COX-2 Promotes Migration and Invasion by the Side Population of Cancer Stem Cell-Like Hepatocellular Carcinoma Cells

Abstract

Cancer stem cells (CSCs) are thought to be responsible for tumor relapse and metastasis due to their abilities to self-renew, differentiate, and give rise to new tumors. Cyclooxygenase-2 (COX-2) is highly expressed in several kinds of CSCs, and it helps promote stem cell renewal, proliferation, and radioresistance. Whether and how COX-2 contributes to CSC migration and invasion is unclear. In this study, COX-2 was overexpressed in the CSC-like side population (SP) of the human hepatocellular carcinoma (HCC) cell line HCCLM3. COX-2 overexpression significantly enhanced migration and invasion of SP cells, while reducing expression of metastasis-related proteins PDCD4 and PTEN. Treating SP cells with the selective COX-2 inhibitor celecoxib down-regulated COX-2 and caused a dose-dependent reduction in cell migration and invasion, which was associated with up-regulation of PDCD4 and PTEN. These results suggest that COX-2 exerts pro-metastatic effects on SP cells, and that these effects are mediated at least partly through regulation of PDCD4 and PTEN expression. These results further suggest that celecoxib may be a promising anti-metastatic agent to reduce migration and invasion by hepatic CSCs.

FIGURE 1

Side population (SP) cell fraction and cyclooxygenase-2 (COX-2) expression in the HCC cell lines Huh7 and HCCLM3. (A) Identification of SP and main population (MP) cells by flow cytometry. (B) The proportion of SP cells differed significantly between HCCLM3 and Huh7 lines. (C) Quantitative real-time PCR (qRT-PCR) showed a significantly higher level of COX-2 mRNA in HCCLM3 cells than in Huh7 cells. Gene expression was normalized to that of GAPDH. Data are shown as mean ± standard deviation. (D) Western blotting showed a higher level of COX-2 protein in HCCLM3 cells than in Huh7 cells. GAPDH served as a loading control. HCC = hepatocellular carcinoma. ^∗∗P < 0.01.

FIGURE 2

SP cells from the HCCLM3 cell line are enriched for cancer stem cell-like cells. (A) Spheroid formation by SP and MP cells after 2-week culture in modified medium was visualized by microscopy (magnification, ×200). SP cells formed larger and more compact spheroids than MP cells. (B) IC₅₀ values of SP and MP cells for 5-fluorouracil (5-Fu) or cisplatin (CDDP) were measured using the CCK8 assay. SP cells were consistently more resistant to 5-Fu and CDDP than MP cells. (C) Differentiation ability was compared between sorted SP and MP cells using Hoechst 33342 staining after 1 week in culture. SP cultures still contained 28.5% Hoechst 33342-negative cells, while MP cultures contained only 0.2% of such cells. (D) SP and MP cells were stained with fluorochrome-conjugated monoclonal antibodies or isotype antibodies and analyzed by flow cytometry. Nearly all SP and MP cells expressed CD44. Expression of ABCG2, CD90, and CD13 was significantly higher in SP cells than in MP cells. MP = main population; SP = side population. ^∗∗P < 0.01.

FIGURE 3

In a transwell assay, (A,B) SP cells from the HCCLM3 line showed significantly greater migration and invasion activity than MP cells. Magnification, 200 ×. Data are shown as mean ± SD. ^∗∗P  < 0.01.

FIGURE 4

COX-2 expression in SP and MP cells from the HCCLM3 line. (A) qRT-PCR showed a significantly higher level of COX-2 mRNA in SP cells than in MP cells. (B) Western blotting showed higher levels of COX-2 protein in SP cells than in MP cells. COX-2 = cyclooxygenase-2; MP = main population; qRT-PCR = quantitative real-time PCR; SP = side population. ^∗∗P < 0.01.

FIGURE 5

COX-2 overexpression enhanced migration and invasion of SP cells in part by down-regulating PDCD4 and PTEN. A stable HCCLM3 cell line constitutively overexpressing COX-2 (HCCLM3-COX-2) was constructed by transducing HCCLM3 cells with a lentiviral COX-2 expression plasmid also encoding green fluorescent protein (LV-COX-2-GFP). In parallel, a negative control cell line (HCCLM3-NC) was prepared by transducing HCCLM3 cells with empty vector (LV-GFP). (A) Flow cytometry showed that SP proportions were significantly higher in the HCCLM3-COX-2 line than in the HCCLM3-NC line. (B) SP cells from HCCLM3-COX-2 were consistently more resistant to 5-Fu and CDDP than SP cells from HCCLM3-NC. (C, D) qRT-PCR and Western blotting showed that COX-2 overexpression was associated with down-regulation of PDCD4 and PTEN mRNA and protein. (E, F) COX-2 overexpression in SP cells increased cell migration and invasion. Magnification, ×200. COX-2 = cyclooxygenase-2; MP = main population; qRT-PCR = quantitative real-time PCR; SP = side population. ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 6

The selective COX-2 inhibitor celecoxib inhibited SP cell migration and invasion and may exert these effects by down-regulating COX-2 and up-regulating PTEN and PDCD4. (A) Celecoxib significantly inhibited SP cell proliferation in a dose- and time-dependent manner. (B) Celecoxib significantly inhibited prostaglandin E2 (PGE2) production in SP cells. (C, D) Celecoxib significantly suppressed SP cell migration and invasion in a dose-dependent manner. Magnification, ×200. (E, F) Analysis by qRT-PCR and Western blotting revealed that celecoxib treatment significantly decreased expression of COX-2 mRNA and protein, while increasing expression of PDCD4 and PTEN mRNA and protein in a dose-dependent manner. COX-2 = cyclooxygenase-2; MP = main population; qRT-PCR = quantitative real-time PCR; SP = side population. ^∗P < 0.05, ^∗∗P < 0.01.