In vitro effect of lysophosphatidic acid on proliferation , invasion and migration of human ovarian cancer cells

Purpose: To evaluate the effect of lysophosphatidic acid (LPA) on the proliferation, invasion and migration ability of 3AO, SKOV3 and CAOV3 human ovarian cancer cell lines. Methods: SKOV3, 3AO and CAOV3 cell lines were respectively treated with LPA. Changes in the proliferation rate of these cell lines were observed after LPA treatment. The cell lines that were not treated with LPA served as control group. Boyden chamber was used to assess cell invasion and migration capability. The expression levels of relevant cytokines related to cell migration in the supernatant of CAOV3 cell line were determined using ELISA following LPA stimulation. Results: The cell proliferation rate of human ovarian cancer cell lines was significantly accelerated after in vitro LPA treatment in a concentration-dependent fashion. Boyden chamber assay data indicate that invasion indices in 3AO and CAOV3 cell lines were significantly higher than those in untreated control cell lines (p < 0.05). However, no statistical significance was noted between 3AO and CAOV3 cell lines (p < 0.05). The expression levels of relevant cytokines in the CAOV3 cell line were significantly upregulated after LPA treatment (p < 0.05). Conclusion: LPA intervention in vitro accelerates cell proliferation rate and also significantly upregulates the expression levels of multiple cytokines related to cell migration in human ovarian cancer cell lines, suggesting that LPA plays a significant role in the invasion and migration of SKOV3, 3AO and CAOV3 cell lines.


INTRODUCTION
Lysophosphatidic acid (LPA) is a bioactive lipid mediator involved in tissue repair and wound healing.It mediates diverse functional effects in fibroblasts, including proliferation, migration and contraction, but less is known about its ability to evoke paracrine signaling to other cell types.In recent years, LPA and its receptor signaling pathways have been shown to be implicated in the incidence, invasion and migration processes of malignant tumors [1][2][3][4][5][6].
Ovarian cancer is a highly metastatic disease which is characterized by ascites formation and adhesion, invasion, and metastasis.Levels of LPA are significantly increased in the plasma of ovarian carcinoma patients, indicating that LPA can promote the incidence of early events in ovarian carcinoma dissemination.LPA is capable of playing a role via its receptors.LPA receptors belong to the endothelial differentiation gene (Edg) family, which consists of 4 categories.In particular, LPA1/Edg-2, LPA2/Edg-4 and LPA3/Edg-7 are frequently investigated.
Recent studies [1,3] have demonstrated that G protein-coupled receptor 23 (GPR23)/p2y9/LPA4 is lowly expressed in the ovarian tissues of most healthy subjects.However, Edg-4 and Edg-7 are highly expressed in human ovarian cancer cell lines.LPA is able of accelerating DNA synthesis after binding with Edg-4 and Edg-7.LPA has been identified to regulate the proliferation, migration and adhesion of cancer cells, the release and activation of matrix metalloproteinase (MMP) and the secretion of angiogenesis factors [7][8][9], indicating that LPA plays a pivotal role in the invasion and metastasis processes of malignant tumor cells.
In this study, human ovarian cancer cell lines including 3AO, CAOV3 and SKOV3 were selected and treated with different concentrations of LPA.Subsequently, the growth, proliferation, invasion and metastasis ability of the ovarian cancer cells were evaluated after LPA treatment.The expression levels of multiple cytokines related to cellular invasion and migration were also quantitatively measured.

EXPERIMENTAL MTT assay
The effect of LPA upon the growth and proliferation of ovarian cancer cell lines was assessed by MTT assay.The ovarian cancer cell lines 3AO, SKOV3 and CAOV3 were cultured in RPMI1640 complete culture solution containing 10 % fetal bovine serum at 37 ℃ and 5 % CO 2 .The ovarian cancer cell lines were inoculated for 24 h and serum-free culture solution was supplemented overnight.The obtained cell lines were inoculated in a 96-well plate at a cell density of 5×10 3 /well, cultured for 24 h using conventional method and co-cultured with different concentrations of LPA (0.2,2,10 and 20 µmol/L).
The culture solution was abandoned at 2, 4, 6, 16 and 24 h after LPA supplement, a portion of 20 mL MTT solution at a concentration of 5 µg/µL was added in each well, continuously cultured for 4 h, and 150 mL DMSO was supplemented in each well.The absorbance value of each well was measured at a wavelength of 490 and 620 nm.
The cell growth curve was drawn.The cell proliferation rate = (Absorbance value LPA groupabsorbance value control group )/absorbance value control group ×100 %.Three replicates were set for each concentration in each well.In the control group, LPA was substituted with the PBS.

Boyden chamber assay
The ovarian cancer cell lines 3AO and CAOV3 were cultured in RPMI1640 complete culture solution containing 10 % fetal bovine serum.In the LPA group, 5 and 20 µmol/L of LPA was added and PBS was used in the control group for 16 h.The obtained cells in logarithmic phase were digested in 0.25% pancreatin and a portion of 80 µL suspension containing 2 × 10 6 of cells was prepared.Artificial matrix gel matrige1 (BD corporation, U.S.) was smeared on polycarbonate millipore filter (Waltman, U.S.) approximately 150 µg for each well and maintained at 37 ℃ for 3 h, air dried at room temperature overnight.
A portion of 20 µL serum-free solution was added in the lower Boyden chamber and pretreated cell suspension was added in these upper Boyden chamber.The obtained cells were cultured at 37 ℃ and 5 % CO 2 for 18 h, fixed in 70 % methanol for 45 min, stained in hematoxylin for 1 -2 min, washed triple times using distilled water, transferred on to a slide, sealed with a cover slip and observed under high-power light microscope.Three replicates were set for each concentration in each well.In the control group, LPA was substituted with PBS.

Cytokine detection after LPA treatment
The CAOV3 cell line was cultured in serum-free solution for 12 h and treated with different concentrations of LPA (5, 20 and 40 µmol/L) for 16 h, and LPA was replaced by PBS in the control group.The treated cells in logarithmic growth phase were digested using 0.25 % pancreatin containing 10 % fetal bovine serum, transferred into the centrifuge tube and added by 100 µL culture solution.The cell suspension at a density of 2 × 10 6 /mL was prepared using 0.9 % cell suspension and the supernatant was preserved at -80 o C.
After LPA treatment at a concentration of (0, 5, 20 and 40 µL/L), CXCL12 (C-X-C motif ligand12), Ecadherin, vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR), epidermal growth factor (EGF), epidermal growth factor receptor (EGFR) were quantitatively measured using ELISA (R & D Systems Inc.).The experimental procedures were performed strictly according to the manufacturer's instructions.Three replicates were set for each concentration in each well.In the control group, LPA was substituted with PBS.

Statistical analysis
SPSS19.0 statistical software was used for statistical analysis and the data are expressed as mean ± standard deviation (SD).Replicate data were statistically analyzed by ANOVA.Group comparison was performed by LSD test.P < 0.05 was considered statistically significant.

Effect of LPA treatment upon ovarian cancer cell growth and proliferation
The cell lines were treated with different concentrations of LPA, the absorbance values in the LPA group were significantly higher compared with those in the control group.At 10 µmol/L LPA treatment for 16 h, the proliferation rate of 3AO, SKOV3 and CAOV3 cell lines was significantly enhanced compared with that in the control group, as illustrated in Figure 1.

Effect of LPA on expression levels of multiple cytokines
The expression levels of CXCL12, E-cadherin, VEGF, VEGFR, EGF and EGFR in the CAOV3 cell supernatant after 5, 20 and 40 µmol/L treatment with LPA are shown in Table 2.No  The expression level of E-cadherin was significantly up-regulated along with increasing concentration of LPA.In the LPA intervention group, the expression level of VEGF ranged from 1.08 to 1.19 ng/mL with a median value of 1.14 ng/mL, significantly higher compared with that in the control group (F = 5.032, p = 0.002).The expression levels of VEGF did not significantly differ among different LPA concentration groups.
Nevertheless, the expression level increased along with elevation of LPA concentration.The expression levels of VEGFR in the different LPA concentration groups were all significantly upregulated compared with that in the control group (F = 5.000, p = 0.010), whereas no statistical significance was observed among different LPA concentration groups.
The expression levels of EGF significantly differed between the control and LPA treatment groups (F = 5.678, p = 0.006), whereas no statistical significance was observed among different LPA concentration groups (all p > 0.05).EGF expression significantly differed between the 5 and 20 µmol/L treatment groups (p = 0.001).No statistical significance was observed between the 5 and 40 µmol/L groups (p = 0.055) in terms of the EGF expression level.
Statistical significance was observed between the 20 and 40 µmo1/L LPA groups (p = 0.000), suggesting that the expression of EGF showed a dose-dependent fashion.No statistical significance was observed in the EGFR expression levels between the LPA treatment and control groups or among different LPA concentration groups (F = 1.754, p = 0.189).

DISCUSSION
Previous studies have demonstrated that LPA receptor is expressed in the human ovarian cancer cell lines 3AO, SKOV3 and CAOV3 [9].LPA possesses multiple biological functions through LPA receptors located on the cell membrane.LPA has been shown to play a vital role in the invasion and migration of malignant tumor cells.The findings in this study further demonstrate that LPA administration is able to enhance the proliferation of ovarian cancer cells, which is consistent with previous investigations [10].
Boyden chamber experiment revealed that the invasion capability of 3AO and CAOV3 cell lines was significantly increased after LPA treatment, which was significantly higher compared with that in the control group.In addition, the invasion ability of ovarian cancer cells was positively correlated with increasing concentration of LPA administration.
However, no statistical significance was observed in the cell invasion between 3AO and CAOV3 cell lines.
The results of this investigation indicate that LPA is capable of binding with tumor cell surface receptors to participate in the growth, proliferation and migration of malignant tumor cells.The supernatant solution of the ovarian cancer cell suspension was collected and demonstrated that LPA can stimulate the growth of CAOV3 cell line and significantly up-regulate the expression level of CXCL12 cytokines.These findings collectively suggest that CAOV3 cell line is able to secrete a certain quantity of CXCL12, which is capable of binding with CXCR4 to induce the formation of actin-related pseudopod and regulate the directional migration, invasion and angiogenesis of tumor cells.CXCL12 is highly expressed in the ascites of ovarian cancer patients, suggesting that CXCLl2/CXCR4 probably plays a role in the metastasis of ovarian cancer.Statistical analysis revealed statistical significance in the E-cadherin expression level in the supernatant after corresponding LPA treatment.The expression level of E-cadherin was significantly up-regulated along with increasing concentration of LPA In vitro experiment demonstrates that VEGF can stimulate endothelial cell mitogenesis and cell migration.VEGF also enhances microvascular permeability and is occasionally referred as vascular permeability factor.The increase in VEGF concentration can up-regulate the expression level of VEGFR, which is considered a vital mechanism underlying the effect of VEGF on the promotion of angiogenesis of tumor tissues [11].
The expression level of VEGFR in the LPA treatment group was significantly up-regulated compared with that in the control group, whereas no statistical significance was observed among different LPA concentration groups.The CAOV3 cancer cells can secrete VEGFR via autocrine or paracrine pathway, which migrate into ascites or extracellular space.The interaction between VEGF and VEGFR is capable of provoking the migration of ovarian cancer cells.
Similarly, the expression level of EGF in the LPA treatment group was significantly up-regulated compared with that in the control group.In addition, the expression level of EGF was increasingly up-regulated along with increasing concentration of LPA, which peaked at 20 µmol/L and subsequently began to decline.The expression level of EGF after 20 µmol/L LPA treatment was significantly higher compared with that following 40 µmol/L LPA treatment.Although the expression level of EGFR is regulated by the LPA in a concentration-dependent manner, no statistical significance was detected among different LPA concentration groups, which is consistent with our previous findings that the expression level of EGFR is low in the chylous ascites of ovarian cancer patients, suggesting that LPA exerts no significant effect upon the production of EGFR by ovarian cancer cells.
The experimental results in this investigation revealed that LPA exerts a nonsynchronous effect upon the production of EGF and EGFR.Previous investigations have demonstrated that CXCLl2 can stimulate the growth and proliferation of ovarian cancer cells through mediating the EGFR [12].Though the content of soluble EGFR is relatively low, the biological role of EGFR should be emphasized.The role of EGF-EGFR interaction in the invasion and metastasis of ovarian cancer remains to be elucidated.
In this investigation, LPA exerts different effects on CXCL12, VEGF and their receptors, whereas it does not affect the level of CXCLl2 secreted by ovarian cancer cells.However, LPA exerts a significant effect upon VEGF and VEGFR, indicate that blockage of CXCR4/CXCL12 biological axis can inhibit tumor growth and suppress the role of VEGF in vascularization, which is consistent with previous reports [13,14].The findings in this investigation may provide alternative evidence for the clinical treatment of ovarian cancer.

CONCLUSION
LPA treatment in vitro enhances cell proliferation rate and significantly up-regulates the expression levels of multiple cytokines associated with cell migration in human ovarian cancer cell lines, indicating that LPA plays a significant role in the invasion and migration of SKOV3, 3AO and CAOV3 cells.

Conflict of Interest
There are no competing interests with regard to this work.

Contribution of authors
We declare that this work was done by the authors named in this article and all liabilities pertaining to claims relating to the content of this article will be borne by the authors.Qingfu Wang, Lixin Zhu and Aiping Qin were responsible for research design and manuscript draft.Tiefeng Chen and Jinpen Li were in charge of study design.Beitai Wu was responsible for data analysis.Yu Xiao, Zongan Lai and Weixiong Xie were in charge of proofreading.All authors have reviewed the final version manuscript.

Figure 1 :Figure 2 :
Figure 1: Effect of different concentrations of LPA on the proliferation rates of 3AO (A), SKOV3 (B) and CAOV3 (C) cell lines

Table 1 :
Effect of different concentrations of LPA on the invasion capability of 3AO and CAOV3 cell lines

Table 2 :
Effect of different concentrations of LPA on the expression levels of multiple cytokines