Hisazumi Ikeda

Hypoxia-inducible factor 1 alpha in oral squamous cell carcinoma and its relation to prognosis

The aim of this study was to investigate the correlation between the expression of hypoxia-inducible factor 1 alpha (HIF-1 alpha) and proliferative activity in tumor cells, lymph node metastasis, as well as prognosis in patients with oral squamous cell carcinoma (OSCC). Fifty-seven biopsy specimens of OSCC were investigated for the expression of HIF-1 alpha and proliferating cell nuclear antigen (PCNA) by immunohistochemistry. None of the patients had received any prior treatments. The percentage of HIF-1 alpha immunopositive area (PHIA) was calculated using computer-assisted image analysis for quantitative assessment of HIF-1 alpha expression. The PCNA labeling index (LI) was evaluated as a proliferation marker. We found that the mean PHIA in all stages was 12.1% in the poor prognosis patients, and it was 6.4% in the good prognosis patients. There was a significant difference of PHIA between poor prognosis and good prognosis patients (P=0.0065). Furthermore, the mean PHIA in the patients who had no metastatic lymph nodes was 7.5%, while it was 11.7% in the patients who had metastatic lymph nodes. There was also a significant difference of PHIA between patients who had no metastatic lymph nodes and those who had metastatic lymph nodes (P=0.0487). On the other hand, significant correlation between PHIA and PCNA LI was not observed. These results provide the clinical data indicating that HIF-1 alpha may play an important role in lymph node metastasis and prognosis in patients with OSCC.

Clinicopathological risk factors for local recurrence in oral squamous cell carcinoma

Local recurrence of oral squamous cell carcinoma (OSCC) after primary surgery has been considered to be a poor prognostic entity in terms of survival rate. The purpose of this study is to evaluate the incidence of local recurrence and to identify significant risk factors for the local recurrence in OSCC. The authors retrospectively reviewed records for 187 patients who underwent radical surgery for OSCC. The local recurrence rate was 16.0% (30/187 patients) in this study. The survival rate of patients with local recurrence was 33.3%, which was significantly lower than that (94.3%) of patients without local recurrence. Pattern of invasion (POI), neoadjuvant chemotherapy (NAC), and the status of the surgical margin were identified as factors influencing local recurrence. In particular, NAC and the status of the surgical margin were independent risk factors by multivariate analysis. The deep margin was resected at a close site in many NAC-treated patients, suggesting that NAC may lead to local recurrence and poor outcomes. No efficacy of NAC was observed, suggesting that the standard treatment of oral cancers is surgery alone.

Inhibitory Effect of Heat Shock Protein 70 on Apoptosis Induced by Photodynamic Therapy In Vitro

We examined the apoptotic effects of photodynamic therapy (PDT) in leukemia cells (HL60) and lymphoma cells (Raji). Moreover, we also investigated the relationship of apoptosis induced by PDT to heat shock protein (HSP) expression. To induce 80% of cell death by PDT, HL60 cells required 6 μg/mL and Raji cells required 9 μg/mL of Photofrin®. PDT induced apoptosis in 77.2% of HL60 and in 0.4% of Raji at lethal dose (LD80) conditions. The cell line in which apoptosis is predisposed may be more susceptible to PDT compared with the cell line in which necrosis is predisposed. Furthermore, HSP‐70 was expressed constitutively in Raji cells but not in HL60 cells. Heat treatment of HL60 cells induced expression of HSP‐70 and resulted in significant reduction of PDT‐mediated apoptosis. From the results of this experiment, it is suggestive that HSP‐70 contributes to inhibition of apoptosis mediated by PDT.

Effect of combined photodynamic and chemotherapeutic treatment on lymphoma cells in vitro

We investigated the cytotoxic and apoptotic effects of a combination of photodynamic therapy (PDT) and cisplatin (CDDP) on L5178Y (LY) cells. Treatment with PDT by photofrin((R)) (5 microg/ml) alone or with CDDP (20 microg/ml) alone killed 41.5+/-8.5% or 42.9+/-6.5% of LY cells, respectively, while a combination of these two treatments killed 99.7+/-0.6%. Apoptotic cell death after combination treatment was also revealed to be 49.6+/-7.8% compared to 12.4+/-3.4% after PDT alone, and 18.8+/-2.6% after CDDP. This study demonstrated that combined treatment of PDT and CDDP results in enhanced apoptotic cell death as well as a cytotoxic effect on LY cells.

Prediction of outcome of patients with oral squamous cell carcinoma using vascular invasion and the strongly positive expression of vascular endothelial growth factors.

Vascular invasion and lymph node metastasis have been used as histopathological prognosticators of cancers including oral squamous cell carcinoma (OSCC). In addition to metastatic potential via blood vessels, tumor-induced angiogenesis might also be associated with prognosis. However, the efficacy of combined evaluation of vascular invasion and angiogenesis-associated molecules for the prognosis of OSCC remains obscure. This is also the case in lymph node metastasis and lymphovasculogenesis-associated molecules. The aim of this study was to examine factors related to prognosis to improve the accuracy of prognostic prediction of OSCC using vasculogenesis-associated markers. Ninety specimens of patients from 1991 to 2002 with previously untreated OSCC, who underwent either biopsy or surgery, were histopathologically and immunohistochemically analyzed using antibodies for vascular endothelial growth factor (VEGF)-A, VEGF-C, cyclooxygenase (COX)-2 and Midkine. The ninety cases were composed of 72 well-differentiated, 12 moderately differentiated and 6 poorly differentiated OSCC. Efficient models of prognostic prediction were evaluated by extensive statistical analyses. The presence of vascular invasion or lymph node metastasis was confirmed to be significantly associated with poor prognosis in the univariate analysis. Multivariate logic regression analysis suggested that patients with the strongly positive expression of either VEGF-A or VEGF-C had a significant association with poor prognosis even in patients without vascular invasion and in early-stage patients. Neither COX-2 nor Midkine contributed to predict the prognosis of the patients. The strongly positive expression of VEGF-A or VEGF-C was suggested to reinforce the histopathological diagnosis of vascular invasion and improve the accuracy and efficacy of prognostic prediction of OSCC.

Engineering Bone Formation from Human Dental Pulp- and Periodontal Ligament-Derived Cells

A robust method for inducing bone formation from cultured dental mesenchymal cells has not been established. In this study, a method for generating bone tissue in vivo from cultured human dental pulp- and periodontal ligament-derived cells (DPCs and PDLCs, respectively) was designed using exogenous bone morphogenetic protein 2 (BMP2). DPCs and PDLCs showed enhanced alkaline phosphatase (ALP) activity and calcified nodule formation in medium containing dexamethasone, β-glycerophosphate, and ascorbic acid (osteogenic medium). However, the addition of recombinant human bone morphogenetic protein 2 (rhBMP2) to osteogenic medium remarkably increased ALP activity and in vitro calcification above the increases observed with osteogenic medium alone. rhBMP2 also significantly upregulated the expression of osteocalcin, osteopontin, and dentin matrix protein 1 mRNA in both cell types cultured in osteogenic medium. Finally, we detected prominent bone-like tissue formation in vivo when cells had been exposed to rhBMP2 in osteogenic medium. In contrast, treatments with osteogenic medium or rhBMP2 alone could not induce abundant mineralized tissue formation. We propose here that treatment with rhBMP2 in osteogenic medium can make dental mesenchymal tissues a highly useful source of cells for bone tissue engineering. In addition, both DPCs and PDLCs showed similar and remarkable osteo-inducibility.

Enhancement of the photodynamic antitumor effect by streptococcal preparation OK-432 in the mouse carcinoma.

Abstract Biological response modifier antitumor effects are enhanced by the activation of the host defense mechanisms. We have investigated the antitumor effect of photodynamic therapy (PDT) and/or local administration of a biological response modifier, the streptococcal preparation OK-432, on transplanted NR-S1 mouse squamous cell carcinoma. Hematoporphyrin oligomers (20 mg/kg body weight) were used to photosensitize PDT. A pulsed Nd:YAG dye laser, tuned at 630 nm, was used as the light source. The laser power was 15 mJ cm−2 pulse−1, and the irradiation time was 40 min. The photosensitizer was injected intraperitoneally 48 h before laser irradiation. Where used, OK-432 was injected into the tumor either 3 h prior to PDT or immediately afterwards. The antitumor effects were evaluated 48 h after each protocol by (a) estimating the area of tumor necrosis (%) in hematoxylin/eosin-stained specimens, and (b) bromodeoxyuridine immunohistochemistry. Furthermore, the tumor sizes were evaluated 3, 7 and 10 days after each protocol, and the survival time after each protocol was evaluated as well. The anti-tumor effect of PDT was enhanced by administration of OK-432 3 h before PDT, whereas the administration of OK-432 immediately after PDT did not potentiate a PDT antitumor effect. Treatment with OK-432 alone had little effect on tumors. Photodynamic therapy in combination with local administration of OK-432 3 h before PDT is considered to be a useful treatment modality.

Tumour destruction and proliferation kinetics following periodic, low power light, haematoporphyrin oligomers mediated photodynamic therapy in the mouse tongue

Photodynamic therapy (PDT) is an experimental modality in the treatment of cancer. It involves photochemical reactions that require the interaction of a photosensitising drug, light and oxygen. The development of an efficient protocol based on assuring oxygen availability through modulation of the incident light power density and its mode of delivery was addressed in this study. An estimated energy dose of 180 J/cm2 of 630 nm light from pulsed Nd:YAG dye laser was delivered 24 h after injection of 10 mg/kg haematoporphyrin oligomers in C3H/HeNCrj mice bearing the transplantable squamous cell carcinoma NR-S1, by either of these light regimens: (1) 5 mJ/cm2/pulse for 30 min, 1 h dark interval, followed by another 30 min exposure to the same power (low power, periodic light regimen) or (2) 15 mJ/cm2/pulse for 20 min (high power, continuous light regimen). Results showed a higher mean percentage area of tumour destruction with the low power, periodic light regimen at 54.34% in contrast to 12.44% of the high power, continuous light regimen 2 days after PDT. Furthermore, the mean bromodeoxyuridine labelling indices of the remaining viable-appearing cancer cells were 27.90 and 42.41, respectively, indicating a smaller tumour growth fraction with the former regimen. These results suggest that use of low power, periodically delivered light increases the antitumour efficacy of PDT.

Expression of the cancer stem cell markers CD44v6 and ABCG2 in tongue cancer: Effect of neoadjuvant chemotherapy on local recurrence

The efficacy of neoadjuvant chemotherapy (NAC) is controversial, and no report supports NAC with a high evidence level. Recently, we showed that a deep surgical margin was resected very close to the tumor site in many NAC-treated oral squamous cell carcinoma patients, suggesting that NAC may lead to local recurrence and poor outcomes. The purpose of this study was to evaluate the effect of NAC on tumor local recurrence using cancer stem cell marker immunohistochemistry. We retrospectively analyzed 89 patients who underwent radical surgery for tongue cancer, and examined the effect of NAC on tumor local recurrence. Cancer stem cell marker (CD44v6 and ABCG2) expression was detected by immunohistochemistry. In our study, the local recurrence rate was 12.4%. CD44v6 and ABCG2 expression was significantly associated with regional lymph node metastasis, pattern of invasion, depth of invasion, perineural invasion and local recurrence, respectively. Tumor local recurrence was a significant independent predictive factor of the 5-year disease specific survival. CD44v6 or ABCG2 positivity in NAC-treated patients was significantly associated with tumor local recurrence. It was suggested that local recurrence in NAC-treated cases is associated with cancer stem-like cells. We propose that NAC leads to the selection and/or residue of more aggressive cancer stem-like cells.

The observational study of delayed wound healing after tooth extraction in patients receiving oral bisphosphonate therapy.

INTRODUCTION In this study, we investigated whether such a discontinuation of oral bisphosphonate (BP) for 3 months might influence the incidence of BP-related osteonecrosis of the jaw (BRONJ) and wound healing after tooth extraction in patients receiving oral BP therapy. MATERIAL AND METHODS There were a total of 434 teeth in 201 patients (18 males and 183 females). The patients were divided into two groups depending on whether or not they underwent a 3-month discontinuation of BP therapy (BP- and BP+) before tooth extraction. In this observational study investigated delayed wound healing after tooth extraction in patients receiving oral BP therapy. RESULTS In all cases of the BP- group, there were no BRONJ although there was delayed wound healing in two cases. However, in one case of the BP+ group, oral BP was continued because it was deemed high risk to discontinue treatment by the patients physician. In this case, an intraoral fistula was still present with bone exposure at 120 weeks after extraction (BRONJ stage 1). CONCLUSION This study supports the idea of a drug holiday and encourages further clinical research on this topic of tooth extraction in patients receiving oral BP therapy.