Masashi Hatori

Inhibitors of cyclooxygenase-2 (COX-2) suppressed the proliferation and differentiation of human leukaemia cell lines

Prostaglandins (PG) are known to play important roles in the proliferation and differentiation of leukaemia cells. The effect of the inhibitors of cyclooxygenase-2 (COX-2), a rate-limiting enzyme for the synthesis of PG, on the proliferation and differentiation of leukaemia cell lines was investigated. COX-2 inhibitors, NS-398 and nabumetone, suppressed the proliferation of U-937 and ML-1 cells by inducing a G0/G1 cell-cycle arrest. Cell-cycle arrest induced by these COX-2 inhibitors was not associated with an upregulation of the cyclin-dependent kinase inhibitors. COX-2 inhibitors also inhibited the differentiation of these cells induced by interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and retinoic acid (RA). Treatment with NS-398 did not suppress the levels of PGs produced by these cells. Although COX-2 antisense oligonucleotide showed a similar inhibitory effect on these cells, its inhibitory effect was smaller than that of NS-398. These results suggest that COX-2 inhibitors may suppress the proliferation and differentiation of leukaemia cells both via COX-2-dependent and -independent pathways.

Hypoxia Induces Resistance to 5-Fluorouracil in Oral Cancer Cells Via G1 Phase Cell Cycle Arrest

Malignant tumors are exposed to various levels of hypoxic condition in vivo. It has been known that tumor cells under hypoxia are resistant to chemotherapies. To clarify the mechanism of the hypoxia-induced chemoresistance, we evaluated the effects of hypoxia on the resistance of oral squamous cell carcinoma (OSCC) cell lines to 5-fluorouracil (5-FU). OSCC cells were divided to two groups by the proliferation activity under hypoxic condition; hypoxia-resistant (HR) and hypoxia-sensitive (HS) cells. Growth of HS cells were inhibited by hypoxia and introduced to G(1) arrest in cell cycle. 5-FU effect on HS cell viability was markedly reduced in hypoxic condition without an induction of chemoresistant related protein, P-glycoprotein. However, proliferation, cell cycle, and 5-FU sensitivity of HR cells were not affected by hypoxia. Hypoxia-inducible factor (HIF)-1alpha was induced by hypoxia in all OSCC cell lines, but diminished in HS cells within 48h. Expression of p21 and p27 was strongly augmented and CyclinD expression was reduced by hypoxia in HS cells. However, the expression of these proteins was constitutive in HR cells during 48h hypoxic culture. Phosphorylation of mammalian target of rapamycin (mTOR) was reduced by hypoxia in HS cells. From these findings, we concluded that HS OSCC cells acquire 5-FU resistance under hypoxia by G(1)/S transition through an upregulation of cell cycle inhibitors.

Identification of a truncated cystatin SA-I as a saliva biomarker for oral squamous cell carcinoma using the SELDI ProteinChip platform

New and more consistent biomarkers of oral squamous cell carcinoma (OSCC) are needed to improve early detection of the disease and to monitor patient management. The aim of this study was to detect new OSCC tumor markers in saliva. Unstimulated saliva, collected from patients with primary stage I OSCC as matched pre-and post-treatment samples, was used in the analysis. A surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF) ProteinChip system was used to screen for differentially expressed proteins in the saliva samples. This analysis revealed 26 proteins with significantly different expression levels in the pre-and post-treatment samples (P<0.05). A 14 kDa protein detected in pre-treatment saliva from the OSCC patients was identified as a truncated cystatin SA-I, with deletion of three amino acids from the N-terminus. The authors propose that ProteinChip analysis may provide a reliable screening test for early diagnosis of OSCC and that truncated cystatin SA-I might be a useful tumor biomarker for OSCC.

Effect of 5-fluorouracil on G1 phase cell cycle regulation in oral cancer cell lines.

5-fluorouracil (5-FU) has been widely used for chemotherapy of head and neck cancer, and is known to affect the cell cycle and induce apoptotic death of cancer cells. However, the molecular actions of 5-FU on the cell cycle regulatory mechanism have not been fully explained. Herein we analyzed the effects of 5-FU on the expression of G1/S-related cell cycle regulators in oral cancer cell lines. In vitro 5-FU treatment of oral cancer cells resulted in an increase in G1/S phase cells. p21 expression was augmented by 5-FU without any notable changes in p53 expression. A remarkable up-regulation of cyclin E and a concomitant down-regulation of cyclin D were observed after 24 h 5-FU treatment. Our results suggest that 5-FU-induced changes in cell cycle regulation of oral cancer cells might associate with an alteration of G1 cyclins expression. p21 was remarkably up-regulated, but it was speculated that its activity might be cancelled by an increased binding to CDK4.

Inhibition of cyclooxygenase-2 suppresses the invasiveness of oral squamous cell carcinoma cell lines via down-regulation of matrix metalloproteinase-2 production and activation.

Increased cyclooxygenase (COX-2) expression in tumors is known to be correlated with tumor invasion, angiogenesis, resistance to apoptosis, and suppression of host immunity. We previously reported that the invasiveness of human oral squamous cell carcinoma (OSCC) cell lines NA and HSC-4 was suppressed by treatment with either NS-398, a selective COX-2 inhibitor, or COX-2 antisense oligonucleotide (AS). In the present study, to explore the effects of COX-2 inhibition on the interaction between cancer cells and fibroblasts, we examined the effects of these anti-COX-2 reagents on the expression of matrix metalloproteinases (MMPs) in fibroblast cell lines WI-38 and MRC-5. Western blotting and enzyme-linked immunosorbent assay revealed that NS-398 and COX-2 AS down-regulated the expression and secretion of MMP-2 and the tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) in human fibroblast cell lines. Furthermore, invasion activity of OSCC cells was down-regulated by the addition of culture supernatant from fibroblasts treated with anti-COX-2 reagents in a Matrigel® invasion assay. These results suggest that selective COX-2 inhibition suppresses the invasion activity of OSCC cells via down-regulation of an MMP-2-activating mechanism involving TIMP-2 and production of the MMP-2 protein by an interaction between cancer cells and stromal fibroblasts. Genetic or pharmacological inhibition of COX-2 may therefore be a beneficial strategy in the treatment of OSCC.

Inhibition of cyclooxygenase-2 suppresses invasiveness of oral squamous cell carcinoma cell lines via down-regulation of matrix metalloproteinase-2 and CD44

Expression of cyclooxygenase-2 (COX-2) in tumors is known to be associated with enhanced angiogenesis, suppression of host immunity, and tumor invasion. In the present study, human oral squamous cell carcinoma (OSCC) cell lines NA and HSC-4 were used to evaluate the effects of NS-398, a selective inhibitor of COX-2, and COX-2 antisense oligonucleotide (COX-2 AS) on the invasion activity of OSCC cells. Matrigel invasion assay revealed that the invasiveness of NA and HSC-4 was suppressed by treatment with either NS-398 or COX-2 AS. These reagents down-regulated the secretion of matrix metalloproteinase-2 (MMP-2) to culture supernatant as well as the expression of MMP-2 mRNA and protein. Membrane-type 1 matrix metalloproteinase (MT1-MMP), an activator of proMMP-2, was also down-regulated by treatment with these reagents. Furthermore, expression of CD44 on the surface of these cells was reduced by treatment with either NS-398 or COX-2 AS. In addition, MMP-2 antisense oligonucleotides reduced the expression of CD44 on the surface of both OSCC cell lines. These findings suggest that NS-398 and COX-2 AS suppress the invasiveness of OSCC cells via down-regulation of MMP-2 and CD44. Genetic or pharmacological inhibition of COX-2 may therefore be a beneficial strategy in the treatment of OSCC patients.

Telomerase-Specific Virotheranostics for Human Head and Neck Cancer

Purpose: Long-term outcomes of patients with squamous cell carcinoma of the head and neck (SCCHN) remain unsatisfactory despite advances in combination of treatment modalities. SCCHN is characterized by locoregional spread and it is clinically accessible, making it an attractive target for intratumoral biological therapies. Experimental Design: OBP-301 is a type 5 adenovirus that contains the replication cassette in which the human telomerase reverse transcriptase promoter drives expression of the E1 genes. OBP-401 contained the replication cassette and the green fluorescent protein (GFP) gene. The antitumor effects of OBP-301 were evaluated in vitro by the sodium 30-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate assay and in vivo in an orthotopic xenograft model. Virus spread into the lymphatics was also orthotopically assessed by using OBP-401. Results: Intratumoral injection of OBP-301 resulted in the shrinkage of human SCCHN tumors orthotopically implanted into the tongues of BALB/c nu/nu mice and significantly recovered weight loss by enabling oral ingestion. The levels of GFP expression following ex vivo infection of OBP-401 may be of value as a positive predictive marker for the outcome of telomerase-specific virotherapy. Moreover, whole-body fluorescent imaging revealed that intratumorally injected OBP-401 could visualize the metastatic lymph nodes, indicating the ability of the virus to traffic to the regional lymphatic area and to selectively replicate in neoplastic lesions, resulting in GFP expression and cell death in metastatic lymph nodes. Conclusions: These results illustrate the potential of telomerase-specific oncolytic viruses for a novel therapeutic and diagnostic approach, termed theranostics, for human SCCHN.

Zygoma implant-supported prosthetic rehabilitation of a patient with a maxillary defect

This clinical report describes the successful management of a patient who underwent extensive resection of a maxillary cancer, by introduction of a maxillary obturator prosthesis using zygoma implants. The patient was a 57-year-old man with cancer of the upper anterior gingiva. The maxillary bone in the affected region had been extensively excised by radical surgery. Owing to loss of teeth retaining the denture, the existing prosthesis was unstable, and the patient experienced severe speech and mastication disorders. Four zygoma implants (two on each side), and two conventional dental implants (one each at both maxillary tuberosities) were used as denture retainers. The obturator prosthesis was stabilized by the implants, and the patients oral function improved. High-level compatibility between the implant and surrounding tissue was obtained by mucosal regeneration around the implant. The results suggest that the combination of zygoma and conventional dental implants improves postoperative oral function by facilitating retention of the obturator prostheses.

Antitumor activity of suberoylanilide hydroxamic acid against human oral squamous cell carcinoma cell lines in vitro and in vivo

It has been reported recently that histone deacetylase inhibitors (HDACIs) can block the growth of a variety of malignant tumor cells by reversing the silencing of the tumor suppressor genes; these will be the anticancer agents of the next generation. In this study, we evaluated the antitumor effects of the HDACI suberoylanilide hydroxamic acid (SAHA) on oral squamous cell carcinoma (OSCC) and investigated its molecular mechanism. SAHA suppressed the in vitro proliferation of OSCC cell lines in a dose- and time-dependent manner. Flow cytometric analyses showed that treatment with SAHA led to G1 phase cell-cycle arrest of OSCC cells, accompanying a decrease in the percentage of S-phase cells. Western blot analyses demonstrated that the expression of p21 protein was remarkably augmented and hyperacetylation of p53 was induced after SAHA treatment. These results suggest that administration of SAHA suppresses OSCC growth through G1 phase arrest. Additionally, we observed that the growth of xenograft SAS tumors in nude mice was significantly blocked by the administration of SAHA without major adverse effects.

Evaluation of trismus after treatment of mandibular fractures

This retrospective study examines the effects of treatment modalities and other contributing factors on the duration of trismus following mandibular fractures. Under a strict protocol, 80 cases involving a total of 120 fractures and their treatment modalities were reviewed. An investigation of duration of trismus was carried out in several ways. Although a relationship was established between age, initial interincisal distance, and duration of trismus, no relationship was found with the number or location of fractures, or reduction method. However, after examining the fixation methods separately, it was found that those cases in which Champy plates were used experienced a shorter duration of trismus compared with those using closed reduction with maxillomandibular fixation.