Myeloid Differentiation Protein Research Articles

Mechanisms underlying reversed TRAIL sensitivity in acquired bortezomib-resistant non-small cell lung cancer cells

Aim: The therapeutic targeting of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) death receptors in cancer, including non-small cell lung cancer (NSCLC), is a widely studied approach for tumor selective apoptotic cell death therapy. However, apoptosis resistance is often encountered. The main aim of this study was to investigate the apoptotic mechanism underlying TRAIL sensitivity in three bortezomib (BTZ)-resistant NSCLC variants, combining induction of both the intrinsic and extrinsic pathways. Methods: Sensitivity to TRAIL in BTZ-resistant variants was determined using a tetrazolium (MTT) and a clonogenic assay. A RT-qPCR profiling mRNA array was used to determine apoptosis pathway-specific gene expression. The expression of these proteins was determined through ELISA assays and western Blotting, while apoptosis (sub-G1) and cytokine expression were determined using flow cytometry. Apoptotic genes were silenced by specific siRNAs. Lipid rafts were isolated with fractional ultracentrifugation. Results: A549BTZR (BTZ-resistant) cells were sensitive to TRAIL in contrast to parental A549 cells, which are resistant to TRAIL. TRAIL-sensitive H460 cells remained equally sensitive for TRAIL as H460BTZR. In A549BTZR cells, we identified an increased mRNA expression of TNFRSF11B [osteoprotegerin (OPG)] and caspase-1, -4 and -5 mRNAs involved in cytokine activation and immunogenic cell death. Although the OPG, interleukin-6 (IL-6), and interleukin-8 (IL-8) protein levels were markedly enhanced (122-, 103-, and 11-fold, respectively) in the A549BTZR cells, this was not sufficient to trigger TRAIL-induced apoptosis in the parental A549 cells. Regarding the extrinsic apoptotic pathway, the A549BTZR cells showed TRAIL-R1-dependent TRAIL sensitivity. The shift of TRAIL-R1 from non-lipid into lipid rafts enhanced TRAIL-induced apoptosis. In the intrinsic apoptotic pathway, a strong increase in the mRNA and protein levels of the anti-apoptotic myeloid leukemia cell differentiation protein (Mcl-1) and B-cell leukemia/lymphoma 2 (Bcl-2) was found, whereas the B-cell lymphoma-extra large (Bcl-xL) expression was reduced. However, the stable overexpression of Bcl-xL in the A549BTZR cells did not reverse the TRAIL sensitivity in the A549BTZR cells, but silencing of the BH3 Interacting Domain Death Agonist (BID) protein demonstrated the importance of the intrinsic apoptotic pathway, regardless of Bcl-xL. Conclusion: In summary, increased sensitivity to TRAIL-R1 seems predominantly related to the relocalization into lipid rafts and increased extrinsic and intrinsic apoptotic pathways.

Gastrodin Attenuates Colitis and Prevents Tumorigenesis in Mice by Interrupting TLR4/MD2/NF-ΚB Signaling Transduction.

Chronic inflammation is one of the causative factors for tumorigenesis. Gastrodin is a main active ingredient isolated from Gastrodia elata Blume, a famous medicinal herb with a long edible history. This study aimed to explore the effects of gastrodin on colitis-associated carcinogenesis (CRC) in mice and to elucidate its potential molecular mechanisms. Balb/c mice were induced with azoxymethane (AOM) and dextran sulfate sodium (DSS) for 12 weeks. Gastrodin (50 mg/kg) was administered via oral gavage three times per week until the end of the experiment. Disease indexes, including body weight, bloody diarrhea, colon length, histopathological score, and tumor size, were measured. Tumor cell proliferation was evaluated by BrdU incorporation assay and tumor cell cytotoxicity was assessed by cell counting kit (CCK-8). The expression levels of toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-ΚB) signaling molecules, NF-ΚB luciferase, and pro-inflammatory cytokines were determined by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), immunoblotting, immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), or reporter gene assays. The binding affinity between gastrodin and myeloid differentiation protein-2 (MD2) was analyzed by molecular docking and cellular thermal shift assay (CETSA). Gastrodin administration was demonstrated to mitigate various CRC-related symptoms in mice, including weight loss, diarrhea, and tissue abnormalities. Notably, gastrodin suppressed tumor cell growth during colitis- associated tumorigenesis, resulting in fewer and smaller adenomas in the colon. Unlike irinotecan, a broadspectrum antitumor drug, gastrodin did not exhibit apparent cytotoxicity in various colorectal adenocarcinoma cell lines. Additionally, gastrodin downregulated TLR4/NF-ΚB signaling molecules and pro-inflammatory mediators in mice and macrophages. Molecular docking and CETSA experiments suggested that gastrodin binds to the MD2 protein, potentially interfering with the recognition of lipopolysaccharide (LPS) by TLR4, leading to NF-ΚB pathway inhibition. This study provides evidence for the first time that gastrodin attenuated colitis and prevented colitisrelated carcinogenesis in mice, at least partially, by diminishing tumor-promoting cytokines through the interruption of TLR4/MD2/NF-ΚB signaling transduction.

Development of recombinant protein-based nanoparticle systems for inducing tumor cell apoptosis: In vitro evaluation of their cytotoxic and apoptotic effects on cancer cells

Myeloid leukemia cell differentiation protein (Mcl-1) is a B-cell lymphoma 2 (Bcl-2) family antiapoptotic protein that regulates cellular apoptosis. Mcl-1 expression is important for cancer cell survival and a prospective target in cancer treatment. Bcl-2 homology 3 (BH3) mimetic peptides, such as the Mcl-1-specific MS1 peptide that targets conserved BH3 domains, are highly specific Mcl-1 inhibitors. However, the application of these peptides is limited due to their metabolic instability, degradation by cellular proteases, and low cell permeability. In this study, it was aimed to form nanostructures to increase the cellular uptake and stability of the Mcl-1 inhibitor MS1 peptide. The Bim BH3 mimetic MS1 peptide was produced recombinantly in fusion with mNeonGreen fluorescent protein and polyhistidine (6xHisTag). Self-assembled protein nanoparticles were fabricated from the recombinant MS1-mNeonGreen protein. Furthermore, the MS1-mNeonGreen protein was encapsulated into polymeric nanoparticles. The cytotoxic effects of these nanoparticles on cancer cells [cervical cancer cell line (HeLa), lung cancer cell line (A549) and breast cancer cell line (MDA-MB-231)] and healthy cell line [Human umbilical vein endothelial cells (HUVEC)] was analyzed by MTT test. The apoptotic effect of nanoparticles on these cells was investigated by determining the antiapoptotic and proapoptotic protein levels. As a result, it was revealed that the nanoparticles have a specific cytotoxic effect against the cells and induce apoptosis.

[Effect and mechanism of Maxing Shigan Decoction on reducing inflammatory response in rats with cough variant asthma via TLR4/MyD88/NF-κB and p38 MAPK signaling pathways].

This study aims to investigate the effect and mechanism of Maxingshigan Decoction on inflammation in the rat model of cough variant asthma(CVA). The SPF-grade SD rats of 6-8 weeks were randomized into normal, model, Montelukast sodium, and low-, medium-, and high-dose Maxing Shigan Decoction groups, with 8 rats in each group. The CVA rat model was induced by ovalbumin(OVA) and aluminum hydroxide sensitization and ovalbumin stimulation. The normal group and model group were administrated with equal volume of normal saline by gavage, and other groups with corresponding drugs by gavage. After the experiment, the number of white blood cells in blood and the levels of interleukin-6(IL-6), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α) in the serum were measured. The lung tissue was stained with hematoxylin-eosin(HE). Western blot was employed to determine the protein levels of nuclear factor-κB(NF-κB), Toll-like receptor 4(TLR4), myeloid differentiation protein(MyD88), and mitogen-activated protein kinase(MAPK) in the lung tissue. Real-time PCR was carried out to measure the mRNA levels of TLR4 and MyD88 in the lung tissue. Compared with the normal group, the model group showed increased white blood cells, elevated IL-6 and TNF-α levels(P<0.01), lowered IL-10 level(P<0.01), up-regulated protein levels of TLR4, MyD88, p-p65/NF-κB p65, and p-p38 MAPK/p38 MAPK(P<0.01) and mRNA levels of TLR4 and MyD88(P<0.01) in the lung tissue. HE staining showed obvious infiltration of inflammatory cells around the airway and cell disarrangement in the model group. Compared with the model group, Montelukast sodium and high-dose Maxing Shigan Decoction reduced the white blood cells, lowered the IL-6 and TNF-α levels(P<0.01), and elevated the IL-10 level(P<0.01). Moreover, they down-regulated the protein levels of TLR4, MyD88, p-p65/NF-κB p65, p-p38 MAPK/p38 MAPK in the lung tissue(P<0.01) and the mRNA levels of TLR4 and MyD88 in the lung tissue(P<0.01). HE staining showed that Montelukast sodium and high-dose Maxing Shigan Decoction reduced inflammatory cell infiltration and cell disarrangement. The number of white blood cells, the levels of IL-10 and TNF-α in the serum, the protein levels of TLR4, MyD88, p-p65/NF-κB p65, and p-p38 MAPK/p38 MAPK, and the mRNA levels of TLR4 and MyD88 in the lung tissue showed no significant differences between the Montelukast sodium group and high-dose Maxing Shigan Decoction group. Maxing Shigan Decoction can inhibit airway inflammation in CVA rats by inhibiting the activation of TLR4/MyD88/NF-κB and p38 MAPK signaling pathways.

Microglial phenotype and proinflammatory gene induction in human prefrontal cortex in Alcohol Use Disorder overlap with Alzheimer’s Disease

AbstractBackgroundAlcohol Use Disorder (AUD) and heavy alcohol abuse are associated with dementia. However, studies focused on Alzheimer’s disease (AD) tend to exclude heavy alcohol drinking patients. We hypothesize microglial phenotype changes in AUD contribute to degeneration that parallels AD.MethodOrbital frontal cortex (OFC) from moderate drinking controls and heavy drinking AUD individuals paired by age were obtained from the New South Wales Tissue Resource Center in Sydney Australia. Tissue blocks were used for RTPCR gene mRNA and paraffin sections for immunohistochemistry assessment of microglial and proinflammatory genes and proteins, respectively. Fluoro‐Jade B, TUNEL stain and neuron marker NeuN were used to assess degeneration.ResultAUD OFC expressed increased levels of proinflammatory HMGB1, RAGE, and multiple Toll‐like receptors (TLR). In addition, AUD OFC increased expression of the TLR adaptor protein myeloid differentiation primary response 88 (MyD88), NFκB, and proinflammatory cytokines, chemokines, and their corresponding receptors. Iba1, a marker of monocytes and microglia, is increased in AUD, but not mRNA levels. Macrophage‐1 antigen, a complement receptor, is increased in AUD cortex as is CX3C chemokine receptor 1 (CX3CR1), the fractalkine receptor. OX42 staining (CD11b) in also increased. Microglial markers P2ry12, and CSF1R also show greater cellular staining in human AUD. The lysosomal scavenger receptor marker CD68 is also increased in AUD cortex. CX3CR1 and CD11b mRNA are increased in AUD cortex, but not CD68. Interestingly, TMEM119, a specific microglial marker, is decreased in AUD, both mRNA and protein staining in contrast to other markers increasing. Assessment of DNA oxidation using 8‐hydroxy‐2'‐deoxyguanosine (8‐OHdG) a biomarker of oxidative DNA/RNA damage 8‐OHdG+IR indicated AUD more than doubled cortical cells with this marker. Induction of HMGB1‐TLR‐MyD88‐NFκB proinflammatory signaling pathways, oxidative stress and microglial activation correlate with markers of neurodegeneration.ConclusionThe increased expression of TLR receptors, MyD88 and NFkB and increased proinflammatory cytokines are consistent with increased microglial activation and oxidative stress induced degeneration. Studies support alcohol activated microglia‐monocytes as driving loss of NeuN and other neurodegeneration markers. The reduced TMEM119 level with other markers increasing may indicate monocytes are replacing microglia in AUD cortex.

2-Pentadecyl-2-oxazoline inhibits lipopolysaccharide-induced microglia activation interfering with TLR4 signaling

Aim2-Pentadecyl-2-oxazoline (PEA-OXA), the oxazoline derivative of N-palmitoylethanolamine, exerts anti-inflammatory activity; however, very little is known about the molecular mechanisms underlying this effect. Here, we tested the anti-neuroinflammatory effect of PEA-OXA in primary microglia and we also investigated the possible interaction of the molecule with the Toll-like receptor 4 (TLR4)–myeloid differentiation protein-2 (MD-2) complex. Main methodsThe anti-inflammatory effect of PEA-OXA was analyzed by measuring the expression and release of pro-inflammatory mediators in primary microglia by real-time PCR and ELISA, respectively. The effect of PEA-OXA on the activation of TLR4 signaling was assessed using two stably TLR4-transfected cell lines (i.e., HEK-293 and Ba/F3 cells). Finally, the putative binding mode of PEA-OXA to TLR4–MD-2 was investigated by molecular docking simulations. Key findingsTreatment with PEA-OXA resulted in the following effects: (i) it down-regulated gene expression of several pro-inflammatory molecules and the secretion of pro-inflammatory cytokines in LPS stimulated microglia cells; (ii) it did not prevent microglia activation after stimulation with TLR2 ligands; (iii) it prevented TLR4/NF-κB activation triggered by LPS in HEK-Blue™ hTLR4 cells; and (iv) it interfered with the binding of LPS to TLR4–MD-2 complex. Furthermore, molecular docking studies suggested that PEA-OXA could bind MD-2 with a 1:3 (MD-2/PEA-OXA) stoichiometry. ConclusionWe show for the first time that the anti-neuroinflammatory effect of PEA-OXA involves its activity against TLR4 signaling, making this molecule a valuable tool for the development of new compounds directed to control neuroinflammation via inhibiting TLR4 signaling.

[Mechanism of myeloid differentiation factor 2 on mediating sepsis-associated encephalopathy].

To investigate the role and underlying mechanism of human myeloid differentiation protein 2 (MD2) in the process of neuronal death induced by lipopolysaccharide (LPS) by establishing an in vitro model of sepsis-associated encephalopathy (SAE) by LPS. Healthy C57BL/6J mice at 14-18 days of gestation were selected, and brain cortical tissue was taken from fetal mice. Neurons were stimulated with 0 (control), 1, 5 and 10 g/L of LPS for 24 hours. The release of lactate dehydrogenase (LDH) was detected and the death of neurons was observed. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors interleukins (IL-6, IL-1β), in order to determine the optimal dose of LPS for establishing an in vitro neuroinflammation model of SAE. The cells were divided into blank control group and LPS group. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) was used to discover apoptosis. Western blotting was used to detect the expression of the relevant protein markers activated caspase-3, necroptosis-associated protein neuronal receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and phosphorylated RIPK3 (p-RIPK3). Immunofluorescence chemical staining was used to detect the expressions of p-RIPK3 and microtubule-associated protein 2 (MAP2) to evaluate the type of cell death and the degree of neuronal death. Western blotting was used to detect MD2 expression. Immunofluorescence chemical staining was performed to observe the expression and distribution of p-RIPK3 and MD2 in neurons to assess whether MD2 was involved in the inflammatory response promoting neuronal death. In addition, the cells were divided into blank control group, LPS group, and MD2 interfering peptide group (LPS+TC group), and the levels of IL-6, IL-1β and LDH were detected to evaluate whether interfering with MD2 can alleviate LPS induced neuroinflammation. 10 g/L LPS induced notable neuronal death, and the release of LDH in neurons stimulated with this concentration for 24 hours was significantly higher than that in the blank control group (relative release: 1.45±0.04 vs. 1.00±0.00, P < 0.01), indicating apoptosis and necroptosis occurred in neurons, and the levels of inflammatory factors IL-6 and IL-1β were remarkable increased [IL-6 (relative level): 1.94±0.04 vs. 1.00±0.00, IL-1β (relative level): 1.53±0.09 vs. 1.00±0.00, both P < 0.01]. Compared with the blank control group, the apoptosis of cells, cleaved-caspase-3 expression, the p-RIPK3/RIPK3 ratio, and p-RIPK3 expression around neurons in the LPS group were significantly increased [cleaved-caspase-3/GAPDH: 1.55±0.10 vs. 1.00±0.00, P < 0.01; p-RIPK3/RIPK3 ratio (relative value): 1.54±0.06 vs. 1.00±0.00, P < 0.05], which suggested that typical apoptosis and necroptosis apoptosis occurred in neurons in the septic environment. Furthermore, MD2 expression was significantly increased in the LPS group compared with the blank control group (MD2/GAPDH: 1.91±0.07 vs. 1.00±0.00, P < 0.01), and MD2 expression around neurons was increased, indicating that LPS-induced MD2 upregulation may play a key role in neuroinflammation and induction of neuronal death in sepsis. In addition, compared with the LPS group, the MD2-interfering peptide could reduce the expression levels of inflammatory factors IL-6 and IL-1β [IL-6 (relative level): 1.16±0.08 vs. 1.94±0.04, IL-1β (relative level): 1.15±0.05 vs. 1.75±0.09, both P < 0.01] and decrease LDH release (relative release: 1.09±0.01 vs. 1.44±0.04, P < 0.05). LPS induced neuronal inflammatory responses via MD2, which ultimately leads to apoptosis and necroptosis. Interfering with MD2 reduces inflammation and inhibits neuronal death.

Nomilin attenuates lipopolysaccharide-induced inflammatory response by binding with myeloid differentiation protein-2.

Nomilin shows anti-inflammatory activity by inhibiting the activation of the Toll-like receptor 4 (TLR 4)/NF-κB pathway. However, the key target of the anti-inflammatory activity of nomilin has not been elaborated and needs further exploration. This study aimed to assess the drug potential of nomilin and its ability to target myeloid differentiation protein 2 (MD-2) as a mechanism underlying the anti-inflammatory activity of nomilin on the lipopolysaccharide (LPS)-TLR4/MD-2-NF-κB signaling pathways. The methods of ForteBio and molecular docking were used to investigate the internation between MD-2 and nomilin. 3-(4,5)-Dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide experiment was performed to test the effect of nomilin on cell viability. Enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and Western blot experiments were carried out to assess the anti-inflammatory activity and possible mechanism of nomilin in vitro. The results indicated that nomilin exhibited binding affinity with MD-2. Nomilin significantly reduced the release and expression of NO, IL-6, TNF-α, and IL-1β induced by LPS in vitro. It inhibited the expression of LPS-TLR4/MD-2-NF-κB signaling pathway proteins, such as TLR4, Myd88, P65, P-P65, and iNOS. Our results suggested that nomilin had therapeutic potential and was bound to MD-2. Nomilin exhibited anti-inflammatory activity by binding to the key protein MD-2 and inhibiting the LPS-TLR4/MD-2-NF-κB signaling pathway.

Arctium lappa L. roots inhibit the intestinal inflammation of dietary obese rats through TLR4/NF-κB pathway

Long-term consumption of Arctium lappa L. roots can lead to weight loss. To explore the relationship between anti-obesity and anti-inflammation, the effects and mechanism of A. lappa L. root powder (ARP) on intestinal inflammation in obese rats were investigated. Dietary obese rats were successfully established by feeding a high-fat and high-sugar diet. The control group (n = 6) consumed a normal diet. The intestines were compared among the groups (each n = 6) with and without the administration of ARP (intragastric 7.5 g/kg·bw/d). Real-time quantitative reverse transcription-polymerase chain reaction and western blotting analysis revealed that ARP effectively inhibited the expression of pro-inflammatory and inflammatory cytokines in the colons of obese rats. These cytokines included interleukin (IL)-1β, IL-8, IL-6, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. The inhibition rates for all these cytokines exceeded 88 %. Moreover, ARP demonstrated the ability to down-regulate key genes involved in Toll-like receptor 4 (TLR4) complexes, namely Tlr4, myeloid differentiation protein-2 (Md2), and myeloid differentiation factor 88 (Myd88), along with downstream signaling molecules such as tumor necrosis factor receptor associated factor 6 (TRAF6) and nuclear factor-κB (NF-κB), with inhibition rates over 81 %. Additionally, ARP was observed to inhibit protein levels of TLR4, NF-κB, IL-1β, and TNF-α in the colons of obese rats, with inhibition rates of 65.6 ± 10.9 %, 84.4 ± 19.9 %, 80.8 ± 14.4 %, and 68.4 ± 17.5 %, respectively. This study confirmed the effectiveness of ARP in inhibiting intestinal inflammation through the blockade of the TLR4/NF-κB signaling pathway. It also suggested that ARP holds potential in improving intestinal health in the context of obesity, implying its possible application in the prevention and treatment of obesity and related metabolic diseases.

Avicularin alleviates acute liver failure by regulation of the TLR4/MyD88/NF-κB and Nrf2/HO-1/GPX4 pathways to reduce inflammation and ferroptosis.

Acute liver failure (ALF) is an inflammation-mediated hepatocyte death process associated with ferroptosis. Avicularin (AL), a Chinese herbal medicine, exerts anti-inflammatory and antioxidative effects. However, the protective effect of AL and the mechanism on ALF have not been reported. Our in vivo results suggest that AL significantly alleviated lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced hepatic pathological injury, liver enzymes, inflammatory cytokines, reactive oxygen species and iron levels and increased the antioxidant enzyme activities (malondialdehyde and glutathione). Our further in vitro experiments demonstrated that AL suppressed inflammatory response in LPS-stimulated RAW 264.7 cells via blocking the toll-like receptor 4 (TLR4)/myeloid differentiation protein-88 (MyD88)/nuclear factor kappa B (NF-κB) pathway. Moreover, AL attenuated ferroptosis in D-GalN-induced HepG2 cells by activating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1)/glutathione peroxidase 4 (GPX4) pathway. Therefore, AL can alleviate inflammatory response and ferroptosis in LPS/D-GalN-induced ALF, and its protective effects are associated with blocking TLR4/MyD88/NF-κB pathway and activating Nrf2/HO-1/GPX4 pathway. Moreover, AL is a promising therapeutic option for ALF and should be clinically explored.

Chemical Modification of Curcumin Increases Its Potency against Hypopharyngeal Carcinoma

Hypopharyngeal carcinoma is notorious for its poor prognosis among all head and neck cancers, posing a persistent challenge in clinical settings. The continuous hyperactivation of the NFκB signaling pathway has been noted in various cancer types, including hypopharyngeal carcinoma. In our quest to develop a novel drug that targets hypopharyngeal cancer via the NFκB pathway, we employed curcumin, a well-known lead compound, and performed chemical modifications to create a mono-carbonyl analog called L42H17. This compound exhibited exceptional stability and displayed an enhanced binding affinity to myeloid differentiation protein 2 (MD2). Consistent with expectations, L42H17 demonstrated the ability to inhibit TNF-α-induced phosphorylation of IKK, prevent IκB degradation, and subsequently impede NFκB-p65 nuclear translocation in hypopharyngeal cancer cells. Additionally, L42H17 exhibited a remarkable capacity to induce cell cycle arrest at the G2-M phase by inactivating the cdc2-cyclin B1 complex. Moreover, it facilitated cell apoptosis by reducing Bcl-2 levels and augmenting the expression of cle-PARP and cle-caspase3. Importantly, we observed a significant enhancement in the anti-cancer efficacy of L42H17 in a patient-derived tumor xenograft (PDTX) model of hypopharyngeal carcinoma. In conclusion, our findings strongly suggest that L42H17 holds promise as a potential candidate drug for the treatment of hypopharyngeal carcinoma in the future.

Synthesis and anti-inflammatory activity evaluation of benzoxazolederivatives as new myeloid differentiation protein 2 inhibitors.

Myeloid differentiation protein 2 (MD2), a key TLR4adaptor protein for sensing LPS, plays animportant role ininflammatoryprocessand has been identified as a promising target for the treatment of avarietyof inflammatory diseases. In our study, aseries of benzoxazolone derivatives weresynthesized, characterized and tested for anti-inflammatory activity in vitro. The compounds 3c, 3dand 3gdemonstrated the greatestanti-inflammatory activityagainst IL-6 with IC50values of 10.14±0.08, 5.43±0.51 and 5.09±0.88µM,respectively. Furthermore, the bis-ANS displacement assay revealed that these compounds competitively inhibited the binding between the probe bis-ANS and the MD2 protein. The most active compound 3g, revealed a directly bind withMD2 proteinvia Arg90binding and a dissociation constant value of 1.52×10-6mol·L-1as determined by the biological layer interference (BLI) assay. Ourfinding suggestedthat compounds3gcould bea promising lead compound as MD2 inhibitor for further anti-inflammatory agent development.

Loss of MD1 Promotes Inflammatory and Apoptotic Atrial Remodelling in Diabetic Cardiomyopathy by Activating the TLR4/NF-κB Signalling Pathway

Introduction: Myeloid differentiation protein 1 (MD1), a negative regulator of toll-like receptor 4 (TLR4), is widely expressed in the heart. Recent studies have shown that MD1 plays an important role in cardiac remodelling. However, the effects and potential mechanisms underlying MD1-mediated atrial remodelling in diabetic cardiomyopathy (DCM) remain unclear. Therefore, this study was designed to explore the role of MD1 in DCM-related atrial remodelling. Methods: MD1 knockout (MD1-KO) mice and wild-type (WT) littermates were injected with streptozotocin (STZ) to establish a diabetic mouse model. These mice were then used to evaluate MD1 expression and its effects on atrial remodelling in vivo. Results: MD1 expression was significantly decreased in STZ-induced diabetic mice. The loss of MD1 aggravated atrial fibrosis, inflammation, and apoptosis in DCM mice and promoted atrial remodelling. MD1-KO diabetic mice also showed higher susceptibility to atrial fibrillation (AF) and worse cardiac function. Mechanistically, the deletion of MD1 promoted the activation of the TLR4/NF-κB signalling pathway, resulting in atrial remodelling in DCM mice via increased p65 phosphorylation. Conclusions: The deletion of MD1 plays an important role in inflammatory and apoptotic atrial remodelling and increases susceptibility to AF in DCM mice, providing a new target for the preventive treatment of DCM-related atrial remodelling.

[Study on the anti-sepsis mechanism of ursolic acid by targeting myeloid differentiation protein-2].

To explore the mechanism of ursolic acid in treating sepsis using myeloid differentiation protein-2 (MD-2) as the research carrier. The affinity of ursolic acid and MD-2 was determined by biofilm interferometry technique, and the bonding mode between ursolic acid and MD-2 was tested with the aid of molecular docking technique. Raw 264.7 cells were cultured in RPMI 1640 medium and subcultured was conducted when the cell density reached 80%-90%. The second-generation cells were used for in the experiment. The effects of 8, 40 and 100 mg/L ursolic acid on cell viability were assessed by methyl thiazolyl tetrazolium (MTT) method. Cells were divided into blank group, lipopolysaccharide (LPS) group (LPS 100 μg/L) and ursolic acid group (100 μg/L LPS treatment after addition of 8, 40 or 100 mg/L ursolic acid). The effect of ursolic acid on the release of cytokines nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukins (IL-6, IL-1β) were evaluated by enzyme-linked immunosorbent assay (ELISA). The influence of ursolic acid on the mRNA expressions of TNF-α, IL-6, IL-1β, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected by reverse transcription-polymerase chain reaction (RT-PCR). The implication of ursolic acid on the protein expressions of LPS-Toll-like receptor 4 (TLR4)/MD-2-nuclear factor-κB (NF-κB) pathway were tested by Western blotting. Ursolic acid could bind to the hydrophobic cavity of MD-2 through hydrophobic bond with the amino acid residues of the protein. Therefore, ursolic acid showed high affinity with MD-2 [dissociation constant (KD) = 1.43×10-4]. The cell viability were decreased slightly, with the concentration of ursolic acid increasing, and the cell viability of 8, 40 and 100 mg/L ursolic acid were 96.01%, 94.32% and 92.12%, respectively, and there was no significant difference compared with the blank group (100%). Compared with the blank group, the cytokine level of the LPS group was significantly increased. The level of cytokines were significantly reduced by the treatment of 8, 40 and 100 mg/L ursolic acid, and the higher the concentration, the more obvious effect [compared between 100 mg/L ursolic acid group and LPS group: IL-1β (μmol/L): 38.018±0.675 vs. 111.324±1.262, IL-6 (μmol/L): 35.052±1.664 vs. 115.255±5.392, TNF-α (μmol/L): 39.078±2.741 vs. 119.035±4.269, NO (μmol/L): 40.885±2.372 vs. 123.405±1.291, all P < 0.01]. Compared with the blank group, the mRNA expressions of TNF-α, IL-6, IL-1β, iNOS and COX-2 in the LPS group were significantly increased, and the protein expressions of MD-2, myeloid differentiation factor 88 (MyD88), phosphorylation NF-κB p65 (p-NF-κB p65) and iNOS in the LPS-TLR4/MD-2-NF-κB pathway were significantly up-regulated. Compared with the LPS group, the mRNA expressions of TNF-α, IL-6, IL-1β, iNOS and COX-2 were significantly reduced by the treatment of 100 mg/L ursolic acid bound with MD-2 protein [TNF-α (2-ΔΔCt): 4.659±0.821 vs. 8.652±0.787, IL-6 (2-ΔΔCt): 4.296±0.802 vs. 11.132±1.615, IL-1β (2-ΔΔCt): 4.482±1.224 vs. 11.758±1.324, iNOS (2-ΔΔCt): 1.785±0.529 vs. 4.249±0.811, COX-2 (2-ΔΔCt): 5.591±1.586 vs. 16.953±1.651, all P < 0.01], and the proteins expressions of MD-2, MyD88, p-NF-κB p65 and iNOS in the LPS-TLR4/MD-2-NF-κB pathway were significantly down-regulated (MD-2/β-actin: 0.191±0.038 vs. 0.704±0.049, MyD88/β-actin: 0.470±0.042 vs. 0.875±0.058, p-NF-κB p65/β-actin: 0.178±0.012 vs. 0.571±0.012, iNOS/β-actin: 0.247±0.035 vs. 0.549±0.033, all P < 0.01). However, there was no difference in protein expression of NF-κB p65 among the three groups. Ursolic acid inhibits the release and expression of cytokines and mediators and regulates LPS-TLR4/MD-2-NF-κB signaling pathway by blocking MD-2 protein, and thus plays an anti-sepsis role.

Effects of TcpC’s NADase Activity on Inhibition of Innate Immune Signaling

Abstract The innate immune system is activated by pattern recognition receptors such as Toll-like receptors (TLRs), which differentiate between self and non-self by recognizing distinct molecular structures on pathogens. As this process threatens pathogen survival in the host, bacteria have developed mechanisms to combat this. Uropathogenic Escherichia coli (UPEC) express the Toll/Interleukin-1 Receptor (TIR) domain-containing protein TcpC, which inhibits the function of TLRs by binding to the signaling adapter protein myeloid differentiation factor 88 (MyD88). Bacterial TIR proteins, including TcpC, recently have been found to also exhibit NADase activity. We hypothesize that the ability of TcpC to block TLR signaling and its NADase function are interrelated and that the TcpC EA mutant, which has been shown to have diminished NADase activity will not bind efficiently to MyD88. We performed in vitro translation assays to first produce both WT and EA mutant versions of TcpC that contain both a streptavidin and histidine tag. Then we conducted pull-down experiments to determine if the TcpC EA mutant could successfully bind to streptavidin beads, as well as MyD88, potentially resulting in clustering with the TIR signaling protein and blocking the signaling pathway entirely. While we have successfully produced tagged WT and EA TcpC proteins and pulled down the proteins using streptavidin beads, we are working to optimize conditions to determine if the NADase-deficient TcpC EA mutant is able to bind MyD88, as has been shown previously for WT TcpC. The resulting information will give us a closer look into TcpC’s effect on virulence and the suppression of the innate immune response via TLR and MyD88 related signaling pathways. This research was partially funded by the USM LSAMP program, supported by NSF LSAMP Award #1619676, the Towson OSPR Seed Funding for Grant Seekers, and the Bridges to Doctorate Scholars Program, NIH-R25GM119970 04.

Demethyleneberberine alleviated the inflammatory response by targeting MD-2 to inhibit the TLR4 signaling.

The colitis induced by trinitrobenzenesulfonic acid (TNBS) is a chronic and systemic inflammatory disease that leads to intestinal barrier dysfunction and autoimmunedisorders. However, the existing treatments of colitis are associated with poor outcomes, and the current strategies remain deep and long-time remission and the prevention of complications. Recently, demethyleneberberine (DMB) has been reported to be a potential candidate for the treatment of inflammatory response that relied on multiple pharmacological activities, including anti-oxidation and antiinflammation. However, the target and potential mechanism of DMB in inflammatory response have not been fully elucidated. This study employed a TNBS-induced colitis model and acute sepsis mice to screen and identify the potential targets and molecular mechanisms of DMB in vitro and in vivo. The purity and structure of DMB were quantitatively analyzed by high-performance liquid chromatography (HPLC), mass spectrometry (MS), Hydrogen nuclear magnetic resonance spectroscopy (1H-NMR), and infrared spectroscopy (IR), respectively. The rats were induced by a rubber hose inserted approximately 8 cm through their anus to be injected with TNBS. Acute sepsis was induced by injection with LPS via the tail vein for 60 h. These animals with inflammation were orally administrated with DMB, berberine (BBR), or curcumin (Curc), respectively. The eukaryotic and prokaryotic expression system of myeloid differentiation protein-2 (MD-2) and its mutants were used to evaluate the target of DMB in inflammatory response. DMB had two free phenolic hydroxyl groups, and the purity exceeded 99% in HPLC. DMB alleviated colitis and suppressed the activation of TLR4 signaling in TNBS-induced colitis rats and LPS-induced RAW264.7 cells. DMB significantly blocked TLR4 signaling in both an MyD88-dependent and an MyD88-independent manner by embedding into the hydrophobic pocket of the MD-2 protein with non-covalent bonding to phenylalanine at position 76 in a pi-pi T-shaped interaction. DMB rescued mice from sepsis shock induced by LPS through targeting the TLR4-MD-2 complex. Taken together, DMB is a promising inhibitor of the MD-2 protein to suppress the hyperactivated TLR4 signaling in inflammatory response.

Alterations of the intestinal mucus layer correlate with dysbiosis and immune dysregulation in human Type 1 Diabetes

In preclinical models of Type 1 Diabetes (T1D) the integrity of the gut barrier (GB) is instrumental to avoid dysregulated crosstalk between the commensal microbiota and immune cells and to prevent autoimmunity. The GB is composed of the intestinal epithelial barrier (IEB) and of the mucus layer containing mucins and antimicrobial peptides (AMPs) that are crucial to maintain immune tolerance. In preclinical models of T1D the alterations of the GB primarily affect the mucus layer. In human T1D increased gut permeability and IEB damage have been demonstrated but the integrity of the mucus layer was never assessed. We evaluated GB integrity by measuring serological markers of IEB damage (serological levels of zonulin) and bacterial translocation such as lipopolysaccharide binding protein (LBP) and myeloid differentiation protein 2 (MD2), and mRNA expression of tight junction proteins, mucins and AMPs in intestinal tissue of T1D patients and healthy controls (HC). Simultaneously, we performed immunological profiling on intestinal tissue and 16S rRNA analysis on the mucus-associated gut microbiota (MAGM). Our data show a GB damage with mucus layer alterations and reduced mRNA expression of several mucins (MUC2, MUC12, MUC13, MUC15, MUC20, MUC21) and AMPs (HD4 and HD5) in T1D patients. Mucus layer alterations correlated with reduced relative abundance of short chain fatty acids (SCFA)-producing bacteria such as Bifidobacterium dentium, Clostridium butyricum and Roseburia intestinalis that regulate mucin expression and intestinal immune homeostasis. In T1D patients we also found intestinal immune dysregulation with higher percentages of effector T cells such as T helper (Th) 1, Th17 and TNF-α+ T cells. Our data show that mucus layer alterations are present in T1D subjects and associated with dysbiosis and immune dysregulation. Research Grants from the Juvenile Diabetes Foundation (Grant 1-INO-2018-640-A-N to MF and 2-SRA-2019-680-S-B to JD) and from the Italian Ministry of Health (Grant RF19-12370721 to MF).

Abstract CT172: A phase 1, open-label, dose-escalation study of PRT1419, a selective induced myeloid leukemia cell differentiation protein (MCL-1) inhibitor, in patients (pts) with advanced/metastatic solid tumors

Abstract Introduction: PRT1419, a selective inhibitor of MCL-1, has demonstrated preclinical activity in solid tumors and hematologic malignancies. We report the results of a phase 1, open-label, multicenter, dose-escalation study of PRT1419 in pts with advanced/metastatic solid tumors (NCT04837677). Methods: Adults with select advanced/metastatic solid tumors were enrolled. Key inclusion criteria were absolute neutrophil count &amp;gt;1.0, platelets &amp;gt;75, hemoglobin &amp;gt;9, and left ventricular ejection fraction ≥50%. Key exclusion criteria were active central nervous system malignancy, significant cardiac disease, and concurrent strong CYP2C8 inhibitors. The study employed a 3+3 dose escalation design. PRT1419 was administered as a once-weekly (QW) infusion. Primary outcome measures were to evaluate dose-limiting toxicities (DLT) and establish the maximum tolerated dose and recommended phase 2 dose (RP2D). Secondary outcome measures included safety, tolerability, pharmacokinetics, pharmacodynamics, and antitumor activity. Results: Pts with melanoma (n=8), sarcoma (n=5), esophageal (n=4), cervical (n=3), head and neck (n=3), non-small cell lung, small cell lung, and triple-negative breast cancer (all n=1) were enrolled. Median age was 59.5 (range, 32-78) years, 61.5% pts were female, and median prior lines of systemic therapy was 3 (range, 1-8). Overall, 26 pts received ≥1 dose of PRT1419 (20 mg/m2 [n=4], 40 mg/m2 [n=4], 80 mg/m2 [n=15], and 120 mg/m2 [n=3]). Most pts (84.6%) discontinued treatment (primary reason: progressive disease, 72.7%). No DLTs, adverse events (AEs) of increased troponin or heart failure, or grade 5 (fatal) AEs were observed. Any grade treatment-related AEs (TRAE) occurred in 80.8% pts; the most common were nausea (50.0%), diarrhea (46.2%), and vomiting (46.2%). Grade 3/4 TRAEs occurred in 11.5% pts; the most common was neutropenia (11.5%), which was dose related. Due to neutropenia observed at doses &amp;gt;80 mg/m2, further dose escalation was not investigated. BAX and caspase 3 activation in peripheral blood monocytes was observed at 80 mg/m2 and 120 mg/m2. Stable disease was the best response observed. Tumor shrinkage was seen in 1 pt with melanoma (10% reduction) and 1 pt with lung cancer (4% reduction) but did not meet the criteria for partial response. At Week 3, mean clearance was 12.6 L/hr and mean half-life was 2.5 hrs; no PRT1419 accumulation was seen with QW dosing. Conclusions: PRT1419 demonstrated acceptable safety and tolerability in pts with advanced/metastatic solid tumors, with primary TRAEs of neutropenia, nausea, diarrhea, and vomiting. No cardiac toxicity was observed. BAX and caspase 3 activation was observed, suggesting potentially successful MCL-1 inhibition. Investigation of PRT1419 in pts with hematologic malignancy in a separate study is ongoing (NCT05107856). Citation Format: Gerald Falchook, Manish Patel, Meredith McKean, Alexander Philipovskiy, Rachel Chiaverelli, William Sun, Michael Fossler, William Novotny, Michael Kurman, Sarah Rowe, Deborah Hunter, Mohammed Milhem. A phase 1, open-label, dose-escalation study of PRT1419, a selective induced myeloid leukemia cell differentiation protein (MCL-1) inhibitor, in patients (pts) with advanced/metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT172.

Effective anti-inflammatory phenolic compounds from dandelion: identification and mechanistic insights using UHPLC-ESI-MS/MS, fluorescence quenching and anisotropy, molecular docking and dynamics simulation

This novel study identifies the effective anti-inflammatory phenolic compounds in dandelion and provides mechanistic insights into their interactions with receptor proteins (toll‐like receptor 4, TLR4; co‐receptor myeloid differentiation protein-2, MD-2) using UHPLC-ESI-MS/MS, lipopolysaccharide (LPS)-stimulated THP-1 cell line, fluorescence quenching and anisotropy, molecular docking (single ligand and multi-ligand docking) and molecular dynamics simulation. A 50 % aqueous methanol extract had a greater anti-inflammatory effect and higher chicoric acid content, compared with the 100 % water and 100 % methanol extracts. Chicoric acid, chlorogenic acid, methylophiopogonone A, caffeic acid, gallic acid monohydrate and 4'-O-demethylbroussonin A had relatively high binding energies and contents in all extracts. Chicoric acid competed with chlorogenic acid, 4'-O-demethylbroussonin A and quercetin for MD-2. Among dandelion’s phenolics, chicoric acid most effectively hindered TLR4-MD-2 complex formation, with a quenching constant of 0.62 × 106 L/mol for MD-2 or TLR4 at 320 K, and binding energies of –6.87 and –5.97 kcal/mol, respectively, for MD-2 and TLR4.

Abstract 6147: MCL1 inhibitor PRT1419 demonstrates anti-tumor activity in PBRM1-altered clear cell renal cancer and synergizes with standard of care agents

Abstract Induced myeloid leukemia cell differentiation protein (MCL1) is a member of the B-cell lymphoma-2 (BCL2) family of apoptosis regulators, which plays a critical role in maintaining cellular homeostasis and promoting cancer cell survival. Increased expression of MCL1 in various cancers has been associated with poor prognosis and resistance to chemotherapeutic and targeted agents. We previously described PRT1419, a novel, potent, selective MCL1 inhibitor that demonstrates anti-tumor efficacy in various preclinical models of solid and hematologic malignancies. PRT1419 is currently under evaluation in Phase I clinical trials in patients with relapsed/refractory hematologic malignancies and advanced solid tumors. To identify novel biomarkers that might predict sensitivity to MCL1 inhibition, we conducted a gene essentiality analysis using publicly available human cancer cell line gene dependency data generated from genome-wide CRISPR/Cas9 cell viability screens. We observed that clear cell renal cancer (ccRCC) cell lines with deleterious alterations in PBRM1 (Polybromo 1) displayed a strong dependency on MCL1. PBRM1, also known as BAF180, is a chromatin-targeting subunit of mammalian pBAF (SWI/SNF-B) complexes. PBRM1 is frequently altered in various human cancers but it has a particularly high alteration rate in ccRCC with ~40% of tumors harboring damaging PBRM1 alterations. We had previously described alterations in other mammalian SWI/SNF factors as biomarkers of MCL1 inhibitor sensitivity.We observed potent inhibition of tumor growth as well as induction of apoptosis by PRT1419 in various preclinical models of PBRM1-mutant ccRCC but not in PBRM1-WT tumor models. Depletion of PBRM1 via RNAi in PBRM1-WT ccRCC induced sensitivity to PRT1419. Mechanistically, PBRM1 depletion coincided with increased expression of pro-apoptotic factors, priming PBRM1-loss cells for caspase-mediated cell death following MCL1 inhibition. Increased MCL1 activity has previously been described as a resistance mechanism to Sunitinib and Everolimus, two approved targeted agents for ccRCC. To investigate if MCL1 inhibition could potentiate the anti-tumor effects of these agents, we evaluated PRT1419 in combination with Sunitinib or Everolimus in PBRM1-loss ccRCC. PRT1419 synergized with both Sunitinib and Everolimus in inhibiting tumor growth in various models. Taken together, these findings suggest PBRM1 loss is associated with sensitivity to MCL1 inhibition in ccRCC and provide rationale for the evaluation of PRT1419 for the treatment for PBRM1-deficient ccRCC Citation Format: Norman Fultang, Brian Vidal, Ashley M. Schwab, Alexander Grego, Diane Heiser, Kris Vaddi, Neha Bhagwat, Peggy Scherle. MCL1 inhibitor PRT1419 demonstrates anti-tumor activity in PBRM1-altered clear cell renal cancer and synergizes with standard of care agents. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6147.