Vitamin D facilitates trophoblast invasion through induction of epithelial-mesenchymal transition.

PROBLEM: Vitamin D deficiency increases the risk of developing pregnancy-related complications, including preeclampsia and small-for-gestational-age infants. Vitamin D was demonstrated to promote the invasiveness of human extravillous trophoblasts (EVTs). However, whether vitamin D induces the epithelial-mesenchymal transition (EMT) of EVTs remains unclear. Therefore, we investigated whether vitamin D promotes EMT and the related signaling pathways. METHOD OF STUDY: In this study, we performed EMT experiments using JAR cells based on the expression of the mesenchymal markers and vitamin D receptor. JAR cells were treated with calcitriol, the active form of vitamin D. Western blotting was performed to evaluate EMT markers and key molecules of signaling pathways. Invasion assays were conducted. Expression and secretion of MMPs were analyzed by real-time PCR and zymography. RESULTS: Calcitriol significantly enhanced EMT and the invasive capability of JAR cells, along with increased expression and secretion of MMP-2 and MMP-9. Moreover, ERK signaling pathway was activated by calcitriol. The effects of calcitriol were neutralized by ERK signaling blocker. CONCLUSION: Calcitriol facilitated EMT induction and expression of MMPs via ERK signaling pathway, which promoted the invasive capability of EVTs. Further studies are warranted to elucidate the potential application of vitamin D in the prevention of pregnancy complications.

Activation of NOD-1/JNK/IL-8 signal axis in decidual stromal cells facilitates trophoblast invasion.

Decidual stromal cells (DSCs) are known to regulate trophoblast invasion via unveiled mechanism yet. And nucleotide-binding oligomerization domain-containing protein 1 (NOD1) may influence on this DSC-trophoblast interaction. We investigated the mechanism underlying the DSC-mediated regulation of trophoblast invasion and the effect of NOD1 on their cross talk. Using human primary DSCs, BeWo cell invasion was measured. Cytokine secretion and MAP kinase signaling were examined in DSCs following treatment with NOD1 agonist, Tri-DAP. DSCs secreted IL-8 and increased trophoblast invasion. Tri-DAP further increased IL-8 secretion from DSCs via JNK pathway and facilitated both MMP-2 production and trophoblast invasion compared with control. Upon cotreatment of IL-8 and anti-IL-8 antibody to BeWo cells, the number of invading trophoblasts and MMP-2 production decreased significantly. These results suggest that IL-8 from DSCs may play a role to increase the invasiveness of trophoblast cells into the decidua via NOD1/JNK pathway.

Emetine inhibits migration and invasion of human non-small-cell lung cancer cells via regulation of ERK and p38 signaling pathways.

Emetine is a natural compound originated from ipecac roots. It was commonly used as anti-protozoal and vomiting agent. The apoptosis-inducing effect of emetine makes it considered as a potential anti-cancer agent for various human cancers. Here in this study, we report that emetine inhibits migration and invasion of human non-small-cell lung cancer (NSCLC) cells. Modulation of three major mitogen-activated protein kinases (MAPKs), ERK, p38 and JNK, is well known to be involved in regulation of matrix metalloproteinases (MMPs), which are essential in tissue remodeling and extracellular matrix (ECM) degradation, for cancer cells to spread out from the origin of tumorigenesis. Emetine regulates two major MAPKs, p38 and ERK. Differential inhibition/stimulation of ERK and p38 induced differential suppressions of ß-catenin and c-myc transcription factors. This leads to the selective down-regulation of MMP-2 and MMP-9, two major gelatinases which can degrade ECM components, and RECK, a negative regulator of MMP-9.

Decursin inhibits osteoclastogenesis by downregulating NFATc1 and blocking fusion of pre-osteoclasts.

Bone sustains its structure through dynamic interaction between osteoblastic cells and osteoclastic cells. But imbalance may lead to osteoporosis caused by overactivated osteoclast cells that have bone-resorbing function. Recently, herbs have been researched as major sources of medicines in many countries. In vitro and in vivo anti-osteoclastogenic activity of Angelica gigas NAKAI have been reported, but the biological activity of decursin, its major component in osteoclast differentiation is still unknown. Therefore, in this study, we explored whether decursin could affect RANKL-mediated osteoclastogenesis. The results showed that decursin efficiently inhibited RANKL-activated osteoclast differentiation by inhibiting transcriptional and translational expression of NFATc1, a major factor in RANKL-mediated osteoclastogenesis. Furthermore, decursin decreased fusion and migration of pre-osteoclasts by downregulating mRNA expression levels of DC-STAMP and ß3 integrin, respectively. In addition, decursin prevents lipopolysaccharide (LPS)-induced bone erosion in vivo. In summary, decursin could prevent osteoclastogenesis and inflammatory bone loss via blockage of NFATc1 activity and fusion and migration of pre-osteoclasts, and it could be developed as a potent phytochemical candidate for treating pathologies of bone diseases.

Inactivation of Src-to-ezrin pathway: a possible mechanism in the ouabain-mediated inhibition of A549 cell migration.

Ouabain, a cardiac glycoside found in plants, is primarily used in the treatment of congestive heart failure and arrhythmia because of its ability to inhibit Na(+)/K(+)-ATPase pump. Recently ouabain has been shown to exert anticancer effects but the underlying mechanism is not clear. Here, we explored the molecular mechanism by which ouabain exerts anticancer effects in human lung adenocarcinoma. Employing proteomic techniques, we found 7 proteins downregulated by ouabain in A549 including p-ezrin, a protein associated with pulmonary cancer metastasis in a dose-dependent manner. In addition, when the relative phosphorylation levels of 39 intracellular proteins were compared between control and ouabain-treated A549 cells, p-Src (Y416) was also found to be downregulated by ouabain. Furthermore, western blot revealed the ouabain-mediated downregulation of p-FAK (Y925), p-paxillin (Y118), p130CAS, and Na(+)/K(+)-ATPase subunits that have been shown to be involved in the migration of cancer cells. The inhibitory effect of ouabain and Src inhibitor PP2 on the migration of A549 cells was confirmed by Boyden chamber assay. Anticancer effects of ouabain in A549 cells appear to be related to its ability to regulate and inactivate Src-to-ezrin signaling, and proteins involved in focal adhesion such as Src, FAK, and p130CAS axis are proposed here.

Praeruptorin A inhibits in vitro migration of preosteoclasts and in vivo bone erosion, possibly due to its potential to target calmodulin.

Excessive activity and/or increased number of osteoclasts lead to bone resorption-related disorders. Here, we investigated the potential of praeruptorin A to inhibit migration/fusion of preosteoclasts in vitro and bone erosion in vivo. Praeruptorin A inhibited the RANKL-induced migration/fusion of preosteoclasts accompanied by the nuclear translocation of NFATc1, a master regulator of osteoclast differentiation. Antimigration/fusion activity of praeruptorin A was also confirmed by evaluating the mRNA expression of fusion-mediating molecules. In silico binding studies and several biochemical assays further revealed the potential of praeruptorin A to bind with Ca(2+)/calmodulin and inhibit its downstream signaling pathways, including the Ca(2+)/calmodulin-CaMKIV-CREB and Ca(2+)/calmodulin-calcineurin signaling axis responsible for controlling NFATc1. In vivo application of praeruptorin A significantly reduced lipopolysaccharide-induced bone erosion, indicating its possible use to treat bone resorption-related disorders. In conclusion, praeruptorin A has the potential to inhibit migration/fusion of preosteoclasts in vitro and bone erosion in vivo by targeting calmodulin and inhibiting the Ca(2+)/calmodulin-CaMKIV-CREB-NFATc1 and/or Ca(2+)/calmodulin-calcineurin-NFATc1 signaling axis.

Repositioning Potential of PAK4 to Osteoclastic Bone Resorption.

Drug repositioning is a rational approach for expanding the use of existing drugs or candidate drugs to treat additional disorders. Here we investigated the possibility of using the anticancer p21-activated kinase 4 (PAK4)-targeted inhibitor PF-3758309 to treat osteoclast-mediated disorders. PAK4 was highly expressed in bone marrow cells and was phosphorylated during their differentiation into osteoclasts, and osteoclast differentiation was significantly inhibited by the dominant negative form of PAK4 and by PF-3758309. Specifically, PF-3758309 significantly inhibited the fusion of preosteoclasts, the podosome formation, and the migration of preosteoclasts. PF-3758309 also had in vivo antiresorptive activity in a lipopolysaccharide-induced bone erosion model and in vitro antiosteoclastogenic activity in the differentiation of human bone marrow-derived cells and peripheral blood mononuclear cells into osteoclasts. These data demonstrate the relevance of PAK4 in osteoclast differentiation and the potential of PAK4 inhibitors for treating osteoclast-related disorders.

Natural polyamines inhibit the migration of preosteoclasts by attenuating Ca2+-PYK2-Src-NFATc1 signaling pathways.

Natural polyamines have numerous biological activities. Several studies have reported their beneficial role in bone metabolism, but their mode of action is not fully understood. Bone diseases such as osteoporosis, which is characterized by impaired bone structure and low bone mass, are caused by an increased number of osteoclasts and/or overactivation of osteoclastogenesis. Osteoclast differentiation is a multi-complex procedure involving the following sequential steps: differentiation-migration-fusion-resorption. In this study, we found that putrescine, spermidine or spermine inhibited the RANKL-mediated migration of preosteoclasts. Furthermore, the RANKL-mediated activation of the Src-PYK2 signaling axis and of transcription factors such as NF-κB and NFATc1 was prevented by each polyamine. Anti-osteoclastogenic and anti-migration activities of polyamines were confirmed by evaluating their potential to downregulate the mRNA expression levels of osteoclastogenesis-related genes such as OSCAR, TRAP, cathepsin K and c-Src, and genes related to fusion and/or migration of preosteoclasts. Moreover, ATP-mediated elevation of cytosolic free Ca(2+) concentration ([Ca(2+)]i) was strongly inhibited by each polyamine, indicating the involvement of [Ca(2+)]i in the anti-fusion activities of polyamines. In conclusion, polyamines could exhibit anti-osteoclastogenic activity by inhibiting the migration of preosteoclasts via the Ca(2+)-PYK2-Src-NFATc1 signaling axis.

Discovery and the structural basis of a novel p21-activated kinase 4 inhibitor.

Functional versatility and elevated expression in cancers have endowed p21-activated kinase 4 (PAK4) as one of the first-in-class anti-cancer drug target. In this study, a novel PAK4 inhibitor, KY-04031 (N(2)-(2-(1H-indol-3-yl)ethyl)-N(4)-(1H-indazol-5-yl)-6-methoxy-1,3,5-triazine-2,4-diamine), was discovered using a high-throughput screening. Analysis of the complex crystal structure illustrated that both indole and indazole of KY-04031 are responsible for PAK4 hinge interaction. Moreover, the molecule's triazine core was found to mimic the ribose of the natural ATP substrate. The cell-based anti-cancer potency of KY-04031 was less effective than the pyrroloaminopyrazoles; however, the unique molecular feature of KY-04031 can be exploited in designing new PAK4 inhibitors.

Anti-osteoclastogenic activity of praeruptorin A via inhibition of p38/Akt-c-Fos-NFATc1 signaling and PLCγ-independent Ca2+ oscillation.

BACKGROUND: A decrease of bone mass is a major risk factor for fracture. Several natural products have traditionally been used as herbal medicines to prevent and/or treat bone disorders including osteoporosis. Praeruptorin A is isolated from the dry root extract of Peucedanum praeruptorum Dunn and has several biological activities, but its anti-osteoporotic activity has not been studied yet. MATERIALS AND METHODS: The effect of praeruptorin A on the differentiation of bone marrow-derived macrophages into osteoclasts was examined by phenotype assay and confirmed by real-time PCR and immunoblotting. The involvement of NFATc1 in the anti-osteoclastogenic action of praeruptorin A was evaluated by its lentiviral ectopic expression. Intracellular Ca(2+) levels were also measured. RESULTS: Praeruptorin A inhibited the RANKL-stimulated osteoclast differentiation accompanied by inhibition of p38 and Akt signaling, which could be the reason for praeruptorin A-downregulated expression levels of c-Fos and NFATc1, transcription factors that regulate osteoclast-specific genes, as well as osteoclast fusion-related molecules. The anti-osteoclastogenic effect of praeruptorin A was rescued by overexpression of NFATc1. Praeruptorin A strongly prevented the RANKL-induced Ca(2+) oscillation without any changes in the phosphorylation of PLCγ. CONCLUSION: Praeruptorin A could exhibit its anti-osteoclastogenic activity by inhibiting p38/Akt-c-Fos-NFATc1 signaling and PLCγ-independent Ca(2+) oscillation.

Anti-osteoclastogenic activity of matairesinol via suppression of p38/ERK-NFATc1 signaling axis.

BACKGROUND: Matairesinol is a plant lignan present in a wide variety of foodstuffs such as seeds, vegetables and fruits. It has various biological functions including anti-angiogenic, anti-cancer and anti-fungal activities, but its anti-osteoporotic activity, if any, is unknown. METHODS: For osteoclast differentiation, primary mouse bone marrow-derived macrophage cells (BMMs) were cultured for 4 days in the presence of RANKL and M-CSF with the vehicle (DMSO) or matairesinol. Cell cytotoxicity was examined by CCK-8 assay. Gene expression of NFATc1, TRAP, OSCAR, v-ATPasev0d2 were observed in the presence or absence of matairesinol (10 µM) for the indicated times. For evaluating the involvement of NFATc1 in the anti-osteoclastogenic action of matairesinol, BMMs were infected with pMX-IRES-GFP or pMX-IRES-CA-NFATc1-GFP for 8 h with polybrene, and then infected BMMs were cultured with M-CSF and RANKL for 4 days in the presence or absence of matairesinol (10 µM). MAPK signaling activation was examined by immunoblotting. For measuring the resorptive activity of mature osteoclasts, osteoclasts and osteoblasts were co-cultured on BioCoat Osteologic MultiTest slides, and treated with matairesinol for 24 h. RESULT: Here we show that matairesinol dose-dependently inhibited the RANKL-induced differentiation of BMMs into osteoclasts by downregulating RANKL-induced expression and activity of NFATc1. Ectopic overexpression of NFATc1 blunted the anti-osteoclastogenic effect of matairesinol implicating NFATc1 in the action of matairesinol. Additionally, matairesinol blocked the RANKL-induced activation of p38 and ERK in BMMs, but had no effect on bone resorption activity in mature osteoclasts. CONCLUSION: Taken together, our results suggest that the anti-osteoporotic activity of matairesinol could arise from its anti-osteoclastogenic potential via p38/ERK-NFATc1 signaling, but not by way of anti-resorptive action.

PF-3758309, p21-activated kinase 4 inhibitor, suppresses migration and invasion of A549 human lung cancer cells via regulation of CREB, NF-κB, and ß-catenin signalings.

Migration and invasion comprise key steps in cancer metastasis. Through the migration and invasion into and out of lymphatic and/or blood vessels, cancer cells can be spread out into the tissues in remote site from the origin. Degradation of extracellular matrix (ECM) must be preceded prior to the metastasis of cancer cells. Matrix metalloproteinases (MMP) can degrade ECM, thus allow cells to migrate from the original site. Among MMPs, two gelatinase MMP-2 and MMP-9 play particularly important roles in ECM degradation. Here, we report that recently developed p21-activated kinase 4 inhibitor PF-3758309 shows anti-metastatic effect in A549 human lung cancer cell. PF-3758309 suppresses CREB, NF-κB, and ß-catenin pathways, which are well known to be closely related with cell migration. This leads to the downregulation of MMP-2/MMP-9 expressions and the inhibition of A549 lung cancer metastasis.

CK2 inhibitor CX4945 induces sequential inactivation of proteins in the signaling pathways related with cell migration and suppresses metastasis of A549 human lung cancer cells.

Casein kinase 2 (CK2) is known to be involved in various cellular processes such as cell cycle, apoptosis and proliferation. It has been reported that the inhibition of CK2 induced by recently developed small molecule CX4945 shows anti-cancer effects including anti-proliferation and anti-angiogenesis in several different cancers including prostate cancer. Here we report that migration and invasion of A549 human lung cancer cells are suppressed by the inhibition of CK2 induced by CX4945. We found that CX4945 sequentially attenuates the proteins in PI3K/Akt and MAPK pathways, two signaling pathways related with cell migration. This sequential control of signal pathways inhibits the expression of membrane type 1-matrix metalloproteinase and this leads to the selective attenuation of one of the gelatinases, MMP-2, which can degrade components of extracellular matrix, and metastasis of A549 human lung cancer cell.

Discovery of non-LBD inhibitor for androgen receptor by structure-guide design.

In this study, we synthesized the BF-3 binding small molecules, a series of pyridazinone-based compounds, as a novel class of non-LBP antiandrogens for treating prostate cancer by inhibiting androgen receptor. The new class compound was discovered to inhibitor the viability of AR-dependent human prostate LNCap cells and AR activity combining with the computational method. It showed a good physicochemical and PK property.

Anti-androgen receptor activity of apoptotic CK2 inhibitor CX4945 in human prostate cancer LNCap cells.

Androgen receptor (AR) is crucial for transcriptional signaling in prostate cancers. The anti-cancer activity of protein kinase CK2 (formerly called casein kinase 2)-specific small molecule inhibitors have been reported in several cancers including prostate cancers. The orally available CX4945, a potent and selective small molecule inhibitor of CK2, has advanced into human clinical trials and has exhibited strong anti-tumor activity. The inhibition of CK2 leads to a down-regulation of the AR-dependent transcription, but the functional relevance of CX4945 to AR-dependent transcription in AR-positive LNCap cells has not been studied yet. Our observation of inhibitory effects of CX4945 on the expression or phosphorylation levels of CK2α, Akt and anti-apoptotic molecules including IAP family members agreed with a previous study showing the effect of CK2 inhibition in cancer cells. This study also provides novel information on the impact of CX4945 in the inhibition of AR-dependent transcriptional activation in LNCap cells via its down-regulation. Pharmacologic inhibition experiment revealed that CX4945 could exhibit its anti-cancer activity in LNCap cells via the independent inhibitions of AR and Akt-survivin signalings.

Thiourea compound AW00178 sensitizes human H1299 lung carcinoma cells to TRAIL-mediated apoptosis.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in a wide variety of cancer cells. Recently, cancer cell resistance to TRAIL-mediated apoptosis has become a challenging issue in the development of TRAIL-based anti-cancer therapies. In this study, we found that 1-(5-chloro-2-methyl-phenyl)-3-[4-(5-trifluoromethyl-pyrazol-1-yl)-phenyl]-thiourea (AW00178) was able to sensitize TRAIL-resistant human lung cancer H1299 cells to TRAIL-mediated apoptosis. Treatment with AW00178, either alone or in combination with TRAIL, induced the expression of CHOP, a protein related to TRAIL sensitivity, and reduced the expression of survivin, an anti-apoptotic protein involved in TRAIL resistance. Additionally, AW00178, alone or in combination with TRAIL, induced the activation of c-Jun and inactivation of Akt. A pharmacologic inhibition study revealed that c-Jun activation and Akt inactivation were strongly related to CHOP induction and survivin down-regulation, respectively. In summary, these results suggested that AW00178 mediated sensitization to TRAIL-mediated apoptosis in H1299 cells by increasing sensitivity and decreasing resistance to TRAIL via the induction of c-Jun-dependent CHOP expression and the reduction of Akt-dependent survivin expression, respectively.

AW00179 potentiates TRAIL-mediated death of human lung cancer H1299 cells through ROS-JNK-c-Jun-mediated up-regulation of DR5 and down-regulation of anti-apoptotic molecules.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in tumor cells, but when used alone, it is not effective at treating TRAIL-resistant tumors. This resistance is challenging for TRAIL-based anti-cancer therapies. In this study, we found that 1-(4-trifluoromethoxy-phenyl)-3-[4-(5-trifluoromethyl-2,5-dihydro-pyrazol-1-yl)-phenyl]-urea (AW00179) sensitized human lung cancer H1299 cells to TRAIL-mediated apoptosis. Even in the absence of TRAIL, AW00179 strongly induced DR5 expression and decreased the expression of anti-apoptotic proteins, suggesting that the sensitizing effect of AW00179 on TRAIL-mediated apoptosis is due to increased levels of DR5 protein and decreased anti-apoptotic molecules. AW00179 also induced the activation of c-Jun and ERK; however, a pharmacologic inhibition study revealed that JNK-c-Jun signaling is involved in the induction of DR5 expression. In addition, reactive oxygen species (ROS) appear to be involved in AW00179 activity. In conclusion, AW00179 has the potential to sensitize H1299 cells to TRAIL-mediated apoptosis through two distinct mechanisms: ROS-JNK-c-Jun-mediated up-regulation of DR5, and down-regulation of anti-apoptotic molecules.

Regulation of the female rat estrous cycle by a neural cell-specific epidermal growth factor-like repeat domain containing protein, NELL2.

NELL2, a protein containing epidermal growth factor-like repeat domains, is predominantly expressed in the nervous system. In the mammalian brain, NELL2 expression is mostly neuronal. Previously we found that NELL2 is involved in the onset of female puberty by regulating the release of gonadotropin-releasing hormone (GnRH), and in normal male sexual behavior by controlling the development of the sexually dimorphic nucleus of the preoptic area (POA). In this study we investigated the effect of NELL2 on the female rat estrous cycle. NELL2 expression in the POA was highest during the proestrous phase. NELL2 mRNA levels in the POA were increased by estrogen treatment in ovariectomized female rats. Blocking NELL2 synthesis in the female rat hypothalamus decreased the expression of kisspeptin 1, an important regulator of the GnRH neuronal apparatus, and resulted in disruption of the estrous cycle at the diestrous phase. These results indicate that NELL2 is involved in the maintenance of the normal female reproductive cycle in mammals.

NELL2 participates in formation of the sexually dimorphic nucleus of the pre-optic area in rats.

Formation of the sexually dimorphic nucleus of the pre-optic area (SDN-POA) in the rat hypothalamus shows a sexually differential development of neurons. Volume of the SDN-POA in males is much bigger than that in females which is because of a neuroprotective effect of estradiol converted from circulating testosterone during a critical period of brain development. We found that neural epidermal growth factor-like like-2 (NELL2), a neural tissue-enriched protein, is a potential downstream target of estrogen. In this study, we examined a possible role of NELL2 in the development of the SDN-POA and in the normalcy of sexual behavior in the male rats. NELL2 was expressed and co-localized with estrogen receptor alpha in the SDN-POA. A blockade of NELL2 synthesis in the brain during postnatal day 0 (d0) to d4 by an intracerebroventricular injection of an antisense NELL2 oligodeoxynucleotide, resulted in a decrease in volume of the SDN-POA in males. Interestingly, it reduced some components of the male sexual behavior such as mounting and intromission, but not the sexual partner preference in adulthood. In vitro study using the hippocampal neuroprecursor HiB5 cells showed that NELL2 has a protective effect from a cell death condition. These data suggest that a relevant expression of NELL2 in the neonatal brain is important for the estrogen-induced normal development of the SDN-POA and the normalcy of sexual behavior in male rats.