JAB1-STAT3 activation loop is associated with recurrence following 5-fluorouracil-based adjuvant chemotherapy in human colorectal cancer.

Jun activation domain-binding protein 1 (JAB1) has been shown to have multiple roles in tumorigenesis, including the degradation of tumor suppressor proteins such as p53, Smad7, Runx3 and the cyclin-dependent kinase inhibitor p27Kip1, and the activation of oncogenic transcription factors, such as c-Jun and hypoxia-inducible factor-1α. In addition, our previous study revealed that JAB1 positively regulates signal transducer and activator of transcription 3 (STAT3) DNA-binding activity in human colon cancer cells. In turn, the oncogenic transcription factor STAT3 positively regulates JAB1 expression, indicative of a positive feedback loop. Furthermore, high JAB1 expression is associated with a poor prognosis in numerous malignant carcinomas. However, the association between JAB1 expression and prognosis in colorectal cancer remains unclear. The aim of the present study was to elucidate the association between JAB1 and STAT3 expression and recurrence in colorectal cancer. In the present study, it was found that high JAB1 expression in primary colorectal cancer tissues is an independent predictor of recurrence following 5-fluorouracil (5-FU)-based adjuvant chemotherapy in colorectal cancer patients, and that high expression of both JAB1 and STAT3 in primary colorectal cancer tissues is associated with a lower recurrence-free survival rate following 5-FU-based adjuvant chemotherapy compared to high expression of only JAB1 or STAT3. Overall, these results suggest that JAB1 is a novel predictive marker of recurrence following 5-FU-based adjuvant chemotherapy in colorectal cancer patients, and that the JAB1-STAT3 activation loop may be a potential therapeutic target in recurrent colorectal cancer following 5-FU-based adjuvant chemotherapy.

The c-MYC-ABCB5 axis plays a pivotal role in 5-fluorouracil resistance in human colon cancer cells.

c-MYC overexpression is frequently observed in various cancers including colon cancer and regulates many biological activities such as aberrant cell proliferation, apoptosis, genomic instability, immortalization and drug resistance. However, the mechanism by which c-MYC confers drug resistance remains to be fully elucidated. In this study, we found that the c-MYC expression level in primary colorectal cancer tissues correlated with the recurrence rate following 5-fluorouracil (5-FU)-based adjuvant chemotherapy. Supporting this finding, overexpression of exogenous c-MYC increased the survival rate following 5-FU treatment in human colon cancer cells, and knockdown of endogenous c-MYC decreased it. Furthermore, c-MYC knockdown decreased the expression level of ABCB5, which is involved in 5-FU resistance. Using a chromatin immunoprecipitation assay, we found that c-MYC bound to the ABCB5 promoter region. c-MYC inhibitor (10058-F4) treatment inhibited c-MYC binding to the ABCB5 promoter, leading to a decrease in ABCB5 expression level. ABCB5 knockdown decreased the survival rate following 5-FU treatment as expected, and the ABCB5 expression level was increased in 5-FU-resistant human colon cancer cells. Finally, using a human colon cancer xenograft murine model, we found that the combined 5-FU and 10058-F4 treatment significantly decreased tumorigenicity in nude mice compared with 5-FU or 10058-F4 treatment alone. 10058-F4 treatment decreased the ABCB5 expression level in the presence or absence of 5-FU. In contrast, 5-FU treatment alone increased the ABCB5 expression level. Taken together, these results suggest that c-MYC confers resistance to 5-FU through regulating ABCB5 expression in human colon cancer cells.

HIF-1α activation under glucose deprivation plays a central role in the acquisition of anti-apoptosis in human colon cancer cells.

A poor vascular network development in a tumor mass leads to poor oxygen and nutrient supply. To adapt to a hypoxic microenvironment, it is well-known that cancer cells activate the transcription factor hypoxia-inducible factor-1α (HIF-1α). HIF-1α plays a central role in hypoxia-induced metabolic switching, anti-apoptosis, angiogenesis and drug resistance. Glucose deprivation, another major stressful microenvironment, protects cancer cells from drug-induced apoptosis. However, the molecular mechanisms by which cancer cells adapt to poor nutrient conditions remain poorly understood. In this study, we focused on HIF-1α, signal transducer and activator of transcription 3 (STAT3) and trans-cription factor 4 (TCF4), which are involved in cell survival, anti-apoptosis and drug resistance. We examined their activities and the relationships among these transcription factors under glucose deprivation. Our results showed that glucose deprivation increased HIF-1α, STAT3 and TCF4 DNA-binding activity, as well as the expression levels of their target genes OCT4, BCL-2 and VEGF. HIF-1α knockdown significantly increased poly(ADP-ribose) polymerase 1 (PARP-1) cleavage at higher levels than STAT3 knockdown under glucose deprivation. Furthermore, HIF-1α knockdown led to a significant decrease in the expression levels of both STAT3 and TCF4, although STAT3 knockdown decreased only HIF-1α expression level. Our data indicated that activation of the HIF-1α signaling pathway under glucose deprivation leads to the acquisition of anti-apoptotic properties in human colon cancer cells, and targeting the HIF-1α signaling pathway may provide an effective avenue for treating cancers resistant to conventional therapy.

Single-incision laparoscopic splenectomy and splenic autotransplantation for an enlarged wandering spleen with torsion.

A wandering spleen is a rare condition in which the spleen is not located in the left upper quadrant, but instead is found in the lower abdomen or in the pelvic region because of the laxity of the peritoneal attachments. The unusually long pedicle is susceptible to twisting, which can lead to ischemia, and eventually to necrosis. We herein report a case of an enlarged wandering spleen with torsion, successfully treated by single-incision laparoscopic splenectomy and autotransplantation. The transplanted splenic tissues could be identified on a spleen scintigram obtained 3 months after the surgery. Howell-Jolly bodies were not observed in blood specimens. This procedure is able to prevent an overwhelming postsplenectomy infection, and leads to satisfactory cosmetic results.

JAB1 regulates unphosphorylated STAT3 DNA-binding activity through protein-protein interaction in human colon cancer cells.

Recent studies have revealed that unphosphorylated STAT3 forms a dimer, translocates to the nucleus, binds to the STAT3 binding site, and activates the transcription of STAT3 target genes, thereby playing an important role in oncogenesis in addition to phosphorylated STAT3. Among signaling steps of unphosphorylated STAT3, nuclear translocation and target DNA-binding are the critical steps for its activation. Therefore, elucidating the regulatory mechanism of these signaling steps of unphosphorylated STAT3 is a potential step in the discovery of a novel cancer drug. However, the mechanism of unphosphorylated STAT3 binding to the promoter of target genes remains unclear. In this study, we focused on Jun activation domain-binding protein 1 (JAB1) as a candidate protein that regulates unphosphorylated STAT3 DNA-binding activity. Initially, we observed that both unphosphorylated STAT3 and JAB1 existed in the nucleus of human colon cancer cell line COLO205 at the basal state (no cytokine stimulation). On the other hand, phosphorylated STAT3 did not exist in the nucleus of COLO205 cells at the basal state. Immunoprecipitation using nuclear extract of COLO205 cells revealed that JAB1 interacted with unphosphorylated STAT3. To investigate the effect of JAB1 on unphosphorylated STAT3 activity, RNAi studies were performed. Although JAB1 knockdown tended to increase nuclear STAT3 expression, it significantly decreased unphosphorylated STAT3 DNA-binding activity. Subsequently, JAB1 knockdown significantly decreased the expression levels of MDR1, NANOG, and VEGF, which are STAT3 target genes. Furthermore, the expression level of nuclear JAB1, but not nuclear STAT3, correlated with unphosphorylated STAT3 DNA-binding activity between COLO205 and LoVo cells. Taken together, these results suggest that nuclear JAB1 positively regulates unphosphorylated STAT3 DNA-binding activity through protein-protein interaction in human colon cancer cell line COLO205.

The mobilization and recruitment of c-kit+ cells contribute to wound healing after surgery.

Delayed wound healing is a serious clinical problem in patients after surgery. A recent study has demonstrated that bone marrow-derived c-kit-positive (c-kit(+)) cells play important roles in repairing and regenerating various tissues and organs. To examine the hypothesis that surgical injury induces the mobilization and recruitment of c-kit+ cells to accelerate wound healing. Mice were subjected to a left pneumonectomy. The mobilization of c-kit+ cells was monitored after surgery. Using green fluorescent protein (GFP(+)) bone marrow-transplanted chimera mice, we investigated further whether the mobilized c-kit+ cells were recruited to effect wound healing in a skin puncture model. The group with left pneumonectomies increased the c-kit(+) and CD34(+) stem cells in peripheral blood 24 h after surgery. At 3 days after surgery, the skin wound size was observed to be significantly smaller, and the number of bone marrow-derived GFP(+) cells and GFP(+)/c-kit+ cells in the wound tissue was significantly greater in mice that had received pneumonectomies, as compared with those that had received a sham operation. Furthermore, some of these GFP(+) cells were positively expressed specific markers of macrophages (F4/80), endothelial cells (CD31), and myofibroblasts (αSMA). The administration of AMD3100, an antagonist of a stromal-cell derived factor (SDF)-1/CXCR4 signaling pathway, reduced the number of GFP(+) cells in wound tissue and completely negated the accelerated wound healing. Surgical injury induces the mobilization and recruitment of c-kit+ cells to contribute to wound healing. Regulating c-kit+ cells may provide a new approach that accelerates wound healing after surgery.

Mucosa-associated lymphoid tissue (MALT) lymphoma arising in the esophagus, stomach, and lung.

A 62-year-old woman was referred to our hospital for further investigation of slow-growing gastrointestinal submucosal tumors (SMTs) and multiple lung nodules. Esophageal SMTs had been identified 6 years earlier, following which lung tumors and gastric SMTs had subsequently developed. Despite repeated endoscopic biopsies, these SMTs could not be diagnosed definitively. Moreover, we were unable to detect any serological abnormalities or radiologic findings such as lymph node swelling. Thoracoscopic excision of a lung nodule led to the definitive diagnosis of mucosaassociated lymphoid tissue (MALT) lymphoma. Cytological findings of aspiration biopsy specimens from the esophagus and stomach were compatible with that of the lung nodule. To our knowledge, this is the first case report of esophageal MALT lymphoma with lung and gastric involvement. We discuss this extremely rare disease with reference to the relevant literature.

Adenoma of the nipple, focusing on the contrast-enhanced magnetic resonance imaging findings: report of a case.

A woman in her 50s was referred to our hospital for an investigation of a right breast tumor. The tumor was palpated below the nipple, but there was no erosion or nipple discharge. Mammography showed a well-defined high-density tumor, measuring 2 cm in diameter, without calcification, and ultrasonography showed a low-echoic mass with a fluid component with posterior echo enhancement and a lateral shadow. Contrast-enhanced magnetic resonance imaging (CE-MRI) demonstrated a 1.3 × 0.8 cm solid component and a gradually increasing time-intensity curve. We performed lumpectomy and the pathological findings were adenoma of the nipple. The pattern of the time-intensity curve might be attributed to moderate fibrosis of the tumor. Contrast-enhanced MRI is therefore considered to be very useful in the diagnosis of breast disease because it can show the nature and extent of the breast lesion; however, we should be aware that various patterns have been observed on CE-MRI for adenoma of the nipple.

Operative injury accelerates tumor growth by inducing mobilization and recruitment of bone marrow-derived stem cells.

BACKGROUND: Although operative injury is thought generally to worsen the prognosis of cancer patients, the relevant mechanisms are not yet understood fully. We tested the hypothesis that operative injury induces mobilization and recruitment of bone marrow stem cells, thereby enhancing angiogenesis and accelerating tumor growth. METHODS: Mice were subjected to an open gastrotomy, and naïve mice were used as controls. The mobilization of bone marrow stem cells was monitored after operation. Using an established tumor model in green fluorescent protein (GFP)(+) bone marrow-transplanted chimera mice, we investigated further whether the mobilized stem cells affected tumor growth. RESULTS: Compared with the control, gastrotomy increased the populations of CD34(+) cells (6.9 ± 4.5 % vs 3.3 ± 0.4%, P < .05) and CD34(+)/Flk-1(+) cells (0.08 ± 0.02% vs 0.05 ± 0.01%, P < .05) in peripheral blood 12 h after operation. Twelve days after operation, the tumor volume almost doubled in mice after gastrotomy compared with control (580 ± 106 mm(3) vs 299 ± 162 mm(3), P < .05). A histologic analysis of tumor tissue revealed that the microvessel density and number of proliferating cells were significantly greater, but those of apoptotic cells were significantly less, in mice after gastrotomy as compared with control. Furthermore, the number of GFP(+) cells found in tumor tissue was significantly greater in mice that underwent gastrotomy than in controls. Some of the stained GFP(+) cells were positive for CD34 and had been incorporated into microvessels. Administration of AMD3100, which is an antagonist of stromal-cell-derived factor (SDF)-1/CXCR4 signaling pathway, inhibited the recruitment of GFP(+) cells and negated completely the acceleration in tumor growth after operation (345 ± 172 mm(3), P < .05). CONCLUSION: Operative injury may induce the mobilization and recruitment of bone marrow stem cells, thereby enhancing angiogenesis and accelerating tumor growth. Inhibition of the SDF-1/CXCR4 signals may represent a new therapeutic strategy for preventing acceleration of tumor growth after operation.

Congenital segmental dilatation of the duodenum: report of a case.

We herein report what, to our knowledge, is only the fourth known case of segmental dilatation of the duodenum. Antenatal ultrasonography revealed an intraabdominal cyst in the fetus, but the exact location of the segmental dilatation was difficult to find preoperatively. Moreover, even using computed tomography, it was not possible to make a diagnosis prior to surgery. The anatomic characteristics of duodenal dilatation made it difficult to perform the usual resection techniques. In fact, the surgical procedure was different from the previously reported cases. We performed a partial resection of the duodenum followed by a tapering procedure to preserve the ampulla of Vater. The infant had an uneventful postoperative course, and sufficient growth and development has been achieved.

DNA damage signaling is activated during cancer progression in human colorectal carcinoma.

PURPOSE: Recent studies have shown that the DNA damage response (DDR) is activated in precancerous lesions, suggesting that neoplastic cells may avoid apoptosis by impairing the DDR which acts as a barrier against tumor progression. To define the role of the DDR pathway in human colorectal carcinoma, we investigated the level of phosphorylated proteins of the DDR pathway. RESULTS: Immunostaining for pATM, gammaH2AX and pChk2 revealed that all were significantly expressed during tumor progression in advanced carcinoma (vs. normal tissue for pATM [p < 0.05]; vs. normal and adenoma for gammaH2AX [p < 0.05]; and vs. normal tissue for pChk2 [p < 0.05]. Western blot analysis of gammaH2AX and pChk2 revealed that their level increased gradually during tumor progression and was maximal in advanced carcinoma (vs. normal tissue; p < 0.05). No apoptotic cells were found in any tissue sample. EXPERIMENTAL DESIGN: Colorectal tissue samples were obtained at the time of surgery, from 55 patients at two hospitals. The tissues were classified into four groups according to pathology: normal mucosa, adenoma, early carcinoma and advanced carcinoma. We evaluated phosphorylated ataxia telangiectasia mutated (pATM), phosphorylated H2AX (gammaH2AX) and Chk2 (pChk2) protein levels by immunohistochemistry and western blot analysis. We also evaluated apoptosis by the TUNEL assay. CONCLUSIONS: The DDR pathway was activated during cancer progression, but no apoptosis was detected, even among the cells with activated DDR. It is likely that activation of DDR was induced by stress signaling as a consequence of oxidative, replication and mechanical stresses occurring during growth and expansion of the colorectal cancer.

Small bowel transplantation tolerance achieved by costimulatory blockade leading to mixed chimerism.

We evaluated mixed chimerism with costimulatory blockade for the achievement murine allogeneic small bowel transplantation (SBTx) tolerance. B6 mice received various combinations of anti-CD8 (day -2) and anti-CD154 mAbs with or without 3Gy total body irradiation (TBI) (day -1), and 20 x 10(6) fully MHC-mismatched B10.A bone marrow cells (BMC, day -1). Heterotopic SBTx was performed on day 0. Chimerism in peripheral blood was followed by flow cytometric (FCM) analysis and the frequency of TCR Vbeta usage was determined by FCM to assess deletion of donor-reactive T cells. All animals without any treatment (n=6) showed acute rejection within 18 days after transplantation. Mice treated with anti-CD8 and anti-CD154 mAbs alone rejected their grafts within 100 days after transplantation (n=10). Mice treated with anti-CD8 and anti-CD154 mAbs, TBI, and BMT achieved long-term multilineage mixed chimerism and accepted small bowel allografts permanently (>350 days) without any evidence of graft-versus-host disease(n=11). There was specific deletion of donor-reactive cells and skin was accepted as allografts from B10.A donors, but 3rd party B10.BR skin was rejected. Donor-specific tolerance was achieved by inducing mixed chimerism with costimulatory blockade in murine SBTx recipients. This approach which provides a reliable method to induce SBTx tolerance, has potential clinical applications.

[A case of multiple lung metastases of hepatocellular carcinoma after resection of liver and tumor thrombi in the inferior vena cava responding to chemotherapy using 5-FU, mitoxantrone and CDDP].

A 58-year-old man, who had a history of chronic type B hepatitis, was diagnosed as hepatocellular carcinoma with tumor thrombi in the inferior vena cava. He underwent resection of central bisegments and tumor thrombi, while postoperative chest CT demonstrated multiple lung metastases. Following 2 courses of chemotherapy using 5-FU, mitoxantrone, and CDDP (FMP therapy), multiple lung nodules disappeared and alpha-fetoprotein returned to the normal level. FMP therapy thus proved effective for a case of distant metastases of hepatocellular carcinoma.

Long-term survival of xenogeneic heart grafts achieved by costimulatory blockade and transient mixed chimerism.

BACKGROUND: Xenotransplantation holds great promise in clinical medicine, but is limited by the vigorous rejection response elicited against solid organs transplanted across species barriers. In this study, we investigated the role of anti-CD40L monoclonal antibody (mAb) in inducing xenogeneic mixed chimerism and donor-specific heart transplantation tolerance. METHODS: One day before heart transplantation, mice were injected intraperitoneally with anti-mouse CD8/NK1.1/Thy1.2 mAbs. On day 0, the mice received 3 Gy total body irradiation (TBI), an intravenous injection of unseparated bone marrow (BM) harvested from F344 rats, and an intraperitoneal injection of hamster antimouse CD40L mAb, MR1. Heart grafts from F344 rats were heterotopically transplanted into the abdomen of B6 mouse recipients. Using flow cytometric analysis of peripheral white blood cells, we assessed donor hematopoiesis at various times after bone marrow transplantation (BMT). RESULTS: Chimerism subsided gradually and disappeared completely 18 weeks after BMT. The cardiac graft survived permanently, even after the mixed chimerism disappeared. To determine if the mice acquired donor-specific tolerance, second rat heart grafts were transplanted 120 days after the first heart transplantation. The second transplanted hearts were also accepted over 60 days. Histological analysis revealed no remarkable vasculopathy in the coronary vessels at any stage. CONCLUSIONS: These findings clearly show that costimulatory blockade plays an important role in inducing xenochimerism, and that transient mixed chimerism can induce permanent acceptance of rat to mouse cardiac xenografts. Transplantation of xenogeneic bone marrow cells under costimulatory blockade at the time of heart transplantation may induce transplantation tolerance.

Ectopic hepatocellular carcinoma arising in the bile duct.

We report a case of ectopic hepatocellular carcinoma arising in the bile duct. A 72-year-old woman was transferred to our hospital with fever, abdominal pain, and jaundice. Contrast-enhanced computed tomography revealed a round mass, measuring 25 mm in diameter, in the bile duct. The mass was causing obstructive jaundice. Endoscopic retrograde cholangiography showed a 27 mm x 21-mm round defect in the superior bile duct. These findings led to a diagnosis of bile duct tumor, and the patient underwent extrahepatic bile duct resection and biliary reconstruction. Gross examination of the tumor showed a fibrous capsule and a stalk arising from the bile duct mucosa. The tumor was diagnosed histopathologically as well-differentiated hepatocellular carcinoma arising in the bile duct.

Expression of intercellular adhesion molecule-1 (ICAM-1) during hypoxia-reoxygenation by sinusoidal endothelial cells (SECs) in an obstructive jaundice model.

BACKGROUND/PURPOSE: The aim of the present study was to investigate the expression of intercellular adhesion molecule-1 (ICAM-1) by sinusoidal endothelial cells (SECs) during hypoxia-reoxygenation in an obstructive jaundice model. METHODS: Male Wistar rats aged 6 to 8 weeks were assigned to an obstructive jaundice group (group OJ; n = 8) and a sham-operated control group (group S; n = 8). Using flow cytometry, we determined the percentage of ICAM-1-positive cells and the mean fluorescence intensity (MFI) of ICAM-1 per cell in each group during hypoxia-reoxygenation. RESULTS: The percentage of ICAM-1-positive cells in group OJ did not change significantly under successive incubation conditions. However, the MFI in group OJ differed significantly between normoxia and 24-h renormoxia. The present study investigated the function of isolated SECs from rats with obstructive jaundice and hypoxia-reoxygenation. The percentage of ICAM-1-positive cells and the MFI of ICAM-1-positive cells showed quite similar changes caused by hypoxia-reoxygenation stress in group S and Group OJ. The ICAM-1 expression increased with hypoxia-reoxygenation in both normal and OJ SECs, and the amount of ICAM-1, on the whole, increased. Obstructive jaundice increased the percentage of ICAM-1-positive cells from the normal state. CONCLUSIONS: The amount of ICAM-1 increased with hypoxia-reoxygenation in obstructive jaundice than normal state.