A549 Non-small Cell Lung Carcinoma Research Articles

Different bonding of triazolopyrimidine to platinum(IV). Structural and in vitro cytotoxicity studies

The reactions of H2PtCl6 with 5,7-dimethyl-1,2,4-triazolo[1,5-a]pyrimidine (dmtp) and 5-methyl-7-isobutyl-1,2,4-triazolo[1,5-a]pyrimidine (ibmtp) yielded two novel Pt(IV) compounds. The structures of both platinum(IV) coordination compounds have been solved by single-crystal X-ray diffraction. The complexes confirmed different bonding modes of heterocycle ligands to platinum(IV). The coordination units of cis-[PtCl4(ibmtp)2] (1) are built from two monodentate ibmtp molecules coordinated via N3 located in equatorial positions and four chloride ions. In contrast, the X-ray crystal structures of (Hdmtp)2[PtI6] (2) confirmed that this platinum(IV) compound is an ionic pair consisting of one octahedral [PtI6]2− anion and two N3-monoprotonated Hdmtp+ cations. In addition, the stable in solution lipophilic complex (1) has been tested for in vitro cytotoxic activity against three human tumour cell lines T47D (breast cancer), A549 (non-small cell lung carcinoma), and LoVo (colon cancer), and one mouse healthy cell line, BALB/3T3 (mouse embryonic fibroblast). The tests indicate significant in vitro cytotoxicity against the non-small cell lung carcinoma (A549) cancer cell line (IC50 = 3.44 μM).

Targeting apoptosis by 1,2-diazole through regulation of EGFR, Bcl-2 and CDK-2 mediated signaling pathway in human non-small cell lung carcinoma A549 cells

Lung cancer is the leading cause of cancer deaths worldwide and non-small cell lung carcinoma (NSCLC), a heterogeneous class of tumors, represents approximately 85% of all new lung cancer diagnosis. Conventional treatment options have limited efficacy because most cases are in the advanced stage at the time of diagnosis. The present study evaluates the anti-cancer activity of 1,2-diazole (pyrazole), a natural compound from mangrove plant Rhizophora apiculata (R.apiculata) on A549 lung carcinoma cells. In the present study the anti-cancer mechanism of pyrazole, was examined by the expression level of proteins Epidermal growth factor receptor (EGFR), Bcl-2-associated X protein (Bax), B-cell lymphoma-2 (Bcl-2) and Cyclin-dependent kinase-2 (CDK-2) which are commonly associated with the cell signaling pathways that control cell survival and apoptosis, that could facilitate to develop a novel target and effective treatment approach for patients with NSCLC. Pyrazole significantly induced cell cycle arrest and initiated apoptosis through inhibition of downstream components of EGFR tyrosine kinase pathway. Pyrazole disrupts the mitochondrial membrane potential and modulated the protein levels of Bax and Bcl-2 which could probably lead to caspase-3 activation. Furthermore, Pyrazole suppresses the expression of CDK-2 resulting in cell cycle arrest at G1 phase and in the G1-S phase transition. Taken together, the current study provides new insight in to the precise molecular mechanisms responsible for the anti-cancer activity of pyrazole in NSCLC, A549 cells. The study opens an avenue for development of a natural compound as a potential therapeutic agent which could target cell signaling pathways to combat human NSCLC.

Regulation of monoamine oxidase A (MAO-A) expression, activity, and function in IL-13–stimulated monocytes and A549 lung carcinoma cells

Monoamine oxidase A (MAO-A) is a mitochondrial flavoenzyme implicated in the pathogenesis of atherosclerosis and inflammation and also in many neurological disorders. MAO-A also has been reported as a potential therapeutic target in prostate cancer. However, the regulatory mechanisms controlling cytokine-induced MAO-A expression in immune or cancer cells remain to be identified. Here, we show that MAO-A expression is co-induced with 15-lipoxygenase (15-LO) in interleukin 13 (IL-13)-activated primary human monocytes and A549 non-small cell lung carcinoma cells. We present evidence that MAO-A gene expression and activity are regulated by signal transducer and activator of transcription 1, 3, and 6 (STAT1, STAT3, and STAT6), early growth response 1 (EGR1), and cAMP-responsive element-binding protein (CREB), the same transcription factors that control IL-13-dependent 15-LO expression. We further established that in both primary monocytes and in A549 cells, IL-13-stimulated MAO-A expression, activity, and function are directly governed by 15-LO. In contrast, IL-13-driven expression and activity of MAO-A was 15-LO-independent in U937 promonocytic cells. Furthermore, we demonstrate that the 15-LO-dependent transcriptional regulation of MAO-A in response to IL-13 stimulation in monocytes and in A549 cells is mediated by peroxisome proliferator-activated receptor γ (PPARγ) and that signal transducer and activator of transcription 6 (STAT6) plays a crucial role in facilitating the transcriptional activity of PPARγ. We further report that the IL-13-STAT6-15-LO-PPARγ axis is critical for MAO-A expression, activity, and function, including migration and reactive oxygen species generation. Altogether, these results have major implications for the resolution of inflammation and indicate that MAO-A may promote metastatic potential in lung cancer cells.

Abstract 2563: Macrophage Toll-like receptor-chimeric antigen receptors (MOTO-CARs) as a novel adoptive cell therapy for the treatment of solid malignancies

Abstract Recent clinical trials using chimeric antigen receptors (CAR) T cells have demonstrated tremendous success in eradicating hematologic malignancies. Notwithstanding the excitement generated by CAR T cell therapy, its clinical efficacy has not been effectively translated to the context of solid tumors; the physical barriers of solid malignancies and the immunosuppressive conditions at the tumor site hinder the efficacy of CAR T cells. Macrophages have the ability to infiltrate almost every tissue and frequently are recruited into tumors. Therefore, macrophages are an attractive vehicle for CAR therapy and could help solve current challenges that CAR T cells face in the treatment of solid tumors. MOTO-CAR cells are monocyte-derived human macrophages that are genetically modified by a lentiviral or adenoviral approach to express a synthetic tumor-targeting receptor and to secrete cytokines, ligands or chemokine receptors. MOTO-CAR receptors are composed of a single-chain variable fragment (ScFv) that binds to a specific tumor target, a hinge to link it to a transmembrane domain, and an engineered Toll/Interleukin-1 receptor (TIR) signaling domain. When the ScFv binds to the tumor cell via its tumor target, an activation signal is transmitted. Myd88 dependent and independent signaling cascades are elicited, activating the macrophage and polarizing it towards a proinflammatory phenotype to eliminate cancer cells in a selective way. We previously reported the expression of Thymidine Kinase 1 (TK1) on the cell membrane of the non-small cell lung carcinoma NCI-H460 and A549 cell lines. The in vitro function of TK1 MOTO-CAR cells was evaluated against these cancer cell lines, using GFP-based phagocytosis and killing assays. Additionally, cell migration and interaction was recorded using time-lapse video with a confocal microscope. Upon co-culturing, with its target TK1 specific MOTO-CARs showed a nearly 4-fold increase in killing activity when compared with the controls (p<0.01). MOTO-CAR cells were produced through a lentiviral approach with around 30% of the cells expressing MOTO-CARs and with an adenoviral approach using the Ad5f35 vector with an efficiency of 70-80% of cells being transduced. Furthermore, after transduction MOTO-CAR cells showed a consistent M1 phenotype expressing high levels of CD14, CD80, CD206 and low levels of CD163. Time-lapse videos showed migration and clustering of MOTO-CAR cells around H460 GFP + cells. Moreover, cell death was observed upon contact of MOTO-CAR cells with target cells as well as phagocytic activity. In vivo testing using an orthotopic NOD scid gamma mice model is in progress. Our preclinical data show evidence that human macrophages are a suitable vehicle for CAR therapy and have the potential to successfully extrapolate the clinical efficacy of CAR therapy to the context of solid tumors. Citation Format: Edwin J. Velazquez, John E. Lattin, Taylor D. Brindley, Zachary Z. Reinstein, Roger Chu, Lu Liu, Evita G. Weagel, Michelle H. Townsend, Kiara V. Whitley, Eliza L. Lawrence, Brandon T. Garcia, Scott Weber, Richard A. Robison, Kim L. O'Neill. Macrophage Toll-like receptor-chimeric antigen receptors (MOTO-CARs) as a novel adoptive cell therapy for the treatment of solid malignancies [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 2563.

Phytochemical naphtho[1,2-b] furan-4,5‑dione induced topoisomerase II-mediated DNA damage response in human non-small-cell lung cancer

BackgroundPhytochemical naphtho[1,2-b] furan-4,5‑dione (NFD) presenting in Avicennia marina exert anti-cancer effects, but little is known regarding about DNA damage-mediated apoptosis in non-small-cell lung carcinoma (NSCLC). PurposeTo examine whether NFD-induced apoptosis of NSCLC cells is correlated with the induction of DNA damage, and to investigate its underlying mechanism. Study designThe anti-proliferative effects of NFD were assessed by MTS Assay Kit FACS assay, and in vivo nude mice xenograft assay. The DNA damage related proteins, the Bcl-2 family and pro-apoptotic factors were examined by immunofluorescence assay, q-PCR, and western blotting. The activity of NF-κB p65 in nuclear extracts was detected using a colorimetric DNA-binding ELISA assay. The inhibitory activity of topoisomerase II (TOPO II) was evaluated by molecular docking and TOPO II catalytic assay. ResultsNFD exerted selective cytotoxicity against NSCLC H1299, H1437 and A549 cells rather than normal lung-embryonated cells MRC-5. Remarkably, we found that NFD activated the hull marker and modulator of DNA damage repairs such as γ-H2AX, ATM, ATR, CHK1, and CHK2 probably caused by the accumulation of intracellular reactive oxygen species (ROS) and inhibition of TOPO II activity. Furthermore, the suppression of transcription factor NF-κB by NFD resulted in significantly decreased levels of pro-survival proteins including Bcl-2 family Bcl-2, Bcl-xL and Mcl-1 and the endogenous inhibitors of apoptosis XIAP and survivin in H1299 cells. Moreover, the nude mice xenograft assay further validated the suppression of H1299 growth by NFD, which is the first report for evaluating the anti-cancer effect of NFD in vivo. ConclusionThese findings provide a novel mechanism indicating the inhibition of TOPO II activity and NF-κB signaling by NFD, leading to DNA damage and apoptosis of NSCLC tumor cells.

Synthesis of novel tetrandrine derivatives and their inhibition against NSCLC A549 cells

A series of novel tetrandrine (Tet) derivatives were synthesized through Suzuki -Miyaura reaction and evaluated for their cytotoxicity against human non-small cell lung carcinoma (NSCLC) A549 cells. Interestingly, most of derivatives showed similar cytotoxicity to Tet against NSCLC A549 cells, and particularly, compounds Y5, Y6, Y9 and Y11 showed the most significant cytotoxic effects with IC50 values ranging from 3.87 to 4.66 mM. The present study is expected to contribute to the future design of more effective anticancer agents in lung cancer chemotherapy.

Evodiamine, a Novel NOTCH3 Methylation Stimulator, Significantly Suppresses Lung Carcinogenesis in Vitro and in Vivo.

Lung cancer is a leading cause of cancer-related deaths worldwide. NOTCH3 signaling is mainly expressed in non-small cell lung carcinoma (NSCLC), and has been proposed as a therapeutic target of NSCLC. While, few agents for preventing or treating NSCLC via targeting NOTCH3 signaling are used in modern clinical practice. Evodiamine (EVO), an alkaloid derived from Euodiae Fructus, possesses low toxicity and has long been shown to exert anti-lung cancer activity. However, the underlying anti-lung cancer mechanisms of EVO are not yet fully understood. In this study, we explored the involvement of NOTCH3 signaling in the anti-lung cancer effects of EVO. Urethane-induced lung cancer mouse model and two NSCLC cell models, A549 and H1299, were used to evaluate the in vivo and in vitro anti-lung cancer action of EVO. A DNA methyltransferase inhibitor was employed to investigate the role of NOTCH3 signaling in the anti-lung cancer effects of EVO. Results showed that EVO potently reduced tumor size and tumor numbers in mice, and inhibited NOTCH3 in the tumors. EVO also dramatically reduced cell viability, induced G2/M cell cycle arrest, inhibited cell migration and reduced stemness in cultured NSCLC cells. Mechanistic studies showed that EVO potently inhibited NOTCH3 signaling by activation of DNMTs-induced NOTCH3 methylation. Importantly, inhibition of NOTCH3 methylation in NSCLC cells diminished EVO’s anti-NSCLC effects. Collectively, EVO, a novel NOTCH3 methylation stimulator, exerted potent anti-lung cancer effects partially by inhibiting NOTCH3 signaling. These findings provide new insight into the EVO’s anti-NSCLC action, and suggest a potential role of EVO in lung cancer prevention and treatment.

Baicalin, a Chinese Herbal Medicine, Inhibits the Proliferation and Migration of Human Non-Small Cell Lung Carcinoma (NSCLC) Cells, A549 and H1299, by Activating the SIRT1/AMPK Signaling Pathway.

BackgroundBaicalin is a flavonoid derived from Scutellaria baicalensis, used in Chinese herbal medicine. Activation of the sirtuin 1 gene (SIRT1) and adenosine monophosphate (AMP)-activated protein kinase gene (AMPK), the SIRT1/AMPK signaling pathway, is associated with human malignant tumors. The aim of this study was to investigate the effects of baicalin on the cell viability, apoptosis, proliferation, and migration of human non-small cell lung cancer (NSCLC) cells, A549 and H1299, in vitro.Material/MethodsHuman NSCLC cells, A549 and H1299, were treated with serial doses of baicalin. Small interfering RNA (siRNA) silencing of the SIRT1 and AMPK genes was performed using cell transfection. The MTT assay was used to determine cell viability, flow cytometry was used to measure cell apoptosis, wound healing and transwell assays were used to assess cell migration of A549 and H1299 cells. Western blotting was used to measure protein expression and phosphorylation levels in untreated A549 and H1299 cells, and cells treated with increasing doses of baicalin.ResultsBaicalin inhibited the viability, migration, and invasion of A549 and H1299 cells, and increased cell apoptosis in a dose-dependent manner. Baicalin activated the SIRT1/AMPK and mechanistic target of rapamycin (mTOR), and SIRT1/AMPK and matrix metalloproteinase (MMP) signaling in A549 and H1299 cells in a dose-dependent manner. siRNA silencing of SIRT1 and AMPK reduced the effects of baicalin on cell proliferation and migration.ConclusionsBaicalin, a flavonoid used in Chinese herbal medicine, inhibited the proliferation and migration of human NSCLC cells, A549 and H1299, by activating the SIRT1/AMPK signaling pathway.

Anti-invasive effect of Cyclamen pseudibericum extract on A549 non-small cell lung carcinoma cells via inhibition of ZEB1 mediated by miR-200c.

Scientists are increasingly focusing attention on natural products of plant origin for use as agents in cancer protection and treatment. Cyclamen L. tuber extracts contain saponin glycosides that have been shown to have anti-cancer and other biological activities. The epithelial-to-mesenchymal transition (EMT) is thought to enhance malignant tumour progress. The transcriptional repressor zinc-finger E-box binding homeobox 1 (ZEB1) is an important inducer of EMT in different human tumours and has recently been shown to boost invasion by tumour cells. In this study, we investigated the effects of endemic Cyclamen pseudibericum (CP) saponin-rich tuber extract on the capacity of non-small cell lung cancer line A549 cells to proliferate, invade and migrate and also examined the expression levels of several invasion-migration-related microRNAs (miRNAs) to identify those which directly targeted ZEB1. The cytotoxicity effect of the CP extract on the A549 cancer cells was determined by the luminometric method. The half-minimal (50%) inhibitory concentration dose in the A549 cells was determined to be 41.64 ± 2.35µg/mL. Using the Matrigel invasion chamber system and the wound healing assay we observed that the CP extract suppressed the invasion and migration capacity of A549 cells, respectively. The expression of miRNAs in A549 cells was evaluated by real-time PCR. Our data showed that overexpression of miRNA miR-200c hindered the EMT by increasing the expression of E-cadherin and decreasing the expression of both N-cadherin and vimentin through the direct targeting of ZEB1. These findings suggest that the saponin-rich tuber extract of CP may have considerable anti-cancer properties in lung cancer. Further studies are required to examine in detail the molecular-based mechanism involved in the EMT process of the extract along with isolation and identification of active saponin components.

Tetrahydrocurcumin‑induced autophagy via suppression of PI3K/Akt/mTOR in non‑small cell lung carcinoma cells.

Lung carcinoma is the leading cause of mortality due to cancer worldwide. Autophagy has a significant role in the development and progression of non‑small cell lung carcinoma (NSCLC). A previous study has revealed that tetrahydrocurcumin (THC), a traditional Chinese medicine isolated from Curcumawenyujin (Chen & Ling, 1981), induces autophagy in human A549 NSCLC cells. The present study evaluated THC‑induced autophagy in A549 cells using various assays, including the Cell Counting Kit‑8, acridine orange staining, flow cytometry, reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR), and western blot analysis of the markers of autophagy. THC inhibited the growth and proliferation of A549 cells (P<0.05). Acridine orange staining and flow cytometry revealed that THC treatment significantly enhanced autophagic cell proliferation inhibition (P<0.05). The RT‑qPCR analysis revealed that THC treatment increased Beclin‑1 expression level and compared with the control group (P<0.05). The light chain 3 (LC3)‑II/LC3‑I ratio was reduced in THC‑treated cells when compared with the control group (P<0.05). Protein expression of various markers of autophagy, including p62, phosphorylated (p)‑mechanistic target of rapamycin (mTOR), phosphoinositide 3‑kinase (PI3K), p‑PI3K, protein kinase B (Akt), and p‑Akt was significantly reduced in THC‑treated cells (P<0.05). In conclusion, the present study revealed the underlying mechanisms associated with THC‑induced autophagy. A promising method of enhancing the therapeutic efficacy of THC against NSCLC cells may include inducing autophagy via inhibition of the PI3K/Akt/mTOR signaling pathway.

Downregulation of Pim-2 induces cell cycle arrest in the G0/G1 phase via the p53-non-dependent p21 signaling pathway.

Pim-2 is a serine/threonine protein kinase that is highly expressed in various types of cancer, with essential roles in the regulation of signal transduction cascades, which promote cell survival and proliferation. The present study demonstrated that Pim-2 was expressed in cells lines derived from hematopoietic tumors and lung cancer. In vitro, downregulation of Pim-2 by short interfering RNA inhibited proliferation and delayed G0/G1 cell cycle progression in K562 leukemia, RPMI-8226 multiple myeloma, and H1299 and A549 non-small cell lung carcinoma cell lines. Furthermore, downregulation of Pim-2 resulted in upregulation of cyclin-dependent kinase (CDK) inhibitor p21, irrespective of the p53 status. In addition, the present study revealed that CDK2 and phosphorylated retinoblastoma (pRb) were significantly downregulated. This finding suggested that inhibition of CDK2 and pRb expression via upregulated p21 was involved in the downregulation of Pim-2-induced G0/G1 cell cycle arrest in lung cancer and hematopoietic malignancy cells. These results suggested that Pim-2 may serve a role in hematopoietic tumors, lung cancer proliferation and cell cycle progression by regulating the p21 signaling pathway. Downregulation of Pim-2 decreased cancer cell proliferation. Therefore, Pim-2 may be a potential therapy target in clinical cancer therapy.

Apatinib resensitizes cisplatin-resistant non-small cell lung carcinoma A549 cell through reversing multidrug resistance and suppressing ERK signaling pathway.

To observe the reversal effect of apatinib on the resistance to cisplatin (DDP) of A549/cisplatin (A549/DDP) cells and its relevant mechanism. A549/DDP cells were treated with the control method, apatinib alone, DDP alone and DDP combined with apatinib. The cell proliferation was detected by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the cell clone formation assay. The cell apoptosis was detected by Hoechst 33258 staining and annexin V and propidium iodide (PI) double labeling. The changes in apoptotic proteins, multidrug resistance protein 1 (MDR1) and extracellular signal-regulated kinase (ERK) signaling pathway proteins in each group after treatment were detected by Western blotting. MTT assay results showed that compared with A549 cells, A549/DDP cells had obvious resistance to DDP. MTT assay and cell clone formation assay revealed that the tumor inhibition rate of the sub-lethal dose of apatinib (10 μM) combined with DDP was higher than that of DDP alone. The apoptosis detection results indicated that the proportion of apoptotic cells in the apatinib (10 μM) combined with DDP group was significantly increased. Western blotting results revealed that compared with that in parental A549 cells, the expression level of MDR1 in A549/DDP cells was significantly increased, and the ERK signaling pathway was activated. In the apatinib combined with DDP group, the levels of cleaved caspase-3, cleaved caspase-9 and B-cell lymphoma-2 (Bcl-2)-associated X (BAX) proteins were significantly upregulated, while the level of Bcl-2 proteins was downregulated. Apatinib could inhibit the expression of MDR1 and the activity of the ERK signaling pathway in a dose-dependent manner. Apatinib can restore the sensitivity of A549/DDP cells to DDP by down-regulating the expression level of MDR1 and inhibiting the activity of the ERK signaling pathway.

Methanolic extract of Euchelus asper exhibits in-ovo anti-angiogenic and in vitro anti-proliferative activities

BackgroundThe marine environment is a rich source of bioactive natural products. Many of the marine bioactive compounds have been derived successfully from molluscs. Euchelus asper is a marine mollusc which is commonly found in the intertidal rocky regions of the Mumbai coast. The present study was focused on evaluating the anti-angiogenic and anti- proliferative activities of methanolic extract of Euchelus asper (EAME).MethodsThe anti-angiogenic activity of EAME (50–800 μg/mL) was assessed by chick chorio-allantoic membrane (CAM) model wherein multiple parameters in the CAM blood vessels were analysed through morphometric and histological investigations. In vitro testing of EAME (5–20 μg/mL) included its cytotoxicity against three different cancer cell lines, its effect on cell proliferation by wound healing assay as well as their relevant molecular mechanisms. Statistical analysis was carried out by two-tailed student’s t test for two unpaired groups.ResultsAnalysis of CAM revealed that the extract is effective in reducing the branching points of the 1st order blood vessels or capillaries of CAM. Histological analysis of CAM showed significant decrease in capillary plexus and compartmentalization along with increase in mesodermal blood vessels, thus establishing its anti-angiogenicity. Further, EAME exhibited moderate but significant cytotoxicity against A549 non-small cell lung carcinoma cell line. We also demonstrated that the cytotoxicity of EAME in A549 was associated with its apoptotic activity by subG1 phase arrest. Lastly, EAME significantly reduced A549 proliferation by reducing the expression of Matrix metalloproteinase-2 (MMP-2) and Matrix metalloproteinase-9 (MMP-9).ConclusionOverall, our study suggested that EAME has potential to inhibit tumour angiogenic and proliferative activity and may be a potential source for development of new anti-cancer pharmaceuticals.

APE1 modulates cellular responses to organophosphate pesticide-induced oxidative damage in non-small cell lung carcinoma A549 cells.

Monocrotophos (MCP) and chlorpyrifos (CP) are widely used organophosphate pesticides (OPPs), speculated to be linked with human pathologies including cancer. Owing to the fact that lung cells are most vulnerable to the environmental toxins, the development and progression of lung cancer can be caused by the exposure of OPPs. The present study investigates the oxidative DNA damage response evoked by MCP and CP in human non-small cell lung carcinoma A549 cells. A549 cells were exposed to MCP and CP; cytotoxicity and reactive oxygen species (ROS) generation were measured to select the non-toxic dose. In order to establish whether MCP and CP can initiate the DNA repair and cell survival signalling pathways in A549 cells, qRT-PCR and Western blotting techniques were used to investigate the mRNA and protein expression levels of DNA base excision repair (BER)-pathway enzymes and transcription factors (TFs) involved in cell survival mechanisms. A significant increase in cell viability and ROS generation was observed when exposed to low and moderate doses of MCP and CP at different time points (24, 48 and 72h) studied. A549 cells displayed a dose-dependent accumulation of apurinic/apyrimidinic (AP) sites after 24h exposure to MCP advocating for the activation of AP endonuclease-mediated DNA BER-pathway. Cellular responses to MCP- and CP-induced oxidative stress resulted in an imbalance in the mRNA and protein expression of BER-pathway enzymes, viz. PARP1, OGG1, APE1, XRCC1, DNA pol β and DNA ligase III α at different time points. The treatment of OPPs resulted in the upregulation of TFs, viz. Nrf2, c-jun, phospho-c-jun and inducible nitric oxide synthase. Immunofluorescent confocal imaging of A549 cells indicated that MCP and CP induces the translocation of APE1 within the cytoplasm at an early 6h time point, whereas it promotes nuclear localization after 24h of treatment, which suggests that APE1 subcellular distribution is dynamically regulated in response to OPP-induced oxidative stress. Furthermore, nuclear colocalization of APE1 and the TF c-jun was observed in response to the treatment of CP and MCP for different time points in A549 cells. Therefore, in this study we demonstrate that MCP- and CP-induced oxidative stress alters APE1-dependent BER-pathway and also mediates cell survival signalling mechanisms via APE1 regulation, thereby promoting lung cancer cell survival and proliferation.

Ginsenosides synergize with mitomycin C in combating human non-small cell lung cancer by repressing Rad51-mediated DNA repair.

The use of ginseng extract as an adjuvant for cancer treatment has been reported in both animal models and clinical applications, but its molecular mechanisms have not been fully elucidated. Mitomycin C (MMC), an anticancer antibiotic used as a first- or second-line regimen in the treatment for non-small cell lung carcinoma (NSCLC), causes serious adverse reactions when used alone. Here, by using both in vitro and in vivo experiments, we provide evidence for an optimal therapy for NSCLC with total ginsenosides extract (TGS), which significantly enhanced the MMC-induced cytotoxicity against NSCLC A549 and PC-9 cells in vitro when used in combination with relatively low concentrations of MMC. A NSCLC xenograft mouse model was used to confirm the in vivo synergistic effects of the combination of TGS with MMC. Further investigation revealed that TGS could significantly reverse MMC-induced S-phase cell cycle arrest and inhibit Rad51-mediated DNA damage repair, which was evidenced by the inhibitory effects of TGS on the levels of phospho-MEK1/2, phospho-ERK1/2 and Rad51 protein and the translocation of Rad51 from the cytoplasm to the nucleus in response to MMC. In summary, our results demonstrate that TGS could effectively enhance the cytotoxicity of MMC against NSCLC cells in vitro and in vivo, thereby revealing a novel adjuvant anticancer mechanism of TGS. Combined treatment with TGS and MMC can significantly lower the required concentration of MMC and can further reduce the risk of side effects, suggesting a better treatment option for NSCLC patients.

Lycorine Induces Apoptosis of A549 Cells via AMPK-Mammalian Target of Rapamycin (mTOR)-S6K Signaling Pathway.

BackgroundThis study was designed to investigate the effect of lycorine (LY) on the AMPK-mTOR-S6K signaling pathway and to clarify its role in autophagy and apoptosis.Material/MethodsVarious concentrations of LY were used to treat non-small cell lung carcinoma A549 cells. The MTT assay was used to measure cell viability and acridine orange staining was used to detect cell morphology changes. Western blot analysis was used to test the effect of LY on the expression levels of LC3, caspase 3, and other proteins involved in the AMPK-mTOR-S6K signaling pathway.ResultsThe half maximal inhibitory concentration (IC50) of LY after 24-h treatment was 8.5 μM, with stronger inhibitory effect of 24-h LY treatment over 12-h LY treatment. Morphological observation showed that lower doses (4 μM and 8 μM) of LY treatment induced A549 cell death mainly caused by autophagy, whereas the higher dose (16 μM) of LY treatment induced A549 cell death, mainly caused by apoptosis. Furthermore, 8 μM LY caused the highest conversion of LC3-II from LC3-I. All LY treatments activated caspase-3. LY treatment also promoted AMPK phosphorylation (Thr172) and inhibited the phosphorylation of mTOR and S6K.ConclusionsLY induced apoptosis of A549 cells by regulating the AMPK-mTOR-S6K signaling pathway. Lower levels (4~8 μM) of LY-induced autophagy contributed to LY-induced apoptosis.

Abstract B38: Radiation enhances intracellular delivery of anti-MGMT oligomers to reduce protein expression in vitro and in a xenograft model

Abstract High levels of DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) in glioblastoma tumor are associated with temozolomide resistance and poor prognosis. MGMT provides a promising therapeutic target to overcome chemo-resistance and improve overall survival in cancer patients. In this study, we investigate the enhanced delivery of anti-MGMT morpholino oligonucleotide (AMON) using a sub-therapeutic dose of radiation to reduce MGMT expression of human cancer cells (T98G glioma and H460 and A549 non-small cell lung carcinoma) in vitro and in vivo. Compared to standard transfection techniques, sub-therapeutic dose of radiation enhanced intracellular AMON delivery and transiently reduced MGMT protein expression at 3 d in vitro. The optimal radiation dosage was cancer cell type dependent and ranged from 1-12 Gy. In addition, AMON delivered using sub-therapeutic dose of radiation increased cytotoxicity of T98G cells in response to temozolomide compared to radiation and temozolomide alone in vitro. In a H460 subcutaneous xenograft tumor model, intravenous AMON administration reduced MGMT protein level by 50% in irradiated tumors but not in contralateral non-irradiated lesions within the same animal. Tumor MGMT protein downregulation by AMON did not alter DNA promoter methylation status. Our results demonstrate for the first time the use of radiation to enhance the intracellular delivery of antisense oligonucleotides to modulate MGMT protein expression in vitro and in vivo. We have previously shown that antisense nucleotides can be delivered to the brain parenchyma using osmotic blood brain barrier disruption. Therefore, we believe that cancer patients with MGMT overexpression, unmethylated DNA promoter and/or resistance to DNA alkylating agents may benefit from this novel antisense therapeutic technology to improve overall survival. Citation Format: Jeffrey Wu, Prakash Ambady, DreeAnna Morris, Michael Pagel, Randy Woltjer, Joshua Walker, Leslie Muldoon, Edward Neuwelt. Radiation enhances intracellular delivery of anti-MGMT oligomers to reduce protein expression in vitro and in a xenograft model [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B38.

PGE2/EP3/SRC signaling induces EGFR nuclear translocation and growth through EGFR ligands release in lung adenocarcinoma cells

Prostaglandin E2 (PGE2) interacts with tyrosine kinases receptor signaling in both tumor and stromal cells supporting tumor progression. Here we demonstrate that in non-small cell lung carcinoma (NSCLC) cells, A549 and GLC82, PGE2 promotes nuclear translocation of epidermal growth factor receptor (nEGFR), affects gene expression and induces cell growth. Indeed, cyclin D1, COX-2, iNOS and c-Myc mRNA levels are upregulated following PGE2 treatment. The nuclear localization sequence (NLS) of EGFR as well as its tyrosine kinase activity are required for the effect of PGE2 on nEGFR and downstream signaling activities. PGE2 binds its bona fide receptor EP3 which by activating SRC family kinases, induces ADAMs activation which, in turn, releases EGFR-ligands from the cell membrane and promotes nEGFR. Amphiregulin (AREG) and Epiregulin (EREG) appear to be involved in nEGFR promoted by the PGE2/EP3-SRC axis. Pharmacological inhibition or silencing of the PGE2/EP3/SRC-ADAMs signaling axis or EGFR ligands i.e. AREG and EREG expression abolishes nEGFR induced by PGE2. In conclusion, PGE2 induces NSCLC cell proliferation by EP3 receptor, SRC-ADAMs activation, EGFR ligands shedding and finally, phosphorylation and nEGFR. Since nuclear EGFR is a hallmark of cancer aggressiveness, our findings reveal a novel mechanism for the contribution of PGE2 to tumor progression.

Polysaccharides isolated from liquid culture broth of Inonotus obliquus inhibit the invasion of human non-small cell lung carcinoma cells

Polysaccharides isolated from Inonotus obliquus (PLIO) have been known to have various pharmacological activities including antioxidant, immunostimulating and anti-tumor activity. However, the anti-metastatic effect of PLIO in human non-small cell lung carcinoma (NSCLC) has not been elucidated. In this study, we investigated the effects of PLIO on the metastatic potential of human NSCLC A549 cells and its underlying mechanisms. PLIO suppressed the invasive potential of A549 cells throughout reducing matrix metalloproteinase (MMP) expression. PLIO treatment inhibited NF-κB nuclear translocation in A549 cells. In addition, PLIO treatment inhibited the phosphorylation of JNK/AKT in A549 cells. These results suggest that PLIO could inhibit human NSCLC invasion via suppression of AKT/NF-κB signaling pathway.

Integrated High Throughput Analysis Identifies GSK3 as a Crucial Determinant of p53-Mediated Apoptosis in Lung Cancer Cells

Background/Aims: p53 dysfunction is frequently observed in lung cancer. Although restoring the tumour suppressor function of p53 is recently approved as a putative strategy for combating cancers, the lack of understanding of the molecular mechanism underlying p53-mediated lung cancer suppression has limited the application of p53-based therapies in lung cancer. Methods and Results: Using RNA sequencing, we determined the transcriptional profile of human non-small cell lung carcinoma A549 cells after treatment with two p53-activating chemical compounds, nutlin and RITA, which could induce A549 cell cycle arrest and apoptosis, respectively. Bioinformatics analysis of genome-wide gene expression data showed that distinct transcription profiles were induced by nutlin and RITA and 66 pathways were differentially regulated by these two compounds. However, only two of these pathways, 'Adherens junction' and 'Axon guidance', were found to be synthetic lethal with p53 re-activation, as determined via integrated analysis of genome-wide gene expression profile and short hairpin RNA (shRNA) screening. Further functional protein association analysis of significantly regulated genes associated with these two synthetic lethal pathways indicated that GSK3 played a key role in p53-mediated A549 cell apoptosis, and then gene function study was performed, which revealed that GSK3 inhibition promoted p53-mediated A549 cell apoptosis in a p53 post-translational activity-dependent manner. Conclusion: Our findings provide us with new insights regarding the mechanism by which p53 mediates A549 apoptosis and may cast light on the development of more efficient p53-based strategies for treating lung cancer.