CL1-5 Cells Research Articles

The inhibition of lung cancer cell migration by AhR-regulated autophagy

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is highly expressed in multiple organs and tissues. Whereas AhR mediates the metabolism of xenobiotic and endogenous compounds, its novel function in cancer epithelial-mesenchymal transition (EMT) remains controversial. Autophagy also participates in tumour progression through its functions in cell homeostasis and facilitates adaptation to EMT progression. In the present study, we found that AhR-regulated autophagy positively modulates EMT in non-small cell lung cancer cells. The motility of A549, H1299, and CL1-5 cells were correlated with different AhR expression levels. Invasive potential and cell morphology also changed when AhR protein expression was altered. Moreover, AhR levels exerted a contrasting effect on autophagy potential. Autophagy was higher in CL1-5 and H1299 cells with lower AhR levels than in A549 cells. Both AhR overexpression and autophagy inhibition decreased CL1-5 metastasis in vivo. Furthermore, AhR promoted BNIP3 ubiquitination for proteasomal degradation. AhR silencing in A549 cells also reduced BNIP3 ubiquitination. Taken together, these results provide a novel insight into the cross-linking between AhR and autophagy, we addressed the mechanistic BNIP3 modulation by endogenous AhR, which affect cancer cell EMT progression.

Chlorella sorokiniana induces mitochondrial-mediated apoptosis in human non-small cell lung cancer cells and inhibits xenograft tumor growth in vivo

BackgroundLung cancer is one of the leading causes of cancer related deaths worldwide. Marine microalgae are a source of biologically active compounds and are widely consumed as a nutritional supplement in East Asian countries. It has been reported that Chlorella or Chlorella extracts have various beneficial pharmacological compounds that modulate immune responses; however, no studies have investigated the anti-cancer effects of Chlorella sorokiniana (CS) on non-small cell lung cancer (NSCLC).MethodsIn this study, we evaluated the anti-cancer effects of CS in two human NSCLC cell lines (A549 and CL1-5 human lung adenocarcinoma cells), and its effects on tumor growth in a subcutaneous xenograft tumor model. We also investigated the possible molecular mechanisms governing the pharmacological function of CS.ResultsOur results showed that exposure of the two cell lines to CS resulted in a concentration-dependent reduction in cell viability. In addition, the percentage of apoptotic cells increased in a dose-dependent manner, suggesting that CS might induce apoptosis in human NSCLC cells. Western blot analysis revealed that exposure to CS resulted in increased protein expression of the cleaved/activated forms of caspase-3, caspase-9, and PARP, except caspase-8. ZDEVD (caspase-3 inhibitor) and Z-LEHD (caspase-9 inhibitor) were sufficient at preventing apoptosis in both A549 and CL1-5 cells, proving that CS induced cell death via the mitochondria-mediated apoptotic pathway. Exposure of A549 and CL1-5 cells to CS for 24 h resulted in decreased expression of Bcl-2 protein and increased expression of Bax protein as well as decreased expression of two IAP family proteins, survivin and XIAP.ConclusionsWe demonstrated that CS induces mitochondrial-mediated apoptosis in NSCLC cells via downregulation of Bcl-2, XIAP and survivin. In addition, we also found that the tumors growth of subcutaneous xenograft in vivo was markedly inhibited after oral intake of CS.

Development of pH-Sensitive Cationic PEGylated Solid Lipid Nanoparticles for Selective Cancer-Targeted Therapy.

Solid lipid nanoparticles (SLNs) are suitable candidates for the delivery of various anti-cancer drugs. However, currently insufficient tumor-permeability and non-specific uptake by the reticuloendothelial system limits the application of SLNs. Here, we developed novel pH-sensitive cationic polyoxyethylene (PEGylated) SLNs (PEG-SLNs+) that could accumulate long-term at various tumor sites to enhance the therapeutic efficiency of camptothecin (CPT). These CPT-loaded PEG-SLNs+ (CPT-PEG-SLNs+) were spherical nanoparticles, with small size (∼52.3±1.7 nm), positive charge (∼34.3±3.5 mV) and high entrapment efficiency (∼99.4±1.7%). Drug release profile indicated the overall released amount of CPT from CPT-PEG-SLNs+ at pH 5.5 was 20.2% more than at pH 7.4, suggesting CPT-PEG-SLNs+ were a pH-sensitive SLNs. This PEG-SLNs+ could be efficiently uptaken into cells to inhibit the proliferation of CL1-5 cells (IC50 = 0.37 ±0.21 ug/ml) or HCC36 cells (IC50 = 0.16±0.43 ug/ml). In living animal, our PEG-SLNs+ could accumulate long-term (for more than 120 hours) in various types of tumor, including human lung carcinoma (NCI-H358, CRL5802, CL1-5), human colon carcinoma (HCT-116) and human hepatocellular carcinoma (HCC36), and CPT-PEG-SLNs+ could efficiently enhance the therapeutic efficiency of CPT to suppress the growth of the HCC36 or CL1-5 tumors. Therefore, Successful development of these pH-sensitive PEGylated cationic SLNs may provide a selective and efficient drug delivery system for cancer therapy.

Human lung adenocarcinoma cells with an EGFR mutation are sensitive to non-autophagic cell death induced by zinc oxide and aluminium-doped zinc oxide nanoparticles.

Lung cancer, mostly non-small cell lung cancer (NSCLC), is the leading cause of cancer deaths; however, efficient treatments for NSCLC remain insufficient. The objective of this study was to investigate the effects of an epidermal growth factor receptor (EGFR) mutation on autophagic cell death in human lung adenocarcinoma cells by 20-nm zinc oxide nanoparticles (ZnONP20) and aluminum-doped ZnONPs (Al-ZnONP20). Two types of human lung adenocarcinoma cells were used throughout the study: wild-type EGFR A549 cells and EGFR-mutated CL1-5 cells. We observed that a significant reduction in cell viability resulting from ZnONP20 and Al-ZnONP20 occurred in A549 and CL1-5 cells after 18 and 24 hr of exposure. A colony formation analysis showed that A549 cells re-grew after exposure to 20 μg/mL Al-ZnONP20. Levels of light chain 3 (LC3) II conversion were activated by ZnONP20 and Al-ZnONP20 in A549 cells, whereas LC3 II was inhibited by ZnONP20 and Al-ZnONP20 in CL1-5 cells. In conclusion, we have shown that human lung adenocarcinoma cells with an EGFR mutation are sensitive to ZnONP20 and Al-ZnONP20, which may have resulted in non-autophagic cell death. ZnONP20 and Al-ZnONP20 may have the potential for personalized therapeutics in NSCLC with an EGFR mutation.

Distinct metabolic changes in human lung cancer cells with differential radiation sensitivities

Background: Radiotherapy is one of the most important modalities for cancer treatment. However, the radio-resistance of malignant tumor cells is the major cause of radiation treatment failure. Although several biochemical pathways are disturbed in response to radiation, the metabolite profiles related to radio-resistance have not been well investigated in malignant non-small cell lung carcinomas (NSCLCs). Malignant lung cancer cells, CL1-5, were more resistant to radiation than were the parental CL1-0 cells. Therefore, the two cell lines provide a suitable cell model to investigate changes in metabolite profiles related to radiation exposure. Methods: Two human lung cancer cell lines, CL1-5 and CL1-0, with differential radiation sensitivities, were irradiated at a dosage of 10 Gy and harvested at 1, 4, and 24 hr after radiation treatment. Proton nuclear magnetic resonance ( 1 H-NMR) was used to analyze the metabolic profiles of the CL1-0 and CL1-5 cell lines. The metabolite profiles of the cell extracts were subjected to principal component analysis (PCA). Both PCA and specific metabolite data were used to examine metabolic differences between the two cell lines. Results: The colony formation assay results demonstrate that CL1-5 cells are more sensitive than CL1-0 cells to irradiation. The PCA score plots for NMR spectra of CL1-0 and CL1-5 cells identified metabolites such as glutathione, creatine phosphate, glutamate, o-phosphocholine, pyroglutamate, taurine, and trimethylamine n-oxide (TMAO) as key molecules with high correlation in response to different irradiation tolerance. The metabolite profiles of the cell lines were inherent different, but following 24-hr irradiation, common metabolite alterations were induced in both cell lines. Moreover, CL1-0 cells showed a larger difference in the elevation of glutathione levels than CL1-5 cells. Conclusions: Our study demonstrates a clear difference in metabolite profiles between irradiation-resistant and irradiation-sensitive cells. These metabolite profile changes could be used to elucidate the possible mechanism of radio-resistance.

Mitomycin C treatment induces resistance and enhanced migration via phosphorylated Akt in aggressive lung cancer cells.

Since 1984, mitomycin C (MMC) has been applied in the treatment of non-small-cell lung cancer (NSCLC). MMC-based chemotherapeutic regimens are still under consideration owing to the efficacy and low cost as compared with other second-line regimens in patients with advanced NSCLC. Hence, it is important to investigate whether MMC induces potential negative effects in NSCLC. Here, we found that the malignant lung cancer cells, CL1-2 and CL1-5, were more resistant to MMC than were the parental CL1-0 cells and pre-malignant CL1-1 cells. CL1-2 and CL1-5 cells consistently showed lower sub-G1 fractions post MMC treatment. DNA repair-related proteins were not induced more in CL1-5 than in CL1-0 cells, but the levels of endogenous and MMC-induced phosphorylated Akt (p-Akt) were higher in CL1-5 cells. Administering a p-Akt inhibitor reduced the MMC resistance, demonstrating that p-Akt is important in the MMC resistance of CL1-5 cells. Furthermore, we revealed that cell migration was enhanced by MMC but lowered by a p-Akt inhibitor in CL1-5 cells. This study suggests that in CL1-5 cells, the activity of p-Akt, rather than DNA repair mechanisms, may underlie the resistance to MMC and enhance the cells' migration abilities after MMC treatment.

Laricitrin ameliorates lung cancer-mediated dendritic cell suppression by inhibiting signal transducer and activator of transcription 3.

Natural polyphenolic compounds of grapes and their seeds are thought to be therapeutic adjuvants in a variety of diseases, including cancer prevention. This study was carried out to investigate the effect of grape phenolic compounds on the regulation of cancer-mediated immune suppression. Laricitrin exhibits the greatest potential to ameliorate the suppressive effects of lung cancer on dendritic cells’ (DCs’) differentiation, maturation and function. Human lung cancer A549 and CL1-5 cells change the phenotype of DCs that express to high levels of IL-10 and prime T cells towards an immune suppression type-2 response (Th2). Laricitrin treatment stimulated DC differentiation and maturation in the condition media of cancer cells, a finding supported by monocyte marker CD14's disappearance and DC marker CD1a's upregulation. Laricitrin decreases expression of IL-10 in cancer-conditioned DCs, and subsequently switches CD4+ T cell response from Th2 to Th1 in vitro and in vivo. Reversal of laricitrin on lung cancer-induced DCs’ paralysis was via inhibiting the phosphorylation of signal transducer and activator of transcription 3 (STAT3). Laricitrin also potentiated the anticancer activity of cisplatin in mouse models. Thus, laricitrin could be an efficacious immunoadjuvant and have a synergistic effect when combined with chemotherapy.

Integrin β3 and CD44 levels determine the effects of the OPN-a splicing variant on lung cancer cell growth.

Osteopontin (OPN), a phosphorylated glycoprotein, is frequently overexpressed in cancer. Among the three OPN isoforms, OPN-a is the most highly expressed in lung cancer cell lines and lung tumors. Overexpression of OPN-a greatly reduced CL1-5 lung adenocarcinoma cell growth, but had no effect on growth in A549 lung adenocarcinoma cells. Examination of the expression of integrins and CD44, which are possible OPN-a receptors, revealed that differences in integrin β3 levels might explain this discrepancy between CL1-5 and A549 cells. When integrin β3 was ectopically expressed in A549 cells, OPN-a inhibited their growth, whereas OPN-a increased cell growth following integrin β3 knockdown in CL1-5 cells. This OPN-a-induced increase in growth appeared to result from activation of the CD44/NFκB pathway. Our results demonstrated that OPN-a inhibits growth of cells with high integrin β3 levels and increases growth via activation of the CD44/NFκB pathway in cells with low integrin β3 levels. Thus, OPN-a, integrin β3, and CD44 interact to affect lung cancer cell growth, and this study may aid in the development of cancer treatment strategies involving these molecules.

Knockdown of Long Noncoding RNA PCAT6 Inhibits Proliferation and Invasion in Lung Cancer Cells.

As a newly identified oncogenic long noncoding RNA (lncRNA), prostate cancer-associated transcript 6 (PCAT6) promoted cellular proliferation and colony formation of prostate cancer. However, the biological function of PCAT6 in lung cancer is still largely unknown. In this study, we found that PCAT6 is significantly increased in cancer tissues compared to normal tissues and positively correlates with metastasis of lung cancer in patients. We then examined PCAT6 expression in lung cancer cell lines and identified that PCAT6 expression was significantly elevated in lung cancer cells compared to normal human bronchial epithelial (NHBE) cells, especially in CL1-5 and H446 cells. PCAT6 knockdown significantly inhibited cellular proliferation and metastasis, as well as induced early apoptosis of lung cancer cells. Molecular analysis revealed that PCAT6 regulated the expression of two pivotal cancer-related proteins, c-Myc and p53, in lung cancer cells. However, PCAT6 was not directly combined with c-Myc and p53 as confirmed by RNA immunoprecipitation. Finally, a retrospective study further revealed that PCAT6 negatively correlates with overall survival of lung cancer patients. In conclusion, these results suggest that PCAT6 could play an oncogenic role in lung cancer progression and may serve as a biomarker for prognosis of lung cancer patients.

Abstract 3392: Lung cancer cell derived extracellular vesicles carrying DNAs, RNAs and proteins that may trigger oncogenic signals and promote angiogenesis

Abstract Extracellular vesicles (EVs) secreted from tumor cells have been implicated in modulating the tumor microenvironment changes. In this study, we demonstrated that EVs secreted by lung cancer cell are loaded with several cargos, such as DNA, RNA, cytokines, and growth factors. After the EVs had been taken up by recipient cells, the cargos might activate oncogenic pathways. We firstly isolated EVs from cell-free medium of lung cancer cells lines (AS2, PC9) and malignant pleural effusion (MPE) from patients with lung cancer. Isolated EVs were confirmed by the morphology and size in transmission electron microscopy (TEM) and the presentation of specific EV markers (Alix, CD63, and Tsg101). We found that several RNA species with enrichment of small RNAs and mutated EGFR DNA in the EVs of PC9 cells. After labeling with the green fluorescent dye Cellvue, the uptake of EVs by AS2 cells was imaged by the fluorescent microscopy. With these data, we further investigated the role of EVs in regulating the tumor microenvironment. We found the content of IL-6 in EVs, secreted from AS2 and CL1-5 cells, increased gradually after changing culture medium. And after uptake of EVs by recipient cells the cargos induced higher phsophorylation of Stat3 and AKT compared to the treatment with serum-free medium. Previously, we have shown that IL-6/Stat3/tissue factor (TF)/VEGF pathway enhances lung cancer angiogenesis, metastasis and the generation of MPE. In the current study, we demonstrated that EVs from lung cancer cells and in the MPE contain high level of VEGF and TF. And treatment with the EVs, containing high VEGF and TF, induced more prominent extravasation than cell free medium did in Miles assays. In conclusion, we successfully isolated EVs not only from culture supernatants but also from MPE of lung cancer patients. The EVs contain many biologically active molecules that may modulate behaviors of cancer cells and cells in tumor microenvironment. Citation Format: Chien-Chung Lin, Wei-Lun Huang, Wu-Chou Su. Lung cancer cell derived extracellular vesicles carrying DNAs, RNAs and proteins that may trigger oncogenic signals and promote angiogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3392.

Abstract 1308: Enhanced antitumor activity of rapamycin and genipin, a UCP-2 inhibitor, in lung cancer

Abstract mTOR is constitutively activated in lung cancer. Deletion of TOR1 increases expression of OXPHOS proteins, and mitochondrial uncoupling proteins 2 (UCP-2) expression promotes chemoresistance. Therefore, we evaluated whether adding genipin, a UCP-2 inhibitor, to rapamycin has better antitumor effect in lung cancer. Combined with genipin and rapamycin have a synergistic effect with more cellular apoptosis in A549, H460 and CL1-0 cells. The similar phenomenon was found in the combination treatment with genipin and everolimus or BGT226 (a dual PI3K/mTOR inhibitor) in this three lung cancer cells. It is said that NrF2 may affect UCP-2 expression, and we found NrF2 was activated in lung cancer A549, H460 and CL1-0 cells treated with rapamycin. Everolimus and dual PI3K/mTOR inhibitor, BGT226, also induced NrF2 nuclear translocation in these lung cancer cells. It is said that under oxidant stress, p62 activates NrF2 through P62 Ser351-phosphorylation. We found that rapamycin induced ROS generation, p62 serine phosphorylation and NrF2 downstream mRNA and protein expression, including UCP-2. ROS inhibitor, NAC, and down-regulation of p62 by siRNA suppressed rapamycin-induced UCP-2 expression. Overexpression of p62 by cDNA transfection enhanced NrF2 activation and then UCP-2 expression in lung cancer cells. Moreover, rapamycin-induced increased UCP-2 expression, leading decreased mitochondrial membrane potential and mild increased oxygen consumption rate. Finally in A549 subcutaneous tumor mode in SCID mice, combined genipin and everolimus also demonstrated the greatest antitumor activity. Therefore, inhibition UCP-2 by genipin enhances anticancer effect of rapamycin in lung cancer. Citation Format: Wen-Pin Su, Jang-Yang Chang, Ching-Chuan Kuo, Wu-Chou Su. Enhanced antitumor activity of rapamycin and genipin, a UCP-2 inhibitor, in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1308.

Abstract 1166: The cytosolic domain of a disintergrin and metalloprotease (ADAM) 15 promotes non-small cell lung cancer (NSCLC) invasion and migration

Abstract Emerging evidence has indicated that proteins of a disintergrin and metalloprotease (ADAM) family contribute to cancer progression and metastasis. One member of this family, ADAM15, has been shown to be upregulated in multiple cancers, including gastric, lung, breast, and prostate cancers, and the enzymatic activities of its extracellular metalloprotrease domain promote breast cancer proliferation and migration through mediating ErbB signaling pathway. The patients with ADAM15 high-expressing lung tumors have shorter survival time and enzymatic activity of ADAM15 extracellular domain activates MMP-9 and promotes NSCLC migration and invasion. We firstly demonstrated other than extracellular enzymetic activity, the longest isoform of ADAM15 (ADAM15 i6), which contains the most cytoplasmic Src homology 3 (SH3) binding motifs, significantly upregulated in primary lung cancer tissues and promoted NSCLC proliferation via growth factor receptor-bound protein 2 (Grb2) and Src homolog 2 domain containing (Shc) association. In this study, we further explore the roles of ADAM15 cytosolic domain in NSCLC invasion and migration. Overexpression of ADAM15 i6 promoted CL1-0 cell invasion and migration according to the transwell and wound healing assay. Ablation of Ras protein activator (RASA)1 and protein tyrosine kinase (PTK)6 attenuated the ADAM15 i6-promoted invasion and migration respectively. Thus, we identified a novel mechanism of the ADAM15 cytoplasmic domain in NSCLC tumor progression, which will shed light on the molecular mechanisms of ADAM proteins, and facilitate development of novel therapy in NSCLC. Citation Format: Hsin-Han Hou, Chong-Jen Yu. The cytosolic domain of a disintergrin and metalloprotease (ADAM) 15 promotes non-small cell lung cancer (NSCLC) invasion and migration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1166.

SPANXA suppresses EMT by inhibiting c-JUN/SNAI2 signaling in lung adenocarcinoma.

SPANXA (Sperm Protein Associated with the Nucleus on the X-chromosome, family members A1/A2) acts as a cancer-testis antigen expressed in normal testes, but dysregulated in various tumors. We found that SPANXA is highly expressed in low-invasive CL1-0 cells compared with isogenous high-invasive CL1-5 cells. SPANXA was preferably expressed in tumor tissues and associated with the prolonged survival of lung adenocarcinomas. SPANXA suppressed the invasion and metastasis of lung cancer cells in vitro and in vivo. By the expression microarray and pathway analysis, we found that the SPANXA-altered genes were enriched in the epithelial–mesenchymal transition (EMT) pathway. SPANXA reduced SNAI2 expression resulted in up-regulating E-cadherin. c-JUN acts as the positive-regulator of EMT. Silencing SPANXA increased c-JUN mRNA expression and blockage of c-JUN led to SNAI2 down-regulation. Our results clearly characterized SPANXA as an EMT inhibitor by suppressing c-JUN-SNAI2 axis in lung adenocarcinoma.

Ling Zhi-8 reduces lung cancer mobility and metastasis through disruption of focal adhesion and induction of MDM2-mediated Slug degradation

We recently reported that recombinant Ling Zhi-8 (rLZ-8), a medicinal mushroom Ganoderma lucidum recombinant protein, effectively prevents lung cancer cells proliferation in vivo mice model. In our current study, we demonstrated that rLZ-8 suppressed tumor metastasis and increased the survival rate in Lewis lung carcinoma cell-bearing mice. The epithelial to mesenchymal transition (EMT) process is regarded as the critical event in tumor metastasis. Herein, we showed that rLZ-8 effectively induced changes in EMT by interfering with cell adhesion and focal adhesion kinase (FAK) functions in lung cancer cells. Slug, a transcription factor, represses E-cadherin transcription and is regarded as a critical event in EMT and tumor metastasis. Functional studies revealed that downregulation of Slug as a result of rLZ-8-induced FAK inactivation enhanced E-cadherin expression and repressed cancer cell mobility. Moreover, we found that rLZ-8 enhanced the ubiquitination proteasome pathway (UPP)-mediated degradation of Slug in CL1-5 cells. Mechanistically, we demonstrated that rLZ-8 promoted the interaction between MDM2 and Slug, resulting in Slug degradation; however, MDM2-shRNA abolished rLZ-8-enhanced Slug degradation. This study is the first to determine anti-metastatic activity of rLZ-8 and its potential mechanism, with how the regulation of EMT and cell mobility is via the negative modulation of FAK, and thereby leading to the ubiquitination and degradation of Slug. Our findings suggest that the targets of FAK play a key role in metastasis. Moreover, rLZ-8 may be useful as a chemotherapeutic agent for treating lung cancer.

Tangeretin derivative, 5-acetyloxy-6,7,8,4′-tetramethoxyflavone induces G2/M arrest, apoptosis and autophagy in human non-small cell lung cancer cells in vitro and in vivo

ABSTRACTTangeretin, a major phytochemicals in tangerine peels - an important Chinese herb, has been found to have anti-carcinogenic properties. To improve bioavailability and increase potency of tangeretin, its derivative, 5-acetyloxy-6,7,8,4′-tetramethoxyflavone (5-AcTMF), has been synthesized and shown potent inhibition of proliferation activity against human breast and leukemia cancer cell lines. In this study, we have further investigated the anticancer effects of 5-AcTMF on CL1-5 non-small cell lung cancer cells (NSCLC) both in vitro and in vivo and demonstrated that 5-AcTMF effectively inhibited cancer cell proliferation, induced G2/M-phase arrest associated with cdc2 and CDC25c and increased in the apoptotic cells associated with caspase activation, down regulation of Bcl-2, XIAP and Survivn, inducing release of cytochrome c into the cytosol and disruption of mitochondrial membrane potential. We also found that 5-AcTMF treatment of CL1-5 activated autophagy, indicated by triggered autophagosome formation and increased LC3-II levels and formation of LC3 puncta. Moreover, we also found that 5-AcTMF lowered phophoatidylinositol 3-kinase/AKT/mTOR signaling pathway. Over-expression of AKT by AKT cDNA transfection decreased 5-AcTMF mediated apoptosis and autophagy, supporting the induction of apoptosis and autophagy by inhibition of AKT pathway. In an animal study, 5-AcTMF effectively delayed tumor growth in a nude mouse model of CL1-5 xenografts without observed adverse effect. Immunohistochemistry Analysis indicated that 5-AcTMF induced CL1-5 cell apoptosis and autophagy in vivo. Taken together, these data demonstrate that 5-AcTMF is a novel small molecule agent that can inhibit NSCLC cell proliferation, and induce G(2)/M phase arrest and via the mitochondrial apoptotic pathway and autophagy.

Using the matrix-induced ion suppression method for concentration normalization in cellular metabolomics studies.

Studies of the cell metabolome greatly improve our understanding of cell biology. Currently, most cellular metabolomics studies control only cell numbers or protein content without adjusting the total metabolite concentration, mainly because of the lack of an effective concentration normalization method for cell metabolites. This study proposed a matrix-induced ion suppression (MIIS) method to measure the total amount of cellular metabolites by utilizing flow injection analysis coupled with electrospray ionization mass spectrometry (FIA-ESI-MS).We used series dilutions of HL-60 cell extracts to establish the relationship between cellular metabolite concentration and the degree of ion suppression of the ion suppression indicator, and a good correlation was obtained between 2- and 12-fold dilutions of cell extracts (R(2) = 0.999). Two lung cancer cells, CL1-0 and CL1-5, were selected as the model cell lines to evaluate the efficacy of the MIIS method and the importance of metabolite concentration normalization. Through MIIS analysis, CL1-0 cells were found to contain metabolites at a concentration 2.1 times higher than in CL1-5, and the metastatic properties of CL1-5 could only be observed after 2.1-fold dilution of CL1-0 before metabolomic analysis. Our results demonstrated that the MIIS method is an effective approach for metabolite concentration normalization and that controlling metabolite concentrations can improve data integrity in cellular metabolomics studies.

Abstract 3065: MicroRNA-137 confers chemoresistance by suppressing CASP3 in lung cancer cells

Abstract Lung cancer is the leading cause of cancer-related death. Although molecular targeting therapy (MTT) could benefit patients with EGFR activating mutations, chemotherapy still is the first choice of EGFR wild type patients, MTT failed ones and patients of developing countries without regarding their EGFR status. However most of these chemotherapy-treated patients will develop drug resistance eventually. Here, we identified miR-137 might contribute to chemo-resistance in lung cancer in part. Our previous study identified 5 miRNAs (let-7a, miR-182*, miR-372, miR-137 and miR-221) whose expression can be an independent marker to predict the outcome of non-small cell lung cancer (NSCLC) patients. Among them, miR-137 acts as a risky miRNA. Caspase-3 (CASP3) was predicted as one of miR-137 potential targets. CASP3 is a critical effector caspase that promotes programmed cell-death when activated in the apoptotic cells both by extrinsic and intrinsic pathways that are highly conserved and related to drug resistance. We found miR-137 could regulate CASP3 through binding to its 3′UTR. Endogenous CASP3 can be up-regulated by over-expressing miR 137 in CL1-5, H1437 and A549 cells and down-regulated by silencing miR-137 in H1299, CL1-0 and A549 cells. Suppression of CASP3 by miR-137 providing lung cancer cells with ability of anti-apoptosis under UV-induced cell death. MiR-137 also increase IC50 of CL1-5, H1437, A549 and H1975 cells under cisplatin treatment to at least 2 folds. Restoration of CASP3 could reverse the effect. Our IHC data also shows the CASP3 protein expression is negative correlated with miR-137 level. In summary, manipulation of miR-137 might provide a potential treatment for EGFR wild-type and MTT failed NSCLC. patients in the future. Citation Format: Te-Jen Shu, Sung-Liang Yu, Jeremy J.W. Chen. MicroRNA-137 confers chemoresistance by suppressing CASP3 in lung cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3065. doi:10.1158/1538-7445.AM2015-3065

Syringetin suppresses osteoclastogenesis mediated by osteoblasts in human lung adenocarcinoma.

Bone metastasis in lung cancer results in an unfavorable outcome for patients by not only impairing the quality of life, yet also increasing the cancer-related death rates. In the present study, we discuss a novel treatment strategy that may benefit these patients. Human CD14+ monocytes treated with macrophage-colony stimulating factor (M-CSF)/receptor activator of nuclear factor κB ligand (RANKL) differentiated into osteoclasts, whereas syringetin (SGN), a flavonoid derivative found in both grapes and wine, suppressed the osteoclastogenesis in vitro in a dose-dependent manner. In addition, SGN inhibited osteoclast formation induced by human lung adenocarcinoma A549 and CL1-5 cells. The associated signaling transduction pathway in osteoclastogenesis and SGN inhibition was found to be via the AKT/mammalian target of rapamycin (mTOR) signaling pathway. Blocking AKT and mTOR by respective inhibitors significantly decreased lung adenocarcinoma-mediated osteoclastogenesis. Moreover, SGN regulated the lung adenocarcinoma-mediated interaction between osteoblasts and osteoclasts by suppressing the stimulatory effect of lung adenocarcinoma on M-CSF and RANKL production in osteoblasts, and reversing the inhibitory effect of the lung adenocarcinoma on OPG production in osteoblasts. The present study has two novel findings. It is the first to illustrate lung adenocarcinoma-mediated interaction between osteoblasts and osteoclasts, leading to osteolytic bone metastasis. It also reveals that SGN, a flavonoid derivative, directly inhibits osteoclastogenesis and reverses lung adenocarcinoma-mediated osteoclastogenesis. In conclusion, the present study suggests that SGN, a natural compound, prevents and treats bone metastasis in patients with lung cancer.

An increase in glucosylceramide synthase induces Bcl-xL-mediated cell survival in vinorelbine-resistant lung adenocarcinoma cells.

Reversing drug resistance with concurrent treatment confers anticancer benefits. In this study, we investigated the potential mechanism of glucosylceramide synthase (GCS)-mediated vinca alkaloid vinorelbine (VNR) resistance in human lung adenocarcinoma cells. Compared with PC14PE6/AS2 (AS2) and CL1-0 cells, apoptotic analysis showed that both A549 and CL1-5 cells were VNR-resistant, while these cells highly expressed GCS at the protein level. VNR treatment significantly converts ceramide to glucosylceramide in VNR-resistant cells; however, pharmacologically inhibiting GCS with (±)-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride (PDMP) induced ceramide accumulation, accompanied by a decrease in glucosylceramide. Under concurrent treatment with VNR and PDMP, an increase in cell apoptosis could be identified; furthermore, genetically silencing GCS confirmed these effects. In VNR-resistant cells, Bcl-xL expression was aberrantly increased, while pharmacologically inhibiting Bcl-xL with ABT-737 sensitized cells to VNR-induced apoptosis. Conversely, enforced expression of Bcl-xL strengthened the survival response of the VNR-susceptible cells AS2 and CL1-0. Without changes in mRNA expression, Bcl-xL was overexpressed independent of β-catenin-mediated transcriptional regulation in VNR-resistant cells. Simultaneous GCS inhibition and VNR treatment caused a decrease in Bcl-xL expression. According to these findings, an increase in GCS caused Bcl-xL augmentation, facilitating VNR resistance in lung adenocarcinoma cells.

Quantitative proteomics reveals a novel role of karyopherin alpha 2 in cell migration through the regulation of vimentin-pErk protein complex levels in lung cancer.

Karyopherin alpha 2 (KPNA2) is overexpressed in various human cancers and is associated with cancer invasiveness and poor prognosis. Herein, to understand the essential role of KPNA2 protein complexes in cancer progression, we applied stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomic strategy combined with immunoprecipitation (IP) to investigate the differential KPNA2 protein complexes in lung adenocarcinoma cell lines with different invasiveness potentials. We found that 64 KPNA2-interaction proteins displayed a 2-fold difference in abundance between CL1-5 (high invasiveness) and CL1-0 (low invasiveness) cells. Pathway map analysis revealed that the formation of complexes containing KPNA2 and cytoskeleton-remodeling-related proteins, including actin, beta tubulin, tubulin heterodimers, vimentin, keratin 8, keratin 18, and plectin, was associated with cancer invasiveness. IP demonstrated that the levels of KPNA2-vimentin-pErk complexes were significantly higher in CL1-5 cells than in CL1-0 cells. The KPNA2-vimentin-pErk complex was also up-regulated in the advanced stage compared with the early-stage lung adenocarcinoma tissues. Importantly, the levels of pErk as well as cell migration ability were significantly reduced in KPNA2-knockdown cells; however, migration was restored by treatment with pErk phosphatase inhibitors. Collectively, our results demonstrate the usefulness of a SILAC-based proteomic strategy for identifying invasiveness-associated KPNA2 protein complexes and provide new insight into the KPNA2-mediated modulation of cell migration.