Inhibition Of DNA Replication Research Articles

WASHC1 interacts with MCM2-7 complex to promote cell survival under replication stress.

WASHC1 is a member of the Wiskott-Aldrich syndrome protein (WASP) family and is involved in endosomal protein sorting and trafficking through the generation of filamentous actin (F-actin) via activation of the Arp2/3 complex. There is increasing evidence that WASHC1 is present in the nucleus and nuclear WASHC1 plays important roles in regulating gene transcription, DNA repair as well as maintaining nuclear organization. However, the multi-faceted functions of nuclear WASHC1 still need to be clarified. We show here that WASHC1 interacts with several components of the minichromosome maintenance (MCM) 2-7 complex by using co-immunoprecipitation and in situ proximity ligation assay. WASHC1-depleted cells display normal DNA replication and S-phase progression. However, loss of WASHC1 sensitizes HeLa cells to DNA replication inhibitor hydroxyurea (HU) and increases chromosome instability of HeLa and 3T3 cells under condition of HU-induced replication stress. Re-expression of nuclear WASHC1 in WASHC1KO 3T3 cells rescues the deficiency of WASHC1KO cells in the chromosomal stability after HU treatment. Moreover, chromatin immunoprecipitation assay indicates that WASHC1 associates with DNA replication origins, and knockdown of WASHC1 inhibits MCM protein loading at origins. Since efficient loading of excess MCM2-7 complexes is required for cells to survive replicative stress, these results demonstrate that WASHC1 promotes cell survival and maintain chromosomal stability under replication stress through recruitment of excess MCM complex to origins.

A ferrocene-containing nucleoside analogue targets DNA replication in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is a disease that remains refractory to existing treatments including the nucleoside analogue gemcitabine. In the current study we demonstrate that an organometallic nucleoside analogue, the ferronucleoside 1-(S,Rp), is cytotoxic in a panel of PDAC cell lines including gemcitabine-resistant MIAPaCa2, with IC50 values comparable to cisplatin. Biochemical studies show that the mechanism of action is inhibition of DNA replication, S-phase cell cycle arrest and stalling of DNA-replication forks, which were directly observed at single molecule resolution by DNA-fibre fluorography. In agreement with this, transcriptional changes following treatment with 1-(S,Rp) include activation of three of the four genes (HUS1, RAD1, RAD17) of the 9-1-1 check point complex clamp and two of the three genes (MRE11, NBN) that form the MRN complex as well as activation of multiple downstream targets. Furthermore, there was evidence of phosphorylation of checkpoint kinases 1 and 2 as well as RPA1 and gamma H2AX, all of which are considered biochemical markers of replication stress. Studies in p53-deficient cell lines showed activation of CDKN1A (p21) and GADD45A by 1-(S,Rp) was at least partially independent of p53. In conclusion, because of its potency and activity in gemcitabine-resistant cells, 1-(S,Rp) is a promising candidate molecule for development of new treatments for PDAC.

Joint toxicity mechanisms of binary emerging PFAS mixture on algae (Chlorella pyrenoidosa) at environmental concentration

Since traditional Per- and polyfluoroalkyl substances (PFAS) were banned in 2009 due to their bioaccumulation, persistence and biological toxicity, the emerging PFAS have been widely used as their substitutes and entered the aquatic environment in the form of mixtures. However, the joint toxicity mechanisms of these emerging PFAS mixtures to aquatic organisms remain largely unknown. Then, based on the testing of growth inhibition, cytotoxicity, photosynthesis and oxidative stress, and the toxicity mechanism of PFAS mixture (Perfluorobutane sulfonate and Perfluorobutane sulfonamide) to algae was explored using the Gene set enrichment analysis (GSEA). The results revealed that all three emerging PFAS treatments had a certain growth inhibitory effect on Chlorella pyrenoidosa (C. pyrenoidosa), but the toxicity of PFAS mixture was stronger than that of individual PFAS and showed a significant synergistic effect at environmental concentration. The joint toxicity mechanisms of binary PFAS mixture to C. pyrenoidosa were related to the damage of photosynthetic system, obstruction of ROS metabolism, and inhibition of DNA replication. Our findings are conductive to adding knowledge in understanding the joint toxicity mechanisms and provide a basis for assessing the environmental risk of emerging PFAS.

Addiction of Merkel cell carcinoma to MUC1-C identifies a potential new target for treatment

Merkel cell carcinoma (MCC) is an aggressive malignancy with neuroendocrine (NE) features, limited treatment options, and a lack of druggable targets. There is no reported involvement of the MUC1-C oncogenic protein in MCC progression. We show here that MUC1-C is broadly expressed in MCCs and at higher levels in Merkel cell polyomavirus (MCPyV)-positive (MCCP) relative to MCPyV-negative (MCCN) tumors. Our results further demonstrate that MUC1-C is expressed in MCCP, as well as MCCN, cell lines and regulates common sets of signaling pathways related to RNA synthesis, processing, and transport in both subtypes. Mechanistically, MUC1-C (i) interacts with MYCL, which drives MCC progression, (ii) is necessary for expression of the OCT4, SOX2, KLF4, MYC, and NANOG pluripotency factors, and (iii) induces the NEUROD1, BRN2 and ATOH1 NE lineage dictating transcription factors. We show that MUC1-C is also necessary for MCCP and MCCN cell survival by suppressing DNA replication stress, the p53 pathway, and apoptosis. In concert with these results, targeting MUC1-C genetically and pharmacologically inhibits MCC self-renewal capacity and tumorigenicity. These findings demonstrate that MCCP and MCCN cells are addicted to MUC1-C and identify MUC1-C as a potential target for MCC treatment.

Validation of a high throughput screening assay to identify small molecules that target the eukaryotic replicative helicase.

Mcm2-7 is the catalytic core of the eukaryotic replicative helicase, which together with CDC45 and the GINS complex unwind parental DNA to generate templates for DNA polymerase. Being a highly regulated and complex enzyme that operates via an incompletely understood multi-step mechanism, molecular probes of Mcm2-7 that interrogate specific mechanistic steps would be useful tools for research and potential future chemotherapy. Based upon a synthetic lethal approach, we previously developed a budding yeast multivariate cell-based high throughput screening (HTS) assay to identify putative Mcm inhibitors by their ability to specifically cause a growth defect in an mcm mutant relative to a wild-type strain[1]. Here, as proof of concept, we used this assay to screen a 1280-member compound library (LOPAC) for potential Mcm2-7 inhibitors. Primary screening and dose-dependent retesting identified twelve compounds from this library that specifically inhibited the growth of the Mcm mutant relative to the corresponding wild-type strain (0.9 % hit rate). Secondary assays were employed to rule out non-specific DNA damaging agents, establish direct protein-ligand interaction via biophysical methods, and verify in vivo DNA replication inhibition via fluorescence activated cell sorter analysis (FACS). We identified one agent (β-carboline-3-carboxylic acid N-methylamide, CMA) that physically bound to the purified Mcm2-7 complex (Kdapp119µM), and at slightly higher concentrations specifically blocked S-phase cell cycle progression of the wild-type strain. In total, identification of Mcm2-7 as a CMA target validates our synthetic lethal HTS assay paradigm as a tool to identify chemical probes for the Mcm2-7 replicative helicase.

High abundance of CDC45 inhibits cell proliferation through elevation of HSPA6.

ObjectivesCDC45 is the core component of CMG (CDC45‐MCMs‐GINS) complex that plays important role in the initial step of DNA replication in eukaryotic cells. The expression level of cdc45 is under the critical control for the accurate cell cycle progression. Loss‐of‐function of cdc45 has been demonstrated to inhibit cell proliferation and leads to cell death due to the inhibition of DNA replication and G1‐phase arrest. An increasing of CDC45 inhibits cell proliferation as well. Nevertheless, a systematic analysis of the effect of high dose of CDC45 on cell physiology and behaviors is unclear. In the present study, we aimed to investigate the effects and mechanisms of high dose of CDC45 on cell behaviors.Materials and MethodsWe overexpressed cdc45 in cultured cell lines, Ciona and Drosophila embryos, respectively. The cell cycle progression was examined by the BrdU incorporation experiment, flow cytometry and PH3 (phospho‐Histone 3) staining. RNA‐sequencing analysis and qRT‐PCR were carried out to screen the affected genes in HeLa cells overexpressing cdc45. siRNA‐mediated knockdown was performed to investigate gene functions in HeLa cells overexpressing cdc45.ResultsWe found that high level of cdc45 from different species (human, mammal, ascidian, and Drosophila) inhibited cell cycle in vitro and in vivo. High dose of CDC45 blocks cells entering into S phase. However, we failed to detect DNA damage and cell apoptosis. We identified hspa6 was the most upregulated gene in HeLa cells overexpressing cdc45 via RNA‐seq analysis and qRT‐PCR validation. Overexpression of Hs‐hspa6 inhibited proliferation rate and DNA replication in HeLa cells, mimicking the phenotype of cdc45 overexpression. RNAi against hspa6 partially rescued the cell proliferation defect caused by high dose of CDC45.ConclusionsOur study suggests that high abundance of CDC45 stops cell cycle. Instead of inducing apoptosis, excessive CDC45 prevents cell entering S phase probably due to promoting hspa6 expression.

Ulipristal-temozolomide-hydroxyurea combination for glioblastoma: in-vitro studies.

Glioblastoma multiforme (GBM) is a brain malignancy with worst survival. Low dose progesterone stimulates GBM growth, while progesterone receptor (PR)-antagonist mifepristone was shown to reduce growth and to enhance temozolomide sensitivity in GBM cells. Mifepristone is not available in all countries due to ethical reasons and may cause adrenal insufficiency and pelvic infections. Ulipristal is also a PR-antagonist used in treatment of uterine leiomyomas with higher biosafety. Ulipristal is demonstrated to suppress growth of breast cancer, yet it is not tested as yet whether it can also block growth and sensitize to temozolomide in glioblastoma as it was previously shown with mifepristone. Our first aim was to detect whether ulipristal exerts antiproliferative and chemotherapy-sensitizing effects in glioblastoma. Hydroxyurea inhibits DNA replication via blocking ribonucleotide reductase (RR) and it was demonstrated to increase temozolomide antineoplasticity in GBM. Progesterone receptor-activation in the uterus enhances RR transcription. Hence, we have hypothesized that PR-inactivation with ulipristal would further enhance hydroxyurea antineoplasticity by shutting down DNA synthesis mechanisms through further suppression of RR. Lastly, there exists no study as yet whether ulipristal, hydroxyurea and temozolomide could exert ternary antineoplastic efficacy, which was our last aim to define. To reveal interactions between ulipristal, hydroxyurea and temozolomide, we treated human U251 GBM cell line with these agents alone and in combination and measured cell proliferation, total antioxidant capacity (TAC) and total oxidant status (TOS) in conditioned medium and cellular cytokine gene expressions. All agents significantly reduced cell proliferation significantly, yet the most significant decrease of GBM cells occured with the triple drug combination at the 96th hour. All agents significantly decreased TAC and increased TOS in culture media, which was mostly relevant for the triple combination at the 96th hour. All these 3 agents tend to reduce the expression of immunosuppressive and/or GBM-growth stimulating cytokines TGFβ, IL-10 and IL-17 while increasing the expression of GBM-growth suppressing cytokine IL-23. Repurposal of these agents in treatment of GBM would be a plausible approach if future studies prove their efficacy.

DLK-Dependent Biphasic Reactivation of Herpes Simplex Virus Latency Established in the Absence of Antivirals.

Understanding the molecular mechanisms of herpes simplex virus 1 (HSV-1) latent infection and reactivation in neurons requires the use of in vitro model systems. Establishing a quiescent infection in cultured neurons is problematic, as any infectious virus released can superinfect the cultures. Previous studies have used the viral DNA replication inhibitor acyclovir to prevent superinfection and promote latency establishment. Data from these previous models have shown that reactivation is biphasic, with an initial phase I expression of all classes of lytic genes, which occurs independently of histone demethylase activity and viral DNA replication but is dependent on the cell stress protein DLK. Here, we describe a new model system using HSV-1 Stayput-GFP, a reporter virus that is defective for cell-to-cell spread and establishes latent infections without the need for acyclovir. The establishment of a latent state requires a longer time frame than previous models using DNA replication inhibitors. This results in a decreased ability of the virus to reactivate using established inducers, and as such, a combination of reactivation triggers is required. Using this system, we demonstrate that biphasic reactivation occurs even when latency is established in the absence of acyclovir. Importantly, phase I lytic gene expression still occurs in a histone demethylase and viral DNA replication-independent manner and requires DLK activity. These data demonstrate that the two waves of viral gene expression following HSV-1 reactivation are independent of secondary infection and not unique to systems that require acyclovir to promote latency establishment. IMPORTANCE Herpes simplex virus-1 (HSV-1) enters a latent infection in neurons and periodically reactivates. Reactivation manifests as a variety of clinical symptoms. Studying latency and reactivation in vitro is invaluable, allowing the molecular mechanisms behind both processes to be targeted by therapeutics that reduce the clinical consequences. Here, we describe a novel in vitro model system using a cell-to-cell spread-defective HSV-1, known as Stayput-GFP, which allows for the study of latency and reactivation at the single neuron level. We anticipate this new model system will be an incredibly valuable tool for studying the establishment and reactivation of HSV-1 latent infection in vitro. Using this model, we find that initial reactivation events are dependent on cellular stress kinase DLK but independent of histone demethylase activity and viral DNA replication. Our data therefore further validate the essential role of DLK in mediating a wave of lytic gene expression unique to reactivation.

Catalytic properties and biological function of a PIWI-RE nuclease from Pseudomonas stutzeri

BackgroundProkaryotic Argonaute (pAgo) proteins are well-known oligonucleotide-directed endonucleases, which contain a conserved PIWI domain required for endonuclease activity. Distantly related to pAgos, PIWI-RE family, which is defined as PIWI with conserved R and E residues, has been suggested to exhibit divergent activities. The distinctive biochemical properties and physiological functions of PIWI-RE family members need to be elucidated to explore their applications in gene editing.ResultsHere, we describe the catalytic performance and cellular functions of a PIWI-RE family protein from Pseudomonas stutzeri (PsPIWI-RE). Structural modelling suggests that the protein possesses a PIWI structure similar to that of pAgo, but with different PAZ-like and N-terminal domains. Unlike previously reported pAgos, recombinant PsPIWI-RE acts as an RNA-guided DNA nuclease, as well as a DNA-guided RNA nuclease. It cleaves single-stranded DNA at temperatures ranging from 20 to 65 °C, with an optimum temperature of 45 °C. Mutation at D525 or D610 significantly reduced its endonuclease activity, confirming that both residues are key for catalysis. Comparing with wild-type, mutant with PIWI-RE knockout is more sensitive to ciprofloxacin as DNA replication inhibitor, suggesting PIWI-RE may potentially be involved in DNA replication.ConclusionOur study provides the first insights into the programmable nuclease activity and biological function of the unknown PIWI-RE family of proteins, emphasizing their important role in vivo and potential application in genomic DNA modification.Graphical

Diclofenac: A Nonsteroidal Anti-Inflammatory Drug Inducing Cancer Cell Death by Inhibiting Microtubule Polymerization and Autophagy Flux.

Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID) used to treat inflammatory diseases induces cellular toxicity by increasing the production of reactive oxygen species (ROS) and impairing autophagic flux. In this study, we investigated whether diclofenac induces cancer cell death and the mechanism by which diclofenac causes cell death. We observed that diclofenac induces mitotic arrest with a half-maximal effective concentration of 170 μM and cell death with a half-maximal lethal dose of 200 µM during 18-h incubation in HeLa cells. Cellular microtubule imaging and in vitro tubulin polymerization assays demonstrated that treatment with diclofenac elicits microtubule destabilization. Autophagy relies on microtubule-mediated transport and the fusion of autophagic vesicles. We observed that diclofenac inhibits both phagophore movement, an early step of autophagy, and the fusion of autophagosomes and lysosomes, a late step of autophagy. Diclofenac also induces the fragmentation of mitochondria and the Golgi during cell death. We found that diclofenac induces cell death further in combination with 5-fuorouracil, a DNA replication inhibitor than in single treatment in cancer cells. Pancreatic cancer cells, which have high basal autophagy, are particularly sensitive to cell death by diclofenac. Our study suggests that microtubule destabilization by diclofenac induces cancer cell death via compromised spindle assembly checkpoints and increased ROS through impaired autophagy flux. Diclofenac may be a candidate therapeutic drug in certain type of cancers by inhibiting microtubule-mediated cellular events in combination with clinically utilized nucleoside metabolic inhibitors, including 5-fluorouracil, to block cancer cell proliferation.

Topoisomerase II poisons inhibit vertebrate DNA replication through distinct mechanisms.

Topoisomerase II (TOP2) unlinks chromosomes during vertebrate DNA replication. TOP2 "poisons" are widely used chemotherapeutics that stabilize TOP2 complexes on DNA, leading to cytotoxic DNA breaks. However, it is unclear how these drugs affect DNA replication, which is a major target of TOP2 poisons. Using Xenopus egg extracts, we show that the TOP2 poisons etoposide and doxorubicin both inhibit DNA replication through different mechanisms. Etoposide induces TOP2-dependent DNA breaks and TOP2-dependent fork stalling by trapping TOP2 behind replication forks. In contrast, doxorubicin does not lead to appreciable break formation and instead intercalates into parental DNA to stall replication forks independently of TOP2. In human cells, etoposide stalls forks in a TOP2-dependent manner, while doxorubicin stallsforks independently of TOP2. However, both drugs exhibit TOP2-dependent cytotoxicity. Thus, etoposide and doxorubicin inhibit DNA replication through distinct mechanisms despite shared genetic requirements for cytotoxicity.

Targeted inhibition of the expression of both MCM5 and MCM7 by miRNA-214 impedes DNA replication and tumorigenesis in hepatocellular carcinoma cells

MicroRNAs are noncoding RNAs with a typical length of 22 nucleotides that post-transcriptionally suppress gene expression by inducing target mRNA degradation and/or impairing translation in eukaryotes. Thousands of miRNA genes in the human genome are involved in various physiological and pathological processes. Each miRNA targets many different mRNAs, while each mRNA may be targeted by various miRNAs. Mini-chromosome maintenance (MCM2-7) protein complex functions as essential components of the pre-replicative complex (pre-RC) and forms a helicase together with other proteins to unwind the DNA duplex in S phase. MCM proteins are overexpressed in all cancer cells, while they are strictly regulated in normal cells, with no expression in non-proliferating normal cells. Here we report that miRNA-214–3p (miR-214) targets both MCM5 and MCM7. The level of miR-214 is lower in HepG2 and Hep3B hepatocellular carcinoma cells than the L-02 normal liver cells. Introduction of miRNA-214 mimic into HepG2 and Hep3B cells reduced the mRNA and protein levels of MCM5/7 and inhibited DNA replication, cell cycle progression, cell proliferation and colony formation. Comparatively, miRNA-214 mimic had little effect in L-02 cells. Importantly, miR-214 mimic can also inhibit the growth of HepG2 xenografts in nude mice. Our data suggest that miRNA-214 regulates DNA replication by targeting MCM5/7 and has the potential to be developed into a liver cancer drug. ImplicationsThis study supports the notion that DNA replication-initiation proteins (DRIPs), including MCM2-7 proteins, are attractive anticancer targets. Furthermore, the potential of miR-214 as an anticancer agent, with activity against liver cancer cells but not normal livre cells, may be of high significance.

Silver Complexes as Anticancer Agents: A Perspective Review

Metal complexes gained more attention in the medical field, particularly cancer treatment, due to their enhanced potential and redox ability. These complexes exhibited more significant antiproliferation potential by triggering the generation of reactive oxygen species (ROS), altering cell membrane, changing cell redox potential and by DNA replication inhibition. Platinum-based metal complex Cisplatin was prepared. It was widely used in treating lung, breast, ovarian and testicular cancers; however, these complexes showed many adverse effects such as renal insufficiency and electrolyte abnormalities, neurotoxicity, ototoxicity, nausea, vomiting and drug resistance. To overcome these side effects and drug resistance mechanisms, silver metal is highly employed as a cytotoxic agent by DNA binding through the formation of disulphide bonds. Moreover, silver complexes exhibited more significant cytotoxicity with the lowest toxicity. This review highlighted silver complexes' anticancer and cytotoxic potential with various bioactive ligands.

ADRV 12L: A Ranaviral Putative Rad2 Family Protein Involved in DNA Recombination and Repair.

The Andrias davidianus ranavirus (ADRV) is a member of the family Iridoviridae and belongs to the nucleocytoplasmic large DNA viruses. Based on genomic analysis, an ADRV-encoding protein, ADRV 12L, and its homologs from other iridoviruses were predicted as Rad2 family proteins based on the conserved amino acids, domains, and secondary structures. Expression analysis showed that the transcription of ADRV 12L started at 4 h post infection, and its expression was not inhibited by a DNA-replication inhibitor. Meanwhile, immunofluorescence localization showed that ADRV 12L mainly localized in viral factories and colocalized with the viral nascent DNA, which hinted at a possible role in DNA replication. Furthermore, a mutant ADRV lacking 12L (ADRV-Δ12L) was constructed. In both luciferase assays based on homologous recombination (HR) and double-strand break repair (DSBR) that followed, ADRV-Δ12L induced less luciferase activity than the wild-type ADRV, indicating that HR and DSBR were impaired in ADRV-Δ12L infected cells. In addition, infection with ADRV-Δ12L resulted in smaller plaque sizes and lower viral titers than that with wild-type ADRV, indicating an important role for 12L in efficient virus infection. Therefore, the results suggest that Rad2 homologs encoded by iridovirus have important roles in HR- and DSBR-process of the viral DNA and, thus, affect virus replication and the production of progeny virions.

Dinitramine induces implantation failure by cell cycle arrest and mitochondrial dysfunction in porcine trophectoderm and luminal epithelial cells

The herbicide market is growing rapidly, as weed control is a significant challenge in agriculture. Many studies have reported the toxicity of herbicides to non-target organisms. Dinitramine is a dinitroaniline herbicide that is particularly toxic to aquatic organisms. However, little is known about the effects of dinitramine on the female reproductive system. Therefore, in the present study, we utilized porcine trophectoderm (pTr) cells and porcine endometrial luminal epithelial (pLE) cells to verify the reproductive toxicity of dinitramine. Dinitramine reduced the viability of both cell types, by triggering cell cycle arrest, especially at the sub-G1 phase, and increasing apoptosis, inhibiting DNA replication. Dinitramine disrupted intracellular calcium homeostasis and induced oxidative stress by producing reactive oxygen species, leading to the loss of mitochondrial membrane potential and alteration of mitochondrial respiration. Mitogen-activated protein kinase pathways were altered, and migration decreased in pTr and pLE cells after dinitramine treatment; the expression of pregnancy-related genes in these cells was decreased. Thus, dinitramine reduced the viability and migratory capacity of both cell types, and this could interrupt the early stages of pregnancy.

A chalcone-syringaldehyde hybrid inhibits triple-negative breast cancer cell proliferation and migration by inhibiting CKAP2-mediated FAK and STAT3 phosphorylation

BackgroundAlthough triple-negative breast cancer (TNBC) accounts for only 15% of breast cancer cases, it is associated with a high relapse rate and poor outcome after standard treatment. Currently, the effective drugs and treatment strategies for TNBC remain limited, and thus, developing effective treatments for TNBC is pressing. Several studies have demonstrated that both chalcone and syringaldehyde have anticancer effect, but their potential anti-TNBC bioactivity are still unknown. PurposeThe present study aimed to synthesize a chalcone-syringaldehyde hybrid (CSH1) and explore its potential anti-TNBC effects and the underlying molecular mechanism. MethodsCell cytotoxicity was determined by 3-(4,5-dimethythiazol)-2,5-diphenyltetrazolium bromide (MTT). The activity of cell proliferation was measured by colony formation assay and 5-ethynyl-2’-deoxyuridine (EdU) staining assay. Cell cycle distribution and cell apoptosis were determined by fluorescence-activated cell sorter (FACS). The situation of DNA damage was observed using fluorescence microscopy. The ability of cell-matrix adhesion, migration and invasion was detected using cell adhesion assay and transwell assay. Transcriptome sequencing was performed to find out the changed genes. Levels of various signaling proteins were assessed by western blotting. ResultsCSH1 treatment triggered DNA damage and inhibited DNA replication, cell cycle arrest, and cell apoptosis via suppressing signal transducer and activator of transcription 3 (STAT3) phosphorylation. Whole genome RNA-seq analysis suggested that 4% of changed genes were correlated to DNA damage and repair, and nearly 18% of changed genes were functionally related to cell adhesion and migration. Experimental evidence indicated that CSH1 treatment significantly affected the distribution of focal adhesion kinase (FAK) and its phosphorylation, resulting in cell-matrix-adhesion reduction and migration inhibition of TNBC cells. Further mechanistic studies indicated that CSH1 inhibited TNBC cell proliferation, adhesion, and migration by inhibiting cytoskeleton-associated protein 2 (CKAP2)-mediated FAK and STAT3 phosphorylation signaling. ConclusionThese results suggest that CKAP2-mediated FAK and STAT3 phosphorylation signaling is a valuable target for TNBC treatment, and these findings also reveal the potential of CSH1 as a prospective TNBC drug.

The Effects of Nanosilver on Bacterial Contamination and Increase Durability Cultivars of Rosa hybrida L. Through of Stenting Method.

The rose flower (Rosa hybrida L.) is one of the world's most popular flowers among ornamental plants and the cut rose flower industry is the most important aspect of the rose culture industry in the world. The stenting method (cutting and grafting) uses a stem containing a leaf and a dormant bud as a scion, which is grafted onto an internode from the rootless rootstock so that the formation of the graft and the induction of the root on the rootstock are carried out simultaneously. In this method, Natal Briar is often used due to its desirable traits such as high rooting, the possibility of faster regeneration after cutting, the ability to adapt to different culture media and high resistance to root diseases. Most imported Natal Briar rootstocks, in addition to increasing the cost of producing hybrid roses, have a percentage of Agrobacterium contamination that can reduce the efficiency of stenting. Therefore, using the tissue culture technique to propagate the base is one of the important solutions. But one of the problems of tissue culture is the control of bacterial contamination so that these factors grow faster than explants and absorbing carbohydrate sources from the culture medium prevents regeneration of Natal Briar rootstocks. Nanotechnology today has expanded the field of application of nanomaterials due to the increase in the surface-to-volume ratio, electron transfer ability and surface reaction capability. Inhibit DNA replication and destroy the cellular structure of Agrobacterium. Therefore, the use of appropriate concentrations of Nanosilver in the culture medium of Natal Briar rootstocks increases the economic efficiency and durability of Rosa hybrida cultivars obtained by the stenting method.

Microarray analysis of breast cancer gene expression profiling in response to 2-deoxyglucose, metformin, and glucose starvation

BackgroundBreast cancer (BC) is the most frequently diagnosed cancer in women. Altering glucose metabolism and its effects on cancer progression and treatment resistance is an emerging interest in BC research. For instance, combining chemotherapy with glucose-lowering drugs (2-deoxyglucose (2-DG), metformin (MET)) or glucose starvation (GS) has shown better outcomes than with chemotherapy alone. However, the genes and molecular mechanisms that govern the action of these glucose deprivation conditions have not been fully elucidated. Here, we investigated the differentially expressed genes in MCF-7 and MDA-MB-231 BC cell lines upon treatment with glucose-lowering drugs (2-DG, MET) and GS using microarray analysis to study the difference in biological functions between the glucose challenges and their effect on the vulnerability of BC cells.MethodsMDA-MB-231 and MCF-7 cells were treated with 20 mM MET or 4 mM 2-DG for 48 h. GS was performed by gradually decreasing the glucose concentration in the culture medium to 0 g/L, in which the cells remained with fetal bovine serum for one week. Expression profiling was carried out using Affymetrix Human Clariom S microarrays. Differentially expressed genes were obtained from the Transcriptome Analysis Console and enriched using DAVID and R packages.ResultsOur results showed that MDA-MB-231 cells were more responsive to glucose deprivation than MCF-7 cells. Endoplasmic reticulum stress response and cell cycle inhibition were detected after all three glucose deprivations in MDA-MB-231 cells and only under the metformin and GS conditions in MCF-7 cells. Induction of apoptosis and inhibition of DNA replication were observed with all three treatments in MDA-MB-231 cells and metformin-treated MCF-7 cells. Upregulation of cellular response to reactive oxygen species and inhibition of DNA repair mechanisms resulted after metformin and GS administration in MDA-MB-231 cell lines and metformin-treated MCF-7 cells. Autophagy was induced after 2-DG treatment in MDA-MB-231 cells and after metformin in MCF-7 cells. Finally, inhibition of DNA methylation were observed only with GS in MDA-MB-231 cells.ConclusionThe procedure used to process cancer cells and analyze their expression data distinguishes our study from others. GS had the greatest effect on breast cancer cells compared to 2-DG and MET. Combining MET and GS could restrain both cell lines, making them more vulnerable to conventional chemotherapy.

Design, synthesis, and biological evaluation of novel (E)-1-arylethan-1-one O-((3-arylisoxazol-5-yl) methyl) oxime derivatives as potent non-nucleoside HBV inhibitors

• Synthesized (E)-1-arylethan-1-one O-((3-arylisoxazol-5-yl)methyl) oximes derivatives • These oxime derivatives as potent HBV inhibitor • Chlorinating at phenyl when excessive NCS reacting with some benzaldehyde oximes • The synthesized compounds interacting to HBV core protein in molecular docking • Crystal structure confirmed the reaction path A series of novel O-((3-arylisoxazol-5-yl) methyl) oxime derivatives were synthesized by highly regioselective dipolar cycloadditions of nitrile oxides with ( E )-1-arylethan-1-one O-prop-2-yn-1-yl oxime substrates. Results showed that in the formation of (E)-N-hydroxy-3,4-dimethoxybenzimidoyl chloride, chlorinating substitution in phenyl occurred when excess N-chlorosuccinimide. The crystal structure of 10 among the series of compounds revealed it was cis-cyclization product and moiety of -C(CH 3 )=N-O- was E configuration. As a triclinic crystal, the twist angle between the isoxazol-5-yl ring ( B ) and phenyl ring ( A ) was 57.43°, at dihedral angles of 25.22°. Screening for anti-HBV activities of these compounds was carried out. The results showed compounds 11, 19 and 12 effectively inhibited HBeAg (IC 50 = 158.28 ± 0.02, 127.51 ± 0.01, and 213.26 ± 0.04 respectively). Compounds 11, 19 and 23 manifested stronger inhibition on HBsAg (IC 50 =12.68 ± 0.06, 61.24 ± 0.02, and 89.59 ± 0.02 µM respectively). Compound 15 was the effctivest inhibition of DNA replication (IC 50 =43.52±0.02 µM) among the compounds. Docking study of bioactive compounds with HBV core protein (3KXS) was carried out to explore their interaction.This study determined a new non-nucleoside analogs with inhibition of HBV.

Narciclasine suppresses esophageal cancer cell proliferation and migration by inhibiting the FAK signaling pathway

Esophageal cancer (EC) is one of the malignant cancer with pool survival due to the limited therapeutic and drug-resistance. Narciclasine, a natural compound from Lycoris sanguinea possesses antitumor and anti-inflammatory properties. However, the mechanisms underlying the growth-inhibitory effect of narciclasine against EC have not yet been elucidated. Experimental evidences indicated that narciclasine treatment significantly affected the distribution of FAK and its phosphorylation, resulting in proliferation inhibition and migration inhibition of EC. Our study also showed that narciclasine treatment triggered DNA damage and inhibited DNA replication, leading to cell cycle arrest and apoptosis. Further mechanistic studies indicated that narciclasine inhibited EC cell proliferation and migration through FAK/JNK and p38 pathway. Altogether, these findings suggest that narciclasine could be a potential novel chemotherapeutic agent for esophageal cancer cell proliferation and migration.