Figure 1.
Effects of I-BOP on MCP-1 expression.
A I-BOP induced transcription of MCP-1. Cells were serum-starved for 24 h before treated with 50 nM I-BOP for the indicated time periods. RNAs were isolated and RT-PCR was carried out as described in Materials and methods. B MCP-1 mRNA induction in A was confirmed by quantitative real-time PCR. **p<0.01 compared with untreated cells. C Time-dependent effects of I-BOP on MCP-1 protein expression. After 24 h serum-starvation, cells were treated with 50 nM I-BOP for the indicated time periods. Media were collected and proteins in each medium were concentrated by trichloroacetic acid (TCA) precipitation as described in Materials and methods. Concentrated samples were quantified by Bio-Rad protein assays to ensure equal protein loading for Western blot analysis. D Dose-dependent effects of I-BOP on MCP-1 protein expression in A549-TPα cells. E I-BOP-induced MCP-1 protein expression in A549-TPβ not A549-GFP cells. F I-BOP-induced MCP-1 protein expression in H157 and H460 cells. G Comparison of I-BOP with other prostanoids on the induction of MCP-1 in A549-TPα and H460 cells. Cells were treated with 50 nM I-BOP, 1 µM PGD2, 1 µM PGE2 or 1 µM PGF2α for 24 h. The concentrations of prostanoids used here were optimized to induce the most expression of MCP-1. Media were collected and assayed as described in C. Densitometric analysis of each band was made, and control untreated time point is normalized to 1. All data are representative of at least three independent experiments.
Figure 2.
I-BOP regulates MCP-1 expression in SP1-dependent manner.
A Effects of TP antagonist and several protein kinases inhibitors on MCP-1 induction by I-BOP stimulation. Cells were treated with TP antagonist SQ29548 (10 µM), PKC inhibitor GF109203X (0.5 µM), and MEK inhibitor U0126 (10 µM) for 30 min before incubation with 50 nM I-BOP for 24 h. Media were collected and assayed as described in Fig. 1C. B Effects of SP1 inhibition on I-BOP-induced MCP-1 expression at protein levels. Cells were treated with indicated concentrations of SP1 inhibitor mithramycin A (MTM) and Hsp90 inhibitor geldanamycin (GA) for 30 min before incubation with 50 nM I-BOP for 24 h. Media were collected and assayed as described in Fig. 1C. C Effects of SP1 inhibition on I-BOP-induced MCP-1 expression at mRNA levels. Cells were treated with above inhibitors for 30 min before incubation with 50 nM I-BOP for 4 h. RNAs were isolated and real-time PCR was performed as described in Materials and methods. *p<0.05 and **p<0.01 compared with I-BOP treated alone. D Protein level of SP1 was increased in the nuclei of A549-TPα cells following I-BOP stimulation. Cells were treated with 50 nM I-BOP for the indicated time periods. Nuclear extract and whole cell lysate were prepared as described in Material and methods. Protein level of SP1 was determined by Western blot. Densitometric analysis of each band was made and control untreated time point was normalized to 1. E Regulation of MCP-1 promoter activity by SP1. A549-TPα cells were transfected with luciferase reporter plasmids containing 500 bp of MCP-1 promoter sequence with either wild type or mutated SP1 binding sites. After 24 h transfection, cells were treated with vehicle control (0.1% ethanol) or 50 nM I-BOP for additional 18 h in serum free medium. Luciferase assay was carried out as described in Materials and methods. **P<0.01. All data are representative of at least three independent experiments.
Figure 3.
Chemotactic effects of A549-TPα conditioned media and MCP-1 on RAW 264.7 macrophages.
A. Conditioned media increased the chemotaxis of RAW cells via MCP-1. CM-C is derived from vehicle-treated cells, and CM-I is derived from I-BOP-treated cells. SFM represents serum free medium. The concentrations of anti-MCP-1 antibodies and isotype control IgG were each at 5 µg/ml original CM. The concentration of RS-102895 was 10 µM. Detailed conditions were as described in Materials and Methods. **P<0.01. B Effect of recombinant MCP-1 on the chemotaxis of RAW cells. MCP-1 at 1, 10 and 100 ng/ml were tested as described in Materials and methods.
Figure 4.
Transwell co-culture of RAW 264.7 macrophages induces metastatic gene expression by A549 cells.
A A transwell co-culture system of RAW 264.7 macrophages and A549 cells was used in this study. RAW 264.7 cells and A549 cells were separately cultured for 24 h and then co-cultured in serum free medium for further 12 h or 24 h as described in Materials and methods. B Control A549 and A549-TPα cells became more scattered and spindle shaped after culture with RAW 264.7 cells. Both types of A549 cells were pictured after 12 h of culture with or without RAW 264.7 cells as described in A. C Co-culture of RAW 264.7 macrophages induced metastatic gene expression by A549 cells. After 12 h of co-culture with macrophages, the expression of several metastatic genes in control A549 and A549-TPα cells, including MMPs, VEGF and MCP-1 mRNA, were examined by RT-PCR as described in Materials and methods. D Co-culture of RAW 264.7 macrophages decreased E-cadherin expression by A549 cells. Control A549 and A549-TPα cells were collected after 24 h of culture with or without RAW 264.7 cells as described in A. Protein level of E-cadherin was determined by Western blot as described in Material and methods. All data are representative of at least three independent experiments.
Figure 5.
Direct co-culture of RAW 264.7 macrophages induces morphological change and invasion of A549 cells.
A. A direct co-culture system of RAW 264.7 macrophages and A549 cells was used in this study. RAW 264.7 cells and A549 cells were plated into the same well of a culture plate. B. A549 cells exhibit elongated shape after culture with RAW 264.7 macrophages. The control A549 cells were plated into a 12-well plate with or without RAW 264.7 cells as described in A. Cells were pictured at the indicated time points. In the lower panel, those small cells exhibiting round shape were RAW 264.7 cells. C. Co-culture of RAW 264.7 macrophages induced invasion of A549 cells. Invasion assay of A549 cells was carried out as described in Material and methods. **P<0.01. Results are representative of three independent experiments.
Figure 6.
Proposed scheme of TP-mediated interactions between cancer cells and macrophages.