Cellular homeostasis and oncology safety of low pulse frequency laser for photobiomodulation in oral and dermal cells.

In response to transforming growth factor beta1 (TGFbeta) stimulation, fibroblasts modify their integrin repertoire and adhesive capabilities to certain extracellular matrix proteins. Although TGFbeta has been shown to increase the expression of specific alphav integrins, the mechanisms underlying this are unknown. In this study we demonstrate that TGFbeta1 increased both beta3 integrin subunit mRNA and protein levels as well as surface expression of alphavbeta3 in human lung fibroblasts. TGFbeta1-induced alphavbeta3 expression was strongly adhesion-dependent and associated with increased focal adhesion kinase and c-Src kinase phosphorylation. Inhibition of beta3 integrin activation by the Arg-Gly-Asp tripeptide motif-specific disintegrin echistatin or alphavbeta3 blocking antibody prevented the increase in beta3 but not beta5 integrin expression. In addition, echistatin inhibited TGFbeta1-induced p38 MAPK but not Smad3 activation. Furthermore, inhibition of the Src family kinases, but not focal adhesion kinase, completely abrogated TGFbeta1-induced expression of alphavbeta3 and p38 MAPK phosphorylation but not beta5 integrin expression and Smad3 activation. The TGFbeta1-induced alphavbeta3 expression was blocked by pharmacologic and genetic inhibition of p38 MAPK- but not Smad2/3-, Sp1-, ERK-, phosphatidylinositol 3-kinase, and NF-kappaB-dependent pathways. Our results demonstrate that TGFbeta1 induces alphavbeta3 integrin expression via a beta3 integrin-, c-Src-, and p38 MAPK-dependent pathway. These data identify a novel mechanism for TGFbeta1 signaling in human lung fibroblasts by which they may contribute to normal and pathological wound healing.

Focal Adhesion Kinase (FAK) activity is controlled by growth factors and adhesion signals in tumor cells. The scaffolding protein RACK1 (receptor for activated C kinases) integrates insulin-like growth factor I (IGF-I) and integrin signaling, but whether RACK1 is required for FAK function is unknown. Here we show that association of FAK with RACK1 is required for both FAK phosphorylation and dephosphorylation in response to IGF-I. Suppression of RACK1 by small interfering RNA ablates FAK phosphorylation and reduces cell adhesion, cell spreading, and clonogenic growth. Peptide array and mutagenesis studies localize the FAK binding interface to blades I-III of the RACK1 beta-propeller and specifically identify a set of basic and hydrophobic amino acids (Arg-47, Tyr-52, Arg-57, Arg-60, Phe-65, Lys-127, and Lys-130) as key determinants for association with FAK. Mutation of tyrosine 52 alone is sufficient to disrupt interaction of RACK1 with FAK in cells where endogenous RACK1 is suppressed by small interfering RNA. Cells expressing a Y52F mutant RACK1 are impaired in adhesion, growth, and foci formation. Comparative analyses of homology models and crystal structures for RACK1 orthologues suggest a role for Tyr-52 as a site for phosphorylation that induces conformational change in RACK1, switching the protein into a FAK binding state. Tyrosine 52 is further shown to be phosphorylated by c-Abl kinase, and the c-Abl inhibitor STI571 disrupts FAK interaction with RACK1. We conclude that FAK association with RACK1 is regulated by phosphorylation of Tyr-52. Our data reveal a novel mechanism whereby IGF-I and c-Abl control RACK1 association with FAK to facilitate adhesion signaling.

Low-level laser therapy (LLLT) exhibited biostimulatory effects on fibroblasts viability. Secretomes can be administered to culture mediums by using bone marrow mesenchymal stem cells conditioned medium (BM-MSCs CM). This study investigated the combined effects of LLLT and human bone marrow mesenchymal stem cell conditioned medium (hBM-MSCs CM) on the cellular viability of human dermal fibroblasts (HDFs), which was cultured in a high-glucose (HG) concentration medium. The HDFs were cultured either in a concentration of physiologic (normal) glucose (NG; 5.5 mM/l) or in HG media (15 mM/l) for 4 days. LLLT was performed with a continuous-wave helium-neon laser (632.8 nm, power density of 0.00185 W/cm(2) and energy densities of 0.5, 1, and 2 J/cm(2)). About 10% of hBM-MSCs CM was added to the HG HDF culture medium. The viability of HDFs was evaluated using dimethylthiazol-diphenyltetrazolium bromide (MTT) assay. A significantly higher cell viability was observed when laser of either 0.5 or 1 J/cm(2) was used to treat HG HDFs, compared to the control groups. The cellular viability of HG-treated HDFs was significantly lower compared to the LLLT + HG HDFs, hBM-MSCs CM-treated HG HDFs, and LLLT + hBM-MSCs CM-treated HG HDFs. In conclusion, hBM-MSCs CM or LLLT alone increased the survival of HG HDFs cells. However, the combination of hBM-MSCs CM and LLLT improved these results in comparison to the conditioned medium.

β1 Integrin Gene Excision in the Adult Murine Cardiac Myocyte Causes Defective Mechanical and Signaling Responses

Pulsed low-intensity laser treatment stimulates wound healing without enhancing biofilm development in vitro.

Triple-Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer with high metastatic potential and increased morbidity and mortality. Although expression of, and signaling by the epidermal growth factor receptor (EGFR) is commonly seen in TNBC, anti-EGFR antibodies such as Cetuximab have had limited therapeutic efficacy, used either alone or in combination with chemotherapy. Primary TNBC tumor growth and metastases require supporting vasculature, which develops through a combination of endothelial angiogenesis and vasculogenic mimicry (VM). Our laboratory previously developed a novel targeted therapy, αEGFR-E-P125A, an antibody-endostatin fusion protein, by linking an anti-EGFR antibody targeting domain to a mutated version of the anti-angiogenic protein endostatin (E-P125A). αEGFR-E-P125A delivers a dimeric E-P125A payload which inhibits TNBC angiogenesis and VM in vitro and in vivo, and decreases metastatic tumor growth (lung metastasis) in both the MDA-MB-231-4175 and MDA-MB 468 TNBC xenograft models. Preliminary studies of the mechanism of αEGFR-E-P125A action indicate that αEGFR-E-P125A downregulates VM through combined inhibition of EGFR and integrin signaling. Phospho-array analysis of αEGFR-E-P125A treated MDA-MB-231-4175 cells demonstrated downregulation of phosphorylation of EGFR at Y1069, a site of aberrant EGFR signaling which promotes TNBC motility. The phospho-array also demonstrated inhibition of focal adhesion kinase (FAK) phosphorylation at Y397. FAK signaling occurs downstream of β1 and β3 integrins, and the Y397 site is implicated in the regulation of endothelial and TNBC motility, VM, and metastatic behavior. We specifically inhibited FAK at the Y397 site using a small molecule inhibitor, PF-573228, and demonstrated that FAK inhibition at Y397 alone inhibited the TNBC VM in vitro. shRNA-mediated knockdown of FAK in MDA-MB-231 TNBC cells confirmed that downregulation of FAK inhibited VM formation in vitro. Immunoprecipitation (IP) of EGFR detected the presence of β1 integrin, and IP of β1 integrin detected EGFR in both MDA-MB-231-4175 and MDA-MB-468 TNBC cells treated with αEGFR-E-P125A. αEGFR-E-P125A treatment increased levels of bound β1 integrin following IP of EGFR relative to untreated or Cetuximab treated TNBC cells. These results indicate that αEGFR-E-P125A is binding to both EGFR and β1 integrin simultaneously, suppressing downstream EGFR and integrin signaling. Simultaneous inhibition of EGFR and β1 integrin signaling by αEGFR-E-P125A fusion is a promising approach to inhibition of TNBC growth and metastases. Citation Format: Ankita P. Sankar, Hyun Mi Cho, Hava Gil-Henn, Sundaram Ramakrishnan, Rathin Das, Christian Elledge, Yu Zhang, Seung-Uon Shin, Joseph D. Rosenblatt. αEGFR-E-P125A antibody-endostatin fusion protein reduces vasculogenic mimicry and metastasis by inhibiting FAK, integrin, and EGFR signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2413.

Low-level laser therapy (LLLT) has several clinical applications; however, its benefits are not universal. Therefore, combination therapy with LLLT and extracts from the guarana (Paullinia cupana) plant may improve its effectiveness as guarana extracts exhibit anti-aging properties. To evaluate the antioxidant, anti-inflammatory, anti-apoptotic, and proliferative effects of combined LLLT and guarana extract therapy on human dermal fibroblasts. Human dermal fibroblasts (HFF-1) were cultured and initially exposed to several concentrations (1, 3, 5, 10, 30µg/mL) of guarana extract. The experimental concentration of guarana extract was selected by analyzing cytokine levels, DNA oxidation, and apoptotic markers in LLLT-exposed (4J/cm2 ) and LLLT-unexposed fibroblast cultures. After 72hours, the cells were analyzed using spectrophotometric, fluorimetric, immunological, and gene expression (qRT-PCR) assays. Flow cytometry was used to evaluate the effect of each treatment on cell cycle. Fibroblasts treated with guarana (5µg/mL) exhibited anti-inflammatory and anti-apoptotic properties been used in complementary protocols. Combined guarana and LLLT treatment significantly decreased protein carbonylation, lipoperoxidation, and DNA oxidation, downregulated the mRNA and protein expression of pro-inflammatory molecules, and upregulated IL-10 gene and protein expression. Guarana plus LLLT also decreased the levels of caspases 1, 3, and 8, increased the percentage of S-phase cells, and decreased FGF-1 and KGF-1 levels. Some of these changes were also observed after treatment with guarana or LLLT alone. Our results suggest that concomitant treatment with guarana and LLLT may promote fibroblast biostimulation and thus is clinically relevant.

Platelet-rich plasma (PRP) and plasma rich in growth factors (PRGF) are autologous preparations widely applied in regenerative medicine because of their potential to enhance tissue repair and cellular activity. Low-level laser therapy (LLLT) has also been reported to promote fibroblast proliferation and mitochondrial activity. This study aimed to investigate the combined effects of PRP, PRGF, and LLLT on human dermal fibroblasts (HDFCs). HDFCs were cultured and treated with varying concentrations of PRP and PRGF. Cells were subsequently exposed to a gallium-aluminum-arsenide (GaAlAs) laser at 830 nm, 10 mW, continuous mode, for 90 s, 3 min, or 6 min. Proliferation, metabolic activity, and oxidative stress were evaluated using MTT, ATP, and ROS assays at 24 h and 48 h. A significant increase (p < 0.001) in proliferation and ATP production was observed in the PRP 30% and PRGF 60% groups after 90 s of LLLT. MTT values were 1.17 ± 0.00 and 1.39 ± 0.05 at 24 h, and 1.40 ± 0.04 and 1.62 ± 0.01 at 48 h, respectively. ATP levels reached 6710.33 ± 59.72 and 18,709 ± 535.29 RLU at 24 h, and 8202 ± 333.39 and 22,272 ± 839.64 RLU at 48 h, compared with control (1005 ± 52.92 RLU). No significant differences in ROS activity were detected among most groups, although PRGF 70% showed elevated ROS with and without laser irradiation. PRP and PRGF synergistically enhanced fibroblast proliferation and metabolism when combined with LLLT. PRGF consistently demonstrated greater effects compared with PRP. Optimal stimulation was achieved with 90 s of LLLT at 10 mW (3.78 J/cm2) combined with 30% PRP or 60% PRGF.

Neuroblastoma (NB) is the most common pediatric extracranial solid tumor. It is derived from the sympatho-adrenal lineage of the neural crest, during development of the sympathetic nervous system, and usually arises in the adrenal gland or sympathetic ganglia. Since &amp;gt;50% of diagnosed cases are metastatic, it is important to know the mechanisms underlying its dissemination. During metastasis, cells migrate, invade, and colonize local and distant organs. Focal Adhesion Kinase (FAK) and Integrin β1 are key regulators of focal adhesion formation and cell migration. FAK induces Integrin β1 activation, focal adhesion turnover, and consequently cell migration, and it has been shown to be upregulated in tumors. FAK binds several upstream receptors including Neogenin-1 (NEO1), which is involved in axonal guidance, angiogenesis, and neuronal cell migration. On the other hand, NEO1 binds to the Netrin family of ligands. Analysis of public databases of NB tumors reveals that high expression of NEO1 correlates with poor patient survival. The aim of this work is to evaluate the role of migrationrelated signaling pathways downstream of NEO1/Netrin-1 (main ligand of the Netrin family). To this end, we focused on FAK and Integrin β1 interaction/activation. Western blot in SK-N-SH (MYCN WT) and LAN-1 (MYCN high) NB cell lines indicated that NEO1 and Netrin-4 are expressed while Netrin-1 is only expressed in LAN-1. Also, NEO1 associated with Netrin-1, as shown by co-immunoprecipitation assays in both cell lines. To evaluate the contribution of NEO1 in cell migration, integrin β1 activation, and metastasis, stable NEO1 knockdown cells (shNEO1) were generated using shRNAs via lentiviral infection. Cell migration was decreased in shNEO1 cells, as shown in transwell assays using Netrin-1 as chemoattractant, which could be due to decreased sensibility to the chemotactic stimulus of the ligand. We also assessed FAK dependency by pharmacologic inhibition with the PF562,271 compound in a spheroid-based cell migration assay. To further evaluate the association between NEO1 with Integrin β1 and FAK, we performed co-immunoprecipitation assays. Our data show that NEO1 associates in a complex with Integrin β1 and FAK in SK-N-SH cells. Immunoprecipitation of activated Integrin β1 with a specific conformational antibody shows that activation of Integrin β1 is less in shNEO1 cells compared to control cells. In an in vivo analysis of metastasis, SK-N-SK shNEO1 and control cells were injected into flank of immunodeficient mice (NSG strain). Metastasis was evaluated via luciferase activity in several organs, revealing that NEO1 is important to NB metastases. Our work suggests that NEO1 is a tumor progression-promoting protein, with an active role in metastasis. We propose a possible mechanism whereby NEO1, via its interaction with Netrin-1, is involved in cell migration and metastasis of NB cells. NEO1 likely plays its role by associating with FAK, allowing its phosphorylation and complex formation with Integrin β1, inducing its activation. Our results provide further information on the potential use of NEO1 as a therapeutic target to reduce metastasis in neuroblastoma. Citation Format: Andrea A. Villanueva, Ernesto Muñoz-Palma, Carlos Cubo, Vicente A. Torres, Pilar Sanchez-Gomez, Veronica Palma. Netrin-1/Neogenin-1 promotes neuroblastoma cell migration via activation of Integrin β1 [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A18.

BackgroundLow-level laser therapy (LLLT) with various types of lasers promotes fibroblast proliferation and migration during the process of wound healing. Although LLLT with a carbon dioxide (CO2) laser was also reported to promote wound healing, the underlying mechanisms at the cellular level have not been previously described. Herein, we investigated the effect of LLLT with a CO2 laser on fibroblast proliferation and migration.Materials and MethodsCultured human dermal fibroblasts were prepared. MTS and cell migration assays were performed with fibroblasts after LLLT with a CO2 laser at various doses (0.1, 0.5, 1.0, 2.0, or 5.0 J/cm2) to observe the effects of LLLT with a CO2 laser on the proliferation and migration of fibroblasts. The non-irradiated group served as the control. Moreover, western blot analysis was performed using fibroblasts after LLLT with a CO2 laser to analyze changes in the activities of Akt, extracellular signal-regulated kinase (ERK), and Jun N-terminal kinase (JNK), which are signaling molecules associated with cell proliferation and migration. Finally, the MTS assay, a cell migration assay, and western blot analysis were performed using fibroblasts treated with inhibitors of Akt, ERK, or JNK before LLLT with a CO2 laser.ResultsIn MTS and cell migration assays, fibroblast proliferation and migration were promoted after LLLT with a CO2 laser at 1.0 J/cm2. Western blot analysis revealed that Akt, ERK, and JNK activities were promoted in fibroblasts after LLLT with a CO2 laser at 1.0 J/cm2. Moreover, inhibition of Akt, ERK, or JNK significantly blocked fibroblast proliferation and migration.ConclusionsThese findings suggested that LLLT with a CO2 laser would accelerate wound healing by promoting the proliferation and migration of fibroblasts. Activation of Akt, ERK, and JNK was essential for CO2 laser-induced proliferation and migration of fibroblasts.

Ganglioside GD3 is widely expressed in human malignant melanoma cell lines and tumors. Previously, we reported that GD3+ cells show stronger tyrosine phosphorylation of focal adhesion kinase (FAK), p130(Cas), and paxillin when treated with fetal calf serum than GD3- cells. In this study, we analyzed the changes in the signals mediated by the interaction between integrins and extracellular matrices (ECM) to clarify how GD3 enhances cell signals in the vicinity of the cell membrane. An adhesion assay with a real time cell electronic sensing system revealed that GD3+ cells had stronger adhesion to all extracellular matrices examined. In particular, GD3+ cells attached more strongly to collagen type I and type IV than controls. Correspondingly, they showed stronger tyrosine phosphorylation of FAK and paxillin during adhesion to collagen type I. In the floating pattern of detergent extracts, a high level of integrin beta1 was found in glycolipid-enriched microdomain (GEM)/rafts in GD3+ cells before adhesion, whereas a smaller amount of integrin beta1 was detected in the GEM/rafts of controls. Some phosphorylated forms of FAK as well as total FAK were found in GEM/rafts during cell adhesion only in GD3+ cells. Another signal consisting of integrin-linked kinase/Akt was also activated during adhesion more strongly in GD3+ cells than in controls. In double stained GD3+ cells, GD3 and integrin beta1 co-localized at the focal adhesion with a punctate pattern. All these results suggested that integrins assembled and formed a cluster in GEM/rafts, leading to the enhanced signaling and malignant properties under GD3 expression.

Migration of Human Polymorphonuclear Leukocytes through a Synovial Fibroblast Barrier Is Mediated by both β2 (CD11/CD18) Integrins and the β1 (CD29) Integrins VLA-5 and VLA-6

Cell shape variations and substratum re-organization during contraction of floating collagen and fibrin lattices seeded with human gingival fibroblasts were determined by computerized image analysis of light and scanning electron microscopic images. Data were compared with those obtained with lattices populated with human dermal fibroblasts. The extent of collagen lattice contraction was similar with both cell types, resulting in a two-fold decrease in the area fractions occupied by collagen fibers. Fibroblasts exhibited a rounded shape (form factors equal to 0.8 and 0.7 for gingival and dermal cells, respectively) at day 1 of culture; they possessed a more elongated appearance (with form factors equal to 0.3 and 0.15 for gingival and dermal cells, respectively) at day 7. Continuous (gingival) and discontinuous (dermal) layers of cells were evidenced at the cortex of lattices. Contractions were associated with a significant reduction of the diameters of collagen fibers. Re-organization of substratum, as analyzed by the "Rose of Directions" technique, was evidenced only at the vicinity of filopodia where fibers ran parallel to these protrusions. Several lysed matrix cavities were observed when fibrin lattices were populated with gingival but not dermal fibroblasts at day 5 of culture. Although cells in fibrin lattices exhibited morphometric parameters comparable with those in collagen lattices, no fibroblast layers could be demonstrated at gel peripheries. Fibrin matrices consisted of an isotropic network of entangled fibrin filaments from the start of culture, and only a slight reduction of the diameters of fibrin fibers could be evidenced in dermal fibroblast-populated lattices. Fibrinolysis at the vicinity of gingival fibroblasts led to an entire re-organization of substratum toward the formation of larger fibers. The differential behavior of gingival vs. dermal fibroblasts inside fibrin but not collagen matrices could therefore partly explain the increased rate of remodeling of gingiva as compared with dermis.

Aim To clinically evaluate the postoperative pain (PP) after using intracanal diode laser (DL) irradiation and low-level laser therapy (LLLT) following single-visit endodontic treatment using visual analogue scale. Patients and methods Thirty-two patients requiring endodontic therapy of asymptomatic vital or nonvital maxillary central or canine teeth were enrolled in this study. Root canal treatment was done in a single-visit using ProTaper Universal rotary system. Irrigation was done using 2.5% sodium hypochlorite (NaOCl). Patients were randomly assigned into four equal groups according to the type of DL used: group I: conventional endodontic method (control group), group II: intracanal laser irradiation, group III: LLLT, group IV: both intracanal laser irradiation and LLLT. PP was assessed after 6, 24, 48 h, 1 and 2 weeks using visual analogue scale. Scores were collected from patients and statistically analyzed. Results Regarding PP, the group of both intracanal laser (group II) and LLLT (group IV) showed the least PP values while the control group showed the highest values, and this difference was statistically significant (P &lt; 0.001). Intracanal laser group and LLLT had lower PP values than the control group, and the difference was statistically significant between each group and the control group (P = 0.041) and (P = 0.009), respectively. In all groups, the intensity of PP decreased over time. Conclusions DL in both ways (intracanal laser irradiation and LLLT) reduced PP in single-visit root canal treatment. Using combination of both intracanal laser and LLLT may be an alternative option over the conventional treatment choice relying on nonsteroidal anti-inflammatory drugs in pain relief.

Different types of lasers are used in various research and medical applications such as therapies, surgical procedures and medical diagnostic. Low level laser therapy (LLT) utilizes low intensity power with various wavelength that are proposed for a wide variety of medical applications for promoting tissue regeneration, reduce inflammation and pain, and wound healing process. This study aimed to investigate the effect of laser irradiation diode-pumped solid state laser (DPSS) violet 405nm on cell viability of human normal and cancer cell lines. Human dermal fibroblasts (HDF) and human breast cancer (MCF7) cells lines were exposed to laser light for 5 minutes of exposure time. The cells viability were analyzed by Trypan Blue Exclusion Assay and morphological changes were evaluated using inverted microscope. Findings revealed that the viability of HDF was not affected by laser irradiation. However, this laser treatment exhibited cytotoxic effect on MCF7. Reduction of cells number and changes in cell morphology were observed in MCF7 after being treated with laser in short duration of time. Thus, the results may suggest that DPSS violet laser at 405nm had inhibition effect on cancer cell lines and did not cause loss of cell viability in normal cell lines.