Epstein-Barr virus and oral squamous cell carcinoma in patients without HIV infection: viral detection by polymerase chain reaction.

In order to test the hypothesis that Epstein-Barr virus (EBV) may be a cofactor for oral squamous cell carcinoma (OSCC) the authors evaluated tumour cells from OSCC of 108 patients without HIV infection, for the presence of EBV DNA by polymerase chain reaction. The sequences of oligonucleotides used in the amplification and hybridization included a set for the DNA polymerase region. The amplification was detected using an ELISA assay with peroxidase. EBV DNA was detected in 17.59% of the tumours. Inhibition studies showed that the ability to detect EBV DNA was not affected by the pathological material, suggesting that the negative PCR results in these samples were not caused by PCR inhibitors in the biopsy. Results revealed that 63.1% of the tumours (12 cases) were DNA positive affecting the lateral margin of the tongue, and were statistically significant (p < 0.001; chi 2). In the pool of tumours with EBV DNA only 26.3% (5 of 19 cases) were well differentiated OSCCs whereas the remaining 73.7% (14 of 19 cases) were moderately and poorly differentiated OSCCs, with a statistical significance of p = 0.08; chi 2. This study suggests a relationship between OSCC and EBV.     

Expression of obestatin and ghrelin in papillary thyroid carcinoma

Ghrelin and obestatin are two peptide hormones with opposing roles in the control of appetite: orexigenic and anorexigenic, respectively. Loss of appetite is a common, serious complication of many forms of malignancy. The goals of this study were to investigate: (i) whether there are differences in ghrelin and obestatin peptide expression in thyroid tissues from a series of papillary carcinoma cases and normal controls, and (ii) whether there are correlations between tissue ghrelin and obestatin levels in series of papillary carcinoma cases and normal controls. Immunohistochemical analysis showed that in sections of benign human thyroid tissue, anti-ghrelin antibody reacted with intense staining in colloid-filled follicles. In benign thyroid tissues, colloids displayed plentiful dispersion in comparison with papillary microcarcinomas, whereas colloids in malignant thyroid tissues were uncommon. We found markedly lower tissue ghrelin levels in thyroid tissue of patients with papillary carcinomas, compared with normal thyroid tissues (P = 0.001). Immunohistochemical analysis also showed that obestatin in papillary carcinoma stained positively to various degrees. Obestatin tissue levels in papillary carcinomas tended to be slightly higher than those in normal thyroid tissue, but this was not statistically significant (P = 0.29). We also report that thyroid tissue of patients with Hashimoto’s thyroiditis produced ghrelin and obestatin at similar levels as in normal thyroid tissue, even though colloid in Hashimoto’s disease is scarce. We conclude that depressed expression of ghrelin, but not obestatin, is specific to papillary carcinoma, and this difference might constitute a diagnostic tool to differentiate papillary carcinoma from normal thyroid tissue. We currently do not know how these peptides are regulated and what factors are involved in papillary carcinoma, which inhibit the expression of ghrelin but not obestatin. This issue warrants further studies.     

Survivin phosphorylation and M-phase promoting factor in oral carcinogenesis

Survivin is a recently described inhibitor of apoptosis and mitotic regulator which is selectively over-expressed in human tumors. Its expression rate is predictive of disease progression, early recurrences and resistance to therapy. Up-regulation of survivin in oral pre-malignant lesions (OPL) and in oral squamous cell carcinoma (OSCC) has already been demonstrated in previous studies. A critical step for activation of survivin has been identified in the phosphorylation on Thr34 by the main mitotic kinase p34cdc2-cyclin B1. The aim of this work was to investigate the relationship between survivin, its phosphorylated active form (p-survivin) and M-phase promoting factor (MPF), p34cdc2-cyclin B1 in oral carcinogenesis. 32 OSCCs and 17 OPLs from surgical specimens were studied for cyclin B1, p-survivin, survivin, and p34cdc2 expression by immunohistochemistry. All cases of OSCC expressed survivin and its expression rate was correlated to p-survivin levels (P<0.05). Cyclin B1 was positive in 80% of cases, while p-34cdc2 was over-expressed in all OSCCs. All OPLs associated with OSCC expressed survivin and its levels were correlated to p-survivin levels (P<0.05). Cyclin B1 was positive in 70% of cases, while p-34cdc2 was positive in all OPLs. In conclusion, this study demonstrated that MPF, survivin and p-survivin are expressed during early and late phase of oral carcinogenesis. MPF proteins, which are co-expressed on mitotic apparatus, could represent a potential target for therapies based on manipulation of survivin phosphorylation, which would induce apoptosis in cancer cells.     

MiR-34a inhibits the proliferation,migration, and invasion of oral squamous cell carcinoma by directly targeting SATB2

In various kinds of carcinomas, the special AT-rich sequence-binding protein 2 (SATB2) with its atypical expression promotes the metastasis and progression of the tumor, though in the oral squamous cell carcinoma (OSCC) its inherent mechanism and the status of SATB2 remain unclear. The role played by the SATB2 expression in the OSCC cell lines and tissue samples in the target of miR-34a downstream is the intended endeavor of this study. In te OSCCs the miR-34a expression was determined by quantitative real-time polymerase chain reaction (q-PCR), while the SATB2 expression in the cell lines and tissue samples in OSCC was analyzed with the q-PCR and the western blot. Studies in both in vitro and in vivo of the effects of miR-34a on the initiation of OSCC were conducted. As a direct target of the miR-34a the SATB2 was verified with the luciferase reporter assay. In cases where the miR-34a levels were low, the SATB2 in OSCCs seemed to be overexpressed. Besides, both in the in vitro and in vivo a suppression of migration, invasion, and cell growth was caused by miR-34a by down regulating the SATB2 expression. The SATB2 being a direct target of miR-34a was confirmed by the cotransfection of miR-34a mimics specifically the decrease in the expression of luciferase of SATB2–3′UTR-wt reporter. As a whole, our study confirmed the inhibition of miR-34a in the invasion, proliferation, and migration of the OSCCs, playing a potential tumor suppressor role with SATB2 as its downstream target.     

微小RNAmiR-21和miR-31在13腔鳞癌组织中的表达及意义

目的：检测口腔鳞癌中微小RNA miR-21和miR-31的表达,探讨其与肿瘤发展的关系。方法：应用实时荧光定量PCR的方法检测72例口腔鳞癌,38例正常口腔粘膜miR-21和miR-31的表达,统计学分析其表达与肿瘤临床分期和病理分型的关系。结果：①口腔鳞癌组织与对照组比较,微小RNA miR-21和miR-31的表达都有显著增高（P〈0.05）,其中miR-21的增高更为显著（P〈0.001）。②统计学分析表明,miR-21的表达在晚期鳞癌组织较早中期鳞癌组织增高更为显著（P〈0.01）,在低分化鳞癌组织较中、高分化鳞癌组织增高更为显著（p〈0.001）;MiR-31的表达水平在不同肿瘤临床分期和病理分型鳞癌组织中无明显差异（P〉0.05）。结论：miR-21和miR-31在口腔鳞癌发生发展过程中表达有明显增高,其中miR-21表达水平可作为潜在的口腔鳞癌临床分期分级和预后的指标。     

Downregulation of FAP suppresses cell proliferation and metastasis through PTEN/PI3K/AKT and Ras-ERK signaling in oral squamous cell carcinoma

It is largely recognized that fibroblast activation protein (FAP) is expressed in cancer-associated fibroblasts (CAFs) of many human carcinomas. Furthermore, FAP was recently also reported to be expressed in carcinoma cells of the breast, stomach, pancreatic ductal adenocarcinoma, colorectum, and uterine cervix. The carcinoma cell expression pattern of FAP has been described in several types of cancers, but the role of FAP in oral squamous cell carcinoma (OSCC) is unknown. The role of endogenous FAP in epithelium-derived tumors and molecular mechanisms has also not been reported. In this study, FAP was found to be expressed in carcinoma cells of OSCC and was upregulated in OSCC tissue samples compared with benign tissue samples using immunohistochemistry. In addition, its expression level was closely correlated with overall survival of patients with OSCC. Silencing FAP inhibited the growth and metastasis of OSCC cells in vitro and in vivo. Mechanistically, knockdown of FAP inactivated PTEN/PI3K/AKT and Ras-ERK and its downstream signaling regulating proliferation, migration, and invasion in OSCC cells, as the inhibitory effects of FAP on the proliferation and metastasis could be rescued by PTEN silencing. Our study suggests that FAP acts as an oncogene and may be a potential therapeutic target for patients with OSCC.     

NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

Recent studies have shown that Notch signaling is involved in many types of cancers, including oral squamous cell carcinomas (OSCCs). However, the role of Notch signaling in the tumor microenvironment is not yet fully understood. In this study, we investigated the roles of NOTCH3 signaling in cancer associated fibroblasts (CAFs) in OSCCs. Immunohistochemical study of 93 human tongue OSCC cases indicated that about one third of OSCCs showed NOTCH3 expression in CAFs, and that this expression significantly correlated with tumor-size. In vitro study showed that OSCC cell lines, especially HO1-N-1 cells stimulated NOTCH3 expression in normal human dermal fibroblasts (NHDFs) through direct cell-to-cell contact. Immunohistochemical and morphometric analysis using human OSCC samples demonstrated that NOTCH3 expression in CAFs significantly correlated with micro-vessel density in cancer stroma. In vitro angiogenesis assays involving co-culture of NHDFs with HO1-N-1 and human umbilical endothelial cells (HUVECs), and NOTCH3 knockdown in NHDFs using siRNA, demonstrated that HO1-N-1 cells significantly promoted tube formation dependent on NOTCH3-expression in NHDFs. Moreover, NOTCH3 expression in CAFs was related to poor prognosis of the OSCC patients. This work provides a new insight into the role of Notch signaling in CAFs associated with tumor angiogenesis and the possibility of NOTCH3-targeted molecular therapy in OSCCs.     

Oral epithelial stem cells—Implications in normal development and cancer metastasis

Oral mucosa is continuously exposed to environmental forces and has to be constantly renewed. Accordingly, the oral mucosa epithelium contains a large reservoir of epithelial stem cells necessary for tissue homeostasis. Despite considerable scientific advances in stem cell behavior in a number of tissues, fewer studies have been devoted to the stem cells in the oral epithelium. Most of oral mucosa stem cells studies are focused on identifying cancer stem cells (CSC) in oral squamous cell carcinomas (OSCCs) among other head and neck cancers. OSCCs are the most prevalent epithelial tumors of the head and neck region, marked by their aggressiveness and invasiveness. Due to their highly tumorigenic properties, it has been suggested that CSC may be the critical population of cancer cells in the development of OSCC metastasis. This review presents a brief overview of epithelium stem cells with implications in oral health, and the clinical implications of the CSC concept in OSCC metastatic dissemination.     

Nicotinamide N-methyltransferase upregulation inversely correlates with lymph node metastasis in oral squamous cell carcinoma

We investigated expression levels of Nicotinamide N-Methyltransferase (NNMT), an enzyme involved in the biotransformation of many drugs and xenobiotic compounds, in oral squamous cell carcinoma (OSCC). Measurements were performed by semi-quantitative RT-PCR and quantitative real-time PCR in tumor and matched adjacent healthy tissue. Interestingly, NNMT was up-regulated in most of the favorable OSCCs, while no marked NNMT expression alterations between tumor and normal mucosa were detected in most of the unfavorable OSCCs. Western blot and immunohistochemical analyses also were performed and the relationship between tumor characteristics and NNMT levels in OSCC were studied to evaluate the effectiveness of NNMT as a prognostic marker in the squamous cell carcinoma of the oral cavity. In summary, the present study suggests that NNMT may have potential as a biomarker and a therapeutic target for OSCC.     

Ghrelin and obestatin can promote human ovarian granulosa cell functions and FSH effects

The aim of the present in-vitro experiments was to examine the direct influence of ghrelin and obestatin on viability, proliferation and progesterone release by human ovarian granulosa cells and their response to FSH administration. Human granulosa cells were cultured in presence of ghrelin or obestatin (both at 0, 1, 10 or 100 ng/ml) alone or in the presence of FSH (10 ng/ml). Cell viability, accumulation of proliferation markers PCNA and cyclin B1 and release of progesterone were analyzed by Trypan blue extrusion test, quantitative immunocytochemistry and ELISA. Ghrelin, obestatin and FSH up-regulated all the measured ovarian cell parameters. Moreover, both ghrelin and obestatin promoted all the stimulatory effects of FSH. The obtained results demonstrate the direct stimulatory action of ghrelin, obestatin and FSH on basic ovarian cell functions, as well as the ability of metabolic hormones to improve FSH action on human ovarian cells.     

Study of feline oral squamous cell carcinoma: potential target for cyclooxygenase inhibitor treatment

Oral squamous cell carcinoma (OSCC) is associated with high morbidity and mortality. A potential target for OSCC treatment is cyclooxygenase-2 (cox-2). Pet cats with naturally occurring OSCC may offer the opportunity to study anticancer activity of cox inhibitors. Cox-2 expression in feline OSCC was determined by immunohistochemistry. High intensity cox-2 immunoreactivity was detected in 6 of 34 (18%) feline OSCC samples. Weak immunoreactivity was noted in 22 OSCCs and in epithelial cells from oral mucosa of clinically normal cats. Pharmacokinetics of a cox inhibitor (piroxicam, 0.3 mg/kg) were studied in carcinoma-bearing cats to confirm a dose for follow-up trials. The average peak serum piroxicam concentration (948 ng/ml, which inhibited cox-2 activity) and serum half-life (15.9 h) were similar to that in normal cats. These results provide information (cox-2 expression as an inclusion criteria, 0.3 mg/kg daily piroxicam) for the design of follow-up trials of cox inhibitor treatment in pet cats with OSCC.     

Obestatin is present in saliva: alterations in obestatin and ghrelin levels of saliva and serum in ischemic heart disease

Ghrelin and obestatin are a single gene products and are a multiple functional peptides that regulates energy homeostasis, and food intake. In the present work, we studied the secretion of ghrelin and its co-secreted peptide obestatin in 44 patients with ischemic heart disease with that of 27 healthy matched controls. Here we first conducted using an immunohistochemistry assay to screen whether human salivary glands have any obestatin immunoreactivity. Then, serum and saliva obestatin and acylated ghrelin levels were determined by using Radioimmunoassay. Our immunohistochemical analysis demonstrated that obestatin was localized in the striated and excretory duct of human salivary gland. We also report for the first time that obestatin, like ghrelin, is present in human salivary gland and saliva. No evidence of the role of obestatin or ghrelin saliva levels in the context of ischemic heart disease was found. Salivary ghrelin and obestatin levels are correlated in controls with the blood levels. Determination of salivary values could represent a non-invasive alternative to serum ones that can be useful in clinical practice.     

Distribution of obestatin and ghrelin in human tissues: immunoreactive cells in the gastrointestinal tract, pancreas, and mammary glands.

Obestatin and ghrelin are two peptides derived from the same prohormone. It is well established that ghrelin is produced by endocrine cells in the gastric mucosa. However, the distribution of human obestatin immunoreactive cells is not thoroughly characterized. A polyclonal antibody that specifically recognizes human obestatin was produced. Using this antibody and a commercial antibody vs ghrelin, the distribution of obestatin and ghrelin immunoreactive cells was determined in a panel of human tissues using immunohistochemistry. The two peptides were detected in the mucosa of the gastrointestinal tract, from cardia to ileum, and in the pancreatic islets. Interestingly, epithelial cells in the ducts of mammary glands showed distinct immunoreactivity for both ghrelin and obestatin. By double immunofluorescence microscopy, it was shown that all detected cells were immunoreactive for both peptides. Furthermore, the subcellular localization of obestatin and ghrelin was essentially identical, indicating that obestatin and ghrelin are stored in the same secretory vesicles.     

Computer-assisted analysis of p53 and PCNA expression in oral lesions infected with human papillomavirus

OBJECTIVE: To carry out a retrospective study to determine whether human papillomavirus (HPV) infection and immunohistochemical expression of p53 and proliferating cell nuclear antigen (PCNA) are related to the risk of oral cancer. STUDY DESIGN: Fifty-seven oral biopsies, consisting of 30 oral squamous papillomas (OSPs) and 27 oral squamous cell carcinomas (OSCCs) were tested for the presence of HPV 6/11 and 16/18 by in situ hybridization using catalyzed signal amplification and in situ hybridization. p53 And PCNA expression was analyzed by immunohistochemistry and evaluated quantitatively by image analysis. RESULTS: Nineteen of the 57 oral lesions (33.3%) were positive for HPV. HPV 6/11 was found in 6 of 30 (20%) OSPs and 1 of 27 (3.7%) OSCCs. HPV 16/18 was found in 10 of 27 (37%) OSCCs and 2 of 30 (6.7%) OSPs. Sixteen of the 19 HPV-positive cases (84.2%) were p53 negative; 5 (9%) were HPV 6/11 and 11 (19%) HPV 16/18, with an inverse correlation between the presence of HPV DNA and p53 expression (P = .017, P < .05). PCNA expression appeared in 18 (94.7%) of HPV positive cases, showing that HPV 16/18 was associated with intensity of PCNA expression and with OSCCs (P = .037, P < .05). CONCLUSION: Quantitative evaluation of p53 by image analysis showed an inverse correlation between p53 expression and HPV presence, suggesting protein degradation. Image analysis also demonstrated that PCNA expression was more intense in HPV DNA 16/18 OSCCs. These findings suggest involvement of high-risk HPV types in oral carcinogenesis.     

Involvement of the metabolic hormones leptin, ghrelin, obestatin, IGF-I and of MAP kinase in control of porcine oocyte maturation

The general aim of our in vitro experiments was to study the role of the metabolic hormones leptin, ghrelin, obestatin and IGF-I and mitogen-activated protein kinase (MAPK)-dependent intracellular mechanisms in the control of nuclear maturation of porcine oocytes. For this purpose, porcine oocytes were isolated from the ovary and cultured in the presence of leptin, ghrelin, obestatin, IGF-I, MAPK blocker PD98059 and the combinations of hormones with PD98059. Proportions of matured oocytes (at metaphase II of meiosis, determined by DAPI staining) and of oocytes containing MAPK/ERK1-2 (determined by immunocytochemistry) were measured before and after culture. It was observed that the majority of oocytes isolated from the ovary before culture were immature and did not contain visible MAPK, but some oocytes were mature, and the majority of these oocytes contained MAPK. Incubation of oocytes resulted in a significant increase in the proportion of matured oocytes and in the percentage of oocytes containing MAPK in both the matured and not matured groups. Addition of IGF-I to the culture medium increased the proportion of matured oocytes, addition of leptin decreased it, and ghrelin and obestatin did not oocyte maturation. Addition of hormones did not affect the expression of MAPK in either immature or mature oocytes. PD98059, when given alone, suppressed the maturation and accumulation of MAPK in both mature and immature oocytes. When given together with hormones, PD98059 was able to reduce the stimulatory effect of IGF-I, to invert the inhibitory action of leptin to stimulatory and to induce the stimulatory action of ghrelin and obestatin on meiosis. IGF-I, ghrelin and obestatin, but not leptin, when given together with PD98059, increased the accumulation of MAPK in both immature and mature oocytes. Association of nuclear maturation and expression of MAPK in oocytes before, but not after culture, as well as the prevention of oocyte maturation by MAPK blocker suggests the involvement of MAPK-dependent intracellular mechanisms in the promotion of reinitiation, but not completion of meiosis. The effect of hormonal additions on meiosis of oocytes suggests that IGF-I is a stimulator, leptin can be an inhibitor, while ghrelin and obestatin probably do not control oocyte maturation. The ability of PD98059 to modify the effect of hormones on oocyte maturation and on MAPK expression suggests possible interference of hormones and MAPK-dependent intracellular mechanisms in oocytes. However, no influence of hormones on MAPK and lack of association between action of hormones and PD98059 on MAPK and meiosis suggest that MAPK is probably not a mediator of effect of IGF-I, leptin, ghrelin and obestatin on porcine oocyte nuclear maturation.     

Expression of the transmembrane protein tyrosine phosphatase RPTPα in human oral squamous cell carcinoma

 Little is known about the role of protein-tyrosine phosphatases (PTPs), the cellular counterparts of protein-tyrosine kinases, both for normal growth regulation and for its dysregulation in cancer. The receptor-like PTPα (RPTPα) may play a positive role in growth regulation and has been shown to be overexpressed in colon carcinoma. An RNA/RNA in situ hybridisation protocol for RPTPα as well as RPTPα immunohistochemistry was developed to evaluate RPTPα expression in oral squamous cell carcinomas (OSCCs) of different histological grade and to reveal the synthetically active cells and their tissue distribution. In well-differentiated OSCC (G1), RPTPα mRNA could be detected by in situ hybridisation exclusively in stroma cells (fibro/myofibroblasts and inflammatory cells). A higher histological grade (G2/G3) was associated with an increased number of RPTPα-synthesising carcinoma cells haphazardly distributed within invading tumour areas. Consistent results were obtained by immunocytochemistry. Thus, both carcinoma dedifferentiation and stroma recruitment and activation seem to be associated with an upregulation of RPTPα expression in OSCC. The results speak in favour of the important role of activation of stroma fibro/myofibroblasts influencing the biological behaviour of epithelial tumours and also suggest that elevated RPTPα expression may be a more general marker for proliferating or dedifferentiated cells. Accepted: 2 February 1999     

Low miR-143/miR-145 Cluster Levels Induce Activin A Overexpression in Oral Squamous Cell Carcinomas,Which Contributes to Poor Prognosis

Deregulated expression of activin A is reported in several tumors, but its biological functions in oral squamous cell carcinoma (OSCC) are unknown. Here, we investigate whether activin A can play a causal role in OSCCs. Activin A expression was assessed by qPCR and immunohistochemistry in OSCC tissues. Low activin A-expressing cells were treated with recombinant activin A and assessed for apoptosis, proliferation, adhesion, migration, invasion and epithelial-mesenchymal transition (EMT). Those phenotypes were also evaluated in high activin A-expressing cells treated with follistatin (an activin A antagonist) or stably expressing shRNA targeting activin A. Transfections of microRNA mimics were performed to determine whether the overexpression of activin A is regulated by miR-143/miR-145 cluster. Activin A was overexpressed in OSCCs in comparison with normal oral mucosa, and high activin A levels were significantly associated with lymph node metastasis, tumor differentiation and poor survival. High activin A levels promoted multiple properties associated with malignant transformation, including decreased apoptosis and increased proliferation, migration, invasion and EMT. Both miR-143 and miR-145 were markedly downregulated in OSCC cell lines and in clinical specimens, and inversely correlated to activin A levels. Forced expression of miR-143 and miR-145 in OSCC cells significantly decreased the expression of activin A. Overexpression of activin A in OSCCs, which is controlled by downregulation of miR-143/miR-145 cluster, regulates apoptosis, proliferation and invasiveness, and it is clinically correlated with lymph node metastasis and poor survival.     

Plasma obestatin levels in normal weight, obese and anorectic women

Obestatin is a recently discovered peptide produced in the stomach, which was originally described to suppress food intake and decrease body weight in experimental animals. We investigated fasting plasma obestatin levels in normal weight, obese and anorectic women and associations of plasma obestatin levels with anthropometric and hormonal parameters. Hormonal (obestatin, ghrelin, leptin, insulin) and anthropometric parameters and body composition were examined in 15 normal weight, 21 obese and 15 anorectic women. Fasting obestatin levels were significantly lower in obese than in normal weight and anorectic women, whereas ghrelin to obestatin ratio was increased in anorectic women. Compared to leptin, only minor differences in plasma obestatin levels were observed in women who greatly differed in the amount of fat stores. However, a negative correlation of fasting obestatin level with body fat indexes might suggest a certain role of obestatin in the regulation of energy homeostasis. A significant relationship between plasma obestatin and ghrelin levels, independent of anthropometric parameters, supports simultaneous secretion of both hormones from the common precursor. Lower plasma obestatin levels in obese women compared to normal weight and anorectic women as well as increased ghrelin to obestatin ratio in anorectic women might play a role in body weight regulation in these pathologies.