Synergistic Inhibitory Effect Research Articles

Phytol and α-Bisabolol Synergy Induces Autophagy and Apoptosis in A549 Cells and Additional Molecular Insights through Comprehensive Proteome Analysis via Nano LC-MS/MS.

Non-Small Cell Lung Cancer (NSCLC) is a malignancy with a significant prevalence and aggressive nature, posing a considerable challenge in terms of therapeutic interventions. Autophagy and apoptosis, two intricate cellular processes, are integral to NSCLC pathophysiology, each affecting the other through shared signaling pathways. Phytol (Phy) and α-bisabolol (Bis) have shown promise as potential anticancer agents individually, but their combined effects in NSCLC have not been extensively investigated. The present study was to examine the synergistic impact of Phy and Bis on NSCLC cells, particularly in the context of autophagy modulation, and to elucidate the resulting differential protein expression using LCMS/ MS analysis. The A549 cell lines were subjected to the patented effective concentration of Phy and Bis, and subsequently, the viability of the cells was evaluated utilizing the MTT assay. The present study utilized real-time PCR analysis to assess the expression levels of crucial apoptotic genes, specifically Bcl-2, Bax, and Caspase-9, as well as autophagy-related genes, including Beclin-1, SQSTM1, Ulk1, and LC3B. The confirmation of autophagy marker expression (Beclin-1, LC3B) and the autophagy-regulating protein SQSTM1 was achieved through the utilization of Western blot analysis. Differentially expressed proteins were found using LC-MS/MS analysis. The combination of Phy and Bis demonstrated significant inhibition of NSCLC cell growth, indicating their synergistic effect. Real-time PCR analysis revealed a shift towards apoptosis, with downregulation of Bcl-2 and upregulation of Bax and Caspase-9, suggesting a shift towards apoptosis. Genes associated with autophagy regulation, including Beclin-1, SQSTM1 (p62), Ulk1, and LC3B, showed significant upregulation, indicating potential induction of autophagy. Western blot analysis confirmed increased expression of autophagy markers, such as Beclin-1 and LC3B, while the autophagy-regulating protein SQSTM1 exhibited a significant decrease. LC-MS/MS analysis revealed differential expression of 861 proteins, reflecting the modulation of cellular processes. Protein-protein interaction network analysis highlighted key proteins involved in apoptotic and autophagic pathways, including STOML2, YWHAB, POX2, B2M, CDA, CAPN2, TXN, ECHS1, PEBP1, PFN1, CDC42, TUBB1, HSPB1, PXN, FGF2, and BAG3, emphasizing their crucial roles. Additionally, PANTHER pathway analysis uncovered enriched pathways associated with the differentially expressed proteins, revealing their involvement in a diverse range of biological processes, encompassing cell signaling, metabolism, and cellular stress responses. The combined treatment of Phy and Bis exerts a synergistic inhibitory effect on NSCLC cell growth, mediated through the interplay of apoptosis and autophagy. The differential protein expression observed, along with the identified proteins and enriched pathways, provides valuable insights into the underlying molecular mechanisms. These findings offer a foundation for further exploration of the therapeutic potential of Phy and Bis in the management of NSCLC.

Determination of cinnamaldehyde, thymol and eugenol in essential oils by LC–MS/MS and antibacterial activity of them against bacteria

Plant essential oils contain many secondary metabolites, some of which can effectively inhibit the growth of pathogenic microorganisms, so it is a very promising antibacterial agent. In this study, a qualitative and quantitative method based on high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) was developed for the simultaneous determination of three bioactive substances, cinnamaldehyde (CNM), thymol (THY), and eugenol (EUG), in the essential oils of plants. Necessary tests for linearity, limit of quantification, recovery, carryover contamination and precision of the method were carried out. Then, the antibacterial activity of 3 bioactive compounds against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was evaluated by minimal inhibitory concentration and the synergistic antimicrobial effect. The results indicated that CNM, THY and EUG had good antibacterial activity. According to the results of fractional inhibitory concentration index (FICI), it is considered that CNM + THY and CNM + THY + EUG has obvious synergistic inhibitory effect on E. coli, and CNM + THY and CNM + EUG has obvious synergistic inhibitory effect on S. aureus. Finally, we analyzed the effect of the bioactive compounds on trace elements in bacteria and found significant changes in magnesium, calcium, copper and iron.

Synergistic corrosion inhibition of rubber seed extract with KI on cold rolled steel in sulfuric acid solution

BackgroundPlant-based inhibitor has emerged an important topic owing to the combing merits of eco-friendly, low cost, rich natural resources as well as easy preparation. Rubber seed is the agricultural waste, and is mainly composed of oil and fatty acids those contain many polar oxygen atoms, and so it can be extracted to prepare the potential corrosion inhibitor. MethodsRubber seed extract (RSE) is prepared using reflux extraction method with the extracting solvent of ethanol aqueous solution. The synergistic inhibition effect of RSE and potassium iodide (KI) on the corrosion of cold rolled steel (CRS) in 0.50 M H2SO4 solution is firstly studied by weight loss, potentiodynamic polarization (PDP) curves and electrochemical impedance spectroscopy (EIS) methods. CRS surface was thoroughly characterized using metallographic microscope, scanning electron microscope (SEM), atomic force microscope (AFM), X-ray photoelectron spectroscopy (XPS) and contact angle measurements. FTIR, UV–vis and liquid chromatography mass spectrometry (LC-MS) compositional analysis of RSE were used to analyze the efficient components in RSE. Significant findingsThe results indicate that individual RSE or KI cannot function as a successful inhibitor to prevent CRS corrosion, but the mixture of RSE/KI performs better at inhibiting, with a maximal inhibition efficacy of up to 96.4% of 200 mg L−1 RSE/100 mg L−1 KI. The synergistic coefficient of the mixture is larger than 1, indicating that a true synergism. RSE/KI can drastically retard both cathodic and anodic processes, and interfacial capacitance falls as the charge transfer resistance of CRS/solution rises. It exhibits remarkable inhibition properties when the capacitor arc grows. There is only a small corrosion response in the inhibited media, as evidenced by the variations in electrical conductivity and ultraviolet spectrum before and after immersion. The hydrophilicity and surface roughness of CRS surface is dropped as a result of the introduction of RSE/KI. A series of metallurgical microscopy, AFM, SEM or contact angle tests indicate a synergistic inhibition effect is more pronounced for RSE/KI. XPS results clearly demonstrate that elements including Fe, S, N, and I are bonded to the surface of CRS. The primary ingredients of oleic acid, polyphenols, phenylpropanoids can also produce a strong synergism with KI.

Inhibition of endocytic uptake of severe acute respiratory syndrome coronavirus 2 and endo-lysosomal acidification by diphenoxylate.

Cell culture-based screening of a chemical library identified diphenoxylate as an antiviral agent against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The observed 50% effective concentrations ranged between 1.4 and 4.9 µM against the original wild-type strain and its variants. Time-of-addition experiments indicated that diphenoxylate is an entry blocker targeting a host factor involved in viral infection. Fluorescence microscopic analysis visualized that diphenoxylate prevented SARS-CoV-2 particles from penetrating the cell membrane and also impaired endo-lysosomal acidification. Diphenoxylate exhibited a synergistic inhibitory effect on SARS-CoV-2 infection in human lung epithelial Calu-3 cells when combined with a transmembrane serine protease 2 (TMPRSS2) inhibitor, nafamostat. This synergy suggested that efficient antiviral activity is achieved by blocking both TMPRSS2-mediated early and endosome-mediated late SARS-CoV-2 entry pathways. The antiviral efficacy of diphenoxylate against SARS-CoV-2 was reproducible in a human tonsil organoids system. In a transgenic mouse model expressing the obligate SARS-CoV-2 receptor, human angiotensin-converting enzyme 2, intranasal administration of diphenoxylate (10 mg/kg/day) significantly reduced the viral RNA copy number in the lungs by 70% on day 3. This study underscores that diphenoxylate represents a promising core scaffold, warranting further exploration for chemical modifications aimed at developing a new class of clinically effective antiviral drugs against SARS-CoV-2.

Iron promotes ovarian cancer malignancy and advances platinum resistance by enhancing DNA repair via FTH1/FTL/POLQ/RAD51 axis

Iron is crucial for cell DNA synthesis and repair, but an excess of free iron can lead to oxidative stress and subsequent cell death. Although several studies suggest that cancer cells display characteristics of ‘Iron addiction’, an ongoing debate surrounds the question of whether iron can influence the malignant properties of ovarian cancer. In the current study, we initially found iron levels increase during spheroid formation. Furthermore, iron supplementation can promote cancer cell survival, cancer spheroid growth, and migration; vice versa, iron chelators inhibit this process. Notably, iron reduces the sensitivity of ovarian cancer cells to platinum as well. Mechanistically, iron downregulates DNA homologous recombination (HR) inhibitor polymerase theta (POLQ) and relieves its antagonism against the HR repair enzyme RAD51, thereby promoting DNA damage repair to resist chemotherapy-induced damage. Additionally, iron tightly regulated by ferritin (FTH1/FTL) which is indispensable for iron-triggered DNA repair. Finally, we discovered that iron chelators combined with platinum exhibit a synergistic inhibitory effect on ovarian cancer in vitro and in vivo. Our findings affirm the pro-cancer role of iron in ovarian cancer and reveal that iron advances platinum resistance by promoting DNA damage repair through FTH1/FTL/POLQ/RAD51 pathway. Our findings highlight the significance of iron depletion therapy, revealing a promising avenue for advancing ovarian cancer treatment.

Synthesis and synergistic effects of antibacterial and corrosion inhibition based on concentrated hydrosols of silver nanoparticles

A highly water-soluble and uniformly dispersed concentrate of silver nanoparticles (Ag NPs) hydrosols were synthesized using a chemical reduction method. The Ag NPs hydrosols exhibited remarkable antibacterial activity against sulfate-reducing bacteria (SRB) and can serve as a biocide for anti-corrosion purposes. The addition of 50 ppm Ag NPs hydrosols reduced the corrosion rate by over 91 % in the presence of SRB biofilm compared to untreated conditions. Numerous Ag NPs adsorbed onto the cell wall, with some internalizing and entering the cell, resulting in cytoplasmic efflux and DNA damage, ultimately leading to cell death.

Inhibition effect of inert gas jet on gas and hybrid explosions caused by thermal runaway of lithium-ion battery

When lithium-ion batteries undergo thermal runaway in a confined space, the released gases and/or vapors or dust particles can lead to an explosion. An 8L cylindrical experimental vessel was constructed to study the inert effect of inert gases on battery-vented gas (BVG) in a confined vessel. Firstly, the inhibition effect and inert mechanism of CO2 and N2 on BVG explosion were analyzed by experiments and simulation software. The results show that the inert effect of CO2 on the BVG is significantly better than that of N2. This is because CO2 is not only better than N2 in physical explosion inhibition, but also participates in the chemical reaction. Secondly, the effect of the release of high-pressure CO2 gas on the explosion of the BVG in a closed container was simulated, showing that when the BVG is near the lower explosive limit, the CO2 jet will significantly increase the rate of explosion pressure rise of the BVG. This suggests that the inhibition effect of the CO2 jet is not as good as in premixed conditions. Finally, it is found that the inhibition effect of CO2 on the hybrid explosion is better than that of CO2 jet on the BVG. Since CO2 makes oxygen insufficient, leading to a reduction in the amount of graphite dust burned. The graphite dust and CO2 have a synergistic inhibition effect on the hybrid explosion. The above findings can provide a reference for the lithium-ion battery explosion inhibition method in the process industry.

Abstract PO5-25-05: Crosstalk between adipocyte and tumor cells with ESR1 mutations via adipsin enhances tumor growth

Abstract While endocrine therapies have shown clinical benefits for breast cancer with ESR1 mutations, resistance development is a significant challenge. Evidence suggests that the complement system is critical to tumor development, and recent studies highlight the role of adipsin in breast cancer growth. In a previous study, we characterized the role of complement pathway-mediated adipsin in ESR1 mutant cell lines (Y537S and D538G), identifying the upregulation of adipsin, C3, and C3aR in ESR1 mutant cells. The interaction between C3a and C3aR enhanced ESR1 mutant cell growth, and inhibiting C3aR abolished cell growth and increased apoptosis. Combining C3aR inhibitors with fulvestrant or CDK4/6 inhibitors had a synergistic inhibitory effect on ESR1 mutant cells. Our findings suggest that the complement pathway plays a role in ESR1 mutant cell proliferation and that C3aR is a potential target to mitigate ESR1 mutant breast cancer growth and metastasis. Additionally, we demonstrated the critical role of the feedback loop of adipsin in ESR1 mutant cell growth in adipocyte crosstalk. Using differentiated mouse 3T3-L1 adipocytes induced with tumor-conditioned media from ESR1 mutant cells, we observed the upregulation of adipsin, C3, and C3a in the adipocyte secretome, which enhanced C3a levels. Blocking the interaction between C3a and C3aR in ESR1 mutant cells using a C3aR inhibitor significantly inhibited ESR1 mutant cell growth compared to wild-type cells and enhanced apoptosis. Our findings suggest that upregulation of adipsin from ESR1 mutant cells and adipocytes in crosstalk increases tumor cell growth. Future studies will investigate the critical role of adipsin's auto- and paracrine effects in breast cancer with ESR1 mutations. Citation Format: Artem Belyakov, Kideok Jin. Crosstalk between adipocyte and tumor cells with ESR1 mutations via adipsin enhances tumor growth [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-25-05.

Filamentation and inhibition of prokaryotic CTP synthase with ligands

AbstractCytidine triphosphate synthase (CTPS) plays a pivotal role in the de novo synthesis of cytidine triphosphate (CTP), a fundamental building block for RNA and DNA that is essential for life. CTPS is capable of directly binding to all four nucleotide triphosphates: adenine triphosphate, uridine triphosphate, CTP, and guanidine triphosphate. Furthermore, CTPS can form cytoophidia in vivo and metabolic filaments in vitro, undergoing regulation at multiple levels. CTPS is considered a potential therapeutic target for combating invasions or infections by viral or prokaryotic pathogens. Utilizing cryo‐electron microscopy, we determined the structure of Escherichia coli CTPS (ecCTPS) filament in complex with CTP, nicotinamide adenine dinucleotide (NADH), and the covalent inhibitor 6‐diazo‐5‐oxo‐ l‐norleucine (DON), achieving a resolution of 2.9 Å. We constructed a phylogenetic tree based on differences in filament‐forming interfaces and designed a variant to validate our hypothesis, providing an evolutionary perspective on CTPS filament formation. Our computational analysis revealed a solvent‐accessible ammonia tunnel upon DON binding. Through comparative structural analysis, we discern a distinct mode of CTP binding of ecCTPS that differs from eukaryotic counterparts. Combining biochemical assays and structural analysis, we determined and validated the synergistic inhibitory effects of CTP with NADH or adenine on CTPS. Our results expand our comprehension of the diverse regulatory aspects of CTPS and lay a foundation for the design of specific inhibitors targeting prokaryotic CTPS.

The combination of ciprofloxacin and dialkyldimethyl ammonium compound synergistically proliferated intracellular resistance genes in nitrifying system

Antibiotics and quaternary ammonium compounds (QACs) usually co-exist in wastewater treatment plants. Hence, three sequencing batch reactors were established and named as R1, R2 and R3, to investigate the effects of individual and combined exposure of different concentrations of ciprofloxacin (CIP) (0.2, 1.0 and 2.0 mg/L) and dialkyldimethyl ammonium compound (DADMAC) (0.4, 2.0 and 4.0 mg/L) on the performance, microbial community structures and resistance genes (RGs) in nitrifying system during 150 days. Results showed that CIP had a slight effect on ammonia oxidation activity, while 2.0 and 4.0 mg/L DADAMAC could obviously inhibit it, and the combination of CIP and DADMAC had a synergistic inhibitory effect. Besides, both CIP and DADMAC caused partial nitrification, and the order of nitrite accumulation rate was ranked as R3 > R2 > R1. The combination of CIP and DADMAC had an antagonistic effect on the increase of sludge particle size and α-Helix/(β-Sheet + Random coil) was lowest in R3 (0.40). The combination of CIP and DADMAC synergistically stimulated most intracellular RGs in sludge, and the relative abundances of target RGs (e.g., qacEdelta1-01, qacH-01 and qnrS) at the end of operation in R3 were increased by 4.61–18.19 folds compared with those in CK, which were 1.34–5.57 folds higher than the R1 and R2. Moreover, the combination of CIP and DADMAC also promoted the transfer of RGs from sludge to water and enriched more potential hosts of RGs, further promoting the spread of RGs in nitrifying system. Thus, the combined pollution of CIP and DADMAC in wastewaters should attract more attentions.

Inhibition of Helicobacter pylori by Lactobacillus rhamnosus L08 in combination with Curcuma longa extracts

Helicobacter pylori (H. pylori) resistance due to antibiotics is becoming increasingly prevalent, and the search for healthy alternatives to drugs has become a popular research topic. Curcuma longa and Lactobacillus rhamnosus are not only inhibitory to H. pylori but also have good compatibility. It was found that acid-tolerant L. rhamnosus had a significant coaggregation effect on H. pylori, but the inhibitory effect was weak. The polyphenol or flavonoid content of Curcuma extract was significantly correlated with the antimicrobial effect, but the inhibition time would be shortened due to its decomposition or destabilization in the digestive system. Both were able to synergistically inhibit H. pylori adhesion and expression of certain genes (SabA, UreE, UreI, and UreG), with no significant synergistic effect in inhibiting urease. The combination of Curcuma extract with L. rhamnosus enhanced the inhibitory effect, solved the problem of drug resistance, and improved the short duration of inhibition of Curcuma extract. This study aimed to combine the antimicrobial effect of Curcuma extract with the function of L. rhamnosus in regulating the gastrointestinal flora and to investigate the synergistic inhibitory effect of the two on H. pylori. The findings provide a theoretical basis for subsequent cellular, animal, or clinical experiments.

The influence of cyclodextrin on hydrophobicity of pipeline and asphalt distribution: A green and efficient corrosion inhibitor

Corrosion inhibitors face significant limitations due to their non-degradability and poor applicability to asphalt systems. The molecular simulation studies show that cyclodextrin (CD) and asphalt can form supramolecular corrosion inhibitors with pipeline hydrophobic modification and corrosion inhibition effects. In particular, the rich hydroxyl structure of CD enables it to stably bind to the pipeline by forming coordination bonds and feedback bonds. When 2 CD molecules are added, the initial wetting time of the emulsion is delayed by 257.81 % compared to Span 80 with the same mass fraction. When 4 CD molecules are added, the emulsion cannot wet the iron surface within the simulation time. Furthermore, the combination of CD and asphalt allows more than 90 % of the asphalt to emulsify and prevent the emulsion from spreading on the pipeline. This results in a synergistic corrosion inhibition effect. These findings suggest that CD-based supramolecular corrosion inhibitors have the potential to be effective and environmentally friendly alternatives to traditional inhibitors in asphalt systems.

Ultrasound-Assisted Extraction of Atractylodes chinensis (DC.) Koidz. Polysaccharides and the Synergistic Antigastric Cancer Effect in Combination with Oxaliplatin.

Oxaliplatin (OXA) is recognized as a first-line drug for gastric cancer. However, low accumulation of the OXA in the target site and the development of drug resistance directly led to treatment failure. In the present study, an ultrasonic extraction method for Atractylodes chinensis (DC.) Koidz. polysaccharides (AKUs) and the combination treatment with OXA in vitro were studied. Results showed that when the pH level was 11, the ultrasound power at 450 W, the solid-liquid ratio was 1:20, and the ultrasound treatment for 30 min, the yield of AKUs was significantly increased to 13.20 ± 0.35%. The molecular weights of the AKUs ranged from 7.21 to 185.94 kDa, and its monosaccharides were mainly composed of arabinose (Ara), galactose (Gal), and glucose (Glu) with a ratio of 58.36, 16.90, and 15.49%, respectively. Cell experiments showed that, compared to OXA alone (2 μg/mL, inhibition rate of 18%), the treatment of OXA with AKUs had a significant synergistic inhibitory effect on MKN45 proliferation, which increased to 33, 41, and 45% with increasing AKUs concentrations (5-50 μg/mL), respectively, representing a 2.5-fold inhibition. Inductively coupled plasma-mass spectrometry (ICP-MS) determination confirmed that AKUs significantly increased the intracellular uptake of OXA by 29%, compared to that of OXA alone. We first demonstrated that the combined synergistic inhibitory effect of AKUs and OXA on gastric cancer cells was mediated by reducing the expression of efflux proteins (MRP1 and MRP2) and increasing the expression of ingested protein (OCT2). As a result of the above, AKUs deserved to be an effective adjuvant combined with chemotherapeutics in a clinical setting.

Aquatic life criteria of hydrothermal liquefaction wastewater via ecotoxicity test and modeling

Wastewater resulting from hydrothermal liquefaction (HTL-AP) of biowaste is gaining attention as an emerging hazardous material. However, there is a lack of specific and systematic ecotoxicity studies on HTL-AP. This study addresses this gap by conducting acute toxicity tests on HTL-AP using typical aquatic species and integrating these results with predicted toxicity values from interspecies correlation estimation models to establish aquatic life criteria. HTL-AP exhibited significant toxicity with LC50 of 956.12–3645.4 mg/L, but demonstrated moderate toxicity compared to common freshwater pollutants like commercial microbicides, personal care products, and insect repellents. The resulting hazardous concentration for 5 % of species (HC5), the criterion maximum concentration, and the short-term water quality criteria for aquatic were 506.0, 253.0, and 168.7 mg/L, respectively. Notably, certain organisms like Misgurnus anguillicaudatus and Cipangopaludina chinensis showed high tolerance to HTL-AP, likely due to their metabolic capabilities on HTL-AP components. The significant decrease in HC5 values for some HTL-AP substances compared to pure compounds could indicate the synergistic inhibition effects among HTL-AP compositions. Furthermore, according to the established criteria, HTL-AP required significantly less diluted water (13 t) than carbendazim (1009 t) to achieve biosafety, indicating a safer release. This research establishes a preliminary water quality criterion for HTL-AP, offering a valuable reference for risk assessment and prediction in the utilization of HTL-AP within environmental contexts.

Characterization and application of in situ curcumin/ZNP hydrogels for periodontitis treatment

BackgroundPeriodontitis is a chronic inflammatory disease that occurs in tooth-supporting tissues. Controlling inflammation and alleviating periodontal tissue destruction are key factors in periodontal therapy. This study aimed to develop an in situ curcumin/zinc oxide (Cur/ZNP) hydrogel and investigate its characteristics and effectiveness in the treatment of periodontitis.MethodsAntibacterial activity and cytotoxicity assays were performed in vitro. To evaluate the effect of the in situ Cur/ZNP hydrogel on periodontitis in vivo, an experimental periodontitis model was established in Sprague‒Dawley rats via silk ligature and inoculation of the maxillary first molar with Porphyromonas gingivalis. After one month of in situ treatment with the hydrogel, we examined the transcriptional responses of the gingiva to the Cur/ZNP hydrogel treatment and detected the alveolar bone level as well as the expression of osteocalcin (OCN) and osteoprotegerin (OPG) in the periodontal tissues of the rats.ResultsCur/ZNPs had synergistic inhibitory effects on P. gingivalis and good biocompatibility. RNA sequencing of the gingiva showed that immune effector process-related genes were significantly induced by experimental periodontitis. Carcinoembryonic antigen-related cell adhesion molecule 1 (Ceacam1), which is involved in the negative regulation of bone resorption, was differentially regulated by the Cur/ZNP hydrogel but not by the Cur hydrogel or ZNP hydrogel. The Cur/ZNP hydrogel also had a stronger protective effect on alveolar bone resorption than both the Cur hydrogel and the ZNP hydrogel.ConclusionThe Cur/ZNP hydrogel effectively inhibited periodontal pathogenic bacteria and alleviated alveolar bone destruction while exhibiting favorable biocompatibility.

Palbociclib inhibits the progression of head and neck cancer and improves the Cetuximab response under specific condition in vitro and in vivo.

In head and neck squamous cell carcinoma (HNSCC) with human papillomavirus (HPV)-negative, deregulation of cell cycle is partly due to inactivation of p16INK4 and overexpression of cyclin D1. Here we investigated the in vitro and in vivo effects of the CDK4/6 inhibitor Palbociclib alone or combined with EGFR inhibitor Cetuximab in HNSCC. CCK-8, soft agar assay, colony formation assay, wound healing assay and transwell assay, β-galactosidase assay, western blotting, and cell-derived xenografts were used to investigated the in vitro and in vivo activity of drugs. Cell viability and colony formation decreased in a dose-dependent manner in Tu686, AMC-HN8, and Fadu cells under Palbociclib treatment. Palbociclib remarkably inhibited migration, invasion and the expression MMP-9 in HNSCC cells. Palbociclib also induced senescence. Palbociclib caused the dephosphorylation of RB but increased the cyclin D1 level in a dose-dependent manner. Moreover, combination with Cetuximab could significantly prevent the induction of cyclin D1 and activation of EGFR signals from Palbociclib treatment. Nevertheless, only within the range of certain concentrations, a synergistic inhibitory effect on cell growth was observed when combined with Palbociclib and Cetuximab. Although the synergistic effect in Palbociclib/Cetuximab combined therapy was comparable to that in traditional chemotherapeutic regimens (cisplatin/Cetuximab) in Fadu tumor xenograft, the combination therapy was less active than Cetuximab monotherapy in Tu686 tumor xenograft. In HPV-negative HNSCC, CDK4/6 inhibitor shows promising anti-tumor effects, which exhibits a synergistic effect when combined with EGFR inhibitor only in specific drug concentration and mouse model.

Harmony in detoxification: Microalgae unleashing the potential of lignocellulosic pretreatment wastewater for resource utilization

Lignocellulosic biomass is a pivotal renewable resource in biorefinery process, requiring pretreatment, primarily chemical pretreatment, for effective depolymerization and subsequent transformation. This process yields solid residue for saccharification and lignocellulosic pretreatment wastewater (LPW), which comprises sugars and inhibitors such as phenols and furans. This study explored the microalgal capacity to treat LPW, focusing on two key hydrolysate inhibitors: furfural and vanillin, which impact the growth of six green microalgae. Chlorella sorokiniana exhibited higher tolerance to furfural and vanillin. However, both inhibitors hindered the growth of C. sorokiniana and disrupted algal photosynthetic system, with vanillin displaying superior inhibition. A synergistic inhibitory effect (Q < 0.85) was observed with furfural and vanillin on algal growth. Furfural transformation to low-toxic furfuryl alcohol was rapid, yet the addition of vanillin hindered this process. Vanillin stimulated carbohydrate accumulation, with 50.48 % observed in the 0.1 g/L furfural + 0.1 g/L vanillin group. Additionally, vanillin enhanced the accumulation of C16: 0 and C18: 2, reaching 21.71 % and 40.36 %, respectively, with 0.1 g/L vanillin. This study proposed a microalgae-based detoxification and resource utilization approach for LPW, enhancing the comprehensive utilization of lignocellulosic components. The observed biomass modifications also suggested potential applications for biofuel production, contributing to the evolving landscape of sustainable biorefinery processes.

Abstract 1359: Rewiring CD8+T cell responses to PD-1 immune checkpoint blockade in PDAC by preventing terminal exhaustion via the inhibitory receptor PSGL-1

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, poorly immunogenic cancer with increasing incidence and a poor 5-year survival rate of 12%. PDAC tumors are refractory to all currently available treatments, including immune checkpoint blockade (ICB) therapies, and demonstrate limited infiltration of cytotoxic CD8+ T cells that kill tumor cells. There is therefore an urgent, unmet need for novel therapeutic options. Analysis of single-cell RNA-sequencing data of patient PDAC revealed high expression of the novel immune checkpoint, PSGL-1, on infiltrating CD8+ T cells. We hypothesize that PSGL-1-mediated immune suppression is a critical barrier to effective anti-tumor immunity in PDAC. To test this, we utilized a preclinical model of PDAC where orthotopic injection of the KPC.4662 tumor cell line into pancreata recapitulates patient tumor responses with limited T cell infiltrates and uncontrolled tumor growth in C57BL/6 (wildtype) control mice and compared to PSGL-1KO (C57BL/6 background) mice. At endpoint, tumor burden in PSGL-1KO mice was reduced by &amp;gt;50% compared to controls, with 45% of PSGL-1KO animals exhibiting &amp;gt;70% reduction. Total immune infiltration (CD45+) was 2-fold greater in tumors from PSGL-1KO mice, with a significant increase in infiltrating CD3+ T cells. Detailed analyses revealed that infiltrating CD8+ T cells from PSGL-1KO mice were not only more abundant, but also less differentiated towards terminal exhaustion with sustained TCT-1 expression compared to controls. Moreover, PSGL-1KO mice showed protection against metastatic disease. Since our data support a critical role for T cell driven anti-tumor immunity to PDAC, we transiently depleted CD4+ T cells, CD8+ T cells, or both just prior to tumor cell implantation. While depletion of CD8+ T cells did not impede tumor control by PSGL-1KO mice, depletion of all T cells or only CD4+ T cells resulted in the loss of PDAC tumor growth inhibition, demonstrating a key role of CD4+ cells in the response. Given our previous studies demonstrating an intrinsic role for PSGL-1 in development of terminal T cell exhaustion in melanoma, including loss of TCF-1+ CD8+ T cells, we hypothesized that PSGL-1-deficiency would promote responsiveness to PD-1 ICB, which is ineffective as a monotherapy in PDAC patients and mouse models. To test this prediction, PD-1 ICB was therapeutically administered to control and PSGL-1KO mice. While treatment with anti-PD-1 had no effect on the tumor growth in the control animals, PD-1 ICB resulted in complete tumor ablation in 85% of PSGL-1KO animals, demonstrating a profound synergistic effect of PD-1 inhibition in the absence of PSGL-1. From these studies, we conclude that PSGL-1 is a critical inhibitor of T cell anti-tumor responses in PDAC. PSGL-1 therefore represents a novel ICB target with high translational potential for promoting immune responses to PDAC tumors. Citation Format: Jennifer L. Hope, Yijuan Zhang, Hannah A. Hetrick, Gabriele Romano, Sreeja Roy, Michelle Lin, Ashley B. Palete, Swetha Maganti, Dennis C. Otero, Cosimo Commisso, Linda M. Bradley. Rewiring CD8+T cell responses to PD-1 immune checkpoint blockade in PDAC by preventing terminal exhaustion via the inhibitory receptor PSGL-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1359.

Abstract 226: Effect of MCL-1 inhibitor on organoids derived from cholangiocarcinoma patients with IDH1 mutation

Abstract Cholangiocarcinomas are highly aggressive malignancies with a poor prognosis. The global incidence of cholangiocarcinoma patients appears to be on the rise. While surgical resection remains the curative option for cholangiocarcinoma patients, the majority of cases are diagnosed at an advanced stage, resulting in poor outcomes. Patients with inoperable cholangiocarcinomas typically undergo chemotherapy regimens, including Gemcitabine, but their efficacy is limited, leading to low 5-year survival rates. Recent advancements in organoid culture technology allow for the in vitro replication of tissue structures by culturing tissue stem cells in three dimensions, offering great promise for personalized medicine. Utilizing patient-derived organoids has proven effective in predicting the outcomes of therapeutic drugs. We have successfully established organoids using cancer tissue derived from cholangiocarcinoma patients, and stably culturing and maintaining them, promoting their application to personalized medicine (Saito Y et al. Cell Rep. 27, 1265, 2019, Saito Y et al. Cancer Cell 40, 226, 2022). IDH1 mutations have been identified in approximately 15% of intrahepatic cholangiocarcinomas and are known to convert α-ketoglutarate (α-KG) in the TCA cycle to produce 2-hydroxyglutarate (2-HG), which is observed in gliomas and acute myeloid leukemia. 2-HG acts as an antagonist to α-KG-dependent dioxygenases, promoting cancer development. This study aims to develop a novel treatment for cholangiocarcinomas with IDH1 mutations. Metabolomic analysis of patient-derived cholangiocarcinoma organoids revealed 2-HG accumulation and reduced ATP production in IDH1-mutated cholangiocarcinoma organoids compared to IDH1 wild-type cholangiocarcinoma organoids. This may suppress the protein synthesis of MCL-1 and mTOR signaling through the activation of AMPK kinase, suggesting that further inhibition of MCL-1 could be lethal in IDH1-mutated cholangiocarcinoma organoids. Indeed, exposure to S63845, an MCL-1 inhibitor, significantly inhibited cell proliferation in IDH1-mutated cholangiocarcinoma organoids, unlike in IDH1 wild-type cholangiocarcinoma organoids. Furthermore, the combination of MCL-1 inhibitor and BCL-XL inhibitor demonstrated a synergistic and potent growth inhibitory effect on cholangiocarcinoma organoids. These findings indicate that the MCL-1 inhibitor holds promise as a new therapeutic agent for cholangiocarcinoma patients with IDH1 mutations. Citation Format: Shiho Suzuki, Juntaro Matsuzaki, Toshihide Muramatsu, Yae Kanai, Yoshimasa Saito. Effect of MCL-1 inhibitor on organoids derived from cholangiocarcinoma patients with IDH1 mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 226.

Abstract 7127: An ATM inhibitor: ZN-B-2262 in combination with radiation/ADCs containing topoisomerase inhibitors for the treatment of solid tumors

Abstract Background: ZN-B-2262 is a best-in-class, IND-approved, small molecule serine/threonine kinase receptor inhibitor targeting Ataxia Telangiectasia Mutated (ATM), which plays a key role in DNA damage response (DDR) associated with double-strand breaks (DSB). Radiotherapy (RT) and topoisomerase inhibition can directly cause DSB. ZN-B-2262 is expected to enhance the therapeutic effect of RT, or antibody drug conjugates (ADCs) containing topoisomerase inhibitors by suppressing DSB DNA repair and blocking checkpoint controls. Methods: The inhibition of isolated recombinant ATM, ATR, mTOR, DNA-PK, PI3Kα, PI3Kβ, PI3Kδ and PI3Kγ enzymes was evaluated using In-Cell-Western assay to determine the effect of ZN-B-2262 on ATM enzyme activity by detecting phosp-KAP1 (Ser824) expression. The inhibition of phospho-ATM (Ser1981) was evaluated by ATM assay on HT29 cells using a High-Content Imaging System (HCS). Therapeutic efficacy, as measured by tumor growth inhibition (TGI), was evaluated using ZN-B-2262 in combination with DS-1062 (Trop2 ADC), irinotecan or RT in MDA-MB-468 triple negative breast cancer (TNBC), SW620 colorectal cancer, and Fadu head and neck squamous cell carcinoma (HNSCC) subcutaneous xenograft murine models, respectively. In addition, ZN-B-2262 was tested for Human Aldehyde Oxidase (AO) activity. Safety margins were determined based on the ratio of unbound AUC obtained from 28-day GLP animal toxicology studies and predicted human PK at efficacious dose. Results: ZN-B-2262 exhibited strong inhibition against ATM with the IC50 at 4.4 nM. No inhibition effect was observed on other IKK family enzymes with IC50 value &amp;gt;10 uM. ZN-B-2262 showed high inhibition activity on phosp-KAP1 with IC50 of 13.25 nM and on phospho-ATM with IC50 of 21.5 nM. In the MDA-MB-468 xenograft efficacy study, ZN-B-2262 combined with a clinically relevant dose of DS-1062 showed significantly superior tumor inhibition effects compared to DS-1062 alone. In the SW620 xenograft efficacy study, in combination with irinotecan, ZN-B-2262 showed synergistic tumor inhibition effects. In the Fadu xenograft efficacy study, ZN-B-2262 demonstrated synergistic anti-tumor activity when combined with RT. Sustained and enhanced anti-tumor efficacy was observed post withdrawal of ZN-B-2262 combined with RT, which was not observed after stopping treatment of RT alone. Based on 28-day GLP tox in rat and dog, the predicted human safety margin is 25- to 50-fold. Conclusion: ZN-B-2262, in combination with ADCs containing topoisomerase inhibitors, (e.g., Dato-Dxd), or RT, is expected to be a best-in-class (possibly first-in-class) compound, for the treatment of solid tumors. In particular, ZN-B-2262 should demonstrate improved PK and minimal inter-patient PK variability compared to current ATM clinical candidates since ZN-B-2262 is not an AO substrate. Citation Format: Ding Zhou, Zheng Wang, Yan (Pita) Liu, Tingting Fu, Ziqiang (Zack) Cheng. An ATM inhibitor: ZN-B-2262 in combination with radiation/ADCs containing topoisomerase inhibitors for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7127.