Molecular Mobility of Polyrotaxane Surfaces Alleviates Oxidative Stress-Induced Senescence in Mesenchymal Stem Cells

Polyrotaxane is a supramolecular assembly consisting of multiple cyclic molecules threaded by a linear polymer. One of the unique properties of polyrotaxane is molecular mobility, cyclic molecules moving along the linear polymer. Molecular mobility of polyrotaxane surfaces affects cell spreading, differentiation, and other cell-related aspects through changing subcellular localization of yes-associated proteins (YAPs). Subcellular YAP localization is also related to cell senescence derived from oxidative stress, which is known to cause cancer, diabetes, and heart disease. Herein, the effects of polyrotaxane surface molecular mobility on subcellular YAP localization and cell senescence following H₂O₂-induced oxidative stress are evaluated in human mesenchymal stem cells (HMSCs) cultured on polyrotaxane surfaces with different molecular mobilities. Oxidative stress promotes cytoplasmic YAP localization in HMSCs on high-mobility polyrotaxane surfaces; however, low-mobility polyrotaxane surfaces more effectively maintain nuclear YAP localization, exhibiting lower senescence-associated β-galactosidase activity and senescence-related gene expression and DNA damage than that seen with the high-mobility surfaces. These results suggest that the molecular mobility of polyrotaxane surfaces regulates subcellular YAP localization, thereby protecting HMSCs from oxidative stress-induced cell senescence. Applying the molecular mobility of polyrotaxane surfaces to implantable scaffolds can provide insights into the prevention and treatment of diseases caused by oxidative stress.     

Stimulation of Microvascular Networks on Sulfonated Polyrotaxane Surfaces with Immobilized Vascular Endothelial Growth Factor

Modulation of material properties and growth factor application are critical in constructing suitable cell culture environments to induce desired cellular functions. Sulfonated polyrotaxane (PRX) surfaces with immobilized vascular endothelial growth factors (VEGFs) are prepared to improve network formation in vascular endothelial cells. Sulfonated PRXs, whereby sulfonated α‐cyclodextrins (α‐CDs) are threaded onto a linear poly(ethylene glycol) chain capped with bulky groups at both terminals, are coated onto surfaces. The molecular mobility of sulfonated PRX surfaces is modulated by tuning the number of threading α‐CDs. VEGF is immobilized onto surfaces with varying mobility. Low mobility and VEGF‐immobilization reinforce cell proliferation, yes‐associated protein activity, and rhoA, pdgf, ang‐1, and pecam‐1 gene expression. Highly mobile surfaces and soluble VEGF weakly affect these cell responses. Network formation is strongly stimulated in vascular endothelial cells only on low‐mobility VEGF‐immobilized surfaces, suggesting that molecular mobility and VEGF immobilization synergistically control cell function.     

Mobility Tuning of Polyrotaxane Surfaces to Stimulate Myocyte Differentiation

Polyrotaxanes, consisting of poly(ethylene glycol) and α‐cyclodextrins, are mechanically interlocked supermolecules. The structure allows α‐cyclodextrins to move along the polymer, referred to as molecular mobility. Here, polyrotaxane‐based triblock copolymers, composed of polyrotaxanes with different degrees of methylation and poly(benzyl methacrylate) at both terminals, are coated on culture surfaces to fabricate dynamic biointerfaces for myocyte differentiation. The molecular mobility increases with the degree of methylation and the contact angle hysteresis of water droplets and air bubbles. When the mouse myoblast cell line C2C12 is cultured on methylated polyrotaxane surfaces, the expression levels of myogenesis‐related genes, myogenin (Myog) and myosin heavy chain (Myhc) are altered by the degree of methylation. Polyrotaxane surfaces with intermediate degrees of methylation promote the highest expression levels among all the surfaces. The polyrotaxane surface provides an appropriate environment for myocyte differentiation by accurately adjusting the degrees of methylation.     

Cisplatin-induced alteration on membrane composition of A549 cells revealed by ToF-SIMS

Cisplatin has been clinically used for treatment of solid tumors such as non–small-cell lung cancer for decades. However, tumor resistance may be acquired with losing the antitumor activity of cisplatin. As cellular membrane is the first barrier that cisplatin has to overcome before its further action inside the cells, the membrane composition must play a vital role in the cisplatin uptake and excretion, which further influences cisplatin sensitivity. In this work, we applied time-of-flight secondary ion mass spectrometry (ToF-SIMS) surface analysis combined with principle component analysis to distinguish the differences of cell membrane composition between non–small-cell lung cancer cells (A549) and its cisplatin resistant counterpart A549/DDP cells. The decreased phosphatidylcholine content and more abundant cholesterol were observed in the drug resistant cell surfaces, indicating the decreased membrane fluidity of A549/DDP cells. Moreover, we further compared membrane composition of A549 and A549/DDP cells after being treated with different concentrations of cisplatin. A higher composition level of proteins was discovered on all groups of A549/DDP cell membranes. The altered surface chemistry of cellular membranes induced by cisplatin indicates the significance of membrane structures in the drug resistance, which deserves further investigations to this regard.     

Ligand accessibility to receptor binding sites enhanced by movable polyrotaxanes

Functionalized polyrotaxanes are utilized to investigate the relation to multivalent interactions between the mannose moiety and Con A immobilized surfaces. According to the results of SPR spectroscopy, the mannose-conjugated polyrotaxanes show a higher response than any other mannose conjugate on both surfaces of high- and low-density Con A. Moreover, the results of the FRET analysis suggest that the mobility of α-cyclodextrins in the polyrotaxane more efficiently contributes to their binding interactions in a multivalent manner. This well-defined polyrotaxane system provides control over ligand density, ligand mobility, and gives an efficient response to the biological interaction receptor, which has not been easy to achieve in covalently bound polymeric systems.     

Effects of concave and convex substrate curvature on cell mechanics and the cytoskeleton

In order to understand how cells respond to concave and convex subcellular surface structures,colloidal crystal array and honeycomb-structured surfaces composed of highly ordered hexagonal units with completely inverse curvature were fabricated via facile self-assembly and breath figure approaches, respectively.The influence of hexagonal surface curvature on cell fate was subsequently investigated. Cells underwent more extensive spreading on the convex colloidal crystal array surface,while adhesive forces were higher on the concave honeycomb surface.The behaviors of cells on the different surfaces were investigated by comparing cell morphology,cellular adhesive force and cytoskeleton structure.The results revealed comprehensive differences in cell behavior between those on concave honeycomb surfaces and convex colloidal crystal arrays.     

High‐Throughput Isolation of Cell Protrusions with Single‐Cell Precision for Profiling Subcellular Gene Expression

Invading cancer cells extend cell protrusions, which guide cancer‐cell migration and invasion, eventually leading to metastasis. The formation and activity of cell protrusions involve the localization of molecules and organelles at the cell front; however, it is challenging to precisely isolate these subcellular structures at the single‐cell level for molecular analysis. Here, we describe a newly developed microfluidic platform capable of high‐throughput isolation of cell protrusions at single‐cell precision for profiling subcellular gene expression. Using this microfluidic platform, we demonstrate the efficient generation of uniform cell‐protrusion arrays (more than 5000 cells with protrusions) for a series of cell types. We show precise isolation of cell protrusions with high purity at single‐cell precision for subsequent RNA‐Seq analysis, which was further validated by RT‐qPCR and RNA FISH. Our highly controlled protrusion isolation method opens a new avenue for the study of subcellular functional mechanisms and signaling pathways in metastasis.     

Imaging live cells grown on a three dimensional collagen matrix using Raman microspectroscopy

Three dimensional collagen gels have been used as matrices for the imaging of live cells by Raman spectroscopy. The study is conducted on a human lung adenocarcinoma (A549) and a spontaneously immortalized human epithelial keratinocyte (HaCaT) cell line. The lateral resolution of the system has been estimated to be <1.5 μm making it possible to access the subcellular organization. Using K-means clustering analysis, it is shown that the different subcellular compartments of individual cells can be identified and differentiated. The biochemical specificity of the information contained in the Raman spectra allows the visualization of differences in the molecular signature of the different sub-cellular structures. Furthermore, to enhance the chemical information obtained from the spectra, principal component analysis has been employed, allowing the identification of spectral windows with a high variability. The comparison between the loadings calculated and spectra from pure biochemical compounds enables the correlation of the variations observed with the molecular content of the different cellular compartments.     

The Mobility of Threaded α-Cyclodextrins in PR Copolymer and Its Influences on Mechanical Properties

The methylated polyrotaxane(Me PR) copolymer was prepared via the methylation of hydroxyl of threaded α-cyclodextrin(α-CDs) in polyrotaxane(PR) copolymer by CH_3I/Na H. Its structure was characterized by GPC, IR and NMR. The WXRD and TGA measurements showed the destruction of channel-like crystalline structure in Me PR copolymer. The sliding of threaded α-CDs along PEG axis in PR and Me PR copolymers was demonstrated by their dielectric spectra that also evidenced the presence of rotating of threaded α-CDs around PEG axis in Me PR copolymer. The frequent and vigorous molecular mobility in Me PR and PR copolymers was also verified by dynamic mechanical analysis(DMA) and rheological measurement, which was possibly assigned to the sliding and rotating of threaded α-CDs. DMA and rheological results showed that the mobility of α-CDs could simultaneously strengthen and toughen PR copolymer proved by stress-stain curves. In this paper, we report the CD mobility in PR and Me PR copolymers. The macroscopic behaviors of PR copolymer, such as mechanical properties in solid state, were also found to be benefited from CD mobility.     

Resistance to anticancer drugs permanently alters electrophoretic mobility of cancer cell lines

Electrophoretic mobility is a physical phenomenon defining the mobility of charged particles in a solution under applied electric field. As charged biological systems, living cells including both prokaryotes and eukaryotes have been assessed in terms of electrophoretic mobility to decipher their electrochemical structure. Moreover, determination of electrophoretic mobility of living cancer cells have promoted the advance exploration of the nature of the cancer cells and separation of cancer cells from normal ones under applied electric field. However, electrophoretic mobility of drug‐resistant cells has not yet been examined. In the present study, we determined the electrophoretic mobility of drug‐resistant cancer cell lines for both suspension and adherent cells and compared with those of drug‐sensitive counterparts. We showed that resistance to anticancer drugs alters the electrophoretic mobility in a permanent manner, even lasting without any exposure to anticancer agents for a long time period. We also studied the cellular morphologies of adherent cells where the cellular invaginations and protrusions were increased in drug‐resistant adherent cells, which could be direct cause of altered surface charge and electrophoretic mobility as a result. These findings could be helpful in terms of understanding the electrophysiological and physicochemical background of drug resistance in cancer cells and developing systems to separate drug‐sensitive cells from drug‐resistant ones.     

Insulin Receptor Substrate 1 Is Involved in the Phycocyanin-Mediated Antineoplastic Function of Non-Small Cell Lung Cancer Cells

Phycocyanin, derived from marine algae, is known to have noteworthy antineoplastic properties. However, the underlying mechanism involved in phycocyanin-mediated anti-growth function on non-small cell lung cancer (NSCLC) cells is still ambiguous. Here, we investigated the mechanism of action of phycocyanin on H1299, A549, and LTEP-a2 cells. According to the results obtained, insulin receptor substrate 1 (IRS-1) expression was reduced by phycocyanin. Cell phenotype tests showed that siRNA knockdown of IRS-1 expression significantly inhibited the growth, migration, colony formation, but promoted the apoptosis of NSCLC cells. Meanwhile, phycocyanin and IRS-1 siRNA treatment both reduced the PI3K-AKT activities in NSCLC cells. Moreover, overexpression of IRS-1 accelerated the proliferation, colony formation, and migration rate of H1299, A549, and LTEP-a2 cells, which was contradicting to the knockdown results. Overall, this study uncovered a regulatory mechanism by which phycocyanin inhibited the growth of NSCLC cells via IRS-1/AKT pathway, laying the foundation for the potential target treatment of NSCLC.     

Panduratin A inhibits the growth of A549 cells through induction of apoptosis and inhibition of NF-kappaB translocation

In the present study we investigated the effects of panduratin A, isolated from Boesenbergia rotunda, on proliferation and apoptosis in A549 human non-small cell lung cancer cells. Cell proliferation and induction of apoptosis was determined by the real-time cellular analyzer (RTCA), MTT assay and High Content Screening (HCS). The RTCA assay indicated that panduratin A exhibited cytotoxicity, with an IC?? value of 4.4 μg/mL (10.8 μM). Panduratin A arrested cancer cells labeled with bromodeoxyuridine (BrdU) and phospho-Histone H3 in the mitotic phase. The cytotoxic effects of panduratin A were found to be accompanied by a dose-dependent induction of apoptosis, as assessed by DNA condensation, nuclear morphology and intensity, cell permeability, mitochondrial mass/ potential, F-actin and cytochrome c. In addition, treatment with an apoptosis-inducing concentration of panduratin A resulted in significant inhibition of Nuclear Factor-kappa Beta (NF-κB) translocation from cytoplasm to nuclei activated by tumor necrosis factor-alpha (TNF-α), as illustrated by the HCS assay. Our study provides evidence for cell growth inhibition and induction of apoptosis by panduratin A in the A549 cell line, suggesting its therapeutic potential as an NF-κB inhibitor.     

Engineered Bacteriorhodopsin May Induce Lung Cancer Cell Cycle Arrest and Suppress Their Proliferation and Migration

Highly expressible bacteriorhodopsin (HEBR) is a light-triggered protein (optogenetic protein) that has seven transmembrane regions with retinal bound as their chromophore to sense light. HEBR has controllable photochemical properties and regulates activity on proton pumping. In this study, we generated HEBR protein and incubated with lung cancer cell lines (A549 and H1299) to evaluate if there was a growth-inhibitory effect with or without light illumination. The data revealed that the HEBR protein suppressed cell proliferation and induced the G₀/G₁ cell cycle arrest without light illumination. Moreover, the migration abilities of A549 and H1299 cells were reduced by ~17% and ~31% after incubation with HEBR (40 μg/mL) for 4 h. The Snail-1 gene expression level of the A549 cells was significantly downregulated by ~50% after the treatment of HEBR. In addition, HEBR significantly inhibited the gene expression of Sox-2 and Oct-4 in H1299 cells. These results suggested that the HEBR protein may inhibit cell proliferation and cell cycle progression of lung cancer cells, reduce their migration activity, and suppress some stemness-related genes. These findings also suggested the potential of HEBR protein to regulate the growth and migration of tumor cells, which may offer the possibility for an anticancer drug.     

Effect of Dai-Bai-Jie on the proliferation and migration of the A549 cells

Lung cancer is the most malignant tumor disease with the highest diagnosis and mortality rate in China.The development of therapeutic drugs is the current research focus.Dai-Bai-Jie is a traditional medicine of the Dai nationality,which is commonly used in the treatment of decreasing swelling,alleviating pain and detoxification.Most of the current researches focused on the component analysis of Dai-Bai-Jie,but few researches studied on its antitumor and pharmacological effect.In this study,we incubated A549 cells with different concentrations of Dai-Bai-Jie.The cell proliferation experiment showed that the DaiBai-Jie solution inhibited the proliferation of A549 cells and caused cell apoptosis.In this work,we confirmed that Dai-Bai-Jie had an inhibitory effect on the proliferation and migration of non-small cell lung cancer A549,which may be used as a novel candidate of anti-tumor therapy for lung cancer patients.     

Synergistic Roles of Curcumin in Sensitising the Cisplatin Effect on a Cancer Stem Cell-Like Population Derived from Non-Small Cell Lung Cancer Cell Lines

Cancer stem cells (CSCs) represent a small subpopulation within a tumour. These cells possess stem cell-like properties but also initiate resistance to cytotoxic agents, which contributes to cancer relapse. Natural compounds such as curcumin that contain high amounts of polyphenols can have a chemosensitivity effect that sensitises CSCs to cytotoxic agents such as cisplatin. This study was designed to investigate the efficacy of curcumin as a chemo-sensitiser in CSCs subpopulation of non-small cell lung cancer (NSCLC) using the lung cancer adenocarcinoma human alveolar basal epithelial cells A549 and H2170. The ability of curcumin to sensitise lung CSCs to cisplatin was determined by evaluating stemness characteristics, including proliferation activity, colony formation, and spheroid formation of cells treated with curcumin alone, cisplatin alone, or the combination of both at 24, 48, and 72 h. The mRNA level of genes involved in stemness was analysed using quantitative real-time polymerase chain reaction. Liquid chromatography-mass spectrometry was used to evaluate the effect of curcumin on the CSC niche. A combined treatment of A549 subpopulations with curcumin reduced cellular proliferation activity at all time points. Curcumin significantly (p < 0.001) suppressed colonies formation by 50% and shrank the spheroids in CSC subpopulations, indicating inhibition of their self-renewal capability. This effect also was manifested by the down-regulation of SOX2, NANOG, and KLF4. Curcumin also regulated the niche of CSCs by inhibiting chemoresistance proteins, aldehyde dehydrogenase, metastasis, angiogenesis, and proliferation of cancer-related proteins. These results show the potential of using curcumin as a therapeutic approach for targeting CSC subpopulations in non-small cell lung cancer.     

Microfluidic three-dimensional biomimetic tumor model for studying breast cancer cell migration and invasion in the presence of interstitial flow

Cell migration and invasion are critical steps in cancer metastasis, which are the major cause of death in cancer patients. Tumor-associated macrophages(TAMs) and interstitial flow(IF) are two important biochemical and biomechanical cues in tumor microenvironment, play essential roles in tumor progression. However, their combined effects on tumor cell migration and invasion as well as molecular mechanism remains largely unknown. In this work, we developed a microfluidic-based 3 D breast cancer model by co-culturing tumor aggregates, macrophages, monocytes and endothelial cells within 3 D extracellular matrix in the presence of IF to study tumor cell migration and invasion. On the established platform, we can precisely control the parameters related to tumor microenvironment and observe cellular responses and interactions in real-time. When co-culture of U937 with human umbilical vein endothelial cells(HUVECs) or MDA-MB-231 cells and tri-culture of U937 with HUVECs and MDA-MB-231 cells, we found that mesenchymal-like MDA-MB-231 aggregates activated the monocytes to TAM-like phenotype macrophages. MDA-MB-231 cells and IF simultaneously enhanced the macrophages activation by the stimulation of colony-stimulating factor 1(CSF-1). The activated macrophages and IF further promoted vascular sprouting via vascular endothelial growth factor(VEGFα) signal and tumor cell invasion. This is the first attempt to study the interaction between macrophages and breast cancer cells under IF condition. Taken together, our results provide a new insight to reveal the important physiological and pathological processes of macrophages-tumor communication. Moreover, our established platform with a more mimetic 3 D breast cancer model has the potential for drug screening with more accurate results.     

An important role for peroxiredoxin II in survival of A549 lung cancer cells resistant to gefitinib

Redox adaptation is an important concept that explains the mechanisms by which cancer cells survive under persistent endogenous oxidative stress and become resistant to certain anticancer agents. To investigate this concept, we determined the expression levels of peroxiredoxins (Prxs), antioxidant enzymes in drug-resistant non-small cell lung carcinoma cells. Prx II was remarkably increased only in A549/GR (gefitinib-resistant) cells compared with A549 cells, consistent with methylation/demethylation. Prx II was highly methylated in the A549 cells but was demethylated in the A549/GR cells. The elevated expression of Prx II resulted in the downregulation of reactive oxygen species (ROS) and cell death and upregulation of cell cycle progression in the A549/GR cells. When Prx II mRNA in the A549/GR cells was knocked down, the levels of ROS and apoptosis were significantly recovered to the levels of the controls. In addition, signaling molecules involved in apoptosis were increased in the A549/GR-shPrx II cells. There was no difference in the expression of MAPK/ERK between the A549/GR cells and A549/GR-shPrx II cells, but the phosphorylation of JNK was increased in the A549/GR cells and was markedly decreased in the A549/GR-shPrx II cells. Colony number and tumor growth were significantly decreased in the A549/GR-shPrx II cells compared with the A549/GR cells. Our findings suggest that Prx II has an important role in cancer cell survival via the modulation of signaling molecules involved in apoptosis and the phosphorylation of JNK by the downregulation of ROS levels in A549/GR cells.     

多壁碳纳米管活化A549细胞核转录子-κB的机制

探讨多壁碳纳米管对人肺上皮细胞A549核转录因子-κB(NF-κB)活性的影响及其活化机制.不同浓度的多壁碳纳米管作用于A549细胞后,用活性氧(ROS)敏感探针2′,7′-二氯荧光素二乙酸酯结合流式细胞仪检测细胞内氧化应激状态;用凝胶电泳迁移率改变这一分析技术检测A549细胞NF-κB DNA结合活性;用蛋白印迹检测A549细胞NF-κB p65蛋白和IκBα蛋白表达;用免疫荧光结合共聚焦显微镜观察A549细胞NF-κB p65蛋白的核转位情况.结果表明,多壁碳纳米管诱导A549细胞内ROS过量产生和NF-κB DNA结合活性;同时伴有p65蛋白核移位和IκBα蛋白胞浆降解.抗氧化剂N-乙酰半胱氨酸(NAC)可抑制多壁碳纳米管诱导的A549细胞内ROS产生、NF-κB DNA结合活性、p65蛋白核移位以及IκBα蛋白降解.结果表明,多壁碳纳米管可以通过诱导A549细胞氧化应激机制从而活化核转录因子NF-κB活性.     

Antiproliferative and Proapoptotic Effects of Phenanthrene Derivatives Isolated from Bletilla striata on A549 Lung Cancer Cells

Lung cancer continues to be the world’s leading cause of cancer death and the treatment of non-small cell lung cancer (NSCLC) has attracted much attention. The tubers of Bletilla striata are regarded as “an excellent medicine for lung diseases” and as the first choice to treat several lung diseases. In this study, seventeen phenanthrene derivatives, including two new compounds (1 and 2), were isolated from the tubers of B. striata. Most compounds showed cytotoxicity against A549 cells. An EdU proliferation assay, a cell cycle assay, a wound healing assay, a transwell migration assay, a flow cytometry assay, and a western blot assay were performed to further investigate the effect of compound 1 on A549 cells. The results showed that compound 1 inhibited cell proliferation and migration and promoted cell apoptosis in A549 cells. The mechanisms might correlate with the regulation of the Akt, MEK/ERK, and Bcl-2/Bax signaling pathways. These results suggested that the phenanthrenes of B. striata might be important and effective substances in the treatment of NSCLC.