Lung Cancer Cell Line H1299 Research Articles

Abstract 2933: Synergistic antitumor effect of ONC201 in combination with radiation therapy

Abstract Radiation therapy is used as the main treatment when surgery is not feasible, before/after surgery, and to palliate symptoms in advanced stage. About 50% of cancer patients receive radiation therapy. 40% of the cure is achieved by radiation therapy. First-in-class small-molecule imipridone ONC201 can cause dual inactivation of ERK and AKT and induce an integrated stress response, proapoptotic TRAIL receptor DR5 activation, cancer stem cell depletion, and cell cycle arrest. ONC201 could synergize with radiation therapy because of its proapoptotic and antimetastasis effects. We tested this hypothesis in colorectal cancer, non-small cell lung cancer and glioblastoma cell lines (HCT116, RKO, LS513, H460, A549 and SNB19). Firstly, CellTiter-Glo® luminescent cell viability assays were performed with single treatment or combination of radiation therapy (0 to 4 Gy) and ONC201 (0 to 20 μM). For combination treatment, cancer cells were pretreated with ONC201 for 24 hours followed by radiation therapy. Next, colony formation assays with single treatment or combination were used to evaluate the effective dose range from cell viability assays. Flow cytometry and Western blot analysis were used for determination of apoptosis. We observed synergism between ONC201 and radiation therapy in several colorectal cancer, non-small cell lung cancer and glioblastoma cell lines. Combination indices are 0.53, 0.59 and 0.68 when non-small cell lung cancer cell line H460 was treated with ONC201 at 5 μM and radiation therapy at 1, 2 and 4 Gy. We are currently pursuing this promising combinational therapy in vivo, using the H460 xenograft model and potential syngeneic lung tumor models with wild-type p53. We plan to pursue the mechanism of this combination from in vivo samples. Our results indicate that the combination of radiation therapy and ONC201 could be an important approach in the application of ONC201 in clinical practice. Citation Format: Lanlan Zhou, Jessica Wagner, Wafik S. El-Deiry. Synergistic antitumor effect of ONC201 in combination with radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2933.

Abstract 1662: Synthesis and biologic evaluation of diethylbenzaldehyde analogues to probe functional activity of aldehyde dehydrogenase activity in cancer

Abstract The human aldehyde dehydrogenases (ALDHs) play a major role in detoxifying highly reactive aldehydes into carboxylic acids. Deregulation of ALDHs has implications in a number of cancers. They play an important role as a cancer stem cell (CSC) marker due to high activity found in CSCs, while high expression is also known to lead to resistance to chemotherapeutic drugs. Although the exact roles of ALDHs are not fully understood, emerging information indicates several isoforms from the ALDH1 family, ALDH3A1 and ALDH7A1, play key roles in several cancer types. To probe the role of ALDH functional activity, we here report on the perturbation of ALDH expression and function using diethylbenzaldehyde (DEAB) analogues. The H1299 lung cancer cell line has negligible amount of ALDH activity, and ALDH-expressing variants were used to study the novel DEAB analogues. H1299 wild-type cells (WT) and cells expressing RFP were used as controls. Expression of the proteins was confirmed by Western blots and ALDH enzymatic activity assay. We used cell culture, colorimetric MTT assay, aldefluor assay and in vitro treatment with DEAB analogues. ALDH-expressing lysates were used to obtain biochemical inhibition data of the DEAB analogues using propionaldehyde as substrate with NAD+ as a co-factor. DEAB has been shown to covalently bind to ALDH7A1, and computational modeling was employed to understand how chemical modifications to DEAB affected the binding mode. Selected ALDH-affinic probe compounds were tested in a panel of colon, lung and prostate cancer cell lines including docetaxel-resistant variant and patient samples to investigate if DEAB analogues had an effect on cell viability as single agents or in combination with docetaxel. In conclusion, the novel DEAB analogues aid us to understand ALDH functional activity and provide a platform for discovering more selective ALDH inhibitors Citation Format: Ali Ibrahim, Maria Sadiq, Daniela Presa, Ioannis Ntafoulis, Ahmad Alkawi, Fiona Frame, Norman J. Maitland, Zoe Cournia, Paul M. Loadman, Jan Moreb, Klaus Pors. Synthesis and biologic evaluation of diethylbenzaldehyde analogues to probe functional activity of aldehyde dehydrogenase activity in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1662.

Overexpressed CDR1as functions as an oncogene to promote the tumor progression via miR-7 in non-small-cell lung cancer.

BackgroundCircular RNA (circRNA) is a novel member of the noncoding RNA and function as efficient microRNA sponges with gene-regulatory potential, especially the circular RNA ciRS-7 (CDR1as)/tumor suppressor miRNA-7 (miR-7) signals. However, the function of CDR1as/miR-7 in non-small cell lung cancer (NSCLC) is unknown.MethodsNormal lung tissues (n=20), adjacent non-tumor tissues (n=60), and NSCLC tissues (n=60) were collected to determine the expression and significance of CDR1as/miR-7. Lung cancer cell lines A549 and H460 were overexpressed or knocked down of CDR1as, miR-7 to determine the tumor growth etc. The CDR1as/miR-7-related pathway were analyzed.ResultsCDR1as levels was robustly increased with the development of NSCLC (P<0.001) and the NSCLC tissues harbored highest expression of CDR1as, which negatively correlated to the expression of miR-7. Patients with high expression of CDR1as had high TNM stage (P=0.004), more lymph nodes metastasis (LNM) (P=0.021) and shorted overall survival time (OS) (P=0.0135). The CDR1as level was an independent prognostic factor for the patients with NSCLC. Overexpression of CDR1as induced increased cell vitalities and growth, which could be abrogated by knockdown of CDR1as or overexpressed miR-7 to induce apoptosis and G1/S arrest. Mechanistically, CDR1as functioned as miR-7 sponges to up-regulate the key target genes of miR-7 including EGFR, CCNE1 and PIK3CD. The results in vivo further confirmed that CDR1as functioned as oncogene to inhibit the anti-tumor effects of tumor suppressor miR-7 by up-regulation of proliferation index Ki-67, EGFR, CCNE1 and PIK3CD levels.ConclusionOverexpressed CDR1as in NSCLC functions promotes the tumor progression via miR-7 signals.

RBE Model-Based Biological Dose Optimization for Proton Radiobiology Studies.

The purpose of the current study was (1) to develop a straightforward and rapid method to incorporate a dose-averaged linear energy transfer (LET d )-based biological effect model into a dose optimization algorithm for scanned protons; and (2) to apply a novel beam delivery strategy with increased LET d within the target, thereby enhancing the biological effect predicted using the selected relative biological effectiveness (RBE) model. We first generated pristine dose Bragg curves in water and their corresponding LET d distributions for 94 groups of proton beams, using experimentally validated Geant4 Monte Carlo simulations. Next, we developed 1-dimensional dose optimization algorithms by using the Python programming language. To calculate the RBE of protons for biological dose optimization, we invoked the McNamara RBE model and applied the radiobiological parameters of the lung cancer H460 cell line with 137Cs reference photons. High-accuracy optimization results were obtained. The relative difference between the delivered dose and the prescribed dose was approximately within ±1.0% in the target. In addition, we obtained the RBE enhancement within the target by applying the LET-painting technique. For example, considering a simple case in which 2 opposed downslope dose fields were superimposed to form a uniform dose in the 5- to 10-cm target region, the center RBE was 1.23 ± 0.01, which was greater than the center RBE of 1.16 ± 0.01 found when using the traditional method of delivering 2 opposed flat dose fields. We have successfully developed an easy-to-implement method to perform the biological dose optimization procedure by invoking the McNamara RBE model in the iteration process using the Python programming language. According to the RBE model predictions, we conclude that the increased target LET d enhances the RBE. The accuracy of the RBE model predictions needs to be validated in radiobiological experiments.

Glass-based microchip with finger electrodes for the formation, collection, and biointeractions of liposomes

A concept using a glass-based microchip with finger electrodes, under a two-step voltage-controlled manner, to accomplish the formation and collection of liposomes is presented. By the actions of electro-osmotic and electrolytic bubbles in low and high applied voltages, respectively, the liposomes were first formed in an electrolyte reservoir; then, they were successfully moved to a collected reservoir, which is helpful for subsequent on-chip processing. This study evaluated the distribution of an electric field for different couple-finger distances using finite-element analysis software. The simulation result shows that the electric field density increases rapidly with the decrease of finger distance, with a quadratic relationship. Experiment results show that, when the applied AC voltage is 1 Vp-p, at a driving frequency of 10 Hz, the liposomes are first formed near the boundary of the electrolyte reservoir, and when the voltage is higher, the mass production of liposomes is achieved. When the voltage is higher than 4 Vp-p, the electrolytic bubbles grow cyclically to separate the liposomes from the electrolyte reservoir and therefore collect in a specific reservoir. Liposomes encapsulate doxorubicin, and their drug release to the lung cancer cell line H1299 was also implemented to verify their biointeraction characteristics. These experimental results were all identified using an inverted fluorescence microscope.

An effective approach to study the biocompatibility of Fe3O4 nanoparticles, graphene and their nanohybrid composite

The present manuscript describes a simple, facile and effective solvothermal route to synthesize Fe3O4 nanoparticles (Fe3O4 NPs), reduced graphene oxide nanosheets (rGO NSs) and Fe3O4/reduced graphene oxide nanohybrid composite (Fe3O4/rGO nanohybrid composite) and subsequently examines their comparative biocompatibilities. The as-obtained Fe3O4 NPs, rGO NSs and Fe3O4/rGO nanohybrid composite have been characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Raman spectroscopy. The XRD studies and scanning electron microscope confirmed the proper phase formation and the surface morphology of the as-synthesized products, respectively. The Raman spectra of Fe3O4 NPs show the strongest peak at 673 cm−1 which can be assigned to A1g peak of bare Fe3O4 NPs and it complements the XRD studies. Furthermore, the increment in the ID/IG ratio in the Fe3O4/rGO nanohybrid composite suggests the creation of defects in graphene sheets due to strain caused by Fe3O4 NPs. The biocompatibility of these samples has been tested using Lung cancer cell line H1299 through MTT assay. The MTT assay reveals that the nanohybrid composite endows more biocompatible and effectiveness than rGO NSs and Fe3O4 NPs individually, as anti-proliferative agent for cancer treatment.

Nrf-2 transcriptionally activates P21Cip/WAF1 and promotes A549 cell survival against oxidative stress induced by H2O2.

Cancer cells possess elevated ROS coupled with increased levels of antioxidant enzymes which render them resistant against cytotoxic chemotherapies. Therefore, an understanding of the interaction between key molecules involved in stress adaptive mechanisms is important to innovate strategies against cancer cell chemoresistance. Here, the lung adenocarcinoma cell line A549 with constitutively expressed Nrf2 was found to be more tolerant to H2O2 (0.1, 0.2, 0.5 and 1 mM) than normal lung cell line L132 or p53 null lung cancer cell line H1299. Maximum cytoprotection was observed at 0.2 mM H2O2 accompanied by a significant increase in p21, Nrf2 and antioxidant enzymes in A549 cells. The increased p21 expression was independent of p53 but dependent on Nrf2 as evident from qPCR, Western blotting and dual luciferase assays after silencing Nrf-2 and p53 genes. Highly conserved Nrf-2 binding sites were identified in p21 promoter by bioinformatics and homology modeling which was further confirmed by ChIP and reporter assay.

Pan-HDAC inhibition by panobinostat mediates chemosensitization to carboplatin in non-small cell lung cancer via attenuation of EGFR signaling

Accumulating evidence has implicated the aberrant regulation of histone deacetylases (HDACs) as a nexus for multiple cancer hallmarks and in mediating tumor adaptation and resistance to genotoxic chemotherapy, suggesting a rational pairing of HDAC inhibitors with DNA damaging chemotherapeutic agents in the treatment of human malignancies. Here we report that panobinostat (LBH589), a potent pan-HDAC inhibitor, effectively curbed the proliferation of non-small cell lung cancer (NSCLC) cell lines A549, Calu-1, H226, H460, H838 and SKMES-1 at IC50 concentrations between 4 and 31 nmol/L via pleiotropic mechanisms, including crosstalk with EGFR signal transduction cascades. Combination therapy with carboplatin elicited rapid tumor cell kill and effectively restrained anchorage-independent clonogenic survival to a considerably greater extent over either monotherapy. The administration of carboplatin and panobinostat at clinically relevant doses to NOD-SCID xenograft mice drastically stalled disease progression by 92% as compared with negative control (P = .0026), which was greater than the 28% and 54% achieved with either carboplatin (P = .220) or panobinostat (P = .017) alone. These data demonstrate that panobinostat has strong anti-NSCLC activity and chemosensitizes tumors to carboplatin, thus justifying further evaluation of this combination approach in clinical trials.

Regulation of Apoptosis and Radiation Sensitization in Lung Cancer Cells via the Sirt1/NF-κB/Smac Pathway

Background/Aims: SirT1, a conserved NAD⁺-dependent deacetylase, has been implicated in modulating cell survival and stress responses, and it appears to play an important role in tumorigenesis and cancer resistance to chemoradiotherapy. The mechanism of SirT1 in cancer chemoradiotherapy remains to be further elucidated, which could provide potential targets for cancer therapy. Methods: We performed colony formation, immunofluorescence microscopy, flow cytometry, RNA interference, and western blotting assays to determine whether SirT1 regulates radiation sensitization and which mechanisms and/or pathways it takes in lung cancer cell lines A549 and H460. Results: Initially, the expression of SirT1 was found to be negatively correlated with radiosensitivity in lung cancer cell lines A549 and H460. RNA interference with siSirT1 against SirT1 specifically reduced SirT1 expression and induced radiosensitivity both in A549 and H460 cell lines. In contrast, the radiosensitivity was significantly reduced once SirT1 was activated by resveratrol. Immunofluorescence assay and apoptosis analysis indicated that the effect of SirT1 on the radiosensitivity observed in the A549 and H460 cell lines was mainly achieved by regulating DNA damage repair and apoptosis processes. Furthermore, the expression of SirT1 negatively modulated the expression of apoptosis-related protein NF-κB and its downstream regulator of Smac. Conclusion: Our results indicate that SirT1 regulates apoptosis and radiation sensitization in lung cancer cell lines A549 and H460 via the SirT1/NF-κB/Smac pathway.

Stepwise synthesis, characterization, DNA binding properties and cytotoxicity of diruthenium oligopyridine compounds conjugated with peptides.

Although the interactions of oligopyridine ruthenium complexes with DNA have been widely studied, the biological activity of similar diruthenium oligopyridine complexes conjugated with peptides has not been investigated. Herein, we report the stepwise synthesis and characterization of diruthenium complexes with the general formula [(La)Ru(tppz)Ru(Lb)]n+ (tppz = 2,3,5,6-tetra(2-pyridyl)pyrazine, La = 2,2':6',2''-terpyridine or 4-phenyl-2,2':6',2''-terpyridine and Lb = 2,2':6',2''-terpyridine-4'-CO(Gly1-Gly2-Gly3-LysCONH2) (5), (6), n = 5; 2,2':6',2''-terpyridine-4'-CO(Gly1-Gly2-Lys1-Lys2CONH2) (7), (8), n = 6; 2,2':6',2''-terpyridine-4'-CO(Ahx-Lys1Lys2CONH2) (9), (10), n = 5, Ahx = 6-aminohexanoic acid). The compounds [(trpy)Ru(tppz)Ru(trpy-CO2H)](PF6)4, (2)(PF6)4, [(ptrpy)Ru(tppz)Ru(trpy-CO2H)](PF6)4, (3)(PF6)4 and [(ptrpy)Ru(tppz)Ru(trpy)](PF6)4, (4)(PF6)4 were also characterized by single crystal X-ray methods. Moreover, the interactions of the chloride salts (5), (6) and (4) with the self-complementary dodecanucleotide duplex d(5'-CGCGAATTCGCG-3')2 were studied by NMR spectroscopic techniques. The results show that complex (4) binds in the central part of the oligonucleotide, from the minor groove through the ligand ptrpy, while the ligand trpy, which was located on the other side of the diruthenium core, does not contribute to the binding. Complex (5) binds similarly, through the ligand ptrpy. However, the induced upfield shifts of the ptrpy proton signals are significantly lower than the corresponding ones in the case of (4), indicating much lower binding affinity. This is clear evidence that the tethered peptide Gly1-Gly2-Gly3-Lys1CONH2 hinders the complex binding, even though it contains groups that are able to assist it (e.g., the positively charged amino group of lysine, the peptidic backbone, the terminal amide). Complex (6) shows a non-specific binding, interacting through electrostatic forces. The chloride salts of (4), (5) and (6) had insignificant effects on the cell cycle distribution and marginal cytotoxicity (IC50 > 750 μM) against human lung cancer cell lines H1299 and H1437, indicating that their binding to the oligonucleotide is not a sufficient condition for their cytotoxicity.

Upregulation of FoxM1 by MnSOD Overexpression Contributes to Cancer Stem-Like Cell Characteristics in the Lung Cancer H460 Cell Line.

Manganese superoxide dismutase promotes migration and invasion in lung cancer cells via upregulation of the transcription factor forkhead box M1. Here, we assessed whether upregulation of forkhead box M1 by manganese superoxide dismutase overexpression mediates the acquisition of cancer stem-like cell characteristics in non-small cell lung cancer H460 cells. The second-generation spheroids from H460 cells were used as lung cancer stem-like cells. The levels of manganese superoxide dismutase, forkhead box M1, stemness markers (CD133, CD44, and ALDH1), and transcription factors (Bmi1, Nanog, and Sox2) were analyzed by Western blot. Sphere formation in vitro and carcinogenicity of lung cancer stem-like cells were evaluated by spheroid formation assay and limited dilution xenograft assays. Knockdown or overexpression of manganese superoxide dismutase or/and forkhead box M1 by transduction with short hairpin RNA(shRNA) or complementary DNA were performed for mechanistic studies. We showed that manganese superoxide dismutase and forkhead box M1 amounts as well as the expression levels of stemness markers and transcription factors sphere formation in vitro, and carcinogenicity of lung cancer stem-like cells were higher than in monolayer cells. Lung cancer stem-like cells transduced with manganese superoxide dismutase shRNA or FoxM1 shRNA exhibited decreased sphere formation and lower amounts of stemness markers and transcription factors. Overexpression of manganese superoxide dismutase or FoxM1 in H460 cells resulted in elevated sphere formation rates and protein levels of stemness markers and transcription factors. Meanwhile, manganese superoxide dismutase knockdown or overexpression accordingly altered forkhead box M1 levels. However, forkhead box M1 knockdown or overexpression had no effect on manganese superoxide dismutase levels but inhibited or promoted lung cancer stem-like cell functions. Interestingly, forkhead box M1 overexpression alleviated the inhibitory effects of manganese superoxide dismutase knockdown in lung cancer stem-like cells. In a panel of non-small cell lung cancer cells, including H441, H1299, and H358 cells, compared to the respective monolayer counterparts, the expression levels of manganese superoxide dismutase and forkhead box M1 were elevated in the corresponding spheroids. These findings revealed the role of forkhead box M1 upregulation by manganese superoxide dismutase overexpression in maintaining lung cancer stem-like cell properties. Therefore, inhibition of forkhead box M1 upregulation by manganese superoxide dismutase overexpression may represent an effective therapeutic strategy for non-small cell lung cancer.

Interferon Consensus Sequence-Binding Protein 8, a Tumor Suppressor, Suppresses Tumor Growth and Invasion of Non-Small Cell Lung Cancer by Interacting with the Wnt/β-Catenin Pathway

Background/Aims: Interferon consensus sequence-binding protein 8 (IRF8) belongs to a family of interferon (IFN) regulatory factors that modulates various important physiological processes including carcinogenesis. As reported by others and our group, IRF8 expression is silenced by DNA methylation in both human solid tumors and hematological malignancies. However, the role of IRF8 in lung carcinoma remains elusive. In this study, we determined IRF8 epigenetic regulation, biological functions, and the signaling pathway involved in non-small cell lung cancer (NSCLC). Methods: IRF8 expression were determined by Q- PCR. MSP and A+T determined promotor methylation. MTS, clonogenic, Transwell assay, Flow cytometry, three-dimensional culture and AO/EB stain verified cell function. In vivo tumorigenesis examed the in vivo effects. By Chip-QPCR, RT-PCR, Western blot and Immunofluorescence staining, the mechanisms were studied. Results: IRF8 was significantly downregulated in lung tumor tissues compared with adjacent non-cancerous tissues. Furthermore, methylation-specific PCR analyses revealed that IRF8 methylation in NSCLC was a common event, and demethylation reagent treatment proved that downregulation of IRF8 was due to its promoter CpG hypermethylation. Clinical data showed that the IRF8 methylation was associated with tumor stage, lymph node metastasis status, patient outcome, and tumor histology. Exogenous expression of IRF8 in the silenced or downregulated lung cancer cell lines A549 and H1299 at least partially restored the sensitivity of lung cancer cells to apoptosis, and arrested cells at the G0/G1 phase. Cell viability, clonogenicity, and cell migration and invasive abilities were strongly inhibited by restored expression of IRF8. A three-dimensional culture system demonstrated that IRF8 changed the cells to a more spherical phenotype. Moreover, ectopic expression of IRF8 enhanced NSCLC chemosensitivity to cisplatin. Furthermore, as verified by Chip-qPCR, immunofluorescence staining, and western blotting, IRF8 bound to the T-cell factor/lymphoid enhancer factor (TCF /LEF) promoter, thus repressing β-catenin nuclear translocation and its activation. IRF8 significantly disrupted the effects of Wnt agonist, bml284, further suggesting its involvement in the Wnt/β-catenin pathway. Conclusion: IRF8 acted as a tumor suppressor gene through the transcriptional repression of β-catenin-TCF/LEF in NSCLC. IRF8 methylation may serve as a potential biomarker in NSCLC prognosis.

Negative Regulation of PTEN by MicroRNA-221 and Its Association with Drug Resistance and Cellular Senescence in Lung Cancer Cells.

Objective Chemotherapy is the routine method for treating many cancers, but long-term treatment may result in developing resistance to the drugs. The aim of this study was to identify whether noncoding RNAs play a role in drug resistance and how they affect drug resistance. Materials and Methods The expression levels of miR-221 in different lung cancer cell lines H226, H1299, and A549 were measured. H1299 and A549 cell lines were transfected to overexpress and downexpress miR-221, and cell viability and cell senescence were determined. The PTEN/Akt pathway was then examined by real-time polymerase chain reaction and Western blot analysis. Results MiR-221 together with proteins MDR1 and ABCG2 was upregulated in Cisplatin-resistant A549 lung cancer cells. Anti-miR-221 inhibits proliferation and induces senescence in lung cancer cells. PTEN/Akt pathway axis was identified as a target of drug resistance induced by miR-221. Conclusion Our results revealed that miR-221 is an important regulator for chemotherapy sensitivity and showed miR-221 as a potential target for drug sensitization.

The inhibitory activity of gallic acid against DNA methylation: application of gallic acid on epigenetic therapy of human cancers.

Epigenome aberrations have been observed in tobacco-associated human malignancies. (−)-epigallocatechin-3-gallate (EGCG) has been proven to modulate gene expression by targeting DNA methyltransferases (DNMTs) through a proposed mechanism involving the gallate moiety of EGCG. We show that gallic acid (GA) changes the methylome of lung cancer and pre-malignant oral cell lines and markedly reduces both nuclear and cytoplasmic DNMT1 and DNMT3B within 1 week. GA exhibits stronger cytotoxicity against the lung cancer cell line H1299 than EGCG. We found that GA reactivates the growth arrest and DNA damage-inducible 45 (GADD45) signaling pathway may through the demethylation of CCNE2 and CCNB1 in H1299 cells. To improve the epigenetic anti-cancer activities of oolong tea, we identified a fungus, Aspergillus sojae which can efficiently increase the GA content in oolong tea via a 2-week fermentation process. The fungus dramatically increased GA up to 44.8 fold in the post-fermentation oolong tea extract (PFOTE), resulting in enhanced demethylation effects and a significant reduction in the nuclear abundances of DNMT1, DNMT3A, and DNMT3B in lung cancer cell lines. PFOTE also showed stronger anti-proliferation activities than oolong tea extract (OTE) and increased sensitivity to cisplatin in H1299 cells. In summary, we demonstrate the potent inhibitory effects of GA on the activities of DNMTs and provide a strong scientific foundation for the use of specialized fermented oolong tea high in GA as an effective dietary intervention strategy for tobacco-associated cancers.

Comparative gene co-expression network analysis of epithelial to mesenchymal transition reveals lung cancer progression stages

BackgroundThe epithelial to mesenchymal transition (EMT) plays a key role in lung cancer progression and drug resistance. The dynamics and stability of gene expression patterns as cancer cells transition from E to M at a systems level and relevance to patient outcomes are unknown.MethodsUsing comparative network and clustering analysis, we systematically analyzed time-series gene expression data from lung cancer cell lines H358 and A549 that were induced to undergo EMT. We also predicted the putative regulatory networks controlling EMT expression dynamics, especially for the EMT-dynamic genes and related these patterns to patient outcomes using data from TCGA. Example EMT hub regulatory genes were validated using RNAi.ResultsWe identified several novel genes distinct from the static states of E or M that exhibited temporal expression patterns or ‘periods’ during the EMT process that were shared in different lung cancer cell lines. For example, cell cycle and metabolic genes were found to be similarly down-regulated where immune-associated genes were up-regulated after middle EMT stages. The presence of EMT-dynamic gene expression patterns supports the presence of differential activation and repression timings at the transcriptional level for various pathways and functions during EMT that are not detected in pure E or M cells. Importantly, the cell line identified EMT-dynamic genes were found to be present in lung cancer patient tissues and associated with patient outcomes.ConclusionsOur study suggests that in vitro identified EMT-dynamic genes capture elements of gene EMT expression dynamics at the patient level. Measurement of EMT dynamic genes, as opposed to E or M only, is potentially useful in future efforts aimed at classifying patient’s responses to treatments based on the EMT dynamics in the tissue.

MiR-5100 increases the cisplatin resistance of the lung cancer stem cells by inhibiting the Rab6.

Cisplatin-based chemotherapy is the most commonly used treatment regimen for lung cancer. Cancer stem cells (CSCs) are postulated to be important promoters of drug resistance. We previously found that miR-5100 is overexpressed in lung cancer, but it is unknown whether and how miR-5100 regulates cisplatin resistance. Here, we demonstrated that miR-5100 was significantly up-regulated in CD44+ CD133+ lung cancer stem cells (LCSCs) compared with non-CSCs. Additionally, over-expression of miR-5100 increased CSC properties, cell growth, and tumor sphere formation in lung cancer cell line A549 or H1299, and that miR-5100 inhibitor significantly increased sensitivity of LCSCs to cisplatin in vitro. Surprisingly, the combination with miR-5100 inhibitor significantly decreased the IC50 of LCSCs to cisplatin. Furthermore, miR-5100 increased CSC properties and cisplatin resistance by inhibiting Rab6, a direct target gene of miR-5100. We demonstrated that miR-5100 overexpression increases the cisplatin resistance of the LCSCs through the mitochondrial apoptosis pathway. In conclusion, our results suggest that miR-5100 increases the cisplatin resistance of the LCSCs by inhibiting the Rab6. This study provides novel insight into the regulation of LCSCs by miRNA.

SR 11302, an AP-1 Inhibitor, Reduces Metastatic Lesion Formation in Ex Vivo 4D Lung Cancer Model

Activator protein (AP) -1 is a transcription factor, plays important role in cell differentiation, proliferation and apoptosis. Analysis of tumor cells grown on ex vivo 4D lung cancer model shows increase in components of AP-1, c-Fos and c-Jun in circulating tumor cells (CTC) compared to primary tumor. Our aim was to determine whether the AP-1 inhibitor SR11302 reduces metastatic lesion formation in the 4D model. Human lung cancer cell lines A549, H1299, and H460 were grown in the 4D model and treated with SR11302 (1 μM). We compared the number of cells in the metastatic site upon SR11302 treatment and number of viable CTCs isolated from the 4D model with parental cells treated/untreated with SR11302 on a petri dish. There were significantly fewer tumor cells per high-power field on metastatic site in 4D model seeded with H460 (p = 0.009), A549 (p = 0.01), or H1299 (p = 0.02) cells treated with SR11302. Furthermore, the CTCs from SR11302 treated 4D models, seeded with H460 (p = 0.04), A549 (p = 0.008), or H1299 (p = 0.01) cells had significantly fewer viable tumor cells after 4 days in culture than the respective untreated control. However, the SR11302 had no impact on the viability of parental H460 (p = 0.87), A549 (p = 0.93), or H1299 (p = 0.25) cells grown on a petri dish (2D). SR11302 reduces metastatic lesion formation in the ex vivo 4D lung cancer model due to the presence of an independent yet common pathway among three cell lines. The ex vivo 4D model may provide a tool to better understand the complex process of metastasis.

Study on Genetype in Lung Squamous Carcinoma by High-throughput of Transcriptome Sequence

背景与目的目前尚无有效的靶向药物用于肺鳞癌的临床治疗，在肺腺癌中有效的靶向药物却无法让鳞癌患者获益，而研究者对肺鳞癌的靶点知之甚少。本研究重点筛选并鉴定肺鳞癌的特异关键基因，为肺鳞癌的治疗提供新的靶点。方法转录组测序检测5例肺鳞癌患者的配对肺癌组织及正常肺组织样本，使用生物信息学分析筛选两组之间的差异编码基因，进而应用实时定量PCR验证关键差异基因在肺癌细胞系的表达情况。结果转录组测序结果显示，与正常肺组织相比，肺鳞癌组织上调的差异基因数目为534条。位于前十位肺癌组织高表达的基因包括GAGE12J、SPRR3、PRAME、SPRR1A、SPRR2E、MAGEA3、SPRR1B、IL36G、TMPRSS11D和SPRR2D。进而于不同生物特征的肺癌细胞系H520、GLC82、A549、H1299及PC9验证SPRR家族表达状态，我们发现在淋巴结转移细胞系H1299处于高表达水平。结论高通量转录组测序筛选到肺鳞癌异常高表达基因SPRR家族，此家族与肺癌淋巴结转移相关，为肺癌的靶向治疗提供了新的思路。

Biological functions of lung cancer cells are suppressed in co-culture with mesenchymal stem cells isolated from umbilical cord.

Stem cell-based therapy serves a key role in clinical treatments, and mesenchymal stem cells (MSCs) have been widely used in clinical tumor therapy trials. In the present study, MSCs were isolated from umbilical cord (UC) and co-cultured with the lung cancer cell line H1299. The effects of UC-derived MSCs (UCMSCs) on H1299 cell invasion and proliferation were evaluated using a Matrigel-based Transwell assay and CCK8 assay, respectively. Apoptosis and cell cycle progression among H1299 cells were detected by flow cytometry, and kinase expression in H1299 cells was detected by western blotting. The results indicated that UCMSCs significantly inhibited H1299 cell invasion and significantly induced apoptosis of H1299 cells, but exhibited no effect on H1299 cell proliferation and cell cycle progression. It was also identified that H1299 cell expression of key kinases (AKT, phosphoinositide 3-kinase, signal transducer and activator of transcription 3 and mechanistic target of rapamycin) was significantly suppressed in the presence of UCMSCs. To the best of our knowledge, the present study demonstrates for the first time that UCMSCs have an anti-tumor effect against lung cancer cells, which may indicate that AKT/phosphoinositide 3-kinase/signal transducer and activator of transcription 3 signaling is important in the UCMSC-mediated regulation of H1299 cell functions.

Knockdown of TBRG4 affects tumorigenesis in human H1299 lung cancer cells by regulating DDIT3, CAV1 and RRM2.

The transforming growth factor β regulator 4 (TBRG4) gene, located on the 7p14-p13 chromosomal region, is implicated in numerous types of cancer. However, the contribution(s) of TBRG4 in human lung cancer remains unknown. In the present study, the expression of TBRG4 mRNA was investigated in the H1299 lung cancer cell line using the quantitative polymerase chain reaction (qPCR) following the knockdown of TBRG4 by a lentivirus-mediated small interfering RNA (siRNA). Results identified that the expression of TBRG4 within H1299 cells was significantly suppressed (P<0.01) by RNA interference, and 586 genes were differentially expressed following TBRG4 silencing. Ingenuity Pathway Analysis (IPA) revealed that these genes were often associated with infectious diseases, organismal injury, abnormalities and cancer functional networks. Further IPA of these networks revealed that TBRG4 knockdown in H1299 cells deregulated the expression of 21 downstream genes, including the upregulation of DNA damage-inducible transcript 3 (DDIT3), also termed CCAAT/enhancer-binding protein homologous protein, and downregulation of caveolin 1 (CAV1) and ribonucleotide reductase regulatory subunit M2 (RRM2). Results were validated using qPCR and western blotting. Furthermore, immunohistochemical staining of TBRG4 protein identified that expression was markedly increased in carcinoma compared with in normal tissue. In conclusion, TBRG4 serves a role in the tumorigenesis of lung cancer via deregulation of DDIT3, CAV1 and RRM2. The results of the present study may be important in contributing to our understanding of TBRG4 as a target for lung cancer treatment.