Synergistic Inhibitory Effect Research Articles

Natural Carrier-Free Binary Small Molecule Self-Assembled Hydrogel Synergize Antibacterial Effects and Promote Wound Healing by Inhibiting Virulence Factors and Alleviating the Inflammatory Response.

Methicillin-resistant Staphylococcus aureus (MRSA)-infected skin wounds have caused a variety of diseases and seriously endanger global public health. Therefore, multidimensional strategies are urgently to find antibacterial dressings to combat bacterial infections. Antibacterial hydrogels are considered potential wound dressing, while their clinical translation is limited due to the unpredictable risks and high costs of carrier excipients. it is found that the natural star antibacterial and anti-inflammatory phytochemicals baicalin (BA) and sanguinarine (SAN) can directly self-assemble through non-covalent bonds such as electrostatic attraction, π-π stacking, and hydrogen bonding to form carrier-free binary small molecule hydrogel. In addition, BA-SAN gel exhibited a synergistic inhibitory effect on MRSA. And its plasticity and injectability allowed it to be applied as a wound dressing. Due to the matched physicochemical properties and synergistic therapeutic effects, BA-SAN gel can inhibit bacterial virulence factors, alleviate wound inflammation, promote wound healing, and has good biocompatibility. The current study not only provided an antibacterial hydrogel with clinical value but also opened up new prospects that carrier-free hydrogels can be designed and originated from clinically used small-molecule phytochemicals.

Feiyanning formula modulates the molecular mechanism of osimertinib resistance in lung cancer by regulating the Wnt/β-catenin pathway.

Feiyanning Formula (FYN), a Chinese herbal formula derived from summarized clinical experience, is proven to have anti-tumor effects in lung cancer patients. Osimertinib, a third-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), can improve progression-free survival and overall survival of patients but drug resistance is inevitable. The current study evaluated the effects of FYN in osimertinib-resistant HCC827OR and PC9OR cells. FYN preferentially inhibited the proliferation and migration of HCC827OR and PC9OR cells. Moreover, FYN and osimertinib exhibited synergistic inhibitory effects on proliferation and migration. Real-time qPCR (RT-qPCR) and western blotting results indicated that FYN downregulated gene and protein levels of GSK3β and SRFS1, which are enriched in the Wnt/β-catenin pathway. Besides, FYN inhibited tumor growth and exhibited synergistic effects with osimertinib in vivo. Collectively, the results suggested that FYN exerted an anti-osimertinib resistance effect via the Wnt/β-catenin pathway.

Effects of matrine combined with LY294002 on proliferation, apoptosis and cell cycle of human myeloid leukemia K562 cells

To investigate the effects of matrine combined with LY294002 on proliferation, apoptosis and cell cycle of human myeloid leukemia K562 cells and explore the underlying mechanism. The effects of different concentrations of matrine alone and in combination with LY294002 on the proliferation of K562 cells were examined with CCK-8 assay. The changes in morphology of K562 cells were observed following treatment for 48 h with 0.4 g/L matrine and 10 μmol/L Y294002, either alone or in combination, and cell apoptosis was detected using flow cytometry with annexin V-FITC/PI double labeling; the changes in cell cycle was detected with PI labeling. Western blotting was performed to examine the effect of matrine combined with LY294002 on expressions of p-mTOR, p-PI3K, Akt, p-Akt, cyclinD1, Bcl-2 and caspase-9 in the cells. Treatment with different concentrations of matrine, both alone and in combination with LY294002, inhibited the proliferation of K562 cells in a time- and concentration-dependent manner. Compared with matrine treatment alone, the combined treatment caused more obvious morphological changes of the cells, significantly increased cell apoptosis (P < 0.01), and induced cell cycle arrest in G0/G1 (P < 0.01). Western blotting showed that the protein expression levels of p-mTOR, cyclinD1, p-PI3K, p-Akt and Bcl-2 in K562 cells increased while the expression level of caspase-9 decreased significantly after the combined treatment (P < 0.01). Matrine combined with LY294002 produces a synergistic inhibitory effect on K562 cells possibly by down-regulating the p-Akt expression in PI3K/Akt signaling pathway, reducing the expressions of p-mTOR, cyclinD1 and Bcl-2, and increasing the expression of caspase-9.

Synergistic effects of PI3K inhibition and pioglitazone against acute promyelocytic leukemia cells.

Although pioglitazone, a well-known anti-diabetic agent, has recently established itself as a pillar of cancer treatment, its therapeutic value could be attenuated by the aberrant activation of the PI3K/Akt pathway. To evaluate whether the PI3K/Akt suppression in leukemic cells could potentiate the anti-leukemic effects of pioglitazone. To assess the anti-leukemic effects of PI3K/Akt inhibitors on anti-leukemic effects of pioglitazone, we used MTT and trypan blue assays. Flow cytometric analysis and qRT-PCR were also applied to evaluate cell cycle and apoptosis. The resulting data revealed that upon PPARγ stimulation in different leukemic cell lines using pioglitazone, the survival and the proliferative capacity of the cells were significantly halted. Then, we evaluated the impact of the PI3K/Akt axis on the effectiveness of the drug in the most sensitive leukemic cell line; NB4 cells. Our results showed that treatment of NB4 cells with the PI3K inhibitors increased the sensitivity of leukemic cells to pioglitazone to the degree that even lower concentrations of the agent succeeded to induce apoptotic as well as the anti-proliferative effects. Moreover, it seems that PI3K inhibition could potentiate the anti-leukemic effect of pioglitazone through induction of p21-mediated sub-G1 cell cycle arrest and altering the balance between the pro-and anti-apoptotic genes. This study sheds light on the significance of the PI3K/Akt pathway in APL cell sensitivity to pioglitazone and proposed that the presence of the PI3K inhibitor in the therapeutic regimen containing pioglitazone could be promising in the treatment of this malignancy.

Synthesis of Glycolysis Inhibitor PFK15 and Its Synergistic Action with an Approved Multikinase Antiangiogenic Drug on Human Endothelial Cell Migration and Proliferation.

Activated endothelial, immune, and cancer cells prefer glycolysis to obtain energy for their proliferation and migration. Therefore, the blocking of glycolysis can be a promising strategy against cancer and autoimmune disease progression. Inactivation of the glycolytic enzyme PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase) suppresses glycolysis level and contributes to decreased proliferation and migration of cancer (tumorigenesis) and endothelial (angiogenesis) cells. Recently, several glycolysis inhibitors have been developed, among them (E)-1-(pyridin-4-yl)-3-(quinolin-2-yl)prop-2-en-1-one (PFK15) that is considered as one of the most promising. It is known that PFK15 decreases glucose uptake into the endothelial cells and efficiently blocks pathological angiogenesis. However, no study has described sufficiently PFK15 synthesis enabling its general availability. In this paper we provide all necessary details for PFK15 preparation and its advanced characterization. On the other hand, there are known tyrosine kinase inhibitors (e.g., sunitinib), that affect additional molecular targets and efficiently block angiogenesis. From a biological point of view, we have studied and proved the synergistic inhibitory effect by simultaneous administration of glycolysis inhibitor PFK15 and multikinase inhibitor sunitinib on the proliferation and migration of HUVEC. Our results suggest that suppressing the glycolytic activity of endothelial cells in combination with growth factor receptor blocking can be a promising antiangiogenic treatment.

Exploring the Inhibition of Quercetin on Acetylcholinesterase by Multispectroscopic and In Silico Approaches and Evaluation of Its Neuroprotective Effects on PC12 Cells.

This study investigated the inhibitory mechanism of quercetin in acetylcholinesterase (AChE) and its neuroprotective effects on β-amyloid25-35-induced oxidative stress injury in PC12 cells. Quercetin inhibited AChE in a reversible mixed manner with an IC50 of 4.59 ± 0.27 µM. The binding constant of quercetin with AChE at 25 °C was (5.52 ± 0.05) × 104 L mol-1. Hydrogen bonding and van der Waals forces were the main interactions in forming the stable quercetin-AChE complex. Computational docking revealed that quercetin was dominant at the peripheral aromatic site in AChE and induced enzymatic allosterism; meanwhile, it extended deep into the active center of AChE and destabilized the hydrogen bond network, which caused the constriction of the gorge entrance and prevented the substrate from entering the enzyme, thus resulting in the inhibition of AChE. Molecular dynamics (MD) simulation emphasized the stability of the quercetin-AChE complex and corroborated the previous findings. Interestingly, a combination of galantamine hydrobromide and quercetin exhibited the synergistic inhibition effect by binding to different active sites of AChE. In a β-amyloid25-35-induced oxidative stress injury model in PC12 cells, quercetin exerted neuroprotective effects by increasing the glutathione level and reducing the malondialdehyde content and reactive oxygen species levels. These findings may provide novel insights into the development and application of quercetin in the dietary treatment of Alzheimer's disease.

Silibinin-Loaded Nanostructured Lipid Carriers for Growth Inhibition of Cisplatin-Resistant Ovarian Cancer Cells.

Cisplatin is the most often used chemotherapy in the treatment of ovarian cancer (OC), however long-term usage leads to drug resistance and treatment failure. Silibinin is a sparingly water-soluble natural compound with well-known anticancer effects. The use of lipid-based delivery systems is a potential approach for enhancing silibinin's water solubility. In this study, nanostructured lipid carriers (NLCs) containing silibinin were prepared and their inhibitory effects were tested in combination with cisplatin against sensitive/resistant A2780 OC cells. Silibinin-loaded NLCs (silibinin-NLCs) were prepared by the hot homogenization method, and their size, shape, zeta potential (ZP), and encapsulation efficiency (EE), as well as their inhibitory effects, were examined in combination with cisplatin against sensitive/resistant A2780 OC cells. Formulation of silibinin-NLCs using cocoa butter led to spherical-shaped NLCs with a size of 95 nm and EE of 98%. The ZP and the dispersion index of the silibinin-NLCs were -27.12 ± 0.13 mv and 0.12 ± 0.04, respectively. The release kinetics of silibinin-NLCs was best fitted with the zero-order model. The combination of cisplatin and silibinin-NLCs sensitized the cisplatin-resistant A2780 OC cells and exhibited a more synergistic inhibitory effect on A2780 cells as compared with the combination of cisplatin and plain silibinin. The optimized silibinin-NLCs can be considered a suitable drug delivery system for the inhibition of cisplatin-resistant OC cells.

Standardized Pomegranate (Pomella®) and Red Maple (Maplifa®) Extracts and Their Phenolics Protect Type I Collagen by the Inhibition of Matrix Metalloproteinases, Collagenase, and Collagen Cross-Linking.

Phenolics enriched pomegranate fruit (Pomella®) and red maple leaf (Maplifa®) extracts and their major phenolic constituents have demonstrated beneficial skin effects through the protection of human skin keratinocytes from oxidative-stress-induced damage. However, their mechanisms of protection of cutaneous collagen are still unclear. Herein, the collagen protective effects of Pomella® and Maplifa®, and their major bioactive phytochemicals, namely, punicalagin (PA) and ginnalin A (GA), respectively, were evaluated using enzymatic assays including collagenase, anti-glycation and cell-based models as well as computational methods. The importance of the modulatory effects was validated at the protein level for type I collagen and matrix metalloproteinases (MMPs) using human-skin-derived keratinocytes. The synergistic collagenase inhibitory effects upon combinations of Pomella® + Maplifa® and PA + GA at a combination ratio of 1:2 and 1:1, respectively, were evaluated using their combination index (CI; a well-established assessment of synergism). Pomella® (50-400 µg/mL), Maplifa® (100-800 µg/mL), PA (50-400 µM), and GA (50-400 µM) dose-dependently inhibited collagenase activity by 26.3-86.3%, 25.7-94.0%, 26.2-94.0%, and 12.0-98.0%, respectively. The CI of the anti-collagenase activity of Pomella® and Maplifa® ranged from 0.53-0.90, while that of PA and GA (12.5/12.5 and 25/25 µM) ranged from 0.66 and 0.69, respectively, suggesting a synergistic inhibitory effect. Interestingly, in the cell-based assays by Western blotting, Pomella® and Maplifa® reduced the protein expression levels of collagen degradation enzymes (MMPs), while simultaneously increasing that of type I collagen in epidermoid carcinoma A431 cells. This is the first report to show that these extracts exert synergistic collagen protective effects. Taken together, these findings provide molecular insights into the usefulness of Pomella® and Maplifa® or their phenolics as bioactive ingredients for skin care products to slow down aging and enhance skin tone.

Strategies for Alleviating Tyrosine Kinase Resistance in Acute Myeloid Leukemia By Modulating p53 Signaling Pathway with Small Molecules

Background:FLT3-ITD can be found in up to 30% of acute myeloid leukemia (AML) patients and confers a poor prognosis. While midostaurin and gilteritinib are FDA-approved for these high-risk patients, they only result in a temporary response without transplantation, suggesting the importance of identifying the acquired resistance. Previously, we demonstrated that cabozantinib, an oral multi-target tyrosine kinase inhibitor, was selectively cytotoxic to AML cells with FLT3-ITD (MV4-11 and Molm13). Subsequently, we established cabozantinib-resistant human leukemic MV4-11 derivative (MV4-11-XR) and Molm13 derivative (Molm13-XR) after gradual escalating concentration of cabozantinib incubation from parental MV4-11 (MV4-11-P) and Molm13 (Molm13-P) cells, respectively. Subsequently, cell cytotoxicity assay showed these two cabozantinib-resistant cells were also resistant to quizartinib, sorafenib and gilteritinib. RNA-seq analysis revealed that MV4-11-XR and Molm13-XR cells shared 87 identical differentially down-regulated genes compared to their respective parental cells. Further Metascape for gene enriched pathway analysis demonstrated the most affected was p53 signaling pathway. Aim: To investigate whether modulation with p53 activator RITA or MDM2 inhibitor RG7388 could alleviate the drug resistance and their underlying molecular mechanism. Materials and Methods: Human leukemia MV4-11 and Molm13 cells were used in this study. The differential expression genes (DEGs) were examined using RNA-seq (Illumina NextSeq-500). Metascape enrichment pathway analyses were preformed to predict the biological functions of DEGs. Cabozantinib, RITA, and RG7388 (Idasanutlin) were used. MTS assay was performed to the effects of various drug on cell growth. Western blotting, flow cytometry and quantitative real-time PCR were applied to elucidate potential molecular mechanism. Fluorescent leukemic cell xenografts in zebrafish and high content imaging system were used for investigating the efficacy of various drugs in vivo. Results: The gene expression profile in MV4-11-P and Molm13-P cells was used as a baseline to establish the up- and down-regulated genes in MV4-11-XR and Molm13-XR cells. The FPKM were estimated with the selection criteria of p value <0.05 and [log2 (fold change)] <1 for significantly differential expression for downregulation. We identified a total of 747 DEGs between the MV4-11-P and -XR cells and 407 DEGs between the Molm13-P and -XR cells. Among them, 87 DEGs were shared by these two cohorts. Metascape enrichment analysis revealed that those down-regulated DEGs were enriched in p53-signaling pathway, in line with decreased expression of P53-associated genes, BBC3, CCNG2, CKDN1A, ZMAT3, confirmed by using q-RT-PCR. IGV (Integrative Genomics Viewer) analysis showed that the variant allele frequency of R248W progressed from 57% in MV4-11-P to 100% in MV4-11-XR cells, a similar phenomenon also appeared in P72R (65% to 98%). Microsatellite analysis confirmed the loss of heterozygosity in the TP53 gene of MV4-11-XR. On the other hand, there were not TP53 variants in both Molm13-P and Molm13-XR cells, but increased level of MDM2 in Molm13-XR cells was noted. Further study revealed that p53 activator RITA with 77.6nM could reduce IC50 of cabozantinib from 1026nM to 310.7nM, coupled with increased gene expression and protein levels of BBC3 (PUMA), CCNG2 (cyclin G2), CDKN1A (p21) and ZMAT3. In addition, RITA had synergistic effect with cabozantinib on cell cycle arrest on G2/M phase, increased apoptosis and decreased colony formation and proliferation. On the other hand, MDM2 inhibitor RG7388 reduced the IC50 of cabozantinib from 517nM to 101nM with only 50.6 nM in Molm13-XR cells. Similarly, RG7388 could increase those P53-associated gene expression and protein levels, and had synergistic effect with cabozantinib on cell cycle arrest on G0/G1 phase, increased apoptosis and decreased colony formation and proliferation. In addition, in vivo zebrafish experiment demonstrated that the combination of RG7388 and cabozantinib had a synergistic and significant inhibitory effect compared with single use. Conclusion: These findings suggested that reactivation of p53 is a potential therapeutic option to alleviate drug resistance in FLT3-mutated AML, provided that AML patients are tested for p53 dysfunction, including TP53 mutational status and downregulation.

Fingolimod in Combination with Bortezomib Demonstrates Synergistic Tumor Inhibition and Bone Anabolic Effects in Myeloma Bone Disease

Fingolimod in Combination with Bortezomib Demonstrates Synergistic Tumor Inhibition and Bone Anabolic Effects in Myeloma Bone Disease

Synergistic antimicrobial effect of the combination of beta-lactam antibiotics and chitosan derivative on multidrug-resistant bacteria

Dissemination of multidrug-resistant (MDR) bacteria with CTX-M-type extended-spectrum β-lactamases (blaCTX-M) has become the greatest challenge in public health care. This study aimed to investigate the synergistic antibacterial potential of N-alkylaminated chitosan nanoparticles (CNPs) combined with conventional β-lactam antibiotics (BLAs) against multidrug-resistant pathogen with blaCTX-M gene. The results of this study showed that the developed nano-formulation resensitized the studied E. coli MDR strain (E001) to ampicillin (AMP) and piperacillin (PIP) by causing a 1000–10,000-fold decrease in their MIC values (5000–50,000 mg/L to 5 mg/L). The conjugation of CNPs with cefoxitin (FOX) and ceftazidime (CAZ) showed a comparatively lower synergistic inhibitory effect owing to the higher susceptibility (MIC value = 0.5 mg/L–5 mg/L) of E001 to these antibiotics. The results indicate that CNPs could be effectively employed as an additive to augment the antibacterial effect of the BLAs for which MDR strains exhibit higher MIC values.

Carbonate and bicarbonate ions impacts on the reactivity of ferrate(VI) for 3,4-dichlorophenol removal.

Carbonate and bicarbonate ions are common constituents found in wastewater and natural water matrices, and their impacts on the reactivity of ferrate(VI) (Fe(VI)) with 3,4-dichlorophenol (3,4-DCP) were investigated by determining second-order rate constants of 3,4-DCP removal by Fe(VI) in the presence of CO32- and/or HCO3-. The second-order rate constants decreased from 41.75 to 7.04M-1s-1 with an increase of [CO32-] from 0 to 2.0mM, indicating that CO32- exhibits an inhibitory effect on 3,4-DCP removal kinetics, and experiments on pH effect, radical quenching, and Fe(VI) stability were conducted to explore possible reasons for its effect. Under identical pH conditions, the rate constant in NaOH medium was always higher than in Na2CO3 medium, suggesting that the inhibitory effect partially comes from an increase in alkalinity. Furthermore, the scavenging of hydroxyl radical by carbonate ion also contributed to the inhibitory effect of CO32-. On the other hand, the enhancement effect of CO32- depending on the increase in Fe(VI) stability was found, but did not exceed its inhibitory effect. In addition, 3,4-DCP removal kinetics was not affected by HCO3-, while synergistically inhibited by CO32-/HCO3-. Moreover, 3,4-DCP removal efficiency was substantially suppressed in the presence of CO32-, while the slight enhancement effect of HCO3- and the synergistic inhibitory effect of CO32-/HCO3- were observed. The experimental results clearly demonstrated that carbonate and bicarbonate ions play an important role in the process of 3,4-DCP removal by Fe(VI) and should not be considered only as scavengers.

Inhibition of CDC7 with Sgr-2921 in AML Models Results in Enhanced DNA Damage and Anti-Leukemic Activity As Monotherapy and in Combination with Standard of Care Agents

Introduction: CDC7 is a protein kinase that is active during the S-phase of the cell cycle. CDC7 plays a critical role in the replication stress response by phosphorylation and activation of the BRCA1 and Cohesin complexes, which protect and restart stalled replication forks. In addition, by maintaining DNA helicase activity during replication fork stalling, CDC7 establishes a platform for ATR checkpoint activation. Acute myeloid leukemia (AML) is typically a rapidly proliferating cancer and is characterized by high replication stress and DNA damage. Agents that target replication stress and DNA damage response pathways represent novel therapeutic opportunities in AML. Here we show that inhibition of CDC7 in AML models results in potent anti-tumor activity by amplifying replication stress and DNA damage. We show that CDC7 inhibition has synergistic anti-proliferative effects when combined with hypomethylating agents and a BCL2 inhibitor. We previously discovered that CDC7 activity drives resistance to FLT3 inhibition (Cancer Cell. 2021 Jul 12;39(7):999-1014.e8), and now show that inhibition of CDC7 overcomes resistance to a variety of FLT3 inhibitor resistant cell lines. CDC7 inhibition is efficacious in multiple AML models resistant to standard of care agents, suggesting that CDC7 inhibitor combinations may be efficacious in the clinic. Results: We identified a potent and selective CDC7 inhibitor, SGR-2921, using large-scale physics-based computational modeling technology. In a panel of ~300 cancer cell lines, AML cell lines were on average the most sensitive to SGR-2921, and AML patient samples were highly sensitive to CDC7 inhibition ex vivo. In vivo, SGR-2921 showed strong anti-tumor growth activity in MV-4-11 and Molm-16 AML xenograft models at tolerated doses. In combination with hypomethylating agents, SGR-2921 increased the level of replication stress, DNA damage and apoptosis markers in vitro. Combination of SGR-2921 with venetoclax (BCL2 inhibitor) showed synergy on anti-tumor activity both in vitro and in vivo. SGR-2921 was highly efficacious in AML cell lines resistant to FLT3 inhibitors, hypomethylating agents and venetoclax, and in multi-agent resistant cell lines. Co-treatment with SGR-2921 partially restored sensitivity to FLT3 inhibition in FLT3 resistance AML cell lines. Conclusions: SGR-2921, a novel, potent CDC7 small molecule inhibitor, demonstrates strong anti-proliferative activity both in vitro in AML cell models and in vivo in AML xenograft models. SGR-2921 showed synergistic inhibitory effects on cell-proliferation and tumor growth in combination with standard of care agents, and was anti-proliferative in AML cell lines and patient samples resistant to standard of care agents. Together, these data show that SGR-2921-mediated CDC7 inhibition is an attractive novel treatment opportunity in AML, with a potential utility in patients with relapsed and refractory AML.

Tyrosine Phosphorylation of CARM1 Promotes Its Enzymatic Activity and Alters Its Target Specificity in Myeloid Neoplasms with Hyperactivated JAK2

Introduction Coactivator-associated arginine methyltransferase 1 (CARM1) catalyzes the asymmetric dimethylation of arginine residues in histone and non-histone substrates, including transcription factors, RNA splicing factors, and the SWI/SNF chromatin remodeling complex. CARM1 has emerged as an important target in acute myeloid leukemia. We have previously shown that Janus kinase 2 (JAK2) affects the activity of epigenetic modifiers. A V617F mutation in JAK2 genes leads to its hyperactivation. This mutation is a common hallmark of the BCR/ABL1-negative myeloproliferative neoplasms, and also found in patients with acute myeloid leukemia and myelodysplastic syndrome. Here, we find that hyperactivated JAK2, mediated by biallelic V617F mutation, triggers CARM1 phosphorylation, increasing its methyltransferase activity and altering its target specificity. Methods We generated two phospho-tyrosine specific antibodies against CARM1 tyrosine-149 (Y139) and -334 (Y334), and two asymmetric dimethylation-arginine specific antibodies against RUNX1 arginine-223 (R223) and -319 (R319). We also generated isogenic HEL cells carrying homozygous CARM1 non-phosphorylatable mutation (Y149F or Y334F single, or Y149F/Y334F double mutation), using the CRISPR/Cas9 nuclease system. Results We first performed cell-free in vitro kinase assays using recombinant CARM1 protein and JAK2 kinase, and mass spectrometry analysis, which identified Y139 and Y334 as the sites of JAK2 phosphorylation in CARM1. We next compared the levels of CARM1-Y149 and -Y334 phosphorylation in 15 myeloid leukemia cell lines and normal human CD34+ cord blood cells. HEL and UKE-1 cells, that harbor bi-allelic JAK2-V617F mutation, showed the highest level of phosphorylated CARM1-Y149 and -Y334, while SET2 cells with mono-allelic JAK2-V617F mutation and the other cell lines showed abundant CARM1 protein but a lower relative amount of phosphorylated CARM1 protein. We found that the JAK2 activation, through transphosphorylation by JAK2 and TYK2, enhances tyrosine kinase activity against CARM1 using HEL cells with knockout of either JAK1 or TYK2. Having previously shown that CARM1 methylates RUNX1 at R223, in an immunoprecipitation assay, WT-CARM1 was able to pull down RUNX1, while the non-phosphorylatable CARM1 mutants were not. Using mass spectrometry and an in vitro methylation assay, we confirmed R223 and R319 as CARM1 target sites. We examined non-phosphorylatable CARM1 mutation knock-in HEL cells and found that mutation of either site reduces the asymmetric dimethylation of RUNX1 as well as known substrates (BAF155 and PABP1). We observed that CARM1 Y149F/Y334F mutant-expressing HEL cells reduced cell proliferation with G2/M cell cycle arrest and an increase in apoptosis. To identify the transcription networks regulated by CARM1 phosphorylation, we performed RNA-seq on WT-CARM1 and double CARM1 Y149F/Y334F mutant-expressing HEL cells. Gene set enrichment analysis identified that gene sets associated with G2/M cell cycle progression and anti-apoptosis were significantly downregulated in Y149F/Y334F mutation knock-in HEL cells. We next performed ChIP-seq analyses using antibodies against total RUNX1 and against asymmetrically dimethylated R319-RUNX1 in HEL cells expressing knock-in of CARM1 non-phosphorylatable mutation. CARM1 non-phosphorylatable mutation decreased the signals of dimethylated RUNX1-R319 within 5 kb of the transcription start sites for RUNX1 target genes, with less effect on total RUNX1. We next assessed the efficacy of the combination with CARM1 inhibitor (EPZ025654) and JAK2 inhibitor (ruxolitinib). Dual targeting inhibition of JAK2 and CARM1 showed a synergistic inhibition effect on HEL and UKE-1 cells (with abundant phosphor-CARM1), but only an additive effect on SET2 cells (with less amount of phosphor-CARM1). Conclusion CARM1 phosphorylation mediated by hyperactivated JAK2 regulates its methyltransferase activity and is required for the maximal proliferation of myeloid neoplasms. We also demonstrated that dual targeting of JAK2 and CARM1 is more effective than monotherapy in cell lines with phosphor-CARM1. Thus, our results reveal a novel role for CARM1 phosphorylation in myeloid neoplasms.

Co-existence effect of copper oxide nanoparticles and ciprofloxacin on simultaneous nitrification, endogenous denitrification, and phosphorus removal by aerobic granular sludge

Nanoparticles and antibiotics are toxic to humans and ecosystems, and they inevitably coexist in the wastewater treatment plants. Hence, the co-existence effects and stress mechanism of copper (II) oxide nanoparticles (CuO NPs) and ciprofloxacin (CIP) on simultaneous nitrification, endogenous denitrification and phosphorus removal (SNEDPR) by aerobic granular sludge (AGS) were investigated here. The co-existence stress of 5 mg/L CuO NPs and 5 mg/L CIP resulted in the synergistic inhibitory effect on nutrient removal. Transformation inhibition mechanisms of carbon (C), nitrogen (N) and phosphorus (P) with CuO NPs and CIP addition were time-dependent. Furthermore, the long-term stress mainly inhibited PO43--P removal by inhibiting phosphorus release process, while short-term stress mainly inhibited phosphorus uptake process. The synergistic inhibitory effect of CuO NPs and CIP may be due to the changes of physicochemical characteristics under the co-existence of CuO NPs and CIP. This further altered the sludge characteristics, microbial community structure and functional metabolic pathways under the long-term stress. Resistance genes analysis exhibited that the co-existence stress of CuO NPs and CIP induced the amplification of qnrA (2.38 folds), qnrB (4.70 folds) and intI1 (3.41 folds) compared with the control group.

EXTH-86. BH3 PROFILING IDENTIFIES ONC201/TIC10 AS A PROMISING PARTNER OF ABT-263 IN MEDULLOBLASTOMA IN VITRO

Abstract OBJECTIVE Medulloblastoma represents one of the most common brain tumors in children. In this study, we identified by BH3 profiling that ONC201/TIC10 sensitizes for Bcl-xL/Bcl-2 inhibition in medulloblastoma and performed a preclinical testing of a combined treatment with ONC201/TIC10 and the Bcl-xL/Bcl-2 inhibitor ABT-263. METHODS BH3 profiling was performed to examine ONC201/TIC10-mediated dependencies on anti-apoptotic Bcl-2 family proteins. The combination treatment with ONC201/TIC10 and ABT-263 was tested in different medulloblastoma cells using MTT assays. Isobolograms were calculated to characterize the drug-drug interaction. Tumor spheroid and chorioallantoic membrane assays were used to examine the effects of the combination therapy in a 3D setting. Annexin V/PI staining and flowcytometry were used to detect pro-apoptotic effects. Western blot analyses and knockdown experiments with siRNA were performed for molecular analysis. Extracellular flux analyses served at examining effects on the tumor cell metabolism. RESULTS BH3 profiling showed that ONC201/TIC10 sensitizes medulloblastoma cells to Bcl-xL/Bcl-2 inhibition. In line with this finding, combined treatment with ONC201/TIC10 and ABT-263 led to a predominantly synergistic inhibitory effect on the cell viability of established (D425, D458, DAOY, HD-MB03), primary cultured (PC322) and stem-like (SC322) medulloblastoma cells in 2D and 3D models. The response towards the combination therapy was independent of baseline c-myc expression. On the molecular level, treatment with ONC201/TIC10 led to a dose-dependent decrease of Mcl-1. Moreover, the combination caused enhanced cleavage of caspases 9 and 3. On the metabolic level, the combination therapy led to a reduction in both, oxidative phosphorylation and the glycolytic rate and a reduced expression of respiratory chain proteins. CONCLUSION Combined treatment with ONC201/TIC10 and ABT-263 had a predominantly synergistic inhibitory effect on the cell viability of medulloblastoma cells. This effect was associated with downregulation of Mcl-1. Moreover, the combination treatment resulted in a metabolic reprogramming which likely creates a state of energy deprivation.

EXTH-48. INHIBITION OF SREBP-1 IS SYNTHETICALLY LETHAL WITH BCL-XL/BCL-2 INHIBITION IN GLIOBLASTOMA IN VITRO

Abstract OBJECTIVE A cellular homeostasis that is shifted away from apoptosis and a reprogramming of the lipid metabolism are both, features that are frequently encountered in glioblastoma. This study aimed at investigating whether interference with the lipid metabolism is synthetically lethal with inhibition of anti-apoptotic Bcl-2 family proteins in glioblastoma in vitro. METHODS Established (U251) and primary-cultured glioblastoma cells (PC38, PC40 and PC128) as well as glioblastoma stem-like cells (SC38 and SC40) were treated with the Bcl-xL/Bcl-2 inhibitor ABT-263 (navitoclax) and/or the SREBP-1 inhibitor Fatostatin. MTT-assays were performed to assess effects of the combination therapy on the cell viability. Isobolograms were calculated to characterize the drug-drug interaction. Spheroids were used to determine anti-proliferative effects in a 3-dimensional setting. Staining with annexin V/propidium iodide and flowcytometric analysis were performed to assess pro-apoptotic effects. For molecular analyses, Western blots and specific knock-down experiments with siRNA were performed. RESULTS Combined targeting of SREBP-1 and Bcl-xL/Bcl-2 led to a synergistic inhibitory effect on the cellular viability of established, primary-cultured and glioblastoma stem-like cells as well as spheroids. This effect was shown to be at least in part mediated by enhanced apoptosis and to occur in a caspase-dependent manner. On the molecular level, treatment with increasing concentrations of Fatostatin led to a downregulation of Mcl-1. CONCLUSION Our study indicates that combined inhibition of Bcl-xL/Bcl-2 and interference with the lipid metabolism targeting SREBP-1 synergistically induces caspase-dependent apoptosis in glioblastoma cells. This effect can also be observed in more complex 3-dimensional glioblastoma cell formations. Further studies will focus on deciphering the drug-induced alterations of the metabolic pathways that are responsible for the synergistic effect of this therapeutic strategy.

CTNI-29. INVESTIGATING SAFETY AND EFFICACY OF TTFIELDS PRIOR AND CONCOMITANT TO RADIOTHERAPY IN NEWLY DIAGNOSED GLIOBLASTOMA - FIRST RESULTS OF THE PRICOTTF PHASE I/II TRIAL

Abstract Background A synergistic inhibiting effect on glioblastoma cell proliferation was reported for the combination of TTFields and radiotherapy. Based on these preclinical findings, we performed the phase I/II PriCoTTF trial in adult nGBM patients to investigate the safety and efficacy of TTFields therapy initiated prior and concomitant to radiochemotherapy. MATERIAL AND METHODS In this phase I/II trial, TTFields therapy was initiated following surgery and continued throughout radiochemotherapy and adjuvant chemotherapy for a total of approximately 9 months. TTFields rechallenge was allowed at recurrence. Radiotherapy was conducted with arrays applied on the patients’ scalp. Safety and tolerance are the study’s primary endpoint, determined by a selection of pre-specified treatment-limiting toxicities (TLTs). RESULTS A total of 33 patients have been enrolled. Patients' characteristics were mostly typical for glioblastoma, except for a rather low fraction of patients with gross total resection (GTR, 22.5%). The distribution of adverse events of ≥ common toxicity criteria (CTC) grade 3 was comparable to that of established glioblastoma trials. Notably, skin toxicity of ≥ CTC grade 3 was uncommon (n = 2, 6%). No patient developed TLTs. Median TTFields treatment duration was 8.4 months. Overall survival data was not mature enough (event rate 48%) to allow for a definite conclusion Notably, on multivariable Cox regression, the number of days with TTFields adherence &amp;gt; 23 hours was independently associated with overall survival (HR 0.96, 95% confidence interval 0.93 - 0.99, p = 0.008). CONCLUSION The PriCoTTF trial met its primary endpoint indicating that combined TTFields and radiotherapy is safe and well tolerated. High-grade skin toxicity was quite uncommon and the patients with high TTFields adherence seem to perform particularly well. An extended follow-up is required to provide first estimates regarding putative efficacy. At that point in time, the reduced overall TTFields duration and fraction of patients with GTR need to be factored in.

Designing a novel GO@AAP reinforced epoxy coating for achieving the long-term corrosion protection of steel substrate

In this study, 4-amino-antipyrine (AAP) was grafted on graphene oxide (GO) via the hydrothermal strategy (GO@AAP) to improve the corrosion resistant property of epoxy coating. Transmission electron microscope (TEM), scanning probe microscope (SPM) and Raman techniques were employed to investigate the morphology and structure of GO@AAP nanohybrid, which also effectively confirmed the successful AAP grafted on GO surface. Electrochemical and salt spray results indicated that incorporating as-synthesized GO@AAP into epoxy coating powerfully enhanced the anti-corrosion property, which was greatly consistent with the mild steel morphology after corrosion experiment. Besides, after 60 days of immersion, the |Z|0.01 Hz was 3.7 × 108 Ω·cm2, which was two order of magnitude higher than that of pure epoxy coating. Local electrochemical impedance spectroscopy (LEIS) was visually demonstrated the synergistic effect of physical shielding and corrosion inhibition of GO@AAP in 3.5 wt% NaCl solution. This work provides a new method to achieve the barrier and inhibition activities of epoxy coatings, which are suitable for heavy corrosion resistant application in harsh environment.

Corrosion inhibition performance of polyolefin smart self-healing composite coatings modified with ZnO@β-Cyclodextrin hybrid particles

Corrosion is a global problem that results in substantial economic loss and is life-threatening as well. Currently, the utilization of self-healing coatings involving smart carriers encapsulated with the appropriate self-healing agents is a topic of great interest. Following this strategy, our work reports the synergistic corrosion inhibition performance of polyolefin-based smart coatings containing modified hybrid-based particles. The hybrid particles (ZnO@β-CD) comprising zinc oxide (ZnO) and β-Cyclodextrin (β-CD) were synthesized and modified with 2-mercaptobenzothiazole (2-MBT). The synthesized unmodified and modified hybrid particles were characterized by numerous techniques. TGA analysis confirmed that 2MBT was effectively loaded into ZnO@β-CD with almost 43% loading. UV-vis spectroscopic analysis results suggested that the self-release behavior of 2MBT is more pronounced in the acidic environment compared to the basic and neutral environment due to the dissociation of ZnO in the acidic environment. In the next stage, the synthesized unmodified (ZnO@β-CD) and modified (ZnO@β-CD-2MBT) hybrid particles were reinforced into a polyolefin matrix, followed by its application on steel substrate via dip-coating technique to formulate smart composite coatings. The Electrochemical impedance spectroscopy (EIS) results conducted on the developed smart composite coatings demonstrated that polyolefin coatings reinforced with modified hybrid particles (ZnO@β-CD-2MBT) displayed improved corrosion inhibition performance (98 GΩ cm2) as compared to the composite coatings (87 GΩ cm2) containing unmodified particles ((ZnO@β-CD) in 0.56 M NaCl solution after 20 days of immersion. This decent improvement in corrosion resistance behavior is attributed to the synergistic corrosion inhibition effect experienced due to the combined corrosion inhibition effect of ZnO and 2MBT.