Soluble EP2 neutralizes prostaglandin E2–induced cell signaling and inhibits osteolytic tumor growth

Abstract

Prostaglandin E 2 (PGE 2 ) plays a key role in osteolytic bone metastasis as well as roles in inflammation, cell growth, and tumor development. PGE 2 exerts its effects by binding and activating E-prostanoid receptor (EP). In this study, we propose a new approach for blocking EP-mediated cell signaling using a soluble chimeric EP2 fragment. Mammalian expression vectors encoding several human EP2 cDNAs were introduced into 293 cells and the culture medium was tested for their function as a decoy receptor for PGE 2 . PGE 2 binding assays revealed that culture medium containing the second extracellular region of EP2 (FuEP2/Ex2) had binding activity. FuEP2/Ex2 neutralized PGE 2 -induced cyclic AMP production, cyclic AMP -responsive element binding protein phosphorylation, and subsequent induction of cyclooxygenase-2, interleukin (IL)-1B, and IL-6 mRNAs. In human osteoblasts, this culture medium neutralized the induction of receptor activator of nuclear factor-KB ligand mRNA. A stable transfectant expressing FuEP2/Ex2 was established from human prostate cancer PC-3 cells (PC3-FuEP2/Ex2). PC3-FuEP2/Ex2 cells grew at similar rates to vector control cells under normal culture conditions, although PGE 2 -induced growth stimulation was suppressed. Intraosseous injection of PC3-FuEP2/Ex2 cells into the tibia of athymic nude mice revealed that the degrees of tumor growth and osteolysis were decreased compared with control cellinjected mice, with decreased osteoclasts and increased apoptotic cells. Furthermore, the cyclooxygenase-2, IL-1B, and IL-6 mRNA levels were reduced in the tumor lesions. These data suggest that FuEP2/Ex2 is useful for treating osteolytic bone metastasis and cancers that depend on EP signaling for their growth and development.

Inhibitory effect of soluble EP2 receptor on ovarian tumor growth in nude mice and utility of TMPRSS4 as a combinatorial molecular target

“…We used the mammalian expression vector encoding FuEP2/Ex2 cDNA, known as pFUSE-FuEP2/Ex2 (9). The mock control vector (pFUSE-hFc2, Invivogen, San Diego, CA) or pFUSE-FuEP2/Ex2 (10 µg per 4x10 5 cells) was transfected using a TransFast TM transfection reagent (Promega, Madison, WI) according to the manufacturer's protocol.…”

“…Aliquots of these total RNAs (1 µg/sample) were subjected to RT-PCR under conditions and procedures reported previously (9). The amplicons were as follows: FuEP2/Ex2-hIgG (covers from 5' end of FuEP2/Ex2 cDNA to 3' end of hIgG Fc sequence in expression vector), IL2ss-hIgG (covers from 5' end of IL2 signal sequence in expression vector to 3' end of hIgG Fc sequence in expression vector), hEP1, hEP2, hEP3, hEP4, hCOX-1, hCOX-2, hmPGES, hcPGES and β-actin.…”

“…We reported previously that the soluble fragment of human EP2, which contains second extracellular loops (FuEP2/Ex2), can bind to PGE 2 , inhibits PGE 2 -induced cellular signaling, and suppresses osteolytic prostate tumor growth and endometrial tumor growth (9,10). Here, we examine the effect of FuEP2/Ex2 in a peritoneal metastasis model of ovarian cancer to determine which genes exhibit altered expression levels in the tumor lesions.…”

Inhibitory effect of soluble platelet‐derived growth factor receptor β on intraosseous growth of breast cancer cells in nude mice

“…Cells were stably transfected using the TransFast transfection reagent (Promega, Madison, WI, USA) as previously described. (39) The transfectants were designated TNBCT ⁄ Bo-mock and TNBCT ⁄ Bo-sPDGFRb. After selection by Zeocin (InvivoGen), the transfectants and their culture media (CM) were examined for expression of sPDGFRbIg1-3 mRNA and secreted protein.…”

“…Aliquots of these RNAs (1 lg ⁄ sample) were subjected to RT-PCR as described previously. (39) b-actin was amplified as an internal standard. The primers used are listed in Table 1.…”