Role of Piezo2 in Schwann Cell Volume Regulation and Its Impact on Neurotrophic Release Regulation.

BACKGROUND/AIMS: Tactile perception relies on mechanoreceptors and nerve fibers, including c-fibers, Aß-fibers and Aδ-fibers. Schwann cells (SCs) play a crucial role in supporting nerve fibers, with non-myelinating SCs enwrapping c-fibers and myelinating SCs ensheathing Aß and Aδ fibers. Recent research has unveiled new functions for cutaneous sensory SCs, highlighting the involvement of nociceptive SCs in pain perception and Meissner corpuscle SCs in tactile sensation. Furthermore, Piezo2, previously associated with Merkel cell tactile sensitivity, has been identified in SCs. The goal of this study was to investigate the channels implicated in SC mechanosensitivity and the release process of neurotrophic factor secretion. METHODS: Immortalized IFRS1 SCs and human primary SCs generated two distinct subtypes of SCs: undifferentiated and differentiated SCs. Quantitative PCR was employed to evaluate the expression of differentiation markers and mechanosensitive channels, including TRP channels (TRPV4, TRPM7 and TRPA1) and Piezo channels (Piezo1 and Piezo2). To validate the functionality of specific mechanosensitive channels, Ca2+ imaging and electronic cell sizing experiments were conducted under hypotonic conditions, and inhibitors and siRNAs were used. Protein expression was assessed by Western blotting and immunostaining. Additionally, secretome analysis was performed to evaluate the release of neurotrophic factors in response to hypotonic stimulation, with BDNF, a representative trophic factor, quantified using ELISA. RESULTS: Induction of differentiation increased Piezo2 mRNA expression levels both in IFRS1 and in human primary SCs. Both cell types were responsive to hypotonic solutions, with differentiated SCs displaying a more pronounced response. Gd3+ and FM1-43 effectively inhibited hypotonicity-induced Ca2+ transients in differentiated SCs, implicating Piezo2 channels. Conversely, inhibitors of Piezo1 and TRPM7 (Dooku1 and NS8593, respectively) had no discernible impact. Moreover, Piezo2 in differentiated SCs appeared to participate in regulatory volume decreases (RVD) after cell swelling induced by hypotonic stimulation. A Piezo2 deficiency correlated with reduced RVD and prolonged cell swelling, leading to heightened release of the neurotrophic factor BDNF by upregulating the function of endogenously expressed Ca2+-permeable TRPV4. CONCLUSION: Our study unveils the mechanosensitivity of SCs and implicates Piezo2 channels in the release of neurotrophic factors from SCs. These results suggest that Piezo2 may contribute to RVD, thereby maintaining cellular homeostasis, and may also serve as a negative regulator of neurotrophic factor release. These findings underscore the need for further investigation into the role of Piezo2 in SC function and neurotrophic regulation.

TRPV4 and chloride channels mediate volume sensing in trabecular meshwork cells.

Aqueous humor drainage from the anterior eye determines intraocular pressure (IOP) under homeostatic and pathological conditions. Swelling of the trabecular meshwork (TM) alters its flow resistance but the mechanisms that sense and transduce osmotic gradients remain poorly understood. We investigated TM osmotransduction and its role in calcium and chloride homeostasis using molecular analyses, optical imaging, and electrophysiology. Anisosmotic conditions elicited proportional changes in TM cell volume, with swelling, but not shrinking, evoking elevations in intracellular calcium concentration [Ca2+]TM. Hypotonicity-evoked calcium signals were sensitive to HC067047, a selective blocker of TRPV4 channels, whereas the agonist GSK1016790A promoted swelling under isotonic conditions. TRPV4 inhibition partially suppressed hypotonicity-induced volume increases and reduced the magnitude of the swelling-induced membrane current, with a substantial fraction of the swelling-evoked current abrogated by Cl- channel antagonists 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS) and niflumic acid. The transcriptome of volume-sensing chloride channel candidates in primary human was dominated by ANO6 transcripts, with moderate expression of ANO3, ANO7, and ANO10 transcripts and low expression of LTTRC genes that encode constituents of the volume-activated anion channel. Imposition of 190 mosM but not 285 mosM hypotonic gradients increased conventional outflow in mouse eyes. TRPV4-mediated cation influx thus works with Cl- efflux to sense and respond to osmotic stress, potentially contributing to pathological swelling, calcium overload, and intracellular signaling that could exacerbate functional disturbances in inflammatory disease and glaucoma.NEW & NOTEWORTHY Intraocular pressure is dynamically regulated by the flow of aqueous humor through paracellular passages within the trabecular meshwork (TM). This study shows hypotonic gradients that expand the TM cell volume and reduce the outflow facility in mouse eyes. The swelling-induced current consists of TRPV4 and chloride components, with TRPV4 as a driver of swelling-induced calcium signaling. TRPV4 inhibition reduced swelling, suggesting a novel treatment for trabeculitis and glaucoma.

Maternal Fluoride Exposure Exerts Different Toxicity Patterns in Parotid and Submandibular Glands of Offspring Rats.

There is currently a controversial and heated debate about the safety and ethical aspects of fluoride (F) used for human consumption. Thus, this study assessed the effects of prenatal and postnatal F exposure of rats on the salivary glands of their offspring. Pregnant rats were exposed to 0, 10, or 50 mg F/L from the drinking water, from the first day of gestation until offspring weaning (42 days). The offspring rats were euthanized for the collection of the parotid (PA) and submandibular (SM) glands, to assess the oxidative biochemistry and to perform morphometric and immunohistochemical analyses. F exposure was associated with a decrease in the antioxidant competence of PA in the 10 mg F/L group, contrasting with the increase observed in the 50 mg F/L group. On the other hand, the antioxidant competence of the SM glands was decreased at both concentrations. Moreover, both 10 and 50 mg F/L groups showed lower anti-α-smooth muscle actin immunostaining area in SM, while exposure to 50 mg F/L was associated with changes in gland morphometry by increasing the duct area in both glands. These findings demonstrate a greater susceptibility of the SM glands of the offspring to F at high concentration in comparison to PA, reinforcing the need to adhere to the optimum F levels recommended by the regulatory agencies. Such findings must be interpreted with caution, especially considering their translational meaning.

Cardiomyocyte BRAF and type 1 RAF inhibitors promote cardiomyocyte and cardiac hypertrophy in mice in vivo.

The extracellular signal-regulated kinase 1/2 (ERK1/2) cascade promotes cardiomyocyte hypertrophy and is cardioprotective, with the three RAF kinases forming a node for signal integration. Our aims were to determine if BRAF is relevant for human heart failure, whether BRAF promotes cardiomyocyte hypertrophy, and if Type 1 RAF inhibitors developed for cancer (that paradoxically activate ERK1/2 at low concentrations: the 'RAF paradox') may have the same effect. BRAF was up-regulated in heart samples from patients with heart failure compared with normal controls. We assessed the effects of activated BRAF in the heart using mice with tamoxifen-activated Cre for cardiomyocyte-specific knock-in of the activating V600E mutation into the endogenous gene. We used echocardiography to measure cardiac dimensions/function. Cardiomyocyte BRAFV600E induced cardiac hypertrophy within 10 d, resulting in increased ejection fraction and fractional shortening over 6 weeks. This was associated with increased cardiomyocyte size without significant fibrosis, consistent with compensated hypertrophy. The experimental Type 1 RAF inhibitor, SB590885, and/or encorafenib (a RAF inhibitor used clinically) increased ERK1/2 phosphorylation in cardiomyocytes, and promoted hypertrophy, consistent with a 'RAF paradox' effect. Both promoted cardiac hypertrophy in mouse hearts in vivo, with increased cardiomyocyte size and no overt fibrosis. In conclusion, BRAF potentially plays an important role in human failing hearts, activation of BRAF is sufficient to induce hypertrophy, and Type 1 RAF inhibitors promote hypertrophy via the 'RAF paradox'. Cardiac hypertrophy resulting from these interventions was not associated with pathological features, suggesting that Type 1 RAF inhibitors may be useful to boost cardiomyocyte function.

Evaluation of human relevance of Nicofluprole-induced rat thyroid disruption.

Nicofluprole is a novel insecticide of the phenylpyrazole class conferring selective antagonistic activity on insect GABA receptors. After repeated daily dietary administration to Wistar rats for 28/90 days, Nicofluprole induced increases in thyroid (and liver) weight, associated with histopathology changes. Nicofluprole did not inhibit thyroid peroxydase nor sodium/iodide symporter, two key players in the biosynthesis of thyroid hormones, indicating the absence of a direct thyroid effect. The results seen in rats suggested a mode of action of Nicofluprole driven by the molecular initiating event of CAR/PXR nuclear receptor activation in livers, with key events of increases in liver weight and hypertrophy, decreasing circulatory thyroid hormones, a compensatory increase in TSH release and follicular cell hypertrophy. To explore the relevance of these changes to humans, well established in vitro rat and human sandwich-cultured hepatocytes were exposed to Nicofluprole up to 7 days. A concentration-dependent CYP3A induction (PXR-activation), an increase in T4-glucuronoconjugation accompanied by UGT1A/2B inductions was observed in rat but not in human hepatocytes. The inductions seen with Nicofluprole in rat (in vivo and in vitro in hepatocytes) that were absent in human hepatocytes represent another example of species-selectivity of nuclear CAR/PXR receptor activators. Importantly, the different pattern observed in rat and human models demonstrate that Nicofluprole-related thyroid effects observed in the rat are with no human relevance.

Effects and Related Mechanisms of the Senolytic Agent ABT-263 on the Survival of Irradiated A549 and Ca9-22 Cancer Cells.

Senolytic agents eliminate senescent cells and are expected to reduce senescent cell-mediated adverse effects in cancer therapy. However, the effects of senolytic agents on the survival of irradiated cancer cells remain unknown. Here, the effects of the senolytic agent ABT-263 on the survival of irradiated A549 and Ca9-22 cancer cells were investigated. ABT-263 was added to the culture medium after irradiation. SA-ß-gal activity and cell size, which are hallmarks of cell senescence, were evaluated using a flow cytometer. The colony-forming assay and annexin V staining were performed to test cell survival. We first confirmed that radiation increased the proportion of cells with high SA-ß-gal activity and that ABT-263 decreased it. Of note, ABT-263 decreased the survival of irradiated cancer cells and increased the proportion of radiation-induced annexin V+ cells. Furthermore, the caspase inhibitor suppressed the ABT-263-induced decrease in the survival of irradiated cells. Intriguingly, ABT-263 decreased the proportion of SA-ß-gal low-activity/large cells in the irradiated A549 cells, which was recovered by the caspase inhibitor. Together, these findings suggest that populations maintaining the ability to proliferate existed among the irradiated cancer cells showing senescence-related features and that ABT-263 eliminated the population, which led to decreased survival of irradiated cancer cells.

Reactivities of a Prostanoid EP2 Agonist, Omidenepag, Are Useful for Distinguishing between 3D Spheroids of Human Orbital Fibroblasts without or with Graves' Orbitopathy.

BACKGROUND: To obtain new insights into the activation of the thyroid-stimulating hormone (TSH) and insulin-like growth factor 1 (IGF-1) receptors in human orbital fibroblasts (n-HOFs), the effects of the prostanoid EP2 agonist, omidenepag (OMD), and a rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, ripasudil (Rip) were evaluated using three-dimension (3D) n-HOFs spheroids in the absence and presence of the recombinant human TSH receptor antibodies, M22 and IGF-1. METHODS: The effects of 100 nM OMD or 10 µM Rip on the physical properties, size, stiffness, and mRNA expression of several extracellular matrix (ECM) molecules, their regulator, inflammatory cytokines, and endoplasmic reticulum (ER) stress-related factors were examined and compared among 3D spheroids of n-HOFs, M22-/IGF-1-activated n-HOFs and GO-related human orbital fibroblasts (GHOFs). RESULTS: The physical properties and mRNA expressions of several genes of the 3D n-HOFs spheroids were significantly and diversely modulated by the presence of OMD or Rip. The OMD-induced effects on M22-/IGF-1-activated n-HOFs were similar to the effects caused by GHOHs, but quite different from those of n-HOFs. CONCLUSIONS: The findings presented herein indicate that the changes induced by OMD may be useful in distinguishing between n-HOFs and GHOFs.

Comparison of Isotonic Activation of Cell Volume Regulation in Rat Peritoneal Mesothelial Cells and in Kidney Outer Medullary Collecting Duct Principal Cells.

In disease states, mesothelial cells are exposed to variable osmotic conditions, with high osmotic stress exerted by peritoneal dialysis (PD) fluids. They contain unphysiologically high concentrations of glucose and result in major peritoneal membrane transformation and PD function loss. The effects of isotonic entry of urea and myo-inositol in hypertonic (380 mOsm/kg) medium on the cell volume of primary cultures of rat peritoneal mesothelial cells and rat kidney outer medullary collecting duct (OMCD) principal cells were studied. In hypertonic medium, rat peritoneal mesothelial cells activated a different mechanism of cell volume regulation in the presence of isotonic urea (100 mM) in comparison to rat kidney OMCD principal cells. In kidney OMCD cells inflow of urea into the shrunken cell results in restoration of cell volume. In the shrunken peritoneal mesothelial cells, isotonic urea inflow caused a small volume increase and activated regulatory volume decrease (RVD). Isotonic myo-inositol activated RVD in hypertonic medium in both cell types. Isotonic application of both osmolytes caused a sharp increase of intracellular calcium both in peritoneal mesothelial cells and in kidney OMCD principal cells. In conclusion, peritoneal mesothelial cells exhibit RVD mechanisms when challenged with myo-inositol and urea under hyperosmolar isotonic switch from mannitol through involvement of calcium-dependent control. Myo-inositol effects were identical with the ones in OMCD principal cells whereas urea effects in OMCD principal cells led to no RVD induction.

Functional drug susceptibility testing using single-cell mass predicts treatment outcome in patient-derived cancer neurosphere models.

Functional precision medicine aims to match individual cancer patients to optimal treatment through ex vivo drug susceptibility testing on patient-derived cells. However, few functional diagnostic assays have been validated against patient outcomes at scale because of limitations of such assays. Here, we describe a high-throughput assay that detects subtle changes in the mass of individual drug-treated cancer cells as a surrogate biomarker for patient treatment response. To validate this approach, we determined ex vivo response to temozolomide in a retrospective cohort of 69 glioblastoma patient-derived neurosphere models with matched patient survival and genomics. Temozolomide-induced changes in cell mass distributions predict patient overall survival similarly to O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and may aid in predictions in gliomas with mismatch-repair variants of unknown significance, where MGMT is not predictive. Our findings suggest cell mass is a promising functional biomarker for cancers and drugs that lack genomic biomarkers.

Empagliflozin Treatment Is Associated With Improvements in Cardiac Energetics and Function and Reductions in Myocardial Cellular Volume in Patients With Type 2 Diabetes.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce the risk of major adverse cardiovascular (CV) events and hospitalization for heart failure (HF) in patients with type 2 diabetes (T2D). Using CV MRI (CMR) and 31P-MRS in a longitudinal cohort study, we aimed to investigate the effects of the selective SGLT2 inhibitor empagliflozin on myocardial energetics and cellular volume, function, and perfusion. Eighteen patients with T2D underwent CMR and 31P-MRS scans before and after 12 weeks' empagliflozin treatment. Plasma N-terminal prohormone B-type natriuretic peptide (NT-proBNP) levels were measured. Ten volunteers with normal glycemic control underwent an identical scan protocol at a single visit. Empagliflozin treatment was associated with significant improvements in phosphocreatine-to-ATP ratio (1.52 to 1.76, P = 0.009). This was accompanied by a 7% absolute increase in the mean left ventricular ejection fraction (P = 0.001), 3% absolute increase in the mean global longitudinal strain (P = 0.01), 8 mL/m2 absolute reduction in the mean myocardial cell volume (P = 0.04), and 61% relative reduction in the mean NT-proBNP (P = 0.05) from baseline measurements. No significant change in myocardial blood flow or diastolic strain was detected. Empagliflozin thus ameliorates the "cardiac energy-deficient" state, regresses adverse myocardial cellular remodeling, and improves cardiac function, offering therapeutic opportunities to prevent or modulate HF in T2D.

Modulation of Inherent Niches in 3D Multicellular MSC Spheroids Reconfigures Metabolism and Enhances Therapeutic Potential.

Multicellular spheroids show three-dimensional (3D) organization with extensive cell-cell and cell-extracellular matrix interactions. Owing to their native tissue-mimicking characteristics, mesenchymal stem cell (MSC) spheroids are considered promising as implantable therapeutics for stem cell therapy. Herein, we aim to further enhance their therapeutic potential by tuning the cultivation parameters and thus the inherent niche of 3D MSC spheroids. Significantly increased expression of multiple pro-regenerative paracrine signaling molecules and immunomodulatory factors by MSCs was observed after optimizing the conditions for spheroid culture. Moreover, these alterations in cellular behaviors may be associated with not only the hypoxic niche developed in the spheroid core but also with the metabolic reconfiguration of MSCs. The present study provides efficient methods for manipulating the therapeutic capacity of 3D MSC spheroids, thus laying solid foundations for future development and clinical application of spheroid-based MSC therapy for regenerative medicine.

Indole glucosinolates exhibit anti-inflammatory effects on Ehrlich ascites carcinoma cells through modulation of inflammatory markers and miRNAs.

BACKGROUND: Nuclear factor-κB (NF-κB) has been identified as the major link between inflammation and cancer. Natural agents that inhibit this pathway are essential in attenuating inflammation induced by cancer or chemotherapeutic drugs. High intake of Brassicaceae vegetables has been determined to modulate essential pathways related to chronic diseases. In this study, we investigated the anti-proliferative and anti-inflammatory effects of the indole glucosinolates; indole-3-carbinol (I3C) and its metabolite 3,3-diindolylmethane (DIM) on the inflammatory biomarkers and miRNAs controlling the NF-κB pathway. METHODS AND RESULTS: In our study, we inoculated Ehrlich ascites carcinoma (EAC) cells in female albino mice, which increased their packed cell volume and induced a significant increase in the levels of several cytokines and inflammatory biomarkers (NF-κB IL-6, IL-1b, TNF-α, and NO). A significant elevation in inflammatory-medicated miRNAs (miR-31 and miR-21) was also noted. Treatment with 5-fluorouracil (5-FU) significantly reduced packed cell volume and viable cell count. However, it was accompanied by a significant increase in the levels of inflammatory markers and expression of miR-31 and miR-21. Nevertheless, although treatment with indoles (I3C and DIM) significantly reduced the packed cell volume and viable cell count, their prominent effect was the marked reduction of all inflammatory biomarkers compared to both the EAC untreated group and the EAC group treated with 5-FU. Moreover, the anti-inflammatory effect of I3C or DIM was accompanied by a significant decrease in the expression of miR-31 and miR-21. CONCLUSION: Our findings have; therefore, revealed that I3C and DIM have strong anti-inflammatory effects, implying that their use as a co-treatment with chemotherapeutic drugs can effectively improve the anti-tumor effect of chemotherapeutic drugs.

RGD-targeted redox responsive nano micelle: co-loading docetaxel and indocyanine green to treat the tumor.

Cancer, also known as a malignant tumor, has developed into a type of disease with the highest fatality rate, seriously threatening the lives and health of people. Chemotherapy is one of the most important methods for the treatment of cancer. However, chemotherapy drugs have some problems, such as low solubility and lack of targeting, which severely limit their clinical applications. To solve these problems, we designed a block copolymer that has a disulfide bond response. The polymer uses RGD peptide (arginine-glycine-aspartic acid) as the active targeting group, PEG (polyethylene glycol) as the hydrophilic end, and PCL (polycaprolactone) as the hydrophobic end. Then we utilized the amphiphilic polymer as a carrier to simultaneously deliver DOC (docetaxel) and ICG (indocyanine green), to realize the combined application of chemotherapy and photothermal therapy. The antitumor efficacy in vivo and histology analysis showed that the DOC/ICG-loaded micelle exhibited higher antitumor activity. The drug delivery system improved the solubility of DOC and the stability of ICG, realized NIR-guided photothermal therapy, and achieved an ideal therapeutic effect.

Diclofenac Alters the Cell Cycle Progression of the Green Alga Chlamydomonas reinhardtii.

The aim of the study was to verify the hypothesis that a potential cause of the phytotoxicity of diclofenac (DCF, a non-steroidal anti-inflammatory drug) is an effect of cell cycle progression. This research was conducted using synchronous cultures of a model organism, green alga Chlamydomonas reinhardtii. The project examined DCF effects on selected parameters that characterize cell cycle progression, such as cell size, attainment of commitment points, DNA replication, number of nuclei formed during cells division and morphology of cells in consecutive stages of the cell cycle, together with the physiological and biochemical parameters of algae cells at different stages. We demonstrated that individual cell growth remained unaffected, whereas cell division was delayed in the DCF-treated groups grown in continuous light conditions, and the number of daughter cells from a single cell decreased. Thus, the cell cycle progression is a target affected by DCF, which has a similar anti-proliferative effect on mammalian cells.

The role of bFGF in preventing the shrinkage of cardiac progenitor cell-engineered conduction tissue by downregulating α-SMA expression.

AIMS: Engineered conduction tissues (ECTs) fabricated from cardiac progenitor cells (CPCs) and collagen sponges were precisely targeted for the treatment of atrioventricular conduction block in our previous studies. However, obvious shrinkage and deformation of ECTs was observed during in vitro culture. According to the literature, it can be speculated that basic fibroblast growth factor (bFGF) may downregulate alpha-smooth muscle actin (α-SMA) produced by CPCs to prevent the shrinkage of CPC-engineered conduction tissues. MAIN METHODS: In this study, culture media with or without bFGF were used for both cell culture and 3D tissue construction. The expression of α-SMA and the size change of engineered tissue were analyzed to evaluate the feasibility of adding bFGF to regulate α-SMA expression and shrinkage of constructs. In addition, cardiac-specific examinations were performed to evaluate the effect of bFGF on cardiac tissue formation. KEY FINDINGS: Supplementation with bFGF efficiently relieved shrinkage of engineered tissue by downregulating the expression of α-SMA at both the cellular and 3D tissue levels. Moreover, bFGF had a positive influence on cardiac tissue formation in terms of cell viability, tissue organization and electrical conduction velocity. SIGNIFICANCE: This study provides a guide for both shape control and quality improvement of CPC-engineered cardiac tissues.

New Insights into Red Blood Cell Microcytosis upon mTOR Inhibitor Administration.

The aim of this study was to evaluate the effect of everolimus, a mammalian target of rapamycin (mTOR) inhibitor, on red blood cell parameters in the context of iron homeostasis in patients with tuberous sclerosis complex (TSC) and evaluate its effect on cell size in vitro. Everolimus has a significant impact on red blood cell parameters in patients with TSC. The most common alteration was microcytosis. The mean MCV value decreased by 9.2%, 12%, and 11.8% after 3, 6, and 12 months of everolimus treatment. The iron level declined during the first 3 months, and human soluble transferrin receptor concentration increased during 6 months of therapy. The size of K562 cells decreased when cultured in the presence of 5 µM everolimus by approximately 8%. The addition of hemin to the cell culture with 5 µM everolimus did not prevent any decrease in cell size. The stage of erythroid maturation did not affect the response to everolimus. Our results showed that the mTOR inhibitor everolimus caused red blood cell microcytosis in vivo and in vitro. This effect is not clearly related to a deficit of iron and erythroid maturation. This observation confirms that mTOR signaling plays a complex role in the control of cell size.

Genetic interaction between F-box motif encoding YDR131C and retrograde signaling-related RTG1 regulates the stress response and apoptosis in Saccharomyces cerevisiae.

The retrograde signaling pathway is well conserved from yeast to humans, which regulates cell adaptation during stress conditions and prevents cell death. One of its components, RTG1 encoded Rtg1p in association with Rtg3p communicates between mitochondria, nucleus, and peroxisome during stress for adaptation, by regulation of transcription. The F-box motif protein encoded by YDR131C  constitutes a part of SCF Ydr131c -E3 ligase complex, with unknown function; however, it is known that retrograde signaling is modulated by the E3 ligase complex. This study reports epistasis interaction between YDR131C and RTG1, which regulates cell growth, response to genotoxic stress, decreased apoptosis, resistance to petite mutation, and cell wall integrity. The cells of ydr131cΔrtg1Δ genetic background exhibits growth rate improvement however, sensitivity to hydroxyurea, itraconazole antifungal agent and synthetic indoloquinazoline-based alkaloid (8-fluorotryptanthrin, RK64), which disrupts the cell wall integrity in Saccharomyces cerevisiae. The epistatic interaction between YDR131C and RTG1 indicates a link between protein degradation and retrograde signaling pathways.

Concomitant attenuation of HMGCR expression and activity enhances the growth inhibitory effect of atorvastatin on TGF-ß-treated epithelial cancer cells.

Epithelial-mesenchymal transition (EMT) in primary tumor cells is a key prerequisite for metastasis initiation. Statins, cholesterol-lowering drugs, can delay metastasis formation in vivo and attenuate the growth and proliferation of tumor cells in vitro. The latter effect is stronger in tumor cells with a mesenchymal-like phenotype than in those with an epithelial one. However, the effect of statins on epithelial cancer cells treated with EMT-inducing growth factors such as transforming growth factor-ß (TGF-ß) remains unclear. Here, we examined the effect of atorvastatin on two epithelial cancer cell lines following TGF-ß treatment. Atorvastatin-induced growth inhibition was stronger in TGF-ß-treated cells than in cells not thusly treated. Moreover, treatment of cells with atorvastatin prior to TGF-ß treatment enhanced this effect, which was further potentiated by the simultaneous reduction in the expression of the statin target enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). Dual pharmacological targeting of HMGCR can thus strongly inhibit the growth and proliferation of epithelial cancer cells treated with TGF-ß and may also improve statin therapy-mediated attenuation of metastasis formation in vivo.

Activation of Wnt signalling reduces the population of cancer stem cells in ameloblastoma.

OBJECTIVES: The treatment of ameloblastoma, an odontogenic epithelial tumour destroying jawbone, mainly depends on radical destructive resections. Other therapeutic options are limited by the characteristics of ameloblastoma, such as high recurrence rates and resistance to radiation and chemotherapy, which implies possible existence of cancer stem cells (CSCs) in ameloblastoma. Here, we identified a putative CSC population in immortalized and primary human ameloblastoma cells and examined possible therapeutic reagents to reduce the CSC population. METHODS: We investigated subpopulations of AM-1 cell line and human ameloblastoma cells using immunocytochemistry and flow cytometry and the effects of Wnt signalling activators on the 2- and 3-dimensional cultured ameloblastoma cells using molecular biological analyses. RESULT: Among heterogenous ameloblastoma cells, small-sized and round-shaped cells were found to be proliferative and expressed a marker of dental epithelial stem cells, SRY-box 2 (Sox2). Exogenous activation of Wnt signalling using glycogen synthase kinase 3ß inhibitors, lithium chloride (LiCl) and valproic acid (VPA), increased the cell size and decreased proliferation of cells and expression of Sox2 in 2 dimensionally cultured AM-1 and human primary ameloblastoma cells. Furthermore, the growth of 3 dimensionally cultured AM-1 cells as suspended or embedded in gel was suppressed by treatment with Wnt signalling activators, VPA and CHIR99021, or antibodies to sclerostin, an antagonist of Wnt signalling. CONCLUSION: We suggest that Wnt signalling activators are potential drug candidates to suppress CSCs in ameloblastoma.

Flow Cytofluorometric Analysis of Molecular Mechanisms of Premature Red Blood Cell Death.

This chapter describes, in detail, the operational principles and experimental design to analyze the premature death of human red blood cells (RBCs; erythrocytes). Necrosis (i.e., hemolysis), eryptosis, and necroptosis are the three types of cell death thus far known to exist in RBCs, and distinctive markers of each are well established. Here, methods based on flow cytometry are presented in an easily reproducible form. Moreover, manipulation of incubation medium to promote or inhibit certain physiological phenomena, along with a step-by-step approach to examine membrane scrambling, cell volume, surface complexity, calcium activity, oxidative stress, and signal transduction pathways are also discussed.