Tomoaki Kahyo

Induction of Endothelial Nitric Oxide Synthase, SIRT1, and Catalase by Statins Inhibits Endothelial Senescence Through the Akt Pathway

Objective—Statins (3-hydroxy-3-methylglutaryl–coenzyme A reductase inhibitors) have pleiotropic vascular protective effects besides cholesterol lowering. Recently, experimental and clinical studies have indicated that senescence of endothelial cells is involved in endothelial dysfunction and atherogenesis. Therefore, the present study was performed to determine whether statins would reduce endothelial senescence and to clarify the molecular mechanisms underlying the antisenescent property of statins. Methods and Results—Senescent human umbilical vein endothelial cells were induced by hydrogen peroxide (H2O2), as judged by senescence-associated &bgr;-galactosidase assay and cell morphological appearance. Atorvastatin, pravastatin, and pitavastatin inhibited the oxidative stress induced-endothelial senescence. These statins phosphorylated Akt at Ser473 and subsequently led to increased expression of endothelial nitric oxide synthase (eNOS), SIRT1, and catalase. Treatment with LY294002 or Akt short interfering RNA decreased the eNOS activation, SIRT1 expression, and antisenescent property of atorvastatin. Moreover, in streptozotocin-diabetic mice, administration of pitavastatin increased eNOS, SIRT1, and catalase expression and decreased endothelial senescence, but levels remained unaltered in Sirt1 knockout mice. Conclusion—Our results indicate that treatment with statins inhibits endothelial senescence and that enhancement of SIRT1 plays a critical role in prevention of endothelial senescence through the Akt pathway, a direct target of statins.

Accumulated phosphatidylcholine (16:0/16:1) in human colorectal cancer; possible involvement of LPCAT4

The identification of cancer biomarkers is critical for target‐linked cancer therapy. The overall level of phosphatidylcholine (PC) is elevated in colorectal cancer (CRC). To investigate which species of PC is overexpressed in colorectal cancer, an imaging mass spectrometry was performed using a panel of non‐neoplastic mucosal and CRC tissues. In the present study, we identified a novel biomarker, PC(16:0/16:1), in CRC using imaging mass spectrometry. Specifically, elevated levels of PC(16:0/16:1) expression were observed in the more advanced stage of CRC. Our data further showed that PC(16:0/16:1) was specifically localized in the cancer region when examined using imaging mass spectrometry. Notably, because the ratio of PC(16:0/16:1) to lyso‐PC(16:0) was higher in CRC, we postulated that lyso‐PC acyltransferase (LPCAT) activity is elevated in CRC. In an in vitro analysis, we showed that LPCAT4 is involved in the deregulation of PC(16:0/16:1) in CRC. In an immunohistochemical analysis, LPCAT4 was shown to be overexpressed in CRC. These data indicate the potential usefulness of PC(16:0/16:1) for the clinical diagnosis of CRC and implicate LPCAT4 in the elevated expression of PC(16:0/16:1) in CRC.

Testosterone Deficiency Accelerates Neuronal and Vascular Aging of SAMP8 Mice: Protective Role of eNOS and SIRT1

Oxidative stress and atherosclerosis-related vascular disorders are risk factors for cognitive decline with aging. In a small clinical study in men, testosterone improved cognitive function; however, it is unknown how testosterone ameliorates the pathogenesis of cognitive decline with aging. Here, we investigated whether the cognitive decline in senescence-accelerated mouse prone 8 (SAMP8), which exhibits cognitive impairment and hypogonadism, could be reversed by testosterone, and the mechanism by which testosterone inhibits cognitive decline. We found that treatment with testosterone ameliorated cognitive function and inhibited senescence of hippocampal vascular endothelial cells of SAMP8. Notably, SAMP8 showed enhancement of oxidative stress in the hippocampus. We observed that an NAD+-dependent deacetylase, SIRT1, played an important role in the protective effect of testosterone against oxidative stress-induced endothelial senescence. Testosterone increased eNOS activity and subsequently induced SIRT1 expression. SIRT1 inhibited endothelial senescence via up-regulation of eNOS. Finally, we showed, using co-culture system, that senescent endothelial cells promoted neuronal senescence through humoral factors. Our results suggest a critical role of testosterone and SIRT1 in the prevention of vascular and neuronal aging.

Detection of kinase amplifications in gastric cancer archives using fluorescence in situ hybridization

To test the feasibility of using bacterial artificial chromosomes (BAC) containing kinases for pathological diagnosis using fluorescence in situ hybridization (FISH), 10 BAC probes containing a gene amplified in 5% or more of a pilot cohort were selected from a previous survey using arbitrarily selected BAC clones harboring 100 kinases. In this report, we describe the prevalence and association with the clinico‐pathological profile of these selected 10 BAC probes in 365 gastric cancer tissues. FISH analyses using these 10 BAC probes containing loci encoding EGFR, ERBB2(HER2), EPHB3, PIK3CA, MET, PTK7, ACK1, STK15, SRC, and HCK showed detectable amplifications in paraffin‐embedded tissue in 2.83% to 13.6% of the gastric cancer tissues. Considerable numbers of the cases showed the co‐amplification of two or more of the probes that were tested. BAC probes located within a genome neighborhood, such as PIK3CA, EPHB3, and ACK1 at 3q26‐29 or HCK, SRC, and STK15 at 20q11‐13.1, were often co‐amplified in the same cases, but non‐random co‐amplifications of genes at distant genomic loci were also observed. These findings provide basic information regarding the creation of a strategy for personalizing gastric cancer therapy, especially when using multiple kinase inhibitors.

A novel tumor-derived SGOL1 variant causes abnormal mitosis and unstable chromatid cohesion

Mitosis is the most conspicuous cell cycle phase, because it is the phase in which the dynamic physical distributions of cellular components into the two daughter cells occur. The separation of sister chromatids is especially important during mitosis, because of the extreme accuracy required for distribution to the next generation of cells. Shugoshin-like 1 (SGOL1) is a key protein in protecting sister chromatids from precocious separation. We have reported finding that chromosome instability is more likely in SGOL1-downregulated colorectal cancers, but it is still unknown whether there is an association between cancer and SGOL1 transcript variation. Here, we identified a novel SGOL1 variant, SGOL1-P1, in human colon cancer. The SGOL1-P1 transcript contains an exon-skip of exon 3 that results in a stop codon occurring within exon 4. Overexpression of SGOL1-P1 in HCT116 cells resulted in an increased number of cells with aberrant chromosome alignment, precociously separated chromatids and delayed mitotic progression, occasionally followed by inaccurate distribution of the chromosomes. These phenotypes, observed when SGOL1-P1 was present, were also observed very frequently in SGOL1-knockdown cells. Furthermore, the overexpression of SGOL1-P1 inhibited the localization of endogenous SGOL1 and cohesin subunit RAD21/SCC1 to the centromere. These results suggest that SGOL1-P1 may function as a negative factor to native SGOL1, and that abundant expression of SGOL1-P1 may be responsible for chromosomal instability.

Clinical significance of PD-L1 and PD-L2 copy number gains in non-small-cell lung cancer

New reliable biomarkers are needed to predict the response to immune checkpoint inhibitors against programmed death-1 (PD-1) and its ligand (PD-L1), because PD-L1 expression on tumor cells has limited power for selecting patients who may benefit from such therapy. Here we investigated the significance of PD-L1 and PD-L2 gene copy number gains using fluorescence in situ hybridization as well as PD-L1 and PD-L2 expression in 654 patients with resected non-small-cell lung cancer. The prevalence of PD-L1 amplification and polysomy was 3.1% and 13.2%, respectively. The PD-L1 gene copy number status was in agreement with both the PD-L2 and Janus kinase 2 gene copy number statuses. PD-L1 and PD-L2 expression was observed in 30.7% and 13.1%, respectively. Both PD-L1 copy number gains and expression were associated with smoking-related tumors. Tumor cells with PD-L1 genomic gains exhibited significantly higher levels of PD-L1 expression than those without, but PD-L2 copy number gains were not related to PD-L2 augmentation. PD-L1 gene amplification and polysomy were independently associated with PD-L1 expression, with high immune infiltrates and EGFR expression in a multivariate logistic regression model. Comparative analysis between primary tumors and synchronous regional lymph node metastases revealed that the PD-L1 gene copy number alterations were highly consistent and reproducible compared with the PD-L1 expression. Both PD-L1 amplification and level of protein expression were predictors of poor survival using Cox univariate analyses. Therefore, we conclude that an increase in PD-L1 gene copy number can be a feasible alternative biomarker for predicting response to anti-PD-1/PD-L1 therapy.

Prognostic impact of CD73 and A2A adenosine receptor expression in non-small-cell lung cancer.

In immune cells, CD73 dephosphorylates and converts extracellular AMP into adenosine, which binds the A2A adenosine receptor (A2AR). Blockade of this interaction, which induces an immunosuppressed niche in the tumor microenvironment, represents a potential novel treatment strategy. The clinical significance of CD73 and A2AR expression in non-small-cell lung cancer (NSCLC), however, has yet to be thoroughly investigated. Here we evaluated CD73 and A2AR protein expression levels using immunohistochemistry in tissue microarrays containing 642 resected NSCLC specimens. Furthermore, we compared the expression profiles of 133 paired primary tumors and lymph node metastases. CD73 and A2AR expression levels were significantly higher in females than in males, in never smokers than in ever smokers, and in adenocarcinomas than in squamous cell carcinomas. Among adenocarcinomas, significantly higher CD73 and A2AR expression was observed in TTF-1-positive and mutant EGFR-positive tumors than in their counterparts. Compared with CD73, A2AR expression was more inconsistent between primary tumors and lymph node metastases. Among NSCLC patients, high CD73 expression was an independent indicator of poor prognosis in multivariate Cox regression analyses for overall survival [hazard ratio (HR), 2.18; 95% confidence interval (CI), 1.38–3.46] and recurrence-free survival (HR, 2.05; 95% CI, 1.42–2.95). In contrast, high A2AR expression was an independent predictor of favorable prognosis for overall survival (HR, 0.70; 95% CI, 0.50–0.98) and recurrence-free survival (HR, 0.74; 95% CI, 0.56–0.97). Together, these findings indicate that CD73 and A2AR have opposing prognostic effects, although cases involving CD73 or A2AR expression share some clinicopathological features.

Robust quantitative assessments of cytosine modifications and changes in the expressions of related enzymes in gastric cancer

BackgroundThe rediscovery of 5-hydroxymethylcytosine, the ten-eleven translocation (TET) family, thymine-DNA glycosylase (TDG) and isocitrate dehydrogenase (IDH) have opened new avenues in the study of DNA demethylation pathways in gastric cancer (GC). We performed a comprehensive and robust analysis of these genes and modified cytosines in gastric cancer.MethodsLiquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS) was used to assess 5-methyldeoxycytidine (5-mC), 5-hydroxymethyldeoxycytidine (5-hmC), 5-formyldeoxycytidine (5-fC) and 5-carboxyldeoxycytidine (5-caC) quantitatively in tumorous and non-tumorous regions of GCs; [D2]-5-hmC was used as an internal standard. Expression levels of the genes TET1, TET2, TET3, TDG, IDH1 and IDH2 were measured using a real-time reverse transcription polymerase chain reaction (RT-PCR) and were compared to the clinical attributes of each case. Using HEK293T cells the effects of introducing plasmids containing full-length TET1,TET2, and TET3 and 7 variants of the TET2 catalytic domain were evaluated in terms of their effect on cytosine demethylation.ResultsLC-MS/MS showed that 5-hmC was significantly decreased in tumorous portions. 5-mC was also moderately decreased in tumors, while 5-fC and 5-caC were barely detectable. The expressions of TET1, TET2, TET3, TDG and IDH2, but not IDH1, were notably decreased in GCs, compared with the adjacent non-tumor portion. TET1 expression and the 5-hmC levels determined using LC-MS/MS had a significantly positive correlation and TET1 protein had a greater effect on the increase in 5-hmC than TET2 and TET3 in HEK293T cells.ConclusionsThe loss of 5-hmC and the down-regulation of TET1-3, TDG and IDH2 were found in GCs. The loss of 5-hmC in GCs was mainly correlated with the down-regulation of TET1.

SGOL1 variant B induces abnormal mitosis and resistance to taxane in non-small cell lung cancers.

Mitosis is the most conspicuous cell cycle phase and Shugoshin-like 1 (SGOL1) is a key protein in protecting sister chromatids from precocious separation during mitosis. We studied the role of SGOL1 and its splice variants in non-small cell lung cancer (NSCLC) using 82 frozen NSCLC tissue samples. SGOL1-B expression was prevalent in smokers, in cases with a wild-type (WT) EGFR status, and in cases with the focal copy number amplification of genes that are known to be important for defining the biological behaviors of NSCLC. The overexpression of SGOL1-B1 in an NSCLC cell line induced aberrant chromosome missegregation, precociously separated chromatids, and delayed mitotic progression. A higher level of SGOL1-B mRNA was related to taxane resistance, while the forced downregulation of SGOL1-B increased the sensitivity to taxane. These results suggest that the expression of SGOL1-B causes abnormal mitosis and taxane resistance in NSCLC cells.

A novel organ culture model of aorta for vascular calcification.

Vascular calcification is a characteristic feature of aging, atherosclerosis, diabetes mellitus, and end-stage renal disease. The use of organ culture provides complementary information that may bridge the gap between traditional cell culture and animal models, and establishes easily controlled experimental conditions. Therefore, we investigated whether organ culture of the aorta could be used as a model of vascular calcification, applying it to animal models of other conditions. Thoracic aortas were dissected from C57BL/6 mice and cultured. To induce vascular calcification, stimulation with inorganic phosphate (Pi) was performed. Morphometric assessment of medial calcium deposition was quantitatively performed, and the amount of dissolved calcium was measured. Pi-stimulation induced calcium deposition in medial layers in a time- and dose-dependent manner. To investigate the phenotypic change of vascular smooth muscle cells (VSMC), the expression of Runx2, osterix, osteocalcin, and ALP activity were determined. Finally, to investigate the influence of Pi-stimulation on the cultured aorta in other models, aortas from streptozotocin (STZ)-induced diabetic mice, aged mice, and Sirt1 knockout (+/-) mice were dissected. These cultures showed a greater tendency for aortic calcification by Pi-stimulation than did control cultures. These results indicate that organ culture of the aorta from mice reflects the state of calcification and suggests that this model will be useful to explore the molecular mechanisms of vascular calcification and the pathology of senescence.