72-h Treatment Research Articles

527 Eschar Removal by Bromelain Based Enzymatic Debridement in Burns: European Consensus Guidelines Update

Abstract Introduction Bromelain based Enzymatic Debridement (ED) has been introduced as an additional concept to the burn surgeon’s armamentarium, and is best indicated for mid-to deep dermal burns with mixed patterns. Increasing evidence has been published on special regions and settings, adressing success and limitations of ED for improved patient care. To better guide ED in view of the increasing experience, there is a need to publish updated consensus guidelines with user-orientated recommendations. Methods A multiprofessional expert panel of plastic surgeons and burn care specialists from 10 European countries was convened, to assist in developing current recommendations for best practices with use of ED. Consensus statements were based on peer-reviewed publications and clinical relevance. For consensus agreement, the methodology employed was an agreement algorithm based on a modification of the Willy and Stellar method. For this study on ED, consensus was considered when there was at least 80 % agreement to each statement. Results The updated consensus guidelines from 2019 refer to a diversity of experience and practice patterns of 1232 summarized patient cases in Europe (2017: 500 cases), reflecting the impact of the published recommendations. Forty-three statements were formulated, addressing the following topics: indications, pain management and anesthesia, large surface indication, timing of application for various indications, preparation and application, post-interventional wound management, skin grafting, outcome, scar and revision management, cost-effectiveness, patient′s perspective, logistic aspects and training strategies. The degree of consensus was remarkably high, with consensus in 42 of 43 statements (97.7%), while no consensus was achieved on the role of outpatient treatment/ED as day cases in minor burns in experienced burn centers. A classifications with regard to timing of application for ED was introduced, with „immediate/ very early“(≤12h), early (12-72h) or delayed (>72h) treatment. All further recommendations are addressed in the presentation. Conclusions The updated guidelines in this paper represent further refinement of the recommended indication, application and post-interventional management for the use of ED. The published statements contain detailed, user-orientated recommendations aiming to align current and future users and prevent unnecessary pitfalls. The significance of this work is reflected by the magnitude of patient experience behind it, larger than the total number of patients treated in all published ED clinical trials. Applicability of Research to Practice These consensus guidelines may serve as refined user-orientated recommendations for implementation and successful application of ED in further countries (e.g. USA) based on the evidence and experience of 1232 summarized patient cases.

Uncarboxylated osteocalcin decreases insulin-stimulated glucose uptake without affecting insulin signaling and regulators of mitochondrial biogenesis in myotubes.

Uncarboxylated osteocalcin (uOC) is a circulating bone matrix protein, which has previously been shown to regulate glucose uptake and systemic metabolism. However, the cellular mechanism by which uOC acts has yet to be elucidated. C2C12 mouse myotubes were treated for 72h with uOC (1-100ng/mL). Cellular metabolism was analyzed using oxygen consumption and extracellular acidification rate. Metabolic gene and protein expression were measured via quantitative real-time polymerase chain reaction and Western blot, respectively. Additionally, C2C12 myotubes were treated with 10ng/mL uOC to examine glucose uptake and activation of insulin signaling with or without insulin resistance. Finally, cellular lipid content was measured via Oil Red O and Nile Red staining. uOC treatment resulted in dose-dependent alterations of oxygen consumption with little effect on regulators of mitochondrial metabolism. Basal expression of regulators of glucose uptake were unaffected by uOC treatment. However, insulin-stimulated glucose uptake was blunted by uOC treatment with no concurrent alterations in insulin signaling. While chronic insulin treatment resulted in suppressed activation of Akt, concurrent uOC treatment was unable to prevent these detrimental effects on insulin signaling. uOC treatment had no effect on markers of lipogenesis and cellular lipid content. These findings suggest that 72-h uOC treatment may alter oxygen consumption without effect on regulators of mitochondrial biogenesis. Additionally, uOC treatment suppressed insulin-stimulated glucose uptake in cultured myotubes but had little effect on insulin signaling or regulators of cellular metabolism and was unable to mitigate insulin resistance.

Effect of dual inhibition of histone deacetylase and phosphatidylinositol-3 kinase in Philadelphia chromosome-positive leukemia cells.

ABL tyrosine kinase inhibitors (TKIs) have demonstrated potency in the treatment of chronic myeloid leukemia (CML) patients. However, resistance to ABL TKIs can develop in CML patients due to BCR-ABL point mutations. Furthermore, CUDC-907 is an oral inhibitor of class I phosphoinositide 3-kinase (PI3K) as well as class I and II histone deacetylase (HDAC) enzymes. In this study, we evaluated the effect of combination therapy of CUDC-907 and ABL TKIs, using BCR-ABL-positive cell lines and primary samples. CUDC-907 treatment for 72h resulted in cell growth inhibition. Over the same period, an increase in histone acetylation and both caspase three and poly (ADP-ribose) polymerase (PARP) enzyme activity was observed. When ABL TKI treatment and CUDC-907 treatment were combined, significantly greater cytotoxicity was observed. Moreover, combined oral therapy with ponatinib (20mg/kg/day) and CUDC-907 (30mg/kg/day) greatly inhibited tumor growth compared to each drug alone. Lastly, CUDC-907 treatment also inhibited the growth of Ba/F3 ponatinib-resistant cells, K562 nilotinib-resistant cells, and T315I mutant primary samples. Taken together, our results indicate that administration of CUDC-907, a dual PI3K and HDAC inhibitor, may be an effective strategy against ABL TKI-resistant cells, including cells harboring the T315I mutation. Moreover, CUDC-907 may enhance the cytotoxic effects of ABL TKI when a combined treatment strategy is used against Philadelphia chromosome-positive leukemia cells.

Macrophage migration inhibitory factor modulates cytokine expression in the trophoblast cell line HTR-8/SVneo.

Extravillous trophoblasts are specific placental cells that invade the uterine stroma and spiral arteries modifying and adjusting them to pregnancy. Many pregnancy pathologies are associated with impairment of this process, including preeclampsia and intrauterine growth restriction, among others. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that is abundant at the fetomaternal interface. Previous results from our group showed that MIF participates in trophoblast invasion and modulates the expression of molecules known to mediate stromal and endovascular trophoblast invasion. In this study we investigated the possibility that MIF could act as a regulator of cytokines known to modulate trophoblast invasion using the normal extravillous trophoblast-derived cell line HTR-8/SVneo. Expression of trophoblast MIF was attenuated by MIF mRNA-specific small interfering RNAs. Cytokine expression was assessed at the mRNA and protein levels using real-time quantitative polymerase chain reaction and flow cytometry respectively. Knockdown of MIF led to a significant decrease in mRNA for IL-1β (IL1B) and IL-8 (CXCL8) and interleukin (IL)-8 protein. The addition of recombinant human MIF to cell culture medium increased IL-6 after 24h treatment and IL-6 and IL-8 after 72h treatment. Cell viability was not affected by MIF silencing or rhMIF treatment. The results of this study imply that at least some of the effects of MIF on trophoblast invasion could be mediated through autocrine or paracrine modulation of trophoblast cytokine production.

The 3D reconstructed skin micronucleus assay: considerations for optimal protocol design.

Implementation of the seventh amendment to the EU Cosmetics Directive has driven much research into suitable in vitro alternative assays to support satisfactory risk assessments. One such assay is the reconstructed skin micronucleus (RSMN) assay. First reported in 2006, further development occurred and a standard protocol was published in 2011. To evaluate and optimise the assay at Covance Laboratories, we tested nine chemicals [4-nitrophenol (4-NP), cyclohexanone (CH), 2-ethyl-1,3-hexanediol (2-EHD), methyl methansulfonate (MMS), mitomycin C (MMC), ethyl nitrosourea (ENU), benzo[a]pyrene (BaP), cyclophosphamide (CPA) and vinblastine (VIN)] using the EpiDerm™ 3D skin model (MatTek Corporation®, IVLSL, Bratislava, Slovakia) and compared the data using the standard 48-h treatment regimen and also an emerging 72-h treatment protocol. The EpiDerm™ tissue has reportedly some metabolic capacity but data using 48-h treatments has provided mixed results. Our investigations demonstrate that the two chemicals requiring metabolic activation (BaP and CPA) were negative following the 48-h protocol but were clearly positive following 72-h treatment. Furthermore, Replication Index (RI) data showed higher RI values in vehicle control treatments (indicating increased cell division) across the treatment set following 72-h treatments. A general greater magnitude of micronucleus (MN) induction was also observed following test chemical treatment. These data suggest that the 72-h treatment protocol is more suitable as a standard approach for the detection of clastogenic, aneugenic and metabolically activated chemicals in the RSMN assay. For further assay optimisation, we compare the statistical power of scoring cells from duplicate or triplicate cultures per treatment concentration and provide recommendations.

Ganoderic acid A/DM-induced NDRG2 over-expression suppresses high-grade meningioma growth.

N-myc downstream-regulated gene 2 (NDRG2) is down-regulated in grade-III meningioma [anaplastic meningioma (AM)] and associated with clinically aggressive behavior. Current therapies in the treatment of high-grade meningioma are lacking with limited success. This study aims to validate the effect of NDRG2-targeted therapy using structurally related bioactive triterpene compounds derived from the edible mushroom Ganoderma lucidum (ganoderic acid A:GA-A/ganoderic acid DM:GA-DM) in human AM in relevant pre-clinical models. Tissue samples from the AM tumor regions of three human patients and control non-tumor samples were used to analyze the expression pattern of NDRG2. In vitro cell culture and in vivo cell-line-derived orthotopic xenograft animal models of AM were utilized to assess efficacy of treatment with GA-A/DM. Downregulation of NDRG2 expression was observed in surgically resected high-grade meningiomas compared to normal brain. These results prompt us to use NDRG2-targeting agents GA-A/DM. In vitro results showed that 72-h treatments of 25µM GA-A/DM induced AM cell death, upregulate NDRG2 protein expression, downregulate NDRG2 promoter methylation in meningioma cells as compared to azacitidine and decitabine, the most commonly used demethylating agents. Our results also demonstrated that GA-A/DM does not have any detrimental effect on normal human neurons and arachnoid cells. GA-A/DM promoted apoptotic factors (Bax) while suppressing MMP-9, p-P13K, p-AKT, p-mTOR, and Wnt-2 protein expression. RNAi-mediated knockdown of NDRG2 protein expression increased tumor proliferation, while forced expression of wt-NDRG2 decreased proliferation in an in vitro model. Magnetic resonance (MR) imaging and Hematoxylin (H&E) staining demonstrated gross reduction of tumor volume in GA-A/DM treated mice at 5weeks when compared with saline-treated orthotopic AM xenografted controls. There was an overall decrease in tumor cell proliferation with increased survival in GA-A/DM-treated animals. Enzyme assays showed that GA-A/DM did not negatively impact hepatic function. GA-A/DM may be a promising natural therapeutic reagent in the treatment of AM by suppressing growth via NDRG2 modulation and altering of intracellular signal pathways. We have shown it could potentially be an effective treatment for AM with decreased cellular proliferation in vitro, decreased tumor volume and increased survival in vivo.

5946The immune protein MD-2 is associated with early death in dilated cardiomyopathy and increases M1 macrophage polarization and recruitment in vitro

Abstract Objective Dilated cardiomyopathy (DCM) is characterized by systolic dysfunction and dilatation of ventricles. Myocardial inflammation and leukocyte activation and recruitment play a major role in the development and progression of disease. Myeloid differentiation factor-2 (MD-2) is the TLR (toll like receptor)-4 co-receptor and has been shown to be an important risk predictor for mortality of DCM patients. It is expressed in various cell types and mediates TLR-4 dependent inflammation/activation processes. Purpose We examined the impact of MD-2 on mortality of DCM patients and on polarization and recruitment of monocytes in vitro. Methods In 77 DCM patients, divided by median time point of death after first hospital admission into early and late death and alive group, MD-2 was quantified by means of ELISA. In THP-1 monocytes, cytokine secretion was quantified by ELISA after 72h treatment with MD-2 (5μg/mL). Bone marrow derived macrophages (BMDM) were generated from MD-2 KO and WT mice. NFkB phosphorylation (10min) and changes in gene expression (4h) of different adhesion molecules was quantified after treatment with 1 or 10ng/mL LPS. In human umbilical vein endothelial cells (HUVEC), protein kinase B (PKB) phosphorylation was quantified after 15min of treatment with 10ng/mL LPS or 5μg/mL MD-2. CCL2 gene expression in lysed cells (4h) and CCL2 secretion in supernatants (48h) were quantified to. Adhesion of monocytes on treated HUVEC was determined by FACS (Fig.1a). Initial HUVEC treatment with MD-2 (5μg/mL) or LPS (10 or 100ng/mL) took place for 48h. Results We found significant increased MD-2 in early (591.3ng/mL; N=18) vs late death (p=0.015) (369.2ng/mL; N=17) and alive (p≤0.0001) (303.2ng/mL; N=42) patients. Treatment of THP-1 cells (N=5) with MD-2 lead to a significantly increased secretion of inflammatory cytokines IL-8 (p=0.012), IP-10 (p=0.029), and MCP-1 (p=0.032) but not of anti-inflammatory cytokines IL-4, IL-10 and IL-13. Treatment of BMDM obtained from MD-2 KO and WT mice with 10ng/mL LPS lead to a increased phosphorylation of NFkB (N=4; p=0.022) and increased gene expression (N=6) of adhesion molecules VLA-4 (p=0.006) and ICAM-1 (p=0.049) in WT mice but not in KO mice. In HUVEC, LPS (p=0.008) and MD-2 induced a comparable increased phosphorylation of PKB (p=0.008) as well as an increase of CCL2 gene expression (p=0.029) and protein amount (p=0.039). Furthermore, treatment of HUVEC with both MD-2 (p=0.015) and LPS (p=0.0001) lead to a significant increase in monocyte adhesion (Fig.1). Conclusion The impact of MD-2 on cardiac inflammation and macrophage recruitment has not been described yet. In this study, we showed that, in DCM, elevated levels of sMD-2 are associated with early death. Furthermore, we could demonstrate that MD-2 enhances the process of HUVEC based monocyte recruitment. Finally, we could show that MD-2 induces inflammatory monocyte activity and triggers polarization of macrophages towards an inflammatory phenotype.

2-Deoxyglucose and Newcastle Disease Virus Synergize to Kill Breast Cancer Cells by Inhibition of Glycolysis Pathway Through Glyceraldehyde3-Phosphate Downregulation.

Targeting cancer cells metabolism is promising strategy in inhibiting cancer cells progression that are known to exhibit increased aerobic glycolysis. We used the glucose analog 2-Deoxyglucose (2-DG) as a competitor molecule of glucose. To further enhance the effectiveness of 2-DG, the Newcastle disease virus (NDV) was used as a combination virotherapy to enhance the anti-tumor effect. Human and mouse-breast cancer cells were treated by NDV and/or 2-DG. The effect was analyzed by study cell viability, apoptosis and level of glyceraldehyde3-phosphate (GAPDH) by ELISA and QPCR assays. Synergistic cytotoxicity was found after a 72-h treatment of human- and mouse-breast cancer cells with 2-DG in combination with NDV at different concentrations. The synergistic cytotoxicity was accompanied by apoptotic cell death and GAPDH downregulation and inhibition to glycolysis product pyruvate. The combination treatment showed significant tumor growth inhibition compared to single treatments in vivo. Our results suggest the effectiveness of a novel strategy for anti-breast cancer therapy through glycolysis inhibition and GAPDH downregulation.

Polyphyllin I induces autophagy and cell cycle arrest via inhibiting PDK1/Akt/mTOR signal and downregulating cyclin B1 in human gastric carcinoma HGC-27 cells

Paris polyphylla. is a traditional medicinal herb that has long been used to prevent cancer in many Asian countries. Polyphyllin I (PPI), an important bioactive constituent of Paris polyphylla, has been found to exhibit a wide variety of anticancer activities in many types of cancer cells. However, the effects of PPI on human gastric carcinoma cells and its mechanism of action remain unclear. In this study, we examined the effective anti-gastric carcinoma activity of PPI and its underlying mechanism of action in HGC-27 cells. In vitro, sub-micromolar concentrations of PPI inhibited HGC-27 cell proliferation with an IC50 of 0.34 ± 0.06 μM after a 72-h treatment. In vivo, 3 mg/kg PPI significantly inhibited proliferation of HGC-27 tumor cells, with a 78.8% inhibition rate compared to paclitaxel, and demonstrated higher safety. Analysis of MDC and mGFP-LC3 fluorescence, Western blotting and flow cytometry indicated that PPI induced cell cycle arrest in HGC-27 cells by promoting the conversion of LC3-I to LC3-II and by downregulating cyclin B1. Furthermore, Western blotting showed that PPI inhibited the autophagy-regulating PDK1/Akt/mTOR signaling pathway in vitro and in vivo. In addition, immunohistochemistry and TUNEL staining revealed that PPI decreased Ki67 expression and increased the percentage of apoptotic cells in HGC-27 xenograft tumors. These data indicate that PPI is an PDK1/Akt/mTOR signaling inhibitor and of therapeutic relevance for gastric cancer treatment and that the rhizome of Paris polyphylla deserves further clinical investigation as an alternative therapy for gastric cancer.

Effect and mechanism of mGluR6 on the biological function of rat embryonic neural stem cells.

Here, we investigated the effects and molecular mechanisms of metabotropic glutamate receptor 6 (mGluR6) on rat embryonic neural stem cells (NSCs). Overexpression of mGluR6 significantly promoted the proliferation of NSCs and increased the diameter of neutrospheres after treatment for 24h, 48h and 72h. Overexpression of mGluR6 promoted G1 to S phase transition, with significantly decreased cell ratio in G1/G0 phase but significantly increased cell ratio in S phase. Additionally, mGluR6 overexpression for 48h decreased the early and late apoptosis significantly. Moreover, overexpression of mGluR6 significantly increased the expression of p-ERK1/2, Cyclin D1 and CDK2, while the expression of p-p38 was significantly decreased. On the contrary, these effects of mGluR6 overexpression were reversed by mGluR6 knockdown. In conclusion, mGluR6 promotes the proliferation of NSCs by activation of ERK1/2-Cyclin D1/CDK2 signaling pathway and inhibits the apoptosis of NSCs by blockage of the p38 MAPK signaling pathway.

Antitumor activity of pazopanib (P) and trametinib (T) in preclinical models of osteosarcoma (OS).

e22509 Background: Receptor tyrosine kinases (RTKs) and their signal transducers are suitable targets for the treatment of advanced OS. We evaluated the antitumor activity of the RTK inhibitor P and the MEK inhibitor T and deeply investigated molecular mechanisms behind their activity and potential escape. Methods: Flow cytometry and western blot analyses were carried out in 7 OS cell lines to study the expression of RTK P targets and the activation of their pathways, respectively. Cell viability and colony growth were evaluated after 72h and 7-day treatment respectively, with scalar doses of both single agents and their constant combination. Cell cycle distribution and apoptosis were evaluated by flow cytometry after 72h. In vivo antitumor activity was studied in NOD/SCID mice bearing MNNG-HOS xenografts after 3 weeks of treatment. Cell migration was studied by scratch assays. The involvement of MAPK-PI3K pathway key transducers was explored by Vantage 3D RNA Panel and Nanostring technology, validated by western blot and confirmed by silencing experiments. Results: P targets are expressed on OS cell lines and their pathways are activated. P+T have synergistic antitumor activity (combination index < 1) in OS cell lines by inducing apoptosis (6/7) and inhibiting both ERK1/2(7/7) and AKT (7/7). Furthermore, in vivo antitumor activity was shown in OS bearing mice (tumor volume: P+T/untreated = 0.036, p = 0.002). P+T significantly down-modulated RTK EphaA2 (mean log2 fold change RNA P+T/untreated = -2.02±0.50) and induced Janus kinase MEK6 (mean log2 fold change RNA P+T/untreated = 2.9±0.51). EphA2 silencing reduced cellular proliferation and migration of OS cells. Impeding MEK6-up-regulation in P+T treated cells significantly increased the antitumor effect (51.5±14.3%) of the studied drugs. Conclusions: P+T exert antitumor activity in OS preclinical models through ERK and AKT inhibition and EphA2 downmodulation. MEK6-upregulation after P+T is likely implied in escape mechanism.

Extrapolating the bypass potential of treated Madre de agua (Trichanthera gigantea Nees) leaf meal as protein source in Rumen-Fistulated Brahman Cattle

This study assessed the effectiveness of formaldehyde, heat, and tannic acid treatments of madre de agua leaf meal (MALM) in reducing the degradation of dry matter (DMD) and crude protein (CPD) in the rumen for increased supply of bypass protein at the intestinal level. The experiment utilized a rumen-fistulated Brahman bull fed with chopped Napier grass soilage (basal diet) and MALM (test diet) at 70:30 basal:test diet ratio. Nylon bags (porosity of ±53μm) containing the treated MALM were incubated in the rumen for 24, 48 and 72 hours following the “sequential addition” method forin situ degradation measurement. Results showed that DMD of MALM was significantly reduced by formaldehyde treatment after 24-h and 48-h period of incubation than the untreated. This was followed by tannic acid treatment, though the reduction was significant only after 48-h incubation than the untreated. At 72-h period of incubation, DMD remained to be significantly lowest with formaldehyde treatment than the untreated or heat and tannic acid treatments. A similar pattern of differences in DMD rate (%/h) was observed as that of DMD (%). The CPD of MALM was also significantly reduced with formaldehyde treatment after 24-h incubation than the untreated. At 48-h incubation, all treatment methods showed significant protection of protein in MALM over that of the untreated. At 72h, formaldehyde and heat treatments significantly reduced the CPD of MALM compared to the untreated. A similar pattern of differences in CPD rate (%/h) was observed as that of CPD (%). Among treatment methods, the use of formaldehyde is the best, followed by tannic acid treatment, in achieving rumen bypass and promoting greater amino acid supply at the intestinal level.

Activity of TP‐0903 in FLT3 inhibitor resistant AML models

BackgroundThe development of tyrosine kinase inhibitors (TKIs) for the treatment of acute myeloid leukemia (AML) with FLT3 internal tandem duplication mutations (FLT3‐ITD+) has been challenging due to intrinsic and acquired drug resistance. Important resistance mechanisms that have been described include: 1) the emergence of secondary FLT3 tyrosine kinase domain (TKD) mutations; 2) compensatory signaling pathways (intrinsic to the leukemia cell or due to tumor‐microenvironment interactions), such as Gas6/Axl upregulation; and 3) the co‐occurrence of somatic mutations with FLT3‐ITD, such as IDH1/2, NRAS and MLL‐PTD. TP‐0903, is a new TKI in development as an Axl inhibitor. Previously, we demonstrated that TP‐0903 is a potent inhibitor of FLT3‐ITD and drug resistant FLT3 TKD mutations in preclinical in vitro and in vivo models (Jeon JY et al. ASH 2017). Here, we present the activity of TP‐0903 in additional TKI‐resistant models of FLT3‐ITD+ AML in comparison to other FLT3 TKIs.MethodsFLT3‐ITD+ MOLM13 cells were seeded in direct co‐culture with or without HS5‐GFP bone marrow stromal cells (MSCs) for 24h followed by 72h treatment with TP‐0903 or 5 other clinical candidate FLT3 TKIs; cell viability of co‐cultured MOLM13 cells was measured via MTT assay. Soluble Gas6 and Axl were measured in co‐cultured media over 24–72h by ELISA. Ex vivo activity of TKIs on the viability of murine primary leukemia cells with FLT3‐ITD+/−/IDH2‐R140Q+/− or FLT3‐ITD+/−/MLL‐PTD co‐occurring mutations was assessed after 72h TKI treatment using a CellTiter Glo assay.ResultsThe effect of TP‐0903 and other FLT3 TKIs on the inhibition of viability of MOLM13 cells cultured with or without MSCs is shown in Table 1. While co‐culture induced a minimal 1.7‐fold shift in the TP‐0903 IC50 value, the other FLT3 TKIs were more resistance in co‐culture, with gilteritinib and sorafenib showing the greatest change in IC50 values (4.9‐ and 5.0‐fold). Soluble Axl and Gas6 levels increased over 24–72h in co‐cultured media with the highest concentrations achieved of 1455 ± 322 pg/mL and 5098 ± 974 pg/mL, respectively. In primary FLT3‐ITD+/−/IDH2‐R140Q+/− leukemia cells, TP‐0903 was 8‐ to 10‐fold more potent than gilteritinib and crenolanib, whereas midostaurin, quizartinib and sorafenib did not show activity in these cells (Table 1). TP‐0903 was also 5‐fold more potent than gilteritinib in primary FLT3‐ITD+/−/MLL‐PTD leukemia cells. Ongoing studies are evaluating the effect of soluble Gas6 on TKI sensitivity in AML cells, as well as the in vivo activity of TP‐0903 in FLT3‐ITD+/−/IDH2‐R140Q+/− or FLT3‐ITD+/−/MLL‐PTD spontaneous mouse models.ConclusionsTP‐0903 retains activity in preclinical models of intrinsic/acquired FLT3 TKI resistance with therapeutic potential to overcome drug resistant FLT3‐ITD+ AML.Support or Funding InformationThis work is supported by Eli Lilly fellowship and NIH grant 5R01CA138744‐09This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Manilkara zapota (L.) P. Royen Leaf Extract Derived Silver Nanoparticles Induce Apoptosis in Human Colorectal Carcinoma Cells Without Affecting Human Lymphocytes or Erythrocytes.

Plant-derived synthesis of silver nanoparticles (AgNPs) has found wide biomedical applications including cancer cure. This report deals with biosynthesis of silver nanoparticles (MZLAgNPs) employing leaf extracts of Manilkara zapota (L.) under optimized conditions. Characterization of MZLAgNPs using UV-Vis spectroscopy, FTIR, XRD, and FESEM analyses revealed that the particles were predominantly spherical averaging 24nm in size. Their cellular effects were assessed by MTT assay, fluorescence, and scanning electron microscopy of cells stained with propidium iodide, acridine orange/ethidium bromide, and annexin V-FITC to visualize signs of apoptosis. Evaluation of cell proliferation by clonogenic assay, wound healing ability by scratch assay and cell cycle distribution by flow-cytometry was also carried out. Apoptosis-related gene expressions were analyzed by RTq-PCR and western blot analysis. MZLAgNPs selectively inhibited growth of colorectal carcinoma HCT116, HeLa, and non-small lung carcinoma A549 cells, dose-dependently with IC50 concentrations of 8, 16, and 29μg/mL respectively, following 72-h treatment, without affecting growth of normal human lymphocytes and erythrocytes. Apoptosis induction was observed by fluorescence and scanning electron microscopy. Overproduction of reactive oxygen species (ROS), reduction of mitochondrial membrane potential, upregulation of apoptotic-related genes - PUMA, cas-3, cas-8, cas-9, and BAX, expression of caspase 3, and occurrence of PARP cleavage were observed in MZLAgNPs/cisplatin treated cells. Taken together, our results clearly demonstrate the therapeutic potential of biogenic MZLAgNPs as an effective agent for killing colorectal carcinoma cells by apoptosis induction.

Inositol hexaphosphate-induced cellular response in myeloid leukemia cells is mediated by nicotinamide adenine dinucleotide phosphate oxidase activation.

The objective of this study was to identify the role of reactive oxygen species (ROS) generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, in inositol hexaphosphate (IP6)-induced metabolic disruption in human leukemia PLB-985 cells. PLB-985 and X chromosome linked gp91-phox gene knockout (X-CGD) cells were treated with 5, 10, or 20mM IP6 for 24 to 72h. Cell growth was assayed using a highly water-soluble tetrazolium salt. The rate of apoptotic and necrotic cell death was determined with an Annexin V-fluorescein isothiocyanate/propidium iodide kit. The expression of CD11b as a marker of monocytic property and of LC3 as an autophagy marker was tested, using flow cytometry combined with fluorescent antibodies. Treatment with 5 and 10mM IP6 for 24h was found to suppress the growth of both cell lines, though the effect was more dramatic in PLB-985 cells. After 6-h treatment with 20mM IP6, the necrosis rate of PLB-985 cells was significantly greater than that of X-CGD cells. Further, after 72-h treatment with 10mM IP6, CD11b expression was observed in PLB-985 cells but inhibited in X-CGD cells. Autophagy monitoring after 6-h treatment with 10mM IP6 revealed that LC3 expression was suppressed in PLB-985 cells, whereas it was somewhat increased in X-CGD cells. Our results suggest that NADPH oxidase activation mediates IP6-induced metabolic disruption associated with necrosis, differentiation, cell growth, and autophagy in PLB-985 cells.

Roles of UGT2B7 C802T gene polymorphism on the efficacy of morphine treatment on cancer pain among the Chinese han population

Morphine is a common analgesic often used to manage chronic pain, especially for patients with pain due to malignancies. Since UGT2B7 plays an important role in the metabolism of morphine, UGT2B7 gene mutation may influence the efficacy of morphine in patients with cancer being treated by this medication. The aim of this study is to investigate the relationship between the polymorphisms of UGT2B7 and the efficacy of morphine treatment on cancer pain among the Chinese Han population. A total of 120 patients with cancer pain were enrolled in this study. Morphine was administrated through patient-controlled analgesia infusion pump, and the visual analog score (VAS) was used for pain assessment at 0.5, 4, 6, 12, 24, 48, and 72-h post morphine treatment, respectively. The plasma concentration of morphine and genetic polymorphism of UGT2B7 C802T and G221T was analyzed, respectively. The frequencies of UGT2B7 C802T were CC: 13.33%, CT: 45% and TT: 41.67%, and the frequencies of UGT2B7 G221T were GG: 76.67%, GT: 22.5% and TT: 0.83%. Moreover, the VAS score of patients with either C802T CT or TT was significantly higher than that in patients with C802T CC. However, no difference of VAS scores was observed between patients carrying G221T GG and patients carrying G221T GT. The plasma concentration of morphine for patients with the C802T CC was significantly lower than that in patients carrying C802T CT or TT, while there was no significant difference in the level of morphine between patients with G221T GG and G221T GT. The polymorphism of UGT2B7 C802T, but not UGT2B7 G221T, has been associated with the efficacy of morphine treatment on cancer pain among Chinese Han population.

Enhanced Anticancer Activity of 5'-DFUR-PCL-MPEG Polymeric Prodrug Micelles Encapsulating Chemotherapeutic Drugs.

The compound 5’-deoxy-5-fluorouridine (5’-DFUR) is a prodrug of the anti-tumor drug 5-fluorouracil (5-FU). Thymidine phosphorylase (TP) is an enzyme that can convert 5’-DFUR to its active form 5-FU and the expression of TP is upregulated in various cancer cells. In this study, 5’-DFUR associated with amphiphilic copolymer poly(ε-caprolactone)-methoxy poly(ethylene glycol) (5’-DFUR-PCL-MPEG) was synthesized, characterized, and self-assembled into functional polymeric micelles. To demonstrate that the prodrug 5’-DFUR could convert into cytotoxic 5-fluorouracil (5-FU) by endogenous TP, HT-29 colorectal cancer cells were treated with 5’-DFUR-PCL-MPEG polymeric micelles for various time periods. Chemotherapeutic drugs doxorubicin (DOX) and 7-ethyl-10-hydroxycamptothecin (SN-38) were also encapsulated separately into 5’-DFUR-PCL-MPEG polymeric micelles to create a dual drug-loaded system. HT-29 cells were treated with DOX or SN-38 encapsulated 5’-DFUR-PCL-MPEG polymeric micelles to examine the efficacy of dual drug-loaded micelles. As a result, HT-29 cells treated with 5’-DFUR-PCL-MPEG polymeric micelles showed up to 40% cell death rate after a 72-h treatment. In contrast, HT-29 cells challenged with DOX or SN-38 encapsulated 5’-DFUR-incorporated polymeric micelles showed 36% and 31% in cell viability after a 72-h treatment, respectively.

Loss of KDM6A Confers Drug Resistance in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive hematologic cancer resulting from the malignant transformation of myeloid progenitors. Despite intensive chemotherapy, relapse caused by intrinsic or acquired drug resistance remains a major hurdle in the treatment of AML. Recently, we found KDM6A as a novel relapse-associated gene in a cohorte of 50 cytogenetically normal AML patients. KDM6A (or UTX) is a histone 3 lysine 27 (H3K27)-specific demethylase and a member of the COMPASS (complex of proteins associated with Set1)-like complex, which is important for chromatin enhancer activation. KDM6A is targeted by inactivating mutations in a variety of cancer types with frequency of occurrence ranging from 0.7 to 4% in AML.In this study, we used matched diagnosis and relapse samples from AML patients, patient-derived xenografts (PDX), and myeloid leukemia cell lines to investigate the status of KDM6A during disease progression and the implications of KDM6A loss regarding chemotherapy resistance. We found three AML patients with enrichment of KDM6A mutations at relapse and mutation-independent, relapse-specific loss of KDM6A expression in three additional AML patients. KDM6A mutations comprise deletions and point mutations and appear to be mainly loss-of-function mutations. In addition, we examined the mutation profile and KDM6A expression in patient-derived xenograft (PDX) samples from 8 relapsed AML patients. In 4/8 samples, KDM6A protein levels were low or completely lost. Due to the fact that all patients had received induction therapy including single or combination treatment with agents such as cytarabine (AraC), daunorubicin (DNR), and 6-thioguanine (6-TG), we hypothesized that loss of KDM6A confers resistance to chemotherapy. To exclude gender-specific effects (KDM6A escapes X inactivation leading to higher levels in females), we compared male KDM6A knockout (KO) with WT leukemia cell lines and found increased AraC resistance in the KDM6A KO cells (unpaired, two-tailed Student's t-test; P=0.0441). In addition, we treated two relapsed PDX AML cells of the same gender, AML 491 (KDM6A WT and strong expression) and AML 393 (KDM6A mutation and weak expression) with AraC for 72h in vitro and found significantly increased AraC resistance in the KDM6A-mutant PDX AML 393 cells (P=0.016). To further investigate whether reduced expression or loss of KDM6A leads to increased resistance towards multiple drugs, we silenced KDM6A expression by shRNA or CRISPR/Cas9 in K562 and MM-1 cells. Compared to control, KDM6A knockdown (KD) and KO K562 cells showed a strong proliferative advantage after AraC and DNR but not 6-TG treatment. A similar drug resistance phenotype was observed in KDM6A KO MM-1 cells.To unravel the mechanism of drug resistance, we performed RNA-Seq analysis in K562 cells treated with siRNA or shRNA against KDM6A under native conditions and after AraC (150nM) treatment for 72h. We compared these differentially expressed genes with known key candidate genes in AraC, DNR, and 6-TG metabolic pathway and found that ENT1 was consistently downregulated in KDM6A KD cells in both siRNA- and shRNA-mediated RNA-Seq screenings. Decreased ENT1 levels were also detected in KDM6A KO K562 single cell clones. ENT1 (also known as SLC29A1) is a membrane transporter relevant for the cellular uptake of nucleosides and its analogues. Competitive inhibition of ENT1 by the small molecule antagonist NBMPR lead to decreased sensitivity towards AraC but not DNR and 6-TG suggesting that increased AraC resistance in KDM6A KO cells is caused, at least partially, by downregulation of ENT1.To elucidate the mechanism of ENT1 regulation by KDM6A, we performed ChIP-seq analysis for H3K27me3 and H3K27ac in the sister cell lines MM-1 (KDM6A WT) and MM-6 (KDM6A KO). ChIP-seq for H3K27me3 showed no enrichment on the ENT1 locus, but we detected differential H3K27ac peaks in the promoter and a putative enhancer region of ENT1 in MM-1 compared to MM-6. These data suggest that increased ENT1 expression may function through direct or indirect effects of KDM6A on enhancer regions, independent of its H3K27 demethylase activity.In conclusion, our results show that mutations in KDM6A are associated with the outgrowth of drug-resistant clones and highlight KDM6A as a novel biomarker of drug resistance in AML. DisclosuresHiddemann:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding. Metzeler:Novartis: Consultancy; Celgene: Consultancy, Research Funding.

Copanlisib, a novel phosphoinositide 3-kinase inhibitor, combined with carfilzomib inhibits multiple myeloma cell proliferation.

Multiple myeloma (MM) is a uniformly fatal disorder of B cells characterized by the accumulation of abnormal plasma cells. Phosphoinositide 3-kinase (PI3K) signaling pathways play a critical regulatory role in MM pathology. Copanlisib, also known as BAY80-6946, is a potent PI3Kα and δ inhibitor. In this study, we investigated the efficacy of copanlisib and a proteasome inhibitor using MM cell lines and primary samples. The p110α and δ catalytic subunits of the class PI3K increased, and carfilzomib activity reduced in the presence of a supernatant from the feeder cell line, HS-5. Phosphorylation of Akt and activation of caspase 3 and poly (ADP-ribose) polymerase (PARP) partially reduced upon carfilzomib treatment in the presence of HS-5. Apoptosis also decreased. Copanlisib treatment for 72h inhibited growth in MM cell lines and induced apoptosis. Combination treatment of MM cells with carfilzomib and copanlisib caused greater cytotoxicity than that caused by either drug alone and increased apoptosis. Caspase 3 activity increased while that of Akt decreased after combination treatment with copanlisib and carfilzomib. Further, copanlisib inhibited vascular endothelial growth factor (VEGF)-mediated angiogenesis in vitro and in vivo. It also inhibited C-X-C motif chemokine 12 (CXCL12)-mediated chemotaxis. The data suggest that administration of the PI3K inhibitor, copanlisib, may be a powerful strategy against stroma-associated drug resistance of MM cells and can enhance the cytotoxic effects of proteasome inhibitors in such residual MM cells.

Concomitant Association of T-DM1 and Radiation: An In Vitro Study on HER2 Positive Cells of Breast Cancer

The better understanding of the EGFR receptors involved in the oncogenesis of HER2 positive breast cancers has led to the emergence of new therapeutic classes: the Antibodies Drug-Conjugate (ADC). Trastuzumab-DM1 (T-DM1) is an ADC combining the anti-HER2 monoclonal antibody trastuzumab with molecules of DM1, a powerful mitotic spindle inhibitor. In HER2 positive breast cancer, T-DM1 is a validated treatment but the effects of concomitant association with radiation are not known. The objective of this study is to determine in vitro the effects of concomitant irradiation with T-DM1 on five different HER2 positive breast cancer cell lines. Five cell lines of human breast cancer with different levels of HER2 expression (HCC-1954, BT-474, SKBR-3, MDA-MB-453, ZR-75). T-DM1 was provided by Roche®/Genetech upon MTA. Expression levels were determined by western blot. T-DM1 toxicity was assessed using the CellTiter-Glo® assay. Cell cycle analysis was performed by flow cytometry after incorporation of BrdU. HER2 cells were irradiated (GSR 137Cs 662 keV) at different dose levels (from 1 to 8 Gy) after exposure to T-DM1. Survival fractions were determined based on clonogenic assays or on cell survival after 5 doubling times. Radiosensitivity parameter (D10 and D37) were calculated using the mean values of a and b determined from the curves drawn for best fit to the experimental data. The action of T-DM1 on HER2 cells showed significant lethality by deprivation of the HER2 signaling pathway and intracellular DM1 action on the cell cycle with significant G2/M phase blocking. After 72h treatment with T-DM1 alone, the median effective dose (ED50) determined for the six cell lines used is function of the expression of the receptor HER2 except for one cell line (BT474). Using sub-toxic doses of T-DM1 and short-term drug exposure (6 or 12h), the cell viability still decreases dramatically after several days. After irradiation alone, D37 and D10 were significantly higher for the three high-HER2 expressing cells lines (HCC1954, BT474 and SKBr3) compared to the low-HER2 expressing cells (MDA-453 and ZR-75-1), with a linear increasing relationship between the radioresistance and the level of HER2 expression (D37: r2=0.9; p<0.0001 and D10: r2=0.7; p<0.0001). In combination with radiation, T-DM1 elicited strictly additive interaction. There was no significant difference on D10 compared to control for all cell lines. HCC1954 : D10 T-DM1 = 6.15±0.64 vs 6.0±0.24 for control; BT-474: D10 T-DM1 = 7.86±0.22 vs 7.6.0±0.43 for control; SKBr3 : D10 T-DM1 = 5.68±0.29 vs 5.43±0.14 for control; MDA-MB-453 : D10 T-DM1 = 4.82±0.07 vs 4.02±0.17 for control and ZR-75-1 = D10 T-DM1 = 3.3±0.3 vs 3.02±0.3 for control. Although HER2 expression is a radioresistant factor, T-DM1 did not have any radiosensitizing impact on HER2 positive breast cancer cells in this in vitro study. The major toxicity of T-DM1 alone could explain this phenomenon but further explorations are needed.