Guideline for gynecological practice in Japan: Japan Society of Obstetrics and Gynecology and Japan Association of Obstetricians and Gynecologists 2023 edition.

Twelve years after the first edition of The Guideline for Gynecological Practice, which was jointly edited by The Japan Society of Obstetrics and Gynecology and The Japan Association of Obstetricians and Gynecologists, the 5th Revised Edition was published in 2023. The 2023 Guidelines includes 5 additional clinical questions (CQs), which brings the total to 103 CQ (12 on infectious disease, 30 on oncology and benign tumors, 29 on endocrinology and infertility and 32 on healthcare for women). Currently, a consensus has been reached on the Guidelines, and therefore, the objective of this report is to present the general policies regarding diagnostic and treatment methods used in standard gynecological outpatient care that are considered appropriate. At the end of each answer, the corresponding Recommendation Level (A, B, C) is indicated.

Decreased intracellular chloride enhances cell migration and invasion via activation of the ERK1/2 signaling pathway in DU145 human prostate carcinoma cells.

Our previous study revealed that changes of the intracellular Cl- concentration ([Cl-]i) affected cell proliferation in cancer cells. However, the role of Cl- on cell migration and invasion in cancer cells remains unanalyzed. Therefore, the aim of the present study is to investigate whether changes of [Cl-]i affects cell migration and invasion of cancer cells. In human prostate cancer DU145 cells, cell migration and invasion were enhanced by culturing in the low Cl- medium (replacement of Cl- by NO3-). We also found that DU145 cells in the low Cl- condition caused significant transient ERK1/2 activation followed by an increase of MMP-1 mRNA levels. Inhibition of ERK1/2 activation in the low Cl- condition reduced enhancement of MMP-1 mRNA levels and decreased cell migration and invasion. These observations indicate that [Cl-]i plays important roles in metastatic function by regulating the ERK1/2 signaling pathway in human prostate cancer cells, and intracellular Cl- would be one of the key targets for anti-cancer therapy.

Guideline for Gynecological Practice in Japan: Japan Society of Obstetrics and Gynecology and Japan Association of Obstetricians and Gynecologists 2020 edition.

Nine years after the first edition of The Guideline for Gynecological Practice, which was jointly edited by The Japan Society of Obstetrics and Gynecology and The Japan Association of Obstetricians and Gynecologists, the 4th Revised Edition was published in 2020. The 2020 Guidelines includes 4 additional clinical questions (CQ), which brings the total to 99 CQ (12 on infectious disease, 29 on oncology and benign tumors, 29 on endocrinology and infertility and 29 on healthcare for women). Currently, a consensus has been reached on the Guidelines, and therefore, the objective of this report is to present the general policies regarding diagnostic and treatment methods used in standard gynecological outpatient care that are considered appropriate. At the end of each answer, the corresponding Recommendation Level (A, B, C) is indicated.

CD44s Induces miR-629-3p Expression in Association with Cisplatin Resistance in Head and Neck Cancer Cells.

Cisplatin (cis-diamminedichloroplatinum II [CDDP] ) is a well-known chemotherapeutic drug that has been used for the treatment of various types of human cancers, including head and neck cancer. Cisplatin exerts anticancer effects by causing DNA damage, replication defects, transcriptional inhibition, cell cycle arrest, and the induction of apoptosis. However, drug resistance is one of the most serious problems with cancer chemotherapy, and it causes expected therapeutic effects to not always be achieved. Here, we analyzed global microRNA (miRNA) expression in CD44 standard form (CD44s)-expressing SAS cells, and we identified miR-629-3p as being responsible for acquiring anticancer drug resistance in head and neck cancer. The introduction of miR-629-3p expression inhibited apoptotic cell death under cisplatin treatment conditions, and it promoted cell migration. Among the computationally predicted target genes of miR-629-3p, we found that a number of gene expressions were suppressed by the transfection with miR-629-3p. Using a xenografting model, we showed that miR-629-3p conferred cisplatin resistance to SAS cells. Clinically, increased miR-629-3p expression tended to be associated with decreased survival in head and neck cancer patients. In conclusion, our data suggest that the increased expression of miR-629-3p provides a mechanism of cisplatin resistance in head and neck cancer and may serve as a therapeutic target to reverse chemotherapy resistance.

CD44 exerts a functional role during EMT induction in cisplatin-resistant head and neck cancer cells.

A number of studies report that epithelial to mesenchymal transition (EMT) supports the generation and maintenance of cancer stem cells (CSCs), which show tumor seeding ability and drug resistance; however, the molecular mechanisms underlying induction of EMT-associated tumor malignancy remain unclear. The present study reports that oral cancer cells switch from expressing the CD44 variant form (CD44v) to expressing the standard form (CD44s) during acquisition of cisplatin-resistance, which resulted in EMT induction. CD44s induced an EMT phenotype in cisplatin resistant cells by up-regulating ZEB1, a transcriptional repressor of E-cadherin. More importantly, CD44s up-regulated ZEB1 by suppressing microRNA-200c, which is a non-coding RNA that directly represses the ZEB1 gene. These results demonstrate the importance of the association between platinum resistance and CD44s during EMT induction in oral cancer cells.

Quercetin is a Useful Medicinal Compound Showing Various Actions Including Control of Blood Pressure, Neurite Elongation and Epithelial Ion Transport.

Quercetin has multiple potential to control various cell function keeping our body condition healthy. In this review article, we describe the molecular mechanism on how quercetin exerts its action on blood pressure, neurite elongation and epithelial ion transport based from a viewpoint of cytosolic Cl- environments, which is recently recognized as an important signaling factor in various types of cells. Recent studies show various roles of cytosolic Cl- in regulation of blood pressure and neurite elongation, and prevention from bacterial and viral infection. We have found the stimulatory action of quercetin on Cl- transporter, Na+-K+-2Cl- cotransporter 1 (NKCC1; an isoform of NKCC), which has been recognized as one of the most interesting, fundamental actions of quercetin. In this review article, based on this stimulatory action of quercetin on NKCC1, we introduce the molecular mechanism of quercetin on: 1) blood pressure, 2) neurite elongation, and 3) epithelial Cl- secretion including tight junction forming in epithelial tissues. 1) Quercetin induces elevation of the cytosolic Cl- concentration via activation of NKCC1, leading to anti-hypertensive action by diminishing expression of epithelial Na+ channel (ENaC), a key ion channel involved in renal Na+ reabsorption, while quercetin has no effects on the blood pressure with normal salt intake. 2) Quercetin also has stimulatory effects on neurite elongation by elevating the cytosolic Cl- concentration via activation of NKCC1 due to tubulin polymerization facilitated through Cl--induced inhibition of GTPase. 3) Further, in lung airway epithelia quercetin stimulates Cl- secretion by increasing the driving force for Cl- secretion via elevation of the cytosolic Cl- concentration: this leads to water secretion, participating in prevention of our body from bacterial and viral infection. In addition to transcellular ion transport, quercetin regulates tight junction function via enhancement of tight junction integrity by modulating expression and assembling tight junction-forming proteins. Based on these observations, it is concluded that quercetin is a useful medicinal compound keeping our body to be in healthy condition.

Cytosolic Cl- Affects the Anticancer Activity of Paclitaxel in the Gastric Cancer Cell Line, MKN28 Cell.

BACKGROUND/AIMS: Our previous study revealed that cytosolic Cl- affected neurite elongation promoted via assembly of microtubule in rat pheochromocytoma PC12D cells and Cl--induced blockade of intrinsic GTPase enhanced tubulin polymerization in vitro. Paclitaxel (PTX) is a microtubule-targeted chemotherapeutic drug and stabilizes microtubules resulting in mainly blockade of mitosis at the metaphase-anaphase transition and induction of apoptosis. In the present study, we tried to clarify whether the cytosolic Cl- affected PTX ability to inhibit cell growth in the gastric cancer cell line, MKN28. METHODS: To clarify the cytosolic Cl- action on PTX-induced cell death and metaphase-anaphase transition in the gastric cancer cell line, MKN28 cell, and PTX-induced tubulin polymerization, we performed cell proliferation assay, cytosolic Cl- concentration measurement, immunofluorescence microscopy, and in vitro tubulin polymerization assay. RESULTS: The decline of cytosolic Cl- weakened the cytotoxic effect of PTX on cell proliferation of MKN28 cells, which could pass through the metaphase-anaphase transition. Moreover, in vitro PTX-induced tubulin polymerization was diminished under the low Cl- condition. CONCLUSIONS: Our results strongly suggest that the upregulation of cytosolic Cl- concentration would enhance the antitumor effect of PTX, and that the cytosolic Cl- would be one of the key targets for anti-cancer therapy.

Loss of microRNA-27b contributes to breast cancer stem cell generation by activating ENPP1.

Cancer stem cells (CSCs) have been identified in various types of cancer; however, the mechanisms by which cells acquire CSC properties such as drug resistance and tumour seeding ability are not fully understood. Here, we identified microRNA-27b (miR-27b) as a key regulator for the generation of a side-population in breast cancer cells that showed CSC properties, and also found that the anti-type II diabetes (T2D) drug metformin reduced this side-population via miR-27b-mediated repression of ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1), which is involved in T2D development. ENPP1 induced the generation of the side-population via upregulation of the ABCG2 transporter. ENPP1 was also identified as a substrate of the 26S proteasome, the activity of which is downregulated in CSCs. Overall, these results demonstrate that a T2D-associated gene plays an important role in tumour development and that its expression is strictly controlled at the mRNA and protein levels.

The Roles of MicroRNAs in Breast Cancer.

MicroRNAs (miRNAs) constitute a large family of small, approximately 20-22 nucleotide, non-coding RNAs that regulate the expression of target genes, mainly at the post-transcriptional level. Accumulating lines of evidence have indicated that miRNAs play important roles in the maintenance of biological homeostasis and that aberrant expression levels of miRNAs are associated with the onset of many diseases, including cancer. In various cancers, miRNAs play important roles in tumor initiation, drug resistance and metastasis. Recent studies reported that miRNAs could also be secreted via small endosome-derived vesicles called exosomes, which are derived from multiple cell types, including dendritic cells, lymphocytes, and tumor cells. Exosomal miRNAs play an important role in cell-to-cell communication and have been investigated as prognostic and diagnostic biomarkers. In this review, we summarize the major findings related to the functions of miRNAs in breast cancer, which is the most frequent cancer in women, and discuss the potential clinical uses of miRNAs, including their roles as therapeutic targets and diagnostic markers.

Lowered extracellular pH is involved in the pathogenesis of skeletal muscle insulin resistance.

Insulin resistance in the skeletal muscle is manifested by diminished insulin-stimulated glucose uptake and is a core factor in the pathogenesis of type 2 diabetes mellitus (DM), but the mechanism causing insulin resistance is still unknown. Our recent study has shown that pH of interstitial fluids was lowered in early developmental stage of insulin resistance in OLETF rats, a model of type 2 DM. Therefore, in the present study, we confirmed effects of the extracellular pH on the insulin signaling pathway in a rat skeletal muscle-derived cell line, L6 cell. The phosphorylation level (activation) of the insulin receptor was significantly diminished in low pH media. The phosphorylation level of Akt, which is a downstream target of the insulin signaling pathway, also decreased in low pH media. Moreover, the insulin binding to its receptor was reduced by lowering extracellular pH, while the expression of insulin receptors on the plasma membrane was not affected by the extracellular pH. Finally, insulin-stimulated 2-deoxyglucose uptake in L6 cells was diminished in low pH media. Our present study suggests that lowered extracellular pH conditions may produce the pathogenesis of insulin resistance in skeletal muscle cells.

The role of microRNAs in the regulation of cancer stem cells.

Cancer stem cells (CSCs) have been reported in many human tumors and are proposed to drive tumor initiation and progression. CSCs share a variety of biological properties with normal somatic stem cells such as the capacity for self-renewal, the propagation of differentiated progeny, and the expression of specific cell surface markers and stem cell genes. However, CSCs differ from normal stem cells in their chemoresistance and tumorigenic and metastatic activities. Despite their potential clinical importance, the regulation of CSCs at the molecular level is not well-understood. MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs that play an important role in the regulation of several cellular, physiological, and developmental processes. Aberrant miRNA expression is associated with many human diseases including cancer. miRNAs have been implicated in the regulation of CSC properties; therefore, a better understanding of the modulation of CSC gene expression by miRNAs could aid the identification of promising biomarkers and therapeutic targets. In the present review, we summarize the major findings on the regulation of CSCs by miRNAs and discuss recent advances that have improved our understanding of the regulation of CSCs by miRNA networks and may lead to the development of miRNA therapeutics specifically targeting CSCs.

A regulatory role of K(+)-Cl(-) cotransporter in the cell cycle progression of breast cancer MDA-MB-231 cells.

K(+)-Cl(-) cotransporter (KCC) has been shown to be involved in cell proliferation as well as cell volume regulation. A regulatory role of KCC in cell cycle progression of breast cancer MDA-MB-231 cells was explored by using synchronized MDA-MB-231 cells and dihydro-indenyloxy-alkanoic acid (DIOA), a potent inhibitor of KCC. MDA-MB-231 cells cultured in the presence of DIOA exhibited an increase in cell volume, a decrease in intracellular Cl(-) concentration, and reduction in cell proliferation with the G0/G1 phase arrest, which was accompanied with down-regulation of cyclin D1 and cyclin E2, and up-regulation of p21. Among these molecules, the expression of cyclin E2, a molecule essential for the transition from G1 to S phase, was markedly suppressed by DIOA treatment. DIOA-mediated up- or down-regulation of these molecules occurred at the transcriptional level. These findings suggest that KCC plays an important role in the early phase of cell cycle progression by regulating the expression of cyclin D1, cyclin E2, and p21, the molecules essential for the cell cycle progression.

microRNA expression profiles in oral squamous cell carcinoma.

microRNAs (miRNAs) are involved in cancer pathogenesis, apoptosis and cell growth, thereby functioning as both tumor suppressors and oncogenes. However, the expression patterns and roles of miRNAs in oral squamous cell carcinoma (OSCC) remain largely unknown. We hypothesized that oral cancer may have a unique miRNA profile, which in turn may play a critical role in oral cancer development, progression, diagnosis and prognosis. We, therefore, investigated the expression profiles of 29 OSCC tumors and 7 normal oral mucosal samples. The miRNA expression patterns in OSCC were examined by TaqMan-based microRNA assays. We were subsequently able to identify the candidates of cancer-related miRNAs through analysis of the miRNA expression profiles. In conclusion, OSCC tissues were shown to have a unique miRNA profile pattern when compared with that in normal tissues. The present study may provide useful information for further investigation of the functional roles of miRNAs in OSCC development, progression, diagnosis and prognosis.

Improvement of insulin resistance, blood pressure and interstitial pH in early developmental stage of insulin resistance in OLETF rats by intake of propolis extracts.

Propolis, a resinous mixture collected from plants by the Apis mellifera bee, contains high level nutrient factors including vitamins, polyphenols, and amino acids that would be expected to improve insulin sensitivity. Insulin resistance would secondarily cause elevation of blood pressure and increase the risk of cardiovascular diseases. The purpose of this study is to investigate the effect of propolis extracts on blood glucose levels and blood pressures in an early developmental stage of insulin resistance in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. OLETF rats (10 weeks old) were divided into three different groups: normal diet, 0.1% propolis diet, and 0.5% propolis diet. After 8 weeks, blood glucose levels, blood pressures, plasma metabolic factors and hormones, and interstitial fluid pH were measured. Casual blood glucose levels were decreased associated with a reduction of plasma insulin levels in both propolis diet groups compared with normal diet group. Propolis decreased systolic blood pressure with no significant changes in plasma aldosterone levels. We also found that interstitial fluid pH in ascites, liver, and skeletal muscle was higher in rats fed propolis diet than rats fed normal diet. These data suggests that dietary propolis improves insulin sensitivity and blood pressures in the early stage of the process in development of insulin resistance, which may be mediated by suppression of metabolic acidosis.

An inhibitor of Na(+)/H(+) exchanger (NHE), ethyl-isopropyl amiloride (EIPA), diminishes proliferation of MKN28 human gastric cancer cells by decreasing the cytosolic Cl(-) concentration via DIDS-sensitive pathways.

BACKGROUND/AIMS: Tumor cells produce a large amount of acidic metabolites due to their high metabolic condition. However, cytosolic pH (pH(c)) of tumor cells is identical to or even slightly higher than that of normal cells. To maintain pH(c) at a normal or higher level, tumor cells would have to have higher expression and/or activity of H(+) transporting systems than normal cells. The purpose of the present study was to identify effects of ethyl-isopropyl amiloride (EIPA, an inhibitor of Na(+)/H(+) exchanger (NHE)) on proliferation of human gastric cancer MKN28 cells. METHODS: Effects of EIPA on proliferation, pH(c), [Cl(-)](c) and expression of proteins regulating cell cycle and MAPKs were studied in MKN28 expressing NHE exposed to EIPA for 48 h. RESULTS: EIPA suppressed proliferation of MKN28 cells by causing G(0)/G(1) arrest without any significant effects on pH(c), but associated with reduction of [Cl(-)](c). Although EIPA alone had no effects on pH(c), EIPA co-applied with DIDS (an inhibitor of Cl(-)/HCO(3)(-) exchangers; i.e., anion exchanger (AE) and Na+-driven Cl(-)/HCO(3)(-) exchanger (NDCBE)) reduced pH(c), suggesting that DIDS-sensitive Cl(-)/HCO(3)(-) transporters such as AE and/or NDCBE keep pH(c) normal by stimulating HCO(3)(-) uptake coupled with Cl(-) release under an NHE-inhibited condition. EIPA-induced lowered [Cl(-)](c) up-regulated expression of p21associated with phosphorylation of MAPKs, suppressing proliferation associated with G(0)/G(1) arrest. CONCLUSIONS: EIPA suppressed proliferation of MKN28 cells through up-regulation of p21 expression via reduction of [Cl(-)](c) as a result from DIDS-sensitive Cl(-)/HCO(3)(-) exchanger-mediated compensation for keeping pH(c) normal under an NHE-inhibited condition. This is the first study revealing that an NHE inhibitor suppressed the proliferation of cancer cells by reducing [Cl(-)](c) but not pH(c).

CLC anion channel regulatory phosphorylation and conserved signal transduction domains.

The signaling mechanisms that regulate CLC anion channels are poorly understood. Caenorhabditis elegans CLH-3b is a member of the CLC-1/2/Ka/Kb channel subfamily. CLH-3b is activated by meiotic cell-cycle progression and cell swelling. Inhibition is brought about by GCK-3 kinase-mediated phosphorylation of S742 and S747 located on a ∼176 amino acid disordered domain linking CBS1 and CBS2. Much of the inter-CBS linker is dispensable for channel regulation. However, deletion of a 14 amino acid activation domain encompassing S742 and S747 inhibits channel activity to the same extent as GCK-3. The crystal structure of CmCLC demonstrated that CBS2 interfaces extensively with an intracellular loop connecting membrane helices H and I, the C-terminus of helix D, and a short linker connecting helix R to CBS1. Point mutagenesis of this interface identified two highly conserved aromatic amino acid residues located in the H-I loop and the first α-helix (α1) of CBS2. Mutation of either residue to alanine rendered CLH-3b insensitive to GCK-3 inhibition. We suggest that the dephosphorylated activation domain normally interacts with CBS1 and/or CBS2, and that conformational information associated with this interaction is transduced through a conserved signal transduction module comprising the H-I loop and CBS2 α1.

Differential regulation of a CLC anion channel by SPAK kinase ortholog-mediated multisite phosphorylation.

Shrinkage-induced inhibition of the Caenorhabditis elegans cell volume and cell cycle-dependent CLC anion channel CLH-3b occurs by concomitant phosphorylation of S742 and S747, which are located on a 175 amino acid linker domain between cystathionine-ß-synthase 1 (CBS1) and CBS2. Phosphorylation is mediated by the SPAK kinase homolog GCK-3 and is mimicked by substituting serine residues with glutamate. Type 1 serine/threonine protein phosphatases mediate swelling-induced channel dephosphorylation. S742E/S747E double mutant channels are constitutively inactive and cannot be activated by cell swelling. S742E and S747E mutant channels were fully active in the absence of GCK-3 and were inactive when coexpressed with the kinase. Both channels responded to cell volume changes. However, the S747E mutant channel activated and inactivated in response to cell swelling and shrinkage, respectively, much more slowly than either wild-type or S742E mutant channels. Slower activation and inactivation of S747E was not due to altered rates of dephosphorylation or dephosphorylation-dependent conformational changes. GCK-3 binds to the 175 amino acid inter-CBS linker domain. Coexpression of wild-type CLH-3b and GCK-3 with either wild-type or S742E linkers gave rise to similar channel activity and regulation. In contrast, coexpression with the S747E linker greatly enhanced basal channel activity and increased the rate of shrinkage-induced channel inactivation. Our findings suggest the intriguing possibility that the phosphorylation state of S742 in S747E mutant channels modulates GCK-3/channel interaction and hence channel phosphorylation. These results provide a foundation for further detailed studies of the role of multisite phosphorylation in regulating CLH-3b and GCK-3 activity.

Regulation of epithelial sodium transport via epithelial Na+ channel.

Renal epithelial Na+ transport plays an important role in homeostasis of our body fluid content and blood pressure. Further, the Na+ transport in alveolar epithelial cells essentially controls the amount of alveolar fluid that should be kept at an appropriate level for normal gas exchange. The epithelial Na+ transport is generally mediated through two steps: (1) the entry step of Na+ via epithelial Na+ channel (ENaC) at the apical membrane and (2) the extrusion step of Na+ via the Na+, K+-ATPase at the basolateral membrane. In general, the Na+ entry via ENaC is the rate-limiting step. Therefore, the regulation of ENaC plays an essential role in control of blood pressure and normal gas exchange. In this paper, we discuss two major factors in ENaC regulation: (1) activity of individual ENaC and (2) number of ENaC located at the apical membrane.

Intracellular chloride regulates cell proliferation through the activation of stress-activated protein kinases in MKN28 human gastric cancer cells.

Recently, we reported that reduction of intracellular Cl(-) concentration ([Cl(-)](i)) inhibited proliferation of MKN28 gastric cancer cells by diminishing the transition rate from G(1) to S cell-cycle phase through upregulation of p21, cyclin-dependent kinase inhibitor, in a p53-independent manner. However, it is still unknown how intracellular Cl(-) regulates p21 expression level. In this study, we demonstrate that mitogen-activated protein kinases (MAPKs) are involved in the p21 upregulation and cell-cycle arrest induced by reduction of [Cl(-)](i). Culture of MKN28 cells in a low Cl(-) medium significantly induced phosphorylation (activation) of MAPKs (ERK, p38, and JNK) and G(1)/S cell-cycle arrest. To clarify the involvement of MAPKs in p21 upregulation and cell growth inhibition in the low Cl(-) medium, we studied effects of specific MAPKs inhibitors on p21 upregulation and G(1)/S cell-cycle arrest in MKN28 cells. Treatment with an inhibitor of p38 or JNK significantly suppressed p21 upregulation caused by culture in a low Cl(-) medium and rescued MKN28 cells from the low Cl(-)-induced G(1) cell-cycle arrest, whereas treatment with an ERK inhibitor had no significant effect on p21 expression or the growth of MKN28 cells in the low Cl(-) medium. These results strongly suggest that the intracellular Cl(-) affects the cell proliferation via activation of p38 and/or JNK cascades through upregulation of the cyclin-dependent kinase inhibitor (p21) in a p53-independent manner in MKN28 cells.