Resistant A549 Cell Line Research Articles

RNAseq reveals extensive metabolic disruptions in the sensitive SF-295 cell line treated with schweinfurthins

The schweinfurthin family of natural compounds exhibit a unique and potent differential cytotoxicity against a number of cancer cell lines and may reduce tumor growth in vivo. In some cell lines, such as SF-295 glioma cells, schweinfurthins elicit cytotoxicity at nanomolar concentrations. However, other cell lines, like A549 lung cancer cells, are resistant to schweinfurthin treatment up to micromolar concentrations. At this time, the precise mechanism of action and target for these compounds is unknown. Here, we employ RNA sequencing of cells treated with 50 nM schweinfurthin analog TTI-3066 for 6 and 24 h to elucidate potential mechanisms and pathways which may contribute to schweinfurthin sensitivity and resistance. The data was analyzed via an interaction model to observe differential behaviors between sensitive SF-295 and resistant A549 cell lines. We show that metabolic and stress-response pathways were differentially regulated in the sensitive SF-295 cell line as compared with the resistant A549 cell line. In contrast, A549 cell had significant alterations in response genes involved in translation and protein metabolism. Overall, there was a significant interaction effect for translational proteins, RNA metabolism, protein metabolism, and metabolic genes. Members of the Hedgehog pathway were differentially regulated in the resistant A549 cell line at both early and late time points, suggesting a potential mechanism of resistance. Indeed, when cotreated with the Smoothened inhibitor cyclopamine, A549 cells became more sensitive to schweinfurthin treatment. This study therefore identifies a key interplay with the Hedgehog pathway that modulates sensitivity to the schweinfurthin class of compounds.

Indole Curcumin Reverses Multidrug Resistance by Reducing the Expression of ABCB1 and COX2 in Induced Multidrug Resistant Human Lung Cancer Cells

Background: Drug resistance by the cancer cells towards current chemotherapeutic approaches poses a great challenge. In the present study, an indole analogue of a well-known plant derived anticancer molecule, curcumin, was tested for its Multidrug Resistance (MDR) reversing potential in induced multi drug resistant A549 cell line. Materials and Methods: Human lung cancer cell line A549 was made Multidrug Resistant (MDR) by prolonged treatment with low dosage of Docetaxel, an established anticancer drug. The MDR induction was confirmed by morphological evidence, Hoechst 33342 staining, MTT assay, Rhodamine123 staining and RT-PCR of ABCB1 gene. Protein expression studies were carried out using western blotting technique Results and Discussions: The induced MDR A549 cells exhibited significant increase in the gene expression of ABCB1 gene at the transcriptional level. Retention and efflux studies with Pglycoprotein (P-gp) substrate Rh123 indicated that indole curcumin inhibited P-gp mediated efflux of Rhodamine. Furthermore, treatment of MDR A549 cells with indole curcumin showed downregulation of gene expression of ABCB1 and COX 2. This was also confirmed from the decreased protein expression of COX 2. Conclusion: The results of the present study indicate that indole curcumin reverses multi drug resistance by downregulating the expression of ABCB1 and COX 2 genes. Thus, indole curcumin may act as a potent modulator for ABCB1 and COX 2 mediated MDR in lung cancer.

Ginsenoside Rd reverses cisplatin resistance in non-small-cell lung cancer A549 cells by downregulating the nuclear factor erythroid 2-related factor 2 pathway.

Clinical drug resistance to platinum-based chemotherapy is considered a major impediment in the successful treatment of non-small-cell lung cancer (NSCLC). The nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway regulates the oxidative stress response, and in many cancer types, the high constitutive expression of NRF2 leads to proliferation and chemoresistance. Ginsenoside Rd (GS-Rd) is the main active component of ginsenosides. Here, GS-Rd was found to inhibit the proliferation of A549 lung cancer cells and induce G0/G1 phase arrest. We established cisplatin (DDP)-resistant A549 cell lines (A549/DDP). The half maximal inhibitory concentrations of DDP, gemcitabine, and adriamycin were much higher in A549/DDP cells than in A549 cells. The A549/DDP cell lines developed multidrug resistance, accompanied by activation of multidrug resistance protein 1 and multidrug resistance-associated protein 1, as well as NRF2 and its target genes. Treatment with GS-Rd inhibited the NRF2 pathway and significantly sensitized A549/DDP cells to therapeutic drugs. In addition, NRF2 knockdown attenuated the synergistic effects of GS-Rd in both A549 and A54/DDP cells. Taken together, these data show that NRF2 plays an important role in acquired drug resistance in NSCLC, and GS-Rd may ameliorate this chemoresistance by downregulating the NRF2 pathway. This study demonstrates that the NRF2 pathway may serve as a therapeutic target in NSCLC, and ginseng compounds may be effective for the treatment of this disease.

Abstract 1418: Brother of regulator of imprinted sites (BORIS) inhibits cisplatin induced DNA damage in non-small-cell lung cancer

Abstract Cisplatin (DDP) treatment is primary modality for non-small-cell lung cancer (NSCLC) chemotherapy. However, patients benefited from DDP treatment limitedly due to the DDP resistance. Brother of Regulator of Imprinted Sites (BORIS, CTCFL) is the paralogue of CCCTC-binding factor (CTCF) and expressed abnormally in most type of cancers and is potential tumor target for breast, cervical carcinoma and lung cancer. However, the funciton of BORIS on carcinoma is unrevealed. In our previous study, we found that BORIS suppressed apoptosis and resisted 5-FU (Fluorouracil) treatment in colorectal cancer. As either 5-FU or DDP induces DNA damage and apoptosis of carcinoma cells, BORIS perhaps resists DDP treatment too. Based on the prevalent expression of BORIS and the high incidence of DDP resistance in NSCLC, we proposed that BORIS contributed to DDP resistance by suppressing DNA damage in NSCLC. We collected RNA sequencing data of 156 NSCLC patients who received DDP chemotherapy from The Cancer Genome Atlas and analyzed the expressions of BORIS. We found high BORIS expression correlated with short survival in NSCLC patients who received DDP chemotherapy. BORIS expression declined from prospective to retrospective collected tissue which responded to DDP chemotherapy. It suggested the probability function of BORIS on DNA damage repair. In cell cultured lung cancer cell lines, BORIS was silenced or over-expressed including DDP resistant cell line. It indicated that A549/DDP (DDP resistant A549 cell line) expressed relative higher BORIS than A549 parental cells and BORISexpression levels correlated with cell viability. H1299 and A549/DDP expressed comparative BORIS and growed comparativly. H460 and A549 expressed relative lower BORIS levels and grew slower than H1299 and A549/DDP. BORIS knockdown inhibited the proliferation of NSCLC cells, enhanced the sensitivity of A549/DDP and H1299 to DDP treatment and induced DNA damage. Reversely DDP induced DNA damage was suppressed by BORIS over-expression. The excision repairing gene ERCC1, which is regulated by BORIS, might be the assistant for DDP resistance. Either high expression of BORIS or ERCC1 indicated short survival rates of NSCLC patients. Our data suggested that BORIS suppressed DNA damage and promoted the progression of NSCLC and DDP resistance, which indicated its potential in NSCLC prognosis and therapy. Citation Format: Yongfei Song, Chao Li, Mengdie Fang, Juan Ren, Jianfei Fang, Xiaoju Wang, Yanmei Zhang. Brother of regulator of imprinted sites (BORIS) inhibits cisplatin induced DNA damage in non-small-cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1418.

The synthetic peptide CIGB-300 modulates CK2-dependent signaling pathways affecting the survival and chemoresistance of non-small cell lung cancer cell lines

BackgroundLung cancer is the most frequently diagnosed cancer and the leading cause of cancer-related deaths worldwide. Up to 80% of cancer patients are classified as non-small-cell lung cancer (NSCLC) and cisplatin remains as the gold standard chemotherapy treatment, despite its limited efficacy due to both intrinsic and acquired resistance. The CK2 is a Ser/Thr kinase overexpressed in various types of cancer, including lung cancer. CIGB-300 is an antitumor peptide with a novel mechanism of action, since it binds to CK2 substrates thus preventing the enzyme activity. The aim of this work was to analyze the effects of CIGB-300 treatment targeting CK2-dependent signaling pathways in NSCLC cell lines and whether it may help improve current chemotherapy treatment.MethodsThe human NSCLC cell lines NCI-H125 and NIH-A549 were used. Tumor spheroids were obtained through the hanging-drop method. A cisplatin resistant A549 cell line was obtained by chronic administration of cisplatin. Cell viability, apoptosis, immunoblotting, immunofluorescence and luciferase reporter assays were used to assess CIGB-300 effects. A luminescent assay was used to monitor proteasome activity.ResultsWe demonstrated that CIGB-300 induces an anti-proliferative response both in monolayer- and three-dimensional NSCLC models, presenting rapid and complete peptide uptake. This effect was accompanied by the inhibition of the CK2-dependent canonical NF-κB pathway, evidenced by reduced RelA/p65 nuclear levels and NF-κB protein targets modulation in both lung cancer cell lines, as well as conditionally reduced NF-κB transcriptional activity. In addition, NF-κB modulation was associated with enhanced proteasome activity, possibly through its α7/C8 subunit. Neither the peptide nor a classical CK2 inhibitor affected cytoplasmic β-CATENIN basal levels. Given that NF-κB activation has been linked to cisplatin-induced resistance, we explored whether CIGB-300 could bring additional therapeutic benefits to the standard cisplatin treatment. We established a resistant cell line that showed higher p65 nuclear levels after cisplatin treatment as compared with the parental cell line. Remarkably, the cisplatin-resistant cell line became more sensitive to CIGB-300 treatment.ConclusionsOur data provide new insights into CIGB-300 mechanism of action and suggest clinical potential on current NSCLC therapy.

Abstract 1787: Identification of factors governing sensitivity of NSCLC cell lines to inhibition of Poly (ADP-Ribose) Polymerase (PARP).

Abstract PARP inhibitors are emerging as important new anticancer agents, especially in cancers defective in homologous recombination (HR) repair, such as those lacking BRCA1 or BRCA2. To date, the potential role of PARP inhibition in NSCLC has not been extensively studied. As previously reported, we examined a panel of 26 genotypically-defined NSCLC cell lines for sensitivity to the PARP-1 inhibitor AG014699 (rucaparib) using colony formation assays. We identified 5 cell lines that are exquisitely sensitive (IC50 ≈ 50nM), 9 cell lines that are moderately sensitive (IC50 ≈ 60-500nM) and 12 cell lines that are resistant (IC50 > 5 μM). These data were confirmed with a second PARP inhibitor, AZD2281 (Olaparib). To determine whether sensitive cell lines have defective HR compared to resistant cell lines, we examined the formation of RAD51 foci. Both sensitive and resistant cell lines demonstrated efficient formation of RAD51 foci as well as gamma-H2AX foci in response to DNA damaging treatments, indicating correct initiation of HR. In resistant cell lines, the repair process following PARP inhibition is completed within 24 hours, with resolution of RAD51 and gamma-H2AX foci. However, in sensitive cell lines, these foci persist greater than 30 hours following DNA damaging treatments. These results suggest that although sensitive lines are able to initiate HR repair, they are unable to complete the process, so that RAD51 foci are not resolved. The persistence of DNA damage in these cell lines is confirmed by persistent gamma-H2AX focus formation as well as S/G2 arrest, the latter representing intact checkpoint control. Our results suggest that proteins required downstream of RAD51, including helicases and Holliday junction resolvases, are lacking in the sensitive lines. Consistent with this hypothesis, NCI-H520 cells, a highly PARP inhibitor-sensitive cell line, lacks expression of WRN, RECQL4 and RECQL5. siRNA-mediated depletion of these proteins individually sensitizes the resistant A549 cell line to PARP inhibition, with reduction in rucaparib IC50 by 100 fold. Our results may define a novel subset of NSCLCs with a late-step defect in HR that can be approached with PARP inhibition as a novel treatment strategy. Citation Format: Naveen Kommajosyula, Ye Cao, Lisa Moreau, Alan D'Andrea, Geoffrey I. Shapiro. Identification of factors governing sensitivity of NSCLC cell lines to inhibition of Poly (ADP-Ribose) Polymerase (PARP). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1787. doi:10.1158/1538-7445.AM2013-1787

Screening metastasis-associated genes from anoikis resistant A549 lung cancer cells by human genome array

背景与目的正常上皮或内皮细胞脱离细胞外基质会发生脱落凋亡，但肿瘤细胞可不依赖细胞基质生长而不发生凋亡，这一现象被称为抗脱落凋亡，目前国内外相关研究均认为抗脱落凋亡是肿瘤发生转移的始动环节。本实验旨在比较抗脱落凋亡及正常贴壁生长肺腺癌A549细胞基因组表达差异，从中筛选肺癌转移相关基因。方法利用多聚羟乙基甲基丙烯酸树脂处理培养皿，致细胞无法贴壁生长，建立抗脱落凋亡肺癌A549细胞系；利用北京博奥生物芯片公司的人类V2.0全基因组寡核苷酸微阵列芯片，检测其与正常贴壁生长A549细胞的基因表达差异性，筛选肺癌转移相关基因。结果共得到表达差异的基因745个，从中筛选出63个与肺癌转移密切相关的基因。结论成功建立抗脱落凋亡肺癌A549细胞系并筛选出转移相关差异表达基因，为进一步研究肺癌转移相关信号转导通路及其它相关研究提供依据。

Establishment of Methotrexate Enantiomers Resistant A549 Cell Lines of Human NSCCL and Its Biological Characteristics

Objective To establish methotrexate(MTX) enantiomers resistant human NSCCL A549 cell line(L-(+)-MTX/A549,D-(-)-MTX/A549) derived from the A549 cell line and observe its biological characters.Methods A549 cells were exposed to intermittently and progressively increasing doses of MTX enantiomers.Two MTX enantiomers(L and D) resistant human NSCLC A549 cell lines were established.The morphology was observed by inverted phase contrast microscope,the sensitivity of the L-(+)-MTX/A549,D-(-)-MTX/A549 cell lines to MTX enantiomers was measured by cytotoxicity test.The cell growth curve was determined by MTT assay,the change of CFSE fluorescence intensity and cell cycle distribution were analyzed by flow cytometry assay.Results The L-(+)-MTX/A549 resistance index was 6.0 and D-(-)-MTX/A549 resistance index was 20.2.Inverted phase contrast microscope showed morphological changes of L-(+)-MTX/A549 and D-(-)-MTX/A549.Cell growth curve demonstrated that the proliferation abilities of L-(+)-MTX/A549 was decreased significantly,while the cell proliferation rate of D-(-)-MTX/A549 was similar to that of A549.The flow cytometry results showed L-(+)-MTX/A549 and D-(-)-MTX/A549 cells at S phase were reduced(P0.05),with an increased at G0/G1 phase(P0.05),the CFSE mean fluorescence intensity of A549 parent cells,L-(+)-MTX/A549 and D-(-)-MTX/A549 were(6.08±0.55),(7.72±0.30),(6.90±0.18).Two enantiomers resistance cell lines have a clear difference in Chirality.Conclusion We established two MTX enantiomers resistant human A549 cell lines and observed their different biological characteristics.It provides a new experimental model for further studying the mechanism of MTX enantiomers resistance.

A highly epothilone B–resistant A549 cell line with mutations in tubulin that confer drug dependence

A 95-fold epothilone B (EpoB)-resistant, but not dependent, A549 human lung carcinoma cell line, A549.EpoB40 (EpoB40), has a Gln to Glu mutation at residue 292 that is situated near the M-loop of betaI-tubulin. Further selection of this cell line with higher concentrations of EpoB produced A549.EpoB480 (EpoB480), which is approximately 900-fold resistant to EpoB. This cell line, like EpoB40, exhibits cross-resistance to Taxol and extreme sensitivity to vinblastine, but in contrast to EpoB40 it is unusually dependent on EpoB, requiring a minimum of 125 nmol/L EpoB to maintain normal growth. Sequence analysis of the beta-tubulin and Kalpha1-tubulin genes in EpoB480 showed that, in addition to the beta292 mutation, beta60 was mutated from Val to Phe and alpha195 was mutated from Leu to Met. Mass spectrometry indicated that both the Val(60)Phe and Leu(195)Met mutations in betaI- and Kalpha1-tubulin, respectively, were expressed at the protein level. Molecular modeling indicated that beta60 is located at the end of the H1-S2 loop that has been implicated as a principal partner of the M-loop for contacts between protofilaments. A mutation at beta60 could inhibit the lateral contacts between protofilaments, thereby destabilizing microtubules. alpha195 is located at the external surface of the microtubule that has been proposed as the domain that interacts with a variety of endogenous proteins, such as stathmin and microtubule-associated protein 4. A mutation at alpha195 could modulate the interactions between tubulin and regulatory proteins. We propose that the betaVal(60)Phe mutation plays a critical role in the drug-dependent phenotype of EpoB480 cells.