MicroRNA-200c-3p Negatively Regulates ATP2A2 and Promotes the Progression of Papillary Thyroid Carcinoma.

microRNA-200c-3p (miR-200c-3p) has emerged as an important tumor growth regulator. However, its function in papillary thyroid carcinoma (PTC) is poorly understood. This study was conducted to investigate the role of miR-200c-3p in the progression of human PTC. The miR-200c-3p expression in human PTC tissues and cell lines was evaluated. The target relationship between miR-200c-3p and candidate genes was predicted through bioinformatic analysis and confirmed with a luciferase reporter assay. miRNA or gene expression was altered using transfection, and cell behavior was analyzed using CCK-8, wound healing, Transwell, and colony formation assays. The tumor-promoting effects of miR-200c-3p were evaluated by xenografting tumors with K1 cells in nude mice. The expression level of miR-200c-3p in human PTC tissues and cell lines markedly increased, and this increased expression was significantly associated with a worse overall survival. When inactivated, miR-200c-3p suppressed K1 cells' malignant behaviors, including decreasing proliferation and attenuating colony formation, migration, and invasion. Its inactivation also attenuated the development of xenografted K1 cells in nude mice. The effects of miR-200c-3p mimics on promoting the malignant behaviors of PTC cells were remarkably reversed by the overexpression of ATP2A2, as a downstream target of miR-200c-3p. miR-200c-3p acts as an oncogenic gene and promotes the malignant biological behaviors of human PTC cells, thereby directly targeting ATP2A2. This regulated axis may be used as a potential therapy of PTC.

Kang-Ai Injection Inhibits Gastric Cancer Cells Proliferation through IL-6/STAT3 Pathway.

OBJECTIVE: To explore the mechanisms underlying the proliferative inhibition of Chinese herbal medicine Kang-Ai injection (KAI) in gastric cancer cells. METHODS: Gastric cancer cell lines MGC803 and BGC823 were treated by 0, 0.3%, 1%, 3% and 10% KAI for 24, 48 and 72 h, respectively. The cell proliferation was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The apoptosis and cell cycle were evaluated by flow cytometry. Interleukin (IL)-6 mRNA and protein expression levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immune sorbent assay (ELISA), respectively. The protein expression levels of cyclin A, cyclin E, cyclin B1, cyclin D1, p21, retinoblastoma (RB), protein kinase B (AKT), extracellular regulated protein kinases (ERK), signal transducer and activator of transcription (STAT) 1 and STAT3 were detected by Western blot. RESULTS: KAI inhibited the proliferation of MGC803 and BGC823 gastric cancer cells in dose- and time-dependent manner. After treated with KAI for 48 h, the proportion of G1 phase was increased, expression level of cyclin D1 and phosphorylation-RB were down-regulated, whereas the expression of p21 was up-regulated (all P<0.01). Furthermore, 48-h treatment with KAI decreased the phosphorylation level of STAT3, inhibited the mRNA and protein expressions of IL-6 (all P<0.01). IL-6 at dose of 10 ng/mL significantly attenuated the proliferative effect of both 3% and 10% KAI, and recovered KAI-inhibited STAT3 phosphorylation and cyclin D1 expression level (all P<0.01). CONCLUSION: KAI exerted an anti-proliferative function by inhibiting IL-6/STAT3 signaling pathway followed by the induction of G1 phase arrest in gastric cancer cells.

Bufalin induces protective autophagy by Cbl-b regulating mTOR and ERK signaling pathways in gastric cancer cells.

Bufalin, a natural small-molecule compound derived from the traditional Chinese medicine Chan su, has shown promising anti-cancer effects against a broad variety of cancer cells through different mechanisms. It has been reported to induce autophagy in gastric cancer cells. However, the molecular mechanism involved is not fully elucidated. In the present study, we aimed to investigate the molecular mechanism by which bufalin induce autophagy in human gastric cancer cells. We found that bufalin induced apoptosis and autophagy in gastric cancer cells, and autophagy prevented human gastric cancer cells from undergoing apoptosis. Bufalin treatment changed the expression of autophagy-related proteins. Moreover, phosphorylated Akt, mTOR, and p70S6K were all significantly decreased, while phosphorylated ERK1/2 was increased by bufalin. Pretreatment of MGC803 cells with the ERK1/2-specific inhibitor PD98059 led to the down-regulation of LC3 II. Further study showed that Cbl-b positively regulated autophagy by suppressing mTOR and enhancing ERK1/2 activation. Therefore, our data provide evidence that bufalin induces autophagy in MGC803 cells via both Akt/mTOR/p70S6K and ERK signaling pathways, and Cbl-b-mediated suppression of mTOR and activation of ERK1/2 might play an important role.

[Expression of nNOS and ultrastructural changes in the penile tissue of rats with prolactinoma-induced erectile dysfunction].

OBJECTIVE: To study the expression of nNOS and ultrastructural changes in the penile tissue of rats with prolactinoma-induced erectile dysfunction (ED). METHODS: We established the model of prolactinoma in 20 male Westar rats by peritoneal injection of diethylstilbestrol (DES) and treated the control rats with normal saline (n = 10) or sterilized arachis oil (n = 10). After 8 weeks, we performed the apomorphine test and measured the weight of the pituitary gland and the levels of serum prolactin (PRL) and testosterone (T) to confirm the successful construction of the prolactinoma-induced ED model. Then we determined the expression of nNOS in the penile tissue by immunohistochemistry and examined the ultrastructural changes of the penile cavernosum under the transmission electron microscope. RESULTS: The prolactinoma-induced ED model was successfully established in 15 rats. The weight of the pituitary gland was significantly increased in the rats treated with DES as compared with the normal saline and sterilized arachis oil controls ([46.7 ± 15.5] vs [11.7 ± 2.4] and [12.4 ± 2.3] mg, both P < 0.05). The level of serum PRL was markedly higher while that of T remarkably lower in the former than in the latter two groups ([1,744.9 ± 304.5] vs [11.5 ± 2.4] and [10.6 ± 1.9] ng/ml, both P < 0.0l; [1.54 ± 0.46] vs [3.11 ± 1.08] and [3.04 ± 1.11] ng/ml, both P < 0.05). The rate of penile erection was significantly reduced in the prolactinoma-induced ED model rats in comparison with the normal saline and arachis oil controls (16.7% vs 100% and 87.5%, both P < 0.05), and so was the expression of nNOS in the penile tissue (0.024 ± 0.011 vs 0.066 ± 0.019 and 0.058 ± 0.021, both P < 0.05). Transmission electron microscopy manifested significant ultrastructural changes in the endothelial and smooth muscle cells of the cavernous tissue in the prolactinoma-induced ED models. CONCLUSION: The ultrastructural changes of the penile cavernous tissue and the reduced expression of nNOS in penile tissue may be the most important mechanisms of prolactinoma-induced ED in rats.

Prostaglandin E2 Inhibits NLRP3 Inflammasome Activation through EP4 Receptor and Intracellular Cyclic AMP in Human Macrophages.

PGE2 is a potent lipid mediator involved in maintaining homeostasis but also promotion of acute inflammation or immune suppression in chronic inflammation and cancer. Nucleotide-binding domain, leucine-rich repeat-containing protein (NLR)P3 inflammasome plays an important role in host defense. Uncontrolled activation of the NLRP3 inflammasome, owing to mutations in the NLRP3 gene, causes cryopyrin-associated periodic syndromes. In this study, we showed that NLRP3 inflammasome activation is inhibited by PGE2 in human primary monocyte-derived macrophages. This effect was mediated through PGE2 receptor subtype 4 (EP4) and an increase in intracellular cAMP, independently of protein kinase A or exchange protein directly activated by cAMP. A specific agonist of EP4 mimicked, whereas its antagonist or EP4 knockdown reversed, PGE2-mediated NLRP3 inhibition. PGE2 caused an increase in intracellular cAMP. Blockade of adenylate cyclase by its inhibitor reversed PGE2-mediated NLRP3 inhibition. Increase of intracellular cAMP by an activator of adenylate cyclase or an analog of cAMP, or a blockade of cAMP degradation by phosphodiesterase inhibitor decreased NLRP3 activation. Protein kinase A or exchange protein directly activated by cAMP agonists did not mimic, and their antagonists did not reverse, PGE2-mediated NLRP3 inhibition. Additionally, constitutive IL-1ß secretion from LPS-primed PBMCs of cryopyrin-associated periodic fever syndromes patients was substantially reduced by high doses of PGE2. Moreover, blocking cytosolic phospholipase A2α by its inhibitor or small interfering RNA or inhibiting cyclooxygenase 2, resulting in inhibition of endogenous PGE2 production, caused an increase in NLRP3 inflammasome activation. Our results suggest that PGE2 might play a role in maintaining homeostasis during the resolution phase of inflammation and might serve as an autocrine and paracrine regulator.

The fish oil ingredient, docosahexaenoic acid, activates cytosolic phospholipase A2 via GPR120 receptor to produce prostaglandin E2 and plays an anti-inflammatory role in macrophages.

Docosahexaenoic acid (DHA) is one of the major ingredients of fish oil and has been reported to have anti-inflammatory properties mediated through the GPR120 receptor. Whether cytosolic phospholipase A2 (cPLA2 ) and lipid mediators produced from cPLA2 activation are involved in the anti-inflammatory role of DHA in macrophages has not been reported. We report here that DHA and the GPR120 agonist, GW9508, activate cPLA2 and cyclooxygenase 2 (COX-2), and cause prostaglandin E2 (PGE2) release in a murine macrophage cell line RAW264.7 and in human primary monocyte-derived macrophages. DHA and GW9508 activate cPLA2 via GPR120 receptor, G protein Gαq and scaffold protein ß-arrestin 2. Extracellular signal-regulated kinase 1/2 activation is involved in DHA- and GW9508-induced cPLA2 activation, but not p38 mitogen-activated protein kinase. The anti-inflammatory role of DHA and GW9508 is in part via activation of cPLA2 , COX-2 and production of PGE2 as a cPLA2 inhibitor or a COX-2 inhibitor partially reverses the DHA- and GW9508-induced inhibition of lipopolysaccharide-induced interleukin-6 secretion. The cPLA2 product arachidonic acid and PGE2 also play an anti-inflammatory role. This effect of PGE2 is partially through inhibition of the nuclear factor-κB signalling pathway and through the EP4 receptor of PGE2 because an EP4 inhibitor or knock-down of EP4 partially reverses DHA inhibition of lipopolysaccharide-induced interleukin-6 secretion. Hence, DHA has an anti-inflammatory effect partially through induction of PGE2.

Low molecular weight hyaluronan activates cytosolic phospholipase A2α and eicosanoid production in monocytes and macrophages.

Hyaluronan (HA) is the major glycosaminoglycan in the extracellular matrix. During inflammation, there is an increased breakdown of HA, resulting in the accumulation of low molecular weight (LMW) HA and activation of monocytes and macrophages. Eicosanoids, derived from the cytosolic phospholipase A2 group IVA (cPLA2α) activation, are potent lipid mediators also attributed to acute and chronic inflammation. The aim of this study was to determine the effect of LMW HA on cPLA2α activation, arachidonic acid (AA) release, and subsequent eicosanoid production and to examine the receptors and downstream mechanisms involved in these processes in monocytes and differently polarized macrophages. LMW HA was a potent stimulant of AA release in a time- and dose-dependent manner, induced cPLA2α, ERK1/2, p38, and JNK phosphorylation, as well as activated COX2 expression and prostaglandin (PG) E2 production in primary human monocytes, murine RAW 264.7, and wild-type bone marrow-derived macrophages. Specific cPLA2α inhibitor blocked HA-induced AA release and PGE2 production in all of these cells. Using CD44, TLR4, TLR2, MYD88, RHAMM or STAB2 siRNA-transfected macrophages and monocytes, we found that AA release, cPLA2α, ERK1/2, p38, and JNK phosphorylation, COX2 expression, and PGE2 production were activated by LMW HA through a TLR4/MYD88 pathway. Likewise, PGE2 production and COX2 expression were blocked in Tlr4(-/-) and Myd88(-/-) mice, but not in Cd44(-/-) mice, after LMW HA stimulation. Moreover, we demonstrated that LMW HA activated the M1 macrophage phenotype with the unique cPLA2α/COX2(high) and COX1/ALOX15/ALOX5/LTA4H(low) gene and PGE2/PGD2/15-HETE(high) and LXA4(low) eicosanoid profile. These findings reveal a novel link between HA-mediated inflammation and lipid metabolism.

A cytosolic phospholipase A2-initiated lipid mediator pathway induces autophagy in macrophages.

Autophagy delivers cytoplasmic constituents to autophagosomes and is involved in innate and adaptive immunity. Cytosolic phospholipase (cPLA(2))-initiated proinflammatory lipid mediator pathways play a critical role in host defense and inflammation. The crosstalk between the two pathways remains unclear. In this study, we report that cPLA(2) and its metabolite lipid mediators induced autophagy in the RAW246.7 macrophage cell line and in primary monocytes. IFN-γ-triggered autophagy involves activation of cPLA(2). Cysteinyl leukotrienes D(4) and E(4) and PGD(2) also induced these effects. The autophagy is independent of changes in mTOR or autophagic flux. cPLA(2) and lipid mediator-induced autophagy is ATG5 dependent. These data suggest that lipid mediators play a role in the regulation of autophagy, demonstrating a connection between the two seemingly separate innate immune responses, induction of autophagy and lipid mediator generation.

Functional characterization of human cysteinyl leukotriene 1 receptor gene structure.

The 5-lipoxygenase pathway has been strongly implicated in the pathogenesis of chronic inflammatory disorders, such as bronchial asthma and atherosclerosis. Cysteinyl leukotrienes (cysLTs), 5-lipoxygenase pathway products, are recognized now not only as important factors in asthmatic inflammation, but also as mediators of cell trafficking and innate immune responses. To study a role of cysLTs in inflammatory reactions we have characterized the gene structure of human cysteinyl leukotriene receptor type I (cysLT(1)R). The cysLT(1)R gene consists of 5 exons that are variably spliced and a single promoter region with multiple transcription start sites. Four different cysLT(1)R transcripts were identified. RT-PCR showed dominant and wide expression of the transcript I, containing exons 1, 4, and 5, with the strongest presence in blood leukocytes, spleen, thymus, lung, and heart. The expression of cysLT(1)R is functionally regulated at the transcriptional level by IL-4 through a STAT6 response element localized to the proximal cysLT(1)R promoter region. IL-4 stimulation increased cysLT(1)R mRNA (real-time PCR) and surface protein expression (flow cytometry) in a time-dependent fashion. CysLTs (LTD(4) and LTC(4)) induced an increased production of a potent monocyte chemoattractant CCL2 (MCP-1) in IL-4-primed THP-1 cells in a dose-dependent manner. This effect was effectively inhibited by the cysLT(1)R-selective antagonist MK571 in a dose-dependent manner and only partially by a nonselective cysLT(1)R/cysLT(2)R inhibitor BAY-u9773, implying a cysLT(1)R-mediated mechanism. Thus, cysLTs signaling through cysLT(1)R might contribute to inflammatory reactions by cooperating with IL-4 in enhanced CCL2 production in human monocytic cells.

Toll-like receptor 4 signaling regulates cytosolic phospholipase A2 activation and lipid generation in lipopolysaccharide-stimulated macrophages.

Inflammatory lipid mediators such as prostaglandins and leukotrienes play crucial roles in the pathogenesis of bacterial lipopolysaccharide (LPS)-induced inflammation. Cytosolic phospholipase A(2) (cPLA(2)) is a key enzyme in the generation of pro-inflammatory lipid mediators. Here, we found that Toll-like receptor 4 (TLR4) is essential for LPS-induced cPLA(2) activation and lipid release. Inhibition of TLR4 protein expression by TLR4 small interfering RNA or neutralization of TLR4 by the specific antibody against TLR4/MD2 blocked cPLA(2) phosphorylation and cPLA(2)-hydrolyzed arachidonic acid release. Furthermore, activation of the TLR4 signaling pathway by LPS regulated cPLA(2) activation and lipid release. cPLA(2) phosphorylation and cPLA(2)-hydrolyzed lipid release were significantly impaired when TLR4 adaptor protein, either MyD88 or TRIF, was knocked down in LPS-stimulated macrophages. Similarly, LPS-induced arachidonate release was inhibited in cells transfected with a dominant-negative MyD88 or TRIF construct. Subsequently, cPLA(2) activation could be suppressed by inhibition of the TLR4 adaptor protein-directed p38 and ERK MAPK pathways. These findings suggest that, in LPS-induced inflammation, the TLR4-mediated MyD88- and TRIF-dependent MAPK pathways result in cPLA(2) activation and production of pro-inflammatory lipid mediators.

DnaK promotes the selective export of outer membrane protein precursors in SecA-deficient Escherichia coli.

Consistent with many other results indicating that SecA plays an essential role in the translocation of presecretory proteins across the Escherichia coli inner membrane, we previously found that a approximately 95% depletion of SecA completely blocks the export of periplasmic proteins in vivo. Surprisingly, we found that about 25% of the outer membrane protein (OMP) OmpA synthesized after SecA depletion was gradually translocated across the inner membrane. In this study we analyzed the export of several other OMPs after SecA depletion. We found that 25-50% of each OMP as well as an OmpA-alkaline phosphatase fusion protein was exported from SecA-deficient cells. This partial export was completely abolished by the SecA inhibitor sodium azide and therefore still required the participation of SecA. Examination of a variety of OmpA derivatives, however, ruled out the possibility that OMPs are selectively translocated in SecA-deficient cells because SecA binds to their N termini with unusually high affinity. Export after SecA depletion was observed in cells that lack SecB, the primary targeting factor for OMPs, but was abolished by partial inactivation of DnaK. Furthermore, OmpA could be isolated in a stable complex with DnaK. The data strongly suggest that OMPs require only a relatively low level of translocase activity to cross the inner membrane because they can be preserved in a prolonged export-competent state by DnaK.