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Most cancer cells rely on glycolysis to generate ATP, even when oxygen is available. However, merely inhibiting the glycolysis is insufficient for the eradication of cancer cells. One main reason for this is that cancer cells have the potential to adapt their metabolism to their environmental conditions. In this study, we investigated how cancer cells modify their intracellular metabolism when glycolysis is suppressed, using PANC-1 pancreatic cancer cells and two other solid tumor cell lines, A549 and HeLa. Our study revealed that glycolytically suppressed cells upregulated mitochondrial function and relied on oxidative phosphorylation (OXPHOS) to obtain the ATP necessary for their survival. Dynamic changes in intracellular metabolic profiles were also observed, reflected by the reduced levels of TCA cycle intermediates and elevated levels of most amino acids. Glutamine and glutamate were important for this metabolic reprogramming, as these were largely consumed by influx into the TCA cycle when the glycolytic pathway was suppressed. During the reprogramming process, activated autophagy was involved in modulating mitochondrial function. We conclude that upon glycolytic suppression in multiple types of tumor cells, intracellular energy metabolism is reprogrammed toward mitochondrial OXPHOS in an autophagy-dependent manner to ensure cellular survival.

Aim： Paeoniflorin has shown to attenuate bleomycin-induced pulmonary fibrosis （PF） in mice. Because the epithelial-mesenchymal transition （EMT） in type 2 lung endothelial cells contributes to excessive fibroblasts and myofibroblasts during multiple fibrosis of tissues, we investigated the effects of paeoniflorin on TGF-β mediated pulmonary EMT in bleomycin-induced PF mice. Methods： PF was induced in mice by intratracheal instillation of bleomycin （5 mg/kg）. The mice were orally treated with paeoniflorin or prednisone for 21 d. After the mice were sacrificed, lung tissues were collected for analysis. An in vitro EMT model was established in alveolar epithelial cells （A549 cells） incubated with TGF-β1 （2 ng/mL）. EMT identification and the expression of related proteins were performed using immunohistochemistry, transwell assay, ELISA, Western blot and RT-qPCR. Results： In PF mice, paeoniflorin （50, 100 mg.kg-1.d-1） or prednisone （6 mg.kg-1.d-1） significantly decreased the expression of FSP-1 and a-SMA, and increased the expression of E-cadherin in lung tissues. In A549 cells, TGF-β1 stimulation induced EMT, as shown by the changes in cell morphology, the increased cell migration, and the increased vimentin and α-SMA expression as well as type I and type Ⅲ collagen levels, and by the decreased E-cadherin expression, in contrast, effects of paeoniflorin on EMT disappeared when the A549 cells were pretreated with TGF-β1 for 24 h. TGF-β1 stimulation markedly increased the expression of Snail and activated Smad2/3, Akt, ERK, JNK and p38 MAPK in A549 cells. Co-incubation with paeoniflorin （1-30 pmol/L） dose-dependently attenuated TGF-β1-induced expression of Snail and activation of Smad2/3, but slightly affected TGF-β1-induced activation of Akt, ERK, JNK and p38 MAPK. Moreover, paeoniflorin markedly increased Smad7 level, and decreased ALK5 level in A549 cells. Conclusion： Paeoniflorin suppresses the early stages of TGF-β mediated EMT in alveolar epithelial cells, likely by decreasing the expression of the transcription factors Snail via a Smad-dependent pathway involving the up-regulation of Smad7.

Pulmonary research requires models that represent the physiology of alveolar epithelium but concerns with reproducibility, consistency and the technical and ethical challenges of using primary or stem cells has resulted in widespread use of continuous cancer or other immortalized cell lines. The A549 'alveolar' cell line has been available for over four decades but there is an inconsistent view as to its suitability as an appropriate model for primary alveolar type II (ATII) cells. Since most work with A549 cells involves short term culture of proliferating cells, we postulated that culture conditions that reduced proliferation of the cancer cells would promote a more differentiated ATII cell phenotype. We examined A549 cell growth in different media over long term culture and then used microarray analysis to investigate temporal regulation of pathways involved in cell cycle and ATII differentiation; we also made comparisons with gene expression in freshly isolated human ATII cells. Analyses indicated that long term culture in Ham's F12 resulted in substantial modulation of cell cycle genes to result in a quiescent population of cells with significant up-regulation of autophagic, differentiation and lipidogenic pathways. There were also increased numbers of up- and down-regulated genes shared with primary cells suggesting adoption of ATII characteristics and multilamellar body (MLB) development. Subsequent Oil Red-O staining and Transmission Electron Microscopy confirmed MLB expression in the differentiated A549 cells. This work defines a set of conditions for promoting ATII differentiation characteristics in A549 cells that may be advantageous for studies with this cell line.

Lung cancer patients face a dismal prognosis mainly due to the low efficacy of current available treatments. Cisplatin is the first-line chemotherapy treatment for those patients, however, resistance to this drug is a common and yet not fully understood phenomenon. Aiming to shed new light into this puzzle, we used established normal and malignant lung cell lines displaying different sensitivity towards cisplatin treatment. We observed a negative correlation between cell viability and DNA damage induction upon cisplatin treatment. Interestingly, drug sensitivity in those cell lines was not due to either difference on DNA repair capacity, or in the amount of membrane ion channel commonly used for cisplatin uptake. Also, we noted that glutathione intracellular levels, and expression and activity of the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) were determinant for cisplatin cytotoxicity. Remarkably, analysis of gene expression in non-small cell lung cancer patients of the TCGA data bank revealed that there is a significant lower overall survival rate in the subset of patients bearing tumors with unbalanced levels of NRF2/KEAP1 and, as consequence, increased expression of NRF2 target genes. Thus, the results indicate that NRF2 and glutathione levels figure as important cisplatin resistance biomarkers in lung cancer.

Infrared fingerprint spectra can reveal the chemical nature of materials down to 20-nm detail, far below the diffraction limit, when probed by scattering-type scanning near-field optical microscopy (s-SNOM). But this was impossible with living cells or aqueous processes as in corrosion, due to water-related absorption and tip contamination. Here, we demonstrate infrared s-SNOM of water-suspended objects by probing them through a 10-nm thick SiN membrane. This separator stretches freely over up to 250 µm, providing an upper, stable surface to the scanning tip, while its lower surface is in contact with the liquid and localises adhering objects. We present its proof-of-principle applicability in biology by observing simply drop-casted, living E. coli in nutrient medium, as well as living A549 cancer cells, as they divide, move and develop rich sub-cellular morphology and adhesion patterns, at 150 nm resolution. Their infrared spectra reveal the local abundances of water, proteins, and lipids within a depth of ca. 100 nm below the SiN membrane, as we verify by analysing well-defined, suspended polymer spheres and through model calculations. SiN-membrane based s-SNOM thus establishes a novel tool of live cell nano-imaging that returns structure, dynamics and chemical composition. This method should benefit the nanoscale analysis of any aqueous system, from physics to medicine.

Background Leaves of the natural plant lotus are used in traditional Chinese medicine and tea production. They are rich in flavonoids. Methods In this study, lotus leaf flavonoids (LLF) were applied to human lung cancer A549 cells and human small cell lung cancer cells H446 in vitro to verify the effect of LLF on apoptosis in these cells through the ROS/p38 MAPK pathway. Results LLF had no toxic effect on normal cells at concentrations up to 500 µg/mL, but could significantly inhibit the proliferation of A549 cells and H446 cells. Flow cytometry showed that LLF could induce growth in A549 cells. We also found that LLF could increase ROS and MDA levels, and decrease SOD activity in A549 cells. Furthermore, qRT-PCR and western blot analyses showed that LLF could upregulate the expression of p38 MAPK (p-p38 MAPK), caspase-3, caspase-9, cleaved caspase-3, cleaved caspase-9 and Bax and downregulate the expression of Cu/Zn SOD, CAT, Nrf2, NQO1, HO-1, and Bcl-2 in A549 cells. Results of HPLC showed that LLF mainly contain five active substances: kaempferitrin, hyperoside, astragalin, phloridzin, and quercetin. The apoptosis-inducing effect of LLF on A549 cells came from these naturally active compounds. Conclusions We have shown in this study that LLF is a bioactive substance that can induce apoptosis in A549 cells in vitro, and merits further research and development.

Claudin-2 (CLDN2), a component of tight junction, is involved in the reduction of anticancer drug-induced toxicity in spheroids of A549 cells derived from human lung adenocarcinoma. Fisetin, a dietary flavonoid, inhibits cancer cell growth, but its effect on chemosensitivity in spheroids is unknown. Here, we found that fisetin (20 μM) decreases the protein level of CLDN2 to 22.3%. Therefore, the expression mechanisms were investigated by real-time polymerase chain reaction and Western blotting. Spheroids were formed in round-bottom plates, and anticancer drug-induced toxicity was measured by ATP content. Fisetin decreased the phosphorylated-Akt level, and CLDN2 expression was decreased by a phosphatidylinositol 3-kinase (PI3K) inhibitor, suggesting the inhibition of PI3K/Akt signal is involved in the reduction of CLDN2 expression. Hypoxia level, one of the hallmarks of tumor microenvironment, was reduced by fisetin. Although fisetin did not change hypoxia inducible factor-1α level, it decreased the protein level of nuclear factor erythroid 2-related factor 2, a stress response factor, by 25.4% in the spheroids. The toxicity of doxorubicin (20 μM) was enhanced by fisetin from 62.8% to 40.9%, which was rescued by CLDN2 overexpression (51.7%). These results suggest that fisetin can enhance anticancer drug toxicity in A549 spheroids mediated by the reduction of CLDN2 expression.

Epirubicin (EPI), an epimer of doxorubicin (DOX), and DOX are anthracycline agents with broad-spectrum antitumor activity. The aim of the present study was to elucidate the transport characteristics of EPI and DOX in human hepatocellular carcinoma HepG2 cells and human non-small cell lung cancer A549 cells, and to examine the relationship of intracellular drug accumulation with their cytotoxic effects. Intracellular concentrations of EPI and DOX were measured using high-performance liquid chromatography (HPLC). Expression level of targeted genes was analyzed by real-time quantitative PCR. Cell viability was evaluated using the MTT assay. Similar to DOX, EPI was taken up into HepG2 and A549 cells by organic cation transporter 6 and passive diffusion; however, the efficiency of saturable and non-saturable uptake of EPI was greater than that of DOX in both cell types. EPI served as a substrate of P-glycoprotein and multidrug associated protein (MRP) 1 and MRP2 similarly to DOX, but the efflux efficiency of each transporter was markedly different between EPI and DOX. The intracellular accumulation of EPI was significantly greater than that of DOX in all cells, and the accumulated level reflected the cytotoxic effects of these drugs. However, the intracellular drug amount did not correspond to the degree of cytotoxicity when compared between HepG2 and A549 cells, which can be explained by the higher expression of Bcl-xl in A549 cells. This study suggested that the transport characteristics are markedly different between EPI and DOX in HepG2 and A549 cells, and that intracellular accumulation is the predominant factor affecting the cytotoxicity of EPI and DOX in individual cells.

Background Resistance to chemotherapy drugs (e.g. taxol) has been a major obstacle in successful cancer treatment. In A549 human lung adenocarcinoma, acquired resistance to the first-line chemotherapy taxol has been a critical problem in clinics. Sphingolipid (SPL) controls various aspects of cell growth, survival, adhesion, and motility in cancer, and has been gradually regarded as a key factor in drug resistance. To better understand the taxol-resistant mechanism, a comprehensive sphingolipidomic approach was carried out to investigate the sphingolipid metabolism in taxol-resistant strain of A549 cell (A549T). Methods A549 and A549T cells were extracted according to the procedure with optimal condition for SPLs. Sphingolipidomic analysis was carried out by using an UHPLC coupled with quadrupole time-of-flight (Q-TOF) MS system for qualitative profiling and an UHPLC coupled with triple quadrupole (QQQ) MS system for quantitative analysis. The differentially expressed sphingolipids between taxol-sensitive and -resistant cells were explored by using multivariate analysis. Results Based on accurate mass and characteristic fragment ions, 114 SPLs, including 4 new species, were clearly identified. Under the multiple reaction monitoring (MRM) mode of QQQ MS, 75 SPLs were further quantified in both A549 and A549T. Multivariate analysis explored that the levels of 57 sphingolipids significantly altered in A549T comparing to those of A549 ( p  < 0.001 and VIP > 1), including 35 sphingomyelins (SMs), 14 ceramides (Cers), 3 hexosylceramides (HexCers), 4 lactosylceramides (LacCers) and 1 sphingosine. A significant decrease of SM and Cer levels and overall increase of HexCer and LacCer represent the major SPL metabolic characteristic in A549T. Conclusions This study investigated sphingolipid profiles in human lung adenocarcinoma cell lines, which is the most comprehensive sphingolipidomic analysis of A549 and A549T. To some extent, the mechanism of taxol-resistance could be attributed to the aberrant sphingolipid metabolism, “inhibition of the de novo synthesis pathway” and “activation of glycosphingolipid pathway” may play the dominant role for taxol-resistance in A549T. This study provides insights into the strategy for clinical diagnosis and treatment of taxol resistant lung cancer.

How a cell changes from one stable phenotype to another one is a fundamental problem in developmental and cell biology. Mathematically, a stable phenotype corresponds to a stable attractor in a generally multi-dimensional state space, which needs to be destabilized so the cell relaxes to a new attractor. Two basic mechanisms for destabilizing a stable fixed point, pitchfork and saddle-node bifurcations, have been extensively studied theoretically; however, direct experimental investigation at the single-cell level remains scarce. Here, we performed live cell imaging studies and analyses in the framework of dynamical systems theories on epithelial-to-mesenchymal transition (EMT). While some mechanistic details remain controversial, EMT is a cell phenotypic transition (CPT) process central to development and pathology. Through time-lapse imaging we recorded single cell trajectories of human A549/Vim-RFP cells undergoing EMT induced by different concentrations of exogenous TGF-β in a multi-dimensional cell feature space. The trajectories clustered into two distinct groups, indicating that the transition dynamics proceeds through parallel paths. We then reconstructed the reaction coordinates and the corresponding quasi-potentials from the trajectories. The potentials revealed a plausible mechanism for the emergence of the two paths where the original stable epithelial attractor collides with two saddle points sequentially with increased TGF-β concentration, and relaxes to a new one. Functionally, the directional saddle-node bifurcation ensures a CPT proceeds towards a specific cell type, as a mechanistic realization of the canalization idea proposed by Waddington. Cells with the same genetic code can take on many different formss, or phenotypes, which have distinct roles and appearances. Sometimes cells switch from one phenotype to another as part of healthy growth or during disease. One such change is the epithelial-to-mesenchymal transition (EMT), which is involved in fetal development, wound healing and the spread of cancer cells. During EMT, closely connected epithelial cells detach from one another and change into mesenchymal cells that are able to migrate. Cells undergo a number of changes during this transition; however, the path they take to reach their new form is not entirely clear. For instance, do all cells follow the same route, or are there multiple ways that cells can shift from one state to the next? To address this question, Wang et al. studied individual lung cancer cells that had been treated with a protein that drives EMT. The cells were then imaged at regular intervals over the course of two to three days to see how they changed in response to different concentrations of protein. Using a mathematical analysis designed to study chemical reactions, Wang et al. showed that the cells transform into the mesenchymal phenotype through two main routes. This result suggests that attempts to prevent EMT, in cancer treatment for instance, would require blocking both paths taken by the cells. This information could be useful for biomedical researchers trying to regulate the EMT process. The quantitative approach of this study could also help physicists and mathematicians study other types of transition that occur in biology.