Genetic and epigenetic alterations in precursor lesions of endometrial endometrioid carcinoma.

The hyperplasia-carcinoma sequence is a stepwise tumourigenic programme towards endometrial cancer in which normal endometrial epithelium becomes neoplastic through non-atypical endometrial hyperplasia (NAEH) and atypical endometrial hyperplasia (AEH), under the influence of unopposed oestrogen. NAEH and AEH are known to exhibit polyclonal and monoclonal cell growth, respectively; yet, aside from focal PTEN protein loss, the genetic and epigenetic alterations that occur during the cellular transition remain largely unknown. We sought to explore the potential molecular mechanisms that promote the NAEH-AEH transition and identify molecular markers that could help to differentiate between these two states. We conducted target-panel sequencing on the coding exons of 596 genes, including 96 endometrial cancer driver genes, and DNA methylome microarrays for 48 NAEH and 44 AEH lesions that were separately collected via macro- or micro-dissection from the endometrial tissues of 30 cases. Sequencing analyses revealed acquisition of the PTEN mutation and the clonal expansion of tumour cells in AEH samples. Further, across the transition, alterations to the DNA methylome were characterised by hypermethylation of promoter/enhancer regions and CpG islands, as well as hypo- and hyper-methylation of DNA-binding regions for transcription factors relevant to endometrial cell differentiation and/or tumourigenesis, including FOXA2, SOX17, and HAND2. The identified DNA methylation signature distinguishing NAEH and AEH lesions was reproducible in a validation cohort with modest discriminative capability. These findings not only support the concept that the transition from NAEH to AEH is an essential step within neoplastic cell transformation of endometrial epithelium but also provide deep insight into the molecular mechanism of the tumourigenic programme. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Genomic features of Helicobacter pylori-naïve diffuse-type gastric cancer.

Helicobacter pylori (HP) is a major etiologic driver of diffuse-type gastric cancer (DGC). However, improvements in hygiene have led to an increase in the prevalence of HP-naïve DGC; that is, DGC that occurs independent of HP. Although multiple genomic cohort studies for gastric cancer have been conducted, including studies for DGC, distinctive genomic differences between HP-exposed and HP-naïve DGC remain largely unknown. Here, we employed exome and RNA sequencing with immunohistochemical analyses to perform binary comparisons between 36 HP-exposed and 27 HP-naïve DGCs from sporadic, early-stage, and intramucosal or submucosal tumor samples. Among the samples, 33 HP-exposed and 17 HP-naïve samples had been preserved as fresh-frozen samples. HP infection status was determined using stringent criteria. HP-exposed DGCs exhibited an increased single nucleotide variant burden (HP-exposed DGCs; 1.97 [0.48-7.19] and HP-naïve DGCs; 1.09 [0.38-3.68] per megabase; p = 0.0003) and a higher prevalence of chromosome arm-level aneuploidies (p < 0.0001). CDH1 was mutated at similar frequencies in both groups, whereas the RHOA-ARHGAP pathway misregulation was exclusive to HP-exposed DGCs (p = 0.0167). HP-exposed DGCs showed gains in chromosome arms 8p/8q (p < 0.0001), 7p (p = 0.0035), and 7q (p = 0.0354), and losses in 16q (p = 0.0167). Immunohistochemical analyses revealed a higher expression of intestinal markers such as CD10 (p < 0.0001) and CDX2 (p = 0.0002) and a lower expression of the gastric marker, MUC5AC (p = 0.0305) among HP-exposed DGCs. HP-naïve DGCs, on the other hand, had a purely gastric marker phenotype. This work reveals that HP-naïve and HP-exposed DGCs develop along different molecular pathways, which provide a basis for early detection strategies in high incidence settings. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Suffruyabiosides A and B, two new monoterpene diglycosides from moutan cortex.

Two new monoterpene diglycosides, suffruyabiosides A and B, and seven known compounds 3-9 were isolated from Moutan Cortex (root cortex of Paeonia suffruticosa Andrews). The structures were elucidated on the basis of 2D NMR spectral data. Suffruyabiosides A and B are rare monoterpene diglycosides, including a cellobiose in the molecules. Salicylpaeoniflorin (4) had a antiproliferation effect similar to paeoniflorin (3) on human lung adenocarcinoma epitherial A549 cells. Galloylpaeoniflorin (8) and salicylpaeoniflorin (4) revealed a more pronounced radical scavenging effect than a-tocopherol (positive control). An increase in the number of phenolic hydroxyl groups produced a more effective radical scavenging effect [8 > mudanpioside E (6) > oxypaeoniflorin (5)]. Comparison of the effects of 4 and 5 showed that o-substitution with a phenolic hydroxyl group was more effective than p-substitution. The results suggest that salicylpaeoniflorin (4) may be useful as a cytotoxic and a radical scavenging agent.

Development of a monoclonal antibody against Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) that can detect EBNA-LP expressed in P3HR1 cells.

A mouse monoclonal antibody, LP4D3, was raised against purified Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) fused to glutathione-S-transferase. The antibody detected endogenous and exogenous EBNA-LP in immunoblotting, immunofluorescence and immunoprecipitation assays, and the epitope of the antibody was mapped in the W2 domain of EBNA-LP. While another monoclonal antibody to EBNA-LP, JF186, which is widely used for analyses of the viral protein, did not react with truncated forms of EBNA-LP expressed in P3HR1 cells, as reported earlier, the LP4D3 antibody did. The LP4D3 antibody will be a useful tool for further studies of EBNA-LP, especially investigations into the phenotypes of mutant EBNA-LP expressed in P3HR1 cells.

Effects of antisense peptide nucleic acid to platelet-derived growth factor A-chain on growth of vascular smooth muscle cells.

To investigate antisense peptide nucleic acid (PNA) as a gene therapy for the arterial proliferative diseases, the authors designed and examined the effects of an antisense PNA targeting platelet-derived growth factor (PDGF) A-chain on expression of PDGF A-chain and growth of vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats. A 15-mer antisense PNA complementary to the initiation codon of rat and human PDGF A-chain mRNA was synthesized and purified by high-performance liquid chromatography. Gel-shift assay and biomolecular interaction analysis (BIAcore) revealed that the antisense PNA bound weakly to the target RNA, whereas it bound strongly to the target DNA. Fluorescein-isothiocyanate-labeled antisense PNA to PDGF A-chain was taken up slowly and maintained in VSMCs for a prolonged period of time. Antisense PNA inhibited expression of PDGF A-chain mRNA and protein as well as DNA synthesis in VSMCs in a dose-independent manner. Inhibition of DNA synthesis by the antisense PNA was greater than that by the antisense DNA at a low concentration (0.5 micromol/L). These results suggest that antisense PNA to PDGF A-chain will be used as a gene therapy for vascular proliferative diseases such as hypertensive vascular diseases, restenosis of coronary arteries after angioplasty, and atherosclerosis.

Determination of donepezil, an acetylcholinesterase inhibitor, in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection.

A simple and sensitive high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantification of donepezil, a centrally and selectively acting acetyleholinesterase inhibitor, in human plasma. After sample alkalinization with 0.5 ml of NaOH (0.1 M), the test compound was extracted from I ml of plasma using isopropanol-hexane (3:97, v/v). The organic phase was back-extracted with 75 microl of HCl (0.1 M) and 50 microl of the acid solution was injected into a C18 STR ODS-II analytical column (5 microm, 150x4.6 mm I.D.). The mobile phase consisted of phosphate buffer (0.02 M, pH 4.6), perchloric acid (6 M) and acetonitrile (59.5:0.5:40, v/v) and was delivered at a flow-rate of 1.0 ml/min at 40 degrees C. The peak was detected using a UV detector set at 315 nm, and the total time for a chromatographic separation was approximately 8 min. The method was validated for the concentration range 3-90 ng/ml. Mean recoveries were 89-98%. Intra- and inter-day relative standard deviations were less than 7.3 and 7.6%, respectively, at the concentrations ranging from 3 to 90 ng/ml. The method shows good specificity with respect to commonly prescribed psychotropic drugs, and it could be successfully applied for pharmacokinetic studies and therapeutic drug monitoring.