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We performed proteomic differential display analysis of human malignant pleural mesothelioma (MPM) cell lines and a human pleural mesothelial cell line by using 2-DE and LC-MS/MS. The human MPM cell lines were NCI-H28, NCI-H2052 and NCI-H2452, and the human pleural mesothelial cell line was MeT-5A. Between MeT-5A and NCI-H2052, we found 38 protein spots whose expression levels were different, from the results of 2-DE; 28 protein spots appeared higher, and 10 other protein spots lower in NCI-H2052 than in MeT-5A. These spots were analyzed by LC-MS/MS analysis and identified by a peptide sequence tag. However, from the results of 2-DE of the other cell lines, there was only one consistently upregulated protein, astrocytic phosphoprotein PEA-15, in all three MPM cell lines. Western blotting using specific antibodies against PEA-15 confirmed the elevated expression level of PEA-15 in all three MPM cell lines compared with MeT-5A cells and normal pleura tissues from patients. PEA-15 was knocked down in NCI-H2052 cells, and the proliferation of PEA-15-silenced NCI-H2052 cells was suppressed 7-15% compared with negative control cells. These results suggest that PEA-15 expression is likely to be associated with the tumorigenesis of MPM.

Oral squamous cell carcinoma (OSCC) has an absolute majority of all oral cancer. We used proteomic technology to analyze the protein expression profile in OSCC tissues and accompanying surrounding normal tissues in four oral locations (buccal mucosa, gingival mucosa, oral floor, and tongue). Ten protein spots were overexpressed more strongly in cancer tissues than normal ones, and were identified as proliferating cell nuclear antigen, 14-3-3 ε, 14-3-3 σ, proteasome subunit α type 5, translationally controlled tumor protein, eukaryotic translation initiation factor 3 subunit, macrophage capping protein, and mitochondrial isocitrate dehydrogenase subunit α. Macrophage capping protein and mitochondrial isocitrate dehydrogenase subunit α had two spots. Especially, we focused on 14-3-3 σ protein, one of the eight identified proteins, and assessed its expression level in four oral locations of OSCC by using differential display methods. The expression level of 14-3-3 σ protein was upregulated in four locations of oral cavity. Eight proteins which we identified in this study may play an important role in OSCC carcinogenesis and progression and could be used as diagnostic biomarkers of OSCC.

Much interest is presently being shown toward identifying markers for the detection of breast cancer. To detect autoantibodies that could represent diagnostic markers for breast cancer, we comprehensively analyzed serum autoantibodies showing immunoreactivity to proteins in tumor tissues of breast cancer. Tumor tissues were obtained from 40 patients with breast cancer, along with sera from 30 other patients with breast cancer and 22 healthy donors. Proteins from tumor tissues were separated by 2-DE. After blotting onto PVDF membranes, tissue proteins were immunoblotted with sera from patients or healthy donors. By comparing each immunoblot pattern, three immunoreactive spots displayed stronger staining intensity with patient sera than with sera from healthy donors. The matched protein spots on 2-DE gels were digested and used for LC-MS/MS analysis, and identified as cyclophilin A (peptidyl-prolyl cis-trans isomerase A), triosephosphate isomerase and ubiquitin-conjugating enzyme E2N. Immunoblot analysis was then performed using commercially available purified proteins, confirming the specificity of anti-cyclophilin A and anti-triosephosphate isomerase antibodies in sera from patients.

Proteomic differential display analysis was performed on human renal cell carcinoma cell SN12C clones having different metastatic potentials by using 2-DE and LC-MS/MS. The SN12C cell clones were SN12C parent cell line, SN12C-clone 2, SN12C-clone 4, and SN12C-PM6. The SN12C parent cell line was established from an HRCC surgical specimen. SN12C-clone 4 has lower, and SN12C-clone 2 and SN12C-PM6 have higher metastatic potential than SN12C parent cells. We found eight protein spots whose expression level was different between low metastatic clones and high metastatic clones. The protein expression of three appeared to be higher in high metastatic clones than low metastatic clones, and that of other five protein spots appeared to be lower in high metastatic clones than low metastatic clones. These spots were selected, digested and analyzed by LC-MS/MS analysis, and they were identified by peptide sequencing tag. In high metastatic potential clones, two isoforms of ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1) were downregulated. These results suggest that UCH-L1 expression seems to be associated with the metastatic potential of HRCC SN12C cell clones.

To identify proteins associated with esophageal carcinogenesis, we performed protein profiling of 16 esophageal squamous cell carcinomas (ESCCs) and paired noncancerous tissues by 2-DE and MS/MS. In cancerous tissues, three spots showed significant up-regulation in the amount of protein, while eight spots were significantly down-regulated. The identities of the spots were determined by PMF with LC-MS/MS and were confirmed by immunoblotting. The up-regulated proteins were tropomyosin alpha 4 chain, transgelin, and pyruvate kinase. The down-regulated proteins were serum albumin precursor, isoforms of annexin A1, tropomyosin beta chain, 14-3-3 protein sigma, and isoforms of serotransferrin precursor. In all 16 cases, up-regulation of the tropomyosin alpha 4 chain was confirmed by immunoblotting. Localization of the tropomyosin alpha 4 chain in ESCC cells and adjacent fibroblasts was confirmed by immunohistochemistry.

To detect autoantibodies that could be diagnostic markers for hepatocellular carcinoma (HCC), we analyzed serum autoantibodies comprehensively that showed immunoreactivity to proteins in tumor tissue obtained from patients with HCC. Fifteen paired samples of HCC tissue and corresponding nontumorous liver tissue as well as five normal liver tissue samples were used in the study. A combination of proteomics and SEREX (serologic analysis of recombinant cDNA expression libraries) technique was used. Tissue proteins were separated by 2-DE, transferred onto PVDF membranes, and immunoblotted with autologous sera. By comparing each immunoblot pattern, we identified four immunoreactive spots with stronger staining intensity in tumorous tissues than in corresponding nontumorous tissues and in normal liver tissues. Matched proteins on 2-DE gels were identified by LC-MS/MS. These immunoreactive proteins were heat shock 70 kDa protein 1 (HSP70), glyceraldehyde 3-phosphate dehydrogenase, peroxiredoxin, and manganese superoxide dismutase (Mn-SOD). In HCC sera, occurrences of autoantibodies against these proteins were 7/15 (46.7%), 5/15 (33.3%), 5/15 (33.3%), and 6/15 (40.0%), respectively, whereas 2/20 (10.0%), 7/20 (35.0%), 0/20 (0.0%), and 2/20 (10.0%) were in control sera. Immunoblot analysis using commercially available purified proteins was performed to confirm the specificity of autoantibodies. By statistical analysis, autoantibodies against HSP70, peroxiredoxin, and Mn-SOD showed significantly high-frequency immunoreaction in HCC sera. The three antibodies were considered patient-specific antibodies in HCC and may be candidate diagnostic biomarkers for HCC.

The effects of various lipoproteins on the growth and the differentiation of cultured normal human keratinocytes were investigated. Primary cultures of human epidermal keratinocytes were obtained from neonatal foreskin, and then added with lipoproteins, very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL). Cell growth potential was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. VLDL and LDL enhanced keratinocytes growth and LDL receptor expression at the plasma membrane level. These effects were more remarkably observed in cells cultured with VLDL than in cells cultured with LDL. Apolipoprotein E (ApoE) was highly increased in VLDL treated cells. These results suggest that VLDL binds with high affinity to cell surface receptors and induces cell proliferation.

Lactoylglutathione lyase (GLO1), a ubiquitously expressed methylglyoxal (MG) detoxification enzyme, is implicated in the progression of various human malignant diseases. However, the role of GLO1 in the development or progression of murine fibrosarcoma is still unclear. We performed proteomic analysis to identify differences in the intracellular proteins of the regressive tumor cell line QR-32 and the inflammatory cell-promoting progressive tumor cell line QRsP-11 of murine fibrosarcoma by 2DE combined with MS. Seven upregulated proteins were identified in QRsP-11 compared to QR-32 cells, namely, GLO1, annexin A1, adenylate kinase isoenzyme 1, transcription factor BTF3, myosin light polypeptide 6, low molecular weight phosphotyrosine protein phosphatase and nucleoside diphosphate kinase B. Heat shock protein beta-1 (HspB1), a methylglyoxal-adducted protein, is concomitantly over-expressed in QRsP-11 as compared to QR-32 cells. We also found out that GLO1 is translocated into the nucleus to a higher extent in QRsP-11 compared to QR-32 cells, which can be reversed by using a MEK inhibitor (U0126). Moreover, U0126 and GLO1 siRNA can inhibit cell proliferation and migration in QRsP-11 cells. Our data suggest that overexpression and nuclear translocation of GLO1 might be associated with tumor progression in murine fibrosarcoma.

To determine intracellular factors influencing the sensitivity of cancer cells to tumor necrosis factor-alpha (TNF-alpha), we studied the expression of intracellular proteins in TNF-alpha-resistant cKDH-8/11 and -sensitive KDH-8/YK rat hepatoma cell lines using the technique of two-dimensional gel electrophoresis (2-DE). From the 2-DE patterns, it was demonstrated that TNF-alpha-resistant cKDH-8/11 cells had increased levels of protein of molecular weight (Mr) 22 500 and isoelectric point (pI) 5.2, compared with TNF-alpha-sensitive KDH-8/YK cells. Therefore, we excised cyanogen bromide (CNBr) fragments of proteins in the spot for N-terminal sequencing. Microsequencing for the CNBr fragments identified the protein as rat phosphatidylethanolamine-binding protein. These findings suggest that the intracellular phosphatidylethanolamine-binding protein could be one of the factors responsible for the resistance of cKDH-8/11 cells to TNF-alpha-induced cell death.