Predictive Value of Neutrophil-Lymphocyte Ratio as a Marker in Antiresorptive Agent-Related Osteonecrosis of the Jaw: A Retrospective Analysis.

Antiresorptive agent-related osteonecrosis of the jaw (ARONJ), a multifactorial disease, can drastically affect a patient's quality of life. Moreover, disease progression to severe acute inflammation can hinder treatment. Therefore, we aimed to investigate the diagnostic value of the neutrophil−lymphocyte ratio (NLR) and platelet−lymphocyte ratio (PLR) in predicting the risk of acute inflammation in patients with ARONJ. In total, 147 patients with ARONJ were enrolled between 1 January 2011 and 31 December 2019. They were divided into two groups according to their baseline NLR (high NLR vs. low NLR) or PLR (high PLR vs. low PLR) to analyze the relationship between NLR and PLR and the outcomes of acute inflammatory events. An optimal NLR cut-off value of 2.83 was identified for hospitalization for an inflammatory event. Logistic regression analysis showed that NLR > 2.83 was associated with an increased risk of hospitalization for an inflammatory event. A PLR cut-off value of 165.2 was identified for hospitalization for an inflammatory event. However, logistic regression analysis showed that PLR > 165.2 was not significantly associated with hospitalization for an inflammatory event. Our study findings suggest that the NLR has diagnostic value in predicting the risk of hospitalization for inflammatory events among patients with ARONJ.

A Case of Old Calcifying Epithelioma Processed Symptomless over 40 Years.

Calcifying epithelioma, a benign tumor derived from the hair apparatus and consisted of hair matrix cells, is relatively prevalent in females. We report a case of right preauricular calcifying epithelioma that was incidentally detected at the examination of multiple facial fractures and became an old lesion without symptoms for 40 years. The patient who was a 42-year-old male visited our department for the first time in October 2011 with a chief complaint of multiple facial fractures. Radiographic imaging demonstrated fracture lines at the anterior and posterior walls of the left maxillary sinus and zygomatic arch and revealed a mass at a right preauricular area. The extraction was performed under general anaesthesia. No recurrence has been observed 15 months after surgery. We also reviewed the literature of calcifying epithelioma.

Characterization of phosphodiesterase 2A in human malignant melanoma PMP cells.

The prognosis for malignant melanoma is poor; therefore, new diagnostic methods and treatment strategies are urgently needed. Phosphodiesterase 2 (PDE2) is one of 21 phosphodiesterases, which are divided into 11 families (PDE1-PDE11). PDE2 hydrolyzes cyclic AMP (cAMP) and cyclic GMP (cGMP), and its binding to cGMP enhances the hydrolysis of cAMP. We previously reported the expression of PDE1, PDE3 and PDE5 in human malignant melanoma cells. However, the expression of PDE2 in these cells has not been investigated. Herein, we examined the expression of PDE2A and its role in human oral malignant melanoma PMP cells. Sequencing of RT-PCR products revealed that PDE2A2 was the only variant expressed in PMP cells. Four point mutations were detected; one missense mutation at nucleotide position 734 (from C to T) resulted in the substitution of threonine with isoleucine at amino acid position 214. The other three were silent mutations. An in vitro migration assay and a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay revealed that suppressing PDE2 activity with its specific inhibitor, erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA), had no impact on cell motility or apoptosis. Furthermore, the cytotoxicity of EHNA, assessed using a trypan blue exclusion assay, was negligible. On the other hand, assessment of cell proliferation by BrdU incorporation and cell cycle analysis by flow cytometry revealed that EHNA treatment inhibited DNA synthesis and increased the percentage of G2/M-arrested cells. Furthermore, cyclin A mRNA expression was downregulated, while cyclin E mRNA expression was upregulated in EHNA-treated cells. Our results demonstrated that the PDE2A2 variant carrying point mutations is expressed in PMP cells and may affect cell cycle progression by modulating cyclin A expression. Thus, PDE2A2 is a possible new molecular target for the treatment of malignant melanoma.

A case of carcinoma ex pleomorphic adenoma in the buccal mucosa: review of the literature.

We present a case of carcinoma ex pleomorphic adenoma on the right buccal mucosa in a 52-year-old Japanese woman. Based on the histopathology, the excised tumor was the non-invasive type, but the majority of the tumor consisted of poorly-differentiated adenocarcinoma cells. We performed proton radiation after the surgery. The patient was well, without evidence of disease, 48 months after surgery. Carcinoma ex pleomorphic adenoma in the buccal mucosa has been reported in only four cases during the past twenty years. Therefore, our case was comparatively rare.

Bax mRNA therapy using cationic liposomes for human malignant melanoma.

BACKGROUND: Bax is a pro-apoptotic molecule that functions as a tumor suppressor and Bax gene therapy has been examined for various cancers. Gene transfer by mRNA lipofection is more efficient than plasmid DNA lipofection and, in the present study, we examined the anti-tumor effects in human malignant melanoma cells (HMGs) using Bax mRNA lipofection. METHODS: Bax protein expression, cell growth inhibition, caspase-3 activity and apoptosis were examined in vitro. Liposome-Bax mRNA was applied locally once every 5 days for a total of five times to peripheral HMG tumors transplanted in nude mice. Tumor growth inhibition was evaluated by measuring the tumor volume and apoptosis was detected using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay. RESULTS: Enhanced expression of Bax protein was observed following Bax mRNA transfer and cell survival was 59.8%. Caspase-3 activity and TUNEL-positive cells increased significantly following Bax mRNA lipofection compared to Bax plasmid transfer. In mice, tumor growth increased only slightly during liposome-Bax mRNA administration and the tumor volume on day 30 (10 days after completion of administration) was 36.7% of that in the saline control group. By contrast, Bax plasmid transfection resulted in little change in tumor growth compared to controls. CONCLUSIONS: Bax mRNA therapy using liposomes has stronger anti-tumor effects than Bax gene therapy using a plasmid, and the results suggest that Bax mRNA lipofection may be a viable treatment for malignant melanoma.

A case of submandibular gland mucocele.

Submandibular Gland Mucocele:The mucocele occuring in the submandibular region is rare, most cases originate in the sublingual gland. Here, we report a rare case of mucocele originating in the submandibular gland. In this report, we present such a case in a 7-year-old boy, who was treated by an extirpation of cyst with submandibular and sublingual gland

Bax gene therapy for human osteosarcoma using cationic liposomes in vivo.

The purpose of this study was to evaluate the anti-tumor effect of human osteosarcoma (HOSM-1) tumor xenografts in nude mice via transfer of the Bax gene using cationic liposomes. The HOSM-1 tumors transplanted into nude mice grew to 5-6 mm in diameter. Following growth of the tumor to this size, liposomes with the Bax plasmid were applied locally to the peripheral tumor (day 0) and were applied 3 times per week for 2 weeks (6 times in total). The tumor growth inhibitory effect was evaluated by measuring the tumor volume up to day 40. The expression of Bax was observed by immunohistochemical analysis and apoptosis was detected using the TUNEL assay. Tumor growth increased only slightly during the administration period, and tumor volume on day 50 was 43% of that in the saline control group. In the tumor margin 48 h after the completion of administration, Bax immunoreactivity was detected and apoptotic cells were clearly increased. Since these results suggested that Bax gene therapy using cationic liposome induced apoptosis in HOSM-1 tumor in vivo, we anticipate that this therapy will be useful for the treatment of osteosarcoma.

G3139 induces cell death by caspase-dependent and -independent apoptosis on human melanoma cell lines.

G3139 is an 18-mer phosphorothioate oligodeoxynucleotide (ODN) which has been targeted on the initiation codon region of the bcl-2 gene. Currently, clinical trials on G3139 for diverse tumors are underway in phase II and phase III. However, basic investigations of bcl-2 antisense ODN (G3139) and reverse ODN (G3622) have not been fully examined. In this report, we investigate cell death caused by G3139 and G3622 and the impact of antisense ODN in melanoma cell lines. We confirmed that G3139 reduced the level of bcl-2 protein and both G3139 and G3622 inhibited cell proliferation and induced apoptosis. G3139 was noted to produce a more intense effect than G3622. Although the general caspase inhibitor, Z-VAD-fmk, prevented apoptosis incompletely, the inhibition ratio of both ODNs was approximately equivalent. Our results suggested that inhibition of cell proliferation by ODNs is produced by apoptosis, but that the apoptotic pathway is not fully induced by the caspase-dependent pathway. Upon examination of the intracellular apoptotic protein dynamics, AIF localized within the mitochondria was translocated to the cytosol within 24 h, and subsequently to the nuclei after 48 h of treatment with G3139. Our results imply the following: the transfection of ODNs can induce apoptosis, the anti-tumor effect of G3139 is better than G3622, and the difference in the anti-tumor effect is specifically based upon the reduction of expression of the target DNA in malignant tumors. We consider that antisense ODNs may be an important tool for anti-tumor chemotherapy and the targeting of specific DNA is important in enhancing the anti-proliferative effect against tumors.

mHFE7A, a newly identified monoclonal antibody to Fas, induces apoptosis in human melanoma cells in vitro and delays the growth of melanoma xenotransplants.

The Fas receptor is a potentially valuable therapeutic target in cancer treatment. However, the clinical application of antibodies directed to this target is hindered by their serious side effects in vivo, including liver toxicity. One murine monoclonal antibody, mHFE7A, binds both to human Fas and murine Fas, without inducing any obvious side effects. However, the potential therapeutic effects of mHFE7A are unclear in human cancer cells or tumors. Here, we determined whether mHFE7A could induce apoptosis in vitro, and assessed its effects on major apoptotic pathways in a human melanoma cell line, MMN9. We also investigated its anti-cancer effects on transplanted melanoma MMN9 tumors in BALB/c nude mice. Treatment of mHFE7A cross-linking with Ig induced cell death similar to CH-11 treatment. Apoptosis induced by mHFE7A was defined by Hoechst 33342 DNA staining and DNA fragmentation assay. Furthermore, mHFE7A-mediated apoptosis was dependent on activation of a caspase signaling cascade involving caspases-8 and -3. Administration of mHFE7A also delayed the growth of melanoma xenografts. Thus, we provide the first evidence that the murine anti-Fas monoclonal antibody, mHFE7A, induces apoptosis of human malignant melanoma cells in vitro and is anti-tumorigenic in vivo.

The influence of oral tumor cell proliferation activity and membrane potential on the transfection efficiency of a cationic liposome.

The transfection efficiency of cationic liposomes varies according to cell type, but the specific cellular characteristics that affect transfection efficiency have not yet been defined. We investigated whether the transfection efficiency of cationic liposomes correlates with cell proliferation activity or cell membrane potential in oral malignant melanoma (HMG) and oral osteosarcoma cell lines (HOSM-1 and HOSM-2). The cell membrane potential was assessed by uptake of a cationic probe. Three oral tumor cell lines were exposed to a cationic liposome complexed with a beta-galactosidase expression plasmid, and beta-galactosidase expression was compared. Cell proliferation was about 2-fold higher in HOSM-1 cells than in HMG cells. The cell membrane potential in HMG and HOSM-1 cells was comparable, while the membrane potential in HOSM-2 cells was 1.6-fold higher. beta-galactosidase expression was measured by X-Gal staining in 7.0% of HMG, 17.0% of HOSM-1 and 11.5% of HOSM-2 cells. The present study demonstrates that gene therapy with cationic liposomes may be a promising new strategy for treatment of oral malignant melanoma and osteosarcoma. In addition, the transfection efficiency of cationic liposomes appears to be influenced by cell proliferation activity, but not cell membrane potential.

Experimental therapy using interferon-gamma and anti-Fas antibody against oral malignant melanoma cells.

The Fas/FasL signalling system plays an important role in chemotherapy-induced apoptosis in several different cell types. After interferon-gamma (IFN-gamma) treatment, we have previously reported a significant increase in Fas expression in oral malignant melanoma cell lines (MMN9, PMP, MAA, HMG) in vitro, and combination therapy using IFN-gamma and anti-Fas antibody (CH-11) has shown a synergistic anti-proliferative effect in MMN9 cells. There have been several in-vitro studies using CH-11, but there are few reports of its anti-tumour effect in vivo. In this study, we investigated experimental therapy using anti-Fas antibody against MMN9 in vivo in a mouse model, and histologically examined tumour tissue removed from BALB/c nude mice. Animals that received both IFN-gamma and CH-11 showed a 53.8% increase in anti-tumour effect (P=0.0018) 20 days after the first administration. In the histological study, the combined administration group tested positive in terminal deoxynucleotidyl transferase-mediated nick end labelling staining, and showed significantly increased levels of Fas expression on immunostaining compared with the vehicle group. These results show the efficacy of anticancer therapy using IFN-gamma and anti-Fas antibody via the modulation of Fas-mediated apoptosis. Moreover, inhibition of IFN-gamma/CH-11-induced apoptosis with a general caspase inhibitor (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) reduced cell death significantly in vitro. Bcl-2 cleavage did not occur under these conditions, suggesting a relationship between caspase activation and Bc1-2 cleavage in MMN9 cells.

Antitumor activity of cationic liposome-mediated Bax gene transfer in osteosarcoma cells: induction of apoptosis and caspase-independent cell death.

The purpose of this study was to evaluate the anti-tumor effects of osteosarcoma (HOSM-1) cells via transfer of the Bax gene using a cationic liposome. We evaluated the levels of Bax, Bcl-xL, Bcl-2 and cytochrome c expression by Western blot analysis, and caspase-9 and -3 activities were determined in a colorimetric assay. Apoptosis was detected using a TUNEL assay, and cell growth inhibition was determined in an MTT assay. Following Bax gene transfer, release of cytochrome c to the cytosol was detected, the activities of caspase-9 and -3 increased, and TUNEL-positive cells (37.5%) were detected. Cell survival rate was 50.8% under these conditions. Induction of apoptosis was inhibited by a caspase inhibitor (zVAD-fmk), but only a slight increase in cell survival rate occurred. Hence, since not only apoptosis but also caspase-independent cell death is induced in HOSM-1 cells, we anticipate that Bax gene therapy with cationic liposomes will be useful for osteosarcoma.

p53 gene therapy of human osteosarcoma using a transferrin-modified cationic liposome.

Gene delivery via transferrin receptors, which are highly expressed by cancer cells, can be used to enhance the effectiveness of gene therapy for cancer. In this study, we examined the efficacy of p53 gene therapy in human osteosarcoma (HOSM-1) cells derived from the oral cavity using a cationic liposome supplemented with transferrin. HOSM-1 cells were exposed to transferrin-liposome-p53 in vitro, and the growth inhibition rate, expression of p53 and bax, and induction of apoptosis were measured 48 hours later. Treatment of HOSM-1 cells with transferrin-liposome-p53 resulted in 60.7% growth inhibition. Wild-type p53 expression and an increase in bax expression were observed following transfection with transferrin-liposome-p53, and 20.5% of the treated HOSM-1 cells were apoptotic. In vivo, the HOSM-1 tumor transplanted into nude mice grew to 5 to 6 mm in diameter. Following growth of the tumor to this size, transferrin-liposome-p53 was locally applied to the peripheral tumor (day 0) and then applied once every 5 days for a total of six times. During the administration period, tumor growth did not occur, and the mean tumor volume on the last day of administration (day 25) was 10.0% of that in the saline control group. These results suggest that p53 gene therapy via cationic liposome modification with transferrin is an effective strategy for treatment of osteosarcoma.

Interferon-gamma and anti-Fas antibody-induced apoptosis in human melanoma cell lines and its relationship to bcl-2 cleavage and bak expression.

Interferon-gamma (IFNgamma) has been shown to induce apoptosis through the induction of the Fas antigen in certain cell lines. In this study, we used four melanoma cell lines (MMN9, PMP, MAA and HMG) to study the antiproliferative effect of exogenous IFNgamma treatment, the expression of IFNgamma-induced Fas antigen, and the combined effect of IFNgamma and anti-Fas antibody (CH-11). We also investigated the relationship between Fas-mediated apoptosis and the expression of the bcl-2 family, measured using Western blotting. IFNgamma increased the mean fluorescence intensity of Fas in MMN9 and PMP cells as measured by flow cytometry. Combined therapy had a significant antiproliferative effect on MMN9 and PMP cells, as measured by the MTT assay. After exposure to IFNgamma and anti-Fas antibody, cleavage of bcl-2 occurred in apoptotic cells, and the signal intensity of both bcl-2 and bak decreased in surviving MMN9 cells, as shown by Western blotting analysis. Our results indicate that IFNgamma induces overexpression of Fas and consequently enhances the sensitivity of melanoma cells to Fas-mediated apoptosis. Furthermore, it is possible that cleavage of bcl-2 correlates with the induction of apoptosis induced by IFNgamma and anti-Fas antibody in melanoma cells. We conclude that combined therapy with IFNgamma and anti-Fas antibody may provide an alternative and more efficient chemotherapeutic approach against melanoma cells by inducing the overexpression of Fas after exposure to IFNgamma.

Expression and role of phosphodiesterase 3 in human squamous cell carcinoma KB cells.

Phosphodiesterase (PDE) 3s have been characterized in human squamous cell carcinoma KB cells. PDE3 activity was detected in homogenates of KB cells. PDE3A and 3B mRNAs were detected by RT-PCR in RNA from KB cells; the nucleotide sequences of the fragments were identical to those of human PDE3A and 3B. Immunoblotting with anti-PDE3 antibodies detected both PDE3A- and 3B-immunoreactive proteins in KB cells. The PDE3-specific inhibitor, cilostamide, inhibited the proliferation of KB cells. Our results indicate that PDE3s may be important regulators of the growth of KB cells. Therefore, PDE3 inhibitors may be potential new drugs for antiproliferative therapies in squamous cell carcinoma in the head and neck.