Lung Epithelial Carcinoma Cells Research Articles

Toxicity effects of short term diesel exhaust particles exposure to human small airway epithelial cells (SAECs) and human lung carcinoma epithelial cells (A549)

In this study, confocal Raman spectroscopy, atomic force microscope (AFM) and multiplex ELISA were applied to analyze the biophysical responses (biomechanics and biospectroscopy) of normal human primary small airway epithelial cells (SAECs) and human lung carcinoma epithelial A549 cells to in vitro short term DEP exposure (up to 2h). Raman spectra revealed the specific cellular biomolecular changes in cells induced by DEP compared to unexposed control cells. Principal component analysis was successfully applied to analyze spectral differences between control and treated groups from multiple individual cells, and indicated that cell nuclei are more sensitive than other cell locations. AFM measurements indicated that 2h of DEP exposure induced a significant decrease in cell elasticity and a dramatic change in membrane surface adhesion force. Cytokine and chemokine production measured by multiplex ELISA demonstrated DEP-induced inflammatory responses in both cell types.

Dual modulation of JNK and Akt signaling pathways by chaetoglobosin K in human lung carcinoma and ras-transformed epithelial cells

Chaetoglobosin K (ChK) is a natural product that inhibits anchorage-dependent and anchorage-independent growth of ras-transformed cells, prevents tumor-promoter disruption of cell-cell communication, and reduces Akt activation in tumorigenic cells. This study demonstrates how ChK modulates the JNK pathway in ras-transformed and human lung carcinoma cells and investigates regulatory mechanisms controlling ChK's effect on the Akt and JNK signaling pathways. Human lung carcinoma and ras-transformed epithelial cell lines treated with ChK or vehicle for varying times were assayed for cell growth or extracted for total proteins for western blot analysis using phosphorylation site-specific antibodies to monitor changes in activation of JNK, Akt, and other signaling enzymes. Results show that ChK inhibited both Akt and JNK phosphorylation at key activation sites in ras-transformed cells as well as human lung carcinoma cells. Downstream effectors of both kinases were accordingly affected. Direct upstream kinases of JNK were not affected by ChK. Wortmannin and LY294002, two PI3 kinase inhibitors, inhibited Akt but not JNK phosphorylation in ras-transformed cells. This report establishes the dual inhibitory effect of ChK on both the Akt and JNK signaling pathways in ras-transformed epithelial and human carcinoma cells. The unique effect of ChK on these two key pathways involved in carcinogenesis earmarks ChK for further studies to determine its molecular target(s) and in vivo anti-tumor potential.

Threonine 2609 Phosphorylation of the DNA-Dependent Protein Kinase Is a Critical Prerequisite for Epidermal Growth Factor Receptor–Mediated Radiation Resistance

The EGF receptor (EGFR) contributes to tumor radioresistance, in part, through interactions with the catalytic subunit of DNA-dependent protein kinase (DNA-PKc), a key enzyme in the nonhomologous end joining DNA repair pathway. We previously showed that EGFR-DNA-PKcs interactions are significantly compromised in the context of activating mutations in EGFR in non-small cell lung carcinoma (NSCLC) and human bronchial epithelial cells. Here, we investigate the reciprocal relationship between phosphorylation status of DNA-PKcs and EGFR-mediated radiation response. The data reveal that both the kinase activity of DNA-PKcs and radiation-induced phosphorylation of DNA-PKcs by the ataxia telangiectasia-mutated (ATM) kinase are critical prerequisites for EGFR-mediated radioresponse. Alanine substitutions at seven key serine/threonine residues in DNA-PKcs or inhibition of DNA-PKcs by NU7441 completely abrogated EGFR-mediated radioresponse and blocked EGFR binding. ATM deficiency or ATM inhibition with KU55933 produced a similar effect. Importantly, alanine substitution at an ATM-dependent DNA-PKcs phosphorylation site, T2609, was sufficient to block binding or radioresponse of EGFR. However, mutation of a DNA-PKcs autophosphorylation site, S2056 had no such effect indicating that DNA-PKcs autophosphorylation is not necessary for EGFR-mediated radioresponse. Our data reveal that in both NSCLCs and human bronchial epithelial cells, activating mutations in EGFR specifically abolished the DNA-PKcs phosphorylation at T2609, but not S2056. Our study underscores the critical importance of a reciprocal relationship between DNA-PKcs phosphorylation and EGFR-mediated radiation response and elucidates mechanisms underlying mutant EGFR-associated radiosensitivity in NSCLCs.

Comparative in vitro cytotoxicity assessment of airborne particulate matter emitted from stationary engine fuelled with diesel and waste cooking oil-derived biodiesel

Biodiesel derived from waste cooking oil (WCO) is gaining increased attention as an alternative fuel due to lower particulate emissions and other beneficial factors such as low cost and utilization of waste oil. However, very little information is available on toxicity of airborne particulate matter (PM) emitted from biodiesel combustion. In this study, PM emitted from WCO-derived biodiesel (B100) was analyzed for its toxic potential together with ultra low sulphur diesel (ULSD) as a reference fuel and their blend (B50). Human lung epithelial carcinoma cells (A549) were used for this comparative toxicity study. Results indicate that cytotoxicity and oxidative stress were higher for B100 relative to ULSD. Furthermore, caspase 3/7 activity indicates that cell death induced by B100 was due to either caspase independent apoptotic process or other programmed cell death pathways. The toxicity was also evaluated for different engine load conditions. It was observed that at lower loads there was no significant difference in the toxicological response of B100 and ULSD. However, with increase in the engine load, B100 and B50 showed significantly higher toxicity and oxidative stress compared to ULSD.

Abstract 4046: Kinetic quantification of caspase-3/7 mediated apoptosis using live-cell imaging

Abstract Apoptosis, the biological process by which cells undergo programmed cell death, is required for normal tissue maintenance and development. However, aberrations in apoptotic signaling networks are implicated in numerous human diseases including neurodegeneration and cancer. Stimulation of either the extrinsic or intrinsic apoptotic pathways triggers a signaling cascade that typically results in the activation of executioner caspase-3. Numerous in vitro assays have been designed to measure the activation of caspase-3. The majority of these assays utilize reagent substrates that incorporate the DEVD motif which is recognized by both activated caspases 3 and 7. Such assays result in single, user-defined time point measurements of caspase-3/7 activity and typically require multiple wash steps or cell lifting prior to data collection; potentially resulting in the loss of cells or critical data in experiments where cells undergo apoptosis at different rates according to treatment conditions. In this study, we optimized an assay system incorporating a bi-functional reagent containing the DEVD motif linked to a DNA dye for use on the IncuCyte FLR imaging system. When added to cell culture growth medium, this relatively inert, non-fluorescent molecule crosses the cell membrane where it is available to be a substrate for activated caspase-3/7. The reaction product is a green fluorescent DNA dye that labels the nuclei of cells undergoing apoptosis. Our data show that this strategy can quantitatively measure the kinetic activation of caspase-3/7 through extrinsic stimulation of human tumor derived MDA-MB-231 breast adenocarcinoma cells, HeLa cervical adenocarcinoma cells, A549 lung epithelial carcinoma cells, and HT 1080 fibrosarcoma cells following treatment with TNFα and cycloheximide. Moreover, we exemplify the use of this strategy in 96-well plate screening protocols using both HT 1080 and MDA-MB-231 cells to measure the induction of caspase-3/7 activity in the presence of staurosporine, a well-known general protein kinase inhibitor and inducer of caspase-3/7 mediated apoptosis with Z’ values α0.6. We provide evidence that this stable, kinetic, mix-and-read assay can provide both quantitative and qualitative assessments of caspase-3/7 activity using fluorescent images and high-definition phase contrast images, respectively. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4046. doi:1538-7445.AM2012-4046

Poly-lactic-glycolic-acid surface nanotopographies selectively decrease breast adenocarcinoma cell functions

The ability of poly(lactic-co-glycolic acid) (PLGA, 50:50 PLG/PGA, wt%) nanotopographies to decrease lung epithelial carcinoma cell functions (including adhesion, proliferation, apoptosis and vascular endothelial growth factor (VEGF) secretion) has been previously reported. Specifically, results demonstrated decreased lung epithelial carcinoma cell VEGF synthesis on 23 nm surface-featured PLGA compared to traditional nanosmooth PLGA. However, clearly, different cell lines could have different behaviors on similar biomaterials. Thus, to investigate the universality of nanopatterned PLGA substrates to inhibit numerous cancer cell functions, here, breast epithelial adenocarcinoma cell (MCF-7) adhesion, proliferation, apoptosis and VEGF secretion were determined on different PLGA nanometer surface topographies. To isolate surface nanotopographical effects from all other surface properties, PLGA surfaces with various nanotopographies but similar chemistry and hydrophobicity were fabricated here. Atomic force microscopy (AFM) verified the varied nanotopographies on the PLGA surfaces prepared in this study. Importantly, results demonstrated for the first time significantly decreased breast adenocarcinoma cell functions (including decreased proliferation rate, increased apoptosis and decreased VEGF synthesis) on 23 nm featured PLGA surfaces compared to all other PLGA surface topographies fabricated (specifically, nanosmooth, 300 and 400 nm surface-featured PLGA surfaces). In contrast, healthy breast epithelial cells proliferated more (24%) on the 23 nm featured PLGA surfaces compared to all other PLGA samples. In summary, these results provided further insights into understanding the role PLGA surface nanotopographies can have on cancer cell functions and, more importantly, open the possibility of using polymer nanotopographies for a wide range of anticancer regenerative medicine applications (without resorting to the use of chemotherapeutics).

Breast Adenocarcinoma Cell Functions on Nanopatterned PLGA Surfaces

ABSTRACTThe effects of poly (lactic-co-glycolic acid) (PLGA) surface properties on lung epithelial carcinoma cell functions were previously identified. Results demonstrated decreased lung epithelial carcinoma cell vascular endothelial growth factor (VEGF) synthesis on 23 nm surface featured PLGA compared to the PLGA nano-smooth substrates. To investigate the universality of nanopatterned PLGA on inhibiting cancer cell functions, here, breast epithelial adenocarcinoma cell, MCF-7, early functions were determined on different PLGA nanometer surface topographies. Moreover, the viability of healthy breast cells was determined by an MTT assay to investigate the effects of nanopatterning on healthy breast cell growth. Atomic force microscopy (AFM) verified the varied nanotopographies on the PLGA surfaces prepared in this study. Importantly, results demonstrated significantly decreased breast adenocarcinoma cell functions (including decreased proliferation rate and decreased VEGF synthesis) on 23 nm featured PLGA surfaces compared to the other PLGA surface topographies (specifically, nano-smooth, 300 nm and 400 nm surface featured PLGA surfaces). In contrast, healthy breast epithelial cell studies indicated a 24% higher cell proliferation on the 23 nm featured PLGA surfaces compared to all other PLGA samples. In summary, these results provided further insights into understanding the role surface nanotopographies can have on selectively inhibiting cancer cell functions for a wide range of anti-cancer applications.

Cytotoxicity of gold(III) Complexes on A549 Human Lung Carcinoma Epithelial Cell Line

We have studied the kinetics of the complex formation of gold(III) complexes, [Au(en)Cl2]+ (dichlorido( ethylendiamine)aurate(III)-ion) [Au(dach)Cl2] (dichloride(1,2-diaminocyclohexane)aurate(III)-ion) and [Au(bipy)Cl2]+ (dichlorido(2,2'-bipyridyl)aurate(III)-ion) with guanosine5'-monophosphate (5'-GMP). It was shown that 5'-GMP have a high affinity for gold(III) complex, which may have important biological implications, since the interactions of Au(III) with DNA are thought to be responsible for the anti-tumor activity. The [Au(bipy)Cl2]+ complex is more reactive than [Au(en)Cl2]+ or [Au(dach)Cl2]+. The activation parameters for all studied reactions suggest an associative substitution mechanism. The cytotoxicity of gold(III) complexes was tested on A549 human lung carcinoma epithelial cell line and was evaluated by cytotoxic (MTT and LDH test) and apoptotic assays. The results showed that all tested gold(III) complexes displayed cytotoxic effect on A549 cells. Among the tested gold (III) complexes, AuBIPY showed the best cytotoxic effects.

Cytotoxic and antibacterial activities of leaf extracts of Astragalus gombiformis Pomel (Fabaceae) growing wild in Tunisia

Many species of the genus Astragalus have been long used in folk medicine because of their biological properties. The aim of this study was to investigate the antibacterial and cytotoxic activities of extracts from the leaves of wild Astragalus gombiformis Pomel. Antibacterial activity was assayed against several common human pathogenic bacterial strains using the paper disk-agar diffusion method. Cytotoxicity was measured against human lung carcinoma cells using the colorimetric MTT assay. The strongest cytotoxic activity against the human A549 lung epithelial carcinoma cell line was detected for dichloromethane extracts at IC_{50} = 85 ± 21.7 µg/mL. The best incubation time for this extract was 48 h. Most of the tested extracts were active against the tested bacterial strains. The methanol extract was found to be the most active against Pseudomonas aeruginosa and Salmonella typhimurium. This study is the first report on the biological activity of leaf extracts of Astragalus gombiformis with respect to antibacterial activity as well as cytotoxicity.

Influence of serum on in situ proliferation and genotoxicity in A549 human lung cells exposed to nanomaterials

In this work in situ proliferation of A549 human lung epithelial carcinoma cells exposed to nanomaterials (NMs) was investigated in the presence or absence of 10% serum. NMs were selected based on chemical composition, size, charge and shape (Lys-SiO2, TiO2, ZnO, and multi walled carbon nanotubes, MWCNTs). Cells were treated with NMs and 4h later, cytochalasin-B was added. 36h later, cell morphology was analyzed under a light microscope. Nuclearity was scored to determine the cytokinesis-block proliferation index (CBPI). CBPI, based on percentage of mono-, bi- and multi-nucleated cells, reflects cell toxicity and cell cycle delay. For some conditions depending on NM type (TiO2 and MWCNT) and serum concentration (0%) scoring of CBPI was impossible due to overload of agglomerated NMs. Moreover, where heavy agglomeration occurs, micronuclei (MN) detection and scoring under microscope was prevented. A statistically significant decrease of CBPI was found for ZnO NM suspended in medium in the absence or presence of 10% serum at 25μg/ml and 50μg/ml, respectively and for Lys-SiO2 NM at 3.5μg/ml in 0% serum. Increase in MN frequency was observed in cells treated in 10% serum with 50μg/ml ZnO. In 0% serum, the concentrations tested led to high toxicity. No genotoxic effects were induced by Lys-SiO2 both in the absence or presence of serum up to 5μg/ml. No toxicity was detected for TiO2 and MWCNTs in both 10% and 0% serum, up to the dose of 250μg/ml. Restoration of CBPI comparable to untreated control was shown for cells cultured without serum and treated with 5μg/ml of Lys-SiO2 NM pre-incubated in 100% serum. This observation confirms the protective effect of serum on Lys-SiO2 NM cell toxicity. In conclusion in situ CBPI is proposed as a simple preliminary assay to assess both NMs induced cell toxicity and feasibility of MN scoring under microscope.

Decreased lung carcinoma cell functions on select polymer nanometer surface features

Biomaterial nanotopographies have been proposed as a means to significantly influence cell functions (including osteoblasts, fibroblasts, endothelial cells, chondrocytes, immune cells, and bacteria). In this study, lung epithelial carcinoma cell functions including adhesion (up to 4 h), proliferation (up to 3 days), apoptosis (up to 5 days), and vascular endothelial growth factor (VEGF) synthesis (up to 5 days) on poly-lactic-co-glycolic (PLGA) films with various nanotopographies were systematically investigated. Importantly, this study created PLGA films with various nanotopographies (specifically, nano-smooth, 23, 300, and 400 nm hemispherical surface features) but similar surface chemistry in order to focus only on the effect of PLGA topography on cancer cell functions. Simple and effective cast-molding and solvent evaporation methods were used to accomplish this. Atomic force microscopy, electron spectroscopy for chemical analysis, and water contact angle measurements verified similar surface chemistry and energy but varied topographies for all of the PLGA films prepared in this study. Lung epithelial carcinoma cell adhesion, proliferation, and morphology results indicated less cell growth and spreading on nano-smooth and 400 nm surface-featured PLGA compared to any other samples. However, results also demonstrated decreased lung epithelial carcinoma cell VEGF (a key growth factor secreted for the vascularization of tumors) synthesis on the 23 nm surface-featured PLGA compared to the nano-smooth substrates for up to 5 days. In summary, these results provided the first insights into understanding the role that PLGA nanotography may play in mediating lung carcinoma cell functions for a wide range of applications in regenerative medicine.

Long‐term monitoring of live cell proliferation in presence of PVP‐Hypericin: a new strategy using ms pulses of LED and the fluorescent dye CFSE

During fluorescent live cell imaging it is critical to keep excitation light dose as low as possible, especially in the presence of photosensitizer drugs, which generate free radicals upon photobleaching. During fluorescent imaging, stress by excitation and free radicals induces serious cell damages that may arrest the cell cycle. This limits the usefulness of the technique for drug discovery, when prolonged live cell imaging is necessary. This paper presents a strategy to provide gentle experimental conditions for dynamic monitoring of the proliferation of human lung epithelial carcinoma cells (A549) in the presence of the photosensitizer Polyvinylpyrrolidone-Hypericin. The distinctive strategy of this paper is based on the stringent environmental control and optimizing the excitation light dose by (i) using a low-power pulsed blue light-emitting diode with short pulse duration of 1.29 ms and (ii) adding a nontoxic fluorescent dye called carboxyfluorescein-diacetate-succinimidyl-ester (CFSE) to improve the fluorescence signals. To demonstrate the usefulness of the strategy, fluorescence signals and proliferation of dual-marked cells, during 5-h fluorescence imaging under pulsed excitation, were compared with those kept under continuous excitation and nonmarked reference cells. The results demonstrated 3% cell division and 2% apoptosis due to pulsed excitation compared to no division and 85% apoptosis under the continuous irradiation. Therefore, our strategy allows live cell imaging to be performed over longer time scales than with conventional continuous excitation.

HSAB principle and nickel(II) ion reactivity towards 1-methyhydantoin

1-Methylhydantoin and its novel nickel(II) complex [Ni(H 2O) 4(1-mhyd) 2] were prepared and identified, by elemental analysis, single crystal X-ray determination and MS methods. In addition, the complex was characterized by spectroscopic (IR, UV–Vis), magnetic and thermal techniques. The ligand reveals an interesting supramolecular architecture with both classical and non-conventional extended HB bonding networks. All rings and chains formed due to this HB bonding are embedded into the undulated pattern. A single crystal X-ray diffraction analysis of the complex shows that the nickel ion is coordinated by deprotonated hydantoin and water ligands in a N 2O 4 tetragonal arrangement. In the [Ni(H 2O) 4(1-mhyd) 2] structure both inter and intramolecular hydrogen bonds are created with the participation of water molecules. The ESI-MS method confirmed mono-nuclearity of the complex while electronic spectroscopy proved the tetragonal and pseudooctahedral geometries around the metal ion in the solid state and solution, respectively. By application of the “average environment rule”, 10Dq parameters were obtained for the hypothetical, hexa-coordinate [Ni(1-mhyd) 6] approximation or rather more realistic [Ni(1-mhyd) 3] chelate. Based on this the mhyd ligand was ranked in the spectrochemical series close to ammonia. The general consideration of the structure of the hydantoin complexes as a function of the metal ion hardness within the framework of the HSAB theory has been provided. Both the ligand and the complex were found to be non-toxic agents against breast (MCF-7), lung carcinoma epithelial (A549) and mouse fibroblasts (Balb/3T3) cancer cell lines.

Cytotoxic and genotoxic effects of defence secretion of Ulomoides dermestoides on A549 cells

Ulomoides dermestoides (Fairmaire, 1893) is a cosmopolitan tenebrionid beetle reared by Argentine people who consume them alive as an alternative medicine in the treatment of different illnesses such as asthma, Parkinson's, diabetes, arthritis, HIV and specially cancer. To evaluate the cytotoxicity and DNA damage of the major volatile components released by Ulomoides dermestoides on human lung carcinoma epithelial cell line A549. The defence compounds of Ulomoides dermestoides were extracted with dichloromethane and analyzed and quantified by capillary gas chromatography. The toxicity effects of the beetle's extract against A549 cell line were evaluated. Cytotoxicity was evaluated by MTT test and Trypan blue assay and genotoxicity was evaluated by the comet assay. The synthetic compounds, individually or combined, were also tested in A549 cells and normal mononuclear human cells. The defence compounds of Ulomoides dermestoides extracted with dichloromethane (methyl-1,4-benzoquinones, ethyl-1,4-benzoquinones and 1-pentadecene as major components) showed cytotoxic activity on A549 cells demonstrated by MTT test and Trypan blue assay, with IC(50) values of 0.26equivalent/ml and 0.34equivalent/ml, respectively (1equivalent=amount of components extracted per beetle). The inhibition of A549 cell proliferation with the synthetic blend (1,4-benzoquinone and 1-pentadecene) or 1,4-benzoquinone alone was similar to that obtained with the insect extract. 1-Pentadecene showed no inhibitory effect. Low doses of insect extract or synthetic blend (0.15equivalent/ml) inhibited mononuclear cell proliferation by 72.2±2.7% and induced significant DNA damage both in tumor and mononuclear cells. Results of this study demonstrated that defence compounds of Ulomoides dermestoides reduced cell viability and induced DNA damage. We also concluded that the insect benzoquinones are primarily responsible for inducing cytotoxicity and genotoxicity in culture cells.

Meningococcal Internalization into Human Endothelial and Epithelial Cells Is Triggered by the Influx of Extracellular l -Glutamate via GltT l -Glutamate ABC Transporter in Neisseria meningitidis

Meningococcal internalization into human cells is likely to be a consequence of meningococcal adhesion to human epithelial and endothelial cells. Here, we identified three transposon mutants of Neisseria meningitidis that were primarily defective in the internalization of human brain microvascular endothelial cells (HBMEC), with insertions occurring in the gltT (a sodium-independent L-glutamate transporter) gene or its neighboring gene, NMB1964 (unknown function). NMB1964 was tentatively named gltM in this study because of the presence of a mammalian cell entry (MCE)-related domain in the deduced amino acid sequences. The null ΔgltT-ΔgltM N. meningitidis mutant was also defective in the internalization into human umbilical vein endothelial cells and the human lung carcinoma epithelial cell line A549, and the defect was suppressed by transcomplementation of the mutants with gltT(+)-gltM(+) genes. The intracellular survival of the ΔgltT-ΔgltM mutant in HBMEC was not largely different from that of the wild-type strain under our experimental conditions. Introduction of a1-bp deletion and amber or ochre mutations in gltT-gltM genes resulted in the loss of efficient internalization into HBMEC. The defect in meningococcal internalization into HBMEC and L-glutamate uptake in the ΔgltT-ΔgltM mutant were suppressed only in strains expressing both GltT and GltM proteins. The efficiency of meningococcal invasion to HBMEC decreased under L-glutamate-depleted conditions. Furthermore, ezrin, a key membrane-cytoskeleton linker, accumulated beneath colonies of the gltT(+)-gltM(+) N. meningitidis strain but not of the ΔgltT-ΔgltM mutant. These findings suggest that l-glutamate influx via the GltT-GltM L-glutamate ABC transporter serves as a cue for N. meningitidis internalization into host cells.

Antimicrobial peptaibols, novel suppressors of tumor cells, targeted calcium-mediated apoptosis and autophagy in human hepatocellular carcinoma cells

BackgroundHepatocellular carcinoma (HCC) is one of the most common cancers in the world which is highly chemoresistant to currently available chemotherapeutic agents. Thus, novel therapeutic targets are needed to be sought for the successful treatment of HCC. Peptaibols, a family of peptides synthesized non-ribosomally by the Trichoderma species and other fungi, exhibit antibiotic activities against bacteria and fungi. Few studies recently showed that peptaibols exerted cytotoxicity toward human lung epithelial and breast carcinoma cells. However, the mechanism involved in peptaibol-induced cell death remains poorly understood.ResultsHere, we showed that Trichokonin VI (TK VI), a peptaibol from Trichoderma pseudokoningii SMF2, induced growth inhibition of HCC cells in a dose-dependent manner. It did not obviously impair the viability of normal liver cells at lower concentration. Moreover, the suppression of cell viability resulted from the programmed cell death (PCD) with characteristics of apoptosis and autophagy. An influx of Ca2+ triggered the activation of μ-calpain and proceeded to the translocation of Bax to mitochondria and subsequent promotion of apoptosis. On the other hand, typically morphological characteristics consistent with autophagy were also observed by punctate distribution of MDC staining and the induction of LC3-II, including extensive autophagic vacuolization and enclosure of cell organelles by these autophagosomes. More significantly, specific depletion of Bak expression by small RNA interfering (siRNA) could partly attenuate TK VI-induced autophagy. However, siRNA against Bax led to increased autophagy.ConclusionTaken together, these findings showed for the first time that peptaibols were novel regulators involved in both apoptosis and autophagy, suggesting that the class of peptaibols might serve as potential suppressors of tumor cells.

Seasonal variations in chemical composition and in vitro biological effects of fine PM from Milan

Fine particulate matter (PM1 and PM2.5) was collected in Milan over the summer (August–September) and winter (January–March) seasons of 2007/2008. Particles were analyzed for their chemical composition (inorganic ions, elements and PAHs) and the effects produced on the human lung carcinoma epithelial cell line A549. In vitro tests were performed to assess cell viability with MTT assay, cytokine release (IL-6 and IL-8) with ELISA, and DNA damage with COMET assay. Results were investigated by bivariate analysis and multivariate data analysis (Principal Component Analysis, PCA) to investigate the relationship between PM chemical composition and the biological effects produced by cell exposure to 12 μg cm −2. The different seasonal chemical composition of PM showed to influence some biological properties. Summer PM samples had a high mass contribution of SO 4 = (13 ± 2%) and were enriched in some elements, like Al, As, Cr, Cu, and Zn, compared to winter PM samples. Cell viability reduction was two times higher for summer PM samples in comparison with winter ones (27 ± 5% and 14 ± 5%, respectively), and the highest correlation coefficients between cell viability reduction and single chemical components were with As ( R 2 = 0.57) and SO 4 = ( R 2 = 0.47). PM1 affected cell viability reduction and induced IL-8 release, and these events were interrelated ( R 2 = 0.95), and apparently connected with the same chemical compounds. PM2.5 fraction, which was enriched in Ca ++ and Mg ++ (from soil dust), and Al, Fe, Zn, Ba Mn, produced cell viability reduction and DNA damage ( R 2 = 0.73).

Glycogen Synthase Kinase-3 and Omi/HtrA2 Induce Annexin A2 Cleavage followed by Cell Cycle Inhibition and Apoptosis

Annexin A2 is involved in multiple cellular processes, including cell survival, growth, division, and differentiation. A lack of annexin A2 makes cells more sensitive to apoptotic stimuli. Here, we demonstrate a potential mechanism for apoptotic stimuli-induced annexin A2 cleavage, which contributes to cell cycle inhibition and apoptosis. Annexin A2 was persistently expressed around the proliferative but not the necrotic region in BALB/c nude mice with human lung epithelial carcinoma cell A549-derived tumors. Knockdown expression of annexin A2 made cells susceptible to either serum withdrawal-induced cell cycle inhibition or cisplatin-induced apoptosis. Under apoptotic stimuli, annexin A2 was time-dependently cleaved. Mechanistic studies have shown that protein phosphatase 2A (PP2A)-activated glycogen synthase kinase (GSK)-3 is essential for this process. Therefore, inhibiting GSK-3 reversed serum withdrawal-induced cell cycle inhibition and cisplatin-induced apoptosis. Furthermore, inhibiting serine proteases blocked apoptotic stimuli-induced annexin A2 cleavage. Bax activation and Mcl-1 destabilization, which is regulated by PP2A and GSK-3, caused annexin A2 cleavage via an Omi/HtrA2-dependent pathway. Taking these results together, we conclude that GSK-3 and Omi/HtrA2 synergistically cause annexin A2 cleavage and then cell cycle inhibition or apoptosis.

Epilysin (MMP‐28) – structure, expression and potential functions

Epilysin (MMP-28) is the newest member of the matrix metalloproteinase (MMP) family of extracellular proteases. Together the MMPs can degrade almost all components of the extracellular matrix (ECM). MMPs also regulate cell behaviour by releasing growth factors and biologically active peptides from the ECM by modulating cell surface receptors and adhesion molecules and by regulating the activity of mediators of the inflammatory pathways. Epilysin differs from most other MMPs as it is expressed in a number of normal tissues, suggestive of functions in tissue homeostasis. The epilysin homologue in Xenopus laevis (XMMP-28) is expressed in neural tissues, where it cleaves the neural cell adhesion molecule. Enhanced expression of epilysin has been observed in basal keratinocytes during wound healing and in different forms of cancer. There are, however, also reports on the downregulation of epilysin in malignant cells. The roles of epilysin in cancer seem to vary based on tumor type and stage of the disease. Importantly, epilysin can induce stable epithelial to mesenchymal transition (EMT) when overexpressed in epithelial lung carcinoma cells. Transforming growth factor beta (TGF-beta) is a crucial mediator of this process, which was characterized by the loss of E-cadherin and increased cell migration and invasion. Current results suggest a plausible interaction between epilysin and TGF-beta also under physiological circumstances, where epilysin activity may not induce EMT but, instead, trigger less permanent changes in TGF-beta signalling and cell motility.

Interferon alfacon 1 inhibits SARS-CoV infection in human bronchial epithelial Calu-3 cells

The primary targets for SARS-CoV infection are the epithelial cells in the respiratory and intestinal tract. The angiotensin-converting enzyme 2 (ACE-2) has been identified as a functional receptor for SARS-CoV. ACE-2 has been shown to be expressed at the apical domain of polarized Calu-3 cells. In this report, interferon alfacon 1 was examined for inhibitory activities against SARS-CoV on human lung carcinoma epithelial Calu-3 cell line and the other three African green monkey kidney epithelial cell lines. Interferon alfacon 1 demonstrated significant antiviral activity in neutral red uptake assay and virus yield reduction assay. The data might provide an important insight into the mechanism of pathogenesis of SARS-CoV allowing further development of antiviral therapies for treating SARS infections.