Naoya Arai

Silencing of Autocrine Motility Factor Induces Mesenchymal-to-Epithelial Transition and Suppression of Osteosarcoma Pulmonary Metastasis

Phosphoglucose isomerase (PGI) is a multifunctional enzyme that functions in glucose metabolism as a glycolytic enzyme catalyzing an interconversion between glucose and fructose inside the cell, while it acts as cytokine outside the cell, with properties that include autocrine motility factor (AMF)-regulating tumor cell motility. Overexpression of AMF/PGI induces epithelial-to-mesenchymal transition with enhanced malignancy. Recent studies have revealed that silencing of AMF/PGI resulted in mesenchymal-to-epithelial transition (MET) of human lung fibrosarcoma cells and breast cancer cells with reduced malignancy. Here, we constructed a hammerhead ribozyme specific against GUC triplet at the position G390 in the human, mouse, and rat AMF/PGI mRNA sequence. Mesenchymal human osteosarcoma MG-63, HS-Os-1, and murine LM8 cells were stably transfected with the ribozyme specific for AMF/PGI. The stable transfectant cells showed effective downregulation of AMF/PGI expression and subsequent abrogation of AMF/PGI secretion, which resulted in morphologic change with reduced growth, motility, and invasion. Silencing of AMF/PGI induced MET, in which upregulation of E-cadherin and cytokeratins, as well as downregulation of vimentin, were noted. The MET guided by AMF/PGI gene silencing induced osteosarcoma MG-63 to terminally differentiate into mature osteoblasts. Furthermore, MET completely suppressed the tumor growth and pulmonary metastasis of LM8 cells in nude mice. Thus, acquisition of malignancy might be completed in part by upregulation of AMF/PGI, and waiver of malignancy might also be controlled by downregulation of AMF/PGI.

CD44 and SSEA-4 positive cells in an oral cancer cell line HSC-4 possess cancer stem-like cell characteristics

BACKGROUND Cancer may be derived from cancer stem-like cells (CSCs), which are tumor-initiating cells that have properties similar to those of stem cells. Identification and isolation of CSCs needs to be improved further. MATERIALS AND METHODS CSCs markers were examined in human oral cancer cell lines by flow cytometry. The stem cell properties of subpopulations expressing different markers were assessed further by in vitro sphere formation assays, expression of stemness genes, drug resistance assays, and the ability to form tumors in nude mice. RESULTS We demonstrated that CSCs could be isolated by the cell surface markers CD44 and stage-specific embryonic antigen-4 (SSEA-4). CD44+SSEA-4+ cells exhibited cancer stem-like properties, including extensive self-renewal into the bulk of cancer cells. In vivo xenograft experiments indicated that CD44+SSEA-4+ cells exhibit the highest tumorigenic capacity compared with the remaining subpopulations and parental cells. Double-positive cells for CD44 and SSEA-4 exhibited preferential expression of some stemness genes and were more resistant to the anticancer drugs, cisplatin and 5-fluorouracil (5-FU). In addition, cells expressing CD44 and SSEA-4 were detected in all tumor specimens analyzed, while coexpression of CD44 and SSEA-4 was not detectable in normal oral mucosa. CONCLUSION Our findings suggest that CD44+SSEA-4+ cells exhibit the characteristics of CSCs in oral squamous cell carcinoma and provide a target for the development of more effective therapies.

Clinical application of a hyperdry amniotic membrane on surgical defects of the oral mucosa.

PURPOSE The aim of this study was to evaluate the usefulness of a hyperdry amniotic membrane (AM), a novel preservable human amnion, as a wound-dressing material for surgical defects of the oral mucosa. MATERIALS AND METHODS A hyperdry AM was used in the treatment of 10 patients who had developed secondary defects in the tongue and buccal mucosa after the surgical removal of cancerous or precancerous lesions. The effectiveness of the hyperdry AM was assessed by scoring its operability during the surgical procedure and by the hemostatic status, pain relief, feeding situation, epithelialization, and scar contracture in the postoperative period. Its usefulness was evaluated by considering its effectiveness and safety based on the absence of wound infection and graft rejection. RESULTS The membrane was found to be easy to handle as an oral-dressing material. It adhered well to the bare connective and muscular tissues. One lingual case showed slight postoperative bleeding, which astriction then stopped. No remarkable adverse effects were observed in the process of wound healing. The average score of the patients was 11.2 points (10 to 13 points) in the present evaluation, with 14 being the highest possible score. CONCLUSIONS This study showed the clinical usefulness of the hyperdry AM as an intraoral wound-dressing material. Although the number of cases was small, the results suggested that the hyperdry AM is biologically acceptable to oral wounds and could be a suitable clinical alternative for the repair of the oral mucosa.

Gemcitabine chemotherapy induces phenotypic alterations of tumor cells that facilitate antitumor T cell responses in a mouse model of oral cancer

OBJECTIVES Gemcitabine (GEM) is a pyrimidine nucleoside analogue that is a new chemotherapeutic agent used for treating various cancers. Because accumulating evidence indicates that GEM may activate host immune responses, its potential as an immune modulator in cancer chemotherapy has generated considerable interest. MATERIALS AND METHODS In the present study, we investigated the antitumor effects of GEM using a mouse oral cancer model using immunological analyses. We examined apoptotic cell death of tumor cells with GEM treatment both in vitro and in vivo. We also investigated whether in vivo administration of GEM affected the distributions of immune cells, tumor-cell surface expression levels of immune accessory molecules and T cell immune responses in tumor-bearing mice. RESULTS GEM induced significant oral cancer-cell apoptosis in vitro, and in vivo GEM administration markedly attenuated established mouse tumor growth. In vivo GEM administration decreased the numbers of both myeloid-derived suppressor cells (MDSCs) and B cells in tumor-bearing mice and enhanced dendritic cell maturation. Moreover, GEM treatment upregulated tumor-cell surface expressions of several immune accessory molecules and adhesion molecules, including CD80, CD86, CD40, ICAM-1, VCAM-1, and P-selectin. Remarkably, these tumor cells augmented tumor specific T-cell responses. CONCLUSION These results suggest that GEM can induce host antitumor immune responses, which would facilitate antitumor effects in the treatment of oral cancer.

Intraoral application of hyperdry amniotic membrane to surgically exposed bone surface

Hyperdry amniotic membrane, a novel preservable material derived from the human amnion, has been introduced clinically in ophthalmology and other fields. This membrane is available as a wound dressing material for surgical wounds of the tongue and buccal mucosa but has not been used on wounds of the alveolar mucosa. This paper reports 2 cases in which intraoral alveolar wounds with bone exposure were successfully treated with the use of hyperdry amniotic membrane: a 74-year-old woman with gingival leukoplakia of the edentulous mandible, and a 43-year-old man who underwent vestibuloplasty of the reconstructed mandible. The results indicate that the hyperdry amniotic membrane is a useful dressing material, not only for soft tissue wounds, but also for exposed bone in the oral cavity.

Constitutive Activation of Caspase-3 in Non-Apoptotic Oral Squamous Cell Carcinoma Cells

Background: Although caspase-3 is a key molecule for apoptosis induction, recent evidence has suggested its protumoral role in various human malignancies. The aim of the present study was to investigate the expression of cleaved caspase-3 (the active form of caspase-3) in both clinical samples and cell lines from oral squamous cell carcinomas (OSCCs) and elaborate on its contribution to the protumor role in oral cancer. Methods: The expression of cleaved caspase-3 was immunohistochemically evaluated in samples from 30 patients with OSCCs. The samples were either from biopsies or surgically-resected specimens with a mix of clinical stages and tumor site origins. The expression of cleaved caspase-3 was further examined in three OSCC cell lines. Results: In addition to apoptotic cancer cells, all the cases of OSCCs demonstrated a surprisingly positive expression of cleaved caspase-3. A diffuse, cytoplasmic pattern was particularly prominent in in situ carcinoma cells, invasive carcinoma cells, and metastatic cancer cells that lacked apoptotic morphology. On the other hand, non-neoplastic, normal epithelial cells were completely negative for cleaved caspase-3. In all the OSCC cell lines studied, cleaved caspase-3 was expressed in the cytoplasm and nucleus of cancer cells. Flow cytometric analysis also confirmed that the activation level of caspase-3 in non-apoptotic cancer cells was relatively lower than that in apoptotic cancer cells. Moreover, caspase-3 inhibition by caspase-3 specific inhibitor decreased the proliferation of OSCC cells. Conclusions: Because cleaved caspase-3 is selectively expressed in non-apoptotic OSCC cells and is associated with cell proliferation, these findings implicate caspase-3 signaling in promoting the progression of oral cancer.

A novel approach to predict cetuximab‐induced hypersensitivity reaction: detection of drug‐specific IgE on basophils

Cetuximab is remarkable for the relatively high rate and severity of hypersensitivity reactions (HR) being reported in the literature. Screening for cetuximab‐specific IgE in serum via immunoassay has been found to be useful in preventing HR; however, these tests are known to have a low positive predictive rate. In an attempt to remedy this, we evaluated the interaction between cetuximab and IgE on basophils for predicting severe cetuximab‐induced HR. Twelve head and neck cancer patients were enrolled in this single‐institution study: four with a history of cetuximab‐induced HR and eight with no such history. Cetuximab‐specific and galactose‐α‐1,3‐galactose (α‐gal) specific IgEs in serum were measured in vitro using an enzyme‐linked immunosorbent assay (ELISA). IgE‐cetuximab binding on basophils was also analyzed to evaluate the decrease in cetuximab molecules on basophils after dissociation of IgE from FcεRI. The positive predictive value associated with the presence of cetuximab‐ or α‐gal‐specific IgE in serum was found to be only 0.67, whereas the negative predictive value was 1.00. On the other hand, in all four patients who developed HR, the cetuximab molecules on basophils were decreased significantly due to the dissociation of IgE from basophils (P < 0.05). However, this was not the case in patients who did not develop HR. In conclusion, our results strongly imply that the IgE‐cetuximab interaction on basophils may be key to developing improved methods for predicting severe cetuximab‐induced HR.

In vitro synergistic effects of zoledronic acid and calcium on viability of human epithelial cells

OBJECTIVE Bisphosphonate-related osteonecrosis of the jaw is a common complication with defective wound healing of oral mucosa and frequently occurs in patients receiving zoledronic acid (ZA). The aim of this in vitro study was to investigate whether ZA has a cytotoxic effect at clinically relevant concentrations on epithelial cells when calcium conditions are altered. METHODS HaCaT human keratinocyte cells were treated with ZA in the presence of various concentrations of calcium. The concentrations of ZA included submicromolar ones, which are comparable with those found in the plasma of patients. Cell viability and apoptosis were assessed using MTT assay and annexin V flow cytometry. RESULTS Under standard culture conditions, cell growth was inhibited at 1 μM of ZA or above, but was unaffected by lower concentrations. However, when calcium concentrations were moderately increased, cell viability was decreased and apoptosis was induced at 0.2-0.3 μM of ZA. Moreover, a 50% reduction in serum in the hypercalcemic medium resulted in a significant decrease in cell viability at a much lower concentration (0.05 μM). CONCLUSION These results suggest that clinically relevant concentrations of ZA, which alone have little effects, can be toxic to the epithelial cells depending on the conditions of extracellular calcium.

Central cystadenocarcinoma of the mandible

Papillary cystadenocarcinoma (PCAC) of the salivary gland is a rare malignant tumour and occurs in major and minor salivary glands. PCAC of the mandible is exceptionally rare; only 2 cases have been reported. In this study, the authors report a case of PCAC within the mandible. The patient presented with a painful right mandibular mass that had gradually increased in size. The lesion appeared radiographically as a well-demarcated multilocular radiolucent area, similar to an odontogenic cystic lesion. The authors present a case of PCAC with reference to the relevant literature.

Extracellular Ca2+-dependent enhancement of cytocidal potency of zoledronic acid in human oral cancer cells

Direct antitumor effects of bisphosphonates (BPs) have been demonstrated in various cancer cells in vitro. However, the effective concentrations of BPs are typically much higher than their clinically relevant concentrations. Oral cancers frequently invade jawbone and may lead to the release of Ca(2+) in primary lesions. We investigated the effects of the combined application of zoledronic acid (ZA) and Ca(2+) on proliferation and apoptosis of oral cancer cells. Human oral cancer cells, breast cancer cells, and colon cancer cells were treated with ZA at a wide range of concentrations in different Ca(2+) concentration environments. Under a standard Ca(2+) concentration (0.6mM), micromolar concentrations of ZA were required to inhibit oral cancer cell proliferation. Increasing extracellular Ca(2+) concentrations greatly enhanced the potency of the ZA cytocidal effect. The ability of Ca(2+) to enhance the cytocidal effects of ZA was negated by the Ca(2+)-selective chelator EGTA. In contrast, the cytocidal effect of ZA was less pronounced in breast and colon cancer cells regardless of whether extracellular Ca(2+) was elevated. In oral cancer cells incubated with 1.6mM Ca(2+), ZA up-regulated mitochondrial Bax expression and increased mitochondrial Ca(2+) uptake. This was associated with decreased mitochondrial membrane potential and increased release of cytochrome c. We suggest that ZA can specifically produce potent cytocidal activity in oral cancer cells in an extracellular Ca(2+)-dependent manner, implying that BPs may be useful for treatment of oral squamous cell carcinoma with jawbone invasion leading to the hypercalcemic state.