Extracellular ATP Mediates Cancer Cell Migration and Invasion Through Increased Expression of Cyclooxygenase 2

Figures

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  FIGURE 1. Extracellular ATP enhances cancer cell migration and invasion. (A) HeLa, IMR-32 and MCF-7 cells were treated with 200 nM doxorubicin for 48 h following which the media was collected for measuring ATP levels (black bars) while the cells were stained with propidium iodide for cell death analysis through flow cytometry (gray bars). 10,000 cells (for HeLa and MCF-7) and 30,000 cells (for IMR-32) were run on a flow cytometer and the % cells dead depicted in gray bars. The amount of ATP released in the media was calculated based on a standard curve and represented here (n = 3 independent experiments). Data depicted mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t-test. (B) Cell migration was assessed by culturing cells on top of a transwell insert in serum-free media while media containing serum was placed in the lower chamber. The same set up was used for invasion studies wherein the cells were instead plated on top of a matrigel in the transwell insert. Migration/invasion was analyzed 24 h after the addition in the lower chamber media from untreated HeLa cells (CM-Ctrl), from HeLa cells treated with 200 nM doxorubicin (CM-Dx), CM-Dx with addition of 10 μM suramin, or…

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  FIGURE 2. P2X7 and P2Y12 receptor antagonists abolish cancer cell migration and invasion. (A) IMR-32 (gray bars) or MCF-7 (black bars) cells were plated on top of a transwell insert and treated with ATP (30 μM for IMR-32 or 100 μM for MCF-7), or 1 μM BzATP, in the presence or absence of 1 μM A740003 (A74), 1 μM oxidized ATP (oATP), or 1 μM PPADS. The number of migrated cells were stained, counted and depicted as mean ± SEM. Student’s t-test shows significance of p < 0.001 with respect to unstimulated (***) or ATP/BzATP-treated ($$$) cells (n = 5 independent experiments). (B) Invasion assay as described in Methods was done in IMR-32 (gray bars) or MCF-7 (black bars) cells stimulated with ATP (30 μM for IMR-32 or 100 μM for MCF-7) or 1 μM BzATP, in the presence or absence of 1 μM A740003 (A74). The number of cells invading through the matrigel were counted and depicted as mean ± SEM. Student’s t-test shows significance of p < 0.001 with respect to unstimulated (***) or ATP/BzATP-treated ($$$) cells. (C) HeLa cells were treated with 1 mM ATP, followed by RNA isolation at 4 and 24 h post-stimulation, for the analysis of P2Y6 and P2Y12 receptors. Fold change was calculated using δδCt method.…

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  FIGURE 3. P2 receptor-mediated cell migration or invasion is dependent on COX-2 expression. (A–C) HeLa (A), IMR-32 (B) or MCF-7 (C) cells were plated on transwell inserts directly (for migration assay) or on top of a layer of matrigel (for invasion assays) and stimulated with ATP, with or without indomethacin (indo), or celecoxib (Cele)—both used at 100 nM. After 24 h, migrated/invaded cells from the bottom of the chamber were stained, counted, and depicted here as average number of cells ±SEM. Significances were calculated with respect to control cells (***p < 0.001) for ATP-treated cells, or with respect to ATP for the antagonists ($ p < 0.05, $$ p < 0.01, $$$ p < 0.001) (n = 5 independent experiments). (D) HeLa cells were stimulated with ATP (1 mM), alone or with suramin (sura, 10 μM), or with ADP (10 μM), alone or with AR-C 69931 (AR-C, 1 μM). Total RNA was isolated for cDNA synthesis followed by real time PCR analysis for COX-2 expression. Fold change was calculated using δδCt method using β-actin as a housekeeping control. Significances were calculated with respect to control cells (**p < 0.01, or ***p < 0.001) for the agonists, or with respect to ATP or ADP in the case of…

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  FIGURE 4. P2 receptor antagonists block eATP-mediated MMP-2 activity. (A) HeLa and IMR-32 cells were stimulated with ATP (1 mM or 30 μM, respectively), 10 μM ADP or 1 μM BzATP (Bz), with or without the inhibitors AR-C 69931 (R, 1 μM) or A740003 (A7, 1 μM). 24 h post-stimulation cell lysates were prepared for the detection of MMP-2 through Western blotting as described in Materials and Methods. β-actin was used as a housekeeping control. (B) MMP-2 activity was observed through an in-gel assay as described in the Materials and Methods with cells stimulated in the same manner as described in (A) and also including the inhibitor suramin (S, 10 μM). Representative zymograph is depicted here. (C) Relative densitometry was made based on three independent gelatin zymography experiments. Student’s t-test was done to calculate significance of agonist-treated cells with their unstimulated counterparts (**p < 0.01) and the antagonists with respect to their agonist counterparts ($ p < 0.05). (D) HeLa and IMR-32 cells were stimulated with ATP (1 mM for HeLa and 30 μM for IMR-32 cells) for 24 h, with or without celecoxib (100 nM) or indomethacin (100 nM). Cell lysates were probed for the detection of…

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  FIGURE 5. P2 receptor stimulation increases p42/44 MAPK and p38 MAPK activity. (A) HeLa or IMR-32 cells were stimulated with ATP (1 mM for HeLa and 30 μM for IMR-32 cells) in the presence or absence of SB202190 (1 μM) or PD98059 (1 μM). The inhibitors were added 30 min prior to the addition of ATP and cells were harvested after 24 h for the detection of COX-2 protein. (B) HeLa cells were stimulated with 1 mM ATP or 10 μM ADP, with or without 1 μM AR-C 69931 (R) for 30 min. Cell lysates were then separated on SDS-PAGE and analyzed for phosphorylated or total forms of p42/44 MAPK or phosphorylated or total p38 MAPK. (C) IMR-32 cells were stimulated with 30 μM ATP or 1 μM BzATP (Bz), with or without 1 μM A740003 (A7), for 30 min. Cell lysates were then analyzed as described for HeLa cells. Representative images of Western blot are shown from a total of five independent experiments. (D,E) Densitometric analysis of phosphorylated p38 or p42/44 MAPK was done with respect to total p38 or p42/44 MAPK, respectively, for HeLa (D) or IMR-32 (E) cells. Significances were calculated with respect to control cells (*p < 0.05, **p < 0.01), or with corresponding agonists ($ p < 0.05, $$ p < 0.01).

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  FIGURE 6. Exogenous ATP mediates increased COX-2 expression leading to cancer cell metastasis. Treatment of tumors with chemotherapeutic agents such as doxorubicin leads to profound cell death and increased levels of exogenous ATP (eATP) in the extracellular milieu. This ATP acts on the P2 receptors of tumor cells in the vicinity through different P2 receptors depending on tumor type such as P2X7 receptors in case of IMR-32 and MCF-7 cells or P2Y12 receptors in case of HeLa cells. Activation of either of these P2 receptors leads to the activation of p38 and p42/44 MAPK which, in turn, induce the transcription of COX-2. Increased COX-2 synthesis has been shown to modulate tumor cell migration and invasiveness, thus contributing toward increased metastasis. P2 receptor-based anti-inflammatory drugs (PBAIDs), which are specific P2 receptor antagonists, have been shown to block the phosphorylation of MAPKs, synthesis of COX-2 and cell migration or invasion.