Hiroshi Mese

Connective tissue growth factor as a major angiogenic agent that is induced by hypoxia in a human breast cancer cell line

Connective tissue growth factor (CTGF) is known to be a potent angiogenic factor. Here, we present the evidence that the hypoxic induction of angiogenesis by human breast cancer cells (MDA-231) can be ascribed at least in part to CTGF. Our results indicate that (i) CTGF is abundantly present in MDA-231 cells in vitro and in vivo, (ii) its secretion is up-regulated by hypoxia, and (iii) its gene expression is enhanced in MDA-231 cells cultured under hypoxic conditions. These data suggest CTGF may stimulate angiogenesis by paracrine mechanisms, thereby contributing to the invasion of breast cancer cells. This is the first evidence that human cancer cells differentially express CTGF protein and mRNA under the control of hypoxic conditions.

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.

Expression of vascular endothelial growth factor-C predicts regional lymph node metastasis in early oral squamous cell carcinoma.

The purpose of this study was to investigate the association of the expression of vascular endothelial growth factor-C (VEGF-C) with regional lymph node metastasis in oral squamous cell carcinoma (OSCC). The expression of VEGF-C in biopsy specimens obtained from 62 patients with OSCC was examined by immunohistochemistry. In the early stages of T1 and T2 (38 cases), VEGF-C expression strongly correlated with lymph node metastasis (P < 0.001), and the percentage of VEGF-C-positive staining was 88.2% for patients with lymph node metastasis (15 of 17 cases) and 28.6% for those without lymph node metastasis (6 of 21 cases). However, in the advanced stages of T3 and T4, no significant correlation between VEGF-C expression and lymph node metastasis was observed. These results indicate that VEGF-C expression in biopsy specimens can be used as a reliable predictor of regional lymph node metastasis, particularly in early OSCC, and may become an important factor in the selection of appropriate treatment.

Specific inhibitor of MEK-mediated cross-talk between ERK and p38 MAPK during differentiation of human osteosarcoma cells

Osteosarcoma is the most common primary malignant bone tumor, accounting for approximately 20% of all primary sarcomas in bone. Although treatment modalities have been improved over the past decades, it is still a tumor with a high mortality rate in children and young adults. Based on histological considerations, osteosarcoma arises from impaired differentiation of these immature cells into more mature types and that correction of this impairment may reduce malignancy and increase the efficiency of chemotherapy. The purpose of this study was to determine the effect of specific inhibitors of MAPK extracellular signaling-regulated kinase (ERK) kinase (MEK) and p38 on the differentiation of human osteosarcoma cell line SaOS-2 cells. We found that PD98059, a specific inhibitor of MEK, inhibited the serum-stimulated proliferation of SaOS-2 cells; whereas SB203580, a specific inhibitor of p38 MAPK, had little effect on it. SB203580 suppressed ALPase activity, gene expression of type I collagen, and expression of ALP and BMP-2 mRNAs; whereas PD98059 upregulated them dose dependently. In addition, immunoblot and immunostaining analysis revealed that phosphorylation of ERK was increased by treatment with SB203580; whereas PD98059 increased the phosphorylation of p38, which implies a seesaw-like balance between ERK and p38 phosphorylation. We suggest that osteosarcoma cell differentiation is regulated by the balance between the activities of the ERK and p38 pathways and that the MEK/ERK pathway negatively regulates osteosarcoma cell differentiation, whereas the p38 pathway does so positively. MEK inhibitor may thus be a good candidate for altering the expression of the osteosarcoma malignant phenotype.

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.

Combination chemotherapy of paclitaxel and cisplatin induces apoptosis with Bcl-2 phosphorylation in a cisplatin-resistant human epidermoid carcinoma cell line.

PurposePaclitaxel (Taxol, TXL) is an antimicrotubule agent that stabilizes microtubules, arrests the cell cycle at the G2/M phase and induces apoptosis. In vitro drug sensitivity assays have shown that the combination of TXL and CDDP is more effective in CDDP-resistant ovarian carcinoma cell lines, with different cytotoxicities depending on the sequence of drug exposure. CDDP also shows poor results in human epidermoid carcinoma particularly of the head and neck region.MethodsWe investigated the effects and the molecular mechanisms of combination chemotherapy with TXL and CDDP in the CDDP-resistant cell line A431/CDDP2, and in its parental human epidermoid cell line A431/P. Drug sensitivity was determined using the MTS assay and cell cycle perturbation was analyzed using flow cytometry. DNA fragmentation was then analyzed and the protein levels of caspase-3 and Bcl-2, and phosphorylated of Bcl-2 were determined by Western blotting.ResultsIn the drug sensitivity assay, exposure to CDDP prior to TXL was more effective than exposure TXL prior to CDDP in A431/P cells. In A431/CDDP2 cells, exposure to TXL prior to CDDP was more effective than exposure to CDDP prior to TXL. Exposure to TXL arrested the cells in the G2/M phase in both cell lines. In A431/CDDP2 cells, exposure to TXL prior to CDDP arrested the cells in the G2/M phase, an effect caused by either CDDP or TXL. Analysis of DNA fragmentation showed similar results to the drug sensitivity assay. Expression of caspase-3 protein active form was detected following exposure to TXL only and to the TXL/CDDP combination in both A431/P and A431/CDDP2 cells, but phosphorylation of Bcl-2 protein was detected only following exposure to TXL and only in A431/CDDP2 cells.ConclusionsThese results indicate that exposure to TXL prior to CDDP plays a key role in circumventing CDDP resistance by phosphorylating Bcl-2 protein in the human epidermoid carcinoma cell line A431/CDDP2.

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.