N-Myc Downstream Regulated Gene-1 May Play an Important Role in the Prognosis of Ovarian Cancer, in Its Association with Beta-Catenin.

NDRG1 is a nickel- and calcium-inducible gene that plays important roles in the primary growth of malignant tumors, as well as in invasion and metastasis. This study investigated the associations of NDRG1 expression with cell adhesion and other clinicopathological factors in ovarian cancer. The clinical records of 123 women who underwent surgery for ovarian cancer in our institute were reviewed retrospectively. The expression of NDRG1, E-cadherin, and beta-catenin in surgical specimens were evaluated immunohistochemically. The NDRG1 expression level was significantly associated with beta-catenin expression, peritoneal metastasis outside the pelvic cavity, lymph node metastasis, and FIGO stages. The Kaplan-Meier analysis showed a significant association between the NDRG1 expression level and progression-free survival: high NDRG1 expression was related to poor survival. Our results suggest that the increased expression of NDRG1 is associated with cell adhesion and may be a poor prognostic indicator in women with ovarian cancer.

Metabolic Oscillations and Glycolytic Phenotypes of Cancer Cells.

Cancer cells show several metabolic phenotypes depending on the cancer types and the microenvironments in tumor tissues. The glycolytic phenotype is one of the hallmarks of cancer cells and is considered to be one of the crucial features of malignant cancers. Here, we show glycolytic oscillations in the concentrations of metabolites in the glycolytic pathway in two types of cancer cells, HeLa cervical cancer cells and DU145 prostate cancer cells, and in two types of cellular morphologies, spheroids and monolayers. Autofluorescence from nicotinamide adenine dinucleotide (NADH) in cells was used for monitoring the glycolytic oscillations at the single-cell level. The frequencies of NADH oscillations were different among the cellular types and morphologies, indicating that more glycolytic cancer cells tended to exhibit oscillations with higher frequencies than less glycolytic cells. A mathematical model for glycolytic oscillations in cancer cells reproduced the experimental results quantitatively, confirming that the higher frequencies of oscillations were due to the higher activities of glycolytic enzymes. Thus, glycolytic oscillations are expected as a medical indicator to evaluate the malignancy of cancer cells with glycolytic phenotypes.

Guidelines for Handling of Cytological Specimens in Cancer Genomic Medicine.

Rapid advances are being made in cancer drug therapy. Since molecularly targeted therapy has been introduced, personalized medicine is being practiced, pathological tissue from malignant tumors obtained during routine practice is frequently used for genomic testing. Whereas cytological specimens fixed mainly in alcohol are considered to be more advantageous in terms of preservation of the nucleic acid quality and quantity. This article is aimed to share the information for the proper handling of cytological specimens in practice for genomic medicine based on the findings established in "Guidelines for Handling of Cytological Specimens in Cancer Genomic Medicine (in Japanese)" published by the Japanese Society of Clinical Cytology in 2021. The three-part practical guidelines are based on empirical data analyses; Part 1 describes general remarks on the use of cytological specimens in cancer genomic medicine, then Part 2 describes proper handling of cytological specimens, and Part 3 describes the empirical data related to handling of cytological specimens. The guidelines indicated proper handling of specimens in each fixation, preparation, and evaluation.

Glycolytic oscillations in HeLa cervical cancer cell spheroids.

Previous studies have unravelled glycolytic oscillations in cancer cells, such as HeLa cervical and DU145 prostate cancer cells, using a monolayer culture system. Here, we demonstrate glycolytic oscillations in HeLa cervical cancer cell spheroids. Experiments revealed that a small number of HeLa cells in spheroids exhibited heterogeneous oscillations with a higher frequency than those in monolayers. Model analyses and our previous experiments indicated that the higher frequencies of oscillations in spheroids were mostly due to the increase in glycolytic enzyme activity in the cells, and to the decrease in glucose concentration by diffusional transport of glucose from the surface to inside the spheroids, as well as the increase in cell density through spheroid formation. These results and our previous studies imply that more malignant cancer cells tend to exhibit glycolytic oscillations with higher frequencies than less malignant cells. Adjacent cells in spheroids oscillated within a 10% difference in frequency, but did not synchronize with each other. This suggests that weak cell-to-cell interactions might exist among HeLa cells connected with cadherins in the spheroid microenvironment; however, the interactions were not strong enough to induce synchronization of glycolytic oscillations.

Oscillations and Dynamic Symbiosis in Cellular Metabolism in Cancer.

The grade of malignancy differs among cancer cell types, yet it remains the burden of genetic studies to understand the reasons behind this observation. Metabolic studies of cancer, based on the Warburg effect or aerobic glycolysis, have also not provided any clarity. Instead, the significance of oxidative phosphorylation (OXPHOS) has been found to play critical roles in aggressive cancer cells. In this perspective, metabolic symbiosis is addressed as one of the ultimate causes of the grade of cancer malignancy. Metabolic symbiosis gives rise to metabolic heterogeneities which enable cancer cells to acquire greater opportunities for proliferation and metastasis in tumor microenvironments. This study introduces a real-time new imaging technique to visualize metabolic symbiosis between cancer-associated fibroblasts (CAFs) and cancer cells based on the metabolic oscillations in these cells. The causality of cellular oscillations in cancer cells and CAFs, connected through lactate transport, is a key point for the development of this novel technique.

Subcategorization of atypical glandular cells is useful to identify lesion site.

BACKGROUND: In the subcategorization of atypical glandular cells (AGCs), origin of cells should be mentioned to estimate lesion sites for diagnosis. However, cases without subcategorization are often encountered due to limited reproducibility. We evaluated whether the subcategorization of AGC based on the Bethesda terminology can estimate lesion sites. METHODS: We retrospectively investigated cases whose cervical smears were interpreted as AGC and underwent pathological assessment at our institution between June 2009 and September 2017. AGC was subcategorized based on the Bethesda System. Not-otherwise-specified (NOS) was subcategorized into endocervical cells (NOS-EC), endometrial cells (NOS-EM), or glandular cells (NOS-G). Favor neoplastic (FN) was subcategorized into endocervical cells (FN-EC) or glandular cells (FN-G). FN-G was further subcategorized into endometrial cells (FN-EM) or unknown origin (FN-UO). Clinicopathological data were retrieved from the medical records. RESULTS: Of 88 AGC cases, there were 30 NOS-EC (34.1%), 2 NOS-EM (2.3%), 25 FN-EC (28.4%), 22 FN-EM (25.0%), and 9 FN-UO (10.2%). A significantly higher proportion of neoplastic lesions occurred in FN than in NOS (P <.001). The concordance of AGC subclass and lesion site was 88.0%, 70.7%, and 77.3% in FN-EC, FN-G, and FN-EM, respectively. The concordance of FN-EM and lesion site increased to 88.9% in patients aged >50 years. Of nine cases of FN-UO, six experienced nonendometrioid endometrial cancer and extrauterine malignancy. CONCLUSION: Subcategorization of NOS and FN would be useful in estimating neoplastic lesions. Further subcategorization into FN-EC, FN-EM, and FN-UO would similarly be beneficial in estimating the lesion site, especially for small endometrial and extrauterine lesions.

Calcium Carbonate Skeletal Material Is Synthesized via Phase Transition of the Calcium Carbonate Cartilaginous Material.

The formation mechanism of calcium carbonate (CC) skeletal tissues in biomineralization has remained poorly understood for a long time. Here, we propose an artificial CC biomineralization system equivalent to the natural one in terms of the primary physicochemical mechanism. Our system is constructed of a polymer gel and a CC solution unsaturated by a dissociated anionic polymer. The gel network consists of proton donor and proton acceptor polymers, which are analogues of polymers in the natural biomineralization system and have affinity for each other through hydrogen bonding interaction. Artificial biomineralization takes place within the polymer gel to produce a monolithic composite of the network and CC, whose powder X-ray diffraction pattern indicates calcite or calcite/vaterite. Scanning electron microscopy and energy-dispersive X-ray spectroscopy observation of the composite during the mineralization process revealed a two-phase structure (network/CC solid solution phase and CC hypercomplex gel phase). As artificial biomineralization proceeds, the solid phase grows in size at the cost of the gel phase as if the latter is substituted with the former, until the solid phase occupies the whole depth of the composite. These results suggest that the hypercomplex gel is the precursor of the resultant network/CC solid solution, and its discontinuous change is a phase transition to the solid solution. Despite minute differences in higher-order structures between our model system and the natural system, the fundamental structure of CC skeletal tissues in the latter can be interpreted as a network/CC solid solution, whereas that of CC cartilaginous tissues as a CC hypercomplex gel. Then, it can be deduced that, in biomineralization, the CC skeletal tissue is in principle formed via a phase transition of the CC cartilaginous tissue.

Effect of tranexamic acid in improving the lifespan of naturally aging mice.

An effective method to improve lifespan is not known. Therefore, in this study, we examined the lifespan-extending effect of tranexamic acid in normal mice. We bred hairless mice without exposure to ultraviolet radiation and psychical stress until they died naturally. During the study period, the mice were orally administered tranexamic acid (12 mg/kg/day) three times weekly. An increase in the lifespan of mice was observed by tranexamic acid administration. Furthermore, age-related diseases of the skin were ameliorated by tranexamic acid administration. Moreover, the blood level of tumor necrosis factor-α, interleukin-6, reactive oxygen species (ROS), and matrix metalloproteinase (MMP)-9 was decreased by tranexamic acid administration. These results indicate that tranexamic acid suppresses the secretion of inflammatory cytokines, MMP-9, and ROS induced by natural aging, ameliorating age-related diseases, and, consequently, extending the lifespan.

Ameliorative effect of tranexamic acid on physiological skin aging and its sex difference in mice.

An effective method to protect the skin from natural aging is unknown. Therefore, in this study, we examined the ameliorative effects of tranexamic acid on natural skin aging. In addition, we examined the sex difference in the effect exhibited by tranexamic acid. We bred hairless mice without ultraviolet ray irradiation and physical stress for 2 years. During the study period, mice were orally administered tranexamic acid (12 mg/kg/day) three times per week. Development of signs of skin aging was found to be ameliorated by tranexamic acid. Furthermore, synthetic inhibition of plasmin was observed following tranexamic acid treatment. The synthetic reinforcement of hyaluronic acid by an increase in the number of epidermal cells and the degradative inhibition of extracellular matrix (ECM) by matrix metalloproteinase (MMP) suppression were observed. These results indicate that natural skin aging was ameliorated by tranexamic acid via the regulation of the plasmin/TGF-ß/epidermal cells/hyaluronic acid and plasmin/MMPs/ECM signal transmission pathways. Taken together, sex difference was observed for the ameliorative effect of tranexamic acid on skin aging, with a stronger effect observed in females than in males. More importantly, we found that the synthesis of hyaluronic acid was stronger in female mice than in male mice.

Modeling studies of heterogeneities in glycolytic oscillations in HeLa cervical cancer cells.

Previous experiments demonstrated that a population of HeLa cells starved of glucose or both glucose and serum exhibited a strong heterogeneity in the glycolytic oscillations in terms of the number of oscillatory cells, periods of oscillations, and duration of oscillations. Here, we report numerical simulations of this heterogeneous oscillatory behavior in HeLa cells by using a newly developed mathematical model. It is simple enough that we can apply a mathematical analysis, but capture the core of the glycolytic pathway and the activity of the glucose transporter (GLUT). Lognormal distributions of the values of the four rate constants in the model were obtained from the experimental distributions in the periods of oscillations. Thus, the heterogeneity in the periods of oscillations can be attributed to the difference in the rate constants of the enzymatic reactions. The activity of GLUT is found to determine whether the HeLa cells were oscillatory or non-oscillatory under the same experimental conditions. Simulation with the log-normal distribution of the maximum uptake velocity of glucose and the four randomized rate constants based on the log-normal distributions successfully reproduced the time-dependent number of oscillatory cells (oscillatory ratios) under the two starving conditions. The difference in the initial values of the metabolites has little effect on the simulated results.

Insulinoma-associated protein 1 expression in pancreatic neuroendocrine tumours in endoscopic ultrasound-guided fine-needle aspiration cytology: An analysis of 14 patients.

BACKGROUND: Insulinoma-associated protein 1 (INSM1) has been reported to be a useful marker for diagnosing pancreatic neuroendocrine tumours (PNETs). However, INSM1 expression in endoscopic ultrasound-guided fine needle aspiration cytology has not been examined. We evaluated INSM1 expression in the cytology of cases diagnosed with PNETs. METHODS: We immunocytochemically stained INSM1 and Ki-67 in 14 PNET cases, and according to the 2017 World Health Organisation criteria, seven PNET Grade 1 cases, four Grade 2 cases and three Grade 3/neuroendocrine carcinoma cases were identified. As a control for INSM1 and Ki-67 expression, we used cytological specimens from 15 cases of pancreatic ductal adenocarcinoma. RESULTS: In PNET cases, INSM1-expressing tumour cells were identified in all cytological specimens of PNET. The median INSM1 expression rate in Grade 1 cases was 49.8% (mean ± standard deviation: 55.1 ± 12.5%, min: 39.3%, max: 74.1%), and in Grade 2 and Grade 3/neuroendocrine carcinoma cases was 81.1% (mean ± standard deviation: 77.6 ± 18.6%, min: 50.3%, max: 100%). However, there was no correlation between INSM1 and Ki-67 expression (r = -0.15). The median expression rate in PNET cases was 64.3%, which was significantly higher than that in pancreatic ductal adenocarcinoma cases (P < 0.0001). CONCLUSION: INSM1 immunocytochemistry of cytological specimens obtained from endoscopic ultrasound-guided fine needle aspiration cytology can accurately diagnose PNETs; therefore, INSM1 could be an important diagnostic tool in assessing therapeutic strategies, including molecular-targeted therapy.

Tranexamic Acid Ameliorates Nonmelanoma Skin Cancer Induced by Long-term Ultraviolet A Irradiation.

To date, there have been no treatments developed to ameliorate nonmelanoma skin cancer induced by long-term exposure to ultraviolet A (UVA) irradiation. In this study, we examined the effects of tranexamic acid (trans-4-aminomethyl cyclohexanecarboxylic acid) on long-term UVA-induced skin cancer. We exposed the dorsal skin of male hairless mice to UVA at a dose of 110 kJ m-2 using an FL20SBLB-A lamp three times weekly for 15 weeks after application of 7,12-dimethylbenz [a] anthracene (DMBA). During the experimental period, the mice were administered tranexamic acid (750 mg kg-1 day-1 ) three times weekly. We found that cancer development was ameliorated by administration of tranexamic acid. Furthermore, tranexamic acid treatment was observed to suppress increases in the plasma levels of matrix metalloproteinase-9 and interleukin (IL)-6, and skin expression of plasmin, CC chemokine 2, macrophages, signal transducer and activator of transcription (STAT)3, cyclin D and vascular endothelial growth factor (VEGF)-A that occurred in mice subjected to long-term UVA irradiation. These results indicated that the nonmelanoma skin cancer induced by DMBA+UVA long-term irradiation is ameliorated by tranexamic acid through regulation of the plasmin/macrophage/IL-6/STAT3/cyclin D signal transmission pathway. In addition, this ameliorative effect against skin cancer may be mediated via inhibition of the IL-6-induced expression of VEGF-A.

Tranexamic acid inhibits the plasma and non-irradiated skin markers of photoaging induced by long-term UVA eye irradiation in female mice.

Photoaging can be induced by long-term ultraviolet (UV)A eye irradiation, but an ameliorating method for such photoaging is not known. In this study, we examined the effects of tranexamic acid (trans-4-aminomethylcyclohexanecarboxylic acid) on photoaging of the skin induced by UVA eye irradiation. We used the C57BL/6 j female mice and locally exposed their eyes to UVA at a dose of 110 kJ/m2 using an FL20SBLB-A lamp multiple times a week for one year. The plasma urocortin 2, ß-endorphin, methionine enkephalin (OGF), and histamine content, as well as the expression of the corticotropin-releasing hormone receptor (CRHR) type 2, µ-opioid receptor, opioid growth factor receptor (OGFR), T-bet, and GATA3 increased in the mice subjected to UVA eye irradiation. However, the increased levels of urocortin 2, methionine enkephalin, histamine, OGFR, T-bet, and GATA3 were suppressed by tranexamic acid treatment. Conversely, the levels of ß-endorphin and µ-opioid receptor increased with tranexamic acid treatment. In addition, the expression of the estrogen receptor-ß on the surface of mast cells was increased by tranexamic acid. These results indicate that photoaging induced by UVA eye irradiation can be ameliorated by tranexamic acid through the regulation of hypothalamo-pituitary hormones. Furthermore, this ameliorative effect on photoaging may be due to an increase in estrogen receptor-ß after tranexamic acid treatment.

Promotion and inhibition of synchronous glycolytic oscillations in yeast by chitosan.

Synchronous rhythmic activities play crucial roles in diverse biological systems. Glycolytic oscillations in yeast cells have been studied for 50 years with the aim of elucidating the mechanisms underlying the intracellular oscillations and their synchronization. We investigated the effects of chemical disturbances on the individual and collective glycolytic oscillations in yeast cells encapsulated in alginate microparticles, and demonstrated that the addition of chitosan, an antimicrobial agent, decreased the duration of these oscillations. In contrast, the periods and the synchronicity states showed two different responses to the chitosan treatments. The periods were shown to be prolonged following the treatment with 5-50 mg·L-1 and shortened at 75 mg·L-1 of chitosan. Collective oscillations became more synchronized at 5 mg·L-1 of chitosan, and desynchronized at 25-75 mg·L-1 of this compound. These findings can be explained by the balance between two chitosan features, increasing cell membrane permeability and acetaldehyde scavenging. At low concentrations, chitosan presumably acts as a synchronization promoter that does not mediate the synchronization itself but induces an increase in intercellular coupling. We believe that our findings may provide new insights into the synchronous rhythmic activities in biological systems.

NMR Biochemical Assay for Oxidosqualene Cyclase: Evaluation of Inhibitor Activities on Trypanosoma cruzi and Human Enzymes.

Oxidosqualene cyclase (OSC), a membrane-associated protein, is a key enzyme of sterol biosynthesis. Here we report a novel assay for OSC, involving reaction in aqueous solution, NMR quantification in organic solvent, and factor analysis of spectra. We evaluated one known and three novel inhibitors on OSC of Trypanosoma cruzi, a parasite causative of Chagas disease, and compared their effects on human OSC for selectivity. Among them, one novel inhibitor showed a significant parasiticidal activity.

The expression of programed death ligand-1 could be related with unfavorable prognosis in salivary duct carcinoma.

BACKGROUND: Salivary duct carcinoma (SDC) is a rare tumor occurring in the salivary gland. SDC is a highly aggressive tumor and its prognosis is extremely poor. Effective treatments in advanced SDC have not yet been established. Recently, immune checkpoint inhibitors have paved the way for the treatment of various malignancies. We examined the expressions of programed death ligand (PD-L) 1/PD-L2 and programed death (PD-1), and the correlation of clinicopathological findings. METHODS: We examined 18 cases of SDC and conducted immunohistochemical staining using formalin-fixed paraffin-embedded full-face sections. RESULTS: The expression of PD-L1 and PD-L2 in tumor cells was observed in nine cases (50%) and 14 cases (78%), respectively. Cases with a high expression of PD-L1 and PD-L2 were found in four (22%) and seven cases (39%), respectively. The cases with a high expression of PD-L1 showed significantly shorter overall survival compared to those with low PD-L1 expression and null expression. We also examined the expression of PD-L1/PD-L2 and PD-1 of tumor-infiltrating mononuclear cells (TIMC) in stroma. The expressions of PD-L1 in tumor cells and stroma had a significant correlation. Association between the expressions of PD-L1 in tumor cells and those of PD-1 in stroma was significant. However, PD-L2 expression in the tumor had no significant correlation with expression in TIMCs. PD-L1, PD-L2 and PD-1 expressions in stroma were not associated with patient prognosis. CONCLUSIONS: High PD-L1 expression in SDC was strongly associated with unfavorable prognosis, indicating that PD-1/PD-L1 inhibitors could be effective in SDC.

Crystal structures of FMN-bound and FMN-free forms of dihydroorotate dehydrogenase from Trypanosoma brucei.

Dihydroorotate dehydrogenase (DHODH) is a flavin-binding enzyme essential for pyrimidine biosynthesis, which converts dihydroorotate to orotate. Three-dimensional structures of cytosolic DHODH of parasitic protozoa are of interest in drug discovery for neglected tropical diseases, especially because these enzymes possess significantly different structural and functional properties from the membrane-associated human enzyme. The existing crystal structures of the flavin mononucleotide (FMN)-bound DHODHs reveal a number of interactions stabilizing FMN. However, to understand the binding mechanism correctly, it is necessary to compare the structures of the FMN-bound and FMN-free forms, because the protein moiety of the former is not necessarily the same as the latter. Here, we prepared the FMN-free DHODH of Trypanosoma brucei using an Escherichia coli overexpression system. Although this apoform lacks enzymatic activity, simple incubation with FMN activated the enzyme. It was stable enough to be crystallized, enabling us to determine its structure by X-ray crystallography at 1.6 Å resolution. We also determined the FMN-bound form at 1.8 Å resolution. Although the two structures have essentially the same scaffold, we observed flipping of a peptide-bond plane in the vicinity of the FMN-binding site, accompanied by an alternative hydrogen-bonding pattern. Comparisons of B factors of the protein main chain revealed that binding of FMN decreased flexibility of most of the residues at the FMN-binding site, but increased flexibility of a lid-like loop structure over the active center. This increase was ascribed to a conformational change in an FMN-contacting residue, Asn195, which induced a rearrangement of a hydrogen-bond network of the residues comprising the lid.