Yoichi Ohiro

Inhibitory effects of epigallocatechin-3 gallate, a polyphenol in green tea, on tumor-associated endothelial cells and endothelial progenitor cells

The polyphenol epigallocatechin‐3 gallate (EGCG) in green tea suppresses tumor growth by direct action on tumor cells and by inhibition of angiogenesis, but it is not known whether it specifically inhibits tumor angiogenesis. We examined the anti‐angiogenic effect of EGCG on tumor‐associated endothelial cells (TEC), endothelial progenitor cells (EPC), and normal endothelial cells (NEC). EGCG suppressed the migration of TEC and EPC but not NEC. EGCG also inhibited the phosphorylation of Akt in TEC but not in NEC. Furthermore, vascular endothelial growth factor‐induced mobilization of EPC into circulation was inhibited by EGCG. MMP‐9 in the bone marrow plasma plays key roles in EPC mobilization into circulation. We observed that expression of MMP‐9 mRNA was downregulated by EGCG in mouse bone marrow stromal cells. In an in vivo model, EGCG suppressed growth of melanoma and reduced microvessel density. Our study showed that EGCG has selective anti‐angiogenic effects on TEC and EPC. It is suggested that EGCG could be a promising angiogenesis inhibitor for cancer therapy. (Cancer Sci 2009; 100: 1963–1970)

Serum p53 antibodies as a prognostic indicator in oral squamous cell carcinoma

The purpose of the present study was to investigate the clinical usefulness of the detection of serum p53 antibodies (p53 Abs) in patients with oral squamous cell carcinoma (SCC). Preoperative values of p53 Abs were measured by enzyme-linked immunosorbent assay in 113 patients with primary oral SCC and seropositive patients were reevaluated postoperatively. The positivity rate of p53 Abs was 16%, and the 5-year survival rate of patients positive for p53 Abs was significantly lower than that of patients negative for p53 Abs (56.2% vs. 80.7%; P = 0.018). The preoperative presence of p53 Abs was found to be an independent prognostic factor in a multivariate analysis (P = 0.028, hazards ratio = 3.34), and its positivity was significantly related to secondary cervical lymph node metastases (P = 0.029). Six of nine patients who remained seropositive for p53 Abs through the disease course and the one with seropositive reversion from temporary negative status developed treatment failure. Therefore, the detection of p53 Abs in the serum of patients with SCC may be a useful prognostic marker.

Cytoplasmic expression of HuR may be a valuable diagnostic tool for determining the potential for malignant transformation of oral verrucous borderline lesions

Oral verrucous carcinoma (OVC) is a low grade variant of oral squamous cell carcinoma, and oral verrucous hyperplasia (OVH) is a benign lesion without malignant features. However, pathologists are sometimes presented with borderline lesions and are indecisive as to diagnose them as benign or malignant. Thus, these lesions are tentatively termed oral verrucous lesions (OVLs). HuR is an ARE mRNA-binding protein, normally localized in the nucleus but cytoplasmic exportation is frequently observed in cancer cells. The present study aimed to elucidate whether expression of the HuR protein facilitates the diagnosis of true malignant lesions. Clinicopathological features were evaluated, and immunohistochemical analysis for p53, Ki67 and HuR proteins was performed in 48 cases of OVH, OVC and OVL, and the outcomes were correlated using appropriate statistical analysis. The association of these three proteins in relation to malignant transformation was analyzed after a 3-year follow-up of 25 OVL cases. The basal characteristics (age, gender and location) of all cases had no significant association with the types of lesions. Gingiva (39.4%) was the common site for all lesions. Distribution of the examined proteins had a significant association with the lesions. As compared with the OVLs, the number of immunostained-positive cells was significantly higher in the OVCs and lower in the OVH cases. During follow-up, 24% of the OVLs underwent malignant transformation for which high HuR expression and a diffuse staining pattern in the epithelium were observed. Taken together, the high degree of HuR expression with diffuse staining pattern in the epithelium may be an effective diagnostic tool that determines the potential of OVLs for malignant transformation.

Does Swallowing Function Recover in the Long Term in Patients With Surgically Treated Tongue Carcinomas

PURPOSE The present study aimed to measure postsurgical swallowing function in patients 5 years after the surgical treatment of tongue carcinoma. PATIENTS AND METHODS Using a retrospective cohort study design, the investigators enrolled postsurgical patients treated for tongue carcinomas in Hokkaido University Hospital. The primary outcome variable was oropharyngeal swallow efficiency (OPSE) determined by videofluoroscopic evaluation, and OPSE at follow-up was compared with that at discharge. Other variables included current nutritional status (body mass index, serum albumin), dietary intake, self-rating of current swallowing function, and occurrence of pneumonia. Statistical analysis used the paired t test and the Spearman rank correlation. RESULTS Swallowing function was assessed in 20 patients (11 men and 9 women) who underwent the surgical treatment of tongue carcinomas; the median age was 70 years (range, 56 to 90 yrs). The mean OPSE values for liquid and paste at follow-up were 26.6 ± 21.2 and 21.9 ± 22.5, respectively. The mean values for the body mass index and serum albumin at presentation were 22.2 ± 3.4 kg/m(2) and 4.5 ± 0.3 g/dL, respectively. All patients had a full oral intake of foods, with a mean self-rated value of 6.4 ± 2.5, a value acceptable to the patients. Pneumonia requiring hospitalization did not occur in these patients. CONCLUSIONS The long-term follow-up of patients after the surgical treatment of tongue carcinomas showed acceptable levels of oral function and nutritional status despite objective measurements of poor swallowing efficiency assessed using videofluoroscopy.

Pim-1 activation of cell motility induces the malignant phenotype of tongue carcinoma.

Pim-1 is a serine/threonine kinase as well as a proto-oncogene that induces T-cell lymphoma. Pim-1 induces cell cycle progression in cooperation with c-Myc and acts as an inhibitor of apoptotic cell death, actions that are involved in blood cell oncogenesis. However, little is known regarding the role of Pim-1 in oral carcinogenesis. We investigated Pim-1 expression in tongue squamous cell carcinoma (SCC) and examined its clinicopathological features. Western blotting was performed in 6 oral SCC cell lines, with Pim-1 being detected in all 6 of the lines. Immunohistochemical detection of Pim-1 was carried out in 39 cases of tongue SCC and analyzed in terms of its associated clinicopathological features. Pim-1 was expressed in 17/39 cases of tongue carcinoma, and was significantly correlated with lymph node metastasis. The role of Pim-1 in cell motility was further examined in HSC3 cells using the GTP-binding assay for Rho family protein, the motility assay and siRNA treatment. Rac1 activation was observed, and cell motility was reduced when Pim-1 was knocked down by siRNA. These results indicate that Pim-1 is involved in the carcinogenesis of oral SCC and is correlated to metastasis, which is in part associated with the enhancement of cell motility.

ALDH1 and podoplanin expression patterns predict the risk of malignant transformation in oral leukoplakia

Oral leukoplakia (OL) is a clinically diagnosed preneoplastic lesion of the oral cavity with an increased oral cancer risk. However, the risk of malignant transformation is still difficult to assess. The objective of the present study was to examine the expression patterns of aldehyde dehydrogenase 1 (ALDH1) and podoplanin in OL, and to determine their roles in predicting oral cancer development. In the present study, the expression patterns of ALDH1 and podoplanin were determined in samples from 79 patients with OL. The association between protein expression and clinicopathological parameters, including oral cancer-free survival, was analyzed during a mean follow-up period of 3.4 years. Expression of ALDH1 and podoplanin was observed in 61 and 67% patients, respectively. Kaplan-Meier analysis demonstrated that the expression of the proteins was correlated with the risk of progression to oral cancer. Multivariate analysis revealed that expression of ALDH1 and podoplanin was associated with 3.02- and 2.62-fold increased risk of malignant transformation, respectively. The malignant transformation risk of OL was considerably higher in cases with expression of both proteins. Point-prevalence analysis revealed that 66% of patients with co-expression of ALDH1 and podoplanin developed oral cancer. Taken together, our data indicate that ALDH1 and podoplanin expression patterns in OL are associated with oral cancer development, suggesting that ALDH1 and podoplanin may be useful biomarkers to identify OL patients with a substantially high oral cancer risk.

Expression of parathyroid hormone-related protein confers malignant potential to mucoepidermoid carcinoma

Parathyroid hormone-related protein (PTHrP) is known to induce bone resorption by activating RANKL as well as PTH. PTHrP plays a central role in humoral hypercalcemia, and its expression has been reported to be closely associated with bone metastasis of breast carcinoma. PTHrP expression in oral squamous carcinoma cell lines was investigated, and PTHrP was expressed in oral squamous cell carcinoma cell lines similar to that in a prostate carcinoma cell line. Mucoepidermoid carcinoma is the most common malignant salivary gland tumor composed of different types of cells including a squamous component. Its clinical behavior is highly variable and ranges from slow-growing and indolent to locally aggressive and highly metastatic. We examined the PTHrP expression in mucoepidermoid carcinoma and assessed the significance of its correlation with clinicopathological features. Immunohistochemical detection of PTHrP was carried out in 21 cases of mucoepidermoid carcinoma in the head and neck region. PTHrP was highly detectable in intermediate and epidermoid cells, and abundant expression of PTHrP in intermediate cells had a significant association with cancer malignancy, including lymph node metastasis and/or tumor recurrence. These results suggest that PTHrP expression can be used as a prognostic factor for mucoepidermoid carcinoma.

Augmentation of vaccination effects of PGE2-producing tumor cells by transfection with cytokine genes

Potentially antigenic tumors often escape from the immune surveillance system by producing immunosuppressive factors. It has previously been reported that a progressor-type murine fibrosarcoma culture line, QRpP cells, produced high levels of PGE2 compared with a regressor-type tumor line, QR-32 cells, and that QRpP cells progressively grew in syngeneic C57BL/6 mice. In order to improve suppressed immunogenicity of QRpP cells with the high levels of PGE2, the author transfected QRpP cells with an effective expression vector containing cDNA for IL-2, IFN-gamma and TNF-alpha. These transfected clones expressed mRNA for IL-2, IFN-gamma and TNF-alpha, respectively, and produced high levels of cytokines in their culture supernatants. Consequentry, QRpP cells transfected with IL-2 (QRpP-IL-2), IFN-gamma (QRpP-IFN-gamma), IL-2 and IFN-gamma (QRpP-IL-2/IFN-gamma), TNF-alpha (QRpP-TNF-alpha) lowered in tumorigenicity. In particular, the mice that had rejected the implantation of QRpP-IL-2 clone also rejected implantation of the parental QRpP cells. The activity of pre-cytotoxic T cells (pre-CTLs) obtained from the mice implanted with QRpP-IL-2 clone was higher than that of the mice implanted with other transfectants. These results suggested that transfection with the gene for IL-2 is an effective strategy against the producing immunosuppressive factors such as PGE2.

Transparent, resin-based, three-dimensional model to help visualise intraosseous tumours.

Three-dimensional computed tomography (CT) and 3imensional models are common in many fields of surgery.1–4 We often use dredging to treat ameloblastoma.5 A ombination of 3-dimensional CT and a 3-dimensional laster-based model provide an image for us to work from, ut the tumour is not easily distinguished from the bone. e have developed a sterilisable, transparent, resin-based, -dimensional model, which we can handle during operaion and which also allows us to colour the tumour and bone eparately to provide a more detailed image for the surgeon. A 49-year-old Japanese woman reported to the dental epartment because of a swelling in her right mandible. rom 1980 to 1991, she had had dredging treatment for an meloblastoma, and had not been back to the clinic since. Initially, we decided to deflate, enucleate, and complete a rst dredging to reduce the size of the tumour and restore he frame of bone. Pathological analysis led to the diagosis of ameloblastoma, and CT showed details of the emaining lesion (Fig. 1). We discussed the design of a esin-based, three-dimensional model and a technician (from PPEAL CO, LTD, Aomori, Japan) created it. The model as printed with an Objet500 Connex3 printer (Stratasys td, Tokyo, Japan), with the tumour printed in red and the one in yellow (Fig. 2). We then sterilised it with orthohthalaldehyde to allow us to touch it for reference while e operated. The red stain was easily distinguishable from the yellow on he model, and the bone that surrounded the tumour appeared range where the colours overlapped (Fig. 2). As a result, perating time was shortened because we were able to find

E1AF expression is closely correlated with malignant phenotype of tongue squamous cell carcinoma through activation of MT1-MMP gene promoters

E1AF is an ets-oncogene family transcription factor that has been shown to up-regulate multiple MMPs whereas MMP-2, a potent extracellular matrix degrading enzyme, is not up-regulated. We investigated the activation mechanism of MMP-2 in oral squamous cell carcinoma. Chloramphenicol acetyltransferase (CAT) assay was utilized to investigate whether E1AF is able to up-regulate membrane type-1 matrix metalloproteinase (MT1-MMP), which is known to activate MMP-2. Expression of the CAT reporter gene under the control of the MT1-MMP promoter was increased approximately 40-fold by co-transfection with the E1AF expression vector. Real-time RT-PCR study was carried out in 25 patients with tongue squamous cell carcinoma, and the mRNA expression level of E1AF and MT1-MMP was synergistically increased. These results indicate that E1AF positively regulates transcription from MT1-MMP genes, which plays an important role in invasion and metastasis of squamous cell carcinoma of the tongue by converting pro-MMP-2 into active-MMP-2.