Flávia Cristina Perillo Rosin

Effect of topical 5-ALA mediated photodynamic therapy on proliferation index of keratinocytes in 4-NQO-induced potentially malignant oral lesions

Fractionation can improve photodynamic therapy (PDT) efficacy for potentially malignant oral lesion treatment. The aim of this study was to demonstrate the apoptosis/proliferation index of oral keratinocytes after two sessions of topical 5-ALA-mediated PDT in 4-Nitroquinoline-1-oxide-induced potentially malignant oral lesion, and to suggest the ideal interval between PDT sessions. Immuno-histochemical tests for proliferating cell nuclear antigen and caspase-3, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were performed at 6h, 24h, 48h, and 72h time intervals after PDT. The number of positive cells showing caspase-3 expression was significantly higher, mainly at 6h after PDT. In the first cycle of PDT, the highest frequency of positive cells for TUNEL was found at 24h. At 72h after PDT, proliferating cell nuclear antigen positive cells increased significantly, indicating that there was an epithelial response in direction towards DNA repair and cell proliferation at this time. Because cell proliferation increases and cell death index decreases at 72h after PDT, it is recommended that the interval between the PDT sessions must not be longer than 2days up to total lesion remission.

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.

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.

Resistance of oral cancer cells to 5-ALA-mediated photodynamic therapy

Photodynamic therapy (PDT) has been indicated for oral squamous cell carcinoma (OSCC) at early stages. Chemo and radio‐resistance are frequently observed in OSCC, but it is unknown whether this tumor can develop resistance to PDT. It was investigated the process of acquiring resistance to multiple cycles of PDT by using OSCC cells. We also analyzed the expression of anti‐apoptotic proteins and those related to Akt/mTOR pathway. Sub‐lethal doses of PDT were applied, consisting of a constant concentration of 5‐aminolevulinic acid (5‐ALA) (1 mM, 4‐h incubation) and increasing irradiation dose with LED (from 5.86 to 10.54 J/cm2). Cell viability, migration capacity, intracellular expression of protoporphyrin IX (PpIX), mitochondrial density, and pro‐survival proteins were investigated in PDT‐resistant cells. Six OSCC cell generations resistant to PDT were isolated. The resistant cells exhibited a survival phenotype characterized by a reduction in the expression of endogenous PpIX, increase in mitochondrial density, increase in migration capacity, and up‐regulation of p‐NFκB, p‐survivin, iNOS, p‐Akt Ser473, cyclin D1, p‐mTOR Ser2481, and p‐mTOR Ser2448. OSCC cells are able to survive doses of 5‐ALA‐PDT by reducing PpIX synthesis and activating signaling pathways related to the inhibition of apoptosis and improvement of cell proliferation. Further studies are necessary to confirm whether this PDT‐resistance phenotype can be clinically present, mainly in OSCC showing clinical recurrences after exposure to different PDT protocols.

Immunohistochemistry profile of p75 neurotrophin receptor in oral epithelial dysplasia and oral squamous cell carcinoma induced by 4-nitroquinoline 1-oxide in rats

OBJECTIVE The 4-nitroquinoline 1-oxide (4-NQO) model for carcinogenesis has been used to investigate cancer stem cells (CSC), but no study has addressed the role of the p75 neurotrophin receptor (p75NTR) in 4-NQO-induced oral dysplasia and oral squamous cell carcinoma (OSCC). The aim of this study was to evaluate the immunohistochemistry profile of the p75NTR during 4-NQO-induced oral carcinogenesis in rats and to verify whether this profile has an association with proliferating cell nuclear antigen (PCNA) immunolabeling. DESIGN For 28 weeks, rats were exposed to 4-NQO, which was diluted in the drinking water. After 3, 5, 7, 16, and 28 weeks, the animals were euthanized and their tongues were histologically analyzed using p75NTR and PCNA immunolabeling. RESULTS In animals without 4-NQO exposure, the p75NTR and PCNA were expressed only in the basal epithelial layer and in a clustered manner. The oral epithelium showed dysplasia and a significant increase in the number of p75NTR- and PCNA-positive cells, which were localized mainly in the basal and suprabasal epithelial layers during weeks 5-16 of 4-NQO exposure. When the epithelium invaded the lamina propria and well-differentiated OSCC began, the p75NTR-positive cell frequency drastically decreased in epithelial cords and nests, showing a negative correlation with PCNA expression. p75NTR immunolabeling during 4-NQO-induced carcinogenesis was similar to that described for human head and neck dysplasia and neoplasia. CONCLUSIONS p75NTR immunolabeling observed in 4-NQO-induced oral dysplastic and OSCC lesions were related to the early phases of oral carcinogenesis and may help predict cell dysplasia and malignant transformation.

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.