We have produced “Multi-PK antibodies” that can detect entire family of protein kinase in order to clarify the intracellular signaling related to various biological phenomena and diseases. In addition, we have developed various methods for the analysis of intracellular protein kinases based on Western blotting using Multi-PK antibodies. By using the Multi-PK antibodies, we have identified protein kinases involved in epigenetics and cancer. In this review, we describe the analysis of protein kinases related to the mechanism of suppression of insulin gene expression in glucotoxicity of type 2 diabetes using Multi-PK antibody.

The existence of the cancer stem cell relates to prognosis and resistance to chemotherapy and radiotherapy. In this study, we aimed at the acquisition of the candidate marker protein for diagnosing lung adenocarcinoma, we established cancer stem cell-like adenocarcinoma cell line using reprogramming factor-induced iPS cells. We focused on NAP1L1 as a result of compared protein profiles with cancer stem cell-like adenocarcinoma cell line and the parent cells to discover biomarker proteins. NAP1L1 is a key regulator of embryonic neurogenesis. In this study, we performed immunohistochemistry with lung cancer tissues to investigate the relationship between NAP1L1 expression and the clinicopathological parameters and prognosis of lung adenocarcinoma patients. NAP1L1 expression was significantly associated with reduced differentiation, higher pathological TNM stages, lymph node metastasis, intrapulmonary metastasis, lymphatic invasion, vascular invasion and poorer prognosis of patients with adenocarcinoma. Moreover, multivariate analyses using the Cox-proportional hazards model confirmed that NAP1L1 expression increased the risk of death after adjusting for other clinicopathological factors. Furthermore, NAP1L1 expression was identified as an independent poor prognostic factor in patients with resectable stage I lung adenocarcinoma.

Electrophoresis is an indispensable technique for research such as separation analysis. A Monoclonal-like peak was discovered in alfa2 region on routine-based capillary zone electrophoresis (CZE) but neither on cellulose acetate membrane electrophoresis nor immunofixation. From clinical records it is strongly indicated the involvement of iopamidol, the radio-contrast medium, administered at CT scan examination. Very high absorbance by the media can be detected at the wavelength of 214 nm on CZE. Other three media used in the hospital were further investigated to show media-specific Monoclonal-like peaks at the constant migrate position from alfa2-beta region with high reproducibility. These include iohexol, ioversol and iopromide. We can newly profile the media in the pattern profile of CZE. Use of a disposable ultrafiltration device can eliminate the nonspecific peak, thus being enable to detect specifically Monoclonal proteins as well as other covered underlying pathophysiologic conditions.

In order to understand the complicated life system which is composed of trillions of cells, highly sensitive and comprehensive analysis system for single cells is needed. For this purpose, I have developed a series of microscale chemical analysis techniques such as sample collection, preparation, separation, and detection. Among them, sensitivity improvement in mass spectrometry (MS) detection has been quite important for the comprehensive bioanalysis. One approach is to perform capillary electrophoresis prior to MS detection, which can reduce the ionization suppression by its excellent separation performance and also can improve the sensitivity by online sample preconcentration methods, such as large-volume dual preconcentration by isotachophoresis and stacking. Consequently, zeptomole-level sensitivity has been realized, which has been successfully applied to single cell metabolome analysis and pharmacokinetic analysis. These results showed great potential of our technologies for contributing to the progress in the biological research fields.

Ovarian cancer is the leading cause of death in women with gynecologic malignancies, and especially, clear cell subtype (OCCCa) shows chemoresistance and clinical outcomes at advanced stages are generally unfavorable. To identify proteins for the novel biomarker in OCCCa, we performed shotgun proteomics analysis using formalin-fixed and paraffin-embedded clinical samples. Lefty may be an excellent OCCCa-specific molecular marker and the functional rule may be the establishment and maintenance of phenotypic characteristics of OCCCa. EBP50 may have great utility in OCCCa recurrence and prognosis through inhibits apoptosis and stabilization of PARP1 activity, a DNA repair enzyme. These results have high significance and could be expected to be developed for clinical application.

Tochigi Cancer Center, which was established in 1986, has been providing medical care for many cancer patients as a prefectural cancer hospital. The importance of building biobanks and databases is increasing in order to respond to the rapid changes and development of cancer diagnosis and treatment accompanying recent scientific and technological innovations. By utilizing biobanks and databases, it is possible to select the optimal treatment for each individual case based on molecular information and to obtain universal information that cannot be obtained from a small number of cases such as rare cancers. It will also be possible to conduct research on refractory and rare cancers. Tochigi Cancer Center opened Tochigi Cancer Biobank in April 2021 to prepare for tests using patient tissues such as cancer genomic medicine, and to promote translational research to develop novel treatment strategy on refractory and rare cancers. We will introduce the purpose and features of Tochigi Cancer biobank.

Rapid advances in next-generation sequencing (NGS) technology over the past decade have led to a paradigm shift in cancer treatment from one-size-fits-all medicine to genomics-based precision medicine that has become more accessible in general clinical practice. It, however, has helped only less than 10% of the cancer patients who have taken genomic profiling tests. Proteomics-based rather than genomics-based approaches can capture biological processes directly contributing to cancer pathogenesis. There is, therefore, a growing need for simultaneous proteomic analysis in the next stage of precision medicine. In the future, it is expected that data from genomic, transcriptomic, proteomic, and epigenomic analyses will be integrated and complemented to more accurately understand the disease states and propose therapeutic strategies. Reverse-phase protein array (RPPA) is a proteomic technology that enables semi-quantitative, accurate, and high throughput signal profiling even in small amounts of samples. This review introduces its current status and the application to precision cancer medicine, and highlights the potential utility of RPPA technology for improving precision cancer medicine.

Phosphorylation is a posttranslational modification of proteins that regulates many biological functions, such as development, morphogenesis, gene expression, metabolism and neuronal activity. Dysregulation of phosphorylation causes many diseases including cancer and Alzheimer’s disease. Then, a lot of research has been performed to reveal the molecular mechanism of phosphorylation; the identification of phosphorylation site(s), protein kinases and phosphatases, and their regulation. Now, in vivo phosphorylation sites can be identified using mass spectrometry and be analyzed by immunoblotting with phospho-specific antibodies. However, it is not easy to grasp the total phosphorylation profile of the target protein, and when the protein is phosphorylated at multiple sites, the quantification of site-specific phosphorylation is still challenging. Phos-tag SDS-PAGE is phospho-affinity SDS-PAGE, which overcomes some of the above problems. Phosphorylated proteins are delayed during electrophoresis and separated from a non-phosphorylated counterpart depending on the number and sites of phosphorylation. We have employed this technique to see the in vivo phosphorylation of many proteins. Here, we introduce our results on the phosphorylation of tau protein and a tau kinase GSK3β and show how useful this technique is in the analysis of in vivo protein phosphorylation.

The progression of autoantibodies (AAbs) to tumor antigens is a humoral immune response that occurs early in tumor development. Serum AAbs are ideal targets for early cancer detection given that AAbs are observed in sera from patients with various cancers before cancer-associated antigens can be detected, moreover, AAbs are found in sera up to five years before symptomatic disease. Although multiple proteomics-based strategies have been developed to identify novel AAbs for early detection and treatment of cancer, only a few targets have satisfied strict clinical requirements. In this paper, we review the trajectory of autoantibody research in terms of cancer diagnosis and then discuss future perspectives on diagnosis and autoantibody-based therapeutics.

Protein phosphorylation is a key process regulating various cellular physiological functions such as proliferation, migration, cell cycle progression, apoptosis, and differentiation. Dysregulated kinase activity occurs in the process of carcinogenesis and cancer progression due to genetic alterations including copy number alterations and point mutations. Moreover, kinome consisting of the dysregulated self-phosphorylation and kinase-to-kinase regulatory relationship is also responsible for the causes of cancer. Aberrantly regulated kinases are considered to be biomarkers and therapeutic targets in oncology. A plenty of kinase inhibitors has been approved for the cancer treatments. The various methods to examine kinome have been developed. Among these methods, a three-dimension peptide array is available to investigate 100 tyrosine kinases simultaneously using a tiny amount of the protein from biopsy samples. We discuss the perspectives of kinome in cancer research and introduce comprehensive kinase activity assay system using three-dimensional peptide array. The comprehensive kinase activity assay system may provide predictive biomarkers and drug targets useful for novel treatments in oncology.

Top-down proteomics, the analysis via mass spectrometry of cellular protein components as intact species without enzymatic digestion, is emerging as a powerful analytical approach to reveal entire proteoform profiles in vivo. To improve the number of proteoforms detected in top-down proteomics, high-resolution proteome fractionation prior to mass spectrometry is crucial, and interest in polyacrylamide gel electrophoresis as a sample pre-fractionation tool has been growing in recent years. This review describes a gel-based proteome fractionation workflow, which facilitates highly efficient protein extraction following polyacrylamide gel electrophoresis, and its application to sample pre-fractionation for top-down proteomics.

Since SDS-PAGE is a powerful tool for separation of proteins according to the apparent molecular weight, the method has been used as a biological tool for more than 50 years. However, SDS-denatured proteins usually lose their native molecular structures and their biological activities. In addition, a 2% SDS can dissociate molecular complexes and aggregates of many proteins, information of the protein-protein interaction may be lost in the SDS-PAGE pattern. Therefore, we employed a low concentration (0% or 0.1%) SDS in the extraction medium for investigating heat-denatured processes of muscle proteins. Bovine muscle pieces were incubated at 40, 50, 60, 70 and 80°C for 30 sec, respectively, homogenized in the extraction medium, and centrifuged at 6,200 rpm for 5 min. The supernatants were subjected to SDS-PAGE. Myosin heavy chain (MHC), collagen, tropomyosin, and actin were aggregated sequentially during heat process, and the respective protein bands were lost in the SDS-PAGE pattern. For food allergy tests, dot blot analysis detected successfully fish egg proteins, however, SDS-PAGE and Western blotting using samples extracted with 0.05% SDS extraction medium failed to detect the fish egg proteins in the Western blot.

The symposium “Research for cancer prevention” was held on the 16th July. Invited speakers were Dr. Futoshi Okada from Tottori University, Dr. Michihiro Mutoh from Kyoto Prefectural University of Medicine, and Dr. Masaru Terasaki from Health Sciences University of Hokkaido. Dr. Okada talked about cancer prevention researches by using experimental inflammation-related carcinogenesis animal model. Dr. Mutoh talked about chemoprevention of colorectal cancers for familial adenomatous polyposis patients. Dr. Terasaki talked about colorectal cancer prevention research of AOM/DSS mice by treatment with fucoxanthin. Their cancer prevention researches were expectable for practical realization of cancer prevention in the future.