Epithelial Lung Cancer Cell Lines Research Articles

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

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

Quantitative and specific detection of cancer-related microRNAs in living cells using surface-enhanced Raman scattering imaging based on hairpin DNA-functionalized gold nanocages.

Variations in the intracellular expression level of cancer-related microRNAs (miRNAs) are connected with worsening tumor progression. A simple, accurate, and sensitive analytical method for the imaging and detection of intracellular miRNA is still a great challenge due to the low abundance of miRNAs and the complexity of intracellular environments. In this work, target miRNA (miRNA)-mediated catalytic hairpin assembly (CHA)-induced gold nanocage (GNC)-hairpin DNA1 (hpDNA1)-hpDNA2-GNC nanostructures were designed for surface-enhanced Raman scattering (SERS) detection and imaging of the specific miR-125a-5p in the normal lung epithelial cell line (BEAS-2B cells) and lung cancer cell line (A549 cells). The finite difference time domain (FDTD) simulations showed that the polymer of GNCs possessed a much stronger electromagnetic field in nanogaps than that of single GNC, theoretically confirming the rational design of the CHA assembly strategy. Using this method, miR-125a-5p can be detected in a wide linear range with a detection limit of 43.96 aM and high selectivity over other miRNAs in vitro. Moreover, SERS imaging successfully detected and distinguished the expression levels of intracellular miR-125a-5p in BEAS-2B cells and A549 cells. The results obtained by the SERS assay were consistent with those obtained by the real-time quantitative polymerase chain reaction (qRT-PCR). This method can offer a powerful strategy for the imaging and quantitative detection of various types of biomolecules in vitro as well as in living cells.

In-vitro anti-cancer activity of organic template-free hierarchical M (Cu, Ni)-modified ZSM-5 zeolites synthesized using silica source waste material

The present paper is focused on a simple and economical route to synthesize the organic template-free hierarchical pure and M (Cu, Ni)-modified ZSM-5 zeolites (1%, 3% and 5%) using hydrothermal treatment in the presence of silica rich rice husk ash and its application studies through the evaluation of their anti-cancer activity on A459 human lung epithelial cancer cell lines. The physical properties of the samples were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, high resolution scanning electron microscopy, high resolution transmission electron microscopy, N2 adsorption/desorption techniques and thermogravimetric analysis respectively. These metals modified hierarchical ZSM-5 zeolites showed considerable in-vitro anticancer efficiency towards human lung cancer (A549) cell lines through (3-(4, 5-dimethyl thiazolyl)-2, 5-diphenyl tetrazolium bromide) MTT assay. Among the all M (Cu, Ni)-modified ZSM-5 zeolites, 5% Cu-ZSM-5 zeolites showed higher potential cytotoxicity against A459 cell lines. The possible mechanism was explored from the fundamental signaling pathways of cell death by hierarchical metal modified ZSM-5 zeolites in A459 human lung epithelial cancer cell lines. Finally, our experimental results revealed that the organic template-free hierarchical pure and M (Cu, Ni)-modified ZSM-5 zeolites have significant anti-cancer mediating action by inducing oxidative stress to intercede DNA damage and can be considered as a potential applicant in the biomedical field.

Doxorubicin and chrysin combination chemotherapy with novel pH-responsive poly [(lactide-co-glycolic acid)-block-methacrylic acid] nanoparticle

A novel pH-responsive poly [(lactide-co-glycolic acid)-co-methacrylic acid] [(PLGA-co-PMAA)] copolymer was synthesized through the combination of radical telomerization and ring opening polymerization method (ROP). A novel strategy to stabilize polymeric nanoparticles interacted on the terminal groups of PLGA segments in (PLGA-co-PMAA) nanoparticles with chrysin (CHS) and it was inverted to (PLGA-co-PMAA)–CHS. The studied copolymers were fully characterized by FTIR and 1HNMR spectroscopy. Nanoparticles were characterized by TEM, dynamic light scattering (DLS) and zeta potential (ξ) measurements. These polymeric nanoparticles were developed with the aim of co-delivering two different anticancer drugs Doxorubicin hydrochloride (DOX) and chrysin. In vitro cytotoxicity and antitumor effect of DOX@CHS-loaded (PLGA-co-PMAA) and DOX-loaded (CHS-PLGA-co-PMAA) were also studied through assessing the survival rate of lung cancer cell line (A549) using MTT and DAPI staining assays. Nanoparticles with homogeneous spherical morphology and an average diameter of around 100 nm were successfully obtained. The viability of human lung epithelial cancer cell lines (A549) was significantly decreased upon interaction DOX@CHS-loaded (PLGA-co-PMAA) nano-formulation.As results, we envision that the novel developed pH-responsive nanoparticle can enhance the efficacy of DOX and Chrysin combination chemotherapy effect and could be applied as an anticancer drug delivery nanosystem for further in vivo uses.

Decitabine Nanoparticles Supplement Docetaxel in the Treatment of Non-Small Cell Lung Cancer (NSCLC)

Docetaxel is one of the most effective chemotherapeutic agents in the treatment of non-small cell lung cancer. Unfortunately, there is altered response to DTX due to resistance. DNA methylation, an epigenetic event, plays a vital role in cancer and chemotherapy drug resistance. Exploiting the gene reactivation by using epigenetically acting agents in combination with cytotoxic therapies, is a strategy of huge clinical relevance. To study the effects of co-administration of epigenetic drug decitabine and docetaxel, we had used human epithelial lung cancer cell lines (A549). A549 cells were cultured and maintained in DMEM, supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin (100,000 U/l penicillin, 100 mg/l streptomycin), at 37 °C in a humidified atmosphere containing 5% carbon dioxide. The cell viability was studied by MTT assay. Decitabine, docetaxwl, decitabine + docetaxel, decitabine nanoparticles + docetaxel were dissolved in the culture medium and added to 96 well plates in different dilutions. The IC50 of DTX and DEC on A549 cells were found to be 0.1 μM and 11.7 μM, respectively. Thereafter, dose dependent response was noted by decreasing the dose of DEC to find a combination dose. The combination of decitabine nanoparticles + docetaxel was found to be more cytotoxic and appears to influence the natural history of NSCLC in a significant manner.

Antiadhesive and cytotoxic effect of Iranian Vipera lebetina snake venom on lung epithelial cancer cells.

Background:Cancer is one of the major health problems worldwide. Hence, finding potent therapeutics from natural sources seems necessary. Snake venom of Vipera lebetina contains potential component with anticancer activities such as antiproliferation, migration, invasion, adhesion, and angiogenesis effect. Evaluation of cytotoxic and antiadhesive effect of V. lebetina venom on lung epithelial cancer tumor cell (TC-1) was the main aim of this study.Materials and Methods:Here, we purified snake venom of V. lebetina by fast protein liquid chromatography (FPLC) using Sephacryl S-200 hr column. The fractions collected and evaluated by SDS-PAGE analysis. The cytotoxicity and antiadhesive effect of crude venom and fractions on TC-1 cells were demonstrated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and adhesion assay, respectively.Results:Our results showed six fractions in FPLC diagram. V. lebetina crude venom and fractions showed dose-dependent cytotoxic effect on TC-1 cells. Fractions 2 and 5 showed high cytotoxic effect with high IC50 value (IC50 = 6 μg/ml for fraction 2 and IC50 = 7.3 μg/ml for fraction 5). Fractions 2 and 5 selected for analysis antiadhesive effect on TC-1 cells. Furthermore, our results showed that both fractions 2 and 5 had antiadhesive effect on TC-1 cells.Conclusion:Because of potent cytotoxic and antiadhesive effect of V. lebetina fractions on lung epithelial cancer cell line, it could be promising tools for further analysis as anticancer therapeutic development.

Pulmonary epithelial cancer cells and their exosomes metabolize myeloid cell-derived leukotriene C4 to leukotriene D4

Leukotrienes (LTs) play major roles in lung immune responses, and LTD4 is the most potent agonist for cysteinyl LT1, leading to bronchoconstriction and tissue remodeling. Here, we studied LT crosstalk between myeloid cells and pulmonary epithelial cells. Monocytic cells (Mono Mac 6 cell line, primary dendritic cells) and eosinophils produced primarily LTC4 In coincubations of these myeloid cells and epithelial cells, LTD4 became a prominent product. LTC4 released from the myeloid cells was further transformed by the epithelial cells in a transcellular manner. Formation of LTD4 was rapid when catalyzed by γ-glutamyl transpeptidase (GGT)1 in the A549 epithelial lung cancer cell line, but considerably slower when catalyzed by GGT5 in primary bronchial epithelial cells. When A549 cells were cultured in the presence of IL-1β, GGT1 expression increased about 2-fold. Also exosomes from A549 cells contained GGT1 and augmented LTD4 formation. Serine-borate complex (SBC), an inhibitor of GGT, inhibited conversion of LTC4 to LTD4 Unexpectedly, SBC also upregulated translocation of 5-lipoxygenase (LO) to the nucleus in Mono Mac 6 cells, and 5-LO activity. Our results demonstrate an active role for epithelial cells in biosynthesis of LTD4, which may be of particular relevance in the lung.

Synthesis and antiproliferative activity of two diastereomeric lignan amides serving as dimeric caffeic acid-l-DOPA hybrids

Two new diastereomeric lignan amides (4 and 5) serving as dimeric caffeic acid-l-DOPA hybrids were synthesized. The synthesis involved the FeCl3-mediated phenol oxidative coupling of methyl caffeate to afford trans-diester 1a as a mixture of enantiomers, protection of the catechol units, regioselective saponification, coupling with a suitably protected l-DOPA derivative, separation of the two diastereomers thus obtained by flash column chromatography and finally global chemoselective deprotection of the catechol units. The effect of hybrids 4 and 5 and related compounds on the proliferation of two breast cancer cell lines with different metastatic potential and estrogen receptor status (MDA-MB-231 and MCF-7) and of one epithelial lung cancer cell line, namely A-549, was evaluated for concentrations ranging from 1 to 256μM and periods of treatment of 24, 48 and 72h. Both hybrids showed interesting and almost equipotent antiproliferative activities (IC50 64–70μM) for the MDA-MB-231 cell line after 24–48h of treatment, but they were more selective and much more potent (IC50 4–16μM) for the MCF-7 cells after 48h of treatment. The highest activity for both hybrids and both breast cancer lines was observed after 72h of treatment (IC50 1–2μM), probably as the result of slow hydrolysis of their methyl ester functions.

Antimicrobial and cytotoxicity effect of silver nanoparticle synthesized by Croton bonplandianum Baill. leaves

Objective(s): For the development of reliable, ecofriendly, less expensive process for the synthesis of silver nanoparticles and to evaluate the bactericidal, and cytotoxicity properties of silver nanoparticles synthesized from root extract of Croton bonplandianum, Baill. Materials and Methods: The synthesis of silver nanoparticles by plant part of Croton bonplandianum was carried out. The formation of nanoparticles was confirmed by Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), XRD and UV-Vis spectrophotometric analysis. The biochemical properties were assayed by antibacterial study, cytotoxicity assay using cancer cell line. Results: The formation of silver nanoparticles was confirmed by UV-VIS spectroscopic analysis which showed absorbance peak at 425 nm. X-ray diffraction photograph indicated the face centered cubic structure of the synthesized AgNPs. TEM has displayed the different dimensional images of biogenic silver nanoparticles with particle size distribution ranging from 15-40 nm with an average size of 32 nm. Silver particles are spherical in shape, clustered. The EDX analysis was used to identify the elemental composition of synthesized AgNPs. Antibacterial activity of the synthesized AgNPs against three Gram positive and Gram negative bacteria strains like Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa carried out showed significant zones of inhibition. The cytotoxicity study by AgNPS also showed cytotoxicity on ovarian cancer cell line PA-1 and lung epithelial cancer cell line A549. Conclusion: The present study confirms that the AgNPs have great promise as antibacterial, and anticancer agent.

Abstract A122: Non-small cell lung cancer cells induce PD-L1 expression in the tumor microenvironment in an orthotopic mouse model of lung cancer

Abstract Background: In many cancers, tumor cells induce local and systemic immunosuppression, inhibiting T-cell-mediated cytotoxic responses to tumor growth. Activation of the programmed cell death 1 (PD-1) pathway has been shown to contribute to immune escape in the setting of lung cancer. Antibody-mediated blockade of PD-1 and programmed death-ligand 1 (PD-L1) induces durable tumor regression and prolongs stabilization of disease in patients with non-small cell lung cancer (NSCLC), which has lead to FDA approval of an anti-PD-1 agent for the treatment of patients with advanced squamous NSCLC. However, patient selection criteria for immunotherapy targeting the PD-1 axis have not been established. Although recent studies have focused on the regulation of PD-L1 expression on lung cancer cells, the role of PD-L1 expression in the lung tumor microenvironment (TME) in mediating responses to anti-PD-1 or anti-PD-L1 therapy has not been defined. The purpose of this study was to determine the relative contributions of PD-L1 expression on cancer cells versus cells in the TME on lung cancer progression. Methods: Two murine lung cancer cell lines derived from C57BL/6 mice were used in an orthotopic model of tumor progression: Lewis Lung Carcinoma (LLC) cells, which are a de-differentiated cell line in which driver mutations have not been identified; and CMT167 cells, which are an epithelial lung cancer cell line that express oncogenic K-Ras. Tumor cells were implanted in the left lungs of wild-type C57BL/6 mice. One week after tumor implantation, mice were treated with anti-PD-1 neutralizing antibody, anti-PD-L1 neutralizing antibody, or isotype control antibodies. The effect of pharmacologic blockade of PD-1 or PD-L1 on tumor progression was determined. In separate experiments, tumor cells were implanted into green fluorescent protein (GFP)-expressing mice, and lungs from tumor-bearing mice were analyzed by flow cytometry to evaluate the expression of PD-L1 on lung cancer cells (GFP-negative cells) and in the TME (GFP-positive cells). Results: Antibody-mediated pharmacologic blockade of PD-1 or PD-L1 suppressed primary tumor growth and liver metastasis of both LLC and CMT167 tumors in our orthotopic model. Whereas there was a modest effect only on LLC tumors, PD-1/PD-L1 antibody blockade had a dramatic effect on CMT167 primary tumor size, with 40% of mice injected with CMT167 cells and treated with anti-PD-L1 antibody having no detectable lung tumors at the time of sacrifice. In contrast to previously published work that has shown epithelial-to-mesenchymal transition can upregulate PD-L1 expression on lung cancer cells, leading to CD8-positive T-cell immunosuppression (Chen et al, Nat Commun 2014), the well-differentiated CMT167 cells were more responsive than LLC cells to PD-L1 inhibition. Moreover, sensitivity to PD-1/PD-L1 antibody blockade did not correlate with expression of PD-L1 on tumor cells. Analysis of lungs from tumor-bearing mice revealed that both LLC cells and CMT167 cells induce PD-L1 expression in the TME (most likely tumor-associated macrophages), and that expression of PD-L1 is higher in the TME than on cancer cells in vivo. Interestingly, CMT167 tumors are associated with higher numbers of both PD-L1-positive cells and PD-1-positive cells in the TME than LLC tumors. Conclusions: In an orthotopic mouse model, lung cancer cells upregulated PD-L1 expression in the TME, which may be an important mechanism whereby tumor cells induce T-cell immunosuppression. The ability to upregulate PD-L1 in the TME may also represent an important factor that predicts sensitivity to PD-1/PD-L1 antibody blockade. Citation Format: Howard Li, Maria McSharry, Eric Clambey, Teresa Nguyen, Eoin McNamee, Mary Weiser-Evans, Raphael Nemenoff. Non-small cell lung cancer cells induce PD-L1 expression in the tumor microenvironment in an orthotopic mouse model of lung cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A122.

Abstract A121: Complement activation is critical for tumor progression in an orthotopic immunocompetent model of lung cancer

Abstract Background: The complement system is an important arm of the innate immune system, and complement proteins are produced by both cancer cells and cells of the TME. A primary function of the complement system is to remove pathogens and injured cells, and it had been assumed that complement activation would result in elimination of cancer cells. However, recent work has revealed new biologic effects of the complement activation fragments C3a and C5a. For example, C3a and C5a directly stimulate the growth of some tissues. Furthermore, inflammatory cells as well as CD4 and CD8 T cells express receptors for both of these activation fragments (C3aR and C5aR), and a recent study reported that C5a reduces the anti-tumor function of CD8+ T cells. Nevertheless, the role of complement activation in the growth and spread of non-small cell lung cancer is incompletely understood. While complement activation can proceed by multiple pathways, a central event is the deposition of C3 on the cell surface. The goal of this study was to examine the role of complement in lung cancer progression employing an immunocompetent orthotopic model which recapitulates many of the features of human lung cancer. Methods: Two murine lung cancer cell lines derived from C57BL/6 mice were used in an orthotopic model of tumor progression: Lewis Lung Carcinoma (LLC) cells, which are a de-differentiated cell line in which driver mutations have not been identified; and CMT167 cells, which are an epithelial lung cancer cell line that express oncogenic K-Ras. Tumor cells were implanted in the left lungs of syngeneic mice WT C57BL/6 mice, or mice which were globally knocked out for C3 (C3-KO). Primary tumor growth and formation of liver metastases were quantitated in the two groups of mice. Complement activation in the plasma was determined by ELISA. Single cell suspensions of the left lung of tumor-bearing mice were prepared and populations of myeloid cells and immune cells quantitated by flow cytometry. Results: Implantation of cancer lead to systemic complement activation as assessed by ELISA in plasma. Primary tumors and liver metastases were decreased in both cell lines implanted into C3-KO mice compared to controls. However, the degree of inhibition was much greater in CMT167 tumors. LLC cells expressed C3 in vitro, whereas CMT167 cells did not, suggesting that autonomous C3 production by cancer cells may contribute to tumor progression. Recruitment of inflammatory myeloid cells was not altered in tumors growing in C3-KO mice. At early time points, we observed equal increases in T cells (CD4, CD8) in the two groups of mice. However, at later time points (3 weeks), the number of T cells had decreased in WT mice, but remained elevated in C3-KO mice. Conclusions: Complement activation occurs in the setting of tumors. C3 plays a critical role in tumor progression, with strong inhibition observed in a subset of cancer cells. The differential response to complement inhibition may depend on the ability of the cancer cells to produce C3. Effects of complement activation may be mediated by inhibiting cytotoxic T cells. These data suggest that complement inhibitors may represent novel immunotherapeutic targets for lung cancer, and suggest interactions between the complement system and checkpoint inhibitors. Citation Format: Jeff Kwak, Howard Li, Jennifer Laskowski, Mary Weiser-Evans, Joshua Thurman, Raphael A. Nemenoff. Complement activation is critical for tumor progression in an orthotopic immunocompetent model of lung cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A121.

Abstract 1426: Lung epithelial cells induce both phenotype alteration and senescence in breast cancer cells

Abstract Purpose: Breast cancer is the most common cancer in women. The lung is one of the most common sites of breast cancer metastasis. While metastatic seeding is often accompanied by a dormancy-promoting mesenchymal to epithelial reverting transitions (MErT), we aimed to determine whether lung epithelial cells can impart this phenotype on aggressive breast cancer cells. Methods: Co-culture experiments of normal lung epithelial cell lines (SAEC, NHBE or BEAS-2B) and breast cancer cell lines (MCF-7 or MDA-MB-231) were conducted. Flow cytometry analysis, immunofluorescence staining for E-cadherin or Ki-67 and senescence associated beta-galactosidase assays assessed breast cancer cell outgrowth and phenotype. Results: Co-culture of the breast cancer cells had different effects on breast cancer cells. The epithelial MCF-7 cells were increased in number but still clustered even if in a slight more mesenchymal spindle morphology. On the other hand, the mesenchymal MDA-MB-231 cells survived but did not progressively grow out in co-culture. These aggressive carcinoma cells underwent an epithelial shift as indicated by cuboidal morphology and increased E-cadherin. Disruption of E-cadherin express in MDA-MB-231 using shRNA prevented this phenotypic reversion in coculture. In addition, we had noted enlarged MDA-MB-231 cells in the co-cultures with SAEC and NHBE but not BEAS-2B cells as suggestion of senescence. A subset of these cells stained with the senescence associated marker β-galactosidase (SA-β-gal). Lung cells limited cancer cell growth kinetics as noted by both (1) some of the cells becoming larger and positive for senescence markers/negative for proliferation marker Ki-67, and (2) Ki-67 positive cells significantly decreasing in MDA-MB-231 and MCF-7 cells after co-culture. Conclusions: Our data indicate that normal lung epithelial cells can drive an epithelial phenotype and suppress the growth kinetics of breast cancer cells by changing their phenotypes.Similar to the hepatocytes, these normal cells may promote the survival, seeding and even dormancy of aggressive mesenchymal carcinoma cells, with phenotypes similar to MDA-MB-231 cells, but also provide progressive signaling when stressed accounting for the spindle morphology and slightly increased growth rates of the epithelial carcinoma cells, such as those MCF-7 represents. In this latter manner, insults the small airway may provoke an outgrowth from dormant breast cancer cells. Citation Format: Masashi Furukawa, Alan Wells, Sarah Wheeler, Amanda M. Clark. Lung epithelial cells induce both phenotype alteration and senescence in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1426. doi:10.1158/1538-7445.AM2015-1426

Lung epithelial cells induce both phenotype alteration and senescence in breast cancer cells.

PurposeThe lung is one of the most common sites of breast cancer metastasis. While metastatic seeding is often accompanied by a dormancy-promoting mesenchymal to epithelial reverting transitions (MErT), we aimed to determine whether lung epithelial cells can impart this phenotype on aggressive breast cancer cells.MethodsCo-culture experiments of normal lung epithelial cell lines (SAEC, NHBE or BEAS-2B) and breast cancer cell lines (MCF-7 or MDA-MB-231) were conducted. Flow cytometry analysis, immunofluorescence staining for E-cadherin or Ki-67 and senescence associated beta-galactosidase assays assessed breast cancer cell outgrowth and phenotype.ResultsCo-culture of the breast cancer cells with the normal lung cells had different effects on the epithelial and mesenchymal carcinoma cells. The epithelial MCF-7 cells were increased in number but still clustered even if in a slightly more mesenchymal-spindle morphology. On the other hand, the mesenchymal MDA-MB-231 cells survived but did not progressively grow out in co-culture. These aggressive carcinoma cells underwent an epithelial shift as indicated by cuboidal morphology and increased E-cadherin. Disruption of E-cadherin expressed in MDA-MB-231 using shRNA prevented this phenotypic reversion in co-culture. Lung cells limited cancer cell growth kinetics as noted by both (1) some of the cells becoming larger and positive for senescence markers/negative for proliferation marker Ki-67, and (2) Ki-67 positive cells significantly decreasing in MDA-MB-231 and MCF-7 cells after co-culture.ConclusionsOur data indicate that normal lung epithelial cells can drive an epithelial phenotype and suppress the growth kinetics of breast cancer cells coincident with changing their phenotypes.

Application of Artificial Neural Network for Modeling and Prediction of MTT Assay on Human Lung Epithelial Cancer Cell Lines

In this paper, a three-layer artificial neural network (ANN) was investigated to predict the inhibitory concentration (IC) values assessed via MTT cell viability assay on the four types of human lung epithelial cancer cell lines. In order to achieve this purpose, a multilayer perceptron (MLP) neural network trained with back-propagation algorithm was employed for developing the ANN model. To develop the model, the input parameters were concentrations and types of cell lines and the outputs were IC10, IC20, IC30, IC40, IC50, IC60, IC70 and IC80 values in the A549, H157, H460 and H1975 cell lines. The proposed ANN model has achieved good agreement with the experimental data and has a small error between the estimated and experimental values. The obtained results show that the proposed ANN model is a useful, reliable, fast and cheap tool to predict the IC values assessed via MTT cell viability assays.

Development and characterization of dexamethasone mesylate anchored on multi walled carbon nanotubes

Dexamethasone conjugated multi wall carbon nanotubes (DEX-MWCNTs) were developed for controlled delivery of a doxorubicin HCl (DOX) with reduced toxicity. The DEX-MWCNTs were prepared by sequential functionalization of MWCNTs. The DOX was physically loaded onto the purified raw MWCNTs and DEX-MWCNTs at pH 7.4 phosphate buffer solutions (PBS) and evaluated for entrapment efficiency, in vitro release, hemolytic toxicity and ex vivo studies on “A-549” lung epithelial cancer cell line. DOX was efficiently loaded into purified raw and DEX-MWCNTs formulation and the highest entrapment of DOX was found to be 92.6 ± 0.5% in the case of DEX-MWCNTS with good dispersion. In-vitro release of DOX was studied from the DOX/DEX-MWCNTs at pH 5.5 and 7.4 (PBS), which displayed an initial faster followed by sustained release up to 200 h. Further, DOX/DEX-MWCNTs were found to be less hemolytic and more cytotoxic as compared to free DOX on “A-549” lung epithelial cancer cell line.

Encapsulation of Alpha-1 antitrypsin in PLGA nanoparticles: In Vitro characterization as an effective aerosol formulation in pulmonary diseases

BackgroundAlpha 1- antitrypsin (α1AT) belongs to the superfamily of serpins and inhibits different proteases. α1AT protects the lung from cellular inflammatory enzymes. In the absence of α1AT, the degradation of lung tissue results to pulmonary complications. The pulmonary route is a potent noninvasive route for systemic and local delivery. The aerosolized α1AT not only affects locally its main site of action but also avoids remaining in circulation for a long period of time in peripheral blood. Poly (D, L lactide-co glycolide) (PLGA) is a biodegradable and biocompatible polymer approved for sustained controlled release of peptides and proteins. The aim of this work was to prepare a wide range of particle size as a carrier of protein-loaded nanoparticles to deposit in different parts of the respiratory system especially in the deep lung. Various lactide to glycolide ratio of the copolymer was used to obtain different release profile of the drug which covers extended and rapid drug release in one formulation.ResultsNonaqueous and double emulsion techniques were applied for the synthesis of nanoparticles. Nanoparticles were characterized in terms of surface morphology, size distribution, powder X-ray diffraction (XRD), encapsulation efficiency, in vitro drug release, FTIR spectroscopy and differential scanning calorimetry (DSC). To evaluate the nanoparticles cytotoxicity, cell cytotoxicity test was carried out on the Cor L105 human epithelial lung cancer cell line.Nanoparticles were spherical with an average size in the range of 100 nm to 1μ. The encapsulation efficiency was found to be higher when the double emulsion technique was applied. XRD and DSC results indicated that α1AT encapsulated in the nanoparticles existed in an amorphous or disordered-crystalline status in the polymer matrix. The lactic acid to glycolic acid ratio affects the release profile of α1AT. Hence, PLGA with a 50:50 ratios exhibited the ability to release %60 of the drug within 8, but the polymer with a ratio of 75:25 had a continuous and longer release profile. Cytotoxicity studies showed that nanoparticles do not affect cell growth and were not toxic to cells.ConclusionIn summary, α1AT-loaded nanoparticles may be considered as a novel formulation for efficient treatment of many pulmonary diseases.

A Strategy in The Design of Micellar Shape for Cancer Therapy

For cancer therapy, optimization of carrier features is necessary to effectively deliver the targeting agents to tumor sites. Biodegradable poly(ether-anhydrides) micelles with filamentous, rod-like, and spherical shapes are fabricated. Their size and morphology are characterized by AFM and TEM. The encapsulation of doxorubicin hydrochloride (DOX) into the micelles does not impact their shape. The effect of micellar shape on the drug loading capacity and encapsulation efficiency, as well as in vitro drug release, is investigated. The cellular uptakes are evaluated using fluorescence microscopy, confocal laser scanning microscopy and flow cytometry on co-cultures of human hepatoblastoma cell line (HepG2), lung epithelial cancer cell line (A549), and human nasopharyngeal epidermoid carcinoma cells (KB) and fibroblast normal cells mixed with the different shapes of DOX-loaded micelles. The results show that the spherical DOX-loaded micelles are more readily taken up by all types of cells. The impact of micellar shape on in vivo antitumor function is also assessed from changes of tumor volume, body weight loss, and survival rate of 4T1-bearing mice and the immunostaining of tumor sections for analysis of tumor cell proliferation. The results reveal that the filamentous DOX-loaded micelles possess the highest safety to body and the best therapeutic effects to artificial solid tumors. Therefore, the filamentous shape is deemed the most suitable morphology for design and engineering of drug vehicles for cancer therapy.

Target-Specific Cellular Uptake of Folate-Decorated Biodegradable Polymer Micelles

For cancer therapy, folate (FA) and β-cyclodextrin (β-CD) decorated micelles based on the biodegradable pluronic F127-b-poly(ε-caprolactone) copolymer were fabricated. These micelles were measured by dynamic light scattering measurements and atomic force microscopy. The in vitro release of doxorubicin hydrochloride (DOX·HCl) from the biodegradable polymer micelles was performed in a phosphate-buffered saline solution at pH 7.4 and acetate buffer solution at pH 5.0 at the temperatures of 4, 25, and 37 °C, and the results show that the release was obviously influenced by the pH and temperature. The material cytotoxicity and the tumor cell growth inhibition assays of DOX·HCl-loaded micelles were studied with the human hepatoblastoma cell line (HepG2), the lung epithelial cancer cell line (A549), and human nasopharyngeal epidermoid carcinoma cells (KB) and fibroblast normal cells using fluorescence microscopy as well as confocal laser scanning microscopy. The cellular uptake was quantitatively analyzed to further evaluate the active targeting behaviors of the micelles by flow cytometry. These quantitative and qualitative results of cellular uptake of the micelles provide evidence for the different targeting efficiencies of FA decoration for HepG2, KB, and A549 tumor cells as well as fibroblast normal cells. It also suggested that FA- and β-CD-decorated doxorubicin-loaded micelles may have great potential as nanocarriers for targeted drug delivery.

Chrysin enhances doxorubicin-induced cytotoxicity in human lung epithelial cancer cell lines: The role of glutathione

We hypothesized that flavonoid-induced glutathione (GSH) efflux through multi-drug resistance proteins (MRPs) and subsequent intracellular GSH depletion is a viable mechanism to sensitize cancer cells to chemotherapies. This concept was demonstrated using chrysin (5–25 μM) induced GSH efflux in human non-small cell lung cancer lines exposed to the chemotherapeutic agent, doxorubicin (DOX). Treatment with chrysin resulted in significant and sustained intracellular GSH depletion and the GSH enzyme network in the four cancer cell types was predictive of the severity of chrysin induced intracellular GSH depletion. Gene expression data indicated a positive correlation between basal MRP1, MRP3 and MRP5 expression and total GSH efflux before and after chrysin exposure. Co-treating the cells for 72 h with chrysin (5–30 μM) and DOX (0.025–3.0 μM) significantly enhanced the sensitivity of the cells to DOX as compared to 72-hour DOX alone treatment in all four cell lines. The maximum decrease in the IC 50 values of cells treated with DOX alone compared to co-treatment with chrysin and DOX was 43% in A549 cells, 47% in H157 and H1975 cells and 78% in H460 cells. Chrysin worked synergistically with DOX to induce cancer cell death. This approach could allow for use of lower concentrations and/or sensitize cancer cells to drugs that are typically resistant to therapy.

Cement‐related particles interact with proinflammatory IL‐8 chemokine from human primary oropharyngeal mucosa cells and human epithelial lung cancer cell line A549

Epidemiological studies have shown that respirable exposure to emitted cement particulate matter is associated with adverse health risk for human. The underlying mechanisms, however, are poorly understood. To examine the effect of cement, nine blinded cement-related particulates (<10 μm) were assessed with regard to their induction of the proinflammatory cytokines IL-6 and IL-8 in human primary epithelial cells (pEC) from oropharyngeal mucosa as well as from nonsmall-cell lung carcinoma (non-SCLC) cells A549. It was demonstrated that the cement specimens did not act cytotoxic as assessed by the lactate dehydrogenase (LDH) assay. The basal and IL-1β-induced IL-8 expression was suppressed, in contrast to an unchanged IL-6. At the transcript level the basal and induced IL-6 and IL-8 gene expression was not influenced by cement dust. To discover the mechanism by which cement influenced the IL-8 expression the following experiments were performed. Submerse exposure experiments have shown that the release of IL-8 was suppressed by cement dust. Furthermore, the incubation of IL-8 with cement-related specimens under cell-free condition led to a loss of immunoreactive IL-8. An immunological masking of IL-8 by free soluble components of respiratory epithelial cells was excluded. Thus, the decrease of IL-8 protein content after cement exposure seems to be a result of the adsorption of IL-8 protein to cement particles and the inhibition of IL-8 release. In conclusion, due to absent cytotoxic and inflammatory effects of cement-related specimens in both human pEC and A549 cell models it remains open how cement exposure may lead to the respiratory adverse effects in humans.