Eigo Otsuji

Circulating microRNAs in plasma of patients with gastric cancers

Background:We examined plasma microRNA (miRNA) concentrations from patients with gastric cancers (GCs) to assess their clinical application for diagnosing and monitoring diseases.Methods:We initially investigated the appropriateness of plasma miRNA assay, and then compared plasma miRNA results with the expressions in cancer tissues from eight GC patients, and also compared plasma miRNAs between pre- and post-operative paired samples from 10 GC patients. Then, plasma miRNAs (miR-17-5p, miR-21, miR-106a, miR-106b and let-7a) were analysed in 69 GC patients and 30 healthy volunteers in total.Results:The initial analysis showed that miRNAs were stable and detectable in all plasma samples, and the plasma miRNA levels reflected the tumour miRNAs in most cases. The levels of these miRNAs were significantly reduced in post-operative samples. In large-scale analysis, the plasma concentrations of miRNAs (miR-17-5p, miR-21, miR-106a, miR-106b) were significantly higher in GC patients than controls (P=0.05, 0.006, 0.008 and <0.001 respectively), whereas let-7a was lower in GC patients (P=0.002). The values of the area under the receiver-operating characteristic curve were 0.721 for the miR-106b assay and 0.879 for the miR-106a/let-7a ratio assay.Conclusion:Detection of circulating miRNAs might provide new complementary tumour markers for GC.

Circulating microRNAs in plasma of patients with oesophageal squamous cell carcinoma

Background:Several recent studies demonstrated that microRNAs (miRNAs) are stably detectable in plasma/serum. We hypothesised that plasma miRNAs concentrations contributed to potential biomarkers in patients with oesophageal squamous cell carcinoma (ESCC).Methods:We selected three oncogenic miRNAs (miR-21, miR-184, miR-221) and one tumour suppressive miRNA (miR-375), which are frequently reported in squamous cell carcinoma, as candidate targets for this plasma miRNA assay. This study was divided into three steps: (1) Determination of appropriate plasma miRNAs in preliminary tests. (2) Evaluation of whether the plasma miRNA assays could monitor tumour dynamics. (3) Validation study on the clinical application of plasma miRNA assays in 50 ESCC patients and 20 healthy volunteers.Results:(1) In preliminary tests, the plasma level of miR-21 was significantly higher (P=0.0218) and that of miR-375 (P=0.0052) was significantly lower in ESCC patients than controls. (2) The high plasma miR-21 levels reflected tumour levels in all cases (100%). The plasma level of miR-21 was significantly reduced in postoperative samples (P=0.0058). (3) On validation analysis, the plasma level of miR-21 tended to be higher in ESCC patients (P=0.0649), while that of miR-375 was significantly lower (P<0.0001) and the miR-21/miR-375 ratio was significantly higher (P<0.0001) in ESCC patients than in controls. The value of the area under the receiver-operating characteristic curve (AUC) was 0.816 for the miR-21/miR-375 ratio assay. Patients with a high plasma level of miR-21 tended to have greater vascular invasion (P=0.1554) and to show a high correlation with recurrence (P=0.0164).Conclusion:Detection of circulating miRNAs might provide new complementary tumour markers for ESCC.

Clinical impact of circulating miR-221 in plasma of patients with pancreatic cancer

Background:Several recent studies have demonstrated that microRNAs (miRNAs) are stably detectable in plasma/serum. We tested miR-221 and miR-375, which are frequently reported to be highly and poorly expressed in pancreatic cancer (PCa), as candidates for plasma biomarkers in PCa.Methods:This study was divided into three parts: (1) Confirmation of higher miR-221 levels in primary PCa tissue and cell lines than normal pancreatic tissues. (2) Evaluation of plasma miR-221 and miR-375 concentrations by comparing results from 47 consecutive PCa patients and 30 healthy volunteers. (3) Evaluation of the assay for monitoring tumour dynamics in PCa patients.Results:(1) Expression of miR-221 was significantly higher in PCa tissues and cell lines than normal pancreatic tissues. (2) Plasma miR-221 concentrations were significantly higher in PCa patients than that in benign pancreatic tumours (P=0.016) and controls (P<0.0005), while plasma miR-375 concentrations tended to be lower in PCa patients (P=0.064), and the miR-221/miR-375 ratio was significantly higher (P<0.0001) in PCa patients than in controls. (3) Plasma miR-221 concentrations were significantly reduced in postoperative samples (P=0.018). Furthermore, PCa patients with high plasma miR-221 concentrations had significant correlation with distant metastasis (P=0.041), and non-resectable status (P=0.021).Conclusion:Plasma miR-221 could be a useful biomarker for cancer detection, monitoring tumour dynamics and predicting malignant outcomes in PCa patients, and may contribute to clinical decision making in PCa treatments.

Detection of gastric cancer-associated microRNAs on microRNA microarray comparing pre- and post-operative plasma.

Background:Recently, it was reported that plasma microRNAs (miRNAs) are low-invasive useful biomarkers for cancer. We attempted to isolate gastric cancer (GC)-associated miRNAs comparing pre- and post-operative paired plasma, thereby excluding the possible effects of individual variability.Methods:This study was divided into four steps: (1) microarray analysis comparing pre- and post-operative plasma; (2) validation of candidate miRNAs by quantitative RT–PCR; (3) validation study of selected miRNAs using paired plasma; and (4) comparison of the levels of selected miRNAs in plasma between healthy controls and patients.Results:From the results of microarray analysis, nine candidate miRNAs the levels of which were markedly decreased in post-operative plasma were selected for further studies. After confirmation of their post-operative marked reduction, two candidate miRNAs, miR-451 and miR-486, were selected as plasma biomarkers, considering the abundance in plasma, and marked decrease in post-operative samples. In validation, the two miRNAs were found to decrease in post-operative plasma in 90 and 93% of patients (both P<0.01). In comparison with healthy controls, the levels of both miRNAs were found to be significantly higher in patients, and the area under the curve values were high at 0.96 and 0.92.Conclusion:Plasma miR-451 and miR-486 could be useful blood-based biomarkers for screening GC.

A combination of indole-3-carbinol and genistein synergistically induces apoptosis in human colon cancer HT-29 cells by inhibiting Akt phosphorylation and progression of autophagy

BackgroundThe chemopreventive effects of dietary phytochemicals on malignant tumors have been studied extensively because of a relative lack of toxicity. To achieve desirable effects, however, treatment with a single agent mostly requires high doses. Therefore, studies on effective combinations of phytochemicals at relatively low concentrations might contribute to chemopreventive strategies.ResultsHere we found for the first time that co-treatment with I3C and genistein, derived from cruciferous vegetables and soy, respectively, synergistically suppressed the viability of human colon cancer HT-29 cells at concentrations at which each agent alone was ineffective. The suppression of cell viability was due to the induction of a caspase-dependent apoptosis. Moreover, the combination effectively inhibited phosphorylation of Akt followed by dephosphorylation of caspase-9 or down-regulation of XIAP and survivin, which contribute to the induction of apoptosis. In addition, the co-treatment also enhanced the induction of autophagy mediated by the dephosphorylation of mTOR, one of the downstream targets of Akt, whereas the maturation of autophagosomes was inhibited. These results give rise to the possibility that co-treatment with I3C and genistein induces apoptosis through the simultaneous inhibition of Akt activity and progression of the autophagic process. This possibility was examined using inhibitors of Akt combined with inhibitors of autophagy. The combination effectively induced apoptosis, whereas the Akt inhibitor alone did not.ConclusionAlthough in vivo study is further required to evaluate physiological efficacies and toxicity of the combination treatment, our findings might provide a new insight into the development of novel combination therapies/chemoprevention against malignant tumors using dietary phytochemicals.

Overexpression of SMYD2 relates to tumor cell proliferation and malignant outcome of esophageal squamous-cell carcinoma

Although we have identified two putative targets, ATF3 and CENPF, for a frequently gained/amplified region around 1q32-q41 in esophageal squamous cell carcinoma (ESCC), it is possible that other amplification targets remain to be identified. In this study, we tested whether SET and MYND domain-containing protein 2 (SMYD2), located between those two genes and encoding a lysine methyltransferase for histone H3K36 and p53K370 that regulates transcription and inhibits transactivation activity, respectively, acts as a cancer-promoting gene through activation/overexpression in ESCC. Frequent overexpression of SMYD2 messenger RNA and protein was observed in KYSE150 cells with remarkable amplification at 1q32-41.1 and other ESCC cell lines (11/43 lines, 25.6%). Overexpression of SMYD2 protein was frequently detected in primary tumor samples of ESCC (117/153 cases, 76.5%) as well and significantly correlated with gender, venous invasion, the pT category in the tumor-lymph node-metastases classification and status of recurrence. Patients with SMYD2-overexpressing tumors had a worse overall rate of survival than those with non-expressing tumors, and SMYD2 positivity was independently associated with a worse outcome in the multivariate analysis. Knockdown of SMYD2 expression inhibited and ectopic overexpression of SMYD2 promoted the proliferation of ESCC cells in a TP53 mutation-independent but SMYD2 expression-dependent manner. These findings suggest that SMYD2 plays an important role in tumor cell proliferation through its activation/overexpression and highlight its usefulness as a prognosticator and potential therapeutic target in ESCC.

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.

Characterization of signet ring cell carcinoma of the stomach.

Background and Objectives: As there is no consensus regarding the prognosis of patients with signet cell carcinoma of the stomach compared with other types of gastric cancer, we retrospectively studied the clinicopathologic features and prognosis of signet cell carcinoma in comparison with other types of gastric cancer.

Raspberry-like assembly of cross-linked nanogels for protein delivery.

Raspberry-like assembly of nanogels (A-CHPNG) with a high potential as a carrier for protein delivery was prepared. Cross-linking of acrylate group-modified cholesterol-bearing pullulan nanogel (CHPANG) with thiol group-modified poly (ethylene glycol) (PEGSH) by Michael addition yielded A-CHPNG with narrow size distribution. The size of A-CHPNGs was controlled in the range of 40-120 nm by changing the concentration of CHPANG and PEGSH. A-CHPNG gradually degraded by hydrolysis under physiological condition and seemed to dissociate back to original nanogel. A-CHPNG encapsulated interleukin-12 (IL-12) efficiently (96%) and stably kept it in the presence of BSA (50 mg/ml). In addition, A-CHPNG had a high potential to maintain a high IL-12 level in plasma after subcutaneous injection in mice. Therefore, A-CHPNG is a promising carrier for long-term medications.

Monoclonal antibody A7-superparamagnetic iron oxide as contrast agent of MR imaging of rectal carcinoma

Superparamagnetic iron oxide (SPIO)-based colloid has been used clinically as a tissue-specific magnetic resonance contrast agent. We coupled monoclonal antibody A7 (Mab A7), which reacts specifically with human colorectal carcinoma, to Ferumoxides (SPIO) and examined the accumulation of this conjugate in xenografted tumours in nude mice. We examined in vitro immunoreactivity of Mab A7 coupled to Ferumoxides and its in vivo distribution in nude mice with human colorectal carcinoma. Magnetic resonance imaging of tumour-bearing nude mice was performed 72 h after injection of A7-Ferumoxides. A7-Ferumoxides retained binding activities that were nearly identical to intact Mab A7. More of the radiolabelled A7-Ferumoxides accumulated in the tumour than normal mouse IgG-Ferumoxides from 12 h onwards after injection (P<0.05). Both A7-Ferumoxides and normal mouse IgG-Ferumoxides disappeared from blood linearly over time. The accumulation levels in normal tissue decreased linearly over time but were lower than levels in tumours from 6 h. In magnetic resonance T2-weighted imaging of the tumour-bearing nude mice, signal intensity was reduced at the margin of the tumour by injection of A7-Ferumoxides. Mab A7 coupled to Ferumoxides is potentially suitable as a magnetic resonance contrast agent for detecting local recurrence of rectal carcinoma.