Lung Cell Lines Research Articles

Immunosuppressants exert differential effects on pan-coronavirus infection and distinct combinatory antiviral activity with molnupiravir and nirmatrelvir.

Immunocompromised populations, such as organ transplant recipients and patients with inflammatory bowel disease (IBD) receiving immunosuppressive/immunomodulatory medications, may be more susceptible to coronavirus infections. However, little is known about how immunosuppressants affect coronavirus replication and their combinational effects with antiviral drugs. This study aims to profile the effects of immunosuppressants and the combination of immunosuppressants with oral antiviral drugs molnupiravir and nirmatrelvir on pan-coronavirus infection in cell and human airway organoids (hAOs) culture models. Different coronaviruses (including wild type, delta and omicron variants of SARS-CoV-2, and NL63, 229E and OC43 seasonal coronaviruses) were used in lung cell lines and hAOs models. The effects of immunosuppressants were tested. Dexamethasone and 5-aminosalicylic acid moderately stimulated the replication of different coronaviruses. Mycophenolic acid (MPA), 6-thioguanine (6-TG), tofacitinib and filgotinib treatment dose-dependently inhibited viral replication of all tested coronaviruses in both cell lines and hAOs. The half maximum effective concentration (EC50) of tofacitinib against SARS-CoV-2 was 0.62μM and the half maximum cytotoxic concentration (CC50) was above 30μM, which resulted in a selective index (SI) of about 50. The anti-coronavirus effect of the JAK inhibitors tofacitinib and filgotinib is dependent on the inhibition of STAT3 phosphorylation. Combinations of MPA, 6-TG, tofacitinib, and filgotinib with the oral antiviral drugs molnupiravir or nirmatrelvir exerted an additive or synergistic antiviral activity. Different immunosuppressants have distinct effects on coronavirus replication, with 6-TG, MPA, tofacitinib and filgotinib possessing pan-coronavirus antiviral activity. The combinations of MPA, 6-TG, tofacitinib and filgotinib with antiviral drugs exerted an additive or synergistic antiviral activity. Thus, these findings provide an important reference for optimal management of immunocompromised patients infected with coronaviruses.

Comparative MS- and NMR-Based Metabolome Mapping of Egyptian Red and White Squill Bulbs F. Liliaceae and in Relation to Their Cytotoxic Effect.

Urginea maritima L. (squill) species is widely spread at the Mediterranean region as two main varieties, i.e., white squill (WS) and red squill (RS), that are recognized for several health potentials. The major secondary metabolite classes of the squill are cardiac glycosides, mainly, bufadienolides, flavonoids, and anthocyanins. Herein, a multiplex MS and NMR metabolomics approach targeting secondary and aroma compounds in WS and RS was employed for varieties classification. Solid-phase micro extraction-gas chromatography/mass spectroscopy (SPME-GC/MS), ultra-high-performance liquid chromatography/mass spectrometry (UPLC/MS), as well as nuclear magnetic resonance (NMR) provided fingerprinting and structural confirmation of the major metabolites for both types of the squill. For comparison of the different platforms' classification potential, multivariate data analysis was employed. While Bufadienolides, viz. "hydroxy-scilliglaucosidin-O-rhamnoside, desacetylscillirosidin-O-rhamnoside and bufotalidin-O-hexoside" as well as oxylipids, were enriched in WS, flavonoids, i.e., dihydro-kaempferol-O-hexoside and its aglycon, taxifolin derivative, were predominant in RS. A cytotoxicity screening against three cancer cell lines, including breast adenocarcinoma (MCF-7), lung (A-549), and ovarian (SKOV-3) cell lines was conducted. Results revealed that WS was more effective on A-549 and SKOV-3 cell lines (WS IC50 0.11 and 0.4 µg/mL, respectively) owing to its abundance of bufadienolides, while RS recorded IC50 (MCF7 cell line) 0.17 µg/mL since is is rich inflavonoids.

Combination Therapy with UV-4B and Molnupiravir Enhances SARS-CoV-2 Suppression.

The host targeting antiviral, UV-4B, and the RNA polymerase inhibitor, molnupiravir, are two orally available, broad-spectrum antivirals that have demonstrated potent activity against SARS-CoV-2 as monotherapy. In this work, we evaluated the effectiveness of UV-4B and EIDD-1931 (molnupiravir's main circulating metabolite) combination regimens against the SARS-CoV-2 beta, delta, and omicron BA.2 variants in a human lung cell line. Infected ACE2 transfected A549 (ACE2-A549) cells were treated with UV-4B and EIDD-1931 both as monotherapy and in combination. Viral supernatant was sampled on day three when viral titers peaked in the no-treatment control arm, and levels of infectious virus were measured by plaque assay. The drug-drug effect interaction between UV-4B and EIDD-1931 was also defined using the Greco Universal Response Surface Approach (URSA) model. Antiviral evaluations demonstrated that treatment with UV-4B plus EIDD-1931 enhanced antiviral activity against all three variants relative to monotherapy. These results were in accordance with those obtained from the Greco model, as these identified the interaction between UV-4B and EIDD-1931 as additive against the beta and omicron variants and synergistic against the delta variant. Our findings highlight the anti-SARS-CoV-2 potential of UV-4B and EIDD-1931 combination regimens, and present combination therapy as a promising therapeutic strategy against SARS-CoV-2.

Design, Synthesis and Antitumor Activity of 1H-indazole-3-amine Derivatives.

A series of indazole derivatives were designed and synthesized by molecular hybridization strategy, and these compounds were evaluated the inhibitory activities against human cancer cell lines of lung (A549), chronic myeloid leukemia (K562), prostate (PC-3), and hepatoma (Hep-G2) by methyl thiazolyl tetrazolium (MTT) colorimetric assay. Among these, compound 6o exhibited a promising inhibitory effect against the K562 cell line with the IC50 (50% inhibition concentration) value of 5.15 µM, and this compound showed great selectivity for normal cell (HEK-293, IC50 = 33.2 µM). Moreover, compound 6o was confirmed to affect apoptosis and cell cycle possibly by inhibiting Bcl2 family members and the p53/MDM2 pathway in a concentration-dependent manner. Overall, this study indicates that compound 6o could be a promising scaffold to develop an effective and low-toxic anticancer agent.

Cell-intrinsic genomic reassortment of pandemic H1N1 2009 and Eurasian avian-like swine influenza viruses results in potentially zoonotic variants

ABSTRACT Influenza A viruses (IAV) cause annual epidemics and occasional pandemics in humans. The most recent pandemic outbreak occurred in 2009 with H1N1pdm09. This virus, which most likely reassorted in swine before its transmission to humans, was reintroduced into the swine population and continues circulating ever since. In order to assess its potential to cause reassortants on a cellular level, human origin H1N1pdm09 and a recent Eurasian avian-like H1N1 swine IAV were (co-)passaged in the newly generated swine lung cell line C22. Co-infection with both viruses gave rise to numerous reassortants that additionally carry different mutations which can partially be found in nature as well. Reassortment most frequently affected the PB1, PA and NA segments with the swine IAV as recipient. These reassortants reached higher titers in swine lung cells and were able to replicate in genuine human lung tissue explants ex vivo, suggesting a possible zoonotic potential. Interestingly, reassortment and mutations in the viral ribonucleoprotein complex influence the viral polymerase activity in a cell type and species-specific manner. In summary, we demonstrate reassortment promiscuity of these viruses in a novel swine lung cell model and indicate a possible zoonotic potential of the reassortants.

Synthesis of Human Antibodies Against HBsAg in Newly Established Chinese Hamster Lung (CHL-YN) Cell Line

Hepatitis B immunoglobulin (HBIG) is an effective treatment for hepatitis B, including postexposure prophylaxis of HBV infection, prevention of HBV reinfection in liver transplant patients, and reducing sexual transmission. This study investigated the synthesis of human IgG antibodies that specifically target HBsAg subtype adr in CHL-YN cells, a newly established cell line that grows faster than CHO-K1. To achieve the synthesis of human IgG antibodies, a plasmid vector encoding DNA sequences for human IgG antibodies against HBsAg was constructed and then transiently transfected into CHL-YN cells. The expression and antigen-binding capacity of the recombinant human IgG antibodies were analyzed using western blot and ELISA. The results showed successful expression and secretion of human IgG antibodies that recognize HBsAg subtype adr in CHL-YN cells. The ELISA test confirmed the specificity of the human IgG antibodies towards HBsAg subtype adr. Thus, this study concluded that human IgG antibodies that target HBsAg subtype adr were transiently expressed in CHL-YN cells.

Synthesis of Human Antibodies Against HBsAg in Newly Established Chinese Hamster Lung (CHL-YN) Cell Line

Hepatitis B immunoglobulin (HBIG) is an effective treatment for hepatitis B, including postexposure prophylaxis of HBV infection, prevention of HBV reinfection in liver transplant patients, and reducing sexual transmission. This study investigated the synthesis of human IgG antibodies that specifically target HBsAg subtype adr in CHL-YN cells, a newly established cell line that grows faster than CHO-K1. To achieve the synthesis of human IgG antibodies, a plasmid vector encoding DNA sequences for human IgG antibodies against HBsAg was constructed and then transiently transfected into CHL-YN cells. The expression and antigen-binding capacity of the recombinant human IgG antibodies were analyzed using western blot and ELISA. The results showed successful expression and secretion of human IgG antibodies that recognize HBsAg subtype adr in CHL-YN cells. The ELISA test confirmed the specificity of the human IgG antibodies towards HBsAg subtype adr. Thus, this study concluded that human IgG antibodies that target HBsAg subtype adr were transiently expressed in CHL-YN cells.

Artemisinin-inspired novel functionalized aryloxy-arylvinyl-1,2,4-trioxanes as potent anticancer agents: Design, synthesis, bioevaluation, SAR and in silico studies

Herein, a range of substituted aryloxy-arylvinyl-1,2,4-trioxanes (8a-8i), inspired from naturally occurring artemisinin, has been synthesized and then evaluated for their in vitro anticancer activity against human lung (A549) and liver (HepG2) cancer cell lines along with immortalized normal lung (BEAS-2B) and liver (LO2) cell lines. Out of the 34 synthesized aryloxy-arylvinyl-1,2,4-trioxanes, six compounds (8b2, 8d1, 8f2, 8f3, 8g1, 8h1) (IC50=11-37.73 µM; SI=1.7-9.6) displays promising in vitro anticancer activity. While these six compounds were proven to be more efficient and selective than those of reference standards such as artemisinin (IC50 = 100 µM) and chloroquine (IC50 = 100 µM), at the same time these were showing comparable in vitro anticancer activity with respect to one of standard reference compound artesunic acid (IC50 = 9.85 µM; SI = 0.76) against (A549) lung cancer cell line. Compound 8d1 from the series of synthesized aryloxy-arylvinyl-1,2,4-trioxanes was found out to be equipotent (IC50 = 11 µM; SI > 9) with respect to standard reference compound artesunic acid and likewise compound 8g1 (IC50 = 17.7 µM; SI > 2.4) also showed promising anticancer activity. In silico molecular docking studies of potent aryloxy-arylvinyl-1,2,4-trioxanes (8d1 & 8g1) along with standard drug molecules against the epidermal growth factor receptor (EGFR; PDB ID: 1M17) revealed a strong virtual interaction.

In vitro and in vivo evaluation of the genotoxicity of titanium dioxide, GST.

Titanium dioxide (TiO2) was used in various applications in a wide range of products including food, cosmetics and photocatalyst. General toxicity studies of titanium dioxide, GST (Green Sludge Titanium) have been investigated in several reports, whereas studies concerning mutagenicity and genotoxicity have not been elucidated. Herein, we investigated the potential mutagenicity and genotoxicity of GST by genetic toxicology testing. The bacterial reverse mutation test was conducted by the pre-incubation method in the presence and absence of metabolic activation system (S9 mixture). The chromosome aberration test was performed using cultured Chinese hamster lung cell line in the absence and presence of S9 mixture. The micronucleus test was performed by using specific pathogen-free male ICR mice. Genotoxicity tests were conducted following the test guidelines of the Organisation for Economic Cooperation and Development with application of Good Laboratory Practice. No statistically significant increases were found in the bacterial reverse mutation test, in vitro chromosome aberration test, and in vivo micronucleus test when tested for induction of genotoxicity in GST. These results suggest that GST did not induce mutagenicity and genotoxicity in both in vitro and in vivo system.

Targeting apoptotic anticancer response with natural glucosinolates from cell suspension culture of Lepidium sativum

BackgroundFinding natural products with anticancer activity is an effective strategy to fight this disease. In this respect, Lepidium sativum or garden cress (family Brassicaceae) has been widely used worldwide for its wide therapeutic application, including anticancer and chemoprotective agents. Plant tissue culture techniques hold great promise for natural product enhancement without any climatic boundaries. In this study, glucosinolates and petroleum ether fractions were isolated from in vitro cell cultures and used against different carcinoma cell lines to investigate their anticancer potential. MethodsIn this study, callus cultures from leaf and root explants were initiated, cell suspension cultures were established, and cell growth and viability profiles were characterized. Different amino acids were added as precursors to the cell suspension cultures to enhance glucosinolates accumulation. Gas chromatography–mass spectrometric analysis (GC–MS) of glucosinolates and petroleum ether fractions was performed, and all fractions were tested against different carcinoma cell lines. ResultsThe findings clarified that the maximum callus initiation percentage was obtained in the medium containing 1.0 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D) + 1.0 mg/l kinetin (Kin) (C1). The viable cell number of cell suspension cultures from leaves and roots increased until it reached the maximum values on day 15. Adding tyrosine and methionine to the cell suspension cultures was the most influential and recorded high glucosinolate percentages. 1H-Cyclopenta (b) pyridine-3-carbonitrile-4,5,6,7-tetrahydro-2-methylthio-4-spirocyclohexane was the main glucosinolate compound found in tyrosine-treated leaf suspension (GLT). Fifteen compounds were detected in the petroleum ether fraction in both cell suspensions initiated from the leaf and root (OL and OR). The major compounds were benzene-1,3,5-trimethyl (12.99%) in root cell suspension (OR), and benzene-2-ethyl-1,4-dimethyl (10.66%) in leaf cell suspension (OL). All glucosinolate extracts demonstrated significant anticancer activity against the prostate (PC3), lung (A-549), colorectal (caco2), and liver (HepG2) cell lines. Glucosinolates extracted from leaf cell suspension (GL) were the most active on the hepatocellular carcinoma cell line (HepG2) among all remaining glucosinolate extracts. Treated hepatocellular carcinoma with an IC50 of GL extract (47.5 ug/ml) upregulates pro-apoptotic BAX and downregulates anti-apoptotic BCL2, which disrupts the BAX/BCL2 ratio, leading to activation of caspase 3 inside treated HepG2 cells. ConclusionsThe anticancer action of the GL extract was validated by the cell cycle study of its glucosinolates, which successfully promoted apoptosis and reduced hepatocellular growth by causing S-phase arrest.

Evaluation of cytotoxic, antifungal, and larvicidal activities of different bis-sulfonamide Schiff base compounds.

Schiff bases (imines or azomethines) are versatile ligands synthesized from the condensation of amino compounds with active carbonyl groups and used for many pharmaceutical and medicinal applications. In our study, we aimed to determine the cytotoxic, antifungal and larvicidal activities of biologically potent bis-sulfonamide Schiff base derivatives that were re-synthesized by us. For this aim, 16 compounds were re-synthesized and tested for their cytotoxic, antifungal and larvicidal properties. Among this series, compounds A1B2, A1B4, A4B2, A4B3, and A4B4 were shown to have cytotoxic activity against tested cancer lung cell line (A549). The most potent antifungal activity was observed in compounds A2B1 and A2B2 against all fungi. A1B1 showed the strongest larvicidal effect at all concentrations at the 72nd h (100% mortality). These obtained results demonstrate that these type of bis-substituted compounds might be used as biologically potent pharmacophores against different types of diseases.

Abstract B004: Multi-omics profiling to identify mechanisms of resistance to KRAS inhibition: A comparative study on colorectal and lung cancer

Abstract Background: Lung adenocarcinoma (LUAD) has a higher objective response rate to single-agent KRASG12C inhibitors (e.g., AMG 510) than colorectal cancer (CRC) patients (32.2% vs 7.1%). It is known that molecular heterogeneities exist between KRASMut LUAD and CRC tissues. We hypothesize that co-mutations and oncogenic pathways may confer resistance to KRAS inhibitors in CRC cells. Methods: Whole-exome and transcriptome profiles of KRASmut LUAD (n=168) and microsatellite stable (MSS) CRC (n=183) patients were downloaded from TCGA. Mutation profiles of KRASmut patients were also obtained from AACR Project-GENIE. Gene essentiality scores derived from the whole genome CRISPR screenings were downloaded from the DepMap to evaluate the relative oncogene addiction to KRAS in lung (n=133) and CRC (n=59) cell lines. Differential gene expressions studies were performed using EBayes, followed by gene set enrichment analysis (GSEA). CRC cell lines SW837, SW1463, SW403, and HCT15 were treated with AMG 510, a KRASG12C inhibitor, and Pictilisib, a PIK3CA inhibitor, and cell viability was measured up to 96 hours after treatment. Results: The KRASmut LUAD cohort had co-mutation clusters remarkable for (i) no co-mutation (59%, 99/168), (ii) KEAP1mut and/or STK11mut (20.2%, 34/168), and (iii) a mixed group with a frequent mutation in other genes (mean=2) but without a clear recurrent pattern (20.8%, 35/168). In CRC, KRASmut tumors formed clusters of (i) no co-mutation (59.6%, 109/183), (ii) PIK3CAmut (26.2%, 48/183), and (iii) a similar cluster with frequent co-mutation (mean=2) without a recurrent pattern (14.2%, 26/183). Similar co-mutation patterns were also observed in AACR Project-GENIE LUAD and CRC cohorts. KRAS oncogene addiction was seen in both KRASMut lung (median scores, -1.1 vs -0.4, p<0.001) and CRC (median scores, -1.3 vs -0.5, p<0.001) cell lines than KRASwt cells. However, KRASmut/PIK3CAmut co-mutated CRC cell lines (n=10) were significantly less dependent on the KRAS gene than KRASmut/PIK3CAwt cells (n=22) (median scores, -1.0 vs -1.4, p=0.03). Two KRASG12C/PIK3CAwt CRC cell lines that are highly dependent on KRAS (SW837 and SW1463 cells) also showed higher sensitivities (IC50<5uM) to AMG 510 and Pictilisib relative to KRASnon-G12C/PIK3CAmut CRC cell lines SW403 and HCT15 (IC50>10uM). GSEA revealed that the Hedgehog, spermatogenesis, and pancreas-beta cells genesets were significantly active (Normalized enrichment scores > 1, FDR<0.05) in KRAS KO-resistant CRC cells, but the same genesets were not enriched in KRAS KO-resistant lung cancer cells. Conclusion: Somatic co-mutations and transcriptomics signatures in KRASmut LUAD and CRC may predict sensitivities to KRAS inhibitors. KRASmut CRC cells with PIK3CAmut or active hedgehog pathway are resistant to KRAS gene KO, suggesting multiple drug resistance mechanisms to KRAS inhibitors exist in KRASmut CRC cells. Future studies should emphasize identifying new drug targets, as optimal drug combinations of KRAS inhibitors may not be same for all KRASmut CRC patients. Citation Format: Saikat Chowdhury, Vinay K. Pattalachinti, Valsala Haridas, Scott Kopetz, John Paul Shen. Multi-omics profiling to identify mechanisms of resistance to KRAS inhibition: A comparative study on colorectal and lung cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B004.

Abstract 611: Effect Of Tumor-derived Extracellular Vesicles On Platelet Function

Tumor-derived extracellular vesicles (EVs) have been described as pro-coagulant particles causing cancer-associated thrombosis. Tumor-derived EVs circulating in the bloodstream may interact with platelets or trigger the coagulation cascade, leading to venous and arterial thrombosis. In order to better understand the interaction of EVs with the hemostatic components, we aim to determine the mechanistic aspects of their interaction with platelets and their potential role in initiating coagulation. Given the wide variation of protein and integrin expression between different cancer cell types and the expectation of varying mechanistic effects, we have selected to study lung (A549), breast (MDA-MB-231) and glioblastoma (LN18) cell lines. We have characterized the effect of EVs derived from these cell lines on platelet function of aggregation and activation. Our data shows that both A549 and MDA-MB-231 EVs at 50 ug/mL significantly increase platelet aggregation as compared to control when incubated with the platelets for 10 min and in the presence of an agonist (ADP 20 uM) (Fig. 1). We confirmed the expression of phosphatidylserine on the surface of EVs using Annexin V and flow cytometry, indicating that they are pro-coagulant in nature. Furthermore, we will be investigating interaction of EVs with platelet receptor GP IIb-IIIa, agonist release, and presence of proteins such as Tissue Factor, as possible mechanisms for EV-induced platelet aggregation. Thus, the understanding of the mechanistic aspects behind the interaction between EVs and the hemostatic components could prove beneficial in the design of next generation agents for cancer-associated thrombosis. Figure 1. Platelet aggregation measured by light transmission aggregometry with ADP 20uM in the presence of A549 and MDA-MB-231 EVs (n=8 to 11)

Abstract PR08: TCR1020 specific for KRAS G12V restricted to HLA-A*11:01 exhibits potent and precise antigen specificity for clinical development

Abstract Background: KRAS mutations drive tumorigenesis in adenocarcinomas of the lung (30-40%), colon (40-50%) and pancreas (>90%). Recent clinical evidence demonstrates potent and durable anti-tumor activity of adoptively transferred KRAS-specific T cells in select patients with KRAS-mutated advanced solid tumors. We have defined mutant KRAS (mKRAS) epitopes restricted to high prevalence HLA class I alleles and validated this oncoprotein as an immunological target amenable to T cell receptor (TCR)-based therapeutic approaches. Here, we describe the pre-clinical development of TCR1020, a human TCRab specific for KRAS G12V restricted to HLA-A*11:01. Methods: In vitro stimulation assays were used to generate CD8+ T cell responses specific for a decamer epitope of KRAS G12V (VVVGAVGVGK) using blood specimens collected from A*11:01+ healthy donors. TCR1020 was identified by TCRab RNA sequencing of flow cytometrically sorted peptide/HLA multimer+ CD8 T cells. Transgenic expression of TCR1020 was attained via lentiviral transduction of reporter cell lines and gene-edited (TCRabnull) primary T cells. TCR1020 antigen specificity, affinity and cross-reactivity was assessed using in vitro coculture, limiting peptide dilution, and combinatorial peptide scanning assays. TCR1020 reactivity to G12V/A*11:01+ cell lines was assessed using in vitro cytokine release and cytotoxicity assays, as well as mouse xenograft tumor models. Results: TCR1020 exhibits specificity to the cognate KRAS G12V epitope with cross-reactivity to G12C but not wild-type KRAS. TCR1020 has a measured functional avidity (EC50) of 262 pM. TCR1020-redirected primary CD8+ T cells promote cytolysis of A*11:01+ tumor cell lines of lung, colonic or pancreatic origin harboring endogenous KRAS G12V mutations. The adoptive transfer of TCR1020-engineered primary CD8+ T cells exhibits potent in vivo antitumor activity in mouse xenograft tumor models of metastatic cancer. TCR1020 demonstrates CD8 co-receptor independence, and CD4+ T cells expressing TCR1020 express cytokines and lyse G12V/A*11:01+ tumor cell lines. In-depth characterization of the TCR1020 peptide recognition motif using alanine/glycine scanning libraries reveals a unique antigen binding motif with 5 critical amino acid residues excluding peptide/HLA anchor residues. Combinatorial peptide library scanning identifies five A*11:01-restricted peptide candidates within the human proteome with potential cross-reactivity to TCR1020, none of which activate TCR1020-engineered T cells over a wide range of concentrations. TCR1020 exhibits no evidence of reactivity against cell lines derived from healthy human tissues nor alloreactivity against a panel of lymphoblastoid cell lines encompassing a wide array of HLA class I alleles. Conclusion: TCR1020 exhibits high potency and exquisite antigen cognate specificity, and it is under clinical development for the treatment of A*11:01+ patients with KRAS G12V+ advanced solid tumors. Citation Format: Adham S. Bear, Rebecca B. Nadler, John Scholler, Robert H. Vonderheide, Gerald P. Linette, Beatriz M. Carreno. TCR1020 specific for KRAS G12V restricted to HLA-A*11:01 exhibits potent and precise antigen specificity for clinical development [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr PR08.

In Vitro Potent Anticancer, Antifungal, and Antioxidant Efficacy of Walnut (Juglans regia L.) Genotypes

Walnuts are one of the healthiest foods in the world because they are one of the best sources of beneficial nutrients, minerals, and antioxidants. They also contain key sources of energy. Despite several traditional uses, the leaves of Juglans regia L. have received little attention regarding phytochemical and pharmacological potential. Thus, the current study intended to find the walnut genotypes with the greatest antioxidant, anticancer, and antifungal activity. The total polyphenolic, flavonoid, and flavanol contents of leaves from 14 walnut genotypes were determined. Genotypes that accumulate flavonoid/flavonol contents (99.8–111.93 mg/g quercetin equivalent (QE) and 101.67–111.83 mg/g QE) showed significantly higher ferric reducing antioxidant potential (FRAP) activity (128.2–148.1 μM Fe2+/g dry weight (DW)] than other genotypes. Maximum divergence in the quercetin content (0.8–1.23 mg/g) of walnut genotypes was obtained by cluster analysis. The active component, quercetin, was measured using RP-HPLC. Moreover, the extracts were investigated for antifungal and anticancer assays. We report the significant antifungal potential of walnut leaf genotypes against Candida glabrata, Candida albicans, and Candida tropicalis, with 57.7–93.6%, 26.8–51.5%, and 26.8–51.5% inhibition, respectively. The most significant antiproliferative effect was shown by Opex Culchry, which exhibited 9.4% cell viability at a concentration of 25 µL (0.75 mg) against lung (A549) cell lines. Chenovo exhibited 2.9, 6.2, and 2.2% cell viability, Opex Culchry exhibited 2, 1.5, and 2.4% cell viability, and Sulieman showed 7.6, 0.9, and 7% cell viability against the colon (HCT116) cell lines. The results showed that walnut leaves possess enormous potential as antioxidants, and as anticancer and antifungal agents.

Design, synthesis, and molecular docking studies of novel quinoxaline derivatives as anticancer agents.

As lung cancer was placed foremost part among other types of cancer in terms of mortality. Recent researches are widely focused on developing multi-targeted and site-specific targeted drug designs. In the present study, we designed and developed a series of quinoxaline pharmacophore derivatives as active EGFR inhibitors for the treatment of non-small cell lung cancer. The compounds were synthesized through a condensation reaction between hexane-3,4-dione and methyl 3,4-diaminobenzoate as a first step. Their structures were confirmed by 1 H-NMR, 13 C-NMR, and HRMS spectroscopic methods. Cytotoxicity (MTT) were applied to determine anticancer activity of the compounds against breast (MCF7), fibroblast (NIH3 T3), and lung (A549) cell lines as EGFR inhibitors. Doxorubicin was used as a reference agent, compound 4i exhibited a significant effect among other derivatives with IC50 = 3.902 ± 0.098 μM value against A549 cell line. The docking study showed that the best position on EGFR receptor could be observed with 4i. From the obtained evaluations of the designed series, compound 4i was a promising agent as EGFR inhibitor for further investigation and evaluation studies in the future.

Viral Fitness of Baloxavir-Resistant Recombinant Influenza B/Victoria- and B/Yamagata-like Viruses Harboring the I38T PA Change, In Vitro, Ex Vivo and in Guinea Pigs

Seasonal influenza A and B viruses may cause severe infections requiring therapeutic interventions. Baloxavir, the latest antiviral drug approved against those infections, targets the endonuclease activity encoded by the polymerase acidic (PA) protein. While appearing effective at cessation of viral shedding, baloxavir demonstrated a low barrier of resistance. Herein, we aimed to assess the impact of PA-I38T substitution, a major marker of baloxavir-resistance, on the fitness of contemporary influenza B viruses. Recombinant wild-type (WT) influenza B/Phuket/2073/13 (B/Yamagata/16/88-like) and B/Washington/02/19 (B/Victoria/2/87-like) viruses and their respective PA-I38T mutants were used to evaluate replication kinetics in vitro, using A549 and Calu3 cells, and ex vivo, using nasal human airway epithelium (HAE) cells. Infectivity was also assessed in guinea pigs. In the B/Washington/02/19 background, there were no major differences between the recombinant WT virus and its I38T mutant when viral replication kinetics were evaluated in human lung cell lines and HAE as well as in nasal washes of experimentally infected guinea pigs. By contrast, the I38T mutation moderately impacted the B/Phuket/2073/13 viral fitness. In conclusion, contemporary influenza B viruses that may acquire baloxavir-resistance through the PA-I38T substitution could retain a significant level of fitness, highlighting the importance of monitoring the emergence of such variant.

Phytochemical screening of Bixa orellana and preliminary antidiabetic, antibacterial, antifibrinolytic, anthelmintic, antioxidant, and cytotoxic activity against lung cancer (A549) cell lines

Bixa orellana (B. Orellana) is a frequently utilized plant that has grown in significance in pharmaceutical applications. The leaves extract of B. orellana was used in the current study for preliminary phytochemical analysis in terms of both quantitative and qualitative, which indicates the presence of phenols, alkaloids, and flavonoids. Furthermore, the extract demonstrated antifungal activity at 30 mm zone of clearance against Candida albicans and antibacterial activity at 16 mm zone of clearance against Bacillus nakamuria by well diffusion method against different strains. The plant extract showed a MIC of 20 µg/mL and MBC of 157.11 µg/mL against E. coli by ELISA and broth dilution method, respectively.To evaluate the phytochemicals in the extract, further purification of the extract by TLC, column chromatography, and component analysis by GC–MS, which reported 18 components, and UV spectrum were performed. A number of therapeutic applicationsfor the extract were observed, including antidiabetic activity (anti-glucosidase) of 98.34% inhibitionat 100 µg/mL, anti-lipase of 100% inhibitionat 100 µg/mL, and anti-amylase activity of 100% inhibitionat 100 µg/mL, antioxidant activity (anti-DPPH activity) of 59.74% inhibitionat 100 µg/mL, anthelmintic at 1 min by 1000 µg/mL, antifibrinolytic at 20 secs by 1000 µg/mL and cytotoxic activity against A549 lung cell lines, wherein, the cell viability was below 40 % at the highest dose (100 µg/mL), with an IC50 value of 39.9 µg/mL.

PLCG2 can exist in eccDNA and contribute to the metastasis of non-small cell lung cancer by regulating mitochondrial respiration

Extrachromosomal circular DNAs (eccDNAs) participate in tumorigenesis and tumor progression. However, the role and mechanism of eccDNAs have yet to be elucidated in non-small cell lung cancer (NSCLC). In our research, three surgically matched NSCLC tissue samples, NSCLC cell lines (H1299, A549, and H460), and a normal lung cell line (MRC-5) were used as study objects. High-throughput eccDNA sequencing and bioinformatics analysis were performed to study the distribution pattern and level of eccDNA expression. The upregulated candidate eccDNA-encoding PLCG2 was validated by routine PCR. Plasmid transfection, RNA interference, qRT‒PCR and western blotting experiments were used to verify the expression level of PLCG2. Our results showed that the chromosome distribution, length distribution, and genomic annotation of the eccDNAs were comparable between the NSCLC and normal groups. Nevertheless, there were no significant differences in eccDNAs between NSCLC tissues and matched normal lung tissues. The eccDNA derived from PLCG2 was upregulated in NSCLC cells. TCGA analysis and immunohistochemistry showed that PLCG2 was highly expressed in lung cancer tissues and tended to be associated with poor outcome. We also demonstrated that PLCG2 can promote metastasis through the regulation of mitochondrial respiration. These results suggested that PLCG2 identified by eccDNA sequencing acts as an oncogene and might be a new biomarker for NSCLC diagnosis and prognosis evaluation.

Abstract 4893: Evaluation of bystander antitumor effect of Dato-DXd in TROP2 (+) and TROP2 (-) lung cell lines admixed tumor xenograft mouse models

Abstract Background: Datopotamab deruxtecan (Dato-DXd) is an antibody-drug conjugate (ADC) consisting of a humanized anti-TROP2 IgG1 monoclonal antibody covalently linked to a highly potent topoisomerase I inhibitor payload (DXd) via a stable, tumor-selective, tetrapeptide-based cleavable linker (Nakada T. et al. Bioorg Med Chem Lett., 2016). Dato-DXd demonstrated encouraging efficacy in clinical studies in TROP2 expressing tumors, including NSCLC and TNBC (NCT03401385). Clinical efficacy of Dato-DXd in heterogeneous tumors is hypothesized to be derived not only via effective selective delivery of DXd to TROP2 (+) tumor cells but also DXd diffusion from TROP2 (+) tumor cells to nearby TROP2 (-) tumor cells due to high membrane permeability of the DXd payload, an effect called the bystander antitumor effect. In this preclinical study, the bystander antitumor effect was tested by establishing tumor xenograft mouse models containing admixed TROP2 (+) and TROP2 (-) lung cell lines. The antitumor activity of Dato-DXd was tested in these models to demonstrate the bystander antitumor effect of Dato-DXd. Materials and Methods: EBC-1 cells and NCI-H526 cells or Calu-6 cells were used as models of TROP2 (+) cells and TROP2 (-) cells, respectively. TROP2 (+) cells and TROP2 (-) cells were mixed in various ratios and inoculated subcutaneously in mice to establish heterogeneous xenograft tumor models with various levels of TROP2 expression. Tumor growth inhibition in each model was evaluated by tumor volume after Dato-DXd or control-ADC administration. Immunohistochemistry (IHC) of DXd and TROP2 were performed in Dato-DXd treated tumors to analyze distribution in TROP2 (+) and (-) tumor cells. Tumor cells that responded to Dato-DXd treatment were identified by IHC assay of γH2AX (an indicator of DNA damage). Results: TROP2 expression analysis showed that various TROP2 positivity tumor models were established by inoculating various ratios of admixed EBC-1 cells with NCI-H526 cells or Calu-6 cells. Dato-DXd did not inhibit growth of tumors consisting of only TROP2 (-) cells, as expected. In contrast, Dato-DXd showed strong tumor growth inhibition effect on all tumors in which TROP2 (+) cells were inoculated in various levels with TROP2 (-) cells. DXd-IHC analysis showed that Dato-DXd distributed to only TROP2 (+) tumor cells in admixed tumors, however, an increase of γH2AX was observed not only in TROP2 (+) tumor cells but also TROP2 (-) tumor cells, suggesting TROP2 (-) tumor cells responded to DXd generated and distributed from Dato-DXd bound to TROP2 (+) tumor cells. Conclusion: Dato-DXd is effective not only in TROP2 (+) tumor cells but also TROP2 (-) tumor cells adjacent to TROP2 (+) tumor cells, thereby demonstrating the bystander antitumor effect of Dato-DXd. This property suggests Dato-DXd should be active in the clinical setting even given potential heterogeneity of TROP2 expression. Citation Format: Satoru Yasuda, Takanori Maejima, Tadashi Toki, Tsuyoshi Karibe, Daisuke Okajima, Penny Phillips. Evaluation of bystander antitumor effect of Dato-DXd in TROP2 (+) and TROP2 (-) lung cell lines admixed tumor xenograft mouse models. [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 4893.