Ultrasonography-Derived Elasticity Estimation of Live Porcine Oral Mucosa.

OBJECTIVES: To investigate the biomechanical properties of porcine oral tissues with in vivo ultrasonography and to compare the difference between oral alveolar mucosa and gingival tissue concerning compressional and tensile mechanical strain. MATERIALS AND METHODS: Sinclair minipigs (6 females and 4 males, 6 to 18 months of age) were anesthetized for ultrasonography. In vivo high-frequency tissue harmonic ultrasound (12/24 MHz) cine-loops were obtained while inducing mechanical tissue stress (0 to 1 N). Post-processing strain analysis was performed in a cardiac speckle tracking software (EchoInsight®). Region of interest (ROI) was placed for gingival and alveolar mucosa tissues for longitudinal (compressional) and tensile strain analyses. A calibrated gel pad was employed to determine the absolute force (pressure) for the measured tissue strain response function. The resulting elasticity data was statistically analyzed using custom Matlab scripts. RESULTS: In total, 38 sonography cine-loops around the third premolars were included in the investigation. The longitudinal strain of alveolar mucosa ε AM L was found to be significantly (P < .05) larger than that of gingiva ε G L . Across the measured force range, ε AM L ~ 1.7 × Îµ G L . Significant differences between alveolar mucosa and gingiva tissues were found for all forces. The tensile strain of the alveolar mucosa ε AM T was found to be ~2 × Îµ G T (on the epithelial surface of the gingiva). Both were statistically significantly different for forces exceeding ~0.08 N. At depth, that is, 500 and 1000 µm below the epithelial surface, the gingiva was found to have less ability to stretch contrary to the alveolar mucosa. Gingival tissue at 500 µm depth has significantly less tensile strain than at its surface and more than at 1000 µm depth. In contrast, the tensile strain of alveolar mucosa is largely independent of depth. CONCLUSION: Ultrasonography can reveal significant differences in oral alveolar mucosal and gingival elastic properties, such as compressional and tensile strain. Under minute forces equivalent to 10 to 40 g, these differences can be observed. As dental ultrasound is a chairside, and noninvasive modality, obtaining real-time images might soon find clinical utility as a new diagnostic tool for the objective and quantitative assessment of periodontal and peri-implant soft tissues in clinical and research realms. As ultrasound is a safe modality with no known bioeffects, longitudinal monitoring of areas of concern would be particularly attractive.

Hypoxic niches are endowed with a protumorigenic mechanism that supersedes the protective function of PTEN.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide and is characterized by a fast-paced growth. Like other solid tumors, the HNSCC growth rate results in the development of hypoxic regions identified by the expression of hypoxia-inducible factor 1α (HIF-1α). Interestingly, clinical data have shown that pharmacological induction of intratumoral hypoxia caused an unexpected rise in tumor metastasis and the accumulation of cancer stem cells (CSCs). However, little is known on the molecular circuitries involved in the presence of intratumoral hypoxia and the augmented population of CSCs. Here we explore the impact of hypoxia on the behavior of HNSCC and define that the controlling function of phosphatase and tensin homolog (PTEN) over HIF-1α expression and CSC accumulation are de-regulated during hypoxic events. Our findings indicate that hypoxic niches are poised to accumulate CSCs in a molecular process driven by the loss of PTEN activity. Furthermore, our data suggest that targeted therapies aiming at the PTEN/PI3K signaling may constitute an effective strategy to counteract the development of intratumoral hypoxia and the accumulation of CSCs.-Nascimento-Filho, C. H. V., Webber, L. P., Borgato, G. B., Goloni-Bertollo, E. M., Squarize, C. H., Castilho, R. M. Hypoxic niches are endowed with a protumorigenic mechanism that supersedes the protective function of PTEN.

BMAL1 Modulates Epidermal Healing in a Process Involving the Antioxidative Defense Mechanism.

The circadian rhythm regulates the physiology and behavior of living organisms in a time-dependent manner. Clock genes have distinct roles including the control over gene expression mediated by the transcriptional activators CLOCK and BMAL1, and the suppression of gene expression mediated by the transcriptional repressors PER1/2 and CRY1/2. The balance between gene expression and repression is key to the maintenance of tissue homeostasis that is disrupted in the event of an injury. In the skin, a compromised epithelial barrier triggers a cascade of events that culminate in the mobilization of epithelial cells and stem cells. Recruited epithelial cells migrate towards the wound and reestablish the protective epithelial layer of the skin. Although we have recently demonstrated the involvement of BMAL and the PI3K signaling in wound healing, the role of the circadian clock genes in tissue repair remains poorly understood. Here, we sought to understand the role of BMAL1 on skin healing in response to injury. We found that genetic depletion of BMAL1 resulted in delayed healing of the skin as compared to wild-type control mice. Furthermore, we found that loss of Bmal1 was associated with the accumulation of Reactive Oxygen Species Modulator 1 (ROMO1), a protein responsible for inducing the production of intracellular reactive oxygen species (ROS). The slow healing was associated with ROS and superoxide dismutase (SOD) production, and pharmacological inhibition of the oxidative stress signaling (ROS/SOD) led to cellular proliferation, upregulation of Sirtuin 1 (SIRT1), and rescued the skin healing phenotype of Bmal1-/- mice. Overall, our study points to BMAL1 as a key player in tissue regeneration and as a critical regulator of ROMO1 and oxidative stress in the skin.

Hypoacetylation of acetyl-histone H3 (H3K9ac) as marker of poor prognosis in oral cancer.

AIMS: Epigenetics refers to changes in cell characteristics that occur independently of modifications to the DNA sequence. Oral carcinogenesis is influenced by modifications in epigenetic mechanisms, including changes in histones, which are proteins that support chromatin remodelling for the dynamic regulation of gene expression and silencing. The dysregulation of histone acetylation can lead to the uncontrolled activity of different genes, thereby triggering events associated with malignant transformation. The aim of this study was to analyse the expression of acetyl-histone H3 at lys9 (H3K9ac) in oral leucoplakia (OL) and oral squamous cell carcinoma (OSCC) in addition to its association with cell proliferation, epithelial-mesenchymal transition (EMT) and clinical-pathological findings. METHODS AND RESULTS: Samples of normal oral mucosa (NOM), OL and OSCC were submitted to immunohistochemical analysis using anti-H3K9ac, Ki67 and vimentin. Slides were submitted to quantitative analysis regarding the percentage of positive cells. OSCC presented less expression of H3K9ac in comparison to OL (P < 0.01), whereas Ki67 and vimentin levels increased from OL to OSCC (P < 0.001 and P = 0.03, respectively). OSCC patients with poor prognosis had less H3K9ac expression (P = 0.04). The Kaplan-Meier cumulative survival curves also revealed lower survival rates in patients with less H3K9ac expression (P < 0.01). CONCLUSIONS: The present findings suggest that changes in H3K9ac occur during the process of oral carcinogenesis along with an increase in cell proliferation and EMT. The results demonstrate that H3K9ac may be a useful novel prognostic marker for OSCC.

Using PDX animal models to identify and stratify adenoid cystic carcinoma patients presenting an enhanced response to HDAC inhibitors.

Adenoid cystic carcinoma (ACC) patients face a highly infiltrative and metastatic disease characterized by poor survival rates and suboptimal response to available therapies. We have previously shown that sensitization of ACC tumors to chemotherapy using histone deacetylase inhibitors (HDACi) constitutes a promising therapeutic strategy to manage tumor growth. Here, we used patient-derived xenografts (PDX) from ACC tumors to evaluate the effects of in vivo administration of the HDAC inhibitor Entinostat combined with Cisplatin over tumor growth. RNA from PDX tumor samples receiving the proposed therapy were analyzed using NanoString technology to identify molecular signatures capable of predicting ACC response to the therapy. We also used an RNAseq dataset from 68 ACC patients to validate the molecular signature identified by the NanoString platform. We found that the administration of Entinostat combined with Cisplatin resulted in a potent tumor growth inhibition (TGI) ranging from 38% to 106% of the original tumor mass. Enhanced response to therapy is consistent with the reactivation of tumor suppressor genes, including SFRP1, and the downregulation of oncogenes like FGF8 and CCR7. Nanostring data from PDX tumors identified a genetic signature capable of predicting tumor response to therapy. We further stratified 68 ACC patients containing RNAseq data accordingly to the activity levels of the identified genetic signature. We found that 23% of all patients exhibit a genetic signature consistent with a high ACC tumor response rate to Entinostat and Cisplatin. Our study provides compelling preclinical data supporting the deployment of a powerful systemic anticancer therapy crafted and explicitly tested for ACC tumors.

Curcuma longa L. Effects on Akt/mTOR Pathway and NF-κB Expression During Skin Wound Healing: An Immunohistochemical Study.

Skin ulcers, wounds, or burns represent a burden for health care worldwide. Our aim was to explore the effects of mucoadhesive formulation with Curcuma longa L. extract mucoadhesive formulation containing curcumin (MFC) on skin healing in Wistar rats. Fifty-four rats were randomly allocated into 3 groups: control, vehicle, and MFC. A full-thickness circular wound was induced on the back of each animal. Two daily applications of the products were performed according to the experimental group. On days 3, 10, and 21, 6 animals in each group were euthanized. Clinical analysis was based on wound area. Histologic analysis was performed in hematoxylin and eosin-stained sections, with re-epithelization and inflammation being assessed by means of semiquantitative scores. To analyze the Akt/mTOR pathway, immunohistochemistry for phospho Akt (pAkt) and phospho ribosomal protein S6 were investigated. In addition, nuclear factor kappa-light-chain-enhancer of activated B cells immunolabeling was performed. Clinical analysis revealed wounds with a smaller area on days 3 and 10 in curcumin-treated animals. Histologically, MFC had a significant impact on inflammatory events on days 3 and 10 and promoted faster re-epithelization, which was evidenced on day 10. MFC-treated wounds exhibited pAkt upregulation on day 10 and both pAkt and phospho ribosomal protein S6 downregulation on day 21. Nuclear factor kappa-light-chain-enhancer of activated B cells expression varied through the evaluation periods; however, no significant difference was observed between groups. Collectively, our results indicate that MFC is efficient in accelerating cutaneous wound repair through modulation of the inflammatory process and stimulus of re-epithelization by an Akt/mTOR-dependent mechanism.

Pharmacological PTEN inhibition: potential clinical applications and effects in tissue regeneration.

Although the human body can heal, it takes time, and slow healing and chronic wounds often occur. Thus, identifying novel therapies to aid regeneration is needed. Here, we conducted a systematic review following the Preferred Reporting Items for Systematic Reviews guidelines and assessed preclinical studies on phosphatase and tensin homolog (PTEN) inhibitors and their effects on tissue repair and regeneration. In conditions associated with neurodegeneration, tissue injury and ischemia, the PTEN-regulated PI3K/AKT signaling pathway is activated. The use of PTEN inhibitors resulted in better tissue response by reducing the healing time and lesion sizes or inducing neuronal regeneration. Notably, all studies included in this systematic review indicated that pharmacological inhibition of PTEN enhanced the repair process of the eye, lung, muscle and nervous system.

Interference with the bromodomain epigenome readers drives p21 expression and tumor senescence.

Head and neck cancer (HNSCC) are one of the most common solid malignancies of the world, being responsible for over 350,000 deaths every year. Much of the complications in managing and treating HNSCC advent from the complex genetic and epigenetic landscape of the disease. Emerging information has shown promising results in targeting BRD4, an epigenetic regulator bromodomain that functions as a scaffold for transcription factors at promoters and super-enhancers. Here we show that by disrupting the interaction between BRD4 and histones using the bromodomain inhibitor JQ1, HNSCC cells undergo cell growth arrest followed by cellular senescence. Mechanistically, JQ1 negatively impacted the phosphorylation levels of SIRT1 along with the acetylation levels of mutant p53 (active). In vivo administration of JQ1 resulted in disruption of HNSCC growth along with the activation of cellular senescence, observed by the accumulation of DNA double-strand breaks, p16ink4, accumulation of senescence-associated beta-galactosidase, and loss of phosphorylated Sirt1ser47. Furthermore, we also demonstrate that JQ1 was efficient in reducing the population of cancer stem cells from HNSCC xenografts.

Interfering with bromodomain epigenome readers as therapeutic option in mucoepidermoid carcinoma.

PURPOSE: Emerging evidence indicates that bromodomains comprise a conserved class of epigenome readers involved in cancer development and inflammation. Bromodomains are associated with epigenetic modifications of gene transcription through interactions with lysine residues of histone tails. Particularly, the bromodomain and extra-terminal domain (BET) family member BRD4 has been found to be involved in the control over oncogenes, including c-MYC, and in the maintenance of downstream inflammatory processes. The objective of this study was to evaluate the effect of pharmacologically displacing BRD4 in mucoepidermoid carcinoma (MEC) cells. METHODS: We assessed the presence of BRD4 levels in a panel of human MEC tissue samples in conjunction with histological grading and clinical information. In vitro studies were carried out using human MEC-derived cell lines. The BET inhibitor iBET762 was administered to MEC cells to assess the impact of disrupted BRD4 signaling on colony forming capacities and cell cycle status. The activation of cellular senescence induced by iBET762 was determined by immunohistochemical staining for p16ink4. Flow cytometry was used to identify populations of cancer stem cells in MEC-derived cell lines. RESULTS: We found that primary human MECs and MEC-derived cell lines are endowed with high BRD4 expression levels compared to those in normal salivary glands. We also found that, by displacing BRD4 from chromatin using the BET inhibitor iBET762, MEC cells lose their colony forming capacities and undergo G1 cell cycle arrest and senescence. Finally, we found that targeted displacement of BRD4 from chromatin results in depletion of cancer stem cells from the overall MEC cell populations. CONCLUSIONS: Our findings indicate that bromodomain-mediated gene regulation constitutes an epigenetic mechanism that is deregulated in MEC cells and that the use of BET inhibitors may serve as a feasible therapeutic strategy to manage MECs.

Overcoming adaptive resistance in mucoepidermoid carcinoma through inhibition of the IKK-ß/IκBα/NFκB axis.

Patients with mucoepidermoid carcinoma (MEC) experience low survival rates and high morbidity following treatment, yet the intrinsic resistance of MEC cells to ionizing radiation (IR) and the mechanisms underlying acquired resistance remain unexplored. Herein, we demonstrated that low doses of IR intrinsically activated NFκB in resistant MEC cell lines. Moreover, resistance was significantly enhanced in IR-sensitive cell lines when NFκB pathway was stimulated. Pharmacological inhibition of the IKK-ß/IκBα/NFκB axis, using a single dose of FDA-approved Emetine, led to a striking sensitization of MEC cells to IR and a reduction in cancer stem cells. We achieved a major step towards better understanding the basic mechanisms involved in IR-adaptive resistance in MEC cell lines and how to efficiently overcome this critical problem.