Cytokeratin 18 & 19 Expression in Normal Mucosa, Lichen Planus and Squamous Cell Carcinoma

Citokeratini (CK) strukturalni su proteini u epitelnim stanicama. Zapažene su promjene u njihovoj ekspresiji tijekom neoplastične transformacije. Svrha: procjena ekspresije citokeratina 18 i 19 u normalnoj sluznici, lichen planusu i karcinomu pločastih stanica. Trideset slučajeva svake od navedenih - normalne sluznice, lichen planusa i karcinomi pločastih stanica - uzeti su iz arhiva Zavoda za oralnu dijagnozu Sveučilišta Khona Kaena. Preparati tkiva obojeni su protutijelima na citokeratin 18 i 19. Obavljena je i evaluacija imunoreaktivnosti prebrojavanjem stanica. Oznaka za obilježavanje CK-a izračunate su dijeljenjem broja pozitivno obojenih stanica x 100 s ukupnim zbrojem izbrojenih stanica. Devet od 30 (30,0%) normalnih sluznica, 5 od 30 (16,7%) lichen planusa i 4 od 30 (13,3%) karcinoma pločastih stanica pokazivali su pozitivno bojenje na CK18. Karcinom pločastih stanica pokazivao je najvišu srednju vrijednost indeksa označavanja CK18 te su zatim u silaznom redoslijedu slijedile normalna sluznica i lichen planus. Dvadeset i tri od 30 (76,7%) normalnih sluznica, 9 od 30 (30,0%) lichen planusa i 1 od 30 (3,33%) karcinoma pločastih stanica postigli su pozitivno bojenje na CK19. Normalna je sluznica pokazala najvišu srednju vrijednost indeksa označavanja sa CK19 te su u silaznom redoslijedu slijedili lichen planus i karcinom pločastih stanica.Ekspresija CK18 u normalnoj sluznici, lichen planusu i karcinomu pločastih stanica niska je i nema statističkih razlika među trima vrstama lezija. Ekspresija CK19 u normalnoj sluznici statistički je viša od one u lichen planusu te karcinomu pločastih stanica. CK19 mogao bi biti dobar za uključivanje u skupinu protutijela za detekciju malignih transformacija oralne sluznice u karcinom pločastih stanica.Cytokeratins (CK) are structural proteins in epithelial cells. Changes in cytokeratin expression have been observed during neoplastic transformation. Objectives: To evaluate cytokeratin 18 and 19 expression in normal mucosa, lichen planus and squamous cell carcinoma. Materials and methods: Thirty cases each of normal mucosa, lichen planus and squamous cell carcinoma were retrieved from the archives of the Department of Oral Diagnosis, Khon Kaen University. Tissue sections were stained with antibodies to cytokeratin 18 and 19. Immunoreactivity was evaluated by counting cells. The CK labeling indeks was calculated by dividing the number of positively stained cells x 100 by total number of cells counted. Results: 9 out of 30 (30.0%) normal mucosa, 5 out of 30 (16.7%) lichen planus and 4 out of 30 (13.3%) squamous cell carcinoma demonstrated positive staining for CK18. Squamous cell carcinoma showed the highest mean CK18 labeling index, followed in descending order by normal mucosa and lichen planus, respectively. Twenty three out of 30 (76.7%) normal mucosa, 9 out of 30 (30.0%) lichen planus and 1 out of 30 (3.33%) squamous cell carcinoma elicited positive staining for CK19. Normal mucosa elicited the highest mean CK19 labeling index, followed in descending order by lichen planus and squamous cell carcinoma, respectively. Conclusions: CK18 expression in normal mucosa, lichen planus and squamous cell carcinoma is low and there is no statistical difference among the three groups of lesions. CK 19 expression in normal mucosa is statistically higher than that of lichen planus and squamous cell carcinoma. CK19 might be a good candidate to be included in a panel of antibodies for detecting malignant transformation of oral mucosa into squamous cell carcinoma

Serum cell-free DNA methylation of OPCML and HOXD9 as a biomarker that may aid in differential diagnosis between cholangiocarcinoma and other biliary diseases

Abstract Background Cholangiocarcinoma (CCA) is a fatal cancer of the bile duct epithelial cell lining. The misdiagnosis of CCA and other biliary diseases may occur due to the similarity of clinical manifestations and blood tests resulting in inappropriate or delayed treatment. Thus, an accurate and less-invasive method for differentiating CCA from other biliary diseases is inevitable. Methods We quantified methylation of OPCML, HOXA9, and HOXD9 in serum cell-free DNA (cfDNA) of CCA patients and other biliary diseases using methylation-sensitive high-resolution melting (MS-HRM). Their potency as differential biomarkers between CCA and other biliary diseases was also evaluated by using receiver operating characteristic (ROC) curves. Results The significant difference of methylation levels of OPCML and HOXD9 was observed in serum cfDNA of CCA compared to other biliary diseases. Assessment of serum cfDNA methylation of OPCML and HOXD9 as differential biomarkers of CCA and other biliary diseases showed the area under curve (AUC) of 0.850 (0.759–0.941) for OPCML which sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were 80.00%, 90.00%, 88.88%, 81.81%, and 85.00%, respectively. The AUC of HOXD9 was 0.789 (0.686–0.892) with sensitivity, specificity, PPV, NPV, and accuracy of 67.50%, 90.00%, 87.09%, 73.46%, and 78.75%, respectively. The combined marker between OPCML and HOXD9 showed sensitivity, specificity, PPV, and NPV of 62.50%, 100%, 100%, and 72.72%, respectively, which may be helpful to prevent a misdiagnosis between CCA and other biliary diseases. Conclusions Our findings suggest the application of serum cfDNA methylation of OPCML and HOXD9 for differential diagnosis of CCA and other biliary diseases due to its less invasiveness and clinically practical method which may benefit the patients by preventing the misdiagnosis of CCA and avoiding unnecessary surgical intervention

The effects of arecoline on cell viability and cell-cycle progression.

<p>Cytotoxicity (A and B) and cell proliferation (C and D) were determined in arecoline-untreated or treated OSCC cell lines at various concentrations for 24 hours using the MTT assay. Statistical significance of the differences of cell viability (%) was analyzed using One-way ANOVA followed by Tukey’s multiple comparison test (*<i>P</i> < 0.05, **<i>P</i> < 0.01 and ***<i>P</i> < 0.001). Cell-cycle phase distribution (E and F) in ORL-48(T) cells treated with 0 and 0.025 μg/ml of arecoline in synchronized condition was analyzed by flow cytometry. The percentages of G0/G1, S and G2/M population (G) of arecoline-treated cells were compared to untreated ORL-48(T) cells as control. Statistical significance of the differences of G2/M population was analyzed using Paired <i>t</i>-test (*<i>P</i> < 0.05).</p

Effects of arecoline on proliferation of oral squamous cell carcinoma cells by dysregulating c-Myc and miR-22, directly targeting oncostatin M

<div><p>Arecoline, the major alkaloid of areca nut, is known to induce oral carcinogenesis, however, its mechanism is still needed to elucidate. This study investigated the effects of arecoline on cell viability and cell-cycle progression of oral squamous cell carcinoma (OSCC) cells as well as a relevant cellular gene expression. The results showed that a low concentration of arecoline (0.025 μg/ml) increased OSCC cell viability, proportion of cells in G2/M phase and cell proliferation. Simultaneously, it induced IL-6, STAT3 and c-Myc expression. Interestingly, c-<i>myc</i> promoter activity was also induced by arecoline. MiR-22 expression in arecoline-treated OSCC cells was suppressed and comparable to an upregulated c-Myc expression. In arecoline-treated OSCC cells, oncostatin M (OSM) expression was significantly upregulated and inversely correlated with miR-22 expression. Likewise, OSM expression and its post-transcriptional activity were significantly decreased in miR-22-transfected OSCC and 293FT cells. This result demonstrated that miR-22 directly targeted OSM. Interestingly, miR-22 played an important role as a tumor suppresser on suppressing cell proliferation, migration and cell-cycle progression of OSCC cells. This result suggested the effect of arecoline to promote cell proliferation and cell-cycle progression of OSCC cells might be involved in induction of c-Myc expression and reduction of miR-22 resulting in OSM upregulation.</p></div

Relative expression levels of miR-22 and OSM.

<p>100 and 500 ng/well of mock control (pIRES2-EGFP vector) and pIRES-miR-22 vectors were transfected into ORL-48(T) and ORL-136(T) cells. At 24 and 48 hours post-transfection, pri-miR-22 (A-B) and OSM (C-D) expression was determined by RT-PCR. Protein levels of OSM (E) were determined in ORL-48(T) and ORL-136(T) cells at 48 hours after transfection with 100 ng of mock control and pIRES-miR-22 vectors. Relative intensity of OSM protein band (F-G) was calculated using ImageJ 1.49v software. Statistical significance of the differences was analyzed using One-way ANOVA followed by Tukey’s multiple comparison test (*<i>P</i> < 0.05, **<i>P</i> < 0.01 and ***<i>P</i> < 0.001).</p

Effect of arecoline on IL-6 and STAT3 in ORL-48(T) and ORL-136(T) cells.

<p>ORL-48(T) and ORL-136(T) cells were treated with 0, 0.025 and 25 μg/ml arecoline for 24 hours. Expression of IL-6 (A and D) and STAT3 (B and E) were investigated by RT-PCR and their amplicons were visualized by 2% agarose gel electrophoresis (C and F). Statistical significance of the differences of relative expression was analyzed using One-way ANOVA followed by Tukey’s multiple comparison test (*<i>P</i> < 0.05 and **<i>P</i> < 0.01).</p

MiR-22 targets OSM and miR-22 functions in cell proliferation, migration and cell-cycle assay.

<p>The construct of the miR-22 targets sequence within the OSM 3′UTR WT and Mut in pGL3-Control vector. The luciferase gene was linked to the 3′UTR WT and Mut of OSM. 293FT cells were co-transfected with 250 ng pIRES-miR-22 and 100 ng pGL3-OSM 3′UTR WT or Mut vectors (A). The normalized luciferase activity in pIRES-miR-22 and pGL3-OSM 3′UTR WT or Mut co-transfected cells was relative to normalized luciferase activity of pIRES2-EGFP and OSM 3′UTR WT or Mut co-transfected cells (B). A green fluorescence expression vector (pEGFP-N3) was transfected for monitoring transfection efficiency. Statistical significance of the differences of luciferase activity was analyzed using Two-way ANOVA (*<i>P</i> < 0.05). Cell proliferation and migration in pIRES-miR-22-transfected ORL-48(T) cells were measured by a hemocytometer and wound healing assay at different incubation time points (C-E). The photograph was taken under 4X objective lens NIS-Elements Advanced Research Imaging Software version 3.0. Statistical significance of the differences of cell viability and wound closure was analyzed using Student's <i>t</i>-test (*<i>P</i> < 0.05 and ***<i>P</i> < 0.001). Cell-cycle assay in miR-22 or mock-transfected ORL-48(T) for 48 hours post-transfection was performed by flow cytometry (F). Statistical significance of the differences of G2/M and G0/G1 population was analyzed using Paired <i>t</i>-test (*<i>P</i> < 0.05 and (**<i>P</i> < 0.01, respectively).</p

Primer sequences.

<p>Primer sequences.</p