Epithelial A549 Research Articles

Abstract 962: A hybrid epithelial-mesenchymal phenotype identifies lung cancer cells with high metastatic potential due to increased stemness properties and escape from NK immunosurveillance

Abstract Introduction: Epithelial to mesenchymal transition (EMT) is essential for cancer dissemination but its role in overt metastasis establishment remains an open issue. Multiple EMT states and great phenotypic plasticity have also been described, indicating the need for in-depth characterization of biological properties associated with different EMT phenotypes. The so-called hybrid phenotype, characterized by presence of both mesenchymal and epithelial markers, might in fact identify cancer cells with enhanced metastatic propensity. Materials and Methods: To assess the metastatic potential of different EMT phenotypes, we exploited two lung cancer cell lines with different properties (A549/epithelial and LT73/hybrid) and generated their respective sublines through four sequential rounds of in vitro migration assay (named M4). TGFβ treatment was also used to induce EMT. With a previously validated score based on the ratio between expression of epithelial (CDH1) or mesenchymal genes (SNAI2), cells were classified as: epithelial (A549)>hybrid (A549-M4, LT73)>mesenchymal (LT73-M4, A549-TGFβ). The disseminating/metastatic properties of the different lines were tested in vivo by subcutaneous and tail-vein injections in SCID mice. Interaction with NK cells (NKs), known to specifically target EMT cancer cells, was also assessed. Results and Discussion: Compared to parental cells, M4 cells showed up-regulation of EMT-associated genes, expansion of the subset of CD133+ lung cancer stem cells (CSC) and displayed in vivo higher ability to disseminate from primary tumors to murine lungs (2-fold change, p=0.004). However, hybrid cells (LT73 and A549-M4) showed an increased ability to generate overt lung metastases in experimental metastasis assays compared to either epithelial (A549) or mesenchymal cells (LT73-M4 and A549-TGFβ). The higher metastatic ability of hybrid cells was correlated with CSCs expansion and reduced NKs infiltration to the metastatic lungs. Expression of IL-8, a cytokine involved in NKs recruitment, was decreased in hybrid cells and they were less prone to chemoattract NKs in vitro compared to both epithelial and mesenchymal cells. Finally, in vivo prevention of NKs recruitment to the lungs using a neutralizing antibody, massively increased metastasis formation and expansion of CSCs in mice injected i.v. with mesenchymal A549-TGFβ (p=0.0006), but had limited effects in groups injected with both epithelial A549 and hybrid A549-M4. Conclusions: Our data indicate that the hybrid phenotype correlates with the highest metastatic potential related to efficient cancer cell dissemination, increased induction of stemness features and ability to avoid NKs recruitment. Conversely, fully mesenchymal cells show limited metastatic potential, possibly due the control exerted by NKs on their CSC subsets. Citation Format: Giulia Bertolini, Monica Parodi, Massimo Milione, Fabio Murianni, Giovanni Centonze, Giulia Taiè, Gabriella Sozzi, Massimo Vitale, Luca Roz. A hybrid epithelial-mesenchymal phenotype identifies lung cancer cells with high metastatic potential due to increased stemness properties and escape from NK immunosurveillance [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 962.

Abstract 1632: An orthotopic model for luciferase-labeled human lung epithelial carcinoma with response to standard of care treatment

Abstract The human lung epithelial carcinoma A549 cell line was first developed in 1972 after culturing pulmonary carcinoma tissue from a 58-year-old Caucasian male. Similar to human non-small cell lung carcinoma (NSCLC), this cell line carries a KRAS mutation making an attractive model for drug discovery/oncology research applications. NSCLC tumors grow slower and less aggressively than small cell lung cancers, yet the 5-year survival rate for NSCLC is 25%. Thus, more relevant preclinical models are still needed to evaluate the potential therapeutic activities of agents that can be used in the clinic. Orthotopic implantation of luciferase-labeled cancer cell lines facilitate the evaluation of disease progression using bioluminescence in vivo imaging. Here we report the development of a novel model of orthotopically implanted Luciferase-labeled A549-luc cells, its tumor growth kinetics and response to standard of care therapy. To establish the orthotopic model, we implanted cells by direct injection into the left lung lobe through a small incision on the lateral thorax between the 6th and 7th rib. Disease progression was evaluated by testing cell implants at multiple cell inoculums and following each group for tumor growth using an IVIS Spectrum CT. In line with the observed slow growth of NSCLC, A549 tumor growth kinetics revealed long latency with a median survival of 69.5-days post-implant. Mean flux values correlated with the number of cells inoculated and clinical observations of respiratory distress. Gross necropsies provided visual evidence of masses in the left lung lobe and thoracic cavity. Following the A549-luc orthotopic model development, we sought to evaluate the survival benefit in response to standard of care agents Paclitaxel and the tyrosine kinase inhibitors Erlotinib and Afatinib. Paclitaxel monotherapy was the most active agent in a 101-day study. A single treatment cycle showed robust decrease tumor burden as determined via imaging during the first few weeks following treatment, but disease progression was apparent despite the overall improved survival over the vehicle control group. To establish durable responses, we evaluated the efficacy of Paclitaxel administered for two dosing cycles. Palbociclib, a cyclin-dependent kinase 4/6 inhibitor that has been recently identified as potential target in KRAS-mutant (NSCLC) was also evaluated. Our preliminary results show significant activity for both Palbociclib and Paclitaxel therapies in the A549-luc lung orthotopic xenograft model. This lung orthotopic model provides a sensible alternative that recapitulates the difficulties of treating NCSL tumors in addition to establishing a more clinically relevant model to evaluate cancer therapies compared to standard subcutaneous implanted models. Histopathological analysis of Ki67, Cyclin D1, and CDK4 will be presented from lungs of treated and control groups. Citation Format: Elizabeth Rainbolt, Andrew Wong, Chassidy Hall, Patrick Wood, Paula L. Miliani De Marval. An orthotopic model for luciferase-labeled human lung epithelial carcinoma with response to standard of care treatment [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 1632.

Abstract 2756: Multiplexed-engineered, iPSC-derived T cells expressing three unique targeting modalities address tumor heterogeneity and antigen escape

Abstract Adoptive T-cell therapy with chimeric antigen receptor (CAR) has shown promising results in cancer treatment, however, antigen escape and tumor heterogeneity are major causes for disease relapse. While CARs are known to trigger an effective immune response through surface antigen recognition many solid tumor cancer antigens are intracellular and presented by MHC molecules recognized by T cell receptors (TCRs). In addition, many therapeutic antibodies have shown clinical efficacy in solid tumor settings. However, antibody-dependent cellular cytotoxicity (ADCC) is mediated by the CD16 Fc receptor naturally expressed on NK cells although its application in T cells is yet not fully appreciated. Utilizing our proprietary induced pluripotent stem cell (iPSC) platform to engineer multiple modalities into a clonal iPSC line, which can serve as the starting cell source for mass production of off-the-shelf, iPSC-derived CAR-T cells (CAR-iT cells), we aimed to study the combination of these three targeting modalities, CAR, TCR, and CD16, to determine whether challenges associated with the treatment of solid tumors, which are heterogeneous and challenging to treat, may be overcome.To test the base line activity of CAR-iT cells in the solid tumor setting, we selected our anti-MICA/B CAR, previously shown to effectively target stress ligands found on transformed cells, to demonstrate effective anti-tumor activity against multiple solid tumor cell lines (72 hrs cytotoxicity: A2058 = 99%; 786-O = 98%; versus non-specific CAR-iT cells: A258 = 13%; 786-O = 17%). To test compatibility of TCR in our iT cell platform, we engineered MR1-TCR in iT cells to show increased cytokine release and degranulation upon stimulated with MR1 positive lung carcinoma epithelial cells line A549 (fold change compared to un-stimulated: IFNg = 210, p = 0.0032; TNFa = 76.9, p = 0.0005; CD107ab = 115.0, p=0.0013). Notably, with the engineering of tumor antigen specific TCR in TCR-less CAR-iT cells, CD3 complex can be re-established to provide an opportunity to combine with bispecific T cell engagers. Lastly, combining CAR-iT cells with MR1-TCR and hnCD16 uniquely demonstrated synergistic tumor growth inhibition and validated our approach to target multiple antigens at once for an effective anti-tumor response (A549 cytotoxicity: tumor only = 3.68±2.04%; effector+TCR = 41.31±2.27%; effector+TCR+ADCC = 90.28±1.87%). In summary, using the unique approach to engineer iPSCs at the clonal level to create a distinct population of engineered iT cells, we successfully demonstrated the compatibility between CAR, TCR, and hnCD16 to mitigate tumor heterogeneity. This approach is an ideal strategy to create off-the-shelf cellular immunotherapy for a promising therapeutic approach to combat heterogeneous and difficult to treat solid tumors, including those that are resistant due to antigen escape. Citation Format: Chia-Wei Chang, Bi-Huei Yang, Eason Lin, Soheila Shirinbak, Wen-I Yeh, Mochtar Pribadi, Helen Chu, Alma Gutierrez, Earl Avramis, Jason ORourke, Tom Lee, Alec Witty, Eigen Peralta, Martin Hosking, Bahram Valamehr. Multiplexed-engineered, iPSC-derived T cells expressing three unique targeting modalities address tumor heterogeneity and antigen escape [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 2756.

Antiviral Activity of a Cyclic Pro-Pro-β3-HoPhe-Phe Tetrapeptide against HSV-1 and HAdV-5.

The core of Cyclolinopeptide A (CLA, cyclo(LIILVPPFF)), responsible for its high immunosuppressive activity, contains a Pro-Pro-Phe-Phe sequence. A newly synthesized cyclic tetrapeptide, cyclo(Pro-Pro-β3-HoPhe-Phe) (denoted as 4B8M) bearing the active sequence of CLA, was recently shown to exhibit a wide array of anti-inflammatory properties in mouse models. In this investigation, we demonstrate that the peptide significantly inhibits the replication of human adenovirus C serotype 5 (HAdV-5) and Herpes simplex virus type-1 (HSV-1) in epithelial lung cell line A-549, applying Cidofovir and Acyclovir as reference drugs. Based on a previously established mechanism of its action, we propose that the peptide may inhibit virus replication by the induction of PGE2 acting via EP2/EP4 receptors in epithelial cells. In summary, we reveal a new, antiviral property of this anti-inflammatory peptide.

Exosomal MicroRNA-328 from Bone Marrow Mesenchymal Stem Cells (BMSCs) Alleviates Acute Lung Injury Through Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase (MAPK/ERK) Pathway

To elucidate the communication between exosomes (exo) derived from BMSCs and injured lung cells. BMSC-exo was isolated and characterized. Lung epithelial cells A549 were incubated with BMSC-exo, and treated by LPS to induce cell damage. CCK-8 assay was carried out to test cell proliferation, flow cytometry was adopted to analyze cell apoptosis, and RT-qPCR as well as Western blot analysis were selected to assess expression of apoptosis- and anti-apoptosis related proteins. Functional experiment was performed to identify the role of microRNA (miRNA)-328 in lung injury. LPS treatment significantly inhibited the viability of A549 cells, induced apoptosis of A549 cells by increasing Bax and casepase-3 levels and reducing Bcl-2 expression, whilst declined expression of miR-328 and suppressed the phosphorylation activation of the MAPK/ERK pathway. Meanwhile, the amount of IL-6, IL-1β and TNF-α were elevated in injured cells, but, the presence of BMSC-exo eliminated the elevation of the contents. Importantly, treatment with BMSC-exo increased miR-328 expression, activated MAPK MAPK/ERK pathway, inhibited apoptosis, and enhanced cell proliferation. However, the effect of BMSC-exo was attenuated when the cells were silenced for miR-328 expression. Collectively, BMSC-exo enriched miR-328 could relieve acute lung injury through MAPK/ERK pathway.

Toxicological assessment of nanocrystalline metal alloys with potential applications in the aeronautical field

The development of new candidate alloys with outstanding characteristics for their use in the aeronautical field is one of the main priorities for the sector. In this context, nanocrystaline (nc) alloys are considered relevant materials due to their special features, such as their exceptional physical and mechanical properties. However, another important point that needs to be considered with newly developed alloys is the potential toxicological impact that these materials may have in humans and other living organisms. The aim of this work was to perform a preliminary toxicological evaluation of three nc metal alloys (WCu, WAl and TiAl) in powder form produced by mechanical alloying, applying different in vitro assays, including a mix of W-Cu powders with standard grain size in the experiments to stablish comparisons. The effects of the direct exposure to powder suspensions and/or to their derived leachates were analysed in three model organisms representative of human and environmental exposures (the adenocarcinomic human alveolar basal epithelial cell line A549, the yeast Saccharomyces cerevisiae and the Gram negative bacterium Vibrio fischeri). Altogether, the results obtained provide new insights about the potential harmful effects of the selected nc alloys, showing that, from a toxicological perspective, nc TiAl is the safest candidate in the model organisms and conditions tested.

Polyhexamethylene guanidine phosphate, chloromethylisothiazolinone, and particulate matter are dispensable for stress granule formation in human airway epithelial cells

ABSTRACT Environmental risk factors are recognized as threats to public health. Stress granules (SGs) are non-membranous assemblies of mRNAs and proteins expressed in response to various stressors to promote cell survival. In this study, SG formation was examined to confirm the effects of polyhexamethylene guanidine phosphate (PHMG), chloromethylisothiazolinone (CMIT), and particulate matter (PM10) in airway epithelial cells, A549, HPAEpiC, and BEAS-2B cells. SGs were not observed after CMIT, PHMG, and PM10 treatments, as determined by immunofluorescence microscopy. Moreover, there was no change in the phosphorylation of the translation initiation factor eIF2αfollowing treatment with PHMG, CMIT, and PM10. Taken together, our findings might help determine the biological hazards of these materials.

Attenuated lncRNA NKILA Enhances the Secretory Function of Airway Epithelial Cells Stimulated by Mycoplasma pneumoniae via NF-κB.

The secretory function of airway epithelial cells is important in the pathogenesis of Mycoplasma pneumoniae pneumonia (MPP). To investigate the regulatory function of NKILA (nuclear factor-κB (NF-κB) interacting long noncoding RNA (lncRNA)) in MPP, we first detected NKILA as well as the concentration of interleukin 8 (IL-8) and tumor necrosis factor-α (TNF-α) in bronchoalveolar lavage fluid of children with MPP. Then, NKILA was knocked down in epithelial cells to investigate its effect on their secretory function. The results suggested that NKILA was downregulated in children with MPP, while IL-8 and TNF-α levels increased. Knockdown of NKILA in vitro promoted the inflammatory effects of Mycoplasma pneumoniae (MP) in epithelial A549 and BEAS-2B cells. Knockdown of NKILA promoted inhibitor of κBα (IκBα) phosphorylation and degradation, and NF-κB p65 nuclear translocation. Furthermore, RNA immunoprecipitation showed that NKILA could physically bind to IκBα in MP-treated A549 cells. Collectively, our data demonstrated that attenuation of NKILA enhances the effects of MP-stimulated secretory functions of epithelial cells via regulation of NF-κB signaling.

Redistribution of EGF Receptor and α5, β1 Integrins in Response to Infection of Epithelial Cells by Serratia proteamaculans

Eukaryotic cells use surface receptors to interact with the environment. Interacting with the cell receptors, pathogenic and opportunistic bacteria can invade cells that normally do not phagocytize. Bacteria Serratia proteamaculans can invade eukaryotic cells, and this ability correlates with the proteolytic activity of the actin-specific protease protealysin (Tsaplina et al., 2009). However, cellular receptors involved in the mechanism by which these bacteria penetrate the cells are not known. Bacterial outer membrane protein OmpX which is one of the substrates of protealysin is known to interact with EGF receptor and fibronectin. Therefore, in this work, changes in the localization of EGF receptors and fibronectin receptors α5, β1 integrins were monitored in the epithelial M-HeLa and A549 cells infected by bacteria S. proteamaculans. It turned out that in response to the infection α5 and β1 integrin subunits accumulate on the surface of M-HeLa cells, and bacteria attach intensively at the sites of the α5 integrin localization. The α5 integrin was not detected in A549 cells. The β1 integrin, localized evenly along the cell perimeter, accumulated in the cytoplasm in response to contact with bacterial cells, while the EGF receptor formed granules in the cytoplasm in response to invasion. A similar redistribution of EGF receptors occurs in response to EGF binding. These results indicate that invasion of S. proteamaculans into the epithelial cells of different origin occurs as a result of their interaction with different surface receptors.

CircRNA_104889 promotes lung adenocarcinoma cell invasion via sponging miR4458

BackgroundLung adenocarcinoma is metastatic cancer with a high mortality rate. Circular RNAs (circRNAs) are a type of noncoding RNA and play a vital role in cancer progression. However, the expression and function of circRNAs in lung adenocarcinoma are still mostly unknown.MethodsIn this study, we screened the differential expression of circRNAs in human bronchial epithelial cells (HBE) and A549 human lung adenocarcinoma cell line (A549) by human circRNA microarray and RT-qPCR. The role of overexpressed circRNA_104889 in A549 cell proliferation, apoptosis, migration, and invasion was studied extensively. Intracellular localization of circRNA_104889 was visualized by FISH assay. MiRNA sponging, ERK1/2 signaling, and caspase-3 expression were analyzed in siRNA-mediated circRNA_104889 knockdowned A549 cells.ResultsCircRNA microarray showed overexpression of circRNA_104889 (> 13-fold) in A459 cells compared to HBE. This finding was further corroborated by the RT-qPCR result. CircRNA_104889 was mainly localized in the cytoplasm of A549 cells. The knockdown of circRNA_104889 in A549 cells by si-RNA mediated RNA interference did not affect cell proliferation and apoptosis but significantly inhibited cell migration and invasion in vitro. Furthermore, knockdown of circRNA_104889 led to an increase of miR4458 expression. Overexpression of miR4458 inhibited A549 cell migration. Both the knockdown of circRNA_104889 and overexpression of miR4458 inhibited the caspase-3 expression and ERK1/2 phosphorylation in A549 cells.ConclusionsCircRNA_104889 promotes lung adenocarcinoma cell migration and invasion by sponging miR4458 and targeting ERK1/2 signaling and caspase-3 expression.

Dexamethasone prevents the Epstein-Barr virus induced epithelial-mesenchymal transition in A549 cells.

Clinical data have shown that pulmonary interstitial fibrosis is likely to occur in the later stages of viral pneumonia. While viral infections are thought to cause chronic pulmonary interstitial inflammation and pulmonary fibrosis, it remains unclear if they promote pulmonary fibrosis by epithelial-mesenchymal transition (EMT). In this study, human epithelial cell line A549 has been used to model the infection of the Epstein-Barr virus (EBV) and the respiratory syncytial virus (RSV). Their differences were compared and the possible infection mechanisms analyzed by randomly assigning cells to one of five treatments. Exposure of the LMP1 is thought to be the key gene during EBV-induced EMT in the A549 cells. Enzyme-linked immunosorbent assay analysis revealed that the EBV infection was associated with the induction of a number of cytokines (interleukin-8 [IL-8], IL-13, tumor necrosis factor-α, and transforming growth factor-β) and dexamethasone (DXM) could significantly prevent the phenotypic changes, and partly the mechanisms related with the IL-13 pathway. Surprisingly, different results were seen with the RSV infection as the A549 cells still displayed an epithelial morphology but the levels of E-cadherin, α-SMA, vimentin, and fibronectin did not change. This is the first study demonstrating the different reactions induced by different viruses, and the protective effects of DXM on the EBV-induced EMT in the A549 cells by partially inhibiting the IL-13 pathway. These findings suggest a novel mechanism, by which DXM or anti-IL-13 may delay the progression of pulmonary fibrosis by preventing the progress of EBV-induced EMT.

Abstract B011: Inhibition of G9a reestablishes the MHC class I loss due to EMT in lung cancer cells

Abstract The epithelial-to-mesenchymal transition (EMT) is an important phenotype for cancer cells to invade and metastasizes. In addition, cancer cells should escape from the immune system during malignant progression. Here, we aimed to demonstrate the potential of G9a inhibition of EMT in cancer cells and most importantly, the reestablishment of MHC class I expression (MHC-I), one of the important factors associated with the loss of antigenicity of cancer cells. The lung epithelial cancer cell line A549 was cultured under controlled conditions in RPMI culture media supplemented with 10% of bovine fetal serum (BFS), 1% of antibiotics (penicillin and streptomycin), 2% of glutamine in incubator at 37oC and air atmosphere containing 5% of CO2. The tumor growth factor beta (TGFb) exposure for 5 days was used to induce EMT. In addition, another group of A549 cells received TGFb plus the UNC0368 an G9a (a histone methyl transferase) inhibitor for 5 days. Then, cells were evaluated for acquiring mesenchymal cell morphology and MHC-I gene expression (relative quantification). After 5 days, A549 cells exposed to TGFb clearly have gone through EMT by changing their morphology to a fibroblast-like phenotype. Also, the MHC-I gene expression decreased significantly (p=0.0036) after EMT. The G9a inhibitor partially impeded the EMT induced changes in morphology of A549 cells and reestablished the MHC-I gene expression to the control level. In conclusion, G9a inhibition seemed a promising therapeutic target to improve the efficacy of immunotherapies depending on neoantigen expression by cancer cells. However, more studies are necessary and are under way in our laboratory. Citation Format: Heidge Fukumasu, Pedro R.L. Pires, Pedro L.P. Xavier. Inhibition of G9a reestablishes the MHC class I loss due to EMT in lung cancer cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B011.

O29 Nrf2/HO-1 pathway alleviates epithelial-mesenchymal transition by inhibiting HMGB1 expression in pulmonary fibrosis

Pulmonary fibrosis (PF) is a chronic, interstitial lung disease with unknown cause, characterized by the activation of accumulated myofibroblasts and deposition of extracellular matrix (ECM). Epithelial-mesenchymal transition (EMT) is considered to be one of the major hypotheses behind the formation of PF. High mobility group box1 (HMGB1) plays an important biological role in infection, inflammation, and immune responses. Nuclear factor E2-related factor 2 (Nrf2) is an important transcription factor for the regulation of oxidative stress. However, there is no direct evidence regarding the relationship between the Nrf2/HO-1 antioxidant system and HMGB1 in lung fibrosis. In our studies, the PF model was induced with bleomycin in both wild type (WT) mice and Nrf2 −/− mice. Rat type II alveolar epithelial cells (RLE-6TN) and the human alveolar epithelial cell line A549 were treated with the Nrf2 activator, sulforaphane, or transfected with Nrf2 and HMGB1 siRNAs to explore their relationship in TGF-β-induced PF. This research showed that bleomycin-induced fibrosis was more severe in Nrf2 −/− mice compared to WT mice. In the in vitro experiments, sulforaphane-treated cells had significantly attenuated TGF-β-induced epithelial-mesenchymal transition accompanied by downregulated expression of HMGB1. In contrast, silencing Nrf2 by siRNA enhanced TGFβ1-induced EMT with increased expression of HMGB1. However, when HMGB1 was silenced by siRNA, sulforaphane could not reduce the progression of EMT in either RLE-6TN cells or A549 cells. These findings suggest that the inhibitory effect of HMGB1 on EMT in PF is regulated by the Nrf2/HO-1 antioxidant pathway.

Adamantane acid esters with alkoxyaryl alcohols: Synthesis, antiproliferative activity, and influence on microtubule network of tumor cells

Adamantaneacetic and adamantanecarboxylic acid esters containing 3-hydroxy-4-methoxybenzyl, 3,4,5-trimethoxybenzyl, or 5-(hydroxymethyl)-2-methoxyphenyl groups were synthesized as unusual analogs of natural antitumor and anti-tubulin agents combretastatin A-4 and 2-methoxyestradiol. The compounds were found to possess noticeable cytotoxicity to epithelial human carcinoma cell line A549 (EC50 = 4.3—81 μmol L–1). An ability to cause complete depolymerization of microtubule network of A549 cells was demonstrated for 5-(hydroxymethyl)-2-methoxyphenyl adamantan-1-ylacetate (6a) at a concentration of 100 μmol L–1. Ester 6a belongs to a new structural type, which is unusual for the ligands of the tubulin colchicine domain, and is an interesting lead compound for further structural optimization.

Abstract 5524: Gene 33/Mig6 regulates apoptosis and the DNA damage response through independent mechanisms

Abstract Gene 33 (Mig6, ERRFI1) is an inducible adaptor/scaffold protein whose expression can be induced by both stress and mitogenic signals. It contains multiple domains for protein-protein interaction and is involved in a broad spectrum of cellular functions including cell proliferation, cell migration, cell apoptosis, and cell senescence. Many functions of Gene 33 are attributable to its ability to bind to the kinase domain of the EGFR family receptor tyrosine kinases thereby inhibiting their activities. Gene 33 also binds and activates tyrosine kinase c-Abl and is involved in other signing pathways such as NF-κB, HGF, and JNK. Growing evidence at the genetic, cellular, and animal levels indicates that Gene 33 functions as a tumor suppressor in the lung. Gene 33 has been shown to promote apoptosis. A recent study has linked this protein to the DNA damage response induced by hexavalent chromium [Cr(VI)]. In the current study we find that ectopic expression of Gene 33 strongly induces apoptosis by both activating the c-Abl/p73 pathway and inhibiting the EGFR/AKT pathways in BEAS-2B lung epithelial cells and A549 lung carcinoma cells. Surprisingly, ectopic expression of Gene 33 also triggers the DNA damage response in an ATM-dependent fashion and through pathways with and without association with apoptosis. We observed striking presence of Gene 33 in the nucleus and chromatin, which is in contrast with the belief that Gene 33 is an exclusively cytoplasmic protein. Our data show that chromatin localization of Gene 33 is partially dependent on the EBD motif of Gene 33, a domain required for interaction with EGFR and c-Abl. Our data also indicate that Gene 33 may regulate chromatin targeting of c-Abl and EGFR. Furthermore we find that both endogenous and ectopically expressed Gene 33 strongly interact with histone H2AX. Our data have revealed potential nuclear/chromatin-associated mechanisms that underline the function of Gene 33 in apoptosis and the DNA damage response. Citation Format: Cen Li, Soyoung Park, Xiaowen Zhang, Leonard Eisenberg, Hong Zhao, Zbigniew Darzynkiewicz, Dazhong Xu. Gene 33/Mig6 regulates apoptosis and the DNA damage response through independent mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5524. doi:10.1158/1538-7445.AM2017-5524

A new indole alkaloid from the traditional Chinese medicine Chansu

A new indole alkaloid N′-formylserotonin (1), along with five known indole alkaloids N′-methylserotonin (2), 5-hydroxy-1H-indole-3-carbaldehyde (3), N-acetylserotonin (4), 6-hydroxy-1-oxo-3,4-dihydro-β-carboline (5), and bufoserotoin C (6), were isolated from the water extract of traditional Chinese medicine Chansu. Their structures were elucidated on the basis of spectral analyses. The cytotoxicities of 1–6 against human lung adenocarcinoma epithelial cells A549 were tested using the MTT method. Compound 6 exhibited stronger cytotoxic effect than 5-FU, and 1–5 showed no cytotoxic effects. Bufoserotonin C is one of the cytotoxic components in water-soluble extract of Chansu.

Differential effects of calcium on PI3K-Akt and HIF-1α survival pathways

Calcium signaling participates in the regulation of numberless cellular functions including cell cycle progression and cellular migration, important processes for cancer expansion. Cancer cell growth, migration, and invasion are typically supported by PI3K/Akt activation, while a hypoxic environment is critical in cancer development. Accordingly, in the present study, we aimed at investigating whether perturbations in calcium homeostasis induce alterations of HIF-1α and activate Akt levels in epithelial A549 and A431 cells. Survival was drastically reduced in the presence of calcium chelator BAPTA-AM and thapsigargin, a SERCA inhibitor inducing store-operated calcium entry, to a lesser extent. Calcium chelation provoked a transient but strong upregulation of HIF-1α protein levels and accumulation in the nucleus, whereas in the presence of thapsigargin, HIF-1α levels were rapidly abolished before reaching and exceeding control levels. Despite cell death, calcium chelation merely inhibited Akt, which was significantly activated in the presence of thapsigargin. Moreover, when store-operated calcium entry was simulated by reintroducing calcium ions in cell suspensions, Akt was rapidly activated in the absence of any growth factor. These data further underscore the growing importance of calcium entry and directly link this elementary event of calcium homeostasis to the Akt pathway, which is commonly deregulated in cancer.

MicroRNA-7-5p regulates human alveolar epithelial sodium channels by targeting the mTORC2/SGK-1 signaling pathway

ABSTRACTPurpose: The aim of this study was to investigate the effect of miRNA-7-5p on human alveolar epithelial sodium channels and clarify the pathway in which miRNA-7-5p regulates the expression of ENaC in ARDS. Materials and Methods: Dual luciferase target gene validation experiments were used to confirm whether mTOR and SGK-1 are the target genes of miRNA-7-5p. Then, we overexpressed and inhibited miRNA-7-5p in the human alveolar epithelial cell line A549, respectively. LPS at a concentration of 100 ng/mL was used to stimulate the cells. The expressions ofmiRNA-7-5p, mTOR, SGK-1, p-Akt-Ser473, α-, β-, and γ-ENaC were detected by quantitative RT polymerase chain reaction (qRT-PCR) and western blotting. Results: In this study, we first confirmed that mTOR and SGK-1 are the target genes of miRNA-7-5p. Then, we found that mRNA expression levels of both mTOR and SGK-1 were downregulated to 0.54- and 0.3-fold, respectively, in the miRNA-7-5p mimic groups than the blank controls (P < 0.01). MiRNA-7-5p overexpression inhibited mTORC2/SGK-1 signaling pathway activity and reduced ENaC expression. The expression of miRNA-7-5p was significantly upregulated in A549 cells stimulated with lipopolysaccharide (LPS) and downregulated mRNA expression levels of both mTOR and SGK-1. After transfection with miRNA-7-5p inhibitors, we found that the mTORC2/SGK-1 pathway activity was restored compared to the group with LPS stimulation only, and the ENaC expression was also obviously increased. Conclusion: Our results demonstrate that miRNA-7-5p can regulate the expression of human alveolar ENaC by targeting the mTORC2/SGK-1 signaling pathway. The inhibition of miRNA-7-5p can enhance the expression of ENaC, which may provide a new target for the treatment of ARDS.

Infectious Spondylitis-Associated &amp;lt;i&amp;gt;Staphylococcus aureus&amp;lt;/i&amp;gt; with Virulence Gene &amp;lt;i&amp;gt;pvl&amp;lt;/i&amp;gt; or &amp;lt;i&amp;gt;tst&amp;lt;/i&amp;gt; Causes More Necrosis than Apoptosis in Human Alveolar Basal Epithelial Cell Line A549

Methicillin-sensitive and resistant Staphylococcus aureus (MSSA and MRSA, respectively) can cause non-tuberculosis infectious spondylitis. In 43 cases of bacterial infectious spondylitis, Mycobacterium tuberculosis and S. aureus were the two major causative pathogens. MRSA caused more anterior operations and thoracic infections, while MSSA caused more posterior infections and lumbar infections. Differences between six S. aureus isolates from infectious spondylitis were characterized. MLST and staphylococcal cassette chromosome mec (SCCmec) analysis identified MSSA ST959 and ST30 isolates, MRSA ST239/SCCmec IIIA isolates 2 and 3, ST59/SCCmec IIIA-like isolate 6, and ST30/SCCmec IV isolate 5. While all of the isolates were resistant to penicillin and ampicillin, the MRSA isolates were more resistant than the MSSA isolates. Carbapenem-resistant MRSA ST239/SCCmec IIIA and ST59/SCCmec IIIA-like isolates of the agr1 type were also resistant to clindamycin and erythromycin. Leukocidin genes (pvl or lukED) and hemolysin genes (hla, hld and hlg) were present in all of the isolates. All six isolates caused more necrosis than apoptosis in the human alveolar basal epithelial cell line A549; however, ST59/SCCmec IIIA-like isolate 6, ST30/ SCCmec IV isolate 5 with pvl genes, and MSSA ST30 isolates with tst caused greater than 40% cell death after the 4-h incubation. Regardless of the MRSA isolate and its SCCmec type or the MSSA isolate, the infectious spondylitis-associated S. aureus isolates differed genetically, and the pvl and tst genes may be important genes for cell necrosis.

Bilberry extract, its major polyphenolic compounds, and the soy isoflavone genistein antagonize the cytostatic drug erlotinib in human epithelial cells.

Erlotinib (Tarceva®) is a chemotherapeutic drug approved for the treatment of pancreatic cancer and non-small cell lung cancer. Its primary mode of action is the inhibition of the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK). Recently, RTK-inhibiting polyphenols have been reported to interact synergistically with erlotinib. Furthermore some anthocyanidins and anthocyanin-rich berry extracts have been reported to inhibit tyrosine kinases, including the EGFR, which raises the question of potential interactions with erlotinib. Polyphenol-rich preparations such as berry- or soy-based products are commercially available as food supplements. In the present study we tested a bilberry extract, its major anthocyanin and potential intestinal degradation products, as well as genistein, with respect to possible interactions with erlotinib. Cell growth inhibition was assessed using the sulforhodamine B assay, while interactions with EGFR phosphorylation were analyzed by SDS-PAGE/western blotting with subsequent immunodetection. Genistein, bilberry extract, delphinidin-3-O-glucoside and delphinidin were found to antagonize erlotinib whereas phloroglucinol aldehyde was found to enhance cytostatic effects of the drug on human epithelial A431 cells. Genistein also antagonized the EGFR inhibitory effects of erlotinib, whereas bilberry anthocyanins showed no significant interactions in this regard. Our data indicate that different polyphenols are potentially able to impair the cytostatic effect of erlotinib in vitro. Genistein interacts via the modulation of erlotinib-mediated EGFR inhibition whereas bilberry anthocyanins modulated the growth-inhibitory effect of erlotinib without affecting EGFR phosphorylation, thus indicating a different mechanism of interference.