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Tissue microarchitecture imposes physical constraints to the migration of individual cells. Especially in cancer metastasis, three-dimensional structural barriers within the extracellular matrix are known to affect the migratory behavior of cells, regulating the pathological state of the cells. Here, we employed a culture platform with micropillar arrays of 2 μm diameter and 16 μm pitch (2.16 micropillar) as a mechanical stimulant. Using this platform, we investigated how a long-term culture of A549 human lung carcinoma cells on the (2.16) micropillar-embossed dishes would influence the pathological state of the cell. A549 cells grown on the (2.16) micropillar array with 10 μm height exhibited a significantly elongated morphology and enhanced migration even after the detachment and reattachment, as evidenced in the conventional wound-healing assay, single-cell tracking analysis, and

Background: STAT3 is a pro-oncogenic transcription factor. Pyrimethamine (PYM) is a STAT3 inhibitor that suppresses the proliferation of some cancer cells through downregulation of STAT3 target proteins. Methodology & Results: We have used structure-based tools to design novel PYM-based compounds. Intracellular target validation studies revealed that representative compounds 11b–d and 15a downregulate STAT3 downstream proteins and inhibit STAT3 DNA binding domain (DBD). Relative to PYM, a cohort of these compounds are >100-fold more cytotoxic to cancer cells with constitutively active (high pSTAT3) and basal (low pSTAT3) STAT3 signaling, suggesting that STAT3 DBD inhibition is deleterious to the proliferation of cancer cells with low and high pSTAT3 levels. Conclusion: These are promising leads for further preclinical evaluation as therapeutic agents for STAT3-dependent cancers.

Chromosome region maintenance protein 1 (CRM1) mediates protein export from the nucleus and is a new target for anticancer therapeutics. Broader application of KPT-330 (selinexor), a first-in-class CRM1 inhibitor recently approved for relapsed multiple myeloma and diffuse large B-cell lymphoma, have been limited by substantial toxicity. We discovered that salicylates markedly enhance the antitumor activity of CRM1 inhibitors by extending the mechanisms of action beyond CRM1 inhibition. Using salicylates in combination enables targeting of a range of blood cancers with a much lower dose of selinexor, thereby potentially mitigating prohibitive clinical adverse effects. Choline salicylate (CS) with low-dose KPT-330 (K+CS) had potent, broad activity across high-risk hematological malignancies and solid-organ cancers ex vivo and in vivo. The K+CS combination was not toxic to nonmalignant cells as compared with malignant cells and was safe without inducing toxicity to normal organs in mice. Mechanistically, compared with KPT-330 alone, K+CS suppresses the expression of CRM1, Rad51, and thymidylate synthase proteins, leading to more efficient inhibition of CRM1-mediated nuclear export, impairment of DNA-damage repair, reduced pyrimidine synthesis, cell-cycle arrest in S-phase, and cell apoptosis. Moreover, the addition of poly (ADP-ribose) polymerase inhibitors further potentiates the K+CS antitumor effect. K+CS represents a new class of therapy for multiple types of blood cancers and will stimulate future investigations to exploit DNA-damage repair and nucleocytoplasmic transport for cancer therapy in general.

Anticancer lipopeptides (ACLPs) are considered promising alternatives to combat resistant cancer cells, but the influence of peptide conformational propensity alone on their selectivity and mechanism remains obscure. In this study, we developed N-palmitoylated MK5E (P1MK5E) and MEK5 (P1MEK5) that have the same composition of 23 residues undergoing the pH-dependent structural alterations but differ in the conformational tendency of their amino acid composites. Nonlipidated peptides were readily accumulated in the A549 cell nucleus by the direct membrane translocation and the heparan sulfate-mediated endocytosis than the lipid-raft-dependent pathway. The increased hydrophobicity favored the amino acid-position-dependent folding of P1MK5E and P1MEK5, respectively, toward the α-helical coiled-coil nanofibrils and amyloidlike β-protofibrils. At the close concentrations (∼7.5 μM) to the toxic effects of doxorubicin (DOX), P1MK5E exhibited (i) an increased anticancer toxicity through a time-dependent S-phase arrest, (ii) enhanced plasma membrane permeability, and (iii) dose-dependent changes in the cell death characteristic features in the A549 cells relative to P1MEK5 that was almost inactive at ∼75 μM. These observations were in accordance with the TNF-α-mediated necroptotic signaling in the c-MYC/PARP1-overexpressed A549 cells exposed to P1MK5E and accompanied by the ultrastructure of plasma membrane protrusions, extensive endoplasmic reticulum (ER) membrane expansion, mitochondrial swelling, and the formation of distinct cytoplasmic vacuolation. The structural results and the bioactivity behaviors, herein, declared the significance of α-helical propensity in the peptide sequence and the nanostructure morphologies of self-assembling ACLPs upon the selectivity and enhanced anticancer effectiveness, which notably holds promise in the design and development of efficient therapeutics for cancer.

Human Microrchidia 4 (MORC4) is associated with acute and chronic pancreatitis, inflammatory disorders and cancer but it remains largely uncharacterized. Here, we describe the structure–function relationship of MORC4 and define the molecular mechanism for MORC4 activation. Enzymatic and binding assays reveal that MORC4 has ATPase activity, which is dependent on DNA-binding functions of both the ATPase domain and CW domain of MORC4. The crystal structure of the ATPaseCW cassette of MORC4 and mutagenesis studies show that the DNA-binding site and the histone/ATPase binding site of CW are located on the opposite sides of the domain. The ATPase and CW domains cooperate in binding of MORC4 to the nucleosome core particle (NCP), enhancing the DNA wrapping around the histone core and impeding binding of DNA-associated proteins, such as transcription factors, to the NCP. In cells, MORC4 mediates formation of nuclear bodies in the nucleus and has a role in the progression of S-phase of the cell cycle, and both these functions require CW and catalytic activity of MORC4. Our findings highlight the mechanism for MORC4 activation, which is distinctly different from the mechanisms of action observed in other MORC family members., Human Microrchidia 4 (MORC4) ATPase has been implicated in acute and chronic pancreatitis, inflammatory disorders and cancer. Here the authors describe the structure–function relationship of MORC4 and define the molecular mechanism for MORC4 activation.

Canine parvovirus (CPV) has been used in cancer control as a drug delivery vehicle or anti-tumor reagent due to its multiple natural advantages. However, potential host cell cycle arrest induced by virus infection may impose a big challenge to CPV associated cancer control as it could prevent host cancer cells from undergoing cell lysis and foster them regain viability once the virotherapy was ceased. To explore CPV-induced cell cycle arrest and the underlying mechanism toward improved virotherapeutic design, we focus on epidermal growth factor receptor (EGFR), a cellular receptor interacting with TfR that mediates CPV-host interactions, and alterations on its tyrosine phosphorylation sites in response to CPV infection. We found that CPV could trigger host G1/S cell cycle arrest via the EGFR (Y1086)/p27 and EGFR (Y1068)/STAT3/cyclin D1 axes, and EGFR inhibitor could not reverse this process. Our results contribute to our understandings on the mechanism of CPV-induced host cellular response and can be used in the onco-therapeutic design utilizing CPV by preventing host cancer cells from entering cell cycle arrest.

Long non‑coding RNA small nucleolar RNA host gene 12 (SNHG12) has been demonstrated to be oncogenic. The aim of the present study was to examine the effects of SNHG12 on the progression of endometrial cancer (EC). The expression levels of SNHG12 and microRNA (miR)‑4429 were assessed in EC cell lines by reverse transcription‑quantitative PCR. Plasmids, including SNHG12 short hairpin RNAs (shRNAs), shRNA negative control (NC), SNHG12 overexpression (OV), OV‑NC, miR‑4429 mimic and mimic‑NC, were transfected into RL95‑2 cells. Post‑transfection, Cell Counting Kit‑8, Transwell Matrigel and wound‑healing assays were performed to assess cell proliferation, invasion and migration, respectively. Cell cycle phase distribution was assessed by flow cytometry. The protein expression levels of matrix metalloproteinase (MMP)2 and MMP9 were detected by western blotting. miR‑4429 target genes were predicted by bioinformatics analysis using target prediction online tools; the findings of this analysis were verified using a dual‑luciferase reporter system. Identified as a target of miR‑4429, SNHG12 was overexpressed in EC cell lines with decreased expression of miR‑4429. Further experiments demonstrated that SNHG12 silencing and overexpression of miR‑4429 markedly suppressed proliferation, migration and invasion of RL95‑2 cells, arrested cells in the G1 phase, and markedly downregulated the expression of MMP2 and MMP9. The opposite effects were observed in miR‑4429 mimic‑transfected RL95‑2 cells after SNHG12 was overexpressed. The findings of the present study established the role of SNHG12 and miR‑4429 in EC. Therefore, targeting the SNHG12/miR‑4429 axis could serve as a potential future therapeutic target for treatment of EC.

Objectives Eurycoma longifolia Jack (Simaroubaceae) is commonly distributed in the Southeast Asia and Indo China, which has been shown to possess antianxiety, antibacterial, anticancer, antifungal, anti-inflammatory, antimalarial and antioxidant biological activities. 14,15β-dihydroxyklaineanone is a diterpene isolated from E. longifolia Jack, which is cytotoxic against human lung cancer and human breast cancer cell lines. However, the effects and underlying mechanisms of 14,15β-dihydroxyklaineanone on hepatocellular carcinoma remain unknown. Methods Cell viability assay and colony formation assay were used to measure HepG2 cell proliferation. Flow cytometry was used to analyse cell cycle and apoptosis. Wound-healing assay and transwell assay were used to observe cells migration. RNA sequencing and the enrichment of differentially expressed genes (DEGs) in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were used to find and determine underlying pathways. Key findings We found that 14,15β-dihydroxyklaineanone inhibited the growth and migration of HepG2 cells but did not induce cell apoptosis. 14,15β-dihydroxyklaineanone induced S cell cycle arrest by downregulating the expression levels of cyclin A, p-CDK2, cyclin B1, p21, E2F-1 and PCNA. In addition, RNA sequencing showed that 14,15β-dihydroxyklaineanone regulated MAPK pathway by increasing the expression levels of phosphor-p38. Downregulating of p38 via both p38 inhibitor (SB203580) and p38-siRNA could antagonize the inhibition of cell proliferation and migration and reverse the changes in p-p38, E-cadherin, N-cadherin and PCNA expression induced by 14,15β-dihydroxyklaineanone treatment. Conclusions 14,15β-dihydroxyklaineanone inhibited cell proliferation and migration through regulating p38 MAPK pathway in HCC cells.

While proteasome inhibitors such as bortezomib showed satisfactory clinical benefits in the initial treatment of multiple myeloma (MM), drug resistance and relapse are unavoidable. Recent studies suggested inhibition of histone deacetylases (HDACs) restored sensitivity of bortezomib-resistant MM. Hence, we designed dual inhibitors targeting both HDACs and proteasomes to address the resistance of bortezomib. The most potent inhibitors, ZY-2 and ZY-13 showed excellent inhibition against proteasome and good selectivity against HDACs. In particular, ZY-2 not only exhibited good antiproliferative activities on the MM cell lines RPMI-8226, U266, and KM3 (IC50 values of 6.66, 4.31, and 10.1 nM, respectively) but also showed more potent antiproliferative activities against the bortezomib-resistant MM cell line KM3/BTZ compared with bortezomib (IC50 values of 8.98 vs. 226 nM, P < 0.01) and even better than the combination of the HDAC inhibitor MS-275 and bortezomib (1:1) (IC50 values of 8.98 vs. 98.0 nM, P < 0.01).

Background: Increased keratinocyte proliferation occurs in the skin of psoriatic patients and is supposed to play a role in the pathogenesis of this disorder. Compounds interfering with keratinocyte proliferation could be useful in the management of psoriatic patients. Aim: To investigate whether albendazole, an anti-helmintic drug that regulates epithelial cell function in various systems, inhibits keratinocyte proliferation in models of psoriasis. Methods: Aldara-treated mice received daily topical application of albendazole. Keratinocyte proliferation and keratin (K) 6 and K16 expression were evaluated by immunohistochemistry and Western blotting and inflammatory cells/mediators were analysed by immunohistochemistry and real-time PCR. In human keratinocytes (HEKa and HaCaT) treated with albendazole, cell cycle and proliferation, keratins and cell cycle-associated factors were evaluated by flow cytometry, colorimetric assay and Western blotting respectively. Results: Aldara-treated mice given albendazole exhibited reduced epidermal thickness, decreased number of proliferating keratinocytes and K6/K16 expression. Reduction of CD3- and Ly6G-positive cells in the skin of albendazole-treated mice associated with inhibition of IL-6, TNF-α, IL-1β, IL-17A, IL-36, CCL17, CXCL1, CXCL2 and CXCL5 expression. Treatment of keratinocytes with albendazole reduced K6/K16 expression and reversibly inhibited cell growth by promoting accumulation of cells in S-phase. This phenomenon was accompanied by down-regulation of CDC25A, a phosphatase regulating progression of cell cycle through S-phase, and PKR-dependent hyper-phosphorylation of eIF2α, an inhibitor of CDC25 translation. In Aldara-treated mice, albendazole activated PKR, enhanced eIF2α phosphorylation and reduced CDC25A expression. Conclusions: Data show that albendazole inhibits keratinocyte proliferation and exerts therapeutic effect in a murine model of psoriasis.

The frequency of esophageal adenocarcinoma is rising despite widespread use of proton pump inhibitors (PPIs), which heal reflux esophagitis but do not prevent reflux of weakly acidic gastric juice and bile in Barrett’s esophagus patients. We aimed to determine if weakly acidic (pH 5.5) bile salt medium (WABM) causes DNA damage in Barrett’s cells. Because p53 is inactivated frequently in Barrett’s esophagus and p38 can assume p53 functions, we explored p38’s role in DNA damage response and repair. We exposed Barrett’s cells with or without p53 knockdown to WABM, and evaluated DNA damage, its response and repair, and whether these effects are p38 dependent. We also measured phospho-p38 in biopsies of Barrett’s metaplasia exposed to deoxycholic acid (DCA). WABM caused phospho-H2AX increases that were blocked by a reactive oxygen species (ROS) scavenger. WABM increased phospho-p38 and reduced bromodeoxyuridine incorporation (an index of S phase entry). Repair of WABM-induced DNA damage proceeded through p38-mediated base excision repair (BER) associated with reduction-oxidation factor 1-apurinic/apyrimidinic endonuclease I (Ref-1/APE1). Cells treated with WABM supplemented with ursodeoxycholic acid (UDCA) exhibited enhanced p38-mediated responses to DNA damage. All of these effects were observed in p53-intact and p53-deficient Barrett’s cells. In patients, esophageal DCA perfusion significantly increased phospho-p38 in Barrett’s metaplasia. WABM exposure generates ROS, causing oxidative DNA damage in Barrett’s cells, a mechanism possibly underlying the rising frequency of esophageal adenocarcinoma despite PPI usage. p38 plays a central role in oxidative DNA damage response and Ref-1/APE1-associated BER, suggesting potential chemopreventive roles for agents like UDCA that increase p38 activity in Barrett’s esophagus. NEW & NOTEWORTHY We found that weakly acidic bile salt solutions, with compositions similar to the refluxed gastric juice of gastroesophageal reflux disease patients on proton pump inhibitors, cause oxidative DNA damage in Barrett’s metaplasia that could contribute to the development of esophageal adenocarcinoma. We also have elucidated a critical role for p38 in Barrett’s metaplasia in its response to and repair of oxidative DNA damage, suggesting a potential chemopreventive role for agents like ursodeoxycholic acid that increase p38 activity in Barrett’s esophagus.

Retrotransposons are genomic DNA sequences that copy them-selves to new genomic locations via RNA intermediates; LINE-1 is the only active and autonomous retrotransposon in the human genome. The mobility of LINE-1 is largely repressed in somatic tissues but is derepressed in many cancers, where LINE-1 retrotransposition is correlated with p53 mutation and copy number alteration (CNA). In cell lines, inducing LINE-1 expression can cause double-strand breaks (DSBs) and replication stress. Reanalyzing multiomic data from breast, ovarian, endometrial, and colon cancers, we confirmed correlations between LINE-1 expression, p53 mutation status, and CNA. We observed a consistent correlation between LINE-1 expression and the abundance of DNA replication complex components, indicating that LINE-1 may also induce replication stress in human tumors. In endometrial cancer, high-quality phosphoproteomic data allowed us to identify the DSB-induced ATM-MRN-SMC S phase checkpoint pathway as the primary DNA damage response (DDR) pathway associated with LINE-1 expres-sion. Induction of LINE-1 expression in an in vitro model led to increased phosphorylation of MRN complex member RAD50, suggesting that LINE-1 directly activates this pathway.

Hinokitiol is a tropolone-related compound isolated from the heartwood of cupressaceous plants. It is known to exhibit various biological functions including antibacterial, antifungal, and antioxidant activities. In the study, we investigated the antitumor activities of hinokitiol against human osteosarcoma cells. The results revealed that hinokitiol treatment inhibited cell viability of human osteosarcoma U-2 OS and MG-63 cells in the MTT assay. Further study revealed that hinokitiol exposure caused cell cycle arrest at the S phase and a DNA damage response with the induction of γ-H2AX foci in both osteosarcoma cell lines. In U-2 OS cells with wild-type tumor suppressor p53, we found that hinokitiol exposure induced p53 expression and cellular senescence, and knockdown of p53 suppressed the senescence. However, in MG-63 cells with mutated p53, a high percentage of cells underwent apoptosis with cleaved-PARP expression and Annexin V staining after hinokitiol treatment. In addition, up-regulated autophagy was observed both in hinokitiol-exposed U-2 OS and MG-63 cells. As the autophagy was suppressed through the autophagy inhibitor chloroquine, hinokitiol-induced senescence in U-2 OS cells was significantly enhanced accompanying more abundant p53 expression. In MG-63 cells, co-treatment of chloroquine increased hinokitiol-induced apoptosis and decreased cell viability of the treated cells. Our data revealed that hinokitiol treatment could result in different cell responses, senescence or apoptosis in osteosarcoma cell lines, and suppression of autophagy could promote these effects. We hypothesize that the analysis of p53 status and co-administration of autophagy inhibitors might provide more precise and efficacious therapies in hinokitiol-related trials for treating osteosarcoma.

We performed a comparative analysis of replication origin activation by genome-wide single-stranded DNA mapping in two common laboratory strains of Saccharomyces cerevisiae challenged by hydroxyurea (HU), an inhibitor of the ribonucleotide reductase. By doing so we gained understanding of the impact on origin activation by three factors: replication checkpoint control, DNA sequence polymorphisms, and relative positioning of origin and transcription unit. Our analysis recapitulated the previous finding that the majority of origins are subject to checkpoint control by the Rad53 kinase when cells were treated with HU. In addition, origins either subject to Rad53 checkpoint control or impervious to it are largely concordant between the two strains. However, these two strains also produced different dynamics of origin activation. First, the W303-RAD53 cells showed a significant reduction of fork progression than A364a-RAD53 cells. This phenotype was accompanied by an elevated level of Rad53 phosphorylation in W303-RAD53 cells. Second, W303-rad53K227A checkpoint-deficient cells activated a greater number of origins accompanied by global reduction of ssDNA across all origins compared to A364a-rad53K227A cells; and this is correlated with lower expression level of the mutant protein in W303 than in A364a. We also show that sequence polymorphism in the consensus motifs of the replication origins plays a minor role in determining origin usage. Remarkably, eight strain-specific origins lack the canonical 11-bp consensus motif for autonomously replicating sequences in either strain background. Finally, we identified a new class of origins that are only active in checkpoint-proficient cells, which we named “Rad53-dependent origins”. The only discernible feature of these origins is that they tend to overlap with an open reading frame, suggesting previously unexplored connection between transcription and origin activation. Our study presents a comprehensive list of origin usage in two diverse yeast genetic backgrounds, fine-tunes the different categories of origins with respect to checkpoint control, and provokes further exploration of the interplay between origin activation and transcription.Author SummaryComparative analysis of origins of replication in two laboratory yeast strains reveals new insights into origin activation, regulation and dependency on the Rad53 checkpoint kinase.

Patients with triple-negative breast cancer have a poor prognosis as only a few efficient targeted therapies are available. Cancer cells are characterized by their unregulated proliferation and require large amounts of nucleotides to replicate their DNA. One-carbon metabolism contributes to purine and pyrimidine nucleotide synthesis by supplying one carbon atom. Although mitochondrial one-carbon metabolism has recently been focused on as an important target for cancer treatment, few specific inhibitors have been reported. In this study, we aimed to examine the effects of DS18561882 (DS18), a novel, orally active, specific inhibitor of methylenetetrahydrofolate dehydrogenase (MTHFD2), a mitochondrial enzyme involved in one-carbon metabolism. Treatment with DS18 led to a marked reduction in cancer-cell proliferation; however, it did not induce cell death. Combinatorial treatment with DS18 and inhibitors of checkpoint kinase 1 (Chk1), an activator of the S phase checkpoint pathway, efficiently induced apoptotic cell death in breast cancer cells and suppressed tumorigenesis in a triple-negative breast cancer patient-derived xenograft model. Mechanistically, MTHFD2 inhibition led to cell cycle arrest and slowed nucleotide synthesis. This finding suggests that DNA replication stress occurs due to nucleotide shortage and that the S-phase checkpoint pathway is activated, leading to cell-cycle arrest. Combinatorial treatment with both inhibitors released cell-cycle arrest, but induced accumulation of DNA double-strand breaks, leading to apoptotic cell death. Collectively, a combination of MTHFD2 and Chk1 inhibitors would be a rational treatment option for patients with triple-negative breast cancer.

A number of point mutations have been identified in reprogrammed pluripotent stem cells such as iPSCs and ntESCs. The molecular basis for these mutations has remained elusive however, which is a considerable impediment to their potential medical application. Here we report a specific stage at which iPSC generation is not reduced in response to ionizing radiation, i.e. radio-resistance. Quite intriguingly, a G1/S cell cycle checkpoint deficiency occurs in a transient fashion at the initial stage of the genome reprogramming process. These cancer-like phenomena, i.e. a cell cycle checkpoint deficiency resulting in the accumulation of point mutations, suggest a common developmental pathway between iPSC generation and tumorigenesis. This notion is supported by the identification of specific cancer mutational signatures in these cells. We describe efficient generation of human integration-free iPSCs using erythroblast cells, which have only a small number of point mutations and INDELs, none of which are in coding regions., Point mutations have been found in induced pluripotent stem cells (iPSCs) but when they arise is unclear. Here, the authors show that a G1/S cell cycle checkpoint deficiency transiently occurs early in genome reprogramming, suggesting a common developmental pathway between iPSC and tumorigenesis, and generate genetic burden-free human iPSCs.

OBJECTIVES Doxorubicin is a DNA-damaging agent used to treat hematological cancers. Unfortunately, drug resistance can occur by defective DNA repair activity coupled with the ability of DNA polymerases to misreplicate unrepaired DNA lesions. This study demonstrates that the efficacy of doxorubicin can be improved by using an artificial nucleoside to efficiently and selectively inhibit this activity. METHODS In vitro studies using acute lymphoblastic leukemia cell lines define the mechanism of cell death caused by combining an artificial nucleoside with doxorubicin. RESULTS Flow cytometry experiments demonstrate that combining an artificial nucleoside with doxorubicin potentiates the cell killing effects of the drug by increasing apoptosis. The potentiation effect correlates with expression of TdT, a specialized DNA polymerase overexpressed in acute lymphoblastic leukemia. Cell cycle experiments demonstrate that this combination blocks cells at S-phase prior to inducing apoptosis. Finally, the unique chemical composition of the nucleoside analog was used to visualize the replication of damaged DNA in TdT-positive cells. This represents a potential diagnostic tool to easily identify doxorubicin-resistant cancer cells. CONCLUSION Studies demonstrate that a novel artificial nucleoside improves the therapeutic efficacy of doxorubicin, thereby reducing the risk of potential side effects caused by the DNA-damaging agent.

Our knowledge of the mechanism of rDNA transcription has benefited from the combined application of genetic and biochemical techniques in yeast. Nomura's laboratory (Nogi, Y., Vu, L., and Nomura, M. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 7026–7030 and Nogi, Y., Yano, R., and Nomura, M. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 3962–3966) developed a system in yeast to identify genes essential for ribosome biogenesis. Such systems have allowed investigators to determine whether a gene was essential and to determine its function in rDNA transcription. However, there are significant differences in both the structures and components of the transcription apparatus and the patterns of regulation between mammals and yeast. Thus, there are significant deficits in our understanding of mammalian rDNA transcription. We have developed a system combining CRISPR/Cas9 and an auxin-inducible degron that enables combining a “genetics-like”approach with biochemistry to study mammalian rDNA transcription. We now show that the mammalian orthologue of yeast RPA49, PAF53, is required for rDNA transcription and mitotic growth. We have studied the domains of the protein required for activity. We have found that the C-terminal, DNA-binding domain (tandem-winged helix), the heterodimerization, and the linker domain were essential. Analysis of the linker identified a putative helix–turn–helix (HTH) DNA-binding domain. This HTH constitutes a second DNA-binding domain within PAF53. The HTH of the yeast and mammalian orthologues is essential for function. In summary, we show that an auxin-dependent degron system can be used to rapidly deplete nucleolar proteins in mammalian cells, that PAF53 is necessary for rDNA transcription and cell growth, and that all three PAF53 domains are necessary for its function.

Translocase of outer mitochondrial membrane 20 (TOMM20) plays an essential role as a receptor for proteins targeted to mitochondria. TOMM20 was shown to be overexpressed in various cancers. However, the oncological function and therapeutic potential for TOMM20 in cancer remains largely unexplored. The purpose of this study was to elucidate the underlying molecular mechanism of TOMM20's contribution to tumorigenesis and to explore the possibility of its therapeutic potential using colorectal cancer as a model. The results show that TOMM20 overexpression resulted in an increase in cell proliferation, migration, and invasion of colorectal cancer (CRC) cells, while siRNA-mediated inhibition of TOMM20 resulted in significant decreases in cell proliferation, migration, and invasion. TOMM20 expression directly impacted the mitochondrial function including ATP production and maintenance of membrane potential, which contributed to tumorigenic cellular activities including regulation of S phase cell cycle and apoptosis. TOMM20 was overexpressed in CRC compared to the normal tissues and increased expression of TOMM20 to be associated with malignant characteristics including a higher number of lymph nodes and perineural invasion in CRC. Notably, knockdown of TOMM20 in the xenograft mouse model resulted in a significant reduction of tumor growth. This is the first report demonstrating a relationship between TOMM20 and tumorigenesis in colorectal cancer and providing promising evidence for the potential for TOMM20 to serve as a new therapeutic target of colorectal cancer. [BMB Reports 2019; 52(12): 712-717].

Clioquinol is recently considered to be the most promising drug for treating cancer and neurodegenerative diseases. However, its mode of action varies from different disease models. In this study, we found that clioquinol inhibited cell growth in human neurotypic SHSY-5Y cells, which was attributed to both S-phase cell-cycle arrest and autophagic cell death. Clioquinol increased the intracellular contents of iron and zinc as well as calcium as measured by ICP-AES. Staining of Fluo-3 confirmed an increase in the level of calcium. Analysis of the metal-binding ability of clioquinol showed that it was not a chelating agent of calcium ions and the elevation of intracellular calcium content is not achieved by clioquinol as an ionophore. CaCl2 could simulate or even aggravate the cytotoxicity of clioquinol and it increased S-phase cell cycle arrest induced by clioquinol in a concentration dependent manner. Staining of acridine orange demonstrated that autophagy induced by clioquinol was not affected by addition of calcium ions. In contrast, the intracellular calcium ion chelator BAPTA-am abolished the clioquinol-induced S phase arrest and reduced the cell death caused by clioquinol. The WB assay of cell cycle-related proteins (CDK2, p21 and p27) further confirmed that S phase arrest is positively correlated with intracellular calcium elevation, which was due to the alterations of the mRNA and protein levels of calcium pumps (SERCA and SPCA). Taken together, these data indicate that clioquinol regulates the level of intracellular calcium ions to induce S-phase cell cycle arrest in human SH-SY5Y cells. Our results demonstrate for the first time that an increase of intracellular calcium content is one of the mechanisms of clioquinol in the inhibition of human neurotypic SHSY-5Y cells.

A large body of evidence suggests that cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT), as well as expression and function of retinoid receptors, are pivotal features of tumor initiation, progression, and chemoresistance. This is also true for pancreatic ductal adenocarcinoma (PDAC), which represents a clinical challenge due to poor prognosis and increasing incidence. Understanding the above features of cancer cells could open new avenues for PDAC treatment strategies. The aim of this study was to investigate the relation between CSCs, EMT, and retinoid receptors in PDAC after treatment with the chemotherapeutic agents - gemcitabine and 5-fluorouracil. First, we demonstrated the difference in the expression levels of CSC and EMT markers and retinoid receptors in the untreated Mia PaCa-2 and Panc1 cells that also differed in the frequency of spontaneous apoptosis and distribution between the cell cycle phases. Chemotherapy reduced the number of cancer cells in the S phase. Gemcitabine and 5-fluorouracil modulated expression of CSC markers, E-cadherin, and RXRβ in Panc1 but not in Mia PaCa-2 cells. We suggest that these effects could be attributed to the difference in the basal levels of expression of the investigated genes. The obtained data could be interesting in the context of future preclinical research.

Background DNA-crosslinking agents like cisplatin and mitomycin C (MMC) are indispensible for the treatment of many solid malignancies. These anticancer drugs generate DNA interstrand crosslinks (ICLs) that cause cell death by blocking replication forks. Many factors counteracting ICL-induced DNA replication stress, including the Fanconi anemia (FA) pathway, are regulated by ubiquitination and, therefore, ubiquitin ligases are potential targets for the sensitization of cancer cells to crosslinking agents. In this study, we investigated the function of the CRL4 ubiquitin ligase in modulating the response of cancer cells to ICL induction. Methods The two cullin paralogs CUL4A and CUL4B, which form the CRL4 ligase scaffold, were depleted in cancer cells by small interfering RNA followed by analysis of the cellular and biochemical responses to ICLs elicited upon cisplatin or MMC treatment. Results We report that the combined depletion of CUL4A and CUL4B weakens an FA pathway-dependent S phase checkpoint response. CRL4 positively stimulates the monoubiquitination of FANCD2 required for the recruitment of XPF-ERCC1, a structure-specific endonuclease that, in turn, contributes to the display of single-stranded DNA (ssDNA) at ICLs. After CRL4 down regulation, the missing ssDNA results in reduced recruitment of RPA, thereby dampening activation of ATR and CHK1 checkpoint kinases and allowing for S phase progression despite ICL induction. Conclusion Our findings indicate that CRL4 promotes cell survival by potentiating an FA pathway-dependent ssDNA-RPA signaling platform installed at ICLs. The anticancer efficacy of crosslinking agents may, therefore, be enhanced by down regulating CRL4 activity.

Budding yeast treated with hydroxyurea (HU) activate the S phase checkpoint kinase Rad53, which prevents DNA replication forks from undergoing aberrant structural transitions and nuclease processing. Rad53 is also required to prevent premature extension of the mitotic spindle that assembles during a HU-extended S phase. Here we present evidence that checkpoint restraint of spindle extension is directly coupled to Rad53 control of replication fork stability. In budding yeast, centromeres are flanked by replication origins that fire in early S phase. Mutations affecting the Zn2+-finger of Dbf4, an origin activator, preferentially reduce centromere-proximal origin firing in HU, corresponding with suppression of rad53 spindle extension. Inactivating Exo1 nuclease or displacing centromeres from origins provides a similar suppression. Conversely, short-circuiting Rad53 targeting of Dbf4, Sld3, and Dun1, substrates contributing to fork stability, induces spindle extension. These results reveal spindle extension in HU-treated rad53 mutants is a consequence of replication fork catastrophes at centromeres. When such catastrophes occur, centromeres become susceptible to nucleases, disrupting kinetochore function and spindle force balancing mechanisms. At the same time, our data indicate centromere duplication is not required to stabilize S phase spindle structure, leading us to propose a model for how monopolar kinetochore-spindle attachments may contribute to spindle force balance in HU.

Glioblastoma is the most aggressive brain tumor. Although miR-141 has been demonstrated to primarily function as a tumor suppressor in numerous malignancies, including glioblastoma, the mechanisms involved remain poorly understood. Here, it is shown that miR-141 is downregulated in glioblastoma cell lines and tissues and may exert its biological function via directly targeting myelin transcription factor 1-like (MYT1L). Using two glioblastoma cell lines that differ from each other by the functionality of DNA-dependent protein kinase (DNAPK), a functional involvement of DNAPK in the miR-141 tumor suppression network was observed. In M059K cells with a normal function of DNAPK, the enforced expression of miR-141 attenuated MYT1L expression and suppressed cell proliferation. Conversely, the inhibition of miR-141 expression promoted cell proliferation; however, in M059J cells with a loss-of-function DNAPK, miR-141 constitutively inhibited cell proliferation upon ectopic overexpression or inhibition. An overexpression of miR-141 suppressed M059J cell migration, while it had no effect on M059K. Furthermore, the ectopic expression of miR-141 induced an S-phase arrest in both cell lines, whereas the inhibition of miR-141 caused a G1 arrest in M059J and accelerated the S phase in M059K. An overexpression and suppression of miR-141 resulted in an aberrant expression of cell-cycle proteins, including p21. Moreover, MYT1L may be a transcription factor of p21 in p53-mutant cells, whereas DNAPK may function as a repressor of MYT1L. The findings revealed the crucial role of DNAPK in miR-141-mediated suppression of gliomagenesis and demonstrated that it may be a target molecule in miR-141-associated therapeutic interventions for glioblastoma.

The BCR-ABL1 fusion gene is the driver oncogene in chronic myeloid leukemia (CML) and Philadelphia-chromosome positive (Ph+) acute lymphoblastic leukemia (ALL). The introduction of tyrosine kinase inhibitors (TKIs) targeting the ABL kinase (such as imatinib) has dramatically improved survival of CML and Ph+ ALL patients. However, primary and acquired resistance to TKIs remains a clinical challenge. Ph+ leukemia patients who achieve a complete cytogenetic (CCR) or deep molecular response (MR) (≥4.5log reduction in BCR-ABL1 transcripts) represent long-term survivors. Thus, the fast and early eradication of leukemic cells predicts MR and is the prime clinical goal for these patients. We show here that the first-in-class inhibitor of the Nedd8-activating enzyme (NAE1) MLN4924 effectively induced caspase-dependent apoptosis in Ph+ leukemia cells, and sensitized leukemic cells for ABL tyrosine kinase inhibitors (TKI) and hydroxyurea (HU). We demonstrate that MLN4924 induced DNA damage in Ph+ leukemia cells by provoking the activation of an intra S-phase checkpoint, which was enhanced by imatinib co-treatment. The combination of MLN4924 and imatinib furthermore triggered a dramatic shift in the expression of MCL1 and NOXA. Our data offers a clear rationale to explore the clinical activity of MLN4924 (alone and in combination with ABL TKI) in Ph+ leukemia patients

Colorectal cancer (CRC) is one of the most common malignancies with high levels of invasiveness, drug resistance and mortality, but the internal mechanisms of CRC are largely unknown. MicroRNAs (miRs) have been reported to be involved in the development of CRC, and numerous studies have demonstrated that the abnormal expression of miR-33a-5p might be associated with CRC. However, the function and downstream mechanism of miR-33a-5p in colorectal cancer (CRC) remains unclear. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), a mitochondrial enzyme involved in folic acid metabolism, interestingly was confirmed to be one of the target genes of miR-33a-5p in the present study. We first confirmed that miR-33a-5p in CRC tissues and cell lines were downregulated (P

MicroRNAs (miRNAs) have emerged as key mediators of posttranscriptional gene silencing in both pathogenic and pathological aspects of ischemic stroke biology. Therefore, the purpose of present study was to explore the effect of microRNA-199b-3p (miR-199b-3p) on the cerebral microvascular endothelial cells (CMECs) in middle cerebral artery occlusion-reperfusion (MCAO-R) mice by regulating MAPK/ERK/EGR1 axis. Mice were used to establish MCAO-R models and to measure the expression of miR-199b-3p and the MAPK/ERK/EGR1 axis-related genes. CMECs were extracted from the MCAO-R mice. A series of mimic or inhibitor for miR-199b-3p, or U0126 (an inhibitor for the MAPK/ERK/EGR1 axis) were introduced to treat these CMECs. The levels of miR-199b-3p and MAPK/ERK/EGR1 axis-related genes in tissues and cells were detected. The effects miR-199b-3p on the process of CMECs, including cell viability, cell cycle and cell apoptosis were evaluated. miR-199b-3p expressed poorly in the brain tissues after MCAO-R, along with activated MAPK/ERK/EGR1 axis and increased CMECs apoptosis. CMECs transfected with miR-199b-3p mimics and U0126 manifested with increased cell viability, more cells arrested at the S stage, and inhibited apoptosis of CMECs. In conclusion, these key results demonstrated up-regulated miR-199b-3p could protect mice against ischemic stroke by inhibiting the apoptosis of CMECs through blockade of MAPK/ERK/EGR1 axis.

DNA replication forks in eukaryotic cells stall at a variety of replication barriers. Stalling forks require strict cellular regulations to prevent fork collapse. However, the mechanism underlying these cellular regulations is poorly understood. In this study, a cellular mechanism was uncovered that regulates chromatin structures to stabilize stalling forks. When replication forks stall, H2BK33, a newly identified acetylation site, is deacetylated and H3K9 trimethylated in the nucleosomes surrounding stalling forks, which results in chromatin compaction around forks. Acetylation-mimic H2BK33Q and its deacetylase clr6-1 mutations compromise this fork stalling-induced chromatin compaction, cause physical separation of replicative helicase and DNA polymerases, and significantly increase the frequency of stalling fork collapse. Furthermore, this fork stalling-induced H2BK33 deacetylation is independent of checkpoint. In summary, these results suggest that eukaryotic cells have developed a cellular mechanism that stabilizes stalling forks by targeting nucleosomes and inducing chromatin compaction around stalling forks. This mechanism is named the “Chromsfork” control: Chromatin Compaction Stabilizes Stalling Replication Forks.

Summary: Histone methylation is essential for regulating gene expression during organogenesis to maintain stem cells and execute a proper differentiation program for their descendants. Here we show that the COMPASS family histone methyltransferase co-factor ASH2L is required for maintaining neural progenitor cells (NPCs) and the production and positioning of projection neurons during neocortex development. Specifically, loss of Ash2l in NPCs results in malformation of the neocortex; the mutant neocortex has fewer neurons, which are also abnormal in composition and laminar position. Moreover, ASH2L loss impairs trimethylation of H3K4 and the transcriptional machinery specific for Wnt-β-catenin signaling, inhibiting the proliferation ability of NPCs at late stages of neurogenesis by disrupting S phase entry to inhibit cell cycle progression. Overexpressing β-catenin after ASH2L elimination rescues the proliferation deficiency. Therefore, our findings demonstrate that ASH2L is crucial for modulating Wnt signaling to maintain NPCs and generate a full complement of neurons during mammalian neocortex development. : Precise orchestration of gene expression dynamics is essential for stem cells to execute a proper differentiation program during organogenesis. Li et al. demonstrate that ASH2L, a core subunit of the COMPASS methyltransferase complex, is critical for maintaining neural progenitor cells and the generation of different neuronal types during mammalian neocortical development. Keywords: ASH2L, COMPASS, Wnt signaling pathway, corticogenesis, H3K4me3, cell cycle, neurogenesis

Selective pharmacologic targeting of cell cycle regulators is a potent anti-cancer therapeutic strategy. Here, we show that caspase-3-mediated p21 cleavage involves p53 independent of triptolide (TPL)-induced S phase arrest in human type 1 nasopharyngeal carcinoma (NPC) cells. Coimmunoprecipitation studies demonstrated that TPL causes S phase cell cycle arrest by suppressing the formation of cyclin A-phosphor (p)-cyclin-dependent kinas 2 (CDK2) (Thr 39) complexes. Ectopic expression of constitutively active protein kinase B1 (Akt1) blocks the induction of S phase arrest and the suppression of cyclin A expression and CDK2 Thr 39 phosphorylation by TPL. Expression of the phosphomimetic mutant CDK2 (T39E) rescues the cells from TPL-induced S phase arrest, whereas phosphorylation-deficient CDK2 (T39A) expression regulates cell growth with significant S phase arrest and enhances TPL-triggered S phase arrest. Treatment with TPL induces an increase in the formation of complexes between unphosphorylated phosphatase and tensin homolog deleted from chromosome 10 (PTEN) and p85α in the plasma membrane. Decreased microRNA (miR)-144 expression and increased PTEN expression after TPL treatment were demonstrated, and TPL-enhanced p85α-PTEN complexes and inhibitory effects on Akt (Ser 473) phosphorylation and S phase arrest were suppressed by ectopic PTEN short hairpin RNA or miR-144 expression. Knockdown of endogenous miR-144 by miR-144 Trap upregulated PTEN expression and accordingly enhanced p85α-PTEN complex formation and S phase arrest. Collectively, the effect of TPL on S phase arrest in human NPC cells is likely to enhance the p85α-PTEN interaction in the plasma membrane by suppressing miR-144 expression, resulting in the attenuation of cyclin A-p-CDK2 (Thr 39) complex formation via Akt inactivation.

Purpose Pancreatic cancer (PC) is one of the most lethal cancers worldwide, but there are currently no effective treatments. The DNA damage response (DDR) is under investigation for the development of novel anti-cancer drugs. Since DNA repair pathway alterations have been found frequently in PC, the purpose of this study was to test the DDR-targeting strategy in PC using WEE1 and ATM inhibitors. Materials and methods We performed in vitro experiments using a total of ten human PC cell lines to evaluate antitumor effect of AZD1775 (WEE1 inhibitor) alone or combination with AZD0156 (ATM inhibitor). We established Capan-1-mouse model for in vivo experiments to confirm our findings. Results In our research, we found that WEE1 inhibitor (AZD1775) as single agent showed anti-tumor effects in PC cells, however, targeting WEE1 upregulated p-ATM level. Here, we observed that co-targeting of WEE1 and ATM acted synergistically to reduce cell proliferation and migration, and to induce DNA damage in vitro. Notably, inhibition of WEE1 or WEE1/ATM downregulated programmed cell death ligand 1 expression by blocking glycogen synthase kinase-3β serine 9 phosphorylation and decrease of CMTM6 expression. In Capan-1 mouse xenograft model, AZD1775 plus AZD0156 (ATM inhibitor) treatment reduced tumor growth and downregulated tumor expression of programmed cell death ligand 1, CMTM6, CD163, and CXCR2, all of which contribute to tumor immune evasion. Conclusion Dual blockade of WEE1 and ATM might be a potential therapeutic strategy for PC. Taken toget.

The antitumor activity of resveratrol, a polyphenolic compound found mainly in grapes, has been studied in several types of cancer. In bladder cancer, its antiproliferative effects have already been demonstrated; however, its mechanism of action is not completely understood. The aim of this study was to evaluate resveratrol antitumor activity (12.5, 25, 50, 100, 150, 200, and 250 μM) and its possible mechanisms of action in bladder tumor cells with different TP53 gene status (RT4, grade 1, TP53 wild type; 5637-grade 2 and T24-grade 3, TP53 mutated). Cell proliferation, clonogenic survival, morphological changes, cell cycle progression, apoptosis rates, genotoxicity, global methylation, immunocytochemistry for p53 and PCNA and relative expression profiles of the AKT, mTOR, RASSF1A, HOXB3, SRC, PLK1, and DNMT1 were evaluated. Resveratrol decreased cell proliferation and induced DNA damage in all cell lines. Regarding the long-term effects, resveratrol reduced the number of colonies in all cell lines; however, TP53 wild type cells were more resistant. Increased rates of apoptosis were found in the TP53 wild type cells and this was accompanied by AKT, mTOR, and SRC downregulation. In addition, the resveratrol antiproliferative effects in wild type TP53 cells were accompanied by modulation of the DNMT1 gene. In the TP53 mutated cells, cell cycle arrest at S phase with PLK1 downregulation was observed. Additionally, there was modulation of the HOXB3/RASSF1A pathway and nuclear PCNA reduction in the highest-grade cells. In conclusion, resveratrol has antiproliferative activity in bladder tumor cells; however, the mechanisms of action are dependent on TP53 status. Environ. Mol. Mutagen., 60:740-751, 2019. © 2019 Wiley Periodicals, Inc.

Musashi RNA‑binding protein 1 (MSI1) is highly expressed in several types of cancer; however, its role in oral squamous cell carcinoma (OSCC) remains unknown. The purpose of this study was to investigate the probable mechanism underlying the involvement of MSI1 in OSCC. The results demonstrated that MSI1 was upregulated in OSCC tissues, but not in adjacent healthy tissues. MSI1 silencing resulted in decreased cell proliferative, invasive and migrative capacity. In addition, MSI1 silencing led to cell cycle arrest at the S phase, downregulation of c‑Myc and cyclin D1, and upregulation of p21 and p27 levels. Additional studies demonstrated that MSI1 suppression inhibited the activation of signal transducer and activator of transcription 3 (STAT3) signaling. Accordingly, the findings of the present study suggested that MSI1 silencing can suppress OSCC cell proliferation and progression, in part by inhibiting the activation of the c‑Myc/STAT3 pathway.

Polyphyllin VI (PP-VI) is one of the major saponins present in Paris polyphylla Sm., a medicinal plant primarily used for cancer treatment in China and India. However, its anti-metastatic activity remains largely unknown. The current study thus investigated the anti-metastatic activity of PP-VI in mouse mammary carcinoma 4T1 and human breast cancer MDA-MB-231 cells. The anti-metastatic effect of PP-VI was investigated at a sub-cytotoxic dose in migration and invasion assays in vitro. Experimental metastasis mouse model was used to examine the anti-metastatic effect of PP-VI in vivo. Additionally, target prediction, real-time PCR, Western blotting and luciferase assay were performed to identify the target gene of a pro-metastatic microRNA, miR-18a in 4T1 cells. The effect of PP-VI on the identified target of miR-18a was further investigated. The results showed that PP-VI impaired the viability of 4T1 and MDA-MB-231 cells. Moreover, when applied at a sub-cytotoxic dose, PP-VI suppressed the metastatic potential of 4T1 and MDA-MB-231 cells. Receptor expressed in lymphoid tissue (RELT)-like 2 (Rell2) was identified as a direct target of miR-18a with anti-metastatic functions in 4T1 and MDA-MB-231 cells. PP-VI treatment resulted in increased expression of Rell2 and decreased level of miR-18a in 4T1 and MDA-MB-231 cells. PP-VI treatment also attenuated miR-18a mimic or Rell2 siRNA-augmented migration of MDA-MB-231 cells. The current work thus demonstrates for the first time that targeted regulation of Rell2 by miR-18a is in part implicated in the anti-metastatic effect of PP-VI in breast cancer cells, providing novel pharmacological insights into the anti-cancer effect of PP-VI.

Studies have identified the potential of chemopreventive effects of sulforaphane (SFN); however, the underlying mechanisms of its effect on breast cancer require further elucidation. This study investigated the anticancer effects of SFN that specifically induces G1/S arrest in breast ductal carcinoma (ZR-75-1) cells. The proliferation of the cancer cells after treatment with SFN was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. DNA content and cell cycle status were analyzed through flow cytometry. Our results demonstrated the inhibition of growth in ZR-75-1 cells upon SFN exposure. In addition, SERTAD1 (SEI-1) caused the accumulation of SFN-treated G1/S-phase cells. The downregulation of SEI-1, cyclin D2, and histone deacetylase 3 suggested that in addition to the identified effects of SFN against breast cancer prevention, it may also exert antitumor activities in established breast cancer cells. In conclusion, SFN can inhibit growth of and induce cell cycle arrest in cancer cells, suggesting its potential role as an anticancer agent.

The Wee1 inhibitor MK1775 (AZD1775) is currently being tested in clinical trials for cancer treatment. Here, we show that the p53 target and CDK inhibitor p21 protects against MK1775-induced DNA damage during S-phase. Cancer and normal cells deficient for p21 (HCT116 p21-/-, RPE p21-/-, and U2OS transfected with p21 siRNA) showed higher induction of the DNA damage marker γH2AX in S-phase in response to MK1775 compared to the respective parental cells. Furthermore, upon MK1775 treatment the levels of phospho-DNA PKcs S2056 and phospho-RPA S4/S8 were higher in the p21 deficient cells, consistent with increased DNA breakage. Cell cycle analysis revealed that these effects were due to an S-phase function of p21, but MK1775-induced S-phase CDK activity was not altered as measured by CDK-dependent phosphorylations. In the p21 deficient cancer cells MK1775-induced cell death was also increased. Moreover, p21 deficiency sensitized to combined treatment of MK1775 and the CHK1-inhibitor AZD6772, and to the combination of MK1775 with ionizing radiation. These results show that p21 protects cancer cells against Wee1 inhibition and suggest that S-phase functions of p21 contribute to mediate such protection. As p21 can be epigenetically downregulated in human cancer, we propose that p21 levels may be considered during future applications of Wee1 inhibitors.

Two transition metal complexes of [M(TMP)2(H2O)2] (TMP-Cu, M = Cu; TMP-Co, M = Co) with (E)-2-(((4H-1,2,4-triazol-4-yl)imino)methyl)-6-methoxyphenol (H-TMP) were first synthesized and characterized by infrared analysis, elemental analysis and single crystal X-ray diffraction analysis. Notably, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay showed that TMP-Cu displayed relatively high cytotoxic activity against Hep-G2 cancer cells, and high selectivity between human hepatocellular carcinoma cells and normal HL-7702 cells, in comparison to TMP-Co and cisplatin. Further studies showed that TMP-Cu and TMP-Co caused cell cycle arrest at S phase through regulation of S phase related protein expressions and induced Hep-G2 cell apoptosis via the mitochondrial pathway.

We synthesized five iron chelator derived from 2,6-diacetylpyridine bis(acylhydrazones) and proved their iron complexes structure by X–ray single crystal diffraction. These ligands have a significant anticancer proliferative activity and low cytotoxicity against normal cells. The Fe(III) complexes show reduced cytotoxic activity compared to the metal–free ligands. Anticancer mechanism studies indicate that ligands with a potential anticancer proliferation activity by inhibiting the activity of ribonucleotide reductase. Ligand rather than iron complexes regulate the expression of cell cycle associated proteins and inhibit cell cycle arrest in S phase. Apoptosis mechanism results showed that both ligand and iron complexes did not significantly promote apoptosis.

Nerve growth factor receptor (NGFR), a member of kinase protein, is emerging as an important target for Glioblastoma (GBM) treatment. Overexpression of NGFR is observed in many metastatic cancers including GBM, promoting tumor migration and invasion. Hydrazones have been reported to effectively interact with receptor tyrosine kinases (RTKs). We report herein the synthesis of 23 arylhydrazones of active methylene compounds (AHAMCs) compounds and their anti-proliferative activity against GBM cell lines, LN229 and U87. Compound R234, 2-(2-(2,4-dioxopentan-3-ylidene)hydrazineyl)benzonitrile, was identified as the most active anti-neoplastic compound, with the IC50 value ranging 87 μM - 107 μM. Molecular docking simulations of the synthesized compounds into the active site of tyrosine receptor kinase A (TrkA), demonstrated a strong binding affinity with R234 and concurs well with the obtained biological results. R234 was found to be a negative regulator of PI3K/Akt/mTOR pathway and an enhancer of p53 expression. In addition, R234 treated GBM cells exhibited the downregulation of cyclins, cyclin-dependent kinases and other key molecules involved in cell cycle such as CCNE, E2F, CCND, CDK6, indicating that R234 induces cell cycle arrest at G1/S. R234 also exerted its apoptotic effects independent of caspase3/7 activity, in both cell lines. In U87 cells, R234 induced oxidative effects whereas LN229 cells annulled oxidative stress. The study thus concludes that R234, being a negative modulator of RTKs and cell cycle inhibitor, may represent a novel class of anti-GBM drugs.