Transcriptome analysis of an AKT inhibitor-resistant endometrial cancer cell line.

BACKGROUND: Drug resistance in endometrial cancer (EC) is a serious problem and a barrier to improving prognosis. The PI3K/AKT/mTOR pathway is highly activated in EC and can serve as a potential therapeutic target. Inhibitors against AKT have been developed, but resistance to these inhibitors is a concern. This study aimed to establish AKT inhibitor resistant cell lines and identify differentially expressed genes (DEGs) between parental and AKT inhibitor resistant cell lines to understand the mechanism of drug resistance to AKT inhibitors in EC. METHODS: The sensitivity of eight EC cell lines to AKT inhibitor was analyzed. One of them was used to establish a drug-resistant cell line. DEGs were examined using RNA sequencing (RNA-seq). Furthermore, DEGs were comprehensively analyzed to identify hub genes. Hub genes were evaluated using quantitative real-time polymerase chain reaction. RESULTS: RNA-seq identified 617 DEGs. Hub genes were selected using bioinformatics analysis. The top 10 hub genes were TNF, CDH1, CCND1, COL1A1, CDH2, ICAM1, CAV1, THBS1, NCAM1, and CDKN2A. Relative mRNA expression was significantly upregulated for TNF, CDH1, CCND1, THBS1, p16INK4a, and p14ARF and significantly downregulated for CDH2, ICAM1, and NCAM1 in borussertib-resistant EC cell line. CONCLUSIONS: Drug resistance to AKT inhibitors may depend on genes related to cell adhesion-mediated resistance and transforming growth factor ß signaling.

Analysis of Autoantibodies Related to Tumor Progression in Sera from Patients with High-grade Non-muscle-invasive Bladder Cancer.

BACKGROUND/AIM: Bladder cancer (BC) has a high recurrence rate and may progress to being a muscle-invasive lesion, that is potentially associated with a poor prognosis. We identified tumor-associated proteins that were recognized by autoantibodies in sera from patients with high-grade non-muscle-invasive bladder cancer (HG-NMIBC) by proteomic analysis. MATERIALS AND METHODS: The serum levels of these autoantibodies against identified proteins were validated by dot blot analysis with sera from 95 patients with BC and 35 healthy controls. The expression of identified proteins was immunohistochemically analyzed in 115 BC tissues. RESULTS: Autoantibody against protein phosphatase 1, catalytic subunit, alpha isoform (PPP1CA) protein was detected in pretreated sera from patients with HG-NMIBC who showed progression. The serum IgG level of anti-PPP1CA autoantibody was significantly correlated with pathological stage, grade, lymphovascular invasion, and prognosis. The immunoreactions for PPP1CA protein in BC was significantly correlated with pathological stage, grade, and lymphovascular invasion. CONCLUSION: PPP1CA is a candidate sero-diagnostic and prognostic marker for patients with BC.

Clinical value of dividing false positive urine cytology findings into three categories: atypical, indeterminate, and suspicious of malignancy.

BACKGROUND: The aim of this study was to evaluate 10 years of false positive urine cytology records, along with follow-up histologic and cytologic data, to determine the significance of suspicious urine cytology findings. MATERIALS AND METHODS: We retrospectively reviewed records of urine samples harvested between January 2002 and December 2012 from voided and catheterized urine from the bladder. Among the 21,283 urine samples obtained during this period, we located 1,090 eligible false positive findings for patients being evaluated for the purpose of confirming urothelial carcinoma (UC). These findings were divided into three categories: atypical, indeterminate, and suspicious of malignancy. RESULTS: Of the 1,090 samples classified as false positive, 444 (40.7%) were categorized as atypical, 367 (33.7%) as indeterminate, and 279 (25.6%) as suspicious of malignancy. Patients with concomitant UC accounted for 105 (23.6%) of the atypical samples, 147 (40.1%) of the indeterminate samples, and 139 (49.8%) of the suspicious of malignancy samples (p<0.0001). The rate of subsequent diagnosis of UC during a 1-year follow-up period after harvesting of a sample with false positive urine cytology initially diagnosed as benign was significantly higher in the suspicious of malignancy category than in the other categories (p<0.001). The total numbers of UCs were 150 (33.8%) for atypical samples, 213 (58.0%) for indeterminate samples, and 199 (71.3%) for samples categorized as suspicious of malignancy. CONCLUSIONS: Urine cytology remains the most specific adjunctive method for the surveillance of UC. We demonstrated the clinical value of dividing false positive urine cytology findings into three categories, and our results may help clinicians better manage patients with suspicious findings.

Detection of tumor-associated antigens in culture supernatants using autoantibodies in sera from patients with bladder cancer.

Secreted proteins play essential roles in the process of tumorigenesis, and the analysis of tumor-secreted proteins has been suggested as a promising strategy for identifying cancer biomarkers. In this study, we performed proteomic analysis to identify proteins secreted from bladder cancer cell lines that are recognized by autoantibodies in sera from patients with bladder cancer. In addition,autoantibodies against the identified proteins were validated using a dot-blot array with sera from patients with bladder cancer and normal controls. As the results, we detected twenty-five and thirty-two immunoreactive spots in sera from patients with high- and low-grade bladder cancer, respectively.In addition, validation analysis revealed that serum IgG levels of anti-calreticulin and matrix metalloproteinase-2 (MMP2) autoantibodies were significantly higher in bladder cancer patients than in normal controls (both P < 0.05). Furthermore, the serum IgG level of anti-MMP2 autoantibody was significantly higher in patients with high- compared to low-grade bladder cancer(P < 0.05). On multivariate analysis, the serum IgG level of anti-MMP2 autoantibody was an independent predictor of cancer-specific survival (P < 0.05). Based on these findings, serum IgG levels of anti-calreticulin and MMP2 autoantibodies may be novel biomarker candidates for bladder cancer and its clinical outcome.

Evaluation of luminal and vessel wall abnormalities in subacute and other stages of intracranial vertebrobasilar artery dissections using the volume isotropic turbo-spin-echo acquisition (VISTA) sequence: a preliminary study.

OBJECTIVE: To evaluate the utility of 3D variable refocusing flip-angle volume isotropic turbo-spin-echo acquisition (VISTA) imaging, using a 1.5-T MRI unit, which can minimize flow artifacts, due to its sequence-endogenous flow-void capability, in the diagnosis of intracranial vertebrobasilar artery dissection (VAD). MATERIAL AND METHODS: The presence of intimal flaps, intramural hematomas, vessel dilatations and abnormal vessel enhancements were evaluated on T1-weighted VISTA images from 18 VAD patients with 20 dissected arteries (15 subacute and five at other stages). Additional gadolinium-enhanced T1VISTA images were available for 13 patients. The frequency of flow artifacts on T1VISTA imaging in 70 non-dissected arteries in VAD patients and 12 control subjects was also evaluated. Furthermore, in 13 and eight patients, contrast-enhanced three-dimensional (CE3D) imaging with spoiled gradient-recalled (SPGR) acquisition in steady state and electrocardiographically gated black-blood (BB) T1-weighted imaging (T1WI) were evaluated to compare visualization of false lumens. RESULTS: Intimal flaps, intramural hematomas and dilatations were identified on T1VISTA images in 65% (13/20), 55% (11/20) and 90% (18/20) of VADs, respectively. Abnormal vessel enhancement was recognized in 100% (15/15) of VADs on contrast-enhanced T1VISTA images. Only four normal arteries showed small, thin, linear artifacts. Compared with CE3D-SPGR imaging, T1VISTA imaging depicted false lumens more conspicuously in seven VADs (P=0.02). T1VISTA also revealed intimal flaps and hematomas as did BB T1WI. CONCLUSION: T1VISTA imaging may be useful for diagnosing VAD at subacute stages, as it can reveal vessel wall and lumen abnormalities with a minimum of flow artifacts.

Comparative image analysis of conventional and thin-layer preparations in endometrial cytology.

We evaluated the differences in cytologic findings between conventional and thin-layer preparations in endometrial cytology to introduce the thin-layer method into routine cytology. Eighty patients who had undergone endometrial cytology and biopsy on the same day were selected and we compared the cytological findings between conventional- and thin-layer preparations (TLP) in endometrial cytology. The numbers of neutrophils and cell clusters in the thin-layer method were lower than those in the conventional smear (CSS) method. The average number of neutrophils in endometrioid adenocarcinoma was significantly higher than that in normal morphology endometrium and endometrial hyperplasia. Regarding the shape of the cell clusters, ball-like patterns and round-edged cell clusters were not identified in CSS. The average number of clusters in CSS was significantly greater than that using the TLP. The average of the nuclear area in CSS was significantly larger than that using the TLP, indicating that the nuclear areas in CSS were more uneven than that using the TLP. In the future, it is expected that liquid-based cytology will be applied to the cytological diagnosis of a variety of lesions. The influence on cells due to fixation is considerable in liquid-based preparations. Therefore, if we strive to pick up the differences between CSS and TLP of endometrial samples, the diagnostic accuracy of the latter could be improved.

Ubiquitin-related proteins in neuronal and glial intranuclear inclusions in intranuclear inclusion body disease.

Recent studies have shown that eosinophilic intranuclear inclusions (INI) in the brain of patients with intranuclear inclusion body disease (INIBD) are immunopositive for ubiquitin and ubiquitin-related proteins (URP). However, the extent and frequency of URP-immunoreactive inclusions in INIBD are uncertain. We immunohistochemically examined the brain, spinal cord and dorsal root ganglia from five patients with INIBD, using a virtual slide system with sequential staining of the same sections with hematoxylin and eosin and by immunolabeling with antibodies against ubiquitin and URP (NEDD8, NUB1, SUMO-1 and SUMO-2). Intranuclear inclusions were widely distributed in neurons and glial cells in all the cases. Sequential staining revealed that 100% of INI in neurons and glial cells were positive for ubiquitin. Moreover, the majority or a significant proportion of INI were positive for NEDD8, NUB1, SUMO-1 and SUMO-2. However, the proportions of NEDD8-, NUB1- and SUMO-1-positive inclusions were significantly higher in neurons than in glial cells (P < 0.05). These findings suggest that proteins related to ubiquitination and proteasomal degradation are involved in the formation of INI in INIBD.

Anti-HuC and -HuD autoantibodies are differential sero-diagnostic markers for small cell carcinoma from large cell neuroendocrine carcinoma of the lung.

Aiming to identify novel sero-diagnostic markers for neuroendocrine carcinomas of the lung, the two-dimensional gel electrophoresis-immunoblot method was used to analyze tumor-associated autoantibodies in patients with small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC). Several autoantigens were revealed and anti-HuC autoantibody was detected only in sera of SCLC patients. Since Hu family proteins including HuC are well-known causes of paraneoplastic encephalomyelitis/sensory neuronopathy (PEM/SN), the expression of HuC as well as HuD mRNAs and their proteins was studied in 11 lung cancer cell lines. The expression of HuC and HuD mRNAs and proteins was only detected in SCLC- and LCNEC-derived cells. To validate the existence of anti-HuC and -HuD auto-antibodies, we studied a large number of sera including those from lung cancer patients employing dot blot analysis. Anti-HuC and -HuD autoantibodies were detected only in SCLC cases with or without PEM/SN, and not in the sera of LCNEC patients. The mechanism leading to different anti-HuC and -HuD autoantibody production between SCLC and LCNEC is unclear; however, the results from the present and previous studies suggest that anti-HuC and -HuD auto-antibodies are novel differential sero-diagnostic markers for SCLC from LCNEC.

Changes of fractional anisotropy and apparent diffusion coefficient in patients with idiopathic normal pressure hydrocephalus.

Since ventricular dilation and periventricular abnormal intensities are commonly seen in patients with idiopathic normal pressure hydrocephalus (INPH) on magnetic resonance imaging (MRI), dysfunction of white matter may have an important role in the mechanism causing symptoms of INPH. To clarify the pathophysiology of INPH, we analyzed axonal water dynamics using diffusion tensor MRI. Thirty-six patients with possible INPH were included. Regional fractional anisotropy (FA) and apparent diffusion coefficient (ADC) were measured in several white matter regions before and 24 h after a cerebrospinal fluid tap test (CSF-TT). The patients were divided into two groups: patients who showed significant improvements in neurological status after the CSF-TT (positive, n = ;17) and those with no neurological improvement (negative, n = 19). After CSF-TT, ADC values were significantly decreased in the frontal periventricular region and the body of the corpus callosum in the positive group (p < 0.05), whereas no significant change was shown in the negative group. FA values were significantly increased in the body of the corpus callosum in both groups after CSF-TT (p < 0.05). After CSF-TT, water molecules at the extracellular space could move to the intraventricular space, resulting in decreased ADC values. This suggests that changes of water dynamics in white matter may have a role in the mechanism causing symptoms of INPH.

Cytological significance of abnormal squamous cells in urinary cytology.

The aim of this study was to evaluate the significance of abnormal squamous cells (ASCs) in urinary cytology to clarify whether finding of ASCs could improve diagnostic accuracy. A total of 3,812 urine specimens were reviewed. We focused on three parameters of ASCs, necrotic debris, and ASC clusters, and linked them to histological diagnosis and clinical information. ASCs were identified in 34 (0.9%) specimens from 21 different patients. The incidence of ASCs was higher in females than in males. The 34 urine specimens were categorized as voided urine (16 cases), bladder-catheterized urine (17 cases), and bladder-washed fluid (1 case). Six (28.6%) of 21 patients were histologically diagnosed as having combined urothelial carcinoma and squamous cell carcinoma (SCC). Eight patients (38.1%) were histologically diagnosed as having SCC originating from sites other than the urinary tract; those urine specimens showed ASCs that were likely to have been exfoliated from malignant lesions. Necrotic debris and ASC clusters were identified in 12 specimens (35.3%) from 11 patients and 4 specimens (11.8%) from 4 patients, respectively, from a total of 34 specimens. Our results indicate that a great amount of care is needed for cytological diagnosis when attempting to recognize ASCs in urine specimens because ASCs were identified in not only SCC of the bladder but also in carcinoma or nonmalignant lesions of nonurinary tracts. Necrotic debris was found not only in patients who had malignant bladder tumors but also in those who had malignant lesions in locations other than the bladder.

Effects of N-[N-(3, 5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) on cell proliferation and apoptosis in Ishikawa endometrial cancer cells.

Endometrial cancer is one of the most common gynecological malignancies in Japan, where the disease shows an increasing morbidity. However, surgical therapy remains the treatment of choice for endometrial cancers that tend to be insensitive to radiation therapy and chemotherapy. Therefore, novel therapeutic strategies are required. The Notch signaling pathway regulates embryogenesis and cellular development, but deregulated Notch signaling may contribute to tumorigenesis in several cancers. Moreover, γ-secretase inhibitors have been shown to be potent inhibitors of the Notch signaling pathway; they suppress cellular proliferation and induce apoptosis in several cancer cells. In the present study, we investigated the effect of N-[N-(3, 5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT, γ-secretase inhibitor) on the cell proliferation and apoptosis in Ishikawa endometrial cancer cells. Real-time PCR detected mRNA derived from NOTCH1 and HES1, which are target genes of the Notch signaling pathway, in Ishikawa endometrial cancer cells. After blocking Notch signaling, cellular proliferation decreased, accompanied by increased expression of p21 mRNA and decreased expression of the cyclin A protein. Furthermore, blockade of Notch signaling induced apoptosis. These results suggest that the Notch signaling pathway may be involved in cell proliferation through cell cycle regulation and apoptosis in Ishikawa endometrial cancer cells. Inhibition of the Notch signaling pathway by γ-secretase inhibitors is expected to be a potential target of novel therapeutic strategies for endometrial cancer.

Immunohistochemical, molecular, and clinicopathological analyses of urothelial carcinoma, micropapillary variant.

The prognosis of urothelial carcinoma, micropapillary variant (MPV), of the bladder has been shown to be worse than that of the conventional urothelial carcinoma (UC). However, it remains to be clarified why the MPV is more aggressive. We therefore here focused on the correlation between clinical features and histological, immunohistochemical and molecular findings for eight MPV and 35 UC, evaluating expression of MUC1, Ki-67, p53, CD147, CD34, D2-40, and extracellular matrix proteins. The Ki-67 labeling index was significantly higher in UC than in MPV but densities of venous and lymphatic tumor emboli were significantly higher in the MPV cases and lymph node metastasis was more frequent, with a poorer prognosis. Tenascin-C and fibronectin also showed significantly greater expression in MPV than in UC at the epithelial-mesenchymal interfaces. Direct sequencing showed point mutations of KRAS exon 1 in three MPV with significantly more frequency compared to UC. Occupation rate of the MPV area in the tumor showed significant inverse correlation with overall survival. Thus our histopathological findings provide clues to explaining why prognosis is poorer in the MPV than UC.

The balance between the expressions of hASH1 and HES1 differs between large cell neuroendocrine carcinoma and small cell carcinoma of the lung.

To clarify the biological differences between small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC), we investigated the expression of two bHLH type transcription factors, human achaete-scute homolog 1 (hASH1) and hairy/enhancer of split 1 (HES1), which positively and negatively regulate the neuroendocrine differentiation of respiratory epithelial cells, respectively. Eighty-eight formalin-fixed and paraffin-embedded pulmonary carcinomas (32 SCLC, 32 LCNEC, 14 adenocarcinomas, and 10 squamous cell carcinomas) and 14 SCLC and 1 LCNEC derived cell lines were used. hASH1 and HES1 mRNA were detected using a highly sensitive in situ hybridization method with digoxigenin-labeled cRNA probes and biotinylated tyramide. The staining results were scored from 0 to 12 by multiplying the staining intensity by the percentage of positive tumor cells. The mean staining score of hASH1 mRNA was significantly higher in SCLC than in LCNEC (p<0.01); conversely, that of HES1 mRNA was lower in SCLC than in LCNEC (p<0.01). These findings reveal that SCLC more strongly expresses the neuroendocrine phenotype, while LCNEC shows characteristics more similar to the ciliated epithelium phenotype, suggesting that the biological characteristics of these two tumors are different.

Nuclear transfer of embryonic cell nuclei to non-enucleated eggs in zebrafish, Danio rerio.

We previously established a novel method for nuclear transfer in medaka (Oryzias latipes) using non-enucleated, diploidized eggs as recipients for adult somatic cell nuclei. Here we report the first attempt to apply this method to another fish species. To examine suitability of using non-enucleated eggs as recipients for nuclear transfer in the zebrafish (Danio rerio), we transferred blastula cell nuclei from a wild-type donor strain to non-enucleated, unfertilized eggs from a golden recipient strain. As a result, 31 of 184 (16.8%) operated eggs developed normally and reached the adult stage. Twenty-eight (15.2%) of these transplants showed wild-type phenotype and the remaining three (1.6%) were golden. Except for one individual that exhibited diploid/tetraploid mosaicism, all of the wild-type nuclear transplants were either triploid or diploid. While all of 19 triploid transplants were infertile, a total of six transplants (21.4%) were fertile (five of the eight diploid transplants and one transplant exhibiting ploidy mosaicism). Except for one diploid individual, all of the fertile transplants transferred both the wild-type golden gene allele (slc24a5) as well as the phenotype, the wild-type body color, to their F(1) and F(2) progeny in a typical Mendelian fashion. PCR analysis of slc24a5 suggested that triploidy originated from a fused nucleus in the diploid donor and haploid recipient nuclei, and that the sole origin of diploidy was the diploid donor nucleus. The results of the present study demonstrated the suitability of using non-enucleated eggs as recipients for nuclear transfer experiments in zebrafish.

Impaired ability to shift weight onto the non-paretic leg in right-cortical brain-damaged patients.

BACKGROUND: : Stroke patients experience postural instability that can impede functional improvements in their gait. However, the precise functions of the dominant and non-dominant hemispheres in controlling static standing posture and weight-bearing remain unclear. OBJECTIVE: : To investigate differences in balancing ability between right-handed patients with right and left hemispheric lesions. METHODS: : Weight shifting was quantitatively evaluated to determine the ability of patients to control their balance in a static posture and during conscious weight shifting onto the paretic or non-paretic leg. Participants were enrolled from a consecutive series of stroke patients attending a rehabilitation program (n=49; 31 male, 18 female; mean age 69.3+/-9.4 years). Age-matched normal controls were recruited as volunteers (n=12; 4 male, 8 female; mean age 67.9+/-4.9 years). RESULTS: : Patients with cortical lesions in the right hemisphere were able to shift less weight onto the non-paretic leg than patients with cortical lesions in the left hemisphere (p<0.05). There were no correlations between the existence of unilateral spatial neglect and the percentage of weight shifted onto the non-paretic leg, static standing posture (r=0.27, p=0.40) or dynamic standing posture (r=-0.37, p=0.24). In contrast, there was a significant correlation between the percentage of weight consciously shifted onto the non-paretic leg and the existence of anosognosia (r=0.74, p=0.006), but not between static standing posture and anosognosia (r=-0.15, p=0.63). CONCLUSION: : Patients with right cortical hemispheric lesions were able to shift less body weight onto their non-paretic leg. These patients should be encouraged to practice shifting their weight towards their non-paretic leg to improve their balance.

Analysis of DNA variations in promoter region of HCNP gene with Alzheimer's disease.

Hippocampal cholinergic neurostimulating peptide (HCNP), which enhances acetylcholine synthesis and induces cholinergic phenotype development of the septohippocampal system, is derived from HCNP precursor protein (HCNPpp), also known as phosphatidylethanolamine binding protein (PEBP) and Raf kinase inhibitor protein (RKIP). Our previous study demonstrated that expression of HCNPpp mRNA was decreased in the hippocampi of autopsied brains of Alzheimer's disease (AD) patients, indicating the association of HCNP with the pathogenesis of AD. To clarify the involvement of gene variations in the promoter region of the gene encoding HCNPpp in this mRNA reduction, we analyzed DNA polymorphisms or mutations within this gene promoter region in AD patients by direct sequencing. The promoter was found to contain a CpG island without a TATA box, an element of housekeeping gene promoters. Moreover, no disease-specific polymorphisms or mutations were identified, suggesting that the decrease of mRNA can be ascribed to transcriptional or posttranscriptional changes in activity.

Adsorption of actives in ophthalmological drugs for over-the-counter on soft contact lens surfaces.

The adsorption of various kinds of ionic/nonionic actives, added in ophthalmological drugs (artificial tear, contact lens wetting solution, eye-drops, and eyewash) for over-the-counter (marketable drugs with no need of any medical prescription) , on soft contact lens (SCL) surfaces has been studied as a function of hydrophobicity of the actives. The common logarithm of the 1-octanol/water partitioning coefficient (AC_log P) has been used in order to normalize the hydrophobicity of the actives employed in this study. No significant adsorption occurs for relatively hydrophilic actives, whereas the adsorption rate is gradually increased with an increase in the hydrophobicity of the actives. This suggests that the adsorption is predominantly governed by the hydrophobic interaction of the actives with the SCL surfaces, although an electrostatic interaction plays an additional role for the adsorption. The most effective adsorption occurs in the following active-lens combinations: cationic actives--the anionic and hydrated lens IV (methacrylic acid-based SCL); anionic actives---the nonionic and hydrated lens II (N-vinyl pyrrolidone-based SCL); and nonionic actives--the anionic and less-hydrated lens III (containing hydrophobic silicone monomers).