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Cyclooxygenase-2 enhances alpha2beta1 integrin expression and cell migration via EP1 dependent signaling pathway in human chondrosarcoma cells.

Liu JF, Fong YC, Chang CS, Huang CY, Chen HT, Yang WH, Hsu CJ, Jeng LB, Chen CY, Tang CH - Mol. Cancer (2010)

Bottom Line: PGE2-mediated migration and integrin up-regulation were attenuated by phospholipase C (PLC), protein kinase C (PKC) and c-Src inhibitor.Activation of the PLCbeta, PKCalpha, c-Src and NF-kappaB signaling pathway after PGE2 treatment was demonstrated, and PGE2-induced expression of integrin and migration activity were inhibited by the specific inhibitor, siRNA and mutants of PLC, PKC, c-Src and NF-kappaB cascades.Our results indicated that PGE2 enhances the migration of chondrosarcoma cells by increasing alpha2beta1 integrin expression through the EP1/PLC/PKCalpha/c-Src/NF-kappaB signal transduction pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung, Taiwan.

ABSTRACT

Background: Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin (PG) synthase, has been implicated in tumor metastasis. Interaction of COX-2 with its specific EP receptors on the surface of cancer cells has been reported to induce cancer invasion. However, the effects of COX-2 on migration activity in human chondrosarcoma cells are mostly unknown. In this study, we examined whether COX-2 and EP interaction are involved in metastasis of human chondrosarcoma.

Results: We found that over-expression of COX-2 or exogenous PGE2 increased the migration of human chondrosarcoma cells. We also found that human chondrosarcoma tissues and chondrosarcoma cell lines had significant expression of the COX-2 which was higher than that in normal cartilage. By using pharmacological inhibitors or activators or genetic inhibition by the EP receptors, we discovered that the EP1 receptor but not other PGE receptors is involved in PGE2-mediated cell migration and alpha2beta1 integrin expression. Furthermore, we found that human chondrosarcoma tissues expressed a higher level of EP1 receptor than normal cartilage. PGE2-mediated migration and integrin up-regulation were attenuated by phospholipase C (PLC), protein kinase C (PKC) and c-Src inhibitor. Activation of the PLCbeta, PKCalpha, c-Src and NF-kappaB signaling pathway after PGE2 treatment was demonstrated, and PGE2-induced expression of integrin and migration activity were inhibited by the specific inhibitor, siRNA and mutants of PLC, PKC, c-Src and NF-kappaB cascades.

Conclusions: Our results indicated that PGE2 enhances the migration of chondrosarcoma cells by increasing alpha2beta1 integrin expression through the EP1/PLC/PKCalpha/c-Src/NF-kappaB signal transduction pathway.

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COX-2-directed migration of human chondrosarcoma cells involves up-regulation of α2β1 integrin. (A) JJ012 cells were incubated with PGE2 for 24 hr, and the cells surface α5, α2, β3, α5β1, αvβ3 and α2β1 integrin was determined using flow cytometry. (B) Cells were incubated with PGE2 for 24 hr, and the mRNA levels of α2 and β1 integrin was determined using qPCR. (C) JJ012 cells were transfected with IPTG/COX-2 expression plasmid or control vector for 24 hr followed by stimulation with IPTG (5 mM) for 24 hr, the mRNA expression of α2 and β1 integrin was determined by qPCR. JJ012 cells were incubated with PGE2 for indicated time intervals, and mRNA and cell surface α2β1 integrin were examined by qPCR (D) and flow cytometry (E). (F) Cells were pretreated with α2β1 monoclonal antibody (3 μg/ml) for 30 min followed by stimulation with PGE2. The in vitro migration activity measured after 24 hr. (E) JJ012 cells were treated with 17-phenyl trinor PGE2 (3 μM), 11-deoxy-PGE1 (10 μM), PGE2, and PGE2 plus SC19220 (10 μM), and cells surface α2β1 integrin was determined using flow cytometry. Results are expressed as the mean ± S.E. *, p < 0.05 compared with control; #, p < 0.05 compared with PGE2-treated group.
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Figure 3: COX-2-directed migration of human chondrosarcoma cells involves up-regulation of α2β1 integrin. (A) JJ012 cells were incubated with PGE2 for 24 hr, and the cells surface α5, α2, β3, α5β1, αvβ3 and α2β1 integrin was determined using flow cytometry. (B) Cells were incubated with PGE2 for 24 hr, and the mRNA levels of α2 and β1 integrin was determined using qPCR. (C) JJ012 cells were transfected with IPTG/COX-2 expression plasmid or control vector for 24 hr followed by stimulation with IPTG (5 mM) for 24 hr, the mRNA expression of α2 and β1 integrin was determined by qPCR. JJ012 cells were incubated with PGE2 for indicated time intervals, and mRNA and cell surface α2β1 integrin were examined by qPCR (D) and flow cytometry (E). (F) Cells were pretreated with α2β1 monoclonal antibody (3 μg/ml) for 30 min followed by stimulation with PGE2. The in vitro migration activity measured after 24 hr. (E) JJ012 cells were treated with 17-phenyl trinor PGE2 (3 μM), 11-deoxy-PGE1 (10 μM), PGE2, and PGE2 plus SC19220 (10 μM), and cells surface α2β1 integrin was determined using flow cytometry. Results are expressed as the mean ± S.E. *, p < 0.05 compared with control; #, p < 0.05 compared with PGE2-treated group.

Mentions: Previous studies have demonstrated significant expression of integrins in human chondrosarcoma cells [27]. We therefore, hypothesized that integrins may be involved in PGE2-directed migration of chondrosarcoma cells. Flow cytometry analysis showed that PGE2 induced the cell surface expression of α2 and α2β1 integrin in JJ012 cells (Fig. 3A). To confirm this finding, expression of mRNAs in the integrins in response to PGE2 was analyzed by qPCR. Treatment of JJ012 cells with PGE2 induced the mRNA expression of α2 and β1 integrins (Fig. 3B). In addition, treatment of IPTG/COX-2-transfected cells with IPTG increased mRNA expression of α2 and β1 integrins (Fig. 3C). Furthermore, compared with normal cartilage, human chondrosarcoma tissues expressed higher levels of α2 and β1 integrin mRNA (Table 1). Therefore, the α2β1 integrin plays an important role in PGE2-induced migration of human chondrosarcoma cells. Stimulation of cells with PGE2 also increased mRNA expression and cell surface expression of α2 and β1 integrin time-dependently (Fig. 3D&3E). Pretreatment of cells for 30 min with anti-α2β1 monoclonal antibody (mAb) (3 μg/ml) markedly inhibited the PGE2-induced cell migration (Fig. 3F). On the other hand, EP1/3 agonist enhanced the cell surface expression of α2β1 integrin (Fig. 3G). Pretreatment of cells with SC19220 reduced PGE2-mediated α2β1 integrin expression (Fig. 3G). These data suggest that PGE2-induced cancer migration may occur via activation of the α2β1 integrin.


Cyclooxygenase-2 enhances alpha2beta1 integrin expression and cell migration via EP1 dependent signaling pathway in human chondrosarcoma cells.

Liu JF, Fong YC, Chang CS, Huang CY, Chen HT, Yang WH, Hsu CJ, Jeng LB, Chen CY, Tang CH - Mol. Cancer (2010)

COX-2-directed migration of human chondrosarcoma cells involves up-regulation of α2β1 integrin. (A) JJ012 cells were incubated with PGE2 for 24 hr, and the cells surface α5, α2, β3, α5β1, αvβ3 and α2β1 integrin was determined using flow cytometry. (B) Cells were incubated with PGE2 for 24 hr, and the mRNA levels of α2 and β1 integrin was determined using qPCR. (C) JJ012 cells were transfected with IPTG/COX-2 expression plasmid or control vector for 24 hr followed by stimulation with IPTG (5 mM) for 24 hr, the mRNA expression of α2 and β1 integrin was determined by qPCR. JJ012 cells were incubated with PGE2 for indicated time intervals, and mRNA and cell surface α2β1 integrin were examined by qPCR (D) and flow cytometry (E). (F) Cells were pretreated with α2β1 monoclonal antibody (3 μg/ml) for 30 min followed by stimulation with PGE2. The in vitro migration activity measured after 24 hr. (E) JJ012 cells were treated with 17-phenyl trinor PGE2 (3 μM), 11-deoxy-PGE1 (10 μM), PGE2, and PGE2 plus SC19220 (10 μM), and cells surface α2β1 integrin was determined using flow cytometry. Results are expressed as the mean ± S.E. *, p < 0.05 compared with control; #, p < 0.05 compared with PGE2-treated group.
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Related In: Results  -  Collection

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Figure 3: COX-2-directed migration of human chondrosarcoma cells involves up-regulation of α2β1 integrin. (A) JJ012 cells were incubated with PGE2 for 24 hr, and the cells surface α5, α2, β3, α5β1, αvβ3 and α2β1 integrin was determined using flow cytometry. (B) Cells were incubated with PGE2 for 24 hr, and the mRNA levels of α2 and β1 integrin was determined using qPCR. (C) JJ012 cells were transfected with IPTG/COX-2 expression plasmid or control vector for 24 hr followed by stimulation with IPTG (5 mM) for 24 hr, the mRNA expression of α2 and β1 integrin was determined by qPCR. JJ012 cells were incubated with PGE2 for indicated time intervals, and mRNA and cell surface α2β1 integrin were examined by qPCR (D) and flow cytometry (E). (F) Cells were pretreated with α2β1 monoclonal antibody (3 μg/ml) for 30 min followed by stimulation with PGE2. The in vitro migration activity measured after 24 hr. (E) JJ012 cells were treated with 17-phenyl trinor PGE2 (3 μM), 11-deoxy-PGE1 (10 μM), PGE2, and PGE2 plus SC19220 (10 μM), and cells surface α2β1 integrin was determined using flow cytometry. Results are expressed as the mean ± S.E. *, p < 0.05 compared with control; #, p < 0.05 compared with PGE2-treated group.
Mentions: Previous studies have demonstrated significant expression of integrins in human chondrosarcoma cells [27]. We therefore, hypothesized that integrins may be involved in PGE2-directed migration of chondrosarcoma cells. Flow cytometry analysis showed that PGE2 induced the cell surface expression of α2 and α2β1 integrin in JJ012 cells (Fig. 3A). To confirm this finding, expression of mRNAs in the integrins in response to PGE2 was analyzed by qPCR. Treatment of JJ012 cells with PGE2 induced the mRNA expression of α2 and β1 integrins (Fig. 3B). In addition, treatment of IPTG/COX-2-transfected cells with IPTG increased mRNA expression of α2 and β1 integrins (Fig. 3C). Furthermore, compared with normal cartilage, human chondrosarcoma tissues expressed higher levels of α2 and β1 integrin mRNA (Table 1). Therefore, the α2β1 integrin plays an important role in PGE2-induced migration of human chondrosarcoma cells. Stimulation of cells with PGE2 also increased mRNA expression and cell surface expression of α2 and β1 integrin time-dependently (Fig. 3D&3E). Pretreatment of cells for 30 min with anti-α2β1 monoclonal antibody (mAb) (3 μg/ml) markedly inhibited the PGE2-induced cell migration (Fig. 3F). On the other hand, EP1/3 agonist enhanced the cell surface expression of α2β1 integrin (Fig. 3G). Pretreatment of cells with SC19220 reduced PGE2-mediated α2β1 integrin expression (Fig. 3G). These data suggest that PGE2-induced cancer migration may occur via activation of the α2β1 integrin.

Bottom Line: PGE2-mediated migration and integrin up-regulation were attenuated by phospholipase C (PLC), protein kinase C (PKC) and c-Src inhibitor.Activation of the PLCbeta, PKCalpha, c-Src and NF-kappaB signaling pathway after PGE2 treatment was demonstrated, and PGE2-induced expression of integrin and migration activity were inhibited by the specific inhibitor, siRNA and mutants of PLC, PKC, c-Src and NF-kappaB cascades.Our results indicated that PGE2 enhances the migration of chondrosarcoma cells by increasing alpha2beta1 integrin expression through the EP1/PLC/PKCalpha/c-Src/NF-kappaB signal transduction pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung, Taiwan.

ABSTRACT

Background: Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin (PG) synthase, has been implicated in tumor metastasis. Interaction of COX-2 with its specific EP receptors on the surface of cancer cells has been reported to induce cancer invasion. However, the effects of COX-2 on migration activity in human chondrosarcoma cells are mostly unknown. In this study, we examined whether COX-2 and EP interaction are involved in metastasis of human chondrosarcoma.

Results: We found that over-expression of COX-2 or exogenous PGE2 increased the migration of human chondrosarcoma cells. We also found that human chondrosarcoma tissues and chondrosarcoma cell lines had significant expression of the COX-2 which was higher than that in normal cartilage. By using pharmacological inhibitors or activators or genetic inhibition by the EP receptors, we discovered that the EP1 receptor but not other PGE receptors is involved in PGE2-mediated cell migration and alpha2beta1 integrin expression. Furthermore, we found that human chondrosarcoma tissues expressed a higher level of EP1 receptor than normal cartilage. PGE2-mediated migration and integrin up-regulation were attenuated by phospholipase C (PLC), protein kinase C (PKC) and c-Src inhibitor. Activation of the PLCbeta, PKCalpha, c-Src and NF-kappaB signaling pathway after PGE2 treatment was demonstrated, and PGE2-induced expression of integrin and migration activity were inhibited by the specific inhibitor, siRNA and mutants of PLC, PKC, c-Src and NF-kappaB cascades.

Conclusions: Our results indicated that PGE2 enhances the migration of chondrosarcoma cells by increasing alpha2beta1 integrin expression through the EP1/PLC/PKCalpha/c-Src/NF-kappaB signal transduction pathway.

Show MeSH
Related in: MedlinePlus