti-Rab5-red (C) and lysosomes with LysoTracker-red (D). Nuclei were counterstained with Hoechst dye (blue).

Fig. 6

Real time cell proliferation of biliary (H69, KKU-100, KKU-M156) and colon (Caco-2) cells induced by Opisthorchis viverrini excretory/secretory products (OvES) in the presence or absence of chlorpromazine (CPZ) using an xCELLigence system. OvES stimulated proliferation of bilary cells (A – C) but not colon cells (D) compared with media control over 96 h of culture. CPZ significantly inhibited growth of all cell types. Growth of each cell type was normalized to its media control without OvES.

Fig. 7

IL6 production. IL6 production in normal human cholangiocytes (H69) and human colon cancer (Caco-2) co-cultured with Opisthorchis viverrini excretory-secretory products (OvES) in several dilutions for 48 h (A). The biliary cells were strongly stimulated by OvES protein, but had no effect in colon cancer cells when compared with control cells. IL6 production from normal human cholangiocyte cell (H69) with and without endocytosis inhibitors (chlorpromazine; CPZ, Filipin and Bafilomycin A1) before OvES stimulation for 48 h using the ELISA technique (B). OvES stimulated IL6 production while all inhibitors of endocytosis significantly suppressed the levels of IL6 secreted. Histograms represent the average of three independent experiments ± S.E.M. of the absorbance at 450 nm measured by a Versamax microplate reader using SoftMax pro V.5 program (*** P < 0.001).