Diagnostic Immunocytostaining with Peroxisome Proliferator-Activated Receptor-Gamma in Urine Cytology Samples.

INTRODUCTION: Urine cytology is a common method for detection of urothelial carcinoma (UC), however, is not high sensitivity. Improvement of the accuracy of cytodiagnosis using immunocytostaining as an auxiliary method is needed. This study aimed to determine the cytodiagnostic usefulness of peroxisome proliferator-activated receptor-gamma (PPAR-γ) immunocytostaining in urine cytology for the detection of UCs, particularly low-grade urothelial carcinomas (LGUC). METHODS: PPAR-γ immunocytostaining was performed for 37 UC cases and 26 benign cases. Among the UC cases, 22 cases were of the papillary proliferation type, not including the mixed type comprising both papillary and flat growth. Fifteen LGUC cases of all papillary proliferation types were included. For comparison, the same samples were also immunocytostained for p53 and Ki-67. RESULTS: Of the UC cases, 25 of 37 were positive for PPAR-γ, while 24 of the 26 benign cases were PPAR-γ-negative. Regardless of histological grading, 13 of the 22 UC cases with papillary proliferation were PPAR-γ-positive. In particular, PPAR-γ immunocytostaining showed higher sensitivity for LGUC cases than that of the other biomarkers. Regarding LGUC specifically, 4 of 10 cases not identified by primary cytology were detected by PPAR-γ immunocytostaining. CONCLUSION: PPAR-γ immunocytostaining enhances the accuracy of urine cytodiagnosis. Furthermore, PPAR-γ is a more useful immunobiomarker in urine cytology than p53 and Ki-67, the commonly used immunobiomarkers for malignant cell detection.

Peroxisome proliferator-activated receptor-α expression is associated with histological type in human gastric carcinoma.

Gastric carcinoma is one of the most common types of cancer worldwide and a leading cause of cancer-related mortality. Gastric carcinoma is histologically subdivided into differentiated and undifferentiated carcinoma, with the latter including poorly differentiated carcinoma and signet ring cell carcinoma (SRCC). Poorly differentiated carcinoma and SRCC have a worse prognosis compared with differentiated carcinoma. Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors and the PPAR-α subtype regulates important cellular functions, including cell proliferation, energy metabolism, oxidative stress, immune responses and cell differentiation. The aim of the present study was to elucidate the associations between clinicopathological factors and PPAR-α expression in patients with gastric carcinoma. The immunohistochemical staining of specimens obtained from 57 patients showed that PPAR-α expression was slightly weaker in undifferentiated carcinoma than in differentiated carcinoma (P<0.01). PPAR-α expression also significantly differed between poorly differentiated carcinoma (both positive and negative: 14/20, 70%) and SRCC (not expressed: 0/7, 0%) (P<0.01). However, PPAR-α expression was not significantly affected by age, lymph node invasion, venous invasion, lymph node metastasis, depth of invasion or stage. Collectively, the present results demonstrated that the downregulated expression of PPAR-α may play a key role in the biological transformation of tumors. Therefore, PPAR-α appears to be an important protein related to histology and may hold promise as a prognostic marker. Further studies with a larger number of subjects are needed to elucidate the relationship between PPAR-α expression and tumor progression and to analyze long-term clinical survival.

Overexpression of the PPAR-γ protein in primary Ta/T1 non-muscle-invasive urothelial carcinoma.

Peroxisome proliferator-activated receptor-γ (PPAR-γ) is a well-known nuclear receptor that is activated in the nucleus to regulate several transcription factors. Its expression patterns have been examined in various types of cancer. The present study investigated the expression patterns of PPAR-γ in non-muscle-invasive urothelial carcinoma. The expression rates of PPAR-γ, p53 and Ki-67 were compared to determine whether PPAR-γ may be considered as an immunobiomarker for bladder cancer. The intensity and extent of PPAR-γ expression were evaluated in 79 cases of non-muscle-invasive urothelial carcinoma (30 cases of papillary carcinoma low-grade, 30 cases of high-grade and 19 cases of carcinoma in situ) and 30 non-malignant cases. The nuclear overexpression of PPAR-γ was frequently observed in non-muscle-invasive urothelial carcinoma (63/79 cases) but was rarely detected in non-malignant cases (2/30 cases). The histological proliferation types of non-muscle-invasive urothelial carcinoma revealed that PPAR-γ was more frequently overexpressed in papillary carcinoma (54/60 cases) than in carcinoma in situ (9/19 cases). Immunohistochemical staining demonstrated that PPAR-γ was more useful as an immunobiomarker than p53 or Ki-67 (diagnostic odds ratios; 55.13, 16.82 and 11.13, respectively). In summary, this study demonstrated that the expression patterns of PPAR-γ were associated with histological proliferation type and that PPAR-γ was expressed in the nuclei of papillary carcinoma cells. These findings suggested that immunohistochemical staining for PPAR-γ may be used to comprehensively detect non-muscle-invasive urothelial carcinoma.

Activation and Expression of Peroxisome Proliferator-Activated Receptor Alpha Are Associated with Tumorigenesis in Colorectal Carcinoma.

Peroxisome proliferator-activated receptor alpha (PPAR-α) belongs to the PPAR family and plays a critical role in inhibiting cell proliferation and tumorigenesis in various tumors. However, the role of PPAR-α in colorectal tumorigenesis is unclear. In the present study, we found that fenofibrate, a PPAR-α agonist, significantly inhibited cell proliferation and induced apoptosis in colorectal carcinoma cells. In addition, PPAR-α was expressed in the nucleus of colorectal carcinoma cells, and the expression of nuclear PPAR-α increased in colorectal carcinoma tissue compared with that of normal epithelium tissue (P<0.01). The correlation between the expression of nuclear PPAR-α and clinicopathological factors was evaluated in human colorectal carcinoma tissues, and the nuclear expression of PPAR-α was significantly higher in well-to-moderately differentiated adenocarcinoma than in mucinous adenocarcinoma (P<0.05). These findings indicate that activation of PPAR-α may be involved in anticancer effects in colorectal carcinomas, and nuclear expression of PPAR-α may be a therapeutic target for colorectal adenocarcinoma treatment.

Qualitative and quantitative cytomorphological features of primary anaplastic lymphoma kinase-positive lung cancer.

OBJECTIVE: Anaplastic lymphoma kinase (ALK) positive (+) lung cancers are predictive for response to crizotinib and alectinib. There are many cases of lung cancer in which surgery cannot be performed, and such cases require diagnosis by cytological specimen or biopsy. Estimating ALK (+) lung cancer from cytomorphology would allow molecular testing to proceed without the waste of a small amount of specimen. The purpose of this study was to assess whether qualitative and quantitative cytomorphological features are sufficient for distinguishing primary ALK (+) from ALK (-) lung cancer. METHODS: We examined eight qualitative cytomorphological parameters and three quantitative nuclear morphometric parameters in 17 cases of primary ALK (+) lung cancer, diagnosed by fluorescence in situ hybridisation (FISH) using histological specimens, and in 41 cases of ALK (-) lung cancer. Quantitative nuclear morphometric parameters were analysed by a computer-assisted image analysis system. RESULTS: In ALK (+) lung cancer, three qualitative parameters (signet ring cells, nuclear grooves and single type nucleoli) and two quantitative parameters (large nuclear area and irregular nuclear shape) were observed in significantly higher proportions. However, in ALK (-) lung cancer, one qualitative parameter (unclear and multiple type nucleoli) was seen significantly more often. CONCLUSIONS: These results show that the cytomorphological features of signet ring cells, nuclear grooves and nucleoli shape can help to triage a small amount of cytological and biopsy specimens for appropriate molecular testing of primary ALK (+) lung cancer.

Nuclear expression of claudin-3 in human colorectal adenocarcinoma cell lines and tissues.

Claudins are members of a large family of transmembrane proteins, which are essential for the formation of tight junctions and have a significant effect on the biological behavior of tumor progression. Previous studies have demonstrated that several claudins show aberrant expression patterns in numerous types of cancer. The present study investigated the expression and localization of claudin-3 and claudin-7 in human colorectal adenocarcinoma cell lines and tissues. The protein expression levels of claudin-3 and claudin-7 were determined using immunocytochemical and immunohistochemical staining. Claudin-3, but not claudin-7, exhibited nuclear localization in the human colorectal adenocarcinoma Caco-2 and SW620 cell lines. Surgically resected colorectal adenocarcinoma tissue specimens were obtained, and the associations between the expression of claudin-3 or claudin-7 and various clinicopathological parameters were analyzed. The membranous expression rates of claudin-3 and claudin-7 were 58.0 and 50.0%, while their nuclear expression rates were 22.0 and 2.0%, respectively. The membranous expression of claudin-3 and claudin-7 was not associated with any clinicopathological factors, whereas the nuclear expression of claudin-3 was associated with histological type and was significantly increased in colorectal mucinous adenocarcinomas compared with that in well- to moderately-differentiated colorectal adenocarcinomas (P<0.01). However, no associations were observed between the nuclear expression of claudin-7 and any clinicopathological parameter. In conclusion, the nuclear expression of claudin-3 in colorectal mucinous adenocarcinoma may be involved in the biological transformation of tumors. The results from the present study indicated that claudin-3 is an important protein associated with histological type and has potential as a prognostic marker. Although the mechanisms underlying the nuclear localization of claudin-3 in tumorigenesis have not yet been elucidated in detail, the present results indicated the potential of claudin-3 as a histopathological biomarker for colorectal adenocarcinomas.

ARHGEF10 directs the localization of Rab8 to Rab6-positive executive vesicles.

The function of ARHGEF10, a known guanine nucleotide exchange factor (GEF) for RhoA with proposed roles in various diseases, is poorly understood. To understand the precise function of this protein, we raised a monoclonal antibody against ARHGEF10 and determined its localization in HeLa cells. ARHGEF10 was found to localize to vesicles containing Rab6 (of which there are three isoforms, Rab6a, Rab6b and Rab6c), Rab8 (of which there are two isoforms, Rab8a and Rab8b), and/or the secretion marker neuropeptide Y (NPY)-Venus in a Rab6-dependent manner. These vesicles were known to originate from the Golgi and contain secreted or membrane proteins. Ectopic expression of an N-terminal-truncated ARHGEF10 mutant led to the generation of large vesicle-like structures containing both Rab6 and Rab8. Additionally, small interfering (si)RNA-mediated knockdown of ARHGEF10 impaired the localization of Rab8 to these exocytotic vesicles. Furthermore, the invasiveness of MDA-MB231 cells was markedly decreased by knockdown of ARHGEF10, as well as of Rab8. From these results, we propose that ARHGEF10 acts in exocytosis and tumor invasion in a Rab8-dependent manner.

Claudin-1, but not claudin-4, exhibits differential expression patterns between well- to moderately-differentiated and poorly-differentiated gastric adenocarcinoma.

Claudins are members of a large family of transmembrane proteins, which are essential in the formation of tight junctions and have previously been associated with the process of tumor progression. Studies have reported the aberrant expression of claudin-1 and claudin-4 in numerous types of cancer. The present study aimed to investigate the expression of claudin-1 and claudin-4 in gastric adenocarcinoma tissue. Surgically resected gastric adenocarcinoma tissue specimens were obtained from 94 patients. Protein expression levels of claudin-1 and claudin-4 were determined using immunohistochemical staining; the association between claudin-1 or claudin-4 expression and various clinicopathological parameters were then analyzed. In gastric adenocarcinoma specimens, the expression rates of claudin-1 and claudin-4 were 43.6 and 87.2%, respectively. Claudin-1 expression demonstrated a significant correlation with histological type (P<0.01) and was significantly higher in well- to moderately-differentiated gastric adenocarcinomas compared with poorly-differentiated tumors. However, no correlation was observed between claudin-4 expression in adenocarcinoma and clinicopathological parameters. In conclusion, downregulation of claudin-1 expression in poorly-differentiated gastric adenocarcinoma may be involved in the biological transformation of tumors. The present findings suggested that claudin-1 may be an important protein associated with histological type and therefore may have potential for use as a prognostic marker for gastric adenocarcinoma. Further studies are required to elucidate the precise mechanism of claudin expression and its involvement in tumor progression.

A New Enzyme Immunoassay for the Quantitative Determination of Classical Autotaxins (ATXα, ATXß, and ATXγ) and Novel Autotaxins (ATXδ and ATXε).

BACKGROUND: Autotaxin (ATX) is a secreted enzyme that converts lysophosphatidylcholine to lysophosphatidic acid, a potent bioactive lipid mediator, through its lysophospholipase D activity. Although five alternative splicing isoforms of ATX have been identified as ATXα, ATXß, ATXγ, ATXδ, and ATXε and the expression patterns of each isoform differ among several tissues, the clinical significance of each isoform remains to be elucidated. METHODS: Anti-ATXß and anti-ATXδ monoclonal antibodies were produced by immunization with recombinant human ATXß and ATXδ expressed using a baculovirus system, respectively. We then developed enzyme immunoassays to measure the serum concentrations of "classical ATX" (ATXα, ATXß, and ATXγ) and "novel ATX" (ATXδ and ATXε) antigens and evaluated the usefulness of these assays using human serum samples. RESULTS: The with-run and between-run precision, interference, detection limit, and linearity studies for the present assay were well validated. In healthy subjects, the serum concentrations of classical ATX and novel ATX were significantly (P < 0.01) higher in women than in men, while the ratios of classical ATX or novel ATX to total ATX were not different between women and men. The concentrations of both classical ATX and novel ATX in normal pregnant subjects and patients with chronic liver diseases or follicular lymphoma were significantly higher than those in healthy subjects, while the ratio of both ATX isoforms to total ATX did not vary among these groups. CONCLUSIONS: We have developed a new enzyme immunoassay to determine the concentrations of classical ATX and novel ATX in human serum. These assays may be helpful for elucidating the distinct functional roles of each ATX isoform, which are largely unknown at present.

Increased serum autotaxin levels in hepatocellular carcinoma patients were caused by background liver fibrosis but not by carcinoma.

BACKGROUND: Controversy exists as to whether autotaxin (ATX) may be importantly associated with pathophysiology of hepatocellular carcinoma (HCC). METHODS: We evaluated serum ATX levels and its mRNA expression in consecutive 148 HCC patients treated with radiofrequency ablation (RFA) and 30 patients with hepatic resection. RESULTS: Although increased serum ATX levels were observed in almost all the patients treated with RFA, they were not reduced after RFA. Furthermore, serum ATX levels were associated not with tumor burden but with the parameters predicting for liver fibrosis, such as liver stiffness values. Then, in surgically-treated patients, there was no significant correlation between serum ATX levels and ATX mRNA expression levels in HCC tissues. Notably, ATX mRNA expression levels in HCC tissues were not higher than those in peri-tumorous tissues. Finally, serum ATX levels in surgically-treated HCC patients were rather correlated with ATX mRNA expression levels in peri-tumorous tissues as well as with liver fibrosis stage. CONCLUSION: The increase in serum ATX levels in HCC patients may not be caused by abundant ATX production in HCC tissues but by fibrosis in the background livers.

Necessity of lysophosphatidic acid receptor 1 for development of arthritis.

OBJECTIVE: Lysophosphatidic acid (LPA) is a bioactive lipid that binds to a group of cell surface G protein-coupled receptors (LPA receptors 1-6 [LPA1-6 ]) and has been implicated as an important mediator of angiogenesis, inflammation, and cancer growth. This study was undertaken to analyze the effects of LPA1 on the development of arthritis. METHODS: Expression of LPA receptors on synovial tissue was analyzed by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction. The effects of abrogation of LPA1 on collagen-induced arthritis (CIA) were evaluated using LPA1 -deficient mice or LPA1 antagonist. Migrating fluorescence-labeled CD11b+ splenocytes, which were transferred into the synovium of mice with CIA, were counted. CD4+ naive T cells were incubated under Th1-, Th2-, or Th17-polarizing conditions, and T helper cell differentiation was assessed. Osteoclast formation from bone marrow cells was examined. RESULTS: LPA1 was highly expressed in the synovium of patients with rheumatoid arthritis (RA) compared with that of patients with osteoarthritis. LPA1 -deficient mice did not develop arthritis following immunization with type II collagen (CII). LPA1 antagonist also ameliorated murine CIA. Abrogation of LPA1 was associated with reductions in cell infiltration, bone destruction in the joints, and interleukin-17 production from CII-stimulated splenocytes. Infiltration of transferred CD11b+ macrophages from LPA1 -deficient mice into the synovium was suppressed compared with infiltration of macrophages from wild-type mice. LPA1 antagonist inhibited the infiltration of macrophages from wild-type mice. Differentiation into Th17, but not Th1 or Th2, and osteoclast formation were also suppressed under conditions of LPA1 deficiency or LPA1 inhibition in vitro. CONCLUSION: Collectively, these results indicate that LPA/LPA1 signaling contributes to the development of arthritis via cellular infiltration, Th17 differentiation, and osteoclastogenesis. Thus, LPA1 may be a promising target molecule for RA therapy.