Ligustilide enhances pregnancy outcomes via improvement of endometrial receptivity and promotion of endometrial angiogenesis in rats.

Ligustilide (LIG) is the main active ingredient of Angelica sinensis (Oliv.) Diels, which could promote focal angiogenesis to exert neuroprotection. However, there was no report that verified the exact effects of LIG on endometrial angiogenesis and the pregnancy outcomes. To explore the effects of LIG on low endometrial receptivity (LER) and angiogenesis, pregnancy rats were assigned into Control (saline treatment), LER (hydroxyurea-adrenaline treatment), LIG 20 mg/kg and LIG 40 mg/kg groups. Hematoxylin and eosin (H&E) staining was performed to evaluate endometrial morphology. Quantitative real-time PCR, immunofluorescence staining, western blot and immunohistochemistry staining were employed to assess the expression of endometrial receptivity factors and angiogenesis-related gene/protein, respectively. RNA sequencing was used to analyze the effects of LIG on LER caused by Kidney deficiency and blood stasis. We found that endometrial thickness and the implanted embryo number were substantially reduced in the hydroxyurea-adrenaline-treated pregnancy rats. At the same time, the gene and protein expressions of ERα, LIF, VEGFA and CD31 in the endometrium were markedly reduced, while the expressions of MUC1, E-cadherin were increased in the LER group. Administration of LIG raised the endometrial thickness and implanted embryos, as well as reversed the expressions of these factors. Collectively, our findings revealed that LIG could facilitate embryo implantation via recovery of the endometrium receptivity and promotion of endometrial angiogenesis.

Protective potential of royal jelly against hydroxyurea -induced hepatic injury in rats via antioxidant, anti-inflammatory, and anti-apoptosis properties.

Hydroxyurea (HDU) is a widely used medication for various malignancies, thalassemia, and sickle cell anemia with reported side effects. The current study investigated HDU- induced hepatic injury and the protective potential of the royal jelly (RJ) against this hepatotoxic effect in the light of hepatic oxidative/ antioxidative status, pro-inflammatory cytokine, apoptosis signaling pathway, and histopathology. Sixty albino rats were used (n = 10/group) for 60 days: control, RJ (100 mg/kg body weight, orally), HDU (225 mg/kg body weight, orally), 2HDU (450 mg/kg body weight, orally), and HDU + RJ groups. HDU-treated rats showed significant elevation of liver function tests as aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase, as well as malondialdehyde and nitric oxide (oxidative biomarkers) and significant decreased hepatic antioxidant molecules (reduced glutathione, superoxide dismutase, and glutathione peroxidase), compared to a control group, that more pronounced in the high dose of HDU. In addition, HDU induced significant upregulation of TNF-α and the Caspase-3 apoptotic pathway. Moreover, the liver of HDU treated groups showed various hepatic lesions from mild to severe necrotic changes related to the HDU dose. However, administration of RJ with HDU improved liver function tests, liver histology, and hepatic oxidative/antioxidative status concerning HDU groups. Furthermore, oral RJ administration with HDU significantly lessens the immune-expression area % of TNF-α and Caspase-3. Thus, the royal jelly has antioxidant, anti-inflammatory, and anti-apoptotic properties against HDU- induced hepatic injury and could be, therefore, used as adjuvant therapy in patients with long-term HDU medication.

The Use of Induced Pluripotent Stem Cells to Study the Effects of Adenosine Deaminase Deficiency on Human Neutrophil Development.

Inherited defects that abrogate the function of the adenosine deaminase (ADA) enzyme and consequently lead to the accumulation of toxic purine metabolites cause profound lymphopenia and severe combined immune deficiency. Additionally, neutropenia and impaired neutrophil function have been reported among ADA-deficient patients. However, due to the rarity of the disorder, the neutrophil developmental abnormalities and the mechanisms contributing to them have not been characterized. Induced pluripotent stem cells (iPSC) generated from two unrelated ADA-deficient patients and from healthy controls were differentiated through embryoid bodies into neutrophils. ADA deficiency led to a significant reduction in the number of all early multipotent hematopoietic progenitors. At later stages of differentiation, ADA deficiency impeded the formation of granulocyte colonies in methylcellulose cultures, leading to a significant decrease in the number of neutrophils generated from ADA-deficient iPSCs. The viability and apoptosis of ADA-deficient neutrophils isolated from methylcellulose cultures were unaffected, suggesting that the abnormal purine homeostasis in this condition interferes with differentiation or proliferation. Additionally, there was a significant increase in the percentage of hyperlobular ADA-deficient neutrophils, and these neutrophils demonstrated significantly reduced ability to phagocytize fluorescent microspheres. Supplementing iPSCs and methylcellulose cultures with exogenous ADA, which can correct adenosine metabolism, reversed all abnormalities, cementing the critical role of ADA in neutrophil development. Moreover, chemical inhibition of the ribonucleotide reductase (RNR) enzyme, using hydroxyurea or a combination of nicotinamide and trichostatin A in iPSCs from healthy controls, led to abnormal neutrophil differentiation similar to that observed in ADA deficiency, implicating RNR inhibition as a potential mechanism for the neutrophil abnormalities. In conclusion, the findings presented here demonstrate the important role of ADA in the development and function of neutrophils while clarifying the mechanisms responsible for the neutrophil abnormalities in ADA-deficient patients.

Investigation into perturbed nucleoside metabolism and cell cycle for elucidating the cytotoxicity effect of resveratrol on human lung adenocarcinoma epithelial cells.

In an effort to understand the molecular events contributing to the cytotoxicity activity of resveratrol (RSV), we investigated its effects on human lung adenocarcinoma epithelial cell line A549 at different concentrations. Cellular nucleoside metabolic profiling was determined by an established liquid chromatography-mass spectrometry method in A549 cells. RSV resulted in significant decreases and imbalances of deoxyribonucleoside triphosphates (dNTPs) pools suppressing subsequent DNA synthesis. Meanwhile, RSV at high concentration caused significant cell cycle arrest at S phase, in which cells required the highest dNTPs supply than other phases for DNA replication. The inhibition of DNA synthesis thus blocked subsequent progression through S phase in A549 cells, which may partly contribute to the cytotoxicity effect of RSV. However, hydroxyurea (HU), an inhibitor of RNR activity, caused similar dNTPs perturbation but no S phase arrest, finally no cytotoxicity effect. Therefore, we believed that the dual effect of high concentration RSV, including S phase arrest and DNA synthesis inhibition, was required for its cytotoxicity effect on A549 cells. In summary, our results provided important clues to the molecular basis for the anticancer effect of RSV on epithelial cells.

Berberine attenuates XRCC1-mediated base excision repair and sensitizes breast cancer cells to the chemotherapeutic drugs.

Berberine (BBR) is a natural isoquinoline alkaloid, which is used in traditional medicine for its anti-microbial, anti-protozoal, anti-diarrhoeal activities. Berberine interacts with DNA and displays anti-cancer activities, yet its effects on cellular DNA repair and on synthetic treatments with chemotherapeutic drugs remain unclear. In this study, we investigated the effects of BBR on DNA repair and on sensitization of breast cancer cells to different types of DNA damage anti-tumoural drugs. We found BBR arrested cells in the cell cycle S phase and induced DNA breaks. Cell growth analysis showed BBR sensitized MDA-MB-231 cells to cisplatin, camptothecin and methyl methanesulfonate; however, BBR had no synergistic effects with hydroxurea and olaparib. These results suggest BBR only affects specific DNA repair pathways. Western blot showed BBR down-regulated XRCC1 expressions, and the rescued XRCC1 recovered the resistance of cancer cells to BBR. Therefore, we conclude that BBR interferes with XRCC1-mediated base excision repair to sensitize cancer cells to chemotherapeutic drugs. These finding can contribute to understanding the effects of BBR on cellular DNA repair and the clinical employment of BBR in treatment of breast cancer.

Effect of zileuton on osteoporotic bone and its healing, expression of bone, and brain genes in rats.

Estrogen deficiency and aging are associated with osteoporosis, impaired bone healing, and lower cognitive performance. Close functional and physical connections occur between bone and the central nervous system. An anti-inflammatory drug, zileuton (which is an inhibitor of arachidonate 5-lipoxygenase), is known to have a positive effect on bone tissue repair and brain ischemia. We studied the effect of zileuton on osteopenic bone and its healing and on the genes considered to be crucial for the cross talks between bone and brain. Three-month-old Sprague-Dawley rats were ovariectomized or left untreated. After 8 wk, bilateral metaphyseal tibia osteotomy with plate osteosynthesis was performed in all rats. Ovariectomized rats were fed with food containing zileuton (1, 10, or 100 mg/kg body wt) for 5 wk. In tibiae, bone volume, callus and cortical volume, and gene expression of osteocalcin and alkaline phosphatase were enhanced by zileuton (10 or 100 mg); biomechanical properties and bone density were not changed. In femur, zileuton enlarged cortical volume distal and trabecular volume proximal, decreasing their density. The expression level of brain Sema3a, known to regulate bone mass positively, was downregulated after ovariectomy. In contrast, bone Sema4d, a negative regulator of bone mass, was upregulated in the tibia callus after ovariectomy, whereas zileuton treatment (10 or 100 mg) resulted in reverse effects. Here, we describe for the first time the expression of Rbbp4 mRNA and its increase in tibia after ovariectomy. Zileuton caused downregulation of Rbbp4 in the hippocampus and had an effect on bone healing, changed the expression of genes involved in cross talk between bones and brain, and may be a potent drug for further examination in estrogen deficiency-related dysfunction(s). NEW & NOTEWORTHY Zileuton, a 5-lipoxygenase inhibitor, increased bone volume, callus and cortical volume in osteotomized tibia, and trabecular and cortical volume in femur. Although the expression of Sema3a (positively regulating bone mass) in brain was downregulated and Sema4d (negatively regulating bone mass) was upregulated in tibia callus after ovariectomy, zileuton could counteract these effects. Rbbp4 (involved in age-related memory loss) was increased in tibia callus after ovariectomy.

Recycling drug screen repurposes hydroxyurea as a sensitizer of glioblastomas to temozolomide targeting de novo DNA synthesis, irrespective of molecular subtype.

Background: Glioblastoma (GBM) is the most common and most aggressive primary malignant brain tumor. Standard-of-care treatment involves maximal surgical resection of the tumor followed by radiation and chemotherapy (temozolomide [TMZ]). The 5-year survival rate of patients with GBM is <10%, a colossal failure that has been partially attributed to intrinsic and/or acquired resistance to TMZ through O6-methylguanine DNA methyltransferase (MGMT) promoter methylation status in the tumor. Methods: A drug screening aimed at evaluating the potential recycling and repurposing of known drugs was conducted in TMZ-resistant GBM cell lines and primary cultures of newly diagnosed GBM with different MGMT promoter methylation status, phenotypic/genotypic background and subtype, and validated with sphere formation, cell migration assays, and quantitative invasive orthotopic in vivo models. Results: We identified hydroxyurea (HU) to synergize with TMZ in GBM cells in culture and in vivo, irrespective of MGMT promoter methylation status, subtype, and/or stemness. HU acts specifically on the S-phase of the cell cycle by inhibiting the M2 unit of enzyme ribonucleotide reductase. Knockdown of this enzyme using RNA interference and other known chemical inhibitors exerted a similar effect to HU in combination with TMZ both in culture and in vivo. Conclusions: We demonstrate preclinical efficacy of repurposing hydroxyurea in combination with TMZ for adjuvant GBM therapy. This combination benefit is of direct clinical interest given the extensive use of TMZ and the associated problems with TMZ-related resistance and treatment failure.

Novel Cell-Killing Mechanisms of Hydroxyurea and the Implication toward Combination Therapy for the Treatment of Fungal Infections.

We have previously reported that an erg11 mutation affecting ergosterol synthesis and a hem13 mutation in the heme synthesis pathway significantly sensitize the fission yeast Schizosaccharomyces pombe to hydroxyurea (HU) (1, 2). Here we show that treatment with inhibitors of Erg11 and heme biosynthesis phenocopies the two mutations in sensitizing wild-type cells to HU. Importantly, HU synergistically interacts with the heme biosynthesis inhibitor sampangine and several Erg11 inhibitors, the antifungal azoles, in causing cell lethality. Since the synergistic drug interactions are also observed in the phylogenetically divergent Saccharomyces cerevisiae and the opportunistic fungal pathogen Candida albicans, the synergism is likely conserved in eukaryotes. Interestingly, our genetic data for S. pombe has also led to the discovery of a robust synergism between sampangine and the azoles in C. albicans Thus, combinations of HU, sampangine, and the azoles can be further studied as a new method for the treatment of fungal infections.

Naja sputatrix Venom Preconditioning Attenuates Neuroinflammation in a Rat Model of Surgical Brain Injury via PLA2/5-LOX/LTB4 Cascade Activation.

Inflammatory preconditioning is a mechanism in which exposure to small doses of inflammatory stimuli prepares the body against future massive insult by activating endogenous protective responses. Phospholipase A2/5-lipoxygenase/leukotriene-B4 (PLA2/5-LOX/LTB4) axis is an important inflammatory signaling pathway. Naja sputatrix (Malayan spitting cobra) venom contains 15% secretory PLA2 of its dry weight. We investigated if Naja sputatrix venom preconditioning (VPC) reduces surgical brain injury (SBI)-induced neuroinflammation via activating PLA2/5-LOX/LTB4 cascade using a partial frontal lobe resection SBI rat model. Naja sputatrix venom sublethal dose was injected subcutaneously for 3 consecutive days prior to SBI. We observed that VPC reduced brain edema and improved neurological function 24 h and 72 h after SBI. The expression of pro-inflammatory mediators in peri-resection brain tissue was reduced with VPC. Administration of Manoalide, a PLA2 inhibitor or Zileuton, a 5-LOX inhibitor with VPC reversed the protective effects of VPC against neuroinflammation. The current VPC regime induced local skin inflammatory reaction limited to subcutaneous injection site and elicited no other toxic effects. Our findings suggest that VPC reduces neuroinflammation and improves outcomes after SBI by activating PLA2/5-LOX/LTB4 cascade. VPC may be beneficial to reduce post-operative neuroinflammatory complications after brain surgeries.

Androgen-mediated sex bias impairs efficiency of leukotriene biosynthesis inhibitors in males.

Proinflammatory leukotrienes (LTs) are produced by 5-lipoxygenase (5-LO) aided by 5-LO-activating protein (FLAP). LT biosynthesis inhibitors are currently under clinical investigation as treatments for respiratory and cardiovascular diseases. Here, we have revealed a sex bias in the efficiency of clinically relevant LT biosynthesis inhibitors, showing that their effects are superior in females. We found that androgens cause these sex differences by impeding the LT-biosynthetic 5-LO/FLAP complex assembly. Lower doses of the FLAP inhibitor MK886 were required to reduce LTB4 levels in exudates of female versus male mice and rats. Following platelet-activating factor-induced shock, MK886 increased survival exclusively in female mice, and this effect was abolished by testosterone administration. FLAP inhibitors and the novel-type 5-LO inhibitors licofelone and sulindac sulfide exhibited higher potencies in human blood from females, and bioactive 5-LO/FLAP complexes were formed in female, but not male, human and murine leukocytes. Supplementation of female blood or leukocytes with 5α-dihydrotestosterone abolished the observed sex differences. Our data suggest that females may benefit from anti-LT therapy to a greater extent than males, prompting consideration of sex issues in LT modifier development.

Effect of the lipoxygenase-inhibitors baicalein and zileuton on the vertebra in ovariectomized rats.

Osteoporosis is one of the most common diseases worldwide. In osteoporosis, vertebral fractures represent a major burden. Lipoxygenase (LOX) inhibitors such as baicalein and zileuton may represent a promising therapeutic option owing to their antioxidative effects and suppression of various inflammatory processes in muscle and bone. The effect of these LOX inhibitors on the spine was studied in osteopenic rats. Female Sprague-Dawley rats were divided two times into five groups: four groups each were ovariectomized (OVX) and one control group was non-ovariectomized (NON-OVX). Eight weeks after ovariectomy, three concentrations of baicalein (1mg/kg body weight [BW], 10mg/kgBW, and 100mg/kgBW) were administered subcutaneously daily in three OVX groups for 4weeks. Similarly, zileuton was administered in three concentrations via food for 5weeks. In vivo computed tomography (pQCT) of the spine was performed before the treatments and at the end of the experiment. Lumbar vertebrae were subjected to a compression test, micro-CT, and ashing analyses. After baicalein treatment, cortical bone mineral density (BMD) was improved; trabecular connectivity and trabecular BMD were diminished at high dose. After zileuton treatment, the total BMD, anorganic weight, trabecular nodes, and trabecular area were improved. The in vivo stress-strain index was increased and alkaline phosphatase activity in serum was enhanced after both treatments. A dose-dependent effect was not clearly observed after both treatments. The treatments using baicalein for 4 and zileuton for 5weeks were not sufficient to change the biomechanical properties and bone volume fraction (BV/TV). Overall, baicalein improved the cortical bone parameters whereas zileuton had a favorable effect on the trabecular structure. Moreover, both treatments increased the bone formation rate. Longer trials, a combination of both LOX inhibitors, and their effect at the cellular and molecular levels should be investigated in further studies.

RANK-Independent Osteoclast Formation and Bone Erosion in Inflammatory Arthritis.

OBJECTIVE: Proinflammatory molecules promote osteoclast-mediated bone erosion by up-regulating local RANKL production. However, recent evidence suggests that combinations of cytokines, such as tumor necrosis factor (TNF) plus interleukin-6 (IL-6), induce RANKL-independent osteoclastogenesis. The purpose of this study was to better understand TNF/IL-6-induced osteoclast formation and to determine whether RANK is absolutely required for osteoclastogenesis and bone erosion in murine inflammatory arthritis. METHODS: Myeloid precursors from wild-type (WT) mice or mice with either germline or conditional deletion of Rank, Nfatc1, Dap12, or Fcrg were treated with either RANKL or TNF plus IL-6. Osteoprotegerin, anti-IL-6 receptor (anti-IL-6R), and hydroxyurea were used to block RANKL, the IL-6R, and cell proliferation, respectively. Clinical scoring, histologic assessment, micro-computed tomography, and quantitative polymerase chain reaction (qPCR) were used to evaluate K/BxN serum-transfer arthritis in WT and RANK-deleted mice. Loss of Rank was verified by qPCR and by osteoclast cultures. RESULTS: TNF/IL-6 generated osteoclasts in vitro that resorbed mineralized tissue through a pathway dependent on IL-6R, NFATc1, DNAX-activation protein 12, and cell proliferation, but independent of RANKL or RANK. Bone erosion and osteoclast formation were reduced, but not absent, in arthritic mice with inducible deficiency of RANK. TNF/IL-6, but not RANKL, induced osteoclast formation in bone marrow and synovial cultures from animals deficient in Rank. Multiple IL-6 family members (IL-6, leukemia inhibitory factor, oncostatin M) were up-regulated in the synovium of arthritic mice. CONCLUSION: The persistence of bone erosion and synovial osteoclasts in Rank-deficient mice, and the ability of TNF/IL-6 to induce osteoclastogenesis, suggest that more than one cytokine pathway exists to generate these bone-resorbing cells in inflamed joints.

Hydroxyurea derivatives of irofulven with improved antitumor efficacy.

Irofulven is a semi-synthetic derivative of Illudin S, a toxic sesquiterpene isolated from the mushroom Omphalotus illudens. Irofulven has displayed significant antitumor activity in various clinical trials but displayed a limited therapeutic index. A new derivative of irofulven was prepared by reacting hydroxyurea with irofulven under acidic conditions. Acetylation of this new compound with acetic anhydride produced a second derivative. Both of these new derivatives displayed significant antitumor activity in vitro and in vivo comparable to or exceeding that of irofulven.

Serum Paraoxonase Activity and Malondialdehyde Serum Concentrations Remain Unaffected in Response to Hydroxyurea Therapy in ß-Thalassemia Patients.

ß-Thalassemia is the most common hereditary disorder characterized by reduced production of ß-globin chains of hemoglobin A (HbA). In recent years, hydroxyurea (HU) has shown promising therapeutic benefits in patients with ß-thalassemia by fetal hemoglobin augmentation. We have analyzed effects of hydroxyurea treatment on oxidative stress in ß-thalassemia patients by assessing activities of paraoxonase (PON) and arylesterase along with malondialdehyde (MDA) and total reactive oxygen species (ROS) concentrations. Blood samples from 159 individuals including 56 HU-treated and 58 untreated ß-thalassemia patients and 45 healthy controls were analyzed. PON activity was found to be highest in healthy individuals (177.76 ± 4.44 U/mL) as compared to treated (52.67 ± 3.65 U/mL) and untreated (55.11 ± 3.26 U/mL) patients. A similar trend was observed in the case of arylesterase activity in normal, ß-thalassemia-treated, and untreated (210.0 ± 11.25 U/mL, 163.03 ± 9.04 U/mL, 139.77 ± 10.10 U/mL) subjects. Serum MDA concentrations (2.59 ± 0.09 nmol/mL, 2.45 ± 0.08 nmol/mL, and 1.15 ± 0.05 nmol/mL) and total ROS concentrations (3.73 ± 0.20 nmol/mL, 3.54 ± 0.23 nmol/mL, and 2.45 ± 0.14 nmol/mL) were significantly elevated in both groups (untreated and treated) as compared to healthy individuals (P < .01). Oxidative stress was found to be markedly elevated in ß-thalassemia patients as compared to healthy controls. Insignificant differences were, however, observed in mean concentrations of PON1 paraoxonase and arylesterase activities, serum MDA concentration and total ROS concentrations between HU-treated and untreated patients. We propose that HU therapy alone seems to be ineffective in managing oxidative stress and is likely to offer a better clinical outcome when supplemented with efficient iron chelation therapy and antioxidants.

Checkpoint Activation of an Unconventional DNA Replication Program in Tetrahymena.

The intra-S phase checkpoint kinase of metazoa and yeast, ATR/MEC1, protects chromosomes from DNA damage and replication stress by phosphorylating subunits of the replicative helicase, MCM2-7. Here we describe an unprecedented ATR-dependent pathway in Tetrahymena thermophila in which the essential pre-replicative complex proteins, Orc1p, Orc2p and Mcm6p are degraded in hydroxyurea-treated S phase cells. Chromosomes undergo global changes during HU-arrest, including phosphorylation of histone H2A.X, deacetylation of histone H3, and an apparent diminution in DNA content that can be blocked by the deacetylase inhibitor sodium butyrate. Most remarkably, the cell cycle rapidly resumes upon hydroxyurea removal, and the entire genome is replicated prior to replenishment of ORC and MCMs. While stalled replication forks are elongated under these conditions, DNA fiber imaging revealed that most replicating molecules are produced by new initiation events. Furthermore, the sole origin in the ribosomal DNA minichromosome is inactive and replication appears to initiate near the rRNA promoter. The collective data raise the possibility that replication initiation occurs by an ORC-independent mechanism during the recovery from HU-induced replication stress.

Hydroxymethylcytosine and demethylation of the γ-globin gene promoter during erythroid differentiation.

The mechanism responsible for developmental stage-specific regulation of γ-globin gene expression involves DNA methylation. Previous results have shown that the γ-globin promoter is nearly fully demethylated during fetal liver erythroid differentiation and partially demethylated during adult bone marrow erythroid differentiation. The hypothesis that 5-hydroxymethylcytosine (5 hmC), a known intermediate in DNA demethylation pathways, is involved in demethylation of the γ-globin gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5 mC) and 5 hmC at a CCGG site within the 5' γ-globin gene promoter region in FACS-purified cells from baboon bone marrow and fetal liver enriched for different stages of erythroid differentiation. Our results show that 5 mC and 5 hmC levels at the γ-globin promoter are dynamically modulated during erythroid differentiation with peak levels of 5 hmC preceding and/or coinciding with demethylation. The Tet2 and Tet3 dioxygenases that catalyze formation of 5 hmC are expressed during early stages of erythroid differentiation and Tet3 expression increases as differentiation proceeds. In baboon CD34+ bone marrow-derived erythroid progenitor cell cultures, γ-globin expression was positively correlated with 5 hmC and negatively correlated with 5 mC at the γ-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced γ-globin promoter DNA methylation and increased γ-globin expression when added alone and in an additive manner in combination with either DNA methyltransferase or LSD1 inhibitors. These results strongly support the hypothesis that the Tet-mediated 5 hmC pathway is involved in developmental stage-specific regulation of γ-globin expression by mediating demethylation of the γ-globin promoter.

RN-1, a potent and selective lysine-specific demethylase 1 inhibitor, increases γ-globin expression, F reticulocytes, and F cells in a sickle cell disease mouse model.

Increased levels of fetal hemoglobin are associated with decreased symptoms and increased lifespan in patients with sickle cell disease (SCD). Hydroxyurea, the only drug currently approved for SCD, is not effective in a large fraction of patients, and therefore, new agents are urgently needed. Recently it was found that lysine demethylase 1, an enzyme that removes monomethyl and dimethyl residues from the lysine 4 residue of histone H3, is a repressor of γ-globin gene expression. In this article, we have compared the ability of tranylcypromine (TCP) and a more potent TCP derivative, RN-1, to increase γ-globin expression in cultured baboon erythroid progenitor cells and in the SCD mouse model. The results indicate that the ability of RN-1 to induce F cells and γ-globin mRNA in SCD mice is similar to that of decitabine, the most powerful fetal hemoglobin-inducing drug known, and greater than that of either TCP or hydroxyurea. We conclude that RN-1 and other lysine demethylase 1 inhibitors may be promising new γ-globin-inducing agents for the treatment of SCD that warrant further studies in other preclinical models, such as nonhuman primates.

The biphasic interphase-mitotic polarity of cell nuclei induced under DNA replication stress seems to be correlated with Pin2 localization in root meristems of Allium cepa.

Long-term treatment of Allium cepa seedlings with low concentration of hydroxyurea (HU) results in a disruption of cell cycle checkpoints, leading root apex meristem (RAM) cells to an abnormal organization of nuclear structures forming interphase (I) and mitotic (M) domains of chromatin at opposite poles of the nucleus. Thus far, both critical cell length and an uneven distribution of cyclin B-like proteins along the nuclear axis have been recognized as essential factors needed to facilitate the formation of biphasic interphase-mitotic (IM) cells. Two new aspects with respect to their emergence are investigated in this study. The first concerns a relationship between the polarity of increasing chromatin condensation (IM orientation) and the acropetal (base→apex) alignment of RAM cell files. The second problem involves the effects of auxin (IAA), on the frequency of IM cells. We provide evidence that there is an association between the advanced M-poles of the IM cell nuclei and the polarized accumulation sites of auxin efflux carriers (PIN2 proteins) and IAA. Furthermore, our observations reveal exclusion regions for PIN2 proteins in the microtubule-rich structures, such as preprophase bands (PPBs) and phragmoplast. The current and previous studies have prompted us to formulate a hypothetical mechanism linking PIN2-mediated unilateral localization of IAA and the induction of bipolar IM cells in HU-treated RAMs of A. cepa.

On the importance of targeting parasite stem cells in anti-echinococcosis drug development.

The life-threatening diseases alveolar and cystic echinococcoses are caused by larvae of the tapeworms Echinococcus multilocularis and E. granulosus, respectively. In both cases, intermediate hosts, such as humans, are infected by oral uptake of oncosphere larvae, followed by asexual multiplication and almost unrestricted growth of the metacestode within host organs. Besides surgery, echinococcosis treatment relies on benzimidazole-based chemotherapy, directed against parasite beta-tubulin. However, since beta-tubulins are highly similar between cestodes and humans, benzimidazoles can only be applied at parasitostatic doses and are associated with adverse side effects. Mostly aiming at identifying alternative drug targets, the nuclear genome sequences of E. multilocularis and E. granulosus have recently been characterized, revealing a large number of druggable targets that are expressed by the metacestode. Furthermore, recent cell biological investigations have demonstrated that E. multilocularis employs pluripotent stem cells, called germinative cells, which are the only parasite cells capable of proliferation and which give rise to all differentiated cells. Hence, the germinative cells are the crucial cell type mediating proliferation of E. multilocularis, and most likely also E. granulosus, within host organs and should also be responsible for parasite recurrence upon discontinuation of chemotherapy. Interestingly, recent investigations have also indicated that germinative cells might be less sensitive to chemotherapy because they express a beta-tubulin isoform with limited affinity to benzimidazoles. In this article, we briefly review the recent findings concerning Echinococcus genomics and stem cell research and propose that future research into anti-echinococcosis drugs should also focus on the parasite's stem cell population.

Modulation of gamma globin genes expression by histone deacetylase inhibitors: an in vitro study.

Induction of fetal haemoglobin (HbF) is a promising therapeutic approach for the treatment of ß-thalassaemia and sickle cell disease (SCD). Several pharmacological agents, such as hydroxycarbamide (HC) and butyrates, have been shown to induce the γ-globin genes (HBG1, HBG2). However, their therapeutic use is limited due to weak efficacy and an inhibitory effect on erythroid differentiation. Thus, more effective agents are needed. The histone deacetylase (HDAC) inhibitors are potential therapeutic haemoglobin (Hb) inducers able to modulate gene expression through pleiotropic mechanisms. We investigated the effects of a HDAC inhibitor, Givinostat (GVS), on erythropoiesis and haemoglobin synthesis and compared it with sodium butyrate and HC. We used an in vitro erythropoiesis model derived from peripheral CD34⁺ cells of healthy volunteers and SCD donors. GVS effects on erythroid proliferation and differentiation and on Hb synthesis were investigated. We found that GVS at high concentrations delayed erythroid differentiation with no specific effect on HBG1/2 transcription. At a low concentration (1 nmol/l), GVS induced Hb production with no effects on cells proliferation and differentiation. The efficacy of GVS 1 mol/l in Hb induction in vitro was comparable to that of HC and butyrate. Our results support the evaluation of GVS as a new candidate molecule for the treatment of the haemoglobinophathies due to its positive effects on haemoglobin production at low and non-toxic concentrations.