Oleanolic Acid Complexation with Cyclodextrins Improves Its Cell Bio-Availability and Biological Activities for Cell Migration.

Wound healing is a complex process to restore skin. Plant-derived bioactive compounds might be a source of substances for the treatment of wounds stalled in a non-resolving stage of wound healing. Oleanolic acid (OA), a pentacyclic triterpene, has shown favorable wound healing properties both in vitro and in vivo. Unfortunately, OA cannot be solubilized in aqueous media, and it needs to be helped by the use of dimethyl sulfoxide (DMSO). In this paper, we have shown that cyclodextrins (CDs) are a good alternative to DMSO as agents to deliver OA to cells, providing better features than DMSO. Cyclodextrins are natural macromolecules that show a unique tridimensional structure that can encapsulate a wide variety of hydrophobic compounds. We have studied the cyclodextrin-encapsulated form of OA with OA/DMSO, comparing their stability, biological properties for cell migration, and cell viability. In addition, detailed parameters related to cell migration and cytoskeletal reorganization have been measured and compared. Our results show that OA-encapsulateds compound exhibit several advantages when compared to non-encapsulated OA in terms of chemical stability, migration enhancement, and preservation of cell viability.

Oleanolic acid rescues critical features of umbilical vein endothelial cells permanently affected by hyperglycemia.

Skin wound healing is a physiological process that involves several cell types. Among them, endothelial cells are required for inflammation resolution and neo-angiogenesis, both necessary for tissue restoration after injury. Primary human umbilical vein endothelial cells (C-HUVECs) are derived from the umbilical cord. When women develop gestational diabetes, chronic exposure to hyperglycemia induces epigenetic modifications in these cells (GD-HUVECs), leading to a permanent pro-inflammatory phenotype and impaired angiogenesis in contrast to control cells. Oleanolic acid (OA) is a bioactive triterpenoid known for its epithelial cell migration promotion stimulation and higher tensile strength of wounds. However, the potentially anti-inflammatory and pro-angiogenic properties of OA are still under investigation. We tested OA on C- and GD-HUVECs under inflammatory conditions induced by low levels of the inflammatory cytokine TNF-α. Reduced expression of adhesion molecules VCAM1, ICAM1, and SELE was obtained in OA-pre-treated C- and GD-HUVECs. Additionally, protein VCAM1 levels were also decreased by OA. Coherently, monocyte adhesion assays showed that a lower number of monocytes adhered to GD-HUVEC endothelium under OA pre-treatment when compared to untreated ones. It is noteworthy that OA improved angiogenesis parameters in both phenotypes, being especially remarkable in the case of GD-HUVECs, since OA strongly rescued their poor tube formation behavior. Moreover, endothelial cell migration was improved in C- and GD-HUVECs in scratch assays, an effect that was further confirmed by focal adhesion (FA) remodeling, revealed by paxillin staining on immunocytochemistry assays. Altogether, these results suggest that OA could be an emergent wound healing agent due to its capacity to rescue endothelial malfunction caused by hyperglycemia.

Oleanolic acid stimulation of cell migration involves a biphasic signaling mechanism.

Cell migration is a critical process for wound healing, a physiological phenomenon needed for proper skin restoration after injury. Wound healing can be compromised under pathological conditions. Natural bioactive terpenoids have shown promising therapeutic properties in wound healing. Oleanolic acid (OA), a triterpenoid, enhances in vitro and in vivo cell migration. However, the underlying signaling mechanisms and pathways triggered by OA are poorly understood. We have previously shown that OA activates epidermal growth factor receptor (EGFR) and downstream effectors such as mitogen-activated protein (MAP) kinase cascade and c-Jun N-terminal kinase (JNK), leading to c-Jun transcription factor phosphorylation, all of which are involved in migration. We performed protein expression or migration front protein subcellular localization assays, which showed that OA induces c-Jun activation and its nuclear translocation, which precisely overlaps at wound-edge cells. Furthermore, c-Jun phosphorylation was independent of EGFR activation. Additionally, OA promoted actin cytoskeleton and focal adhesion (FA) dynamization. In fact, OA induced the recruitment of regulator proteins to FAs to dynamize these structures during migration. Moreover, OA changed paxillin distribution and activated focal adhesion kinase (FAK) at focal adhesions (FAs). The molecular implications of these observations are discussed.

Effect of the Human Amniotic Membrane on the Umbilical Vein Endothelial Cells of Gestational Diabetic Mothers: New Insight on Inflammation and Angiogenesis.

One of the most relevant diabetes complications is impaired wound healing, mainly characterized by reduced peripheral blood flow and diminished neovascularization together with increased inflammation and oxidative stress. Unfortunately, effective therapies are currently lacking. Recently, the amniotic membrane (AM) has shown promising results in wound management. Here, the potential role of AM on endothelial cells isolated from the umbilical cord vein of gestational diabetes-affected women (GD-HUVECs), has been investigated. Indeed, GD-HUVECs in vivo exposed to chronic hyperglycemia during pregnancy compared to control cells (C-HUVECs) have shown molecular modifications of cellular homeostasis ultimately impacting oxidative and nitro-oxidative stress, inflammatory phenotype, nitric oxide (NO) synthesis, and bioavailability, thus representing a useful model for studying the mechanisms potentially supporting the role of AM in chronic non-healing wounds. In this study, the anti-inflammatory properties of AM have been assessed using a monocyte-endothelium interaction assay in cells pre-stimulated with tumor necrosis factor-α (TNF-α) and through vascular adhesion molecule expression and membrane exposure, together with the AM impact on the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-kB) pathway and NO bioavailability. Moreover, GD-HUVEC migration and tube formation ability were evaluated in the presence of AM. The results showed that AM significantly reduced TNF-α-stimulated monocyte-endothelium interaction and the membrane exposure of the endothelial vascular and intracellular adhesion molecules (VCAM-1 and ICAM-1, respectively) in both C- and GD-HUVECs. Strikingly, AM treatment significantly improved vessel formation in GD-HUVECs and cell migration in both C- and GD-HUVECs. These collective results suggest that AM positively affects various critical pathways in inflammation and angiogenesis, thus providing further validation for ongoing clinical trials in diabetic foot ulcers.

Chronic Wound Healing by Amniotic Membrane: TGF-ß and EGF Signaling Modulation in Re-epithelialization.

The application of amniotic membrane (AM) on chronic wounds has proven very effective at resetting wound healing, particularly in re-epithelialization. Historically, several aspects of AM effect on wound healing have been evaluated using cell models. In keratinocytes, the presence of AM induces the activation of mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK) pathways, together with the high expression of c-Jun, an important transcription factor for the progression of the re-epithelialization tongue. In general, the levels of transforming growth factor (TGF)-ß present in a wound are critical for the process of wound healing; they are elevated during the inflammation phase and remain high in some chronic wounds. Interestingly, the presence of AM, through epidermal growth factor (EGF) signaling, produces a fine-tuning of the TGF-ß signaling pathway that re-conducts the stalled process of wound healing. However, the complete suppression of TGF-ß signaling has proven negative for the AM stimulation of migration, suggesting that a minimal amount of TGF-ß signaling is required for proper wound healing. Regarding migration machinery, AM contributes to the dynamics of focal adhesions, producing a high turnover and thus speeding up remodeling. This is clear because proteins, such as Paxillin, are activated upon treatment with AM. On top of this, AM also produces changes in the expression of Paxillin. Although we have made great progress in understanding the effects of AM on chronic wound healing, a long way is still ahead of us to fully comprehend its effects.

The FDA-Approved Antiviral Raltegravir Inhibits Fascin1-Dependent Invasion of Colorectal Tumor Cells In Vitro and In Vivo.

BACKGROUND: Fascin1 is the key actin-bundling protein involved in cancer invasion and metastasis whose expression is associated with bad prognosis in tumor from different origins. METHODS: In the present study, virtual screening (VS) was performed for the search of Fascin1 inhibitors and RAL, an FDA-approved inhibitor of human immunodeficiency virus-1 (HIV-1) integrase, was identified as a potential Fascin1 inhibitor. Biophysical techniques including nuclear magnetic resonance (NMR) and differential scanning fluorimetry (DSF) were carried out in order to confirm RAL as a Fascin1 blocker. The effect of RAL on actin-bundling activity Fascin1 was assessed by transmission electron microscopy (TEM), immunofluorescence, migration, and invasion assays on two human colorectal adenocarcinoma cell lines: HCT-116 and DLD-1. In addition, the anti-metastatic potential of RAL was in vivo evaluated by using the zebrafish animal model. RESULTS: NMR and DSF confirmed in silico predictions and TEM demonstrated the RAL-induced disorganization of the actin structure compared to control conditions. The protrusion of lamellipodia in cancer cell line overexpressing Fascin1 (HCT-116) was abolished in the presence of this drug. By following the addition of RAL, migration of HCT-116 and DLD-1 cell lines was significantly inhibited. Finally, using endogenous and exogenous models of Fascin1 expression, the invasive capacity of colorectal tumor cells was notably impaired in the presence of RAL in vivo assays; without undesirable cytotoxic effects. CONCLUSION: The current data show the in vitro and in vivo efficacy of the antiretroviral drug RAL in inhibiting human colorectal cancer cells invasion and metastasis in a Fascin1-dependent manner.

New role of the antidepressant imipramine as a Fascin1 inhibitor in colorectal cancer cells.

Serrated adenocarcinoma (SAC) is more invasive, has worse outcomes than conventional colorectal carcinoma (CRC), and is characterized by frequent resistance to anti-epidermal growth factor receptor (EGFR) and overexpression of fascin1, a key protein in actin bundling that plays a causative role in tumor invasion and is overexpressed in different cancer types with poor prognosis. In silico screening of 9591 compounds, including 2037 approved by the Food and Drug Administration (FDA), was performed, and selected compounds were analyzed for their fascin1 binding affinity by differential scanning fluorescence. The results were compared with migrastatin as a typical fascin1 inhibitor. In silico screening and differential scanning fluorescence yielded the FDA-approved antidepressant imipramine as the most evident potential fascin1 blocker. Biophysical and different in vitro actin-bundling assays confirm this activity. Subsequent assays investigating lamellipodia formation and migration and invasion of colorectal cancer cells in vitro using 3D human tissue demonstrated anti-fascin1 and anti-invasive activities of imipramine. Furthermore, expression profiling suggests the activity of imipramine on the actin cytoskeleton. Moreover, in vivo studies using a zebrafish invasion model showed that imipramine is tolerated, its anti-invasive and antimetastatic activities are dose-dependent, and it is associated with both constitutive and induced fascin1 expression. This is the first study that demonstrates an antitumoral role of imipramine as a fascin1 inhibitor and constitutes a foundation for a molecular targeted therapy for SAC and other fascin1-overexpressing tumors.

Novel anti-invasive properties of a Fascin1 inhibitor on colorectal cancer cells.

Tumor invasion and metastasis involve processes in which actin cytoskeleton rearrangement induced by Fascin1 plays a crucial role. Indeed, Fascin1 has been found overexpressed in tumors with worse prognosis. Migrastatin and its analogues target Fascin1 and inhibit its activity. However, there is need for novel and smaller Fascin1 inhibitors. The aim of this study was to assess the effect of compound G2 in colorectal cancer cell lines and compare it to migrastatin in in vitro and in vivo assays. Molecular modeling, actin-bundling, cell viability, inmunofluorescence, migration, and invasion assays were carried out in order to test anti-migratory and anti-invasive properties of compound G2. In addition, the in vivo effect of compound G2 was evaluated in a zebrafish model of invasion. HCT-116 cells exhibited the highest Fascin1 expression from eight tested colorectal cancer cell lines. Compound G2 showed important inhibitory effects on actin bundling, filopodia formation, migration, and invasion in different cell lines. Moreover, compound G2 treatment resulted in significant reduction of invasion of DLD-1 overexpressing Fascin1 and HCT-116 in zebrafish larvae xenografts; this effect being less evident in Fascin1 known-down HCT-116 cells. This study proves, for the first time, the in vitro and in vivo anti-tumoral activity of compound G2 on colorectal cancer cells and guides to design improved compound G2-based Fascin1 inhibitors. KEY MESSAGES: • Fascin is crucial for tumor invasion and metastasis and is overexpressed in bad prognostic tumors. • Several adverse tumors overexpress Fascin1 and lack targeted therapy. • Anti-fascin G2 is for the first time evaluated in colorectal carcinoma and compared with migrastatin. • Filopodia formation, migration activity, and invasion in vitro and in vivo assays were performed. • G2 blocks actin structures, migration, and invasion of colorectal cancer cells as fascin-dependent.

SIRT1 and Estrogen Signaling Cooperation for Breast Cancer Onset and Progression.

Breast cancer remains a significant female mortality cause. It constitutes a multifactorial disease for which research on environmental factors offers little help in predicting onset or progression. The pursuit for its foundations by analyzing hormonal changes as a motive for disease development, indicates that increased exposure to estrogens associates with increased risk. A prevalent number of breast cancer cases show dependence on the increased activity of the classic nuclear estrogen receptor (ER) for cell proliferation and survival. SIRT1 is a Type III histone deacetylase which is receiving increasing attention due to its ability to perform activities over relevant non-histone proteins and transcription factors. Interestingly, concomitant SIRT1 overexpression is commonly found in ER-positive breast cancer cases. Both proteins had been shown to directly interact, in a process related to altered intracellular signaling and aberrant transcription, then promoting tumor progression. Moreover, SIRT1 activities had been also linked to estrogenic effects through interaction with the G-protein coupled membrane bound estrogen receptor (GPER). This work aims to summarize present knowledge on the interplay between SIRT1 and ER/GPER for breast cancer onset and progression. Lastly, evidences on the ability of SIRT1 to interact with TGFß signaling, a concurrent pathway significantly involved in breast cancer progression, are reported. The potential of this research field for the development of innovative strategies in the assessment of orphan breast cancer subtypes, such as triple negative breast cancer (TNBC), is discussed.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing.

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Amniotic membrane stimulates cell migration by modulating transforming growth factor-ß signalling.

Keratinocyte migration is a mandatory aspect of wound healing. We have previously shown that amniotic membrane (AM) applied to chronic wounds assists healing through a process resulting in the overexpression of c-Jun at the wound's leading edge. We have also demonstrated that AM modifies the genetic programme induced by transforming growth factor-ß (TGF-ß) in chronic wounds. Here we used a scratch assay of mink lung epithelial cells (Mv1Lu) and a spontaneously immortalized human keratinocyte cell line (HaCaT) cells to examine the influence of AM application on the underlying signalling during scratch closure. AM application induced c-Jun phosphorylation at the leading edge of scratch wounds in a process dependent on MAPK and JNK signalling. Strikingly, when the TGF-ß-dependent Smad-activation inhibitor SB431542 was used together with AM, migration improvement was partially restrained, whereas the addition of TGF-ß had a synergistic effect on the AM-induced cell migration. Moreover, antagonizing TGF-ß with specific antibodies in both cell lines or knocking out TGF-ß receptors in Mv1Lu cells had similar effects on cell migration as using SB431542. Furthermore, we found that AM was able to attenuate TGF-ß-Smad signalling specifically at the migrating edge; AM treatment abated Smad2 and Smad3 nuclear localization in response to TGF-ß in a process dependent on mitogen-activated protein kinase kinase 1 (MEK1) activation but independent of EGF receptor or JNK activation. The involvement of Smad signalling on AM effects on HaCaT keratinocytes was further corroborated by overexpression of either Smad2 or Smad3 and the use of Smad phosphorylation-specific inhibitors, revealing a differential influence on AM-induced migration for each Smad. Thus, AM TGF-ß-Smad signalling abating is essential for optimal cell migration and wound closure.

Sirt1 interaction with active Smad2 modulates transforming growth factor-ß regulated transcription.

BACKGROUND: The simplicity of Transforming Growth Factor ß (TGFß) signaling pathway, linear and non-amplified, hardly sustains its variety of responses. This is often justified by the complex regulation showed by Smad proteins, TGFß signaling intracellular transducers, object of post-translational modifications that modulate TGFß-dependent transcription. Protein acetylation is emerging as a compelling mechanism affecting the activities of significant transcription factors, including p53, FOXO or NF-kB. Smad proteins might be controlled by this mechanism, implying that accessory factors capable of altering Smads-transcriptional complexes acetylation status and hence regulate TGFß responses remain to be identified. Understanding this interaction may help in the assessment of TGFß signaling outcomes, extending from healthy physiology to pathological conditions and cancer. METHODS: A two-hybrid chimera interacting system allowed to identify Sirt1, a NAD+ dependent type III histone deacetylase, as a novel Smad2 interactor. Several well stablished cellular models were applied to characterize this interaction by means of co-immunoprecipitation of tagged proteins and immuno-fluorescence staining. The occurrence of the interaction at Smad2 driven transcriptomic complexes was studied by means of DNA-pull-down and chromatin immunoprecipitation (ChIP), while its effects were assessed by protein over-expression and siRNA applied into a TGFß-dependent reporter gene assay. RESULTS: The interaction was confirmed and observed to be enhanced upon Smad2 acetylation, a known feature of active and nuclear Smad2. However, Sirt1 did not play a major role in Smad2 deacetylation. Anti-Sirt1 ChIP showed increased recovery of promoter regions corresponding to Smad2-driven genes after TGFß-stimulation, while its occurrence at Smad2-dependent transcriptomic complexes on DNA was found to effectively modulate gene expression. CONCLUSIONS: Sirt1 presence on Smad2-driven TGFß-dependent regulatory elements was detected and found to increase after TGFß treatment. Moreover, Sirt1 overexpression resulted in a decrease of the activity of a Smad2-driven TGFß-dependent reporter gene, while Sirt1 interference increased its activity. This would confirm the relevance of the discovered Sirt1-Smad2 interaction for the regulation of TGFß-dependent gene transcription.

Oleanolic acid induces migration in Mv1Lu and MDA-MB-231 epithelial cells involving EGF receptor and MAP kinases activation.

During wound healing, skin function is restored by the action of several cell types that undergo differentiation, migration, proliferation and/or apoptosis. These dynamics are tightly regulated by the evolution of the extra cellular matrix (ECM) contents along the process. Pharmacologically active flavonoids have shown to exhibit useful physiological properties interesting in pathological states. Among them, oleanolic acid (OA), a pentacyclic triterpene, shows promising properties over wound healing, as increased cell migration in vitro and improved wound resolution in vivo. In this paper, we pursued to disclose the molecular mechanisms underlying those effects, by using an in vitro scratch assay in two epithelial cell lines of different linage: non-malignant mink lung epithelial cells, Mv1Lu; and human breast cancer cells, MDA-MB-231. In every case, we observed that OA clearly enhanced cell migration for in vitro scratch closure. This correlated with the stimulation of molecular pathways related to mitogen-activated protein (MAP) kinases, as ERK1,2 and Jun N-terminal kinase (JNK) 1,2 activation and c-Jun phosphorylation. Moreover, MDA-MB-231 cells treated with OA displayed an altered gene expression profile affecting transcription factor genes (c-JUN) as well as proteins involved in migration and ECM dynamics (PAI1), in line with the development of an epithelial to mesenchymal transition (EMT) status. Strikingly, upon OA treatment, we observed changes in the epidermal growth factor receptor (EGFR) subcellular localization, while interfering with its signalling completely prevented migration effects. This data provides a physiological framework supporting the notion that lipophilic plant extracts used in traditional medicine, might modulate wound healing processes in vivo through its OA contents. The molecular implications of these observations are discussed.

Amniotic Membrane Modifies the Genetic Program Induced by TGFß, Stimulating Keratinocyte Proliferation and Migration in Chronic Wounds.

BACKGROUND: Post-traumatic large-surface or deep wounds often cannot progress to reepithelialisation because they become irresponsive in the inflammatory stage, so intervention is necessary to provide the final sealing epidermis. Previously we have shown that Amniotic Membrane (AM) induced a robust epithelialisation in deep traumatic wounds. METHODS AND FINDINGS: To better understand this phenomenon, we used keratinocytes to investigate the effect of AM on chronic wounds. Using keratinocytes, we saw that AM treatment is able to exert an attenuating effect upon Smad2 and Smad3 TGFß-induced phosphorylation while triggering the activation of several MAPK signalling pathways, including ERK and JNK1, 2. This also has a consequence for TGFß-induced regulation on cell cycle control key players CDK1A (p21) and CDK2B (p15). The study of a wider set of TGFß regulated genes showed that the effect of AM was not wide but very concrete for some genes. TGFß exerted a powerful cell cycle arrest; the presence of AM however prevented TGFß-induced cell cycle arrest. Moreover, AM induced a powerful cell migration response that correlates well with the expression of c-Jun protein at the border of the healing assay. Consistently, the treatment with AM of human chronic wounds induced a robust expression of c-Jun at the wound border. CONCLUSIONS: The effect of AM on the modulation of TGFß responses in keratinocytes that favours proliferation together with AM-induced keratinocyte migration is the perfect match that allows chronic wounds to move on from their non-healing state and progress into epithelialization. Our results may explain why the application of AM on chronic wounds is able to promote epithelialisation.

Fibroin and sericin from Bombyx mori silk stimulate cell migration through upregulation and phosphorylation of c-Jun.

Wound healing is a biological process directed to the restoration of tissue that has suffered an injury. An important phase of wound healing is the generation of a basal epithelium able to wholly replace the epidermis of the wound. A broad range of products derived from fibroin and sericin from Bombyx mori silk are used to stimulate wound healing. However, so far the molecular mechanism underlying this phenomenon has not been elucidated. The aim of this work was to determine the molecular basis underlying wound healing properties of silk proteins using a cell model. For this purpose, we assayed fibroin and sericin in a wound healing scratch assay using MDA-MB-231 and Mv1Lu cells. Both proteins stimulated cell migration. Furthermore, treatment with sericin and fibroin involved key factors of the wound healing process such as upregulation of c-Jun and c-Jun protein phosphorylation. Moreover, fibroin and sericin stimulated the phosphorylation of ERK 1/2 and JNK 1/2 kinases. All these experiments were done in the presence of specific inhibitors for some of the cell signalling pathways referred above. The obtained results revealed that MEK, JNK and PI3K pathways are involved in fibroin and sericin stimulated cells migration. Inhibition of these three kinases prevented c-Jun upregulation and phosphorylation by fibroin or sericin. Fibroin and sericin were tested in the human keratinocyte cell line, HaCaT, with similar results. Altogether, our results showed that fibroin and sericin initiate cell migration by activating the MEK, JNK and PI3K signalling pathways ending in c-Jun activation.