Artemisinin reduces human melanoma cell migration by down-regulating αVβ3 integrin and reducing metalloproteinase 2 production

Artemisinin and its derivatives are well known antimalarial drugs, particularly useful after resistance to traditional antimalarial pharmaceuticals has started to occur in Plasmodium falciparum. In recent years, anticancer activity of artemisinin has been reported both in vitro and in vivo. Artemisinin has inhibitory effects on cancer cell growth and anti-angiogenetic activity. In the present investigation, we analyzed the inhibitory effects of artemisinin on migratory ability of melanoma cell lines (A375P and A375M, low and medium metastatic properties, respectively). We demonstrate that artemisinin induces cell growth arrest in A375M, and affects A375P cells viability with cytotoxic and growth inhibitory effects, while it was not effective in contrasting proliferation of other tumor cell lines (MCF7 and MKN). In addition, artemisinin affected the migratory ability of A375M cells by reducing metalloproteinase 2 (MMP-2) production and down-regulating αvβ3 integrin expression. These findings introduce a potential of artemisinin as a chemotherapeutic agent in melanoma treatment.     

Anoikis induction and metastasis suppression by a new integrin αvβ3 inhibitor in human melanoma cell line M21

Integrin αvβ3 plays a critical role in the survival and metastasis process of cancer cells. It is therefore desirable to develop new types of small molecule inhibitors of integrin αvβ3. IH1062 (3, 5-dichloro-phenylbiguanide) is a novel small molecule inhibitor of integrin αvβ3 that we have recently discovered. In this study, we investigated the induction effects of anoikis in human melanoma cell line M21 by IH1062, by detecting caspase activity, measuring the expression levels of apoptosis-related proteins, and performing the AnnexinV/PI apoptosis assay. Furthermore, we established a melanoma pulmonary metastasis mouse model in order to evaluate the suppression of metastasis by IH1062 in vivo. Our results demonstrate that IH1062 triggered human melanoma M21 cells to undergo anoikis by interrupting the attachment of M21 cells to extracellular matrix, reducing the phosphorylation of focal adhesion kinase, decreasing survivin and the ratio of Bcl-2/Bax proteins, and activating caspase cascades in vitro. Additionally, IH1062 showed markedly anti-metastatic effects in the pulmonary metastasis model in vivo, which makes it a promising lead to develop new drugs for anti-metastasis therapies.     

Effect of fibronectin on expression of integrin α5β1 in trabecular meshwork cell in patients with primary open-angle glaucoma

目的 探讨纤维连接蛋白(FN)对体外培养的原发性开角型青光眼(POAG)患者小梁网细胞整合素α5β1表达及细胞存活的影响.方法 体外培养POAG患者小梁网细胞,分别加入不同浓度的FN(0、5、10、20、40、100μg/ml)培养.采用免疫细胞化学法观察各组细胞整合素α5β1的表达及细胞存活情况.结果 随着FN浓度增加,整合素α5β1表达增强.在FN低浓度(0-40 μg/ml)范围内,阳性细胞面积增多;而在FN 100μg/ml时阳性细胞面积减少.结论 FN影响小梁网细胞外基质,参与眼内压的调节.     

Protective effect of kobophenol A on nitric oxide-induced cell apoptosis in human osteoblast-like MG-63 cells: involvement of JNK, NF-κB and AP-1 pathways

Nitric oxide (NO) is a multifunctional signaling molecule and the cytotoxic species responsible for a variety of pathologic disorders including bone destruction. High NO levels induce the apoptosis of osteoblasts and decrease the bone mineral density. We investigated the influence of kobophenol A (kob A) on apoptosis in cultured human osteoblast-like MG-63 cells. Direct NO donor sodium nitroprusside (SNP) that has been recognized as an inducer of apoptosis in various cell lines significantly induced cell death and NO production in MG-63 cells. Coincubation of kob A in SNP-treated MG-63 cells resulted in a significant protection against NO-induced cell death. This is associated with increase in intracellular reactive oxygen species (ROS) scavenging activity and the inhibition of decrease in mitochondrial membrane potential (MMP) by kob A. We also found that kob A inhibited the down-regulation of Bcl-2 and Bcl-X(L), whereas the level of Bax expression was decreased by kob A treatment in SNP-treated MG-63 cells. Furthermore, kob A inhibited SNP-induced phosphorylation of JNK and c-Jun, and SNP-induced reduction in NF-κΒ and AP-1 activities, implicating that protective effect of kob A may occur through the regulation of JNK, NF-κΒ and AP-1 signaling pathways. Together, these findings suggest that kob A has a protective effect against NO-mediated osteoblast apoptosis and might be a plausible candidate for treatment of inflammatory bone diseases relevant to osteoblast cell death.     

Modulation of 5 -HT2C receptor on transcription of neprilysin gene and secretion of neprilysin in human glioma cell line

Neprilysin （NEP） , a member of zinc metallopeptidase family, has recently been focused in pharmacological research. Besides cleaving peptide signaling molecules in cardiovascular, inflammatory and immune system such as atrial natriuretic peptide, bradykinin, tachykinin, substance P and enkephalin, it has been proved to be a principal enzyme capable of degrading extracellular Aβin the brain. Since the results of cell and animal experiments suggested that stimulation of serotonin receptor subtype 5 - HT2C can increase secretion of soluble amyloid precursor protein （sAPP） and reduce accumulation of Aβpeptide both in vitro and in vivo, it is not known whether activation of 5 - HT2C receptor can also -affect NEP expression. We investigated NEP expression in human glioma cell Line U251. U251 was incubated for 24h with different concentrations of m - CPP （5 - HT2C receptor agonist） or RS 102221 （5 - HT2C receptor antagonist）. Real time quantitative PCR （RT - QPCR） was used to measure the mRNA level of neprilysin and western blot was used to analyse the protein level of neprilysin. Compared with the control group, a significant dose - dependent increase in NEP mRNA level was observed ＇after treated with m - CPP., On the contrary, RS 102221 caused NEP mRNA dose - dependent reduction. Relative levels of neprilysin protein generally paralleled those of the mRNA. These results suggest that activation of 5 - HT2C receptor can increase NEP expression in human glioma cell line, which might be of value in the exploration of etiology of and therapy of AD.     

Recombinant viral protein promotes apoptosis and suppresses invasion of ovarian adenocarcinoma cells by targeting α5β1 integrin to down-regulate Akt and MMP-2

BACKGROUND AND PURPOSE

As prognosis for patients with metastatic ovarian cancer is generally poor, advances in treatment are needed. Here, we studied the mechanism of action of a recombinant viral capsid protein (rVP1) and explored its effect against ovarian tumour growth and metastasis in vivo.

EXPERIMENTAL APPROACH

The human ovarian cancer cell line SKOV3 and BALB/cAnN-Foxn1 female nude mice were used. Effects of rVP1 on the viability, invasive ability, matrix metalloproteinase (MMP)-2 activity and cancer cell proliferation and metastasis were determined by cell proliferation assay, Matrigel invasion assay, gelatin zymographic analysis, as well as bioluminescence imaging and immunohistological analysis in xenograft mouse models respectively. Levels of total and phosphorylated focal adhesion kinase (FAK), PKB/Akt, phosphatase and tensin homologue (PTEN) and glycogen synthase kinase-3β (GSK-3β) were detected by Western blotting.

KEY RESULTS

rVP1 promoted apoptosis and decreased invasion of human ovarian cancer cells. This effect of rVP1 was accompanied by activation of PTEN and GSK-3β as well as down-regulation of FAK, Akt and MMP-2. Anti-integrin antibodies or overexpression of constitutively active Akt reversed the cellular effects of rVP1. Orthotopic and intraperitoneal xenograft mouse models demonstrated that rVP1 attenuated survival and metastasis of human ovarian cancer SKOV3 cell line in vivo through selective regulation of Akt and GSK-3β activity as shown by bioluminescence imaging of mice and immunohistochemical analysis.

CONCLUSION AND IMPLICATIONS

These results indicate that negative regulation of Akt signalling and MMP-2 by rVP1 may have the potential to suppress ovarian tumour growth and metastasis in vivo.     

Pharmacokinetics and metabolism of TR-14035, a novel antagonist of α4β1/α4β7 integrin mediated cell adhesion,in rat and dog

The pharmacokinetics and disposition of N-(2,6-dichlorobenzoyl)-4-(2,6-dimethoxyphenyl)-L-phenylalanine (TR-14035), a novel α4β1/α4β7 antagonist, were investigated in the rat and dog. Results indicate extensive clearance of TR-14035 and low oral bioavailability, 17% and 13% in the rat and dog, respectively, at an oral dose of 10?mg/kg. At least 63% of the oral dose was absorbed from the gastrointestinal tract in the rat, and about one-third of the intravenous dose was excreted into bile as unchanged drug in the rat and dog. These data indicate that the oral bioavailability of TR-14035 was limited due to significant first-pass metabolism and biliary excretion in the liver. A species-dependent difference in metabolism was observed. The principal metabolite, O-desmethyl TR-14035, observed in rat, dog and probably human, was further conjugated with sulfate in the rat, but never in dog and human, based on in vitro metabolism and in vivo metabolite profile studies. Urinary excretion was a minor elimination route, but an interesting species difference was recognized. TR-14035 was reabsorbed from the rat renal proximal tubules, and by contrast, secreted into the tubules in the dog, probably via active transport systems.     

The integrin α2β1 agonist,aggretin, promotes proliferation and migration of VSMC through NF-kB translocation and PDGF production

Background and purpose:

During the development of atherosclerotic plaques, vascular smooth muscle cells (VSMCs) migrate from the media to the intima through the basement membrane and interstitial collagenous matrix, and proliferate to form neointima. Here, we investigate the mechanism of VSMC migration and proliferation caused by aggretin, a snake venom integrin α2β1 agonist.

Experimental approach:

Cultures of rat and human VSMCs were treated with aggretin and the signal transduction pathways induced by this agonist were examined by Western blotting, immunoprecipitation and electrophoretic mobility shift assay techniques.

Key results:

Aggretin-induced VSMC proliferation was blocked by a monoclonal antibody (mAb) against integrin α2 (AII2E10) or against the platelet-derived growth factor receptor (PDGFR)-β. Proliferation was also blocked by inhibition of the tyrosine kinase Src with PP2, phospholipase C (PLC) with {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, extracellular signal-regulated kinase (ERK) with PD98059 or nuclear factor-kappa B (NF-kB) activation with pyrrolidine dithiocarbamate (PDTC). VSMC migration towards immobilized aggretin was increased in a modified Boyden chamber and this effect was blocked by α2β1-Src-PLC-MAPK axis inhibitors, but not by PDTC, PDGFR-β mAb, or a phosphoinositide-3 kinase inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Aggretin stimulated the phosphorylation of PDGFR-β, Src and ERK in a time-dependent manner. NF-kB translocation and platelet-derived growth factor (PDGF)-BB production were also observed. The ERK activation, NF-kB translocation and PDGF-BB production were blocked by PP2, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 and PD98059.

Conclusions and implications:

Aggretin induces VSMC proliferation and migration mainly through binding to integrin α2β1, and subsequently activates Src, PLC and ERK pathways, inducing NF-kB activation and PDGF production.     

The neurosteroid 5β-pregnan-3α-ol-20-one enhances actions of etomidate as a positive allosteric modulator of α1β2γ2L GABAA receptors

Background and Purpose

Neurosteroids potentiate responses of the GABA_A receptor to the endogenous agonist GABA. Here, we examined the ability of neurosteroids to potentiate responses to the allosteric activators etomidate, pentobarbital and propofol.

Experimental Approach

Electrophysiological assays were conducted on rat α1β2γ2L GABA_A receptors expressed in HEK 293 cells. The sedative activity of etomidate was studied in Xenopus tadpoles and mice. Effects of neurosteroids on etomidate-elicited inhibition of cortisol synthesis were determined in human adrenocortical cells.

Key Results

The neurosteroid 5β-pregnan-3α-ol-20-one (3α5βP) potentiated activation of GABA_A receptors by GABA and allosteric activators. Co-application of 1 μM 3α5βP induced a leftward shift (almost 100-fold) of the whole-cell macroscopic concentration–response relationship for gating by etomidate. Co-application of 100 nM 3α5βP reduced the EC₅₀ for potentiation by etomidate of currents elicited by 0.5 μM GABA by about three-fold. In vivo, 3α5βP (1mg kg^-1) reduced the dose of etomidate required to produce loss of righting in mice (ED₅₀) by almost 10-fold. In tadpoles, the presence of 50 or 100 nM 3α5βP shifted the EC₅₀ for loss of righting about three- or ten-fold respectively. Exposure to 3α5βP did not influence inhibition of cortisol synthesis by etomidate.

Conclusions and Implications

Potentiating neurosteroids act similarly on orthosterically and allosterically activated GABA_A receptors. Co-application of neurosteroids with etomidate can significantly reduce dosage requirements for the anaesthetic, and is a potentially beneficial combination to reduce undesired side effects.Tables of Links

Establishment of stable cell lines with high expression of heterodimers of human 4F2hc and human amino acid transporter LAT1 or LAT2 and delineation of their differential interaction with α-alkyl moieties

System L is a major transport system for cellular uptake of neutral amino acids. Among system L transporters, L-type amino acid transporter 1 (LAT1) is responsible for the nutrient uptake in cancer cells, whereas L-type amino acid transporter 2 (LAT2) is a transporter for non-cancer cells. In this study, we have established HEK293 cell lines stably expressing high levels of human LAT1 and LAT2 forming heterodimers with native human 4F2hc of the cells. We have found that L-[(14)C]alanine is an appropriate substrate to examine the function of LAT2, whereas L-[(14)C]leucine is used for LAT1. By using L-[(14)C]alanine on LAT2, we have for the first time directly evaluated the function of human LAT2 expressed in mammalian cells and obtained its reliable kinetics. Using α-alkyl amino acids including α-methyl-alanine and α-ethyl-L-alanine, we have demonstrated that α-alkyl groups interfere with the interaction with LAT2. These cell lines with higher practical advantages would be useful for screening and analyzing compounds to develop LAT1-specific drugs that can be used for cancer diagnosis and therapeutics. The strategy that we took to establish the cell lines would also be applicable to the other heterodimeric transporters with important therapeutic implications.     

UVB exposure enhanced benzanthrone-induced inflammatory responses in SKH-1 mouse skin by activating the expression of COX-2 and iNOS through MAP kinases/NF-κB/AP-1 signalling pathways

This study was conducted to explore the role of UVB on benzanthrone (BA)-induced skin inflammation and its mechanism/s. SKH-1 hairless mice were topically exposed with BA (25 and 50 mg/kg b.wt) either alone or along with UVB (50 mJ/cm²) for 24 h and estimation of ROS, histopathological analysis, myeloperoxidase (MPO) activity, mast cell staining, immunohistochemistry for COX-2 and iNOS as well as western blotting for MAPKs, p-NF-κB, c-jun, c-fos COX-2 and iNOS were carried out. Enhanced ROS generation, increased epidermal thickness, mast cell number, MPO activity, enhanced expression of COX-2 and iNOS, MAPKs, c-jun, c-fos, NF-κB were found in BA either alone or when followed by UVB treatment, compared to the control groups. Expression of COX-2, iNOS and phosphorylation of ERK1/2 were found to be more enhanced in BA and UVB- exposed group compared to BA and UVB only group, while phosphorylation of JNK1/2, p38, NF-κB and expression of c-jun and c-fos were comparable with BA and UVB only groups. In summary, we suggest that UVB exposure enhanced BA-induced SKH-1 skin inflammation possibly via oxidative stress-mediated activation of MAPKs-NF-κB/AP-1 signalling, which subsequently increased the expression of COX-2 and iNOS and led to inflammation in SKH-1 mouse skin.     

Kinetics of desensitization and recovery from desensitization for human α4β2-nicotinic acetylcholine receptors stably expressed in SH-EP1 cells

Aim： Studies were conducted to define the kinetics of the onset of and recovery from desensitization for human α4β2- nicotinic acetylcholine receptors （nAChR） heterologously expressed in the SH-EP1 human epithelial cell line. Methods： Whole-cell patch clamp recordings were performed to evaluate α4β2-nAChR currents. Results： Application of 0.1μmol/L nicotine or 1 mmol/L acetylcholine （ACh） for I s or longer induced two phases, with time constants of -70 and -700 ms, for the onset of α4β2-nAChR desensitization. For a given duration of agonist exposure, recovery from desensitization induced by nicotine was slower than recovery from ACh-induced desensitization. Comparisons with published reports indicate that time constants for the recovery of α4β2-nAChRs from desensitization are smaller than those for the recovery of human muscle-type nAChRs from desensitization produced by the same concentrations and durations of exposure to an agonist. Moreover, the extent of human α4β2-nAChR desensitization and rate of recovery are the same, regardless of whether they are measured using whole-cell recording or based on published findings using isotopic ion flux assays; this equality demonstrates the equivalent legitimacy of these techniques in the evaluation of nAChR desensitization. Perhaps most significantly, recovery from desensitization also was best fit to a biphasic process. Regardless of whether it was fit to single or double exponentials, however, half-times for recovery from desensitization grew progressively longer with an increased duration of agonist exposure during the desensitizing pulse. Conclusion： These findings indicate the existence of α4β2-nAChRs in many distinctive states of desensitization, as well as the induction of progressively deeper states of desensitization with the increased duration of agonist exposure.     

Withanolide B promotes osteogenic differentiation of human bone marrow mesenchymal stem cells via ERK1/2 and Wnt/β-catenin signaling pathways

BackgroundThe treatment of bone defects has always been a problem for clinicians. In recent years, research on human bone mesenchymal stem cells (hBMSCs) has found that promoting their osteogenic differentiation could be a useful therapeutic strategy for bone healing. Previous studies have been reported that Withania somnifera Dunal inhibits osteoclastogenesis by inhibiting the NF-κB signaling pathway. Withanolide B is an active component of W. somnifera Dunal, but its role in osteogenic differentiation of hBMSCs remains unknown. Here, we performed a preliminary study on the role of Withanolide B in promoting osteogenic differentiation and its possible mechanism.MethodsWe investigated the effect of Withanolide B on osteogenic differentiation of hBMSCs in vitro and in vivo. The effect of Withanolide B on the activity of hBMSCs was verified by CCK-8 assay and quantitative Real-time polymerase chain reaction (qPCR) and Western blotting analysis were used to verify the effect of Withanolide B on osteogenic differentiation-specific genes and proteins. The effect of Withanolide B on ALP activity and mineral deposition was verified by ALP and ARS staining. We then used a rat tibial osteotomy model to observe the effect of Withanolide B on bone healing.ResultsWithanolide B is noncytotoxic to hBMSCs and can effectively promote their osteogenic differentiation. Moreover, we found that Withanolide B can regulate the osteogenic differentiation of hBMSCs through the ERK1/2 and Wnt/β-catenin signaling pathways. When inhibitors of the ERK1/2 and Wnt/β-catenin signaling pathways were used, the enhancement of osteogenic differentiation induced by Withanolide B was attenuated. Withanolide B also effectively promoted bone healing in the rat tibial osteotomy model.ConclusionsOur results suggest that Withanolide B can promote the osteogenic differentiation of hBMSCs through the ERK1/2 and Wnt/β-catenin signaling pathways and can effectively promote bone defect healing.     

In vitro wound healing activity of 1-hydroxy-5,7-dimethoxy-2-naphthalene-carboxaldehyde (HDNC) and other isolates of Aegle marmelos L.: Enhances keratinocytes motility via Wnt/β-catenin and RAS-ERK pathways

Wound healing is a complex process in which injured skin and tissues repaired by interaction of a complex cascade of cellular events that generates resurfacing, reconstitution and restoration of the tensile strength of injured skin. It follows β-catenin, extracellular signal regulated kinase (ERK) and Akt signaling pathways. Aegle marmelos L., generally known as bael is found to act as anti-inflammatory, antioxidant and anti-ulcer agent. Furthermore, studies have demonstrated that this Indian traditional medicinal plant, A. marmelos flower extract (AMF) was used for wound injury. Henceforth, the current study was investigated to ascertain the effect of its active constituents in vitro wound healing with mechanism involve in migration of cells and activation of β-catenin in keratinocytes, inhibition of PGE₂ in macrophages and production of collagen in fibroblasts. We have taken full thickness wound of rats and applied AMF for 2 weeks. Cutaneous wound healing activity was performed using HaCaT keratinocytes, Hs68 dermal fibroblasts and RAW264.7 macrophages to determine cell viability, nitric oxide production, collagen expression, cell migration and β-catenin activation. Results shows that AMF treated rats demonstrated reduced wound size and epithelisation was improved, involved in keratinocytes migration by regulation of Akt signaling, beta-catenin and extracellular signal-regulated kinase (ERK) pathways. AMF and its active constituent’s increased mRNA expression, inhibited nitric oxide, PGE₂ release, mRNA expression of mediators in RAW 264.7 macrophages and enhances the motility of HaCaT keratinocytes in vitro wound healing of rats.     

Sodium arsenite-induced abnormalities in expressions of Caveolin-1, eNOS,IKKβ, and COX-2 in SV-40 immortalized human uroepithelial cells and in urothelial carcinomas

Arsenic, a known human carcinogen, is found throughout the crust of the earth. Prolonged arsenic exposure is a known cause of urothelial carcinoma (UC) and blackfoot disease (BFD). The aim of this study was to determine the effect of sodium arsenite on Caveolin-1 and downstream signaling molecules (eNOS, IKKβ and COX-2) expression in human urothelial cells (SV-HUC-1). Immunohistochemical (IHC) staining of Caveolin-1, eNOS, IKKβ, and COX-2 was also compared between UC patients from endemic and non-endemic areas of BFD in Taiwan. Immunocytochemical staining and Western blotting results revealed increased expression of Caveolin-1, IKKβ, and COX-2 but decreased eNOS in SV-HUC-1 cells treated with low concentration of arsenite. Additionally, MEK inhibitor (U0126) significantly attenuated arsenite-induced expression of Caveolin-1, IKKβ and COX-2 while reducing eNOS expression. The IHC staining of UCs revealed that expressions of Caveolin-1, IKKβ, and COX-2 were significantly higher in patients from endemic areas of BFD compared to patients from non-endemic areas (p = 0.011, p = 0.002, p = 0.0001) whereas eNOS was significantly lower (p = 0.0001). The correlation observed between Caveolin-1 and downstream signaling molecule expression may be an important mechanism of arsenic-induced urothelial carcinogenesis.