Effect of chronic ethanol ingestion on dendritic cell population during oral mucosal repair: An experimental study.

The aim of this study was to analyze the effect of chronic ethanol ingestion on dendritic cell repopulation during the repair process of rat oral mucosa and in the rat spleen by analyzing the immunohistochemical expression of dendritic cell markers. Wistar rats ingested 20% ethanol solution for 28 days; a surgical wound was performed on the rat tongue after this period. The repair process and the number of CD1a+, CD11c+, and CD207+ cells in the regions adjacent to the wound were determined at day 1, 3, and 7 following the wound as well as in the rat spleen. The wound-only group (no ethanol exposure) had complete reepithelization after 7 days, but this did not occur in the ethanol + wound group at this time point. The inflammatory infiltrate was significantly reduced in animals exposed to ethanol, which also showed significantly lower counts of CD1a+, CD11c+, and CD207+ cells than the wound-only group at all experimental time points. In addition, ethanol exposure also resulted in lower densities of CD11c+ and CD207+ cells in the rat spleen. In conclusion, chronic ethanol intake had a negative impact on dendritic cell numbers, a fact that may contribute to delay in oral mucosa repair.

Does Photobiomodulation Affects CK10 and CK14 in Oral Mucositis Radioinduced Repair?

The mechanisms of action of photobiomodulation (PBM) in oral mucositis (OM) are not completely elucidated. To enlighten the role of PBM in the evolution of epithelial maturity in OM ulcers, the present study evaluated the effect of PBM with red (λ) wavelength of 660 nanometers (nm) and infrared of 780 nm in radio-induced OM wounds on the tongue of rats, eight and twenty days after irradiation with single dose of 20 Gy. The percentage area corresponding to positive staining for cytokeratin 10 (CK10) and 14 (CK14) proteins was evaluated in the epithelial area of the lesions, using an immunohistochemical technique (IHC), 8 and 20 days after the induction of lesions, and compared with an untreated control group. CK10 was significantly more expressed in the group treated with 660 nm PBM. CK14 did not show quantitative differences between the groups evaluated. However, whereas in the groups treated with PBM, CK14 was already restricted to the basal layer of the epithelium, as expected in healthy epithelia, in control group it was also expressed in upper layers of the epithelium. In this work, PBM was able to improve epithelial maturity of the repaired OM wound, especially in the 660 nm group.

Vascular alterations after photodynamic therapy mediated by 5-aminolevulinic acid in oral leukoplakia.

Impairment of vascular functions after photodynamic therapy (PDT) is frequently associated with tumor remission and is considered one of the main antineoplastic PDT effects. Vascular alterations in oral leukoplakia (OL) treated with PDT have not yet been described. The aim of this study was to evaluate the effect of topical 5-ALA-mediated PDT on the vascular network of 4NQO-induced OL in rats. After applying 4NQO topically on the tongue during 16 weeks, there was induction of dysplastic lesions, which were treated with two PDT sessions (with an interval of 72 h between them), using topical application of 5-ALA and posterior irradiation with a laser (90 J/cm2 fluency). Histological sections of the tongues were obtained and analyzed concerning plasmatic exudation and microvessel density after immunolabeling with CD31 and CD34 vessel markers. There was intense plasmatic exudation after 6 h of the first PDT session; at 6 h of the second PDT session, there was a significant reduction in the density of CD31- and CD34-positive microvessels in comparison to controls (p < 0.05). In the PDT intervals, there was an increase in the density of CD31 and CD34 microvessels, suggesting angiogenesis. Topical application of 5-ALA-mediated PDT caused an immediate deleterious effect on the vascular network, increasing vessel permeability and reducing vessel density, mainly after two sessions of the treatment. However, secondary angiogenesis emerged in these lesions during intervals of the PDT session. This fact may be considered during the adoption of a PDT protocol, to avoid OL resistance and recurrence after the treatment.

Systemic toxic effects during early phases of topical 4-NQO-induced oral carcinogenesis in rats.

BACKGROUND: Most studies have demonstrated 4-NQO toxicity to oral epithelium during oral carcinogenesis induction, but systemic toxicity has been poorly addressed. The aim of this study was to describe the systemic effect of 4-NQO topical application during early phases of oral cancer induction. METHODS: A 4-NQO propylene glycol ointment was topically applied on the rat tongue three times a week for 16 weeks. Local and systemic 4-NQO toxicity was evaluated by body weight gain, hematology, and serum chemistry analyses, histopathology, and proliferating cell nuclear antigen (PCNA) immunohistochemistry. RESULTS: Significant reduction in body weight gain and in white blood cell count as well as significant increase in serum ALT and AST was observed after 16 weeks of 4-NQO topical application. Focal hepatic lobular necrosis, renal tubular degeneration, and decreased cellularity in the splenic white pulp were also detected. CONCLUSIONS: 4-NQO topical application on the tongue of rats for 16 weeks seems to have caused hepatic, renal, and splenic toxicity. Potential systemic toxicity should be considered to monitor for variables that could interfere in topical oral carcinogenesis experiments.

Poly-ε-caprolactone/poly(rotaxane) seeded with human dental pulp stem cells or osteoblasts promotes angiogenesis: a chorioallantoic membrane assay.

Biomaterials used for tissue regeneration should ideally provide a favorable environment for cell proliferation and differentiation. Angiogenesis is crucial for supplying oxygen and nutrients necessary for cellular survival at implantation sites. The aim of this study was to evaluate the overall angiogenesis response of a poly ε-caprolactone/poly (rotaxane) blend (poly-blend) carried by human dental pulp stem cells (hDPSCs) or osteoblasts (OB) seeded in the chorioallantoic membranes (CAM) of fertilized chicken eggs on embryonic day 7. They were classified into the following intervention groups: (a) poly(polymeric blend disks free of cells); (b) hDPSC seeded onto CAM; (c) poly/hDPSC (where hDPSCs were seeded onto poly-blend); (d) poly/OB (where osteoblasts were seeded onto poly); (e) OB (where hDPSCs differentiated into osteoblasts were seeded onto CAM); and (f) a negative control when a sterilized silicone ring free of cells or polymer was inserted into CAM. On embryonic day 14, the quantitative and qualitative characteristics of the blood vessels in the CAMs were analyzed macroscopically and microscopically. Macroscopic examination showed that the Poly/hDPSC samples exhibited an increased medium vessel density. Additionally, microscopic observations showed that the Poly/hDPSC group and poly alone resulted in a large lumen area of vascularization. Thus, poly ε-caprolactone/poly (rotaxane) did not impair angiogenesis. Furthermore, poly-blend carried by stem cells of dental pulp origin shows a better vasculogenic potential, which is essential for regenerative therapies.

Photobiomodulation Therapy Minimizes the DNA Damage in 5FU-Treated Gingival Fibroblasts.

5-Fluoroufacil (5FU) is a chemotherapeutic agent indicated for solid tumors but causes oral mucositis, which can be prevented and treated using photobiomodulation therapy (PBMT). It is unknown whether PBMT modifies DNA damage induced by 5FU in oral cells. The aim of this study was to investigate the effect of PBMT on DNA damage and repair and on oxidative stress in gingival fibroblasts exposed to 5FU. Primary gingival fibroblasts were exposed to 5FU and then treated with a laser (660 nm, 100 mW, 1 W cm-2 , 0.09 cm2 spot area) for three different irradiation times (6, 10 or 20 s). Six-second irradiation decreased DNA damage and lipidic peroxidation. All irradiated groups showed low H2AX levels and increased p53 expression. Ten-second irradiation showed a trend to induce high lipidic peroxidation levels and DNA damage than other irradiation groups. In conclusion, the PBMT effect on DNA damage and repair was dependent on the time irradiation: 6 s-time irradiation (6.6 J cm-2 ) protect gingival fibroblasts from 5FU-related genotoxicity and oxidative stress, whereas 10s- and 20s-time irradiations (11.1 J cm-2 and 22.2 J cm-2 , respectively) increased the risk of DNA damage after the 5FU exposure.

Effects of serum-free culture media on human apical papilla cells properties.

OBJECTIVE: Aiming at more effective and safer cell therapies, the objective of this study was to evaluate the biological properties of human apical papilla cells cultured in the absence of serum supplementation in comparison to cells cultured with fetal bovine serum (FBS). DESIGN: Two apical papilla cell populations were isolated from third molars with incomplete rhizogenesis, and cultured in four different media: minimum essential Eagle medium - alpha modification (alpha-MEM); alpha-MEM supplemented with FBS (alpha-MEM + FBS); Dulbecco's modified Eagle medium/nutrient mixture F-12 (DMEM/F12); and DMEM/F12 supplemented with FBS (DMEM/F12 + FBS). We evaluated their proliferation, clonogenicity, and in vitro osteogenic and chondrogenic differentiation potential. RESULTS: Apical papilla cells cultured in DMEM/F12 + FBS and alpha-MEM + FBS were more proliferative than those grown in serum-free media, and also exhibited greater efficiency in colony cell formation. Despite this, all study groups showed immunostaining for the marker of mitosis anti-PHH3. Also, alpha-MEM + FBS, alpha-MEM, and DMEM/F12 + FBS exhibited higher amount of mineralized deposits in vitro than DMEM/F12, while only cells cultured with FBS were able to form spheres in chondrogenic differentiation assay. CONCLUSIONS: Our results showed that, although the cultivation of apical papilla cells in a serum-free medium has reduced the properties of cell proliferation and differentiation, these cells are still capable of maintaining their desirable characteristics.

Keratin expression in gingival tissue and primary cultured gingival keratinocytes: Are there differences?

OBJECTIVE: To investigate whether the process of primary gingival keratinocytes culture obtained from normal human gingiva modifies the expression of keratins (K) 10, K14, and K19. DESIGN: Human gingival fragments were collected from healthy individuals in the same oral site. One part of the samples underwent an immunohistochemistry assay for K10, K14, and K19. The labeling in the epithelium was quantified using a semiautomated method. Another part was used for primary gingival keratinocytes isolation and growth in two-dimensional culture. These cells were also stained for K10, K14, and K19 using immunofluorescence and immunocytochemistry. Positive cells were counted, and the nuclei and cytoplasmatic labeling areas were quantified. RESULTS: In the gingival tissue, a higher expression was found for K14 versus K10 (p < 0.001); K19 was negative in all samples. In gingival keratinocytes culture, K14 (89.6 %) had the highest expression with significant differences in relation to K10 (76.9 %, p < 0.01) and K19 (9.9 %, p < 0.01). The cells positive for K14 exhibited larger nuclei in comparison with K10 (p < 0.05) and K19 (p < 0.05), suggesting a more undifferentiated phenotype. K19 cells showed the largest cytoplasmatic labeling in relation to K10- (p < 0.05) and K14-positive (p < 0.05) cells. CONCLUSION: The process of growth in culture of gingival keratinocytes maintained the expression pattern of K10 and K14 observed in gingival tissues. However, this method induces the expression of K19, suggesting a potential transformation of the keratin network presented in the gingival keratinocytes during the formation of a monolayer in vitro. This reflects the dynamics of cell differentiation.

Photodynamic Therapy Mediated by 5-aminolevulinic Acid Promotes the Upregulation and Modifies the Intracellular Expression of Surveillance Proteins in Oral Squamous Cell Carcinoma.

Expression of proteins related to cell surveillance has been described in tumors presenting resistance to photodynamic therapy (PDT). The aim of this study was to verify whether there was upregulation of proteins related to resistance in oral squamous cell carcinoma (OSCC) after PDT. OSCC was chemically induced in rats and treated after one cycle of PDT mediated by 5-aminolevulinic acid (5-ALA-PDT). Immunolabeling of p-NFκB, Bcl-2, survivin, iNOS, p-Akt, p-mTOR and cyclin D1 was performed after the treatment. There was increased expression of Bcl-2 (P = 0.008), iNOS (P = 0.020), p-Akt (P = 0.020) and p-mTOR (P = 0.010) by surviving neoplastic cells after PDT when compared to the control. In conclusion, after one cycle of 5-ALA-mediated PDT, Bcl-2, p-Akt, p-mTOR and iNOS were upregulated in neoplastic cells of OSCC, suggesting an activation of antiapoptosis and cell proliferation pathways. This fact must be considered in the establishment of PDT protocols for OSCC treatment, mainly those in which PDT will be combined with chemotherapy drugs targeted at the studied proteins.

Analysis of Interleukin 17A in periapical abscess and granuloma lesions.

Interleukin 17A (IL-17A) is a proinflammatory cytokine responsible for the initiation and propagation of inflammation. One of its actions is the recruitment of neutrophils to the site of infection. The aim of this study was to investigate whether there is association between IL-17A expression and neutrophil infiltration in periapical abscesses and periapical granulomas, as well as to find which type of T lymphocyte effector (CD4+ or CD8+) expresses IL-17A in these lesions. Elastase, CD4, CD8, and IL-17A were analyzed by immunohistochemistry and immunofluorescence, in the biopsies of periapical lesions. Abscess lesions exhibited the highest labeling area for IL-17A (p = 0.011). During double immunofluorescence staining, there were significantly more CD4+/IL-17A+ cells compared to CD8+/IL-17A+ cells, both in the abscesses (p = 0.025) and granulomas (p = 0.011). In conclusion, IL-17A was intensively expressed in periapical abscesses rich in neutrophils. The high percentage of IL-17A in these cases suggests the participation of this cytokine particularly in the acute stages of the inflammatory process of the periapical lesions.

Effect of 5-ALA-mediated photodynamic therapy on mast cell and microvessels densities present in oral premalignant lesions induced in rats.

Acute inflammatory response after photodynamic therapy is frequently described, and increase on mast cell degranulation is also present during this process. The mast cell activation may improve angiogenesis, and this fact has been associated with progression of oral premalignant lesions (OPL). The aim of this study was to evaluate whether photodynamic therapy (PDT) increases mast cell density (MCD) and microvessels density (MVD) in 4-nitroquinoline-1-oxide(4NQO)-induced OPL in rats. 4NQO-induced OPL were treated or not with 5-ALA followed by laser irradiation (PDT group and 4NQO groups, respectively). Mast cells and CD34+ microvessels were counted. Both PDT and 4NQO groups had MCD and MVD that were higher than normal mucosa (p b 0.05). The 4NQO group had the lowest number of non-degranulated MCD in comparison to experimental periods of PDT (PDT 6 h ­ p=0.020; 24 h ­ p=0.016; 48 h ­ p=0.003; 72 h ­ p=0.033). Only in the PDT group did MCD and MVD have a significant correlation (r= 0.6219, p = 0.010). 5-ALA-mediated PDT modified the MCD and MVD in the induced OPL, leading to degranulation of mast cells and angiogenesis. A PDT protocol with an efficient eradication of the OPL must be adopted considering the angiogenesis potential associated with the mast cell activation after the therapy.

Identification of myeloid-derived suppressor cells and T regulatory cells in lung microenvironment after Urethane-induced lung tumor.

Tumor growth occurs by the imbalance between cells with effector function and cells with suppressor/regulatory functions. To investigate this scenario we administered the chemical carcinogen Urethane in BALB/c mice and followed these animals during 120 days to observe lung tumor development. In another set of experiments the same protocol was performed with the harvest of spleen, lung and blood at 20 and 30 days after Urethane injection. The lung was used for histology, spleen cells were evaluated for IFN-γ production, and serum nitrite was measured as an indirect form of nitric oxide (NO) evaluation. The spleen and lung-infiltrating cells were evaluated by flow cytometry for CD11b+Gr-1+ myeloid suppressor cells and CD4+FoxP3+ T regulatory cells. Urethane led to lung nodules development after 120 days and the time point evaluation showed that splenocytes stimulated ex vivo expressed higher levels of IFN-γ 20 days after the chemical injection. Also, the level of nitric oxide in serum was higher after 20 days of Urethane injection. There was no statistical difference in spleen cells percentages for CD11b+Gr-1+ and CD4+Foxp3+ in all groups. However, lung-infiltrating cells presented early (20 days) a higher expression of CD11b+Gr-1+ suggesting suppression at this site. In conclusion, it was possible to observe two distinct events at the very early time point after Urethane injection. In periphery there was an increase at the effector immune response (as depicted by IFN-γ-producing cells) and in tumor development site there was an increase at the suppressor cell (CD11b+Gr-1+) phenotype. Suppressor/regulatory cells are targets for cancer therapy.

Analysis of Interleukin 17A in periapical abscess and granuloma lesions

Abstract Interleukin 17A (IL-17A) is a proinflammatory cytokine responsible for the initiation and propagation of inflammation. One of its actions is the recruitment of neutrophils to the site of infection. The aim of this study was to investigate whether there is association between IL-17A expression and neutrophil infiltration in periapical abscesses and periapical granulomas, as well as to find which type of T lymphocyte effector (CD4+ or CD8+) expresses IL-17A in these lesions. Elastase, CD4, CD8, and IL-17A were analyzed by immunohistochemistry and immunofluorescence, in the biopsies of periapical lesions. Abscess lesions exhibited the highest labeling area for IL-17A (p = 0.011). During double immunofluorescence staining, there were significantly more CD4+/IL-17A+ cells compared to CD8+/IL-17A+ cells, both in the abscesses (p = 0.025) and granulomas (p = 0.011). In conclusion, IL-17A was intensively expressed in periapical abscesses rich in neutrophils. The high percentage of IL-17A in these cases suggests the participation of this cytokine particularly in the acute stages of the inflammatory process of the periapical lesions.

Resistência de células de carcinoma epidermóide bucal à terapia fotodinâmica mediada pelo ácido 5-aminolevulínico / Resistance of oral squamous cell carcinomas to 5-aminolevulinic acidmediated photodynamic therapy

O carcinoma epidermóide bucal (CEC) é uma neoplasia maligna com alta morbidade e mortalidade e de difícil tratamento. O tratamento convencional para o CEC inclui cirurgia e radioterapia, seguida ou não de quimioterapia. Apesar de serem amplamente difundidos, esses tratamentos podem ser ineficazes para alguns CECs resistentes. A terapia fotodinâmica (PDT) oncológica tem sido utilizada para o tratamento adjuvante do CEC bucal, principalmente nos casos menos invasivos e que necessitam de redução do tumor para a ressecção cirúrgica. Contudo, semelhantemente aos tratamentos convencionais, a PDT pode também induzir o aparecimento de populações celulares resistentes, fato já descrito para carcinoma cutâneo, adenocarcinoma de cólon e adenocarcinoma mamário. A hipótese de que células de CEC bucal possam desenvolver resistência à PDT ainda não foi testada. Portanto, o objetivo deste trabalho foi verificar se células de CEC bucal (SCC9) desenvolvem resistência a ciclos repetidos de PDT mediada pelo ácido 5- aminolevulínico (5-ALA-PDT) e avaliar se nesse processo ocorre modificação da expressão de marcadores relacionados a sobrevivência celular (NF?B, Bcl-2, iNOS, mTOR e Akt). Foi utilizada linhagem de células de CEC bucal (SCC9), submetida às seguintes condições: 1) Controle - células cultivadas sem nenhum tratamento; 2) ALA - células incubadas com 5-ALA (1mM durante 4 horas); 3) LED - tratadas com iluminação LED (630nm, 5,86J/cm2, 22,5J, 150mW, 150s); 4) PDT - tratadas com 5- ALA-PDT, com os protocolos do grupo ALA e LED combinados, gerando dose letal de 90%. Inicialmente foi realizado somente um ciclo de PDT, sendo avaliada a viabilidade celular em todos os grupos após 24, 48, 72 e 120h da irradiação. Também foi realizado ensaio de detecção da fragmentação de DNA (TUNEL) e análise por imunofluorescência da expressão das proteínas NF?B, Bcl-2, iNOS, pmTOR e pAkt nas células viáveis.

Oral squamous cell carcinoma (SCC) is a malignant tumor with high morbidity and mortality rates, and it is difficult to treat. Conventional treatment for oral SCCs includes surgery and radiotherapy that may be followed by chemotherapy. Although these treatments are widely used, they are ineffective against some resistant tumors. Oncologic photodynamic therapy (PDT) has been used as an adjuvant treatment for oral SCCs, especially in less invasive cases that require tumor reduction before surgical resection. However, like conventional treatments, PDT can induce the occurrence of resistant cell populations such as cutaneous carcinomas and colon and breast adenocarcinomas. The hypothesis that oral SCCs develop resistance to PDT has not yet been tested. Therefore, the aims of this study were to investigate whether oral SCCs (SCC9) develop resistance to several cycles of 5-aminolevulinic acidmediated PDT (5-ALA-PDT) and to determine whether the expression of markers associated with cell survival (NF?B, Bcl-2, iNOS, mTOR, and Akt) is altered during this process. An oral SCC (SCC9) cell line was used, which was subjected to the following conditions: 1) Control: cultured without any treatment; 2) ALA: incubated with 5-ALA (1 mM for 4 h); 3) LED: treated with LED light (630 nm, 5.86 J/cm2, 22.5 J, 150 mW, 150 s); and 4) PDT: treated with 5-ALA-PDT (with the protocols of the ALA and LED groups combined) generating a lethal dose of 90%. Initially, only one cycle of PDT was administered, and cell viability was determined in all groups 24, 48, 72, and 120 h after irradiation. Subsequently, the DNA fragmentation detection assay (TUNEL) and immunofluorescence analysis of the expression of proteins NF?B, Bcl-2, iNOS, pmTOR, and pAkt were performed on viable cells. The fraction of cells that survived the first treatment with 5-ALA-PDT exhibited intense staining for pmTOR and growth potential during the testing period.

Resistência de células de carcinoma epidermóide bucal à terapia fotodinâmica mediada pelo ácido 5-aminolevulínico / Resistance of oral squamous cell carcinomas to 5-aminolevulinic acidmediated photodynamic therapy

O carcinoma epidermóide bucal (CEC) é uma neoplasia maligna com alta morbidade e mortalidade e de difícil tratamento. O tratamento convencional para o CEC inclui cirurgia e radioterapia, seguida ou não de quimioterapia. Apesar de serem amplamente difundidos, esses tratamentos podem ser ineficazes para alguns CECs resistentes. A terapia fotodinâmica (PDT) oncológica tem sido utilizada para o tratamento adjuvante do CEC bucal, principalmente nos casos menos invasivos e que necessitam de redução do tumor para a ressecção cirúrgica. Contudo, semelhantemente aos tratamentos convencionais, a PDT pode também induzir o aparecimento de populações celulares resistentes, fato já descrito para carcinoma cutâneo, adenocarcinoma de cólon e adenocarcinoma mamário. A hipótese de que células de CEC bucal possam desenvolver resistência à PDT ainda não foi testada. Portanto, o objetivo deste trabalho foi verificar se células de CEC bucal (SCC9) desenvolvem resistência a ciclos repetidos de PDT mediada pelo ácido 5- aminolevulínico (5-ALA-PDT) e avaliar se nesse processo ocorre modificação da expressão de marcadores relacionados a sobrevivência celular (NF?B, Bcl-2, iNOS, mTOR e Akt). Foi utilizada linhagem de células de CEC bucal (SCC9), submetida às seguintes condições: 1) Controle - células cultivadas sem nenhum tratamento; 2) ALA - células incubadas com 5-ALA (1mM durante 4 horas); 3) LED - tratadas com iluminação LED (630nm, 5,86J/cm2, 22,5J, 150mW, 150s); 4) PDT - tratadas com 5- ALA-PDT, com os protocolos do grupo ALA e LED combinados, gerando dose letal de 90%. Inicialmente foi realizado somente um ciclo de PDT, sendo avaliada a viabilidade celular em todos os grupos após 24, 48, 72 e 120h da irradiação. Também foi realizado ensaio de detecção da fragmentação de DNA (TUNEL) e análise por imunofluorescência da expressão das proteínas NF?B, Bcl-2, iNOS, pmTOR e pAkt nas células viáveis...

Oral squamous cell carcinoma (SCC) is a malignant tumor with high morbidity and mortality rates, and it is difficult to treat. Conventional treatment for oral SCCs includes surgery and radiotherapy that may be followed by chemotherapy. Although these treatments are widely used, they are ineffective against some resistant tumors. Oncologic photodynamic therapy (PDT) has been used as an adjuvant treatment for oral SCCs, especially in less invasive cases that require tumor reduction before surgical resection. However, like conventional treatments, PDT can induce the occurrence of resistant cell populations such as cutaneous carcinomas and colon and breast adenocarcinomas. The hypothesis that oral SCCs develop resistance to PDT has not yet been tested. Therefore, the aims of this study were to investigate whether oral SCCs (SCC9) develop resistance to several cycles of 5-aminolevulinic acidmediated PDT (5-ALA-PDT) and to determine whether the expression of markers associated with cell survival (NF?B, Bcl-2, iNOS, mTOR, and Akt) is altered during this process. An oral SCC (SCC9) cell line was used, which was subjected to the following conditions: 1) Control: cultured without any treatment; 2) ALA: incubated with 5-ALA (1 mM for 4 h); 3) LED: treated with LED light (630 nm, 5.86 J/cm2, 22.5 J, 150 mW, 150 s); and 4) PDT: treated with 5-ALA-PDT (with the protocols of the ALA and LED groups combined) generating a lethal dose of 90%. Initially, only one cycle of PDT was administered, and cell viability was determined in all groups 24, 48, 72, and 120 h after irradiation. Subsequently, the DNA fragmentation detection assay (TUNEL) and immunofluorescence analysis of the expression of proteins NF?B, Bcl-2, iNOS, pmTOR, and pAkt were performed on viable cells. The fraction of cells that survived the first treatment with 5-ALA-PDT exhibited intense staining for pmTOR and growth potential during the testing period...