Proliferation Of HEK293 Cells Research Articles

Functionalization of graphene oxide as a way to increase biocompatibility

Introduction. Now, one of the most promising areas for the use of graphene-based materials, in particular graphene oxide, is biomedicine. Due to the wide variety of functional groups and the possibility of chemical modification of graphene oxide, the creation of composite materials for biomedical use is promising. These nanomaterials have a unique structure and properties, which determines their use for creating targeted drug delivery systems, in tissue engineering, bioimaging, as well as for creating new materials with antimicrobial and antiviral properties.The objective was to perform synthesis and identification of graphene oxide and its conjugate with glycine, and to study the biocompatibility of the obtained nanomaterials: the effect on haemolysis and platelet aggregation, genotoxicity and cytotoxicity.Methods and materials. Graphene oxide was synthesized from graphite using the modified Hummers and Offeman method, after which the graphene oxide-glycine conjugate was also obtained. Identification was carried out using nuclear magnetic resonance spectroscopy. Estimation of biocompatibility of the obtained nanomaterials included the study of their hemolytic activity, effect on collagen-induced platelet aggregation, cyto- and genotoxicity.Results. Graphene oxide and its conjugate with glycine were synthesized. Identification with using nuclear magnetic resonance spectroscopy confirmed the structure and composition of the substances. The study of the biocompatibility of the obtained nanomaterials showed the absence of hemolytic activity (the degree of hemolysis did not exceed 2.5% at the studied concentration range); the presence of antiplatelet properties (at C=10–100 mg·ml–1); the absence of geno- and cytotoxicity (graphene oxide at C=0.25–25 mg·L–1 does not affect the viability of HEK293 cells; in turn, the conjugate with glycine at C=100–200 mg·L–1 causes a dose-dependent increase proliferation of HEK293 cells).Conclusion. The study demonstrates that functionalization of the graphene surface with oxygen-containing groups and amino acids leads to increased hemocompatibility, as well as to the production of nanomaterials that do not exhibit genoand cytotoxicity.

Counteractions of a Novel Hydroalcoholic Extract from Lens Culinaria against the Dexamethasone-Induced Osteoblast Loss of Native Murine Cells.

The cytoprotective effects of a novel hydroalcoholic extract (0.01–5 mg/mL) from Lens culinaria (Terre di Altamura Srl) were investigated within murine native skeletal muscle fibers, bone marrow cells, and osteoblasts, and in cell lines treated with the apoptotic agent staurosporine (2.14 × 10−6 M), the alkylating drug cisplatin (10−4 M), the topoisomerase I inhibitor irinotecan (10−4 M), the antimitotic pro-oxidant doxorubicin (10−6 M), and the immunosuppressant dexamethasone (2 × 10−6 M). An amount of 10g of plant material was used to obtain a 70% ethanol/water product, following two-step extraction, evaporation, lyophilization, and storage at −20 °C. For the murine osteoblasts, doxorubicin reduced survival by −65%, dexamethasone by −32% and −60% after 24 and 48 h of incubation time, respectively. The extract was effective in preventing the osteoblast count-reduction induced by dexamethasone; it was also effective at preventing the inhibition of mineralization induced by dexamethasone. Doxorubicin and cisplatin caused a significant reduction in cell growth by −77% for bone marrow cells, −43% for irinotecan, and −60% for dexamethasone, but there was no evidence for the cytoprotective effects of the extract in these cells. Staurosporine and doxorubicin caused a fiber death rate of >−40% after 18 and 24 h of incubation, yet the extract was not effective at preventing these effects. The extract was effective in preventing the staurosporine-induced reduction of HEK293 proliferation and colony formation in the crystal violet DNA staining and the clonogenic assays. It was also effective for the cisplatin-induced reduction in HEK293 cell proliferation. The extract, however, failed to protect the SHSY5Y neurons against cisplatin and irinotecan-induced cytotoxicity. A UV/VIS spectroscopy analysis showed three peaks at the wavelengths of 350, 260, and 190 nm, which correspond to flavonoids, proanthocyanins, salicylates, and AA, constituting the extract. These data suggest the possible development of this extract for use against dexamethasone-induced bone loss and renal chemotherapy-induced damage.

Effects of a Novel Hydroalcoholic Extract of Lens Culinaria Against the Dexamethasone‐Induced bone loss and Chemotherapy Cytotoxicity in Murine and Human Cells

Lens culinaria showed nutritional health benefit in humans. Here, we evaluated the effects of a novel hydroalcoholic extract (70% ethanol, 1E030521DIPFARMTDA) from Lens culinaria (Terre di Altamura SRL, Altamura BA) to prevent cytotoxic damage from chemotherapeutic agents such as staurosporine, a non‐selective inhibitor of tyrosine kinases, cisplatin the antineoplastic alkylating agent, doxorubicin as antimitotic, and the glucocorticoid dexamethasone targeting osteoblast.On renal Hek293, staurosporine caused a significant reduction from ‐20% to ‐60% (p <0.05) in cell proliferation and the extract was significantly effective in bringing these values between 70% and 100% (p <0.05) with comparable activity to that of diazoxide. Also, in the clonogenic assay, the extract was effective to prevent the reduction of colonies numbers caused by staurosporine.Cisplatin caused a slight reduction of ‐10% in Hek293 cell proliferation and a significant reduction of ‐30% in SHSY5Y cell proliferation respectively after 48 and 72 hours of incubation time. In both cases, the extract showed a significant cytoprotective effect. On the primary culture of murine osteoblasts, doxorubicin reduced survival by ‐30%, dexamethasone reduced it by ‐15%. The extract was significantly effective in preventing cell count‐reduction induced by dexamethasone but not by doxorubicin.A UV/VIS spectroscopy analysis was carried out. We obtained three peaks at the wavelength of 350, 260 and 190 nm which correspond to families of compounds such as flavonoids, proanthocyanins and salicylates.Therefore, this novel extract prevented the cytotoxic damage induced by cisplatin and staurosporine on the renal cell line, and by cisplatin also on the neuronal cell line by clonogenic assay and DNA staining. The extract was significantly effective in preventing the reduction of cell proliferation induced by dexamethasone in murine osteoblasts suggesting of potential use in bone loss diseases. It is not yet clear which of these families molecules composing the extract is responsible for these effects or if there is a synergistic action.

MicroRNA-150 targets major epigenetic repressors and inhibits cell proliferation

The Polycomb Repressive Complex (PRC) proteins, EZH2 and EZH1 regulate many biological processes by generating the repressive H3K27me3 modifications in the chromatin. However, the factors that regulate the EZH1/EZH2 functions are poorly studied. We identify that the 3′UTRs of EZH2 and EZH1 mRNAs contain the binding sites for the miRNA, miR-150. MicroRNA-150 (miR-150) controls numerous biological processes including cell proliferation, differentiation and pathogenesis of a variety of diseases including cancer. We find that miR-150 regulates the levels of EZH1 and EZH2 through various experimental investigations. Since EZH2 is known to form a repressive complex with other epigenetic repressors especially DNMT3A and DNMT3B, we investigated whether miR-150 also regulates the DNMT3A and DNMT3B levels. We report that miR-150 regulates DNMT3A and DNMT3B levels through direct and indirect mechanisms respectively. Since these epigenetic repressors promote cell proliferation, we investigated the effect of miR-150 perturbation on HEK293 cell proliferation. We found that miR-150 inhibits cell proliferation and induces S-phase arrest by increasing the levels of tumor suppressors and decreasing the cell cycle regulators. Collectively, our study shows that miR-150 act as a tumor suppressor by down-regulating the oncogenic major epigenetic repressors and controls cell proliferation.

Graphene oxide enriched with oxygen-containing groups: on the way to an increase of antioxidant activity and biocompatibility

The article is dedicated to the comprehensive biocompatibility investigation of synthesised graphene oxide (GO) enriched with oxygen-containing functional groups (⁓85%). GO was synthesised through a modified Hummers and Offeman’s method and characterised using 13C NMR, Raman, and IR spectroscopy, XRD, HRTEM, along with size dimensions and ζ-potentials in aqueous dispersions. Biocompatibility study included tests on haemocompatibility (haemolysis, platelet aggregation, binding to human serum albumin and its esterase activity), antioxidant activity (2,2-diphenyl-1-picrylhydrazyl reaction, NO-radical uptake, Radachlorin photobleaching, photo-induced haemolysis), genotoxicity using DNA comet assay, as well as metabolic activity and proliferation of HEK293 cells.

Preparation of Silk Fibroin/Carboxymethyl Chitosan Hydrogel under Low Voltage as a Wound Dressing.

At present, silk fibroin (SF) hydrogel can be prepared by means of electrodeposition at 25 V in direct current (DC) mode. Reducing the applied voltage would provide benefits, including lower fabrication costs, less risk of high voltage shocks, and better stability of devices. Here, a simple but uncommon strategy for SF-based hydrogel preparation using 4 V in DC mode is discussed. SF was mixed and cross-linked with carboxymethyl chitosan (CMCS) through hydrogen bonding, then co-deposited on the graphite electrode. The thickness, mass, and shape of the SF/CMCS hydrogel were easily controlled by adjusting the electrodeposition parameters. Morphological characterization of the prepared hydrogel via SEM revealed a porous network within the fabricated hydrogel. This structure was due to intermolecular hydrogen bonding between SF and CMCS, according to the results of thermogravimetric analysis and rheological measurements. As a potential wound dressing, SF/CMCS hydrogel maintained a suitable moisture environment for wound healing and demonstrated distinct properties in terms of promoting the proliferation of HEK-293 cells and antibacterial activity against Escherichia coli and Staphylococcus aureus. Furthermore, histological studies were conducted on a full-thickness skin wound in rats covered with the SF/CMCS hydrogel, with results indicating that this hydrogel can promote wound re-epithelization and enhance granulation tissue formation. These results illustrate the feasibility of using the developed strategy for SF-based hydrogel fabrication in practice for wound dressing.

Antimicrobial and Wound Healing Properties of FeO Fabricated Chitosan/PVA Nanocomposite Sponge.

Diabetic and anemia-associated diabetic wounds increase the considerable morbidity and mortality in people, as reported by clinical studies. However, no anemia-associated diabetic wound dressing materials have been developed until now. Hence, this study aimed to develop a nanocomposite scaffold composed of chitosan (CS), poly (vinyl alcohol) (PVA), and phytogenic iron oxide nanoparticles (FeO NPs), for accelerated anemia-associated diabetic wound healing. The aqueous leaves extract of Pinus densiflora (PD) was utilized for the synthesis of iron oxide nanoparticles (FeO NPs). TEM and elemental analysis confirmed smaller size PD-FeO NPs (<50 nm) synthesis with the combination of iron and oxide. In addition, in vitro biological studies displayed the moderate antioxidant, antidiabetic activities, and considerable antibacterial activity of PD-FeO NPs. Further, the different concentrations of PD-FeO NPs (0.01, 0.03, and 0.05%) incorporated CS/PVA nanocomposites sponges were developed by the freeze-drying method. The porous structured morphology and the presence of PD-FeO NPs were observed under FE-SEM. Among nanocomposite sponges, PD-FeO NPs (0.01%) incorporated CS/PVA sponges were further chosen for the in vitro wound-healing assay, based on the porous and water sorption nature. Furthermore, the in vitro wound-healing assay revealed that PD-FeO NPs (0.01%) incorporated CS/PVA has significantly increased the cell proliferation in HEK293 cells. In conclusion, the CS/PVA-PD-FeO NPs (0.01%) sponge would be recommended for diabetic wound dressing after a detailed in vivo evaluation.

Ectopically Expressed Olfactory GPCRs OR51E1/2: Ligand Specificity, Signal Transduction, and Cytostatic and Cytotoxic Activity

Olfactory receptors (ORs) are the largest family of G protein-coupled receptors: there are ~380 ORs in humans and ~1,000 in mice. This family was originally cloned from the nasal epithelium, but several years ago, expression of many OR genes was detected in the airways, gut, blood vessels, brain, and other organs. These GPCRs are referred to as ectopic ORs (eORs). Understanding eOR function has lagged behind that of other GPCRs, and the majority of ORs remain to be orphan receptors. One reason for this delay is that the expression of functionally active ORs has not been technically possible for many years. In contrast to the most other Class A GPCRs, which can be produced using standard methods such as transient transfection, stability and trafficking ORs requires special chaperones and/or modification with unique signal peptides. Difficulties with detection and functional analysis of both endogenous and overexpressed eORs caused many controversies, and overall, this vast and potentially interesting family of GPCRs has been grossly understudied. Here, we investigated two eORs, OR51E1 and OR51E2, which due to their marked upregulation in prostate cancer are also known as “prostate-specific G protein-coupled receptors” (PSGR2 and PSGR, respectively). To investigate their signaling and effects on proliferation and survival we developed an inducible expression system in a prostate cancer cell line LNCaP. The robust expression and trafficking of ORs allowed us to analyze their function using cAMP, Ca2+, protein phosphorylation and other assays. Consistent with previous studies, OR51E1 stimulated adenylyl cyclase in response to treatment by short- to medium-chain organic acids (C3-C9) an some branched aliphatic acids. OR51E2 responded to acetate and propionate, but not to the longer chain organic acids. Other putative ligands mentioned in the literature did not have activity. Stimulation of LNCaP cells with butyrate inhibited their growth, and the knockdown of the endogenous OR51E1 negated this cytostatic effect. Interestingly, induced overexpression of OR51E1 or OR51E2 suppressed LNCaP cell proliferation. Overexpression of OR2AT4, beta2-adrenergic receptor or treatment of cells with forskolin did not suppress cell proliferation, indicating that rise in cAMP is not sufficient to induce cytostasis. These findings indicate that like other GPCRs, eORs can signal via more than one G protein pathway. Overexpression of OR51E1 caused upregulation of cytostatic and cell death markers including p27, p21, and p53, strongly increased annexin V staining and stimulated ERK1/2. OR51E1 had no effect on proliferation of HEK293 cells, indicating that cytotoxicity of OR51E1/2 is specific for LNCaP cells. Elucidating this mechanism will contribute to understanding etiology of prostate cancer and may illuminated these two and possibly other eORs as future therapeutic targets.

Small molecule inhibitor of nicotinamide N-methyltransferase shows anti-proliferative activity in HeLa cells

The anti-proliferative effects of 5-methylquinolinium (5MQ) of nicotinamide N-methyltransferase (NNMT) have not been previously investigated on a cervical cancer cell line. NNMT is a metabolic enzyme that is correlated with tumour progression and metastasis. 5MQ is a small molecule inhibitor of NNMT. 0.1–500 μM of 5MQ was tested on the HeLa epithelial cervical cancer cell line. Cell viability was assessed with the MTT test. TWIST, ZEB1, SERPIN1, SIRT1, CD16, mRNA and various protein expression levels were analysed with Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and Western Blotting, respectively. 5MQ significantly inhibited HeLa cell proliferation in a concentration and time-dependent manner. Increased cell shrinkage, loss of cellular adhesions and apoptotic bodies were observed in HeLa cells after 5MQ treatment. Following treatment with 5MQ, ZEB1, SIRT1, CD16 mRNA levels were increased while TWIST and SERPIN1 mRNA levels were reduced. Expressions of oncogenic proteins phospho-Akt and SIRT1 were decreased. 5MQ can effectively inhibit HeLa cell proliferation without apparently affecting HEK-293 cell proliferation. IMPACT STATEMENT What is already known on this subject? NNMT is a cytosolic enzyme involved in tumour progression, metastasis and treatment resistance. It was overexpressed in many human malignancies. 5-amino-1-methylquinolinium (5MQ) is a novel small molecule inhibitor of NNMT that has shown promising results in the treatment of obesity and in senescent muscle regeneration. 5MQ has not been tested on the HeLa cervical cancer cell line, previously. What do the results of this study add? In this study, 5MQ was tested on the HeLa cervical cancer cell line for the first time and the molecular changes associated with 5MQ treatment were analysed. 5MQ demonstrated significant anti-proliferative activity on HeLa cells, which displayed morphological signs of apoptosis. Treatment of HeLa cells with 5MQ led to an increase in ZEB1, SIRT1 mRNA while TWIST mRNA was decreased. Phospho-Akt and Sirtuin1 protein expressions were decreased. What are the implications of these findings for clinical practice and/or further research? 5MQ can effectively inhibit HeLa cell proliferation without apparently affecting HEK-293 cell proliferation. 5MQ treatment was associated with a decrease in the expression of phospho-Akt and Sirtuin1 proteins, both of which have been reported to maintain tumour progression. 5MQ can further be investigated and modified for anti-cancer therapy.

Comprehensive Split TEV Based Protein-Protein Interaction Screening of the Hippo Signaling Pathway Identifies TAOK2 As Central Modulator

The conserved Hippo signaling pathway plays an important role in limiting tissue growth by controlling proliferation rates. The core of the pathway is composed of STK3/4 and LATS1/2 kinases, which restrict the activity of YAP1, the main downstream effector. Upstream, the pathway is controlled by polarity proteins, other kinases, receptors, and scaffolding proteins. We conducted a split TEV-based protein-protein interaction screen to evaluate physical interactions among major Hippo pathway components and potential upstream modulators. TAOK2 was identified as key modulator of Hippo signaling, binding to pathway core kinase cassette components. Especially, TAOK2 binds to and phosphorylates LATS1, regulating YAP1 phosphorylation states, YAP1 transcriptional activity, and the proliferation of HEK293 cells. In a subset of human cancers, reduced TAOK2 expression correlates with reduced patient survival time supporting TAOK2’s role as tumor suppressor gene. As TAOK2 is a principally druggable kinase, it represents an attractive pharmacological target to modulate growth.

BK Polyomavirus Activates HSF1 Stimulating Human Kidney Hek293 Cell Proliferation.

Objectives Some DNA viruses, such as BKPyV, are capable of inducing neoplastic transformation in human tissues through still unclear mechanisms. The goal of this study is to investigate the carcinogenic potential of BK polyomavirus (BKPyV) in human embryonic kidney 293 (Hek293) cells, dissecting the molecular mechanism that determines the neoplastic transformation. Materials and Methods BKPyV, isolated from urine samples of infected patients, was used to infect monolayers of Hek293 cells. Subsequently, intracellular redox changes, GSH/GSSH concentration by HPLC, and reactive oxygen/nitrogen species (ROS/RNS) production were monitored. Moreover, to understand the signaling pathway underlying the neoplastic transformation, the redox-sensitive HFS1-Hsp27 molecular axis was examined using the flavonoid quercetin and polishort hairpin RNA technologies. Results The data obtained show that while BKPyV replication is closely linked to the transcription factor p53, the increase in Hek293 cell proliferation is due to the activation of the signaling pathway mediated by HSF1-Hsp27. In fact, its inhibition blocks viral replication and cell growth, respectively. Conclusions The HSF1-Hsp27 signaling pathway is involved in BKPyV infection and cellular replication and its activation, which could be involved in cell transformation.

Depletion of TRDMT1 affects 5-methylcytosine modification of mRNA and inhibits HEK293 cell proliferation and migration

Human TRDMT1 is a transfer RNA (tRNA) methyltransferase for cytosine-5 methylation and has been suggested to be involved in the regulation of numerous developmental processes. However, little is known about the molecular mechanisms or their biological significance. In this study, we investigated the effects of CRISPR-based TRDMT1 knockdown on phenotypes, mRNA m5C modifications and gene expression changes in HEK293 cells. We found that knockdown of TRDMT1 significantly inhibited cell proliferation and migration but had no effect on clonogenic potential. The inhibitory effects could be attenuated by re-expression of TRDMT1 in HEK293 cells. RNA sequencing (RNA-Seq) and RNA bisulfite sequencing (RNA-BisSeq) were performed in TRDMT1 knockdown and wild-type HEK293 cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that the differentially expressed genes were associated with the cell cycle, RNA transport, and RNA degradation and were enriched in cancer and Notch signaling pathways. We also found that TRDMT1 knockdown could change mRNA methylation levels. For the first time, these findings clarify the role of TRDMT1 in regulating mRNA methylation and inhibiting the proliferation and migration of HEK293 cells. These results provide new insights into a new function of TRDMT1 and elucidate the molecular mechanisms of aberrant RNA m5C during tumorigenesis.

COMMD1 regulates cell proliferation and cell cycle progression by modulating p21 Cip1 levels.

Copper metabolism MURR1 domain-containing 1 (COMMD1) is a protein that participates in multiple cellular processes, including copper homeostasis and nuclear factor kappa B (NF-κB) and hypoxia-inducible factor 1α (HIF-1α) signaling. The COMMD1 upstream regulators X-linked inhibitor of apoptosis protein (XIAP) and p300 and downstream targets such as NF-κB and HIF-1α are involved in the regulation of cell proliferation and cell cycle progression. However, whether COMMD1 regulates cell proliferation and the cell cycle remains unclear. In the present study, we demonstrated that both overexpression and knockdown of COMMD1 affected the proliferation of HEK293 cells, and the cell cycle assay revealed that ectopic expression of COMMD1 arrested the cell cycle at the G1 phase. Furthermore, western blot analysis showed that COMMD1 affected p21 Cip1 levels. Taken together, these results suggest that COMMD1 regulates cell proliferation and cell cycle progression by modulating p21 Cip1 levels. Abbreviations COMMD1: Copper metabolism MURR1 domain containing 1; XIAP: X chromosome-linked inhibitor of apoptosis protein; FCS: Fetal calf serum; WCE: Whole cell extracts; RT-PCR: Reverse transcription-polymerase chain reaction; HEK293: Human embryonic kidney 293; ShRNA: Short hairpin RNA; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells; ARF: Alternate reading frame protein product of the CDKN2A locus.

Bone-Mimicking Injectable Gelatine/Hydroxyapatite Hydrogels

Bioactive synthetic hydrogels have emerged as promising materials because they&lt;br /&gt; can provide molecularly tailored biofunctions and adjustable mechanical properties. To&lt;br /&gt; mimic the mineralogical and organic components of the natural bone, hydroxyapatite and&lt;br /&gt; a tyramine conjugate of gelatine were combined in this study. The effect of various amounts of in situ synthesized hydroxyapatite in gelatine-tyramine on the morphology&lt;br /&gt; and physical properties of injectable hydrogels was investigated. Mineralogical identification confirmed successful precipitation of in situ formed hydroxyapatite. Better distribution of hydroxyapatite crystal agglomerates within modified gelatine was found at 5 % of hydroxyapatite, which could be responsible for increased storage modulus with respect to pure gelatine hydrogel. Prepared composite hydrogels are non-toxic and support&lt;br /&gt; the proliferation of Hek293 cells.

Effect of C-Terminus Modification in Salmonella typhimurium FliC on Protein Purification Efficacy and Bioactivity.

Recombinant flagellin (FliC) has shown low efficacy in purification because of inclusion bodies formation and aggregation. We hypothesized preserving TLR5 binding site of FliC and removing some amino acids could be responsible for aggregation and solubility improvement. Hence, a bioinformatics study was performed to find hotspots in aggregate formation. Protein modeling was carried out by SWISS-MODEL and I-TASSER servers and models were compared by MATRAS server and Chimera 1.11.2. Gene modification was carried out based on bioinformatics studies. Genes, (truncated modified fliC (tmFliC) and full-length fliC (flFliC)), were cloned and expressed in pET-21a vector. Protein purification was carried out using HIS-Tag method. Proliferation assay and also induction of IL-8 in HEK293 cells were performed to confirm bioactivity function of tmFliC. Bioinformatics results showed that partial deletion of C-terminus may increase solubility without unfavorable effect on TLR5 recognition. Also, model comparison showed that this protein may preserve 3D structure. In addition, GlobPlot server demonstrated that tmFliC formed its globular domains which were important in TLR5 recognition. As we expected, high purification efficacy for tmFliC compared with flFliC was also obtained in experimental studies and a proper function for tmFliC was observed. The tmFliC enhanced cell proliferation in HEK293 cells compare with control after 24h. Also, IL-8 level was increased with stimulation by tmFliC after 24h. In conclusion, reducing hydrophobicity in C-terminus and deleting necessary amino acids for filament formation may increase protein solubility.

Small-molecule screening yields a compound that inhibits the cancer-associated transcription factor Hes1 via the PHB2 chaperone

The transcription factor Hes family basic helix-loop-helix transcription factor 1 (Hes1) is a downstream effector of Notch signaling and plays a crucial role in orchestrating developmental processes during the embryonic stage. However, its aberrant signaling in adulthood is linked to the pathogenesis of cancer. In the present study, we report the discovery of small organic molecules (JI051 and JI130) that impair the ability of Hes1 to repress transcription. Hes1 interacts with the transcriptional corepressor transducing-like enhancer of split 1 (TLE1) via an interaction domain comprising two tryptophan residues, prompting us to search a chemical library of 1,800 small molecules enriched for indole-like π-electron-rich pharmacophores for a compound that blocks Hes1-mediated transcriptional repression. This screening identified a lead compound whose extensive chemical modification to improve potency yielded JI051, which inhibited HEK293 cell proliferation with an EC50 of 0.3 μm Unexpectedly, using immunomagnetic isolation and nanoscale LC-MS/MS, we found that JI051 does not bind TLE1 but instead interacts with prohibitin 2 (PHB2), a cancer-associated protein chaperone. We also found that JI051 stabilizes PHB2's interaction with Hes1 outside the nucleus, inducing G2/M cell-cycle arrest. Of note, JI051 dose-dependently reduced cell growth of the human pancreatic cancer cell line MIA PaCa-2, and JI130 treatment significantly reduced tumor volume in a murine pancreatic tumor xenograft model. These results suggest a previously unrecognized role for PHB2 in the regulation of Hes1 and may inform potential strategies for managing pancreatic cancer.

SUMO Modification of the RNA-Binding Protein La Regulates Cell Proliferation and STAT3 Protein Stability.

The cancer-associated RNA-binding protein La is posttranslationally modified by phosphorylation and sumoylation. Sumoylation of La regulates not only the trafficking of La in neuronal axons but also its association with specific mRNAs. Depletion of La in various types of cancer cell lines impairs cell proliferation; however, the molecular mechanism whereby La supports cell proliferation is not clearly understood. In this study, we address the question of whether sumoylation of La contributes to cell proliferation of HEK293 cells. We show that HEK293 cells stably expressing green fluorescent protein (GFP)-tagged wild-type La (GFP-LaWT) grow faster than cells expressing a sumoylation-deficient mutant La (GFP-LaSD), suggesting a proproliferative function of La in HEK293 cells. Further, we found that STAT3 protein levels were reduced in GFP-LaSD cells due to an increase in STAT3 ubiquitination and that overexpression of STAT3 partially restored cell proliferation. Finally, we present RNA sequencing data from RNA immunoprecipitations (RIPs) and report that mRNAs associated with the cell cycle and ubiquitination are preferentially bound by GFP-LaWT and are less enriched in GFP-LaSD RIPs. Taken together, results of our study support a novel mechanism whereby sumoylation of La promotes cell proliferation by averting ubiquitination-mediated degradation of the STAT3 protein.

WWC2 is an independent prognostic factor and prevents invasion via Hippo signalling in hepatocellular carcinoma.

WWC family proteins negatively regulate HEK293 cell proliferation and organ growth by suppressing the transcriptional activity of Yes‐associated protein (YAP), a major effector of the Hippo pathway. The function of the scaffolding protein WWC1 (also called KIBRA) has been intensively studied in cells and animal models. However, the expression and clinicopathologic significance of WWC2 in cancer are poorly characterized. This study aimed to clarify the biological function and mechanism of action of WWC2 in hepatocellular carcinoma (HCC). Retrospective analysis revealed WWC2 was significantly down‐regulated in 95 clinical HCC tissues compared to the paired adjacent non‐cancerous tissues. Moreover, loss of WWC2 expression was significantly associated with advanced clinicopathological features, including venous infiltration, larger tumour size and advanced TNM stage. Positive WWC2 expression was associated with significantly better 5‐year overall survival, and WWC2 was an independent prognostic factor for overall survival in HCC. Moreover, we confirmed WWC2 inhibits HCC cell invasive ability in vitro. Elevated YAP expression was also observed in the same cohort of HCC tissues. Pearson's correlation coefficient analysis indicated WWC2 expression correlated inversely with nuclear YAP protein expression in HCC. Mechanistically, we confirmed overexpression of WWC2 suppresses the invasive and metastatic potential of HCC cells by activating large tumour suppressor 1 and 2 kinases (LATS1/2), which in turn phosphorylates the transcriptional co‐activator YAP. Overall, this study indicates WWC2 functions as a tumour suppressor by negatively regulating the Hippo signalling pathway and may serve as a prognostic marker in HCC.

MiR-181a-2 downregulates the E3 ubiquitin ligase CUL4A transcript and promotes cell proliferation.

MiR-181a-2 plays a major role in cell proliferation both positively and negatively depending on tissue type by targeting several regulators 3'UTR regions. We have predicted several targets for miR-181a-2 through computational approaches and characterized one its interesting target, CUL4A, an E3 ubiquitin ligase. CUL4A regulates diverse functions in the cells including DNA repair, DNA replication, cell cycle, genomic stability through polyubiquitination of target proteins. Deregulation of both miR-181a-2 and CUL4A are reported in many cancerous cells, but the functional link between them is unknown. We show that miR-181a-5p binds to 3'UTR of CUL4A and regulates its transcripts levels in HEK293 cells through overexpression studies. In addition, by using MTT and Neutral red assays, we showed that miR-181a-2 overexpression increased the proliferation in HEK293 cells. Moreover, cell cycle analysis using flow cytometer revealed that an increase in S-phase cells upon the overexpression of miR-181a-2. Though several miRNAs are known to downregulate the CUL4A levels, here we show that miR-181a-2 also participates in the downregulation of CUL4A. Taken together, our data demonstrated that miR-181a-2 increases the cell proliferation in HEK293 cells possibly through the downregulation of CUL4A.

H2S does not regulate proliferation via T-type Ca2+ channels

T-type Ca2+ channels (Cav3.1, 3.2 and 3.3) strongly influence proliferation of various cell types, including vascular smooth muscle cells (VSMCs) and certain cancers. We have recently shown that the gasotransmitter carbon monoxide (CO) inhibits T-type Ca2+ channels and, in so doing, attenuates proliferation of VSMC. We have also shown that the T-type Ca2+ channel Cav3.2 is selectively inhibited by hydrogen sulfide (H2S) whilst the other channel isoforms (Cav3.1 and Cav3.3) are unaffected. Here, we explored whether inhibition of Cav3.2 by H2S could account for the anti-proliferative effects of this gasotransmitter. H2S suppressed proliferation in HEK293 cells expressing Cav3.2, as predicted by our previous observations. However, H2S was similarly effective in suppressing proliferation in wild type (non-transfected) HEK293 cells and those expressing the H2S insensitive channel, Cav3.1. Further studies demonstrated that T-type Ca2+ channels in the smooth muscle cell line A7r5 and in human coronary VSMCs strongly influenced proliferation. In both cell types, H2S caused a concentration-dependent inhibition of proliferation, yet by far the dominant T-type Ca2+ channel isoform was the H2S-insensitive channel, Cav3.1. Our data indicate that inhibition of T-type Ca2+ channel-mediated proliferation by H2S is independent of the channels’ sensitivity to H2S.