Immunostimulatory Effect of Postbiotics Prepared from Phellinus linteus Mycelial Submerged Culture via Activation of Spleen and Peyer's Patch in C3H/HeN Mice.

Medicinal mushrooms are an important natural resource promoting health benefits. Herein, Phellinus linteus mycelia were prepared under submerged cultivation, the mycelium-containing culture broth was extracted as a whole to obtain the postbiotic materials (PLME), and its effect on the immune system was evaluated in normal C3H/HeN mice. Oral administration of PLME for 4 weeks was well tolerated and safe. In the PLME-administered groups, in addition to the production of immunostimulatory cytokines, such as interferon gamma (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6), the mitogenic activity was significantly increased. PLME administration also significantly increased the levels of serum immunoglobulin G (IgG) and IgA in the small intestinal fluid and Peyer's patches and enhanced Peyer's patch-mediated bone marrow cell proliferation activity and cytokine production (IL-2, IL-6, and IFN-γ). Histomorphometric analyses showed an increase in immune cells in the spleen and small intestinal tissues of mice administered PLME, supporting the rationale for its immune system activation. PLME mainly contained neutral sugar (969.1 mg/g), comprising primarily of glucose as a monosaccharide unit. The ß-glucan content was 88.5 mg/g. Data suggest that PLME effectively promote immune function by stimulating the systemic immune system through the spleen and intestinal immune tissues. PLME can thus be developed as a functional ingredient to enhance immune functions.

Developing a Mathematical Model of Intracellular Calcium Dynamics for Evaluating Combined Anticancer Effects of Afatinib and RP4010 in Esophageal Cancer.

Targeting dysregulated Ca2+ signaling in cancer cells is an emerging chemotherapy approach. We previously reported that store-operated Ca2+ entry (SOCE) blockers, such as RP4010, are promising antitumor drugs for esophageal cancer. As a tyrosine kinase inhibitor (TKI), afatinib received FDA approval to be used in targeted therapy for patients with EGFR mutation-positive cancers. While preclinical studies and clinical trials have shown that afatinib has benefits for esophageal cancer patients, it is not known whether a combination of afatinib and RP4010 could achieve better anticancer effects. Since TKI can alter intracellular Ca2+ dynamics through EGFR/phospholipase C-γ pathway, in this study, we evaluated the inhibitory effect of afatinib and RP4010 on intracellular Ca2+ oscillations in KYSE-150, a human esophageal squamous cell carcinoma cell line, using both experimental and mathematical simulations. Our mathematical simulation of Ca2+ oscillations could fit well with experimental data responding to afatinib or RP4010, both separately or in combination. Guided by simulation, we were able to identify a proper ratio of afatinib and RP4010 for combined treatment, and such a combination presented synergistic anticancer-effect evidence by experimental measurement of intracellular Ca2+ and cell proliferation. This intracellular Ca2+ dynamic-based mathematical simulation approach could be useful for a rapid and cost-effective evaluation of combined targeting therapy drugs.

Identification of a Putative Enhancer RNA for EGFR in Hyper-Accessible Regions in Esophageal Squamous Cell Carcinoma Cells by Analysis of Chromatin Accessibility Landscapes.

Abnormal genetic and epigenetic modifications play a key role in esophageal cancer. By Assay for Transposase-Accessible Chromatin by sequencing (ATAC-seq), this study compared chromatin accessibility landscapes among two esophageal squamous cell carcinoma (ESCC) cell lines, KYSE-30 and KYSE-150, and a non-cancerous esophageal epithelial cell line, HET-1A. Data showed that hyper-accessible regions in ESCC cells contained genes related with cancer hallmarks, such as epidermal growth factor receptor (EGFR). Multi-omics analysis and digital-droplet PCR results demonstrated that several non-coding RNAs in EGFR upstream were upregulated in ESCC cells. Among them, one appeared to act as an enhancer RNA responsible for EGFR overexpression. Further motif analysis and pharmacological data suggested that AP-1 family transcription factors were able to bind the hyper-accessible regions and thus to regulate cancer cell proliferation and migration. This study discovered a putative enhancer RNA for EGFR gene and the reliance of ESCC on AP-1 transcription factor.

Mn2+ Quenching Assay for Store-Operated Calcium Entry.

Store-operated Ca2+ entry (SOCE) pathway plays important roles in many cellular processes, which is largely studied by using fluorescent Ca2+ indicator, Fura-2. Extracellular Mn2+ is able to cross the plasma membrane through SOCE and quenches the fluorescence signals from Fura-2. Thus, the fluorescence quenching rate by Mn2+ composes a convenient assay to monitor the extent of SOCE. This chapter describes an experimental method of Mn2+ quenching assay for both cultured esophageal epithelial and skeletal muscle cells. It also explains how to perform a quantitative analysis of graded SOCE.

Targeting Orai1-mediated store-operated calcium entry by RP4010 for anti-tumor activity in esophagus squamous cell carcinoma.

Esophageal cancer (EC) is the 6th leading cause of cancer mortality worldwide with poor prognosis, hence more effective chemotherapeutic drugs for this deadly disease are urgently needed. We previously reported that high expression of Orai1, a store-operated Ca2+entry (SOCE) channel, was associated with poor survival rate in EC patients and Orai1-mediated intracellular Ca2+ oscillations regulated cancer cell proliferation. Previous studies suggested that Orai1-mediated SOCE is a promising target for EC chemotherapy. Here, we evaluated the anti-cancer effect of a novel SOCE inhibitor, RP4010, in cultured EC cells and xenograft models. Compared to other previously reported SOCE channel inhibitors, RP4010 is more potent in blocking SOCE and inhibiting cell proliferation in EC and other cancer cells. Treatment with RP4010 resulted in reduction of intracellular Ca2+ oscillations, caused cell cycle arrest at G0/G1 phase in vitro, decreased nuclear translocation of nuclear factor kappa B (NF-κB) in vivo and in vitro, and inhibited tumor growth in vivo. Taken together, data demonstrated the therapeutic potential of RP4010 in EC patients via inhibition of SOCE-mediated intracellular Ca2+ signaling.

Clinical significance of pancreatic intraepithelial neoplasia in resectable pancreatic cancer on survivals.

PURPOSE: Noninvasive precursor lesions for pancreatic adenocarcinoma include pancreatic intraepithelial neoplasia (PanIN), intraductal papillary mucinous neoplasm, and mucinous cystic neoplasm. PanIN is often found synchronously adjacent to resected pancreatic ductal adenocarcinoma (PDAC) tumors. However, its prognostic significance on outcome after PDAC resection is unknown. The purpose of the current study was to determine if the presence of PanIN has a prognostic or predictive effect on survival after resection for PDAC with curative intent. METHODS: We retrospectively reviewed the clinicopathologic data of patients who underwent pancreatectomy for PDAC from January 2002 to January 2013. Intraductal papillary mucinous lesions and mucinous cystic neoplasms were excluded. All available postoperative imaging and clinical follow-up data were reviewed. RESULTS: There were 95 patients who underwent pancreatectomy. Tumors were most commonly located in the pancreas head and as such pancreaticoduodenectomy was the most commonly performed operation. The median tumor size was 3.2 cm. An absence of PanIN lesions was identified in 39 patients (41%). Of the patients with PanIN lesions, high-grade PanIN (grade 3) was the most common type (64.3%) followed by grade 2 (28.6%). There was no significant difference in overall survival or disease-free survival between the non-PanIN and PanIN groups. CONCLUSION: The presence or absence of PanIN lesions did not affect survival in patients undergoing resection for pancreatic cancer. However, patients with high-grade PanINs tended to have better overall survival. Larger studies with longer follow up are needed to accurately determine its clinical significance.

Selective inhibitory effects of zinc on cell proliferation in esophageal squamous cell carcinoma through Orai1.

Zinc, an essential micronutrient, has a cancer preventive role. Zinc deficiency has been shown to contribute to the progression of esophageal cancer. Orai1, a store-operated Ca2+ entry (SOCE) channel, was previously reported to be highly expressed in tumor tissues removed from patients with esophageal squamous cell carcinoma (ESCC) with poor prognosis, and elevation of its expression contributes to both hyperactive intracellular Ca2+ oscillations and fast cell proliferation in human ESCC cells. However, the molecular basis of cancer preventive functions of zinc and its association with Orai1-mediated cell proliferation remains unknown. The present study shows that zinc supplementation significantly inhibits proliferation of ESCC cell lines and that the effect of zinc is reversible with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine, a specific Zn2+ chelator, whereas nontumorigenic esophageal epithelial cells are significantly less sensitive to zinc treatment. Fluorescence live cell imaging revealed that extracellular Zn2+ exerted rapid inhibitory effects on Orai1-mediated SOCE and on intracellular Ca2+ oscillations in the ESCC cells. Knockdown of Orai1 or expression of Orai1 mutants with compromised zinc binding significantly diminished sensitivity of the cancer cells to zinc treatment in both SOCE and cell proliferation analyses. These data suggest that zinc may inhibit cell proliferation of esophageal cancer cells through Orai1-mediated intracellular Ca2+ oscillations and reveal a possible molecular basis for zinc-induced cancer prevention and Orai1-SOCE signaling pathway in cancer cells.-Choi, S., Cui, C., Luo, Y., Kim, S.-H., Ko, J.-K., Huo, X., Ma, J., Fu, L.-W., Souza, R. F., Korichneva, I., Pan, Z. Selective inhibitory effects of zinc on cell proliferation in esophageal squamous cell carcinoma through Orai1.

Zinc transporters and dysregulated channels in cancers.

As a nutritionally essential metal ion, zinc (Zn) not only constitutes a structural element for more than 3000 proteins but also plays important regulatory functions in cellular signal transduction. Zn homeostasis is tightly controlled by regulating the flux of Zn across cell membranes through specific transporters, i.e. ZnT and ZIP family proteins. Zn deficiency and malfunction of Zn transporters have been associated with many chronic diseases including cancer. However, the mechanisms underlying Zn regulatory functions in cellular signaling and their impact on the pathogenesis and progression of cancers remain largely unknown. In addition to these acknowledged multifunctions, Zn modulates a wide range of ion channels that in turn may also play an important role in cancer biology. The goal of this review is to propose how zinc deficiency, through modified Zn homeostasis, transporter activity and the putative regulatory function of Zn can influence ion channel activity, and thereby contribute to carcinogenesis and tumorigenesis. This review intends to stimulate interest in, and support for research into the understanding of Zn-modulated channels in cancers, and to search for novel biomarkers facilitating effective clinical stratification of high risk cancer patients as well as improved prevention and therapy in this emerging field.

Single-port laparoscopic distal pancreatectomy: initial experience.

INTRODUCTION: Laparoscopic distal pancreatectomy has become the standard treatment of choice for pancreatic tail cystic and solid tumors when technically feasible. Technological advances have led to the development of single-port laparoscopic surgery, a safe alternative procedure. We present our experiences with single-port laparoscopic distal pancreatectomy. MATERIALS AND METHODS: We retrospectively reviewed clinical records and compared clinical outcomes in 40 patients diagnosed with a pancreatic tail mass between 2007 and 2013 who received either conventional laparoscopic (n=28) or single-port laparoscopic distal pancreatectomy (n=12). RESULTS: The mean surgery time in the single-port group (279.8±53.0 minutes) was significantly longer than in the conventional group (186.9±86.6 minutes) (P=.001). The mean duration of postoperative hospital stay in the single-port group (12.2±5.4 days) was also significantly longer than in the conventional group (8.3±4.7 days) (P=.028). The spleen was preserved more in the conventional group (60.7%) than in the single-port group (33.3%), but the difference was not significant (P=.112). There were no significant differences in intraoperative blood loss, tumor size, conversion rate, or postoperative complications between the two groups. CONCLUSIONS: Blood loss and postoperative complications of single-port laparoscopic distal pancreatectomy are similar to those of conventional laparoscopic distal pancreatectomy. Single-port laparoscopic distal pancreatectomy can be performed safely and effectively in select patients with pancreas tail neoplasms, but is associated with a longer surgery time and postoperative hospital stay.

Comparison of hepatocellular carcinoma in American and Asian patients by tissue array analysis.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Although some epidemiologic and etiologic differences between Asian and Western HCC are known, detailed comparative studies with pathologic correlations have not been performed. METHODS: Paraffin sections of resected HCC specimens from Memorial Sloan-Kettering Cancer Center and Korea University Medical Center were used to construct tissue microarrays. Immunohistochemical staining of microarray sections was performed using antibodies against markers of proliferation and regulators of cell cycle. Patient data were correlated with staining results. RESULTS: When comparing both cohorts, significant differences were found in expression of p53 and MDM2. In the Asian group, more frequent positive staining for p53 (24%) was observed compared with the American group (9%; P = 0.037). For MDM2, 26% of American cases stained positive compared with 2% of Asian cases (P = 0.0003). No significant differences were found in expression of Ki67, p21, p27, cyclin D1, or bcl2. Female gender, vascular invasion, and lack of viral hepatitis infection correlated with positive MDM2 staining. CONCLUSION: These data likely correlate with differences in molecular pathogenesis of HCC based on racial and regional differences. These findings may have implications in choice of molecular targeted therapies based on patient ethnicity.

Hepatic differentiation from human embryonic stem cells using stromal cells.

OBJECTIVE: The derivation of hepatocytes from human embryonic stem (hES) cells is of value both in the study of early human liver organogenesis and in the creation of an unlimited source of donor cells for hepatocyte transplantation therapy. Here, we report the generation of hepatocyte-like cells derived from hES cells. METHODS: Hepatic endoderm cells were generated by adding activin A for 5 d- to 1-d-old embryoid bodies formed from hES cells. The hepatic endoderm cells were cocultured with mitomycin treated 3T3-J2 feeder cells. RESULTS: After co-culture with mitomycin treated 3T3-J2 feeder cells, these hepatic endodermal cells yielded hepatocyte-like cell colonies, which possessed the proliferation potential to be cultured for an extended period of more than 30 d. With extensive expansion, they co-expressed the hepatic marker AFP and albumin, indicating that they were hepatocyte-like cells. CONCLUSIONS: We report the generation of proliferative hepatocyte-like cells from hES cells. These hES cell derived hepatic cells can effectively be used as in vitro model for studying the mechanisms of hepatic stem/progenitor cell origin, self-renewal and differentiation.

Single-incision multiport laparoscopic cholecystectomy: things to overcome.

OBJECTIVES: To report on our initial experience with single-incision multiport laparoscopic cholecystectomy, together with its clinical outcomes. DESIGN: Nonrandomized prospective study. SETTING: University department of surgery. PATIENTS: Sixty-four patients with gallstones and gallbladder polyps were enrolled after providing informed consent. Based on our experience, we excluded patients with acute cholecystitis, concomitant choledocholithiasis, a history of previous upper abdominal surgery, and a suspicion of gallbladder cancer. MAIN OUTCOME MEASURES: We analyzed the outcomes and complications, based on our experience, according to the clinicopathologic and operative factors. We also compared patients who underwent single-incision multiport laparoscopic cholecystectomy with those who were converted to conventional laparoscopic cholecystectomy. RESULTS: There were 2 bile duct injuries and 4 surgical site infections. We had difficulties in visualizing the Calot triangle in 22 patients. Higher levels of inflammatory markers, longer operation times, and more frequent bile juice spillage were significantly observed in those patients. Ten patients were converted to conventional laparoscopic cholecystectomy. The mean age of patients who underwent conversion surgery was significantly older than that of the no-conversion group. The more the body mass index increased, the more the conversion rate increased. CONCLUSIONS: Experienced laparoscopic surgeons can safely perform cholecystectomy using conventional and curved laparoscopic instruments in selected patients. We recommend that you consider performing conventional laparoscopic cholecystectomy or that you use additional retraction devices for patients with a higher body mass index or acute cholecystitis.

Expression and clinical significance of cell cycle regulatory proteins in gallbladder and extrahepatic bile duct cancer.

Disruption of cell cycle controls is a pathognomonic feature of all malignant cells. Therefore, we immunohistochemically investigated the relationship between cell cycle regulatory proteins and clinicopathologic features in order to identify the biomarkers related to the outcome of patients with biliary tract cancer (BTC). A cohort of paraffin-embedded specimens were selected from 36 patients, including 18 gallbladder and 18 extrahepatic bile duct cancers, who underwent curative or palliative surgical resection at Korea University Medical Center from June 1998 to December 2004. Tissue microarrays were used to investigate the immunohistochemical staining for p21, p27, p53, cyclin D1, bcl2, and Ki-67. Univariate and multivariate survival analyses were performed to determine the prognostic significance of each protein expression. Absence of p21 expression independently predicted poor outcome in all cases. Well-differentiated tumor was found to be an independent good prognostic factor in gallbladder cancer. Absence of p21 expression and moderately to poorly differentiated tumor were found to be an independent poor prognostic factor in patients with negative for neural invasion. Absence of p21 and bcl2 were found to be an independent poor prognostic factor in patients with no lymph node metastasis. Absence of p21 expression was a significant independent poor prognostic factor in BTC, partly in patients with biologically less aggressive phenotypes. This finding suggests that determination of p21 expression in surgically resected specimens may provide prognostic information in addition to conventional pathologic findings for patients with BTC, especially those who have biologically less aggressive phenotypes.

Laparoscopic resection of a hepatocellular carcinoma arising from an ectopic liver.

Ectopic livers are rarely seen intra-abdominal lesions. Ectopic hepatocellular carcinoma (HCC) can be defined as an HCC arising from hepatic parenchyma located in an extrahepatic organ or tissue. The authors report a case of a primary, well-differentiated HCC arising from ectopic liver tissue in the left subphrenic space at the upper portion of the gastrorenal ligament that was successfully treated by laparoscopic resection. A 59-year-old man was referred to our department for the management of an intra-abdominal mass, which was incidentally found in a follow-up abdominal computed tomography scan for splenic laceration. The preoperative diagnosis suggested that it was a nonspecific stomach mass of maximal diameter 4.5 cm, such as, a gastrointestinal stromal tumor, located between the diaphragm and spleen. A computed tomography scan identified no mass in the liver. Laparoscopic resection was performed, and the final pathologic result confirmed that it was a HCC. The patient's postoperative course was unremarkable. This is the first reported case of a laparoscopically treated ectopic HCC. Moreover, laparoscopic resection was found to be safe and reliable in this case.

Peutz-Jeghers Syndrome with multiple genital tract tumors and breast cancer: a case report with a review of literatures.

We report here on the multiple genital tract neoplasms in a 41-yr-old Korean woman with Peutz-Jeghers Syndrome (PJS). The patient presented with lower abdominal pain. Her previous medical history was PJS and breast cancer. Pelvic ultrasound showed a multilocular cyst at the right adnexal region, diagnosed as bilateral ovarian mucinous borderline tumors. An ovarian sex cord tumor with annular tubules was incidentally diagnosed together with a minimal deviation adenocarcinoma of the uterine cervix and mucinous metaplasia of both the Fallopian tubal mucosa and the endometrium. Although the cases of multiple genital tract tumors with PJS has rarely been reported, the present case appears to be the first in Korea in which the PJS syndrome was complicated by multiple genital tract tumors and infiltrating carcinoma of the breast. The clinical significance of the multiple genital tract tumors and breast cancer associated with PJS is reviewed.

Mesenteric Castleman's disease.

We report here a rare case of mesenteric Castleman's disease presenting as a mesenteric mass. A 13-year-old female child was admitted to our hospital complaining of intermittent vague abdominal pain. She had hypochromic anemia, thrombocytosis and an elevated erythrocyte sedimentation rate (ESR). Ultrasonography and computed tomography indicated an intra- abdominal mass might represent a lymphoma or gastrointestinal stromal tumor or leiomyoma, but the definitive preoperative diagnosis couldn't be confirmed. The surgical resection of the mass revealed the mesenteric hyaline vascular-type Castleman's disease.