Normal Lung Fibroblasts Research Articles

New spiro-indeno[1,2-b]quinoxalines clubbed with benzimidazole scaffold as CDK2 inhibitors for halting non-small cell lung cancer; stereoselective synthesis, molecular dynamics and structural insights

Despite the crucial role of CDK2 in tumorigenesis, few inhibitors reached clinical trials for managing lung cancer, the leading cause of cancer death. Herein, we report combinatorial stereoselective synthesis of rationally designed spiroindeno[1,2-b]quinoxaline-based CDK2 inhibitors for NSCLC therapy. The design relied on merging pharmacophoric motifs and biomimetic scaffold hopping into this privileged skeleton via cost‐effective one-pot multicomponent [3 + 2] cycloaddition reaction. Absolute configuration was assigned by single crystal x-ray diffraction analysis and reaction mechanism was studied by Molecular Electron Density Theory. Initial MTT screening of the series against A549 cells and normal lung fibroblasts Wi-38 elected 6b as the study hit regarding potency (IC50 = 54 nM) and safety (SI = 6.64). In vitro CDK2 inhibition assay revealed that 6b (IC50 = 177 nM) was comparable to roscovitine (IC50 = 141 nM). Docking and molecular dynamic simulations suggested that 6b was stabilised into CDK2 cavity by hydrophobic interactions with key aminoacids.

Tumor-derived exosomal linc00881 induces lung fibroblast activation and promotes osteosarcoma lung migration

Osteosarcoma (OS) commonly metastasizes to the lung, yet the underlying molecular mechanisms remain poorly understood. Exosomes play a crucial role in tumor migration, including OS lung migration. However, the underlying mechanism by which exosome-derived long non-coding RNAs (lncRNAs) contribute to lung migration in osteosarcoma (OS) remains unclear. This study presents a newly discovered lncRNA, linc00881, derived from OS exosomes. Our study shows that linc00881 promotes the migration of OS cells to the lung and induces the conversion of normal lung fibroblasts into cancer-associated fibroblasts (CAFs). Subsequently, we found that exosomal linc00881 secreted by OS cells can regulate the expression of matrix metalloproteinase 2 (MMP2) in HFL-1 cells by sponging miR-29c-3p, thereby activating the NF-κB signaling in lung fibroblasts. Finally, we discovered that pro-inflammatory cytokines, namely IL-1β, IL-6, and IL-8, were secreted through the linc00881/miR-29c-3p/MMP2 axis. These results suggest that OS-derived exosomes can mediate the intercellular crosstalk between OS cells and lung fibroblasts, ultimately impacting OS lung migration. Our study provides a potential target for the treatment of OS lung migration.Graphical

Investigation of the Impact of L-Phenylalanine and L-Tyrosine Pre-Treatment on the Uptake of 4-Borono-L-Phenylalanine in Cancerous and Normal Cells Using an Analytical Approach Based on SC-ICP-MS.

Boron has gained significant attention in medical research due to its B-10 isotope's high cross section for the reaction with thermal neutrons, generating ionizing particles that can eliminate cancer cells, propelling the development of boron neutron capture therapy (BNCT) for cancer treatment. The compound 4-borono-L-phenylalanine (BPA) has exhibited potential in BNCT clinical trials. Enhancing BPA uptake in cells involves proposing L-amino acid preloading. This study introduces a novel analytical strategy utilizing ICP-MS and single cell ICP-MS (SC-ICP-MS) to assess the effectiveness of L-tyrosine and L-phenylalanine preloading on human non-small cell lung carcinoma (A549) and normal Chinese hamster lung fibroblast (V79-4) models, an unexplored context. ICP-MS outcomes indicated that L-tyrosine and L-phenylalanine pre-treatment increased BPA uptake in V79-4 cells by 2.04 ± 0.74-fold (p = 0.000066) and 1.46 ± 0.06-fold (p = 0.000016), respectively. Conversely, A549 cells manifested heightened BPA uptake solely with L-tyrosine preloading, with a factor of 1.24 ± 0.47 (p = 0.028). BPA uptake remained higher in A549 compared to V79-4 regardless of preloading. SC-ICP-MS measurements showcased noteworthy boron content heterogeneity within A549 cells, signifying diverse responses to BPA exposure, including a subset with notably high BPA uptake. This study underscores SC-ICP-MS's utility in precise cellular boron quantification, validating cellular BPA uptake's heterogeneity.

Rh(III) and Ru(II) complexes with phosphanyl-alkylamines: inhibition of DNA synthesis induced by anticancer Rh complex.

Aim: This investigation was designed to synthesize half-sandwich Rh(III) and Ru(II) complexes and study their antiproliferative activity in human cancer cell lines. Materials & methods: Nine compounds were prepared and tested by various assays for their anticancer activity and mechanism of action. Results: Hit Rh(III) complex 6 showed low-micromolar potency in cisplatin-sensitive (A2780) and -resistant (A2780cis) ovarian carcinoma cell lines, promising selectivity toward these cancer cells over normal lung fibroblasts and anunprecedented mechanism of action in the treated cells. DNA synthesis was decreased andCDKN1A expression was upregulated, but p21 expression was not induced. Conclusion: Rh complex 6 showed high antiproliferative activity, which is induced through a p21-independent mechanism of action.

Persistent hypoxia promotes myofibroblast differentiation via GPR-81 and differential regulation of LDH isoenzymes in normal and idiopathic pulmonary fibrosis fibroblasts.

Hypoxia, a state of insufficient oxygen availability, promotes cellular lactate production. Lactate levels are increased in lungs from patients with idiopathic pulmonary fibrosis (IPF), a disease characterized by excessive scar formation, and lactate is implicated in the pathobiology of lung fibrosis. However, the mechanisms underlying the effects of hypoxia and lactate on fibroblast phenotype are poorly understood. We exposed normal and IPF lung fibroblasts to persistent hypoxia and found that increased lactate generation by IPF fibroblasts was driven by the FoxM1-dependent increase of lactate dehydrogenase A (LDHA) coupled with decreased LDHB that was not observed in normal lung fibroblasts. Importantly, hypoxia reduced α-smooth muscle actin (α-SMA) expression in normal fibroblasts but had no significant impact on this marker of differentiation in IPF fibroblasts. Treatment of control and IPF fibroblasts with TGF-β under hypoxic conditions did not significantly change LDHA or LDHB expression. Surprisingly, lactate directly induced the differentiation of normal, but not IPF fibroblasts under hypoxic conditions. Moreover, while expression of GPR-81, a G-protein-coupled receptor that binds extracellular lactate, was increased by hypoxia in both normal and IPF fibroblasts, its inhibition or silencing only suppressed lactate-mediated differentiation in normal fibroblasts. These studies show that hypoxia differentially affects normal and fibrotic fibroblasts, promoting increased lactate generation by IPF fibroblasts through regulation of the LDHA/LDHB ratio and promoting normal lung fibroblast responsiveness to lactate through GPR-81. This supports a novel paradigm in which lactate may serve as a paracrine intercellular signal in oxygen-deficient microenvironments.

Novel 3′-diindolylmethane nanoformulation induces apoptosis, and reduces migration and angiogenesis in liver cancer cells

Liver cancer (LC) ranks as the second most prevalent cause of cancer-related deaths. Herbaceous plants are valuable sources of complementary, adjuvant, or alternative anti-tumor therapy as they contain natural active ingredients with anti-cancer potential. Although the clinical use of 3, 3′-Diindolylmethane (DIM) has been established, its low chemical stability and bioavailability, limits its therapeutic applications. Increasing effort has been undertaken to improve DIM’s biological activity including nanoformulations. Here, we evaluated the efficacy of DIM nanoparticles (DIM-NPs) coated with PEG/chitosan for the treatment of liver cancer and elucidated the underlying molecular mechanisms contributing to its anti-tumor activity. DIM-PLGA-PEG/chitosan NPs were synthesized and characterized using dynamic light scattering (DLS). The effect of newly synthesized DIM-NPs was evaluated in HepG-2 and HUH-7 hepatocarcinoma cells and compared to THLE-2 immortal normal liver cells and WI-38 (normal lung fibroblast cells). These cells were treated with different non-cytotoxic concentrations of DIM-NPs and MTT assay and other functional assays were performed. Compared to normal cells, DIM-NPs induced cytotoxicity in HepG-2 cells at 6.25 µg/mL after 48 h of treatment. Treatment of HepG-2 cells with the 50 % inhibitory concentration (IC50) 12.5 µg/mL of DIM-NPs inhibited cell migration (p < 0.001). Treatment of chicken embryo with 5ug/ml DIM-NPs reduced (p < 0.001) angiogenesis at day 4. Notably, at the molecular level, DIM-NPs upregulated Bax and p53 and downregulated Bcl-2 in a dose-dependent manner. DIM-NPs also induced cell apoptosis in HepG-2 cells. Treatment of hepatic cells with DIM-NPs decreased cell proliferation, migration and angiogenesis, and induced cell death via up-regulation of Bax and p53, and down-regulation of Bcl-2 in HepG-2 cells. Further investigations are necessitated to determine the pharmacokinetics of DIM-NPs using a preclinical cancer model.

A New Cu/Fe Layer Double Hydroxide Nanocomposite Exerts Anticancer Effects against PC-3 Cells by Inducing Cell Cycle Arrest and Apoptosis.

Prostate cancer treatment poses significant challenges due to its varying aggressiveness, potential for metastasis, and the complexity of treatment options. Balancing the effectiveness of therapies, minimizing side effects, and personalizing treatment strategies are ongoing challenges in managing this disease. Significant advances in the use of nanotechnology for the treatment of prostate cancer with high specificity, sensitivity, and efficacy have recently been made. This study aimed to synthesize and characterize a novel Cu/Fe layer double hydroxide (LDH) nanocomposite for use as an anticancer agent to treat prostate cancer. Cu/Fe LDH nanocomposites with a molar ratio of 5:1 were developed using a simple co-precipitation approach. FT-IR, XRD, SEM, TEM, TGA, and zeta potential analyses confirmed the nanocomposite. Moreover, the MTT cell viability assay, scratch assay, and flow cytometry were utilized to examine the prospective anticancer potential of Cu/Fe LDH on a prostate cancer (PC-3) cell line. We found that Cu/Fe LDH reduced cell viability, inhibited cell migration, induced G1/S phase cell cycle arrest, and triggered apoptotic effect in prostate cancer cells. The findings also indicated that generating reactive oxygen species (ROS) formation could improve the biological activity of Cu/Fe LDH. Additionally, Cu/Fe LDH showed a good safety impact on the normal lung fibroblast cell line (WI-38). Collectively, these findings demonstrate that the Cu/Fe LDH nanocomposite exhibited significant anticancer activities against PC-3 cells and, hence, could be used as a promising strategy in prostate cancer treatment.

A Practical Nanoplasmonic SERS Substrate for Differential Diagnosis of Lung Normal and Cancer Cells through Multivariate Statistical Analysis

AbstractLung cancer ranks first for cancer‐related mortalities primarily due to late diagnosis. Though Surface‐Enhanced Raman spectroscopy (SERS) is a popular bioanalytical technique, its direct application to diagnosis is impeded by low data reproducibility. Colloidal nanoparticles suffer from SERS intensity fluctuations due to unavoidable aggregation, and Brownian and diffusion motions in biological samples. The processes for solid‐state SERS substrates are either sophisticated or difficult to reproduce. Herein, we revisit the well‐established thermal evaporation process for the easy and reproducible preparation of silver nanoparticles loaded SERS glass substrates. The static mode of thermal evaporation yielded closely packed and uniformly distributed silver nanoparticles. The properties of these nanoparticles are tuned for the best performance by controlling the thermal evaporation process. And SERS substrate exhibited a reasonably good enhancement factor of ~105 with uniformity and reproducibility &lt;6 % RSD over a large area. It was utilized for label‐free SERS fingerprinting of lung adenocarcinoma cells A549 and normal lung fibroblast cells, WI‐38. The obtained data shows a slight distinction of Raman fingerprints in terms of certain biomolecules like nucleic acids, proteins, and lipids. Further multivariate statistical tools have been utilized which ensures a clear divergence between the cancerous cells and normal cells.

Designed novel nuclear localizing anticancer peptide targets p53 negative regulator MDM2 protein.

Intracellular protein-protein interactions provide a major therapeutic target for the development of peptide-based anticancer therapeutic agents. MDM2 is the 491-residue protein encoded by the MDM2 oncogene. Being a ubiquitin-protein ligase, MDM2 represses the transcription ability of the tumor suppressor p53 by proteasome-mediated degradation. Under typical cellular circumstances, a sustained p53 expression level is maintained by negative regulation of MDM2, whereas under stress conditions, this is alleviated to increase the p53 level. Modulation of MDM2-p53 interaction via fabrication of an MDM2-interacting peptide could be a useful strategy to inhibit subsequent proteasomal degradation of p53 and initiation of p53 signaling leading to the initiation of p53-mediated apoptosis of tumor cells. Here, in this research work, a novel anticancer peptide mPNC-NLS targeting the nucleus and the MDM2 protein (p53 negative regulator) was designed to promote the p53 protein activity for the prevention of cancer. It induces effective apoptosis in both A549 and U87 cells and remains non-cytotoxic to normal lung fibroblast cells (WI38). Further, immunocytochemistry and Western blot results confirm that the designed mPNC-NLS peptide induces the apoptotic death of lung cancer cells via activation of p53 and p21 proteins and remarkably stifled the in vitro growth of 3D multicellular spheroids composed of A549 cells.

Validation of Broth Macrodilution Volatilization Method for Testing of Essential Oils in Liquid and Vapor Phase: Chemical Composition, Cytotoxicity, and Antibacterial Effect of Indian Medicinal Plants against Pneumonia-Causing Pathogens.

Essential oils (EOs) have great potential in inhalation therapy for the treatment of respiratory infections. However, innovative methods for evaluation of antimicrobial activity of their vapors are still needed. The current study reports validation of the broth macrodilution volatilization method for assessment of the antibacterial properties of EOs and shows the growth-inhibitory effect of Indian medicinal plants against pneumonia-causing bacteria in liquid and vapor phase. Among all samples tested, Trachyspermum ammi EO exhibits the strongest antibacterial effect against Haemophilus influenzae, with minimum inhibitory concentrations of 128 and 256 µg/mL in the liquid and vapor phases, respectively. Furthermore, Cyperus scariosus EO is found to be nontoxic to normal lung fibroblasts assessed by modified thiazolyl blue tetrazolium bromide assay. Chemical analysis performed using gas chromatography-mass spectrometry identified α-citral, cyperotundone, and thymol as the main constituents of Cymbopogon citratus, C. scariosus, and T. ammi EOs, respectively. In addition, β-cymene is identified as the major compound of T. ammi EO vapors when analyzed using solid-phase microextraction and gas-tight syringe sampling techniques. This study demonstrates the validity of the broth macrodilution volatilization method for antimicrobial screening of volatile compounds in the vapor phase and suggests the therapeutic potential of Indian medicinal plants in inhalation therapy.

Benzylisoquinoline alkaloids inhibit lung fibroblast activation mainly via inhibiting TGF-β1/Smads and ERK1/2 pathway proteins

BackgroundsLiensinine (Lien), Neferine (Nef), Isoliensinine (Iso) and Tetrandrine (Tet), benzylisoquinoline alkaloids (BIAs), have been shown inhibitory effects on pulmonary fibrosis (PF) through anti-inflammatory, anti-oxidative activities, inhibition of cytokines and NF-κB. Effects of other similar BIAs, Dauricine (Dau), Papaverine (Pap) and lotusine (Lot), on PF remain unclear. Here, we explored the effects of five bisbenzylisoquinoline (Lien, Nef, Iso, Tet and Dau) and two monobenzylisoquinoline (Pap, Lot) alkaloids on normal and PF fibroblasts. MethodsPrimary normal and PF lung fibroblasts were cultured and treated with these alkaloids. Proliferation, activation, migration and apoptosis changes were detected by MTT, wound healing assay, flow cytometry. Protein level was analyzed by Western blot. ResultsAll BIAs inhibited proliferation of normal and PF lung fibroblasts induced by TGF-β. α-SMA protein level in normal and PF lung fibroblasts decreased after Lien, Nef, Iso, Tet and Dau treatment. Pap and Lot had no influence on α-SMA expression. Dau showed the strongest inhibitory effects on proliferation and activation among alkaloids. The migration rates of normal and PF lung fibroblasts were inhibited by Lien, Nef, Iso, and Dau. Lien, Nef, Iso and Dau significantly promoted apoptosis, while Tet had no effect on apoptosis. Pap and Lot had no influence on activation, migration and apoptosis. Dau significantly inhibited Smad3/4 and p-ERK1/2 protein overexpression induced by TGF-β1. ConclusionsBisbenzylisoquinoline alkaloids had stronger effects on inhibiting lung fibroblasts than monobenzylisoquinoline alkaloids. Dau expressed the strongest inhibitory effects, which may be related to its inhibition of TGF-β1/Smad3/4 and p-ERK1/2 pathway proteins.

Inositol possesses antifibrotic activity and mitigates pulmonary fibrosis

BackgroundMyo-inositol (or inositol) and its derivatives not only function as important metabolites for multiple cellular processes but also act as co-factors and second messengers in signaling pathways. Although inositol supplementation has been widely studied in various clinical trials, little is known about its effect on idiopathic pulmonary fibrosis (IPF). Recent studies have demonstrated that IPF lung fibroblasts display arginine dependency due to loss of argininosuccinate synthase 1 (ASS1). However, the metabolic mechanisms underlying ASS1 deficiency and its functional consequence in fibrogenic processes are yet to be elucidated.MethodsMetabolites extracted from primary lung fibroblasts with different ASS1 status were subjected to untargeted metabolomics analysis. An association of ASS1 deficiency with inositol and its signaling in lung fibroblasts was assessed using molecular biology assays. The therapeutic potential of inositol supplementation in fibroblast phenotypes and lung fibrosis was evaluated in cell-based studies and a bleomycin animal model, respectively.ResultsOur metabolomics studies showed that ASS1-deficient lung fibroblasts derived from IPF patients had significantly altered inositol phosphate metabolism. We observed that decreased inositol-4-monophosphate abundance and increased inositol abundance were associated with ASS1 expression in fibroblasts. Furthermore, genetic knockdown of ASS1 expression in primary normal lung fibroblasts led to the activation of inositol-mediated signalosomes, including EGFR and PKC signaling. Treatment with inositol significantly downregulated ASS1 deficiency-mediated signaling pathways and reduced cell invasiveness in IPF lung fibroblasts. Notably, inositol supplementation also mitigated bleomycin-induced fibrotic lesions and collagen deposition in mice.ConclusionThese findings taken together demonstrate a novel function of inositol in fibrometabolism and pulmonary fibrosis. Our study provides new evidence for the antifibrotic activity of this metabolite and suggests that inositol supplementation may be a promising therapeutic strategy for IPF.

Novel hybrids of sclareol and 1,2,4-triazolo[1,5-a]pyrimidine show collateral sensitivity in multidrug-resistant glioblastoma cells

The synthesis of 24 hybrid molecules, consisting of naturally occurring sclareol (SCL) and synthetic 1,2,4-triazolo[1,5-a]pyrimidines (TPs), is described. New compounds were designed with the aim of improving the cytotoxic properties, activity, and selectivity of the parent compounds. Six analogs (12a-f) contained 4-benzylpiperazine linkage, while 4-benzyldiamine linkage was present in eighteen derivatives (12g-r and 13a-f). Hybrids 13a-f consist of two TP units. After purification, all hybrids (12a-r and 13a-f), as well as their precursors (9a-e and 11a-c), were tested on human glioblastoma U87 cells. More than half of the tested synthesized molecules, 16 out of 31, caused a significant reduction of U87 cell viability (more than 75% reduction) at 30 µM. The concentration-dependent cytotoxicity of these 16 compounds was also examined on U87 cells, corresponding multidrug-resistant (MDR) U87-TxR cells with increased P-glycoprotein (P-gp) expression and activity, and normal lung fibroblasts MRC-5. Importantly, 12l and 12r were active in the nanomolar range, while seven compounds (11b, 11c, 12i, 12l, 12n, 12q, and 12r) were more selective towards glioblastoma cells than SCL. All compounds except 12r evaded MDR, showing even better cytotoxicity in U87-TxR cells. In particular, 11c, 12a, 12g, 12j, 12k, 12m, 12n, and SCL showed collateral sensitivity. Hybrid compounds 12l, 12q, and 12r decreased P-gp activity to the same extent as a well-known P-gp inhibitor - tariquidar (TQ). Hybrid compound 12l and its precursor 11c affected different cellular processes including the cell cycle, cell death, and mitochondrial membrane potential, and changed the levels of reactive oxygen and nitrogen species (ROS/RNS) in glioblastoma cells. Collateral sensitivity towards MDR glioblastoma cells was caused by the modulation of oxidative stress accompanied by inhibition of mitochondria.

New Nanostructured Materials Based on Mesoporous Silica Loaded with Ru(II)/Ru(III) Complexes with Anticancer and Antimicrobial Properties.

A new series of nanostructured materials was obtained by functionalization of SBA-15 mesoporous silica with Ru(II) and Ru(III) complexes bearing Schiff base ligands derived from salicylaldehyde and various amines (1,2-diaminocyclohexane, 1,2-phenylenediamine, ethylenediamine, 1,3-diamino-2-propanol, N,N-dimethylethylenediamine, 2-aminomethyl-pyridine, and 2-(2-aminoethyl)-pyridine). The incorporation of ruthenium complexes into the porous structure of SBA-15 and the structural, morphological, and textural features of the resulting nanostructured materials were investigated by FTIR, XPS, TG/DTA, zeta potential, SEM, and N2 physisorption. The ruthenium complex-loaded SBA-15 silica samples were tested against A549 lung tumor cells and MRC-5 normal lung fibroblasts. A dose-dependent effect was observed, with the highest antitumoral efficiency being recorded for the material containing [Ru(Salen)(PPh3)Cl] (50%/90% decrease in the A549 cells' viability at a concentration of 70 μg/mL/200 μg/mL after 24 h incubation). The other hybrid materials have also shown good cytotoxicity against cancer cells, depending on the ligand included in the ruthenium complex. The antibacterial assay revealed an inhibitory effect for all samples, the most active being those containing [Ru(Salen)(PPh3)Cl], [Ru(Saldiam)(PPh3)Cl], and [Ru(Salaepy)(PPh3)Cl], especially against Staphylococcus aureus and Enterococcus faecalis Gram-positive strains. In conclusion, these nanostructured hybrid materials could represent valuable tools for the development of multi-pharmacologically active compounds with antiproliferative, antibacterial, and antibiofilm activity.

Dazzling green luminescent and biocompatible Tb3+ -activated lanthanum tungstate nanophosphors for group-III evaluation of latent fingerprints and anticancer applications

Dazzling green emission exhibiting Tb3+ ions doped lanthanum tungstate (LWO) nanophosphors were synthesized by a facile hydrothermally assisted solid-state reaction method. The nanophosphors were determined to have an orthorhombic structure using XRD analysis. In the optimized Tb3+: LWO nanophosphor, FE-SEM revealed aggregated non-uniform nanorods. TEM, FTIR, and XPS studies were performed to examine the material properties. A predominant excitation band was recorded at 488 nm (7F6→5D4) for the Tb3+ doped LWO nanophosphors, along with a moderately intense excitation band at 378 nm (7F6→5G6). Ravishing green emission at 544 nm (5D4→7F5) was obtained from Tb3+ ions containing LWO nanophosphors under excitation at 378 nm and 488 nm. We improved the green emission intensity by increasing the Tb3+ ion concentration up to 14 mol%, which was reduced after 14 mol% due to concentration quenching. The energy-level diagram shows the green radiative transition pathway. The CIE coordinates and color purity were computed for Tb3+: LWO nanophosphors from their emission profiles, and the evaluated coordinates were centralized in the strong green region. A high color purity of 90.1% was obtained appreciably from the optimized Tb3+: LWO nanophosphor. The lifetime of the green emission transition was systematically evaluated based on decay dynamics. The visualization of fingerprints was examined using the optimized green phosphor with an in-depth analysis of the Group-III level details. The non-cytotoxicity of Tb3+ (14 mol%): LWO nanophosphor was confirmed in normal WI-38 lung fibroblast cells. Moreover, these phosphors exhibited strong cytotoxicity against human breast cancer cells of MCF-7. From these results, the synthesized Tb3+ (14 mol%): LWO non-cytotoxic nanophosphor material is considered the best green luminescent material for latent fingerprints in forensic investigation, photonic and anticancer applications.

Calotropis gigantea stem bark extract activates HepG2 cell apoptosis through ROS and its effect on cytochrome P450

The 95% ethanolic extract of the dry powder of Calotropis gigantea (C. gigantea) stem bark was separated by fractionation with different solutions to yield 4 fractions: dichloromethane (CGDCM), ethyl acetate (CGEtOAc), and water (CGW). This research focused on CGDCM-induced apoptosis in HepG2 cells with IC50 and above-IC50 values, which provide useful information for future anticancer applications. CGDCM had lower cytotoxicity on normal lung fibroblast IMR-90 cells than on HepG2 cells. Apoptotic induction of CGDCM was mediated by decreased fatty acid and ATP synthesis while increasing reactive oxygen species production. The effects of the four extracts on the activity of the four major CYP450 isoforms (CYP1A2, CYP2C9, CYP2E1 and CYP3A4) were determined using the CYP-specific model activity of each isoform. All four fractions of the extract were shown to be poor inhibitors of CYP1A2 and CYP2E1 (IC50 > 1000 μg/mL) and moderate inhibitors of CYP3A4 (IC50 = 56.54–296.9 μg/mL). CGDCM and CGW exerted moderate inhibition activities on CYP2C9 (IC50 = 59.56 and 46.38 μg/mL, respectively), but CGEtOH and CGEtOAc exhibited strong inhibition activities (IC50 = 12.11 and 20.43 μg/mL, respectively). It is proposed that C. gigantea extracts at high doses have potential for further studies to develop alternative anticancer applications. Inhibiting CYP2C9 activity may also lead to drug-herb interactions.

Acyclic enediynes fused to triazole and benzothiophene containing propargylamine moieties

Synthesis of 3-(5-triazolyl)- and 3-(3-benzothienyl)-propargylamines includes the Mannich reaction of the corresponding ethynyl derivatives with diaminomethanes. The MTT test of the synthesized compounds toward the viability of NCI-H460 lung carcinoma cells and WI-26 VA4 normal lung fibroblast cells reveals one of them possessing a notable cytotoxic activity.

Cloning a profibrotic stem cell variant in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible, and rapidly fatal interstitial lung disease marked by the replacement of lung alveoli with dense fibrotic matrices. Although the mechanisms initiating IPF remain unclear, rare and common alleles of genes expressed in lung epithelia, combined with aging, contribute to the risk for this condition. Consistently, single-cell RNA sequencing (scRNA-seq) studies have identified lung basal cell heterogeneity in IPF that might be pathogenic. We used single-cell cloning technologies to generate "libraries" of basal stem cells from the distal lungs of 16 patients with IPF and 10 controls. We identified a major stem cell variant that was distinguished from normal stem cells by its ability to transform normal lung fibroblasts into pathogenic myofibroblasts in vitro and to activate and recruit myofibroblasts in clonal xenografts. This profibrotic stem cell variant, which was shown to preexist in low quantities in normal and even fetal lungs, expressed a broad network of genes implicated in organ fibrosis and showed overlap in gene expression with abnormal epithelial signatures identified in previously published scRNA-seq studies of IPF. Drug screens highlighted specific vulnerabilities of this profibrotic variant to inhibitors of epidermal growth factor and mammalian target of rapamycin signaling as prospective therapeutic targets. This profibrotic stem cell variant in IPF was distinct from recently identified profibrotic stem cell variants in chronic obstructive pulmonary disease and may extend the notion that inappropriate accrual of minor and preexisting stem cell variants contributes to chronic lung conditions.

Abstract 3757: miR-200 inhibits the activation of cancer-associated fibroblasts in lung tumor microenvironment

Abstract Cancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment and promote cancer progression and metastasis. CAFs are regulated by diverse processes including regulation by miRNAs. To identify miRNAs involved in the regulation of CAF activation, we profiled miRNA expression within mouse lung normal fibroblasts (LFs) and CAFs using the NanoString nCounter method. Among numerous miRNAs differentially expressed between CAFs and LFs, miR-200 was selected for further investigation, which is more highly downregulated in CAFs. Overexpression of miR-200 in CAFs suppressed the migration and invasion of lung cancer cells in co-culture systems and inhibited metastasis in a mouse model. In addition, we verified that lung cancer patients with high miR-200 levels showed a good prognosis than those with low miR-200 levels. Next, we tried to identify novel miR-200 targets based on RNA sequencing data, lung adenocarcinoma TCGA, and TargetScan. NRP2 was confirmed to be a target of miR-200 by 3΄-UTR assay. NRP2 knockdown by siRNAs in CAFs suppressed cancer cell invasion, and NRP2 addback in CAF-200 restored cancer cell invasion inhibited by miR-200. NRP2 functions as a co-receptor with VEGFR, thus, CAF-200 was less responsive to VEGF than control CAFs. Collectively, this study suggests that downregulation of miR-200 in CAFs promotes lung cancer progression in tumor microenvironment; therefore, miR-200 will be a good therapeutic target or a biomarker in lung adenocarcinoma. Citation Format: Inyoung Cheon, Sieun Lee. miR-200 inhibits the activation of cancer-associated fibroblasts in lung tumor microenvironment. [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 3757.

Abstract 5843: miR-224 activates cancer-associated fibroblasts to promote cancer cell invasion in lung tumor microenvironment

Abstract Cancer-associated fibroblasts (CAFs) promote EMT (epithelial-mesenchymal transition), invasion, and metastasis of cancer cells in the tumor microenvironment. To identify microRNAs (miRNAs) associated with the reprogramming of normal lung fibroblasts (LFs) into CAFs, global miRNA expression was profiled in LFs and CAFs through Nanostring nCounter. miR-224 was one of the increased miRNAs in CAFs, and its expression was higher in tumors than in normal tissues in dbDEMC and TCGA_lung adenocarcinoma data. miR-224 levels were then checked by qRT-PCR in paraffine-embedded tumor samples from patients with lung adenocarcinoma. Kaplan-Meier survival analysis showed that high miR-224 expression was related to poor survival. These data show that lung cancer progression is promoted by miR-224. To discover the roles of increased miR-224 in CAFs, miR-224 was overexpressed by lentiviral infection in LFs. LF_miR-224 overexpression enhanced the expression of CAF activation markers including Fap, S100a4, and Vim. In spheroid invasion assay using co-culture of LFs and cancer cells, as an experimental model of the tumor microenvironment, LF_miR-224 promoted invasion of lung cancer cells compared with control LFs. In transwell migration assay, LF_miR-224 stimulated migration of lung cancer cells more than control LFs. Collectively, these results indicate that CAFs reprogrammed by miR-224 promote the migration and invasion of lung cancer cells. Citation Format: Seonyeong Oh, Inyoung Cheon, Young-Ho Ahn. miR-224 activates cancer-associated fibroblasts to promote cancer cell invasion in lung tumor microenvironment. [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 5843.