Kenya Okumura

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.

Bax mRNA therapy using cationic liposomes for human malignant melanoma.

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.

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.

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-&ggr;) 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-&ggr; 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-&ggr; 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-&ggr; and anti-Fas antibody via the modulation of Fas-mediated apoptosis. Moreover, inhibition of IFN-&ggr;/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.

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.

A case of tongue carcinoma associated with chronic graft‐versus‐host disease after allogeneic haematopoietic stem cell transplantation

Graft-versus-host disease (GVHD) can occur at various sites, including the oral mucosa, where it is associated with a high risk of head and neck cancer. We report the case of a 46-year-old woman with tongue cancer that developed following Hodgkins lymphoma and chronic GVHD, and we discuss the possible causes of cancer development.

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.