NCI-H460 Cells Research Articles

CircTMEM30A/hsa-miR-130a-3p regulates TNFα and promotes the malignant progression of COPD with primary lung cancer.

Chronic obstructive pulmonary disease (COPD) and lung cancer is the most common respiratory diseases among smokers. Lung cancer is one of the most common malignancy. COPD patients are at high risk of lung cancer, but the specific pathogenesis is still unclear. In recent years, circRNAs have become the focus of lung cancer research. This study investigated the effect of CircTMEM30A on COPD and lung cancer and its possible molecular mechanisms. QPCR was used to detect the expression of CircTMEM30A in COPD patients, lung cancer patients, and COPD patients with lung cancer. CircTMEM30A was transfected with PPF, HFL1, A549 and NCI-H446 cells, and the transfection efficiency was verified by real-time quantitative PCR. The effects of CircTMEM30A on the proliferation, migration and invasion of fibroblasts and lung cancer cells were detected by MTT, Transwell and scratch tests. The target gene of CircTMEM30A was verified by luciferase reporter assay. Real-time quantitative PCR was used to detect the expression level of CircTMEM30A target gene. CircTMEM30A was the most highly expressed in COPD patients with lung cancer. Overexpression of CircTMEM30A enhanced the effects of proliferation, migration and invasion of fibroblasts and lung cancer cells, down-regulated the expression of E-cadherin, up-regulated the expression of Vimentin, and promoted EMT. After knocking down CircTMEM30A, the migration and invasion ability of lung cancer cells was significantly reduced. Bioinformatics retrieval and luciferase reporter gene confirmed that CircTMEM30A could be bind to miR-130a, while TNFα gene was the target gene of miR-130a. Further studies showed that overexpression of miR-130a and knockdown of TNFα reversed the promoting effect of CircTMEM30A on lung cancer cells. CircTMEM30A was highly expressed in COPD patients with lung cancer, and miR-130a was low expressed in lung cancer cells. CircTMEM30A regulated the expression of TNFα through miR-130a, thereby affecting the progression of COPD and lung cancer. CircTMEM30A may be a therapeutic target for COPD patients with lung cancer.

MicroRNA-130b functions as an oncogene and is a predictive marker of poor prognosis in lung adenocarcinoma

AbstractLung cancer is an aggressive disease and the leading cause of cancer-related deaths worldwide. In the past several decades, the incidence of adenocarcinoma has significantly increased, and accounts for ~40% of all lung cancer cases. In the present study, we investigated the clinicopathologic significance of microRNA-130b (miR-130b) in lung adenocarcinoma and analyzed its cancer-specific functions. RNA was extracted from formalin-fixed paraffin-embedded specimens of 146 lung adenocarcinoma cases, and miR-130b expression was analyzed using quantitative real-time polymerase chain reaction. NCI-H1650 cells were transfected with miR-130b mimic and inhibitor to determine its effects on tumor cell proliferation, migration, and invasion. The expression of miR-130b in lung adenocarcinoma tissues was classified into two groups according to the median value. High expression of miR-130b was associated with higher histological grade, advanced pathologic T stage, lymph node metastasis, and lymphovascular invasion. Moreover, survival analysis showed that high miR-130b expression was significantly associated with unfavorable prognosis. In addition, miR-130b upregulation promoted cell migration and invasion, while its downregulation resulted in decreased cell proliferation, migration, and wound healing in in vitro experiments. In conclusion, these findings suggest that miR-130b promotes tumor progression and serves as a biomarker of poor prognosis for patients with lung adenocarcinoma. Hence, targeting miR-130b may serve as a potential therapeutic strategy for lung cancer.The authors investigated the clinicopathologic significance of microRNA-130b expression and analyzed its cancer-specific functions using a cancer cell line. High microRNA-130b expression was associated with adverse clinicopathologic parameters and poor patient outcomes. In vitro, downregulation of microRNA-130b caused a decrease in cell proliferation, migration, and invasion. Hence, miR-130b is a potential therapeutic target for lung cancer.

LncRNA WT1-AS/miR-494-3p Regulates Cell Proliferation, Apoptosis, Migration and Invasion via PTEN/PI3K/AKT Signaling Pathway in Non-Small Cell Lung Cancer.

BackgroundNon-small cell lung cancer (NSCLC) is one of the most common malignancies with the highest morbidity and mortality worldwide. Long non-coding RNAs (lncRNAs) are recently recognized as noteworthy regulators of different tumors, counting NSCLC. However, the biological functions and regulatory mechanism of lncRNA WT1-AS in NSCLC progression still stay uninvestigated.MethodsWT1-AS and miR-494-3p levels in NSCLC cell lines were detected by real-time quantitative polymerase chain reaction (RT-qPCR). In the current study, the regulatory effects of WT1-AS/miR-494-3p axis on cellular behaviors of NSCLC cell lines (A549 and NCI-H1975) were evaluated by a variety of methods. Cell counting kit-8 (CCK-8) and EDU assays were adopted to assess NSCLC cell proliferation. Tunnel staining and flow cytometry assay were applied to determine cell apoptosis and cell cycle distribution. Besides, cell migration and invasion abilities were analyzed by performing wound healing and transwell assays. Meanwhile, the levels of key proteins related to NSCLC cell apoptosis and PTEN/PI3K/AKT pathway were examined using Western blot assay. In addition, luciferase reporter assays were used to determine the interaction between WT1-AS and miR-494-3p or miR-494-3p and PTEN.ResultsVisibly downregulated WT1-AS in NSCLC cell lines was obtained from Broad Institute Cancer Cell Line Encyclopedia (CCLE) database and further verified by performing RT-qPCR. Besides, miR-494-3p was the downstream target gene of WT1-AS and obviously upregulated miR-494-3p in NSCLC cell lines was confirmed. WT1-AS overexpression suppressed cell proliferation, migration and invasion abilities while enhanced cell apoptosis of A549 and NCI-H1975 cells. Furthermore, upregulation of miR-494-3p distinctly reversed these inhibitory effects of WT1-AS overexpression on the tumorigenesis and progression of NSCLC. In addition, WT1-AS promoted PTEN expression and thereby inhibited activation of PI3K/AKT pathway by sponging miR-494-3p.ConclusionTo conclude, lncRNA WT1-AS impeded cell proliferation, migration, invasion but accelerated cell apoptosis via negatively regulating miR-494-3p to mediate PTEN/PI3K/AKT pathway in NSCLC.

Blocking podoplanin inhibits platelet activation and decreases cancer-associated venous thrombosis

BackgroundPatients with cancer are at a high risk of venous thromboembolism (VTE), studies have shown that high expression of podoplanin (PDPN) in tumors is associated with increased risk of VTE. MethodsTwo human malignant cell lines (NCI-H226 and C8161) expressing high levels of PDPN were selected to explore the role of platelet in cancer-associated venous thrombosis in vitro and in vivo. Immunohistochemical staining using anti-PDPN antibody was performed in the pulmonary carcinoma patients. ResultsBoth NCI-H226 and C8161 cells expressing high PDPN triggered platelet activation via CLEC-2 in vitro, which was abrogated by an anti-PDPN antibody SZ-168. Furthermore, the in vivo study revealed that injection of CHO-PDPN or C8161 in two mouse model of venous thrombosis activated platelets, increased platelet counts and enhanced thrombosis. More importantly, PDPN-enhanced thrombosis was reduced in mice treated with SZ168. A total of 63.3% tumor specimens stained positive for PDPN. High PDPN expression was associated with an increased risk of VTE and poor prognosis. ConclusionsPDPN expression in tumors induced platelet activation and was related to a high risk of VTE via platelet activation. SZ168 inhibited PDPN-induced platelet activation in vitro and decreased the incidence of VTE in mice.

Design, synthesis and evaluation of novel ErbB/HDAC multitargeted inhibitors with selectivity in EGFRT790M mutant cell lines.

Acquired resistance leads to the failure of EGFR TKIs in NSCLC treatment. A novel series of hydroxamic acid-containing 4-aminoquinazoline derivatives as irreversible ErbB/HDAC multitargeted inhibitors for NSCLC therapy had been designed and synthesized, which displayed weak anti-proliferative activity in several EGFR wild-type cancer cell lines (NCI-H838, SK-BR-3, A549, A431) yet retained moderate activity to EGFRT790M resistance mutation harboring NCI-H1975cells. The mechanistic studies revealed that the representative compound 11e was able to inhibit the phosphorylation of EGFR, up-regulate hyperacetylation of histone H3 and even reduce the expression of EGFR and Akt in NCI-H1975cells. In further assays, compound 11e also showed moderate anti-proliferative activity in other EGFRT790M harboring tumor cell lines (NCI-H820, Ba/F3_EGFR_Del19-T790M-C797S) and low toxicities in normal cell lines (HL-7702, FHC). This selectivity of designed multitargeted compounds could serve as a potential strategy to circumvent multiple mechanisms of acquired resistance to EGFR-targeted therapy without severe toxicities and side effects resulting from broad inhibition.

Immuno-PET imaging of 68Ga-labeled nanobody Nb109 for dynamic monitoring the PD-L1 expression in cancers.

The checkpoint blockade immunotherapy has become a potent treatment strategy for cancers, and programmed death ligand-1 (PD-L1) is a prominent checkpoint ligand that is highly expressed in some cancers. The identification of immune checkpoint marker PD-L1 is critical for improving the success of immunotherapy. Accordingly, the binding specificity and dynamic monitoring property of a non-blocking nanobody tracer 68Ga-NOTA-Nb109 to PD-L1 were assessed in this study. The endogenous expression level of PD-L1 in several cancer cells was measured by flow cytometry, Western blot, and cellular uptake assay. Sensitivity and specificity of 68Ga-NOTA-Nb109 in monitoring the expression of PD-L1 in vivo were evaluated by PET imaging of different tumor-bearing models (U87, high PD-L1 expression; HCT 116, medium PD-L1 expression; and NCI-H1299, low PD-L1 expression). In vivo PET imaging results agreed well with those detected in vitro. In addition, PET imaging of PD-L1 expression in U87 and NCI-H1299 xenografts using 18F-FDG was also performed for comparison. The maximum tumor-to-muscle uptake ratio of 68Ga-NOTA-Nb109 was more than twofold that of 18F-FDG in U87 xenograft. The change of PD-L1 expression in NCI-H1299 cells and xenografts induced by cisplatin (CDDP) was sensitively monitored by 68Ga-NOTA-Nb109. This study demonstrated the feasibility of tracer 68Ga-NOTA-Nb109 for specifically targeting endogenous PD-L1 and dynamic monitoring the change of PD-L1 expression, and could guide the immunotherapy and immunochemotherapy for refractory cancers.

Thermal treatment decreases resistance to osimertinib in non-small cell lung cancer through the EGFR/PI3K/AKT pathway.

Osimertinib (OSI) resistance commonly occurs during the treatment of non-small-cell lung cancer (NSCLC). This study aims to investigate whether the thermal effects of radiofrequency ablation (RFA) can increase the sensitivity of OSI-resistant NSCLC to OSI treatment and whether OSI effectively inhibits the recurrence of OSI-resistant NSCLC following RFA treatment and improve survival of NSCLC patients. In vitro, OSI-resistant NCI-H1975 (NCI-H1975/OSIR) cells and thermotolerant NCI-H1975/OSIR (NCI-H1975/OSIR-a-h) cells were established using human NSCLC cell line NCI-H1975. Cell viability, apoptosis, sensitivity to OSI, threonine-methionine amino acid substitution at position 790 (T790M) mutation levels, and protein expression of epidermal growth factor receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), hypoxia-inducible factor-1 alpha (HIF-1a) were detected using different methods. In vivo, a nude mouse model of metastatic human lung cancer was developed and subjected to RFA treatment. The tumor growth, apoptosis, sensitivity to OSI, expression of EGFR/PI3K/AKT/HIF-1a, and CD34 levels were detected in the micrometastases of the transition zone (TZ) around the central ablation zone, and the reference zone (RZ) far from central ablation zone. NCI-H1975/OSIR and thermotolerant NCI-H1975/OSIR cell models were successfully established. Thermotolerant NCI-H1975/OSIR cells show higher sensitivity to OSI than NCI-H1975/OSIR cells and NCI-H1975 cells. OSI treatment can inhibit the EGFR/PI3K/AKT pathway and induce apoptosis in both NCI-H1975 cells and thermotolerant NCI-H1975/OSIR cells, but not in NCI-H1975/OSIR cells. In vivo, RFA treatment increases sensitivity to OSI in NCI-H1975/OSIR cell micrometastases in the TZ but not in the RZ. OSI intervention effectively inhibits the over-proliferation of micrometastases and activation of the EGFR/PI3K/AKT pathway, and induces apoptosis of micrometastases in the TZ, but shows little effects on the micrometastases in the RZ. The thermal effects can increase the sensitivity of OSI-resistant NSCLC cells to OSI through the EGFR/PI3K/AKT/HIF-1a signaling pathway, indicating that RFA combined with OSI might be a clinically effective and comprehensive therapy for the treatment of OSI-resistant NSCLC.

The effects of Benjakul extract and its isolated compounds on cell cycle arrest and apoptosis in human non-small cell lung cancer cell line NCI-H226.

Background and purpose:Benjakul, a traditional Thai formulation for cancer treatment, is composed of five plants. This study aimed to assess the cytotoxicity of Benjakul, its five plants, and its isolated compounds against non-small cell lung cancer (NSCLC) by the sulforhodamine B (SRB) assay.Experimental approach:Analyses of cell cycle and membrane asymmetry changes were performed with different fluorescent dyes and analyzed by flow cytometry in NCI-H226 cells. Activation of caspase-3 was measured using a caspase-3 colorimetric assay kit. The pan-caspase inhibitor Z-VAD-FMK was used in analyses of cell cycle and caspase-3 activity.Findings/Results:Benjakul exhibited cytotoxicity against NSCLC with IC50 between 5.56-5.64 μg/mL. Among its five ingredients, Benjakul displayed the highest selectivity with selectivity index values ranging from 2.93 to 6.88, with the exception of Plumbago indica, indicating its protective effects. Plumbagin and 6- shogaol displayed the highest cytotoxicity and underwent molecular studies in NCI-H226 cells. Flow cytometry analysis revealed that Benjakul and 6-shogaol dose-dependently induced G2/M phase arrest, and plumbagin dose-dependently induced S-G2/M phase arrest with the highest percentage in early incubation time (12-24 h). At the highest doses, Benjakul extract, 6-shogaol, and plumbagin time-dependently increased the population of sub-G1 apoptotic cells with the highest percentage in longer incubation time (60-72 h). Similarly, membrane asymmetry changes showed time-dependent increases in the percentage of early and late apoptotic cells. Moreover, the apoptosis-inducing effect of Benjakul, 6-shogaol, and plumbagin at the highest dose, via the caspase cascade was confirmed by time-dependent induction of caspase-3 activity, followed by its complete reduction and abolished sub-G1 peaks upon addition of Z-VAD-FMK.Conclusion and implication:Our findings demonstrated for the first time the effects of Benjakul and its compounds on S-G2/M or G2/M phase arrest and caspase-dependent apoptosis in lung cancer cells.

β-elemene enhances the antitumor activity of erlotinib by inducing apoptosis through AMPK and MAPK pathways in TKI-resistant H1975 lung cancer cells.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) significantly improve the outcome of non-small-cell lung cancer (NSCLC) patients with EGFR mutations, however, most TKI-treated patients will develop resistance to TKIs. β-elemene, extracted from Curcuma aromatica Salisb., has been widely used to treat various malignant tumors, including TKI-resistant NSCLC, but, the effects and the molecular mechanisms remain unclear. In this study, the NCI-H1975 cell line harboring double mutations L858R/T790M was treated with varying concentrations of β-elemene and/or erlotinib. The effects of β-elemene on cell proliferation, migration, apoptosis, and the expression of relevant proteins of NCI-H1975 cells were evaluated. The results revealed that β‑elemene significantly inhibited the growth, colony formation capacity, wound healing ability of NCI-H1975 cells, and improved the sensitivity of NCI-H1975 cells to erlotinib. Compared with erlotinib alone, β-elemene plus erlotinib significantly promoted the apoptosis of NCI-H1975 cells, accompanied by the down-regulated expression of P-mTOR, P-EGFR, CHOP proteins and up-regulated expression of P-AMPKα and Bax proteins. Taken together, these findings demonstrate that β-elemene suppresses the proliferation and migration of TKI-resistant H1975 cells, and enhances the antitumor activity of erlotinib by inducing apoptosis through AMPK and MAPK pathways in TKI-resistant H1975 lung cancer cells, indicating that β-elemene is a promising anti-cancer therapeutic candidate for TKI-resistant NSCLC.

Format (2D vs 3D) and media effect target expression and response of patient-derived and standard NSCLC lines to EGFR inhibitors

Three patient-derived NSCLC lines and three well-established NSCLC lines with varied EGFR gene status were compared for expression of EGFR protein, proliferation and epithelial and mesenchymal markers in monolayer, simple spheroid and complex spheroid cultures. The effects of diverse culture conditions and exposure time on the response of the six NSCLC lines to the EGFR inhibitors erlotinib, afatinib, lapatinib, and osimertinib were examined. The clinical Cmax was used as the test concentration to determine whether cells were responsive or resistant to each agent. Among the patient-derived lines, LG0703-F948, which has an EGFR L858R mutation, was responsive to each of the four EGFR inhibitor when grown as spheroids but resistant when grown in monolayer. The HCC827 line, which carries an EGFR E746-A750 deletion, was responsive to each of the four EGFR inhibitors when grown as spheroids or monolayers. NCI-H1975 cells which have an EGFR T790M mutation and an EGFR L858R mutation, were sensitive to osimertinib when propagated as spheroids but not when grown in monolayer. The results suggest that the expression of cell surface targets and response to drugs targeting cell surface proteins varies depending upon cell culture format. These findings may help to explain, in part, the concordance or discordance between cell culture and in vivo findings in experimental systems.

Ophiopogonin B inhibits migration and invasion in non-small cell lung cancer cells through enhancing the interaction between Axin and β-catenin.

Ophiopogonin B (OP-B), a kind of saponin compound that exists in Radix Ophiopogonis is frequently adopted for the treatment of lung disease as traditional Chinese medicine. The present work aimed to explore the anti-tumor activity of OP-B on non-small cell lung carcinoma (NSCLC) and its possible mechanism. We found that OP-B-treated cells suppressed the viability and proliferation of cells depending on its concentration, as assayed by MTT and Alamar Blue (IC50 were 14.22 ± 1.94, 12.14 ± 2.01, and 16.11 ± 1.83 μM in A549, NCI-H1299, and NCI-H460 cells, respectively). Then, the suppressive effect of OP-B on the invasion and migration of NSCLC was observed through wound healing and Transwell assays, and the epithelial-mesenchymal transition (EMT) markers was detected by immunofluorescence and western blotting. In addition, a dose-dependent reduction of β-catenin both within cytoplasm and nucleus was observed, and the downstream proteins cyclin D1 and c-Myc of Wnt/β-catenin pathway were also reduced. We further constructed β-catenin-overexpression cell models to reveal the underlying mechanism. The results showed that 10 μM of OP-B notably reduced β-catenin protein levels, as well as cell migration and invasion. In spite of the increasement of β-catenin, activation of Wnt pathway and EMT progression, knockdown of Axin leaded to de-function of OP-B on cell metastasis. Taken together, OP-B reduced NSCLC migration and invasion by strengthening the Axin/β-catenin interaction and reducing β-catenin protein translocation.

Physalis alkekengi var. franchetii Extracts Exert Antitumor Effects on Non-Small Cell Lung Cancer and Multiple Myeloma by Inhibiting STAT3 Signaling.

BackgroundPhysalis alkekengi var. franchetii is an herb that possesses various ethnopharmacological applications. Herein, our current study focuses on the antitumor effect of a combination of physalins, which are regarded as the most representative secondary metabolites from calyces of Physalis alkekengi var. franchetii.Materials and MethodsWe mainly investigated the antitumor activity of the physalins extracted from Physalis alkekengi var. franchetii on both solid and hematologic cancers. The main cells used in this study were NCI-H1975 and U266 cells. The major assays used were the CCK-8 assay, Western blot analyses, immunofluorescence assay and Annexin V assay, and a xenograft mouse model was used.ResultsThe results showed that physalins exhibited a strong antitumoural effect on both non-small cell lung cancer (NSCLC) and multiple myeloma (MM) cells by suppressing constitutive STAT3 activity and further inhibiting the downstream target gene expression induced by STAT3 signaling, which resulted in the enhanced apoptosis of tumor cells. Moreover, physalins significantly reduced tumor growth in xenograft models of lung cancer.ConclusionCollectively, these findings demonstrated that the physalins from Physalis alkekengi var. franchetii may potentially act as cancer preventive or chemotherapeutic agents for NSCLC and MM by inhibiting the STAT3 signaling pathway. The present study served as a promising guide to further explore the precise mechanism of Physalis alkekengi var. franchetii in cancer treatment.

Traditional Chinese Medicine Brucea Javanica Oil Enhances the Efficacy of Anlotinib in a Mouse Model of Liver-Metastasis of Small-cell Lung Cancer

Small-cell lung cancer (SCLC) is a recalcitrant disease with liver and other metastasis. The present study evaluated the efficacy of the traditional Chinese medicine Brucea javanica oil (BJO) combined with anlotinib, a multi-tyrosine kinase inhibitor with anti-angiogenic activity, on a nude-mouse model of SCLC liver metastasis. The mouse model was established by injecting NCI-H446 cells (1×106) in Matrigel (20 μl) into the upper liver lobe. All animals were randomized and assigned to three groups: Control (n=8); anlotinib alone (n=8; 3 mg/kg, qd×14+7-day interval with two cycles, oral); anlotinib plus BJO (n=8; 3 mg/kg anlotinib qd×14+7-day interval with two cycles, orally; BJO: 1 g/kg, qd×6 weeks, orally). Body weight was determined every week. Six weeks after initial treatment, tumors were collected for analysis of angiogenesis using immunohistochemistry. The combination of anlotinib and BJO significantly inhibited growth of SCLC liver metastases and angiogenesis more than anlotinib monotherapy (p=0.043). In addition, BJO alleviated body-weight loss associated with anlotinib therapy, including general mouse condition. The results of the present study indicate that the combination of anlotinib with BJO is promisingly active against liver metastases of SCLC, and has clinical potential.

Inhibition potency of disulphides and trisulphides on various tumor cell lines growth

Tumor is one of the leading diseases of today. Chemotherapy and radiation therapy healing chances are limited, so some alternative methods are resorted to. Many papers indicated that garlic and organosulphur compounds diallyldisulphide (DADS) and diallyltrisulphide, which are main components of garlic decrease the cancer risk and inhibit the cell proliferation. In this paper inhibition potency of disulphides (DADS and its synthetic analogues) and trisulphides with different alkyl and phenyl substituents on growth of various tumor (Non-Small cell Lung Cancer, Colon cancer, CNS Cancer, Melanoma, Ovarian Cancer, Renal Cancer, Prostate Cancer, Breast Cancer, Leukemia) cell lines was investigated. Concentration of each compound (DADS, diethyl disulphide, dipropyl disulphide, diphenyl disulphide, tetraethylthiuram disulphide (TETUDS), dimetil trisulphide, dipropyl trisulphide and metilpropyl trisulphide (MPTS) was 10 µmol/L. It was found that DADS has an inhibitory effect on the growth of several cancer cell lines, but synthetic analogues TETUDS and MPTS exhibit stronger effect on certain cells lines. MPTS inhibits the growth of even 12 cell lines for more than 10%, especially leukemia cell line SR and NCI-H522 cell line of Non-Small cell Lung Cancer (reduction of growth for 24% and 47%, resp.). Trisulphide analogues exhibit little higher inhibitory effects in comparison to disulphide ones.

The Acidic Tumor Microenvironment Affects Epithelial-Mesenchymal Transition Markers as Well as Adhesion of NCI-H358 Lung Cancer Cells.

Epithelial-mesenchymal transition (EMT), which is involved in metastasis formation, requires reprogramming of gene expression mediated by key EMT transcription factors. However, signals from the cellular microenvironment, including hypoxia, can also modulate the process of EMT. Hypoxia is often associated with a reduction in the extracellular pH of the tumor microenvironment (acidosis). Whether acidosis alone has an impact on the expression of the EMT markers E-cadherin, N-cadherin, and vimentin was studied in NCI-H358 lung cancer cells. Reducing extracellular pH decreased E-cadherin mRNA, while vimentin and N-cadherin mRNA were doubled. However, at the protein level, E-cadherin and N-cadherin were both reduced, and only vimentin was upregulated. E-cadherin and N-cadherin expression at the cell surface, which is the relevant parameter for cell-cell and cell-matrix interaction, decreased too. The reduction of cell surface proteins was due to diminished protein expression and not changes in cellular localization, since localization of EMT markers in general was not affected by acidosis. Acidosis also affected NCI-H358 cells functionally. Adhesion was decreased when the cells were primed in an acidic medium before measuring cell adherence, which is in line with the reduced expression of cadherins at the cell surface. Additionally, migration was decreased after acidic priming. A possible mechanism for the regulation of EMT markers involves the action of microRNA-203a (miR-203a). In NCI-H358 lung cancer cells, miR-203a expression was repressed by acidosis. Since a decrease in the level of miR-203a has been shown to induce EMT, it might be involved in the modulation of EMT marker expression, adhesion, and migration by the acidic tumor microenvironment in NCI-H358 lung cancer cells.

Establishment and Characterization of a Topotecan Resistant Non-small Cell Lung Cancer NCI-H460/TPT10 Cell Line

While topotecan (TPT) is a first- and second-line chemotherapeutic drug in treating lung cancer, the development of drug resistance in tumors still reserves as a major obstacle to chemotherapeutic success. Therefore, a better understanding of the mechanisms of topotecan resistance is critical. In this study, the first topotecan-resistant human non-small cell lung cancer (NSCLC) cell line, termed NCI-H460/TPT10, was established from the parental NCI-H460 cell line. NCI-H460/TPT10 cells exhibited a 394.7-fold resistance to TPT, and cross-resistance to SN-38, mitoxantrone, and doxorubicin, compared to parental NCI-H460 cells. Overexpression of ABCG2 localized on the cell membrane, but not ABCB1 or ABCC1, was found in NCI-H460/TPT10 cells, indicating that ABCG2 was likely to be involved in topotecan-resistance. This was confirmed by the abolishment of drug resistance in NCI-H460/TPT10 cells after ABCG2 knockout. Moreover, the involvement of functional ABCG2 as a drug efflux pump conferring multidrug resistance (MDR) was indicated by low intracellular accumulation of TPT in NCI-H460/TPT10 cells, and the reversal effects by ABCG2 inhibitor Ko143. The NCI-H460/TPT10 cell line and its parental cell line can be useful for drug screening and developing targeted strategies to overcome ABCG2-mediated MDR in NSCLC.

Screening and Identification of the Peptides Specifically Binding to Human Non-small Cell Lung Cancer NCI-H1299 Cells

背景与目的非小细胞肺癌（non-small cell lung cancer, NSCLC）是最常见的肺癌组织学类型，也是病死率最高的恶性肿瘤之一。多肽作为光学分子成像探针的主体可以实现肿瘤的早期筛查及诊断，提高患者存活率。本研究旨在利用体内噬菌体展示技术筛选与人NSCLC细胞NCI-H1299高度结合的小分子多肽并通过体外实验鉴定其结合特异性。方法制备NCI-H1299细胞荷瘤裸鼠模型，用噬菌体展示环七肽库进行3轮体内筛选后随机挑取噬菌体克隆，免疫组织化学法及酶联免疫吸附法（enzyme-linked immunosorbent assay, ELISA）鉴定噬菌体克隆对NCI-H1299细胞的亲和力。提取阳性单克隆噬菌体DNA测序获得外源多肽氨基酸序列，将重复率最高的序列化学合成多肽并进行异硫氰酸荧光素（fluorescein, FITC）标记，制备光学分子探针，初步鉴定其对NCI-H1299细胞的特异性。结果经3轮体内筛选后噬菌体富集率是首轮的341.3倍；免疫组织化学染色显示随着筛选次数增加，肿瘤组织中结合的噬菌体不断增加，且结合量明显高于正常组织；ELISA结果显示随机挑取的30个噬菌体克隆中20个为阳性克隆，经测序后将重复率最高的序列合成多肽并命名为NSP1；四甲基偶氮唑盐比色法（methyl thiazolyl tetrazolium assay, MTT）、细胞划痕实验表明NSP1不会影响细胞增殖、迁移；流式细胞术、细胞免疫荧光结果表明NSP1可与NCI-H1299细胞特异性结合。结论利用体内噬菌体展示技术成功得到了与肺癌NCI-H1299细胞特异性结合的多肽NSP1，为NSCLC的早期诊断及靶向治疗奠定了研究基础。

Immunomodulatory effects of flavonoids: An experimental study on natural-killer-cell-mediated cytotoxicity against lung cancer and cytotoxic granule secretion profile

Background: A new approach involving immune-cell-mediated cancer therapy has been adopted extensively for the sake of lung cancer treatments by utilizing natural killer (NK) cells. NK cell activity can be enhanced with certain agents, and among them are flavonoids. Thus, this study was conducted to investigate the immunomodulatory roles of apigenin, luteolin and quercetin on NK cell activity against lung cancer cells and on the secretions of perforin and granulysin profile. Methods: The NK-92 cells were grown in complete α-Minimum Essential Medium (MEM). NCI-H460 lung cancer cells were cultured in Roswell Park Memorial Institute 1640 media. NK cell activity against lung cancer cells were done using MTT(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The secretions of perforin and granulysin profiles were then analysed using enzyme-linked immunosorbent assay. Results: Apigenin, luteolin and quercetin significantly increased the NK-cell-mediated cytotoxic activity against lung cancer cells at concentrations 12.5 µg/ml and 25µg/ml ( P < 0.001). The secretion levels of perforin and granulysin from NK cells were also significantly enhanced with apigenin and luteolin treatment but not with quercetin. Conclusions: All three flavonoid compounds possessed some significant immunomodulatory actions on NK cell cytotoxic activity and granule secretion profiles towards lung cancer therapy.

Inhibition of the FGF/FGFR System Induces Apoptosis in Lung Cancer Cells via c-Myc Downregulation and Oxidative Stress.

Lung cancer represents an extremely diffused neoplastic disorder with different histological/molecular features. Among the different lung tumors, non-small-cell lung cancer (NSCLC) is the most represented histotype, characterized by various molecular markers, including the expression/overexpression of the fibroblast growth factor receptor-1 (FGFR1). Thus, FGF/FGFR blockade by tyrosine kinase inhibitors (TKi) or FGF-ligand inhibitors may represent a promising therapeutic approach in lung cancers. In this study we demonstrate the potential therapeutic benefit of targeting the FGF/FGFR system in FGF-dependent lung tumor cells using FGF trapping (NSC12) or TKi (erdafitinib) approaches. The results show that inhibition of FGF/FGFR by NSC12 or erdafitinib induces apoptosis in FGF-dependent human squamous cell carcinoma NCI-H1581 and NCI-H520 cells. Induction of oxidative stress is the main mechanism responsible for the therapeutic/pro-apoptotic effect exerted by both NSC12 and erdafitinib, with apoptosis being abolished by antioxidant treatments. Finally, reduction of c-Myc protein levels appears to strictly determine the onset of oxidative stress and the therapeutic response to FGF/FGFR inhibition, indicating c-Myc as a key downstream effector of FGF/FGFR signaling in FGF-dependent lung cancers.

Long non‑coding RNA LINC00887 promotes progression of lung carcinoma by targeting the microRNA‑206/NRP1 axis

Long non-coding RNAs (lncRNAs) have been reported to participate in multiple biological processes, including tumorigenesis. In the current study, the function of a novel lncRNA LINC00887 was investigated in lung carcinoma. For this purpose, LINC00887 expression was assessed by reverse-transcription quantitative PCR. Cell viability was determined by the CCK-8 and EdU assays. Cell invasion, migration were assessed by the transwell and wound healing assays, respectively. A dual luciferase assay was used for analysis of the interaction between LINC00887 and miR-206, as well as the relationship of miR-206 with NRP1. A tumor xenograft study was performed to investigate the LINC00887-miR-206-NRP1 axis in vivo. The expression levels of LINC00887 were upregulated in lung carcinoma tissues and cells compared with adjacent tissues or normal cells (BEAS-2B). Knockdown LINC00887 significantly inhibited the proliferation, migration and invasion of lung carcinoma A549 and NCI-H460 cells. Furthermore, LINC00887 was identified as a competing endogenous RNA and to directly interact with miR-206. Mechanistically, miR-206 was demonstrated to regulate neuropilin-1 (NRP1) expression by targeting the NRP1 3′-untranslated region. The results of the present study suggested that the LINC00887-miR-206-NRP1 axis served a critical role in regulating lung carcinoma cell proliferation, migration and invasion. In addition, xenograft tumor model experiments revealed that silencing LINC00887 suppressed lung carcinoma tumor growth of in vivo. In summary, our results suggest that LINC00887 may serve an oncogenic role in lung carcinoma by targeting the miR-206/NRP1 axis, providing a potential therapeutic target for patients with lung carcinoma.