Kei Ukon

Synergistic effects of docetaxel and S‐1 by modulating the expression of metabolic enzymes of 5‐fluorouracil in human gastric cancer cell lines

We have recently demonstrated in a Phase I/II study that combination chemotherapy with docetaxel (TXT) and S‐1 is active against metastatic gastric carcinomas. To elucidate the mechanisms underlying the synergistic effects of these drugs, both the growth inhibitory effects and the expression profiles of enzymes involved in fluorouracil (5‐FU) metabolism were examined in vitro and in vivo. TXT alone and in combination with 5‐FU inhibited the growth of each of the 5 gastric cancer cell lines that we examined (TMK‐1, and MKN‐1, ‐28, ‐45 and ‐74), in a time‐ and dose‐dependent manner. Moreover, striking synergistic effects were observed in TMK‐1 cells in vitro with IC50 values of between 4.73 and 0.61 nM 5‐FU. Furthermore, in TMK‐1 xenografts, 5‐FU/TXT cotreatments exhibited synergistic antitumor effects. The combination of S‐1 and TXT, however, exhibited greater growth‐inhibitory effects than the 5‐FU/TXT cotreatments. The mechanisms underlying these synergistic effects of S‐1 and TXT were examined by expression and activity analyses of the 5‐FU metabolic enzymes. The expression of thymidylate synthase (TS), and dihydropyrimidine dehydrogenase (DPD) were decreased 50 and 73% of control levels, respectively, and that of orotate phosphorybosyl transferase (OPRT) was increased by 3.9‐fold at the protein level. These findings suggested that biochemical modulation of the 2 drugs had occurred, which was further confirmed by the results of the activity assays. These data strongly indicate that a combination chemotherapy of TXT and S‐1 is effective against gastric carcinomas and is therefore a good candidate as a standard chemotherapeutic strategy in treating these tumors.

Aberrant methylation of DPYD promoter, DPYD expression, and cellular sensitivity to 5-fluorouracil in cancer cells.

Purpose: Dihydropyrimidine dehydrogenase (DPD), the initial rate-limiting enzyme in the degradation of 5-fluorouracil (5-FU), is known to be a principal factor in clinical responses to the anticancer agent 5-FU, and various reports have clearly demonstrated that DPD activity is closely correlated to mRNA levels. However, the regulatory mechanisms of DPD gene (DPYD) expression remain unclear. In this study, the regulatory mechanisms have been intensively studied. Experimental Design and Results: A subcloned 3.0-kb fragment of the 5′ region of DPYD contains a total of 60 CpG sites, suggesting that methylation status may affect the repression of DPYD. The clone showed various promoter activities that were largely correlated with mRNA levels in most cell lines, except HSC3 and HepG2. Bisulfite sequencing analysis revealed that various CpG sites around the transcription start site were abnormally methylated in cells with low DPYD expression: Reversal of hypermethylation by 5-azacytidine treatment significantly increased DPYD expression in HSC3 and HepG2 cells that showed strong promoter activity. In HepG2, in vitro methylation of the DPYD promoter directly decreased promoter activity, and 5-azacytidine treatment restored higher DPYD expression in a dose- and time-dependent manner, along with decreased sensitivity to 5-FU. Conclusions: We found that DPD activity was controlled, at least in part, at the transcription level of DPYD and that aberrant methylation of the DPYD promoter region acted as one of the repressors of DPYD expression and affected sensitivity to 5-FU in cancer cells. Our new results could lead to a more precise understanding of the molecular basis of 5-FU response.

Sivelestat, a specific neutrophil elastase inhibitor, suppresses the growth of gastric carcinoma cells by preventing the release of transforming growth factor-α

Neutrophil elastase is a neutral serine proteinase produced by polymorphonuclear leukocytes and monocytes/macrophages, especially under surgical stress. In the present study, we investigated whether NE promotes cell growth by activation of EGFR to elucidate whether surgical stress induces tumor proliferation and progression. Furthermore, we examined the antitumor effect of a specific NE inhibitor, sivelestat. Cell growth assays were carried out in vitro and in vivo using TMK‐1 gastric cancer cells. TMK‐1 cell growth was stimulated to 118% of that of the control cells after 48 h stimulation with 1 µg/mL NE according to an MTT assay. Sivelestat inhibited cell growth to 23.4 and 58.0% of control values at concentrations of 100 and 1000 µg/mL, respectively. NE rapidly phosphorylated EGFR in only 5 min and triggered the ERK1/2–mitogenic signaling pathway in TMK‐1. It was further demonstrated that NE‐induced EGFR phosphorylation was transactivated through TGF‐α, using ELISA. NE increased the cleavage of TGF‐α from the cell surface 30‐fold compared with the cells without treatment. Interestingly, sivelestat significantly reduced NE‐induced EGFR phosphorylation and ERK1/2 activation and completely blocked the release of TGF‐α from the TMK‐1 cell surface. In a xenograft study, the addition of ventrotomy as a surgical stress promoted tumor growth. Sivelestat significantly suppressed the tumor growth induced by surgical stress. These results indicate that sivelestat suppresses the growth of gastric cancer cells by inhibiting the release of TGF‐α stimulated by NE, which often occurs after surgical stresses. (Cancer Sci 2006; 97: 1037–1043)

Kidney metastasis of resected early gastric carcinoma: report of a case.

We report a rare case of kidney metastasis of resected early gastric cancer in a 67-year-old man. We performed distal gastrectomy with D2 lymph node dissection for early gastric cancer, which was histologically diagnosed as moderately differentiated adenocarcinoma (T1N0M0, stage IA). Preoperatively, his serum carcinoembryonic antigen (CEA) level was 9.5 ng/ml, and this dropped to 7.0 ng/ml postoperatively. However, 1 year 10 months after the operation, we performed partial kidney resection and the lesion was confirmed to be a metastasis of the gastric cancer. Unfortunately, 5 months later, multiple liver metastasis was found, accompanied by a further increase in the serum CEA level to 2 650.8 ng/ml. This case illustrates the poor prognosis associated with a high preoperative serum CEA level, even if early gastric cancer is resected curatively.