Overexpressing A549 Cells Research Articles

Sulforaphane inhibits the migration and invasion of BPDE-induced lung adenocarcinoma cells by regulating NLRP12

This study aims to explore the impact and underlying mechanism of sulforaphane (SFN) intervention on the migration and invasion of lung adenocarcinoma induced by 7, 8-dihydroxy-9, 10-epoxy-benzo (a) pyrene (BPDE). Human lung adenocarcinoma A549 cells were exposed to varying concentrations of BPDE (0.25, 0.50, and 1.00 μM) and subsequently treated with 5 μM SFN. Cell viability was determined using CCK8 assay, while migration and invasion were assessed using Transwell assays. Lentivirus transfection was employed to establish NLRP12 overexpressing A549 cells. ELISA was utilized to quantify IL-33, CXCL12, and CXCL13 levels in the supernatant, while quantitative real-time PCR (qRT-PCR) and Western Blot were used to analyze the expression of NLRP12 and key factors associated with canonical and non-canonical NF-κB pathways. Results indicated an increase in migratory and invasive capabilities, concurrent with heightened expression of IL-33, CXCL12, CXCL13, and factors associated with both canonical and non-canonical NF-κB pathways. Moreover, mRNA and protein levels of NLRP12 were decreased in BPDE-stimulated A549 cells. Subsequent SFN intervention attenuated BPDE-induced migration and invasion of A549 cells. Lentivirus-mediated NLRP12 overexpression not only reversed the observed phenotype in BPDE-induced cells but also led to a reduction in the expression of critical factors associated with both canonical and non-canonical NF-κB pathways. Collectively, we found that SFN could inhibit BPDE-induced migration and invasion of A549 cells by upregulating NLRP12, thereby influencing both canonical and non-canonical NF-κB pathways.

Clobetasol propionate, a Nrf-2 inhibitor, sensitizes human lung cancer cells to radiation-induced killing via mitochondrial ROS-dependent ferroptosis.

Combining radiotherapy with Nrf-2 inhibitor holds promise as a potential therapeutic strategy for radioresistant lung cancer. Here, the radiosensitizing efficacy of a synthetic glucocorticoid clobetasol propionate (CP) in A549 human lung cancer cellswas evaluated. CP exhibited potent radiosensitization in lung cancer cells via inhibition of Nrf-2 pathway, leading to elevation of oxidative stress. Transcriptomic studies revealed significant modulation of pathways related to ferroptosis, fatty acid and glutathione metabolism. Consistent with these findings, CP treatment followed by radiation exposure showed characteristic features of ferroptosis in terms of mitochondrial swelling, rupture and loss of cristae. Ferroptosis is a form of regulated cell death triggered by iron-dependent ROS accumulation and lipid peroxidation. In combination with radiation, CP showed enhanced iron release, mitochondrial ROS, and lipid peroxidation, indicating ferroptosis induction. Further, iron chelation, inhibition of lipid peroxidation or scavenging mitochondrial ROS prevented CP-mediated radiosensitization. Nrf-2 negatively regulates ferroptosis through upregulation of antioxidant defense and iron homeostasis. Interestingly, Nrf-2 overexpressing A549 cells were refractory to CP-mediated ferroptosis induction and radiosensitization. Thus, this study identifiedanti-psoriatic drug clobetasol propionate can be repurposed as a promising radiosensitizer for Keap-1 mutant lung cancers.

Multi-omics sequencing revealed endostar combined with cisplatin treated non small cell lung cancer via anti-angiogenesis

BackgroundEndostar, an anti-angiogenic drug, has been approved for treating non-small cell lung cancer (NSCLC). At present, endostar combined with radiotherapy or chemotherapy has achieved ideal results in the treatment of some tumors, but there is a lack of application and study in NSCLC. This study investigated the therapeutic effect and potential mechanism of endostar combined with cisplatin (EC) in NSCLC.MethodsHE staining, TUNEL staining, immunofluorescence, colony formation ability, and cell migration ability were used to evaluate the anti-tumor activity of EC. The expressions of FMOD, VEGF, FGF-2, and PDGF-B were detected by western blotting and qPCR. The target of combination therapy was analyzed by m6A sequencing and RNA sequencing. METTL3 knockdown and overexpressed A549 cells were constructed and co-cultured with HUVECs to further evaluate the effect of METLL3 on combination therapy.ResultsCombination therapy significantly reduced the colony formation and migration ability of NSCLC cells, induced cell apoptosis, and inhibited the tube formation ability of HUVECs. The results of m6A sequencing and RNA sequencing showed that the EC could down-regulate the expression level of FMOD in tumor tissues, which might be related to the reduction of its m6A methylation modification regulatory enzyme METTL3. Restricting FMOD expression could reduce the expression of FGF2, TGF-β1, VEGF and PDGF-B. Moreover, overexpression of METTLE almost abolished the anti-tumor effect of EC and promoted angiogenesis.ConclusionsEndostar combined with cisplatin might exert anti-tumor effects by down-regulating the expression of METTL3 and FMOD.

C-C Motif Ligand 21/C-C Motif Chemokine Receptor 7 Modulated Tumor Properties for Non-Small Cell Lung Cancer Brain Metastasis Patients

The non-small cell lung cancer is a common malignancy with high metastasis capacity. The brain is the primary site for non-small cell lung cancer patients with unclear mechanism. Here, in this study, we hypothesized an innovative signaling cascade for the disorder and systematically investigated the underlying mechanism. Their differentially gene expression analysis was based on 13 conventional nonsmall cell lung cancer and 15 non-small cell lung cancer brain metastasis patients respectively. The nonsmall cell lung cancer cell line, adenocarcinomic human alveolar basal epithelial cells-A549 and human bronchial epithelial cell line-BEAS-2B were established for in vitro study. The syngeneic mouse tumor models injected with 5×105 cells of control, C-C motif ligand 21 overexpressing, C-C motif chemokine receptor 7 overexpressing A549 cells were generated for in vivo analysis. Compared with conventional non-small cell lung cancer patients, the non-small cell lung cancer brain metastasis patients showed 352 differentially expressed genes, as 212 down-regulated ones and 140 up-regulated ones. Moreover, the C-C motif ligand 21 and C-C motif chemokine receptor 7 were prominently inhibited in non-small cell lung cancer brain metastasis patients. In A549 cells, C-C motif ligand 21 overexpressing could inhibit epithelial-mesenchymal transition progression. C-C motif ligand 21 modulated epithelial-mesenchymal transition and migration plus invasion ability but not proliferation capacity of non-small cell lung cancer cells through C-C motif chemokine receptor 7 dependent signaling pathway, as Janus kinase 2/signal transducer and activator of transcription 3 was a potential downstream signaling pathway. Moreover, C-C motif ligand 21/C-C motif chemokine receptor 7 signaling axis could modulate tumor growth and survival time in vivo. Collectively, these data initiated a C-C motif ligand 21/C-C motif chemokine receptor 7-Janus kinase 2/signal transducer and activator of transcription 3 signaling for non-small cell lung cancer patients with brain metastasis, which shed various beneficial insights for future study.

Interleukin-17D promotes lung cancer progression by inducing tumor-associated macrophage infiltration via the p38 MAPK signaling pathway.

Cancer immunoediting is defined as the integration of the immune system’s dual host-protective and tumor-promoting roles, including three phases: elimination, equilibrium, and escape. Immune selective pressure causes tumor cells to lose major histocompatibility complex expression or acquire immunosuppressive gene expression, which promotes tumor immune evasion and tumor progression. Interleukin-17D (IL-17D), a member of the IL-17 family of cytokines, plays an important role in the host defense against infection and inflammation. However, the role of IL-17D in the progression of lung cancer remains unclear. In this study, we found that IL-17D was highly expressed in human lung cancer, and increased IL-17D expression was associated with tumor stage and short overall survival. IL-17D overexpression significantly promoted tumor growth in subcutaneous xenograft mouse models but only slightly affected cell proliferation in vitro. Using flow cytometry, we found that IL-17D overexpression enhances the recruitment of tumor-associated macrophages to the tumor microenvironment. Based on the expression profile of IL17D–overexpressing A549 cells, we found that IL-17D increased the expression levels of macrophage polarization– and recruitment–related genes through the MAPK signaling pathway. Moreover, inhibition of the p38 pathway blocked macrophage infiltration induced by IL-17D. These results suggest that IL-17D regulates the tumor immune microenvironment via the p38 MAPK signaling pathway, highlighting IL-17D as a potential therapeutic target for lung cancer.

Abstract 1090: Repurposing an overactive bladder drug to treat chemoresistant lung cancer

Abstract Background: Non-small cell lung cancer (NSCLC) is one of the most common types of cancer diagnosis among men and women leads the most cancer-related deaths in the United States. This is largely due to treatment failure which arises from therapy-resistant tumors. The current study investigates the role of muscarinic acetylcholine receptor 3 (CHRM3) in the development and treatment of NSCLC chemotherapy-resistance and the potential of the overactive bladder CHRM3 antagonist darifenacin to treat this lethal form of cancer. Methods: Human NSCLC cell cultures were used to harvest protein for Western blot analyses of protein expression and activity of intracellular pathways. The chemotherapy drug docetaxel’s (DTX) ability to induce apoptosis was measured with crystal violet survival assay and colony formation assay. Results: CHRM3 expression was measured in chemonaive NSCLC cells (A549) and chemoresistant cells (A549R), which showed increased expression in A549R cells. Chemoresistant A549R were pretreated with the CHRM3 antagonist darifenacin (Dari, 10 μM, 24 hrs) before treatment with DTX (48 hrs). Crystal violet survival assay and colony formation assays showed a significant reduction in cell survival in the Dari + DTX combination treatment group suggesting that CHRM3 signaling is necessary in a chemoresistant state. Chemonaive A549 cells and CHRM3 overexpressing A549 cells (A549 M3OE) were pretreated with carbachol (10 µM, 24 hrs) to activate CHRM3 before treatment with a set of serial dilutions of DTX (48 hrs). Crystal violet survival assay results showed that carbachol was not able to protect either cell line from DTX-induced cell death, suggesting that CHRM3 signaling alone is not sufficient to induce a chemoresistant state. Conclusion: CHRM3 overexpression is observed in chemoresistant forms of NSCLC. While activation of CHRM3 does not instill a resistant state in chemonaive cells, its signaling is essential for maintenance of the chemoresistant state. The repurposing of the CHRM3 antagonist darifenacin shows strong potential as a therapeutic to treat lethal chemoresistant forms of NSCLC.The project described was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant #P20GM103408 Citation Format: Joslyn Bassett, Tyler Bland. Repurposing an overactive bladder drug to treat chemoresistant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1090.

Self-Assembly Cascade Reaction Platform for CD44 Positive Lung Cancer Therapy.

Lung cancer, as one of the most fatal cancers around the world, is responsible for the death of millions every year. Among various types of lung cancers, the ones overexpressing CD44 is usually associated higher cell proliferation with poorer prognosis. Therefore, finding a way to effectively treat CD44 positive lung cancer is urgently needed. Here in this study, negatively charged ultrasmall prussian blue nanoparticles (UPBNPs) was firstly synthesized and adsorbed to polyethyleneimine (PEI) together with glucose oxidase (Gox). Afterwards, the PEI was further complexed with hyaluronic acid (HA) to give a cascade reaction platform (HP/UPB-Gox) for CD44 positive lung cancer therapy. The HP/UPB-Gox with HA shell was able to positively target CD44 overexpressed A549 cells. Upon arriving at the tumor tissue, the Gox catalyzed the glucose of tumor to create H₂O₂, which further served as the substrate of UPBNPs, a peroxidase mimic, to finally give highly toxic hydroxyl radical (OH) for cancer therapy. Therefore, the cascade reaction formed between UPBNPs and Gox was expected to realize effective treatment on CD44 overexpressed lung cancer.

CacyBP promotes the proliferation and invasion of non-small cell lung cancer

Objective: To investigate the effects and the mechanism of Calcyclin-binding protein (CacyBP) on the proliferation and invasion of non-small cell lung cancer (NSCLC) cells. Methods: Six lung cancer tissues and paired normal lung tissues were collected from NSCLC patients who underwent surgical treatment in Jinan Central Hospital during 2016. The expression of CacyBP in these tissues was examined by western blot. The protein and mRNA expression of CacyBP in human bronchial epithelial cells (16HBE), NSCLC cell lines including A549, H1299, H460 and H1975 were examined by western blot and reverse transcription-polymerase chain reaction (RT-PCR), respectively. RNAi and shRNA against negative control (NC) or CacyBP were transfected into A549 cell which were denoted as siNC group, siCacyBP-1 group, sicacyBP-2 group, shNC group and shCacyBP group, respectively. Control and Flag-CacyBP plasmids were transfected into A549 cells which were denoted as NC group and Flag-CacyBP group, respectively. Cell counting kit-8 (CCK-8), plate clone formation assay and flow cytometry assay were used to assess cell proliferation ability and cycle of A549. Wound healing assay and transwell assay were used to assess abilities of A549 cells migration and invasion. The protein expressions of epithelial-mesenchymal transition (EMT) markers including E-cadherin, N-cadherin, Snail1, Vimentin, and phosphorylation of protein kinase B (p-Akt) were examined in CacyBP depleted or overexpressed A549 cells. Results: The CacyBP protein level in NSCLC tissues was 0.41±0.23, significantly higher than 0.11±0.04 in normal lung tissues (P<0.05). The CacyBP protein expression levels in different NSCLC cell lines including A549, H1299, H460 and H1975 were 0.35±0.01, 0.38±0.01, 0.32±0.01 and 0.41±0.01, respectively, which were significantly higher than 0.03±0.01 in 16HBE cells (P<0.05). The result of RT-PCR was consistent with that of western blot. Compared with siNC group (absorbance was 1.54±0.03), siCacyBP-1 group and siCacyBP-2 group showed decreased cell proliferation (absorbances were 1.38±0.04 and 1.34±0.03, P<0.05). The number of cell colony in shNC group was 41.33±3.21, significantly higher than 22.00±3.61 in shCacyBP group (P<0.05). The proportion of G(1) phase in shCacyBP group was (61.35±5.45)%, higher than (49.61±1.54) % in shNC group (P<0.05). The proportion of S phase was (25.41±3.21)%, which was lower than (38.68±0.46)% of shNC group (P<0.05). The cell migration rate of shCacyBP group was (12.67±0.71)%, which was significantly lower than (35.50±2.07)% of shNC group (P<0.05). The numbers of cell migration and invasion in shNC group were 406.33±7.37 and 92.33±8.50, respectively, which were significantly higher than 224.67±10.01 and 66.00±7.94 in shCacyBP group (P<0.05). Compared with siNC group, the expression of epithelial marker E-cadherin was up-regulated, while the expressions of mesenchymal markers including N-cadherin, Vimentin, Snail1 and p-Akt were down-regulated in CacyBP depleted A549 cells. Compared with NC group, overexpression of CacyBP inhibited E-cadherin expression while promoted the expressions of N-cadherin, Snail1, Vimentin and p-Akt, which could be restored by LY294002. Conclusion: CacyBP may promote the proliferation and invasion of NSCLC cells by regulating Akt signal pathway.

Corrigendum] Ski prevents TGF‑β‑induced EMT and cell invasion by repressing SMAD‑dependent signaling in non‑small cell lung cancer.

Following the publication of the above paper, an interested reader drew to our attention apparent anomalies associated with Figs.2, 3 and 4; essentially, these three figures contained panels exhibiting overlapping data, such that data purportedly relating to different experiments were apparently drawn from the same original sources. [Specifically, the Ski, +TGF‑β1 data panel in Fig.2B, the Mock, +TGF‑β1 data panel in Fig.3A, and the +TGF‑β1, +SIS3 data panel in Fig.4B in the original figures were chosen incorrectly.] Upon investigating this matter with the authors, the authors have realized that they made errors in the compilation of the affected figures. The errors were made inadvertently, and the authors have been able to identify the correct data for each of the figures concerned. The corrected versions of these figures are shown opposite and on the next page. Note that these errors did not affect the overall conclusions reported in the study. The authors are grateful to the Editor of Oncology Reports for allowing them the opportunity to publish this Corrigendum; furthermore, the authors apologize for any inconvenience caused to the readership of the Journal. [the original article was published in Oncology Reports 34: 87-94, 2015; DOI: 10.3892/or.2015.3961].

Overexpression of Calcium Activated Chloride Channel A4 Suppressed the Proliferation, Invasion and Gemcitabine Resistance of Non-Small Cell Lung Cancer Cells

Lung cancer is one kind of the malignant tumor with high mortality. And non-small cell lung cancer is the main subtype of lung cancer. And the proteins of CLCA family (CLCA1, CLCA2 and CLCA4) played an inhibitory role in the occurrence and development of multiple types of tumors. However, the effect of CLCA4 on non-small cell lung cancer cells remains unclear. In our study, we used the lentivirus to establish the overexpressed CLCA4 A549 cells. Next, the CCK-8 and clone formation assays were performed to detect the changes of proliferation of A549 cells. The wound healing and transwell assays were performed to determine the changing of the migration and invasion of A549 cells. Then gemcitabine was used to treat these cells and the CCK-8, wound healing and transwell assays were carried out to detect the effect of the combination of gemcitabine and the overexpression of CLCA4 on the proliferation, migration and invasion of A549 cells. After the overexpression of CLCA4, the clone formation and mobility of A549 cells was enhanced. Furthermore, the overexpression of CLCA4 induced the apoptosis of A549 cells and promoted the expression of apoptosis related proteins. The combination of gemcitabine and the overexpression of CLCA4 further suppressed the proliferation, migration and invasion of A549 cells. CLCA4 inhibited the proliferation, migration and invasion of non-small cell lung cancer cells. CLCA4 also strengthened the sensitivity of non-small cell lung cancer cells for gemcitabine.

Synthesis, structure-activity relationship studies and biological characterization of new [1,2,4]triazolo[1,5-a]pyrimidine-based LSD1/KDM1A inhibitors

The histone lysine specific demethylase 1 (LSD1/KDM1A) is implicated in the development of cancers, targeting LSD1 has been recognized as a promising strategy for cancer therapy. To date, some small-molecule inhibitors are currently being investigated in clinical trials. Herein we report the design, synthesis and biochemical characterization of [1,2,4]triazolo[1,5-a]pyrimidine derivatives as new LSD1 inhibitors. Of these compounds, compound C26 inhibited LSD1 in a reversible manner (IC50 = 1.72 μM) and showed selectivity to LSD1 over MAO-A/B. Besides, compound C26 displayed FAD-competitive binding to LSD1. Interestingly, C26 did not inhibit horseradish peroxidase (HRP) and quench H2O2, thus excluding the possibility that LSD1 inhibition by C26 was due to the HRP inhibition and consumption of H2O2. In LSD1 overexpressed A549 cells, compound C26 concentration-dependently induced accumulation of H3K4me1/me2 and H3K9me2 and showed cellular target engagement to LSD1. Additionally, compound C26 significantly inhibited migration of A549 cells in a concentration-dependent manner, further western blot analysis showed that C26 increased expression levels of epithelial cell markers E-Cadherin and Claudin-1, down-regulated mesenchymal cell marker N-Cadherin and the upstream transcription factors Snail and Slug. Docking studies were also performed to rationalize the potency of C26 toward LSD1. To conclude, the [1,2,4]triazolo[1,5-a]pyrimidine could serve as a promising scaffold for the development of new LSD1 inhibitors.

Experience-based discovery (EBD) of aryl hydrazines as new scaffolds for the development of LSD1/KDM1A inhibitors

Phenelzine was first employed to design new aryl hydrazine-based LSD1 inhibitors based on the experience-based discovery (EBD) strategy. Among these compounds, D8 potently inhibited LSD1 (IC50 = 882.30 nM) in a reversible manner. Compound D8 was selective to LSD1 over MAO-A/B and showed H3K4me2 competitive binding to LSD1. The interaction between H3K4me2 and LSD1 was also confirmed by the Co-IP assay. In LSD1 overexpressed A549 cells, compound D8 dose-dependently induced accumulation of LSD1 substrates H3K4me1/2 and H3K9me1/2, showed cellular target engagement to LSD1 and significantly inhibited cell migration of A549 cells. Docking studies suggested that compound D8 occupied the peptide binding region and therefore blocked the access of the peptide substrate to the FAD, finally leading to the demethylase activity inhibition of LSD1. The findings indicate that aryl hydrazines are new scaffolds for the design of LSD1 inhibitors, the identification of D8 provides further evidence for our previously proposed general principle that fused heterocycles with an amine group are potentially active toward LSD1 by competitive binding to LSD1 with H3 peptide substrates.

Dual-targeting liposomes for enhanced anticancer effect in somatostatin receptor II-positive tumor model.

We developed octreotide-modified magnetic liposomes (OMlips) as dual-targeting drug carriers to enhance the drug accumulation in tumor site. Octreotide acts as a modified ligand for receptor-mediated targeting and the coated Fe3O4 nanoparticles offer the magnetic targeting property. SSTR2 overexpressed A549 cells and S180 cells were chosen to explore the targeting ability and antitumor effect of the oleanolic acid (OA)-loaded OMlips in vitro and in vivo. The OMlips platform significantly improves the targeting, penetrating and accumulation of OA at the SSTR2 overexpressed cells and SSTR2-positive tumor-bearing mice. The OA-loaded OMlips have better antitumor effect and lower systemic toxicity. Such a receptor-mediated and magnetically-orienting dual-targeting drug nanocarriers may have great potentials in clinical practice.

Bexarotene inhibits the viability of non-small cell lung cancer cells via slc10a2/PPAR\u03b3/PTEN/mTOR signaling pathway

BackgroundThirty to 40 % of non-small cell lung cancer (NSCLC) patients developed higher hypertriglyceridemia in the process of treatment with bexarotene. And bioinformatics studies discovered that the expression of slc10a2 was increased in high-grade hypertriglyceridemia patients. So, we will explore the mechanism which may involve in this process.MethodsWe constructed slc10a2 overexpressed A549 cells and H1299 cells as cell models, normal A549 cells and H1299 cells as control. Then we explored the effects of slc10a2 on A549 cells and H1299 cells behaviors, including proliferation, invasion and apoptosis. The expression of apoptotic related genes and anti-cancer genes also been detected.ResultsWe found that the proliferation and migration were inhibited and the apoptosis of NSCLC cells was accelerated by bexarotene. In addition, overexpressed slc10a2 in NSCLC cells can further suppress the proliferation and migration, and promote apoptosis under the treatment of bexarotene. On the contrary, the opposite results were obtained after slc10a2 gene was silenced in NSCLC cells treated with bexarotene. Moreover, the expression of caspase 3, caspase 7, PTEN, P21, P53, LKB1, TSC2 were increased and the expression of Bcl-2, cyclin D1, c-FLIP were declined in NSCLC cells and slc10a2 overexpressed NSCLC cells with the treatment of bexarotene, and the opposite situations were seen after slc10a2 gene was silenced in NSCLC cells. The further studies revealed the increased expression of slc10a2 activated the expression of peroxisome proliferator-activated receptor γ (PPARγ), then up-regulated PTEN expression and down-regulated mTOR expression.ConclusionThese results suggest that bexarotene inhibits the viability of lung cancer cells via slc10a2/PPARγ/PTEN/mTOR signaling pathway.

Profiling and characterization of microRNAs responding to sodium butyrate treatment in A549 cells.

Butyrate inhibits growth of lung cancer. However, the molecular mechanism is still unclear. Here we profiled miRNAs that responded to sodium butyrate(NaB) stimulation in A549 cells, a non-small cell lung cancer cell line, using microarray. We found 33 up-regulated microRNAs and 22 down-regulated microRNAs (log2 ≥1.5 folds, P-value <0.05). The expression of miR-3935, miR-574-3p, and miR-494-3p was confirmed by realtime qPCR. Then,we explored their potential targets of miR-3935 and miR-494-3p using long noncoding RNA(LncRNA) microarray. Using cell expressing negative microRNA as control, we found 103 up-regulated transcripts (including 69 mRNA and 34 LncRNA), and 36 down-regulated transcripts (including 34 mRNAs and 2 LncRNA), in miR-3935 over-expressing A549 cells; 128 up-regulated transcripts (121 mRNAs, 7 LncRNAs) and 180 down-regulated transcripts (169 mRNAs, 11 LncRNAs) in mir-494-3p, respectively (log2 Fold change ≥ 1 & P < 0.05). The expression of RNF115, NTRK3, SLC39A6, and USB1 was confirmed with qPCR. Immunoblotting was adopted to detect RNF115 expression in miR-3935 overexpressed A549 cells. Then, using a luciferase reporter assay system, we found that miR-3935 overexpression significantly decreased 3UTR of RNF115 mediated luciferase expression .In addition, we also observed that the proliferation and migration of A549 cells was obviously prevented by miR-3935 overexpression. Finally, we showed miR-3935 and miR-494-3p induced interferon stimulated gene 15(ISG15) expression through activating its promoter transcription. Together, we profiled microRNAs that responded to NaB treatment and characterized their biological functions in A549 cells. Those results provided new clue for the future treatment of non small cell lung cancer.

Abstract P318: Signaling Pathways Triggered by the Mas Receptor

Mas receptor (MasR) is a class A Orphan G-protein-coupled receptor (GPCR). Although angiotensin-(1-7) [Ang-(1-7)] has been reported as its putative ligand, the intracellular signaling pathways activated by the MasR remain only partially characterized. In this study we examined MasR-dependent activation of G protein-mediated and ERK mediated signaling pathways. With that aim in human lung carcinoma A549 cells we evaluated Gq-coupled activity by monitoring changes in intracellular Ca 2+ levels after Ang(1-7) stimulation. Incubation with 100nM Ang-(1-7) failed to increase Ca 2+ levels either in endogenously expressing or MasR overexpressing A549 cells. On the other hand, transfection of HEK293T cells with a wild-type MasR (wt-MasR) construct resulted in a significant decrease in basal cAMP levels (p&lt;0.05) that depended on the amount of wt-MasR protein expressed but was not observed when increasing amounts of mutant MasR lacking the PDZ binding motif were expressed. Pretreatment of wt-MasR expressing cells with pertussis toxin restored basal cAMP levels (p&lt;0.05) whereas no effect was observed in mock-transfected cells. Also, cAMP production after forskolin stimulation was lower in cells expressing wt-MasR than in control cells. These results indicate a high level of constitutive receptor activity towards cAMP modulation involving Gαi-protein. Treatment with Ang-(1-7) increased p-ERK levels in a concentration-dependent manner in both wt-MasR and in mock-transfected HEK293T cells. In view of these results we are analyzing whether ERK activation is mediated through Ang-(1-7) binding to endogenously expressed receptors different from MasR. As MasR has been suggested to participate in cardiovascular and renal functions, comprehensive pharmacological characterization of MasR signaling is essential for developing clinical therapeutics targeting MasR function.

Hyaluronic acid and carbon dots-gated hollow mesoporous silica for redox and enzyme-triggered targeted drug delivery and bioimaging

In this work, a redox and enzyme dual-stimuli responsive drug delivery system (DDS) with tracking function (HMSN-SS-CDPEI@HA) based on carbon dots capped hollow mesoporous silica nanoparticles (HMSN) has been developed for targeted drug delivery. The positively charged CDPEI nanoparticles prepared by polyethylenimine (PEI) were grafted on the pore openings of HMSN through disulfide bonds and were used as “gatekeepers” to trap the drugs within the hollow cavity. The hyaluronic acid (HA), a natural polysaccharide, was further grafted on the surface of HMSN to realize targeted drug delivery, controlled drug release and improved the stability. Doxorubicin (DOX) was chosen as a model drug due to its wide clinical application. In vitro drug release profiles demonstrated that DOX-loaded HMSN-SS-CDPEI@HA exhibited redox and enzyme dual-responsive drug release property. In addition, the prepared HMSN-SS-CDPEI@HA exhibited excellent fluorescent properties and biocompatibility. Confocal laser scanning microscope (CLSM) and flow cytometry (FCM) illustrated that HMSN-SS-CDPEI@HA exhibited a higher cellular uptake via the CD44 receptor-mediated endocytosis by CD44-receptor over-expressed A549 cells than NIH-3T3 (receptor-negative) cells, leading to higher cytotoxicity against A549 cells than NIH 3T3 cells. This work suggested an exploration of dual-stimuli responsive as well as real-time imaging targeted drug delivery system based on HMSN and the prepared HMSN-SS-CDPEI@HA could be a promising platform for cancer therapy.

489. Tumor-Targeted Hursirna-Nanoparticle Delivery Inhibits Lung Tumor Growth In Vitro and In Vivo

HuR, an mRNA binding protein regulates the stability of many oncoproteins associated with cell survival, proliferation, migration and angiogenesis. HuR overexpression is a marker for poor prognosis in patients diagnosed with cancer of lung, ovary, breast and colon. We hypothesized that the silencing of HuR using small interfering RNA (siRNA) could be a promising approach for lung cancer therapy. To test our hypothesis, we developed a tumor-targeted nanoparticle (NP) system that is targeted to transferrin receptor (TfR) for delivering HuRsiRNA (HuR-TfNP) in human lung cancer cells. Human lung cancer cells (A549, HCC827) and normal lung fibroblast (MRC-9) cell lines expressing varying levels of TfR were used in the present study. TfR expression was highest in A549, moderate in HCC827, and low to undetectable in MRC9 cells. In vitro studies demonstrated enhanced uptake of Tf-NP (51%) in TfR overexpressing A549 cells, compared to the non-targeted NP. Specificity studies using desferrioxamine (DFO; 100 µM), a stimulator of TfR, showed a two-fold increased uptake of Tf-NP whereas blocking TfR with exogenous transferrin (1 µg/well) reduced the uptake by 3 fold in A549 cells. Further, HuR-TfNP treatment reduced HuR expression and significantly suppressed cell proliferation at 24h and 48h compared to control siRNA containing NP (C-Tf-NP) in tumor cells but not in normal cells. Greatest inhibition was observed in A549 cells (23% and 30% inhibition at 24 and 48 h respectively) compared to 15% and 25% in HCC827. In MRC-9 cells, only 4% inhibition was observed. HuR-TfNP induced G1 cell-cycle arrest in tumor cells that correlated with marked reduction in Cyclin D1, and Cyclin E protein expression. Further, tumor cell migration and invasion was significantly inhibited in HuR-TfNP treated tumor cells compared to C-TfNP treatment (p<0.001). In-vivo, Tf-NP bio-distribution studies using indocyanine green (ICG) showed accumulation of the NP in tumor tissues over time with maximum accumulation at 24 h post NP injection. Efficacy studies in A549 tumor model demonstrated that systemic administration of HuR-TfNP significantly inhibited growth of both subcutaneous tumor growth and experimental lung metastasis compared to C-TfNP treatment (P<0.05). Further, tumor growth delay was sustained over 70 days when compared to control groups. A marked reduction in the expression of HuR and HuR-regulated oncoproteins (Bcl2, Cyclin D1 and Cyclin E) with a concomitant increase in p27 expression was observed in HuR-TfNP-treated tumors compared to control tumors. Our study results demonstrate HuR-TfNP therapy suppressed lung tumor growth both in vitro and in vivo and is therapeutic target for lung cancer treatment.

Ski prevents TGF-β-induced EMT and cell invasion by repressing SMAD-dependent signaling in non-small cell lung cancer.

Epithelial-mesenchymal transition (EMT) is a key event in cancer metastasis, which confers cancer cells with increased motility and invasiveness, and EMT is characterized by loss of epithelial marker E-cadherin and gain of mesenchymal marker N-cadherin. Transforming growth factor-β (TGF-β) signaling is a crucial inducer of EMT in various types of cancer. Ski is an important negative regulator of TGF-β signaling, which interacts with SMADs to repress TGF-β signaling activity. Although there is accumulating evidence that Ski functions as a promoter or suppressor in human types of cancer, the molecular mechanisms by which Ski affects TGF-β-induced EMT and invasion in non-small cell lung cancer (NSCLC) are not largely elucidated. In the present study, we investigated the mechanistic role of Ski in NSCLC metastasis. Ski was significantly reduced in metastatic NSCLC cells or tissues when compared with non-metastatic NSCLC cells or tissues. Moreover, following TGF-β stimulation Ski-silenced A549 cells had more significant features of EMT and a higher invasive activity when compared with A549 cells overexpressing Ski. Mechanistically, Ski-silenced and overexpressed A549 cells showed an increase and a reduction in the SMAD3 phosphorylation level, respectively. This was supported by plasminogen activator inhibitor-1 (PAI-1) promoter activity obtained in Ski-silenced and overexpressed A549 cells. However, after treatment of SIS3 (inhibitor of SMAD3 phosphorylation) followed by TGF-β1 stimulation, we did not observe any effect of Ski on TGF-β-induced EMT, and invasion in Ski-silenced and overexpressed A549 cells. In conclusion, our findings suggest that Ski represses TGF-β-induced EMT and invasion by inhibiting SMAD-dependent signaling in NSCLC.

Hsp27 Inhibition with OGX-427 Sensitizes Non-Small Cell Lung Cancer Cells to Erlotinib and Chemotherapy.

Non-small cell lung cancer (NSCLC) is the most frequent cause of death from cancer worldwide. Despite the availability of active chemotherapy regimens and EGFR tyrosine kinase inhibitors, all advanced patients develop recurrent disease after first-line therapy. Although Hsp27 is a stress-induced chaperone that promotes acquired resistance in several cancers, its relationship to treatment resistance in NSCLC has not been defined. Understanding adaptive responses of acquired resistance will help guide new strategies to control NSCLC. Hsp27 levels were evaluated in an HCC827 erlotinib-resistant-derived cell line (HCC-827Resistant), and sensitivity to erlotinib was examined in Hsp27-overexpressing A549 cells. The role of Hsp27 in both erlotinib and cytotoxic treatment resistance was evaluated in HCC-827 and A549 NSCLC cells using the Hsp27 antisense drug OGX-427. The effect of OGX-427 in combination with erlotinib was also assessed in mice bearing A549 xenografts. Hsp27 is induced by erlotinib and protects NSCLC cells from treatment-induced apoptosis, whereas OGX-427 sensitizes NSCLC cells to erlotinib. Interestingly, increased resistance to erlotinib was observed when Hsp27 was increased either in HCC827 erlotinib-resistant or overexpressing A549 cells. Combining OGX-427 with erlotinib significantly enhanced antitumor effects in vitro and delayed A549 xenograft growth in vivo. OGX-427 also significantly enhanced the activity of cytotoxic drugs used for NSCLC. These data indicate that treatment-induced Hsp27 contributes to the development of resistance, and provides preclinical proof-of-principle that inhibition of stress adaptive pathways mediated by Hsp27 enhances the activity of erlotinib and chemotherapeutics.