| ObjectiveLung cancer is the most common cancer and the leading cause ofcancer-related death worldwide. Thus diagnosis of lung cancer is of greatimportance for successful therapy. Serological biomarkers are importantin cancer screening, and important in monitoring of cancer progression,treatment response and surveillance for recurrence. However, serumprotein biomarkers, such as carcinoembryonic antigen (CEA),carbohydrate antigen 125 (CA-125), cytokeratin 19 fragment marker(CYFRA 21-1) are not so good in detection of lung cancer due to theirlow specificity and/or sensitivity. For more effective in lung cancerdiagnosis, we need to find new biomarkers.Secreted proteins of cancer cells can be shed into blood. It may beemerged in serum in an early stage of cancer, and increasing levels inserum may be expected alone with the proliferation of cancer cells and/oroverexpression of the secreted proteins. As a result, secreted proteins maybe supposed as potential serum biomarkers of cancer. Some serumbiomarkers are known as secreted proteins. These include AFP for livercancer; CEA, prostasin and human kallikrein 10 for ovarian cancer;prostate-specific antigen (PSA) and cysteine-rich secretory protein-3 forprostate cancer; cytokine colony-stimulating factor-1 (CSF-1) for endometrial carcinoma; and tissue inhibitor of metalloproteinase-1(TIMP-1) for pancreatic adenocarcinoma. It can be inferred that analysisof the secreted proteins of lung cancer cells may be an effective approachfor delineating potential serum biomarkers.Lung cancer is divided clinically into small-cell lung cancer (SCLC)and non-SCLC (NSCLC), and NSCLC accounts for more than 80% oflung cancers. In this study, human NSCLC cell line A549 was culturedand the secreted proteins in conditioned medium were analyzed bytwo-dimensional polyacrylamide gel electrophoresis (2D-PAGE) andmatrix assisted laser desorption/ionization-time of flight mass(MALDI-TOF MS). Secreted protein dihydrodiol dehydrogenase (DDH)was identified and confirmed. Overexpression of DDH was detected inNSCLC cancer tissues. The level of DDH was evaluated in sera fromNSCLC patients, benign lung tumor patients and healthy individualsusing enzyme-linked immunosorbent assay (ELISA).MethodsPatient population and clinical specimensFifteen newly identified NSCLC tumors samples and distantnonmalignant lung samples (distance to the edge of tumor＞5cm) of thesame patient were obtained at the time of surgery at the Department ofcardiothoracic surgery, Xiangya Hospital, China, and stored at -80℃.These were used for RT-PCR and immunoblotting. Samples used for immunohistochemical staining were fixed in 10% buffered formalin andembedded in paraffin. There were 5 adenocarcinomas and 10 squamouscarcinomas. The median age was 55 years (range 45-72 y); 5 werefemale and 10 were male. 84 serum samples were collected whichincluded 34 adenocarcinomas, 30 squamous carcinomas and 20 benignlung tumor patients. Sera were obtained from the Department ofcardiothoracic surgery, Xiangya Hospital. The control group for ELISAconsisted of 20 apparently healthy blood donors (ages 45-66 years; mean51 years; 10 females and 10 males). Blood samples (5 mL) were collected,clotted at 4℃, centrifuged at 3000 rpm for 10 min, and then stored at-80℃.Cell culturing and sample preparationA549 cells were grown in RPMI 1640 supplemented with 5% fetalbovine serum (FBS). Then conditioned medium (about 5ml) of A549 wascollected and treated with DOC-TCA (deoxycholate-tricarboxylic acid)method to precipitated the protein: Added 0.1ml 0.15%DOC (w/v) intoper milliliter supernatant and mixed well, incubated on ice for 10min,then added 50μl 100% TCA (v/v) per milliliter to the mixture,centrifuged at 20000g for 10min, carefully recovered the supernatant, andair dry at last. Medium of RPMI-1640 with 5% FBS was treated in thesame way as the conditioned medium and used for control. Precipitatedprotein samples were resuspended in cell lysis buffer and the supernatant was used for proteomic analysis. Protein concentration was determinedwith the BCA Protein Assay Kit. All samples were stored at -70℃untiluse.Two-dimensional polyacrylamide gel electrophoresis (2DE-PAGE):The first dimensional isoelectric focusing (IEF) was performed on24cm immobilized pH 3-10 gradient (IPG) strips at 20℃. Samplescontaining 1200μg protein which were diluted to 450μl with rehydrationsolution were applied to the IPG strips (pH 3-10L, 24cm) by in-gelrehydration using IPGphor under constant 30 V for 14h.Isoelectrofocusing was carried out succeedingly for 1 h at 500V, 1h at1000V and 8.5h at 8000V. Isoelectrofocused IPG gel strips wereincubated in the equilibration buffer containing 1% DTT for 15min, andthen incubated in the same buffer containing 3% idoacetamide (IAA) foranother 15min. SDS-polyacrylamide gel electrophoresis (SDS-PAGE)was performed using 12.5% polyacrylamide gels. SDS-PAGE was carriedout at constant power (2.5 W/gel for 40 min and 15 W/gel for 6h) andtemperature (20℃) in the running buffer using Ettan DaltⅡsystem.Protein visualization and image analysis:Gels were stained with "blue silver": Stained with 0.12% Coomassieblue G-250, 10% ammonium sulfate, 10% phosphoric acid, and 20%methanol for 24h, and the background was destained with 10% methanoland 10% acetic acid. The stained 2-DE gels were scanned using the Immageseanner and the digitalized gel images were normalized andcomparatively analyzed using software PDQuest. Molecular masses weredetermined by running standard protein markers covering the range of10-100 kDa and pI values were used as given by the supplier of theimmobilized pH gradient strips. The specific protein spots in gels ofconditioned medium of were detected.Protein identification:The specific protein spots in gels of conditioned medium wereexcised. The spots were then subjected to discoloration, in-gel reductionwith DTT and alkylation with IAA. Enzymatic digestion was performedwith TPCK- trypsin (100μg/mL) for 16h at 37℃. Digested peptides wereextracted and condensed. After mixed with 0.5% TFA and CCA, samples(1μl) were loaded on plate and dried for crystallization. Masses ofpeptides were determined using a Voyager-DE STR MALDI-TOF(matrix assisted laser desorption/ionization-time of flight) massspectrometer. Database was searched by Mascot software with thefollowing parameters: mammalian species and one missed cleavage site.Carbomidomethylation of cysteine and oxidation of methionine wereconsidered as fixed modification or possible partial modification. Theprobability scores calculated by software were used as criterion forcorrect identification.Reverse transcription-polymerase chain reaction (RT-PCR) analysis: mRNA expression of PPIA, PEBP, PGAM1, PDX1, ENO1, DDHand PGP9.5 were analyzed. Total RNA was isolated from tumors andpaired lung tissues respectively using TRIzol reagent. One microgram oftotal RNA was reverse transcribed. To ensure that an equal amount ofRNA was used for each RT-PCR reaction, primers forglyceraldehyde-6-phosphate dehydrogenase (GAPDH) were employed asan internal control. All of the PCR reactions used a volume of 20ul.Theabundance of the PCR product (5ul) was quantified by densitometricscanning of the ethidium bromide-stained agarose gels. The observedPCR product intensity was normalized against the PCR product intensityobserved with the GAPDH.Western blot analysis of DDH in conditioned mediumTo verify the results obtained from MALDI-TOF MS, proteins(60μg/lane) of A549 cells extraction, conditioned medium and controlmedium were separated in a 10% polyacrylamide gel with 5% stackinggel. The resolved proteins were transferred onto PVDF membrane usingBio-Rad wet transfer unit, and the blots were blocked with 5% (w/v)nonfat dry milk in TBS-T solution (25 mmol/L Tris, pH 7.5, 150 mmol/LNaCl, 0.05% (w/v) Tween 20). After washing in TBS-T, the blots wereincubated for 1 h with mouse anti-human DDH antibodies at 1:500dilution, followed by horseradish peroxidase (HRP)-conjugatedanti-mouse secondary antibodies diluted 1:2000. Immunoreactive band was visualized by exposing the membrane to an X-film with enhancedchemiluminescent reagent.Western blot analysis of DDH in NSCLC:Tissue specimens about 100mg were ground into powder in liquidnitrogen and lysed in 400μl cell lysis buffer. The lysates were incubatedat 37℃for 30min, and followed by centrifugation at 20,000g for 30 minat 4℃. Protein concentration of the supernatants was determined and thesupernatants were used for immunoblotting analysis. The procedure ofimmunoblotting analysis of DDH was same as above, andimmunoblotting of GAPDH was adopted and serve as internal control.The intensities of DDH and GAPDH bands were quantified bydensitometry and the ratio of the DDH/GAPDH from each sample wasexamined.Immunohistochemical stainningImmunohistochemical stainning for DDH was carried out using theSuperPicTureTM Polymer Detection kit according to the manufacturer'sinstructions. Four-μm sections were made from paraffin tissue blocks,and the slides were dried at 60℃for 30 minutes, treated with xylenes,and then dehydrated in alcohol. Endogenous peroxidase was blocked with3% H2O2. Microwave treatment was performed for 15 min in 0.01 mol/Lcitrate buffer (pH 8.0). After incubating the DDH antibody on the tissueat 1:200 dilutions, the HRP polymer was added, and after a wash step, the DAB chromogen is then added to visualize the antibody binding. Slideswere counterstained with hematoxylin.Slide evaluation.Slides were read by two independent observers. The slides wereevaluated including membrane or cytoplasmic staining and percentage oftumor cells that stained positive. A specimen was considered positive ifmore than 10% of cancer cells were positively stained, and negative ifless than 10% positively stained.ELISA detection of DDH in serumSerum levels of human DDH were determined by enzyme-linkedimmunosorbent assay (ELISA). Microtiter plates (96-well) were washedwith 0.01mol/L PBS (pH7.4) and coated with serum (100μL per well)overnight at 4℃. The plates were then washed with PBS and blockedwith 200μL of ovalbumin (1 mg/mL in PBS) for 2h. Subsequently, DDHantibody at 1:1000 dilutions in PBS (100μL /well) were applied andincubated for 1 h. After washing, 100μL of HRP-conjugated goatantimouse IgG (diluted 5000-fold in PBS, Sigma) was added andincubated for 1 h. 3,5,3',5'-tetramethylbenzidine (TMB) was used as thechromogen and read at 450 nm with a plate reader. All samples wereassayed in triplicate. Sample diluent was used as a background control,wells coated with BSA and first antibody with anti-BSA were used aspositive control, and wells coated with sera and first antibody was alternate by PBS were serve as negative control.Statistical analysisMean values (Mean±SD) between the groups were compared usingthe Student's unpaired two-tailed t-test. The X2 test was used to examinethe association between the DDH expression status andclinicopathological features. All statistical tests were two-sided, anddifferences were considered, significant when p≤0.05. Statistical analysiswas performed using SPSS statistical software.ResultsIdentification of proteins in conditioned mediumIn this study, A549 was cultured in RPMI-1640 mediumsupplemented with low concentration (5%) of fetal bovine serum. It wasbetter for cells propagation and cells secreting function. However, itwould bring bovine protein in the conditioned medium protein samples.To identify the potential secreted protein spots, gels of conditionedmedium and control medium were compared, and the specific proteinspots in the conditioned medium gel were picked out. These protein spotswere either secreted proteins of A549 or degraded products of bovineproteins. In order to identify A549 secreted proteins, we confined thetaxonomy to mammalian species in database searching, so that proteinswhich belonged to Homo sapiens were secreted by A549 and others werebovine proteins. In area of MW lower than 66kD, a total of 248 well resolved spots were detected in A549. There were 66 specific spots inA549. Peptide mass fingerprinting (PMF) of these 66 specific spots wereobtained by MALDI-TOF-MS. The PMF data were used to search theSWISS-PROT and MSDB databases with MASCOT software. Theconfidence of identification was indicated by Mascot score (＞63) and theprotein sequence coverage by the matching peptides. A total of 45 proteinspots were identified and 28 belonged to bovine proteins. Seventeenprotein spots were identified as human proteins which included 14proteins: beta-actin, gamma-actin, Peptidyl-prolyl cis-trans isomerase A(PPIA), manganese superoxide dismutase (MnSOD), Peroxiredoxin 1(PDX1), phosphatidylethanolamine binding protein (PEBP), glutathioneS-transferase P (GSTP1-1), glucose-dependent insulinotropic proteinreceptor (GIPR), Ubiquitin carboxyl-terminal hydrolase isozyme L1(PGP9.5), Alpha enolase (ENO1), Sequence 299 from PatentWO0222660, Dihydrodiol dehydrogenase (DDH), Phosphoglyceratemutase 1 (PGAM1)and Galectin-1 (GAL1).On the gel of conditioned medium, the specific protein spot with anapparent pI 7.1 and molecular mass of≈32 kDa was identified as DDH bymass spectrometry. Since it is known that DDH was located in cytoplasmand augmented expression of DDH was detected in NSCLC, DDH wasselected for further analysis.RT-PCR Analysis The expression levels were compared between the tumors and thematched distant lung tissues. PPIA, PDX1, PGP9.5, PGAM1, ENO1,PEBP and DDH were confirmed to be overexpressed in cancer tissues bymore than 2.8-23.6 folds. ENO1 and PEBP were revealed as novelbiomarkers of NSCLC.Verification of secreted protein DDH by Western blotTo verify the results obtained from MALDI-TOF MS, proteinsamples of conditioned medium and protein extract of A549 cells weresubjected to Western blot analysis. Protein in the control medium wasexamined at the same time as control. DDH were detected in conditionedmedium and A549 cells, and no positive band was detected in the controlmedium.Highly Expressed DDH in NSCLC TissuesDifferentially expression of DDH between cancer tissues and distantnonmalignant lung tissue in NSCLC patients was studied using Westernblot. Highly expressed DDH gene was detected in cancer tissues(p＜0.05). Same as DDH gene, it was found that DDH protein was highlyexpressed in cancer tissues (p＜0.05).Immunohistochemical study of DDHThe tissue samples were immunostained to examine the differentialexpression of DDH in cancer tissues and lung tissues, and to evaluate thecellular distributions of DDH. Positive immunostainnings were found in all cancer tissues (n=15), and no immunoreactivity was found in the lungtissues. The immunoreactivity against DDH was strong in the cytoplasmand cell membrane.ELISA analysis for serum DDHThe relative levels of DDH in sera collected from adenocarcinomapatients (n=34), squamous carcinoma patients (n=30), benign lungtumor patients (n=20) and healthy controls (n=20) were examinedusing the ELISA system. The serum levels of DDH were significantlyhigher in NSCLC patients than in healthy controls (p＜0.05) and benignlung tumor patients (p＜0.05). There was no significantly differencebetween lung adenocarcinoma patients and squamous carcinoma patients.And no significantly differences were detected between healthy controlsand benign lung tumor patients. These results indicated that DDH mightbe potential serum biomarkers for NSCLC.Conclusions1. For the first time, sectrome of lung cancer cell was identified in ourstudy. Fourteen secreted proteins of A549 cell were identified, whichincluded beta-actin, gamma-actin, PPIA, MnSOD, PDX1, PEBP,GSTP1-1, GIPR, PGP9.5, ENO1, Sequence 299 from PatentWO0222660, DDH, PGAM1 and GAL1.2. PPIA, PDX1, PGP9.5, PGAM1, ENO1, PEBP and DDH were revealedto be overexpressed in NSCLC cancer tissues according to RT-PCR analysis. ENO1 and PEBP were revealed as novel biomarkers ofNSCLC3. DDH was first verified as a secreted protein of A549. In addition,strong espression of DDH were found in cytoplasm and cell membranein cancer cells of NSCLC tissue.4. Same as previous studies, DDH was detected as tumor marker ofNSCLC. DDH was highly expressed in lung cancer tissues comparedwith paired distant lung tissues.5. DDH was a novel serum biomarker of NSCLC.6. The results presented here would provide clues to identify newserological biomarkers of NSCLC, and identification of secretedproteins could be a feasible and effective strategy to search potentialserum biomarkers of cancer. |
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