Combination of Two Kinds of Medicated Microparticles Based on Hyaluronic Acid or Chitosan for a Wound Healing Spray Patch

Olive leaves extract (OLE) has been extensively studied as antioxidant and antibiotic and these characteristics make it particularly interesting for use on wounds. For this reason, the aim of this study was to introduce OLE in microparticles (MP) of hyaluronic acid (MPHA-OLE) or chitosan (MPCs-OLE) to obtain a spray patch for the treatment of wounds in anatomical areas that are difficult to protect with traditional patches. The MP were characterized for particle size and ability to protect OLE from degradation, to absorb water from wound exudate, to control OLE release from MP. The MPHA and MPCs medicated or not and mixtures of the two types in different proportions were studied in vitro on fibroblasts by the scratch wound healing assay. The MP size was always less than 5 µm, and therefore, suitable for a spray patch. The MPCs-OLE could slow down the release of OLE therefore only about 60% of the polyphenols contained in it were released after 4 h. Both MPHA and MPCs could accelerate wound healing. A 50% MPHA-OLE-50% MPCs-OLE blend was the most suitable for accelerating wound healing. The MPHA-OLE-MPCs-OLE blends studied in this work were shown to have the characteristics suitable for a spray patch, thus giving a second life to the waste products of olive growers.

Modeling and Predicting the Cell Migration Properties from Scratch Wound Healing Assay on Cisplatin-Resistant Ovarian Cancer Cell Lines Using Artificial Neural Network

The study of artificial neural networks (ANN) has undergone a tremendous revolution in recent years, boosted by deep learning tools. The presence of a greater number of learning tools and their applications, in particular, favors this revolution. However, there is a significant need to deal with the issue of implementing a systematic method during the development phase of the ANN to increase its performance. A multilayer feedforward neural network (FNN) was proposed in this paper to predict the cell migration assay on cisplatin-sensitive and cisplatin-resistant (CisR) ovarian cancer (OC) cell lines via scratch wound healing assay. An FNN training algorithm model was generated using the MATLAB fitting function in a MATLAB script to accomplish this task. The input parameters were types of cell lines, times, and wound area, and outputs were relative wound area, percentage of wound closure, and wound healing speed. In addition, we tested and compared the initial accuracy of various supervised learning classifier and support vector regression (SVR) algorithms. The proposed ANN model achieved good agreement with the experimental data and minimized error between the estimated and experimental values. The conclusions drawn demonstrate that the developed ANN model is a useful, accurate, fast, and inexpensive method to predict cancerous cell migration characteristics evaluated via scratch wound healing assay.

The Role of Hypericum Lydium Boiss. (Hypericaceae) in Soft Tissue Healing and its Capacity to Prevent Infections after Dental Extraction

The genus Hypericum sp. has a number of uses in traditional medicine like curing the burns, ulcers, haemorrhoids and wound healing. The species Hypericum lydium Boiss. (Hypericaceae), however, has not been known to have any properties related to the healing of injuries or antimicrobial working against the oral microorganisms. The present study was aimed to evaluate the efficiency of H. lydium in soft tissue healing and its capacity to prevent infections after dental extraction. H. lydium was extracted with ethanol and the obtained extract was tested for its inhibition ability on extracellular matrix-degrading enzymes; collagenase, hyaluronidase and elastase. To determine the cytotoxicity and wound healing capacity of the extract, MTT and in vitro scratch wound healing assay were performed using the NIH-3T3 fibroblast cells, respectively. Antimicrobial activity was investigated by microdilution method against oral pathogenic microorganisms. The highest enzyme inhibition activity was determined against elastase (80.27±0.1%). According to the cytotoxic activity results, the IC50 value of the H. lydium was found to be 82.20±4.05 μg/mL. Scratch wound healing assay of the extract exhibited a significant enhancement at 24 h with a closed wound area when compared with the control. The extract showed potent antimicrobial properties against oral pathogenic microorganisms. The results of the study revealed out that H. lydium can be considered as a natural compound for dental industry to improve soft tissue healing and to prevent the possible infections after dental extraction.

An image J plugin for the high throughput image analysis of in vitro scratch wound healing assays

In vitro scratch wound healing assay, a simple and low-cost technique that works along with other image analysis tools, is one of the most widely used 2D methods to determine the cellular migration and proliferation in processes such as regeneration and disease. There are open-source programs such as imageJ to analyze images of in vitro scratch wound healing assays, but these tools require manual tuning of various parameters, which is time-consuming and limits image throughput. For that reason, we developed an optimized plugin for imageJ to automatically recognize the wound healing size, correct the average wound width by considering its inclination, and quantify other important parameters such as: area, wound area fraction, average wound width, and width deviation of the wound images obtained from a scratch/ wound healing assay. Our plugin is easy to install and can be used with different operating systems. It can be adapted to analyze both individual images and stacks. Additionally, it allows the analysis of images obtained from bright field, phase contrast, and fluorescence microscopes. In conclusion, this new imageJ plugin is a robust tool to automatically standardize and facilitate quantification of different in vitro wound parameters with high accuracy compared with other tools and manual identification.

Characterization of Mesenchymal Stem Cells Derived from Patients with Cerebellar Ataxia: Downregulation of the Anti-Inflammatory Secretome Profile

Mesenchymal stem cell (MSC) therapy is a promising alternative approach for the treatment of neurodegenerative diseases, according to its neuroprotective and immunomodulatory potential. Despite numerous clinical trials involving autologous MSCs, their outcomes have often been unsuccessful. Several reports have indicated that MSCs from patients have low capacities in terms of the secretion of neurotrophic or anti-inflammatory factors, which might be associated with cell senescence or disease severity. Therefore, a new strategy to improve their capacities is required for optimal efficacy of autologous MSC therapy. In this study, we compared the secretory potential of MSCs among cerebellar ataxia patients (CA-MSCs) and healthy individuals (H-MSCs). Our results, including secretome analysis findings, revealed that CA-MSCs have lower capacities in terms of proliferation, oxidative stress response, motility, and immunomodulatory functions when compared with H-MSCs. The functional differences were validated in a scratch wound healing assay and neuron-glia co-cultures. In addition, the neuroprotective and immunoregulatory protein follistatin-like 1 (FSTL1) was identified as one of the downregulated proteins in the CA-MSC secretome, with suppressive effects on proinflammatory microglial activation. Our study findings suggest that targeting aspects of the downregulated anti-inflammatory secretome, such as FSTL1, might improve the efficacy of autologous MSC therapy for CA.

MiR-124-5p Inhibits the Progression of Gastric Cancer by Targeting MIEN1

Objective: To observe the effect of miR-124-5p on progression of gastric cancer (GC) and explore the targeting mechanism. Methods: After collecting the specimens, we used real-time fluorescence quantitative PCR to detect the miR-124-5p level of GC tissue and corresponding adjacent tissue. Then MTT test and scratch wound-healing assay were hired to evaluate the influence of miR-124-5p in GC cell (SGC-803 and SGC7901) migration and proliferation ability. The binding of miR-124-5p to migration and invasion enhancer 1 (MIEN1) was detected through dual luciferase reporter gene experiment and western blot was utilized to assay the protein level of MIEN1. Results: Compared with adjacent tissues, miR-124-5p level in GC tissues was lower significantly. MiR-124-5p mimic inhibited the metastasis and proliferation ability of SGC7901 cells and miR-124-5p inhibitor promoted the migration and proliferation ability of SGC803 cells. In addition, miR-124-5p targeted MIEN1 and negatively modulated the MIEN1 expression in SGC-803 and SGC7901 cells. Silencing MIEN1 negatively regulated the metastasis and proliferation ability of SGC7901 cells. Conclusion: MiR-124-5p inhibited the GC cell proliferation and metastasis phenotypes through MIEN1, which probably becomes a novel molecular target for clinical GC treatment.

Μελέτη της επίδρασης της οικογένειας του EGFR στον καρκίνο του μαστού

Εισαγωγή: Ο human epidermal growth factor receptor 2 (HER-2) υπερεκφράζεται στο 20-30% των περιπτώσεων καρκίνου του μαστού (ΚΜ) και σχετίζεται με κακή πρόγνωση. Σηματοδοτικά μονοπάτια καθοδικά του υποδοχέα receptor activator of nuclear factor-κB (RANK) παίζουν κεντρικό ρόλο στον ΚΜ. Το σηματοδοτικό μονοπάτι RANKL/RANK εμπλέκεται στον HER-2(+) ΚΜ. Ο μοριακός μηχανισμός της συμμετοχής του στην καρκινογένεση παραμένει αδιερεύνητος. Σκοπός: Σκοπός της παρούσας διδακτορικής διατριβής είναι η διερεύνηση της εμπλοκής του RANK υποδοχέα, και του σηματοδοτικού μονοπατιού του NF-κB στην ογκογένεση των HER-2(+) καρκινωμάτων μαστού (ΚΜ). Επιπλέον στόχος είναι η αποσαφήνιση του μοριακού μηχανισμού ρύθμισης της RANKL/RANK σηματοδότησης από μέλη της οικογένειας EGFR μέσω πιθανής φυσικής αλληλεπίδρασης, επηρεάζοντας βασικές κυτταρικές ιδιότητες. Τελικός στόχο της μελέτης αποτελεί η in vitro αξιολόγηση της συνδυαστικής αναστολής των RANK και EGFR υποδοχέων στην εξέλιξη των καρκινικών κυττάρων γεγονός που συμβάλει σε νέες πιθανές θεραπευτικές προτάσεις. Υλικά και Μέθοδοι: Χρησιμοποιήθηκαν οι ανθρώπινες κυτταρικές σειρές ΚΜ, SKBR3, BT474, MCF7, MDA-MB-453. Μελετήθηκε η έκφραση των RANK και RANKL, μέσω RT-PCR, Western-Blot και τεχνικές ανοσοφθορισμού. Η αλληλεπίδραση του RANK με τον HER-2 ανιχνεύθηκε μέσω της μεθόδου Proximity Ligation Assay (PLA), η οποία επιτρέπει την οπτικοποίηση πρωτεϊνικών αλληλεπιδράσεων. Η ενεργοποίηση του NF-κB μονοπατιού μελετήθηκε μέσω Western-Blot. Χρησιμοποιήθηκαν οι αναστολείς trastuzumab (T), pertuzumab (P), denosumab (D) των δύο μελετώμενων μονοπατιών. Εφαρμόστηκαν λειτουργικές δοκιμασίες μετανάστευσης, απόπτωσης και πολλαπλασιασμού. Ο κυτταρικός πολλαπλασιασμός μελετήθηκε εκτιμώντας τη βιωσιμότητα των κυττάρων με τη δοκιμασία ΧTT και του clonogenic assay, το μεταναστευτικό δυναμικό με τη βιοδοκιμή προσομοίωσης τραύματος δι’ αμυχής (Scratch-Wound Healing Assay), ενώ η απόπτωση μέσω FACS έπειτα από χρώση των κυττάρων με ανεξίνη V. Αποτελέσματα: Η πρωτεϊνική έκφραση του υποδοχεα RANK και του συνδέτη του RANKL εντοπίστηκε σε όλες τις κυτταρικές σειρές. Ο υποδοχέας RANK βρέθηκε ότι διμερίζεται με τα μέλη της οικογένειας EGFR. Ο αριθμός των διμερών RANK/ HER-2 ανά κύτταρο φαίνεται να εξαρτάται από την έκφραση του HER-2 (SKBR3:5.4,BT474:8.2,MCF7:0.7,MDA-MB-453:0.3). Τα διμερή RANK/ HER-2 μειώνονται παρουσία των αναστολέων Denosumab (D), Trastuzumab (T) και Pertuzumab (P), ενώ αυξάνονται παρουσία του RANKL (R) στα κύτταρα SKBR3 (m:5.4, D:1.2, T:1.9, DT:0.6, TP:1, DTP:0.4, R:11.8) και BT474 (m:8.2, D:3.1, T:4.3, DT:0.7, TP:3.4, DTP:3.2, R:11.6). Η συνδυαστική επώαση των κυττάρων SKBR3 με τους αναστολείς μειώνει περαιτέρω την ενεργοποίηση του μονοπατιού NF-κB σε σχέση με τη μονή στόχευση. Η αναστολή των RANKL και HER-2 στη κυτταρική σειρά SKBR3 έχει ως αποτέλεσμα τον μειωμένο κυτταρικό πολλαπλασιασμό, αυξημένη απόπτωση και χαμηλότερο μεταναστευτικό δυναμικό συγκριτικά με τα κύτταρα ελέγχου (m) ενώ αντίθετες τιμές παρατηρήθηκαν παρουσία RANKL. Η συνδυαστική επώαση των κυττάρων SKBR3 με D, T και P πλεονεκτεί έναντι της μονής στόχευσης στις λειτουργικές δοκιμασίες. Το denosumab κατέστειλε την NF-κΒ σηματοδότηση και ελάττωσε το ρυθμό πολλαπλασιασμού στα καρκινικά κύτταρα MDA-MB-453. Η κυτταρική σειρά MCF7 δεν ανταποκρίθηκε στους αναστολείς. Συμπεράσματα: Τα αποτελέσματα μας καταδεικνύουν μια νέα φυσική και μοριακή συσχέτιση μεταξύ των HER-2 και RANK μονοπατιών, η οποία επηρεάζει την εξέλιξη των HER-2(+) ΚΜ. Επιπλέον, παρουσιάζονται ενδείξεις που καθιστούν τη συνδυαστική στόχευση των RANKL, HER-2 ως ωφέλιμη θεραπευτική στρατηγική, η οποία θα πρέπει να ελεγχθεί περαιτέρω σε συγκεκριμένους ασθενείς με ΚΜ.

P07.02 Enhanced glycolysis in glioblastomas is associated with tumor cells migration

BACKGROUND Glioblastoma is the most common primary brain tumor. Its prognosis remains poor even with the standard treatment - the Stupp protocol.The classic Warburg effect in cancers leads to increased glycolysis which causes acidification of the tumor environment. This phenomenon may favor migration of tumor cells as already reported in pancreatic ductal adenocarcinoma. We therefore hypothesized that enhanced glycolysis in glioblastomas could favor the tumor cell migration. MATERIAL AND METHODS We measured glycolysis by the extracellular acidification rate (ECAR) of several human glioblastomas cell lines (LN229, LN18, T98-G, U87-MG, U373-MG, U118-MG) with the Seahorse Analyzer. To confirm these results, we also measured the intracellular cAMP rates using the Cayman’s Elisa kit and we analyzed by RT-PCR the expression of the main genes coding for enzymes involved in glycolysis in these glioblastomas cell lines. Cell migration was measured with a scratch wound healing assay during 24 hours. RESULTS U118-MG was the glioblastoma cell line with the highest glycolysis rate, the highest production of cAMP and showed a strong expression of glycolysis-associated genes. LN229 was the glioblastoma cell line with the less important glycolysis rate, the lower production of cAMP and showed a weaker expression of glycolysis-associated genes. According to the scratch wound healing assay, U118-MG cells showed a more important migration than LN229 cells at 24 hours. CONCLUSION Glycolysis may be an attractive target to prevent effectively tumor cell migration in glioblastomas. Coupling the evaluation of glycolysis with histomolecular characterization of glioblastomas, could help to identify patients to whom adjuvant therapies that inhibit glycolysis such as fenofibrate could be proposed.

MicroRNA-21 Regulates Non-Small Cell Lung Cancer Cell Invasion and Chemo-Sensitivity through SMAD7

Background/Aims: SMAD7 is a key inhibitor of transforming growth factor β (TGFβ) receptor signaling, which regulates the alteration of cancer cell invasiveness through epithelial-mesenchymal cell conversion. Carboplatin is a commonly used drug in the chemotherapy for non-small cell lung cancer (NSCLC). Nevertheless, the molecular mechanisms underlying its suppressive effects on the NSCLC cell invasion are not completely understood. In the current study, we addressed this question by analyzing the effects of Carboplatin on microRNA-regulated SMAD7. Methods: We used Carboplatin to treat NSCLC cell lines. We performed bioinformatics analyses on the binding of microRNA-21 (miR-21) to the 3'-UTR of SMAD7 mRNA, and verified the biological effects of this binding using promoter luciferase reporter assay. The effects of Carboplatin or miR-21-modification on NSCLC cell invasion were evaluated in either a transwell cell invasion assay, or a scratch wound healing assay. Results: We found that Carboplatin inhibited the NSCLC cell invasion, in either a transwell cell invasion assay, or a scratch wound healing assay. Moreover, Carboplatin increased the levels of SMAD7 protein, but not mRNA, in NSCLC cells, suggesting presence of post-transcriptional control of SMAD7 by Carboplatin. Furthermore, expression of miR-21 was found to be inhibited by Carboplatin, and bioinformatics analyses showed that miR-21 targeted the 3'-UTR of SMAD7 mRNA to inhibit its translation, which was confirmed by luciferase reporter assay. Conclusion: Carboplatin may upregulate SMAD7 through suppression of miR-21 to inhibit TGFβ receptor signaling mediated NSCLC cell invasion.