Tetsuji Yoshikawa

Intrathoracic esophageal replacement by in situ tissue-engineered esophagus

OBJECTIVE This study aimed to evaluate in situ tissue-engineered esophagus in a canine model after experimental resection and replacement of a full circumferential defect of the intrathoracic esophagus. METHODS Two types of scaffolding were fabricated. In the KF(+) group (n = 6), oral keratinocytes and fibroblasts cultured on human amniotic membrane were sheeted on polyglycolic acid felt with smooth muscle tissue and were then rolled around tubes. In the KF(-) group (n = 6), the same procedure was followed, but the keratinocytes and fibroblasts were omitted. Both scaffolds were wrapped in omentum and implanted in the abdomen. In the KF(+) group, at 3 weeks after implantation, the scaffold developed into a tube with a well-differentiated lumen of stratified squamous cells surrounded by a thick smooth muscle-like tissue (in situ tissue-engineered esophagus). A part of the esophagus was resected and replaced by the graft in the same dogs. RESULTS In the KF(-) group, strictures developed after esophageal replacement, with almost complete obstruction within 2 to 3 weeks. In contrast, in the KF(+) group, the in situ tissue-engineered esophagus showed good distensibility and the dogs remained without feeding problems through 420 days. Esophageal peristalsis transferred food to the stomach, despite the absence of peristaltic activity in the in situ tissue-engineered esophagus itself. The thickness of the squamous epithelial layer and the smooth muscle layer of the in situ tissue-engineered esophagus were similar to that of the adjacent native esophagus. CONCLUSION The in situ tissue-engineered esophagus can successfully replace the intrathoracic esophagus, and this procedure may offer a promising surgical approach to esophageal diseases.

Frequent downregulation of the runt domain transcription factors RUNX1, RUNX3 and their cofactor CBFB in gastric cancer

Our previous studies suggest that lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer, but potential roles of other members of the RUNX family genes have not yet been reported. We examined the expression of 3 Runt‐related (RUNX) genes, RUNX1, RUNX2 and CBFB, in gastric cancer cell lines and primary gastric cancer specimens and compared them to those of RUNX3 reported earlier in conjunction with clinicopathologic factors. Expression of RUNX family genes in 9 gastric cancer cell lines, 56 primary gastric cancer specimens and surrounding normal gastric mucosa were estimated by Northern blot analysis, quantitative RT‐PCR and in situ hybridization. Northern blot analysis in gastric cancer cell lines showed downregulation of RUNX1 and RUNX3 in 67% and 78% of the cell lines tested, respectively. The ratio of the average RUNX mRNA/β‐actin mRNA ratio (×103) for RUNX1 was 48.0 ± 21.1 vs. 21.4 ± 8.1; RUNX2, 1.1 ± 0.3 vs. 1.0 ± 0.2; RUNX3, 9.2 ± 6.3 vs. 3.1 ± 1.3 and CBFB, 42.0 ± 19.4 vs. 21.0 ± 8.4 (normal vs. tumor, respectively, average ±SD). The basal RUNX2 expression was very weak, and there was no significant change in gastric cancers. Both RUNX1 and RUNX3 showed remarkable downregulation in 62% and 69%, respectively, of surgically resected specimens compared to surrounding mucosa analyzed by quantitative RT‐PCR (p < 0.01). Furthermore, CBFB, the gene encoding the cofactor of RUNX1, ‐2, ‐3, was also downregulated in significant fraction (32%, p < 0.05). The percentage of downregulation of RUNX1, RUNX3 and CBFB increased as the cancer stage progressed. Tricostatin A and 5′‐azacitidin reactivate RUNX3 expression, but they could not reactivate expression of RUNX1 and CBFB in gastric cancer cells, suggesting that the downregulation was due to mechanisms other than methylation of the promoter region. These findings suggest that RUNX1 and CBFB in addition to RUNX3 play some roles in gastric cancers and that roles of RUNX gene family in gastric cancer are more widespread and complex than previously realized.

Possible Involvement of RUNX3 Silencing in the Peritoneal Metastases of Gastric Cancers

Purpose: Our previous results suggested that a lack of RUNX3 function contributed to human gastric carcinogenesis, but the role of RUNX3 in progression and metastasis remains unclear. We examined RUNX3 expression in clinical samples of peritoneal metastases in gastric cancers. Changes in metastatic potential were assessed in animal experiments using stable RUNX3 transfectants of gastric cancer cells. Finally, global expression changes were analyzed using a cDNA microarray. Experimental Design and Results: Significant down-regulation of RUNX3 through methylation on the promoter region was observed in primary tumors (75%) as well as in all clinical peritoneal metastases of gastric cancers (100%) compared with normal gastric mucosa. Stable transfection of RUNX3 inhibited cell proliferation slightly, and modest transforming growth factor-β (TGF-β)–induced antiproliferative and apoptotic effects were observed. Interestingly, it strongly inhibited peritoneal metastases of gastric cancers in animal model (P < 0.01). Furthermore, we did globally analyzed expression profiles of ∼21,000 genes in parent cells and stable transfectant of RUNX3 using a cDNA microarray. Microarray analysis identified ∼28 candidate genes under the possible downstream control of RUNX3, some of these genes were considered to be possibly involved in peritoneal metastases, which were related to signal transduction (vav3, TOLL-like receptor, MAPKK, MET, S1 00A1 1, and cathepsin E), apoptosis (caspase 9), immune responses (CD55 and TLR1O), and cell adhesion (sialyltransferase 1 and galectin 4). Some of the genes are involved in the TGF-β signaling pathway. Conclusion: These results indicate that silencing of RUNX3 affects expression of important genes involved in aspects of metastasis including cell adhesion, proliferation, apoptosis, and promoting the peritoneal metastasis of gastric cancer. Identification of such genes could suggest new therapeutic modalities and therapeutic targets.

Regeneration of skeletal muscle using in situ tissue engineering on an acellular collagen sponge scaffold in a rabbit model.

Because of the limited ability of skeletal muscle to regenerate, resection of a large amount of muscle mass often results in incomplete recovery due to nonfunctional scar tissue. The aim of this study was to regenerate skeletal muscle using in situ tissue engineering in a rabbit model. In 18 male rabbits, a muscle defect (1.0 × ∼1.0 × ∼0.5 cm) was created in the vastus lateralis of both legs. A piece of cross-linked atelocollagen sponge was then inserted into the defect in one leg, whereas the defect in the other leg was left untreated. Both defects were finally covered with fascia. Twenty-four weeks after surgery, the defect that had been filled with the cross-linked atelocollagen sponge scaffold showed mild concavity and slight adhesion to the fascia, while the control side showed severe scar formation and shrinkage. Histologically, the regenerating myofibers at the site containing the collagen sponge were greater in number, diameter, and length than those at the control site. These results indicate that cross-linked atelocollagen sponge has the potential to act as a scaffold for muscle tissue regeneration.

Frequent silencing of RUNX3 in esophageal squamous cell carcinomas is associated with radioresistance and poor prognosis

Radiotherapy is an effective treatment for some esophageal cancers, but the molecular mechanisms of radiosensitivity remain unknown. RUNX3, a novel tumor suppressor of gastric cancer, functions in transforming growth factor (TGF)-β-dependent apoptosis. We obtained paired samples from 62 patients with advanced esophageal cancers diagnosed initially as T3 or T4 with image diagnosis; one sample was obtained from a biopsy before presurgical radiotherapy, and the other was resected in surgical specimens after radiotherapy. RUNX3 was repressed in 67.7% cases of the pretreatment biopsy samples and 96.7% cases of the irradiated, resected samples. The nuclear expression of RUNX3 was associated with radiosensitivity and a better prognosis than cytoplasmic or no RUNX3 expression (P<0.003); cytoplasmic RUNX3 expression was strictly associated with radioresistance. RUNX3 was downregulated and its promoter was hypermethylated in all radioresistant esophageal cancer cell lines examined. Stable transfection of esophageal cancer cells with RUNX3 slightly inhibited cell proliferation in vitro, enhanced the antiproliferative and apoptotic effects of TGF-β and increased radiosensitivity in conjunction with Bim induction. In contrast, transfection of RUNX3-expressing cells with a RUNX3 antisense construct or a Bim-specific small interfering RNA induced radioresistance. Treatment with 5-aza-2′-deoxycytidine restored RUNX3 expression, increased radiosensitivity and induced Bim in both control and radioresistant cells. These results suggest that RUNX3 silencing promotes radioresistance in esophageal cancers. Examination of RUNX3 expression in pretreatment specimens may predict radiosensitivity, and induction of RUNX3 expression may increase tumor radiosensitivity.