Hideaki Tsuzuki

Expression of p27 is associated with Bax expression and spontaneous apoptosis in oral and oropharyngeal carcinoma

p27Kip1, a cyclin‐dependent kinase inhibitor, is a negative regulator of the cell cycle, and apoptosis is a genetically encoded program of cell death. To clarify the relationship between the cell cycle and apoptosis, we investigated expression of p27, cyclin D1 and apoptosis‐related proteins (p53, Bax, Bcl‐2 and c‐Myc) in 60 cases of oral and oropharyngeal squamous‐cell carcinoma (SCC) using an immuno‐histochemical approach, and evaluated spontaneous apoptosis in vivo. Our most notable finding was that spontaneous apoptosis in the p27‐positive group was significantly higher than that in the p27‐negative group (p = 0.028). In addition, the percentage of p27‐positive cells was clearly correlated with that of Bax‐positive cells (γ = 0.288, p = 0.028) and with that of cyclin D1‐positive cells (γ = 0.416, p = 0.002). Expression of p27 was inversely associated with the clinical stage of total tumor progression (p = 0.027). However, no correlation was found between p27 expression and the following parameters: gender, tumor size, lymph node metastasis, overall survival and disease‐free survival. Our results give evidence that the action of the cell‐cycle regulator p27 is closely linked with apoptosis in clinical samples from patients and indicate that over‐expression of p27 might induce apoptosis in cancer cells through elevation of Bax expression, thereby acting on tumor progression. Int. J. Cancer (Pred. Oncol.) 84:315–320, 1999.

Inhibition of N-linked glycosylation by tunicamycin enhances sensitivity to cisplatin in human head-and-neck carcinoma cells.

Tunicamycin (TM), a naturally occurring antibiotic, blocks the first step in the biosynthesis of N‐linked oligosaccharides in cells. In this study, we investigated whether changes in N‐linked glycosylation affect the sensitivity of head‐and‐neck carcinoma cell lines to cis‐diaminedichloroplatinum(II) (cisplatin) in vitro and in vivo. In vitro treatment of the IMC‐3 and KB cell lines with TM significantly decreased the 50% inhibitory concentration (IC50) of cisplatin, as determined by the MTT assay (24.15 to 10.97 μg/ml, p < 0.05). In addition, TM significantly decreased the IC50 of cisplatin against established cisplatin‐resistant IMC‐3/CR cells (>100 to 14.4 μg/ml, p < 0.05) to levels similar to those against parental IMC‐3 cells. TM treatment decreased the number of Con A‐ and L‐PHA‐binding sites on the surface of tumor cells but had no effect on the intracellular platinum concentration. Induction of apoptosis in vitro by TM plus cisplatin in combination was increased compared with that by cisplatin alone. Furthermore, in vivo administration of TM plus cisplatin in combination significantly inhibited local tumor growth in the cisplatin‐resistant in vivo C3H/He mouse model as compared with the control group (p < 0.05) and increased in vivo apoptosis of tumor cells. Our results suggest that the manipulation of glycosylation by TM in tumor cells might be a useful therapeutic strategy for successful chemotherapy using cisplatin against head‐and‐neck cancer. Int. J. Cancer80:279–284, 1999.

IL-10 expression is associated with the expression of platelet- derived endothelial cell growth factor and prognosis in oral and oropharyngeal carcinoma

We investigated the expression of IL-10 in oral and oropharyngeal squamous cell carcinoma (SCC) specimens by an immunohistochemical technique. Of 58 SCC, 13 (22%) and 35 (60%) cases showed intense and moderate positive staining of IL-10, respectively. There was no association between the staining of IL-10 and clinicopathological features. However, the patients with intense staining of IL-10 had a significantly lower overall survival rate than those with moderate or negative staining of IL-10 (P = 0.019). In addition, the patients with intense staining of IL-10 had the highest score of platelet-derived endothelial cell growth factor (PD-ECGF), which is established as a poor prognostic indicator (P = 0.0105). These results suggested that IL-10 contributes to the clinical outcome of oral and oropharyngeal SCC.

Granulocyte‐colony‐stimulating factor enhances invasive potential of human head‐and‐neck‐carcinoma cell lines

Granulocyte-colony-stimulating factor (G-CSF), a hematopoietic cytokine, regulates the proliferation and differentiation of granulocytic progenitor cells and functionally activated mature neutrophils. G-CSF also affects nonhematopoietic tumor cells by the binding of G-CSF to its specific receptor (G-CSFR) on the cells. In this study, we investigated the effect of G-CSF on the invasive potential of head-and-neck carcinoma cells, and explored the intracellular events initiated by the binding of G-CSF in tumor cells. In vitro treatment of head-and-neck-carcinoma cell lines, IMC-2, IMC-3, KB, Ca9-22, SCCKN and SCCTF, with recombinant G-CSF (rG-CSF) significantly augmented their invasive potential in dose- and time-dependent manners. Among these cancer cells, IMC-2, IMC-3, KB and Ca9-22 cells produced little G-CSF, while large amounts of G-CSF were produced by SCCKN and SCCTF cell lines. Anti-G-CSF antibody (Ab) abrogated the rG-CSF-enhanced invasiveness to the control level of that in untreated cancer cell lines. Immunocytochemical staining and Western blotting using anti-G-CSFR monoclonal antibody (MAb) revealed the expression of G-CSFR on head-and-neck-cancer cell lines exhibiting the enhancement of invasive activity by rG-CSF. IMC-2 cells, having the highest invasive ability among the cell lines used, showed augmentation of G-CSFR expression on stimulation with rG-CSF. Furthermore, stimulation of IMC-2 cells with rG-CSF induced rapid activation of tyrosine-phosphorylated JAK1, suggesting that the G-CSF signal may be transduced into the cells through G-CSFR. Moreover, the gelatinolytic activity of IMC-2 cells was enhanced by stimulation of rG-CSF, and the enhanced invasiveness was inhibited on addition of the tissue inhibitors of metalloproteinases (TIMPs). These results suggest that exogenous rG-CSF may increase the risk of metastasis and/or local recurrence in patients with G-CSFR-positive head-and-neck squamous-cell carcinoma, via an invasive mechanism.

Decreased expression of Bax is correlated with poor prognosis in oral and oropharyngeal carcinoma

We investigated the expression of apoptosis-related factors, p53, Bax, Bcl-2, and spontaneous apoptosis in 57 cases of oral and oropharyngeal squamous cell carcinoma (SCC) by immunochemical staining and ApopTag kit. Positive expression of Bax was inversely associated with advanced tumor stage (P = 0.0225), lymph node metastasis (P = 0.0225), clinical stage (P = 0.0083) and poor prognosis (P = 0.0478). Positive expression of p53 was related to poor prognosis (P = 0.0445) and was associated with negative expression of Bax (P = 0.0439). The apoptosis index did not correlate with clinical outcome. These results suggest that abnormality of Bax expression plays an important role in tumor progression in oral and oropharyngeal SCC.

Expression of protein p27 is associated with progression and prognosis in laryngeal cancer

Objective: A cyclin‐dependent kinase inhibitor, p27kip1, is recognized as a negative regulator of the cell cycle. To clarify whether immunohistochemical detection of p27 might provide prognostic information, we investigated the expression of p27 in laryngeal squamous cell carcinoma (SCC). Study Design: A retrospective study of patients was performed in 109 cases of laryngeal SCC. In addition, we investigated the expression of p53 and granulocyte colony‐stimulating factor receptor (GCSF‐R) to examine the prognostic significance of them in the same samples. Methods: Immunohistochemical staining by specific monoclonal antibodies was performed using the avidin‐biotin‐peroxidase complex technique. Results: Advanced tumor size and clinical stage and the occurrence of lymph node metastasis were associated with the absence of p27 expression, but not correlated with p53 expression and GCSF‐R expression. The overall 5‐year survival rate in the p27‐positive group was significantly higher than that in the p27‐negative group. In the Cox proportional hazard model, p27 was demonstrated to be the most powerful prognostic factor among gender, tumor size, lymph node metastasis, stage of disease, and p53 and GCSF‐R expression. Conclusions: We concluded that assessment of p27 expression is useful as a prognostic factor for laryngeal SCC and of value in selecting patients with laryngeal SCC for aggressive therapy.

Apoptosis‐promoting gene (bax) transfer potentiates sensitivity of squamous cell carcinoma to cisplatin In vitro and In vivo

Modulation of apoptosis may potentiate the sensitivity of tumor cells to chemotherapeutic agents, thus improving the clinical outcome of cancer treatment. Bax, an apoptosis‐promoting member of the bcl‐2 family, may be a key factor influencing the chemosensitivity of tumor cells, however, its involvement in cellular sensitivity to anti‐cancer drugs remains uncertain in squamous cell carcinoma (SCC). To investigate the role of bax gene expression in modulating cisplatin (CDDP)‐induced apoptosis in vitro, an established CDDP‐resistant human head and neck SCC (IMC‐3 cell line) was transfected with bax gene‐bearing mammalian expression vector. Overexpression of the bax gene in CDDP‐resistant IMC‐3 cells elevated the CDDP susceptibility of tumor cells to a level similar to that of the parental IMC‐3 cells. In an in vivo study, percutaneous transfer of apoptosis‐promoting bax gene by particle‐mediated (gene gun) delivery caused overexpression of Bax in SCC, which was confirmed by immunohistochemical staining, and inhibited the growth of mouse CDDP‐resistant SCC. Furthermore, combination therapy with bax gene transfer and subcutaneous administration of CDDP at 3‐day intervals markedly inhibited the growth of mouse SCC. Thus, overexpression of bax in SCC by a gene gun system appears to be a rational approach to improving the efficacy of chemotherapy and treatment outcome. We suggest that exogenous bax expression may have therapeutic applications for enhancing chemotherapy in SCC. Int. J. Cancer 82:860–867, 1999.

Gene Expression Analysis of Human Middle Ear Cholesteatoma Using Complementary DNA Arrays

Objective To identify genes regulated in human cholesteatoma compared with normal skin tissue using complementary DNA arrays.

Expression of hMSH2 correlates with in vitro chemosensitivity to CDDP cytotoxicity in oral and oropharyngeal carcinoma

We investigated the expression of hMSH2, a human mutS homologue from chromosome 2p, in oral and oropharyngeal squamous cell carcinoma (SCC) by an immunohistochemical technique and performed tumor in vitro chemosensitivity testing. In 58 oral and oropharyngeal SCC, the hMSH2 positive score was inversely associated with tumor size, but not with other clinical parameters. Among five anticancer drugs (cisplatin (CDDP), 5-FU, peplomycin, mitomycin C and doxorubicin), only for CDDP was sensitivity to cytotoxicity correlated with the hMSH2 positive score. The susceptibility of hMSH2-positive tumors to CDDP killing was significantly higher than that of hMSH2-negative tumors. Immunohistochemical results regarding hMSH2 are promising in the evaluation of the sensitivity of cancer cells to CDDP cytotoxicity and enable one to select patients for adjuvant chemotherapy for oral and oropharyngeal SCC.

In vitro Effect of Hyperthermia on Chemoenhancement and Uptake of Cisplatin in Human Pharyngeal Carcinoma KB Cells

The purpose of this study was to assess the efficacy of hyperthermia (42 or 44 degrees C) as a modifier of cis-diamminedichloroplatinum (II) (CDDP) cytotoxicity and platinum incorporation in human pharyngeal carcinoma KB cells. To maximize the interactive effect, we examined the time sequence of high (above 43 degrees C) or low (below 43 degrees C) hyperthermia and CDDP. Within the dose range of CDDP studied, there was a marked synergism between the effects of heating at 44 degrees C and subsequent CDDP exposure for 5 h. Pretreatment at 44 degrees C for 30 min or at 42 degrees C for 4 h enhanced CDDP cytotoxicity more than posttreatment at 44 degrees C for 30 min or at 42 degrees C for 4 h. However, the chemoenhancement ratio of pretreatment at 44 degrees C for 30 min was higher then that of pretreatment at 42 degrees C for 4 h, although the thermal isotoxic dose decreased the cell count to 60% under both conditions. There was a significant increase in CDDP uptake after hyperthermia at 44 degrees C. These results indicate that high hyperthermia effectively enhances subsequent CDDP cytotoxicity in human pharyngeal carcinoma KB cells.