Lung Adenocarcinoma Cell Line SPC-A1 Research Articles

Super enhancer associated RAI14 is a new potential biomarker in lung adenocarcinoma.

PurposeTyrosine kinase inhibitors (TKIs) are widely used to treat lung adenocarcinoma patients with EGFR mutations or ALK-fusions. However, patients with wild-type genes or TKIs-resistant mutations lack effective therapeutic targets. Extensive studies reveal that super enhancer (SE), a large cis-regulatory element, is associated with key oncogenes in a variety of cancers. By comparing the effect of SE on lung adenocarcinoma cell lines with normal cell line, this work attempts to find new biomarkers and potential therapeutic targets for lung adenocarcinoma.Experimental DesignChromatin Immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of H3K27ac (acetylation on lysine 27 of histone 3) was performed in lung adenocarcinoma cell lines SPC-A1 and SCH-1153. The differences in SE distribution were then analyzed among SPC-A1, SCH-1153, A549 and normal human lung fibroblasts (NHLF) to identify SE-associated oncogenes. The expression of SE-associated oncogenes was then detected by RNA-seq and further verified in 71 patients by real-time PCR.ResultsSE associated with many new oncogenes in lung adenocarcinoma, among which, RAI14 was up-regulated in A549 and 31 of 71 patients. High expression of RAI14 could inhibit cell proliferation, indicating its potential as a new biomarker for lung adenocarcinoma.

Sanguinarine-induced apoptosis in lung adenocarcinoma cells is dependent on reactive oxygen species production and endoplasmic reticulum stress

Sanguinarine (SAN), an alkaloid isolated from plants of the Papaveraceae family, is a compound with multiple biological activities. In the present study, we explored the anticancer properties of SAN in lung cancer using the human lung adenocarcinoma cell line SPC-A1. Our results revealed that SAN inhibited SPC-A1 cell growth and induced apoptosis in a dose-dependent manner. We found that SAN triggered reactive oxygen species (ROS) production, while elimination of ROS by N-acetylcysteine (NAC) reversed the growth inhibition and apoptosis induced by SAN. SAN-induced endoplasmic reticulum (ER) stress resulted in the upregulation of many genes and proteins involved in the unfolded protein response (UPR) pathway, including glucose-regulated protein 78 (GRP78), p-protein kinase R (PKR)-like ER kinase (PERK), p-eukaryotic translation initiation factor 2α (eIF2α), activating transcription factor 4 (ATF4) and CCAAT/enhancer binding protein homologous protein (CHOP). Blocking ER stress with tauroursodeoxycholic acid (TUDCA) markedly reduced SAN-induced inhibition of growth and apoptosis. Furthermore, TUDCA decreased SAN-induced ROS production, and NAC attenuated SAN-induced GRP78 and CHOP expression. Overall, our data indicate that the anticancer effects of SAN in lung cancer cells depend on ROS production and ER stress and that SAN may be a potential agent against lung cancer.

Wnt signaling regulates the stemness of lung cancer stem cells and its inhibitors exert anticancer effect on lung cancer SPC-A1 cells.

Wnt signaling plays an important role in regulating the activity of cancer stem cells (CSCs) in a variety of cancers. In this study, we explored the role of Wnt signaling in the lung cancer stem cells (LCSCs). LCSCs were obtained by sphere culture, for which human lung adenocarcinoma cell line SPC-A1 was treated with IGF, EGF and FGF-10. The stemness of LCSCs was confirmed by immunofluorescence, and pathway analysis was performed by functional genome screening and RT-PCR. The relationship between the identified signaling pathway and the expression of the stemness genes was explored by agonist/antagonist assay. Moreover, the effects of different signaling molecule inhibitors on sphere formation, cell viability and colony formation were also analyzed. The results showed that LCSCs were successfully generated as they expressed pluripotent stem cell markers Nanog and Oct 4, and lung distal epithelial markers CCSP and SP-C, by which the phenotype characterization of stem cells can be confirmed. The involvement of Wnt pathway in LCSCs was identified by functional genome screening and verified by RT-PCR. The expression of Wnt signaling components was closely related to the expression of the Nanog and Oct 4. Furthermore, targeting Wnt signaling pathway by using different signaling molecule inhibitors can exert anticancer effects. In conclusion, Wnt signaling pathway is involved in the stemness regulation of LCSCs and might be considered as a potential therapeutic target in lung adenocarcinoma.

The Study on Newly Developed McAb NJ001 Specific to Non-Small Cell Lung Cancer and Its Biological Characteristics

Monoclonal antibody (McAb) is the key tool for cancer immunodiagnosis and immunotherapy. McAb-based immunotherapy that targets tumor antigens has had great achivement. In this study, a cell clone which kept secreting high-titer IgG1-type McAb named NJ001 against human non-small cell lung cancer (NSCLC) cells was obtained. The titer of purified NJ001 was 2×106. The antigen named SP70 of NSCLC specifically identified by NJ001 was proved to be a protein with the relative molecular mass (Mr) of 70 kDa. The results of immunohistochemical staining indicated that NJ001 could positively react to NSCLC, but weak positively or negatively react to human small-cell lung cancer (SCLC), pulmonary pseudotumor and other epithelial tumors. In soft agar assay, the colony formation efficiency in NJ001 groups decreased in a dose-dependent manner. For the concentration of 100 µg/ml, 200 µg/ml and 400 µg/ml, the inhibition ratio of colony formation was 23.4%, 62.5% and 100% respectively. Meanwhile, NJ001 caused significant reduction in tumor volume and tumor weight compared to control mice in lung cancer xenograft model. The tumor growth inhibition ratio in 200 µg, 400 µg and 800 µg NJ001 groups was 10.44%, 37.29% and 44.04%, respectively. NJ001 also led to cytomorphological changes and induced the apoptosis of human lung adenocarcinoma cell line SPC-A1 significantly. The newly developed NJ001 selectively reacted to NSCLC and exhibited anti-tumor activity both in vitro and in vivo. NJ001 is of great value concerning immunodiagnostics and immunotherapy for NSCLC and holds promise for further research regarding the mechanism underlying tumor progression of NSCLC.

Nimotuzumab Increases Chemosensitivity of Human Lung Adenocarcinoma Cell Lines to Docetaxel

Overexpression of epidermal growth factor receptor (EGFR) is common in non-small-cell lung cancer (NSCLC) and has been recently shown to contribute to cancer chemoresistance. It has been reported that the EGFR antibodies such as cetuximab in combination with chemotherapy could lead to an absolute benefit of overall survival (OS) compared with chemotherapy alone. In this study, we investigated the effects of nimotuzumab (h-R3), a humanized anti-EGFR antibody, in combination with docetaxel (DTX), on DTX-resistant human lung adenocarcinoma cell line SPC-A1 (SPC-A1/DTX) both in vitro and in vivo. Immunohistochemistry and FCM assays demonstrated that SPC-A1/DTX cells had a relatively higher rate of EGFR overexpression than SPC-A1 cells. Accordingly, SPC-A1/DTX cells were approximately 13.7 times resistant to DTX than SPC-A1 cells. The combined therapy of h-R3 and DTX showed strong synergistic suppressive effect on cell proliferation of SPC-A1/DTX cells in vitro. The synergistic antitumor effect was also observed in SPC-A1/DTX xenograft-bearing nude mice. Further study showed that h-R3 could lead to a significant cell arrest at G1 phase of cell cycle in both SPC-A1DTX and SPC-A1 cells. A dramatic increase of apoptosis rate was detected in h-R3-treated SPC-A1/DTX but not SPC-A1 cells. Moreover, when combined with DTX, h-R3 brought higher apoptosis rate in SPC-A1/DTX cells rather than in SPC-A1 cells. In conclusion, our results suggested that h-R3 could significantly enhance chemosensitivity of human lung adenocarcinoma cells to DTX, at least partially by induction of G1 phase arrest and cell apoptosis.

Orthogonal test design for optimization of suitable conditions to separate C-phycocyanin from Spirulina platensis by high-speed counter-current chromatography using reverse micelle solvent system

High-speed counter-current chromatography (HSCCC) was applied to separate C-phycocyanin (C-PC) from Spirulina platensis in the article. The suitable conditions were optimized by an orthogonal test design (L(9)(3)(3)), including the stationary phase of reverse micelle solvent system (0.10 g/mL cetyltrimethylammonium bromide [CTAB]/isooctane-hexylalcohol), mobile phase A (0.05 mol/L sodium phosphate buffer, pH 4.0, containing 0.2 mol/L KCl) and mobile phase B (0.05 mol/L sodium phosphate buffer, pH 8.0, containing 0.4 mol/L KCl). Under the selected conditions, 78.7 mg protein was purified from 200 mg crude extract of S. platensis, and the purity of the product was 4.25 based on the absorbance ratio of A(620)/A(280) , which was increased 6.85 times compared with the crude extract. Then, the protein was identified to be C-PC by MALDI-TOF/TOF-MS and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis compared with the standard. The application of HSCCC used in the separation of C-PC from S. platensis was first reported in the article. Furthermore, three kinds of tumor cell lines including human hepatoma cell line SMMC-7721, human ovarian carcinoma cell line ES-2, and human lung adenocarcinoma cell line SPCA-1 were used to evaluate the anticancer activities of the separated product, and the results showed that the separated C-PC had excellent anti-tumor actions with the IC(50) values at 2.998, 4.854, and 8.423 μg/mL, respectively, for 48 h treatment. The outcome indicates that an effective method for C-PC purification by HSCCC has been established.

Inhibitory effects of the survivin siRNA transfection on human lung adenocarcinoma cells SPCA1 and SH77

背景与目的Survivin是IAP家族的新成员，具有抑制凋亡和调节细胞增殖的双重作用，是迄今发现最强的凋亡抑制因子。本项研究的目的是通过将survivin的小干扰RNA（small interfering RNA, siRNA）表达载体分别转染至肺癌细胞SPCA1和SH77，分析survivin siRNA对不同肺癌细胞的增殖抑制作用。方法构建靶向survivin的siRNA表达载体与pSi scrambled，经由脂质体法分别转染至肺癌细胞SPCA1和SH77。通过MTT法检测细胞增殖抑制，用流式细胞仪检测细胞凋亡率和细胞周期，利用半定量RT-PCR和Western blot印迹法分别检测细胞survivin mRNA及蛋白表达水平。结果Survivin siRNA干预后，SPCA1和SH77细胞均出现凋亡，G0/G1期阻滞。实验组Survivin mRNA水平和蛋白表达水平比脂质体对照组明显减少。结论Survivin基因特异性RNA干扰可以明显抑制肺癌SPCA1和SH77细胞的体外增殖并诱导明显的细胞凋亡。

Mechanism of drug resistance identified in human lung adenocarcinoma cell line SPC-A1 selected for resistance to docetaxel

To investigate the mechanism of resistance to docetaxel in human lung cancer. Human lung carcinoma SPC-A1/Docetaxel cells were derived from parental SPC-A1 cells by continuous exposure to increasing concentration of docetaxel. The drug sensitivity was measured by MTT assay in vitro. The cDNA microarray identified a set of differentially expressed genes, and some genes were confirmed by RT-PCR. P-glycoprotein level was measured by flow cytometry analysis. The results of drug sensitivity measured by MTT assay showed that SPC-A1/Docetaxel cells were 13.2-fold resistant to docetaxel and cross-resistant at varying levels to other drugs. The cDNA microarray results identified a set of differentially expressed genes, which showed 428 genes that were up-regulated and 506 genes that were down-regulated in SPC-A1/Docetaxel cells, and some genes were confirmed by RT-PCR. Flow cytometry analysis suggests expression of P-glycoprotein (P-gp) was more abundant in SPC-A1/Docetaxel cells than in the parental cells and docetaxel selection reduces the apoptotic response. The results suggest that docetaxel selection led to changes in gene expression that contribute to the multidrug resistance phenotype.

The anti-tumor effects of CIK cells combined with docetaxel against drug-resistant lung adenocarcinoma cell line SPC-A1/DTX in vitro and in vivo

Objective:To investigate the inhibitory effects of cytokine induced-killer(CIK) cells combined with docetaxel(DTX) on the expansion of lung adenocarcinoma cell line SPC-A1/DTX in vitro and in vivo.Methods:Peripheral blood mononuclear cells(PBMC) from healthy donors were incubated in vitro to induce CIK cells in the presence of interferon-gamma(IFN-γ),IL-2 and anti-CD3 monoclonal antibody.MTT assay was employed to evaluate the cytotoxic activity of DTX,CIK cells and their combination against SPC-A1/DTX cells in vitro.SPC-A1/DTX lung adenocarcinoma cells were injected subcutaneously into nude mice.On the 14 th day,normal saline(control group),docetaxel(DTX 1 mg/kg in 0.2 ml,DTX group),CIK cells(1×107,CIK group),and CIK cells combined with docetaxel(CIK+DTX group) were administered intraperitoneally respectively.All the nude mice were sacrificed at day 15 after treatment and the tumor were weighted out.Results:MTT assay showed that the IC50 to docetaxel in SPC-A1/DTX cells was 110.5 μg/ml and 8.5 μg/ml in wild SPC-A1 cells.CIK cells possessed a higher anti-tumor cytotoxic activity in SPC-A1/DTX cells in vitro than in wild SPC-A1 cells(P0.05).The killing activity of CIK cells combined with DTX was 1.4 times more than that of separate use of CIK cells,and 6.1 times more than that of separate use of DTX.Increasing the E∶T ratio and the concentration of docetaxel directly correlated with higher mean percent specific lysis.In addition,CIK cells plus DTX had a stronger suppressive effect on the tumor growth in nude mice bearing SPC-A1/DTX tumor in vivo.Compared to other groups,in the combined therapy group of mice,the tumor grew slowly,with more marked tissue necrosis and more infiltrated lymphocyte.Conclusion:CIK cells combined with docetaxel can significantly inhibit the growth of MDR cells of lung cancer in vitro and in vivo.The result provides an experimental basis for CIK combined with chemotherapy for clinical application in treatment of MDR lung cancer.

The antitumor effects of CIK cells combined with docetaxel against drug-resistant lung adenocarcinoma cell line SPC-A1/DTX in vitro and in vivo

3039 Background: Cytokine-induced killer (CIK) cells transfer is now being considered the most promising adoptive cellular therapy strategy. Multidrug resistance (MDR) phenomenon is a major hindrance to the successful chemotherapy of cancer. In this study, the anti-tumor activity of CIK cells combined with docetaxel (DTX) against multidrug resistance SPC-A1/DTX cell line was evaluated in vitro and in vivo. Methods: MTT assay was employed to evaluate the cytotoxic activity of DTX, CIK cells, and DTX plused CIK cells against SPC-A1/DTX cells in vitro. In vivo assay, SPC-A1/DTX cells were injected to nude mice subcutaneously to establish tumor-bearing mice model. On the 14th day, normal saline, docetaxel, CIK cells, and CIK cells combined with docetaxel were administered intraperitoneally respectively. All the nude mice were sacrificed at day 15 after treatment and the tumor were weight out. Results: MTT assay showed that CIK cells possessed a higher antitumor cytotoxic activity against SPC-A1/DTX cells than SPC-A1 cells in vitro (p <0.05). The synergetic anti-tumor activity positively correlated with the E:T ratio and the concertration of docetaxel. The animal data also suggested that CIK cells combined with DTX had a stronger suppressive effect on the tumor growth in vivo. Conclusions: CIK cells plused with docetaxel demonstrated a prominent augmentation of anti-tumor activity against MDR lung adenocarcinoma cell lines both in vitro and in vivo. No significant financial relationships to disclose.

Paclitaxel-loaded Pluronic P123/F127 mixed polymeric micelles: Formulation, optimization and in vitro characterization

The objective of this study was to optimize and characterize a novel polymeric mixed micelle composed of Pluronic P123 and F127 loaded with paclitaxel (PTX). A Doehlert matrix design was utilized to investigate the effect of four variables, namely P123 mass fraction, amount of water, feeding of PTX and hydration temperature on the responses including drug-loading coefficient (DL %), encapsulation ratio (ER %) and the percentage of PTX precipitated from the drug-loaded mixed micelles after 48 h at 37 (PTX precipitated %) for improvement of drug solubilization efficiency and micelle stability. PTX-loaded P123/F127 mixed micelles were prepared by thin-film hydration method. The optimized formulation showed a particle size of about 25 nm with ER % > 90%, and a sustained release behavior compared to Taxol. Micelle formation was confirmed by NMR spectroscopy. The mixed micelles had a low CMC of 0.0059% in water. In addition, micelle stability studies implied that introduction of Pluronic F127 (33 wt%) into P123 micelle system significantly increased the stability of PTX-loaded micelles. More importantly, in vitro cytotoxicity was assessed using human lung adenocarcinoma cell lines SPC-A1 and A-549 and was compared to Taxol and the free drug. The cell viability assay against A-549 cells exhibited the 50% inhibition concentration (IC50) of PTX-loaded P123/F127 mixed micelles (0.1 μg/ml) was much lower than those of Taxol injection (0.4 μg/ml) and the free PTX (1.7 μg/ml). Therefore, PTX-loaded P123/F127 mixed micelles may be considered as an effective anticancer drug delivery system for cancer chemotherapy.

The Antitumor Effects of CIK Cells Combined with Docetaxel Against Drug-Resistant Lung Adenocarcinoma Cell Line SPC-A1/DTXin Vitroandin Vivo

The aim of this study was to investigate the inhibitory effects of cytokine-induced killer (CIK) cells combined with docetaxel (DTX) on the growth of drug-resistant lung adenocarcinoma cell line SPC-A1/DTX in vitro and in vivo. The MTT assay was employed to evaluate the cytotoxic activity of DTX, CIK cells, and DTX plused CIK cells against SPC-A1/DTX cells in vitro. For the in vivo assay, SPC-A1/DTX cells were injected into nude mice subcutaneously to establish a tumor-bearing mice model. On the day 14, normal saline, docetaxel, CIK cells, and CIK cells combined with docetaxel were administered intraperitoneally, respectively. All the nude mice were sacrificed at day 15 after treatment and the tumors were weighed out. The MTT assay showed that CIK cells possessed a higher antitumor cytotoxic activity against SPC-A1/DTX cells than SPC-A1 cells in vitro (p < 0.05). The synergetic antitumor activity positively correlated with the E:T ratio and the concentration of docetaxel. The animal data also suggested that CIK cells combined with DTX had a stronger suppressive effect on tumor growth in vivo. CIK cells plused with docetaxel demonstrated a prominent augmentation of antitumor activity against multidrug resistance lung adenocarcinoma cell lines both in vitro and in vivo.

Polycation nanostructured lipid carrier, a novel nonviral vector constructed with triolein for efficient gene delivery

A novel nonviral gene transfer vector was developed by modifying nanostructured lipid carrier (NLC) with cetylated polyethylenimine (PEI). Polycation nanostructured lipid carrier (PNLC) was prepared using the emulsion–solvent evaporation method. Its in vitro gene transfer properties were evaluated in the human lung adenocarcinoma cell line SPC-A1 and Chinese Hamster Ovary (CHO) cells. Enhanced transfection efficiency of PNLC was observed after the addition of triolein to the PNLC formulation and the transfection efficiency of the optimized PNLC was comparable to that of Lipofectamine™2000. In the presence of 10% serum the transfection efficiency of the optimal PNLC was not significantly changed in either cell line, whereas that of Lipofectamine™2000 was greatly decreased in both. Thus, PNLC is an effective nonviral gene transfer vector and the gene delivery activity of PNLC was enhanced after triolein was included into the PNLC formulation.

Relationship between methylation status of multi-drug resistance protein(MRP) and multi-drug resistance in lung cancer cell lines

To study the relationship between the methylation status of multi-drug resistance protein (MRP) gene and the expression of its mRNA and protein in lung cancer cell lines. Human embryo lung cell line WI-38, lung adenocarcinoma cell line SPCA-1 and its drug-resistant cells induced by different concentrations of doxorubicin were treated with restriction endonuclease Eco47III. The methylation status of MRP was examined by PCR, and the expressions of its mRNA and protein were evaluated by in situ hybridization and immunohistochemistry. MRP gene promoter region of WI-38 cells was in hypermethylation status, but the promoter region of MRP in SPCA-1 cells and their resistant derivatives induced by different concentrations of doxorubicin were in hypomethylation status. There were significant differences in the expression of MRP mRNA among WI-38 cell line, SPCA-1 cells and their drug-resistant derivatives induced by different concentration of doxorubicin. Consistently, MRP immunostaining presented similar significant differences. The promoter region of MRP in SPCA-1 lung adenocarcinoma cells was in hypomethylation status. The hypomethylation status of 5′ regulatory region of MRP promoter is an important structural basis that can increase the activity of transcription and results in the development of drug resistance in lung cancer.

Screening differential expression of docetaxel-resistance related genes of human lung adenocarcinoma cell line SPC-A1 by cDNA microarray

Docetaxel is one of effective chemotherapeutics in the last few years, however, it is interfered by drug resistance in its further application. The aim of this study is to screen differentially expressed genes of docetaxel resistant cell line SPC-A1/Docetaxel and its parent cell line SPC-A1 with gene chip technique. The cDNA retro-transcribed from equal quantity mRNA derived from SPC-A1/Docetaxel and SPC-A1 cell lines. The mixed probes were hybridized with Affymetrix GeneChip HG-U133A2.0. The acquired image was analyzed by Affymetrix GeneChip Operating Software Version 1.0. Then, part of these results were verified by RT-PCR. A total of 934 differentially expressed genes were screened out, in which up-and down-regulated genes were 428 and 506 respectively. These genes involved in ABC transporter, apoptosis regulator, tubulin, signal transducer, enzyme and so on. These differentially expressed genes may be related to the mechanisms of docetaxel resistance in SPC-A1/Docetaxel cell line.

Expression of hnRNP A2/B1 in human lung cancer cell lines

To explore the mRNA expression of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 in human lung cancer cell lines. Expression of hnRNP A2/B1 and hnRNP B1 mRNA was detected by RT-PCR using specific primers. Expression of hnRNP B1 protein was examined by immunocytochemistry using specific anti-hnRNP B1 antibody. RT-PCR indicated that the hnRNP A2/B2 and hnRNP B1 mRNA existed in the lung adenocarcinoma cell lines SPC-A1 and Y-90, squamous cell carcinoma cell line A431 and small cell lung cancer cell line NCI-H446. According to immunocytochemistry results, the expression of hnRNP B1 was observed in lung cancer cell lines, but the expression in squamous cell carcinoma cell line A431 and small cell lung cancer cell line NCI-H446 was markedly higher than that in adenocarcinoma cell lines SPC-A1 and Y-90. The results suggest that the human lung cancer cell lines detected have overexpression of hnRNP A2/B1 and hnRNP B1.