Human Lung Adenocarcinoma Cell Research Articles

Folic acid-decorated PEGylated magnetite nanoparticles as efficient drug carriers to tumor cells overexpressing folic acid receptor

The improved drug delivery systems (DDS) are needed for the targeted delivery of their therapeutic cargo (biologically active protein/peptide molecules, nucleic acids, vaccines, etc.) to diseased cells. Thus, we aimed to develop magnetite nanoparticles (Fe3O4), stabilized with polyethylene glycol (PEG) and decorated (surface-functionalized) with folic acid (FA) (Fe3O4@PEG@FA) to ensure targeted internalization in cells expressing the folic acid receptors (FR). The Fe3O4@PEG@FA nanoparticles were synthesized by co-precipitation in a one-pot methodology. Curcumin (Curc), a polyphenol with anti-tumoral activity, was loaded on the nanoparticles, and FA-targeted (Fe3O4@PEG@FA@Curc) and non-targeted (Fe3O4@PEG@Curc) systems were obtained. The internalization of Fe3O4@PEG@FA@Curc and Fe3O4@PEG@Curc nanoparticles was determined in two tumor cell lines, the FR-positive MCF-7 human breast carcinoma cell line and A549 human lung adenocarcinoma cell line, expressing a low level of FR. The results showed that MCF-7 cells internalize FA-functionalized nanoparticles to a greater extent than non-targeted ones and also than A549 cells. The competitive studies performed in the presence of FA in excess suggested that internalization is an FR-dependent process. The increased internalization of Fe3O4@PEG@FA@Curc nanoparticles in MCF-7 cells is correlated with increased cytotoxicity in this cell line compared to A549 cells. In conclusion, the FA-functionalized magnetic systems can ensure a better internalization of the nanoparticles and can be used to deliver various therapeutic agents, both in cancer treatment and also in the treatment of other inflammation-associated diseases such as rheumatoid arthritis, systemic lupus erythematosus, osteoarthritis, Crohn's disease or atherosclerosis.

Ligand-independent integrin β1 signaling supports lung adenocarcinoma development.

Integrins — the principal extracellular matrix (ECM) receptors of the cell — promote cell adhesion, migration, and proliferation, which are key events for cancer growth and metastasis. To date, most integrin-targeted cancer therapeutics have disrupted integrin-ECM interactions, which are viewed as critical for integrin functions. However, such agents have failed to improve cancer patient outcomes. We show that the highly expressed integrin β1 subunit is required for lung adenocarcinoma development in a carcinogen-induced mouse model. Likewise, human lung adenocarcinoma cell lines with integrin β1 deletion failed to form colonies in soft agar and tumors in mice. Mechanistically, we demonstrate that these effects do not require integrin β1–mediated adhesion to ECM but are dependent on integrin β1 cytoplasmic tail-mediated activation of focal adhesion kinase (FAK). These studies support a critical role for integrin β1 in lung tumorigenesis that is mediated through constitutive, ECM binding–independent signaling involving the cytoplasmic tail.

Design, synthesis and biological evaluation of rhein-piperazine-dithiocarbamate hybrids as potential anticancer agents

A series of novel rhein-piperazine-dithiocarbamate hybrids 3 were efficiently synthesized from rhein through a catalyst-free and one-pot, three-step sequence involving chlorination and N-acylation followed by dithiocarbamate formation. Hybrids 3 were evaluated for their in vitro cytotoxic potency by MTT assay against several human cancer and non-cancer cells. Five of the hybrids were more cytotoxic to human lung cancer cell line A549 than the parent rhein and the reference, cytarabine (CAR). Structure-activity relationship (SAR) analysis indicated that cytotoxicity was significantly enhanced when ester groups were incorporated into the hybrids (3h–j). In particular, hybrid 3h (IC50 = 10.93 μg/mL), containing a long-chain alkyl ester, was the most potent compound toward A549 tumor cells, being 7- and 5-fold more toxic than rhein (IC50 = 77.11 μg/mL) and CAR (IC50 = 49.27 μg/mL), respectively. Additionally, hybrid 3h was less toxic to the corresponding normal human lung fibroblast cell line, WI-38, with a higher selectivity index (SI, WI-38/A549 ≈ 5) than doxorubicin (DOX, SI ≈ 0), CAR (SI ≈ 2) and rhein (SI ≈ 1). Furthermore, hybrid 3h displayed more toxicity against four types of lung cancer cells (A549, Calu-1, PC-9, and H460; IC50 = 10.81–23.78 μg/mL) than against six other types of cancer cells (Huh-7, 786-O, HCT116, Hela, SK-BR-3, and SK-OV-3; IC50 = 23.85–51.98 μg/mL). Further mechanistic studies showed that hybrid 3h induced apoptosis in a concentration-dependent manner in human lung adenocarcinoma cell line PC-9. In vivo safety studies showed that hybrid 3h had no acute toxicity to the major organs of mice and did not lead to blood biochemical index changes. Our results exhibit prominent anti-cancer cell inhibition ability and no obvious systemic toxicity to normal organs, indicating that hybrid 3h has promising potential for further applications in anti-lung cancer drug development.

MiRNA-338-3p inhibits the migration, invasion and proliferation of human lung adenocarcinoma cells by targeting MAP3K2.

Objective: This study aimed to investigate the effects of micro ribonucleic acid (miR)-338-3p on the migration, invasion and proliferation of lung adenocarcinoma (LUAD) cells.Methods: Bioinformatics analysis was employed to evaluate the function and expression of related genes in lung cancer. Human A549 and NCI-H1299 cells cultured to logarithmic growth stage were assigned to negative control (NC) mimic group, miR-338-3p mimic group (miR-mimic group), NC inhibitor group and miR-338-3p inhibitor group (miR-inhibitor group) treated with or without MAP3K2 overexpression (OE)-lentivirus, or TBHQ or FR180204. Transwell assay, cell colony formation assay, Western blotting and cell-cycle analysis were carried out.Results: Bioinformatics results manifested that miR-338 and MAP3K2 were involved in LUAD. The expression levels of MAP3K2, p-ERK1/2, MMP-2, MMP-3, MMP-9, cyclin A2 and cyclin D1 were increased after addition of miR-338-3p inhibitor, consistent with the raised amount of LUAD cells in migration and invasion experiments and number of colonies formed, as well as the cell cycle, but miR-338-3p mimic reversed these results. Moreover, MAP3K2 overexpression elevated the level of p-ERK1/2. Meanwhile, after treatment with TBHQ or FR180204, the influence of miR-338-3p inhibitor or mimic was also verified.Conclusions: MiR-338-3p overexpression can modulate the ERK1/2 signaling pathway by targeting MAP3K2, thus inhibiting the migration, invasion and proliferation of human LUAD cells.

Study on the molecular mechanisms of tetrandrine against pulmonary fibrosis based on network pharmacology, molecular docking and experimental verification

AimsThis study aims to screen the potential targets of tetrandrine (Tet) against pulmonary fibrosis (PF) based on network pharmacological analysis, molecular docking and experimental verification. Main methodsThe network pharmacology methods were employed to predict targets, construct Tet-PF-intersection target-pathway networks, and screen the candidate targets. The molecular docking was performed using AutoDockTools1.5.6. TGF-β1-induced human lung adenocarcinoma A549 cells were used as an in vitro experimental verification model, taking dexamethasone (Dex) as the positive control, to verify the effects of Tet on the mRNA expression of the candidate targets. Key findingsSix candidate targets were predicted based on network pharmacology and molecular docking, namely PIK3CA, PDPK1, RAC1, PTK2, KDR, and RPS6KB1. The experimental verification results showed that Dex and Tet presented quite different pharmacological effects. Specifically, compared with the model group, both Dex and Tet (5 μΜ) significantly increased the mRNA expression of PIK3CA and KDR (P < 0.001). Dex up-regulated the mRNA expression of PDPK1 and RAC1, while Tet (1.25 μΜ) down-regulated (P < 0.001). Dex up-regulated the mRNA expression of PTK2, but Tet had no effect. Dex down-regulated RPS6KB1 mRNA expression, while Tet (5 μΜ) up-regulated (P < 0.01). SignificanceCombined with the results of theoretical calculation and experimental verification, and considering the roles of these targets in the pathogenesis of PF, Tet might antagonize PF by acting on PDPK1 and RAC1. The results of this study will provide scientific reference for the prevention and clinical diagnosis and treatment of PF.

S-palmitoylation of PCSK9 induces sorafenib resistance in liver cancer by activating the PI3K/AKT pathway.

Sorafenib is currently the first-line treatment for advanced hepatocellular carcinoma (HCC). However, sorafenib resistance remains a significant challenge. Aberrant AKT signaling activation is a crucial mechanism driving sorafenib resistance in HCC. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a vital role in antitumor immune responses. In this study, we demonstrate that aberrant PCSK9 upregulation promotes cell proliferation and sorafenib resistance in HCC by inducing AKT-S473 phosphorylation. After palmitoylation at cysteine 600, the binding affinity between PCSK9 and tensin homolog (PTEN) is dramatically increased, inducing lysosome-mediated PTEN degradation and subsequent AKT activation. We identify zinc finger DHHC-type palmitoyltransferase 16 (ZDHHC16) as a palmitoyltransferase that promotes PCSK9 palmitoylation at cysteine 600. We also develop a biologically active PCSK9-derived peptide that competitively inhibits PCSK9 palmitoylation, suppressing AKT phosphorylation and augmenting the antitumor effects of sorafenib in HCC.

Enhanced Inhibitory Effect of DC-CIK Cells on Lung Adenocarcinoma via Anti-Tim-3 Antibody and Antiprogrammed Cell Death-1 Antibody and Possible Mechanism.

Objective To investigate the effect and mechanism of blocking the signaling pathways of the T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) and programmed death protein 1 (PD-1) in dendritic cell-cytokine induced killer (DC-CIK) cells on human lung adenocarcinoma A549 cells. Methods Peripheral blood mononuclear cells (PBMCs) were isolated and induced into mature DC-CIK cells by cytokines in vitro. After blocking the Tim-3 and PD-1 signaling transduction pathways with anti-Tim-3 and anti-PD-1 antibodies, DC-CIK cells were coincubated with A549 cells. The killing effect of DC-CIK cells against A549 cells was measured by a CCK-8 assay. The impact of DC-CIK cells on the invasion and migration ability of A549 cells was detected by the Transwell test. The apoptosis rate of DC-CIK cells and the ratio of CD4+, CD8+, and DC-CIK cell subsets were determined by flow cytometry. The cell proliferation of DC-CIK was detected by the CCK-8 assay. Results The antibodies of anti-Tim-3 antibody and anti-PD-1 could block Tim-3+ and PD-1+ DC-CIK cells and could significantly increase the killing effect of DC-CIK cells on A549 cells. The number of A549 cells under the microporous membrane of the Transwell chamber was reduced considerably in invasion and migration tests. Anti-Tim-3 and anti-PD-1 antibodies significantly reduced apoptosis of DC-CIK cells. No significant differences were observed in the ratios of CD4+ and CD8+ DC-CIK cell subsets or the proliferation capacity of DC-CIK cells in each group. Conclusion Blocking the Tim-3 and PD-1 signaling pathways of DC-CIK cells with antibodies can enhance the killing ability of DC-CIK cells in A549 cells and significantly suppress the invasion and migration ability of A549 cells. The potential mechanism may be related to reduced apoptosis of DC-CIK cells.

New Guanidinium and Aminoguanidinim Salts of 2-Hydroxypyridine-3-carboxylic acid: Preparation and spectral, structural, thermal, ADMET, biological, and molecular docking studies

The reaction of guanidine carbonate and aminoguanidine bicarbonate with 2-hydroxypyridine-3-carboxylic acid in various mole ratios (1:1, 1:2, and 2:1) resulted in the formation of the salts [GunH][C6H4NO3]▪H2O (1), [GunH][C12H9N2O6] ▪H2O (2), [GunH]2[C6H3NO3]▪H2O (3), [AmguH][C6H4NO3]▪H2O (4), [AmguH][C12H9N2O6] (5), and [AmguH]2[C6H3NO3]▪H2O (6) [where GunH+- guanidinium (+1) and AmguH+- aminoguanidinium (+1) salts]. Elemental analysis, FT-IR, 1H NMR, and TG-DTA studies were used to identify and characterize the synthesized simple salts. All compounds were polycrystalline in nature, except (1), which was obtained as single crystal. Single-crystal X-ray diffraction studies confirmed the structure of (1), which crystallizes in a monoclinic system with the P21/n space group, with molecular formula C7H12N4O4. FT-IR spectra of the aminoguanidinium salts reveal NN stretching frequencies in the range of 989–914 cm−1, indicating the presence of aminoguanidinium moiety. The NH2 protons of the guanidinium/aminoguanidinium moiety are assigned to the 7–7.5 δ signals in the 1H NMR signals for the simple salts (1)–(6). The thermal reactivity and stability of the as-prepared salts were investigated in air using simultaneous TG-DTA analysis. Except for compounds (5) and (6), the TG-DTA of all the salts showed dehydration followed by continuous decomposition to produce gaseous products. All the salts underwent endothermic decomposition followed by exothermic combustion in the temperatures range from 120 to 450 °C. Furthermore, the salts have appreciable drug-likeness characters according to Lipinski's regulations, as predicted by the in silico ADMET properties. Molecular docking studies of salts also revealed good binding affinity toward 3MIW, 4WM8, and 5IBV proteins. Among the six salts prepared, the diguanidinium salts showed good anticancer activity against human lung adenocarcinoma and human breast adenocarcinoma cell lines. The disk diffusion method was adopted to investigate the antibacterial activities of compounds (1)–(6) in vitro against two bacterial strains. All the salts displayed potential antibacterial activity against Escherichia coli and Staphylococcus aureus.

Influence of the Extraction Solvent and of the Altitude on the Anticancer Activity of Lebanese Eucalyptus camaldulensis Extract Alone or in Combination with Low Dose of Cisplatin in A549 Human Lung Adenocarcinoma Cells

Background: Lung cancer is the second most common cancer worldwide. Eucalyptus plant extract has been shown to have anti-neoplastic effects. We investigated the antitumor effect of ethanolic and aqueous extracts of Eucalyptus camaldulensis collected at different altitudes on A549. In addition, we evaluated the additive effect of its combination with low-dose cisplatin (CDDP). Methods: Qualitative and quantitative analyses of secondary metabolites present in the plants were carried out. The antioxidant and cytotoxic activities of the different extracts on A549 were evaluated using the 2.2-diphenyl-1-picrylhydrazyl radical scavenging activity and neutral red assay, respectively. The cytotoxic effect of the combination of certain extract concentrations with low-dose CDDP on A549 cells was studied. Results: In the Ethanoic extract, a higher number of active substances and antioxidant activities were observed. The four E. camaldulensis extracts showed cytotoxic activity on A549 cells, with a higher cytotoxicity for the Ethanoic extract and the sea-level altitude species. Moreover, the dual exposure of cells to both E. camaldulensis extracts and a low dose of Cisplatin showed an additional cytotoxic effect on A549 cells in certain concentrations. Conclusions: This study opens novel therapeutic options in combinational therapies of Eucalyptus camaldulensis with low-dose CDDP for the treatment of adenocarcinoma cells of human lungs.

Magnolol Induces Apoptosis Through Extrinsic/intrinsic Pathways and Attenuates NF-κB/STAT3 Signaling in Non-small-cell Lung Cancer Cells.

Non-small-cell lung cancer (NSCLC) is the most common type of lung cancer worldwide, and treatment outcomes are still poor. Magnolol, a hydroxylated biphenyl isolated from Magnolia officinalis, was found to be effective against hepatocellular carcinoma via inactivating nuclear-factor-kappa B (NF-B) signaling. However, whether magnolol targets not only NF-B but also other factors in NSCLC and may contribute to the suppression of tumor progression is unclear. Cell viability, flow cytometry, and western blotting assays were used to identify the mechanism of magnolol action in human lung adenocarcinoma cell lines A549 and CL1-5-F4. Our results indicated that magnolol induced cytotoxicity through extrinsic/intrinsic apoptosis signaling and suppressed phosphorylation of signal transducer and activator of transcription 3 (STAT3)/NF-B and expression of their downstream proteins. Magnolol not only induced extrinsic and intrinsic apoptosis signaling but also inactivated STAT3/NF-B and attenuated their signaling of epithelial-mesenchymal transition and metastasis-related protein expression in NSCLC.

Development, characterisation, and in vitro anti-tumor effect of self-microemulsifying drug delivery system containing polyphyllin I.

Polyphyllin I (PPI), an effective active ingredient in Paris polyphylla, has a diverse set of pharmacological properties. However, due to its poor solubility and oral absorption, its application and development are limited. In the study, we were committed to improving the solubility of PPI by developing a self-microemulsifying drug delivery system of PPI (PPI-SMEDDS), screening the best preparation process, and evaluating the quality and the in vivo pharmacokinetics of PPI, and PPI-SMEDDS following oral administration to rats were also studied. In addition, the pharmacological activities against human lung adenocarcinoma cell A549 in vitro were assessed. The best formulation had 15.89% ethyl oleate, 47.38% Cremophor RH40, and 36.73% 1,2 propylene glycol. The produced PPI-SMEDDS was clear and transparent, with an average particle size of 24.51nm and a zeta potential of -17.54 ± 0.51mV. In vitro, the cumulative release rate of PPI-SMEDDS was nearly 80% within 2h. PPI-SMEDDS had a substantially greater area under the curve than PPI following oral treatment in rats, and the relative bioavailability of PPI in rats was 278.99%. More importantly, the anti-tumor effect of PPI-SMEDDS in vitro was significantly greater than that of PPI. These findings suggested that PPI-SMEDDS has the potential to improve the solubility, oral bioavailability of PPI, and anti-tumor effect, laying the groundwork for future research on the new PPI dosage form.

Alcohol Extract of YFXJ Decoction Reverses the Drug Resistance of Human Lung Adenocarcinoma Cell Line A549/DDP.

Objective Lung cancer is the wide and common tumor. This study was designed to explore the effect of YFXJ formula on non-small-cell lung cancer (NSCLC) cell lines. Methods YFXJ formula (mainly composed of Astragalus membranaceus, Atractylodes macrocephala, Radix Saposhnikoviae, Radix Glehniae, coix seed, Herba Sabina chinensis, Hedyotis diffusa, Pericarpium Citri Reticulatae, sarcophagus martensii, Prunella vulgaris, Meretrix meretrix, and oyster) was extracted with 75% ethanol. We performed MTT, FACS, TUNEL, and mass spectrometry to study the effect of YFXJ formula on A549/DDP cells. Results The results showed that YFXJ could inhibit the growth of A549/DDP cells, and it can reverse the sensitivity of A549/DDP cells to cisplatin. YFXJ inhibits the expression of MDR1, MRP1, and LRP genes in A549/DDP cells. Conclusion YFXJ formula can reverse the drug resistance of A549/DDP cell, which could be through activation of autophagy.

Arisaema heterophyllum Blume Monomer Stigmasterol Targets PPARγ and Inhibits the Viability and Tumorigenicity of Lung Adenocarcinoma Cells NCI-H1975.

To clarify the regulatory effect and molecular mechanism of Arisaema heterophyllum Blume (AhBl) monomer stigmasterol on lung adenocarcinoma in human lung adenocarcinoma cells NCI-H1975 cultured in vitro and in nude mice. Oil red O staining, free fatty acid detection, adenosine triphosphate (ATP), and NADPH were applied to elucidate the regulatory effect of stigmasterol on the energy metabolism of NCI-H1975 cells. Simultaneously, colony formation assay and nude mouse tumorigenesis were performed to clarify the underlying mechanisms of stigmasterol on the proliferation and tumorigenesis of NCI-H1975 cells. Furthermore, peroxisome proliferator-activated receptor gamma (PPARγ) inhibitor GW9662 was supplemented to determine the expression changes of cyclins to clarify the regulation mechanism of stigmasterol. The results revealed that stigmasterol administration markedly inhibited the viability but promoted lipid deposition of NCI-H1975 cells. Meanwhile, the reduction of cell energy metabolism affected cell proliferation and colony formation. qPCR and western blot assays indicated that stigmasterol played a role in regulating the expression of cyclins and PPARγ signaling pathway proteins. Nude mouse tumorigenesis suggested that tumor size and weight in the stigmasterol-treated group were apparently lower as compared with the control group. Tumor tissue cells developed varying degrees of degeneration and large areas of ischemic necrosis presented in the central and peripheral cells. Immunohistochemistry results revealed that Ki67 expression in the stigmasterol group was substantially inhibited, while PPARγ expression was greatly elevated as compared with the control. GW9662 could mediate the inhibitory effect of stigmasterol on NCI-H1975 cells. The current study demonstrated that stigmasterol targeted PPARγ and inhibited the viability and tumorigenicity of lung adenocarcinoma cells NCI-H1975.

Synthesis of vildagliptin loaded acrylamide-g-psyllium/alginate-based core-shell nanoparticles for diabetes treatment

Diabetes mellitus has become a major public health concern all over the world. Vildagliptin is one of the antidiabeticdrug that can overcome the existing problem of this prevalent disease. Present study aims to synthesize and investigate the role of vildagliptin-loaded core-shell nanoparticle of grafted psyllium and alginate (VG@P/A-NPs) in anti-diabetes application. FTIR, SEM, XRD, 13CNMR and zeta analyzer were used for characterization of the core-shell nanoparticles (VG@P/A-NPs). The synthesized acrylamide-grafted-psyllium was also optimized through varying grafting parameters such as acrylamide and ceric ammonium nitrate (CAN) concentration, time and temperature to obtain the maximum yield of acrylamide-grafted-psyllium. Rheological analysis of pure psyllium, grafted psyllium and alginate were also performed. For biological studies, the first cytotoxicity of grafted psyllium and VG@P/A-NPs were examined on human lung adenocarcinoma cell line A549 in which it was observed that VG@P/A-NPs did not exhibited any toxicity. The antidiabetic potential of VG@P/A-NPs was investigated by glucose uptake assay, using TNF-α induced insulin resistance skeletal cell model using mouse muscle L6 cell line. The insulin signaling impaired cell line displayed a highly significant (p < 0.0001) dose-dependent increase in glucose uptake after treatment with increasing doses of VG@P/A-NPs.The drug release behavior of VG@P/A-NPs was examined at various pH and the highest drug release (98 %) was obtained at pH (7.4). The drug release kinetic data was following the Higuchi (R2 = 0.9848) kinetic model, suggesting the release of drug from vildagliptin-loaded grafted psyllium-alginate core-shell nanoparticles (VG@P/A-NPs) as a square root of time-dependent process and diffusion controlled. This study provides an economical and environment-friendly approach towards the synthesis of VG@P/A-NPs with antidiabetes applications.

Protein corona formation on silver nanoparticles under different conditions

The surfaces of nanoparticles become covered by biomolecules in biological fluids. This protein ‘corona’ modifies materials’ characteristics and biological activity. The composition of the protein corona is dynamic, abundant biomolecules that bind first are subsequently replaced by less abundant but more tightly bound ones. Here, we explore the formation of the silver nanoparticle protein corona on exposure to cell culture media containing 10 % fetal bovine serum supplemented Dulbecco's Modified Eagle's medium. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry analysis were used to monitor how different parameters such as incubation time, heating duration, cell culture medium, incubation temperature, and the number of washes affect the nanoparticle–protein corona complex. silver nanoparticles with and without bound proteins were characterized by electron microscopy, dynamic light scattering, and ultraviolet-visible-near-IR spectroscopy. The tetrazolium-based MTT assay was used to determine viability of A549 human lung adenocarcinoma cells treated with silver nanoparticles. Characterization of the nanoparticles before and after protein binding provided insights into their changing morphology on corona formation. Our results confirmed that the physiological environment directly affects protein corona formation on nanoparticle surfaces. In particular, incubation condition-dependent differences in the amount of bound proteins were observed. This work highlights the importance of environmental drivers of protein adsorption, which should be considered when predicting and/or controlling protein targets of silver nanoparticles.

Depleting hsa_circ_0000567 suppresses acquired gefitinib resistance and proliferation of lung adenocarcinoma cells through regulating the miR-377-3p / ZFX axis: an in vitro and in vivo study.

Circular RNAs (circRNAs) expression profile has been reported in lung adenocarcinoma (LUAD) cells resistant to gefitinib, and hsa_circ_0000567 was abnormally upregulated. However, its precise role in gefitinib resistance remains unclarified. Levels of hsa_circ_0000567, microRNA (miR)-377-3p and zinc finger protein X-linked (ZFX) were detected by real-time quantitative PCR. Direct attachment was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay. Gefitinib resistance was measured by MTT assay, colony formation assay, flow cytometry, western blotting, and xenograft in mice. Expression of hsa_circ_0000567 was upreguated in human gefitinib-resistant human LUAD tissues and cells (HCC827/GR and PC9/GR). Clinically, higher hsa_circ_0000567 correlated with advanced tumor burden. Blockage of hsa_circ_0000567 suppressed cell viability, half maximal inhibitory concentration (IC₅₀) value, colony formation ability, and expression of Bcl-2 and proliferating cell nuclear antigen (PCNA) in HCC827/GR and PC9/GR cells under gefitinib treatment or not, accompanied with enhanced apoptosis rate and Bax expression. In vivo, tumor growth of PC9/GR cells untreated and treated with gefitinib was restrained by silencing hsa_circ_0000567. miR-377-3p was directly regulated by hsa_circ_0000567, and then targeted ZFX. Similar to hsa_circ_0000567 knockdown, overexpressing miR-377-3p inhibited chemoresistance in genitinib-resistant LUAD cells in vitro, whereas, depleting miR-377-3p was able to promote gefitinib resistance in spite of hsa_circ_0000567 knockdown. Moreover, restoring ZFX abrogated miR-377-3p-mediated chemosensitivity in genitinib-resistant LUAD cells in vitro. The hsa_circ_0000567/miR-377-3p/ZFX axis might contribute to acquired gefitinib resistance in LUAD cells both in vitro and in vivo, suggesting hsa_circ_0000567 as a novel therapeutic target in treatment of gefitinib-resistant LUAD.

Dimethoxycurcumin Acidifies Endolysosomes and Inhibits SARS-CoV-2 Entry

The pandemic of coronavirus disease 2019 (COVID-19) caused by infection by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) continues to take a huge toll on global health. Although improving, currently there are only limited therapies against SARS-CoV-2. Curcumin, a natural polyphenol, exerts antiviral effects against a wide variety of viruses and can inhibit SARS-CoV-2 entry. However, undesirable physicochemical and pharmacokinetic properties of curcumin limit its clinical application. Here, we determined the effects of dimethoxycurcumin (DiMC), a methylated analog of curcumin with improved bioavailability, on the entry of SARS-CoV-2. DiMC blocked entry of pseudo-SARS-CoV-2 into Calu-3 human non-small cell lung adenocarcinoma cells and Vero E6 green monkey kidney epithelial cells. Mechanistically, DiMC acidified lysosomes, enhanced lysosome degradation capabilities, and promoted lysosome degradation of angiotensin converting enzyme 2 (ACE2), a major receptor for SARS-CoV-2 entry, as well as pseudo-SARS-CoV-2 and the SARS-CoV-2 S1 protein. Furthermore, other lysosome acidifying agents, including the TRPML1 agonist ML-SA1 and the BK channel activator NS1619, also blocked the entry of pseudo-SARS-CoV-2. Thus, the anti-SARS-CoV-2 potential of DiMC and lysosome acidifying agents might be explored further as possible effective therapeutic strategies against COVID-19.

Cucurbitacin B Down-Regulates TNF Receptor 1 Expression and Inhibits the TNF-α-Dependent Nuclear Factor κB Signaling Pathway in Human Lung Adenocarcinoma A549 Cells.

Pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), induce the expression of intracellular adhesion molecule-1 (ICAM-1) by activating the nuclear factor κB (NF-κB) signaling pathway. In the present study, we found that cucurbitacin B decreased the expression of ICAM-1 in human lung adenocarcinoma A549 cells stimulated with TNF-α or interleukin-1α. We further investigated the mechanisms by which cucurbitacin B down-regulates TNF-α-induced ICAM-1 expression. Cucurbitacin B inhibited the nuclear translocation of the NF-κB subunit RelA and the phosphorylation of IκBα in A549 cells stimulated with TNF-α. Cucurbitacin B selectively down-regulated the expression of TNF receptor 1 (TNF-R1) without affecting three adaptor proteins (i.e., TRADD, RIPK1, and TRAF2). The TNF-α-converting enzyme inhibitor suppressed the down-regulation of TNF-R1 expression by cucurbitacin B. Glutathione, N-acetyl-L-cysteine, and, to a lesser extent, L-cysteine attenuated the inhibitory effects of cucurbitacin B on the TNF-α-induced expression of ICAM-1, suggesting that an α,β-unsaturated carbonyl moiety is essential for anti-inflammatory activity. The present results revealed that cucurbitacin B down-regulated the expression of TNF-R1 at the initial step in the TNF-α-dependent NF-κB signaling pathway.

Antitumor and Immunomodulatory Properties of the Bulgarian Endemic Plant Betonica bulgarica Degen et Neič. (Lamiaceae).

Background: Extracts obtained from different Betonica species have been shown to possess important biological properties. The present study aimed to investigate the cytotoxicity, antitumor and immunomodulatory potential of the endemic plant Betonica bulgarica (Lamiaceae) and thus, reveal new aspects of its biological activity. Methods: Methanolic extract obtained from inflorescences was analyzed for cytotoxicity against mammalian cell lines. The antitumor potential of the sample was determined using human cervical and lung adenocarcinoma cells (HeLa and A549). Programmed cell death-inducing effects against HeLa cells and peripheral blood lymphocytes, as well as immunomodulatory properties of the extract were determined by flow cytometry analysis. Results: The research results demonstrated that the extract has significant inhibitory potential against HeLa cells (mean IC50 value 119.2 μg/mL). The sample selectively induced apoptotic death in tumor cells. Cytotoxic effects towards mouse cell lines were detected following treatment with high concentrations of Betonica bulgarica extract (200 and 250 μg/mL). Twenty-four-hour ex vivo incubation of peripheral blood leucocytes in growth medium containing plant extract induced prominent effects in distinct immune cell populations. They included elevated levels of CD25+ and CD56+ T cells’ lymphocytes, particularly CD4+CD25+ and CD8+CD56+ cells. Conclusions: The present study demonstrates that Betonica bulgarica inflorescence extract possesses potential beneficial antitumor and immunomodulatory activity and could serve as a source of bioactive compounds with biomedical application.

Abstract 6108: In vivo CRISPR screens identified dual function of MEN1-MLL1 in regulating tumor-microenvironment interactions

Abstract INTRODUCTION: CRISPR functional genomic screens have been widely adopted to identify essential genes and potential drug targets in cell line models. However, it is well known that cell line models studied in vitro do not fully capture the biology in patient tumors due to the lack of the tumor microenvironment. The primary objective of this study was to perform functional genomic screens in in vivo models to identify clinically relevant epigenetic vulnerabilities. METHODS: We designed an EpiDrug sgRNA library that target 357 epigenetic regulators in human, and applied this targeted sgRNA library for essentiality screen in 2D cell culture and 3D xenograft models. We subsequently selected targets that can only be identified in vivo for validation. We also investigated the underlying mechanism of the in vivo specific phenotype caused by target knockouts. Finally, we explored the clinical significance of treating selected target with a small molecule inhibitor. RESULTS: We identified MEN1 as the top hit that confers differential essentialities between in vitro and in vivo models. Knockout of MEN1 has no impact on cell proliferation in 2D cell culture but profoundly promotes tumor growth in human lung and colorectal adenocarcinoma cell line derived xenograft models. In syngeneic colon cancer model CT26, knockout of Men1 resulted in faster tumor growth in immune deficient mice, but reduced tumor growth in immune competent mice. Mechanistically, knockout of MEN1/Men1 can cause the redistribution of its interaction partner MLL1/Mll1, a histone methyl transferase, to repeat regions in chromatin and stimulate the expression of double stranded RNA. This resulted in viral mimicry response, which activates a group of genes involved in cytokine-cytokine receptor interaction. Single-cell RNA-seq and CyTOF analysis revealed that activation of the cytokines induces tumor promoting neutrophil and tumor suppressing CD8+ T cell infiltration in immunodeficient and immunocompetent mice, respectively. Using patient data from TCGA, we identified that the expression level of MEN1 is negatively correlated with neutrophil and CD8+ T cell tumor infiltration in most cancer types including lung and colorectal adenocarcinoma. A small molecular inhibitor targeting Men1-Mll1 interaction dramatically reduced CT26 tumor growth in immunocompetent mice, but its phenotype was reversed by CD8 neutralizing antibody. Finally, we demonstrated the strong synergy of the inhibitor with anti-PD-L1 antibody. CONCLUSION: Our study demonstrated the utility of in vivo CRISPR screen in identifying therapeutic targets that modulate tumor-microenvironment interactions, and identified MEN1 as a promising therapeutic targets alone or in combination with immunotherapy. Citation Format: Peiran Su, Yin Liu, Ming-Sound Tsao, Housheng H. He. In vivo CRISPR screens identified dual function of MEN1-MLL1 in regulating tumor-microenvironment interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6108.