Junichi Kaganoi

Nicotine induces the fragile histidine triad methylation in human esophageal squamous epithelial cells

The fragile histidine triad (FHIT) gene has been proposed to have an important role in very early carcinogenesis. Methylation of the FHIT gene is associated with transcriptional inactivation in esophageal squamous cell carcinoma, and FHIT inactivation has been linked to smoking‐related carcinogenesis. In this study, we confirmed methylation of the FHIT gene in human esophageal squamous epithelial cells (HEECs) and examined whether nicotine induced alteration of FHIT. Methylation status in the promoter region of the FHIT gene and p16INK4A gene was determined by methylation‐specific PCR in HEECs exposed to nicotine under various conditions. Methylation status of the FHIT gene was confirmed by DNA‐sequencing analysis. Protein expression of Fhit and the DNA methyltransferases (DNMTs) DNMT1 and DNMT3a were assessed by immunoblot analysis. In the absence of nicotine, methylation of the FHIT gene and attenuation of Fhit protein were not detected in HEECs. Nicotine induced the methylation of FHIT gene and attenuated Fhit protein in association with increased expression of DNMT3a. Reexpression of Fhit protein in HEECs was found after cessation of moderate‐ to long‐term exposure to nicotine. Our results show that nicotine induces methylation of the FHIT gene followed by loss of Fhit protein expression in HEECs. Continuous smoking may thus increase the risk of esophageal cancer.

Detection of circulating oesophageal squamous cancer cells in peripheral blood and its impact on prognosis.

Many studies have attempted to detect cancer cells using the reverse transcription–polymerase chain reaction (RT–PCR) for specific mRNAs. None has examined the correlation between the presence of circulating oesophageal cancer cells in peripheral blood and long‐term outcome.

Detection of lymph node metastasis of oesophageal cancer by RT-nested PCR for SCC antigen gene mRNA

With recent development in molecular biology, reverse transcriptase polymerase chain reaction (RT-PCR) has been applied to detect occult lymph node metastasis, but there have been few reports concerning oesophageal cancer. The objective of this study is to investigate the usefulness of the squamous cell carcinoma (SCC) antigen gene as a marker with RT-nested PCR to detect occult lymph node metastases of oesophageal cancer. The SCC antigen has been widely used as a serum tumour marker and was reported as a target gene to detect tumour cells in peripheral blood in cervical cancer. In this study, 620 lymph nodes from 14 oesophageal cancer patients were analysed. The results of RT-nested PCR were compared with that of pathological and immunohistochemical examinations. In the test of sensitivity, the RT-nested PCR detected 101 of SCC antigen producing cells in 107 peripheral blood mononucleocytes and was not found in 43 control lymph nodes. The pathological examination, immunohistochemical examination and the RT-nested PCR detected 36, 45 and 65 nodes respectively. The RT-nested PCR detected statistically more lymph nodes than the pathological or immunohistochemical examination. The sensitivity and specificity seem higher in squamous cell carcinoma cases. The SCC antigen gene is one of the more useful markers for RT-nested PCR to detect occult lymph node metastases of oesophageal cancer.

Histological response of cisplatin predicts patients' survival in oesophageal cancer and p53 protein accumulation in pretreatment biopsy is associated with cisplatin sensitivity

The aim of this study was to evaluate whether cisplatin sensitivity relates to patients prognosis in oesophageal squamous cell carcinoma and to find a useful chemosensitivity molecular marker. 59 oesophageal squamous cell carcinoma (SCC) patients received cisplatin 30 mg/m2/week treatment of two to five cycles, followed by oesophagectomy. We analysed retrospectively whether the histological effect was related to patients prognosis. Furthermore, to evaluate the relationship between the effect of preoperative cisplatin treatment and p53 and cyclin D1 expression, we investigated p53 and cyclin D1 expression in pretreatment biopsy samples using an immunohistochemical analysis and compared the results with the histological effect to cisplatin in the resected oesophagus. The cases that showed immunohistochemical p53 staining in the pretreatment biopsy samples were resistant to cisplatin (P = 0.032). However, there was no relationship between cyclin D1 expression and histological effect (P = 0.230). Nevertheless, combined analysis of p53 and cyclin D1 can predict histological effect (P = 0.032). The prognosis of cisplatin-sensitive cases was significantly better than that of cisplatin-resistant cases (P = 0.041). Coxs multivariate analysis revealed that the histological effect was an independent prognostic factor. In contrast, p53 protein accumulation and cyclin D1 were not. Histological response after neoadjuvant cisplatin treatment is a prognostic factor for oesophageal SCC and cisplatin chemotherapy may be selected according to the findings of p53 and cyclin D1 expression.

Clinicopathological Significance of Human Macrophage Metalloelastase Expression in Esophageal Squamous Cell Carcinoma

Objective: Human macrophage metalloelastase is referred to as matrix metalloproteinase (MMP-12), its function in tumors is contradictory. The current study was undertaken to investigate the role of MMP-12 in esophageal squamous cell carcinoma (SCC). Patients and Methods: We analyzed the levels of MMP-12 mRNA expression in 67 patients with primary esophageal SCC by Northern blot analysis and the tissues were subjected to in situ hybridization analysis for MMP-12. Immunohistochemical staining was performed to detect the macrophages infiltrated in esophageal SCCs. Results: MMP-12 mRNA was detected in 27 of 67 esophageal SCC samples by Northern blot analysis. In situ hybridization and immunohistochemical staining revealed that MMP-12 mRNA signals are located mainly in tumor cells. The frequency of lymph node metastasis was significantly higher in the MMP-12-positive (MMP-12(+)) subgroup than MMP-12-negative (MMP-12(–)) subgroup (p < 0.05); furthermore, invasion was significantly deeper in the MMP- 12(+) subgroup than in the MMP-12(–) subgroup (p < 0.01). MMP-12 mRNA was inversely correlated with prognosis (p < 0.05). However, Cox multivariate analysis revealed that upregulation of MMP-12 was not related to prognosis. Conclusions: MMP-12 gene expression was associated with the progression of esophageal SCC; however, it was not an independent prognostic factor.

Significance of nerve growth factor overexpression and its autocrine loop in oesophageal squamous cell carcinoma

Nerve growth factor (NGF) is overexpressed not only in nervous system, but also in several types of cancers. However, the role of NGF in oesophageal squamous cell carcinoma (OESCC) remains unclear. Here, we show the first evidence of NGF-TrkA autocrine loop and clinical significance of NGF overexpression in OESCC. Immunohistochemical study of 109 OESCC specimens revealed that NGF overexpression, found in 63 out of 109 patients (57.8%), was associated with lymph node metastasis, distant metastasis, higher TNM stage, poorer tumour differentiation, and poorer survival. NGF overexpression was also associated with strong expression of TrkA and negative expression of low-affinity neurotrophin receptor (p75NTR). Semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) of 19 surgical specimens showed upregulation of NGF mRNA in 17 out of 19 (89%) patients. All five OESCC cell lines tested in vitro secreted detectable NGF in enzyme-linked immunosorbent assay, and expressed TrkA and p75NTR on RT–PCR and Western blot. The motility of HSA/c, one of the OESCC cell lines overexpressing NGF, was significantly decreased by either neutralising anti-NGF antibody, an inhibitor of TrkA, or NGF-small interfering RNA in transwell migration assay. Our findings suggest that NGF is of potential interest not only as a prognostic factor, but also as a novel therapeutic target in OESCC.

Spontaneous perforation of the rectum with possible stercoral etiology: Report of a case and review of the literature

Stercoral perforation of the colon or rectum is a rare cause of acute abdomen, with fewer than 70 cases documented in the literature. We report herein the case of a 60-year-old man who presented with anuria and epigastric pain with physical signs of peritonitis. An abdominal X-ray showed bilateral subphrenic free air accumulation, and an emergency laparotomy subsequently revealed perforation of the rectum, suggestive of a stercoral cause, which was treated by simple closure after debridement. Following an uneventful postoperative course, he was discharged from the hospital 3 weeks after his operation and is now doing well without having suffered any further gastrointestinal problems. The clinical features, diagnosis, and treatment of the disease are reviewed following the presentation of this case. Surgeons should be aware of the possibility of this fatal disease, despite its rare incidence. Furthermore, it is important to recognize the condition at an early stage because it has a significantly high mortality if not treated early. Conversely, the surgical outcome is satisfactory provided surgery is performed in due time.

Paratracheal lymph node metastasis is associated with cervical lymph node metastasis in patients with thoracic esophageal squamous cell carcinoma.

Background: We determined which lymph node metastases were associated with cervical lymph node metastases of thoracic esophageal squamous cell carcinoma.Methods: A total of 6464 lymph nodes derived from 155 consecutive patients with thoracic esophageal squamous cell carcinoma were stained by immunohistochemistry (antibody: AE1/AE3). Lymph node metastases were mapped according to the mapping scheme of the American Thoracic Society, as modified by Casson et al. (Ann Thorac Surg 1994;58:1569–70). Patients were divided into two groups: those with and without cervical lymph node metastasis (CLNM). Mapping data were examined by uni- and multivariate analysis.Results: Hematoxylin and eosin-positive and AE1/AE3-positive lymph node metastases were found in 59% and 77% of patients, respectively. Twenty-one (55%) of 38 patients in the CLNM(+) group and 30 (26%) of 117 patients in the CLNM(−) group had AE1/AE3-positive lymph node metastasis in the thoracic paratracheal lymph node. Paratracheal lymph node metastasis is only one independent factor for (CLNM), whereas upper thoracic paraesophageal lymph node and pulmonal hilar lymph node status were also significant in univariate analysis. Three (43%) of seven patients with cervical jumping metastasis from the thoracic esophagus had micrometastasis in the paratracheal lymph node.Conclusions: The paratracheal lymph node is most associated with (CLNM) of thoracic esophageal squamous cell carcinoma.

Growth inhibition through activation of peroxisome proliferator-activated receptor γ in human oesophageal squamous cell carcinoma

Peroxisome proliferator-activated receptor gamma (PPARgamma) heterodimerises with retinoid X receptor alpha (RXRalpha) and is thought to be a novel therapeutic target for human malignancies. We evaluated the ability of troglitazone (TRO) alone or in combination with 9-cis retinoic acid (9CRA), ligands of PPARgamma and RXRalpha, respectively, to inhibit the growth of oesophageal squamous cell carcinoma (OSCC). All 10 tested OSCC cell lines of a KYSE series expressed PPARgamma and RXRalpha at both the mRNA and protein levels. In four tested cell lines, TRO inhibited growth, and a synergistic effect was observed with simultaneous 9CRA application. In KYSE 270 cells, a luciferase reporter assay showed that the simultaneous application of TRO and 9CRA to the cells increased the relative luciferase activity approximately 20-fold compared with the controls without TRO or 9CRA application. In this cell line, flow cytometry demonstrated that combined treatment with TRO and 9CRA greatly increased the sub-G1 phase, and Hoechst 33342/propidium iodide (PI) staining showed that apoptotic cell death was mainly induced through ligand treatment. In addition, implanted tumours in nude mice showed significant inhibition of tumour growth when treated with TRO. These results suggest that the PPARgamma/RXRalpha heterodimer may be a new therapeutic target for OSCC.

Chenodeoxycholic acid stimulates the progression of human esophageal cancer cells : A possible mechanism of angiogenesis in patients with esophageal cancer

Bile acids are known to promote the growth of gastrointestinal cancer. However, the underlying mechanism remains unclear. We examined whether bile acids induce tumor growth via the cyclooxygenase (COX)‐2 angiogenic pathway. In vitro, esophageal squamous cell carcinoma (ESCC) cells and esophageal adenocarcinoma cells were studied. Production of prostaglandin E2 (PGE2) and vascular endothelial growth factor (VEGF) in response to treatment with chenodeoxycholic acid (CDCA) was assessed by enzyme‐linked immunosorbent assay (ELISA). COX‐2 protein and VEGF protein were measured by immunoblot analysis, and COX‐2 activity was measured by ELISA. In vivo, CDCA was administered to ESCC cell‐bearing mice. Tumor tissues were analyzed immunohistochemically, and microvessel density was evaluated. Clinically, 134 patients with ESCC who underwent esophagectomy were studied. In vitro, CDCA induced the production of PGE2 and VEGF in dose‐ and time‐dependent manners, and these effects were attenuated by a selective COX‐2 inhibitor, mitogen‐activated protein kinases inhibitor, or epidermal growth factor receptor inhibitor. CDCA‐induced COX‐2 in the cell lysate increased the secretion of VEGF into the culture medium. In vivo, CDCA markedly enhanced tumor growth and increased vascularization. Clinically, patients whose tumors expressed both COX‐2 and VEGF had poor outcomes. Our results suggest that bile acids, important constituents of duodenal fluid, stimulate the development of human esophageal cancer by promoting angiogenesis via the COX‐2 pathway.