7056 MOLECULAR MEDICINE REPORTS 16: 7056-7063, 2017 Detection of RACK1 and CTNNBL1‑induced activation of mouse splenocytes using an immunoprecipitation‑based technique BOHAN DONG1,2, GUANGLI DAI3, LEI XU1,2 and DAMIN SHI1 1Department of Biochemistry; 2Anhui Key Laboratory of Active Biological Macro‑Molecules, Wannan Medical College, Wuhu, Anhui 241002; 3Department of Gynecology and Obstetrics, Traditional Chinese Medical Hospital of Wuhu, Wuhu, Anhui 241000, P.R. China Received November 17, 2016; Accepted July 4, 2017 DOI: 10.3892/mmr.2017.7485 Abstract. Tumor cell lysates (TCLs) have been reported to or tumor-associated proteins and peptides (2). Tumor cell induce antitumor immunity; however, it remains unclear which lysates (TCLs) have been reported to induce antitumor immu- elements serve a role in this process. The present study identi- nity, since all antigens and signal transduction factors that are fied 768 proteins that were upregulated in TCL prepared from present in tumor cells exist in the TCL (3,4); thus, they may Lewis lung cancer cells compared with the lysate from type II have potential to be used in antitumor vaccines. In a dendritic alveolar epithelial cells. Among the proteins that were upregu- cell (DC)‑based antitumor study, lung cancer TCL was used lated in TCL, receptor for activated C kinase 1 (RACK1) and to activate mouse DCs in vitro; subsequently, the activated catenin β‑like 1 (CTNNBL1) are closely associated with cell DCs were transfused back into the mice to treat the tumor (5). proliferation and the inhibition of apoptosis. To determine the In addition, our previous study combined Lewis lung cancer role of these proteins in TCL, a protein extraction method was TCL with heat shock protein 65 to activate immunocytes in designed, which was based on immunoprecipitation. Using mice; the results demonstrated that the activated immunocytes this method, RACK1 and CTNNBL1 were extracted, whereas induced anti‑lung cancer immunity in vivo (6). Although TCLs the other proteins within the TCL were not affected. The have been used in antitumor immunotherapy, the key proteins modified TCL exhibited a stronger ability to induce spleno- involved in antitumor immunity remain unknown. Receptor cyte apoptosis, whereas the ability to promote cell activation for activated C kinase 1 (RACK1) is expressed in numerous was reduced. These findings suggested that the TCL depends tumor types, including lung cancer, colorectal cancer and on RACK1 and CTNNBL1 to activate mouse immunocytes, hepatocellular carcinoma (7). This factor is able to activate including monocytes and B lymphocytes, and inhibit apop- the Sonic hedgehog signaling pathway, which promotes tumor tosis. Therefore, the present study may provide information cell proliferation and inhibits apoptosis (8). Similarly, catenin regarding the composition of TCLs and their positive regula- β‑like 1 (CTNNBL1) is a protein that has been detected in tory effect on immunocytes. colorectal cancer, osteosarcoma and B lymphocytes (9-12). Although the function of this protein has yet to be determined, Introduction it has been identified as a putative regulator of the canonical Wnt signaling pathway using RNA interference technology, Tumor incidence is associated with impaired antitumor immu- where it acts upstream of, or in parallel to β-catenin, particu- nity in the patients. Effectively stimulating the immune system larly in colorectal cancer cell proliferation (9). RACK1 and to kill tumor cells is a critical factor for the success or failure CTNNBL1 are expressed in numerous tumor cell types to of antitumor immunotherapy (1). Efforts have been made to transduce the Sonic hedgehog cell proliferation signaling construct antitumor vaccines, aiming to induce antitumor pathway; therefore, it is hypothesized that these proteins immunity using tumor cells, genes that express tumor antigens must also exist in TCLs. In the present study, RACK1 and CTNNBL1 were detected in TCL prepared from Lewis lung cancer cells, and their roles in the activation of mouse spleno- cytes were determined. Correspondence to: Dr Bohan Dong, Department of Biochemistry, Wannan Medical College, 22 Wenchang West Road, Wuhu, Materials and methods Anhui 241002, P.R. China E-mail: [email protected] Animals and cell lines. Female C57BL/6 mice (n=9; age, 6‑8 weeks; weight, 18‑22 g) were purchased from Anhui Key words: tumor cell lysate, vaccine, mouse splenocytes, Medical University Laboratory Animal Center (Hefei, immunocyte, apoptosis, lung cancer, receptor for activated C China) and were maintained in microisolator cages under kinase 1, catenin β‑like 1 pathogen‑free conditions, at a temperature of 20‑26˚C under a 12‑h light/dark cycle. Food and water was autoclaved before feeding and provided ad libitum. The mice were evenly DONG et al: RACK1 AND CTNNBL1 INDUCE ACTIVATION OF MOUSE SPLENOCYTES 7057 divided into 3 groups; a PBS group, whole TCL group and 12,000 x g overnight at 4˚C. Acidified with 50% CF3COOH, TCL RACK1‑/CTNNBL1‑group. Experimental manipulation the peptides were separated using linear gradient elution; the of the mice was undertaken in accordance with the National mobile phases were comprised as follows: Mobile phase A, Institutes of Health Guide for the Care and Use of Laboratory 5% ACN and 0.1% formic acid (FA); mobile phase B, 80% Animals (https://www.ncbi.nlm.nih.gov/books/NBK54050), ACN and 0.1% FA. The gradient was increased from 5 to 90% with the approval of the Scientific Investigation Board of mobile phase B in 50 min at a flow rate of 300 nl/min. The Science and Technology of Anhui Province and the animal tandem mass spectrometry (MS/MS) analysis was performed experiments of the present study were also approved by the using a Q Exative system (Thermo Fisher Scientific, Inc.) Ethics Committee of Wannan Medical College. The mouse in Information‑Dependent Mode. Briefly, MS spectra were Lewis lung cancer cell line was purchased from Wuhan acquired across the mass range of 350‑1800 m/z in high Boster Biological Technology, Ltd. (Wuhan, China). The cells resolution mode (>70,000 full width at half maximum) using were cultured in high‑glucose Dulbecco's modified Eagle's a 40 msec accumulation time per spectrum. A maximum of medium (Hyclone; GE Healthcare Life Sciences, Logan, UT, 20 precursors per cycle were chosen for fragmentation from USA) supplemented with 10% fetal calf serum (FCS; Gibco; each MS spectrum with 60 msec minimum accumulation Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml time for each precursor and dynamic exclusion for 20 sec. penicillin and 100 µg/ml streptomycin (Sigma‑Aldrich; Merck Tandem mass spectra were recorded in high sensitivity KGaA, Darmstadt, Germany). The present study was approved mode (resolution>17,500) with rolling collision energy on by the Ethics Committee of Wannan Medical College (Wuhu, and iTRAQ reagent collision energy adjustment on. For China). protein identification, the MS/MS spectra were processed using ProteinPilot software version 5.0 (Sciex, Framingham, Preparation of TCL. To prepare the TCL, cultured Lewis cells MA, USA). Differentially abundant proteins were examined were lysed using a freeze‑thaw cycle in PBS solution between using QuickGO (https://www.ebi.ac.uk/QuickGO/) for Gene ‑70˚C and 37˚C. After five cycles, the prepared TCL was Ontology annotation and enrichment analysis. stored at ‑70˚C until further use. The TCL was observed under a microscope (Olympus Corporation, Tokyo, Japan) using Reverse transcription‑quantitative polymerase chain reaction trypan blue staining (Sigma‑Aldrich; Merck KGaA) to ensure (RT‑qPCR). Total RNA was extracted from mouse Lewis lung all of the cells were lysed. cancer cell whole TCL and reverse transcribed into cDNA using an RNAprep pure kit (Tiangen Biotech Co. Ltd., Beijing, Preparation of mouse type II alveolar epithelial cell lysate. China) and FastQuant RT kit (with gDNase; Tiangen Biotech To prepare the lysate, cultured type II alveolar epithelial cells Co. Ltd.), respectively. Oligo dT mixed with total RNA was were lysed using a freeze‑thaw cycle in PBS solution between and placed in a warm bath at 70˚C for10 min, and then into ‑70˚C and 37˚C. After five cycles, the prepared lysate was ice‑water mixture. dNTP\RTase\Rnase free H2O was added stored at ‑70˚C until further use. The lysate was observed to prepare the reverse transcription reaction system. The under a microscope (Olympus Corporation, Tokyo, Japan) system was warmed at 42˚C for 1 h, and then was heated to using trypan blue staining (Sigma‑Aldrich; Merck KGaA) to 70˚C for10 min to produce cDNA. Subsequently, qPCR was ensure all of the cells were lysed. performed to detect the mRNA expression levels of RACK1, CTNNBL1, Cullin 3, B‑cell lymphoma 2‑associated tran- Isobaric tags for relative and absolute quantitation (iTRAQ) scription factor 1 (BCLAF1), ribosomal protein S6 (RPS6), analysis. TCL was prepared from Lewis lung cancer cells superoxide dismutase 1 (SOD1), high mobility group box or mouse type II alveolar epithelial cell using the above 1 (HMGB1), inhibitor of growth family member 1 (ING1), method. Protein concentration of TCL was determined ERCC excision repair 3, TFIIH core complex helicase subunit using a bicinchoninic acid (BCA) assay using bovine serum (ERCC3) and GAPDH in mouse Lewis lung cancer cell TCL albumin as standard (BCA Assay kit; Beyotime Institute of using a SuperReal PreMix Plus kit with SYBR Green (Tiangen Biotechnology, Shanghai, China). Subsequently, 100 µg TCL Biotech Co. Ltd.). Thermocycling conditions were 30 sec at protein was digested with trypsin (Promega Corporation, 95˚C, 2 step PCR at 95˚C for 0.5 sec and at 60˚C for 30 sec, Madison, WI, USA) overnight at 37˚C. Digested samples were for 40 cycles. Following dissociation was 1 cycle at 95˚C for labeled using the 8‑plex iTRAQ kit (Applied Biosystems; 15 sec, at 60˚C for 30 sec and at 95˚C for 15 sec successively.