UV Radiation-induced Impairment of Cellular Morphology and Motility is Enhanced by DUSP3/VHR Loss and FAK Activation.

DUSP3 is a phosphatase expressed and active in several tissues that dephosphorylates tyrosine residues in many regulatory proteins of cellular activities such as proliferation, survival, and cell death. Recently, two new independent functions were assigned to this enzyme: dephosphorylation of focal adhesion kinase (FAK) and regulation of nucleotide-excision repair (NER) pathway. Genotoxic stress by UV radiation is known to affect cell morphology, adhesion, and migration for affecting, for example, the Rho GTPases that regulate actin cytoskeleton. This work investigated the intersection of DUSP3 function, XPA protein activity, and UV toxicity by examining cell migration, FAK, and SRC kinase phosphorylation status, in addition to cell morphology, in fibroblast cells proficient (MRC-5) or deficient (XPA) of the NER pathway. DUSP3 loss reduced cell migration of normal cells, which was stimulated by the genotoxic stress, effects evidenced in presence of serum mitogenic stimulus. However, NER-deficient cells migration response was the opposite since DUSP3 loss increased migration, especially after cells being exposed to UV stress. The levels of pFAK(Y397) peaked 15 min and 1 h after UV radiation in normal cells, but only slightly increased in repair-deficient cells. However, the DUSP3 knockdown strongly raised pFAK(Y397) levels in both cells, but especially in XPA cells as supported by the higher SRC activity. These effects impacted on the dynamics of actin-based structures formation, such as stress fibres, apparently dependent on DUSP3 and DNA-repair (NER) proficiency of the cells. Altogether our findings suggest this dual-phosphatase is bridging gaps between the complex regulation of cell morphology, motility, and genomic stability.

γ-irradiation from radiotherapy improves the virulence potential of Candida tropicalis.

AIM: To evaluate if radiation used in radiotherapy can cause changes in the virulence potential of Candida tropicalis ATCC 750. MATERIALS & METHODS: C. tropicalis was exposed in vitro to identical dose and scheme of irradiation would be used in patients with head and neck cancer. Some virulence parameters were analyzed before and after irradiation. RESULTS: Colony morphologies were irreversibly affected by irradiation. Increase in growth rate, filamentation, adhesion on cell lines and phagocytosis process were also observed. Overall the irradiated C. tropicalis cells became more efficient at causing systemic infection in mice. CONCLUSION: γ-radiation induced important changes in C. tropicalis increasing its virulence profile, which could directly affect the relationship between yeasts and hosts.

Endothelial cell responses to castor oil-based polyurethane substrates functionalized by direct laser ablation.

Surface-induced thrombosis and lack of endothelialization are major drawbacks that hamper the widespread application of polyurethanes for the fabrication of implantable cardiovascular devices. Endothelialization of the blood-contacting surfaces of these devices may avoid thrombus formation and may be implemented by strategies that introduce micro and submicron patterns that favor adhesion and growth of endothelial cells. In this study, we used laser radiation to directly introduce topographical patterns in the low micrometer range on castor oil-based polyurethane, which is currently employed to fabricate cardiovascular devices. We have investigated cell adhesion, proliferation, morphology and alignment in response to these topographies. Reported results show that line-like and pillar-like patterns improved adhesion and proliferation rate of cultured endothelial cells. The line-like pattern with 1 µm groove periodicity was the most efficient to enhance cell adhesion and induced marked polarization and alignment. Our study suggests the viability of using laser radiation to functionalize PU-based implants by the introduction of specific microtopography to facilitate the development of a functional endothelium on target surfaces.

The role of cytoskeleton and adhesion proteins in the resistance to photodynamic therapy. Possible therapeutic interventions.

It is known that Photodynamic Therapy (PDT) induces changes in the cytoskeleton, the cell shape, and the adhesion properties of tumour cells. In addition, these targets have also been demonstrated to be involved in the development of PDT resistance. The reversal of PDT resistance by manipulating the cell adhesion process to substrata has been out of reach. Even though the existence of cell adhesion-mediated PDT resistance has not been reported so far, it cannot be ruled out. In addition to its impact on the apoptotic response to photodamage, the cytoskeleton alterations are thought to be associated with the processes of metastasis and invasion after PDT. In this review, we will address the impact of photodamage on the microfilament and microtubule cytoskeleton components and its regulators on PDT-treated cells as well as on cell adhesion. We will also summarise the impact of PDT on the surviving and resistant cells and their metastatic potential. Possible strategies aimed at taking advantage of the changes induced by PDT on actin, tubulin and cell adhesion proteins by targeting these molecules will also be discussed.

Effects of low level laser therapy on attachment, proliferation, and gene expression of VEGF and VEGF receptor 2 of adipocyte-derived mesenchymal stem cells cultivated under nutritional deficiency.

Low-level laser therapy (LLLT) has been shown to increase the proliferation of several cell types. We evaluated the effects of LLLT on adhesion, proliferation, and gene expression of vascular endothelial growth factor (VEGF) and type 2 receptor of VEGF (VEGFR2) at mesenchymal stem cells (MSCs) from human (hMSCs) and rat (rMSCs) adipose tissues on nutritional deficiencies. A dose-response curve was performed with cells treated with laser Ga-Al-As (660 nm, 30 mW) at energy of 0.7 to 9 J. Cell adhesion and proliferation were quantified 20, 40, and 60 min after LLLT and 24, 72, and 120 h after cultivation. Gene expression was verified by RT-PCR after 2 h of LLLT. A minor nutritional support caused a significant decrease in proliferation and adhesion of hMSCs and rMSCs. However, at the lowest LLLT dose (0.7 J), we observed a higher proliferation in hMSCs at standard condition shortly after irradiation (24 h). Adhesion was higher in hMSCs cultivated in controlled conditions at higher LLLT doses (3 and 9 J), and rMSCs show a reduction in the adhesion on 1.5 to 9 J. On nutritional deprivation, a 9 J dose was shown to reduce proliferation with 24 h and adhesion to all culture times in rMSCs. VEGF and VEGFR2 were increased after LLLT in both cell types. However, hMSCs under nutritional deprivation showed higher expression of VEGF and its receptor after irradiation with other laser doses. In conclusion, LLLT on human and rat MSCs might upregulate VEGF messenger RNA (mRNA) expression and modulate cell adhesion and proliferation distinctively.

Cytostatic in vitro effects of DTCM-glutarimide on bladder carcinoma cells.

Bladder cancer is a common malignancy worldwide. Despite the increased use of cisplatin-based combination therapy, the outcomes for patients with advanced disease remain poor. Recently, altered activation of the PI3K/ Akt/mTOR pathway has been associated with reduced patient survival and advanced stage of bladder cancer, making its upstream or downstream components attractive targets for therapeutic intervention. In the present study, we showed that treatment with DTCM-glutaramide, a piperidine that targets PDK1, results in reduced proliferation, diminished cell migration and G1 arrest in 5637 and T24 bladder carcinoma cells. Conversely, no apoptosis, necrosis or autophagy were detected after treatment, suggesting that reduced cell numbers in vitro are a result of diminished proliferation rather than cell death. Furthermore previous exposure to 10 µg/ml DTCM- glutarimide sensitized both cell lines to ionizing radiation. Although more studies are needed to corroborate our findings, our results indicate that PDK1 may be useful as a therapeutic target to prevent progression and abnormal tissue dissemination of urothelial carcinomas.

Effect of Er,Cr:YSGG and Er:YAG laser irradiation on the adhesion of blood components on the root surface and on root morphology

The aim of this study was to conduct an in vitro evaluation, by scanning electron microscopy (SEM), of the adhesion of blood components on root surfaces irradiated with Er,Cr:YSGG (2.78 µm) or Er:YAG (2.94 µm) laser, and of the irradiation effects on root surface morphology. Sixty samples of human teeth were previously scaled with manual instruments and divided into three groups of 20 samples each: G1 (control group) - no treatment; G2 - Er,Cr:YSGG laser irradiation; G3 - Er:YAG laser irradiation. After performing these treatments, blood tissue was applied to 10 samples of each group, whereas 10 samples received no blood tissue application. After performing the laboratory treatments, the samples were observed under SEM, and the resulting photomicrographs were classified according to a blood component adhesion scoring system and root morphology. The results were analyzed statistically (Kruskall-Wallis and Mann Whitney tests, α= 5%). The root surfaces irradiated with Er:YAG and Er,Cr:YSGG lasers presented greater roughness than those in the control group. Regarding blood component adhesion, the results showed a lower degree of adhesion in G2 than in G1 and G3 (G1 × G2: p = 0.002; G3 × G2: p = 0.017). The Er:YAG and Er,Cr:YSGG laser treatments caused more extensive root surface changes. The Er:YAG laser treatment promoted a greater degree of blood component adhesion to root surfaces, compared to the Er,Cr:YSGG treatment.

Effect of Er,Cr:YSGG and Er:YAG laser irradiation on the adhesion of blood components on the root surface and on root morphology.

The aim of this study was to conduct an in vitro evaluation, by scanning electron microscopy (SEM), of the adhesion of blood components on root surfaces irradiated with Er,Cr:YSGG (2.78 µm) or Er:YAG (2.94 µm) laser, and of the irradiation effects on root surface morphology. Sixty samples of human teeth were previously scaled with manual instruments and divided into three groups of 20 samples each: G1 (control group) - no treatment; G2 - Er,Cr:YSGG laser irradiation; G3 - Er:YAG laser irradiation. After performing these treatments, blood tissue was applied to 10 samples of each group, whereas 10 samples received no blood tissue application. After performing the laboratory treatments, the samples were observed under SEM, and the resulting photomicrographs were classified according to a blood component adhesion scoring system and root morphology. The results were analyzed statistically (Kruskall-Wallis and Mann Whitney tests, α= 5%). The root surfaces irradiated with Er:YAG and Er,Cr:YSGG lasers presented greater roughness than those in the control group. Regarding blood component adhesion, the results showed a lower degree of adhesion in G2 than in G1 and G3 (G1 × G2: p = 0.002; G3 × G2: p = 0.017). The Er:YAG and Er,Cr:YSGG laser treatments caused more extensive root surface changes. The Er:YAG laser treatment promoted a greater degree of blood component adhesion to root surfaces, compared to the Er,Cr:YSGG treatment.

Changes in actin and E-cadherin expression induced by 5-aminolevulinic acid photodynamic therapy in normal and Ras-transfected human mammary cell lines.

Photodynamic therapy (PDT) is an anticancer treatment based on light-induced destruction of photosensitised malignant cells. It has been reported that PDT strongly affects cell-cell and cell-substrate adhesion through the reorganization of some cytoskeletal and adhesion proteins. The aim of the present work was to study the changes induced by PDT employing aminolevulinic acid (ALA), on the cytoskeleton actin network and E-cadherin expression. We employed the normal mammary HB4a cell line and its tumor counterpart transfected with the oncogene H-Ras, which has been shown to be resistant to PDT. Ras insertion induces per se disorganization of both F-actin and E-cadherin distribution. ALA-PDT induces on HB4a cells a dramatic disorganization of actin stress fibers, resembling normal Ras-transfected cells. After 48h some features of disorganization remain present. In HB4a-Ras cells, F-actin exhibits signals of photodamage, but distribution is recovered 24h after treatment. On the other hand, PDT did not impact on E-cadherin distribution, other than a transient disorganization, which was recovered at 24h. Moreover, E-cadherin disorganization did not favoured cell-cell detachment after PDT of HB4a-Ras cells. Actin but not E-cadherin constitutes in this model an important target of PDT. The fact that some features of microfilament disorganization remain present in HB4a surviving cells but not in Ras-transfected cells, suggests that cytoskeletal structures such as F-actin may be involved in the mechanisms of resistance to PDT.

Effect of low-level laser therapy on proliferation, differentiation, and adhesion of steroid-treated osteoblasts.

There has recently been constant effort to evaluate therapies that may have a positive effect on bone regeneration. However, there are few studies in the literature on the effects of low-level laser therapy (LLLT) involving tissues treated with anabolic steroids. The present study evaluated the effects of LLLT (AsGaAl 780 nm, 3 J/cm(2), 10 mW, beam spot of 0.04 cm(2), total energy 0.12 J) on the proliferation, adhesion, and differentiation of osteoblasts cultured in the presence of nandrolone decanoate (ND). The MTT method was employed to evaluate cell proliferation and adhesion. Cell differentiation was evaluated by measuring alkaline phosphatase activity. There was a significant decrease in cell proliferation in the irradiated group treated with 50 µM ND when compared to the control group, after 48 h. After 72 h, cell proliferation was significantly greater in the control group than in the irradiated groups treated with the steroid at concentrations of 10, 25, and 50 µM. With regard to cell differentiation, alkaline phosphatase activity was significantly higher in the irradiated group treated with 50 µM ND than in the control group, irradiated non-treated group, and irradiated group treated with 25 µM ND. After 60 min of plating, the irradiated non-treated group and irradiated groups treated with the steroid at concentrations of 5, 10, and 25 µM exhibited a significant increase in cell adhesion compared to the control group. LLLT in combination with a high concentration of steroid inhibited cell proliferation, possibly by inducing cell differentiation, while irradiation combined with lower concentrations of the steroid induced an increase in cell adhesion.

Efectos de la luz UV sobre placas de titanio para la adhesión osteoblástica / The effects of ultraviolet irradiation of titanium plates on osteoblastic adhesion

Objetivo: investigar los efectos de la irradiación ultravioleta (UV) sobre placas de titanio (Ti) para la adhesión osteoblástica por medio de un método colorimétrico simple y reproducile para determinar la densidad celular. Materiales y métodos: dos diferentes tamaños (10x10x0.5 mm y 20x20x0.5 mm) de placas (n=10 c/gp) fueron obtenidas de una hoja de Ti puro y divididas en dos grupos (n=5/gp) para cada tamaño de placa. La superficie de las placas de Ti fue pulida, observada con microscopía electrónica de barrido (MEB) y estimada la rugosidad de la superficie pulida. Para el grupo experimental, las placas de Ti fueron irradiadas a una longitud de onda de 253.7 nm con luz UV durante 5, 20, 40, 60 minutos ó 4 y 6 horas. Células odontoblásticas de ratón MC3T3-E1 fueron cultivadas en medio alfa mínimo esencial (-MEM) e inoculadas sobre cada placa de Ti. El número de células adheridas y proliferadas fue determinado por medio del método MTT y el consumo de animoácidos. Resultados: 20 minutos de irradiación UV de las placas de Ti fueron suficientes para incrementar significativamente (p<0.05) la adhesión y proliferación celular, acompañado de un mayor consumo de aminoácidos. Conclusiones: la irradiación UV sobre placas de Ti incrementó significatiamente la adhesión celular por medio del método MTT, confirmando la potencialidad de la luz UV.

In vitro photodynamic inactivation of Candida spp. growth and adhesion to buccal epithelial cells.

In this study, photodynamic inactivation (PDI) was used to inhibit in vitro growth and adhesion of different Candida isolates to buccal epithelial cells (BEC). Experimental conditions were optimized and 25muM toluidine blue O (TBO) and 15min of irradiation time by light emitting diode (LED) (energy density of 180J/cm(2)) were selected due to higher reductions in cellular viability obtained after treatment. Reduction media of Log(10) 3.41 in viable cellular growth and media of 55% in the inhibition of adhesion to buccal epithelial cells were obtained. Two fluconazole resistant isolates were susceptible to PDI (Log(10) 3.54 in IB05 and Log(10) 1.95 in CG09) and a second session of this treatment for CG09 isolate inhibited cellular viability in 100%, without producing heat. The results permit to conclude that photodynamic inactivation under these experimental conditions would be a possible alternative approach to inhibit Candida spp. cellular growth and adhesion to buccal epithelial cells.

Attenuation of genotoxicity under adhesion-restrictive conditions through modulation of p53, gamma H2AX and nuclear DNA organization.

Interactions between tumor cells and their substratum influence cancer progression by modulating cell proliferation and survival. We now investigated whether signaling responses to UV irradiation differ on adhesion-permissive or restrictive substrates. The latter conditions diminished spreading and proliferation of neo 6.3/C8161 melanoma in which metastasis is suppressed by introduction of neo-tagged chromosome 6, but permitted proliferation of human metastatic C8161 melanoma. Apoptosis-associated PARP cleavage and DNA fragmentation induced by UV irradiation were diminished on the restrictive substrate in C8161 melanoma. Genotoxic responses to UV irradiation like persistent increases in the phosphorylation of histone H2AX, induction of the tumor suppressor p53 protein and greater binding of this protein to its DNA consensus sequence, were all decreased on the restrictive substrate. The latter also promoted a 2 fold increase of DNA condensation in chromatin and enhanced activation of the survival - and invasion-associated MMP-9 gelatinase B, preferentially in metastatic C81261 melanoma. Our data suggest that adaptation to restrictive substrates in metastatic C8161 melanoma decreases UV-induced apoptosis, partly through attenuation of DNA damage signaling responses and changes in genomic organization.

Loss of E-cadherin mediated cell-cell adhesion as an early trigger of apoptosis induced by photodynamic treatment.

Photodynamic treatment with different photosensitizers (PSs) can result in the specific induction of apoptosis in many cell types. It is commonly accepted that this apoptotic response depends on the mitochondrial accumulation of the PS. Accumulation in other cellular organelles, such as lysosomes or the Golgi complex, and subsequent photodamage resulting in an apoptotic process has been also described. However, the role played by cell adhesion in apoptosis induced in epithelial cells after photodynamic treatment is not well characterized. Here, we have used a murine keratinocyte line, showing a strong dependence on E-cadherin for cell-cell adhesion and survival, to analyze the relevance of this adhesion complex in the context of zinc(II)-phthalocyanine (ZnPc) photodynamic treatment. We report that under apoptotic conditions, ZnPc phototreatment induces a rapid disorganization of the E-cadherin mediated cell-cell adhesion, which largely preceded both the detachment of cells from the substrate, via beta-1 integrins and the induction of apoptotic mitochondrial markers. Therefore, the alteration in E-cadherin, alpha- and beta-catenins adhesion proteins preceded the release of cytochrome c (cyt c) from mitochondria to the cytosol and the activation of caspase 3. In addition, blocking E-cadherin function with a specific antibody (Decma-1) induced apoptosis in this cell system. These results strongly suggest that the E-cadherin adhesion complex could be the primary target of ZnPc phototreatment, and that loss of E-cadherin mediated cell adhesion after early photodamage triggers an apoptotic response.

Photo-induced cytomorphologic changes in an advanced cancer phase I clinical trial.

BACKGROUND AND OBJECTIVES: The aim of this study was to investigate whether the application of an Infrared Pulsed Laser Device (IPLD) photo-induced significant cytomorphologic changes during the monitoring of advanced cancer patients participating in a phase I clinical trial. MATERIALS AND METHODS: Patients were irradiated with an IPLD (904 nm pulsed at 3 MHz) under a one-dose, one-schedule, and one-procedure design. Total daily dose consisted of a Radiant Exposure of 4.5x10(5) J/m(2). Thirty-one tissue samples from eleven patients with progressive solid neoplastic diseases (TNM IV, UICC) were obtained at three intervals: Time 0 (15-90 days pre-treatment, n=11); Time I (2-5 months post-treatment; n=11); Time II (6-12 months post-treatment, n=09). Three blinded pathologists evaluated samples; scores were determined by consensus. Data were evaluated by using the Wilcoxon matched-pairs signed-rank test and Spearman rank correlation coefficient. The level of statistical significance was alpha=0.05. RESULTS: Increased apoptosis (Time I, P<0.003; Time II, P<0.007), necrosis (Time I, NS; Time II, P<0.01), cytoplasmic vacuoles (Time I, P<0.03; Time II, P<0.02), and nuclear vacuoles (Time I, NS; Time II, P<0.01), reduced cell size (Time I, P<0.007; Time II, P<0.01) and intercellular adhesion (Time I, P<0.01; Time II, P<0.02) were present in neoplastic cells after IPLD treatment. No apparent changes were noted in non-neoplastic cells. The Spearman rank correlation coefficient between apoptosis, necrosis, nuclear vacuoles, cytoplasmatic vacuoles, intercellular adhesion, and cell size was positive and highly significant (P<0.006). CONCLUSIONS: Although further research is necessary, our preliminary results support the novel possibility that the IPLD photo-induces chaotic dynamics that modulate complex physiologically reparative bioeffects.

Schistosoma mansoni: inflammatory foci around larvae in the peritoneal cavity of naive mice is radiosensitive.

Innate attack to Schistosoma mansoni cercariae was evaluated in irradiated mice. It was observed that 70% of the larvae from mice sacrificed one day after whole body irradiation with 400 or 800 rads were surrounded by cluster reactivities, without difference from controls. Differences were apparent on day 5 after irradiation with sub lethal (400 rads) or lethal doses (800 rads) suggesting that innate defence to infection take at least 5 days to be affected by low dose whole-body radiation.

Schistosoma mansoni: inflammatory foci around larvae in the peritoneal cavity of naive mice is radiosensitive

Innate attack to Schistosoma mansoni cercariae was evaluated in irradiated mice. It was observed that 70 percent of the larvae from mice sacrificed one day after whole body irradiation with 400 or 800 rads were surrounded by cluster reactivities, without difference from controls. Differences were apparent on day 5 after irradiation with sub lethal (400 rads) or lethal doses (800 rads) suggesting that innate defence to infection take at least 5 days to be affected by low dose whole-body radiation

Laminin-induced PC-12 cell differentiation is inhibited following laser inactivation of cellular prion protein.

Prions, the etiological agents for infectious degenerative encephalopathies, act by inducing structural modifications in the cellular prion protein (PrPc). Recently, we demonstrated that PrPc binds laminin (LN) and that this interaction is important for the neuritogenesis of cultured hippocampal neurons. Here we have used the PC-12 cell model to explore the biological role of LN-PrPc interaction. Antibodies against PrPc inhibit cell adhesion to LN-coated culture plaques. Furthermore, chromophore-assisted laser inactivation of cell surface PrPc perturbs LN-induced differentiation and promotes retraction of mature neurites. These results point out to the importance of PrPc as a cell surface ligand for LN.