Normal Squamous Cell Research Articles

The Notch inhibitor, FLI-06, increases the chemosensitivity of head and neck Squamous cell carcinoma cells to taxanes-based treatment

Aberration of Notch signaling is one of the key events involved in the development and progression of head and neck squamous cell carcinoma (HNSCC). The Notch pathway controls the tissue-specific differentiation of normal squamous epithelial cells and is frequently altered in squamous carcinomas, thus affecting their proliferation, growth, survival, and chemosensitivity or resistance against anti-cancer agents. In this study, we show that the use of novel, small-molecule inhibitors of Notch signaling, such as FLI-06, can have a beneficial effect on increasing the chemosensitivity of HNSCC to taxane-based chemotherapy. Inhibition of Notch signaling by FLI-06 alone virtually blocks the proliferation and growth of HNSCC cells in both 2D and 3D cultures and the zebrafish model, which is accompanied by down-regulation of key Notch target genes and proteins. Mechanistically, FLI-06 treatment causes cell cycle arrest in the G1-phase and induction of apoptosis in HNSCC, which is accompanied by increased c-JunS63 phosphorylation. Combining FLI-06 with Docetaxel shows a synergistic effect and partially blocks the cell growth of aggressive HNSCC cells via enhanced apoptosis and modification of c-JunS243 phosphorylation via GSK-3β inhibition. In conclusion, inhibition of Notch signaling in HNSCC cells that retain active Notch signaling significantly supports taxane-based anticancer activities via modulation of both the GSK-3β and the c-Jun.

Transmission of Genetic Material and Malignant Cells of Cutaneous Origin via Surgical Sutures.

Seeding of skin cancer cells following diagnostic or therapeutic surgical procedures can occur and might cause local recurrences. As current preferred therapy for skin malignancy is surgical excision, seeding of tumour cells by manipulating malignant tissue or suturing can be another factor leading to recurrences. To evaluate whether genetic material and malignant cells adhere to standard suture materials. This prospective study included patients who underwent excision of skin lesions. Monofilament and braided sutures were examined. Sutures were passed through the observed tumour or healthy skin margins and were examined for DNA material and cells by cytological analysis, cell culture and characterization, and DNA analysis. Twenty-two patients and 148 sutures were included. DNA quantification showed DNA material on all sutures, with no significant difference between braided and monofilament sutures. Cytological analysis showed that all slides prepared from cell blocks contained normal squamous and atypical cells. Cell culture and characterization showed viable cells adhering to the sutures under direct light microscopy. Cell cultures showed rapid proliferation of epithelial cells from squamous cell carcinoma specimens. Suture materials carry DNA material and cells, including malignant cells of cutaneous origin and may seed them at distant sites.

Exploring the Role of EZH2 and BCL2 in Demarcating Oral Verrucous Hyperplasia and Verrucous Carcinoma

Introduction Oral verrucous hyperplasia (OVH) and verrucous carcinoma (OVC) are precursors of oral squamous cell carcinoma exhibiting overlapping histopathological picture which warrants distinction. EZH2 is an epigenetic marker possessing multifaceted function in cellular proliferation, migration, and malignant transformation, whereas BCL2 is an integral part of the antiapoptotic mechanism regulating cellular homeostasis. Aim The aim was to distinguish OVH and OVC by analysis of immunohistochemical expression of EZH2 and BCL2. Material and Methods The study sample consisted of 79 formalin-fixed paraffin-embedded tissue sections of normal oral mucosa (10), OVH (10), oral OVC (27), and oral squamous cell carcinoma (32). Immunohistochemical analysis of EZH2 and BCL2 was done and labeling indices were calculated. Additionally, six histopathological parameters were assessed in OVH and OVC. Statistical analysis was done using Kruskal–Wallis test, Tukey honest significant difference test, and Spearman's correlation. Receiver operating characteristic curve was plotted and sensitivity, specificity, and cutoff score of each marker were calculated. Result and Discussion Labeling indices of EZH2 and BCL2 depicted a gradual incline from normal mucosa to oral squamous cell carcinoma. Significant difference of EZH2 and nonsignificant difference in BCL2 expression between OVH and OVC were noted. Out of the six histopathological parameters, keratin plugging, juxtaepithelial lymphocytic response, and frank endophytic growth yielded a significant difference. EZH2 serves as a superior marker than BCL2 to differentiate OVH and OVC. Juxtaepithelial lymphocytic response can also serve as a histopathological parameter in distinguishing OVH and OVC.

Oral mucosa - an examination map for confocal laser endomicroscopy within the oral cavity: an experimental clinical study

ObjectivesConfocal laser endomicroscopy (CLE) is an optical method that enables microscopic visualization of oral mucosa. Previous studies have shown that it is possible to differentiate between physiological and malignant oral mucosa. However, differences in mucosal architecture were not taken into account. The objective was to map the different oral mucosal morphologies and to establish a “CLE map” of physiological mucosa as baseline for further application of this powerful technology.Materials and methodsThe CLE database consisted of 27 patients. The following spots were examined: (1) upper lip (intraoral) (2) alveolar ridge (3) lateral tongue (4) floor of the mouth (5) hard palate (6) intercalary line. All sequences were examined by two CLE experts for morphological differences and video quality.ResultsAnalysis revealed clear differences in image quality and possibility of depicting tissue morphologies between the various localizations of oral mucosa: imaging of the alveolar ridge and hard palate showed visually most discriminative tissue morphology. Labial mucosa was also visualized well using CLE. Here, typical morphological features such as uniform cells with regular intercellular gaps and vessels could be clearly depicted. Image generation and evaluation was particularly difficult in the area of the buccal mucosa, the lateral tongue and the floor of the mouth.ConclusionA physiological “CLE map” for the entire oral cavity could be created for the first time.Clinical relevanceThis will make it possible to take into account the existing physiological morphological features when differentiating between normal mucosa and oral squamous cell carcinoma in future work.

Oncostatin M [OSM] protein immuno-expression in oral squamous cell carcinoma – A prospective study

BackgroundAn important factor in the progression of tumours is the formation of blood vessels in the tumour stroma. When there is an upregulation of angiogenesis stimulators or a downregulation of angiogenesis inhibitors, the tumour may manifest its capacity for angiogenesis. Immune cells secrete a substance called Oncostatin M (OSM), which has been linked to cancer angiogenesis and subsequent pathogenesis. This study is aimed to assess OSM expression in oral squamous cell carcinoma. ObjectiveTo compare the intensity of expression of Oncostatin M [OSM] in normal oral mucosa and Oral Squamous Cell Carcinoma and to analyse their role in the disease progression by enhancing angiogenesis and metastasis. Materials and methodsThe study analysed the expression of OSM with the immunohistochemical aid. The samples were treated with Anti-OSM polyclonal antibody and immunohistochemical staining was done using NovoLinkTM NovocastraTM Polymer Detection System (Leica Microsystems). Stained samples were categorized based on the staining percentage and staining intensity. ResultsThe comparison of staining percentage between the epithelium of control group and the OSCC group revealed that it was statistically significant [p value = 0.002685]. Similarly, the comparison of staining intensity [p value = 0.001202] and staining index [p value = 0.001817] between the control group and the OSCC group revealed that it was statistically significant. ConclusionThe expression of oncostatin M in normal epithelium and OSCC group with the given immunohistochemical staining percentage, intensity and staining index suggests strong diagnostic potential of oncostatin M which can also be used for further therapeutic.

Characterization of the skin microbiome in normal and cutaneous squamous cell carcinoma affected cats and dogs.

The progression of precancerous actinic keratosis lesions (AK) to cutaneous squamous cell carcinoma (SCC) is poorly understood in humans and companion animals, despite causing a significant burden of disease. Recent studies have revealed that the microbiota may play a significant role in disease progression. Staphylococcus aureus has been found in high abundance on AK and SCC lesions, where it secretes DNA-damaging toxins, which could potentiate tumorigenesis. Currently, a suitable animal model to investigate this relationship is lacking. Thus, we examined the microbiome of cutaneous SCC in pets, revealing similarities to humans, with increased staphylococci and reduced commensals on SCC lesions and peri-lesional skin compared to normal skin. Two genera that were in abundance in SCC samples have also been found in human oral SCC lesions. These findings suggest the potential suitability of pets as a model for studying microbiome-related skin cancer progression.

VAV2 orchestrates the interplay between regenerative proliferation and ribogenesis in both keratinocytes and oral squamous cell carcinoma

VAV2 is an activator of RHO GTPases that promotes and maintains regenerative proliferation-like states in normal keratinocytes and oral squamous cell carcinoma (OSCC) cells. Here, we demonstrate that VAV2 also regulates ribosome biogenesis in those cells, a program associated with poor prognosis of human papilloma virus-negative (HPV−) OSCC patients. Mechanistically, VAV2 regulates this process in a catalysis-dependent manner using a conserved pathway comprising the RAC1 and RHOA GTPases, the PAK and ROCK family kinases, and the c-MYC and YAP/TAZ transcription factors. This pathway directly promotes RNA polymerase I activity and synthesis of 47S pre-rRNA precursors. This process is further consolidated by the upregulation of ribosome biogenesis factors and the acquisition of the YAP/TAZ-dependent undifferentiated cell state. Finally, we show that RNA polymerase I is a therapeutic Achilles’ heel for both keratinocytes and OSCC patient-derived cells endowed with high VAV2 catalytic activity. Collectively, these findings highlight the therapeutic potential of modulating VAV2 and the ribosome biogenesis pathways in both preneoplastic and late progression stages of OSCC.

Abstract IA010: Unraveling mutation and selection to better understand early cancer evolution

Abstract Molecular variants drive tumorigenesis. The degree by which specific molecular variants drive cell proliferation varies between tissues, is dependent upon mutational background within tissues, and also varies along the progression from normal tissue development through cancer growth. Knowledge regarding the magnitude by which variants drive cell proliferation, quantifiable as the scaled selection coefficient or cancer effect size of variants, throughout the trajectory from healthy tissue into cancer informs early cancer evolution, detection, and diagnosis—and also has direct relevance to clinical trials, targeted therapeutics, and basic research prioritization. However, in order to quantify these scaled selection coefficients one must disentangle the prevalence of fixed substitutions from the rate at which they occur within cells—the mutation rate. These mutation rates also vary between tissues, throughout genomes, and over stages of tissue development and carcinogenesis within the same tissue lineage. Here, we present a novel methodology to quantify scaled selection coefficients in a stepwise manner over several discrete steps in tissue evolution and tumorigenesis. We highlight results from ongoing work in two tissue types: the esophagus and the endometrium. Within the normal esophagus and esophageal squamous cell carcinoma we find that mutation rates vary substantially along the trajectory from embryogenesis through normal tissue and from normal tissue through tumorigenesis. Accordingly, we also find that selective advantages vary between these steps of evolution, with variants of NOTCH1, NOTCH2, and FAT1 having measurable proliferative advantage within normal tissues but no detectable proliferative advantage during tumorigenesis, and variants of PIK3CA, TP53, NFE2L2, and FBXW7 being selected to fix within both normal and tumorigenic tissues. Intriguingly, we complement this analysis with a quantification of pairwise epistasis and find that mutation of NOTCH1 decreases the selective advantage of variants within TP53 and other drivers, informing recent research suggesting NOTCH1 substitutions may actively inhibit tumorigenesis. We similarly deconvolute mutation and selection between embryogenesis through atypical hyperplasia of the endometrium and atypical hyperplasia through Stage 1 endometrial carcinoma and find that variants within CTNNB1 and PTEN have higher selective advantage within hyperplasia, whereas variants within ARID1A and PIK3R1 are more highly selected through Stage 1 tumorigenesis. Overall, we demonstrate how accounting for differences in mutational processes and rates among discrete steps in tumorigenesis and epistatic interactions allows one to determine which variants are contributing to clonal expansion within normal tissues, which variants are contributing to tumorigenesis, which variants are important to both, and in certain instances which variants may impede the progression of a tumor. Citation Format: Vincent L. Cannataro, Kira A. Glasmacher, Mary Summers, Jeffrey D. Mandell, J. Nic Fisk, Mia Jackson, Sem Asmelash, Jeffrey P. Townsend. Unraveling mutation and selection to better understand early cancer evolution [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr IA010.

Mitochondrial antioxidants in normal oral epithelial and oral squamous cell carcinoma cell lines

Mitochondrial antioxidants play an important role in regulation of oral cancer progression. In our study we profiled the expression levels of various mitochondrial antioxidants such as reduced glutathione, lipid peroxides, mitochondrial catalase, mitochondrial superoxide dismutase 2 (SOD2), and mitochondrial glutaredoxin 2( GLRX2) in normal oral epithelial cell line and oral squamous cell carcinoma cell (OSCC) line. Our comparative study on these cell lines using UV-Vis Spectrophotometry and Immunoblotting reveal there is an altered profile of the mitochondrial antioxidants in oral squamous cell carcinoma. While the expression levels of mitochondrial Catalase, mitochondria Lipid Peroxide, mitochondrial Superoxide Dismutase 2 and mitochondrial Glutaredoxin 2 decrease in oral squamous cell carcinoma cell line as compared to oral epithelial cell line, the levels of reduced glutathione increase. Our results indicate there is a complex interplay of mitochondrial antioxidants in regulation of oral squamous cell carcinoma.

Labeled-free quantitative proteomic analysis of cervical squamous cell carcinoma identifies potential protein biomarkers.

Cervical cancer remains a prevalent cancer among women, and reliance on surgical and radio-chemical therapies can irreversibly affect patients' life span and quality of life. Thus, early diagnosis and further exploration into the pathogenesis of cervical cancer are crucial. Mass spectrometry technology is widely applied in clinical practice and can be used to further investigate the protein alterations during the onset of cervical cancer. Employing labeled-free quantitative proteomics technology and bioinformatics tools, we analyzed and compared the differential protein expression profiles between normal cervical squamous cell tissues and cervical squamous cell cancer tissues. GEPIA is an online website for analyzing the RNA sequencing expression data of tumor and normal tissue data from the TCGA and the GTEx databases. This approach aided in identifying qualitative and quantitative changes in key proteins related to the progression of cervical cancer. Compared to normal samples, a total of 562 differentially expressed proteins were identified in cervical cancer samples, including 340 up-regulated and 222 down-regulated proteins. Gene ontology functional annotation, and KEGG pathway, and enrichment analysis revealed that the differentially expressed proteins mainly participated in metabolic pathways, spliceosomes, regulation of the actin cytoskeleton, and focal adhesion signaling pathways. Specifically, desmoplakin (DSP), protein phosphatase 1, regulatory (inhibitor) subunit 13 like (PPP1R13L) and ANXA8 may be involved in cervical tumorigenesis by inhibiting apoptotic signal transmission. Moreover, we used GEPIA database to validate the expression of DSP, PPP1R13L and ANXA8 in human cancers and normal cervix. In this study, we identified 562 differentially expressed proteins, and there were three proteins expressed higher in the cervical cancer tissues. The functions and signaling pathways of these differentially expressed proteins lay a theoretical foundation for elucidating the molecular mechanisms of cervical cancer.

The effect of synergistic Candida albicans infection with P63 protein expression on the development of oral carcinom

Oral cancer accounts for 2-4% of all cancer cases. There is compelling evidence that suggest that Candida may influence the development of oral cancer by interfering with the expression of the Tp63 gene, which is crucial for tumor progression, and promotes proliferation. The goal was to assess the p63 expression, compare it qualitatively and quantitatively in a normal buccal mucosa, epithelial dysplasia, leukoplakia, and oral squamous cell carcinoma (OSCC), and also determine how does it relate to the Candida albicans infection. A retrospective cross-sectional study of 100 Formalin-Fixed Paraffin-Embedded (FFPE) cases and controls were studied, and they were divided into four groups: Group I, included 40 controls with normal oral mucosa; Group II, included 20 cases of dysplasia; Group III, included 20 cases of leukoplakia, and Group IV, included 20 cases of OSCC. C. albicans was examined by Real-Time PCR based on Erg11 gene and the immunohistochemical expression of p63 protein. Significant overexpression of p63 and positive PCR results of C. albicans were seen in noninvasive lesions (leukoplakia, epithelial dysplasia) and invasive OSCCs with an increased suprabasal expression in cases of leukoplakia and epithelial dysplasia. The mean of labeling index (LI) of p63 with C. albicans positive cases were found to be in increasing order from normal oral mucosa than leukoplakia and dysplasia to OSCC. These findings point to p63 association with oral carcinogenesis and candidal infection, and they also imply that dysplastic and leukoplakia lesions may have the ability to serve as a signal for oral premalignancy due to increased LI and higher suprabasal expression of this gene. 
 KEY WORDS: Candida albicans; epithelial dysplasia; Erg11; leukoplakia; p63 protein.

Vocal Cord Leukoplakia Classification Using Siamese Network Under Small Samples of White Light Endoscopy Images.

Accurate vocal cord leukoplakia classification is instructive for clinical diagnosis and surgical treatment. This article introduces a reliable very deep Siamese network for accurate vocal cord leukoplakia classification. A study of a classification network based on a retrospective database. Academic university and hospital. The white light image datasets of vocal cord leukoplakia used in this article were classified into 6 classes: normal tissues, inflammatory keratosis, mild dysplasia, moderate dysplasia, severe dysplasia, and squamous cell carcinoma. The classification performance was assessed by comparing it with 6 classical deep learning models, including AlexNet, VGG Net, Google Inception, ResNet, DenseNet, and Vision Transformer. Experiments show the superior classification performance of our proposed network compared to state-of-the-art methods. The overall accuracy is 0.9756. The values of sensitivity and specificity are very high as well. The confusion matrix provides information for the 6-class classification task and demonstrates the superiority of our proposed network. Our very deep Siamese network can provide accurate classification results of vocal cord leukoplakia, which facilitates early detection, clinical diagnosis, and surgical treatment. The excellent performance obtained in white light images can reduce the cost for patients, especially those living in developing countries.

A Deep Learning Framework with an Intermediate Layer Using the Swarm Intelligence Optimizer for Diagnosing Oral Squamous Cell Carcinoma.

One of the most prevalent cancers is oral squamous cell carcinoma, and preventing mortality from this disease primarily depends on early detection. Clinicians will greatly benefit from automated diagnostic techniques that analyze a patient's histopathology images to identify abnormal oral lesions. A deep learning framework was designed with an intermediate layer between feature extraction layers and classification layers for classifying the histopathological images into two categories, namely, normal and oral squamous cell carcinoma. The intermediate layer is constructed using the proposed swarm intelligence technique called the Modified Gorilla Troops Optimizer. While there are many optimization algorithms used in the literature for feature selection, weight updating, and optimal parameter identification in deep learning models, this work focuses on using optimization algorithms as an intermediate layer to convert extracted features into features that are better suited for classification. Three datasets comprising 2784 normal and 3632 oral squamous cell carcinoma subjects are considered in this work. Three popular CNN architectures, namely, InceptionV2, MobileNetV3, and EfficientNetB3, are investigated as feature extraction layers. Two fully connected Neural Network layers, batch normalization, and dropout are used as classification layers. With the best accuracy of 0.89 among the examined feature extraction models, MobileNetV3 exhibits good performance. This accuracy is increased to 0.95 when the suggested Modified Gorilla Troops Optimizer is used as an intermediary layer.

Vocal cord leukoplakia classification using deep learning models in white light and narrow band imaging endoscopy images.

Accurate vocal cord leukoplakia classification is critical for the individualized treatment and early detection of laryngeal cancer. Numerous deep learning techniques have been proposed, but it is unclear how to select one to apply in the laryngeal tasks. This article introduces and reliably evaluates existing deep learning models for vocal cord leukoplakia classification. We created white light and narrow band imaging (NBI) image datasets of vocal cord leukoplakia which were classified into six classes: normal tissues (NT), inflammatory keratosis (IK), mild dysplasia (MiD), moderate dysplasia (MoD), severe dysplasia (SD), and squamous cell carcinoma (SCC). Vocal cord leukoplakia classification was performed using six classical deep learning models, AlexNet, VGG, Google Inception, ResNet, DenseNet, and Vision Transformer. GoogLeNet (i.e., Google Inception V1), DenseNet-121, and ResNet-152 perform excellent classification. The highest overall accuracy of white light image classification is 0.9583, while the highest overall accuracy of NBI image classification is 0.9478. These three neural networks all provide very high sensitivity, specificity, and precision values. GoogLeNet, ResNet, and DenseNet can provide accurate pathological classification of vocal cord leukoplakia. It facilitates early diagnosis, providing judgment on conservative treatment or surgical treatment of different degrees, and reducing the burden on endoscopists.

Jaw Bone Invasion of Oral Squamous Cell Carcinoma Is Associated with Osteoclast Count and Expression of Its Regulating Proteins in Patients and Organoids.

Oral squamous cell carcinoma (OSCC) frequently invades the jaw. The exact mechanism of bone invasion remains unclear. This study investigates (premature) osteoclasts and the expression of its differentiation regulating proteins RANKL, OPG and RANK in patients with OSCC. Resection specimens from OSCC patients were divided into NI group (No Invasion), E group (Erosion) or I group (bone Invasion). Tissue sections were stained with Cathepsin K (osteoclast-counting), RANKL, OPG and RANK. The staining intensity was scored on different regions of the tumor: front, center, back and normal mucosa. Immunohistochemistry and qPCR for RANKL/OPG/RANK were performed on five head and neck squamous cell carcinoma (HNSCC) organoids. The mean number of osteoclasts (I group) and premature osteoclasts (E group) was significantly higher compared to the NI group (p = 0.003, p = 0.036). RANKL expression was significantly higher in the tumor front and tumor center compared to normal mucosa (all groups). In the I group, RANKL and RANK expression was significantly higher in the tumor front compared to the tumor back and there was a trend of higher RANKL expression in the tumor front compared to the E group and NI group. qPCR showed a 20-43 times higher RANKL mRNA expression in three out of five tumor organoids compared to a normal squamous cell organoid line. There was no correlation between protein and mRNA expression in the HNSCC organoids. These findings suggest that OSCCs induce bone invasion by stimulating osteoclast activation by regulating the production of RANKL and RANK proteins.

Improving Mobile-Based Cervical Cytology Screening: A Deep Learning Nucleus-Based Approach for Lesion Detection

Liquid-based cytology (LBC) plays a crucial role in the effective early detection of cervical cancer, contributing to substantially decreasing mortality rates. However, the visual examination of microscopic slides is a challenging, time-consuming, and ambiguous task. Shortages of specialized staff and equipment are increasing the interest in developing artificial intelligence (AI)-powered portable solutions to support screening programs. This paper presents a novel approach based on a RetinaNet model with a ResNet50 backbone to detect the nuclei of cervical lesions on mobile-acquired microscopic images of cytology samples, stratifying the lesions according to The Bethesda System (TBS) guidelines. This work was supported by a new dataset of images from LBC samples digitalized with a portable smartphone-based microscope, encompassing nucleus annotations of 31,698 normal squamous cells and 1395 lesions. Several experiments were conducted to optimize the model’s detection performance, namely hyperparameter tuning, transfer learning, detected class adjustments, and per-class score threshold optimization. The proposed nucleus-based methodology improved the best baseline reported in the literature for detecting cervical lesions on microscopic images exclusively acquired with mobile devices coupled to the µSmartScope prototype, with per-class average precision, recall, and F1 scores up to 17.6%, 22.9%, and 16.0%, respectively. Performance improvements were obtained by transferring knowledge from networks pre-trained on a smaller dataset closer to the target application domain, as well as including normal squamous nuclei as a class detected by the model. Per-class tuning of the score threshold also allowed us to obtain a model more suitable to support screening procedures, achieving F1 score improvements in most TBS classes. While further improvements are still required to use the proposed approach in a clinical context, this work reinforces the potential of using AI-powered mobile-based solutions to support cervical cancer screening. Such solutions can significantly impact screening programs worldwide, particularly in areas with limited access and restricted healthcare resources.

A Comparative Study of Morphometric Analysis of Nucleolar Organizer Regions in Oral Leukoplakia and Oral Squamous Cell Carcinoma and Significance of AgNOR as a Diagnostic Tool.

Background Silver stainable nucleolar organizer regions (AgNORs) have proven to exhibit utmost importance due to their higher occurrence in the nucleus especially in malignant cells than in normal. Thus, they assist in the examination of nucleolar structures and variations in nucleolar activity. Aim Quantitative and qualitative analysis in relation to the number and area of AgNOR in tissue sections of the normal oral mucosa (NOM), oral leukoplakia (OL), and oral squamous cell carcinoma (OSCC) was the main aim of the study. Materials & method A total of 50 cases comprising 20 OL with dysplasia, 20 OSCC cases, and 10 samples of normal oral mucosa were taken. Silver nitrate (Sol A) & gelatin (Sol B) solutions were freshly prepared for staining the lesional slides. Results The mean value of nuclear profile area (A Nuc) was comparatively higher in oral leukoplakia i.e. 41.97 and in oral squamous cell carcinoma i.e. 62.36 in comparison to the control group where it was 36.19. The mean value of a single AgNOR profile area per nucleus (A NOR) was found to be comparatively lower in both study groups i.e. oral leukoplakia (2.76) and oral squamous cell carcinoma (1.61) in comparison to the control group (3.45) . The mean value of total AgNOR profile area per nucleus (TA NOR) and the number of profiles of AgNORs per nucleus (n NOR) were found higher in both study groups (oral leukoplakia and oral squamous cell carcinoma) as compared to normal oral mucosa of the control group. However, the findings of all four parameters of morphometric analysis were found to be significantly associated with disorder of oral mucosa i.e. cases of oral leukoplakia and oral squamous cell carcinoma (P value<0.01). Conclusion It can thus be suggested that the mean AgNOR count displayed a higher value in OSCC. Hence, the number of AgNORs in nuclei increases as epithelial cells undergo malignant transformation which is designated that mean AgNOR count may contribute to establishing the prognosis of a lesion.

Molecular biomarkers of progression from Barrett’s esophagus to esophageal adenocarcinoma

IntroductionBarrett’s esophagus (BO) is a pre-malignant condition for esophageal adenocarcinoma (OAC), the incidence rate of which has risen dramatically over the last four decades in the Western world. The 5-year survival rate of OAC is poor, and one of the ways to improve it would be by focusing on identifying high-risk Barrett’s patients through a surveillance program. Currently, histologic dysplasia is the only recognized marker of progression to OAC. Molecular biomarkers found in tissue samples that predict which patients have a higher risk of progression to OAC may act as a reliable tool for the stratification of patients with BO.AimTo determine whether molecular biomarkers have a potential use in predicting which patients with BO have a higher risk of progression to OAC.MethodsImmunohistochemistry was performed on 25 tissue samples obtained from the endoscopic biopsies of 19 patients with confirmed BO. Hematoxylin and eosin (H&amp;amp;E) staining was used to confirm the presence of BO and dysplasia. Staining was performed in an external independent laboratory. Statistical analysis using the Mann–Whitney U test was performed using R Studio® statistical software.ResultsOf the 19 patients sampled, three had low-grade dysplasia (LGD), and all had confirmed metaplasia diagnostic of BO. Expression of cyclin D1 was noted to be elevated in patients with LGD compared with those with metaplasia only (p = 0.042). Expression of Sox2 was elevated in metaplastic BO cells compared with normal squamous cells within the same stain (p = 0.046). Of all eight biomarkers tested, β-catenin had the greatest overall expression (p &amp;lt; 0.004).ConclusionsIsolating elevated cyclin D1 in patients with LGD highlights its potential use as a biomarker in identifying BO patients at risk of developing dysplasia, and, in turn, their possible progression to OAC. Elevated levels of both Sox2 and β-catenin may also serve as markers for disease progression when overexpressed in BO patients. Both conclusions, however, would need long-term follow-up to fully establish their prognostic usefulness, as at the time of writing no patients in this study had gone on to develop OAC. Although only a small sample size was present for this study, and follow-up was limited, it serves as a strong pilot for further research into the use of novel biomarkers in predicting which BO patients are at high risk of developing dysplasia and progressing to OAC.

KLF5 and p53 comprise an incoherent feed-forward loop directing cell-fate decisions following stress

In response to stress, cells make a critical decision to arrest or undergo apoptosis, mediated in large part by the tumor suppressor p53. Yet the mechanisms of these cell fate decisions remain largely unknown, particularly in normal cells. Here, we define an incoherent feed-forward loop in non-transformed human squamous epithelial cells involving p53 and the zinc-finger transcription factor KLF5 that dictates responses to differing levels of cellular stress from UV irradiation or oxidative stress. In normal unstressed human squamous epithelial cells, KLF5 complexes with SIN3A and HDAC2 repress TP53, allowing cells to proliferate. With moderate stress, this complex is disrupted, and TP53 is induced; KLF5 then acts as a molecular switch for p53 function by transactivating AKT1 and AKT3, which direct cells toward survival. By contrast, severe stress results in KLF5 loss, such that AKT1 and AKT3 are not induced, and cells preferentially undergo apoptosis. Thus, in human squamous epithelial cells, KLF5 gates the response to UV or oxidative stress to determine the p53 output of growth arrest or apoptosis.

Ginsenoside Rh2 attenuates the progression of non-small cell lung cancer by sponging miR-28-5p/STK4 axis and inactivating Wnt/β-catenin signaling.

Ginsenoside Rh2 (G-Rh2) exerts anti-tumor activity in non-small cell lung cancer (NSCLC). microRNAs (miRNAs, miRs) play pivotal roles in NSCLC. We aimed to investigate whether G-Rh2 inhibited NSCLC progression by targeting miRNA. Cell viability, apoptosis and cycle were determined by Cell Counting Kit-8, 6-diamidino-2-phenylindole (DAPI) staining and flow cytometry. The potential target miRNAs of G-Rh2 were screened by real-time quantitative polymerase chain reaction (RT-qPCR). The difference in miR-28-5p expression between lung adenocarcinoma (LUAD) tissues and normal tissues or lung squamous cell carcinoma (LUSC) tissues and normal tissues was retrieved from TCGA-LUAD and TCGA-LUSC, respectively. Kaplan-Meier Plotter was conducted to analyze the survival rate for different serine/threonine-protein kinase 4 (STK4) expressions with different prognostic risks. immunohistochemistry of STK4 expression in non-tumor and tumor tissues was analyzed from the HPA database. RT-qPCR and Western blot were adopted for detecting mRNA and protein expression. TargetScan V7.2, miRanda and PITA were adopted for predicting targets of miR-28-5p, overlapped genes were subjected to GO analysis. The interactions of miR-28-5p-Wnt and miR-28-5p-STK4 were detected by TOP/FOP luciferase reporter assay and dual luciferase reporter assay, respectively. Current study observed that G-Rh2 reduced miR-28-5p expression in NSCLC cells dose-dependently. miR-28-5p was upregulated in NSCLC tissues and cells. The target genes of miR-28-5p were enriched in negative regulation of Wnt signaling. miR-28-5p inhibitor inactivated Wnt signaling, inhibited cell viability and cell cycle, while enhanced cell apoptosis of NSCLC cells by targeting STK4. G-Rh2 exerted the similar effects with miR-28-5p inhibitor by reducing miR-28-5p. G-Rh2 and miR-28-5p inhibitor exerted a synergistic effect on inhibiting NSCLC tumor growth. In conclusion, G-Rh2 attenuates NSCLC development by affecting miR-28-5p/STK4 axis and inactivating Wnt signaling. Taken together, we project out a novel therapeutic target for NSCLC.