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Few studies have investigated the relationship between blood type and trauma outcomes according to the type of injury. We conducted a retrospective multicenter observational study in twelve emergency hospitals in Japan. Patients with isolated severe abdominal injury (abbreviated injury scale for the abdomen ≥ 3 and that for other organs p p = 0.012). Furthermore, type O was associated with significantly higher cause-specific mortalities, fewer VFD, and larger transfusion volumes. Blood type O was associated with significantly higher mortality and larger transfusion volumes in patients with isolated severe abdominal trauma.

The incidence of rectal cancer with a pelvic abscess is rare; hence, treatment strategies are difficult because both malignant and infectious inflammation need to be addressed. Here, we report the case of a 53-year-old man diagnosed with rectal cancer accompanied by a pelvic abscess. We performed transrectal drainage of the abscess, and a transanal rectal drainage tube was inserted into the abscess cavity. His symptoms rapidly improved, and computed tomography showed that the pelvic abscess had disappeared. Six weeks after drainage, radical laparoscopic Hartmann’s procedure with resection of the rectal cancer and incision drainage scar was performed. After adjuvant chemotherapy, laparoscopic stoma closure was performed a year after the operation. The patient showed no evidence of cancer recurrence 1.5 years after radical surgery. Transrectal drainage followed by laparoscopic radical resection can be a less invasive and effective treatment for rectal cancer accompanied by a pelvic abscess.

BACKGROUND:We sought to investigate the efficacy of perioperative tight glycemic control (TGC) in reducing of postoperative infectious complications (POICs) and study its impact on early inflammatory mediators in patients who underwent pancreaticoduodenectomy., METHODS:In this non-randomized trial, the artificial pancreas (AP) group received TGC (target glucose range of 80-110mg/dL; n=14), while the control group received conventional glycemic control (range of 80-180mg/dL; n=15). The primary endpoint was POICs., RESULTS:The AP group had a markedly decreased POIC rate (28.6% vs. 73.3%; P= 0.027), mean glycemic variability (13.5±3.5% vs. 16.4±5.9%; P=0.038), and plasma interleukin-6 level (26.3±33.8 vs 98.3±89.1pg/ml; P=0.036) compared to the control group, but insulin dosage (27.0±13.4 vs. 10.2±16.2 U; P=0.002) and the adiponectin ratio (i.e., postoperative/preoperative adiponectin; 0.8±0.2 vs. 0.6±0.3; P=0.021) were markedly higher in the AP group., CONCLUSIONS:Among patients undergoing PD with impaired glucose tolerance, AP facilitated strict glycemic control and resulted in a reduction of anti-inflammatory mediators and POICs., SUMMARY:Perioperative hyperglycemia increases postoperative infectious complications; however, tight glycemic control using artificial pancreas can reduce them via a dual effect. Artificial pancreas facilitates strict and safe glycemic control while reducing anti-inflammatory mediators, including adiponectin, following pancreaticoduodenectomy.

A Bochdalek hernia (BH) is a congenital abnormality with incomplete closure of the diaphragm. It is usually manifested in infants but rarely in adults. Here, we report an adult patient with gastric volvulus and giant BH that were safely repaired by endoscopic reduction and elective laparoscopic surgery, respectively. A 79-year-old woman presented with left upper abdominal pain but no history of trauma. CT revealed a giant BH with gastric volvulus. After emergency endoscopic reduction of the volvulus, elective laparoscopic repair of the BH was performed. The 8 × 8-cm defect was repaired with interrupted nonabsorbable sutures and a mesh. The patient's postoperative course was uneventful, and no complications or recurrence were observed in the 6 months that followed.

Postoperative pancreatic fistula (POPF) is a serious complication of pancreaticoduodenectomy. However, the criteria for prompting drainage have not been clarified yet. We evaluated 80 patients who underwent pancreaticoduodenectomy between 2011 and 2016. Clinically relevant POPF (International Study Group of Postoperative Pancreatic Fistula grade B or C) was evaluated on the basis of the following parameters: changes in pancreatic thickness between preoperation and postoperative day (POD) 4 identified via enhanced CT, drain amylase level, laboratory data, and operative factors. POPF occurred in 21 patients (26.3%). The median change in pancreatic thickness before and after operation was 8.33 mm in the POPF-positive group, which was significantly larger than that in the POPF-negative group (3.79 mm, P

Introduction Even if laparoscopic cholecystectomy (LC) has lower invasiveness through small incisions compared with laparotomy, postoperative pain control is important. Methods This prospective, randomized, single-blinded, interventional, single-center study was conducted from December 2016 to March 2018 at the Shiga University of Medical Science Hospital in Japan. Enrolled patients were assigned to either a rectus sheath block (RSB) group or an infiltrative local anesthesia (LA) group. After LC, the RSB group received bilateral RSB with 10 mL of 0.375% ropivacaine and the LA group received subcutaneous and fascial injection with 10 mL of 0.75% ropivacaine at the umbilical wound. The primary endpoint was a visual analog scale (VAS) score on postoperative day (POD) 1. Results This study enrolled 62 patients (RSB group = 31, LA group = 31). On POD1, the mean VAS scores were 36.4 ± 18.9 and 29.4 ± 15.4 in the RSB group and LA groups, respectively, showing that the LA group tended to describe lesser postoperative pain than the RSB group (P = 0.062). Conclusions VAS scores on POD1 were not different between the groups. LC patients might obtain postoperative pain control via long-acting local analgesia.

Background: A rectovaginal fistula (RVF) after low anterior resection for rectal cancer is troublesome and refractory. Although various surgical procedures have been previously described, no definitive procedure has shown satisfactory outcomes. We present two consecutive Japanese patients who underwent successful surgery for a RVF after low anterior resection. Case presentation: The patients were a 61-year-old woman and a 64-year-old woman. They were admitted to our hospital with a chief complaint of fecal discharge from the vagina after low anterior resection using the double-stapling technique for rectal cancer. They were diagnosed with RVF. Local surgical procedures, including diverting ileostomy, were unsuccessful in previous hospitals. Therefore, we performed laparoscopy-assisted repair of the RVF. In both the patients, laparoscopically robust pelvic adhesions were dissected, and the sigmoid colon was transected just before the RVF. Thereafter, in combination with a perineal approach, the rectum along with a previous anastomosis and fistula were completely removed. Surgeries were completed after vaginal repair, redo coloanal anastomosis, and interposition of the dissected connective tissue. In both the patients, the postoperative courses were uneventful. They have complained of neither recurrence of any RVF nor fecal incontinence 1 year and 10 months respectively after diverting stoma closure.Conclusions: A laparoscopy-assisted procedure with reanastomosis and interposition of the perineal connective tissue can be an effective treatment for an RVF after low anterior resection for rectal cancer.

Retroperitoneal liposarcoma is a relatively rare disease, with a high recurrence rate and poor prognosis. We encountered 8 patients with retroperitoneal liposarcoma who underwent surgery in Shiga University of Medical Science Hospital. We often encounter elderly male patients without symptoms. Of the 8 patients, 6 received extensive resection that included the surrounding organs or tissues; however, 3 patients demonstrated positive surgical margins, which resulted in liposarcoma recurrence. Despite the additional resection in the 3 recurrent cases, all the patients had a tumor relapse. One patient with an unresectable tumor received chemotherapy. The other patients received surgical treatment 3 times. One patient developed an unresectable relapse after receiving chemotherapy. Another patient attained long-term survival by adjuvant chemoradiotherapy combined with 3 surgeries. Aggressive surgical resection to achieve a negative surgical margin and careful postoperative follow-up seem important for the treatment of retroperitoneal liposarcoma. This study suggests that postoperative adjuvant therapy may contribute to the improvement of prognosis. Further findings must be accumulated to clarify the significance of postoperative adjuvant therapies in the future.

Adoptive cell transfer (ACT) is an emerging and promising cancer immunotherapy that has been improved through various approaches. Here, we described the distinctive characteristics and functions of tumor Ag-specific effector CD8+ T-cells, co-cultured with a tumor-specific peptide and a stimulatory anti-OX40 antibody, before being used for ACT therapy in tumor-bearing mouse recipients. Splenic T-cells were obtained from wild-type FVB/N mice that had been injected with a HER2/neu (neu)-expressing tumor and a neu-vaccine. The cells were then incubated for 7 days in vitro with a major histocompatibility complex (MHC) class I peptide derived from neu, in the presence or absence of an agonistic anti-OX40 monoclonal antibody, before CD8+ T cells were isolated for use in ACT therapy. The proliferative ability of OX40-driven tumor Ag-specific effector CD8+ T-cells in vitro was less than that of non-OX40-driven tumor Ag-specific effector CD8+ T-cells, but they expressed significantly more early T-cell differentiation markers, such as CD27, CD62L and CCR7, and significantly higher levels of Bcl-2, an anti-apoptotic protein. These OX40-driven tumor Ag-specific effector CD8+ T-cells, when transferred into tumor-bearing recipients, demonstrated potent proliferation capability and successfully eradicated the established tumor. In addition, these cells exhibited long-term antitumor function, and appeared to be established as memory T-cells. Our findings suggest a possible in vitro approach for improving the efficacy of ACT, which is simple, requires only a small amount of modulator, and can potentially avoid several toxicities associated with co-stimulation in vivo.

An 80s man presenting with general malaise and anorexia was referred for treatment of abdominal tumor. Abdominal contrast-enhanced CT revealed a tumor in the left renal cavity. The inside of the tumor coexisted with a fat component and a solid component having a contrast effect. In addition, 2 solid tumors were found to be in contact with the stomach, away from the primary lesion. Based on these findings, retroperitoneal liposarcoma with intraperitoneal metastases was diagnosed. The patient underwent excision of the retroperitoneal tumor and local gastrectomy. The size of the main tumor was 21×18 cm, and the weight was 2.0 kg. Histopathology of the resected specimen showed dedifferentiated liposarcoma and its metastases. The resected margin of the excised tumor was negative. Liposarcoma has a high local recurrence rate, and the status of a resected margin of the tumor is an important factor for prognosis. Here, we report a case of dedifferentiated liposarcoma with metastatic lesions that could be completely resected.

We evaluated the effectiveness of palliative stomas created to resolve symptoms of bowel obstruction.We retrospectively evaluated patient characteristics and outcomes in 16 cases of palliative enterostomy performed to resolve malignant bowel obstruction.The median age of the patients was 64 years, and the most common original cancer was colorectal cancer(11 cases, 69%). Oral intake was recovered in 15 patients after the surgery(94%), and the colorectal obstruction scoring system(CROSS)score improved from 2 to 4. Postoperative complications(Clavien-Dindo grade Ⅱ or higher)were observed in 8 cases(50%), in which serum albumin levels were less than 3.5 g/dL(p=0.077), resulting in longer hospital stays(p=0.041). The median overall survival time was 107 days, and longer survival time was achieved in patients aged younger than 70 years, with CA19-9 levels C37.7 U/mL and postoperative CROSS score of 4, who were administered postoperative chemotherapy. The chemotherapy was feasible after surgery when CROSS scores improved to 4(p=0.019).Palliative stomas contributed to the improvement in oral intake of patients with terminal cancer. This improvement may allow patients to receive postoperative chemotherapy, which leads to longer survival times.

The aim of this study was to assess the relationship between the computed tomography (CT) pancreatic parenchyma attenuation value and clinically relevant postoperative pancreatic fistula (POPF).The medical records of 115 patients who underwent pancreaticoduodenectomy and preoperative dynamic CT were retrospectively reviewed. The CT attenuation values of the nonenhanced (N), arterial (A), portal venous (P), and late (L) phase in the pancreatic parenchyma were determined via CT, and the A/N, A/P, and P/L ratios were calculated. The CT attenuation values and value ratios were compared between the POPF and non-POPF groups.Thirty-two patients (28%) were categorized in the POPF group. On univariate analysis, the A/P ratio (P0.001) and P/L ratio (P = 0.018) were significantly higher in the POPF group. On receiver operating characteristic curve analysis, the A/P and P/L ratio cutoff values for predicting POPF were 1.19 and 1.17, respectively. Of the preoperative evaluable factors, A/P ratio of 1.19 or greater (P0.001; odds ratio, 10.3) and P/L ratio of 1.17 or greater (P = 0.049; odds ratio, 3.23) were independent predictive factors for POPF, and the combination of the 2 ratios was useful in detecting POPF preoperatively.The enhancement pattern of the pancreatic parenchyma is associated with the development of clinically relevant POPF.

Recently, accelerometers measuring physical activity level have been available to the public. In the present study, it was examined whether the accelerometer could evaluate postoperative outcomes for 12 patients subjected to hepatic resection from August-November 2016. The association was evaluated between the changing pattern of activity level until the postoperative day (POD) 7 and the occurrence of postoperative complications. The median age of patients was 79 years (range, 58-85). Postoperative complications were identified in 6 patients. The activity level in patients with complications was low from POD 1 and was significantly lower than patients without complications following POD 6. The changing pattern of activity level with all included patients could be divided into the following 3 types: Increase type, bell curve type and flat type. Patients without complications exhibited an accelerated increase of postoperative activity level, categorized as increase type. Bell curve type and flat type demonstrated delay of recovery in postoperative activity levels, and were suggested to be associated with the occurrence of postoperative complications. These findings may provide rationale for larger sample studies to evaluate whether physical activity level measured via accelerometer may be a surrogate marker for postoperative complications.

Interferon (IFN) has been identified to suppress carcinogenesis when used for treating hepatitis C virus (HCV) infections. Treatment with IFN-free direct-acting antiviral agents (DAAs) is an acceptable alternative, even in elderly patients or patients who have been treated for hepatocellular carcinoma (HCC), because it has a lower incidence of side effects and higher sustained virological response (SVR) rate compared with IFN treatment. However, the suppression of carcinogenesis by DAAs is unclear. In the present study, 19 patients who underwent DAA treatment following treatment for HCC between January 2015 and March 2017 were retrospectively investigated. The clinical data were compared between 9 patients with HCC recurrence following DAA treatment (recurrence group) and 10 patients without HCC recurrence (no-recurrence group). The 1-year cumulative recurrence rate of HCC following SVR was as high as 50.2%. Age and sex did not significantly differ between the two groups, and the average number of HCC treatments prior to DAA treatment was also not significantly different between the recurrence and no-recurrence groups (3.2 and 2.2, respectively). The median interval between the final HCC treatment and the commencement of DAA treatment was 88 days in the recurrence group, which was significantly less compared with 790 days in the no-recurrence group (P=0.018). An interval of 120 days or more from final HCC treatment to the commencement of DAA treatment was a significant independent factor of no HCC recurrence following DAA treatment (P=0.028). A high HCC recurrence rate was identified following DAA treatment in patients with a history of HCC treatment. Therefore, there should be at least a 4-month interval from the final HCC treatment to the commencement of DAA treatment to ensure no HCC recurrence.

A 70's man presenting with a chief complaint of stomachache was found to have advanced gastric cancer with a deep ulcer and some lymph-node metastases. We decided performing a curative operation after 2 courses of S-1 plus cisplatin. On the first course day 13 of chemotherapy, he complained of severe epigastralgia, and we diagnosed as generalized peritonitis due to perforation of gastric cancer. We performed an urgent laparoscopic operation, which made perforation simple closure and omentopexy. Curative distal gastrectomy with D2 lymph node dissection was successfully performed on postoperative day 16.

A 70-year-old woman presenting with abdominal pain was admitted to our hospital. Abdominal contrast CT revealed a small intestine tumor of 10 cm with active bleeding and performed partial resection of the small intestine including tumor. Pathological findings were high risk GIST of the small intestine because of spindle cells and c-kit positive. Imatinib 400mg/day as adjuvant chemotherapy was administered. However administration was stopped for 15 days because of the Grade 4 erythema multiforme. Recurrence of peritoneal dissemination was observed in 2 years after surgery and tumor resection was performed, but complete resection was difficult. Within 5 years after surgery, tumor resection was performed on a total of 5 times peritoneal disseminative recurrences, and it was possible to avoid the appearance of symptoms due to tumor augmentation.

Adoptive cell transfer (ACT) is an emerging and promising cancer immunotherapy that has been improved through various approaches. Here, we described the distinctive characteristics and functions of tumor Ag-specific effector CD8

Hepatocyte growth factor (HGF) has an inhibitory effect on human HepG2 hepatoma cell proliferation. Previously, it was shown that HGF treatment downregulated Id1 and upregulated p16INK4a in an ERK-dependent manner, leading to the inhibition of cellular proliferation. Here, new insight suggests that Skp2, an SCF complex component and potential prognosticator in cancer, is downregulated by injection of HGF into established HepG2 xenograft tumors. The downregulation was evident at both the mRNA and protein level and in an ERK-dependent manner. Critically, high expression of Skp2 restored HGF-inhibited cell proliferation, indicating that the inhibitory effect of HGF required the downregulation of Skp2. However, downregulation was not involved in the HGF-induced upregulation of a CDK inhibitor, p27Kip1, a known SCF-Skp2 target. Instead, data revealed that Skp2 regulated Myc activity, which has oncogenic potential in the generation of hepatocellular carcinoma. Elevated expression of Skp2 or a mutant that is unable to associate with the SCF complex was capable of activating Myc, suggesting that Skp2 does not act on Myc as a component of the SCF complex, and thus functions as an activator of Myc independent of its role in ubiquitination. Furthermore, Skp2 regulated Id1 expression by regulating Myc activity, and the regulation of Skp2 is involved in the activity of p16 promoter through regulation of Id1 expression. Overall, these mechanistic findings provide the first evidence that ERK-dependent downregulation of Skp2 reduced Myc activity, leading to HGF-induced inhibition of cell proliferation through decreased Id1 expression. Implications: This study elucidates the molecular details of HGF-induced inhibition of cellular proliferation in liver cancer cells. Mol Cancer Res; 11(11); 1437–47. ©2013 AACR.

An 80's woman was diagnosed with eccrine porocarcinoma of the head in 2010.T he tumor was removed surgically but relapsed in the cervical and axillary lymph nodes 2 years later.The patient underwent surgery, and received systemic chemotherapy and radiation.Chest CT after treatment revealed an irregular mass and thickened skin in the left breast.Core needle biopsy specimens were used to diagnose metastasis of eccrine porocarcinoma.A wide excision with a 1 cm margin was performed under local anesthesia.After surgery, supraclavicular lymph node recurrence was detected.The patient received palliative care because there was no effective treatment available.Eccrine porocarcinoma is a rare malignant tumor of the intraepidermal sweat duct.Breast metastasis from malignant disease is also rare.To our knowledge, breast metastasis of eccrine porocarcinoma has not been reported.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a Kunitz-type transmembrane serine protease inhibitor initially identified as a potent inhibitor of hepatocyte growth factor activator (HGFA), a serine protease that converts pro-HGF to the active form. HAI-1 also has inhibitory activity against serine proteases such as matriptase, hepsin and prostasin. In this study, we examined effects of HAI-1 on the protease activity and proteolytic activation of human airway trypsin-like protease (HAT), a transmembrane serine protease that is expressed mainly in bronchial epithelial cells. A soluble form of HAI-1 inhibited the protease activity of HAT in vitro. HAT was proteolytically activated in cultured mammalian cells transfected with its expression vector, and a soluble form of active HAT was released into the conditioned medium. The proteolytic activation of HAT required its own serine protease activity. Co-expression of the transmembrane full-length HAI-1 inhibited the proteolytic activation of HAT. In addition, full-length HAI-1 associated with the transmembrane full-length HAT in co-expressing cells. Like other target proteases of HAI-1, HAT converted pro-HGF to the active form in vitro. These results suggest that HAI-1 functions as a physiological regulator of HAT by inhibiting its protease activity and proteolytic activation in airway epithelium.

Hepatocyte growth factor (HGF) is a multifunctional molecule that acts as mitogen, motogen, and/or morphogen in a variety of cells. MET, a specific receptor tyrosine kinase for HGF, is upregulated in various tumors including squamous cell carcinoma of the human head and neck (HNSCC), but how HGF affects the expression of downstream functional genes has not yet been elucidated in detail. In the present study, we examined the expression of microRNA (miRNA), non-coding small RNA that regulate cell proliferation and functions by interfering with the translation of target mRNA, with or without HGF stimulation in HNSCC cell line HSC3. Among several miRNAs, in which the expression was altered after HGF stimulation, we focused on miR-200c and miR-27b, both of which were drastically downregulated after HGF stimulation. Expression of ZEB1, a target mRNA for miR-200c, was upregulated 3 and 6 h after HGF stimulation, and that of E-cadherin, a downstream molecule of ZEB1, was downregulated 12 h after HGF stimulation. Expression of ST14/matriptase, an enzyme for extracellular matrix (ECM) degradation and HGF activation and a target mRNA for miR-27b, was drastically upregulated in the protein level after HGF stimulation, although it was not statistically altered in the mRNA level. These results suggest that miR-200c and miR-27b downregulated by HGF might play an important role in epithelial–mesenchymal transition mediated by ZEB1/E-cadherin and ECM degradation and HGF autoactivation mediated by ST14/matriptase, respectively. Altered expression of miRNA directly regulated by HGF might contribute enhanced progressive and invasive characteristics of HNSCC by regulating the translation of HGF-induced functional molecules. (Cancer Sci 2011; 102: 2164–2171)

The complete mechanism of labor induction in eutherian mammals remains unclear. Although important roles for the fetus and placenta in triggering labor have been proposed, no gene has been shown to be required in the fetus/placenta for labor induction. Here we show that Nrk, an X-linked gene encoding a Ser/Thr kinase of the germinal center kinase family, is essential in the fetus/placenta for labor in mice. Nrk was specifically expressed in the spongiotrophoblast layer, a fetus-derived region of the placenta, and Nrk disruption caused dysregulated overgrowth of the layer. Due to preferential inactivation of the paternally derived X chromosome in placenta, Nrk heterozygous mutant placentas exhibited a similar defect to that in Nrk-null tissues when the wild-type allele was paternally derived. However, the phenotype was weaker than in Nrk-null placentas due to leaky Nrk expression from the inactivated X chromosome. Crossing of Nrk-null females to wild-type and Nrk-null males, as well as uterine transfer of Nrk-null fetuses to wild-type females, revealed that pregnant mice exhibit a severe defect in delivery when all fetuses/placentas are Nrk-null. In addition, Nrk was not expressed in female reproductive tissues such as the uterus and ovary, as well as the fetal amnion and yolk sac, in pregnant mice. Progesterone and estrogen levels in the maternal circulation and placenta, which control the timing of labor, were unaffected upon Nrk disruption. We thus provide evidence for a novel labor-inducing fetoplacental signal that depends on the X chromosome and possibly arises from the placenta.

鼠径ヘルニアを有する慢性腎不全患者は一般的に腹膜透析療法を選択されない．一方，腎不全患者の高齢化に伴い，鼠径ヘルニアを合併することが多くなっている．われわれは，Kugel法によるヘルニア修復を行った後，腹膜透析を行った経験を報告する．Kugel法は腹膜前腔にパッチを挿入して，1枚のパッチで内鼠径輪・外鼠径輪・大腿輪を同時にカバーし，鼠径部の全てのタイプのヘルニアに同時に対応する方法である．従来のMesh plug法に比べ理論上強度が強く，本法を行うことで鼠径ヘルニア修復と安全な腹膜透析が同時に可能と考えられる．

Type II transmembrane serine proteases (TTSPs) are structurally defined by the presence of a transmembrane domain located near the N-terminus and a C-terminal extracellular serine protease domain. The human TTSP family consists of 17 members. Some members of the family have pivotal functions in development and homeostasis, and are involved in tumorigenesis and viral infections. The activities of TTSPs are regulated by endogenous protease inhibitors. However, protease inhibitors of most TTSPs have not yet been identified. In this study, we investigated the inhibitory effect of hepatocyte growth factor activator inhibitor type 1 (HAI-1), a Kunitz-type serine protease inhibitor, on several members of the TTSP family. We found that the protease activity of a member, TMPRSS13, was inhibited by HAI-1. A detailed analysis revealed that a soluble form of HAI-1 with one Kunitz domain (NK1) more strongly inhibited TMPRSS13 than another soluble form of HAI-1 with two Kunitz domains (NK1LK2). In addition, an in vitro protein binding assay showed that NK1 formed complexes with TMPRSS13, but NK1LK2 did not. TMPRSS13 converted single-chain pro-hepatocyte growth factor (pro-HGF) to a two-chain form in vitro, and the pro-HGF converting activity of TMPRSS13 was inhibited by NK1. The two-chain form of HGF exhibited biological activity, assessed by phosphorylation of the HGF receptor (c-Met) and extracellular signal-regulated kinase, and scattered morphology in human hepatocellular carcinoma cell line HepG2. These results suggest that TMPRSS13 functions as an HGF-converting protease, the activity of which may be regulated by HAI-1.

e16191Background: To elucidate recurrence pathophysiology after gastrointestinal cancer surgery, the prognostic effect of peritoneal cancer cell spillage during surgery was evaluated in patients (p...

Hepatocyte growth factor (HGF), an antimitogenic factor for HepG2 cells, increased mRNA and protein levels of UGT1A1 and CYP2B6, as well as the endogenous cyclin-dependent kinase (CDK) inhibitors p16, p21, and p27 in HepG2 cells but not in HuH6, Caco2, or MCF7 cells. Treatment with 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) (an extracellular signal-regulated kinase inhibitor) suppressed the HGF-induced expression of UGT1A1 and CYP2B6, as well as p16, p21, and p27 in HepG2 cells. The CDK inhibitor roscovitine also enhanced the expression of UGT1A1, CYP2B6, and CYP3A4. Transfection of anti-CDK2 siRNA led to elevated levels of UGT1A1, CYP2B6, and CYP3A4 in HepG2 and SW480 cells, whereas anti-CDK4 small interfering RNA (siRNA) did not significantly enhance the expression of these enzymes. In fact, CDK2 activity was decreased in HGF-treated HepG2 cells. In cells arrested in S phase by a thymidine block and then released into a synchronous cell cycle, there was a clear dissociation among the activation of CDK2 and the expression of UGT1A1, CYP2B6, and CYP3A4. Furthermore, the induction of CYP3A4 but not UGT1A1 or CYP2B6 mRNA expression by roscovitine was repressed in pregnane X receptor (PXR) siRNA-transfected HepG2 cells. Transfection with constitutive androstane receptor siRNA or PXR siRNA in HepG2 cells did not repress the HGF-stimulated expression of UGT1A1 mRNA. Taken together, our results show that the expression of UGT1A1 and CYP2B6 is negatively regulated through a CDK2 signaling pathway linked to cell cycle progression in HepG2 and SW480 cells, the mechanism of which may differ from that of CYP3A4 expression through PXR phosphorylated by CDK2.

Regulatory T cells (Tregs) play an important role in maintaining immunological tolerance that is one of the main obstacles to overcome for improving antitumor immunity. Recently, the Treg has been shown to constitutively express OX40 (CD134), which is a member of the TNF-receptor family that is transiently expressed on effector T cells after TCR triggering, and through which the signal enhances effector T-cell proliferation and memory T-cell development. However, little is known about the role of OX40 costimulation to Tregs in tumor immunology. Here we show that OX40 signaling modulates the function of naturally occurring Tregs in vitro and in vivo. Foxp3 expression on Tregs was reduced by OX40 costimulation, but not by IL-2 stimulation. Tregs suppressed the proliferation of naïve CD4(+) CD25(-) T cells after TCR triggering, in contrast, OX40 costimulated Tregs that reduced Foxp3 expression reversed the suppressive function. In addition, Tregs inhibited the proliferation of TCR-stimulated (primed) CD4(+) T cells and naïve CD8(+) T cells after TCR-mediated activation, however, Tregs with OX40 costimulation lost their suppressive function. Interestingly, Tregs minimally suppressed the proliferation or the cytokine secretion of Ag-specific CD8(+) T cells after Ag-restimulation. Furthermore, Tregs suppressive function to the antitumor effect was reversed by OX40 costimulation in vivo. Our data indicate that, in addition to controlling effector T-cell function, OX40 costimulation directly controls Treg-mediated suppression in tumor immunity.

Hepatocyte growth factor (HGF) inhibits the proliferation of several tumor cell lines and tumor growth in vivo. We showed previously that HGF induces cell cycle arrest at G1 in a human hepatoma cell line, HepG2, by up-regulating the expression of p16INK4a through strong activation of extracellular signal-regulated kinase (ERK). However, although essential, the activation was not sufficient for the up-regulation of p16. In this study, we examined regulatory mechanisms of p16 expression through a transcription factor, Ets, which has been shown previously to bind to the promoter. The treatment of HepG2 cells with HGF induced ERK-dependent phosphorylation of Ets, which leads to its activation, before the up-regulation of p16, suggesting that another factor suppresses Ets activity. We found that HGF reduces the amount of Id1, which is a dominant-negative inhibitor of Ets, leading to a decrease in Ets associated with Id1. Id1 was down-regulated via transcriptional regulation not via the ubiquitin-proteasome-mediated pathway. Inhibition of the HGF-induced high-intensity ERK activity had a modest effect on the Id1 down-regulation, and inhibition of the phosphatidylinositol 3-kinase pathway had no effect, showing that Id1 is regulated by ERK-dependent and -independent pathways other than the phosphatidylinositol 3-kinase pathway. Exogenously expressed Id1 suppressed the up-regulation of p16 by HGF and the antiproliferative effect of HGF. Knockdown of Id1 significantly enhanced the activity of the p16 promoter coordinately with the activation of ERK. Our results indicated that down-regulation of Id1 plays a key role in the inhibitory effect of HGF on cell proliferation and provides a molecular basis for cancer therapy with HGF. (Mol Cancer Res 2009;7(7):1179–88)

The nucleolus is a subnuclear compartment and the site of ribosome biogenesis. Previous studies have implicated protein ubiquitylation in nucleolar activity. Here we show that USP36, a deubiquitylating enzyme of unknown function, regulates nucleolar activity in mammalian cells. USP36 localized to nucleoli via the C-terminal region, which contains basic amino acid stretches. Dominant-negative inhibition of USP36 caused the accumulation of ubiquitin-protein conjugates in nucleoli, suggesting that nucleoli are the site of USP36 action. USP36 deubiquitylated the nucleolar proteins nucleophosmin/B23 and fibrillarin, and stabilized them by counteracting ubiquitylation-mediated proteasomal degradation. RNAi-mediated depletion of cellular USP36 resulted in reduced levels of rRNA transcription and processing, a less-developed nucleolar morphology and a slight reduction in the cytoplasmic ribosome level, which eventually led to a reduced rate of cell proliferation. We conclude that by deubiquitylating various nucleolar substrate proteins including nucleophosmin/B23 and fibrillarin, USP36 plays a crucial role in regulating the structure and function of nucleoli.

Constitutive androstane receptor (CAR) is a transcription factor to regulate the expression of several genes related to drug-metabolism. Here, we demonstrate that CAR protein accumulates during G1 in human SW480 and HepG2 cells. After the G1/S phase transition, CAR protein levels decreased, and CAR was hardly detected in cells by the late M phase. CAR expression in both cell lines was suppressed by RNA interference-mediated suppression of CDK4. Depletion of CAR by RNA interference in both cells and by hepatocyte growth factor treatment in HepG2 cells resulted in decreased MDM2 expression that led to p21 upregulation and repression of HepG2 cell growth. Thus, our results demonstrate that CAR expression is an early G1 event regulated by CDK4 that contributes to MDM2 expression; these findings suggest that CAR may influence the expression of genes involved in not only the metabolism of endogenous and exogenous substances but also in the cell proliferation.

Strong activation of the ERK signal is required for hepatocyte growth factor (HGF) to inhibit proliferation of the human hepatocellular carcinoma cell line HepG2. However, it is still to be elucidated whether the activation alone is sufficient to induce the inhibitory effect. In this study, we constructed HepG2 cell clones expressing a high level of epidermal growth factor receptor (EGFR), and examined the effect of the strong activation of ERK on the proliferation of the cell clones. EGF treatment of the cell clones induced strong activation of ERK similar to HGF treatment, but did not inhibit cell proliferation. HGF treatment of the cell clones up-regulated the expression of a Cdk inhibitor p16INK4a, which has previously been shown to be required to inhibit the proliferation of HepG2 cells, but EGF treatment did not. Furthermore, EGF treatment of the cell clones did not induce the up-regulation of another Cdk inhibitor p21CIP1, whereas HGF treatment did. Knockdown of p21 by siRNA restored the proliferation of HepG2 cells inhibited by HGF, and restored Cdk2 activity suppressed in HGF-treated HepG2 cells. These results suggest that strong activation of ERK alone is not sufficient, and some other pathway(s), which is activated through the HGF receptor but not through EGFR, is also required to induce the up-regulation of p16 and p21 expression, and also suggest that in addition to the up-regulated expression of p16, that of p21 contributes to the suppression of Cdk2 activity leading to the inhibition of proliferation of HGF-treated HepG2 cells. J. Cell. Biochem. 104: 176–188, 2008. © 2007 Wiley-Liss, Inc.

Activation of the extracellular signal-regulated kinase (ERK) pathway is a key factor in the regulation of cell proliferation by growth factors. Hepatocyte growth factor (HGF)-induced cell cycle arrest in the human hepatocellular carcinoma cell line HepG2 requires strong activation of the ERK pathway. In this study, we investigated the molecular mechanism of the activation. We constructed a chimeric receptor composed of the extracellular domain of the NGF receptor and the cytoplasmic domain of the HGF receptor (c-Met) and introduced a point mutation (N1358H) into the chimeric receptor, which specifically abrogates the direct binding of Grb2 to c-Met. The mutant chimeric receptor failed to mediate the strong activation of ERK, up-regulation of the expression of a Cdk inhibitor p16(INK4a) and inhibition of HepG2 cell proliferation by ligand stimulation. Moreover, the mutant receptor did not induce tyrosine phosphorylation of the docking protein Gab1. Knockdown of Gab1 using siRNA suppressed the HGF-induced strong activation of ERK and inhibition of HepG2 cell proliferation. These results suggest that coupling of Grb2 to Gab1 mediates the HGF-induced strong activation of the ERK pathway, which is required for the inhibition of HepG2 cell proliferation.

The association of hepatocyte growth factor (HGF) with its high-affinity receptor (c-Met) has been shown to induce mitogenesis, motogenesis and morphogenesis in a variety of cell types. Various point mutations in c-Met have been identified in hereditary and sporadic papillary renal carcinomas as well as in other carcinomas. In the present study, we examined the effects of c-Met point mutations on the morphology of a porcine aortic endothelial (PAE) cell line. When cultured in three-dimensional collagen gel, PAE cells formed branching tubule structures, and HGF treatment caused breakdown of the structures and induced a scattered morphology. The exogenous expression of c-Met point mutants inhibited the formation of tubules. HGF treatment induced the formation of tubules by PAE cells expressing some c-Met mutants, but it induced the scattering of PAE cells expressing other c-Met mutants. The presence of a low concentration of a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor cancelled the inhibitory effect of the c-Met point mutations on the formation of tubules. These results suggest that c-Met point mutations affect the extracellular signal-regulated kinase (ERK) signaling required for the formation of tubules by PAE cells, and HGF binding changes the conformation of c-Met mutants, leading to the different signals required for formation of tubules and cell scattering.

Monoubiquitination of endocytosed cell surface receptors serves as a sorting signal for their trafficking from endosomes to lysosomes. The sorting of ubiquitinated proteins is executed by concerted actions of class E vacuolar protein sorting (Vps) proteins. Some proteins in the sorting machinery undergo monoubiquitination, suggesting that their functions are also regulated by ubiquitination. The Hrs-STAM complex, a class E Vps protein complex essential for the initial step of the sorting pathway, binds two deubiquitinating enzymes, UBPY and AMSH. Here we examined the effects of inactivating UBPY on protein ubiquitination at endosomes. Overexpression of a catalytically inactive UBPY mutant or depletion of UBPY by RNA interference resulted in the accumulation of ubiquitinated proteins on morphologically aberrant endosomes. Electron microscopy showed that they are aggregates of multivesicular endosomes. Among the sorting machinery proteins that undergo ubiquitination, Eps15 was monoubiquitinated at an elevated level in UBPY-inactivated cells. UBPY also deubiquitinated Eps15 in vitro, suggesting that Eps15 is a cellular substrate for UBPY. Furthermore, inactivation of UBPY caused the accumulation of Eps15 on the endosomal aggregates. These results suggest that UBPY regulates the level of protein ubiquitination on endosomes, which is required for maintaining the morphology of the organelle.

Cell surface receptor proteins that have undergone endocytosis are transported to the endosome. From the endosome, ligand-activated receptor tyrosine kinases are further transported to the lysosome for degradation, a process called "receptor downregulation." By contrast, nutrient receptors, such as those for low-density lipoprotein and transferrin, are recycled back to the plasma membrane. Sorting of these two types of receptors occurs at the endosome, where ubiquitination of receptor proteins serves as the sorting signal. Namely, ubiquitinated receptors are incorporated into the lysosomal degradation pathway, whereas those that are not ubiquitinated are returned to the cell surface. Hrs and STAM are proteins that form a complex on the endosomal membrane. Recent studies have shown that the Hrs/STAM complex binds ubiquitin moieties and acts as sorting machinery that recognizes ubiquitinated receptors and transfers them to further sequential lysosomal sorting/trafficking processes.

Background & Aims: Hepatocyte growth factor activator (HGFA) is a serum proteinase that specifically converts an inactive single-chain form of hepatocyte growth factor (HGF) into an active 2-chain form. HGFA is produced in its precursor form and then activated in injured tissues. To address the precise role of HGFA and to investigate the mechanisms of HGF activation in injured tissues, we generated mice deficient in HGFA. Methods: HGFA-deficient mice were generated using targeted gene disruption. The regenerating process of intestinal mucosa damaged by oral administration of dextran sodium sulfate (DSS) or by rectal administration of acetic acid was examined in both HGFA-deficient and control mice. HGF processing activity was analyzed using Western blotting and an HGF activation assay. Results: Homozygous mutant mice were viable and fertile without obvious abnormalities. When mice were treated with 3% DSS in drinking water for 6 days followed by distilled water without DSS, 72% of HGFA-deficient mice died through day 12 while 75% of control mice survived injury. Similar results were also observed in the acetic acid-induced intestinal injury; the survival rate was 36.6% in HGFA-deficient mice and 84.2% in control mice. In HGFA-deficient mice, the injured mucosa was not sufficiently covered by regenerated epithelium and the activation of HGF was impaired in the injured colon. Conclusions: These results indicate that HGFA is required for repair of injured intestinal mucosa but is not essential for normal development during embryogenesis or after birth.

c-Met is a high-affinity receptor for hepatocyte growth factor (HGF) and plays a crucial role in embryonic development, as well as in the process of tissue repair. Overexpression and amplification of c-Met are often observed in various cancer tissues, especially in gastric carcinoma. It has, however, been unclear whether the overexpression leads to activation of the c-Met receptor. To address this point, we prepared an antibody (anti-phospho-Met) which specifically recognizes c-Met that is phosphorylated at Y1235, a major phosphorylation site of c-Met. Normal as well as cancerous gastric tissue was positive for anti-total-Met staining, whereas only cancerous tissue was strongly positive for anti-phospho-Met staining; cells near the basal layer were moderately positive, and the proliferative zone in normal tissue was only weakly positive. Among cancerous tissues from seven patients examined in the present study, those from six patients were strongly positive for phospho-Met staining. These results indicate that c-Met is actually activated in gastric carcinoma tissue, and may trigger proliferation/anti-apoptotic signals.

Metastatic pancreas tumors from breast cancer are comparatively uncommon and patients with this tumor usually remain asymptomatic during their life. A 55-year-old woman presented with obstructive jaundice following mastectomy for invasive ductal carcinoma. We diagnosed obstructive jaundice due to a pancreatic tumor demonstrated on computed tomography and performed percutaneous transhepatic cholangio-drainage. Although the patient recovered from the jaundice, she had exacerbation of pneumonia from which she died. At autopsy, invasive ductal carcinoma was found in the pancreas tumor. Immunohistochemical staining was performed to confirm whether the pancreatic tumor was primary or secondary. Human milk fat globules 1 and 2 and gross cystic disease fluid protein-15, which characteristically exist in normal breast tissue or breast carcinoma, were expressed both in the primary breast tumor and the pancreatic tumor. In contrast, both the anti-estrogen receptor and anti-progesterone receptor antibodies stained positively in the primary breast cancer; however, neither of them was positive in the metastatic pancreatic tumor. We report a rare case of a patient who presented with obstructive jaundice from a pancreatic tumor metastasizing from breast cancer and in whom immunohistochemical staining using the antibodies unique to the mammary gland was effective for the diagnosis of this secondary tumor.

Nck-interacting kinase (NIK)-related kinase (NRK)/NIK-like embryo-specific kinase (NESK) is a protein kinase that belongs to the germinal center kinase family, and activates the c-Jun N-terminal kinase (JNK) signaling pathway. In this study, we examined the effect of NRK/NESK on actin cytoskeletal organization. Overexpression of NRK/NESK in COS7 cells induced accumulation of polymerized actin at the perinuclear. Phosphorylation of cofilin, an actin-depolymerizing factor, was increased in NRK/NESK-expressing HEK 293T cells. In addition, in vitro phosphorylation of cofilin was observed on NRK/NESK immunoprecipitates from HEK 293T cells expressing the kinase domain of NRK/NESK. The cofilin phosphorylation occurred at the serine residue of position 3 (Ser-3). Since the phosphorylation at Ser-3 inactivates the actin-depolymerizing activity of cofilin, these results suggest that NRK/NESK induces actin polymerization through cofilin phosphorylation. The cofilin phosphorylation did not appear to be mediated through activation of LIM-kinasel, a cofilin-phosphorylating kinase, or through the activation of JNK. Thus, cofilin is likely to be a direct substrate of NRK/NESK. NRK/NESK is predominantly expressed in skeletal muscle during the late stages of mouse embryogenesis. Thus, NRK/NESK may be involved in the regulation of actin cytoskeletal organization in skeletal muscle cells through cofilin phosphorylation.

The cleavage cycle, which is initiated by fertilization, consists of only S and M phases, and the gap phases (G1 and G2) appear after the midblastula transition (MBT) in the African clawed frog, Xenopus laevis. During early development in Xenopus, we examined the E2F activity, which controls transition from the G1 to S phase in the somatic cell cycle. Gel retardation and transactivation assays revealed that, although the E2F protein was constantly present throughout early development, the E2F transactivation activity was induced in a stage-specific manner, that is, low before MBT and rapidly increased after MBT. Introduction of the recombinant dominant negative E2F (dnE2F), but not the control, protein into the 2-cell stage embryos specifically suppressed E2F activation after MBT. Cells in dnE2F-injected embryos appeared normal before MBT, but ceased to proliferate and eventually died at the gastrula. These cells contained decreased cdk activity with enhanced inhibitory phosphorylation of Cdc2 at Tyr15. Thus, E2F activity is required for cell cycle progression and cell viability after MBT, but not essential for MBT transition and developmental progression during the cleavage stage.

Hepatocyte growth factor (HGF) plays a crucial role in the recovery of injured liver. Liver functions are mostly impaired in patients with liver diseases including cirrhosis. However, a significant amount of inactive HGF precursor (proHGF) is reported in the plasma of these patients. proHGF is proteolytically converted to an active form (mature HGF) by HGF-activator. Thus conversion of proHGF into mature HGF presumably contributes to the recovery of liver functions. In this study, rats with a partial hepatectomy were used, as proHGF is accumulated in the remnant liver. Recombinant human HGF-activator was administered via the portal vein to investigate the effect on molecular forms of HGF and its biological signaling. rhHGF-activator promptly converted proHGF into mature HGF, reaching maximal levels at 5–10 min after the injection, while the decreased proHGF was quickly recovered to the initial levels in the liver. The HGF receptor/c-Met was found to be autophosphorylated in the liver treated with rhHGF-activator. Further, the proliferating cell nuclear antigen labeling index and the liver regeneration rate were significantly higher in rhHGF-activator group than in control animals. These results indicate that exogenously administered HGF-activator produces a biologically active HGF from its precursor form and increases the potential for liver regeneration in vivo.

Hepatocyte growth factor activator inhibitor type 2 (HAI-2) is a serine proteinase inhibitor containing two Kunitz-type inhibitor domains, initially identified as a potent inhibitor of hepatocyte growth factor activator (HGFA). In a previous study (Biochem. Biophys. Res. Commun. 255, 740-748, 1999), we reported that a predominant transcript of mouse HAI-2 is a splicing variant lacking the first Kunitz domain (KD-1). Since KD-1 was reported to be responsible for the inhibition of HGFA in human HAI-2 and the second Kunitz domain (KD-2) of human HAI-2 was much less inhibitory against HGFA, it has been suggested that most of mouse HAI-2 may be ineffective in inhibiting HGFA. In this study, we have performed functional characterization of Kunitz domains in mouse HAI-2 by using recombinant proteins synthesized by Chinese hamster ovary cells without or with point mutation in the putative reactive site of each Kunitz domain. The results revealed that, unlike human HAI-2, KD-2 of mouse HAI-2 efficiently inhibits HGFA. Therefore, the major mouse HAI-2 protein that consists only of KD-2 can be a potent inhibitor of HGF activation in vivo.