Key structural requirements of benzamide derivatives for histone deacetylase inhibition: design, synthesis and biological evaluation.

Background: Histone deacetylase inhibitors (HDACIs) are important as anticancer agents. Objective: This study aimed to investigate some key structural features of HDACIs via the design, synthesis and biological evaluation of novel benzamide-based derivatives. Methods: Novel structures, designed using a molecular modification approach, were synthesized and biologically evaluated. Results: The results indicated that a subset of molecules with CH3/NH2 at R2 position possess selective antiproliferative activity. However, only those with an NH2 group showed HDACI activity. Importantly, the shorter the molecule length, the stronger HDACI. Among all, 7j was the most potent HDAC1-3 inhibitor and antiproliferative compound. Conclusion: The results of the present investigation could provide valuable structural knowledge applicable for the development of the HDACIs and benzamide-based antiproliferative agents in the future.

The inhibition of ADAM17 in cord blood stem cell-derived CD16+ NK cells to enhance their cytotoxicity against acute lymphoblastic leukemia cells.

Fortunately, ample efforts are being made to find the best strategy to improve the anti-leukemia capacity of NK cells for treating different types of cancer. Despite the favorable ADCC capacity of functional CD16 + NK cells for immunotherapy, when NK cells face leukemia cells, the CD16 receptor is cleaved during the process mediated by a disintegrin and metalloproteinase-17(ADAM17). Reduced CD16 expression on NK cells weakens their cytotoxicity against leukemia cells. In addition, the expression of the CD47 receptor is high in acute lymphoblastic leukemia (ALL) compared to normal cells and can be correlated with poor prognosis. In the present study, ADAM17 was inhibited in cord blood-derived CD16 + NK cells, and their activity against ALL cell lines was evaluated following blockage with anti-CD47 antibody. As the results showed, the CD16 expression was reduced in the NK cells co-cultured with ALL cell lines. However, the ADAM17 inhibition increased the CD16 expression on the NK cells. This enhanced the cytotoxicity of those cells as well as cytokine production was evaluated by measuring expression of CD107-a expression, and IFN-γ production. Moreover, the presence of the ADAM17 inhibitor increased the apoptosis effect of the generated NK cells in response to ALL cells. Therefore, the inhibition of ADAM17 is useful for the activity of CD16 + NK cells against cancer cells.

Targeting TdT gene expression in Molt-4 cells by PNA-octaarginine conjugates.

Peptide nucleic acid (PNA) is an amide based structural nucleic acid mimic with potential applications in gene therapeutic drug discovery. In the present study, we evaluated and compared the effects on gene expression, cell viability and apoptosis of two antisense PNA-d-octaarginine conjugates, targeting sequences at the AUG translation start site or the 5'-UTR of the TdT (terminal deoxynucleotidyl transferase) gene, as well as a sense oligomer corresponding to the 5'-UTR-antisense, in Molt-4 cells. The protein level of TdT was determined by flow cytometry, and qPCR was used for mRNA expression analysis. Mismatch PNAs were used as control to address the sequence/target spcifity of the biological effects. The results showed that treatment with the AUG- and to slightly lesser extent with the 5'-UTR-antisense PNAs reduced the TdT mRNA as wel as the protein level, whereas only very low effect was observed for the 5'-UTR-sense PNA. A parallel effect was observed on reduced cell survival and increased rate of apoptosis. Our findings suggest that antisense PNAs can inhibit expression of the TdT gene and induce apoptosis in Molt-4 cells.

Effects of vitamin D supplementation on follicular development, gonadotropins and sex hormone concentrations, and insulin resistance in induced polycystic ovary syndrome.

OBJECTIVE: Polycystic ovary syndrome (PCOS) as a reproductive disorder disturbs ovarian follicular development, vitamin D stimulated insulin activity, and sex hormone concentrations. This study aimed to examine the effects of vitamin D on ovarian follicular development, insulin resistance, and sex hormone changes in rats with induced PCOS. MATERIALS AND METHODS: Forty female Wistar rats were randomly divided into four groups: (1) control, (2) induced PCOS, (3) vitamin D-treated non-PCOS (sham group), (4) vitamin D treated PCOS groups. All rats were then sacrificed under anesthesia and ovarian tissue samples were evaluated histomorphometrically. Blood samples were collected for analyzing the serum concentrations of sex hormones and insulin resistance. RESULTS: The number of atretic follicles at different stages of development increased in the PCOS ovaries (p<0.001). Vitamin D treatment significantly increased the normality of follicles in rats with PCOS (p<0.001). The serum concentration of follicle stimulating hormone and the estradiol significantly increased in rats with PCOS, whereas the testosterone and luteinizing hormone concentrations, glucose, insulin, and insulin resistance concentrations significantly decreased during vitamin D treatment (p<0.001). CONCLUSION: This study indicated that vitamin D treatment may protect ovarian tissue from the negative effect of PCOS by improving insulin activity and gonadotropin concentrations.

Telomere shortening as a hallmark of stem cell senescence.

Stem cells, especially mesenchymal stem cells (MSCs)-based therapies have been greatly attentioned in regenerative medicine through multi-lineage differentiation, self-renewal properties, etc. Despite the above advantages of MSCs, the defined properties of these cells are strongly affected by aging. Thus, the use of MSCs from older donors is lower than younger one, which limits clinical applications in cell therapy. According to the theories of aging, it is determined that aging is most likely caused by telomere shortening and telomere shortening is considered hallmarks of aging. Finding out the most mechanisms of these changes will probably reveal novel therapeutic targets for prolonging human health and for ameliorating age-associated phenotypes. This review focuses on prevalent knowledge about the mechanisms of stem cell senescence by telomere shortening and the molecular mechanism details involved in it.

NK cells: An attractive candidate for cancer therapy.

Natural killer (NK) cells have significant capability in tumor immune-surveillance. The ability of lyse transformed cells immediately in an antigen-independent manner make them an attractive candidate for cancer cell therapy. Despite employment of NK cells in cancer immunotherapy, clinical trials are faced with serious limitations such as trouble with the penetration of NK cells in tumor sites, limited in vivo persistence, and tumor microenvironment interference. Taken together, the NK-cell cancer therapy is still infant scenario that has a long way to be translated in clinic. Current article first reviews characteristic features of NK lymphocytes. Then, it discusses about important disruptive barriers and motivator in the developmental stages of NK cells like as tumor microenvironment. Finally, some revolutionary approaches are highlighted utilizing of NK cells in cancer therapy.

Advances in nanomaterial based optical biosensing and bioimaging of apoptosis via caspase-3 activity: a review.

Caspase-3 plays a vital role in intrinsic and extrinsic pathways of programed cell death and in cell proliferation. Its detection is an important tool for early detection of some cancers and apoptosis-related diseases, and for monitoring the efficacy of pharmaceuticals and of chemo- and radiotherapy of cancers. This review (with 72 references) summarizes nanomaterial based methods for signal amplification in optical methods for the determination of caspase-3 activity. Following an introduction into the field, a first large section covers optical assays, with subsections on luminescent and chemiluminescence, fluorometric (including FRET based), and colorimetric assays. Further section summarize methods for bioimaging of caspase-3. A concluding section covers current challenges and future perspectives. Graphical Abstract ᅟ.

Regulation and modulation of PTEN activity.

PTEN (Phosphatase and tensin homolog deleted on chromosome ten) is a tumor suppressor that is frequently mutated in most human cancers. PTEN is a lipid and protein phosphatase that antagonizes PI3K/AKT pathway through lipid phosphatase activity at the plasma membrane. More recent studies showed that, in addition to the putative role of PTEN as a PI(3,4,5)P3 3-phosphatase, it is a PI(3,4)P2 3-phosphatase during stimulation of class I PI3K signaling pathway by growth factor. Although PTEN tumor suppressor function via it's lipid phosphatase activity occurs primarily in the plasma membrane, it can also be found in the nucleus, in cytoplasmic organelles and extracellular space. PTEN has also shown phosphatase independent functions in the nucleus. PTEN can exit from the cell through exosomal export or secretion and has a tumor suppressor function in adjacent cells. PTEN has a critical role in growth, the cell cycle, protein synthesis, survival, DNA repair and migration. Understanding the regulation of PTEN function, activity, stability, localization and its dysregulation outcomes and also the intracellular and extracellular role of PTEN and paracrine role of PTEN-L in tumor cells as an exogenous therapeutic agent can help to improve clinical conceptualization and treatment of cancer.

Farnesiferol C induces cell cycle arrest and apoptosis mediated by oxidative stress in MCF-7 cell line.

Farnesiferol C is one of the major compounds, isolated from Ferula asafoetida (a type of coumarins) and used for cancer treatment as a folk remedy. Treatment of many cancers depends on oxidative stress situation. In this study, we sought the hypothesis that oxidative stress induced by Farnesiferol C contribute to anticancer property and induce apoptosis in MCF-7, human breast cancer cell line. We investigated the effect of Farnesiferol C on oxidative stress by measurement of some enzymes activity including catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), as well as some parameters such as total thiol and ROS levels. Also we evaluated Farnesiferol C effects on the cell cycle and apoptosis induction by using flow cytometry analysis. Our findings demonstrated that Farnesiferol C significantly induced apoptosis mediated by increasing in the cellular ROS levels. This compound increased cellular SOD and CAT activities in 24 and 48 h and reduced activity of these enzymes after 72 h exposure. Furthermore, MDA and total thiol levels were increased and decreased, respectively in the cells treated with Farnesiferol C after 24-72 h. G0/G1 phase cell cycle arrest followed by induction of apoptosis was also observed in MCF-7 cells after treatment with Farnesiferol C. According to these data, Farnesiferol C has a therapeutic effect on MCF-7 cells and can be suitable candidate for breast cancer treatment; however it is necessary for further experiments.

L-carnitine contributes to enhancement of neurogenesis from mesenchymal stem cells through Wnt/ß-catenin and PKA pathway.

The identification of factors capable of enhancing neurogenesis has great potential for cellular therapies in neurodegenerative diseases. Multiple studies have shown the neuroprotective effects of L-carnitine (LC). This study determined whether neuronal differentiation of rat adipose tissue-derived mesenchymal stem cells (ADSCs) can be activated by LC. In this study, protein kinase A (PKA) and Wnt/ß-catenin pathways were detected to show if this activation was due to these pathways. The expression of LC-induced neurogenesis markers in ADSCs was characterized using real-time PCR. ELISA was conducted to assess the expression of cyclic adenosine monophosphate (cAMP) and PKA. The expression of ß-catenin, reduced dickkopf1 (DKK1), low-density lipoprotein receptor-related protein 5 (LRP5), Wnt1, and Wnt3a genes as Wnt/ß-catenin signaling members were used to detect the Wnt/ß-catenin pathway. It was observed that LC could promote neurogenesis in ADSCs as well as expression of some neurogenic markers. Moreover, LC causes to increase the cAMP levels and PKA activity. Treatment of ADSCs with H-89 (dihydrochloride hydrate) as PKA inhibitor significantly inhibited the promotion of neurogenic markers, indicating that the PKA signaling pathway could be involved in neurogenesis induction. Analyses of real-time PCR data showed that the mRNA expressions of ß-catenin, DKK1, LRP5c-myc, Wnt1, and Wnt3a were increased in the presence of LC. Therefore, the present study showed that LC promotes ADSCs neurogenesis and the LC-induced neurogenic markers could be due to both the PKA and Wnt/ß-catenin signaling pathway. Impact statement Neural tissue has long been believed as incapable of regeneration and the identification of cell types and factors capable of neuronal differentiation has generated intense interest. Mesenchymal stem cells (MSCs) are considered as potential targets for stem cell-based therapy. L-carnitin (LC) as an antioxidant may have neuroprotective effects in oxidative damage and possibly in neurodegenerative disorders. We have tried to evaluate the effect of LC as an antioxidant on the neurogenic differentiation of ADSCs in order to further elucidate the simultaneous effects on the capability of the neural regeneration. In this study, PKA and Wnt/ß-catenin signaling pathways were detected to see if LC could also activate these pathways. The results of this study showed that 200 µM LC promoted ADSCs neurogenic differentiation, and that it was correlated with the PKA and Wnt/ß-catenin signaling pathways.

Platelet-Derived Ectosomes Reduce NK Cell Function.

Platelet (PLT) transfusions are potentially life saving for individuals with low PLT numbers; however, previous work revealed that PLT transfusions are associated with increased infection risk. During storage, PLT intended for transfusion continuously shed ectosomes (Ecto) from their surface, which express immunomodulatory molecules like phosphatidylserine or TGF-ß1. Recently, PLT-Ecto were shown to reduce proinflammatory cytokine release by macrophages and to favor the differentiation of naive T cells toward regulatory T cells. Whether PLT-Ecto modify NK cells remains unclear. We exposed purified NK cells and full PBMCs from healthy donors to PLT-Ecto. We found a reduced expression of several activating surface receptors (NKG2D, NKp30, and DNAM-1) and decreased NK cell function, as measured by CD107a expression and IFN-γ production. Pretreatment of PLT-Ecto with anti-TGF-ß1 neutralizing Ab restored surface receptor expression and NK cell function. We further observed a TGF-ß1-mediated upregulation of miR-183, which, in turn, reduced DAP12, an important protein for stabilization and downstream signaling of several activating NK cell receptors. Again, these effects could antagonized, in part, when PLT-Ecto were preincubated with anti-TGF-ß1 Ab. Erythrocyte Ecto did not affect NK cells. Polymorphonuclear cell Ecto expressed MHC class I and inhibited NK cell function. In addition, they induced the secretion of TGF-ß1 by NK cells, which participated in an auto/paracrine manner in the suppressive activity of polymorphonuclear cell-derived Ecto. In sum, our study showed that PLT-Ecto could inhibit NK cell effector function in a TGF-ß1-dependent manner, suggesting that recipients of PLT transfusions may experience reduced NK cell function.

Potent anti-angiogenic and cytotoxic effect of conferone on human colorectal adenocarcinoma HT-29 cells.

BACKGROUND: Cancer is one of the leading causes of death worldwide, both in developed and developing countries. Of note, colorectal adenoma encompasses a high rate of gastrointestinal-associated cancer death in human being. Today, different strategies, including surgery approaches, photodynamic therapy, radiation and particularly natural compounds have been extensively used to manage tumor behavior in human body. METHODS: The objective of the present study was to elucidate the multilateral effects of conferone on HT-29 cell lines. In addition to cell cytotoxicity, the extent of lipid peroxidation, MDA formation, catalase, superoxide dismutase and intracellular ROS levels, as markers of oxidative stress, were also studied. P-glycoprotein-mediated cellular efflux effectiveness, anti-angiogenic and finally anti-migratory capacities of conferone-exposed HT-29 cells were monitored over a course of 72 h. RESULTS: It was found that, conferone mediated cell proliferation arrest and induced cell death through both apoptosis and necrosis phenomena. HT-29 cells, exposed to 20 µM conferone, under gone oxidative stress and total content of reactive oxygen species was increased in a time-dependent manner. Intracellular accumulation of rhodamine 123 and cell's swelling under iso- and hypo-osmotic conditions could be related to P-glycoprotein incorrect performance in the presence of conferone. A significant reduction in CD31 positive cells population and in vitro tubulogenesis of endothelial cells was also observed after incubation with conditioned medium collected from 72 h conferone-treated HT-29 cells. Conferone also precluded angiogenesis capability of treated HT-29 cells through an altered secretome profile, including vascular endothelial growth factor, Angiopoietin-1 and -2 factors. In addition to anti-angiogenic properties of conferone, a profound decrease in migration capability of HT-29 cells was also evident.

Quantity of HLA-C surface expression and licensing of KIR2DL+ natural killer cells.

Natural killer (NK) cells require interaction of inhibitory surface receptors with human leukocyte antigen (HLA) ligands during development to acquire functional competence in a process termed "licensing." The quantity of HLA required for this process is unknown. Two polymorphisms affecting HLA-C surface expression (rs9264942 and rs67384697) have recently been identified, and shown to influence progression of HIV infection. We typed a cohort of healthy donors for the two HLA-C-related polymorphisms, KIR2DL1 and KIR2DL3, and their respective HLA-C ligands and analyzed how HLA ligands influenced licensing status of killer cell immunoglobulin-like receptor (KIR)+ NK cells in terms of degranulation and cytokine production in response to HLA-deficient target cells. The presence of respective HLA class I ligands increased the function of KIR2DL1+ and KIR2DL3+ NK cells in a dose-dependent manner. In contrast, neither of the HLA-C-related polymorphisms nor the quantity of cell surface HLA-C had any significant effect on NK cell function. Interestingly, HLA-Cw7-an HLA-C allele with low surface expression-licensed KIR2DL3+ NK cells more strongly than any other KIR2DL3 ligand. The quantity of cell surface HLA-C does not appear to influence licensing of NK cells, and the HLA-C-related polymorphisms presumably influence HIV progression through factors unrelated to NK cell education.

Emergence of NK-cell progenitors and functionally competent NK-cell lineage subsets in the early mouse embryo.

The earliest stages of natural killer (NK)-cell development are not well characterized. In this study, we investigated in different fetal hematopoietic tissues how NK-cell progenitors and their mature NK-cell progeny emerge and expand during fetal development. Here we demonstrate, for the first time, that the counterpart of adult BM Lin(-)CD122(+)NK1.1(-)DX5(-) NK-cell progenitor (NKP) emerges in the fetal liver at E13.5. After NKP expansion, immature NK cells emerge at E14.5 in the liver and E15.5 in the spleen. Thymic NK cells arise at E15.5, whereas functionally competent cytotoxic NK cells were present in the liver and spleen at E16.5 and E17.5, respectively. Fetal NKPs failed to produce B and myeloid cells but sustained combined NK- and T-lineage potential at the single-cell level. NKPs were also found in the fetal blood, spleen, and thymus. These findings show the emergence and expansion of bipotent NK/T-cell progenitor during fetal and adult lymphopoiesis, further supporting that NK/T-lineage restriction is taking place prethymically. Uncovering the earliest NK-cell developmental stages will provide important clues, helping to understand the origin of diverse NK-cell subsets, their progenitors, and key regulators.

FLT3 receptor and ligand are dispensable for maintenance and posttransplantation expansion of mouse hematopoietic stem cells.

Originally cloned from hematopoietic stem cell (HSC) populations and its ligand being extensively used to promote ex vivo HSC expansion, the FMS-like tyrosine kinase 3 (FLT3; also called FLK2) receptor and its ligand (FL) were expected to emerge as an important physiologic regulator of HSC maintenance and expansion. However, the role of FLT3 receptor and ligand in HSC regulation remains unclear and disputed. Herein, using Fl-deficient mice, we establish for the first time that HSC expansion in fetal liver and after transplantation is FL independent. Because previous findings in Flk2(-/-) mice were compatible with an important role of FLT3 receptor in HSC regulation and because alternative ligands might potentially interact directly or indirectly with FLT3 receptor, we here also characterized HSCs in Flk2(-/-) mice. Advanced phenotypic as well as functional evaluation of Flk2(-/-) HSCs showed that the FLT3 receptor is dispensable for HSC steady-state maintenance and expansion after transplantation. Taken together, these studies show that the FLT3 receptor and ligand are not critical regulators of mouse HSCs, neither in steady state nor during fetal or posttransplantation expansion.

Distinct and overlapping patterns of cytokine regulation of thymic and bone marrow-derived NK cell development.

Although bone marrow (BM) represents the main site for postnatal NK cell development, recently a distinct thymic-dependent NK cell pathway was identified. These studies were designed to investigate the role of cytokines in regulation of thymic NK cells and to compare with established regulatory pathways of BM-dependent NK cell compartment. The common cytokine receptor gamma-chain (Il2rg) essential for IL-15-induced signaling, and FMS-like tyrosine kinase 3 (FLT3) receptor ligand (Flt3l) were previously identified as important regulatory pathways of the BM NK cell compartment based on lack of function studies in mice, however their complementary action remains unknown. By investigating mice double-deficient in Il2rg and Flt3l (Flt3l(-/-) Il2rg(-/-)), we demonstrate that FLT3L is important for IL2Rg-independent maintenance of both immature BM as well as peripheral NK cells. In contrast to IL-7, which is dispensable for BM but important for thymic NK cells, IL-15 has a direct and important role in both thymic and BM NK cell compartments. Although thymic NK cells were not affected in Flt3l(-/-) mice, Flt3l(-/-)Il2rg(-/-) mice lacked detectable thymic NK cells, suggesting that FLT3L is also important for IL-2Rg-independent maintenance of thymic NK cells. Thus, IL-2Rg cytokines and FLT3L play complementary roles and are indispensable for homeostasis of both BM and thymic dependent NK cell development, suggesting that the cytokine pathways crucial for these two distinct NK cell pathways are largely overlapping.