Akiyoshi T

Nuclear factor-κB contributes to hedgehog signaling pathway activation through sonic hedgehog induction in pancreatic cancer

The hedgehog (Hh) signaling pathway, which functions as an organizer in embryonic development, is implicated in the development of various tumors. In pancreatic cancer, pathway activation is reported to result from aberrant expression of the ligand, sonic Hh (Shh). However, the details of the mechanisms regulating Shh expression are not yet known. We hypothesized that nuclear factor-kappaB (NF-kappaB), a hallmark transcription factor in inflammatory responses, contributes to the overexpression of Shh in pancreatic cancer. In the present study, we found a close positive correlation between NF-kappaB p65 and Shh expression in surgically resected pancreas specimens, including specimens of chronic pancreatitis and pancreatic adenocarcinoma. We showed that blockade of NF-kappaB suppressed constitutive expression of Shh mRNA in pancreatic cancer cells. Further activation of NF-kappaB by inflammatory stimuli, including interleukin-1beta, tumor necrosis factor-alpha, and lipopolysaccharide, induced overexpression of Shh, resulting in activation of the Hh pathway. Overexpression of Shh induced by these stimuli was also suppressed by blockade of NF-kappaB. NF-kappaB-induced Shh expression actually activated the Hh pathway in a ligand-dependent manner and enhanced cell proliferation in pancreatic cancer cells. In addition, inhibition of the Hh pathway as well as NF-kappaB suppressed the enhanced cell proliferation. Our data suggest that NF-kappaB activation is one of the mechanisms underlying Shh overexpression in pancreatic cancer and that proliferation of pancreatic cancer cells is accelerated by NF-kappaB activation in part through Shh overexpression.

Clinical significance of tissue inhibitor of metalloproteinase expression in gastric carcinoma.

Tissue inhibitor of metalloproteinase (TIMP) has been reported to inhibit tumour invasion through an inactivation of matrix metalloproteinase (MMP) both in vitro and in vivo. Among the TIMP family, TIMP-1 possesses not only proteinase inhibitory activity but also a growth-promoting function. However, the significance of the expression of TIMP-1 in human gastric carcinoma tissue has yet to be clarified. In 50 examined cases of gastric carcinoma, 44 (88%) cases showed a higher expression of TIMP-1 mRNA in the biopsy samples from the tumour tissue (T) than in the biopsy samples from the corresponding normal tissue (N), as determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). In a multivariate analysis, the T/N ratio of TIMP-1 mRNA was found to be an independent factor influencing the depth of tumour invasion and was the second most important factor in determining the prognosis of patients. As RT-PCR assay can be performed on biopsy specimens obtained before surgery, an evaluation of the TIMP-1 expression in biopsy specimens by RT-PCR may thus provide useful preoperative information on tumour aggressiveness.

Gli1, downregulated in colorectal cancers, inhibits proliferation of colon cancer cells involving Wnt signalling activation

Background: Early events in the progression of 90% of sporadic colorectal cancers depend on constitutive activation of Wnt signalling. Recent data also indicate a close association between the Hedgehog (Hh) and Wnt pathways in colonic epithelial cell differentiation. Aims: To investigate whether expression of Gli1, a transactivator of Hh signalling, can suppress Wnt signalling and inhibit proliferation of human colorectal cancer cells. Methods: Gli1 and nuclear β-catenin expression were examined in a series of 40 human colorectal cancers by immunohistochemistry. We quantified Gli1 and nuclear β-catenin staining as markers of Hh and Wnt pathway activation, respectively. Two human colon cancer cell lines, SW480 and HCT116, with mutations in APC and β-catenin, respectively, were used. The effects of Gli1 overexpression on Wnt transcriptional activity, β-catenin subcellular distribution, and proliferation in these cells were analysed. Results: Nuclear accumulation of β-catenin and the Gli1 staining level were inversely associated in the 40 human colorectal cancers. Wnt transcriptional activity was reduced in Gli1 transfected cells. These effects were observed even in Gli1 transfected cells cotransfected with mutated β-catenin. Furthermore, nuclear accumulation of β-catenin was diminished compared with that in empty vector transfected cells, and downregulated transcription of c-Myc was observed in Gli1 transfected cells. Proliferation of Gli1 transfected cells was also significantly suppressed compared with that in empty vector transfected cells. Conclusions: Our data suggest that Gli1 plays an inhibitory role in the development of colorectal cancer involving Wnt signalling, even in cases with the stabilising mutation of β-catenin.

γ-Secretase Inhibitors Enhance Taxane-Induced Mitotic Arrest and Apoptosis in Colon Cancer Cells

BACKGROUND & AIMS Colorectal cancers are resistant to conventional chemotherapeutic treatments, including taxanes. gamma-Secretase is a multimeric membrane protein complex responsible for the intramembrane proteolysis of various type I transmembrane proteins, including amyloid beta-precursor protein and Notch. gamma-Secretase inhibitors have attracted increasing interest as anticancer drugs because of their ability to inhibit Notch signaling. However, the therapeutic usefulness of gamma-secretase inhibitors against colorectal cancers remains unclear. METHODS The effects of gamma-secretase inhibitors on growth and apoptosis induced by various chemotherapeutic agents in colon cancer cells were evaluated using Hoechst 33342 staining, colony formation assay, and cell cycle analysis. The effect of gamma-secretase inhibitors on taxane-induced mitotic arrest was evaluated using the cyclin B1-associated histone H1 kinase assay and MPM-2 reactivity. The involvement of Notch signaling was evaluated by the silencing of Notch/CBF1 signaling by RNA interference. RESULTS gamma-Secretase inhibitors enhanced taxane-induced mitotic arrest and apoptosis of colon cancer cells both in vitro and in vivo, although gamma-secretase inhibitors alone did not affect growth and apoptosis of colon cancer cells. We also showed that this effect by gamma-secretase inhibitors was restricted to taxanes and colon cancer cells. Silencing of Notch/CBF1 signaling failed to affect paclitaxel-induced mitotic arrest and apoptosis. CONCLUSIONS These data suggest that gamma-secretase inhibitors could be a new therapeutic modality for overcoming resistance to taxanes in colorectal cancers.

Gli1 contributes to the invasiveness of pancreatic cancer through matrix metalloproteinase‐9 activation

The hedgehog (Hh) signaling pathway has been reported to be associated with the growth of pancreatic cancer, but its role in the invasive phenotype is poorly understood. Therefore, we investigated the role of the Hh pathway in pancreatic cancer cell invasiveness using a Matrigel invasion assay. Blockade of the Hh pathway by cyclopamine inhibited pancreatic cancer cell invasion in association with a decreased expression of matrix metalloproteinase (MMP)‐9. By contrast, activation of the Hh pathway by the addition of exogenous Sonic hedgehog increased cell invasion and MMP‐9 expression. Stable transfection of pancreatic cancer cells with Gli1 increased their invasiveness, which was associated with activation of MMP‐9. We also showed that inhibition of MMP‐9 by small interfering RNA blocked the increased invasiveness of Gli1‐transfected cells. Furthermore, inhibition of Gli1 by small interfering RNA suppressed the invasiveness and MMP‐9 expression of pancreatic cancer cells. Taken together, these findings suggest that members of the Hh pathway, especially Gli1, play an important role in the invasiveness of pancreatic cancer cells through the regulation of MMP‐9 expression. (Cancer Sci 2008; 99: 1377–1384)

Crosstalk of hedgehog and Wnt pathways in gastric cancer

Morphogenic signals like Hedgehog (Hh) and Wnt are reported to play critical roles in the progression of gastric cancer. We aimed to assess the relationship between Hh and Wnt signaling pathways. In 58 gastric cancer specimens, Wnt pathway activation was inversely correlated with Hh pathway activation. When AGS gastric cancer cells, in which Wnt signaling was constitutively active, were used as a target cell line, Gli1 overexpression suppressed Wnt transcriptional activity, nuclear beta-catenin accumulation and proliferation of AGS cells. Knock-down of beta-catenin by siRNA suppressed Wnt pathway activity and proliferation of AGS cells. Our data may provide some clues for the treatment of gastric cancer associated with Wnt signaling activation.

Cystadenocarcinoma of the liver without mesenchymal stroma : possible progression from a benign cystic lesion suspected by follow-up imagings

We herein report a 64-year-old Japanese woman with cystadenocarcinoma of the liver without mesenchymal stroma which had been followed up by imagings for 12 years. A small round cyst grew from 1cm to 4 cm in diameter during 10 years. Thereafter, solid components were first detected in the unilocular cystic mass, which showed very rapid growth. Extended right lobectomy with right caudate lobectomy was performed. The histopathological diagnosis was cystadenocarcinoma of the liver without mesenchymal stroma. In this communication, we describe the progressive morphologic changes, shown on imagings, from a benign hepatic cyst to cystadenocarcinoma of the liver without mesenchymal stroma, and we briefly discuss the carcinogenesis of cystadenocarcinoma of the liver.

Lymphokine-activated killer cell function of peripheral blood mononuclear cells, spleen cells and regional lymph node cells in gastric cancer patients.

Lymphokine‐activated killer (LAK) cells generated by culture of peripheral blood mononuclear cells (PBMC). spleen cells (SPC) and regional lymph node cells (LNC) with IL‐2 for 4 days were examined for their functional capabilities m 29 patients with gastric carcinoma. The cytotoxic activity of LAK cells induced from LNC was significantly lower than that from either PBMC or SPC. although there was no difference between PBMC or SPC. The induction of mRNA of interferon‐gamma (IFN‐γ) or tumour necrosis factor‐alpha (TNF‐α) and the production of these cytokines in the non‐adherent LAK cells from LNC were also significantly reduced compared with those from PBMC or SPC. Further, the LAK cells from LNC secreted significantly lower levels of these cytokines when stimulated with tumour target, Raji cells, although the production of these cytokines was markedly increased by stimulation with the targets in all three cell populations. Phenotypic analysis of each cell population revealed a decreased proportion of the cells mediating natural killer (NK) activity, including CD16+. CD56+, and CD57+ cells in LNC either before or after culture, although OKIal+ and CD25+ cells were uniformly increased in all ceil populations after culture. Changes in subpopulations of CD4+ and CD8+ cells in LNC were not apparently different from PBMC or SPC. These results indicated the differential reactivity of each lymphocyte population to IL‐2 and the reduced LAK cell function of LNC compared with PBMC or SPC in patients with gastric carcinoma.

Expression of pyrimidine nucleoside phosphorylase mRNA plays an important role in the prognosis of patients with oesophageal cancer

SummaryTo clarify the significance of the expression of pyrimidine nucleoside phosphorylase (PyNPase) mRNA as a predictive factor for the prognosis of patients with oesophageal carcinoma, the PyNPase mRNA in the tumours and normal tissues from 55 resected cases of oesophageal carcinoma was examined by a reverse transcription polymerase chain reaction (RT-PCR). As a result, a positive correlation was observed between the tumour/normal (T/N) ratio of the expression of PyNPase mRNA by RT-PCR and that of the enzyme activity of PyNPase based on the findings of an enzyme linked immunosolvent assay (r = 0.594, P = 0.009). The T/N ratio of the expression of PyNPase mRNA was significantly higher in the cases with lymph vessel invasion (P = 0.013), lymph node metastasis (P = 0.0016), and an advanced stage of the disease (P = 0.021) than those without these factors. The patients with a higher T/N ratio of PyNPase mRNA showed significantly worse prognosis than those with a lower T/N ratio (P = 0.023 with log-rank tests). A multivariate analysis for the cumulative survival rates revealed that a high T/N ratio of the expression of PyNPase mRNA was independently related to a poor prognosis. These findings suggested that the determination of PyNPase mRNA by RT-PCR thus appears to be a new useful parameter for identifying both a poor prognosis and a highly malignant potential of oesophageal carcinoma.

Estrogen receptor status and effects of endocrine ablative surgery in ethyl methanesulphonate-induced rat mammary carcinoma

An experimental model of rat mammary carcinoma induced by oral administration of ethyl methanesulphonate (EMS) was characterized with reference to estrogen receptors (ER) and hormone dependency. After the administration of EMS, the mammary carcinomas developed more rapidly in the 4-week old rats than in the 16-week old rats. The developed mammary carcinomas were related to the ER status. Tumor development was prevented by ablative oophorectomy prior to EMS administration, but only partially, by adrenalectomy. These inhibitory effects on tumor development were more obvious when ablative surgery was performed on younger rats at age 4 weeks. In rats subjected to oophorectomy at after EMS administration at age 16 weeks, tumor induction was retarded but not completely prevented. Thus, it appears that mammary tumors induced by EMS are dependent on ovarian hormones and that mammary glands of the younger female rats are more sensitive to carcinogenic actions of EMS.