Fig 1.
Colon cancer cells present different LPA receptor expression profiles.
Representative western blotting and densitometric analyses of Caco-2, HT-29 and HCT-116 cells using specific antibodies against LPAR1-3 (a-c, respectively). Caco-2 cells express high levels of LPA2, HT-29 cells express high levels of LPA1-3 and HCT-116 cells express high levels of LPA2-3. The average scores ± SEM. for three independent experiments are shown.
Fig 2.
LPA increased proliferation in HCT-116 cells but did not induce cell death.
Colon cancer cells were FBS starved for 24 h and then treated with LPA (10 μM) for 24, 48 or 72 h. The relative cell number was evaluated through crystal violet staining (a), and apoptosis was followed by the Annexin-V/PI double staining method (b). a) LPA increased the relative number of HCT-116 cells, but not Caco-2 or HT-29 cells. Statistical analyses were performed using two-way ANOVA with post-hoc Bonferroni test. ** p< 0.01; *** p<0.001. Average scores± SEM. for three independent experiments are shown. b) FACs analysis via Annexin V-FITC/PI staining showed that LPA did not reduce cell death in HCT-116 cells. Four different cell populations were detected after the Annexin V/PI staining of HCT-116 cells. Alive cells are grouped in the lower left part of the panel, early apoptotic cells are grouped in the lower right part of the panel, late apoptotic cells are grouped in the higher right part of the panel and necrotic cells are grouped in the higher left part of the panel. FL1-H, Annexin V; FL2-H, PI. c) Data obtained from the flow cytometric analyses are plotted in a graph. There was no increased percentage of LPA-mediated apoptosis.
Fig 3.
Effect of LPA on cell cycle progression in HCT-116 cells.
The cells were FBS starved for 24 h (cont, control), treated with LPA at the indicated times, stained with PI and analyzed using FACS. a) The proportion of cells in the G2/M phase was significantly increased after 12 and 16 h with LPA treatment. M1: cells in G1; M2: cells in the S phase; M3: cells in G2/M. b) The graph indicates the percentage of S and G2/M cells in relation to the control group. The proportion of DNA in the S phase was calculated using ModfitLT Software. Data are shown as the mean ± SEM from three independent experiments. Statistical analyses were performed using one-way ANOVA (*** p<0.001). PI, propidium iodide; FACS, fluorescence-activated cell sorting.
Fig 4.
LPA mediates cell proliferation through RhoA-ROCK activation.
Subconfluent cell monolayers were FBS depleted for 24 h and treated with LPA at the indicated times. a) Crystal violet staining of HCT-116 showed that the ROCK inhibitor Y-27632 (10 μM) prevented an LPA-mediated increase in the relative cell number after 48 h of treatment. Statistical analyses were performed using two-way ANOVA with post-hoc Bonferroni test. *** p<0.001, vs control; ### p<0.001, vs LPA. Average scores± SEM. for three independent experiments are shown. b) The FACS analysis via PI staining showed that ROCK inhibition with Y-27632 prevented the LPA-induced increase in the proportion of cells in the S-G2/M phase. The statistical analyses were performed using one-way ANOVA with post-hoc Bonferroni test. Data are presented as mean ± SEM. (*** p<0.001, vs control; ## p<0.01, vs LPA).
Fig 5.
LPA mediates cell proliferation through STAT-3 activation.
Subconfluent monolayers of HCT-116 cells were FBS depleted overnight and treated with LPA at the indicated times. a) Total lysates were obtained and prepared for western blotting using a specific antibody against the phosphorylated form of STAT-3 at tyrosine 705 (p-STAT3). The band images were quantified by optical density, and the score was calculated using the ratio between p-STAT3, STAT3 and α-tubulin. LPA increased STAT-3 phosphorylation after 5–15 min of treatment. b) Immunofluorescence of HCT-116 cells corroborated the increase in pSTAT-3 after LPA treatment (green, arrows) and displayed its nuclear location (nucleus, blue). Scale bar, 20 μm. c) The relative cell number of LPA-treated HCT-116 cells was evaluated using crystal violet staining. STAT-3 inhibition using STA 21 prevented the increase in cell numbers after 48 h of LPA treatment. Statistical analyses were performed using two-way ANOVA with post-hoc Bonferroni test (*** p<0.001, vs control; ### p<0.001, vs. LPA). d) Cell cycle analyses through PI staining indicated that STAT-3 inhibition reduced the number of cells at the S-G2/M phase after LPA treatment. Statistical analyses were performed using one-way ANOVA (*** p<0.001, vs. control; ### p<0.001, vs. LPA). Data are presented as mean ± SEM.
Fig 6.
RhoA-ROCK and STAT-3 signaling pathways cooperate to control LPA-mediated cell proliferation.
HCT-116 cells were pre-treated with the ROCK inhibitor Y-27632 for 1 h and then treated with LPA as indicated. a) Western blotting against the phosphorylated form of STAT-3 at tyrosine 705 showed that ROCK inhibition did not prevent STAT-3 phosphorylation mediated by LPA. O.D., optical density. Data are presented as mean ± SEM. b) Confocal images indicating that ROCK inhibition did not prevent the LPA induction of pSTAT3 (green) translocation to the nucleus (blue). The STAT-3 inhibitor, STA 21, was used as a positive control. Cont: control. Scale bar, 20 μm. c) Cell cycle analyses through PI staining indicated that the dual inhibition of STAT-3 and ROCK using both STA 21 and Y-27632 significantly reduced the number of cells in the S-G2/M phase after LPA treatment. Statistical analyses were performed using one-way ANOVA. Average scores ± SEM. for three independent experiments are shown (*** p<0.001, vs. control; ### p<0.001; ## p<0.01, vs. LPA; * p<0.05, LPA+Y-27632+STA21 vs. LPA + Y-27632).
Table 1.
Upregulated genes related to cell cycle of LPA-treated cells.
Fig 7.
LPA regulates cyclin E1, A2 and B1 expression through Rho-ROCK and STAT-3 signaling.
a) HCT-116 cells were treated with LPA at the indicated times, and the total lysates were obtained and prepared for western blotting. LPA increased cyclin E1, A2 and B1 expression. The numbers represent the ratio of the optical density of LPA-treated to untreated cells normalized by GAPDH. b) HCT-116 cells were pre-treated for 1 h with the respective chemical inhibitors prior to LPA treatment at the indicated times. The immunoblotting analysis indicated that either ROCK or STAT-3 inhibition significantly decreased cyclin A2 LPA-mediated expression; however, the concomitant inhibition of these proteins reduced LPA-induced cyclin E1, A2 and B1 expression. The bar graphs are normalized as the percentage of protein expression in which the control group is 1, and the GAPDH protein was used as a loading control. Statistical analyses were performed using one-way ANOVA (*** p<0.001, ** p<0.01, * p<0.05, vs. control; ### p<0.001, ## p<0.01, # p<0.05, vs. LPA; ♦♦ p<0.01 vs. LPA+Y-27632; ★ p<0.05 vs.LPA+STA21). O.D., optical density. Average scores± SEM for three independent experiments are shown.
Fig 8.
A model for the regulation of the LPA-induced cell cycle.
LPA, through its G protein-coupled receptors, induces both Rho-ROCK and STAT-3 signaling pathways to mediate cyclin E1, A2 and B1 expression and to regulate the HCT-116 cell cycle.