Morichika Takita

Prostaglandin E receptor EP4 antagonist suppresses osteolysis due to bone metastasis of mouse malignant melanoma cells

We examined the effects of prostaglandin E (PGE) receptor subtype EP4 antagonist on bone metastasis of cancer to clarify PGEs role in bone metastasis. Metastatic regions were detected in femurs accompanying severe bone loss in mice injected with B16 malignant melanoma cells. Administration of EP4 antagonist restored the bone loss induced by B16 melanoma. Adding B16 cells induced osteoclast formation in the coculture of bone marrow cells and osteoblasts without any exogenous bone‐resorbing factor, and EP4 antagonist completely suppressed the osteoclast formation induced by B16 cells. Therefore, EP4 antagonist is a possible candidate for the therapy of bone metastasis of cancer.

A novel carborane analog, BE360, with a carbon-containing polyhedral boron-cluster is a new selective estrogen receptor modulator for bone

Carboranes are a class of carbon-containing polyhedral boron-cluster compounds with globular geometry and hydrophobic surface that interact with hormone receptors. Estrogen deficiency results in marked bone loss due to increased osteoclastic bone resorption in females, but estrogen replacement therapy is not generally used for postmenopausal osteoporosis due to the risk of uterine cancer. We synthesized a novel carborane compound BE360 to clarify its anti-osteoporosis activity. BE360 showed a high binding affinity to estrogen receptors (ER), ERalpha and ERbeta. In ovariectomized (OVX) mice, femoral bone volume was markedly reduced and BE360 dose-dependently restored bone loss in OVX mice. However, BE360 did not exhibit any estrogenic activity in the uterus. BE360 also restored bone loss in orchidectomized mice without androgenic action in the sex organs. Therefore, BE360 is a novel selective estrogen receptor modulator (SERM) that may offer a new therapy option for osteoporosis.

Hyaluronan inhibits bone resorption by suppressing prostaglandin E synthesis in osteoblasts treated with interleukin-1.

Hyaluronan (HA), a large glycosaminoglycan, is a component of the extra-cellular matrix in various tissues. HA is essential for matrix assembly and fluid viscosity in cartilage, but the roles of HA in bone are unclear. Bone resorption associated with inflammation is closely related to prostaglandin E (PGE) synthesis by osteoblasts induced by cytokines such as interleukin-1 (IL-1). In mouse calvarial cultures, HA inhibited osteoclastic bone resorption and PGE production induced by IL-1. In mouse osteoblasts, HA suppressed IL-1-induced expression of cyclooxygenase(COX)-2 and membrane-bound PGE synthase (mPGES)-1 mRNAs, and PGE2 production. Matrix metalloproteinases (MMPs), including MMP-2 and MMP-13, were produced by osteoblasts in response to IL-1, and were clearly suppressed by HA. In osteoblasts, HA suppressed the NFkappaB-dependent transcription in a luciferase assay. Therefore, HA acts on osteoblasts to suppress the production of PGE2 and MMPs, and inhibits bone resorption, suggesting critical roles of HA in pathological bone loss with inflammation.

Direct Melanoma Cell Contact Induces Stromal Cell Autocrine Prostaglandin E2-EP4 Receptor Signaling That Drives Tumor Growth, Angiogenesis, and Metastasis

Background: Prostaglandin E2 (PGE2) is an inflammatory mediator produced in cancer. Results: B16 melanoma cells injected into mice metastasized to bone and soft tissues by activating PGE2-EP4 signaling in stromal cells, inducing osteoclast activation, angiogenesis, and cancer cell proliferation. Conclusion: Stromal cell PGE2 is a key mediator of melanoma tumorigenesis and metastasis. Significance: EP4 receptor blockade is a new potential therapy for tumor metastasis. The stromal cells associated with tumors such as melanoma are significant determinants of tumor growth and metastasis. Using membrane-bound prostaglandin E synthase 1 (mPges1−/−) mice, we show that prostaglandin E2 (PGE2) production by host tissues is critical for B16 melanoma growth, angiogenesis, and metastasis to both bone and soft tissues. Concomitant studies in vitro showed that PGE2 production by fibroblasts is regulated by direct interaction with B16 cells. Autocrine activity of PGE2 further regulates the production of angiogenic factors by fibroblasts, which are key to the vascularization of both primary and metastatic tumor growth. Similarly, cell-cell interactions between B16 cells and host osteoblasts modulate mPGES-1 activity and PGE2 production by the osteoblasts. PGE2, in turn, acts to stimulate receptor activator of NF-κB ligand expression, leading to osteoclast differentiation and bone erosion. Using eicosanoid receptor antagonists, we show that PGE2 acts on osteoblasts and fibroblasts in the tumor microenvironment through the EP4 receptor. Metastatic tumor growth and vascularization in soft tissues was abrogated by an EP4 receptor antagonist. EP4-null Ptger4−/− mice do not support B16 melanoma growth. In vitro, an EP4 receptor antagonist modulated PGE2 effects on fibroblast production of angiogenic factors. Our data show that B16 melanoma cells directly influence host stromal cells to generate PGE2 signals governing neoangiogenesis and metastatic growth in bone via osteoclast erosive activity as well as angiogenesis in soft tissue tumors.

Novel vitamin D3 analogs, 1α, 25(OH)2D3-26, 23-lactam (DLAMs), antagonize bone resorption via suppressing RANKL expression in osteoblasts

Novel vitamin D analogs, 1alpha, 25-dihydroxyvitamin D(3)-26, 23-lactam (DLAMs) with a lactam moiety in the side chain, were synthesized and examined for their function in bone. In computer docking simulation, DLAM-1P binds to vitamin D receptor (VDR), and its lactam moiety may interfere with VDR helix-12 folding. In co-cultures of mouse bone marrow cells and osteoblasts, (23S,25S)-DLAM-1P dose-dependently suppressed osteoclast differentiation induced by 1alpha, 25-dihydroxyvitamin D(3) [1alpha, 25(OH)(2)D(3)]. Its stereoisomer (23R,25R)-DLAM-1P did not affect the osteoclast differentiation. In osteoblasts, (23S,25S)-DLAM-1P suppressed 1alpha, 25(OH)(2)D(3)-induced mRNA expression of the receptor activator of NF-kappaB ligand (RANKL). In an organ culture using mouse calvaria, bone-resorbing activity induced by 1alpha, 25(OH)(2)D(3) was clearly suppressed by (23S,25S)-DLAM-1P. The other analog, (23S,25S)-DLAM-2P, showed a similar activity to (23S,25S)-DLAM-1P. Therefore, DLAMs act on osteoblasts as an antagonist of 1alpha, 25(OH)(2)D(3) to suppress RANKL-dependent osteoclast formation, suggesting them as a novel candidate for the treatment of pathological bone loss.

Paradoxical counteraction by imatinib against cell death in myeloid progenitor 32D cells expressing p210BCR-ABL

Chronic myeloid leukemia (CML) is believed to be caused by the tyrosine kinase p210BCR-ABL, which exhibits growth-promoting and anti-apoptotic activities. However, mechanisms that allow cell differentiation in CML still remain elusive. Here we established tetracycline (Tet)-regulatable p210BCR-ABL-expressing murine 32D myeloid progenitor (32D/TetOff-p210) cells to explore p210BCR-ABL-induced cell death and differentiation. Tet-regulatable overexpression of p210BCR-ABL induced cell death due to the activation of both caspase-1 and caspase-3, coincident with the differentiation from myeloid progenitors into CD11b+Ly6C+Ly6G+ cells with segmented nuclei, exemplified as granulocytic myeloid-derived suppressor cells (G-MDSC), and the ability to secrete IL-1β, TNF-α, and S100A8/A9 into the culture supernatant. Treatment with imatinib almost completely abrogated all these phenotypes. Moreover, overexpression of a sensor of activated caspase-1 based on fluorescence resonance energy transfer (FRET) probe enabled us to detect activation of caspase-1 in a human CML cell line, K562. Furthermore, increased numbers of splenic G-MDSC associated with enhancement of S100A8/A9 production were observed in transgenic mice expressing p210BCR-ABL compared with that in wild-type mice. We also propose the novel mode of cell death in this 32D/TetOff-p210 system termed as myeloptosis.

C1D is not directly involved in the repair of UV-damaged DNA but protects cells from oxidative stress by regulating gene expressions in human cell lines

&NA; A small nuclear protein, C1D, has roles in various cellular processes, transcription regulation, genome stability surveillance, DNA repair and RNA processing, all of which are required to maintain the host life cycles. In the previous report, C1D directly interacts with XPB, a component of the nucleotide excision repair complex, and C1D knockdown reduced cell survival of 27–1 cells, CHO derivative cells, after UV irradiation. To find out the role of C1D in UV‐damaged cells, we used human cell lines with siRNA or shRNA to knockdown C1D. C1D knockdown reduced cell survival rates of LU99 and 786‐O after UV irradiation, although C1D knockdown did not affect the efficiency of the nucleotide excision repair. Immunostaining data support that C1D is not directly involved in the DNA repair process in UV‐damaged cells. However, H2O2 treatment reduced cell viability in LU99 and 786‐O cells. We also found that C1D knockdown upregulated DDIT3 expression in LU99 cells and downregulated APEX1 in 786‐O cells, suggesting that C1D functions as a co‐repressor/activator. The data accounts for the reduction of cell survival rates upon UV irradiation.