The Oncological Effect of Mutant p53 on the Metastatic Phenotype of Gastric Cancer Cells.

BACKGROUND/AIM: P53 is the most frequently mutated tumor suppressor gene among all cancers. In human cancers, specific residues of p53 are mutated at a high frequency, and those mutations are known as hotspot mutations. Mutant p53 promotes tumor progression through the gain-of-function (GOF) mechanism. However, its biological characteristics, especially its metastatic potential, owing to different hotspot mutations in gastric cancer remain unclear. In the present study, we investigated the p53-depended metastatic phenotype. MATERIALS AND METHODS: This study examined the differences in the metastatic potential of wild-type, mutant-p53-R175H, and mutant-p53-R273H NUGC-4 gastric cancer cells in vitro and in vivo. RESULTS: NUGC-4-mutant-p53-R175H cells showed significant cell proliferation, healing and invasive abilities in proliferation, wound healing and invasion assay, respectively, compared to wild-type and mutant-p53-R273H cells. Both NUGC-4-mutant-p53 cell types expressed epithelial-mesenchymal transition (EMT)-related proteins. Furthermore, NUGC-4-mutant-p53-R175H cells showed less attachment to the extracellular matrix and greater expression of EMT-related proteins than NUGC-4-mutant-p53-R273H cells. Regarding the peritoneal dissemination model, NUCG-4-mutant-p53-R175H and NUCG-4-mutant-p53-R273H cells demonstrated less frequent formation of dissemination nodules than NUGC-4-empty cells. In contrast, liver metastases were more frequent and greater in number in NUCG3-mutant-p53-R175H than in the other cell lines. CONCLUSION: Our results suggest that differences in the p53 status, even in the hotspot mutation site, affect not only the characteristics of the cells but also the metastatic ability of gastric cancer.

The analysis of fecal calprotectin as a diagnostic marker for anastomotic leakage after rectal cancer surgery: a pilot study.

A prospective pilot study was conducted on 11 patients with rectal cancer to investigate fecal calprotectin (FC) as a diagnostic tool for detecting anastomotic leakage (AL) after low anterior resection. Among the 11 patients, 1 patient (9.1%) experienced AL (Clavien-Dindo Grade IIIa). During the post-operative course until post-operative day (POD) 5, the white blood cell count of the patient with AL was within the normal range. The C-reactive protein level in the AL and non-AL groups showed a similar time course. On the other hand, the FC level in patient with AL dramatically increased on POD5, while the FC level of the non-AL group remained relatively stable. There was no significant correlation between the preoperative FC level and the tumor circumference rate, tumor size, depth of invasion or stage. This pilot study showed the possibility of FC as a useful diagnostic tool for the detection of AL after low anterior resection for rectal cancer.

Risk factors of incisional hernia at the umbilical specimen extraction site in patients with laparoscopic colorectal cancer surgery.

Purpose: Incisional hernia (IH) is a frequent complication following laparoscopic colorectal surgery. The present study investigated the risk factors for IH after laparoscopic surgery for colorectal cancer. Methods: A retrospective study was conducted on 202 patients who underwent laparoscopic surgery for colorectal cancer. Univariate and multivariate analyses were performed to determine the clinicopathological factors associated with IH. Results: The overall incidence of IH was 25.7% (52 of 202). The univariate analysis showed that female sex (P=0.004), a high body mass index (P<0.001), non-current smoking habit (P=0.043), low level of hemoglobin (P=0.035), high subcutaneous fat area (P<0.001), high visceral fat area (P=0.006), low skeletal muscle area (P=0.001), long distance between the inner edges of the rectus abdominis muscle (P=0.001), long protrusion of the peritoneum at the umbilical site (P<0.001), and lymph node metastasis (P=0.007) were significantly more frequent in the group with IH than in the group without it. The multivariate logistic regression analysis revealed an older age (10-year increments: odds ratio [OR], 1.576; 95% confidence interval [CI], 1.027-2.419; P=0.037), lymph node metastasis (OR, 2.384; 95% CI, 1.132-5.018; P=0.022) and lengthy protrusion of the peritoneum at the umbilical site (10-mm increments: OR, 5.555; 95% CI, 3.058-10.091; P<0.001) were independent risk factors for IH. Conclusion: Our findings suggest that older age, lymph node metastasis, and lengthy protrusion of the peritoneum at the umbilical site are risk factors for IH after laparoscopic surgery for colorectal cancer. An assessment using these factors before the operation and the implementation of countermeasures might help prevent IH.

Postoperative hiatal hernia after minimally invasive esophagectomy for esophageal cancer.

BACKGROUND: Minimally invasive esophagectomy (MIE) can reduce various complications compared with conventional thoracotomic esophagectomy. However, several reports suggested that MIE promoted incidence of post-operative hiatal hernia (HH). In current reports, we retrospectively analyzed incidence and risk factors of HH development after MIE. METHODS: A total of 113 patients undergoing MIE (McKeown esophagectomy) at our institute from April 2009 to December 2015 were included in this study. Patients with clinical stage II and III received neoadjuvant chemotherapy (NAC). RESULTS: Eleven of 113 patients (9.7%) undergoing MIE developed HH. Four of them were female and the ratio of female among the patient with HH was higher than that among the patient without HH after MIE (36.4% vs. 13.7%, P=0.05). Sixty-six patients (58.4%) during the study period were administered NAC and 10 of 11 patients with HH (90.9%) received NAC according to the clinical stage, which was significantly more than in the non-HH group (P=0.02). Type and route of graft organ were not related to HH development. Moreover, the fixation of the conduit organ at the hiatus does not contribute to post-operative HH. CONCLUSIONS: In the current study, we showed that NAC was a major risk factor of HH development after MIE.

Hypoxia-induced ANGPTL4 sustains tumour growth and anoikis resistance through different mechanisms in scirrhous gastric cancer cell lines.

Patients with scirrhous gastric cancer (SGC) frequently develop peritoneal dissemination, which leads to poor prognosis. The secreted protein angiopoietin-like-4 (ANGPTL4), which is induced by hypoxia, exerts diverse effects on cancer progression. Here, we aimed to determine the biological function of ANGPTL4 in SGC cells under hypoxia. ANGPTL4 levels were higher in SGC cells under hypoxia than in other types of gastric cancer cells. Hypoxia-induced ANGPTL4 mRNA expression was regulated by hypoxia-inducible factor-1α (HIF-1α). Under hypoxic conditions, monolayer cultures of ANGPTL4 knockdown (KD) 58As9 SGC (58As9-KD) cells were arrested in the G1 phase of the cell cycle through downregulation of c-Myc and upregulation of p27, in contrast to control 58As9-SC cells. Moreover, the ability of 58As9-KD xenografts to form tumours in nude mice was strongly suppressed. When 58As9-KD cells were cultured in suspension, hypoxia strongly increased their susceptibility to anoikis through suppression of the FAK/Src/PI3K-Akt/ERK pro-survival pathway, followed by activation of the apoptotic factors caspases-3, -8 and -9. The development of peritoneal dissemination by 58As9-KD cells was completely inhibited compared with that by 58As9-SC cells. In conclusion, ANGPTL4 is uniquely induced by hypoxia in cultured SGC cells and is essential for tumour growth and resistance to anoikis through different mechanisms.

Regulation and clinical significance of the hypoxia-induced expression of ANGPTL4 in gastric cancer.

Solid tumors are often exposed to hypoxia. Hypoxia inducible factor (HIF)-1α upregulates numerous target genes associated with the malignant behavior of hypoxic cancer cells. A member of the angiopoietin family, angiopoietin-like protein 4 (ANGPTL4) is a hypoxia-inducible gene. The present study aimed to clarify whether ANGPTL4 is regulated by HIF-1α in gastric cancer cells. The study also assessed whether ANGPTL4 expression is associated with clinicopathological factors or HIF-1α expression in gastric cancer tissues. Hypoxia-induced ANGPTL4 expression was quantitatively analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in 10 gastric cancer cell lines. RT-qPCR was further employed to investigate the HIF-1α dependency of ANGPTL4 expression using HIF-1α-knockdown transfectant 58As9-KD and control 58As9-SC gastric cancer cells. The HIF-1α and ANGPTL4 expression levels were immunohistochemically analyzed in 170 gastric cancer tissue specimens and were assessed for any correlations with the clinicopathological factors and/or patient survival. Subsequently, hypoxia-induced ANGPTL4 expression was observed in 7 out of 10 gastric cancer cell lines. The hypoxic induction of ANGPTL4 was almost preserved in the 58As9-KD cells compared with that observed in the 58As9-SC cells, while the induction of known HIF-1α target gene, carbonic anhydrase 9, was completely suppressed in the 58As9-KD cells. In the gastric cancer tissues, ANGPTL4 expression was inversely correlated with the tumor depth, whereas HIF-1α expression was positively correlated with venous invasion. A survival analysis revealed that the expression of ANGPTL4 was significantly correlated with a longer survival time, whereas that of HIF-1α was correlated with a shorter survival time. In conclusion, the present findings indicate that hypoxia-induced ANGPTL4 expression is independent of HIF-1α in hypoxic gastric cancer cells. ANGPTL4 may be a favorable marker for predicting a long survival time, whereas HIF-1α predicts a poor prognosis, in gastric cancer patients. The hypoxic environment independently induces ANGPTL4 and HIF-1α, which are believed to exhibit adverse effects on tumor progression.

The Apoptotic Effect of HIF-1α Inhibition Combined with Glucose plus Insulin Treatment on Gastric Cancer under Hypoxic Conditions.

Gastric cancer grows under a hypoxic environment. HIF-1α is known to play an important role in controlling the production of reactive oxygen species (ROS) in the mitochondria under hypoxic conditions. We previously established HIF-1α knockdown (KD) cells and control (SC) cells in the 58As9 gastric cancer cell line. In this study, we revealed that KD cells, but not SC cells, induced apoptosis under conditions of hypoxia (1% O2) due to excessive production of ROS. A quantitative RT-PCR analysis demonstrated that the expressions of ten genes, which are involved in the control mechanisms of ROS (including the Warburg effect, mitophagy, electron transport chain [ETC] modification and ROS scavenging), were regulated by HIF-1α. Moreover, the promotion of glucose uptake by glucose plus insulin (GI) treatment enhanced the apoptotic effect, which was accompanied by further ROS production in hypoxic KD cells. A Western blot analysis showed that the membranous expression of GLUT1 in KD cells was elevated by glucose and/or insulin treatments, indicating that the GI-induced glucose uptake is mediated by the increased translocation of GLUT1 on the cell membrane. Finally, the anti-tumor effect of HIF-1α knockdown (KD) plus GI was evaluated using a tumor xenograft model, where a hypoxic environment naturally exists. As a result, the GI treatment strongly inhibited the growth of the KD tumors whereby cell apoptosis was highly induced in comparison to the control treatment. In contrast, the growth of the SC tumors expressing HIF-1α was not affected by the GI treatment. Taken together, the results suggest that HIF-1α inhibition plus GI may be an ideal therapy, because the apoptosis due to the destruction of ROS homeostasis is specifically induced in gastric cancer that grows under a hypoxic environment, but not in the normal tissue under the aerobic conditions.

Pure Robotic Surgery for Intraluminally Growing Gastrointestinal Stromal Tumors around the Esophagogastric junction or Pyloric Ring.

BACKGROUND/AIMS: Laparoscopic resection of gastrointestinal stromal tumors has become wide-spread as a minimally invasive surgical method. However, the limitations of laparoscopic surgery for GISTs are well recognized. METHODOLOGY: We developed a local resection by pure robotic surgical procedure to treat intraluminally growing GISTs located in sites that are unsuitable for laparoscopic surgery. Using articulated robotic arms, the GIST is completely excised with a safe margin while employing a unique technique to provide a good operative view and to prevent the intra-abdominal dissemination of the tumor from the cut edge created by robotic excision. The defect created after excision of the tumor is closed using robotic sewing. RESULTS: Four patients were successfully treated with pure robotic surgery without conversion to laparoscopic or open surgery or changing in the method of gastrectomy. CONCLUSIONS: Using robotic surgery, intraluminally growing GISTs located in sites unsuitable for conventional laparoscopic surgery can be treated with minimally invasive procedures.

The value of trefoil factor 3 expression in predicting the long­term outcome and early recurrence of colorectal cancer.

The trefoil factor (TFF) family comprises three thermo-stable and protease-resistant proteins (TFF1, TFF2 and TFF3) and plays an essential role in gastrointestinal mucosa protection and regeneration, and TFFs have recently been found to be involved in the development and progression of various types of cancer. However, the clinical significance of TFFs in colorectal cancer (CRC) patients remains unclear. The present study determined the relationship between TFF expression and clinicopathological findings, as well as long-term outcome in CRC patients. The mRNA expression levels of TFFs were examined in the excised CRC specimens obtained from 154 consecutive CRC patients who underwent surgical resection between 2005 and 2007 at our institution. TFF3 expression was significantly associated with the presence of distant metastasis (p=0.017), although neither TFF1 nor TFF2 expression was associated with the clinicopathological features. Survival rate of the patients with positive TFF3 was significantly worse compared to those with negative TFF3 (p=0.011). A multivariate analysis revealed that the expression of TFF3, lymph node metastasis, and vascular invasion were independent prognostic factors for disease-specific survival. Furthermore, among 134 patients with no clinical findings of metastasis at surgery, the patients with positive TFF3 experienced recurrence within one year more frequently than those with negative TFF3 (p=0.039). In conclusion, TFF3 is not only a useful biomarker for a long-term surgical result in CRC patient, but also may be a risk factor of early recurrence.

Thoracoscopic esophagectomy using prone positioning.

Thoracotomic esophagectomy followed by cervical and abdominal procedures has been conventionally performed as the best curable operative procedure for treating invasive thoracic esophageal carcinoma. Despite improvements in the survival rate, the procedure is associated with significant operative morbidity and mortality rates due to the extreme invasiveness of an extensive dissection of the lymph nodes. Minimally invasive esophagectomy (MIE) was developed to reduce surgical invasiveness. Recently, the use of thoracoscopic esophagectomy performed in the prone position has stimulated new interest in minimally invasive approaches. However, the advantages and disadvantages of this technique are not well known. In this review, the literature to date, including series and comparative studies of minimally invasive esophagectomy performed in the prone position, is summarized, and the various lessons learned and controversies surrounding this technique are addressed.

HIF-1α is a crucial factor in the development of peritoneal dissemination via natural metastatic routes in scirrhous gastric cancer.

The molecular mechanisms underlying the peritoneal dissemination of gastric cancer remain unclear. Using in vivo metastatic models, this study attempted to clarify the role of hypoxia inducible factor (HIF)-1α in the development of peritoneal dissemination of gastric cancer. HIF-1α knockdown (KD) cells were established in the scirrhous gastric cancer cell line 58As9. Using KD and control (SC) cells, the presence of peritoneal dissemination was assessed in orthotopic implantation (o.i.) and intraperitoneal injection (i.p.) models. A series of in vitro analyses were also conducted. Finally, tumor angiogenesis was immunohistochemically analyzed. In the o.i. model, peritoneal dissemination was more frequently observed in the SC mice (93%) compared to the KD mice (13%) (P<0.001). In the i.p. model, peritoneal dissemination occurred at a high rate in both types of mice; however, a greater number of nodules was observed in the KD mice (P=0.017). The in vitro assays showed that HIF-1α exerts unfavorable effects on anoikis resistance and adhesion to extracellular matrix. Angiogenesis and vascular invasion were more aggressive in the SC gastric tumors. Vascular invasion was present in the intratumoral regions of the disseminated nodules in the SC o.i., but not the i.p., mice. HIF-1α was found to be crucial for the development of peritoneal dissemination in o.i. model, which mimics natural metastasis. In contrast, HIF-1α played an inhibitory role in suppressing peritoneal dissemination in the i.p. model. These results indicate that peritoneal dissemination in o.i. mice may not act through a seeding mechanism. An immunohistochemical analysis demonstrated HIF-1α-activated angiogenesis and vascular invasion in stomach tumors. Furthermore, the results showed that the disseminated nodules observed in SC o.i. mice were formed via extravasation of cancer cells. We provide a possible mechanism in which peritoneal dissemination of gastric cancer develops via a vascular network whereby HIF-1α activates tumor angiogenesis.

Loss of trefoil factor 1 is regulated by DNA methylation and is an independent predictive factor for poor survival in advanced gastric cancer.

Trefoil factor 1 (TFF1) is considered to be a tumor suppressor gene in gastric cancer. However, the role of TFF1 expression and its regulation in gastric cancer patients remain unclear. The aims of this study were to clarify the clinical significance of TFF1 and to determine its regulatory mechanisms. We assessed the immunohistochemical expression of TFF1 in 182 gastric cancer patients and examined whether or not TFF1 is associated with the clinicopathological factors and patient survival. In vitro study using TFF1 knockdown gastric cancer cells evaluated the role of TFF1 in cancer invasion. Bisulfite sequencing was performed to assess DNA methylation of TFF1 in cells and resected tissues. Patients with low expression of TFF1 showed a significantly deeper invasion of the tumor than those with high expression (p=0.037). Low expression of TFF1 was also associated with a poor survival (p=0.029) in 108 patients who were treated by surgery alone. Both TFF1 expression and lymph node metastasis are independent predictive factors for disease-specific survival in a multivariate analysis. In an in vitro study, invasive power of the cells was significantly increased in the TFF1­deficient cells compared with the control cells. Bisulfate sequencing showed that TFF1 expression is strongly dependent on DNA methylation in both gastric cancer cells and tissues. Interestingly, methylation status of two specific CpG sites, which are located close to a TATA box and hypoxia response element (HRE), determined the TFF1 expression in the resected tissues. TFF1 expression is silenced by DNA methylation and is associated with tumor invasion and a poor survival in gastric cancer patients. The expression and̸or methylation status of TFF1 may, therefore, serve as a useful biomarker for predicting survival in patients with advanced gastric cancer.

Immunohistochemical and molecular genetic analyses of multiple sporadic gastrointestinal stromal tumors.

A 77-year-old Japanese male patient was admitted to our hospital complaining of general fatigue and melena. A gastroduodenal endoscopic examination revealed no definitive localized lesions. However, both a large amount of cruor and blood flow from the small intestine into the ascending colon was observed during the colonoscopic examination. At least three tumors, believed to originate from the small intestine, were detected by abdominal computed tomography. Based on these findings, multiple and hemorrhagic small intestinal tumors were diagnosed and surgical treatment of the tumors planned. During the celiotomy, twelve tumors were found in the small intestine. Intestinal wedge or partial resection was applied. All excised specimens demonstrated morphology of a submucosal tumor and the largest tumor had a delle with coagulation on the mucosal face. In the histological findings, hematoxylin and eosin staining showed spindle cell morphology. The immunohistochemical examination revealed that the tumor cells were diffusely positive for KIT and CD34. The myenteric plexus layer of the small intestine was focal-positive for KIT and showed no intestinal cells of Cajal hyperplasia. The tumor sequencing results revealed an identical missense mutation in codon 642 of c-kit exon 13 leading to the replacement of lysine by glutamic acid and a silent germ-line mutation in exon 12 of the PDGFRA gene concerning whole blood, normal mucosa and tumors. We concluded that the current subject was categorized as having multiple sporadic-type gastrointestinal stromal tumor with identical mutational types. Although the patient did not receive any adjuvant chemotherapy, there has been no sign of recurrence over the 3 years since the surgery.

A hepatocellular neoplasm accompanied with massive histiocyte infiltration.

This report presents the case of a unique hepatocellular nodule occurring in a 73-year-old Japanese male with diabetes mellitus and mild obesity. The nodule consisted of hepatocyte-like tumor cells and abundant foam cell type histiocytes that filled up a sinusoid-like space and formed central loose fibrosis. The superficial area did not contain as many histiocytes and showed hepatocellular adenoma character, but there was a focal hepatocellular carcinoma-like lesion, thus suggesting hepatocellular adenoma with malignant transformation. The background liver showed an almost normal histology except for mild steatosis with no specific infiltration of macrophages. These macrophages contained abundant fat droplets, whereas the tumor cells had no fat droplets. The expressions of monocyte chemoattractant protein-1 and macrophage colony-stimulating factor were significantly higher in the tumor than in the background liver. These findings suggested such macrophage infiltration induced by the tumor cells and these macrophages probably phagocytosed surplus fat at the intercellular space of this unique tumor.