Takanori Nishimori

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.

A novel mechanism of keratin cytoskeleton organization through casein kinase Iα and FAM83H in colorectal cancer.

Summary Keratin filaments form cytoskeletal networks in epithelial cells. Dynamic rearrangement of keratin filament networks is required for epithelial cells to perform cellular processes such as cell migration and polarization; however, the mechanism governing keratin filament rearrangement remains unclear. Here, we describe a novel mechanism of keratin cytoskeleton organization mediated by casein kinase I&agr; (CK-1&agr;) and a newly identified keratin-associated protein, FAM83H. Knockdown of FAM83H induces keratin filament bundling, whereas overexpression of FAM83H disassembles keratin filaments, suggesting that FAM83H regulates the filamentous state of keratins. Intriguingly, keratin filament bundling is concomitant with the dissociation of CK-1&agr; from keratin filaments, whereas aberrant speckle-like localization of CK-1&agr; is observed concomitantly with keratin filament disassembly. Furthermore, CK-1&agr; inhibition, similar to FAM83H knockdown, causes keratin filament bundling and reverses keratin filament disassembly induced by FAM83H overexpression, suggesting that CK-1&agr; mediates FAM83H-dependent reorganization of keratin filaments. Because the N-terminal region of FAM83H interacts with CK-1&agr; and the C-terminal region interacts with keratins, FAM83H might tether CK-1&agr; to keratins. Colorectal cancer tissue also shows keratin filament disassembly accompanied with FAM83H overexpression and aberrant CK-1&agr; localization, and FAM83H-overexpressing cancer cells exhibit loss or alteration of epithelial cell polarity. Importantly, knockdown of FAM83H inhibits cell migration accompanied by keratin cytoskeleton rearrangement in colorectal cancer cells. These results suggest that keratin cytoskeleton organization is regulated by FAM83H-mediated recruitment of CK-1&agr; to keratins, and that keratin filament disassembly caused by overexpression of FAM83H and aberrant localization of CK-1&agr; could contribute to the progression of colorectal cancer.

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.

Identification of a novel SEREX antigen family, ECSA, in esophageal squamous cell carcinoma

BackgroundDiagnosis of esophageal squamous cell carcinoma (SCC) may improve with early diagnosis. Currently it is difficult to diagnose SCC in the early stage because there is a limited number of tumor markers available.ResultsFifty-two esophageal SCC SEREX antigens were identified by SEREX (serological identification of antigens by recombinant cDNA expression cloning) using a cDNA phage library and sera of patients with esophageal SCC. Sequence analysis revealed that three of these antigens were similar in amino acid sequences, and they were designated as ECSA (e sophageal c arcinoma S EREX a ntigen)-1, -2 and -3. The ECSA family was also similar to an EST clone, hepatocellular carcinoma-associated antigen 25a (HCA25a). Serum antibody levels to ECSA-1, -2 and -3 were significantly higher in patients with esophageal SCC than in healthy donors. Based on the conserved amino acid sequences, three peptides were synthesized and used for enzyme-linked immunosorbent assays (ELISA). The serum antibody levels against one of these peptides were significantly higher in patients with esophageal SCC. This peptide sequence was also conserved in FAM119A, GOSR1 and BBS5, suggesting that these are also ECSA family members. Reverse transcription followed by quantitative PCR analysis showed that the mRNA expression levels of ECSA-1, -2 and -3 and FAM119A but not of HCA25a, GOSR1 and BBS5 were frequently elevated in esophageal SCC tissues.ConclusionsWe have identified a new gene family designated ECSA. Serum antibodies against the conserved domain of the ECSA family may be a promising tumor marker for esophageal SCC.

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.

Serum Periplakin as a Potential Biomarker for Urothelial Carcinoma of the Urinary Bladder

The objectives of this study were to examine serum periplakin expression in patients with urothelial carcinoma of the urinary bladder and in normal controls, and to examine relationships with clinicopathological findings. Detection of serum periplakin was performed in 50 patients and 30 normal controls with anti-periplakin antibodies using the automatic dot blot system, and a micro-dot blot array with a 256 solid-pin system. Levels in patients with urothelial carcinoma of the urinary bladder were significantly lower than those in normal controls (0.31 and 5.68, respectively; p<0.0001). The area under the receiver-operator curve level for urothelial carcinoma of the urinary bladder was 0.845. The sensitivity and specificity, using a cut-off point of 4.045, were 83.7% and 73.3%, respectively. In addition, serum periplakin levels were significantly higher in patients with muscle-invasive cancer than in those with nonmuscle-invasive cancer (P=0.03). In multivariate Cox proportional hazards regression analysis, none of the clinicopathological factors was associated with an increased risk for progression and cancer-specific survival. Examination of the serum periplakin level may play a role as a non- invasive diagnostic modality to aid urine cytology and cystoscopy.

Usefulness of microRNA‑375 as a prognostic and therapeutic tool in esophageal squamous cell carcinoma.

The aim of this study was to clarify the importance of microRNA‑375 (miR‑375) expression in patients with esophageal squamous cell carcinoma (ESCC) and to examine the in vivo antitumor effects of miR‑375 in a model of ESCC using a non‑viral delivery system. We estimated the miR‑375 and LDHB and AEG‑1/MTDH mRNA expression of the ESCC tumors from 85 patients. The correlation between the miR‑375 expression and clinicopathological features, including the prognosis, were evaluated. The presence of high miR‑375 expression was associated with lymphatic vessel invasion, while a low expression of miR‑375 significantly correlated with a poor prognosis for the 85 ESCC patients. We also found that there was a significant inverse correlation between the expression of miR‑375 and that of LDHB. Before the examination of miR‑375 in the in vivo assay, we confirmed that atelocollagen prolonged the accumulation of miRNA by using fluorescently‑labeled miRNA and an in vivo imaging system. We injected the miR‑375/atelocollagen complex or a control‑miRNA/atelocollagen complex into mice bearing TE2 and T.Tn xenografts via subcutaneous (s.c.) injections. The growth of both the TE2 and T.Tn tumors in the miR‑375 groups was significantly suppressed compared with that in the control‑miRNA groups. In addition, The LDHB mRNA expression of TE2 xenografts was significantly downregulated after miR‑375 treatment. In conclusion, it might be possible for the level of miR‑375 expression to be a utilized as a prognostic indicator for ESCC patients. The administration of miR‑375 using a non‑viral delivery might represent a powerful new treatment for ESCC.

Suture Granuloma With False-Positive Findings on FDG-PET/CT Resected via Laparoscopic Surgery

A 61-year-old woman who had undergone total hysterectomy 16 years previously exhibited a pelvic tumor on computed tomography (CT). F-18 fluorodeoxyglucose (FDG) combined positron emission tomography (PET)/CT imaging revealed a solitary small focus of increased FDG activity in the pelvis. A gastrointestinal stromal tumor originating in the small intestine or another type of tumor originating in the mesentery (desmoid, schwannoma, or foreign body granuloma) was suspected; therefore, laparoscopic resection was conducted. A white, hard tumor was found to originate from the mesentery of the sigmoid colon and adhered slightly to the small intestine. The tumor was resected with a negative margin, and the pathologic diagnosis was suture granuloma. The possibility of suture granuloma should be kept in mind in cases of tumors with positive PET findings and a history of surgery close to the lesion. However, it is difficult to preoperatively diagnose pelvic tumors using a biopsy. Therefore, considering the possibility of malignancy, it is necessary to achieve complete resection without exposing the tumor.

Screening of Alternative Drugs to the Tumor Suppressor miR-375 in Esophageal Squamous Cell Carcinoma Using the Connectivity Map

Objective: The aim of this study was to identify alternative compounds to the tumor suppressor miR-375 using the connectivity map (CMAP) and to validate the antitumor effects of the identified drugs in esophageal squamous cell carcinoma (ESCC). Methods: Gene profiling of miR-375-treated TE2 and T.Tn cells was applied in order to search the CMAP database. Among the compounds identified using the CMAP, we focused on 8 drugs [(-)-epigallocatechin-3-gallate, metformin, rosiglitazone among others], excluding 2 drugs among the top 10 compounds. We evaluated whether these compounds possess tumor-suppressive functions in ESCC. Results: A cytotoxicity assay showed that the sensitivity of TE2 and T.Tn cells treated with the 8 compounds was evaluated based on IC50 values of 42.9 µM to 3.8 mM. A cell cycle analysis revealed that the percentage of TE2 and T.Tn cells incubated with 6 compounds in the G0/G1 phase or the G2/M phase increased by approximately 40-80%. A TUNEL assay showed that the percentages of apoptotic cells treated with almost all compounds were significantly increased (p < 0.05) compared with the control cells. Conclusion: The CMAP database is a useful tool for identifying compounds affecting the same molecular pathways, particularly products that are difficult to apply via practical approaches, such as microRNAs.