Haruhito Sakata

Histone Deacetylase Inhibitor FK228 Activates Tumor Suppressor Prdx1 with Apoptosis Induction in Esophageal Cancer Cells

Purpose: The histone deacetylase inhibitor FK228 shows strong activity as a potent antitumor drug but its precise mechanism is still obscure. The purpose of this study is to reveal the effect of FK228 on gene expression in the cell and to determine the mechanism of the antitumor activity of FK228 for further clinical applications. Experimental Design and Results: Microarray analysis was applied to verify the gene expression profiles of 4,608 genes after FK228 treatment using human esophageal squamous cell cancer cell lines T.Tn and TE2. Among them, peroxiredoxin 1 (Prdx1), a member of the peroxiredoxin family of antioxidant enzymes having cell growth suppression activity, as well as p21WAF1, were significantly activated by FK288. In addition, FK228 strongly inhibited the cell growth of T.Tn and TE2 by the induction of apoptosis. Further, chromatin immunoprecipitation analysis revealed that FK228 induced the accumulation of acetylated histones H3 and H4 in Prdx1 promoter, including the Sp1-binding site. In mouse xenograft models of T.Tn and TE2 cells, FK228 injection resulted in significant tumor regression as well as activated Prdx1 expression in tumor tissues. Prdx1 suppression by RNA interference hindered the antitumor effect of FK228. Conclusion: Our results indicate that the antitumor effect of FK228 in esophageal cancer cells is shown at least in part through Prdx1 activation by modulating acetylation of histones in the promoter, resulting in tumor growth inhibition with apoptosis induction.

The molecular role of Fra-1 and its prognostic significance in human esophageal squamous cell carcinoma

The expression of Fra‐1 (Fos related antigen 1) involves tumor progression and invasion, and its gene ablation could suppress the invasive phenotypes of human tumor cells. The authors investigated the significance of Fra‐1 expression in esophageal squamous cell carcinoma (ESCC) and studied the effect of its down‐regulation on cell proliferation, motility, and invasion.

CHAP31 Induces Apoptosis Only via the Intrinsic Pathway in Human Esophageal Cancer Cells

Objective: The antitumor mechanism of histone deacetylase (HDAC) inhibitors differs from conventional antitumor agents. HDAC inhibitors may be effective as novel therapeutic agents for esophageal squamous cell carcinoma (ESCC). This study describes the antiproliferative activity of CHAP31, a novel HDAC inhibitor. Furthermore, the molecular mechanism of CHAP31-induced apoptosis was investigated in ESCC. Methods/Results: The antitumor activity of CHAP31 was tested in esophageal cancer cell lines (T.Tn and TE2), and potent antitumor activity was observed in vitro and in vivo. In addition, CHAP31 induced apoptosis in esophageal cancer cells. Next, the mechanisms of CHAP31-induced apoptosis were examined using quantitative real-time RT-PCR and Western blotting. No processing of caspase 8 was observed, but CHAP31 induced the cleavage of caspase 9 and up-regulation of the Bax/Bcl-2 protein ratio. Conclusion: This study provides new and important information on the potent antitumor activity of CHAP31 and the apoptotic pathway induced by CHAP31 in human esophageal cancer cell lines T.Tn and TE2. In contrast to previous reports showing that apoptosis induced by HDAC inhibitors includes the extrinsic pathway, in our study, apoptosis induced by CHAP31 in the human esophageal cell lines T.Tn and TE2 involved only the intrinsic pathway.

Combined Effects of p53 Gene Therapy and Leptomycin B in Human Esophageal Squamous Cell Carcinoma

Background: p53 gene therapy has been examined in several clinical trials, however, the results of those trials have mostly been unsatisfactory due to the low efficacy of this therapy. Leptomycin B (LMB) is an antibiotic originally isolated from Streptomyces that has the ability to inhibit the export of proteins containing a nuclear export signal from the nucleus to the cytoplasm. Currently, it has been shown that p53 protein has a nuclear export signal. In this study, we assessed whether LMB augments the transduced p53 gene effect. Methods: Antiproliferative effect of LMB was assessed in human esophageal squamous cancer cell lines. Accumulation of p53 protein into the nucleus by LMB was observed by fluorescence microscopy. The combined effect of p53 and LMB was evaluated in in vitro experiments. Results: LMB induced cell death in a dose-dependent manner and p53 drastically accumulated in the nucleus after LMB treatment. The combinatory treatment of p53 gene and LMB significantly increases the efficiency compared to either agent alone. Conclusions: Our findings suggest that LMB has a potent ability to augment the effect of the tumor suppressor p53 in esophageal squamous cancer cell lines and that it is a promising component in p53 gene therapy.

ZNF750 Expression as a Novel Candidate Biomarker of Chemoradiosensitivity in Esophageal Squamous Cell Carcinoma

Objective: ZNF750, an epidermal differentiation regulator, has been suggested to act as a tumor suppressor of esophageal squamous cell carcinoma (ESCC). Although a correlation between the epidermal differentiation gene and resistance to chemoradiotherapy (CRT) has been posited, no data regarding the ZNF750 status in ESCC have been reported. The aim of the present study was to evaluate the relationship between ZNF750 expression and response to CRT in ESCC. Methods: Eighty-seven patients who had been pathologically diagnosed with ESCC were evaluated in the present study. All patients underwent neoadjuvant CRT, followed by curative esophagectomy. The expression of ZNF750 in pretreatment biopsy samples was immunohistochemically investigated and compared to the histopathological effectiveness of CRT in surgical specimens. Results: High expression of ZNF750 was closely correlated with good sensitivity to CRT (p = 0.016). A univariate analysis showed that high/intermediate expression of ZNF750 was a significant predictive factor for good sensitivity to CRT (p = 0.006). High/intermediate expression of ZNF750 (30% or more) remained an independent predictive factor for sensitivity to CRT in a multivariate analysis (p = 0.033). Conclusions: ZNF750 expression predicts sensitivity to CRT and can be a biomarker that reliably predicts the response of ESCC to CRT.

Antitumor effects of metformin are a result of inhibiting nuclear factor kappa B nuclear translocation in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is an intractable digestive organ cancer that has proven difficult to treat despite multidisciplinary therapy, and a new treatment strategy is demanded. Metformin is used for type 2 diabetes mellitus and its antitumor effects have been reported recently. Metformin exerts antitumor effects in various respects, such as inhibiting inflammation, tumor growth and epithelial‐mesenchymal transition (EMT). However, few reports have described the efficacy of metformin on ESCC, and their findings have been controversial. We analyzed the antitumor effects of metformin and clarified its effects on anti‐inflammation, growth suppression and EMT inhibition. Activation of nuclear factor kappa B (NF‐κB), the major transcription factor induced by inflammation, was investigated by immunostaining. We found that localization of NF‐κB in the nucleus was reduced after metformin treatment. This suggests that metformin inhibited the activation of NF‐κB. Metformin inhibited tumor growth and induced apoptosis in ESCC cell lines. Associated with EMT, we examined cell motility by a wound healing assay and the epithelial marker E‐cadherin expression of various ESCC cell lines by western blotting. Metformin inhibited cell motility and induced E‐cadherin expression. In conclusion, metformin showed multiple antitumor effects such as growth suppression, invasion inhibition, and control of EMT by inhibiting NF‐κB localization on ESCC. Further exploration of the marker of treatment efficacy and combination therapy could result in the possibility for novel treatment to use metformin on ESCC.

ZNF750 Expression Is a Potential Prognostic Biomarker in Esophageal Squamous Cell Carcinoma

Objective: ZNF750, a transcriptional regulator of epidermal differentiation, has been identified as a tumor suppressor in esophageal squamous cell carcinoma (ESCC). The aim of the present study was to investigate the clinical and prognostic significance of ZNF750 expression and to evaluate the effect of ZNF750 knockdown on cell proliferation, migration, and invasion in ESCC. Methods: A total of 124 patients with ESCC who underwent curative esophagectomy were evaluated in this study. The expression of ZNF750 in surgical specimens was immunohistochemically assessed and used in the analysis of clinicopathological features and overall survival (OS). The molecular role of ZNF750 was investigated by ZNF750 knockdown using small interfering RNA (siRNA) in ESCC cell lines. Results: Low ZNF750 expression had a significant correlation with positive lymph node metastasis (p = 0.028). Furthermore, there was a significant relationship between low expression of ZNF750 in ESCC and a poor OS, and a multivariate analysis showed that low ZNF750 expression was an independent prognostic factor (p = 0.020). The cell growth, migration, and invasion were significantly increased by downregulation of ZNF750. Conclusions: The low expression of ZNF750 was significantly associated with a poor prognosis, and ZNF750 expression may, therefore, be a reliable prognostic biomarker in ESCC.