H1299 Non-small Cell Lung Cancer Cells Research Articles

2-Aminoethoxydiphenylborane sensitizes anti-tumor effect of bortezomib via suppression of calcium-mediated autophagy

Non-small-cell lung cancer (NSCLC) accounts for most lung cancer cases. Therapeutic interventions integrating the use of different agents that focus on different targets are needed to overcome this set of diseases. The proteasome system has been demonstrated clinically as a potent therapeutic target for haematological cancers. However, promising preclinical data in solid tumors are yet to be confirmed in clinics. Herein, the combinational use of Bortezomib (BZM) and 2-aminoethoxydiphenylborane (2-APB) toward NSCLC cells was studied. We confirmed that BZM-triggered cytoprotective autophagy that may counteract with the cytotoxic effects of the drug per se. 2-APB was selected from screening of a commercial natural compounds library, which potentiated BZM-induced cytotoxicity. Such an enhancement effect was associated with 2-APB-mediated autophagy inhibition. In addition, we revealed that 2-APB suppressed calcium-induced autophagy in H1975 and A549 NSCLC cells. Interestingly, BZM [0.3 mg/kg/3 days] combined with 2-APB [2 mg/kg/day] significantly inhibited both primary (around 47% tumor growth) and metastatic Lewis lung carcinoma after a 20-day treatment. Our results suggested that BZM and 2-APB combination therapy can potentially be developed as a novel formulation for lung cancer treatment.

Combination of S-1 and gefitinib increases the sensitivity to radiotherapy in lung cancer cells.

To investigate the potential radiosensitization of S-1 and gefitinib in human non-small cell lung cancer (NSCLC) in vitro and in vivo. The impact of radiation, 5-fluorouracil (5-Fu), and gefitinib on the proliferation and apoptosis of human NSCLC A549, H1299, H1975, and HCC827 cells was examined by MTT and flow cytometry. The effect of radiation, 5-Fu, and gefitinib on the clonogenicity of H1975 and HCC827 cells was determined by colony formation assay. The effect of radiation, 5-fluorouracil (5-Fu), and gefitinib on the EGFR, AKT, and ERK1/2 activation in H1975 cells was determined by Western blot. The therapeutic efficacy of radiation, S-1, and gefitinib in the growth of implanted H1975 tumors and the AKT activation in the tumors were examined in vivo and immunohistochemistry, respectively. Combination of radiation, 5-Fu, and gefitinib significantly inhibited the proliferation of H1975 cells and triggered their apoptosis, but not other NSCLC cells tested. The combination therapy significantly mitigated the clonogenicity and attenuated the activation of EGFR and AKT signaling in H1975 cells. Furthermore, combination of S-1, gefitinib, and radiation significantly inhibited the growth of implanted H1975 tumors in mice and remarkably reduced the AKT phosphorylation in the tumors. Our data indicated that combination of S-1 and gefitinib significantly increased radiosensitivity of H1975 cells. The triple combination therapies may benefit patients with the EGFR T790M mutant NSCLC.

Bu-Fei decoction and modified Bu-Fei decoction inhibit the growth of non-small cell lung cancer, possibly via inhibition of apurinic/apyrimidinic endonuclease 1.

Human apurinic/apyrimidinic endonuclease 1 (APE1) is a ubiquitous multifunctional protein, which possesses DNA repair and redox activities. High levels of APE1 are associated with chemo- and radioresistance, and poor prognosis in various types of cancer, including non-small cell lung cancer (NSCLC). Bu-Fei decoction (BFD) is a traditional Chinese herbal formula, which is believed to supplement Qi, clear away heat and nourish the lungs. BFD and modified Bu-Fei decoction (MBFD) have been used in China to treat patients with lung cancer. The present study aimed to evaluate the potential antitumor effects of BFD and MBFD on NSCLC in vitro and in vivo. In addition, the possible contribution of APE1 was examined. MTT assay was used to investigated the anti-tumor activity of BFD and MBFD on H1975 and H292 NSCLC cell lines. The DNA damage of cells in the control and the experimental groups was detected using comet assay. The in vivo anti-tumor effects of BFD and MBFD were evaluated in a NSCLC tumor nude mouse xenograft model. Polymerase chain reaction (PCR), reverse transcription-quantitative PCR (RT-qPCR) analysis and western blot analysis were applied to analyze the mRNA and protein expression levels of APE1 in H1975 and H292 cells, so as to the xenograft tumor tissues. The concentration of APE1 in mice plasma was determined using enzyme linked immunosorbent assay (ELISA). In vitro, BFD and MBFD inhibited the growth of cultured H1975 and H292 NSCLC cells. The results of a comet assay revealed that BFD and MBFD increased DNA damage. Furthermore, the expression levels of APE1 were decreased in response to BFD and MBFD at the mRNA and protein levels. In mice carrying NSCLC xenografts, BFD and MBFD inhibited tumor growth and decreased APE1 expression. In addition, in normal human lung bronchial epithelial BEAS-2B cells, the half maximal inhibitory concentrations of BFD and MBFD were much higher compared with in NSCLC cells, and they had no effect on DNA damage. These results suggested that BFD and MBFD may inhibit the growth of NSCLC, possibly by inhibiting APE1 expression.

RETRACTED ARTICLE: Dual roles of miR-374a by modulated c-Jun respectively targets CCND1-inducing PI3K/AKT signal and PTEN-suppressing Wnt/\u03b2-catenin signaling in non-small-cell lung cancer

MiR-374a appears to play a complex role in non-small-cell lung cancer (NSCLC). Here, we demonstrate a dual role for miR-374a in NSCLC pathogenesis. The effects and modulatory mechanisms of miR-374a on cell growth, migration, invasion, and in vivo tumorigenesis and metastasis in nude mice were also analyzed. The expression of miR-374a was examined in NSCLC and non-cancerous lung tissues by quantitative real-time reverse transcription-PCR (qRT-PCR), and in situ hybridization, respectively. miR-374a directly targets CCND1 and inactivates PI3K/AKT and Ras-mediated cell cycle signalings, as well as epithelial–mesenchymal transition (EMT). This not only dramatically suppressed cell growth, migration, invasion,and metastasis, but also elevated A549 and pc-9 NSCLC cell sensitivity to cisplatin (DDP) while increasing survival time of tumor-bearing mice. Interestingly, miR-374a serves an inverse function in SPCA-1 and H1975 NSCLC cells by directly targeting PTEN to activate Wnt/β-catenin and Ras signalings and its downstream cascade signals. Surprisingly, transcription factor c-Jun bound to the promoter region of human miR-374a and suppressed miR-374a in A549 and pc-9 cells while inducing it in SPCA-1 and H1975 cells. Increased levels of miR-374a appeared to serve a protective role by targeting CCND1 in early-stage NSCLC (Stages I and II). Inversely, increased miR-374a was an unfavorable factor when targeting PTEN in more advanced staged NSCLC patients. Our studies are the first to demonstrate that miR-374a plays divergent roles in NSCLC pathogenesis at different stages of the disease and implicate the potential application of miR-374a targeting for cancer therapy.

Synergistic Anticancer Activity of Combined Use of Caffeic Acid with Paclitaxel Enhances Apoptosis of Non-Small-Cell Lung Cancer H1299 Cells in Vivo and in Vitro

Background/Aims: Caffeic acid (CA) is known to possess multiple biological activities including anti-cancer activities. However, the molecular mechanisms underlying these activities in non-small-cell lung cancer (NSCLC) cells are not fully understood. We attempted to clarify whether CA could enhance paclitaxel (PTX)-induced cytotoxicity in H1299 cells. Methods: First, we tested the cytotoxic effects in both H1299 cells and normal human Bease-2b cells by cell proliferation experiments. Next, we use Annexin V/propidium iodide apoptosis analysis and flow cytometric analysis to investigate apoptosis and cell cycle arrest under the treatments mentioned above. To further pinpoint changes in apoptosis, we tested the caspase-associated apoptotic pathway, which involves the activities of caspase-3 and caspase-9. Moreover, apoptosis-related proteins and MAPK pathway proteins were examined by western blot. An H1299 xenograft nude mice model was used to further evaluate the tumor-suppressing effects of CA and PTX in vivo. Results: Combination treatment with low-dose CA and PTX decreased the proliferation of NSCLC H1299 cells but not normal Beas-2b cells. Flow cytometry showed that H1299 cells were arrested in the sub-G1 phase and apoptosis was significantly increased in H1299 cells after CA treatment. Caspase-3 and caspase-9 activities were both increased after CA treatment. Furthermore, CA increased the PTX-induced activation of Bax, Bid, and downstream cleaved PARP, and phosphorylation of extracellular signal regulated kinase1/2 and c-Jun NH2-terminal protein kinase1/2. An in vivo tumor-suppression assay demonstrated that CA and PTX combined treatment exerted a more effective suppressive effect on tumor growth in H1299 xenografts without causing significant adverse effects. Conclusions: Our results indicated that CA inhibited NSCLC H1299 cell growth by inducing apoptosis and CA and PTX combined produced a synergistic anti-cancer effect in H1299 cells.

DMH4, a VEGFR2 inhibitor, effectively suppresses growth and invasion of lung cancer cells

Non-small-cell lung cancer (NSCLC), the most common type of lung cancer, remains the leading cause of cancer death worldwide. Blocking vascular endothelial growth factor (VEGF) signalling is an effective approach to the treatment of NSCLC. Small molecules have been proven to be good resource for discovery of inhibitors of VEGF signalling. DMH4 is a small molecule that we previously developed and demonstrated to have the property of selectively inhibiting VEGF signalling by targeting VEGF receptor 2 (VEGFR2). In this study, we reported that DMH4 can effectively block phosphorylation of VEGFR2 in both H460 and A549 NSCLC cells, which resulted in significant reduction of NSCLC cell viability in a dose-dependent manner, and the growth inhibition (GI50) of DMH4 against H460 and A549 cell lines were 13.27 and 2.75 mm respectively at 24h. Our further studies demonstrated that DMH4 significantly suppressed migration and invasion of A549 and H460 cells, and induced apoptosis in those cells. Therefore, DMH4 as a small molecular VEGFR2 inhibitor may represent a new valuable drug lead for NSCLC treatment.

Effect of miR‑146a‑5p on tumor growth in NSCLC using chick chorioallantoic membrane assay and bioinformatics investigation.

Our previous study demonstrated that the expression of miR-146a-5p was downregulated in non-small cell lung cancer (NSCLC) tissue, which affected the progression and prognosis of patients with NSCLC. Thus, the present study was conducted to investigate the functional mechanism of miR-146a-5p in tumorigenesis and angiogenesis in NSCLC. Following the construction of a H460 NSCLC cell line in which miR-146a-5p was overexpressed via lentivirus transduction, the NSCLC chick embryo chorioallantoic membrane (CAM) model was established by transplanting miR-146a-5p-overexpressing NSCLC cells into the CAM. Then, the size of the neoplasms within the CAM was measured, the vessel ratio was calculated, and the cellular morphology, metastasis and inflammation of tumor cell was observed using hematoxylin and eosin staining. The target genes of miR-146a-5p were predicted by 12 online software programs; these genes were then subjected to Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway annotations using the Database for Annotation, Visualization and Integrated Discovery 6.7 as well as constructed into a protein interaction network using protein-protein interaction from Search Tool for the Retrieval of Interacting Genes/Proteins. The xenograft tumor size and angiogenesis conditions of the miR-146a-5p-overexpressing group (volume 6.340±0.066 mm3, vessel ratio 9.326±0.083) was obviously restricted (P<0.001) when compared with the low expression group (volume 30.13±0.06 mm3, vessel ratio 16.94±0.11). In addition, marked necrosis along with inflammatory cell infiltration was observed with the HE-stained slices from the miR-146a-5p low expression group. Regarding the results of the target gene prediction, cancer and toll-like receptor signaling were the two most significant pathways represented among the target genes, while JUN, EGFR and RAC1 were the most relevant proteins among the selected potential targets of miR-146a-5p. In a CAM xenograft tumor model, overexpression of miR-146a-5p inhibited the tumorigenesis and angiogenesis of an NSCLC cell line. miR-146a-5p may act as a tumor suppressor gene in NSCLC and have moderate prognostic value in lung cancer.

CHAF1A, the largest subunit of the chromatin assembly factor?1 complex, regulates the growth of H1299 human non‑small cell lung cancer cells by inducing G0/G1 cell cycle arrest

Chromatin assembly factor 1 subunit A (CHAF1A) is the largest subunit of the chromatin assembly factor 1 (CAF-1) complex that is implicated in the assembly of nucleosomes on newly synthesized DNA. The aim of the present study was to determine its expression and biological function in non-small cell lung cancer (NSCLC). The current study examined the levels of CHAF1A expression in 22 samples of NSCLC and corresponding normal lung tissues. Subsequently, endogenous CHAF1A expression in H1299 NSCLC cells was knocked down via lentiviral delivery of CHAF1A-targeting short hairpin RNA (shRNA), and cell proliferation, colony formation and cell cycle distribution were measured. The results demonstrated that levels of CHAF1A mRNA level were ~3-fold greater in NSCLC samples compared with adjacent normal tissues (P<0.05). shRNA-mediated silencing of CHAF1A significantly inhibited the proliferation and colony formation of H1299 cells, compared wirh the delivery of control shRNA (P<0.05). Furthermore, CHAF1A shRNA-transduced cells exhibited a significant increase in the percentage of S-phase cells and a significant decrease in the percentage of cells at the G0/G1 and G2/M phases, compared with control cells (P<0.05). Additionally, CHAF1A knockdown significantly decreased the expression of cyclin D1, cyclin-dependent kinase 2 and S-phase kinase-associated protein 2, and increased the expression of p21 and p27. This indicates that CHAF1A is upregulated in NSCLC and that its silencing suppresses the proliferation and colony formation of NSCLC cells, potentially by inducing G0/G1 cell cycle arrest. CHAF1A may therefore represent a potential therapeutic target to treat NSCLC.

Analysis of Chromatin Opening in Heterochromatic Non-Small Cell Lung Cancer Tumor-Initiating Cells in Relation to DNA-Damaging Antitumor Treatment

We previously reported that sphere-forming non-small cell lung cancer (NSCLC) tumor-initiating cells (TICs) have an altered activation of DNA damage response- and repair proteins and are refractory to DNA-damaging treatments. We analyzed whether chromatin organization plays a role in the observed refractoriness. Bulk cells and TICs from the NSCLC H23 and H1299 cell lines were examined using cell viability, clonogenic survival, Western blot, short interfering RNA analysis, and micronucleus assay. NSCLC TICs displayed elevated heterochromatin markers trimethylated lysine 9 of histone H3 and heterochromatin protein 1γ relative to bulk cells and reduced cell viability upon histone deacetylase inhibition (HDACi). Vorinostat and trichostatin A increased the euchromatin markers acetylated lysine 9/14 of histone H3 and lysine 8 of histone H4, and HDACi pretreatment increased the phosphorylation of the DNA damage response proteins ataxia telangiectasia mutated and DNA-dependent protein kinase, catalytic subunit, upon irradiation in TICs. HDACi sensitized TICs to cisplatin and to some extent to ionizing irradiation. The protectiveness of a dense chromatin structure was indicated by an enhanced frequency of micronuclei in TICs following irradiation, after knockdown of heterochromatin protein 1γ. Although confirmatory studies in additional NSCLC model systems and with respect to analyses of other DNA damage response proteins are needed, our data point toward a heterochromatic structure of NSCLC TICs, such that HDACi can sensitize TICs to DNA damage.

HuR-targeted small molecule inhibitor exhibits cytotoxicity towards human lung cancer cells

Human antigen (Hu) R is an RNA-binding protein whose overexpression in human cancer correlates with aggressive disease, drug resistance, and poor prognosis. HuR inhibition has profound anticancer activity. Pharmacologic inhibitors can overcome the limitations of genetic inhibition. In this study, we examined the antitumor activity of CMLD-2, a small-molecule inhibitor directed against HuR, using non-small cell lung cancer (NSCLC) as a model. CMLD-2 efficacy was tested in vitro using H1299, A549, HCC827, and H1975 NSCLC cells and MRC-9 and CCD-16 normal human fibroblasts. Treatment of NSCLC cells with CMLD-2 produced dose-dependent cytotoxicity, caused a G1 phase cell-cycle arrest and induced apoptosis. CMLD-2 decreased HuR mRNA and the mRNAs of HuR-regulated proteins (Bcl2 and p27) in tumor cells. Additionally, reduction in the expression of HuR, Bcl2, cyclin E, and Bcl-XL with increased expression of Bax and p27 in CMLD-2-treated NSCLC cells were observed. CMLD-2-treated normal cells, HuR-regulated mRNAs and proteins albeit showed some reduction were less compared to tumor cells. Finally, CMLD-2 treatment resulted in greater mitochondrial perturbation, activation of caspase-9 and -3 and cleavage of PARP in tumor cells compared to normal cells. Our proof-of concept study results demonstrate CMLD-2 represents a promising HuR-targeted therapeutic class that with further development could lead to advanced preclinical studied and ultimately for lung cancer treatment.

Lamin B2 binding to minichromosome maintenance complex component 7 promotes non-small cell lung carcinogenesis.

We investigated the role of lamin B2 in non-small cell lung cancer (NSCLC). We detected higher lamin B2 expression in 20 NSCLC tumor tissues obtained from The Cancer Genome Atlas than in adjacent normal lung tissues. LMNB2-RNAi knockdown in A549 and H1299 NSCLC cells inhibited colony formation, cell proliferation and G1-S cell cycle progression while increasing apoptosis. LMNB2 overexpression had the opposite effects. Tumor xenograft experiments showed diminished tumor growth with LMNB2 knockdown H1299 cells than with controls. Yeast two-hybrid studies revealed minichromosome maintenance complex component 7 (MCM7) to be a binding partner of lamin B2, which was confirmed by co-immunoprecipitation and co-localization studies. Lamin B2 binding enhanced DNA binding and helicase activities of MCM7. Deletion analysis with MCM7-N, MCM7-M or MCM7-C mutant proteins showed that lamin B2 binds to the C-terminus of MCM7, and competes with the binding of the tumor suppressor retinoblastoma (RB) protein. Immunohistochemical analysis of 150 NSCLC patient samples revealed that both lamin B2 and MCM7 levels positively correlated with histological grade and tumor TNM stage. Moreover, high lamin B2 and MCM7 levels correlated with shorter overall survival of NSCLC patients. In sum, these results show that lamin B2 interaction with MCM7 promotes NSCLC progression.

Abstract 5484: Structural domains of βIII-Tubulin regulate multiple stress responses and influence cell growth and survival in glucose-deprived non-small cell lung cancer

Abstract BACKGROUND: Non-small Cell Lung Cancer (NSCLC) survival rates are dismal and elevated βIII-tubulin expression is associated with chemotherapy resistance and tumor aggressiveness1. βIII-tubulin modulates glucose metabolism and stress response signaling to support cancer cell survival and proliferation in glucose starvation conditions in the tumor microenvironment2. βIII-Tubulin shares sequence similarity with other β-tubulin isotypes, differing in the C-terminal tail region of the protein, however the function of βIII-tubulin structural domains remain uncharacterized. This study investigated the importance of the βIII-tubulin body and tail structural domains in conferring survival advantage to glucose-starved NSCLC cells. METHODS: H460 NSCLC cells were gene-edited at the endogenous βIII-tubulin gene using zinc finger nuclease technology to replace the endogenous βIII-tubulin protein with either the full-length or truncated βIII-tubulin protein, or replacement of the βIII-tubulin body or tail with the βI-tubulin sequence, each fused to a GFP tag. Clones expressing the modified βIII-tubulin proteins were cultured in glucose-free conditions for 48h to 10 days and cell growth and viability were measured by Trypan Blue Dye Exclusion and Annexin-V apoptosis assay. The effect of the βIII-tubulin modifications on the endoplasmic reticulum (ER) stress response, autophagy and Akt signaling were examined by western blotting. RESULTS: Characterization of the gene-edited clones revealed that loss of the βIII-tubulin C-terminal tail or substitution of the βIII-tubulin body or tail sequence with that of the βI-tubulin sequence did not alter the microtubule architecture, cellular proliferation rate or viability in normal growth conditions. However, loss of the βIII-tubulin C-terminal tail significantly reduced the viability of H460 cells by 37 ± 0.05% in acute glucose starvation. Substitution of the βIII-tubulin body with the βI-tubulin body significantly reduced cell number by 53 ± 0.01% during prolonged glucose starvation. Loss of the βIII-tubulin C-terminal tail increased Akt activation and LC3B-II expression in glucose-rich conditions and potentiated the increase in ER stress response markers GRP78, ATF4 and CHOP in response to glucose starvation. These results indicate that the βIII-tubulin body and tail differentially confer a survival advantage to glucose-starved H460 cells and modulate multiple stress response signaling pathways in an isotype-dependent and independent manner. CONCLUSION: This study provides insight into the importance of βIII-tubulin structural domains in conferring βIII-tubulin function in NSCLC, thereby advancing our understanding of the role of tubulin isotypes in cell biology and the mechanisms contributing to poor patient outcome in this disease. 1 Kavallaris, Nat Rev Cancer 2010 2Parker et al., Carcinogenesis 2016 Citation Format: Amelia L. Parker, Wee S. Teo, Joshua A. McCarroll, Maria Kavallaris. Structural domains of βIII-Tubulin regulate multiple stress responses and influence cell growth and survival in glucose-deprived non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5484. doi:10.1158/1538-7445.AM2017-5484

Abstract 4037: High throughput kinase inhibitor screen reveals novel inhibitor combinations acting in synergy with TUBB3/βIII-tubulin suppression in non-small cell lung cancer

Abstract Background: Non-Small Cell Lung Cancer (NSCLC) survival rates remain dismal and this malignancy is poorly responsive to current therapy. The mechanisms contributing to this resistance phenotype and aggressive behavior of this disease are not well defined. TUBB3/βIII-tubulin is aberrantly expressed in NSCLC and its expression correlates negatively with disease free survival of patients. Our group has previously shown that βIII-tubulin plays a functional role in chemotherapy resistance and tumorigenesis in NSCLC via modulation of the PTEN/AKT signaling pathway1, 2. This study aimed to identify kinase inhibitors that synergize with βIII-tubulin inhibition in NSCLC using a high-throughput cell-based screen. Methods: A high-throughput cell-based screen of a kinase inhibitor library was performed against our previously described1, 2 NSCLC H460 cells, expressing either βIII-tubulin shRNA or control non-targeting shRNA. The primary screen consisted of a total of 210 kinase inhibitors tested at a single, fixed concentration and cell viability determined using the Alamar blue assay. Secondary screens were performed using a five point dose response. Further validation of the kinase inhibitors was performed against two independent NSCLC, H460 and A549 expressing either βIII-tubulin shRNA or control non-targeting shRNA. Results: A number of kinase inhibitors that act on key signalling cascades were identified to act in synergy with TUBB3/βIII-tubulin knockdown to inhibit NSCLC cell growth. Interestingly, several different inhibitors which target mTOR were identified as top candidates for acting in combination with βIII-tubulin knockdown to inhibit NSCLC cell growth. Treatment of cells with clinically approved mTOR inhibitors, Temsirolimus and Rapamycin, resulted in a significant decrease in cell viability in the βIII-tubulin knockdown cells compared to control. Notably, mTOR acts downstream of AKT signalling and further highlights a potential connection with βIII-tubulin and the AKT signal transduction cascade. Other kinase inhibitors that act on key signaling cascades including Amuvatinib-Multi Kinase Inhibitor (Flt3, Met, Kit, c-Ret &amp; PDGFR); Mubritinib-HER-2 inhibitor; and Neratinib HER2, EGFR, also acted in combination with βIII-tubulin knockdown to inhibit NSCLC cell growth. Conclusions: Our results suggest that clinically approved therapies designed to target mTOR combined with inhibition of TUBB3/βIII-tubulin may offer a novel therapeutic strategy for the treatment of NSCLC patients.

Lung cancer suppressor gene GPRC5A mediates p53 activity in non‑small cell lung cancer cells invitro.

Cellular tumor antigen p53 (p53) functions to maintain genomic stability and regulate cell apoptosis, while G protein‑coupled receptor class C group 5 memberA (GPCR5A) is a lung cancer suppressor gene whose expression is induced by retinoids. The present invitro study assessed the effects of p53 on the regulation of GPRC5A expression and on non‑small cell lung cancer (NSCLC) cells. Human NSCLC H1299 (p53‑null) and A549 (wild‑type p53) cell lines were subjected to p53 cDNA and small interfering (si)RNA transfection, respectively. GPRC5A expression was analyzed by reverse transcription‑quantitative polymerase chain reaction and western blotting, and cell behavior was analyzed using cell viability and apoptosis assays. The results of the present study demonstrated that knockdown of GPRC5A expression markedly upregulated tumor cell viability and reduced tumor cell apoptosis, while p53 overexpression in H1299 cells significantly increased the expression level of GPRC5A. p53 overexpression and GPRC5A induction markedly inhibited tumor cell viability and induced apoptosis, while knockdown of p53 resulted in a decrease in GPRC5A expression, inhibited tumor cell apoptosis and increased tumor cell viability. In serum‑free culture conditions, GPRC5A expression was decreased in the two cell lines; this decrease was less marked in p53 cDNA‑transfected H1299 cells and more marked in p53 siRNA‑transfected A549 cells. The results of the present study indicated that p53 antitumor activity may be mediated by GPRC5A in NSCLC cells.

MicroRNA-17-5p inhibits proliferation and triggers apoptosis in non-small cell lung cancer by targeting transforming growth factor β receptor 2.

MicroRNAs (miRs) are small non-coding RNAs that suppress gene expression by directly binding to the 3′-untranslated region of their target mRNAs. Specific miRs serve key roles in the development and progression of non-small cell lung cancer (NSCLC). The aim of the present study was to determine the mechanism of miR-17-5p in the regulation of NSCLC cell survival and proliferation. Reverse transcription-quantitative polymerase chain reaction data indicated that miR-17-5p was significantly downregulated in 28 NSCLC tissues compared with 7 non-tumorous lung tissues. Furthermore, lower miR-17-5p expression was associated with a higher pathological stage in NSCLC patients. Lower miR-17-5p expression was also observed in several common NSCLC cell lines, including SK-MES-1, A549, SPCA-1, H460, H1229 and HCC827, compared with the bronchial epithelium cell line, BEAS-2B. Additionally, overexpression of miR-17-5p significantly inhibited proliferation while inducing the apoptosis of NSCLC H460 cells. Subsequently, transforming growth factor β receptor 2 (TGFβR2) was identified as a direct target of miR-17-5p using a luciferase reporter assay. Western blot analysis confirmed that miR-17-5p negatively mediated the expression of TGFβR2 in NSCLC cells. Furthermore, small interfering RNA-induced downregulation of TGFβR2 also suppressed the proliferation of H460 cells while triggering apoptosis. Therefore, the results of the current study suggest that miR-17-5p may inhibit proliferation and trigger apoptosis in NSCLC H460 cells at least partially by targeting TGFβR2.

MicroRNA-338-3p suppresses cell proliferation and induces apoptosis of non-small-cell lung cancer by targeting sphingosine kinase 2

BackgroundLung cancer is the major cause of cancer-related death worldwide, and 80% patients of lung cancer are non-small-cell lung cancer (NSCLC) cases. MicroRNAs are important gene regulators with critical roles in diverse biological processes, including tumorigenesis. Studies indicate that sphingosine kinase 2 (SphK2) promotes tumor progression in NSCLC, but how this occurs is unclear. Thus, we explored the effect of miR-338-3p targeting SphK2 on proliferation and apoptosis of NSCLC cells.MethodsExpression of miR-338-3p and SphK2 in NSCLC A549 and H1299 cell lines was measured using qRT-PCR and Western blot. CCK-8 and colony formation assays were used to assess the effect of miR-338-3p on NSCLC cell line proliferation. Flow cytometry was used to study the effect of miR-338-3p on NSCLC apoptosis. Luciferase reporter assay and Western blot were used to confirm targeting of SphK2 by miR-338-3p. Finally, in vivo tumorigenesis studies were used to demonstrate subcutaneous tumor growth.ResultsmiR-338-3p expression in 34 NSCLC clinical samples was downregulated and this was correlated with TNM stage. miR-338-3p significantly suppressed proliferation and induced apoptosis of NSCLC A549 and H1299 cells in vitro. SphK2 was a direct target of miR-338-3p. Overexpression of miR-338-3p significantly inhibited SphK2 expression and reduced luciferase reporter activity containing the SphK2 3′-untranslated region (3′-UTR) through the first binding site. SphK2 lacking 3′-UTR restored the effects of miR-338-3p on cell proliferation inhibition. miR-338-3p significantly inhibited tumorigenicity of NSCLC A549 and H1299 cells in a nude mouse xenograft model.ConclusionsCollectively, miR-338-3p inhibited cell proliferation and induced apoptosis of NSCLC cells by targeting and down-regulating SphK2, and miR-338-3p could inhibit NSCLC cells A549 and H1299 growth in vivo, suggesting a potential mechanism of NSCLC progression. Therapeutically, miR-338-3p may serve as a potential target in the treatment of human lung cancer.

Plumbagin reduces osteopontin-induced invasion through inhibiting the Rho-associated kinase signaling pathway in A549 cells and suppresses osteopontin-induced lung metastasis in BalB/c mice

Lung cancer is the second most commonly diagnosed cancer and the leading cause of cancer deaths in both men and women in the United States. It has been recently demonstrated that osteopontin (OPN) effectively inhibits cofilin activity through the focal adhesion kinase (FAK)/AKT/Rho-associated kinase (ROCK) pathway to induce the invasion of human non-small cell lung cancer (NSCLC) cells. Plumbagin was isolated from the roots of the medicinal plant Plumbago zeylanica L. and has been reported to possess anticancer activities. However, the molecular mechanisms by which plumbagin inhibits the invasion of cancer cells is still unclear. In this study, the anti-invasive and anti-metastatic mechanisms of plumbagin were investigated in OPN-treated NSCLC A549 cells. OPN effectively induced the motility and invasion of NSCLC A549 cells and H1299 cells, which was strongly suppressed by plumbagin with no evidence of cytotoxicity. In addition, lamellipodia formation at the leading edge of cells by OPN was dramatically decreased in plumbagin-treated cells. Plumbagin caused an effective inhibition in OPN-induced the expression of ROCK1 as well as the phosphorylation of LIM kinase 1 and 2 (LIMK1/2), and cofilin. OPN-induced the phosphorylation of FAK and AKT was impaired without affecting their total forms by plumbagin treatment. OPN facilitated metastatic lung colonization, which was effectively suppressed in plumbagin-treated mice. Taken together, these results suggest that plumbagin reduces OPN-induced the invasion of NSCLC A549 cells, which resulted from inhibiting the ROCK pathway mediated by the FAK/AKT pathway and suppresses lung metastasis in vivo.

Ulmus davidiana Nakai induces apoptosis and autophagy on non-small cell lung cancer cells

Ethnopharmacological relevanceUlmus davidiana Nakai (UDN) is frequently used in the treatment of cancer in traditional oriental medicine. Although several reports indicate that UDN has inhibitory effects in some cancers, there has been no report on the inhibitory effects of UDN via both autophagy and apoptosis. Materials and methodsCytotoxicity induced by UDN in human non-small cell lung cancer (NSCLC) H-1299 and H-460 cell lines was evaluated using the 2, 3-Bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay and trypan blue exclusion assay. Induction of apoptosis was also investigated using Hoechst staining and annexin-V binding assay and was confirmed with western blot analysis. Induction of autophagy was investigated through observation of autophagy vacuoles under inverted phase-contrast microscopy and was confirmed by observing the formation of autophagy vacuoles under a fluorescence microscope using monodansylcadaverine (MDC) staining and western blot analysis. The in vivo anti-tumorigenic effect of UDN was investigated in an athymic nude mouse xenograft model using H-1299 NSCLC cells. ResultsUDN exhibited a marked inhibitory effect on cell growth in H-1299 and H-460 human NSCLC cell lines in a dose- and time-dependent manner in vitro and in vivo. It induced not only apoptosis, but also autophagy in both H-1299 and H-460 cells in a dose-dependent manner. UDN-mediated autophagy led to the accumulation of autophagosome, resulting in apoptosis induction and cell death. ConclusionsFrom our current knowledge, we are the first to demonstrate that UDN has the potential to induce both autophagy and apoptosis in H-1299 and H-460 human NSCLC cell lines. We suggest that UDN can be considered a potential candidate for lung cancer-specific chemotherapy with efficacy as a cytotoxic agent.

Dual roles of extracellular signal-regulated kinase (ERK) in quinoline compound BPIQ-induced apoptosis and anti-migration of human non-small cell lung cancer cells

Background2,9-Bis[2-(pyrrolidin-1-yl)ethoxy]-6-{4-[2-(pyrrolidin-1-yl)ethoxy] phenyl}-11H-indeno[1,2-c]quinoline-11-one (BPIQ), is a synthetic quinoline analog. A previous study showed the anti-cancer potential of BPIQ through modulating mitochondrial-mediated apoptosis. However, the effect of BPIQ on cell migration, an index of cancer metastasis, has not yet been examined. Furthermore, among signal pathways involved in stresses, the members of the mitogen-activated protein kinase (MAPK) family are crucial for regulating the survival and migration of cells. In this study, the aim was to explore further the role of MAPK members, including JNK, p38 and extracellular signal-regulated kinase (ERK) in BPIQ-induced apoptosis and anti-migration of human non-small cell lung cancer (NSCLC) cells.MethodsWestern Blot assay was performed for detecting the activation of MAPK members in NSCLC H1299 cells following BPIQ administration. Cellular proliferation was determined using a trypan blue exclusion assay. Cellular apoptosis was detected using flow cytometer-based Annexin V/propidium iodide dual staining. Cellular migration was determined using wound-healing assay and Boyden’s chamber assay. Zymography assay was performed for examining MMP-2 and -9 activities. The assessment of MAPK inhibition was performed for further validating the role of JNK, p38, and ERK in BPIQ-induced growth inhibition, apoptosis, and migration of NSCLC cells.ResultsWestern Blot assay showed that BPIQ treatment upregulates the phosphorylated levels of both MAPK proteins JNK and ERK. However, only ERK inhibitor rescues BPIQ-induced growth inhibition of NSCLC H1299 cells. The results of Annexin V assay further confirmed the pro-apoptotic role of ERK in BPIQ-induced cell death of H1299 cells. The results of wound healing and Boyden chamber assays showed that sub-IC50 (sub-lethal) concentrations of BPIQ cause a significant inhibition of migration in H1299 cells accompanied with downregulating the activity of MMP-2 and -9, the motility index of cancer cells. Inhibition of ERK significantly enhances BPIQ-induced anti-migration of H1299 cells.ConclusionsOur results suggest ERK may play dual roles in BPIQ-induced apoptosis and anti-migration, and it would be worthwhile further developing strategies for treating chemoresistant lung cancers through modulating ERK activity.

Synthesis and biological assay of erlotinib analogues and BSA-conjugated erlotinib analogue

A series of erlotinib analogues that have structural modification at 6,7-alkoxyl positions is efficiently synthesized. The in vitro anti-tumor activity of synthesized compounds is studied in two non-small cell lung cancer (NSCLC) cell lines (A549 and H1975). Among the synthesized compounds, the iodo compound 6 (ETN-6) exhibits higher anti-cancer activity compared to erlotinib. An efficient method is developed for the conjugation of erlotinib analogue-4, alcohol compound, with protein, bovine serum albumin (BSA), via succinic acid linker. The in vitro anti-tumor activity of the protein attached erlotinib analogue, 8 (ETN-4-Suc-BSA), showed stronger inhibitory activity in both A549 and H1975 NSCLC cell lines.