Shigeru Matsukawa

The activation of Proteinase-Activated Receptor-1 (PAR1) mediates gastric cancer cell proliferation and invasion

BackgroundIn addition to regulating platelet function, the G protein-coupled sub-family member Proteinase-activated receptor-1 (PAR1) has a proposed role in the development of various cancers, but its exact role and mechanism of action in the invasion, metastasis, and proliferation process in gastric cancer have yet to be completely elucidated. Here, we analyzed the relationship between PAR1 activation, proliferation, invasion, and the signaling pathways downstream of PAR1 activation in gastric cancer.MethodsWe established a PAR1 stably transfected MKN45 human gastric cancer cell line (MKN45/PAR1) and performed cell proliferation and invasion assays employing this cell line and MKN28 cell line exposed to PAR1 agonists (α-thrombin and TFLLR-NH2). We also quantified NF-κB activation by electrophoretic mobility shift assay (EMSA) and the level of Tenascin-C (TN-C) expression in conditioned medium by ELISA of MKN45/PAR1 following administration of α-thrombin. A high molecular weight concentrate was derived from the resultant conditioned medium and subsequent cultures of MKN45/PAR1 and MKN28 were exposed to the resultant concentrate either in the presence or absence of TN-C-neutralizing antibody. Lysates of these subsequent cells were probed to quantify levels of phospholyrated Epidermal Growth Factor Receptor (EGFR).ResultPAR1 in both PAR1/MKN45 and MKN28 was activated by PAR1 agonists, resulting in cell proliferation and matrigel invasion. We have shown that activation of NF-κB and EGFR phosphorylation initially were triggered by the activation of PAR1 with α-thrombin. Quantitative PCR and Western blot assay revealed up-regulation of mRNA and protein expression of NF-κB target genes, especially TN-C, a potential EGFR activator. The suppressed level of phosphorylated EGFR, observed in cells exposed to concentrate of conditioned medium in the presence of TN-C-neutralizing antibody, identifies TN-C as a putative autocrine stimulatory factor of EGFR possibly involved in the sustained PAR1 activation responses observed.ConclusionOur data indicate that in gastric carcinoma cells, PAR1 activation can trigger an array of responses that would promote tumor cell growth and invasion. Over expression of NF-κB, EGFR, and TN-C, are among the effects of PAR1 activation and TN-C induces EGFR activation in an autocrine manner. Thus, PAR1 is a potentially important therapeutic target for the treatment of gastric cancer.

Interferons upregulate thymidine phosphorylase expression via JAK-STAT-dependent transcriptional activation and mRNA stabilization in human glioblastoma cells

SummaryOverexpression of the angiogenic enzyme thymidine phosphorylase (TP) in tumor cells and/or infiltrating macrophages correlates with increased microvessel density and poor prognosis in various tumor types including glioma. The present study examined how the TP gene expression is regulated by different types of interferons (IFNs) in human T98G and A172 glioblastoma cells. Both type I (α, β) and type II (γ) IFNs upregulated TP mRNA and protein expression while inhibiting cell proliferation. IFN-induced TP mRNA accumulation was not inhibited by the protein synthesis inhibitor cycloheximide, but was strongly blocked by the transcription inhibitor actinomycin D, as well as by transcription factor decoy oligodeoxynucleotides containing the putative IFN response element or the γ-activated sequence in the TP promoter. The Janus kinase (JAK) inhibitor AG-490 blocked both IFN-induced STAT1 (signal transducers and activators of transcription 1) phosphorylation and TP expression. All IFNs increased the stability of TP mRNA as well. In addition, IFN-evoked TP enzyme activity enhanced the cytotoxicity of 5-fluorouracil (5-FU). These findings indicate that TP expression may be upregulated by IFNs via the JAK-STAT signaling pathway␣and both transcriptional and posttranscriptional mechanisms. Combined treatment with IFN and 5-fluorouracil may be a useful therapeutic strategy for malignant gliomas.

Usefulness of metyrapone treatment to suppress cancer metastasis facilitated by surgical stress

BACKGROUND One causative factor of tumor metastasis enhanced by surgical stress is thought to be hypersecretion of endogenous glucocorticoids. This study evaluated the effectiveness of metyrapone treatment and adrenalectomy in preventing the harmful effects of glucocorticoids in the enhancement of tumor metastasis resulting from surgical stress. METHODS The effect of dexamethasone on pulmonary metastasis of MRMT-1 cells and on the number of peripheral lymphocytes was evaluated in rats. To evaluate the suppressive effect of adrenalectomy and metyrapone treatment on operation-induced enhancement of metastasis, several parameters such as induction of pulmonary metastasis, serum corticosterone levels, and the number of blood lymphocytes and apoptotic thymocytes were determined. RESULTS With dexamethasone treatment, the number of peripheral lymphocytes rapidly decreased; in contrast, pulmonary metastasis increased. The serum corticosterone level was doubled at 1 hour, apoptotic thymocyte numbers were increased about sevenfold at 3 hours and about fourfold at 6 hours, and blood lymphocyte numbers were decreased at 3 hours after laparotomy, which facilitated about a 10-fold increase in the pulmonary metastasis. These changes were almost completely suppressed by preoperative adrenalectomy and metyrapone treatment. CONCLUSIONS Preoperative metyrapone treatment, which suppresses hypersecretion of endogenous glucocorticoids as a result of operation, modulates the enhancement of cancer metastases and may be an effective treatment.

Clinical Significance of Combined Immunohistochemical Detection of CD44v and Sialyl Lex Expression for Colorectal Cancer Patients Undergoing Curative Resection

To evaluate their prognostic value, the expressions of CD44v and sialyl LeX (SLX) in colorectal cancers were studied immunohistochemically. Tissue specimens were reacted with monoclonal antibodies (mAb) CD44-1V and CSLEX-1. Of the 145 colorectal cancer patients undergoing curative resection, 59 (40.7%) were positive for mAb CD44-1V, and 40 (27.6%) were positive for mAb CSLEX-1. There was a significant correlation between the combined expression of SLX and CD44v8–10 and lymph node metastasis. The patients with tumors negative for CD44v8–10 and SLX had the most favorable prognoses. Conversely, the patients with tumors positive for both CD44v8–10 and SLX had a high recurrence rate and the poorest prognoses. In a multivariate analysis using the Cox regression model, the combined expression of SLX and CD44v8–10 emerged as an independent prognostic indicator. These results suggested that the combined expression of CD44v8–10 and SLX may be a biologic marker of prognostic significance.

The activation of proteinase-activated receptor-1 (PAR1) promotes gastric cancer cell alteration of cellular morphology related to cell motility and invasion

Cell motility proceeds by cycles of edge protrusion, adhesion and retraction. Whether these functions are coordinated by biochemical or biomechanical processes is unknown. Tumor invasion and metastasis is directly related to cell motility. We showed that stimulation of proteinase-activated receptor-1 (PAR1) can trigger an array of responses that would promote tumor cell growth and invasion. Thus, we examined aspects of PAR1 activation related to cell morphological change that might contribute to cell motility. We established a PAR1 stably transfected MKN45 gastric cancer cell line (MKN45/PAR1). We examined morphological changes, Rho family activation and overexpression of cytoskeletal protein in cells exposed to PAR1 agonists (α-thrombin and TFLLR-NH2). MKN45/PAR1 grows with an elongated and polarized morphology, extending pseudopodia at the leading edge. However, in the presence of PAR1 antagonist, MKN45/PAR1 did not show any changes in cell shape upon addition of either α-thrombin or TFLLR-NH2. Activated PAR1 induced RhoA and Rac1 phosphorylation, and subsequent overexpression of myosin IIA and filamin B which are stress fiber components that were identified by PMF analysis of peptide mass data obtained by MALDI-TOF/MS measurement. Upon stimulation of MKN45/PAR1 for 24 h with either α-thrombin or TFLLR-NH2, the distribution of both myosin IIA and filamin B proteins shifted to being distributed throughout the cytoplasm to the membrane, with more intense luminescence signals than in the absence of stimulation. These results demonstrate that PAR1 activation induces cell morphological change associated with cell motility via Rho family activation and cytoskeletal protein overexpression, and has a critical role in gastric cancer cell invasion and metastasis.

Cell-specific but p53-independent regulation of vascular endothelial growth factor expression by interferons in human glioblastoma cells.

SummaryVascular endothelial growth factor (VEGF) is a key mediator of tumor angiogenesis. Interferons (IFNs) have been widely used in the treatment of malignant or recurrent gliomas with only marginal benefit. The association between IFNs and VEGF expression remains unclear and should be an intensively investigated subject. The present study therefore examined the effects of different types of IFNs on VEGF expression in human T98G, A172 and U251 glioblastoma cells by quantitative RT-PCR and ELISA. Both type I (α, β) and type II (γ) IFNs upregulated VEGF expression in a cell-specific but p53-independent manner. Actinomycin D experiments demonstrated that IFNs did not alter VEGF mRNA stability. In contrast, induction of VEGF mRNA by IFNs was blocked by the protein synthesis inhibitor cycloheximide. Interestingly, cycloheximide also blocked IFN-induced activation of the p44/p42 mitogen-activated protein kinase, which was partially required for induction of VEGF by IFNs. These findings suggest that VEGF might be an indirect target gene of IFNs, and might provide insights into therapeutic applications of IFNs against angiogenesis-dependent tumors.

Nanoparticle-Assisted Laser Desorption/Ionization Mass Spectrometry (Nano-PALDI MS) with Py-Tag for the Analysis of Small Molecules

We compared two ionization methods, matrix assisted laser desorption/ionization (MALDI) and nanoparticle assisted laser desorption/ionization (Nano-PALDI) mass spectrometry (MS), for the analysis of amino acids derivatized with Py-Tag™ that consists pyrylium-based compound. Py-Tag is a useful stable derivatization reagent due to wide mass differences (using 13C as the sole stable labelling isotope). For Py-Tag labelled lysine, sensitive signals that showed less noise with a ten times higher sensitivity, showed a wider mass difference by Nano-PALDI MS compared to MALDI MS.