Biomedical subjects

Miki Shitashige

Publications and source records attributed to Miki Shitashige.

5 recordsLinked to original sources

PubMed2006

Mass spectrometry analysis of the native protein complex containing actinin-4 in prostate cancer cells.

Actinin-4 was originally identified as an actin-binding protein associated with cell motility and cancer invasion and metastasis. However, actinin-4 forms complexes with a large number of different partner proteins and is speculated to have several distinct functions depending on its partner. The level of actinin-4 expression was found to be significantly lower in prostate cancer cells than in non-cancerous basal cells, and restoration of actinin-4 expression inhibited cell proliferation by prostate cancer cell line 22RV1. Immunoprecipitation and mass spectrometry analysis revealed that actinin-4 forms native complexes with several partner proteins in 22RV1 cells, including with beta/gamma-actin, calmodulin, the clathrin heavy chain, non-muscular myosin heavy chain, heterogeneous nuclear ribonucleoprotein A1, and Ras-GTPase-activating protein SH3 domain-binding protein. Clathrin is a coat protein that covers the internalized membrane pit that forms during early endocytosis. We found that other clathrin-related and unrelated cargo proteins, including dynamin, adaptin-delta, beta subunit of neuronal adaptin-like protein, and p47A, also interact with actinin-4. Immunofluorescence microscopy revealed that dynamin and clathrin co-localized with actinin-4 at the sites of membrane ruffling, and transfection of actinin-4 cDNA facilitated the transport of transferrin into perinuclear endosomes. Endocytosis terminates signaling evoked by cell surface receptors and regulates the recycling of receptors and ligands. We identified a panel of proteins whose expression and/or subcellular localization was regulated by actinin-4 by performing organelle fractionation and ICAT-LC-MS/MS. The decreased expression of actinin-4 protein in prostate cancer cells may cause aberrations in the intracellular trafficking of various cell surface molecules and contribute to carcinogenesis.

Actinin↗

PubMed2006

Label-free quantitative proteomics using large peptide data sets generated by nanoflow liquid chromatography and mass spectrometry.

We developed an integrated platform consisting of machinery and software modules that can apply vast amounts of data generated by nanoflow LC-MS to differential protein expression analyses. Unlabeled protein samples were completely digested with modified trypsin and separated by low speed (200 nl/min) one-dimensional HPLC. Mass spectra were obtained every 1 s by using the survey mode of a hybrid Q-TOF mass spectrometer and displayed in a two-dimensional plane with m/z values along the x axis, and retention time was displayed along the y axis. The time jitter of nano-LC was adjusted using newly developed software based on a dynamic programming algorithm. The comprehensiveness (60,000-160,000 peaks above the predetermined threshold detectable in 60-microg cell protein samples), reproducibility (average coefficient of variance of 0.35-0.39 and correlation coefficient of over 0.92 between duplicates), and accurate quantification with a wide dynamic range (over 10(3)) of our platform warrant its application to various types of experimental and translational proteomics.

Actinin↗

PubMed2006

[Early detection of pancreatic cancer by novel proteomic technique].

Pancreatic cancer is the fifth leading cause of cancer-related mortality in Japan. Early detection in pancreatic cancer is one of the most feasible strategies to improve outcome. We compared plasma proteome between pancreatic cancer patients and healthy controls using surface-enhanced laser desorption/ionization coupled with hybrid quadrupole time-of-flight mass spectrometry. Proteomic spectra were generated from a total of 245 plasma samples obtained from two institutes. A discriminating proteomic pattern was built from training cohort using machine learning algorithm and was applied two validation cohorts. This set discriminating cancer patients in the first validation cohort with a sensitivity of 90.9% and a specificity of 91.9%, and was further validated in an independent cohort at a second institution. When combined with CA19-9, 100% tumor of pancreatic cancers, including early stage tumors, were detected. In this report, we describe a possible detection of early pancreatic cancer using novel proteomic technique.

Biomarkers, Tumor↗

PubMed2002

Enhancement by cyclosporin A of taxol-induced apoptosis of human urinary bladder cancer cells.

Taxol is a microtubule-stabilizing agent which induces apoptosis in various cancer cells. In this study, we found that T24 cells derived from high grade human urinary bladder cancer were relatively resistant to taxol and that the IC50 value determined by a colorimetric WST-1 assay was 406.0 nM. Interestingly, cyclosporin A (CsA), an immunosuppressive drug, dramatically enhanced sensitivity to taxol, and the IC50 value was decreased to 47.5 nM in the presence of 1 microM CsA. KK47 cells derived from low grade human urinary bladder cancer showed high sensitivity to taxol with an IC50 value of 78.8 nM which decreased to 14.4 nM in the presence of 1 microM CsA. FK506, another immunosuppressive drug, also enhanced sensitivity to taxol. Furthermore, a concomitant loss of calcineurin activity was observed after the treatment of both cell lines with both CsA and FK506. Taxol induced apoptosis of the cells, as assessed by Hoechst 33258 staining and by the measurement of caspase 3 activity. Immunoblot analysis with an antibody against Bcl-2 phosphorylated at serine 70 demonstrated that taxol induced the phosphorylation of Bcl-2 with its enhancement in the presence of CsA. In addition, treatment of the cells with CsA significantly decreased the expression of Bcl-2 at both the protein and mRNA levels. These results suggest that the enhancement of taxol-induced apoptosis by immunosuppressive drugs is at least partly due to the inhibition of calcineurin activity and the loss of the antiapoptotic function of Bcl-2 via the enhancement of phosphorylation and the reduction of expression.

Antineoplastic Agents, Phytogenic↗

PubMed2002

Binary regulation of interleukin (IL)-6 production by EP1 and EP2/EP4 subtypes of PGE2 receptors in IL-1beta-stimulated human gingival fibroblasts.

Prostaglandin E2 (PGE2) exerts its biological actions via EP receptors, which are divided into four subtypes of EP1, EP2, EP3 and EP4. In the present study, we investigated whether PGE2 regulated interleukin (IL)-6 production in human gingival fibroblasts (HGF) stimulated with IL-1beta and if so, which subtype(s) of PGE2 receptors were involved. Indomethacin, a cyclooxygenase inhibitor, significantly enhanced IL-1beta-induced IL-6 production by HGF, although it completely inhibited IL-1beta-induced PGE2 production. Exogenous PGE2 suppressed the IL-1beta-induced IL-6 production. Reverse transcription-polymerase chain reaction analysis demonstrated that mRNA of EP1, EP2 and EP4, but not EP3 mRNA, was expressed in unstimulated and IL-1beta-stimulated HGF. 11-deoxy-PGE1, a selective EP2/EP3/EP4 agonist, and butaprost, a selective EP2 agonist, inhibited IL-1beta-induced IL-6 production, although butaprost was less potent than 11-deoxy-PGE1. 17-phenyl-omega-trinor PGE2, an EP1 agonist, enhanced IL-1beta-induced IL-6 production. Based on these data, we suggest that PGE2 can up- or downregulate IL-1beta-induced IL-6 production via EP1 receptors or via EP2/EP4 receptors in HGF, respectively. Expression and function of EP1, EP2 and EP4 receptors in HGF may play critical roles in controlling inflammatory periodontal conditions.

Alprostadil↗