Therapeutic potential of sulforaphane: modulation of NRF2 mediated PI3/AKT/mTOR pathway in oral fibrosis / Potencial terapêutico do sulforafano: modulação do NRF-2 mediado o pela via PI3/AKT/mTOR na fibrose oral

Oral Submucous Fibrosis is a potentially malignant disorder caused by habitual areca nut chewing, which contributes to the dispersion of active alkaloids into subepithelial tissues, stimulating excessive extracellular matrix deposition. Various treatment modalities are available; however, their efficacy in inhibiting fibrosis progression remains limited. Sulforaphane (SFN), an isothiocyanate found abundantly in cruciferous plants, is known to have effective antifibrotic properties. Objective: The present study investigated the antifibrotic effect of SFN via phosphatidylinositol 3 kinase (PI3K), Serine/Threonine Kinase 1 (AKT-1), mammalian target of rapamycin (mTOR) pathway in arecoline (AER) induced fibrosis in human gingival fibroblasts [HGFs]. Material and Methods: MTT assay determined the half-maximal inhibitory concentration of AER and SFN at 24h in the HGF cell line. Expression levels of transforming growth factor ß1 (TGFß1), collagen type 1 alpha 2 (COL1A2), hydroxyproline (HYP), PI3, AKT, mTOR, and nuclear factor erythroid 2­related factor 2 (NRF2) were assessed post-AER and SFN treatment using qPCR and western blot analysis. Results: The findings of the study revealed that AER elicited a stimulatory effect, upregulating TGFß1, COL1A2, HYP, PI3K, AKT, and mTOR and downregulating NRF2 expression. Conversely, SFN treatment significantly upregulated NRF2, inhibiting TGFß1 mediated PI3/AKT/mTOR pathway. Conclusion: These observations suggest that SFN can be used as a promising synergistic antifibrotic agent to combat fibrogenesis via the non-Smad pathway (AU)

Fibrose submucosa oral é uma desordem potencialmente maligna causada pelo habito de mascar a noz da areca, o que contribui para a dispersão de alcalóides ativos nos tecidos subepiteliais, estimulando a deposição excessiva de matriz extracelular. Há várias modalidades terapêuticas, no entanto, com eficácia limitada no controle da progressão da fibrose. O sulforafano (SFN), isotiocianato encontrado abundantemente em plantas crucíferas, é conhecido por suas propriedades antifibróticas. Objetivo: Investigar os efeitos antifibróticos do SFN na via fosfatidilinositol3-quinase (PI3K), via quinase serina/treonina 1 (AKT-1), via do alvo da rapamicina em mamíferos (mTOR), na fibrose induzida por arecolina (AER) em fibroblastos gengivais de humanos (HGFs). Material e Métodos: A meia concentração inibitória mínima de AER e SFN em 24 horas nas células HGFs foi determinada por MTT. Os níveis de expressão de ß1 (TGFß1), colágeno tipo 1 alfa 2 (COL1A2), hidroxiprolina (HYP), PI3K, AKT, mTOR, fator nuclear eritroide 2 relacionado ao fator 2 (NRF2) foram analisados após tratamento com ERA e SFN através de qPCR e western blot. Resultados: O ERA apresentou efeito estimulatório aumentando a expressão de TGFß1, COL1A2, HYP, PI3K, AKT e mTOR e diminuindo a expressão de NRF2. Por outro lado, tratamento com SFN aumentou significativamente a expressão de NRF2, inibindo a liberação de TGFß1 mediada pela via PI3/AKT/mTOR. Conclusão: Esses achados sugerem que o SFN pode ser um agente antifibrótico promissor no combate à fibrogênese decorrente da via não-Smad (AU)

Molecular pathways of oral submucous fibrosis and its progression to malignancy.

OBJECTIVE: The review aims to comprehend various factors engaged in the alteration of molecular events resulting in Oral submucous fibrosis (OSMF) and its malignant transformation. DESIGN: Literature pertinent to pathways involved in OSMF were explored in databases such as PubMed, Scopus and Google Scholar. The relevant literature was reviewed and critically appraised in this narrative review. RESULTS: Areca nut components influence myriad of cellular molecules such as cytokines, growth factors, myofibroblasts, non-coding RNAs and alter their expression. These aberrantly expressed molecules drive the progression of OSMF from localized inflammation to fibrosis of buccal mucosa. The oral tissue suffers from oxidative stress, hypoxia, autophagy, aberration of cell cycle and DNA damage. Apoptosis of epithelial layer results in its atrophy facilitating deeper penetration of areca nut elements. With the advance of disease, epithelial-mesenchymal transition eventuates and promotes dysplasia. The jeopardized expression of various cellular molecules, suppressed apoptosis, along with increased genetic alterations and neovascularization favors the malignant transformation. CONCLUSION: OSMF is a progressive disorder with complex mechanism of pathogenesis initiated by inflammation of oral mucosa. Continuous habit of areca nut chewing and the resulting insult to the tissues prevents healing process and is destined to debilitating disease which affects the quality of life with a higher probability of progression to malignancy.

In vitro anticancer activity of a pentacyclic triterpenoid via the mitochondrial pathway in bone-invasive oral squamous cell carcinoma.

CONTEXT: Oral cancer is the most dreadful cancer worldwide with a 5-year survival rate of approximately 50%. Anticancer therapies such as chemotherapy and radiotherapy result in severe side effects. AIM: We aimed to evaluate the in vitro anticancer activity of Asiatic acid (AA) on bone-invasive oral squamous cell carcinoma (BHY) cell line. SETTINGS AND DESIGN: This was an in vitro laboratory setting. MATERIALS AND METHODS: BHY cell lines were used for the experiment. Confocal microscopy was used to observe cellular alterations. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to determine the IC50 concentration of AA and flow cytometry to analyze the percentage of cells in each phase of the cell cycle post treatment. Immunoblot assays and semiquantitative reverse transcriptase-polymerase chain reaction (rt-PCR) were used to study the expression level of genes involved. STATISTICAL ANALYSIS USED: Student's t-test and one-way analysis of variance were used for statistical analysis. RESULTS: IC50 concentration of AA was 15.6 µM. On flow cytometry analysis, treatment with 15.6 µM and 31.25 µM of AA for 24 h increased the percentage of cells in the G2/M phase to 45.63% and 53.12%, respectively, compared to 9.62% in control group. Immunoblot analysis and semiquantitative rt-PCR demonstrated an upregulation of p53, cyclin-dependent kinase inhibitors (p21 and p27), caspase-3, caspase-9, cytochrome c and Bax in a time-dependent manner and downregulation of cyclins and anti-apoptotic protein Bcl-2 (**P < 0.05, ***P < 0.001 versus control) post AA treatment. CONCLUSION: AA induces apoptosis via the mitochondrial-dependent pathway and causes cell cycle arrest at the G2/M phase in BHY cell line.

Antifibrotic effect of Centella asiatica Linn and asiatic acid on arecoline-induced fibrosis in human buccal fibroblasts.

AIM: The aim of the present study was to investigate the in vitro antifibrogenic effects of Centella asiatica Linn (CA) and its bioactive triterpene aglycone asiatic acid (AA) on arecoline-induced fibrosis in primary human buccal fibroblasts (HBF). METHODS: An ethanolic extract of CA was prepared, and AA was purchased commercially. High-performance thin-layer chromatography (HPTLC) was performed to quantify AA in the CA extract; colorimetric assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was performed to determine an half-maximal inhibitory concentration. HBF were cultured and stimulated with arecoline. The inhibitory effects of CA and AA at different concentrations were assessed using gene-expression studies on fibrosis-related markers: transforming growth factor-ß1, collagen 1 type 2, and collagen 3 type 1. The stimulatory effect of arecoline and the inhibitory effect of AA on fibroblast morphology and extracellular matrix were assessed qualitatively using Masson trichrome stain. RESULTS: The HPTLC analysis determined 1.2% AA per 100 g of CA extract. Arecoline produced a concentration-dependent increase in the fibrotic markers, treatment with CA significantly downregulated fibrotic markers at higher concentrations, and AA downregulated at lower concentrations. Arecoline altered fibroblast morphology and stained strongly positive for collagen, and AA treatment regained fibroblast morphology with faint collagen staining. CONCLUSION: CA and AA can be used as antifibrotic agents.