Effect of the LPA-mediated CXCL12-CXCR4 axis in the tumor proliferation, migration and invasion of ovarian cancer cell lines

Abstract

Ovarian cancer is the most fatal gynecological cancer, with a 5-year survival rate of only 30%. Lysophosphatidic acid (LPA), which possesses growth factor-like functions, is a major regulatory factor in the peritoneal metastasis of ovarian cancer. LPA stimulates the expression of numerous genes that are associated with angiogenesis and metastasis. Ovarian epithelial carcinoma specifically expresses chemotactic factor C-X-C motif chemokine ligand 12 (CXCL12) and its receptor, CXC receptor 4 (CXCR4). The CXCL12-CXCR4 axis directly contributes to ovarian cancer cell proliferation, migration and invasion. The present study investigated the regulation of LPA on the CXCL12-CXCR4 axis and the effect of the LPA-mediated CXCL12-CXCR4 axis on the tumor proliferation, migration and invasion of ovarian cancer cell lines. The CXCR4 proteins expressed in the cell membrane and the cytoplasm of ovarian cancer cells, CAOV3 and SKOV3, were detected by immunocytochemistry. The expression of CXCR4 and CXCL12 was increased in the ovarian cancer cells in a dose- and time-dependent manner when treated with LPA compared with the control groups (P<0.05), as determined by reverse transcription polymerase chain reaction and flow cytometry. LPA (20 μM) and CXCL12 (100 ng/ml) enhanced the proliferation, migration and invasion of the ovarian cancer cells, CAOV3 and SKOV3, as identified by MTT, Transwell and Matrigel assays following co-treatment for 24 h. LPA promoted invasiveness of ovarian cancer by upregulating CXCL12-CXCR4 axis expression.

Keywords: CXCL12-CXCR4 axis; lysophosphatidic acid; metastasis; ovarian neoplasm.

Figure 1

CXCR4 protein expression in SKOV3 and CAOV3 cell lines, as determined by immunocytochemistry (streptavidin-perosidase staining; magnification, ×400). CXCR4, C-X-C motif chemokine receptor 4.

Figure 2

Effects of LPA at different doses on CXCR4 mRNA expression in ovarian cancer cells by RT-PCR. 1, CAOV3 control group; 2, CAOV3 5-μM LPA 24-h group; 3, CAOV3 20-μM LPA 24-h group; 4, SKOV3 20-μM LPA 24-h group; 5, SKOV3 5-μM LPA 24-h group; and 6, SKOV3 control group. RT-PCR, reverse transcritpion polymerase chain reaction; LPA, lysophosphatidic acid; CXCR4, C-X-C motif chemokine receptor 4.

Figure 3

Effects of LPA with different times on CXCR4 mRNA expression in ovarian cancer cells by RT-PCR. 1, SKOV3 control group; 2, SKOV3 20-μM LPA 12-h group; 3, SKOV3 20-μM LPA 24-h group; 4, CAOV3 20-μM LPA 24-h group; 5, CAOV3 20-μM LPA 12-h; and 6, CAOV3 control group. RT-PCR, reverse transcription polymerase chain reaction; LPA, lysophosphatidic acid; CXCR4, C-X-C motif chemokine receptor 4.

Figure 4

Expression of CXCL12 protein in ovarian cancer cell lines, CAOV3 and SKOV3, in groups treated with different doses of LPA, as determined by ELISA. 1, SKOV3 control group; 2, SKOV3 5-μM LPA 24-h group; 3, SKOV3 20-μM LPA 24-h group; 4, CAOV3 control group; 5, CAOV3 5-μM LPA 24-h group; and 6, CAOV3 20-μM LPA 24-h group. ^*P<0.05 vs. control group; and ^#P<0.05 vs. 5-μM LPA 24-h group. LPA, lysophosphatidic acid; CXCL12, C-X-C motif chemokine ligand 12.

Figure 5

Expression of CXCL12 protein in ovarian cancer cell lines, SKOV3 and CAOV3, in groups treated for different times with LPA, as determined by ELISA. 1, SKOV3 control group; 2, SKOV3 20-μM LPA 12-h group; 3, SKOV3 20-μM LPA 24-h group; 4, CAOV3 control group; 5, CAOV3 20-μM LPA 12-h group; and 6, CAOV3 20-μM LPA 24-h group. ^*P<0.05 vs. control; and ^# P<0.05 vs. 20-μM LPA 12-h group. LPA, lysophosphatidic acid; CXCL12, C-X-C motif chemokine ligand 12.

Figure 6

Cell proliferation of CAOV3 and SKOV3 cells in the different groups, as determined by MTT. 1, Control group; 2, 20-μM LPA group; 3, 100-ng/ml CXCL12 group; 4, 20-μM LPA + 100-ng/ml CXCL12 group; and 5, 20-μM LPA + 100-ng/ml CXCL12 + 100-ng/ml PTX group. LPA, lysophosphatidic acid; CXCL12, C-X-C motif chemokine ligand 12; PTX, pertussis toxin.

Figure 7

Migration of CAOV3 cells in the different groups, as observed by HE staining. 1, Control group; 2, 20-μM LPA group; 3, 100-ng/ml CXCL12 group; 4, 20-μM LPA + 100-ng/ml CXCL12 group; and 5, 20-μM LPA + 100-ng/ml CXCL12 + 100-ng/ml PTX group. ^*P<0.01 vs. control group; ^# P<0.01 vs. 100-ng/ml CXCL12 group; and ^**P<0.01 vs. 20-μM LPA + 100-ng/ml CXCL12 group. HE, hematoxylin and eosin; LPA, lysophosphatidic acid; CXCL12, C-X-C motif chemokine ligand 12; PTX, pertussis toxin.

Figure 8

Migration of SKOV3 cells in the different groups, as determined by MTT. 1, Control group; 2, 20-μM LPA group; 3, 100-ng/ml CXCL12 group; 4, 20-μM LPA + 100-ng/ml CXCL12 group; and 5, 20-μM LPA + 100-ng/ml CXCL12 + 100-ng/ml PTX group. ^*P<0.05 vs. control group; ^#P<0.01 vs. 100-ng/ml CXCL12 group; and ^** P<0.05 vs. 20-μM LPA + 100-ng/ml CXCL12 group. LPA, lysophosphatidic acid; CXCL12, C-X-C motif chemokine ligand 12.