Maki Kitagawa

Calpain‐mediated regulation of the distinct signaling pathways and cell migration in human neutrophils

We studied the mechanisms underlying calpain inhibition‐mediated human neutrophil migration. MAPKs, including ERK, p38, and JNK, MEK1/2, MAPK kinase 3/6 (MKK3/6), PI‐3K/Akt, c‐Raf, and p21‐activated kinase (PAK; an effector molecule of Rac) were rapidly (within 30 s) activated in neutrophils upon exposure to calpain inhibitors (PD150606 and N‐acetyl‐Leu‐Leu‐Nle‐CHO) but not PD145305 (inactive analog of PD150606). Following activation of these pathways, neutrophils displayed active migration (chemotaxis), which was sustained for more than 45 min. The studies with pharmacological inhibitors suggest that calpain inhibition‐mediated neutrophil migration is mediated by activation of MEK/ERK, p38, JNK, PI‐3K/Akt, and Rac. NSC23766 (Rac inhibitor) and pertussis toxin (PTX) suppressed calpain inhibitor‐induced phosphorylation of distinct signaling molecules (PAK, c‐Raf, MEK1/2, ERK, MKK3/6, p38, JNK, and Akt) as well as cell migration, suggesting that the PTX‐sensitive G protein and Rac axis may be a possible key target of calpain inhibitors. Differentiated neutrophil‐like HL‐60 cells but not undifferentiated cells displayed cell migration and activation of MAPKs and PI‐3K/Akt on calpain inhibition. These findings suggest that constitutively active calpain negatively regulates activation of the distinct signaling pathways and cell migration in resting neutrophils, and this regulatory system develops during differentiation into mature neutrophils.

Calpain inhibition induces activation of the distinct signalling pathways and cell migration in human monocytes

We have recently reported that constitutively active calpain negatively regulates activation of the distinct signalling pathways and cell migration in human neutrophils. Here, we report that a similar regulatory system is also functioning in human monocytes, but not lymphocytes. Calpain was constitutively active in resting human monocytes, but not lymphocytes. Mitogen‐activated protein kinases, including extracellular signal‐regulated kinase (ERK), p38 and c‐Jun N‐terminal kinase (JNK), phosphatidylinositol 3‐kinase (PI3K)/Akt and p21‐activated kinase (PAK, an effector molecule of Rac) were rapidly (within 1 min) activated in monocytes, but not lymphocytes, upon exposure to calpain inhibitors (PD150606 and N‐acetyl‐Leu‐Leu‐Nle‐CHO), but not PD145305 (the inactive analogue of PD150606). Following activation of these signalling pathways, monocytes displayed active migration within 5 min after exposure to calpain inhibitors, and active migration was sustained for more than 45 min. The micropipette method revealed that calpain inhibition‐mediated monocyte migration was chemotaxis, not random migration. The studies with pharmacological inhibitors suggest that calpain inhibition‐mediated monocyte migration is mediated by activation of ERK, p38, JNK, PI3K/Akt and Rac. NSC23766 (Rac inhibitor) and pertussis toxin (PTX) suppressed calpain inhibitor‐induced phosphorylation of distinct signalling molecules (PAK, ERK, p38, JNK and Akt) as well as cell migration, suggesting that the PTX‐sensitive G protein and Rac axis may be a possible key target of calpain inhibitors. These findings suggest that constitutively active calpain negatively regulates activation of the distinct signalling pathways and cell migration in resting monocytes, but not lymphocytes.

Distinct Role of c-Jun N-Terminal Kinase Isoforms in Human Neutrophil Apoptosis Regulated by Tumor Necrosis Factor-α and Granulocyte-Macrophage Colony-Stimulating Factor

We studied the role of c-Jun N-terminal kinase (JNK) in human neutrophils stimulated by tumor necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Stimulation of neutrophils with TNF-alpha and GM-CSF caused phosphorylation of p54 or p46 JNK or both. The phosphorylated p46 JNK band in TNF-alpha-stimulated neutrophils mobilized faster than that in GM-CSF-stimulated cells. The JNK isoform transcripts expressed in neutrophils were JNK1beta1, JNK1beta2, JNK2alpha1, and JNK2alpha2. The JNK isoforms phosphorylated by TNF-alpha and GM-CSF stimulation were found to be JNK1 and JNK2, respectively, on the basis of the molecular mass and the capture assay. TNF-alpha-induced JNK phosphorylation was sustained in the presence of cycloheximide, which was accompanied by accelerated neutrophil apoptosis. The JNK inhibitors (SP600125 and TAT-TI-JIP(153163)) suppressed neutrophil apoptosis induced by TNF-alpha plus cycloheximide, whereas they attenuated the GM-CSF-mediated antiapoptotic effect on neutrophils. The JNK inhibitor did not affect the levels of Mcl-1 and XIAP (antiapoptotic molecules), which were regulated by TNF-alpha plus cycloheximide and GM-CSF. The JNK inhibitor markedly suppressed TNF-alpha-induced and GM-CSF-induced superoxide release. These findings suggest that JNK1 and JNK2 are involved in TNF-alpha-induced neutrophil apoptosis and GM-CSF-mediated antiapoptotic effect on neutrophils, respectively, and both JNK isoforms are involved in TNF-alpha-induced and GM-CSF-induced superoxide release.

A regulatory role of K+–Cl− cotransporter in the cell cycle progression of breast cancer MDA-MB-231 cells

K(+)-Cl(-) cotransporter (KCC) has been shown to be involved in cell proliferation as well as cell volume regulation. A regulatory role of KCC in cell cycle progression of breast cancer MDA-MB-231 cells was explored by using synchronized MDA-MB-231 cells and dihydro-indenyloxy-alkanoic acid (DIOA), a potent inhibitor of KCC. MDA-MB-231 cells cultured in the presence of DIOA exhibited an increase in cell volume, a decrease in intracellular Cl(-) concentration, and reduction in cell proliferation with the G0/G1 phase arrest, which was accompanied with down-regulation of cyclin D1 and cyclin E2, and up-regulation of p21. Among these molecules, the expression of cyclin E2, a molecule essential for the transition from G1 to S phase, was markedly suppressed by DIOA treatment. DIOA-mediated up- or down-regulation of these molecules occurred at the transcriptional level. These findings suggest that KCC plays an important role in the early phase of cell cycle progression by regulating the expression of cyclin D1, cyclin E2, and p21, the molecules essential for the cell cycle progression.

Secondary Torsion of Vermiform Appendix with Mucinous Cystadenoma

Torsion of the vermiform appendix is a rare disorder, which causes abdominal symptoms indistinguishable from acute appendicitis. We report a case (a 34-year-old male) of secondary torsion of the vermiform appendix with mucinous cystadenoma. This case was characterized by mild inflammatory responses, pentazocine-resistant abdominal pain, and appendiceal tumor, which was not enhanced by the contrast medium on computed tomography presumably because of reduced blood flow by the torsion. These findings may be helpful for the preoperative diagnosis of secondary appendiceal torsion.

Calpain inhibition delays neutrophil apoptosis via cyclic AMP-independent activation of protein kinase A and protein kinase A-mediated stabilization of Mcl-1 and X-linked inhibitor of apoptosis (XIAP)

Human neutrophils underwent spontaneous apoptosis, which was accompanied with proteasome-mediated degradation of Mcl-1 and X-linked inhibitor of apoptosis (XIAP). Calpain inhibitors (PD150606 and N-acetyl-Leu-Leu-Nle-CHO) prevented spontaneous neutrophil apoptosis and degradation of Mcl-1 and XIAP, and the effects of calpain inhibitors on neutrophils were resistant to cycloheximide. Calpain inhibitors induced protein kinase A (PKA) activation, which was unaccompanied with an increase in intracellular cyclic AMP. Calpain inhibition-mediated delayed neutrophil apoptosis, stabilization of Mcl-1 and XIAP, and phosphorylation of PKA substrates were suppressed by H-89 (specific PKA inhibitor). These findings suggest that calpain inhibition delays neutrophil apoptosis via cyclic AMP-independent activation of PKA and PKA-mediated stabilization of Mcl-1 and XIAP.

Efficacy of a Hypotonic Treatment for Peritoneal Dissemination from Gastric Cancer Cells: An In Vivo Evaluation

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

Gastrointestinal Stromal Tumor in a Patient with Neurofibromatosis: Abscess Formation in the Tumor Leading to Bacteremia and Seizure.

A 66-year-old woman with neurofibromatosis type 1 (NF1) was brought to the emergency room with seizures and high-grade fever. Seizure in adult NF1 patients raises concern for intracranial lesions. However, neurological examination and central nervous system imaging failed to detect any causative intracranial lesions for her seizure. Gram-positive cocci, Streptococcus anginosus, were detected by blood cultures. Abdominal computed tomography revealed a well-defined round mass 7 cm in diameter, which was found to be a small intestinal gastrointestinal stromal tumor (GIST) containing an abscess. There was fistula formation between the intestinal lumen and the abscess, in which there were numerous Gram-positive cocci. The seizure may have been caused by hypoosmolality (hyponatremia and hypoproteinemia), which may result from decreased food intake associated with high-grade fever and general malaise. In this case GIST originating from the small intestine was invaded by S. anginosus through a fistula, leading to abscess formation, bacteremia, high-grade fever, and seizure, which was the first clinical manifestation.

Noncontrast and contrast enhanced computed tomography for diagnosing acute appendicitis: A retrospective study for the usefulness

Abdominal computed tomography (CT) provides great benefits for the differential diagnosis in patients complaining of acute abdominal pain. However, the use of diagnostic X-rays is associated with the cumulative risk of cancer development. In order to determine the relative usefulness of noncontrast and enhanced CT with intravenous contrast material for diagnosing acute appendicitis, the retrospective analysis was performed using 247 patients (46 children and 201 adults) with clinically suspected appendicitis, who were admitted to our hospital from 2002 to 2006 and underwent noncontrast or combined noncontrast and enhanced CT examination. Of 185 patients who were diagnosed to have acute appendicitis with appendiceal thickening (167 cases) or normal-sized appendix (18 cases), 73 cases underwent noncontrast CT alone and these 73 cases could be retrospectively diagnosed to have appendicitis on noncontrast CT. On the other hand, 112 cases of these 185 patients underwent noncontrast CT followed by enhanced CT, and vermiform appendix was detected in 86 cases of them (86/112, 76.8%) on noncontrast CT. These 86 cases could be retrospectively diagnosed to have acute appendicitis on noncontrast CT, whereas enhanced CT was required to detect vermiform appendix and to obtain the final diagnosis of appendicitis in the remaining 26 cases (26/112, 23.2%). Enhanced CT was superior to noncontrast CT in diagnosing appendicitis in all age and any gender groups. We suggest that enhanced, but not noncontrast, CT should be primarily performed for diagnosing acute appendicitis in all patients to minimize the radiation exposure unless intravenous administration of contrast material is contraindicated.

Tumor necrosis factor-α-induced apoptosis of gastric cancer MKN28 cells: Accelerated degradation of the inhibitor of apoptosis family members

The role of the inhibitor of apoptosis (IAP) family members in tumor necrosis factor-α (TNF-α)-induced apoptosis of human gastric cancer MKN28 cells was explored. TNF-α induced up-regulation of cIAP2, whereas cycloheximide (CHX) induced down-regulation of XIAP and survivin. Degradation of cIAP1 and XIAP, but not survivin, was accelerated by co-treatment of cells with TNF-α and CHX, and TNF-α-induced up-regulation of cIAP2 was inhibited by BMS-345541 (NF-κB inhibitor). Treatment of MKN28 cells with TNF-α plus CHX induced degradation of survivin and activation of caspase-8 and -3, followed by degradation of cIAP1 and XIAP and apoptosis. Proteasome inhibitors (MG132 and epoxomicin) suppressed TNF-α plus CHX-induced degradation of survivin, cIAP1, and XIAP as well as apoptosis. A caspase inhibitor (z-VAD-fmk) suppressed TNF-α plus CHX-induced apoptosis, but allowed degradation of survivin, cIAP1 and XIAP. TNF-α receptor 1 and 2 were expressed on MKN28 cells. The magnitude of apoptosis induced by TNF-α plus BMS-345541 was much less than that induced by TNF-α plus CHX. These findings suggest that TNF-α plus CHX-induced apoptosis of gastric cancer MKN28 cells may be caused by accelerated degradation of the IAP family members (survivin, cIAP1, and XIAP), in addition to inhibition of NF-κB-dependent synthesis of anti-apoptotic molecules.