Lung Cancer Cell Line H1299 Research Articles

Long non-coding RNA maternally expressed gene 3 expression in non-small cell lung cancer and its role in proliferation and migration

Objective To evaluate the maternally expressed gene 3 (MEG3) expression in non-small cellu luang cancer and its role proliferation and migration. Methods Fourty eight paties diagnosed of non-small cell lung cancer were included in this study. The MEG3 expression of the cancer tissue and normal lung tissue for the 48 cases were tested by real-time quantitative polymerase chain reaction (Real-time PCR) assay. The lung cancer cell line of A549 and H460 were transfected with long non-coding RNA MEG3 over expression plasmid (Pc-DNA3.1-MEG3). After transfection of Pc-DNA3.1-MEG3, the ablity of prolifieration and migration were tested by methyl thiazol tetrazolium (MTT) and wound assay. The associated protein fore prolifieration and migration were tested by Western blotting assay. Results Compared with normal lung tissue, 25 (52.08%) cancer tissue were showed low expression of MEG3 with statistical difference (P<0.05); The expression of MEG3 were 0.48±0.04, 0.62±0.05 and 10.36±1.78, 11.66±2.01 in A549 AND H460 lung cancer cell line for transfection of pcDNA3.1 and pcDNA3.1-MEG3 with statistical difference (P<0.05); The proliferation and migration ability was significant decreased after transfection of pcDNA3.1-MEG3 for A549 and H460 lung cancer cells (P<0.05). And the cyclin D1 protein expression was significant inhibit after transfection of pcDNA3.1-MEG3 (P<0.05). Conclusion MEG3 was relative low expressed in non-small cell lung cancer (NSCLC) and it inhibits proliferation and migration through inbibiting Cyclin D1 expression. Key words: Non-samll cell lung cancer; Long non-coding RNA; Maternally expressed gene 3; Proliferation; Migration

Inhibitory effect of trans-ferulic acid on proliferation and migration of human lung cancer cells accompanied with increased endogenous reactive oxygen species and β-catenin instability.

BackgroundTrans-ferulic (FA) acid exhibits antioxidant effects in vitro. However, the underlying mechanism of trans-FA activity in cellular physiology, especially cancer physiology, remains largely unknown. This study investigated the cellular physiological effects of trans-FA on the H1299 human lung cancer cell line.MethodsThe 2,2-diphenyl-1-picrylhydrazyl assay was used to determine free radical scavenging capability. Assessment of intracellular reactive oxygen species (ROS) was evaluated using oxidized 2ʹ,7ʹ-dichlorofluorescin diacetate and dihydroethidium staining. Trypan blue exclusion, colony formation, and anchorage-independent growth assays were used to determine cellular proliferation. Annexin V staining assay was used to assess cellular apoptosis by flow cytometry. Wound healing and Boyden’s well assays were used to detect the migration and invasion of cells. Gelatin zymography was used to detect matrix metalloproteinase (MMP-2 and MMP-9) activity. Western blotting was used to detect expression levels of various signaling pathway proteins.ResultsDPPH assay results indicated that trans-FA exerted potent antioxidant effects. However, trans-FA increased intracellular ROS levels, including hydrogen peroxide and superoxide anion, in H1299 cells. Trans-FA treatment inhibited cellular proliferation and induced moderate apoptotic cell death at the highest concentration used (0.6 mM). Furthermore, trans-FA moderately inhibited the migration of H1299 cells at the concentrations of 0.3 and 0.6 mM and attenuated MMP-2 and MMP-9 activity. Trans-FA caused the phosphorylation of β-catenin, resulting in proteasomal degradation of β-catenin. Conversely, trans-FA treatment increased the expression of pro-apoptotic factor Bax and decreased the expression of pro-survival factor survivin.ConclusionVarious concentrations (0.06–0.6 mM) of trans-FA exert both anti-proliferation and anti-migration effects in the human lung cancer cell line H1299.Electronic supplementary materialThe online version of this article (doi:10.1186/s13020-016-0116-7) contains supplementary material, which is available to authorized users.

Identification of a novel transcript isoform of the TTLL12 gene in human cancers.

Tubulin tyrosine ligase like 12 (TTLL12), a member of the tubulin tyrosine ligase (TTLL) family, has not been completely characterized to date. It is reported that histone methylation, tubulin modifications, mitotic duration and chromosome ploidy play crucial roles in a variety of cancers, and are related to tumorigenesis and cancer progression. A recent study showed that TTLL12 may be a pseudo-enzyme which has a SET-like domain and a TTL-like domain. In the present study, we first used 3′-rapid amplification of cDNA ends (3′-RACE) to amplify the transcripts of the TTLL12 gene from a human lung cancer cell line H1299, and unexpectedly discovered a new transcript isoform characterized with an additional 108-bp nucleotide sequence inserted at the location from 902 to 903 bases of the TTLL12 coding sequence (CDS), where it also locates between exons 5 and 6. Next, utilizing RT-PCR and Sanger sequencing, we further confirmed the existence of such a new transcript isoform of TTLL12 in more human cancer cells including lung cancer cells and other cancer cells. Moreover, several lung cancer cell lines were found to display a much higher proportion of the new isoform compared with TTLL12 wild-type transcript. These results suggest that the new TTLL12 isoform may be of importance for proper maintenance of lung cancer cells. Therefore, the new isoform of TTLL12, with the inserted sequences probably acting as a disordered region, provides a novel perspective regarding TTLL12 functions in human cancers including lung cancer.

Cordycepin Induces Apoptosis and Inhibits Proliferation of Human Lung Cancer Cell Line H1975 via Inhibiting the Phosphorylation of EGFR.

Cordycepin is an active component of the traditional Chinese medicine Cordyceps sinensis and Cordyceps militaris with notable anticancer activity. Though the prominent inhibitory activity was reported in different kinds of cancer cell lines, the concrete mechanisms remain elusive. It was reported that cordycepin could be converted into tri-phosphates in vivo to confuse a number of enzymes and interfere the normal cell function. For the inhibitory mechanism of EGFR inhibitors and the structure similarity of ATP and tri-phosphated cordycepin, human lung cancer cell line H1975 was employed to investigate the inhibitory effect of cordycepin. The results showed that cordycepin could inhibit cell proliferation and induce apoptosis in a dose-dependent manner. Cell cycle analysis revealed that H1975 cells could be arrested at the G0/G1 phase after cordycepin treatment. The expression levels of apoptosis-related protein Caspase-3 and Bcl-2 and phosphorylated expression levels of EGFR, AKT and ERK1/2 were all decreased compared with the control group stimulated with EGF. However, the protein expression levels of proapoptotic protein Bax and cleaved caspase-3 were increased. These results implied that cordycepin could inhibit cell proliferation and induce apoptosis via the EGFR signaling pathway. Our results indicated that there was potential to seek a novel EGFR inhibitor from cordycepin and its chemical derivatives.

IL-24 modulates the high mobility group (HMG) A1/miR222 /AKT signaling in lung cancer cells.

Interleukin (IL)-24, a novel tumor suppressor/cytokine exhibits antitumor activity against a broad-spectrum of human cancer cells. In a recent study, we showed that IL-24 inhibited AKT in lung cancer cells. However, the molecular mechanism of AKT inhibition by IL-24 remains elusive.The high mobility group (HMG) A1 a member of the non-histone chromosomal proteins and commonly referred to as architectural transcription factor, regulates transcription of various genes involved in cell growth and survival. Overexpression of HMGA1 has been shown to be associated with tumor progression and metastasis in several cancers, including human lung cancer. A recent study demonstrated that HMGA1 activates AKT function by reducing the activity of the protein phosphatase, phosphatase 2A subunit B (PPP2R2A) via the oncogenic micro (mi) RNA-222. Based on this report we hypothesized that IL-24-mediated AKT inhibition involved the HMGA1/miR-222 axis.To test our hypothesis, in the present study we used a H1299 lung cancer cell line that expressed exogenous human IL-24 when induced with doxycycline (DOX). Induction of IL-24 expression in the tumor cells markedly reduced HMGA1 mRNA and protein levels. Using a mechanistic approach, we found that IL-24 reduced miR-222-3p and -5p levels, as determined by qRT-PCR. Associated with HMGA1 and miR-222 inhibition was a marked increase in PPP2R2A, with a concomitant decrease in phosphorylated AKTT308/S473 expression. SiRNA-mediated knockdown of HMGA1 in combination with IL-24 significantly reduced AKT T308/S473 protein expression and greatly reduced cell migration and invasion compared with individual treatments. Further combination of IL-24 and a miR-222-3p inhibitor significantly increased PPP2R2A expression.Our results demonstrate for the first time that IL-24 inhibits AKT via regulating the HMGA1/miR-222 signaling node in human lung cancer cells and acts as an effective tumor suppressor. Thus, a therapy combining IL-24 with HMGA1 siRNA or miR-222-3p inhibitor should present effective treatment of lung cancer.

Abstract 1041: Glutamine drives glutathione synthesis and contributes to radiation sensitivity of A549 and H460 lung cancer cell lines

Abstract Increased glutamine uptake is known to drive cancer cell proliferation, making tumor cells glutamine-dependent. Glutamine provides additional carbon and nitrogen sources for cell growth. The first step in glutamine utilization is its conversion to glutamate by glutaminase (GLS). Glutamate is a precursor for glutathione synthesis, and we investigated the hypothesis that glutamine drives glutathione synthesis and thereby contributes to cellular defense systems. The importance of glutamine for glutathione synthesis was studied in H460 and A549 lung cancer cell lines using glutamine-free medium and BPTES a GLS inhibitor. Metabolic activities were determined by targeted mass spectrometry. A significant correlation between glutamine consumption and glutathione excretion was demonstrated in H460 and A549 tumor cells. Culturing in the presence of [13C5]glutamine demonstrated that by 12 hrs &amp;gt;50% of excreted glutathione is derived from glutamine. Culturing in glutamine-free medium or treatment with Bis-2-(5-phenyl-acetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a glutaminase (GLS)-specific inhibitor, reduced cell proliferation and viability, and abolished glutathione excretion. Treatment with glutathione-ester prevented BPTES induced cytotoxicity. Inhibition of GLS markedly radiosensitized the lung tumor cell lines, suggesting an important role of glutamine-derived glutathione in determining radiation sensitivity. We demonstrate here for the first time that a significant amount of extracellular glutathione is directly derived from glutamine. This finding adds yet another important function to the already known glutamine dependence of tumor cells and probably tumors as well. Citation Format: Daniel Sappington, Rosalind Penney, Eric Siegel, Gunnar Boysen. Glutamine drives glutathione synthesis and contributes to radiation sensitivity of A549 and H460 lung cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1041.

Abstract 1376: Ultra-sensitive picodroplet digital PCR assay for multiplex genotyping of mutations in epidermal growth factor receptor (EGFR) in non-small cell lung cancer patients

Abstract BACKGROUND: The epidermal growth factor receptor (EGFR) mutations have been used as a reliable predictor of response to EGFR tyrosine kinase inhibitor (TKI) treatment. Two common EGFR mutations (L858R, and exon19 deletion) and EGFR T790M mutation are clinically important for decision-making in clinical practice. Picodroplet digital PCR (ddPCR) has recently emerged as an accurate and ultra-sensitive method for tumor genotyping and have a great advantage over conventional mutation detection methods. We have previously reported far more frequent incidence of pretreatment EGFR T790M mutation than previous reports utilizing ddPCR method. Here, we developed a multiplex ddPCR assay to detect three clinically relevant EGFR mutations in one reaction. METHODS: Positive and negative control plasmids for the EGFR mutation assay were prepared by cloning DNA fragments containing wild-type or mutant EGFR. Genomic DNAs from lung cancer cell lines H1975, PC-9/ZD, A549 and wild-type human genomic DNA were digested with CviQ1, and then were used to quantitatively assess each EGFR mutant sequence in the multiplex assay panels. We then evaluated the utility of multiplex ddPCR to detect the three clinically relevant mutations in EGFR from FFPE samples of patients with non-small cell lung cancer. 45 FFPE samples were also genotyped for EGFR mutations by conventional qPCR-based methods for validation. The concordance between duplex and multiplex ddPCR assay was also evaluated. RESULTS: Serial dilutions experiments using genomic DNA harboring EGFR mutations revealed a linear performance with an analytical sensitivity of approximately 0.01% for each mutation. All of the 33 EGFR-activating mutations detected in FFPE samples by conventional qPCR-based method were also detected by multiplex ddPCR assay. Owing to its ultra-high sensitivity, additional T790M mutation at ultra-low allele frequency (&amp;lt;0.1%) was also detected in the same reaction. The regression analysis between duplex and multiplex assay demonstrated a correlation coefficient (R2) of 0.9986 for L858R, 0.9844 for exon19 deletion, and 0.9959 for T790M, respectively. CONCLUSIONS: Using ddPCR technology, we established multiplex and ultra-sensitive genotyping platform for three clinically relevant EGFR mutations. Results of a proof-of-principle study using clinical samples indicated that the clinical utility of multiplex ddPCR assay to screen for multiple EGFR-activating mutations concurrently with low-frequency T790M mutation. Citation Format: Yasuhiro Koh, Tomoya Kawaguchi, Masaru Watanabe, Shun-ichi Isa, Masahiko Ando, Akihiro Tamiya, Akihito Kubo, Hideo Saka, Sadanori Takeo, Hirofumi Adachi, Tsutomu Tagawa, Seiichi Kakegawa, Motohiro Yamashita, Kazuhiko Kataoka, Yukito Ichinose, Yukiyasu Takeuchi, Kazuhiro Sakamoto, Akihide Matsumura. Ultra-sensitive picodroplet digital PCR assay for multiplex genotyping of mutations in epidermal growth factor receptor (EGFR) in non-small cell lung cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1376.

Abstract 2021: Identification of novel brachyury target genes in lung cancer

Abstract Lung cancer is the leading cause of cancer-related death worldwide. Previous studies have shown that the T-box transcription factor brachyury is commonly expressed in advanced lung cancer and is associated with a poor prognosis. The activation of brachyury promotes lung cancer progression, aggressiveness and resistance to conventional antitumor therapies. However, the mechanism by which brachyury drives lung cancer is unknown. Here we have sought to identify novel gene targets of brachyury to better understand the molecular pathogenesis of lung cancer. We used a bioinformatics approach and found a shortlist of putative potential brachyury target genes. We subsequently explored the modulation of candidate brachyury target genes in lung cancer. RNA interference analysis for lung cancer cell line H460 overexpressing brachyury demonstrated that fibroblast growth factor 8 (FGF8), MAS1 Proto-Oncogene G Protein-Coupled Receptor (MAS1) and Ran GTPase Activating Protein 1 (RANGAP1) played essential roles in cell growth, whereas transmembrane protein 37 (TMEM37) was involved in cell migration and invasion. ChIP-qPCR experiments confirmed the strong binding of brachyury to these functional target genes. Analysis of datasets derived from patients with lung cancer and a panel of lung cancer cell lines revealed that brachyury expression was positively correlated with FGF8, MAS1, RANGAP1 and TMEM37. In addition, the expression levels of FGF8, MAS1 and RANGAP1 were significantly associated with non-metastasis (early-stage and locally advanced lung cancer), whereas TMEM37 was associated with metastasis. Taken together, our findings suggest that brachyury may influence lung cancer progression and/or metastasis via direct regulation of FGF8, MAS1, RANGAP1 and TMEM37 expression. Identification of potential brachyury transcriptional targets provides novel insights into the molecular pathobiology of lung cancer. Citation Format: Yunping Hu, Wesley Hsu. Identification of novel brachyury target genes in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2021.

Abstract 2793: ADP- dependent glucokinase enhances hypoxia- inducible factor-α target gene transactivation through modulation of ROS levels in hypoxic human cancer cells

Abstract ADP- dependent glucokinase (ADPGK) is a glycolytic enzyme catalyzing the reaction of glucose→glucose 6-P by using ADP instead of ATP. ADPGK is remarkably conserved across various phyla from archaea to humans. Despite an important role priming glycolysis in conditions of metabolic stress in extremophile organisms, human ADPGK does not contribute to glycolysis. Recent work shows that ADPGK is essential for ROS generation during T-cell activation. Intriguingly, ADPGK protein levels are upregulated across a panel of cancer cell lines and primary tumors. Hereby we hypothesized that the increase of cellular ROS in response to mild (∼1% O2) hypoxia is dependent on ADPGK therefore contributing to the stabilization of hypoxia- inducible factor (HIF)-1α, a central regulator of the transcriptional response of cancer cells to hypoxia. We achieved ADPGK loss-of-function (LOF) utilizing lentiviral shRNA- mediated knockdown (&amp;gt;75%) in HCT-116 and HT-29 (colon); or H460 (lung) cancer cell lines. Intracellular ROS levels under hypoxia (1% O2) were analyzed by CM-H2DCFDA fluorescence and flow cytometry. Protein levels of HIF-1α (and -2α), and selected HIF-α targets were assessed by immunoblot assays. HIF-1α (and -2α) transcriptional activity was measured through a RT-qPCR array developed in house targeting 84 hypoxia- responsive transcripts in HCT-116 cells exposed to 20, 10, 5, 1, .2 and &amp;lt;.02% O2 for 24 h. Baseline non-hypoxic oxygen consumption and glycolytic activities were measured in real-time with an automated bioanalyzer (Seahorse Biosciences) whereas 3D spheroid growth assays and HCT-116 xenografts were used to assess the effect of ADPGK LOF on tumor mass. ADPGK LOF decreased ROS and HIF-1α (-and 2α) protein levels in a cell type- and O2- dependent manner in HCT-116, HT-29 and H460 cells. Results from our RT-qPCR array show a blunted transcriptional response to hypoxia with maximal inhibition at .2% O2 (∼15 mmHg). Immunoblots confirmed a striking impairment of CA9 hypoxic induction in addition to a significant decrease of the HIF-α targets IGFBP3, ERO-1α and DDIT4. 3D spheroid assays showed a significant growth rate increase after ADPGK knockdown. Addition of the antioxidant N-Ac-Cysteine during the spheroid formation phase mimicked the effect of ADPGK LOF in control cells whereas it further enhanced cell growth in ADPGK knockdown. However, baseline O2 consumption and glycolysis were not affected. Consistently with results of 3D spheroid assays, we observed enhanced tumor growth of HCT-116 xenografts bearing ADPGK LOF. Our work uncovers a hitherto unknown function of ADPGK in cancer cells whereby it enhances ROS- dependent HIF-1α (and -2α) hypoxic stabilization and transactivation therefore limiting tumor growth. Further work will be focused on the mitochondrial origin of ADPGK- dependent hypoxic ROS generation as suggested by previous studies in primary T-lymphocytes. Citation Format: Sergio Rey, Luana Schito, Marianne Koritzinsky, Bradly G. Wouters. ADP- dependent glucokinase enhances hypoxia- inducible factor-α target gene transactivation through modulation of ROS levels in hypoxic human cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2793.

Actin bundling by dynamin2 and cortactin is implicated in cell migration by stabilizing filopodia in human non-small cell lung carcinoma cells.

The endocytic protein dynamin participates in the formation of actin-based membrane protrusions such as podosomes, pseudopodia, and invadopodia, which facilitate cancer cell migration, invasion, and metastasis. However, the role of dynamin in the formation of actin-based membrane protrusions at the leading edge of cancer cells is unclear. In this study, we demonstrate that the ubiquitously expressed dynamin 2 isoform facilitates cell migration by stabilizing F-actin bundles in filopodia of the lung cancer cell line H1299. Pharmacological inhibition of dynamin 2 decreased cell migration and filopodial formation. Furthermore, dynamin 2 and cortactin mostly colocalized along F-actin bundles in filopodia of serum-stimulated H1299 cells by immunofluorescent and immunoelectron microscopy. Knockdown of dynamin 2 or cortactin inhibited the formation of filopodia in serum-stimulated H1299 cells, concomitant with a loss of F-actin bundles. Expression of wild-type cortactin rescued the punctate-like localization of dynamin 2 and filopodial formation. The incubation of dynamin 2 and cortactin with F-actin induced the formation of long and thick actin bundles, with these proteins colocalizing at F-actin bundles. A depolymerization assay revealed that dynamin 2 and cortactin increased the stability of F-actin bundles. These results indicate that dynamin 2 and cortactin participate in cell migration by stabilizing F-actin bundles in filopodia. Taken together, these findings suggest that dynamin might be a possible molecular target for anticancer therapy.

Evaluations of thyme extract effects in human normal bronchial and tracheal epithelial cell lines and in human lung cancer cell line

Thyme (Thymus vulgaris) is used traditionally to prepare herbal remedies possessing expectorant, mucolytic, antitussive and antispasmodic properties.The aim of the present study was to investigate the effects of a standardized hydroalcoholic extract of thyme on primary human airway (bronchial/tracheal) epithelial cell lines in a model of lung inflammation induced by LPS. In addition, the effects of thyme extract on human lung cancer cell line (H460) were analysed. Thyme extract showed significant anti-inflammatory properties by reducing the NF-κB p65 and NF-κB p52 transcription factors protein levels followed by the decrease of pro-inflammatory cytokines (IL-1 beta and IL-8), and Muc5ac secretion in human normal bronchial and tracheal epithelial cells. Moreover, the extract showed cytotoxic effects on H460 cancer cells, modulated the release of IL-1 beta, IL-8 and down-regulated NF-κB p65 and NF-κB p52 proteins.Taken together, these results substantiated the traditional uses of thyme in the treatment of respiratory diseases. Thyme extract might be an effective treatment of chronic diseases based on inflammatory processes when hypersecretion of mucus overwhelms the ciliary clearance and obstructs airways, causing morbidity and mortality. Moreover thyme extract, evaluated in H460 lung cancer cell line, demonstrated to induce cell cytotoxicity in addition to reduce inflammatory cell signals.

Folate receptor-targeted nanoparticle delivery of HuR-RNAi suppresses lung cancer cell proliferation and migration.

BackgroundHuman antigen R (HuR) is an RNA binding protein that is overexpressed in many human cancers, including lung cancer, and has been shown to regulate the expression of several oncoproteins. Further, HuR overexpression in cancer cells has been associated with poor-prognosis and therapy resistance. Therefore, we hypothesized that targeted inhibition of HuR in cancer cells should suppress several HuR-regulated oncoproteins resulting in an effective anticancer efficacy. To test our hypothesis, in the present study we investigated the efficacy of folate receptor-α (FRA)-targeted DOTAP:Cholesterol lipid nanoparticles carrying HuR siRNA (HuR-FNP) against human lung cancer cells.ResultsThe therapeutic efficacy of HuR-FNP was tested in FRA overexpressing human H1299 lung cancer cell line and compared to normal lung fibroblast (CCD16) cells that had low to no FRA expression. Physico-chemical characterization studies showed HuR-FNP particle size was 303.3 nm in diameter and had a positive surface charge (+4.3 mV). Gel retardation and serum stability assays showed that the FNPs were efficiently protected siRNA from rapid degradation. FNP uptake was significantly higher in H1299 cells compared to CCD16 cells indicating a receptor-dose effect. The results of competitive inhibition studies in H1299 cells demonstrated that HuR-FNPs were efficiently internalized via FRA-mediated endocytosis. Biologic studies demonstrated HuR-FNP but not C-FNP (control siRNA) induced G1 phase cell-cycle arrest and apoptosis in H1299 cells resulting in significant growth inhibition. Further, HuR-FNP exhibited significantly higher cytotoxicity against H1299 cells than it did against CCD16 cells. The reduction in H1299 cell viability was correlated with a marked decrease in HuR mRNA and protein expression. Further, reduced expression of HuR-regulated oncoproteins (cyclin D1, cyclin E, and Bcl-2) and increased p27 tumor suppressor protein were observed in HuR-FNP-treated H1299 cells but not in C-FNP-treated cells. Finally, cell migration was significantly inhibited in HuR-FNP-treated H1299 cells compared to C-FNP.ConclusionsOur results demonstrate that HuR is a molecular target for lung cancer therapy and its suppression using HuR-FNP produced significant therapeutic efficacy in vitro.

WE‐H‐BRA‐05: Investigation of LET Spectral Dependence of the Biological Effects of Therapeutic Protons

Purpose:To investigate the dependence of biologic effect (BE) of therapeutic protons on LET spectra by comparing BEs with equal dose‐averaged LET (LETd) derived from different LET spectra using high‐throughput in vitro clonogenic survival assays.Methods:We used Geant4 to design the relevant experimental setups and perform the dose, LETd, and LET spectra calculations for spot‐scanning protons. The clonogenic assay was performed using the H460 lung cancer cell line cultured in 96‐well plates. In the first experimental setup (S1), cells were irradiated using 127.4 MeV protons with a 93.22 mm Lucite buildup resulting in a LETd value of 3.4 keV/µm in the cell layer. In the second experimental setup (S2), cells were irradiated by a combination of 127.4 MeV and 136.4 MeV protons with a 96.61 mm Lucite buildup. The LETd values in the cell layer were 11.4 keV/µm and 1.5 keV/µm respectively, but an average LETd of 3.4 keV/µm was obtained by adjusting the relative fluence of each beam. Ten discrete dose levels with 0.5 Gy increments were delivered.Results:In the two setups, the energies or LET spectra were different but resulted in identical LETd values. We quantified the dose contributions from high‐LET (≥10 keV/µm, threshold determined by previous experiments) events in the LET spectra separately for these two setups as 3.2% and 10.5%. The biologic effects at each identical dose level yielded statistically significant different survival curves (extra sum‐of‐squares F‐test, P&lt;0.0001). The second setup with a higher contribution from high‐LET events exhibited the higher biologic effect with a dose enhancement factor of 1.17±0.03 at 0.10 surviving fraction.Conclusion:The dose‐averaged LET may not be an accurate indicator of the biological effects of protons. Detailed LET spectra may need to be considered explicitly to accurately quantify the biologic effects of protons.Funding Support: U19 CA021239‐35, R21 CA187484‐01 and MDACC‐IRG

Preparation and preliminary evaluation of alginate crosslinked microcapsules as potential drug delivery system (DDS) for human lung cancer therapy

Human lung cancer is the main cancer cause of death in men and the third cancer cause in women. As it is well-known, current antitumor treatments involve chemotherapy which mostly entails aggressive side effects. This study describes a drug delivery targeted strategy based in bioconjugated polymeric microcapsules with a size of around 30 μm. Alginate microcapsules, produced using a fan-jet nozzle, are used due to its biodegradability and biocompatibility and will be tested in vitro. Firstly, microparticles are prepared by ionic gelation of an alginate core sprayed on a barium chloride solution. Afterwards, epidermal growth factor is attached onto the surface of these microcapsules to specifically target non-small lung cancer cells (NSCLC). This bioconjugation is performed together with an anticancer agent, cisplatin. The in vitro studies of cytotoxicity has been tested with H460 lung cancer cell line and with controls on systems loaded with free drugs. In vitro validation tests were carried out in Petri plates and the cells viability were checked by MTT assay. These experiments showed that drug delivery treatment allows a faster mortality in H460 lung cancer cell plates. To sum up, this study reports the development of an advanced drug delivery system that improves the specificity issue on drug delivery in addition and in vitro confirmation in cell mediums containing NSCLC.

TH‐CD‐209‐12: Spatial Mapping of Scanned Proton Biologic Effect Using the High‐Throughput Technique, Continued

Purpose:To investigate the biologic effects of scanned protons by evenly sampling dose‐averaged LET (LETd) values.Methods:Our previous high‐throughput clonogenic study demonstrated a distinct relationship between RBE and LETd. However, our initial experimental design resulted in over‐sampling the low LETd values in the plateau region of the Bragg curve while under‐sampling in the region proximal to the Bragg peak as well as the high LETd values in the distal edge of the Bragg curve. To further examine the relationship between RBE and LETd, we refined the experimental design to more evenly sample proton LETd values from 1 to 20 keV/µm by optimizing the thicknesses of the irradiation jig steps. We used the clonogenic survival as the biological endpoint for the H460 lung cancer cell line cultured in 96‐well plates (12 columns by 8 rows). In the irradiation, the 8 wells in each column received a uniform dose‐LETd pair. The dose‐LETd pairs of the 12 different columns were sampled along the Bragg curve of 81.4 MeV scanned protons. Five peak dose levels from 1.5 Gy to 7.5 Gy were delivered with an increment of 1.5 Gy in the preliminary test. Two 96‐well plates were irradiated simultaneously to decrease the statistical uncertainties.Results:In the proximal region, for LETd = 5 keV/µm and 8 keV/µm, we did not observe any distinct differential biologic effects between the survival curves. At the Bragg peak (LETd = 9.5 keV/µm) and in the distal edge, irradiation with increasing LET values resulted in decreasing cell survival.Conclusion:The survival curves from the new experimental design support our previous findings that below 10 keV/µm, the LET effect in cell kill is obscured, but above 10 keV/µm, the biologic effects increase with LETd.Funding Support: U19 CA021239‐35 and R21 CA187484‐01

Inhibition of JNK-mediated autophagy enhances NSCLC cell sensitivity to mTORC1/2 inhibitors.

As the activation of autophagy contributes to the efficacy of many anticancer therapies, deciphering the precise role of autophagy in cancer therapy is critical. Here, we report that the dual mTORC1/2 inhibitors PP242 and OSI-027 decreased cell viability but did not induce apoptosis in the non-small cell lung cancer (NSCLC) cell lines H460 and A549. PP242 induced autophagy in NSCLC cells as demonstrated by the formation of massive vacuoles and acidic vesicular organelles and the accumulation of LC3-II. JNK was activated by PP242, and PP242-induced autophagy was blocked by inhibiting JNK pathway with SP600125 or JNK siRNA, suggesting that JNK activation is required for the mTORC1/2 inhibitor-mediated induction of autophagy in NSCLC cells. Inhibiting JNK or autophagy increased the sensitivity of H460 cells to mTORC1/2 inhibitors, indicating that JNK or autophagy promoted survival in NSCLC cells treated with mTORC1/2 inhibitors. Together, these data suggest that combining mTORC1/2 inhibitors with inhibitors of JNK or autophagy might be an effective approach for improving therapeutic outcomes in NSCLC.

A natural small molecule, catechol, induces c-Myc degradation by directly targeting ERK2 in lung cancer.

Various carcinogens induce EGFR/RAS/MAPK signaling, which is critical in the development of lung cancer. In particular, constitutive activation of extracellular signal-regulated kinase 2 (ERK2) is observed in many lung cancer patients, and therefore developing compounds capable of targeting ERK2 in lung carcinogenesis could be beneficial. We examined the therapeutic effect of catechol in lung cancer treatment. Catechol suppressed anchorage-independent growth of murine KP2 and human H460 lung cancer cell lines in a dose-dependent manner. Catechol inhibited ERK2 kinase activity in vitro, and its direct binding to the ERK2 active site was confirmed by X-ray crystallography. Phosphorylation of c-Myc, a substrate of ERK2, was decreased in catechol-treated lung cancer cells and resulted in reduced protein stability and subsequent down-regulation of total c-Myc. Treatment with catechol induced G1 phase arrest in lung cancer cells and decreased protein expression related to G1-S progression. In addition, we showed that catechol inhibited the growth of both allograft and xenograft lung cancer tumors in vivo. In summary, catechol exerted inhibitory effects on the ERK2/c-Myc signaling axis to reduce lung cancer tumor growth in vitro and in vivo, including a preclinical patient-derived xenograft (PDX) model. These findings suggest that catechol, a natural small molecule, possesses potential as a novel therapeutic agent against lung carcinogenesis in future clinical approaches.

Pyrrolopyrimidine Derivatives as Novel Inhibitors of Multidrug Resistance-Associated Protein 1 (MRP1, ABCC1).

Five series of pyrrolo[3,2-d]pyrimidines were synthesized and evaluated with respect to potency and selectivity toward multidrug resistance-associated protein 1 (MRP1, ABCC1). This transport protein is a major target to overcome multidrug resistance in cancer patients. We investigated differently substituted pyrrolopyrimidines using the doxorubicin selected and MRP1 overexpressing small cell lung cancer cell line H69 AR in a calcein AM and daunorubicin cell accumulation assay. New compounds with high potency and selectivity were identified. Piperazine residues at position 4 bearing large phenylalkyl side chains proved to be beneficial for MRP1 inhibition. Its replacement by an amino group led to decreased activity. Aliphatic and aliphatic-aromatic variations at position 5 and 6 revealed compounds with IC50 values in high nanomolar range. All investigated compounds had low affinity toward P-glycoprotein (P-gp, ABCB1). Pyrrolopyrimidines with small substituents showed moderate inhibition against breast cancer resistance protein (BCRP, ABCG2).

ER stress and autophagy are involved in the apoptosis induced by cisplatin in human lung cancer cells.

Cisplatin [cis-diamminedichloroplatinum II (CDDP)] is one of the most classical and effective chemotherapeutic drugs for the treatment of cancers including lung cancer. However, the presence of cisplatin resistance in cancer lowers its curative effect and limits its usage in the clinic. The aim of the present study was to investigate the underlying mechanisms of cisplatin resistance in lung cancer involving endoplasmic reticulum (ER) stress and autophagy. In the present study, we detected the effect of cisplatin on cell viability, ER stress and autophagy in lung cancer cell lines A549 and H460. We also tested the effects of ER stress and autophagy on apoptosis induced by cisplatin. The results showed that cisplatin induced apoptosis, ER stress and autophagy in lung cancer cell lines. In addition, the inhibition of ER stress by 4-phenylbutyric acid (4-PBA) or tauroursodeoxycholic acid sodium (TUDC) enhanced cisplatin-induced apoptosis in the human lung cancer cells. Meanwhile, combination treatment with the autophagic inhibitor 3-methyladenine (3-MA) or chloroquine (CQ) further increased the apoptosis induced by cisplatin in the human lung cancer cells. The present study provides a novel treatment strategy - cisplatin in combination with an autophagic inhibitor or an ER stress inhibitor leads to increased apoptosis in human lung cancer cells.

Transcription factor CCAAT/enhancer binding protein alpha up-regulates microRNA let-7a-1 in lung cancer cells by direct binding.

AimsThe transcription factor CCAAT/enhancer binding protein α (C/EBPα) and microRNA (miRNA) let-7a-1 act as tumor suppressors in many types of cancers including lung cancer. In the present study, we aim to investigate whether let-7a-1 is a novel important target of C/EBPα in lung cancer cells.MethodsThe DNA sequence of the 2.1 kb let-7a-1 promoter was analyzed with MatInspector 4.1 (http://www.genomatix.de). Human lung cancer cell lines A549 and H1299, and human cervical cancer cell line Hela were used for transfection. Total RNA was extracted from cells using Trizol reagent and pri-let-7a-1 mRNA expression was measured using quantitative real-time polymerase chain reaction. Western blotting was performed to detect C/EBPα protein expression. To test whether C/EBP-α could up-regulate the expression level of let-7a at transcription level, dual-luciferase reporter gene assay was carried out. To determine whether C/EBPα could bind let-7a-1 promoter, electrophoretic mobility shift assay was employed. To further confirm the direct targeting let-7a-1 promoter by C/EBPα, chromatin immunoprecipitation was used.ResultsBoth C/EBPα and let-7a-1 were down-regulated in lung cancer A549 and H1299 cells, but up-regulated in Hela cells. Transfection and reporter gene assay showed that C/EBPα increased the expression of let-7a-1 at transcription level. Bioinformatics assay identified four putative C/EBP elements within let-7a-1 promoter. Dual-luciferase reporter gene, electrophoretic mobility shift assay and chromatin immunoprecipitation assays demonstrated that these four elements mediated the up-regulation effect of C/EBPα on let-7a-1.ConclusionsThe present study reveals that decreased C/EBPα contributes to the down-regulation of miRNA let-7a-1 in lung cancer cells.