bafilomycin-a1 and Carcinoma--Non-Small-Cell-Lung

The novel SARS-CoV-2 Omicron variant (B.1.1.529), first found in early November 2021, has sparked considerable global concern and it has >50 mutations, many of which are known to affect transmissibility or cause immune escape. In this study, we sought to investigate the virological characteristics of the Omicron variant and compared it with the Delta variant which has dominated the world since mid-2021. Omicron variant replicated more slowly than the Delta variant in transmembrane serine protease 2 (TMPRSS2)-overexpressing VeroE6 (VeroE6/TMPRSS2) cells. Notably, the Delta variant replicated well in Calu3 cell line which has robust TMPRSS2 expression, while the Omicron variant replicated poorly in this cell line. Competition assay showed that Delta variant outcompeted Omicron variant in VeroE6/TMPRSS2 and Calu3 cells. To confirm the difference in entry pathway between the Omicron and Delta variants, we assessed the antiviral effect of bafilomycin A1, chloroquine (inhibiting endocytic pathway), and camostat (inhibiting TMPRSS2 pathway). Camostat potently inhibited the Delta variant but not the Omicron variant, while bafilomycin A1 and chloroquine could inhibit both Omicron and Delta variants. Moreover, the Omicron variant also showed weaker cell-cell fusion activity when compared with Delta variant in VeroE6/TMPRSS2 cells. Collectively, our results suggest that Omicron variant infection is not enhanced by TMPRSS2 but is largely mediated via the endocytic pathway. The difference in entry pathway between Omicron and Delta variants may have an implication on the clinical manifestations or disease severity.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Chlorocebus aethiops; Chloroquine; COVID-19; Endocytosis; Esters; Guanidines; Humans; Immune Evasion; Lung Neoplasms; Macrolides; Recombinant Proteins; SARS-CoV-2; Serine Endopeptidases; Vero Cells; Virus Cultivation; Virus Internalization; Virus Replication; Whole Genome Sequencing

Sulforaphane-N-acetyl-cysteine (SFN-NAC) is a potential drug to inhibit human non-small cell lung cancer (NSCLC), but the underlying mechanisms are elusive. Here, we uncovered that SFN-NAC induced apoptosis via flow cytometer assay and transmission electron microscopy. Further, SFN-NAC increased LC3 II/LC3 I and the number of LC3 punctas, but Western blot showed that SFN-NAC inhibited cell autophagy in response to a co-treatment of Bafilomycin A1 and SFN-NAC. Furthermore, immunofluorescence staining and Western blot showed that SFN-NAC triggered microtubule disruption causing apoptosis via downregulating α-tubulin and phosphorylated ERK1/2-mediated Stathmin-1. Besides, SFN-NAC upregulated Hsp70 via phosphorylating ERK1/2. Confocal microscopy and immunoprecipitation assay showed that SFN-NAC promoted the colocalization and interaction of Hsp70 and α-tubulin; knockdown of Hsp70 enhanced SFN-NAC-induced microtubule disruption, lowered LC3 II/LC3 I and promoted apoptosis. Interestingly, tissue microarray analysis showed that the increased expression of either α-tubulin or Hsp70 correlated to NSCLC malignant grading, indicating that microtubule and Hsp70 are two key targets for SFN-NAC. These results will give us a new insight into SFN-NAC-induced apoptosis so that we develop more efficient therapeutics to treat NSCLC.

Topics: Acetylcysteine; Aged; Anticarcinogenic Agents; Apoptosis; Autophagy; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cell Survival; Female; HSP70 Heat-Shock Proteins; Humans; Isothiocyanates; Lung Neoplasms; Macrolides; Male; Microtubules; Middle Aged; Phenotype; Stathmin; Sulfoxides; Tissue Array Analysis; Tubulin

Studies of radiation interaction with tumor cells often focus on apoptosis as an end point; however, clinically relevant doses of radiation also promote autophagy and senescence. Moreover, functional p53 has frequently been implicated in contributing to radiation sensitivity through the facilitation of apoptosis. To address the involvement of apoptosis, autophagy, senescence and p53 status in the response to radiation, the current studies utilized isogenic H460 non-small cell lung cancer cells that were either p53-wild type (H460wt) or null (H460crp53). As anticipated, radiosensitivity was higher in the H460wt cells than in the H460crp53 cell line; however, this differential radiation sensitivity did not appear to be a consequence of apoptosis. Furthermore, radiosensitivity did not appear to be reduced in association with the promotion of autophagy, as autophagy was markedly higher in the H460wt cells. Despite radiosensitization by chloroquine in the H460wt cells, the radiation-induced autophagy proved to be essentially nonprotective, as inhibition of autophagy via 3-methyl adenine (3-MA), bafilomycin A1 or ATG5 silencing failed to alter radiation sensitivity or promote apoptosis in either the H460wt or H460crp53 cells. Radiosensitivity appeared to be most closely associated with senescence, which occurred earlier and to a greater extent in the H460wt cells. This finding is consistent with the in-depth proteomics analysis on the secretomes from the H460wt and H460crp53 cells (with or without radiation exposure) that showed no significant association with radioresistance-related proteins, whereas several senescence-associated secretory phenotype (SASP) factors were upregulated in H460wt cells relative to H460crp53 cells. Taken together, these findings indicate that senescence, rather than apoptosis, plays a central role in determination of radiosensitivity; furthermore, autophagy is likely to have minimal influence on radiosensitivity under conditions where autophagy takes the nonprotective form.

Topics: Apoptosis; Autophagy; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Chromatography, High Pressure Liquid; Genes, p53; Humans; Lung Neoplasms; Macrolides; Radiation Tolerance; Tandem Mass Spectrometry

Ursolic acid (UA) is a pentacyclic triterpenoid with recognized anticancer properties. We prepared a series of new A-ring cleaved UA derivatives and evaluated their antiproliferative activity in non-small cell lung cancer (NSCLC) cell lines using 2D and 3D culture models. Compound 17, bearing a cleaved A-ring with a secondary amide at C3, was found to be the most active compound, with potency in 2D systems. Importantly, even in 3D systems, the effect was maintained albeit a slight increase in the IC50. The molecular mechanism underlying the anticancer activity was further investigated. Compound 17 induced apoptosis via activation of caspase-8 and caspase-7 and via decrease of Bcl-2. Moreover, induction of autophagy was also detected with increased levels of Beclin-1 and LC3A/B-II and decreased levels of mTOR and p62. DNA synthetic capacity and cell cycle profiles were not affected by the drug, but total RNA synthesis was modestly but significantly decreased. Given its activity and mechanism of action, compound 17 might represent a potential candidate for further cancer research.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Chemistry Techniques, Synthetic; DNA; Drug Screening Assays, Antitumor; Humans; Lung Neoplasms; Protein Biosynthesis; RNA; Triterpenes; Ursolic Acid

Previous studies have shown that patients with schizophrenia have a lower incidence of cancer than the general population, and several antipsychotics have been demonstrated to have cytotoxic effects on cancer cells. However, the mechanisms underlying these results remain unclear. The present study aimed to investigate the effect of clozapine, which is often used to treat patients with refractory schizophrenia, on the growth of non-small cell lung carcinoma cell lines and to examine whether autophagy contributes to its effects.. A549 and H1299 cells were treated with clozapine, and cell cytotoxicity, cell cycle and autophagy were then assessed. The autophagy inhibitor bafilomycin A1 and siRNA-targeted Atg7 were used to determine the role of autophagy in the effect of clozapine.. Clozapine inhibited A549 and H1299 proliferation and increased p21 and p27 expression levels, leading to cell cycle arrest. Clozapine also induced a high level of autophagy, but not apoptosis, in both cell lines, and the growth inhibitory effect of clozapine was blunted by treatment with the autophagy inhibitor bafilomycin A1 or with an siRNA targeting atg7.. Clozapine inhibits cell proliferation by inducing autophagic cell death in two non-small cell lung carcinoma cell lines. These findings may provide insights into the relationship between clozapine use and the lower incidence of lung cancer among patients with schizophrenia.

Topics: Apoptosis; Autophagy; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Clozapine; Humans; Macrolides; RNA, Small Interfering; Schizophrenia

Despite excellent initial clinical responses of non-small cell lung cancer (NSCLC) patients to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), many patients eventually develop resistance. According to a recent report, vacuolar H+ ATPase (vATPase) is overexpressed and is associated with chemotherapy drug resistance in NSCLC. We investigated the combined effects of EGFR TKIs and vATPase inhibitors and their underlying mechanisms in the regulation of NSCLC cell death. We found that combined treatment with EGFR TKIs (erlotinib, gefitinib, or lapatinib) and vATPase inhibitors (bafilomycin A1 or concanamycin A) enhanced synergistic cell death compared to treatments with each drug alone. Treatment with bafilomycin A1 or concanamycin A led to the induction of Bnip3 expression in an Hif-1α dependent manner. Knock-down of Hif-1α or Bnip3 by siRNA further enhanced cell death induced by bafilomycin A1, suggesting that Hif-1α/Bnip3 induction promoted resistance to cell death induced by the vATPase inhibitors. EGFR TKIs suppressed Hif-1α and Bnip3 expression induced by the vATPase inhibitors, suggesting that they enhanced the sensitivity of the cells to these inhibitors by decreasing Hif-1α/Bnip3 expression. Taken together, we conclude that EGFR TKIs enhance the sensitivity of NSCLC cells to vATPase inhibitors by decreasing Hif-1α/Bnip3 expression. We suggest that combined treatment with EGFR TKIs and vATPase inhibitors is potentially effective for the treatment of NSCLC.

Topics: Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; ErbB Receptors; Erlotinib Hydrochloride; Gefitinib; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Lapatinib; Lung Neoplasms; Macrolides; Membrane Proteins; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Quinazolines; RNA Interference; Transfection; Vacuolar Proton-Translocating ATPases

The biochemical mechanisms of cell death by oleifolioside B (OB), a cycloartane-type triterpene glycoside isolated from Dendropanax morbifera Leveille, were investigated in A549 human lung carcinoma cells. Our data indicated that exposure to OB led to caspase activation and typical features of apoptosis; however, apoptotic cell death was not prevented by z-VAD-fmk, a pan-caspase inhibitor, demonstrating that OB-induced apoptosis was independent of caspase activation. Subsequently, we found that OB increased autophagy, as indicated by an increase in monodansylcadaverine fluorescent dye-labeled autophagosome formation and in the levels of the autophagic form of microtubule-associated protein 1 light chain 3 and Atg3, an autophagy-specific gene, which is associated with inhibiting phospho-nuclear factor erythroid 2-related factor 2 (Nrf2) expression. However, pretreatment with bafilomycin A1, an autophagy inhibitor, attenuated OB-induced apoptosis and dephosphorylation of Nrf2. The data suggest that OB-induced autophagy functions as a death mechanism in A549 cells and OB has potential as a novel anticancer agent capable of targeting apoptotic and autophagic cell death and the Nrf2 signaling pathway.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Autophagy; Autophagy-Related Proteins; Carcinoma, Non-Small-Cell Lung; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 3; Caspase 8; Caspase 9; Caspase Inhibitors; Cell Line, Tumor; Enzyme Inhibitors; Humans; Inhibitor of Apoptosis Proteins; Lung Neoplasms; Macrolides; Microtubule-Associated Proteins; NF-E2-Related Factor 2; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Saponins; Survivin; Ubiquitin-Conjugating Enzymes

Selenium in the form of sodium selenite has been reported to exert anti-tumor effects in several cancer cell types by inducing autophagic cell death and apoptosis mediated by reactive oxygen species (ROS). However, the exact molecular pathways underlying these effects have not been fully established. The present study used A549 human lung carcinoma cells for further investigation of the anti-cancer mechanism of sodium selenite. We showed that sodium selenite modulated both the extrinsic and intrinsic apoptotic pathways, which were interconnected by Bid truncation. We used z-VAD-fmk, a pan-caspase inhibitor, to demonstrate that sodium selenite-induced apoptosis was dependent on the activation of caspases. Sodium selenite also increased autophagy, as indicated by an increase in microtubule-associated protein light chain-3 (LC3) puncta, accumulation of LC3II, and elevation of autophagic flux. Pretreatment with bafilomycin A1 enhanced sodium selenite-induced apoptosis, indicating that sodium selenite-induced autophagy functioned as a survival mechanism. Sodium selenite treatment also resulted in generation of ROS, which abrogated mitochondrial membrane potential (MMP) and regulated both apoptosis and autophagy. Phospho-nuclear factor erythroid 2-related factor 2 (p-Nrf2) showed a ROS-dependent translocation to the nucleus, which suggested that Nrf2 might increase cell survival by suppressing ROS accumulation and apoptosis mediated by oxidative stress. Sodium selenite treatment of A549 cells therefore appeared to trigger both apoptosis and cytoprotective autophagy, which were both mediated by ROS. The data suggest that regulation of ROS generation and autophagy can be a potential strategy for treating lung cancer that is resistant to pro-apoptotic therapeutics.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Inhibitors; Humans; Macrolides; Membrane Potential, Mitochondrial; NF-E2-Related Factor 2; Reactive Oxygen Species; Sodium Selenite

Neodymium, a rare earth element, was known to exhibit cytotoxic effects and induce apoptosis in certain cancer cells. Here we show that nano-sized neodymium oxide (Nano Nd2O3) induced massive vacuolization and cell death in non-small cell lung cancer NCI-H460 cells at micromolar equivalent concentration range. Cell death elicited by Nano Nd2O3 was not due to apoptosis and caspases were not involved. Electron microscopy and acridine orange staining revealed extensive autophagy in the cytoplasm of the cells treated by Nano Nd2O3. Autophagy induced by Nano Nd2O3 was accompanied by S-phase cell cycle arrest, mild disruption of mitochondrial membrane potential, and inhibition of proteasome activity. Bafilomycin A1, but not 3-MA, induced apoptosis while inhibiting autophagy. Our results revealed a novel biological function for Nano Nd2O3 and may have implications for the therapy of non-small cell lung cancer.

Topics: Antineoplastic Agents; Autophagy; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Humans; Lung Neoplasms; Macrolides; Membrane Potentials; Mitochondria; Neodymium; Oxides; Proteasome Endopeptidase Complex; S Phase; Vacuoles