Abstract
Prostaglandin E2 (PGE2) plays a key role in osteolytic bone metastasis as well as roles in inflammation, cell growth, and tumor development. PGE2 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 PGE2. PGE2 binding assays revealed that culture medium containing the second extracellular region of EP2 (FuEP2/Ex2) had binding activity. FuEP2/Ex2 neutralized PGE2-induced cyclic AMP production, cyclic AMP-responsive element binding protein phosphorylation, and subsequent induction of cyclooxygenase-2, interleukin (IL)-1beta, and IL-6 mRNAs. In human osteoblasts, this culture medium neutralized the induction of receptor activator of nuclear factor-kappaB 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 PGE2-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 cell-injected mice, with decreased osteoclasts and increased apoptotic cells. Furthermore, the cyclooxygenase-2, IL-1beta, 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.
Highlights
Prostaglandins are autacoid substrates produced from arachidonic acid and play important roles in physiologic homeostasis as paracrine factors
reversetranscription PCR (RT-PCR) analyses revealed that all transfectants successfully expressed mRNAs encoding the individual human EP2 (hEP2) fragments (Fig. 1B)
ELISA for hIgG showed that 293-Fumock, 293-FuEP2/N+Ex1, 293-FuEP2/ Ex2, and 293-FuEP2/Ex3+C cells secreted hEP2 fragments into the culture medium (CM), whereas no hIgG was detected in the CM from parent HEK293 cells (Fig. 1B)
Summary
Prostaglandins are autacoid substrates produced from arachidonic acid and play important roles in physiologic homeostasis as paracrine factors. Prostaglandin E2 (PGE2) is closely associated with inflammation, cell growth, tumor development, and tumor metastasis [1]. PGE2 is a key molecule for bone metabolism. PGE2 is produced in bone by osteoblasts and acts as a stimulator of bone resorption. This effect is closely associated with osteolytic cancer bone metastasis, which involves in bone destruction [2]. This knowledge is further supported by reports that administration of cyclooxygenase (COX) inhibitors suppresses the bone metastasis of breast and prostate cancer cells [3, 4]
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