ORIGINAL ARTICLE

Low-level laser therapy promotes proliferationand 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 tobe effective in promoting cell proliferation. There is specula-tion that the biostimulatory effect of LLLT causes undesirableenhancement of tumor growth in neoplastic diseases sincemalignant 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 cellline (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 curvesand cell cycle analysis, whereas the invasion of cells wasevaluated using a Matrigel cell invasion assay. Expression ofcyclin D1, E-cadherin, β-catenin, and MMP-9 was analyzedby immunofluorescence and flow cytometry and associatedwith the biological activities studied. LLLT induced signifi-cantly the proliferation of SCC25 cells at 1.0 J/cm2, whichwas accomplished by an increase in the expression of cyclin

D1 and nuclear β-catenin. At 1.0 J/cm2, LLLT significantlyreduced E-cadherin and induced MMP-9 expression, promot-ing SCC25 invasion. The results of this study demonstratedthat LLLT exerts a stimulatory effect on proliferation andinvasion 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 acceleraterepair processes in soft and hard tissues due to itsbiomodulatory effects, activating or inhibiting physiological,biochemical, and metabolic processes. Its capacity to acceler-ate wound healing is related to increased cell proliferationsince evidence indicates that LLLT stimulates the respiratorychain in mitochondria, increasing the production of adenosinetriphosphate (ATP) and, consequently, the synthesis of DNA,RNA, and proteins [1, 2].

The effect of LLLT on the metabolism of benign cells hasbeen extensively studied, mainly in an attempt to better un-derstand its mechanism of action [1, 2]. In the case of benigncells, LLLT has beneficial effects since, by increasing cellproliferation, it contributes to wound healing, bone repair,and muscle and neural regeneration. In addition, laser therapycould be important for advances in tissue engineering usingstem cells [3, 4]. However, in malignant cells that exhibitgenomic instability, laser-induced proliferation may increasethe number of genomically altered cells with higher prolifer-ative activity, thus indirectly accelerating the gain of addition-al mutations during the natural process of carcinogenesis.

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

Á. C. Gomes Henriques : F. Ginani :C. A. Galvão Barboza :R. de Almeida FreitasDepartment of Dentistry, Federal University of Rio Grande do Norte,Natal, RN, Brazil

R. M. Oliveira : T. S. L. Keesen :H. A. Oliveira RochaDepartment of Biochemistry, Federal University of Rio Grande doNorte, Natal, RN, Brazil

J. F. L. de CastroDepartment of Clinics and Preventive Dentistry, Federal Universityof Pernambuco, Recife, PE, Brazil

R. Della ColettaDepartment of Oral Diagnosis, Campinas University, Piracicaba, SP,Brazil

R. de Almeida Freitas (*)Av. Senador Salgado Filho, 1787, Lagoa Nova, Natal, Rio Grande doNorte, Brazil 59056-000e-mail: roseanafreitas@hotmail.com

Lasers Med Sci (2014) 29:1385–1395DOI 10.1007/s10103-014-1535-2

of proteins related to cell cycle regulation, apoptosis, celladhesion and migration, extracellular matrix degradation,and angiogenesis. Therefore, the unintended use of LLLTduring the development and progression of a neoplastic pro-cess may favor biological activities that are determinant fortumorigenesis, such as cell proliferation and migration. As aconsequence, the identification of alterations in these cellularactivities may restrict the use of LLLT in any clinical situationwith a potential of malignant transformation or when thetumor is located near the field of irradiation.

In an attempt to better understand the mechanisms of actionof laser therapy on malignant cells, the present study investi-gated the effect of LLLT on potential of proliferation andinvasion of a tongue squamous carcinoma cell line and ana-lyzed its effects in the expression of proteins related to tumorgrowth 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 ahuman tongue squamous cell carcinoma (ATCC, Manassas,VA, USA), were cultured in a 1:1 mixture of Dulbecco’smodified 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 emptywells between seeded wells in order to prevent unintentionallight scattering during laser application. After 24 h, cells werestimulated 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 spotsize of 0.03 cm2 and area of 1.0 cm2 were applied at 0 and48 h. The wells were randomly divided into a control group(C) not submitted to irradiation and two treated groups, oneirradiated with a dose of 0.5 J/cm2 (L0.5) and irradiance of0.03W/cm2 for 16 s (0.48 J) and another group irradiated witha dose of 1.0 J/cm2 (L1.0) and irradiance of 0.03 W/cm2 for33 s (0.99 J). The choice of the laser irradiation parameterswas based on previous in vitro studies, in which energydensities of 0.5 to 4.0 J/cm2 had a positive biostimulatoryeffect on cell proliferation [5, 6]. Additionally, the distancebetween the laser beam and the cell monolayer was kept

constant at 0.5 cm. Laser irradiation was carried out in partialdarkness, without influence from light sources other than thelaser.

Cell growth assay

Trypan blue assay was used to evaluate the number of cells inthe culture after LLLT. The cells were cultured in 24-wellplates at a density of 3×104 cells/well. Cell counts wereobtained from all groups at 0, 24, 48 and 72 h after the firstlaser application. The number of cells is reported as median ofindependent experiments carried out in quadruplicate.

Cell cycle analysis

Cells in the S/G2/M (proliferating) and G0/G1 phases wereanalyzed by flow cytometry and compared between the con-trol, L0.5 and L1.0 groups. The cells were serum-deprived for48 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 firstlaser application, washed with cold phosphate-buffered saline(PBS), and fixed in 2 % paraformaldehyde at room tempera-ture for at least 30min. Next, the cells were washed twice withcold PBS, incubated in 200 μL of a solution containing0.01 % saponin and 0.2 mg/mL RNAase at 37 °C for 1 h,and stained with propidium iodide (50 μg/mL) for 15 min inthe dark at 4 °C. Fluorescence emitted from the propidium–DNA complex after excitation of the dye was quantified byflow cytometry (FACSCANTOII, Becton Dickinson, SanJose, CA, USA). At least 30,000 events were acquired persample and the data were analyzed using appropriate software(FlowJo-Tree Star). The experiments were carried out intriplicates.

Immunofluorescence

Cells grown on glass coverslips in 24-well plates at a densityof 3×104 cells/well were fixed in 4 % paraformaldehyde inPBS for 10 min, rinsed in PBS, and incubated in Tris-bufferedsaline (TBS) solution/0.5 % Triton X-100 (Sigma) in PBS for30 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 immunofluorescenceprotocol to detect cyclin D1 (A-12), E-cadherin (G-10), β-catenin (E-5), andMMP-9 (2C3). All primary antibodies weremouse 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 andthe MMP-9 antibody was diluted 1:25. Alexa Fluor®488 F(ab')2 (Invitrogen) was used as the secondary antibodyat a final concentration of 1:500 in 1 % BSA and TBS. Allsamples were incubated for 60 min at 37 °C for the primaryantibody and at 4 °C for the secondary antibody. Cells were

1386 Lasers Med Sci (2014) 29:1385–1395

mounted with Fluormount-G (Vector Laboratories Inc.,Burlingame, CA, EUA) and then examined under aphotomicroscope equipped with epifluorescence (ZeissAxiophot, Carl Zeiss, Oberköchen, Germany). To generatefluorescent labeled images, cells were excited at 480/40 nmwith a 527/30 band pass filter. Cells untreated with primaryantibodies were used as negative controls. The experimentswere carried out in duplicate.

Flow cytometry

Protocols of intracellular staining of cyclin D1, β-catenin, andMMP-9 and membrane staining of E-cadherin, β-catenin andMMP-9 were adopted. The cells were cultured in six-wellplates at a density of 2×105 cells/well. Cells were collectedat 0, 24, 48, and 72 h after the first laser application, washedwith cold PBS, and incubated with the primary antibodiesdiluted in antibody dilution buffer for 60 min at 37 °C. Thesame dilutions as described earlier were adopted for the pri-mary antibodies, except for MMP-9 (1:10). After washing incold PBS, the secondary antibody (Alexa Fluor® 488) wasadded at a final concentration of 1:20 in antibody dilutionbuffer and the samples were incubated for 45 min at 4 °C.The intracellular protocol required fixation of the samples in2 % 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 thepercentage of positive cells was analyzed using the FlowJoprogram. 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 culturemicroplates. The transwell chambers were covered with a thinlayer of Matrigel (BD Biosciences) at a concentration of 1 μg/μL in DMEM/F-12 without FBS. Two milliliters of culturemedium with 10 % FBS was added to the bottom well. Apolyethylene membrane (pore size 8 μm) was placed betweenthe bottomwell and the top well. Unstimulated and stimulatedcells were resuspended in culture medium without FBS and2×105 cells were added to the top well of the transwellchambers. After incubation for 72 h at 37 °C in a 5 % CO2

atmosphere, the cells that had not migrated were removedfrom the upper compartment of the polyethylene membranewith 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 andimages were captured with a high-resolution digital cameraat×100 magnification. Invasion was determined by countingthe 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 wastaken as the number of invading cells per group.

Statistical analysis

The results are expressed as median and were comparedbetween groups by the nonparametric Kruskal–Wallis andMann–Whitney tests. A level of significance of 5 % wasadopted.

Results

Effect of LLLT on cell proliferation

Proliferation curve

The highest proliferation rate was observed for SCC25 cellsirradiated with 1.0 J/cm2 after 24 h of culture when comparedto the control group and the group irradiated with 0.5 J/cm2

(p=0.019) (Fig. 1). Although not significant (p>0.05), theL0.5 group tended to show a higher growth rate than thecontrol 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 Sand G2/M phases was observed in all groups after 24 h ofculture. This difference was more pronounced in the L1.0

group (p=0.027), with 17 % and 46 % of cells in the S andG2/M phases, respectively. These percentages were lower forthe control and L0.5 groups, with the control group presentingthe 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

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reduction of cells in G2/M were observed after 48 h in thecontrol group and in the L0.5 and L1.0 groups, indicating adecrease of cell division rates. Separate analysis of each timeinterval showed that the proportion of cells in the S and G2/Mphases was generally constant or slightly higher in the laser-irradiated groups when compared to control. In the L1.0 group,the highest proportion of cells in the S phase was observed at24 and 48 h of culture (p=0.027). In addition, this grouppresented the highest proportion of cells in the G2/M phasethroughout the experiment (p=0.027), except after 48 h whenthe percentage of cells was similar to that of the control andL0.5 groups (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 L1.0 group(Fig. 3a–d). Flow cytometry analysis confirmed that the ex-pression 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 intracellularand membrane (Fig. 3e–g). Comparison between laser-irradiated and control groups showed higher intracellular ex-pression of this protein in the L1.0 group at 48 and 72 h of

culture (Fig. 4b), with the difference being significant for theperiod of 48 h (p=0.027). Expression of this adhesion mole-cule in the cytoplasmic membrane was detected in less than6.1 % of cells in all groups (Fig. 4c). The L0.5 and L1.0 groupsexhibited the lowest expression of β-catenin when comparedto control at all time points analyzed (p=0.027), particularly at48 and 72 h. Figure 3e, f shows the nuclear and/or perinuclearstaining for β-catenin detected in the groups studied. Thestaining pattern of β-catenin in the cytoplasmic membrane isshown in Fig. 3g. Most cells, especially in the irradiatedgroups, 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 of β-catenin, expression of E-cadherin wasdetected in less than 5 % of cells of the control, L0.5, and L1.0

groups. Expression of this protein was lower in irradiated cellscompared to control cells at the time points analyzed, partic-ularly at 24 h (p=0.027) (Fig. 4d).

MMP-9

The immunofluorescence staining pattern ofMMP-9 is shownin Fig. 3i. Intracellular expression ofMMP-9was significantlyhigher in the L0.5 and L1.0 groups at the beginning of theexperiment (0 h) when compared to the control group butslightly decreased or increased by about 1 % at the subsequenttime 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 theL0.5 group almost throughout the experiment (p=0.027), ex-cept at 24 h (Fig. 4f).

Figure 5 shows the dot plots and histograms correspondingto membrane and intracellular protein expression, respective-ly, obtained for the control, L0.5, and L1.0 groups.

Effect of LLLT on cell invasion potential

A significantly higher invasion potential was observed forSCC25 cells irradiated with 1.0 J/cm2 when compared to thecontrol group and the group irradiated with 0.5 J/cm2

(p<0.001). Figure 6a illustrates 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 themembrane was higher in group L1.0 when compared to theL0.5 and control groups (Fig. 6b).

Discussion

In vitro studies have shown an increase in the proliferationrates of different normal and neoplastic cells after laser ther-apy [5, 7–10]. In the present investigation, LLLT increased theproliferation of epithelial cells derived from an oral squamouscell 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 irradiatedwith 0.5 J/cm2 and the control group after 24 and 72 h ofculture. Considering that the duration of the cell cycle ofhuman cells is approximately 24 h [11], it can be assumedthat the second irradiation (48 h) was necessary to furtherincrease cell proliferation since cells of the L1.0 group present-ed a significant increase of proliferative activity only between24 and 72 h when compared to the other groups. Cell viabilitydid 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 cellshave also been reported in other studies [5, 7, 9, 12]. Incontrast, some reports have shown inhibitory effects of lasertherapy on hepatoma [13], melanoma [8], glioblastoma [14],and oral squamous cell carcinoma cells [10]. However, theseinhibitory effects were observed with the use of light of theinfrared or red spectrum and high irradiation doses [8, 10, 13,14], whereas the studies demonstrating an increase in theproliferation of malignant cells used red lasers and low energydensities [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 andirradiation parameters used. A recent systematic review [6]suggests that a dose range of 0.5 to 4.0 J/cm2 and wavelengthof 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 beused to illustrate the dose response of cells to irradiation: ifinsufficient energy is applied, no response is observed becausethe minimum threshold is not met; if more energy is appliedand the threshold is exceeded, biostimulation is achieved.However, if the energy applied is very high, an inhibitoryeffect occurs. Taken together, the results of previous studiesand the present findings suggest that LLLT contributes to cellgrowth, whereas high doses interfere negatively with the cellcycle, inhibiting proliferation. LLLT has been suggested toinduce the phosphorylation of tyrosine kinase receptors, acti-vating the MAPK/ERK signaling cascade [2]. On the otherhand, Huang et al. [15] showed that high doses increase therelease of reactive oxygen species that mediate inhibitoryeffects and induce cell death. In addition, high-power lasertherapy 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 thepresent study to further support the possible effects of lasertherapy on cell proliferation. According to Schartinger et al.[10], an increase in the proportion of cells in the S and G2/Mphases and a concomitant reduction of cells in the G0/G1phase indicate enhanced cell cycle progression from G1 to Sand G2/M. This change was observed in the present study24 h after the first irradiation and was more pronounced ingroup L1.0. However, an increase in the proportion of cells inthe G0/G1 phase and a decrease in the proportion of cells inthe G2/M phase were observed in the control group and in theirradiated groups (L0.5 and L1.0), indicating a reduction in celldivision after 48 h of culture. These findings might be ex-plained by the fact that part of the cells duplicated during thefirst 24 h progressed to the G1 phase of a new cycle, suggest-ing prolongation of this phase, whereas the other part enteredthe resting stage (G0). This reduction in cell cycle velocitywas probably due to the fact that confluence was reachedalready within 48 h. The proliferative activity of the cells didnot continue to increase until 72 h because of the relativedecrease of nutrients in the culture medium as a result of theincrease in the number of cells during the experiment. Thecells probably needed to adapt to the new conditions of themicroenvironment, and cell cycle velocity was therefore re-duced. Nevertheless, higher proportions of cells in the S andG2/M phases were observed in the L1.0 group at all time pointswhen 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 D1regulates the transition from the G1 to the S phase throughbinding to CDK4 and CDK6, promoting the phosphorylation

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Fig. 5 Dot plots and histograms corresponding to membrane and intracellular protein expression obtained for the control, L0.5, and L1.0 groups

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of protein RB and subsequent release of the E2F transcriptionfactor. Cyclin D1 is a useful marker of proliferation and itsdysregulation is essential for the genesis and progression ofcancer [18].

In the present study, analysis of protein expression by flowcytometry showed high levels of cyclin D1 at all time pointsanalyzed. Expression was higher in the L1.0 group, followedby the L0.5 group and control group, confirming the higherproliferation rates demonstrated in group L1.0. In all groups,expression of this protein was higher at the beginning of theexperiment. A slight decrease was observed after 24 h andexpression again increased at 72 h. This finding may have alsocontributed to the reduction in the number of cells in the S andG2/M phases and the increase of cells in G0/G1 observed after48 h of culture. Shefer et al. [19] also found higher expressionof cyclin D1 in irradiated cells during the first 24 h, which wasassociated with progression from the G1 to the S phase of thecell cycle. In the study of Taniguchi et al. [20], cells irradiatedwith 660 nm exhibited low nuclear expression of p15, amember of the INK4 family that regulates the cell cycle inthe G1 phase, inhibiting CDK4 and CDK6. Using microarraysand PCR, Wu et al. [21] identified overexpression of the Akt,cyclin D1, and PI3K genes in irradiated mesenchymal stemcells. On the basis of scientific evidence, we suggest that theincreased expression of cyclin D1 observed in the present

study is related to the activation of components of cellularsignaling 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 moleculesinvolved in cell adhesion and migration since these biologicalactivities are directly associated with cell proliferation inmalignant neoplastic processes. In this respect, membraneexpression of E-cadherin was low in SCC25 cells, in agree-ment with Gasparoni et al. [23]. This finding can be easilyexplained by the malignant phenotype of these cells.However, lower staining was observed in the groups submit-ted to laser therapy, particularly the group treated with a doseof 1.0 J/cm2 at 24 and 72 h. The reduced or absent expressionof E-cadherin and of associated cellular adhesion moleculesresults 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 growthcurve, cell cycle distribution, and expression of cyclin D1,showing a growth advantage in the L1.0 group.

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

Fig. 6 Light microscopy results showing the percentage of invading cells (a, b)

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(FGF) [25–28]. The activation of these factors seems to berelated to DNA hypermethylation in the E-cadherin gene andthe induction of transcriptional repressors which inhibit tran-scription of the gene [29]. Interestingly, studies using benigncells demonstrated the capacity of LLLT to induce the expres-sion of these growth factors [30, 31]. It is therefore likely thatin 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 thisprotein was also low in SCC25 cells in the groups studied,with the lowest expression being observed in the L0.5 and L1.0

groups. In contrast, marked nuclear expression of β-cateninwas detected, which increased throughout the experiment.Gasparoni et al. [32] also found weak membrane and strongnuclear expression of this protein in SCC25 cells and corre-lated this finding with higher proliferative activity. In thepresent study, the L1.0 group 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 inthe membrane or nucleus, where it is involved in cell adhesionor cell growth, respectively. The aberrant expression of thisprotein in the nucleus has been shown to be associated withelevated 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 of β-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 theaxin/APC/GSK3β complex which is necessary for degrada-tion of β-catenin in the cytoplasm. β-Catenin, in turn, istranslocated to the nucleus, increasing the transcription ofthe CCND1 gene [34].

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

A previous study demonstrated that transfection of the E-cadherin gene into an E-cadherin-negative prostate carcinomacell line resulted in lower production of MMP-2 and MMP-9and a concomitant decrease in the invasion capacity of thesecells [36]. The present results are consistent with these obser-vations since the reduction of E-cadherin expression in theSCC25 cell line was accompanied by high levels of MMP-9.Furthermore, in vitro studies have shown that the inhibition ofMMP-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 potentialin the group irradiated with a dose of 1.0 J/cm2 when com-pared to the control group and the group irradiated with 0.5 J/cm2. This finding can be explained by the higher proliferativeactivity of SCC25 cells of the L1.0 group as well as the higherexpression ofMMP-9 associated with high levels of cyclin D1and nuclearβ-catenin and the loss of cell–cell adhesion. Otherstudies also highlighted the capacity of LLLT to facilitate cellinvasion [38, 39].

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

The effects of LLLT on the proliferation and invasioncapacity of SCC25 cells observed in the present studysuggest that application of LLLT to oral mucositis lesionslocated close to head and neck tumors may favor tumorgrowth if malignant cells are located in the irradiation field.LLLT has been used successfully for the prevention of oralmucositis in patients receiving high-dose chemotherapy andin patients with head and neck cancer undergoing radio-therapy. In these cases, laser therapy should be used withcaution due to the risk of exposure of malignant cells to theirradiation field. Furthermore, field cancerization in the oralmucosa is a contraindication for laser therapy since thismethod may favor the proliferation of cells that carry initialmolecular alterations contributing to the clonal expansion ofthese cells and the possibility of developing malignantlesions.

In conclusion, the present results show that LLLT at660 nm using low doses facilitates the proliferation and inva-sion of SCC25 cells by influencing the expression of cyclinD1, 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 cellproliferation and invasion can disturb the balance betweencells and tissues.

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

Lasers Med Sci (2014) 29:1385–1395 1393

provide professionals with the perfect laser combination inclinical practice and to better understand the molecular eventsinduced by laser therapy in different cell types. In this respect,we assume that the real mechanisms of action of LLLTwill beclarified, providing evidence of the biological effects derivedfrom its use in oral lesions of different types.

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

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