RhoA/ROCKs signaling is increased by treatment with TKI-258 and leads to increased apoptosis in SCC-4 oral squamous cell carcinoma cell line.

BACKGROUND: This study evaluated the effect of treatment with TKI-258 on apoptosis, involving Rho GTPases and their effectors in SCC-4 cells of oral squamous cell carcinoma. METHODS: Markers of cell death and apoptosis were analyzed in control and TKI-258-treated SCC-4 cells by flow cytometry. The involvement of Rho GTPases and effectors in the induction of apoptosis by TKI-258 was evaluated by quantification of cleaved PARP. Also, gene expression analysis of those proteins was performed. RESULTS: The treatment with TKI-258 led to a significant increase in cell death (7-AAD) and apoptosis (annexin V and cleaved PARP). When Rho GTPases were stimulated with LPA and inhibited with toxin A Clostridium difficile, the percentage of apoptotic cells increased and decreased, respectively. A similar effect was found when the treatment was with TKI-258 combined with LPA and toxin A. Treatment with TKI-258 significantly increased RhoA gene expression, while RhoB, RhoC, Rac1, and Cdc42 decreased significantly. ROCKs inhibitors (Y-27632 and HA-1077) reduced apoptosis compared with control. TKI-258 combined with Y-27632 or HA-1077 led to an increase in apoptosis compared with inhibitors only. Treatment with TKI-258 led to an increase in ROCK1 and ROCK2 gene expression, and a decrease in PAK1 and PAK2 gene expression. CONCLUSIONS: TKI-258 stimulates apoptosis in SCC-4 cells of oral squamous cell carcinoma. Possibly, RhoA GTPase and their effectors ROCKs participate in the signaling pathway inhibited by TKI-258. CLINICAL RELEVANCE: Therapies with multi-target inhibitors, such as TKI-258, may be promising alternatives for the clinical treatment of oral squamous cell carcinoma.

Effect of radiofrequency on patellar ligament repair of Wistar rats.

This study aimed to evaluate the effects of radiofrequency (RF) on patellar ligament repair through the analysis of type I and III collagens and immunostaining for TGF-ß3. To evaluate the effect of RF on patellar ligament repair of Wistar rats, cross-sectional incision (60% of the width - grade I) was performed in patellar ligaments of the groups: lesion (L, n = 7), treated with RF on the 5-day (5RF, n = 7) and 7-day (7RF, n = 7) post injury were compared to control group (C, n = 7). Histological evaluation, immunohistochemistry, morphometry and statistical analysis were performed. At 10 days post injury, ligament rupture were observed only in L. Active fibroblasts, type 3 collagen and TGF-ß3 in L, 5RF and 7RF was significantly (p < 0.05) higher than control (C). Type 1 collagen was significantly (p < 0.05) higher in C than L, 5RF and 7RF. A positive correlation (p < 0.05) was observed: TGF-ß3 vs active fibroblasts and TGF-ß3 vs type 3 collagen; otherwise, negative correlation (p < 0.05): type I collagen vs TGF-ß3. These results suggest that RF seemed to accelerate the wound healing process of the patellar ligament and may be used as a non-invasive treatment of partial ligament injuries.

Tyrosine kinase inhibitor TKI-258 inhibits cell motility in oral squamous cell carcinoma in vitro.

BACKGROUND: Oral squamous cell carcinoma is extremely invasive, and this behavior is regulated by binding of extracellular molecules to the cell membrane receptors. The TKI-258 inhibits phosphorylation of FGFRs VEGFRs and PDGFRs. Our aim was to analyze the effect of TKI-258 treatment in cell movement using SCC-4 cell line from human oral squamous cell carcinoma. METHODS: F-actin was stained with rhodamine phalloidin, and confocal analysis was performed. The migration and invasion (membrane covered with Matrigel™ ) three-dimensional assays were performed, and control and cells treated with TKI-258 that migrated through the membrane were counted after 24 h. RESULTS: Control cells presented abundant cytoplasm with F-actin wide distributed and evident cell cortex; however, treated (1, 5 and 10 µM TKI-258) cells showed round morphology, scanty cytoplasm, F-actin disorganized and preserved cell cortex. TKI-258 (1, 5, and 10 µM) treatment inhibits migrating cells (ANOVA, F = 97.749, d.f. = 3, 10; P < 0.0001), and it was concentration dependent. Invading cell treated with 5 µM TKI-258 was significantly lower (t = 6.708, d.f. = 5, P < 0.001). CONCLUSIONS: These results suggest that the tyrosine kinase inhibitor TKI-258 has an inhibitory effect on cell motility, affecting F-actin, cell migration, and cell invasion, and probably, these processes are regulated by signaling pathways FGFRs and/or PDGFRs and/or VEGFRs.

HA-1077 inhibits cell migration/invasion of oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most malignant lesion occurring in the head and neck. The Rho-kinases (ROCKs), effectors of Rho proteins, are involved in actin cytoskeletal organization, cell migration, and maintenance cortex. The HA-1077 inhibits the ROCKs. This study aimed to evaluate the effect of treatment with HA-1077 on cell motility in SCC-4 cells, a cell line originating from human OSCC. F-actin of SCC-4 cells treated or not with HA-1077 (1, 50 and 100 µmol/l), and also HA-1077 50 µmol/l and/or inhibitors Y-27632 30 µmol/l was stained with rhodamine-conjugated phalloidin and analyzed by confocal microscopy. Approximately 1×10 cells/well, control and treated with HA-1077 (25, 50, and 100 µmol/l) were added to the migration plate assay. In addition, 1×10 cells/well, control and treated with HA-1077 50 µmol/l, were tested by invasion assays (plate coated with Matrigel). The inhibition of ROCKs with HA-1077 and/or Y-27632 leads to morphological changes, affecting the organization of the actin. The inhibitory effect of HA-1077 (P<0.0001) was dose dependent as the number of cells migrated at 100 µmol/l was statistically different: 25 µmol/l (P<0.0001) and 50 µmol/l (P<0.01). The number of cells treated with HA-1077 50 µmol/l decreased compared with control cells that invaded through Matrigel (P<0.0001). This study shows an inhibitory effect of HA-1077 on cell migration and invasion, suggesting that the use of HA-1077 can be a potential therapy for OSCC.

Depressive behavior induced by social isolation of predisposed female rats.

Depression is a mood disorder that is more prevalent in women and has been closely associated with chronic stress. Many models of depression have been suggested that consider different forms of stress. In fact, stress is present in the life of every human being, but only a few develop depression. Accordingly, it seems wrong to consider all stressed animals to be depressed, emphasizing the importance of predisposition for this mood disorder. Based on this finding, we evaluated a predisposition to depressive behavior of female rats on the forced swim test (FST), and the more immobile the animal was during the FST, the more predisposed to depression it was considered to be. Then, animals were subjected to the stress of social isolation for 21 days and were re-evaluated by the FST. The Predisposed/Isolated rats presented higher immobility times. Once all the rats had prior experience in the FST, we calculated an Index of Increase by Isolation, confirming the previous results. Based on this result, we considered the Predisposed/Isolated group as presenting depressive behavior ('Depressed') and the Nonpredisposed/Nonisolated group as the control group ('Nondepressed'). The animals were distributed into 4 new groups: Nondepressed/Vehicle, Nondepressed/Amitriptyline, Depressed/Vehicle, Depressed/Amitriptyline. After 21 days of treatment, only the Depressed/Vehicle group differed from the other 3 groups, demonstrating the efficacy of amitriptyline in treating the depressive behavior of the Depressed animals, validating the model. This study shows that conducting an FST prior to any manipulation can predict predisposition to depressive behavior in female rats and that the social isolation of predisposed animals for 21 days is effective in inducing depressive behavior. This behavior can be considered real depressive behavior because it takes into account predisposition, chronic mild stress, and the prevalent gender.

Comparison of the effects of carboxytherapy and radiofrequency on skin rejuvenation.

BACKGROUND: Carboxytherapy (CA) refers to the cutaneous and subcutaneous administration of CO(2) for therapeutic purposes. Radiofrequency (RF) is a method that uses electric current for heating layers of the skin. Both techniques are indicated for the treatment of skin laxity. OBJECTIVE: The aim of this study was to compare the effects of CA and RF on human skin. METHODS: After eight patients underwent abdominoplasty, each of them received a single treatment of CA and a single treatment of RF on the right and left infra-umbilical regions, respectively. In the infra-umbilical region, CA was performed on the right and RF was performed on the left side. Untreated skin was used as a control. The sample collection period lasted 120 days. CA was administered at a velocity of 40 mL/min, and the total quantity of CO(2) infused was approximately 20 mL. RF was carried out at a temperature higher than 40°C on the epidermis for 5 min. RESULTS: CA and RF led to collagen remodeling; however, this result was more evident and lasted longer with RF. With CA an increase in elastic fibers was observed, whereas with RF no alteration was observed. CONCLUSION: Our results suggest that RF is more efficient than CA in stimulating collagen synthesis.

Immunolocalization of RhoA and RhoB GTPases in pleomorphic adenoma of the parotid.

The pleomorphic adenoma of the parotid (PA) is characterized by the high tissues diversity. Rho GTPases participate in signal transduction pathways that regulate several biological processes, including cell differentiation. A quantitative analysis of RhoA and RhoB GTPases immunoexpression was performed in healthy parotids and in 23 PA cases, predominantly epithelial (PE) or mesenchymal (PM), followed by Student's t test. In PE cases, RhoA immunoexpression was higher in sheets and trabeculae (p < 0.05), whereas RhoB only in sheets (p < 0.05). In normal parotids, RhoA and RhoB were not detected in acinar cells. Ducts have expressed RhoA and RhoB in normal parotids and PA. RhoB was detected in myxoid and chondromyxoid cells. Normal parotids do not express RhoA and RhoB proteins in acinar cells, indicating a lack of function in secretory cells. Despite RhoA and RhoB GTPases are different in their biological roles, no significant difference in immunoexpression of the RhoA and RhoB GTPases in epithelial and mesenchymal structures of PA.

Effect of indomethacin on surface treatment and intracanal dressing of replanted teeth in dogs.

PURPOSE: This study evaluated the healing process of teeth replanted after root treatment and intracanal dressing with indomethacin alone or indomethacin with calcium hydroxide (Ca[OH]2). MATERIALS AND METHODS: Through a case-control study, 24 teeth of 6 adult dogs were extracted, dried, and divided into 4 groups according to the root surface treatment protocols performed before replantation and the intracanal medication used after replantation. In group 1 (negative control), root surfaces were treated by immersion in a 0.9% saline solution and then replanted. In the other groups, the roots were immersed for 10 minutes in Ca(OH)2 (group 2), indomethacin (group 3), or a solution of indomethacin and Ca(OH)2 (group 4). After 2 weeks, group 1 teeth were subjected to single-visit root canal treatment and obturation with gutta-percha and sealer consisting of zinc oxide and eugenol. The teeth in the other groups were subjected to intracanal dressing with the same material used for immersion. After an additional period of 28 weeks, the animals were euthanized and the jaws containing the replanted teeth were processed for histologic analysis. Histometric values were statistically analyzed, with significance set at a P value less than or equal to .05. RESULTS: Group 1 exhibited significantly more normal periodontium than group 4 (P = .02). Total resorption was greater in group 4 than in group 1 (P = .02). No statistically significant difference in the percentage of surface resorption or in total inactive resorption was observed between the groups. CONCLUSIONS: The findings of this study suggest that intracanal dressing and topical root treatment with Ca(OH)2 with or without indomethacin is not recommended for teeth dried for 50 minutes, but the use of indomethacin alone as root surface treatment for delayed tooth replantation deserves further study using longer drying periods. In addition, the present results suggest that a single-visit root canal, performed up to 2 weeks after replantation, might be indicated for teeth dried for up to 50 minutes.

Effects of microdermabrasion on skin rejuvenation.

BACKGROUND: Microdermabrasion is a surface treatment, noninvasive, which uses a negative pressure and drives programmable inert microcrystals on the skin, causing an exfoliation. OBJECTIVE: The aim of this study was to evaluate the effects of application of microdermabrasion in human skin rejuvenation. METHODS: Eleven women who were undergoing abdominoplasty were considered. An area of 25 cm² in the umbilicus to the right was conditioned with microcrystals of Al2O3 in maximum flow, negative pressure of 200 mmHg and total of 8 past, the left side being used as control. The number of sessions ranged from one to five, with weekly intervals, and timing of sample collection ranged from 0 to 132 days. Samples were fixed in 10% formaldehyde in phosphate buffer and were evaluated histologically. RESULTS: A mild to marked hyperpigmentation was observed and remained for a variable period. Histological findings suggest an improvement in the epidermal layer with increased thickness and reestablishing their interdigitations in the dermis initially observed an increase in collagen synthesis. The analysis showed a late stay of epidermal changes, which did not occur in the dermis. CONCLUSION: Under the conditions and parameters used in this work, the microdermabrasion had a positive skin structure, showing that a viable resource in promoting skin rejuvenation.

Immunohistochemical expression of Rho GTPases in ameloblastomas.

Rho GTPases are proteins that regulate cell cycle, shape, polarization, invasion, migration, and apoptosis, which are important characteristics of normal and neoplastic cells. Rho GTPases expression has been reported in normal tooth germ and several pathologies; however, it has not been evaluated in ameloblastomas. The aim of this study was to analyze the expression and distribution of RhoA, RhoB, Rac1, and Cdc42 Rho GTPases in solid and unicystic ameloblastomas. Three-micrometer sections from paraffin-embedded specimens were evaluated by using an avidin-biotin immunohistochemical method with antibodies against the proteins mentioned above. RhoA and RhoB staining was observed in a high number of cells (P < 0.05) and greater intensity in non-polarized ones. Rac1 was not observed, and Cdc42 did not show any statistical differences between the number of non-polarized and basal positive cells (P > 0.05). Upon comparing the studied ameloblastomas, a higher number of positive cells in the unicystic variant was observed than that in the solid one (P < 0,05). The results obtained suggest that these GTPases could play a role in the ameloblastoma neoplastic epithelial cell phenotype determination (polarized or non-polarized), as well as in variant (solid or unicystic) and subtype (follicular or plexiform) determination. Furthermore, they could participate in solid ameloblastoma invasion mechanisms.

GTPases RhoA and Rac1 are important for amelogenin and DSPP expression during differentiation of ameloblasts and odontoblasts.

Morphogenesis and cytodifferentiation are distinct processes in tooth development. Cell proliferation predominates in morphogenesis; differentiation involves changes in form and gene expression. The cytoskeleton is essential for both processes, being regulated by Rho GTPases. The aim of this study was to verify the expression, distribution, and role of Rho GTPases in ameloblasts and odontoblasts during tooth development in correlation with actin and tubulin arrangements and amelogenin and dentin sialophosphoprotein (DSPP) expression. RhoA, Rac1, and Cdc42 were strongly expressed during morphogenesis; during cytodifferentiation, RhoA was present in ameloblasts and odontoblasts, Rac1 and its effector Pak3 were observed in ameloblasts; and Cdc42 was present in all cells of the tooth germ and mesenchyme. The expression of RhoA mRNA and its effectors RockI and RockII, Rac1 and Pak3, as analyzed by real-time polymerase chain reaction, increased after ameloblast and odontoblast differentiation, according to the mRNA expression of amelogenin and DSPP. The inhibition of all Rho GTPases by Clostridium difficile toxin A completely abolished amelogenin and DSPP expression in tooth germs cultured in anterior eye chamber, whereas the specific inhibition of the Rocks showed only a partial effect. Thus, both GTPases are important during tooth morphogenesis. During cytodifferentiation, Rho proteins are essential for the complete differentiation of ameloblasts and odontoblasts by regulating the expression of amelogenin and DSPP. RhoA and its effector RockI contribute to this role. A specific function for Rac1 in ameloblasts remains to be elucidated; its punctate distribution indicates its possible role in exocytosis/endocytosis.