Low-level laser therapy promotes proliferation and invasion of oral squamous cell carcinoma cells

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 / Published online: 14 February 2014 # Springer-Verlag London 2014

Abstract Low-level laser therapy (LLLT) has been shown to be effective in promoting cell proliferation. There is specula-tion 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, β-catenin, 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 signifi-cantly the proliferation of SCC25 cells at 1.0 J/cm2, which was accomplished by an increase in the expression of cyclin

D1 and nuclearβ-catenin. At 1.0 J/cm2, LLLT significantly reduced E-cadherin and induced MMP-9 expression, promot-ing 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 alter-ations 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 acceler-ate wound healing is relacceler-ated 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 un-derstand 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 prolifer-ative activity, thus indirectly accelerating the gain of addition-al mutations during the naturaddition-al process of carcinogenesis.

Strong evidences suggest that laser therapy enhances the growth of neoplastic cells as a result of the altered expression

Á. C. Gomes Henriques

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Department of Dentistry, Federal University of Rio Grande do Norte, Natal, RN, Brazil

R. M. Oliveira

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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

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 pro-cess 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 investi-gated the effect of LLLT on potential of proliferation and invasion of a tongue squamous carcinoma cell line and ana-lyzed its effects in the expression of proteins related to tumor growth and invasion, including cyclin D1, E-cadherin, β-catenin, 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. Laser irradiation

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 cm2and area of 1.0 cm2were 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/cm2for 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/cm2for 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

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 con-trol, L0.5and L1.0groups. The cells were serum-deprived for 48 h and cultured in six-well plates at a density of 2×105cells/ 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 tempera-ture 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 (50μg/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.

Immunofluorescence

Cells grown on glass coverslips in 24-well plates at a density of 3×104cells/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), β-catenin (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, andβ-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

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 2×105cells/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 pri-mary 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 anti-body. 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 cham-bers (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 1μg/ 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 8μm) 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 com-partment of the polyethylene membrane were fixed in 10 % formaldehyde and stained with toluidine blue. The polyethyl-ene 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/cm2after 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 con-comitant 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 L0.5groups, 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 irradi-ated with 0.5 J/cm2

reduction of cells in G2/M were observed after 48 h in the control group and in the L0.5and L1.0groups, 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 laser-irradiated groups when compared to control. In the L1.0group, 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 L0.5groups (p=0.06) (Fig.2).

Effect of LLLT on protein expression Cyclin D1

Immunofluorescence analysis revealed a clear nuclear stain-ing for cyclin D1, which was more intense in the L1.0group (Fig.3a–d). Flow cytometry analysis confirmed that the ex-pression of cyclin D1 was significantly higher in L1.0and 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 ofβ-catenin was found in both intracellular and membrane (Fig. 3e–g). Comparison between laser-irradiated and control groups showed higher intracellular ex-pression of this protein in the L1.0group at 48 and 72 h of

culture (Fig.4b), with the difference being significant for the period of 48 h (p=0.027). Expression of this adhesion mole-cule in the cytoplasmic membrane was detected in less than 6.1 % of cells in all groups (Fig.4c). The L0.5and L1.0groups exhibited the lowest expression ofβ-catenin when compared to control at all time points analyzed (p=0.027), particularly at 48 and 72 h. Figure3e, f shows the nuclear and/or perinuclear staining for β-catenin detected in the groups studied. The staining pattern ofβ-catenin in the cytoplasmic membrane is shown in Fig. 3g. Most cells, especially in the irradiated groups, were negative for this antibody.

E-cadherin

Immunofluorescence staining revealed the absence of mem-brane expression in most cells (Fig.3h). Similar to the mem-brane expression ofβ-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, partic-ularly 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. 3 Results of immunofluorescence analysis (a–i)

was higher in irradiated cells at 0, 24, and 72 h (Fig.4f). Expression of MMP-9 was higher in the L1.0group than in the L0.5group almost throughout the experiment (p=0.027), ex-cept at 24 h (Fig.4f).

Figure5shows the dot plots and histograms corresponding to membrane and intracellular protein expression, respective-ly, obtained for the control, L0.5, and L1.0groups.

Effect of LLLT on cell invasion potential

A significantly higher invasion potential was observed for SCC25 cells irradiated with 1.0 J/cm2when compared to the control group and the group irradiated with 0.5 J/cm2 (p<0.001). Figure6aillustrates the median number of invad-ing cells after 72 h. Light microscopy showed that the per-centage of invading cells on the lower compartment of the membrane was higher in group L1.0when compared to the L0.5and control groups (Fig.6b).

Discussion

In vitro studies have shown an increase in the proliferation rates of different normal and neoplastic cells after laser ther-apy [5,7–10]. In the present investigation, LLLT increased the proliferation of epithelial cells derived from an oral squamous cell carcinoma line in a dose- and time-dependent manner. The group irradiated with an energy density of 1.0 J/cm2 exhibited higher proliferative activity than the group irradiated with 0.5 J/cm2and the control group after 24 and 72 h of culture. Considering that the duration of the cell cycle of human cells is approximately 24 h [11], it can be assumed that the second irradiation (48 h) was necessary to further increase cell proliferation since cells of the L1.0group present-ed a significant increase of proliferative activity only between 24 and 72 h when compared to the other groups. Cell viability did not differ significantly over the 72 h of the experiment, with cell growth being observed over time in all groups.

Positive biomodulatory effects of LLLT on malignant cells have also been reported in other studies [5, 7, 9, 12]. In contrast, some reports have shown inhibitory effects of laser therapy on hepatoma [13], melanoma [8], glioblastoma [14], and oral squamous cell carcinoma cells [10]. However, these inhibitory effects were observed with the use of light of the infrared or red spectrum and high irradiation doses [8,10,13,

14], whereas the studies demonstrating an increase in the proliferation of malignant cells used red lasers and low energy densities [5,7,9,12]. The results of in vitro studies investi-gating the effects of laser therapy on tumor cells are contro-versial because of differences in the tumor cell lines and irradiation parameters used. A recent systematic review [6] suggests that a dose range of 0.5 to 4.0 J/cm2and wavelength of 600 to 700 nm are effective in increasing cell proliferation.

On the basis of these findings, we used a visible laser (660 nm) and low doses (0.5 and 1.0 J/cm2).

According to Huang et al. [15], a biphasic curve can be used to illustrate the dose response of cells to irradiation: if insufficient energy is applied, no response is observed because the minimum threshold is not met; if more energy is applied and the threshold is exceeded, biostimulation is achieved. However, if the energy applied is very high, an inhibitory effect occurs. Taken together, the results of previous studies and the present findings suggest that LLLT contributes to cell growth, whereas high doses interfere negatively with the cell cycle, inhibiting proliferation. LLLT has been suggested to induce the phosphorylation of tyrosine kinase receptors, acti-vating the MAPK/ERK signaling cascade [2]. On the other hand, Huang et al. [15] showed that high doses increase the release of reactive oxygen species that mediate inhibitory effects and induce cell death. In addition, high-power laser therapy can induce the apoptosis of cells through the activa-tion of the mitochondrial caspase-3 pathway [16] and induc-tion of GSK3β, which activates the pro-apoptotic Bax protein [17].

Cell cycle distribution of SCC25 cells was analyzed in the present study to further support the possible effects of laser therapy on cell proliferation. According to Schartinger et al. [10], an increase in the proportion of cells in the S and G2/M phases and a concomitant reduction of cells in the G0/G1 phase indicate enhanced cell cycle progression from G1 to S and G2/M. This change was observed in the present study 24 h after the first irradiation and was more pronounced in group L1.0. However, an increase in the proportion of cells in the G0/G1 phase and a decrease in the proportion of cells in the G2/M phase were observed in the control group and in the irradiated groups (L0.5and L1.0), indicating a reduction in cell division after 48 h of culture. These findings might be ex-plained by the fact that part of the cells duplicated during the first 24 h progressed to the G1 phase of a new cycle, suggest-ing prolongation of this phase, whereas the other part entered the resting stage (G0). This reduction in cell cycle velocity was probably due to the fact that confluence was reached already within 48 h. The proliferative activity of the cells did not continue to increase until 72 h because of the relative decrease of nutrients in the culture medium as a result of the increase in the number of cells during the experiment. The cells probably needed to adapt to the new conditions of the microenvironment, and cell cycle velocity was therefore re-duced. Nevertheless, higher proportions of cells in the S and G2/M phases were observed in the L1.0group at all time points when compared to the other groups.

According to Gao and Xing [2], an increase in the expres-sion of cell cycle proteins, including cyclin D1 and PCNA, contributes to faster progression of the cycle. Cyclin D1 regulates the transition from the G1 to the S phase through binding to CDK4 and CDK6, promoting the phosphorylation

of protein RB and subsequent release of the E2F transcription factor. Cyclin D1 is a useful marker of proliferation and its dysregulation is essential for the genesis and progression of cancer [18].

In the present study, analysis of protein expression by flow cytometry showed high levels of cyclin D1 at all time points analyzed. Expression was higher in the L1.0group, followed by the L0.5group and control group, confirming the higher proliferation rates demonstrated in group L1.0. In all groups, expression of this protein was higher at the beginning of the experiment. A slight decrease was observed after 24 h and expression again increased at 72 h. This finding may have also contributed to the reduction in the number of cells in the S and G2/M phases and the increase of cells in G0/G1 observed after 48 h of culture. Shefer et al. [19] also found higher expression of cyclin D1 in irradiated cells during the first 24 h, which was associated with progression from the G1 to the S phase of the cell cycle. In the study of Taniguchi et al. [20], cells irradiated with 660 nm exhibited low nuclear expression of p15, a member of the INK4 family that regulates the cell cycle in the G1 phase, inhibiting CDK4 and CDK6. Using microarrays and PCR, Wu et al. [21] identified overexpression of the Akt, cyclin D1, and PI3K genes in irradiated mesenchymal stem cells. On the basis of scientific evidence, we suggest that the increased expression of cyclin D1 observed in the present

study is related to the activation of components of cellular signaling cascades such as MAPK [22] and PI3K/Akt [2,19,

21], which are also activated after laser therapy.

This study also investigated the expression of molecules involved in cell adhesion and migration since these biological activities are directly associated with cell proliferation in malignant neoplastic processes. In this respect, membrane expression of E-cadherin was low in SCC25 cells, in agree-ment with Gasparoni et al. [23]. This finding can be easily explained by the malignant phenotype of these cells. However, lower staining was observed in the groups submit-ted to laser therapy, particularly the group treasubmit-ted with a dose of 1.0 J/cm2at 24 and 72 h. The reduced or absent expression of E-cadherin and of associated cellular adhesion molecules results in the easy disintegration of cells and has been corre-lated with higher proliferation and a greater invasion potential [24]. This finding is consistent with the results of the growth curve, cell cycle distribution, and expression of cyclin D1, showing a growth advantage in the L1.0group.

According to the literature, several growth factors can induce the loss of expression and function of E-cadherin. These factors include interleukin 6 (IL-6), transforming growth factor beta (TGF-β), hepatocyte growth factor and its receptor (HGF, HGFR), epidermal growth factor (EGF), insulin-like growth factor (IGF), and fibroblast growth factor

(FGF) [25–28]. The activation of these factors seems to be related to DNA hypermethylation in the E-cadherin gene and the induction of transcriptional repressors which inhibit tran-scription of the gene [29]. Interestingly, studies using benign cells demonstrated the capacity of LLLT to induce the expres-sion of these growth factors [30,31]. It is therefore likely that in malignant cells LLLT influences the expression of E-cadherin through the activation of the growth factors men-tioned earlier.

With respect to β-catenin, membrane expression of this protein was also low in SCC25 cells in the groups studied, with the lowest expression being observed in the L0.5and L1.0 groups. In contrast, marked nuclear expression ofβ-catenin was detected, which increased throughout the experiment. Gasparoni et al. [32] also found weak membrane and strong nuclear expression of this protein in SCC25 cells and corre-lated this finding with higher proliferative activity. In the present study, the L1.0group exhibited higher nuclear expres-sion ofβ-catenin after 48 h and also a higher proliferation rate. The functions ofβ-catenin are determined by its location in the membrane or nucleus, where it is involved in cell adhesion or cell growth, respectively. The aberrant expression of this protein in the nucleus has been shown to be associated with elevated levels of cyclin D1 in oral epithelial dysplasias [33]. In the present study, high levels of cyclin D1 were accompa-nied by high nuclear expression ofβ-catenin. Evidence sug-gests the simultaneous up-regulation of cyclin D1 and β-catenin through activation of the PI3K/Akt pathway and con-sequent inhibition of GSK3β, preventing the formation of the axin/APC/GSK3β complex which is necessary for degrada-tion of β-catenin in the cytoplasm. β-Catenin, in turn, is translocated to the nucleus, increasing the transcription of the CCND1 gene [34].

In the present study, SCC25 cells expressed high levels of MMP-9, particularly those irradiated with a dose of 1.0 J/cm2. Studies have demonstrated a relationship between higher MMP-9 expression and tumor aggressiveness, poor differen-tiation, and higher proliferation, invasion, and metastasis [35]. Expression of MMP-9 was higher in the L1.0group at most time points analyzed (0, 48, and 72 h). This higher expression was associated with higher proliferation rates, increased ex-pression of cyclin D1, redistribution ofβ-catenin and reduced E-cadherin expression, conferring a more aggressive behavior to cells of this group.

A previous study demonstrated that transfection of the E-cadherin gene into an E-E-cadherin-negative prostate carcinoma cell line resulted in lower production of MMP-2 and MMP-9 and a concomitant decrease in the invasion capacity of these cells [36]. The present results are consistent with these obser-vations since the reduction of E-cadherin expression in the SCC25 cell line was accompanied by high levels of MMP-9. Furthermore, in vitro studies have shown that the inhibition of MMP-2 and MMP-9 activity in cell cultures derived from

tongue squamous cell carcinoma (SCC4) reduced the migra-tion and invasion capacity of these cells [37].

The cell invasion assay showed a higher invasion potential in the group irradiated with a dose of 1.0 J/cm2when com-pared to the control group and the group irradiated with 0.5 J/ cm2. This finding can be explained by the higher proliferative activity of SCC25 cells of the L1.0group as well as the higher expression of MMP-9 associated with high levels of cyclin D1 and nuclearβ-catenin and the loss of cell–cell adhesion. Other studies also highlighted the capacity of LLLT to facilitate cell invasion [38,39].

The increased invasion capacity of cancer cells is related to the phenomenon of epithelial–mesenchymal transition, a re-versible biological process characterized by the plasticity of epithelial cells that acquire fibroblastic properties during mi-gration and invasion and return to the epithelial phenotype once they are installed at the metastatic site. Some of the molecular mechanisms underlying this process demonstrated by Krisanaprakornkit and Iamaroon [40] were observed in the present study: loss of E-cadherin expression, redistribution of β-catenin, higher MMP expression, and evidence of activa-tion of the PI3K/Akt and MAP/ERK pathways. Although these pathways were not investigated in this study, overex-pression of cyclin D1, an effector molecule of these signaling cascades, was detected. Taken together, the results suggest that the altered expression of these molecules is related to a greater cell invasion potential.

The effects of LLLT on the proliferation and invasion capacity of SCC25 cells observed in the present study suggest that application of LLLT to oral mucositis lesions located close to head and neck tumors may favor tumor growth if malignant cells are located in the irradiation field. LLLT has been used successfully for the prevention of oral mucositis in patients receiving high-dose chemotherapy and in patients with head and neck cancer undergoing radio-therapy. In these cases, laser therapy should be used with caution due to the risk of exposure of malignant cells to the irradiation field. Furthermore, field cancerization in the oral mucosa is a contraindication for laser therapy since this method may favor the proliferation of cells that carry initial molecular alterations contributing to the clonal expansion of these cells and the possibility of developing malignant lesions.

In conclusion, the present results show that LLLT at 660 nm using low doses facilitates the proliferation and inva-sion of SCC25 cells by influencing the expresinva-sion of cyclin D1,β-catenin, E-cadherin, and MMP-9. These findings sug-gest that the use of laser therapy, particularly a dose of 1.0 J/ cm2, should be avoided in situations in which increased cell proliferation and invasion can disturb the balance between cells and tissues.

Further studies are needed to standardize the most effective parameters of LLLT that promote desired effects in order to

provide professionals with the perfect laser combination in clinical practice and to better understand the molecular events induced by laser therapy in different cell types. In this respect, we assume that the real mechanisms of action of LLLT will be clarified, providing evidence of the biological effects derived from its use in oral lesions of different types.

Acknowledgments The authors acknowledge partial support from the National Council for Scientific and Technological Development (CNPq), Brazil.

References

1. Karu T (1999) Primary and secondary mechanisms of action of visible to near-IR radiation on cells. J Photochem Photobiol B 49: 17–24

2. Gao X, Xing D (2009) Molecular mechanisms of cell proliferation induced by low power laser irradiation. J Biomed Sci 9:1–16 3. Eduardo FP, Bueno DF, de Freitas PM, Marques MM, Passos-Bueno

MR, Eduardo Cde P, Zatz M (2008) Stem cell proliferation under low intensity laser irradiation: a preliminary study. Lasers Surg Med 40: 433–438

4. Yazdani SO, Golestaneh AF, Shafiee A, Hafizi M, Omrani HA, Soleimani M (2012) Effects of low level laser therapy on prolifera-tion and neurotrophic factor gene expression of human Schwann cells in vitro. J Photochem Photobiol B 107:9–13

5. Castro JL, Pinheiro AL, Werneck CE, Soares CP (2005) The effect of laser therapy on the proliferation of oral KB carcinoma cells: an in vitro study. Photomed Laser Surg 23:586–589

6. Alghamdi KM, Kumar A, Moussa NA (2012) Low-level laser ther-apy: a useful technique for enhancing the proliferation of various cultured cells. Lasers Med Sci 27:237–249

7. Renno AC, McDonnell PA, Parizotto NA, Laakso EL (2007) The effects of laser irradiation on osteoblast and osteosarcoma cell prolif-eration and differentiation in vitro. Photomed Laser Surg 25:275–280 8. Frigo L, Luppi JS, Favero GM, Maria DA, Penna SC, Bjordal JM, Bensadoun RJ, Lopes-Martins RA (2009) The effect of low-level laser irradiation (In-Ga-Al-AsP—660 nm) on melanoma in vitro and in vivo. BMC Cancer 9:404–411

9. Pinheiro AL, Carneiro NS, Vieira AL, Brugnera A Jr, Zanin FA, Barros RA, Silva OS (2002) Effects of low-level laser therapy on malignant cells: in vitro study. J Clin Laser Med Surg 20:23–26 10. Schartinger VH, Galvan O, Riechelmann H, Dudás J (2001)

Differential responses of fibroblasts, non-neoplastic epithelial cells, and oral carcinoma cells to low-level laser therapy. Support Care Cancer 20:523–529

11. Bernard S, Herzel H (2006) Why do cells cycle with a 24 hour period? Genome Inform 17:72–79

12. Powell K, Low P, McDonnell PA, Laakso EL, Ralph SJ (2010) The effect of laser irradiation on proliferation of human breast carcinoma, melanoma, and immortalized mammary epithelial cells. Photomed Laser Surg 28:115–123

13. Liu YH, Cheng CC, Ho CC, Pei RJ, Lee KY, Yeh KT, Chan Y, Lai YS (2004) Effects of diode 808 nm GaAlAs low-power laser irradi-ation on inhibition of the proliferirradi-ation of human hepatoma cells in vitro and their possible mechanism. Res Commun Mol Pathol Pharmacol 115:185–201

14. Murayama H, Sadakane K, Yamanoha B, Kogure S (2012) Low-power 808-nm laser irradiation inhibits cell proliferation of a human-derived glioblastoma cell line in vitro. Lasers Med Sci 27:87–93

15. Huang YY, Chen ACH, Carroll JD, Hamblin MR (2009) Biphasic dose response in low level light therapy. Dose Response 7:358–383

16. Wu S, Xing D, Gao X, Chen WR (2009) High fluence low-power laser irradiation induces mitochondrial permeability transition medi-ated by reactive oxygen species. J Cell Physiol 218:603–611 17. Huang L, Wu S, Xing D (2011) High fluence low-power laser

irradiation induces apoptosis via inactivation of Akt/GSK3b signal-ing pathway. J Cell Physiol 226:588–601

18. Donnellen R, Chetty R (1998) Cyclin D1 and human neoplasia. J Clin Pathol 51:1–7

19. Shefer G, Barash I, Oron U, Halevy O (2003) Low-energy laser irradiation enhances de novo protein synthesis via its effects on translation-regulatory proteins in skeletal muscle myoblasts. Biochim Biophys Acta 1593:131–139

20. Taniguchi D, Dai P, Hojo T, Yamaoka Y, Kubo T, Takamatsu T (2009) Low-energy laser irradiation promotes synovial fibroblast proliferation by modulating p15 subcellular localization. Lasers Surg Med 41:232–239

21. Wu YH, Wang J, Gong DX, Gu HY, Hu SS, Zhang H (2012) Effects of low-level laser irradiation on mesenchymal stem cell proliferation: a microarray analysis. Lasers Med Sci 27:509–519

22. Miyata H, Genma T, Ohshima M, Yamaguchi Y, Hayashi M, Takeichi O, Ogiso B, Otsuka K (2006) Mitogen-activated protein kinase/extracellular signal-regulated protein kinase activation of cul-tured human dental pulp cells by low-power gallium aluminium arsenic laser irradiation. Int Endod J 39:238–244

23. Gasparoni A, Fonzi L, Schneider GB, Wertz PW, Johnson GK, Squier CA (2004) Comparison of differentiation markers between normal and two squamous cell carcinoma cell lines in culture. Arch Oral Biol 49:653–664

24. Wang X, Zhang J, Fan M, Zhou Q, Deng H, Aisharif MJ, Chen X (2009) The expression of E-cadherin at the invasive tumor front of oral squamous cell carcinoma: immunohistochemical and RTPCR analysis with clinicopathological correlation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 107:547–554

25. Zavadil J, Böttinger EP (2005) TGF-β and epithelial to mesenchymal transitions. Oncogene 24:5764–5774

26. Lo HW, Hsu SC, Xia W, Cao X, Shih JY, Wei Y, Abbruzzese JL, Hortobagyi GN, Hung MC (2007) Epidermal growth fac-tor recepfac-tor cooperates with signal transducer and activafac-tor of transcription 3 to induce epithelial-mesenchymal transition in cancer cells via up-regulation of TWIST gene expression. Cancer Res 67:9066–9076

27. Graham TR, Zhau HE, Odero-Marah VA, Osunkoya AO, Kimbro KS, Tighiouart M, Liu T, Simons JW, O'Regan RM (2008) Insulin-like growth factor-I-dependent up-regulation of ZEB1 drives epithelial-to-mesenchymal transition in human prostate cancer cells. Cancer Res 68:2479–2488

28. Yadav A, Kumar B, Datta J, Teknos TN, Kumar P (2011) IL-6 promotes head and neck tumor metastasis by inducing epithelial– mesenchymal transition via the JAK-STAT3-SNAIL signaling path-way. Mol Cancer Res 9:1658–1667

29. Yang X, Pursell B, Lu S, Chang TK, Mercurio AM (2009) Regulation ofβ4-integrin expression by epigenetic modifications in the mammary gland and during the epithelial to mesenchymal transition. J Cell Sci 122:2473–2480

30. Safavi SM, Kazemi B, Esmaeili M, Fallah A, Modarresi A, Mir M (2008) Effects of low-level He–Ne laser irradiation on the gene expression of IL-1β, TNF-α, IFN-γ, TGF-β, bFGF, and PDGF in rat’s gingival. Lasers Med Sci 23:331–335

31. Saygun I, Nizam N, Ural AU, Serdar MA, Avcu F, Tözüm TF (2012) Low-level laser irradiation affects the release of basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I), and re-ceptor of IGF-I (IGFBP3) from osteoblasts. Photomed Laser Surg 30: 149–154

32. Gasparoni A, Chaves A, Fonzi L, Johnson GK, Schneider GB, Squier CA (2002) Subcellular localization of beta-catenin in malignant cell lines and squamous cell carcinomas of the oral cavity. J Oral Pathol Med 31:385–394

33. Ishida K, Ito S, Wada N, Deguchi H, Hata T, Hosoda M, Nohno T (2007) Nuclear localization of beta-catenin involved in precancerous change in oral leukoplakia. Mol Cancer 9:6–62

34. Klein EA, Assoian RK (2008) Transcriptional regulation of the cyclin D1 gene at a glance. J Cell Sci 121:3853–3857

35. Liu CJ, Chang KW, Lin SC, Cheng HW (2009) Presurgical serum levels of matrix metalloproteinase-9 and vascular endothelial growth factor in oral squamous cell carcinoma. Oral Oncol 45:920–925 36. Luo J, Lubaroff DM, Hendrix MJ (1999) Suppression of prostate

cancer invasive potential and matrix metalloproteinase activity by E-cadherin transfection. Cancer Res 59:3552–3556

37. Ho YT, Yang JS, Li TC, Lin JJ, Lin JG, Lai KC, Ma CY, Wood WG, Chung JG (2009) Berberine suppresses in vitro migration and inva-sion of human SCC-4 tongue squamous cancer cells through the inhibitions of FAK, IKK, NF-kappaB, u-PA and MMP-2 and -9. Cancer Lett 279:155–162

38. Chen CH, Hung HS, Hsu SH (2008) Low-energy laser irradiation increases endothelial cell proliferation, migration, and eNOS gene expression possibly via PI3K signal pathway. Lasers Surg Med 40: 46–54

39. Tsai WC, Hsu CC, Pang JH, Lin MS, Chen YH, Liang FC (2012) Low-level laser irradiation stimulates tenocyte migration with up-regulation of dynamin II expression. PLoS One 7:e38235

40. Krisanaprakornkit S, Iamaroon A (2012) Epithelial–mesenchymal transition in oral squamous cell carcinoma. ISRN Oncol 681469. doi:10.5402/2012/681469