Shuko Nakayama

The E-cadherin gene is silenced by CpG methylation in human oral squamous cell carcinomas

Reduction of E‐cadherin strongly relates to invasiveness and metastasis in vitro. To clarify CpG methylation around the promoter region of the E‐cadherin gene in oral squamous cell carcinoma (SCC), we examined the DNA samples of various human SCC cell lines and primary oral SCC tissues by methylation‐specific polymerase chain reaction (MSP). CpG methylation of the E‐cadherin gene markedly correlated to the reduction of E‐cadherin expression in human oral SCC cell lines. In primary oral SCC tissues, only 1 of 5 preserved E‐cadherin‐expressing tissues was methylated, whereas methylation was found in 17 (94.4%) of 18 E‐cadherin‐reduced tissues. Our results suggest that reduction of E‐cadherin expression is associated with CpG methylation of the E‐cadherin gene promoter. We recently established two cell lines with high and low metastatic potential, UM1 and UM2, from SCC primary tongue tissue of a patient. E‐cadherin expression of high‐metastatic UM1 was clearly lower than that of low‐metastatic UM2, and MSP results showed CpG methylation in the UM1 but not the UM2 cell line. To investigate whether demethylation of CpG methylation of the E‐cadherin gene could restore expression and function of E‐cadherin, we treated UM1 with the demethylating agent 5‐azacytidine (5‐aza) and found that E‐cadherin expression was indeed restored by demethylation. Moreover, in the demethylated UM1, invasion of the collagen gel was clearly suppressed compared with the untreated UM1. These results suggested that inactivation of E‐cadherin expression resulted from CpG methylation of the gene promoter; a correlation between CpG methylation of the E‐cadherin gene promoter and invasive potential was also suggested.

Establishment of High and Low Metastasis Cell Lines Derived from a Human Tongue Squamous Cell Carcinoma

Malignant tumors are composed of cells with different phenotypic properties and only certain cell subpopulations present metastatic potential. The establishment of cell lines with high or low metastatic potential is necessary to investigate the molecular mechanisms of the metastatic process. However, human oral squamous cell carcinoma (SCC) cell lines that are suitable for the above investigation are scarce. High and low metastatic cells were obtained from a primary lesion of a patient with tongue carcinoma who had not received any therapy. Two distinct cell lines were selected, UM1 with a scattered growth pattern and loose cell-cell adhesion, and UM2 with a colony-formed growth pattern and firm cell-cell adhesion. The expression of E-cadherin in UM1 was clearly lower than that in UM2. UM1 exhibited a higher motility, invasive and metastatic activity than UM2 in vivo and in vitro. A low invasive and a metastatic oral SCC cell line, useful to investigate invasion and metastasis mechanisms, have been established.

Inhibition of Telomerase activity as a measure of tumor cell killing by cisplatin in squamous cell carcinoma cell line

Background: Telomerase is a ribonucleoprotein enzyme which is involved in the maintenance of chromosome ends. Telomerase activity is frequently associated with malignant phenotypes, and it can be considered a ubiquitous tumor marker. In this study we describe an approach for developing in vitro chemosensitivity assays based on the assessment of telomerase activity in squamous cell carcinoma to treatment with anticancer drugs. Methods: We used A431/CDDP1 and A431/CDDP2, two previously established cisplatin (CDDP)-resistant A431 sublines. These cell lines were not multidrug resistant but specifically resistant to the CDDP drug family. The telomeric repeat amplification protocol (TRAP assay) was used to measure telomerase activity in CDDP-resistant cell lines and to examine the effect of CDDP and other anticancer drugs on enzyme levels. We next analyzed the relations between telomerase activity and cytotoxic effects of CDDP in human squamous cell carcinoma (HSC2, HSC3, HSC4 and KB cell lines). Results: The telomerase activity of A431/CDDP1 and A431/CDDP2 was significantly higher than that of the parent A431 cell (A431/P) treated with CDDP and carboplatin at IC50. On the other hand, telomerase activity in these cell lines was not influenced by treatment with 5-fluorouracil, bleomycin, Adriamycin or taxol. The regression line derived from the quantitative analysis of the telomeric ladder versus IC50 of CDDP concentrations was analyzed. Telomerase activity was found to be positively correlated with the IC50 values for CDDP. Conclusions: The results of the present studies on in vitro squamous cell carcinoma cell lines indicate that telomerase activity inhibition can be used as a marker of tumor cell killing by CDDP. Therefore, the present investigation provides a rational basis for an in vitro chemosensitivity assay based on telomerase activity evaluation.

Regulation of Apoptosis Reduction in the Cisplatin-Resistant A431 Cell Line by Bcl-2 and CPP32

Cisplatin (cis-diamminedichloroplatinum(II), CDDP) is one of the most important chemotherapeutic agents; however, the mechanisms of resistance to this drug are still unknown. Recent reports have demonstrated that chemotherapy can induce apoptosis in some cancer cells, indicating that apoptosis may play a very important role in cancer therapy. Therefore, we used a CDDP-resistant cell line from the human epidermoid carcinoma cell line A431 to investigate whether the modulation of apoptosis influences CDDP resistance. In the CDDP-resistant cell, the cell cycle was not perturbed after CDDP treatment. DNA gel electrophoresis and ELISA of the CDDP-resistant cell showed reduced apoptosis when compared with A431 cells treated with CDDP. We determined the p53, Bcl-2, Bax and CPP32 protein levels by Western blotting. This analysis demonstrated a marked increase in Bcl-2 protein levels and a reduction in CPP32 protein levels in CDDP-resistant cells. Our results indicate that the reduction of apoptosis was one of the CDDP-resistant mechanisms, and that reduced apoptosis in CDDP-resistant cells was influenced by Bcl-2 and CPP32 proteins.

The role of caspase family protease, caspase-3 on cisplatin-induced apoptosis in cisplatin-resistant A431 cell line

Purpose: Cisplatin (cis-diamminedichloroplatinum(II), CDDP) has been reported to induce apoptosis in cancer cells, the mechanism of the apoptosis in cancer cells induced by CDDP is still unclear. Recent studies have revealed that caspase family of cystine proteases play an important role in the regulation of several apoptotic processes. In this study, whether apoptosis induced by CDDP could be mediated by the activation of caspase-3, a caspase family protease, was investigated. Methods: The CDDP-resistant subline A431/CDDP2 from the previously established human epidermoid carcinoma cell line A431 was used. The parent A431 cells (A431/P) and the A431/CDDP2 were exposed to CDDP with or without a caspase family protease inhibitor (Z-Asp-CH2-DCB), and cellular sensitivity to CDDP was determined. DNA fragmentation was then analyzed, and the caspase-3 protein levels determined by Western blotting following exposure of the cells to CDDP with or without Z-Asp-CH2-DCB. Results: In the A431/P cells, the cytotoxicity of CDDP was clearly reduced by Z-Asp-CH2-DCB compared with its cytotoxicity in A431/CDDP2 cells. Furthermore, quantitative analysis of DNA fragmentation revealed that Z-Asp-CH2-DCB inhibited DNA fragmentation induced by CDDP in A431/P cells, but not in A431/CDDP2 cells. Western blotting analysis demonstrated a marked reduction in procaspase-3 protein levels in A431/P cells treated with Z-Asp-CH2-DCB. In the A431/CDDP2 cells, procaspase-3 protein levels were no different with and without Z-Asp-CH2-DCB. Conclusions: These findings suggest that caspase-3 may mediate apoptosis induced by CDDP, and its induction could represent a novel approach to the effective treatment of malignant tumors.

Effects of bisphosphonate on experimental jaw metastasis model in nude mice

The mechanism of osteolysis associated with metastatic cancer of the jaws is essentially osteoclast-mediated. Therefore, it is likely that potent osteoclastic bone resorption inhibitors such as bisphosphonates would be efficacious for the treatment of jaw metastasis. We examined the effects of a third generation bisphosphonate, YM175, in a nude mice jaw metastasis model with intracardiac injection of a human breast cancer cell line, MDA-MB-231. The metastatic lesions in untreated mice were radiographically observed at the body and angle of the mandible. Histology of the mandible of untreated mice revealed that most of the bone marrow cavities had been occupied by the metastatic tumor with active osteoclasts along the trabecular bone. The experimental group showed that YM175 markedly reduced the size of tumor and the number of osteoclasts. These results suggest that YM175 may suppress metastasis formation and tumor growth in jaw through inhibition of osteoclastic bone resorption.

Establishment and Characterization of Cisplatin-Resistant Human Epidermoid Carcinoma Cell Line, A431 Cell

Cisplatin, cis-diamminedichloroplatinum(II) (CDDP) is one of the most important anticancer agents, initially producing good responses in various tumors. However, resistance to this drug often develops in various tumors, and additional administration decreases its chemotherapeutic efficacy. The precise mechanism of acquisition of resistance to this drug is still uncertain. However in the present study, we established two CDDP-resistant sublines A431/CDDP1 and A431/CDDP2 from human epidermoid carcinoma cell line A431. These resistant sublines were constituted by exposing A431 cells to a gradually increasing dose of CDDP (A431/CDDP1), and by mutagenic induction with mutagen (A431/CDDP2). A431/CDDP1 and A431/CDDP2 have developed 3.1 and 2.7 times more resistance to CDDP than the original A431 cell in terms of IC50. The two CDDP-resistant sublines showed cross-resistance to the CDDP analogue, carboplatin (CBDCA), but not to other chemotherapeutic drugs such as Adriamycin (ADR) and 5-fluorouracil (5-FU). These CDDP-resistant sublines were transplanted into nude mice to demonstrate the resistance to CDDP treatment in vivo. According to the in vitro assay, the mechanism of resistance in A431/CDDP1 and A431/CDDP2 seems to be based on a reduction of intracellular accumulation of CDDP, because their platinum concentration, which is the major component of CDDP, significantly declined. The established CDDP-resistant sublines may be used in further trials to improve the understanding of the mechanisms of resistance to CDDP.