Synergistic Inhibitory Effect Research Articles

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 >50% compared to controls, with 45% of PSGL-1KO animals exhibiting >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 >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.

Abstract 6490: Pt1a and sorafenib combination treatment exerts synergistic anti-hepatocellular carcinoma effect through RNA m6A methylation modification

Abstract Hepatocellular carcinoma (HCC) is the most common type of liver cancer and has a poor prognosis. The survival benefits of sorafenib in patients with advanced HCC are modest owing to acquired resistance. Accumulating evidence has demonstrated that cancer stemness is an important cause of drug resistance and the root cause of tumor recurrence. [PtII(C^N^N)(NHC2Bu)]PF6 (Pt1a) is a newly developed platinum-based drug containing N-heterocyclic carbene with proven anticancer properties against a range of tumor types. However, its role in targeting cancer stemness and its ability to sensitize cancer cells to overcome molecular-targeted therapy drug resistance in HCC remains elusive. Herein, we demonstrate the synergistic inhibitory effect of sorafenib and Pt1a combination treatment on HCC through RNA m6A methylation modification. In cultured HCC cells and patient-derived organoids, the drug combination synergistically reduced the cell viability and promoted cell apoptosis compared to single-drug treatment. Combination treatment also attenuated self-renewal, as evidenced by a marked decrease in tumor-initiating cancer cell frequency, as measured by limiting dilution spheroid formation assay. These findings were further validated in HCC patient-derived xenograft mouse models and a mouse HCC model induced by hydrodynamic tail vein delivery of NRAS+AKT coactivation plasmids. Notably, the combination treatment with sorafenib and Pt1a, but not a single treatment, led to a significant reduction in tumor burden. The combination treatment was also effective in decreasing tumor-initiating cell frequency, as measured by subjecting the harvested tumor cells for limiting dilution spheroid formation assay ex vivo. Mechanistically, integrative RNA-sequencing and thermal proteome profiling analyses both indicated the enrichment of m6A-related pathways in combination drug treatment. An increased global m6A level upon combination treatment was validated using m6A dot blotting, confirming that the synergistic effect may be mediated by the elevated global m6A level in HCC cells. Collectively, our study demonstrated that Pt1a and sorafenib combination treatment synergistically inhibited the viability and stemness of HCC cells by increasing global RNA m6A level. These findings supported the potential of Pt1a as a therapeutic option for HCC and overcoming sorafenib drug resistance. Ongoing studies are focused on identifying m6A regulators and downstream targets involved in combination treatment. Citation Format: Yinjia Huang, Yalu Cui, Ianto Bosheng Huang, Yanyan Wang, Yang Xuan, Pui Ki Wan, Chun Nam Lok, Chi Ming Che, Stephanie Ma. Pt1a and sorafenib combination treatment exerts synergistic anti-hepatocellular carcinoma effect through RNA m6A methylation modification [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 6490.

Abstract 432: The noncanonical mechanism of metabolic enzyme LDHA in cancer progression

Abstract Metabolic reprogramming is a hallmark of cancer cells and a key contributor to cancer progression, which is a promising target for cancer therapies. The enhanced glycolysis, also known as the Warburg effect, represents the most widely recognized metabolic alteration in cancer, wherein the majority of cancer cells undergoes reprogramming to heavily rely on glycolysis for their accelerated growth and proliferation. The glycolytic enzyme lactate dehydrogenase A (LDHA) catalyzes the conversion of pyruvate to lactate in the final stage of glycolysis. LDHA is frequently overexpressed in cancers, which plays a critical role in promoting glycolysis and cancer progression. To deepen our understanding of the oncogenic mechanism of LDHA, we screened for LDHA-interacting proteins in human breast cancer cells by liquid chromatography mass spectrometry (LC/MS) analysis. Through this strategy, small GTPase Rac1 was identified as an LDHA-interacting protein. LDHA interacted with the active form of Rac1, Rac1-GTP, and impaired the interaction of Rac1-GTP with its negative regulators, GTPase-activating proteins, leading to Rac1 activation independently of the glycolytic enzyme activity of LDHA. In addition, LDHA overexpression was significantly associated with increased Rac1 activity in clinical breast cancer specimens. Inhibiting Rac1 significantly attenuated the oncogenic effect of LDHA. Combining small-molecule inhibitors targeting LDHA enzyme activity and Rac1 activity displayed a synergistic inhibitory effect on breast cancers with LDHA overexpression in mouse models. Thus, our results revealed a novel and critical noncanonical mechanism of LDHA in promoting cancer progression through its direct interaction and activation of Rac1. Given that LDHA is frequently overexpressed in cancer, LDHA overexpression constitutes an important mechanism contributing to aberrant Rac1 activation in cancer. Importantly, our findings also suggest that pharmacologically targeting LDHA enzyme activity and Rac1 activity simultaneously is a promising strategy to treat breast cancers with LDHA overexpression. Citation Format: Juan Liu, Wenwei Hu, Zhaohui Feng. The noncanonical mechanism of metabolic enzyme LDHA in cancer progression [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 432.

Fabrication of Protective Organic Layer Using Schiff-Base Metal Complex Responsible for Excellent Corrosion Performance: Experimental and Theoretical Perspectives.

The effectiveness of a copper(II) complex with a Schiff base derived from 2-amino-4-phenyl-5-methylthiazole and salicylaldehyde (APMS) as a corrosion inhibitor for XC18 steel in an HCl solution was investigated. Experimental findings indicated a slight negative correlation between inhibition efficiencies in 1 M HCl and temperature but a positive correlation with both inhibitor concentration and immersion time, respectively. The weight loss measurement revealed that APMS achieved a maximum inhibition rate of 92.07% at 303 K. A fitting analysis demonstrated that APMS adheres to the Langmuir adsorption isotherm. The electrochemical results revealed an enhanced inhibitive performance of APMS, with the efficiency increasing as concentrations increased, ultimately reaching a peak of 94.47% at 5 × 10-3 mol L-1. Potentiodynamic polarization measurements revealed that APMS acted as a mixed-type inhibitor without affecting the corrosion mechanism. Scanning electron microscopy investigations of the metal surfaces corroborated the presence of an adsorbed organic layer. Advanced theoretical calculations utilizing density functional theory and first-principles density-functional tight-binding were conducted to gain insights into the behavior of APMS on the metal surface. APMS derives its advantages from crucial inter- and intramolecular interactions, resulting in the formation of a resilient adsorption layer, in line with the experimental findings. It is found that the presence of the APMS-based inhibitor exhibits a significant synergistic corrosion inhibition effect. The current study offers a design direction for enhancing the structural characteristics of Schiff base metal complexes, laying the groundwork for multifunctional frameworks to minimize corrosion rates with considerations for real-world use and cost-efficiency. The ability to replace harmful, expensive constituents with sustainable, and cost-effective organic alternatives represents a significant outcome of this study.

Exosome-Inhibiting Polymeric Sonosensitizer for Tumor-Specific Sonodynamic Immunotherapy.

Combination cancer immunotherapy based on electromagnetic energy and immunotherapy shows potent anti-cancer efficacy. However, as a factor that mediates tumor metastasis and immune suppression, the impact of tumor exosomes on therapy under electromagnetic energy stimulation remains unclear. Herein, findings indicate that sonodynamic therapy (SDT) increases serum exosome levels by inducing apoptotic exosomes and loosening the tumor extracellular matrix, promoting lung metastasis. To address this problem, an exosome-inhibiting polymeric sonosensitizer (EIPS) selectively inhibiting tumor exosome generation in response to the tumor biomarker is synthesized. EIPS consists of a semiconducting polymer backbone capable of inducing SDT and a poly(ethylene glycol) layer conjugated with a tumor-specific enzyme-responsive exosome inhibitor prodrug. After being cleaved by tumor Cathepsin B, EIPS releases active exosome inhibitors, preventing tumor exosome-mediated immune suppression and lung metastasis. As a result, EIPS elicits robust antitumor effects through the synergistic effect of SDT and tumor exosome inhibition, completely preventing lung metastasis and establishing a long-term immune memory effect. This is the first example showing that combining SDT with tumor-specific exosome inhibition can elicit a potent immune response without the help of typical immune agonists.

Securinine inhibits the tumor growth of human bladder cancer cells by suppressing Wnt/β-catenin signaling pathway and activating p38 and JNK signaling pathways

Bladder cancer (BC) is the most common malignant tumor in urinary system. Although chemotherapy is one of the most important adjuvant treatments for BC, drug resistance, non-specific toxicity and severe side effects are the major obstacles to BC chemotherapy. Natural products have always been a leading resource of antitumor drug discovery, with the advantages of excellent effectiveness, low toxicity, multi-targeting potency and easy availability.In this study, we evaluated the potential anti-tumor effect of securinine (SEC), a natural alkaloid from Securinega suffruticosa, on BC cells in vitro and in vivo, and delineated the underlying mechanism. We found that SEC inhibited the proliferation, migration and invasion, induced the apoptosis of BC cells in vitro, and retarded the xenograft tumor growth of BC cell in vivo. Notably, SEC had a promising safety profile because it presented no or low toxicity on normal cells and mice. Mechanistically, SEC inactivated Wnt/β-catenin signaling pathway while activated p38 and JNK signaling pathway. Moreover, β-catenin overexpression, the p38 inhibitor SB203580 and the JNK inhibitor SP600125 both mitigated the inhibitory effect of SEC on BC cells. Furthermore, we demonstrated a synergistic inhibitory effect of SEC and gemcitabine (GEM) on BC cells in vitro and in vivo.Taken together, our findings suggest that SEC may exert anti-BC cell effect at least through the activation of p38 and JNK signaling pathways, and the inhibition of Wnt/β-catenin signaling pathway. More meaningfully, the findings indicate that GEM-induced BC cell killing can be enhanced by combining with SEC.

New insights into the all-testis differentiation in zebrafish with compromised endogenous androgen and estrogen synthesis.

The regulatory mechanism of gonadal sex differentiation, which is complex and regulated by multiple factors, remains poorly understood in teleosts. Recently, we have shown that compromised androgen and estrogen synthesis with increased progestin leads to all-male differentiation with proper testis development and spermatogenesis in cytochrome P450 17a1 (cyp17a1)-/- zebrafish. In the present study, the phenotypes of female-biased sex ratio were positively correlated with higher Fanconi anemia complementation group L (fancl) expression in the gonads of doublesex and mab-3 related transcription factor 1 (dmrt1)-/- and cyp17a1-/-;dmrt1-/- fish. The additional depletion of fancl in cyp17a1-/-;dmrt1-/- zebrafish reversed the gonadal sex differentiation from all-ovary to all-testis (in cyp17a1-/-;dmrt1-/-;fancl-/- fish). Luciferase assay revealed a synergistic inhibitory effect of Dmrt1 and androgen signaling on fancl transcription. Furthermore, an interaction between Fancl and the apoptotic factor Tumour protein p53 (Tp53) was found in vitro. The interaction between Fancl and Tp53 was observed via the WD repeat domain (WDR) and C-terminal domain (CTD) of Fancl and the DNA binding domain (DBD) of Tp53, leading to the K48-linked polyubiquitination degradation of Tp53 activated by the ubiquitin ligase, Fancl. Our results show that testis fate in cyp17a1-/- fish is determined by Dmrt1, which is thought to stabilize Tp53 by inhibiting fancl transcription during the critical stage of sexual fate determination in zebrafish.

Scavenging Glyoxal and Methylglyoxal by Synephrine Alone or in Combination with Neohesperidin at High Temperatures.

α-Dicarbonyl compounds, such as glyoxal (GO) and methylglyoxal (MGO), are a series of chemical hazards that exist in vivo and in vitro, posing a threat to human health. We aimed to explore the scavenging effects on GO/MGO by synephrine (SYN) alone or in combination with neohesperidin (NEO). First, through LC-MS/MS, we confirmed that both SYN and NEO could effectively remove GO and form GO adducts, while NEO could also clear MGO by forming MGO adducts, and its ability to clear MGO was stronger than that of GO. Second, a synergistic inhibitory effect on GO was found when SYN and NEO were used in combination by using the Chou-Talalay method; on the other hand, SYN could promote NEO to clear more MGO, although SYN could not capture MGO. Third, after synthesizing four GO/MGO-adducts (SYN-GO-1, SYN-GO-3, NEO-GO-7, and NEO-MGO-2) and identifying their structure through NMR, strict correlations between the GO/MGO-adducts and the GO/MGO-clearance rate were found when using SYN and NEO alone or in combination. Furthermore, it was inferred that the synergistic effect between SYN and NEO stems from their mutual promotion in capturing more GO by the quantitative analysis of the adducts in the combined model. Finally, a study was conducted on flowers of Citrus aurantium L. var. amara Engl. (FCAVA, an edible tea) rich in SYN and NEO, which could serve as an effective GO and MGO scavenger in the presence of both GO and MGO. Therefore, our study provided well-defined evidence that SYN and NEO, alone or in combination, could efficiently scavenge GO/MGO at high temperatures, whether in the pure form or located in FCAVA.

Long-term toxicity assessment of antibiotics against Vibrio fischeri: Test method optimization and mixture toxicity prediction

The current luminescent bacteria test for acute toxicity with short contact time was invalid for antibiotics, and the non-uniformed contact times reported in the literature for long-term toxicity assessment led to incomparable results. Herein, a representative long-term toxicity assessment method was established which unified the contact time of antibiotics and Vibrio fischeri within the bioluminescence increasing period (i.e. 10–100% maximum luminescence) of control samples. The effects of excitation and detoxification of antibiotics such as β-lactams were discovered. Half maximal inhibitory concentration (IC50) of toxic antibiotics (0.00069–0.061 mmol/L) obtained by this method was 2–3 orders of magnitude lower than acute test, quantifying the underestimated toxicity. As antibiotics exist in natural water as mixtures, an equivalent concentration addition (ECA) model was built to predict mixture toxicity based on physical mechanism rather than mathematical method, which showed great fitting results (R2 = 0.94). Furthermore, interaction among antibiotics was investigated. Antibiotics acting during bacterial breeding period had strong synergistic inhibition (IC50 relative deviation from 0.1 to 0.6) such as macrolides and quinolones. Some antibiotics produced increasing synergistic inhibition during concentration accumulation, such as macrolides. The discharge of antibiotics with severe long-term toxicity and strong synergistic inhibition effect should be seriously restricted.

Carrimycin inhibits coronavirus replication by decreasing the efficiency of programmed –1 ribosomal frameshifting through directly binding to the RNA pseudoknot of viral frameshift-stimulatory element

The pandemic of SARS-CoV-2 worldwide with successive emerging variants urgently calls for small-molecule oral drugs with broad-spectrum antiviral activity. Here, we show that carrimycin, a new macrolide antibiotic in the clinic and an antiviral candidate for SARS-CoV-2 in phase III trials, decreases the efficiency of programmed –1 ribosomal frameshifting of coronaviruses and thus impedes viral replication in a broad-spectrum fashion. Carrimycin binds directly to the coronaviral frameshift-stimulatory element (FSE) RNA pseudoknot, interrupting the viral protein translation switch from ORF1a to ORF1b and thereby reducing the level of the core components of the viral replication and transcription complexes. Combined carrimycin with known viral replicase inhibitors yielded a synergistic inhibitory effect on coronaviruses. Because the FSE mechanism is essential in all coronaviruses, carrimycin could be a new broad-spectrum antiviral drug for human coronaviruses by directly targeting the conserved coronaviral FSE RNA. This finding may open a new direction in antiviral drug discovery for coronavirus variants.

Electrochemical and surface analysis investigation of corrosion inhibition performance: 6-Thioguanine, benzotriazole, and phosphate salt on simulated patinas of bronze relics

Organic heterocyclic corrosion inhibitors play a crucial role in the non-destructive protection of excavated bronze relics exposed to the atmosphere. However, the current selection of corrosion inhibitors, additives, and dosage requires further investigation. This study involves a comprehensive preparation simulation of electrode materials and atmospheric corrosion electrolytes. The corrosion inhibition performance of 6-thioguanine (6-Tg) and traditional benzotriazole (BTA) inhibitors at pH = 7.5 is explored. Analytical techniques such as potentiodynamic polarization, EIS, XRD, XPS, and SEM-EDS were employed to analyze the characteristics of the research electrode materials. Surface analysis reveals that 6-Tg forms a protective corrosion inhibition film on the surface of the covered patina electrode, influencing the transition of Cu2(OH)3Cl to the more stable Cu2(OH)2CO3 crystal. Electrochemical results demonstrate that, after 600 h in the corrosion solution containing 0.1 g/L of 6-Tg, the corrosion potential and corrosion current reach 0.244 V and 4.9 μA cm−2, respectively. Upon adding 1.2 g/L of NaH2PO4, the polarization resistance and inhibiting efficiencies reach 15.6 kΩ cm2 and 99%, resulting in a synergistic corrosion inhibition effect and passivation potential shift from −0.6 V to 0.2 V region.

Tumor Treating Fields (TTFields) combined with the drug repurposing approach CUSP9v3 induce metabolic reprogramming and synergistic anti-glioblastoma activity in vitro

BackgroundGlioblastoma represents a brain tumor with a notoriously poor prognosis. First-line therapy may include adjunctive Tumor Treating Fields (TTFields) which are electric fields that are continuously delivered to the brain through non-invasive arrays. On a different note, CUSP9v3 represents a drug repurposing strategy that includes 9 repurposed drugs plus metronomic temozolomide. Here, we examined whether TTFields enhance the antineoplastic activity of CUSP9v3 against this disease.MethodsWe performed preclinical testing of a multimodal approach of TTFields and CUSP9v3 in different glioblastoma models.ResultsTTFields had predominantly synergistic inhibitory effects on the cell viability of glioblastoma cells and non-directed movement was significantly impaired when combined with CUSP9v3. TTFields plus CUSP9v3 significantly enhanced apoptosis, which was associated with a decreased mitochondrial outer membrane potential (MOMP), enhanced cleavage of effector caspase 3 and reduced expression of Bcl-2 and Mcl-1. Moreover, oxidative phosphorylation and expression of respiratory chain complexes I, III and IV was markedly reduced.ConclusionTTFields strongly enhance the CUSP9v3-mediated anti-glioblastoma activity. TTFields are currently widely used for the treatment of glioblastoma patients and CUSP9v3 was shown to have a favorable safety profile in a phase Ib/IIa trial (NCT02770378) which facilitates transition of this multimodal approach to the clinical setting.

Synergistic inhibition effects of andrographolide and baicalin on coronavirus mechanisms by downregulation of ACE2 protein level

The SARS-CoV-2 virus, belonging to the Coronavirus genus, which poses a threat to human health worldwide. Current therapies focus on inhibiting viral replication or using anti-inflammatory/immunomodulatory compounds to enhance host immunity. This makes the active ingredients of traditional Chinese medicine compounds ideal therapies due to their proven safety and minimal toxicity. Previous research suggests that andrographolide and baicalin inhibit coronaviruses; however, their synergistic effects remain unclear. Here, we studied the antiviral mechanisms of their synergistic use in vitro and in vivo. We selected the SARS-CoV-2 pseudovirus for viral studies and found that synergistic andrographolide and baicalein significantly reduced angiotensin-converting enzyme 2 protein level and viral entry of SARS-CoV-2 into cells compared to singal compound individually and inhibited the major protease activity of SARS-CoV-2. This mechanism is essential to reduce the pathogenesis of SARS-CoV-2. In addition, their synergistic use in vivo also inhibited the elevation of pro-inflammatory cytokines, including IL-6 and TNF-α—the primary cytokines in the development of acute respiratory distress syndrome (the main cause of COVID-19 deaths). In conclusion, this study shows that synergistic andrographolide and baicalein treatment acts as potent inhibitors of coronavirus mechanisms in vitro and in vivo—and is more effective together than in isolation.

Activity of zinc oxide and zinc borate nanoparticles against resistant bacteria in an experimental lung cancer model.

Recent research indicates a prevalence of typical lung infections, such as pneumonia, in lung cancer patients. Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii stand out as antibiotic-resistant pathogens. Given this, there is a growing interest in alternative therapeutic avenues. Boron and zinc derivatives exhibit antimicrobial, antiviral, and antifungal properties. This research aimed to establish the effectiveness of ZnO and ZB NPs in combating bacterial infections in lung cancer cell lines. Initially, this study determined the minimal inhibitory concentration (MIC) and fractional inhibitory concentration (FIC) of zinc oxide nanoparticles (ZnO NPs) and zinc borate (ZB) on chosen benchmark strains. Subsequent steps involved gauging treatment success through a lung cancer-bacteria combined culture and immunohistochemical analysis. The inhibitory impact of ZnO NPs on bacteria was charted as follows: 0.97µg/mL for K. pneumoniae 700603, 1.95µg/mL for P. aeruginosa 27853, and 7.81µg/mL for Acinetobacter baumannii 19,606. In comparison, the antibacterial influence of zinc borate was measured as 7.81µg/mL for Klebsiella pneumoniae 700603 and 500µg/mL for both P. aeruginosa 27853 and A.baumannii 19606. After 24h, the cytotoxicity of ZnO NPs and ZB was analyzed using the MTT technique. The lowest cell viability was marked in the 500µg/mL ZB NPs group, with a viability rate of 48.83% (P < 0.001). However, marked deviations appeared at ZB concentrations of 61.5µg/mL (P < 0.05) and ZnO NPs at 125µg/mL. A synergistic microbial inhibitory effect was observed when ZnO NP and ZB were combined against the bacteria under investigation.

Synergistic inhibitory effects of EGCG and chitosan or sodium alginate on 5-hydroxymethylfurfural formation in biscuits

Heat-induced hazard 5-hydroxymethylfurfural (5-HMF) is detected in various baked foods. Although catechins and polysaccharides could inhibit 5-HMF, their inhibition efficiency was limited when used alone. Therefore, this study aimed to explore the synergistic inhibitory effect of catechins and polysaccharides on 5-HMF in biscuits. Epigallocatechin gallate (EGCG), chitosan, and sodium alginate were screened out due to their superior maximum inhibition rates (55.90%, 67.20%, and 66.62%, respectively), and their synergism were investigated by isobologram analysis. The interaction index (γ) varies inversely with the synergism. When γ < 1 or >1, it represented synergism or antagonism, respectively. The results showed that the synergism between EGCG and chitosan in the ratio of 1:1 (γ = 0.221) was better than that of 1:2 and 1:3 (γ = 0.671 and 0.523, respectively). Moreover, EGCG and sodium alginate only showed synergism in the ratio of 1:1 (γ = 0.358), while antagonism was observed in the ratios of 1:2 and 1:3 (γ = 1.12 and 1.25, respectively).The optimum inhibition rate (54.57%) on 5-HMF was reached when EGCG (0.1 g/kg) combined with sodium alginate (0.1 g/kg). Compared with those used alone, the co-addition of EGCG and chitosan could decrease the dosage of EGCG to 1/5 and that of chitosan to 1/7, indicating a significant improvement in inhibit efficiency. Finally, the concentration of inhibitors correlated negatively with 5-HMF content and browning index of biscuits, suggesting they might inhibit 5-HMF by hindering Maillard reaction or caramelization reaction. This study provided an efficient and cost-effective solution for 5-HMF control in baking products.

Enhancement of the antitumor effect of cisplatin and ginsenoside Rg3 by encapsulation in polylactic-co-glycolic acid nanoparticles

Purpose: To develop dual-loaded polylactic-co-glycolic acid (PLGA) nanoparticles (NPs) with two chemotherapeutic agents, i.e., cisplatin (DDP) and 20(R)-ginsenoside Rg3(Rg3), and to evaluate the drug release profiles and synergistic inhibitory effects of the nanoparticles on lung cancer A549 cells.Methods: The dual-laden PLGA NPs were synthesized using modified emulsion and solvent vaporization procedures. Drug loading (DL) and efficacy of co-encapsulation (EE) were determined with a modification of column elution technique. The cytotoxic and inhibitory effects of individual and combined drugs on A549 lung cancer cells were evaluated using MTT assay.Results: A sustained pattern of drug release was shown by PLGA/DDP, PLGA/Rg3, and PLGA/DDP+Rg3. The formulated PLGA and unbound drugs exerted dose- and time-reliant cytotoxic effects on A549 cells. At all concentrations tested, drugs encapsulated in PLGA nanoparticles were more effective in killing the cells than the free drugs (p &lt; 0.05). In particular, PLGA/DDP + Rg3 formulation produced a synergistic inhibitory effect on the proliferation of A549 cells.Conclusion: Single- and dual-load PLGA groups display variabilities in profiles of drug release and in vitro cytotoxic effect. Furthermore, dual-load PLGA group produces significantly enhanced treatment effect. These results suggest that PLGA formulations hold great potential for future drug delivery.

Comparative Inhibitory Effects of Tacrolimus, Cyclosporine, and Rapamycin on Human Anti-Pig Xenogeneic Mixed Lymphocyte Reactions.

Long-term immunosuppressive maintenance therapy is necessary to prevent the rejection of xenografts. However, it is still unclear which oral immunosuppressant is most suitable for pig-to-human xenotransplantation . A xenogeneic mixed lymphocyte reaction (MLR) system was established using peripheral blood mononuclear cells (PBMCs) isolated from wildtype (WT) or GTKO/CMAHKO/β4GalNT2KO (TKO) pigs as stimulator cells and human PBMCs as responder cells. Various concentrations of tacrolimus (Tac), cyclosporine (CsA), or rapamycin (Rapa) were added to the MLR system as interventions. The inhibitory effects of the three immunosuppressants on the proliferation and cytokine production of human T cells were studied and compared. The inhibitory effect of anti-CD154 mAb alone or in combination with Tac/CsA/Rapa on xenoreactive MLR was also investigated. PBMCs from both WT and TKO pigs stimulated significant proliferation of human T cells. Tac had a strong inhibitory effect on human T-cell proliferation stimulated by pig PBMCs. CsA inhibited human T-cell proliferation in a typical dose-dependent manner. When Tac and CsA concentrations reached 5 and 200ng/mL, respectively, the proliferation rates of CD3+/CD4+/CD8+ T cells were reduced almost to a negative level. Even at high concentrations, Rapa had only a moderate inhibitory effect on xenogeneic MLR. The inhibitory effects of these three immunosuppressants on xenogeneic T-cell responses were further confirmed by the detection of CD25 expression and supernatant cytokines (IL-2, IL-6, IFN-γ, TNF-α, IL-4, IL-10, and IL-17). Although anti-CD154 mAb monotherapy showed only moderate inhibitory effects on xenoreactive T-cell proliferation, low-dose anti-CD154 mAb combined with low-dose Tac, CSA, or Rapa could produce significant synergistic inhibitory effects. Tac is more efficient than CsA or Rapa in inhibiting xenogeneic T-cell responses in vitro. If used in combination with anti-CD154 mAb, all the three immunosuppressants can achieve satisfactory synergistic inhibitory effects.

An angiotensin system inhibitor (losartan) potentiates antitumor efficacy of cisplatin in a murine model of non–small cell lung cancer

ObjectivePrevious studies have demonstrated synergistic antitumor effects of angiotensin system inhibition (ASI) combined with cisplatin therapy in pancreatic cancer. This study examines whether or not synergistic antitumor effects occur with combination ASI and cisplatin treatment in lung cancer, and whether or not ASI-induced changes in epithelial-mesenchymal transition play a role in the mechanism of this antitumor phenomenon. MethodsA set of lung cancer cell lines representing a spectrum of epithelial to mesenchymal phenotypes were identified and characterized. Response of epithelial-mesenchymal transition markers to losartan was characterized. Cell culture models of lung cancer were next treated with losartan, cisplatin, or combination of both. Markers of epithelial-mesenchymal transition or surrogates of other signaling pathways (AKT, Stat3, and programmed death-ligand), and cell viability were quantified. Findings were confirmed in both allogenic and syngeneic in vivo murine flank tumor models. ResultsLosartan treatment significantly increased E-cadherin and reduced vimentin in human lung cancer cell lines. Combination treatment with losartan and cisplatin enhanced epithelial markers, reduced mesenchymal markers, inhibited promesenchymal signaling mediators, and reduced cell viability. Findings were confirmed in vivo in a murine flank tumor model with transition from mesenchymal to epithelial phenotype and reduced tumor size following combination losartan and cisplatin treatment. ConclusionsCombination losartan and cisplatin treatment attenuates the epithelial-mesenchymal transition pathway and enhances the cytotoxic effect of chemotherapy with in vitro and in vivo models of non–small cell lung cancer. This study suggests an important role for ASI therapy in the treatment of lung cancer.