A549 Human Non-small Cell Lung Research Articles

The Effect of 1'-acetoxychavicol Acetate on A549 Human Non-small Cell Lung Cancer

Lung cancer, a global leading cause of cancer mortality, requires safer and more effective treatments. 1'-acetoxychavicol acetate (ACA) is notable for its wide-ranging therapeutic properties, spanning anticancer, anti-inflammatory, and anti-obesity effects. While its efficacy has been explored in various cell lines, its potential against lung cancer remains few reported. This research aimed to investigate the impact of ACA on cytotoxicity, anti-proliferation, and antimigration potential in A549 human non-small cell lung cell lines. The investigation utilized MTT assays to assess cell viability and determined the IC50 values of ACA at 24, 48, and 72 hours to be 50.42 µM, 33.22 µM, and 21.66 µM, respectively. These results reveal a concentration- and time-dependent reduction in cell viability following ACA treatment. In addition, anti-proliferation was performed using colony-forming assays, and ACA showed notable efficacy in significantly decreasing colony formation, highlighting its strong impact on cell proliferation and viability. In the final part, ACA had promising inhibitory effects on migration, which varied depending on the dose used in the scratch assay. In conclusion, ACA highlighted cytotoxic and anti-proliferation properties of A549 cell line, supporting its potential role in lung cancer therapy pending further investigation and development.

Synthesis and anticancer activity evaluation of some novel imidazo[1,2-a]pyridine based heterocycles containing S-alkyl/aryl moiety

Here we report the synthesis and anticancer activities of fourteen novel imidazo[1,2-a]pyridine derivatives (6a-6n) containing S-alkyl/aryl moiety. The target compounds were obtained via a six-step synthetic route. 1-(4-Aminophenyl)ethan-1-one was the starting material. In the last step, 2-chloro-N-[4-(imidazo[1,2-a]pyridin-2-yl)phenyl]acetamide (5) was allowed to react with various aliphatic/aromatic thiol derivatives, affording the final compounds 6a-6n. The cytotoxicity of the compounds was evaluated against A549 (human non-small cell lung cancer), C6 (rat glioma), MCF-7 (human breast carcinoma) and HepG2 (human liver carcinoma) tumor cells and NIH/3T3 (mouse embryonic fibroblast cells) healthy cells. First step MTT assay reported that compounds 6a, 6d, 6e and 6i exhibited antiproliferative activity against all tested tumor lines. Second step BrdU cell proliferation assay and flow cytometric analysis revealed that compounds 6d and 6i inhibited DNA synthesis on HepG2 cell line time-dependently by apoptotic pathway. Molecular docking study of compounds 6d and 6i with caspase-3 and caspase-9 revealed their binding interactions with the enzyme’s active site, confirming the experimental findings.

Genome-guided discovery of two undescribed 6,6-spiroketal polyketides and stereochemical correction of bafilomycins P and Q from the marine-derived Streptomyces sp. SCSIO 66814

Bafilomycins are macrocyclic polyketides with intriguing structures and therapeutic value. Genomic analysis of Streptomyces sp. SCSIO 66814 revealed a type I polyketide synthase biosynthetic gene cluster (BGC), namely blm, which encoded bafilomycins and featured rich post-modification genes. The One strain many compounds (OSMAC) strategy led to the discovery of six compounds related to the blm BGC from the strain, including two previously undescribed 6,6-spiroketal polyketides, streptospirodienoic acids D (1) and E (2), and four known bafilomycins, bafilomycins P (3), Q (4), D (5), and G (6). The structures of 1 and 2 were determined by extensive spectroscopic analysis, quantum calculation, and biosynthetic analysis. Additionally, the absolute configurations of the 6/5/5 tricyclic ring moiety containing six consecutive chiral carbons in the putative structures of 3 and 4 were corrected through NOE analysis, DP4+ calculation, and single-crystal X-ray diffraction data. Bioinformatic analysis uncovered a plausible biosynthetic pathway for compounds 1–6, indicating that both streptospirodienoic acids and bafilomycins were derived from the same blm BGC. Additionally, sequence analysis revealed that the KR domains of module 2 from blm BGC was B1-type, further supporting the configurations of 1–4. Notably, compounds 3 and 4 displayed significant cytotoxic activities against A-549 human non-small cell lung cancer cells and HCT-116 human colon cancer cells.

Abstract 510: Flow cytometry evaluation of cell cycle arrest in A549 human non-small cell lung cancer cells and tumors following treatment with cell cycle inhibitors: A multifaceted approach to targeting cancer

Abstract The contemporary landscape of cancer treatment has witnessed significant progress with FDA-approved targeted drugs. The FDA's Project Optimus has underscored the growing need for innovative pharmacodynamic endpoints to determine the optimal biological dose in clinical development. Flow cytometry, a potent analytical tool in clinical and preclinical labs, now plays a leading role in cancer research, enabling the evaluation of new drug effects on cell cycle regulation and consideration of rational drug combinations with approved drugs. At TD2, we strive to enhance the translatability of in vitro induced cell cycle arrest studies to in vivo biomarker proof of concept and efficacy. We employ a comprehensive approach, utilizing flow cytometry to assess cell cycle dynamics in cultured cells and in vivo tumor models after treatment with approved and novel drugs. This abstract focuses on the translational relevance of combined in vitro and in vivo testing of well-known drugs that disrupt the cell cycle, laying the foundation for optimized treatment strategies and potentially personalized cancer therapies. Human A549 non-small cell lung carcinoma cells were treated with either thymidine (2.0mM) or the microtubule-depolymerizing drug nocodazole (100ng/ml) for 18 hours, then examined by flow cytometry. Thymidine induced a greater than 85% S-phase arrest, while nocodazole induced a greater than 88% G2/M-phase arrest. A549 cells (5 × 106 cells/mouse) were subcutaneously implanted in SCID mice, followed by treatment with an FDA-approved cell cycle inhibitor, Palbociclib, or cisplatin chemotherapy. Palbociclib, a CDK4/6 inhibitor, and low-dose cisplatin treatment resulted in an increased accumulation of cells in the G0/G1 phase, greater than 80% for each. Both treatments exhibited promising anti-cancer activity, with tumor growth inhibition (TGI) of 69% and 87%, respectively. Our findings highlight flow cytometry's multifaceted utility in assessing cell cycle arrest, providing a comprehensive view of agent effects on cancer cells in culture and within the complex in vivo tumor microenvironment. Determining the impact of any new drug on specific cell cycle checkpoints could allow for a more strategic plan for combination therapy. Integrating flow cytometry could enable early biomarker monitoring of tumor cell cycle changes, offering insights into the therapeutic potential of new agents alone or in combination. This multidimensional evaluation contributes to understanding cancer biology and has the potential to guide the development of novel therapeutic strategies, optimize treatment regimens to identify optimal biological doses, and improve patient outcomes in the era of precision medicine. Citation Format: Erin E. Trachet, Emily Cluff, Ana Lemos de Matos, Damaris Diaz, Chelsea Holmquist, Paul Gonzales. Flow cytometry evaluation of cell cycle arrest in A549 human non-small cell lung cancer cells and tumors following treatment with cell cycle inhibitors: A multifaceted approach to targeting cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 510.

A near-infrared emitting “off-on” fluorescent probe for bioimaging of Pd(Ⅱ) ions in living cells and mice

BackgroundThe surging consumption of palladium in modern industry has given rise to its accumulation in the ecosystem, posing conspicuous toxicity to aquatic organisms and human health. The investigation of palladium in biological systems is highly demanded for the in-depth understanding of its dynamics and behaviors. Fluorescence imaging serves as a powerful approach to assess palladium species in biological systems, and currently most of the sensing probes are applicable to living cells. Effective tracking of palladium species in living organisms is challenging, which requires sufficient hydrophilicity and imaging depth of the probes. ResultsBased on an intramolecular charge transfer (ICT) mechanism, a distyryl boron dipyrromethene (BODIPY) derivative (DISBDP-Pd) has been prepared for the near-infrared (NIR) fluorescence imaging of Pd2+ ions. Two additional methoxy triethylene glycol (TEG) chains could serve as flexible and hydrophilic moieties to enhance the aqueous solubility and cell permeability of the extended conjugate. Solution studies revealed that DISBDP-Pd exhibited a NIR fluorescence enhancement signal exclusively to Pd2+ ions (detection limit as low as 0.85 ppb) with negligible interference from Pd0 species and other closely related metal ions. Computational calculations have been performed to rationalize the binding mode and the mechanism of action. Fluorescence imaging assays have been conducted on A549 human non-small cell lung carcinoma cells and mouse models. Exhibiting negligible cytotoxicity, DISBDP-Pd demonstrated concentration-related fluorescence enhancement signals in response to Pd2+ ions in living cells and mice. SignificanceDISBDP-Pd exhibits advantages over many small molecule palladium probes in terms of satisfactory aqueous solubility, high sensitivity and selectivity, and biocompatible NIR emission property, which are particularly favorable for the sensing application in biological environments. The design strategy of this probe can potentially be adopted for the functionalization of other BODIPY probes implemented for NIR fluorescence bioimaging.

Cisplatin Dependent Secretion of Immunomodulatory High Mobility Group Box 1 (HMGB1) Protein from Lung Cancer Cells.

High mobility group box 1 (HMGB1) is secreted from activated immune cells, necrotic cells, and certain cancers. Previous studies have reported that different patterns of post-translational modification, particularly acetylation and oxidation, mediate HMGB1 release and confer distinct extracellular HMGB1 signaling activity. Here we report that cisplatin but not carboplatin induces secretion of HMGB1 from human A549 non-small cell lung cancer (NSCLC) cells. Cisplatin-mediated HMGB1 secretion was dose-dependent and was regulated by nuclear exportin 1 (XPO1) also known as chromosomal maintenance 1 (CRM1) rather than adenosine diphosphate (ADP)-ribosylation, acetylation, or oxidation. HMGB1, as well as lysine acetylation and cysteine disulfide oxidation of secreted HMGB1, were monitored by sensitive and specific assays using immunoprecipitation, stable isotope dilution, differential alkylation, and nano liquid chromatography parallel reaction monitoring/high-resolution mass spectrometry (nano-LC-PRM/HRMS). A major fraction of the HMGB1 secreted by low-dose cisplatin treatment of A549 NSCLC cells was found to be in the fully reduced form. In contrast, mainly oxidized forms of HMGB1 were secreted by dimethyl sulfoxide (DMSO)-mediated apoptosis. These findings suggest that inhibition of XPO1 could potentiate the anti-tumor activity of cisplatin by increasing the nuclear accumulation of HMGB1 protein, an inhibitor of cisplatin DNA-adduct repair. Furthermore, low-dose cisplatin therapy could modulate the immune response in NSCLC through the established chemokine activity of extracellular reduced HMGB1. This could potentially enhance the efficacy of subsequent immunotherapy treatment.

Design, synthesis, and anticancer activity of novel 4,6-dimorpholinyl-1,3,5-triazine compounds

Abstract A series of novel 4,6-dimorpholinyl-1,3,5-triazine derivatives 6a–6r were obtained through N-substitution and Claisen-Schmidt condensation. 1H NMR, 13C NMR, and mass spectrometry were used to characterize the molecular structures of the derivatives. The in vitro antiproliferation activity of derivatives was evaluated using the MTT assay against SW620 (human colon cancer cells), A549 (human nonsmall cell lung cancer cells), HeLa (human cervical cancer cells), and MCF-7 (human breast cancer cells). Compound 6o bearing a pyridyl group exhibited good cytotoxicity against four cancer cells, with IC50 values of 8.71, 9.55, 15.67, and 21.77 μM, sequentially. In addition, compound 6a showed some selectivity against SW620.

Abstract 4092: Development of nucleotide metabolic reprogrammed CAR T cell therapy suitable for solid tumors

Abstract CAR T cell therapy has represented an exciting breakthrough in the treatment of patients with hematologic malignancies. In contrast, the antitumor activity of CAR T cell therapy in solid tumors has been modest so far in clinical studies. The suboptimal clinical efficacy is a result of multiple factors including limited T cell trafficking, persistence, and the immunosuppressive tumor microenvironment (TME). While current strategies utilize costimulatory molecules and cytokines to activate CAR T cells, a key but largely overlooked problem of CAR T cell therapy is nutrient competition between tumor cells and T cells in the nutrient-poor TME. In our previous studies, a metabolic screen identified inosine as an alternative fuel for T cells which can support T cell growth and function in the absence of glucose. T cells could metabolize inosine into hypoxanthine and phosphorylated ribose by purine nucleoside phosphorylase. Moreover, we demonstrated that T cells have advantage over cancer cells in utilizing inosine, and supplementation with inosine enhances the antitumor efficacy of PD-1 mAb and adoptive T cell transfer in mouse models. In this study, we therefore engineered CAR T cells to express CD26 on the cell surface and a secreted adenosine deaminase (ADA) fused with an anchor. These metabolic reprogrammed CAR (designated as MRCAR) T cells exerts their activities through different mechanisms. ADA irreversibly converts adenosine to inosine, overcoming adenosine-mediated immunosuppression and providing inosine for CAR T cell growth, while CD26 induces a rich chemokine receptor profile enabling CAR T cells to traffic to solid tumors. We demonstrated that CD26-overexpressed CAR T cells displayed superior migration capacity and resisted TGF-beta suppression, while the CD26 expression on unmodified CAR T cells was significantly down-regulated by TGF-beta. We further proved that ADA is conditionally secreted in stress condition (i.e., low pH, lack of nutrient and oxygen) based on ADA’s biological characteristics, suggesting that ADA activates CAR T cells as a trans-signaling to the CAR construct in a tumor specific manner. Remarkably, we observed that the MRCAR T cells inhibit tumor growth more effectively than unmodified CAR T cells in both Huh7 human hepatocellular carcinoma (GPC3-MRCAR) and A549 human non-small cell lung cancer (HER2-MRCAR) mouse models. Thus, MR-CAR T cell therapy represents a promising approach to improve CAR T cell therapy against solid tumors. Citation Format: Yue Hu, Abhijit Sarkar, Kevin Song, Magnus Hook, Andras Heczey, Xiaotong Song. Development of nucleotide metabolic reprogrammed CAR T cell therapy suitable for solid tumors. [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 4092.

Effect of Extraction Solvent and Harvest Time on Recovery of Bioactive Com-pounds, Antioxidant and Cancer Cell Growth Inhibition Activities of Korean Ca-mellia mistletoe

This study aimed to determine the phytochemical profiles,in vitroantioxidant and antiproliferative properties of KoreanCamellia mistletoe(Korthalsella japon-ica(Thunb.) Engl.) depending on the harvest time (August and November) and sol-vent (100% methanol, 70% ethanol, and hot water). TheCamellia japonicaL. mistle-toe extracts were analyzed for total phenol, flavonoid, carotenoid, and L-ascorbic acid contents and antioxidant properties such as scavenging capacities (1,1-diphenyl-2-pic-rylhydrazyl (DPPH) and NO•),ferrous ion chelating and reducing power. Concur-rently, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) as-say was used to assess the antiproliferative properties against human cancer cell lines; MCF (human breast cancer cells), HeLa (human cervical cancer cells), A375 (human malignant melanoma cells), HCT116 (human colon cancer cells), HepG2 (human liver cancer cells) and A549 (human non-small cell lung adenocarcinoma cells). The results showed that extraction solvent and harvest time had significant impacts on antioxidant and anticancer activities and selectivity for free phenolic compounds inCamellia ja-ponicaL. mistletoe. Among all the tested extracts, the highest amounts of total phe-nolic and total flavonoids content were found in ethanol extracts ofCamellia japon-icaL. mistletoe harvested in November, while the methanol extracts ofCamellia ja-ponicaL. mistletoe collected in August showed the highest contents of total carote-noids and L-ascorbic acidscompared to the other tested extracts. Additionally, the highest NO•radical scavenging activity was found in ethanol extracts, whereas the strongest DPPH radical scavenging activity was found in methanol extracts ofCamel-lia japonicaL. mistletoe harvested in November. Furthermore, methanol extracts showed much higher antiproliferative activity against all human cancer cells than eth-anol extracts ofCamellia japonicaL. mistletoe harvested in November. In conclusion, the antioxidant and anticancer properties ofCamellia japonicaL. mistletoe showed significant dependence on the extraction solvent type and harvest time. With optimum harvest time and extraction solvent, they boast a wide range of promising medical, pharmaceutical, and food applications

Cytotoxicity and acute toxicity evaluation of hydrogen-rich Gynostemma pentaphyllum Makino distillate

Hydrogen-rich Gynostemma pentaphyllum Makino distillate (HRGD) consists of Gynostemma pentaphyllum Makino steam distillate with hydrogen gas. Although both G. pentaphyllum Makino and hydrogen-rich water are well known for their biological and medical benefits, there is a lack of information on their safety and toxicity in vivo acute oral toxicity test and in vitro cytotoxicity method. The current study aimed to assess the cytotoxicity and acute oral toxicity of HRGD as a part of a safety evaluation using rat and human cell models. HRGD was administered orally once by gavage to male and female Sprague-Dawley rats at doses of 0, 2500, and 5000 mg/kg. Cytotoxicity assay was conducted in vitro at various concentrations in 10 different human normal and cancer cell lines; TK6 (human normal lymphomablastoid cells), Chang (human hepatic cells), 16HBE14o- (human bronchial epithelial cells), URotsa (human urothelium cells), MCF (human breast cancer cells), Hela (human cervical cancer cells), A375 (human malignant melanoma cells), HCT116 (human colon cancer cells), HepG2 (human liver cancer cells) and A549 (human non-small cell lung adenocarcinoma cells). From a 14-day study in rats, we observed no compound-related changes in mortality, clinical signs, body weight, food/water consumption, organ weight and gross pathology in all dose group. The result of in vivo acute toxicity shows that no observed adverse effect level of HRGD was below 5000 mg/kg for both sexes of rats, and the minimal lethal dose was considered to be more than 5000 mg/kg. HRGD also had no in vitro cytotoxicity against all tested cells. The present study data indicated that HRGD may contain bioactive compounds of potential therapeutic significance that are relatively safe from toxic effects.

TM2, a novel semi-synthetic taxoid, exerts anti-MDR activity in NSCLC by inhibiting P-gp function and stabilizing microtubule polymerization

Taxane agents are of particular interest in non-small cell lung carcinomas (NSCLC) treatment, while multidrug resistance (MDR) mediated by P-glycoprotein (P-gp) limits their clinical efficacy. TM2, a chemically semi-synthesized taxane derivative, exerted significant anti-cancer efficacy in vitro and in vivo, especially against vincristine-resistant and adriamycin-resistant cancer cells. In this study, the anti-cancer effect of TM2 on drug-resistant NSCLC was evaluated both in vitro and in vivo, and the mechanism underlying its anti-MDR activity was further clarified. It was found that TM2 was significantly cytotoxic to cisplatin- and paclitaxel-resistant A549 (human non-small cell lung cancer) cells that overexpressing P-gp, resulting in IC50 values of 0.19 µM and 0.12 µM. TM2 micelles (5mg/kg, 10mg/kg, 20mg/kg, i.v., 21 days) inhibited the growth of MDR xenograft with the maximal inhibitory rate up to 80.4%. Moreover, TM2 caused cell cycle arrest in the G2-M phase and apoptosis in drug-resistant cells through promoting tubulin polymerization, which acted in a way similar to taxane agents. Notably, TM2 acted as a P-gp inhibitor with high binding affinity, which resulted in impaired efflux function through forming H-bonds and ATP hydrolysis to induce P-gp conformational alterations. These findings indicated that TM2 displays anti-MDR activity with the potential for the treatment of NSCLC, which can inhibit P-gp function and stabilize microtubule polymerization.

Exploring the interaction of sesamol as an antilung cancer compound with albumin through spectroscopic and bioinformatic analyses and the mechanism of anticancer effect

Sesamol has moved into biomedical research in recent years. However, its interactions with blood proteins and cancer cells have not been fully explored. Therefore, we aimed to investigate the interaction of sesamol with human serum albumin (HSA), A549 human nonsmall cell lung cancer (NSCLC) cell line, and Raw 264.7 macrophage. The interaction of HSA with sesamol was explored via application of fluorescence and circular dichroism (CD) spectroscopy studies as well as molecular docking analysis. Then, the cytotoxic effects of sesamol on A549 lung cancer cells and Raw 264.7 macrophages were evaluated by qPCR analysis. It was found that sesamol spontaneously (ΔG˚=-45.89 kJ/mol) binds with HSA having a high affinity (log K b = 8.05, n = 1.70, T = 298 K ) and form a static complex trough contribution of hydrogen bonds and van der Waals interactions (ΔH˚=-409.43 kJ/mol, TΔS˚=-363.54 kJ/mol) which was supported by molecular docking study. Furthermore, by using CD and synchronous fluorescence spectroscopy analyses it was found that sesamol induced some minor secondary and tertiary structural changes, respectively in HSA structure. Cellular assays displayed that sesamol triggered selective cytotoxicity against A549 lung cancer cells through regulation of intrinsic apoptosis pathway mediated by mitigation of mitochondrial membrane potential, elevation of ROS generation, downregulation of Bax, and up regulation of caspase-9, −3. In conclusion, it was found that sesamol could show high affinity with HSA and mediate intrinsic apoptosis pathway through ROS generation in the A549 lung cancer cell lines. These data indicate that the biochemical and anticancer mechanisms of sesamol can be further investigated in future studies to integrate it in the biomedical platforms.

Pennogenin-3-O-α-L-Rhamnopyranosyl-(1→2)-[α-L-Rhamnopyranosyl-(1→3)]-β-D-Glucopyranoside (Spiroconazol A) Isolated from Dioscorea bulbifera L. var. sativa Induces Autophagic Cell Death by p38 MAPK Activation in NSCLC Cells.

In our previous study, we reported the isolation of pennogenin-3-O-α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→3)]-β-D-glucopyranoside (spiroconazol A), a steroidal saponin, from the flowers of Dioscorea bulbifera L. var. sativa. In the present study, we aimed to investigate the effects of spiroconazol A on autophagy and its underlying mechanisms in A549 and NCI-H358 human non-small cell lung cancer (NSCLC) cells. Spiroconazol A inhibited the proliferation of NSCLC cells in a concentration- and time-dependent manner. To determine the type of programmed cell death induced by spiroconazol A, we performed a characterization of apoptosis in spiroconazol A-treated A549 cells. Our results showed that spiroconazol A significantly suppressed A549 cell viability but did not influence cell apoptosis because phosphatidylserine and caspase activation were not detected. Furthermore, spiroconazol A treatment upregulated the expression of LC3-II and autophagy-related Beclin-1 protein, suggesting that spiroconazol A induces autophagy in A549 cells. Moreover, spiroconazol A activated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) but did not affect the phosphorylation of Janus kinase or ERK1/2. Notably, SB203580, a p38 MAPK inhibitor, had a significant inhibitory effect on spiroconazol A-induced autophagic cell death in A549 cells. Our results indicated that spiroconazol A-induced autophagy is dependent on p38 MAPK signaling and has potential as a therapeutic target in NSCLC.

Effects of miRNA-149-5p and Platelet-Activating Factor-Receptor Signaling on the Growth and Targeted Therapy Response on Lung Cancer Cells

Accumulating evidence indicates that microRNAs (miRs) play critical roles in essentially all biological processes and their altered expression has been documented in various disease conditions, including human malignancies. Although several cellular mechanisms have been identified in mediating the effects of miRs, the involvement of G-protein-coupled, platelet-activating factor-receptor (PAFR) signaling in miR-149-5p-induced effects on lung cancer growth and therapeutic potential has not been studied. To that end, we first evaluated the functional significance of PAFR and miR-149-5p in A549 and H1299 human non-small cell lung cancer (NSCLC) cell lines. We observed that these tumor lines express endogenous PAFR and miR-149-5p and that PAFR activation by PAF agonist (CPAF) significantly increased, whereas miR-149-5p mimic transfection inhibited cell proliferation in a dose-dependent manner. Interestingly, miR-149-5p mimic significantly attenuated CPAF-mediated increased proliferation of NSCLC cells, as confirmed by miR-149-5p, cyclin D1, and forkhead box protein M1 (FOXM1) expression analysis via qPCR. Our next studies examined PAFR- and miR-149-5p-mediated effects on targeted therapy (i.e., erlotinib and gefitinib) responses. We observed that erlotinib and gefitinib inhibited A549 and H1299 cell survival in a dose- and time-dependent manner, and CPAF significantly blocked this effect. These findings indicate that miR-149-5p blocks PAFR-mediated increased cell proliferation, and PAFR activation attenuates the cytotoxic effects of targeted therapy.

Spherical Invasion Assay: A Novel Method to Measure Invasion of Cancer Cells.

The invasion of tumor cells into the neighboring blood vessels and lymph nodes is a vital step for distant metastasis. Traditionally, the invasive activity of growth factors (or the anti-invasive activity of drugs) is measured with the Boyden chamber assay. However, this assay has a few disadvantages like poor physiological relevance of transwell inserts and an inability to control chemokine gradients. The Boyden chamber assay is one of the most prevalent methods to measure the invasion of cancer cells. It would be advantageous to develop another assay that could validate the results of the Boyden chamber assay. With this in mind, our laboratory developed the spherical invasion assay (SIA) to measure the pro-invasive activity of human cancer cells. The SIA also circumvents some of the drawbacks of the Boyden chamber assay. The present manuscript measures the anti-invasive activity of the Src kinase inhibitor PP2 in A549 human non-small cell lung carcinoma (NSCLC) cells using the SIA. The SIA protocol is comprised of two steps. In the first step, A549 human NSCLC cells (treated or not with PP2) were mixed with Matrigel and seeded in the middle of an eight-well chamber slide. After 24 h, a second layer of Matrigel was overlaid over the first layer. Over the course of the next 24 h, the A549 cells invade from the primary to the secondary Matrigel layers. Subsequently, the cells are visualized by phase-contrast microscopy and the images obtained are quantified using ImageJ to calculate the anti-invasive activity of PP2 in A549 cells. The results of the SIA correlate well with Boyden chamber assays. The SIA may be adapted for multiple experimental designs, such as drug screening (to combat invasion and metastasis), measuring the pro-invasive activity of growth factors, and elucidating the signaling pathways underlying the pro-invasive/anti-invasive activity of biological modifiers. Graphic abstract: Diagrammatic illustration of the spherical invasion assay ( Hurley et al., 2017 ) . A. The first layer is comprised of human cancer cells mixed in a 1:1 suspension with Phenol Red containing Matrigel (represented as LAYER 1 in the figure). After 24 h, the cancer cells grow and extend up to the boundary of this first layer. B. A second layer of 1:1 solution Phenol Red-free Matrigel, in Phenol Red-free RPMI (represented as LAYER 2 in the figure) is added on top of the first Matrigel spot. The cells are incubated for 24 h at 37°C. C. Over these 24 h, the cancer cells invade from the primary layer into the secondary Matrigel layer. The chamber slides are observed by phase-contrast microscopy. D. A representative photograph of the images obtained by the SIA is shown. The black arrow indicates the cancer cells invading into the second layer of Matrigel. The dotted line represents the interface between the two layers. The distance to which the cells have traveled (into the secondary Matrigel layer) is measured at ten sites (for each photograph) in a randomized double-blind fashion by three independent observers, using NIH ImageJ Version 1.47. This process is repeated for three separate photographic fields per sample.

Three-dimensional prints from 3-dimensional cell culture aggregates of human cancer cell lines

Objectives: Three-dimensional (3D) printing has gained significance for human health-care applications in recent years. Some of these applications include obtaining models which mimic anatomical parts. One other parallel development in the biological research area is the development of 3D cell cultures. Such cultures are now becoming the material of choice for in vitro experiments, fast replacing the traditional adherent/monolayer 2D culture approaches. We present here, a method to obtain 3D prints of 3D aggregates of three human cancer cell lines. Such 3D prints can be useful models to understand solid tumor morphologies and also as effective teaching models. Materials and Methods: Photomicrographs of the 3D aggregates of the human cancer cell lines SiHa, MCF-7, and A549 (human cervical cancer, breast cancer, and non-small cell lung cancer cell lines, respectively) were obtained using inverted phase contrast microscopy. Conversion of normal jpeg images into 3D files was performed using the lithophane method and CAD files obtained. The CAD files thus generated were used to print the objects using the Stratasys Polyjet J750 3D Printer. Results: We could obtain 3D prints of SiHa, MCF-7, and A549 (human cervical cancer, breast cancer, and non-small cell lung cancer cell lines, respectively) 3D aggregates/spheroids. Conclusion: It is hoped that this approach will be useful for studying solid tumor morphologies in finer details. Furthermore, other benefits of such 3D prints would be in them being excellent models for teaching purposes.

Fish Oil, Plant Polyphenols, and Their Combinations Have No Tumor Growth Promoting Effects on Human Lung and Colon Carcinoma Xenograft Mice

The goal of this study was to evaluate if combinations of ingredients with known anti-cachexia benefits (Fish oil-FO with either curcumin or Green tea extract-GTE), have adverse effects on tumor growth, using human carcinoma xenograft mice models. FO (EPA/DHA 360 mg/kg bw), GTE (90 mg/kg bw), and curcumin (180 mg/kg bw) were administered orally, alone or in combination, to nude mice bearing either A549 human non-small cell lung carcinoma or SW620 human colon carcinoma tumors. Bodyweight, tumor growth, survival time, and other clinical endpoints were assessed. The ingredients either alone or in combinations were well tolerated in both lung and colon tumor-bearing mice. There were no significant group differences between individual or combination treatments for tumor growth (A549 or SW620) as measured by the median time in days to endpoint of tumor volume (TTE). TTE results indicate that these ingredients (alone or combinations) did not adversely impact tumor growth. No significant differences in body weights or survival were observed between controls and treatment groups indicating no adverse health effects of the ingredients. In conclusion, FO, GTE or curcumin administered as monotherapies and in combination were well tolerated and displayed no adverse effects on tumor growth in mouse xenograft models of lung and colon cancer.

M6A-seq analysis of microRNAs reveals that the N6-methyladenosine modification of miR-21–5p affects its target expression

In eukaryotes, N6-methyladenosine (m6A) is one of the most abundant modifications on RNAs, and it plays important roles in many biological processes and diseases such as cancer. While most m6A researches focus on message RNAs and long non-coding RNAs, recent studies have reported the presence of m6A in small RNAs. Nevertheless, current knowledge about m6A prevalence in mature microRNAs (miRNA) is extremely limited and the functional significance of m6A methylation in miRNAs remains to be elucidated. Here, we demonstrated cell-specific m6A profiles of miRNAs in A549 human non-small cell lung cancer (NSCLC) cells and HEK293A cells by using miRNA m6A immunoprecipitation sequencing and constructed the consensus motif in m6A-enriched miRNAs de novo. We found that miR-21–5p, an oncogenic miRNA, showed the highest m6A enrichment in NSCLC cells. Depletion of the demethylase ALKBH5 did not change the expression level of miR-21–5p, but altered the m6A abundance of miR-21–5p, thereby changing the expression levels of its target gene. We further synthesized m6A modified miR-21–5p mimics in vitro and demonstrated that in NSCLC cells, m6A marks in mature miR-21–5p could directly affect its silencing potency towards target genes, which finally impaired its promotion to proliferation and motility. Together, our findings reveal the landscape of m6A modification in mature miRNAs, and provide the first evidence that it may contribute to the mRNA responses to cancer-related miRNAs.

Biomimetic Recognition-Based Bioorthogonal Host–Guest Pairs for Cell Targeting and Tissue Imaging in Living Animals

Biomimetic Recognition-Based Bioorthogonal Host–Guest Pairs for Cell Targeting and Tissue Imaging in Living Animals

The Cardenolide Glycoside Acovenoside A Interferes with Epidermal Growth Factor Receptor Trafficking in Non-Small Cell Lung Cancer Cells.

Cardenolide glycosides are natural compounds known to inhibit the ion pumping function of the Na+/K+-ATPase in cellular systems. Interestingly, various cancer cell types are highly susceptible to cardenolide glycosides. Herein, we explore the cardenolide glycoside Acovenoside A (AcoA) with respect to its influences on human A549 non-small cell lung cancer (NSCLC) cells. We found that exposure to AcoA, digoxin and ouabain increases intracellular sodium and ATP levels indicating that the ion pumping function of the transmembrane Na+/K+-ATPase is effectively inhibited. Like digoxin and ouabain, AcoA inhibits transcription factor NF-κB activation and induces apoptotic cell death in NSCLC cells. This was confirmed by a preclinical in vivo model in which AcoA treatment of NSCLC xenografts grown on chick chorioallantoic membranes inhibited the expression of proliferation antigen Ki-67 and induced apoptotic DNA strand breaks. We aimed to elucidate the underlying mechanisms. The Na+/K+-ATPase transmembrane complex contains Src kinase and epidermal growth factor receptor (EGFR). Indeed, we found that AcoA activates Src kinase in A549 cells, but not in a cell-free assay using recombinant Src kinase. Src kinase is a downstream target of EGFR, and correlation analysis using the NCI60 database pointed to a role of EGFR in cardenolide glycoside-induced cancer cell death. Accordingly, NSCLC cells expressing hyperphosphorylated EGFRmut exhibited resistance to AcoA. To investigate the interaction between cardenolide glycosides and EGFR in detail, we performed immunoblotting studies: Whereas ligand binding and EGFR phosphorylation were not significantly affected, ubiquitinated EGFR accumulated after prolonged incubation with AcoA. To visualize EGFR trafficking we used A549 cells transfected with a fluorescent biosensor which binds to activated EGFR. Pretreatment with AcoA and digoxin induced accumulation of EGFR in endosomal compartments thus inhibiting EGF-induced EGFR degradation comparable to the Na+ ionophore monensin, a known inducer of EGFR endosomal arrest. Intracellular Na+ concentrations regulate EGFR trafficking and signaling. Na+ homeostasis is maintained by the Na+/K+-ATPase, which might account for its close interaction with the EGFR. Cardenolide glycosides inhibit the ATP-dependent Na+/K+ exchange through the Na+/K+-ATPase resulting in higher intracellular Na+ levels. Our data provide first evidence that this impedes efficient EGFR trafficking at the endosomal compartment.