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Purpose Hydrogen sulphide (H2S) is an important signalling molecule involved in the regulation of several physiological and pathophysiological processes. The objective of this study was to investigate the feasibility of transdermal delivery of ADT-OH, a H2S donor, by investigating the transdermal flux of aqueous gels loaded with penetration enhancers or liposomes. Furthermore, we explored the ability of permeated ADT-OH to promote angiogenesis and mitochondrial bioenergetics in HUVEC cells. Methods Aqueous hypromellose gels (5% w/v) were prepared with up to 10% v/v propylene glycol (PG) or deformable liposomes with 0.025% w/w ADT-OH. ADT-OH permeation from formulations across excised murine skin into PBS was quantified over 24 h using HPLC-UV detection. Media was collected and applied to HUVEC cells to evidence ADT-OH functionality following permeation. Tube formation assays were performed as indicative of angiogenesis and mitochondrial oxygen consumption was evaluated using a Seahorse XF24. Results Increasing the loading of PG caused an increase in ADT-OH permeation rate across skin and a decrease in dermal drug retention whereas liposomal gels produced a slow-release profile. Treatment of HUVEC’s using conditioned media collected from the ADT-OH loaded permeation studies enhanced tube formation and the basal oxygen consumption rates after 30 min of treatment. Conclusions These findings demonstrate that transdermal delivery of ADT-OH may provide a promising approach in the treatment of impaired vascular function. Gels prepared with 10% v/v PG have the potential for use in conditions requiring rapid H2S release whereas liposomal loaded gels for treatment requiring sustained H2S release.

Vascular supply of tumors is one of the main targets for cancer therapy. Here, we investigated if plocabulin (PM060184), a novel marine-derived microtubule-binding agent, presents antiangiogenic and vascular-disrupting activities., info:eu-repo/semantics/published

Acquired resistance during BRAF inhibitor therapy remains a major challenge for melanoma treatment. Accordingly, we evaluated the phenotypical and molecular changes of isogeneic human V600E BRAF-mutant melanoma cell line pairs pre- and post-treatment with vemurafenib. Three treatment na&iuml, ve lines were subjected to in vitro long-term vemurafenib treatment while three pairs were pre- and post-treatment patient-derived lines. Molecular and phenotypical changes were assessed by Sulforhodamine-B (SRB) assay, quantitative RT-PCR (q-RT-PCR), immunoblot, and time-lapse microscopy. We found that five out of six post-treatment cells had higher migration activity than pretreatment cells. However, no unequivocal correlation between increased migration and classic epithelial&ndash, mesenchymal transition (EMT) markers could be identified. In fast migrating cells, the microphthalmia-associated transcription factor (MITF) and epidermal growth factor receptor (EGFR) mRNA levels were considerably lower and significantly higher, respectively. Interestingly, high EGFR expression was associated with elevated migration but not with proliferation. Cells with high EGFR expression showed significantly decreased sensitivity to vemurafenib treatment, and had higher Erk activation and FRA-1 expression. Importantly, melanoma cells with higher EGFR expression were more resistant to the EGFR inhibitor erlotinib treatment than cells with lower expression, with respect to both proliferation and migration inhibition. Finally, EGFR-high melanoma cells were characterized by higher PD-L1 expression, which might in turn indicate that immunotherapy may be an effective approach in these cases.

Estrogen promotes growth of estrogen receptor-positive (ER+) breast tumors. However, epidemiological studies examining the prognostic characteristics of breast cancer in postmenopausal women receiving hormone replacement therapy reveal a significant decrease in tumor dissemination, suggesting that estrogen has potential protective effects against cancer cell invasion. Here, we show that estrogen suppresses invasion of ER+ breast cancer cells by increasing transcription of the Ena/VASP protein, EVL, which promotes the generation of suppressive cortical actin bundles that inhibit motility dynamics, and is crucial for the ER-mediated suppression of invasion in vitro and in vivo. Interestingly, despite its benefits in suppressing tumor growth, anti-estrogenic endocrine therapy decreases EVL expression and increases local invasion in patients. Our results highlight the dichotomous effects of estrogen on tumor progression and suggest that, in contrast to its established role in promoting growth of ER+ tumors, estrogen has a significant role in suppressing invasion through actin cytoskeletal remodeling., Whilst estrogen is known to be tumorigenic in some breast cancer, in some contexts it can be protective against invasion and dissemination. Here, the authors show estrogen can promote generation of Suppressive Cortical Actin Bundles that can inhibit motility dynamics through EVL-mediated actin cytoskeletal remodeling.

Mushrooms comprise an unlimited source of active compounds that have beneficial health effects without known negative side effects and can potentially be used as important therapeutic products against cancer, which is the leading cause of death worldwide. In this study we investigated the cytotoxic, antiproliferative, apoptotic, and anti-invasion effects of Macrolepiota procera, which is valued as an edible and medicinal mushroom, on A549 lung cancer cells. The cytotoxic effect of the M. procera extract was determined by using the XTT method. Total RNA was isolated from cells with TRI Reagent to determine the apoptotic effect of the extract, after which complementary DNA was synthesized. Expression profiles of the target genes were determined by quantitative reverse-transcriptase polymerase chain reaction, and protein changes were determined by using Western blotting. We used the TUNEL assay to evaluate the apoptotic effects of the M. procera extract. Effects of M. procera on cell invasion were investigated by using a Matrigel chamber assay. The half-maximal inhibitory concentration of the M. procera extract was determined to be 2 mg/mL against A549 lung cancer cells at 72 hours. According to our results, expression of Cyclin Dl, CDK4, CDK6, Bcl-2, Akt, and NOXA genes significantly decreased and that of Bax, Caspase-3, Caspase-9, PTEN, PUMA, p21, and p53 increased in cells from the dose group compared with their expression in control cells. According to the results of the TUNEL assay, 28 ± 3.6% of cells were apoptotic in the dose group. The M. procera extract also reduced invasion in A549 cancer cells. The results suggest that M. procera has an antiproliferative effect in a dose- and time-dependent manner.

Ruthenium-based compounds show strong potential as anti-cancer drugs and are being investigated as alternatives to other well-established metal-based chemotherapeutics. The organometallic compound [Ru(η6-p-cymene)Cl2(pta)], where pta = 1,3,5-triaza-7-phosphaadamantane (RAPTA-C) exhibits broad acting anti-tumor efficacy with intrinsic angiostatic activity. In the search for an optimal anti-angiogenesis drug combination, we identified synergistic potential between RAPTA-C and the epidermal growth factor receptor (EGFR) inhibitor, erlotinib. This drug combination results in strong synergistic inhibition of cell viability in human endothelial (ECRF24 and HUVEC) and human ovarian carcinoma (A2780 and A2780cisR) cells. Additionally, erlotinib significantly enhances the cellular uptake of RAPTA-C relative to treatment with RAPTA-C alone in human ovarian carcinoma cells, but not endothelial cells. Drug combinations induce the formation of chromosome bridges that persist after mitotic exit and delay abscission in A2780 and A2780cisR, therefore suggesting initiation of cellular senescence. The therapeutic potential of these compounds and their combination is further validated in vivo on A2780 tumors grown on the chicken chorioallantoic membrane (CAM) model, and in a preclinical model in nude mice. Immunohistochemical analysis confirms effective anti-angiogenic and anti-proliferative activity in vivo, based on a significant reduction of microvascular density and a decrease in proliferating cells.

Squamous cell carcinoma of the skin (SCC) represents one of the most common cancers in the general population and is associated with a substantial risk of metastasis. Previous work uncovered the functional role of CYFIP1 in epithelial tumors as an invasion inhibitor. It was down-regulated in some cancers and correlated with the metastatic properties of these malignant cells. We investigated its role and expression mechanisms in SCC. We analyzed the expression of CYFIP1 in patient derived SCC, primary keratinocytes and SCC cell lines, and correlated it to the differentiation and NOTCH1 levels. We analyzed the effects of Notch1 manipulation on CYFIP1 expression and confirmed the biding of Notch1 to the CYFIP1 promoter. CYFIP1 expression was down-regulated in SCC and correlated inversely with histological differentiation of tumors. As keratinocyte differentiation depends on Notch1 signaling, we investigated the influence of Notch1 on CYFIP1 expression. CYFIP1 mRNA was highly increased in human Notch1-overexpressing keratinocytes. Further manipulation of the Notch1 pathway in keratinocytes impacted CYFIP1 levels and chromatin immunoprecipitation assay confirmed the direct binding of Notch1 to the CYFIP1 promoter. CYFIP1 may be a link between loss of differentiation and invasive potential in malignant keratinocytes of cutaneous squamous cell carcinoma.

Neuroblastoma is one of the most common types of pediatric tumors that can spread quickly in neuronal tissues. Oleuropein which is active compound of olive leaves, belongs to polyphenols group and has antioxidant, anti-microbial, anti-inflammatory, anti-hypertensive and anti-carcinogenic effects. The aim of the study is to determine the therapeutic effects of oleuropein on cell proliferation, invasion, colony formation, cell cycle and apoptotic mechanisms in SH-SY5Y neuroblastoma cell line under in vitro conditions. The effect of oleuropein on cell viability was determined by XTT method. 84 cell cycle control and 84 apoptosis related genes were evaluated by RT-PCR. Effects of oleuropein on apoptosis were researched by TUNEL assay. Protein expressions were determined by western blot analysis. Effects of oleuropein on cell invasion, colony formation and migration were detected by matrigel-chamber, colony formation assay and wound-healing assay, respectively. IC50 value of oleuropein in SH-SY5Y cells was detected as 350 μM at 48th hours. It is determined that oleuropein causes cell cycle arrest by down-regulating of CylinD1,CylinD2,CyclinD3,CDK4,CDK6 and up-regulating of p53 and CDKN2A, CDKN2B, CDKN1A gene expressions. Oleuropein also induces apoptosis by inhibiting of Bcl-2 and activating of Bax,caspase-9 and caspase-3 gene expressions. Apoptotic cell ratio was found 36.4 ± 3.27% in oleuropein dose group. Oleuropein decreased invasion in SH-SY5Y cells and suppressed colony numbers in ratio of 53.6 ± 4.71%.Our results demonstrated that oleuropein can be a therapeutic agent in the treatment of neuroblastoma.

Exendin-4 is a GLP-1 analog used for the treatment of type 2 diabetes mellitus in its synthetic form. As women with diabetes have higher breast cancer incidence and mortality, we examined the effect of the incretin drug exendin-4 on breast cancer cells. The aim of the study is to investigate anticancer mechanism of exendin-4 in MCF-7 breast cancer cells. Cytotoxic effects of exendin-4 were determined by XTT assay. IC50 dose in MCF-7 cells were detected as 5 μM at 48th hour. Gene messenger RNA (mRNA) expressions were evaluated by real-time PCR. According to results, caspase-9, Akt, and MMP2 expression was reduced in dose group cells, compared with the control group cells. p53, caspase-3, caspase-8, caspase-10, BID, DR4, DR5, FADD, TRADD, PARP, PTEN, PUMA, NOXA, APAF, TIMP1, and TIMP2 expression was increased in dose group cells, compared with the control group cells. Effects of exendin-4 on cell invasion, colony formation, and cell migration were detected by Matrigel chamber, colony formation assay, and wound-healing assay, respectively. To conclude, it is thought that exendin-4 demonstrates anticarcinogenesis activity by effecting apoptosis, invasion, migration, and colony formation in MCF-7 cells. Exendin-4 may be a therapeutic agent for treatment of breast cancer as single or in combination with other agents. More detailed researches are required to define the pathways of GLP-1 effect on breast cancer cells because of the molecular biology of breast cancer that involves a complex network of interconnected signaling pathways that have role in cell growth, survival, and cell invasion.

Objective— Arterial injury stimulates remodeling responses that, when excessive, lead to stenosis. These responses are influenced by integrin signaling in vascular smooth muscle cells (VSMCs). Microfibrillar-associated protein 4 (MFAP4) is an integrin ligand localized to extracellular matrix fibers in the vascular wall. The role of MFAP4 in vascular biology is unknown. We aimed to test the hypothesis that MFAP4 would enhance integrin-dependent VSMC activation. Approach and Results— We produced Mfap4 -deficient ( Mfap4 −/− ) mice and performed carotid artery ligation to explore the role of MFAP4 in vascular biology in vivo. Furthermore, we investigated the effects of MFAP4 in neointimal formation ex vivo and in primary VSMC and monocyte cultures in vitro. When challenged with carotid artery ligation, Mfap4 −/− mice exhibited delayed neointimal formation, accompanied by early reduction in the number of proliferating medial and neointimal cells, as well as infiltrating leukocytes. Delayed neointimal formation was associated with decreased cross-sectional area of ligated Mfap4 −/− carotid arteries resulting in lumen narrowing 28 days after ligation. MFAP4 blockade prohibited the formation of neointimal hyperplasia ex vivo. Moreover, we demonstrated that MFAP4 is a ligand for integrin α V β 3 and mediates VSMC phosphorylation of focal adhesion kinase, migration, and proliferation in vitro. MFAP4-dependent VSMC activation was reversible by treatment with MFAP4-blocking antibodies and inhibitors of focal adhesion kinase and downstream kinases. In addition, we showed that MFAP4 promotes monocyte chemotaxis in integrin α V β 3 –dependent manner. Conclusions— MFAP4 regulates integrin α V β 3 –induced VSMC proliferation and migration, as well as monocyte chemotaxis, and accelerates neointimal hyperplasia after vascular injury.

Purpose: Vascular adhesion protein (VAP)-1 is an adhesion molecule elucidated as a mediator of the leukocyte recruitment cascade. The purpose of this study was to investigate the role of VAP-1 in ocular inflammatory neovascularization using a mouse laser-induced choroidal neovascularization (CNV) model. Methods: CNV was induced with 532 nm laser irradiation in C57BL/6 mice, and production of VAP-1 protein in the retinal pigment epithelium (RPE) choroid during CNV formation was examined. CNV animals were treated with the specific VAP-1 inhibitor U-V002 or vehicle solution, and the volume of CNV tissue was evaluated with volumetric measurements. Macrophage infiltration into the CNV lesions was evaluated using two different techniques, flatmount staining and real-time polymerase chain reaction (PCR) for F4/80. The protein levels of intercellular adhesion molecule (ICAM)-1, monocyte chemoattractant protein (MCP)-1, P-selectin, and vascular endothelial growth factor (VEGF) in the RPE-choroid were measured with enzyme-linked immunosorbent assay (ELISA). Results: VAP-1 inhibition significantly suppressed CNV formation in a dose-dependent manner and reduced macrophage infiltration into CNV lesions. Furthermore, VAP-1 blockade decreased the expression of ICAM-1 and MCP-1, both of which play a pivotal role in macrophage recruitment. Conclusions: Our data suggest VAP-1 has an important role during ocular inflammatory neovascularization through leukocyte recruitment. VAP-1 inhibition may be a novel and potent therapeutic strategy in treating CNV formation.

International audience; IL-34 is a challenging cytokine sharing functional similarities with M-CSF through M-CSFR activation. It also plays a singular role that has recently been explained in the brain, through a binding to the receptor protein tyrosine phosphatase RPTPβ/ζ. The aim of this paper was to look for alternative binding of IL-34 on other cell types. Myeloid cells (HL-60, U-937, THP-1) were used as cells intrinsically expressing M-CSFR, and M-CSFR was expressed in TF-1 and HEK293 cells. IL-34 binding was studied by Scatchard and binding inhibition assays, using 125I-radiolabelled cytokines, and surface plasmon resonance. M-CSFR activation was analysed by Western blot after glycosaminoglycans abrasion, syndecan-1 overexpression or repression and addition of a blocking anti-syndecan antibody. M-CSF and IL-34 induced different patterns of M-CSFR phosphorylations, suggesting the existence of alternative binding for IL-34. Binding experiments and chondroitinase treatment confirmed low affinity binding to chondroitin sulphate chains on cells lacking both M-CSFR and RPTPβ/ζ. Amongst the proteoglycans with chondroitin sulphate chains, syndecan-1 was able to modulate the IL-34-induced M-CSFR signalling pathways. Interestingly, IL-34 induced the migration of syndecan-1 expressing cells. Indeed, IL-34 significantly increased the migration of THP-1 and M2a macrophages that was inhibited by addition of a blocking anti-syndecan-1 antibody. This paper provides evidence of alternative binding of IL-34 to chondroitin sulphates and syndecan-1 at the cell surface that modulates M-CSFR activation. In addition, IL-34-induced myeloid cell migration is a syndecan-1 dependent mechanism.

International audience; IL-34 is a challenging cytokine sharing functional similarities with M-CSF through M-CSFR activation. It also plays a singular role that has recently been explained in the brain, through a binding to the receptor protein tyrosine phosphatase RPTPβ/ζ. The aim of this paper was to look for alternative binding of IL-34 on other cell types. Myeloid cells (HL-60, U-937, THP-1) were used as cells intrinsically expressing M-CSFR, and M-CSFR was expressed in TF-1 and HEK293 cells. IL-34 binding was studied by Scatchard and binding inhibition assays, using 125I-radiolabelled cytokines, and surface plasmon resonance. M-CSFR activation was analysed by Western blot after glycosaminoglycans abrasion, syndecan-1 overexpression or repression and addition of a blocking anti-syndecan antibody. M-CSF and IL-34 induced different patterns of M-CSFR phosphorylations, suggesting the existence of alternative binding for IL-34. Binding experiments and chondroitinase treatment confirmed low affinity binding to chondroitin sulphate chains on cells lacking both M-CSFR and RPTPβ/ζ. Amongst the proteoglycans with chondroitin sulphate chains, syndecan-1 was able to modulate the IL-34-induced M-CSFR signalling pathways. Interestingly, IL-34 induced the migration of syndecan-1 expressing cells. Indeed, IL-34 significantly increased the migration of THP-1 and M2a macrophages that was inhibited by addition of a blocking anti-syndecan-1 antibody. This paper provides evidence of alternative binding of IL-34 to chondroitin sulphates and syndecan-1 at the cell surface that modulates M-CSFR activation. In addition, IL-34-induced myeloid cell migration is a syndecan-1 dependent mechanism.

To improve well-known titanium implants, pores can be used for increasing bone formation and close bone-implant interface. Selective Laser Melting (SLM) enables the production of any geometry and was used for implant production with 250-microm pore size. The used pore size supports vessel ingrowth, as bone formation is strongly dependent on fast vascularization. Additionally, proangiogenic factors promote implant vascularization. To functionalize the titanium with proangiogenic factors, polycaprolactone (PCL) coating can be used. The following proangiogenic factors were examined: vascular endothelial growth factor (VEGF), high mobility group box 1 (HMGB1) and chemokine (C-X-C motif) ligand 12 (CXCL12). As different surfaces lead to different cell reactions, titanium and PCL coating were compared. The growing into the porous titanium structure of primary osteoblasts was examined by cross sections. Primary osteoblasts seeded on the different surfaces were compared using Live Cell Imaging (LCI). Cross sections showed cells had proliferated, but not migrated after seven days. Although the cell count was lower on titanium PCL implants in LCI, the cell count and cell spreading area development showed promising results for titanium PCL implants. HMGB1 showed the highest migration capacity for stimulating the endothelial cell line. Future perspective would be the incorporation of HMGB1 into PCL polymer for the realization of a slow factor release. DFG/299/11-1

BACKGROUND: Interleukin-8 (IL-8, CXCL8) is readily produced by human malignant cells. Dendritic cells (DC) both produce IL-8 and express the IL-8 functional receptors CXCR1 and CXCR2. Most human colon carcinomas produce IL-8. IL-8 importance in malignancies has been ascribed to angiogenesis promotion. PRINCIPAL FINDINGS: IL-8 effects on human monocyte-derived DC biology were explored upon DC exposure to recombinant IL-8 and with the help of an IL-8 neutralizing mAb. In vivo experiments were performed in immunodeficient mice xenografted with IL-8-producing human colon carcinomas and comparatively with cell lines that do not produce IL-8. Allogenic T lymphocyte stimulation by DC was explored under the influence of IL-8. DC and neutrophil chemotaxis were measured by transwell-migration assays. Sera from tumor-xenografted mice contained increasing concentrations of IL-8 as the tumors progress. IL-8 production by carcinoma cells can be modulated by low doses of cyclophosphamide at the transcription level. If human DC are injected into HT29 or CaCo2 xenografted tumors, DC are retained intratumorally in an IL-8-dependent fashion. However, IL-8 did not modify the ability of DC to stimulate T cells. Interestingly, pre-exposure of DC to IL-8 desensitizes such cells for IL-8-mediated in vitro or in vivo chemoattraction. Thereby DC become disoriented to subsequently follow IL-8 chemotactic gradients towards malignant or inflamed tissue. CONCLUSIONS: IL-8 as produced by carcinoma cells changes DC migration cues, without directly interfering with DC-mediated T-cell stimulation.

BACKGROUND: Posttranslational modification of chemokines is one of the mechanisms that regulate leukocyte migration during inflammation. Multiple natural NH(2)-terminally truncated forms of the major human neutrophil attractant interleukin-8 or CXCL8 have been identified. Although differential activity was reported for some CXCL8 forms, no biological data are available for others. METHODOLOGY/PRINCIPAL FINDINGS: Aminopeptidase-cleaved CXCL8(2-77) and CXCL8(3-77), the product of alternative cleavage of the signal peptide CXCL8(-2-77) and the previously studied forms containing 77 and 72 amino acids, CXCL8(1-77) and CXCL8(6-77), were prepared by solid-phase peptide synthesis, purified and folded into active proteins. No differences in binding and calcium signaling potency were detected between CXCL8(1-77), CXCL8(-2-77), CXCL8(2-77) and CXCL8(3-77) on cells transfected with one of the human CXCL8 receptors, i.e. CXCR1 and CXCR2. However, CXCL8(-2-77) was more potent compared to CXCL8(1-77), CXCL8(2-77) and CXCL8(3-77) in signaling and in vitro chemotaxis of peripheral blood-derived human neutrophils. Moreover, CXCL8(-2-77) was less efficiently processed by plasmin into the more potent CXCL8(6-77). The truncated forms CXCL8(2-77) and CXCL8(3-77) had higher affinity for heparin than CXCL8(1-77), a property important for the presentation of CXCL8 on endothelial layers. Upon intraperitoneal injection in mice, elongated, truncated and intact CXCL8 were equally potent to recruit neutrophils to the peritoneal cavity. CONCLUSIONS: In terms of their ability to induce neutrophil recruitment in vivo, the multiple CXCL8 forms may be divided in three groups. The first group includes CXCL8 proteins consisting of 75 to 79 amino acids, cleaved by aminopeptidases, with intermediate activity on neutrophils. The second group, generated through proteolytic cleavage (e.g. by Ser proteases), contains 69 to 72 amino acid forms which are highly potent neutrophil attractants in vivo. A third category is generated through the modification of the arginine in the NH(2)-terminal region into citrulline by peptidylarginine deiminases and has weak potency to induce neutrophil extravasation.

Background Activating transcription factor-3 (ATF3) is involved in the complex process of cellular stress response. However, its exact role in cancer is discussed controversially because both tumor suppressive and oncogenic effects have been described. Here we followed-up on our previous observation that inhibition of Hsp90 may increase ATF3 expression and sought to determine the role of ATF3 in colon cancer. Methods Regulation of ATF3 was determined in cancer cells using signaling inhibitors and a heat-shock protein-90 (Hsp90) antagonist. Human HCT116 cancer cells were stably transfected with an ATF3-shRNA or a luciferase-shRNA expression plasmid and alterations in cell motility were assessed in migration assays. The impact of ATF3 down-regulation on cancer growth and metastasis were investigated in a subcutaneous tumor model, a model of hepatic tumor growth and in a model of peritoneal carcinomatosis. Human colon cancer tissues were analyzed for ATF3 expression. Results The results show that therapeutic Hsp90 inhibition substantially up-regulates the expression of ATF3 in various cancer cells, including colon, gastric and pancreatic cancer. This effect was evident both in vitro and in vivo. RNAi mediated knock-down of ATF3 in HCT116 colon cancer cells significantly increased cancer cell migration in vitro. Moreover, in xenogenic mouse models, ATF3 knock-down promoted subcutaneous tumor growth and hepatic metastasis, as well as peritoneal carcinomatosis. Importantly, ATF3 expression was lower in human colon cancer specimens, as compared to corresponding normal surrounding tissues, suggesting that ATF3 may represent a down-regulated tumor suppressor in colon cancer. Conclusion In conclusion, ATF3 down-regulation in colon cancer promotes tumor growth and metastasis. Considering that blocking Hsp90 induces ATF3 expression, Hsp90 inhibition may represent a valid strategy to treat metastatic colon cancer by up-regulating this anti-metastatic transcription factor.

Raf kinase inhibitor protein (RKIP) regulates a number of cellular processes, including cell migration. Exploring the role of RKIP in cell adhesion, we found that overexpression of RKIP in Madin-Darby canine kidney (MDCK) epithelial cells increases adhesion to the substratum, while decreasing adhesion of the cells to one another. The level of the adherens junction protein E-cadherin declines profoundly, and there is loss of normal localization of the tight junction protein ZO-1, while expression of the cell-substratum adhesion protein beta1 integrin dramatically increases. The cells also display increased adhesion and spreading on multiple substrata, including collagen, gelatin, fibronectin and laminin. In three-dimensional culture, RKIP overexpression leads to marked cell elongation and extension of long membrane protrusions into the surrounding matrix, and the cells do not form hollow cysts. RKIP-overexpressing cells generate considerably more contractile traction force than do control cells. In contrast, RNA interference-based silencing of RKIP expression results in decreased cell-substratum adhesion in both MDCK and MCF7 human breast adenocarcinoma cells. Treatment of MDCK and MCF7 cells with locostatin, a direct inhibitor of RKIP and cell migration, also reduces cell-substratum adhesion. Silencing of RKIP expression in MCF7 cells leads to a reduction in the rate of wound closure in a scratch-wound assay, although not as pronounced as that previously reported for RKIP-knockdown MDCK cells. These results suggest that RKIP has important roles in the regulation of cell adhesion, positively controlling cell-substratum adhesion while negatively controlling cell-cell adhesion, and underscore the complex functions of RKIP in cell physiology.

Nicotine, one of the major components in tobacco, is at high concentrations in the bloodstream of cigarette smokers. However, the mechanisms of how nicotine affects tumor development and whether nicotine is a potential carcinogen for malignancies induced by secondhand smoking are not fully understood yet. Here, we investigate the signaling pathways by which nicotine potentiates tumorigenesis in human mammary epithelial-like MCF10A or cancerous MCF7 cells. We show that human MCF10A and MCF7 cells both express four subunits of nicotine acetylcholine receptor (nAChR). The treatment of these cells with nicotine enhances the activity of protein kinase C (PKC) α without altering the expression level of this kinase. Nicotine also stimulates [3H]thymidine incorporation into the genome of these cells as well as forces serum-starved cells to enter S phase of the cell cycle, resulting in growth promotion. Importantly, on nicotine treatment, the mobility of MCF10A and MCF7 cells is enhanced, which can be blocked by the addition of nAChR or PKC inhibitor. Experiments using small interfering RNA knockdown or ectopic expression of cdc42 showed that cdc42 functions as a downstream effector of PKC and is crucial in the regulation of nicotine-mediated migratory activity in the cells. Together, our findings suggest that nicotine, through interacting with its receptor, initiates a signaling cascade that involves PKC and cdc42 and consequently promotes migration in mammary epithelial or tumor cells. [Cancer Res 2008;68(20):8473–81]

SummaryAmple evidence suggests that many of the in vivo anti-metastatic effects by heparins reflect their actions on P-selectin-mediated binding. We hypothesized that the ability of widely used heparins and derivatives to interfere with P-selectin-dependent tumour cell interactions under flow in vitro could be used to identify anticoagulants with advanced inhibitory functions on experimental blood-borne metastasis in vivo. To test this assumption, the impact of unfractionated heparin, the low-molecular-weight heparins (LMWH) nadroparin and enoxaparin, and the synthetic pentasaccharide fondaparinux on P-selectin-dependent tumour interactions in vitro and metastasis formation in vivo were evaluated. Our data revealed that these commonly used anticoagulants widely differ in their potential to interfere with P-selectinmediated cell binding. Importantly, the superior inhibitory capacity on P-selectin function of unfractionated heparin and LMWH nadroparin as opposed to LMWH enoxaparin and synthetic heparin pentasaccharide fondaparinux strongly correlated to the inhibitory potency of each in inhibiting experimental lung metastasis in vivo. Hence, P-selectin inhibition may constitute a valuable feature to identify anticoagulants that are suitable for anticancer therapy.

The tumor suppressor function of activin A, together with our findings that activin A is an inhibitor of angiogenesis, which is down-regulated by the N-MYC oncogene, prompted us to investigate in more detail its role in the malignant transformation process of neuroblastomas. Indeed, neuroblastoma cells with restored activin A expression exhibited a diminished proliferation rate and formed smaller xenograft tumors with reduced vascularity, whereas lung metastasis rate remained unchanged. In agreement with the decreased vascularity of the xenograft tumors, activin A inhibited several crucial angiogenic responses of cultured endothelial cells, such as proteolytic activity, migration, and proliferation. Endothelial cell proliferation, activin A, or its constitutively active activin receptor-like kinase 4 receptor (ALK4T206D), increased the expression of CDKN1A (p21), CDKN2B (p15), and CDKN1B (p27) CDK inhibitors and down-regulated the expression of vascular endothelial growth factor receptor-2, the receptor of a key angiogenic factor in cancer. The constitutively active forms of SMAD2 and SMAD3 were both capable of inhibiting endothelial cell proliferation, whereas the dominant-negative forms of SMAD3 and SMAD4 released the inhibitory effect of activin A on endothelial cell proliferation by only 20%. Thus, the effects of activin A on endothelial cell proliferation seem to be conveyed via the ALK4/SMAD2-SMAD3 pathways, however, non-SMAD cascades may also contribute. These results provide novel information regarding the role of activin A in the malignant transformation process of neuroblastomas and the molecular mechanisms involved in regulating angiogenesis thereof. Cancer Res

Integrins are a major family of heterodimeric surface glycoproteins that act as adhesion molecules, have a spectrum of extracellular matrix (ECM) molecules as their ligands, and regulate a variety of cellular functions. Integrins are known to be critical to embryonic brain development, and recent studies have indicated their essential role in adult brain function, although their role in postnatal brain development and function has not been examined. Here, we used the organotypic slice culture system to investigate the role of integrins in postnatal hippocampal development by exposing the tissue to either an integrin competitive antagonist, the peptide GRGDSP containing Arg-Gly-Asp (RGD) attachment site, or to function-blocking beta(1)-integrin antibodies to disrupt integrin interactions. These experiments revealed that beta(1)-integrin antibodies interfered with spreading of the culture, resulting in a rapid and marked diminution of slice area. beta(1)-integrin antibodies and RGD peptide disrupted cell adhesion, causing cell detachment and migration of glial cells from the explant. The majority of the detached cells were of macroglial origin and switched to expression of the intermediate filament proteins vimentin and nestin, suggesting a developmental regression. The organotypic organization of slice cultures was not affected, although exposure to either integrin antagonist or antibody resulted in a statistically significant reduction in the number of synapses measured in the apical dendrites of CA1 pyramidal neurons. The results demonstrate that integrins markedly affect postnatal CNS development, in both ultrastructural construction and organizational processes.

Stromal cell-derived factor-1 (SDF-1) is a chemoattractant involved in hematopoietic progenitor cell (HPC) trafficking to the bone marrow. We studied the role of bone marrow endothelial proteoglycans (PGs) in SDF-1-mediated migration of HPC using a transwell assay. A subclone of progenitor cell line KG-1 (KG-1v) was used, displaying CXCR4-dependent transmigration. Cell surface PGs on bone marrow endothelial cell line 4LHBMEC did not mediate SDF-1-induced transendothelial migration. In contrast, transwell filters precoated with various glycosaminoglycans (GAGs) enhanced migration toward SDF-1. SDF-1-induced migration was reduced by degradation of heparan sulfate in subendothelial matrix produced by 4LHBMEC. The stimulating effect of GAGs was caused by the formation of a stable haptotactic SDF-1 gradient, as SDF-1 bound to immobilized GAGs and triggered migration. Soluble heparan sulfate enhanced SDF-1-induced migration dose-dependently, suggesting that SDF-1-heparan sulfate complexes optimized SDF-1 presentation. In conclusion, we provide evidence that PGs in the subendothelial matrix establish an SDF-1 gradient guiding migrating HPC into the bone marrow.

Specific interference with molecular mechanisms guiding tissue localization of leukocytes may be of great utility for selective immunosuppressive therapies. We have discovered and characterized efomycines, a new family of selective small-molecule inhibitors of selectin functions. Members of this family significantly inhibited leukocyte adhesion in vitro. Efomycine M, which was nontoxic and showed the most selective inhibitory effects on selectin-mediated leukocyte-endothelial adhesion in vitro, significantly diminished rolling in mouse ear venules in vivo as seen by intravital microscopy. In addition, efomycine M alleviated cutaneous inflammation in two complementary mouse models of psoriasis, one of the most common chronic inflammatory skin disorders. Molecular modeling demonstrated a spatial conformation of efomycines mimicking naturally occurring selectin ligands. Efomycine M might be efficacious in the treatment of human inflammatory disorders through a similar mechanism.

Recent studies have shown that airway inflammation dominated by neutrophils, ie, polymorphonuclear cells (PMN) was observed in infants and children with cystic fibrosis (CF) even in the absence of detectable infection. To assess whether there is a CF-related anomaly of PMN migration across airway epithelial cells, we developed an in vitro model of chemotactic migration across tight and polarized CF(15) cells, a CF human nasal epithelial cell line, seeded on porous filters. To compare PMN migration across a pair of CF and control monolayers in the physiological direction, inverted CF(15) cells were infected with increasing concentrations of recombinant adenoviruses containing either the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, the DeltaF508 CFTR cDNA, or the beta-galactosidase gene. The number of PMN migrating in response to N-formyl-Met-Leu-Phe across inverted CF(15) monolayers expressing beta-galactosidase was similar to that seen across CF(15) monolayers rescued with CFTR, whatever the proportion of cells expressing the transgene. Moreover, PMN migration across monolayers expressing various amounts of mutated CFTR was not different from that observed across matched counterparts expressing normal CFTR. Finally, PMN migration in response to adherent or Pseudomonas aeruginosa was equivalent across CF and corrected monolayers. The possibility that mutated CFTR may exert indirect effects on PMN recruitment, via an abnormal production of the chemotactic cytokine interleukin-8, was also explored. Apical and basolateral production of interleukin-8 by polarized CF cells expressing mutated CFTR was not different from that observed with rescued cells, either in baseline or stimulated conditions. CF(15) cells displayed a CF phenotype that could be corrected by CFTR-containing adenoviruses, because two known CF defects, Cl(-) secretion and increased P. aeruginosa adherence, were normalized after infection with those viruses. Thus, we conclude that the presence of a mutated CFTR does not per se lead to an exaggerated inflammatory response of CF surface epithelial cells in the absence or presence of a bacterial infection.