Diallyl Trisulfide, a Major Bioactive Constituent of Garlic, Promotes Colonic Mucosal Healing in Ulcerative Colitis through Accelerating Focal Adhesion Assembly and Consequent Epithelial Cell Migration via the Rab21-Integrin ß1-Fak Pathway.

SCOPE: Colonic mucosal healing is the terminal goal for the treatment of ulcerative colitis (UC), but there is currently no specific drug available. This study investigates the beneficial effect of diallyl trisulfide (DATS) on the colonic mucosal healing. METHODS AND RESULTS: Dextran sulfate sodium (DSS) is used to induce colitis in female C57BL/6 mice, and DATS is orally administered during the recovery period. DATS hardly impacts the inflammation of the colonic tissues, but significantly promotes the mucosal repair. DATS promotes the migration but not proliferation of colonic epithelial cells in the colitis mice. In addition, DATS accelerates the wound healing, cell migration, focal adhesion assembly, and phosphorylation of focal adhesion kinase (FAK) of colonic epithelial cells in vitro, which are evidently reversed by combined use of FAK inhibitor PF-573228. Similar results are shown in colitis mice. Mechanically, DATS promotes the binding of Rab21 to integrin ß1 and accelerates the endocytosis of integrin ß1, which is significantly attenuated by the knockdown of Rab21. CONCLUSIONS: DATS promotes the binding of Rab21 to integrin ß1 and the endocytosis of integrin ß1, thereby increases FAK phosphorylation and focal adhesion assembly, finally accelerates the migration of colonic epithelial cells and mucosal healing.

Exploring the mechanisms of anti-ovarian cancer of Hedyotis diffusa Willd and Scutellaria barbata D. Don through focal adhesion pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Hedyotis diffusa Willd and Scutellaria barbata D.Don (HD-SB) pairing were widely used as traditional medicine known for their anti-tumor effects. However, the inhibitory effect of HD-SB on ovarian cancer and its potential mechanisms were still not clear. AIM OF THE STUDY: Our study identified the anti-tumor effect of HD-SB on ovarian cancer and analyzed the potential mechanisms by the network pharmacology and molecular docking method. MATERIALS AND METHODS: The inhibitory effect of HD-SB combination on the growth and migration of ovarian cancer was detected by MTT and transwell assays. The effective ingredients of HD-SB and their potential targets were obtained from the Traditional Chinese Medicines for Systems Pharmacology Database (TCMSP), the GeneCards database, and the UniProt database. The relationships between active ingredients of HD-SB and potential targets or pathways of ovarian cancer were analyzed by String database, Cytoscape 3.7.2 software, and David 6.7 online database. The anti-ovarian cancer targets of HD-SB in the focal adhesion pathway were identified by RT-qPCR and molecular docking. RESULTS: HD-SB combination significantly inhibited the proliferation and migration of ovarian cancer cells. We observed that the 1:2 ratio of HD-SB had the lowest IC50 value. 60 gene targets of 33 active ingredients in HD-SB were selected by pharmacokinetic parameters. The network pharmacological analysis showed that quercetin, luteolin, and baicalein might be the important anti-ovarian cancer ingredients in HD-SB, and the inhibitory effects of these three ingredients on the proliferation of ovarian cancer cells were verified respectively. Functional enrichment results suggested that HD-SB inhibited ovarian cancer growth and migration mainly through the focal adhesion pathway and the potential targets were EGFR, MAPK1, VEGFA, and PIK3CG. CONCLUSIONS: HD-SB pairing significantly inhibited the proliferation and migration of ovarian cancer. Using network pharmacological methods and validation experiments, we found that HD-SB might, at least partially, inhibit ovarian cancer through the focal adhesion pathway. We believed that the HD-SB combination could be a potential therapeutic drug for the treatment of ovarian cancer patients.

Integrative analysis of epigenomics, transcriptomics, and proteomics to identify key targets and pathways of Weining granule for gastric cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Weining granule (WNG) is a "Qi-Enriching and Kidney-Tonifying, Spleen-Reinforcing and Stasis-Removing" formula for gastric cancer (GC). Past research we noted WNG inhibited cell growth and raised apoptosis in GC. However, the underlying mechanism of WNG for GC have yet to be systematically clarified. AIM OF THE STUDY: We sought to characterize the molecular landscape of GC cells in vitro after WNG treated, to identify the molecular targets and pathways that were associated with WNG for inducing the apoptosis of GC cells, and further to clarify underlying molecular mechanism of WNG for GC. MATERIALS AND METHODS: We performed the techniques of RNA sequencing, tandem mass tags (TMT) based quantitative proteomics, and reduced representation bisulfite sequencing (RRBS) in WNG-treated/or untreated SGC-7901 GC cells to gain a comprehensive molecular portrait of WNG treatment. Then we integrated methylomics, transcriptomics, and proteomics data to carry out the bioinformatics analysis, and constructed the protein-protein interaction (PPI) network to identify molecular targets, and to discover the underlying signaling pathways associated with WNG for GC by network analysis. Besides, we verified the candidate target genes by Kaplan-Meier plotter database. RESULTS: We identified 1249 significant differentially expressed genes (DEGs) from RNA expression datasets, 191 significant differentially abunabundant proteins (DAPs) from proteomics datasets, and 8293 significant differentially methylated regions (DMRs) from DNA methylation datasets. GO and KEGG analysis showed DEGs, DAPs, and DMRs enriched in the cancer-related biological processes of calcium signaling pathway, pathways in cancer, metabolic pathways, MAPK signaling pathway, PI3K-Akt signaling pathway, and transcriptional misregulation in cancer. We integrated three profile datasets and performed network analysis to distinguish the hub genes, and finally the genes of SOD2, HMOX1, MMP1, SRXN1, NOTCH1, MAPK14, TXNIP, VEGFA, POLR2F, and HSPA9 were identified. The Kaplan-Meier plotter confirmed that SOD2, MMP1, SRXN1, NOTCH1, MAPK14, TXNIP, VEGFA, and HSPA9 were significantly correlated with OS in GC patients (P < 0.01), while HMOX1 and POLR2F expression were not significantly relevant to survival of GC patients (P > 0.01). CONCLUSIONS: SOD2, MMP1, SRXN1, NOTCH1, MAPK14, TXNIP, VEGFA, and HSPA9 were the predictive pharmaceutical targets of WNG for GC. The anticancer function of WNG was significantly associated with the pathways of focal adhesion pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, and Wnt signaling pathway.

Linarin and its aglycone acacetin abrogate actin ring formation and focal contact to bone matrix of bone-resorbing osteoclasts through inhibition of αvß3 integrin and core-linked CD44.

BACKGROUND: Since enhanced bone resorption due to osteoclast differentiation and activation cause skeletal diseases, there is a growing need in therapeutics for combating bone-resorbing osteoclasts. Botanical antioxidants are being increasingly investigated for their health-promoting effects on bone. Edible Cirsium setidens contains various polyphenols of linarin, pectolinarin, and apigenin with antioxidant and hepatoprotective effects. PURPOSE: This study aimed to determine whether linarin present in Cirsium setidens water extracts (CSE) and its aglycone acacetin inhibited osteoclastogenesis of RANKL-exposed RAW 264.7 murine macrophages for 5 days. METHODS: This study assessed the osteoprotective effects of CSE, linarin and acacetin on RANKL-induced differentiation and activation of osteoclasts by using MTT assay, TRAP staining, Western blot analysis, bone resorption assay actin ring staining, adhesion assay and immunocytochemical assay. This study explored the underlying mechanisms of their osteoprotection, and identified major components present in CSE by HPLC analysis. RESULTS: Linarin and pectolinarin were identified as major components of CSE. Nontoxic linarin and acacetin as well as CSE, but not pectolinarin attenuated the RANKL-induced macrophage differentiation into multinucleated osteoclasts, and curtailed osteoclastic bone resorption through reducing lacunar acidification and bone matrix degradation in the osteoclast-bone interface. Linarin and acacetin in CSE reduced the transmigration and focal contact of osteoclasts to bone matrix-mimicking RGD peptide. Such reduction was accomplished by inhibiting the induction of integrins, integrin-associated proteins of paxillin and gelsolin, cdc42 and CD44 involved in the formation of actin rings. The inhibition of integrin-mediated actin ring formation by linarin and acacetin entailed the disruption of TRAF6-c-Src-PI3K signaling of bone-resorbing osteoclasts. The functional inhibition of c-Src was involved in the loss of F-actin-enriched podosome core protein cortactin-mediated actin assembly due to linarin and acacetin. CONCLUSION: These observations demonstrate that CSE, linarin and acacetin were effective in retarding osteoclast function of focal adhesion to bone matrix and active bone resorption via inhibition of diffuse cloud-associated αvß3 integrin and core-linked CD44.

Chlorogenic acid-enriched extract of Ilex kudingcha C.J. Tseng tea inhibits neutrophil recruitment in injured zebrafish by promoting reverse migration via the focal adhesion pathway.

Neutrophil-regulated inflammation plays crucial roles in tissue damage and repair. Dysregulation of the neutrophil response system can contribute to diseases such as cancer. Clearance of excessive neutrophils at the site of inflammation by reverse migration provides a promising strategy to mitigate the negative effects. Chlorogenic acid treatment of injured zebrafish embryos showed low-developmental toxicity. Using a transgenic zebrafish Tg (mpx: egfp) model, chlorogenic acid-enriched kudingcha extract promoted neutrophil reverse migration via phosphorylation of ERK and AKT. Using i-TRAQ analysis, differentially expressed proteins involved in focal adhesion were identified, such as: Cdc42, SRC, MLC, ITGA, and Calpain. In support of this, ERK and AKT proteins are involved in the focal adhesion pathway. Real time qPCR determined that CGA downregulates genes associated with cancer metastasis, such as: HSPA5, YWHAZ, RP17, and ITGAV. Together, these results suggest that CGA-enriched Kudingcha extract may have potential as an anticancer or anti-inflammatory therapeutic agent. PRACTICAL APPLICATIONS: Ilex kudingcha C.J Tseng, commonly referred to as the large-leaved kudingcha, is a tea variety naturally rich in chlorogenic acid. Chlorogenic acid, the ester of caffeic and quinic acids, has antioxidant, antibacterial, anticancer, and anti-inflammatory, activities. Kudingcha has several known biological functions, including: anticancer, anti-inflammatory, antidiabetic, and hypolipidemic effects. Treatment with kudingcha extract reduces the recruitment of neutrophils, potentially by inhibiting focal adhesion, and activation of cancer metastasis-related genes. Importantly, kudingcha extract could be used as an alternative nutritional supplement for anticancer or anti-inflammation via its ability to suppress neutrophil recruitment.

Acacetin and Pinostrobin Inhibit Malignant Breast Epithelial Cell Adhesion and Focal Adhesion Formation to Attenuate Cell Migration.

Naturally occurring flavonoids, such as acacetin and pinostrobin, disrupt a wide range of processes during tumor progression, such as cell proliferation, apoptosis, and angiogenesis. Although the antiproliferative and antiapoptotic effects of acacetin and pinostrobin have been studied using various cell lines, relatively little is known about the effects of acacetin and pinostrobin on cancer cell migration and metastasis. For instance, it is unclear whether acacetin or pinostrobin have any effect on breast cancer cell migration or adhesion. In this study, we assessed the effects of acacetin and pinostrobin on malignant MDA-MB-231 and T47D breast epithelial cells and non-tumorigenic MCF10A breast epithelial cells. Our results demonstrate that both acacetin and pinostrobin selectively inhibit the migration of both MDA-MB-231 and T47D cells in a dose-dependent manner while exhibiting blunted effects on MCF10A cells. Interestingly, neither compound had an effect on cell proliferation in any of the 3 cell lines. Furthermore, both acacetin and pinostrobin inhibit MDA-MB-231 and T47D cell adhesion, cell spreading, and focal adhesion formation, but have no significant effect on MCF10A cells. Collectively, these results suggest that both acacetin and pinostrobin selectively inhibit malignant breast epithelial cell migration through attenuation of cell adhesion and focal adhesion formation. These findings indicate that both acacetin and pinostrobin may serve as potential therapeutic options to target breast tumor cell migration during late-stage tumor progression.

RHBDD2 overexpression promotes a chemoresistant and invasive phenotype to rectal cancer tumors via modulating UPR and focal adhesion genes.

The current standard of care for locally advanced rectal cancer (RC) is neoadjuvant radio-chemotherapy (NRC) with 5-fluorouracil (5Fu) as the main drug, followed by surgery and adjuvant chemotherapy. While a group of patients will achieve a pathological complete response, a significant percentage will not respond to the treatment. The Unfolding Protein Response (UPR) pathway is generally activated in tumors and results in resistance to radio-chemotherapy. We previously showed that RHBDD2 gene is overexpressed in the advanced stages of colorectal cancer (CRC) and that it could modulate the UPR pathway. Moreover, RHBDD2 expression is induced by 5Fu. In this study, we demonstrate that the overexpression of RHBDD2 in CACO2 cell line confers resistance to 5Fu, favors cell migration, adhesion and proliferation and has a profound impact on the expression of both, the UPR genes BiP, PERK and CHOP, and on the cell adhesion genes FAK and PXN. We also determined that RHBDD2 binds to BiP protein, the master UPR regulator. Finally, we confirmed that a high expression of RHBDD2 in RC tumors after NRC treatment is associated with the development of local or distant metastases. The collected evidence positions RHBDD2 as a promising prognostic biomarker to predict the response to neoadjuvant therapy in patients with RC.

Danggui Buxue Tang promotes the adhesion and migration of bone marrow stromal cells via the focal adhesion pathway in vitro.

ETHNOPHARMACOLOGY RELEVANCE: Danggui Buxue Tang has been used in China to treat clinical anemia for more than 800 years. However, there is no scientific report on its effect on bone marrow stromal cells. AIM OF THE STUDY: Here, we aimed to explore the effect of Danggui Buxue Tang on bone marrow stromal cell adhesion and migration. MATERIALS AND METHODS: Bone marrow stromal cells were used as a model to evaluate the effect of Danggui Buxue Tang on the adhesion and migration of bone marrow stromal cells. RNA-sequencing, quantitative polymerase chain reaction, and western blotting were used to detect and confirm the expression of genes related to the focal adhesion pathway before and after drug delivery. RESULTS: Danggui Buxue Tang significantly increased the number of bone marrow stromal cells. After 12 days of 16 mg/mL Danggui Buxue Tang treatment, bone marrow stromal cells were significantly increased (by 0.527 ±â€¯0.008 fold; p < 0.001) as compared to the control group (0.180 ±â€¯0.019). The effect was not due to enhanced cell proliferation, as there was no difference in the cell cycle (p > 0.05). The adhesion area of a single cell was doubled by Danggui Buxue Tang treatment (p < 0.001), and the time required for cell adhesion to a Petri dish was shortened. Thus, Danggui Buxue Tang increases the number of bone marrow stromal cells by promoting adhesion. Danggui Buxue Tang also significantly promoted bone marrow stromal cell migration (p <  0.001). Transcript analysis revealed that the focal adhesion and PI3K-Akt signaling pathways were activated. Expression analysis confirmed that the gene and protein expression of focal adhesion-related factors were upregulated. CONCLUSION: Danggui Buxue Tangaffects bone marrow stromal cell adhesion and migration by enhancing the focal adhesion pathway in vitro, and bone marrow stromal cells are a target of DBT-regulated hematopoiesis, and the active ingredients of DBT involved in the effects require further investigation.

Suppression of lung cancer progression by isoliquiritigenin through its metabolite 2, 4, 2', 4'-Tetrahydroxychalcone.

BACKGROUND: Licorice is an herb extensively used for both culinary and medicinal purposes. Various constituents of licorice have been shown to exhibit anti-tumorigenic effect in diverse cancer types. However, majority of these studies focus on the aspect of their growth-suppressive role. In this study, we systematically analyzed known licorice's constituents on the goal of identifying component(s) that can effectively suppress both cell migration and growth. METHODS: Effect of licorice's constituents on cell growth was evaluated by MTT assay while cell migration was assessed by both wound-healing and Transwell assays. Cytoskeleton reorganization and focal adhesion assembly were visualized by immunofluorescence staining with labeled phalloidin and anti-paxillin antibody. Activity of Src in cells was judged by western blot using phosphor-Src416 antibody while Src kinase activity was measured using Promega Src kinase assay system. Anti-tumorigenic capabilities of isoliquiritigenin (ISL) and 2, 4, 2', 4'-Tetrahydroxychalcone (THC) were investigated using lung cancer xenograft model. RESULTS: Using a panel of lung cancer cell lines, ISL was identified as the only licorice's constituent capable of inhibiting both cell migration and growth. ISL-led inhibition in cell migration resulted from impaired cytoskeleton reorganization and focal adhesion assembly. Assessing the phosphorylation of 141 cytoskeleton dynamics-associated proteins revealed that ISL reduced the abundance of Tyr421-phosphorylation of cortactin, Tyr925- and Tyr861-phosphorylation of FAK, indicating the involvement of Src because these sites are known to be phosphorylated by Src. Enigmatically, ISL inhibited Src in cells while displayed no effect on Src activity in cell-free system. The discrepancy was explained by the observation that THC, one of the major ISL metabolite identified in lung cancer cells abrogated Src activity both in cells and cell-free system. Similar to ISL, THC deterred cell migration and abolished cytoskeleton reorganization/focal adhesion assembly. Furthermore, we showed both ISL and THC suppressed in vitro lung cancer cell invasion and in vivo tumor progression. CONCLUSION: Our study suggests that ISL inhibits lung cancer cell migration and tumorigenesis by interfering with Src through its metabolite THC. As licorice is safely used for culinary purposes, our study suggests that ISL or THC may be safely used as a Src inhibitor.

Identification of miR-29b targets using 3-cyanovinylcarbazole containing mimics.

MicroRNAs (miRNAs) are highly conserved ∼22 nt small noncoding RNAs that bind partially complementary sequences in target transcripts. MicroRNAs regulate both translation and transcript stability, and play important roles in development, cellular homeostasis, and disease. There are limited approaches available to agnostically identify microRNA targets transcriptome-wide, and methods using miRNA mimics, which in principle identify direct miRNA:transcript pairs, have low sensitivity and specificity. Here, we describe a novel method to identify microRNA targets using miR-29b mimics containing 3-cyanovinylcarbazole (CNVK), a photolabile nucleoside analog. We demonstrate that biotin-tagged, CNVK-containing miR-29b (CNVK-miR-29b) mimics are nontoxic in cell culture, associate with endogenous mammalian Argonaute2, are sensitive for known targets and recapitulate endogenous transcript destabilization. Partnering CNVK-miR-29b with ultra-low-input RNA sequencing, we recover ∼40% of known miR-29b targets and find conservation of the focal adhesion and apoptotic target pathways in mouse and human. We also identify hundreds of novel targets, including NRAS, HOXA10, and KLF11, with a validation rate of 71% for a subset of 73 novel target transcripts interrogated using a high-throughput luciferase assay. Consistent with previous reports, we show that both endogenous miR-29b and CNVK-miR-29b are trafficked to the nucleus, but find no evidence of nuclear-specific miR-29b transcript binding. This may indicate that miR-29b nuclear sequestration is a regulatory mechanism in itself. We suggest that CNVK-containing small RNA mimics may find applicability in other experimental models.

The roles and mechanisms of homogalacturonan and rhamnogalacturonan I pectins on the inhibition of cell migration.

Our previous paper reported the structure of ginseng pectic polysaccharides related to the cell migration inhibitory effects, but the underlying mechanisms are poorly understood. In this manuscript, rhamnogalacturonan I (RGI)-rich pectins prepared from ginseng pectin were investigated for their effect on cell migration. The results indicated that the combination of homogalacturonan (HG) and RGI-rich pectins exerted stronger effects than either HG- or RGI-rich pectin alone. Further studies revealed that the effects of HG- and RGI-rich pectins were dependent on pretreatment, which caused alterations in cell morphologies such as cell size and shape, focal adhesion, and the organization of actin filaments, suggesting that HG and RGI pectins exert synergistic effects on cell migration, likely through different ways. Morphological data and quantitative cell adhesion and spreading assays showed that HG- and RGI-rich pectin treatment decreased cell adhesion and cell spreading on the substratum, suggesting that HG- and RGI-rich pectins may exert their effects on cell migration via decreasing cell adhesion and cell spreading. Additionally, we showed that L-929 cells expressed little galectin-3 (Gal-3) and that lactose, an inhibitor of Gal-3 did not block the activities of HG- and RGI-rich pectins, implicating that cell migration inhibited by pectin did not correlate to Gal-3.

Black rice-derived anthocyanins inhibit HER-2-positive breast cancer epithelial-mesenchymal transition-mediated metastasis in vitro by suppressing FAK signaling.

This study aimed to investigate the role of focal adhesion kinase (FAK) signaling in the inhibitory effects of black rice anthocyanins (BRACs) on human epidermal growth factor receptor-2 (HER-2)-positive human breast cancer cell metastasis, using the MCF-10A, MCF-7 and MDA-MB-453 cells. BRACs exerted an anti-metastatic effect on the HER-2-positive breast cancer cells. The effects of BRACs on the proliferation of the MDA-MB-453 cells were examined by cell counting kit-8 assay. A wound-healing assay was used to examine the effects of BRACs on the migration of the breast cancer cells. BRACs interrupted migration and invasion. BRACs decreased the migration distance of the HER-2-positive human breast cancer cells, MDA-MB-453, by 37% compared with the cells in the untreated group. They also reduced the number of invading MDA-MB-453 cells by 68%. In addition, BRACs exerted an inhibitory effect on epithelial-mesenchymal transition. Western blot analysis revealed that BRACs decreased the phosphorylation of FAK, cSrc and p130Cas. The FAK inhibitor, Y15, was also used to further evaluate the role of FAK signaling in the anti-metastatic effects of BRACs on MDA-MB-453 cells. The results of western blot analysis revealed that BRACs increased the expression of the epithelial marker, E-cadherin, and decreased the expression of the mesenchymal markers, fibronectin and vimentin, in the MDA-MB­453 cells. In addition, BRACs decreased the interaction between HER-2 and FAK, FAK and cSrc, cSrc and p130Cas, and between FAK and p130Cas. These results suggest that BRACs suppress the metastasis of HER-2-positive breast cancer in vitro, and that the cSrc/FAK/p130Cas pathway plays a vital role in this inhibitory effect.

SRVF, a novel herbal formula including Scrophulariae Radix and Viticis Fructus, disrupts focal adhesion and causes detachment-induced apoptosis in malignant cancer cells.

When cells lose adhesion, they undergo detachment-induced apoptosis, known as anoikis. In contrast, tumor cells acquire resistance to anoikis, enabling them to survive, even after separating from neighboring cells or the ECM. Therefore, agents that restore anoikis sensitivity may serve as anti-cancer candidates. In this study, we constructed a novel herbal formula, SRVF, which contains Scrophulariae Radix (SR) and Viticis Fructus (VF). SRVF rapidly decreased cell adhesion, altered the cell morphology to round, and induced cell death; however, SR, VF, or their co-treatment did not. SRVF arrested HT1080 cells in G2/M phase, increased the levels of pro-apoptotic proteins, and decreased the levels of anti-apoptotic proteins. Furthermore, SRVF efficiently reduced cell-cell and cell-ECM interactions by disrupting the F-actin cytoskeleton and down-regulating the levels of focal adhesion-related proteins, suggesting that SRVF efficiently triggers detachment-induced apoptosis (i.e., anoikis) in malignant cancer cells. In xenograft mouse models, daily oral administration of 50 or 100 mg/kg SRVF retarded tumor growth in vivo, and repeated administration of SRVF did not cause systemic toxicity in normal mice. These data collectively indicate that SRVF induces cancer cell death by restoring anoikis sensitivity via disrupting focal adhesion. Therefore, SRVF may be a safe and potent anti-cancer herbal decoction.

Inkjet printed periodical micropatterns made of inert alumina ceramics induce contact guidance and stimulate osteogenic differentiation of mesenchymal stromal cells.

Bioinert high performance ceramics exhibit detrimental features for implant components with direct bone contact because of their low osseointegrating capability. We hypothesized that periodical microstructures made of inert alumina ceramics can influence the osteogenic differentiation of human mesenchymal stromal cells (hMSC). In this study, we manufactured pillared arrays made of alumina ceramics with periodicities as low as 100µm and pillar heights of 40µm employing direct inkjet printing (DIP) technique. The response of hMSC to the microstructured surfaces was monitored by measuring cell morphology, viability and formation of focal adhesion complexes. Osteogenic differentiation of hMSCs was investigated by alkaline phosphatase activity, mineralization assays and expression analysis of respective markers. We demonstrated that MSCs react to the pillars with contact guidance. Subsequently, cells grow onto and form connections between the microstructures, and at the same time are directly attached to the pillars as shown by focal adhesion stainings. Cells build up tissue-like constructs with heights up to the micropillars resulting in increased cell viability and osteogenic differentiating properties. We conclude that periodical micropatterns on the micrometer scale made of inert alumina ceramics can mediate focal adhesion dependent cell adhesion and stimulate osteogenic differentiation of hMSCs.

System biology analysis of long-term effect and mechanism of Bufei Yishen on COPD revealed by system pharmacology and 3-omics profiling.

System pharmacology identified 195 potential targets of Bufei Yishen formula (BYF), and BYF was proven to have a short-term therapeutic effect on chronic obstructive pulmonary disease (COPD) rats previously. However, the long-term effect and mechanism of BYF on COPD is still unclear. Herein, we explored its long-term effect and underlying mechanism at system level. We administered BYF to COPD rats from week 9 to 20, and found that BYF could prevent COPD by inhibiting the inflammatory cytokines expression, protease-antiprotease imbalance and collagen deposition on week 32. Then, using transcriptomics, proteomics and metabolomics analysis, we identified significant regulated genes, proteins and metabolites in lung tissues of COPD and BYF-treated rats, which could be mainly attributed to oxidoreductase-antioxidant activity, focal adhesion, tight junction or lipid metabolism. Finally, based on the comprehensive analysis of system pharmacology target, transcript, protein and metabolite data sets, we found a number of genes, proteins, metabolites regulated in BYF-treated rats and the target proteins of BYF were involved in lipid metabolism, inflammatory response, oxidative stress and focal adhension. In conclusion, BYF exerts long-term therapeutic action on COPD probably through modulating the lipid metabolism, oxidative stress, cell junction and inflammatory response pathways at system level.

Quantitative proteomic analysis of milk fat globule membrane (MFGM) proteins in human and bovine colostrum and mature milk samples through iTRAQ labeling.

Milk fat globule membrane (MFGM) proteins have many functions. To explore the different proteomics of human and bovine MFGM, MFGM proteins were separated from human and bovine colostrum and mature milk, and analyzed by the iTRAQ proteomic approach. A total of 411 proteins were recognized and quantified. Among these, 232 kinds of differentially expressed proteins were identified. These differentially expressed proteins were analyzed based on multivariate analysis, gene ontology (GO) annotation and KEGG pathway. Biological processes involved were response to stimulus, localization, establishment of localization, and the immune system process. Cellular components engaged were the extracellular space, extracellular region parts, cell fractions, and vesicles. Molecular functions touched upon were protein binding, nucleotide binding, and enzyme inhibitor activity. The KEGG pathway analysis showed several pathways, including regulation of the actin cytoskeleton, focal adhesion, neurotrophin signaling pathway, leukocyte transendothelial migration, tight junction, complement and coagulation cascades, vascular endothelial growth factor signaling pathway, and adherens junction. These results enhance our understanding of different proteomes of human and bovine MFGM across different lactation phases, which could provide important information and potential directions for the infant milk powder and functional food industries.

Cell shape and the presentation of adhesion ligands guide smooth muscle myogenesis.

The reliable generation of smooth muscle cells is important for a number of tissue engineering applications. Human mesenchymal stem cells (MSCs) are a promising progenitor of smooth muscle, with high expression of smooth muscle markers observed in a fraction of isolated cells, which can be increased by introduction of soluble supplements that direct differentiation. Here we demonstrate a new micropatterning technique, where peptides of different ligand affinity can be microcontact printed onto an inert background, to explore MSC differentiation to smooth muscle through controlled biochemical and biophysical cues alone. Using copper-catalyzed alkyne-azide cycloaddition (CuAAC), we patterned our surfaces with RGD peptide ligands-both a linear peptide with low integrin affinity and a cyclic version with high integrin affinity-for the culture of MSCs in shapes with various aspect ratios. At low aspect ratio, ligand affinity is a prime determinant for smooth muscle differentiation, while at high aspect ratio, ligand affinity has less of an effect. Pathway analysis reveals a role for focal adhesion turnover, Rac1, RhoA/ROCK, and calpain during smooth muscle differentiation of MSCs in response to cell shape and the affinity of the cell adhesion interface. Controlling integrin-ligand affinity at the biomaterials interface is important for mediating adhesion but may also prove useful for directing smooth muscle myogenesis. Peptide patterning enables the systematic investigation of single to multiple peptides derived from any protein, at different densities across a biomaterials surface, which has the potential to direct multiple MSC differentiation outcomes without the need for soluble supplements.

In Vitro and In Vivo Anti-Melanoma Effects of Pituranthos tortuosus Essential Oil Via Inhibition of FAK and Src Activities.

A large number of plants used in traditional medicines have been shown to possess antitumor activities. The aims of this study were to evaluate any anticancer effect of the essential oil (EO) extracted from P. tortuosus against B16F10 melanoma cancer cells in vitro as well as in vivo. In vitro, EO was shown to induce apoptosis and to inhibit migration and invasion processes. Further investigation revealed that EO decreased focal adhesion and invadopodia formation which was accompanied by a drastic downregulation of FAK, Src, ERK, p130Cas and paxillin. Moreover, EO treatment decreased the expression level of p190RhoGAP, and Grb2, which impair cell migration and actin assembly. Mice bearing the melanoma cells were used to confirm any in vivo effectiveness of the EO as an anti-tumor promoting agent. In mice dosed with 100 mg EO/kg/d (for 27 days), tumor weight was inhibited by 98% compared to that in mice that did not receive the product. In conclusion, these data suggested to us that an EO of P. tortuosus could evolve to be a potential medicinal resource for use in the treatment of cancers.

Promotion of human mesenchymal stem cell osteogenesis by PI3-kinase/Akt signaling, and the influence of caveolin-1/cholesterol homeostasis.

INTRODUCTION: Stem cells are considered an important resource for tissue repair and regeneration. Their utilization in regenerative medicine will be aided by mechanistic insight into their responsiveness to external stimuli. It is likely that, similar to all other cells, an initial determinant of stem cell responsiveness to external stimuli is the organization of signaling molecules in cell membrane rafts. The clustering of signaling molecules in these cholesterol-rich membrane microdomains can affect the activity, specificity, cross-talk and amplification of cell signaling. Membrane rafts fall into two broad categories, non-caveolar and caveolar, based on the absence or presence, respectively, of caveolin scaffolding proteins. We have recently demonstrated that caveolin-1 (Cav-1) expression increases during, and knockdown of Cav-1 expression enhances, osteogenic differentiation of human bone marrow derived mesenchymal stem cells (MSCs). The increase in Cav-1 expression observed during osteogenesis is likely a negative feedback mechanism. We hypothesize that focal adhesion signaling pathways such as PI3K/Akt signaling may be negatively regulated by Cav-1 during human MSC osteogenesis. METHODS: Human bone marrow MSCs were isolated from femoral heads obtained after total hip arthroplasty. MSCs were incubated in standard growth medium alone or induced to osteogenically differentiate by the addition of supplements (ß-glycerophosphate, ascorbic acid, dexamethasone, and 1,25-dihydroxyvitamin D3). The activation of and requirement for PI3K/Akt signaling in MSC osteogenesis were assessed by immunoblotting for phosphorylated Akt, and treatment with the PI3K inhibitor LY294002 and Akt siRNA, respectively. The influences of Cav-1 and cholesterol membrane rafts on PI3K/Akt signaling were investigated by treatment with Cav-1 siRNA, methyl-ß-cyclodextrin, or cholesterol oxidase, followed by cellular sub-fractionation and/or immunoblotting for phosphorylated Akt. RESULTS: LY294002 and Akt siRNA inhibited MSC osteogenesis. Methyl-ß-cyclodextrin, which disrupts all membrane rafts, inhibited osteogenesis. Conversely, Cav-1 siRNA and cholesterol oxidase, which displaces Cav-1 from caveolae, enhanced Akt signaling induced by osteogenic supplements. In control cells, phosphorylated Akt began to accumulate in caveolae after 10 days of osteogenic differentiation. CONCLUSIONS: PI3K/Akt signaling is a key pathway required for human MSC osteogenesis, and it is likely that localization of active Akt in non-caveolar and caveolar membrane rafts positively and negatively contributes to osteogenesis, respectively.

Preclinical evaluation of the supercritical extract of azadirachta indica (neem) leaves in vitro and in vivo on inhibition of prostate cancer tumor growth.

Azadirachta indica, commonly known as neem, has gained worldwide prominence because of its medical properties, namely antitumor, antiviral, anti-inflammatory, antihyperglycemic, antifungal, and antibacterial activities. Despite these promising results, gaps remain in our understanding of the molecular mechanism of action of neem compounds and their potential for use in clinical trials. We investigated supercritical extract of neem leaves (SENL) for the following: molecular targets in vitro, in vivo efficacy to inhibit tumor growth, and bioactive compounds that exert antitumor activity. Treatment of LNCaP-luc2 prostate cancer cells with SENL suppressed dihydrotestosterone-induced androgen receptor and prostate-specific antigen levels. SENL inhibited integrin ß1, calreticulin, and focal adhesion kinase activation in LNCaP-luc2 and PC3 prostate cancer cells. Oral administration of SENL significantly reduced LNCaP-luc2 xenograft tumor growth in mice with the formation of hyalinized fibrous tumor tissue, reduction in the prostate-specific antigen, and increase in AKR1C2 levels. To identify the active anticancer compounds, we fractionated SENL by high-pressure liquid chromatography and evaluated 16 peaks for cytotoxic activity. Four of the 16 peaks exhibited significant cytotoxic activity against prostate cancer cells. Mass spectrometry of the isolated peaks suggested the compounds with cytotoxic activity were nimbandiol, nimbolide, 2',3'-dihydronimbolide, and 28-deoxonimbolide. Analysis of tumor tissue and plasma samples from mice treated with SENL indicated 28-deoxonimbolide and nimbolide as the bioactive compounds. Overall, our data revealed the bioactive compounds in SENL and suggested that the anticancer activity could be mediated through alteration in androgen receptor and calreticulin levels in prostate cancer.