Identification of Up- and Down-Regulated Proteins in Pemetrexed

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Identification of up- and down-regulated proteins in pemetrexed-resistant human lung adenocarcinoma: flavin reductase and calreticulin play key roles in the development of pemetrexed-associated resistance Hsiu-Chuan Chou, Jing-Yi Chen, Dai-Ying Lin, Yueh-Feng Wen, Chi-Chen Lin, Sheng-Hao Lin, ChingHsiung Lin, Ting-Wen Chung, En-Chi Liao, Ying-Jen Chen, Yu-Shan Wei, Yi-Ting Tsai, and Hong-Lin Chan J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00794 • Publication Date (Web): 09 Oct 2015 Downloaded from http://pubs.acs.org on October 21, 2015

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Journal of Proteome Research

Identification of up- and down-regulated proteins in pemetrexed-resistant human lung adenocarcinoma: flavin reductase and calreticulin play key roles in the development of pemetrexed-associated resistance Hsiu-Chuan Chou1,*, Jing-Yi Chen2,*, Dai-Ying Lin2,*, Yueh-Feng Wen2,3,*, Chi-Chen Lin4,5,6,7, Sheng-Hao Lin4,7, Ching-Hsiung Lin7,8,9, Ting-Wen Chung2, En-Chi Liao2, Ying-Jen Chen2, Yu-Shan Wei2, Yi-Ting Tsai2, and Hong-Lin Chan2, # 1

Department of Applied Science, National Hsinchu University of Education, Hsinchu, Taiwan

2

Department of Medical Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan 3

Department of Internal Medicine, National Taiwan University Hospital Hsinchu Branch, Hsinchu, Taiwan 4 Institute of Biomedical Science, National Chung-Hsing University, Taichung, Taiwan 5 Institute of Biomedical Science, and Rong Hsing Research Center for Translational Medicine, National Chung Hsing University 6 Department of Medical Research and Education, Taichung Veterans General Hospital, Taichung, Taiwan 7 Division of Chest Medicine, Department of Internal Medicine, Changhua Christian Hospital, Changhua, Taiwan 8 Department of Respiratory Care, College of Health Sciences, Chang Jung Christian University, Tainan 9 School of Medicine, Chung Shan Medical University, Taichung, Taiwan *

Equal contribution of these authors.

#

Correspondence to: Dr. Hong-Lin Chan, Institute of Bioinformatics and Structural Biology & Department of Medical Sciences, National Tsing Hua University, No.101, Kuang-Fu Rd. Sec.2, Hsin-chu, 30013, Taiwan. Tel: 886-3-5742476; Fax: 886-3-5715934; E-mail: [email protected] Running Title: Proteomic Analysis of Pemetrexed Resistance Related Proteins Abbreviations: DIGE, differential gel electrophoresis; PEM, pemetrexed

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Abstract Drug resistance is one of the major causes of cancer chemotherapy failure. In the current study, we used a pair of lung adenocarcinoma cell lines, A549 and the pemetrexed-resistant A549/PEM cells, as a model to monitor resistance-dependent cellular responses and identify potential therapeutic targets. By means of two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), we investigated the global protein expression alterations induced by pemetrexed treatment and resistance. The proteomic result revealed that pemetrexed exposure obviously altered the expression of 81 proteins in the A549 cells, whereas no significant response was observed in the similarly treated A549/PEM cells, hence implying an association between these proteins and the drug-specific response. Moreover, 72 proteins including flavin reductase and calreticulin demonstrated differential expression between the A549 and A549/PEM cells, indicating baseline resistance. Additional tests employed siRNA silencing, protein over-expression, cell viability analysis, and analysis of apoptosis to examine and confirm the potency of flavin reductase and calreticulin proteins in the development of pemetrexed resistance. To sum up, By using a proteomic approach, we identified numerous proteins, including flavin reductase and calreticulin, involved in pemetrexed drug resistance-developing mechanisms. Our results provide useful diagnostic markers and therapeutic candidates for pemetrexed-resistant lung cancer treatment.

Key Words: proteomics, DIGE, pemetrexed, resistance, lung adenocarcinoma


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Introduction Drug resistance reduces the effectiveness of medication used in the treatment of diseases such as cancer. In clinical practice, drug resistance has been recognized as a severe problem when the anticancer-drug concentrations, administered at dosages required for eradicating tumor cells, reach toxic levels. Biochemical mechanisms correlated with drug resistance have been described through the enhanced activities of plasma membrane drug-efflux transporter molecules such as ABC-transporters, the activation of drug targeting molecules responsible for DNA repair as well as detoxification and the modulation of cellular signal transduction pathways leading to cell death 1. Pemetrexed is a chemically analogous of folic acid and is currently used as a chemotherapeutic drug against various tumors including advanced nonsquamous non-small cell lung cancer (NSCLC) as well as malignant pleural mesothelioma. It functions by inhibiting three enzymes in one-carbon transfer pathway: thymidylate synthase, dihydrofolate reductase and glycinamide ribonucleotide formyltransferase used in purine and pyrimidine synthesis, thus further preventing the synthesis of DNA and RNA, which are essential for the survival and growth of normal and cancer cells 2. In addition, pemetrexed is used to combine with platinum-based chemotherapy for advanced NSCLC and malignant peritoneal mesothelioma 3,4. In NSCLC, the average thymidylate synthase expression is relatively lower in adenocarcinomas rather than in squamous cell carcinomas and the similar results are also observed in clinical trials 5. Hence, advanced nonsquamous NSCLC has a higher pemetrexed response rate than the other lung adenocarcinomas. Conversely, malignant pleural mesothelioma, a rare and a highly lethal tumor, is almost unresponsive to conventional radiotherapy or chemotherapy, but shows a significant survival benefit by treatment with pemetrexed 6. However, pemetrexed resistance has been widely reported in cancer research on colon



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cancer, leukemia and lung cancer

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7-10

. This resistance is a major barrier to the

successful treatment of cancer patients receiving chemotherapy. Thus, a clearer understanding of the molecular mechanisms of pemetrexed resistance is essential to facilitate effective pemetrexed usage. In this study, to investigate pemetrexed resistance mechanisms in lung cancer in vitro, increase our understanding of the molecular processes involved, and identify potential resistance disease markers with possible diagnostic or therapeutic applications, we established

a

pair

of

lung

adenocarcinoma

cell

lines,

A549

and

the

pemetrexed-resistant partners, A549/PEM cells, as a model system. We investigated pemetrexed-resistance-associated protein alterations by conducting a quantitative proteomic analysis using 2D-DIGE and MALDI-TOF MS. Moreover, we performed tests involving siRNA silencing and protein over-expression against the selected identified proteins, flavin reductase and calreticulin, to monitor and evaluate their potencies against pemetrexed chemotherapy resistance.


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Materials and Methods Chemical and Reagents Generic chemicals were purchased from USB corporation company (Santa Clara, CA, USA), while reagents for 2D-DIGE were purchased from GE Healthcare (Uppsala, Sweden). All primary antibodies were purchased from Genetex (Hsinchu, Taiwan) and anti-rabbit secondary antibodies were purchased from GE Healthcare (Uppsala, Sweden). All the chemicals and biochemicals used in this study were of analytical grade. Cell lines and cell cultures The lung adenocarcinoma line, A549, was purchased from American Type Culture Collection, (Manassas, Virginia, USA) and cultured in DMEM medium containing 10% fetal bovine serum, L-glutamine (2 mM), streptomycin (100 µg/mL), penicillin (100 IU/mL) (all from Gibco-Invitrogen Corp., Paisley, UK). The pemetrexed resistance lines A549/PEM was derived from A549 through stepwise increasing the pemetrexed concentrations in medium followed by long-term cultured in the same medium and supplement with 0.4 µM pemetrexed. All cells were incubated at 37oC in a humidified atmosphere containing 5% CO2.

MTT cell viability assay The detailed MTT experimental procedure has been described in our previous study 11

.

2D-DIGE, gel image analysis, protein staining, in-gel digestion and MALDI-TOF MS analysis The detailed experimental procedures for 2D-DIGE analysis and MALDI-TOF MS analysis have been described in our previous study


12-14

. For peptide mass


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fingerprinting

analysis,

the

target

proteins

were

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searched

against

the

Swiss-Prot/TrEMBL database (2013_08) with 540732 entries using Mascot software v2.4.01 (Matrix Science, London, UK). The following parameters were used for the search: Homo sapiens; tryptic digest with a maximum of 1 missed cleavage; carbamidomethylation of cysteine, partial protein N-terminal acetylation, partial methionine oxidation and partial modification of glutamine to pyroglutamate and a mass tolerance of 50 ppm. Identification was accepted based on significant MASCOT Mowse scores (p

Identification of Up- and Down-Regulated Proteins in Pemetrexed

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