Chloride intracellular channel 1 as a switch among tumor behaviors in human esophageal squamous cell carcinoma.

Background: Recent studies have reported important roles for chloride intracellular channel 1 (CLIC1) in various cancers; however, its involvement in esophageal squamous cell carcinoma (ESCC) remains unclear. The aim of the present study was to investigate the role of CLIC1 in human ESCC. Methods: CLIC1 expression in human ESCC cell lines was analyzed by Western blotting. Knockdown experiments were conducted with CLIC1 siRNA, and their effects on cell proliferation, the cell cycle, apoptosis, migration, and invasion were analyzed. The gene expression profiles of cells were analyzed using a microarray analysis. An immunohistochemical analysis was performed on 61 primary tumor samples obtained from ESCC patients who underwent esophagectomy. Results: ESCC cells strongly expressed CLIC1. The depletion of CLIC1 using siRNA inhibited cell proliferation, induced apoptosis, and promoted cell migration and invasion. The results of the microarray analysis revealed that the depletion of CLIC1 regulated apoptosis via the TLR2/JNK pathway. Immunohistochemistry showed that CLIC1 was present in the cytoplasm of carcinoma cells, and that the very strong or very weak expression of CLIC1 was an independent poor prognostic factor. Conclusions: The present results suggest that the very strong expression of CLIC1 enhances tumor survival, while its very weak expression promotes cellular movement. The present study provides an insight into the role of CLIC1 as a switch among tumor behaviors in ESCC.

[A Case That Was Complicated with Pulmonary Artery Thrombosis during Chemotherapy of the Regimen of CapeOX plus Bevacizumab against Recurrence of Colon Cancer].

An 80-year-old woman with peritoneal dissemination after laparoscopic right hemicolectomy for appendicular carcinoma (mucinous adenocarcinoma, pT4aN1M0, stage Ⅲa)underwent CapeOX plus bevacizumab chemotherapy. The patient achieved stable disease over 5 courses of the treatment. Subjective and objective symptoms were not observed; however, chest computed tomography findings revealed a thrombus in the pulmonary artery that was considered to be associated with bevacizumab. Chemotherapy was discontinued, and anticoagulation therapy was initiated with heparin and then switched to apixaban. The thrombus resolved with treatment, but the patient died following an increase in peritoneal dissemination. CapeOX plus bevacizumab is a recommended colon cancer treatment, and even though it is generally considered safe, the side effects of bevacizumab include relatively rare occurrences of gastrointestinal perforation or thrombosis. The frequency of pulmonary embolism associated with bevacizumab is approximately 0.1%, and fatalities have been reported. The possibility of asymptomatic thrombosis warrants regular monitoring of this serious side effect in patients receiving bevacizumab. Early detection and prompt antithrombotic treatment are necessary to ensure patient safety and continued disease management.

[A Case of Orange-Induced Small Bowel Diverticular Obstruction Treated with Laparoscopically-Assisted Surgery].

We report a case of orange-induced small bowel diverticular obstruction treated with laparoscopically-assisted surgery. A 64-year-old man was seen at the hospitalbecause of abdominalpain and vomiting after dinner. Abdominalcomputed tomography( CT)showed a small intestinal ileus. We performed laparoscopically-assisted surgery on the same day for definitive diagnosis and treatment. The postoperative course was uneventful. The pathological diagnosis was orange-induced small boweldiverticul ar obstruction. Food-induced smallbowelobstruction is rare disease, but often requires surgery. Laparoscopic surgery is an effective option for surgery of food-induced smallbowel obstruction.

[A Giant Malignant Lymphoma of the Ileocecum Treated with Laparoscopic Surgery].

We experienced a case ofa giant malignant lymphoma ofthe ileocecum treated with laparoscopic surgery. A 78-year-old man presented with right flank pain. Lower endoscopy and abdominal computed tomography revealed a giant tumor in ileocecum. Biopsy results suggested malignant lymphoma or adenocarcinoma. We performed a laparoscopic ileocecal resection for definite diagnosis and treatment. The postoperative course was uneventful. The pathological diagnosis was malignant diffuse large B-cell lymphoma. The patient underwent chemotherapy and is being followed. Laparoscopic surgery can be considered useful to resect gastrointestinal malignant lymphoma.

[A Giant Appendiceal Mucinous Neoplasm Treated with Laparoscopic Surgery].

We encountered a case of giant appendiceal mucinous neoplasm that was treated with laparoscopic surgery. The patient was a 77-year-old man with constipation. Lower endoscopy demonstrated a giant SMT-like tumor in the cecum, and abdominal computed tomography revealed a giant appendiceal mucinous neoplasm and nearby lymph nodes swelling. Hematological examination showed an elevated serum CEA level. We performed laparoscopic ileocecal resection for a definite diagnosis and treatment. The postoperative course was uneventful. The pathological diagnosis was low-grade appendicealmucinous neoplasm(LAMN). Laparoscopic surgery can be considered safe for the resection of appendicealmucinous neoplasm when it is performed with a carefulsurgicalapproach.

Evaluation of the efficacy of peritoneal lavage with distilled water in colorectal cancer surgery: in vitro and in vivo study.

BACKGROUND: Peritoneal lavage with distilled water has been performed during colorectal cancer surgery. This study investigated the cytocidal effects of hypotonic shock in vitro and in vivo in colorectal cancer cells. METHODS: Three human colorectal cancer cell lines, DLD1, HT29, and CACO2, were exposed to distilled water, and morphological changes were observed under a differential interference contrast microscope connected to a high-speed digital video camera. Cell volume changes were assessed using a high-resolution flow cytometer. Re-incubation experiments were performed to investigate the cytocidal effects of distilled water. In the in vivo experiment, cancer cells after hypotonic shock were injected intraperitoneally into mice and the degree of established peritoneal metastasis was subsequently evaluated. The effects of the blockade of Cl(-) channels on these cells during hypotonic shock were also analyzed. RESULTS: Morphological observations revealed a rapid cell swelling followed by cell rupture. Measurements of cell volume changes showed that mild hypotonic shock induced regulatory volume decrease (RVD) while severe hypotonic shock broke cells into fragments. Re-incubation experiments demonstrated the cytocidal effects of hypotonicity. In vivo experiments revealed the absence of peritoneal dissemination in mice in the distilled water group, and its presence in all mice in the control group. The blockade of Cl(-) channels increased cell volume by inhibiting RVD and enhanced cytocidal effects during mild hypotonic shock. CONCLUSIONS: These results clearly support the efficacy of peritoneal lavage with distilled water during colorectal cancer surgery and suggest that regulating of Cl(-) transport may enhance the cytocidal effects of hypotonic shock.

Claudin 1 mediates tumor necrosis factor alpha-induced cell migration in human gastric cancer cells.

AIM: To investigate the role of claudin 1 in the regulation of genes involved in cell migration and tumor necrosis factor alpha (TNF-α)-induced gene expression in human gastric adenocarcinoma cells. METHODS: Knockdown experiments were conducted with claudin 1 small interfering RNA (siRNA), and the effects on the cell cycle, apoptosis, migration and invasion were analyzed in human gastric adenocarcinoma MKN28 cells. The gene expression profiles of cells were analyzed by microarray and bioinformatics. RESULTS: The knockdown of claudin 1 significantly inhibited cell proliferation, migration and invasion, and increased apoptosis. Microarray analysis identified 245 genes whose expression levels were altered by the knockdown of claudin 1. Pathway analysis showed that the top-ranked molecular and cellular function was the cellular movement related pathway, which involved MMP7, TNF-SF10, TGFBR1, and CCL2. Furthermore, TNF- and nuclear frctor-κB were the top-ranked upstream regulators related to claudin 1. TNF-α treatment increased claudin 1 expression and cell migration in MKN28 cells. Microarray analysis indicated that the depletion of claudin 1 inhibited 80% of the TNF-α-induced mRNA expression changes. Further, TNF-α did not enhance cell migration in the claudin 1 siRNA transfected cells. CONCLUSION: These results suggest that claudin 1 is an important messenger that regulates TNF-α-induced gene expression and migration in gastric cancer cells. A deeper understanding of these cellular processes may be helpful in establishing new therapeutic strategies for gastric cancer.

Efficacy of a hypotonic treatment for peritoneal dissemination from gastric cancer cells: an in vivo evaluation.

The aim of the present study was to determine the efficacy of a hypotonic treatment for peritoneal dissemination from gastric cancer cells using an in vivo model. We firstly evaluated the toxicity of a peritoneal injection of distilled water (DW) (2 mL for 3 days) in mice. Macroscopic and microscopic examinations revealed that the peritoneal injection of DW did not severely damage the abdominal organs of these mice. MKN45 gastric cancer cells preincubated with NaCl buffer or DW for 20 minutes in vitro were then intraperitoneally injected into nude mice, and the development of dissemination nodules was analyzed. The total number, weight, and volume of the dissemination nodules were significantly decreased by the DW preincubation. We then determined whether the peritoneal injection of DW inhibited the establishment of peritoneal dissemination. After a peritoneal injection of MKN45 cells into nude mice, NaCl buffer or DW was injected into the abdominal cavity for 3 days. The total volume of dissemination nodules was significantly lower in DW-injected mice than in NaCl-injected mice. In conclusion, we demonstrated the safeness of a peritoneal injection of DW. Furthermore, the development of dissemination nodules from gastric cancer cells was prevented by a preincubation with or peritoneal injection of DW.

Role of the Na ⁺/K ⁺/2Cl⁻ cotransporter NKCC1 in cell cycle progression in human esophageal squamous cell carcinoma.

AIM: To investigate the role of Na(+)/K(+)/2Cl(-) cotransporter 1 (NKCC1) in the regulation of genes involved in cell cycle progression and the clinicopathological significance of its expression in esophageal squamous cell carcinoma (ESCC). METHODS: An immunohistochemical analysis was performed on 68 primary tumor samples obtained from ESCC patients that underwent esophagectomy. NKCC1 expression in human ESCC cell lines was analyzed by Western blotting. Knockdown experiments were conducted using NKCC1 small interfering RNA, and the effects on cell cycle progression were analyzed. The gene expression profiles of cells were analyzed by microarray analysis. RESULTS: Immunohistochemical staining showed that NKCC1 was primarily found in the cytoplasm of carcinoma cells and that its expression was related to the histological degree of differentiation of SCC. NKCC1 was highly expressed in KYSE170 cells. Depletion of NKCC1 in these cells inhibited cell proliferation via G2/M phase arrest. Microarray analysis identified 2527 genes with altered expression levels in NKCC1depleted KYSE170. Pathway analysis showed that the top-ranked canonical pathway was the G2/M DNA damage checkpoint regulation pathway, which involves MAD2L1, DTL, BLM, CDC20, BRCA1, and E2F5. CONCLUSION: These results suggest that the expression of NKCC1 in ESCC may affect the G2/M checkpoint and may be related to the degree of histological differentiation of SCCs. We have provided a deeper understanding of the role of NKCC1 as a mediator and/or a biomarker in ESCC.

xCT, component of cysteine/glutamate transporter, as an independent prognostic factor in human esophageal squamous cell carcinoma.

BACKGROUND: xCT is a component of the cysteine/glutamate transporter, which plays a key role in glutathione synthesis. The objectives of the present study were to investigate the role of xCT in the regulation of genes involved in cell cycle progression and the clinicopathological significance of its expression in esophageal squamous cell carcinoma (ESCC). METHODS: xCT expression in human ESCC cell lines was analyzed by Western blotting and immunofluorescent staining. Knockdown experiments were conducted with xCT siRNA, and the effect on cell cycle was analyzed. The cells' gene expression profiles were analyzed by microarray analysis. An immunohistochemical analysis of 70 primary tumor samples obtained from ESCC patients that had undergone esophagectomy was performed. RESULTS: xCT was highly expressed in TE13 and KYSE170 cells. In these cells, the knockdown of xCT using siRNA inhibited G1-S phase progression. Microarray analysis identified 1652 genes whose expression levels in TE13 cells were altered by the knockdown of xCT. Pathway analysis showed that the top-ranked canonical pathway was the G1/S checkpoint regulation pathway, which involves TP53INP1, CDKN1A, CyclinD1/cdk4, and E2F5. Immunohistochemical staining showed that xCT is mainly found in the nuclei of carcinoma cells, and that its expression is an independent prognostic factor. CONCLUSIONS: These observations suggest that the expression of xCT in ESCC cells might affect the G1/S checkpoint and impact on the prognosis of ESCC patients. As a result, we have a deeper understanding of the role played by xCT as a mediator and/or biomarker in ESCC.

[Esophagectomy after endoscopic submucosal dissection (ESD)].

Herein, we report 9 patients who underwent esophagectomy after endoscopic submucosal dissection (ESD) between April 2003 and December 2013. All patients were men, with a mean age of 65 years. En bloc ESD was performed, and no complications arose in any patient. The mean surgical time of esophagectomy was 323 minutes, and mean blood loss was 295 mL. Postoperative complications were present in 5 patients(anastomotic leakage in 3, pulmonary complications in 2, and recurrent laryngeal nerve palsy in 1). In a patient diagnosed with pT1b-SM1 disease after ESD, a residual tumor(pT1a-MM, N0) was detected after esophagectomy. In another patient diagnosed with pT1b-SM2 disease, lymph node metastasis was detected after esophagectomy. In all patients, curative resection was performed, and no recurrences have been observed to date. This highlights the importance of additional esophagectomy after ESD for patients with pT1b disease. Esophagectomy after ESD can be considered a valid treatment because it provides high curative rates with acceptable safety.

E2F5 as an independent prognostic factor in esophageal squamous cell carcinoma.

BACKGROUND: E2F Transcription Factor 5 Protein (E2F5) is considered to act primarily as a transcriptional repressor in the cell cycle. However, its expression and role in esophageal squamous cell carcinoma (ESCC) have not been investigated. We examined whether the expression of E2F5 is related to the clinicopathological features and prognosis of patients with ESCC. MATERIALS AND METHODS: The expression of E2F5 was analyzed by immunohistochemistry in 64 primary tumor samples obtained from patients with ESCC who had undergone curative esophagectomy between 1998 and 2009. According to the expression of E2F5 in tumor cells, cases were divided into E2F5-positive (27 cases) and -negative groups (37 cases). The relationship of various clinicopathological features and prognosis with the E2F5 status, were analyzed. RESULTS: In the clinicopathological analysis, the proportion of poorly-differentiated tumors was significantly higher in the E2F5-positive group than in the E2F5-negative group (p=0.027). The 5-year survival rate of the E2F5-positive group was 39.3%, which was significantly poorer than that of the E2F5-negative group (83.8%) (p=0.006). In multivariate analysis, the expression of E2F5 was one of the most important independent prognostic factors after radical esophagectomy. CONCLUSION: The expression of E2F5 in ESCC may be correlated with a worse prognosis of patients with ESCC.

Prognostic significance of p21 expression in patients with esophageal squamous cell carcinoma.

BACKGROUND: Although p21 is an important component that regulates cell-cycle progression, no consensus has been reached about its clinicopathological significance in esophageal squamous cell carcinoma (ESCC). In the present study, we investigated its prognostic significance and correlation with cyclin-D1 (CCND1) expression in ESCC. MATERIALS AND METHODS: The p21 labeling index (LI) was calculated by immunohistochemistry for 69 primary tumor samples obtained from patients with ESCC who had undergone curative esophagectomy, and correlations between p21 LI and various clinicopathological features, prognosis, and CCND1 LI were studied. RESULTS: The p21 LI of these tumors ranged from 2.0% to 57.0% (median=28.4%, mean±SD=27.3% ± 13.0). p21 LI was positively correlated with CCND1 LI. When patients were divided into two groups using a p21 LI cut-off value of 30%, the 5-year survival rate of patients with p21 LI of ≥ 30% was 80.0%, which was significantly higher than that of patients with p21 LI of <30% (55.5%). Furthermore, when patients were divided into four groups according to p21 and CCND1 expression, the 5-year survival rate of patients with p21 LI of <30% and CCND1 LI of ≥ 45% was the lowest (44.4%). Multivariate analysis demonstrated that venous invasion, lymphatic invasion, and p21 LI were independent prognostic factors. CONCLUSION: Our results indicate that p21 LI is correlated with CCND1 LI and can be used as an independent prognostic factor for patients with ESCC following selection of a suitable cut-off value.

[A case of esophageal squamous cell carcinoma with submucosal tumor-like tumor due to direct invasion of the gastric wall].

We encountered a case of esophageal squamous cell carcinoma (ESCC) with submucosal tumor (SMT)-like tumor in the cardia of the stomach, formed by direct invasion of the gastric wall. The patient was a 72-year-old woman with dysphagia. Upper endoscopy showed a giant SMT-like tumor in the cardia of the stomach, and imaging studies revealed metastasis to the nearby and distant lymph nodes. Although squamous cell carcinoma was diagnosed by biopsy of the SMT-like tumor in the gastric cardia, gastric involvement was suspected via metastasis to the perigastric lymph nodes or direct invasion of the gastric wall by the ESCC, because the primary lesion of ESCC could not be identified during the examinations. FP chemotherapy (days 1-5, 800 mg/m2 5-fluorouracil[5-FU]; day 1, 80 mg/m2 cisplatin[CDDP]) was initiated for the treatment of ESCC. Considering the risk of invasion, we discontinued the chemotherapy and performed lower esophagectomy and cardiectomy. Pathologically, the primary lesion of the ESCC was detected in the abdominal esophagus, and the SMT-like tumor in the gastric cardia was determined to result from direct invasion of the gastric wall by the ESCC. We herein report a rare case of ESCC with an SMT-like tumor in the gastric cardia formed by direct invasion of the gastric wall.

Enhancement of the cytocidal effects of hypotonic solution using a chloride channel blocker in pancreatic cancer cells.

BACKGROUND: Tumor cells exfoliated during surgery for pancreatic cancer can cause peritoneal recurrence. Peritoneal lavage with distilled water has been performed during surgery, but there have been no systematic studies for its efficacy and no experimental data demonstrating the cytocidal effects of distilled water on pancreatic cancer cells. This study investigated the cytocidal effects of hypotonic shock and enhancement using chloride channel blocker in pancreatic cancer cells. METHODS: Three human pancreatic cancer cell lines, KP4-1, PK-1, and PK45-H, were exposed to distilled water, and the resultant morphological changes were observed under a differential interference contrast microscope connected to a high-speed video camera. Analysis of cell volume changes was performed using a high-resolution flow cytometer. To investigate the cytocidal effects of water, re-incubation of cells was performed after exposure to hypotonic solution. Additionally, the effects of 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), a Cl(-) channel blocker, on cells during exposure to hypotonic solution were analyzed. RESULTS: Video recordings demonstrated that hypotonic shock induced cell swelling followed by cell rupture. Measurement of cell volume changes indicated that severe hypotonicity increased broken fragments of cancer cells within 5 min. Re-incubation experiments demonstrated the cytocidal effects of hypotonic shock. In all cell lines, treatment with NPPB increased cell volume by inhibiting regulatory volume decreases, which are observed during hypotonic shock, and enhanced the cytocidal effects of hypotonic solution. CONCLUSIONS: These findings support the efficacy of peritoneal lavage with distilled water for pancreatic cancer and suggest that regulation of Cl(-) transport enhances the cytocidal effects of hypotonic shock.