Lasers Med Sci DOI 10.1007/s10103-014-1535-2
ORIGINAL ARTICLE
Low-level laser therapy promotes proliferation and invasion of oral squamous cell carcinoma cells Águida Cristina Gomes Henriques & Fernanda Ginani & Ruth Medeiros Oliveira & Tatjana Souza Lima Keesen & Carlos Augusto Galvão Barboza & Hugo Alexandre Oliveira Rocha & Jurema Freire Lisboa de Castro & Ricardo Della Coletta & Roseana de Almeida Freitas
Received: 10 October 2013 / Accepted: 28 January 2014 # Springer-Verlag London 2014
Abstract Low-level laser therapy (LLLT) has been shown to be effective in promoting cell proliferation. There is speculation that the biostimulatory effect of LLLT causes undesirable enhancement of tumor growth in neoplastic diseases since malignant cells are more susceptible to proliferative stimuli. This study evaluated the effects of LLLT on proliferation, invasion, and expression of cyclin D1, E-cadherin, βcatenin, and MMP-9 in a tongue squamous carcinoma cell line (SCC25). Cells were irradiated with a diode laser (660 nm) using two energy densities (0.5 and 1.0 J/cm2). The proliferative potential was assessed by cell growth curves and cell cycle analysis, whereas the invasion of cells was evaluated using a Matrigel cell invasion assay. Expression of cyclin D1, E-cadherin, β-catenin, and MMP-9 was analyzed by immunofluorescence and flow cytometry and associated with the biological activities studied. LLLT induced significantly the proliferation of SCC25 cells at 1.0 J/cm2, which was accomplished by an increase in the expression of cyclin Á. C. Gomes Henriques : F. Ginani : C. A. Galvão Barboza : R. de Almeida Freitas Department of Dentistry, Federal University of Rio Grande do Norte, Natal, RN, Brazil R. M. Oliveira : T. S. L. Keesen : H. A. Oliveira Rocha Department of Biochemistry, Federal University of Rio Grande do Norte, Natal, RN, Brazil J. F. L. de Castro Department of Clinics and Preventive Dentistry, Federal University of Pernambuco, Recife, PE, Brazil R. Della Coletta Department of Oral Diagnosis, Campinas University, Piracicaba, SP, Brazil R. de Almeida Freitas (*) Av. Senador Salgado Filho, 1787, Lagoa Nova, Natal, Rio Grande do Norte, Brazil 59056-000 e-mail: [email protected]
D1 and nuclear β-catenin. At 1.0 J/cm2, LLLT significantly reduced E-cadherin and induced MMP-9 expression, promoting SCC25 invasion. The results of this study demonstrated that LLLT exerts a stimulatory effect on proliferation and invasion of SCC25 cells, which was associated with alterations on expression of proteins studied. Keywords Cell cycle . Cell proliferation . Flow cytometry . Low-level laser therapy . Squamous cell carcinoma
Introduction Low-level laser therapy (LLLT) has been used to accelerate repair processes in soft and hard tissues due to its biomodulatory effects, activating or inhibiting physiological, biochemical, and metabolic processes. Its capacity to accelerate wound healing is related to increased cell proliferation since evidence indicates that LLLT stimulates the respiratory chain in mitochondria, increasing the production of adenosine triphosphate (ATP) and, consequently, the synthesis of DNA, RNA, and proteins [1, 2]. The effect of LLLT on the metabolism of benign cells has been extensively studied, mainly in an attempt to better understand its mechanism of action [1, 2]. In the case of benign cells, LLLT has beneficial effects since, by increasing cell proliferation, it contributes to wound healing, bone repair, and muscle and neural regeneration. In addition, laser therapy could be important for advances in tissue engineering using stem cells [3, 4]. However, in malignant cells that exhibit genomic instability, laser-induced proliferation may increase the number of genomically altered cells with higher proliferative activity, thus indirectly accelerating the gain of additional mutations during the natural process of carcinogenesis. Strong evidences suggest that laser therapy enhances the growth of neoplastic cells as a result of the altered expression
Lasers Med Sci
of proteins related to cell cycle regulation, apoptosis, cell adhesion and migration, extracellular matrix degradation, and angiogenesis. Therefore, the unintended use of LLLT during the development and progression of a neoplastic process may favor biological activities that are determinant for tumorigenesis, such as cell proliferation and migration. As a consequence, the identification of alterations in these cellular activities may restrict the use of LLLT in any clinical situation with a potential of malignant transformation or when the tumor is located near the field of irradiation. In an attempt to better understand the mechanisms of action of laser therapy on malignant cells, the present study investigated the effect of LLLT on potential of proliferation and invasion of a tongue squamous carcinoma cell line and analyzed its effects in the expression of proteins related to tumor growth and invasion, including cyclin D1, E-cadherin, βcatenin, and MMP-9.
Materials and methods Cell culture The SCC25 cells, a tumorigenic cell line originated from a human tongue squamous cell carcinoma (ATCC, Manassas, VA, USA), were cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s media and Ham’s F12 media (DMEM/F12; Invitrogen, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS; Cultilab, Campinas, Brazil), 400 ng/mL hydrocortisone (Sigma, St. Louis, MO, USA), and 1 % antibiotic–antimycotic solution (Gibco, Carlsbad, CA USA) at 37 °C in a humidified atmosphere of 5 % CO2.
constant at 0.5 cm. Laser irradiation was carried out in partial darkness, without influence from light sources other than the laser. Cell growth assay Trypan blue assay was used to evaluate the number of cells in the culture after LLLT. The cells were cultured in 24-well plates at a density of 3×104 cells/well. Cell counts were obtained from all groups at 0, 24, 48 and 72 h after the first laser application. The number of cells is reported as median of independent experiments carried out in quadruplicate. Cell cycle analysis Cells in the S/G2/M (proliferating) and G0/G1 phases were analyzed by flow cytometry and compared between the control, L0.5 and L1.0 groups. The cells were serum-deprived for 48 h and cultured in six-well plates at a density of 2×105 cells/ well. Cells were collected at 0, 24, 48, and 72 h after the first laser application, washed with cold phosphate-buffered saline (PBS), and fixed in 2 % paraformaldehyde at room temperature for at least 30 min. Next, the cells were washed twice with cold PBS, incubated in 200 μL of a solution containing 0.01 % saponin and 0.2 mg/mL RNAase at 37 °C for 1 h, and stained with propidium iodide (50 μg/mL) for 15 min in the dark at 4 °C. Fluorescence emitted from the propidium– DNA complex after excitation of the dye was quantified by flow cytometry (FACSCANTOII, Becton Dickinson, San Jose, CA, USA). At least 30,000 events were acquired per sample and the data were analyzed using appropriate software (FlowJo-Tree Star). The experiments were carried out in triplicates.
Laser irradiation Immunofluorescence Cells (2×105) were plated in six-well plates, allowing empty wells between seeded wells in order to prevent unintentional light scattering during laser application. After 24 h, cells were stimulated with an indium–gallium–aluminum phosphide (InGaAlP) diode laser (Kondortech, São Carlos, Brazil). Two sessions of irradiation consisting of visible red light (30 mW, 660 nm) in the continuous mode with a beam spot size of 0.03 cm2 and area of 1.0 cm2 were applied at 0 and 48 h. The wells were randomly divided into a control group (C) not submitted to irradiation and two treated groups, one irradiated with a dose of 0.5 J/cm2 (L0.5) and irradiance of 0.03 W/cm2 for 16 s (0.48 J) and another group irradiated with a dose of 1.0 J/cm2 (L1.0) and irradiance of 0.03 W/cm2 for 33 s (0.99 J). The choice of the laser irradiation parameters was based on previous in vitro studies, in which energy densities of 0.5 to 4.0 J/cm2 had a positive biostimulatory effect on cell proliferation [5, 6]. Additionally, the distance between the laser beam and the cell monolayer was kept
Cells grown on glass coverslips in 24-well plates at a density of 3×104 cells/well were fixed in 4 % paraformaldehyde in PBS for 10 min, rinsed in PBS, and incubated in Tris-buffered saline (TBS) solution/0.5 % Triton X-100 (Sigma) in PBS for 30 min, followed by incubation in 5 % bovine serum albumin (BSA) (Sigma) in TBS for 60 min at room temperature. Next, the cells were subjected to a standard immunofluorescence protocol to detect cyclin D1 (A-12), E-cadherin (G-10), βcatenin (E-5), and MMP-9 (2C3). All primary antibodies were mouse monoclonal antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) diluted in 1 % BSA in TBS. Cyclin D1, E-cadherin, and β-catenin antibodies were diluted 1:50 and the MMP-9 antibody was diluted 1:25. Alexa Fluor® 488 F(ab')2 (Invitrogen) was used as the secondary antibody at a final concentration of 1:500 in 1 % BSA and TBS. All samples were incubated for 60 min at 37 °C for the primary antibody and at 4 °C for the secondary antibody. Cells were
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mounted with Fluormount-G (Vector Laboratories Inc., Burlingame, CA, EUA) and then examined under a photomicroscope equipped with epifluorescence (Zeiss Axiophot, Carl Zeiss, Oberköchen, Germany). To generate fluorescent labeled images, cells were excited at 480/40 nm with a 527/30 band pass filter. Cells untreated with primary antibodies were used as negative controls. The experiments were carried out in duplicate. Flow cytometry Protocols of intracellular staining of cyclin D1, β-catenin, and MMP-9 and membrane staining of E-cadherin, β-catenin and MMP-9 were adopted. The cells were cultured in six-well plates at a density of 2×105 cells/well. Cells were collected at 0, 24, 48, and 72 h after the first laser application, washed with cold PBS, and incubated with the primary antibodies diluted in antibody dilution buffer for 60 min at 37 °C. The same dilutions as described earlier were adopted for the primary antibodies, except for MMP-9 (1:10). After washing in cold PBS, the secondary antibody (Alexa Fluor® 488) was added at a final concentration of 1:20 in antibody dilution buffer and the samples were incubated for 45 min at 4 °C. The intracellular protocol required fixation of the samples in 2 % paraformaldehyde and subsequent incubation with 0.01 % saponin for 15 min prior to incubation with the primary antibody. At least 30,000 events were acquired per sample and the percentage of positive cells was analyzed using the FlowJo program. The experiments were carried out in triplicate. Invasion assay The cell invasion assay was performed using transwell chambers (BD Biosciences, San Jose, CA, USA) in six-well culture microplates. The transwell chambers were covered with a thin layer of Matrigel (BD Biosciences) at a concentration of 1 μg/ μL in DMEM/F-12 without FBS. Two milliliters of culture medium with 10 % FBS was added to the bottom well. A polyethylene membrane (pore size 8 μm) was placed between the bottom well and the top well. Unstimulated and stimulated cells were resuspended in culture medium without FBS and 2×105 cells were added to the top well of the transwell chambers. After incubation for 72 h at 37 °C in a 5 % CO2 atmosphere, the cells that had not migrated were removed from the upper compartment of the polyethylene membrane with cotton swabs and those that migrated to the lower compartment of the polyethylene membrane were fixed in 10 % formaldehyde and stained with toluidine blue. The polyethylene membranes were removed and mounted on glass slides. Histological fields were examined by light microscopy and images were captured with a high-resolution digital camera at×100 magnification. Invasion was determined by counting the total number of cells using the Image J program. The assay
was repeated six times for each group (control, L0.5, and L1.0). The median value of cells that invaded the six membranes was taken as the number of invading cells per group. Statistical analysis The results are expressed as median and were compared between groups by the nonparametric Kruskal–Wallis and Mann–Whitney tests. A level of significance of 5 % was adopted.
Results Effect of LLLT on cell proliferation Proliferation curve The highest proliferation rate was observed for SCC25 cells irradiated with 1.0 J/cm2 after 24 h of culture when compared to the control group and the group irradiated with 0.5 J/cm2 (p=0.019) (Fig. 1). Although not significant (p>0.05), the L0.5 group tended to show a higher growth rate than the control group after 24 h. Cell cycle distribution A reduction in the number of cells in the G0/G1 phase concomitant with an increase in the proportion of cells in the S and G2/M phases was observed in all groups after 24 h of culture. This difference was more pronounced in the L1.0 group (p=0.027), with 17 % and 46 % of cells in the S and G2/M phases, respectively. These percentages were lower for the control and L0.5 groups, with the control group presenting the lowest proportion of cells in the S and G2/M phases (p= 0.027). An increase in the proportion of cells in G0/G1 and a
Fig 1 Proliferation rate for SCC25 cells irradiated with 1.0 J/cm2 after 24 h of culture vs. the control group and the group irradiated with 0.5 J/cm2
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reduction of cells in G2/M were observed after 48 h in the control group and in the L0.5 and L1.0 groups, indicating a decrease of cell division rates. Separate analysis of each time interval showed that the proportion of cells in the S and G2/M phases was generally constant or slightly higher in the laserirradiated groups when compared to control. In the L1.0 group, the highest proportion of cells in the S phase was observed at 24 and 48 h of culture (p=0.027). In addition, this group presented the highest proportion of cells in the G2/M phase throughout the experiment (p=0.027), except after 48 h when the percentage of cells was similar to that of the control and L0.5 groups (p=0.06) (Fig. 2).
culture (Fig. 4b), with the difference being significant for the period of 48 h (p=0.027). Expression of this adhesion molecule in the cytoplasmic membrane was detected in less than 6.1 % of cells in all groups (Fig. 4c). The L0.5 and L1.0 groups exhibited the lowest expression of β-catenin when compared to control at all time points analyzed (p=0.027), particularly at 48 and 72 h. Figure 3e, f shows the nuclear and/or perinuclear staining for β-catenin detected in the groups studied. The staining pattern of β-catenin in the cytoplasmic membrane is shown in Fig. 3g. Most cells, especially in the irradiated groups, were negative for this antibody. E-cadherin
Effect of LLLT on protein expression Cyclin D1 Immunofluorescence analysis revealed a clear nuclear staining for cyclin D1, which was more intense in the L1.0 group (Fig. 3a–d). Flow cytometry analysis confirmed that the expression of cyclin D1 was significantly higher in L1.0 and L0.5 (p=0.027), particularly in the L1.0 group at 0 h (18.6 % compared to control), 24 h (4.7 %), and 72 h (7.8 %) (Fig. 4a). Beta-catenin The expression of β-catenin was found in both intracellular and membrane (Fig. 3e–g). Comparison between laserirradiated and control groups showed higher intracellular expression of this protein in the L1.0 group at 48 and 72 h of
Immunofluorescence staining revealed the absence of membrane expression in most cells (Fig. 3h). Similar to the membrane expression of β-catenin, expression of E-cadherin was detected in less than 5 % of cells of the control, L0.5, and L1.0 groups. Expression of this protein was lower in irradiated cells compared to control cells at the time points analyzed, particularly at 24 h (p=0.027) (Fig. 4d). MMP-9 The immunofluorescence staining pattern of MMP-9 is shown in Fig. 3i. Intracellular expression of MMP-9 was significantly higher in the L0.5 and L1.0 groups at the beginning of the experiment (0 h) when compared to the control group but slightly decreased or increased by about 1 % at the subsequent time points (Fig. 4e). On the cell surface, MMP-9 expression
Fig. 2 Proportion of cells in the G1, S and G2/M phase throughout the experiment
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Fig. 3 Results of immunofluorescence analysis (a–i)
Fig. 4 Results of flow cytometry analysis (a–f)
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was higher in irradiated cells at 0, 24, and 72 h (Fig. 4f). Expression of MMP-9 was higher in the L1.0 group than in the L0.5 group almost throughout the experiment (p=0.027), except at 24 h (Fig. 4f). Figure 5 shows the dot plots and histograms corresponding to membrane and intracellular protein expression, respectively, obtained for the control, L0.5, and L1.0 groups. Effect of LLLT on cell invasion potential A significantly higher invasion potential was observed for SCC25 cells irradiated with 1.0 J/cm2 when compared to the control group and the group irradiated with 0.5 J/cm 2 (p
ORIGINAL ARTICLE
Low-level laser therapy promotes proliferation and invasion of oral squamous cell carcinoma cells Águida Cristina Gomes Henriques & Fernanda Ginani & Ruth Medeiros Oliveira & Tatjana Souza Lima Keesen & Carlos Augusto Galvão Barboza & Hugo Alexandre Oliveira Rocha & Jurema Freire Lisboa de Castro & Ricardo Della Coletta & Roseana de Almeida Freitas
Received: 10 October 2013 / Accepted: 28 January 2014 # Springer-Verlag London 2014
Abstract Low-level laser therapy (LLLT) has been shown to be effective in promoting cell proliferation. There is speculation that the biostimulatory effect of LLLT causes undesirable enhancement of tumor growth in neoplastic diseases since malignant cells are more susceptible to proliferative stimuli. This study evaluated the effects of LLLT on proliferation, invasion, and expression of cyclin D1, E-cadherin, βcatenin, and MMP-9 in a tongue squamous carcinoma cell line (SCC25). Cells were irradiated with a diode laser (660 nm) using two energy densities (0.5 and 1.0 J/cm2). The proliferative potential was assessed by cell growth curves and cell cycle analysis, whereas the invasion of cells was evaluated using a Matrigel cell invasion assay. Expression of cyclin D1, E-cadherin, β-catenin, and MMP-9 was analyzed by immunofluorescence and flow cytometry and associated with the biological activities studied. LLLT induced significantly the proliferation of SCC25 cells at 1.0 J/cm2, which was accomplished by an increase in the expression of cyclin Á. C. Gomes Henriques : F. Ginani : C. A. Galvão Barboza : R. de Almeida Freitas Department of Dentistry, Federal University of Rio Grande do Norte, Natal, RN, Brazil R. M. Oliveira : T. S. L. Keesen : H. A. Oliveira Rocha Department of Biochemistry, Federal University of Rio Grande do Norte, Natal, RN, Brazil J. F. L. de Castro Department of Clinics and Preventive Dentistry, Federal University of Pernambuco, Recife, PE, Brazil R. Della Coletta Department of Oral Diagnosis, Campinas University, Piracicaba, SP, Brazil R. de Almeida Freitas (*) Av. Senador Salgado Filho, 1787, Lagoa Nova, Natal, Rio Grande do Norte, Brazil 59056-000 e-mail: [email protected]
D1 and nuclear β-catenin. At 1.0 J/cm2, LLLT significantly reduced E-cadherin and induced MMP-9 expression, promoting SCC25 invasion. The results of this study demonstrated that LLLT exerts a stimulatory effect on proliferation and invasion of SCC25 cells, which was associated with alterations on expression of proteins studied. Keywords Cell cycle . Cell proliferation . Flow cytometry . Low-level laser therapy . Squamous cell carcinoma
Introduction Low-level laser therapy (LLLT) has been used to accelerate repair processes in soft and hard tissues due to its biomodulatory effects, activating or inhibiting physiological, biochemical, and metabolic processes. Its capacity to accelerate wound healing is related to increased cell proliferation since evidence indicates that LLLT stimulates the respiratory chain in mitochondria, increasing the production of adenosine triphosphate (ATP) and, consequently, the synthesis of DNA, RNA, and proteins [1, 2]. The effect of LLLT on the metabolism of benign cells has been extensively studied, mainly in an attempt to better understand its mechanism of action [1, 2]. In the case of benign cells, LLLT has beneficial effects since, by increasing cell proliferation, it contributes to wound healing, bone repair, and muscle and neural regeneration. In addition, laser therapy could be important for advances in tissue engineering using stem cells [3, 4]. However, in malignant cells that exhibit genomic instability, laser-induced proliferation may increase the number of genomically altered cells with higher proliferative activity, thus indirectly accelerating the gain of additional mutations during the natural process of carcinogenesis. Strong evidences suggest that laser therapy enhances the growth of neoplastic cells as a result of the altered expression
Lasers Med Sci
of proteins related to cell cycle regulation, apoptosis, cell adhesion and migration, extracellular matrix degradation, and angiogenesis. Therefore, the unintended use of LLLT during the development and progression of a neoplastic process may favor biological activities that are determinant for tumorigenesis, such as cell proliferation and migration. As a consequence, the identification of alterations in these cellular activities may restrict the use of LLLT in any clinical situation with a potential of malignant transformation or when the tumor is located near the field of irradiation. In an attempt to better understand the mechanisms of action of laser therapy on malignant cells, the present study investigated the effect of LLLT on potential of proliferation and invasion of a tongue squamous carcinoma cell line and analyzed its effects in the expression of proteins related to tumor growth and invasion, including cyclin D1, E-cadherin, βcatenin, and MMP-9.
Materials and methods Cell culture The SCC25 cells, a tumorigenic cell line originated from a human tongue squamous cell carcinoma (ATCC, Manassas, VA, USA), were cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s media and Ham’s F12 media (DMEM/F12; Invitrogen, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS; Cultilab, Campinas, Brazil), 400 ng/mL hydrocortisone (Sigma, St. Louis, MO, USA), and 1 % antibiotic–antimycotic solution (Gibco, Carlsbad, CA USA) at 37 °C in a humidified atmosphere of 5 % CO2.
constant at 0.5 cm. Laser irradiation was carried out in partial darkness, without influence from light sources other than the laser. Cell growth assay Trypan blue assay was used to evaluate the number of cells in the culture after LLLT. The cells were cultured in 24-well plates at a density of 3×104 cells/well. Cell counts were obtained from all groups at 0, 24, 48 and 72 h after the first laser application. The number of cells is reported as median of independent experiments carried out in quadruplicate. Cell cycle analysis Cells in the S/G2/M (proliferating) and G0/G1 phases were analyzed by flow cytometry and compared between the control, L0.5 and L1.0 groups. The cells were serum-deprived for 48 h and cultured in six-well plates at a density of 2×105 cells/ well. Cells were collected at 0, 24, 48, and 72 h after the first laser application, washed with cold phosphate-buffered saline (PBS), and fixed in 2 % paraformaldehyde at room temperature for at least 30 min. Next, the cells were washed twice with cold PBS, incubated in 200 μL of a solution containing 0.01 % saponin and 0.2 mg/mL RNAase at 37 °C for 1 h, and stained with propidium iodide (50 μg/mL) for 15 min in the dark at 4 °C. Fluorescence emitted from the propidium– DNA complex after excitation of the dye was quantified by flow cytometry (FACSCANTOII, Becton Dickinson, San Jose, CA, USA). At least 30,000 events were acquired per sample and the data were analyzed using appropriate software (FlowJo-Tree Star). The experiments were carried out in triplicates.
Laser irradiation Immunofluorescence Cells (2×105) were plated in six-well plates, allowing empty wells between seeded wells in order to prevent unintentional light scattering during laser application. After 24 h, cells were stimulated with an indium–gallium–aluminum phosphide (InGaAlP) diode laser (Kondortech, São Carlos, Brazil). Two sessions of irradiation consisting of visible red light (30 mW, 660 nm) in the continuous mode with a beam spot size of 0.03 cm2 and area of 1.0 cm2 were applied at 0 and 48 h. The wells were randomly divided into a control group (C) not submitted to irradiation and two treated groups, one irradiated with a dose of 0.5 J/cm2 (L0.5) and irradiance of 0.03 W/cm2 for 16 s (0.48 J) and another group irradiated with a dose of 1.0 J/cm2 (L1.0) and irradiance of 0.03 W/cm2 for 33 s (0.99 J). The choice of the laser irradiation parameters was based on previous in vitro studies, in which energy densities of 0.5 to 4.0 J/cm2 had a positive biostimulatory effect on cell proliferation [5, 6]. Additionally, the distance between the laser beam and the cell monolayer was kept
Cells grown on glass coverslips in 24-well plates at a density of 3×104 cells/well were fixed in 4 % paraformaldehyde in PBS for 10 min, rinsed in PBS, and incubated in Tris-buffered saline (TBS) solution/0.5 % Triton X-100 (Sigma) in PBS for 30 min, followed by incubation in 5 % bovine serum albumin (BSA) (Sigma) in TBS for 60 min at room temperature. Next, the cells were subjected to a standard immunofluorescence protocol to detect cyclin D1 (A-12), E-cadherin (G-10), βcatenin (E-5), and MMP-9 (2C3). All primary antibodies were mouse monoclonal antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) diluted in 1 % BSA in TBS. Cyclin D1, E-cadherin, and β-catenin antibodies were diluted 1:50 and the MMP-9 antibody was diluted 1:25. Alexa Fluor® 488 F(ab')2 (Invitrogen) was used as the secondary antibody at a final concentration of 1:500 in 1 % BSA and TBS. All samples were incubated for 60 min at 37 °C for the primary antibody and at 4 °C for the secondary antibody. Cells were
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mounted with Fluormount-G (Vector Laboratories Inc., Burlingame, CA, EUA) and then examined under a photomicroscope equipped with epifluorescence (Zeiss Axiophot, Carl Zeiss, Oberköchen, Germany). To generate fluorescent labeled images, cells were excited at 480/40 nm with a 527/30 band pass filter. Cells untreated with primary antibodies were used as negative controls. The experiments were carried out in duplicate. Flow cytometry Protocols of intracellular staining of cyclin D1, β-catenin, and MMP-9 and membrane staining of E-cadherin, β-catenin and MMP-9 were adopted. The cells were cultured in six-well plates at a density of 2×105 cells/well. Cells were collected at 0, 24, 48, and 72 h after the first laser application, washed with cold PBS, and incubated with the primary antibodies diluted in antibody dilution buffer for 60 min at 37 °C. The same dilutions as described earlier were adopted for the primary antibodies, except for MMP-9 (1:10). After washing in cold PBS, the secondary antibody (Alexa Fluor® 488) was added at a final concentration of 1:20 in antibody dilution buffer and the samples were incubated for 45 min at 4 °C. The intracellular protocol required fixation of the samples in 2 % paraformaldehyde and subsequent incubation with 0.01 % saponin for 15 min prior to incubation with the primary antibody. At least 30,000 events were acquired per sample and the percentage of positive cells was analyzed using the FlowJo program. The experiments were carried out in triplicate. Invasion assay The cell invasion assay was performed using transwell chambers (BD Biosciences, San Jose, CA, USA) in six-well culture microplates. The transwell chambers were covered with a thin layer of Matrigel (BD Biosciences) at a concentration of 1 μg/ μL in DMEM/F-12 without FBS. Two milliliters of culture medium with 10 % FBS was added to the bottom well. A polyethylene membrane (pore size 8 μm) was placed between the bottom well and the top well. Unstimulated and stimulated cells were resuspended in culture medium without FBS and 2×105 cells were added to the top well of the transwell chambers. After incubation for 72 h at 37 °C in a 5 % CO2 atmosphere, the cells that had not migrated were removed from the upper compartment of the polyethylene membrane with cotton swabs and those that migrated to the lower compartment of the polyethylene membrane were fixed in 10 % formaldehyde and stained with toluidine blue. The polyethylene membranes were removed and mounted on glass slides. Histological fields were examined by light microscopy and images were captured with a high-resolution digital camera at×100 magnification. Invasion was determined by counting the total number of cells using the Image J program. The assay
was repeated six times for each group (control, L0.5, and L1.0). The median value of cells that invaded the six membranes was taken as the number of invading cells per group. Statistical analysis The results are expressed as median and were compared between groups by the nonparametric Kruskal–Wallis and Mann–Whitney tests. A level of significance of 5 % was adopted.
Results Effect of LLLT on cell proliferation Proliferation curve The highest proliferation rate was observed for SCC25 cells irradiated with 1.0 J/cm2 after 24 h of culture when compared to the control group and the group irradiated with 0.5 J/cm2 (p=0.019) (Fig. 1). Although not significant (p>0.05), the L0.5 group tended to show a higher growth rate than the control group after 24 h. Cell cycle distribution A reduction in the number of cells in the G0/G1 phase concomitant with an increase in the proportion of cells in the S and G2/M phases was observed in all groups after 24 h of culture. This difference was more pronounced in the L1.0 group (p=0.027), with 17 % and 46 % of cells in the S and G2/M phases, respectively. These percentages were lower for the control and L0.5 groups, with the control group presenting the lowest proportion of cells in the S and G2/M phases (p= 0.027). An increase in the proportion of cells in G0/G1 and a
Fig 1 Proliferation rate for SCC25 cells irradiated with 1.0 J/cm2 after 24 h of culture vs. the control group and the group irradiated with 0.5 J/cm2
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reduction of cells in G2/M were observed after 48 h in the control group and in the L0.5 and L1.0 groups, indicating a decrease of cell division rates. Separate analysis of each time interval showed that the proportion of cells in the S and G2/M phases was generally constant or slightly higher in the laserirradiated groups when compared to control. In the L1.0 group, the highest proportion of cells in the S phase was observed at 24 and 48 h of culture (p=0.027). In addition, this group presented the highest proportion of cells in the G2/M phase throughout the experiment (p=0.027), except after 48 h when the percentage of cells was similar to that of the control and L0.5 groups (p=0.06) (Fig. 2).
culture (Fig. 4b), with the difference being significant for the period of 48 h (p=0.027). Expression of this adhesion molecule in the cytoplasmic membrane was detected in less than 6.1 % of cells in all groups (Fig. 4c). The L0.5 and L1.0 groups exhibited the lowest expression of β-catenin when compared to control at all time points analyzed (p=0.027), particularly at 48 and 72 h. Figure 3e, f shows the nuclear and/or perinuclear staining for β-catenin detected in the groups studied. The staining pattern of β-catenin in the cytoplasmic membrane is shown in Fig. 3g. Most cells, especially in the irradiated groups, were negative for this antibody. E-cadherin
Effect of LLLT on protein expression Cyclin D1 Immunofluorescence analysis revealed a clear nuclear staining for cyclin D1, which was more intense in the L1.0 group (Fig. 3a–d). Flow cytometry analysis confirmed that the expression of cyclin D1 was significantly higher in L1.0 and L0.5 (p=0.027), particularly in the L1.0 group at 0 h (18.6 % compared to control), 24 h (4.7 %), and 72 h (7.8 %) (Fig. 4a). Beta-catenin The expression of β-catenin was found in both intracellular and membrane (Fig. 3e–g). Comparison between laserirradiated and control groups showed higher intracellular expression of this protein in the L1.0 group at 48 and 72 h of
Immunofluorescence staining revealed the absence of membrane expression in most cells (Fig. 3h). Similar to the membrane expression of β-catenin, expression of E-cadherin was detected in less than 5 % of cells of the control, L0.5, and L1.0 groups. Expression of this protein was lower in irradiated cells compared to control cells at the time points analyzed, particularly at 24 h (p=0.027) (Fig. 4d). MMP-9 The immunofluorescence staining pattern of MMP-9 is shown in Fig. 3i. Intracellular expression of MMP-9 was significantly higher in the L0.5 and L1.0 groups at the beginning of the experiment (0 h) when compared to the control group but slightly decreased or increased by about 1 % at the subsequent time points (Fig. 4e). On the cell surface, MMP-9 expression
Fig. 2 Proportion of cells in the G1, S and G2/M phase throughout the experiment
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Fig. 3 Results of immunofluorescence analysis (a–i)
Fig. 4 Results of flow cytometry analysis (a–f)
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was higher in irradiated cells at 0, 24, and 72 h (Fig. 4f). Expression of MMP-9 was higher in the L1.0 group than in the L0.5 group almost throughout the experiment (p=0.027), except at 24 h (Fig. 4f). Figure 5 shows the dot plots and histograms corresponding to membrane and intracellular protein expression, respectively, obtained for the control, L0.5, and L1.0 groups. Effect of LLLT on cell invasion potential A significantly higher invasion potential was observed for SCC25 cells irradiated with 1.0 J/cm2 when compared to the control group and the group irradiated with 0.5 J/cm 2 (p
