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J. Biol. Chem., Vol. 280, Issue 9, 8260-8265, March 4, 2005

This Article Abstract Full Text (PDF) All Versions of this Article: 280/9/8260    most recent M410054200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nam, M. J. Articles by Hanash, S. M. Search for Related Content PubMed PubMed Citation Articles by Nam, M. J. Articles by Hanash, S. M. Social Bookmarking                      What's this?

Identification of Defensin 6 as a Potential Biomarker in Colon Adenocarcinoma^*

Myeong J. Nam, Mee K. Kee, Rork Kuick¶, and Samir M. Hanash¶

From the National Institute of Health, Seoul 122-701, Korea and the ^¶Department of Pediatrics and Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109

Received for publication, September 1, 2004 , and in revised form, December 16, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
There is substantial interest in the identification of circulating human tumor-derived proteins in serum for the purposes of early cancer diagnosis. We have implemented an approach based on the analysis of microarray data for the identification of tumor proteins that may have utility as biomarkers in colon cancer. Expression analysis of microarray data obtained from a variety of 283 tumors and normal tissues revealed that defensin 6 was maximally expressed in colon cancer. These findings were corroborated by reverse transcription-PCR, in which the colon cancer cell lines LoVo, Caco2, HCT-15, SW480, and SW620 showed significantly higher levels of defensin 6 expression than did non-colon cancer cell lines. Moreover, our data were concordant with data obtained from the NCI, National Institutes of Health Cancer Genome Anatomy Project. To evaluate defensin 6 as a potential biomarker of colon cancer, a preliminary "training" set of serum from 91 healthy donors and 109 colon cancer patients was analyzed by enzyme-linked immunosorbent assay. The data pattern was confirmed by an independent set of 67 masked serum samples: 18 from healthy donors and 49 from colon cancer patients. This result yielded a sensitivity of 69.4% (95% CI 54.6–81.8), specificity of 83.3% (58.6–96.4), and positive predictive value of 91.9% (78.1–98.3). These findings justify a prospective assessment of serum defensin 6 protein as a screening tool for colon cancer.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Colorectal cancer is the second leading cause of cancer death in the United States, with 135,000 new cases diagnosed each year and an overall 5-year survival rate of 50%. Most colorectal cancers develop slowly, beginning as small benign colorectal adenomas that progress over several decades to larger and more dysplastic lesions, eventually becoming malignant. This gradual progression provides ample opportunity for prevention and intervention. Diagnostic screening methods are at present suboptimal; therefore, new approaches are needed (1). In an effort to identify potential molecular markers of colorectal tumors, we have implemented an approach based on the analysis of microarray data for the identification of tumor proteins that may have utility as biomarkers in colon cancer. Expression analysis of microarray data obtained from a variety of 283 tumors and normal tissues revealed that defensin 6 was maximally expressed in colon cancer.

Human defensins comprise a family of closely related, cationic polypeptides 29–42 amino acids in length. The peptides contain 6 conserved cysteines linked in disulfide bonds that stabilize the molecules as triple-stranded amphiphilic -sheet structures (2, 3). In vitro, human defensins exhibit antimicrobial activity against some bacteria, fungi, enveloped viruses, and parasites (4, 5). Two classes of human defensins, termed "-defensins" and "-defensins," have been identified that differ with respect to their localization and linkage of cysteine residues, precursor peptide structure, and pattern of tissue expression. Whereas -defensins are most abundant in epithelial cells of the lung (6, 7), skin (8), and urogenital tract (9), the -defensins were first found in human polymorphonuclear leukocytes and intestinal Paneth cells (10, 11).

Although defensins seem to have diverse functional activities in innate antimicrobial immunity, a few reports have also indicated the presence of several defensins in epithelial tumors (12–14). There is much interest in identification of circulating tumor-derived proteins that may serve as biomarkers for the early detection of colon cancer. We demonstrated that the defensin 6 protein is expressed at higher levels in serum from colon cancer patients relative to serum from non-cancer controls. As such, defensin 6 may have utility as a biomarker for colon cancer.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Sera—Sera representing the predetermined training set were obtained at the time of diagnosis from 91 healthy individuals and 109 colon cancer patients following informed consent. For the independent test, 67 masked serum samples consisting of 18 from healthy donors and 49 from colon cancer patients were obtained in the same way. The experimental protocol was approved by The University of Michigan Institutional Review Board prior to serum collection.

Cell Lines and Cell Culture—The LoVo, HCT116, SW480, and SW620 colon adenocarcinoma cell lines, the Hs1 testis cancer cell line, and the SH-SY5Y neuroblastoma cell line were each cultured (5% CO₂, 37 °C) in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 units/ml), all purchased from Invitrogen.

Microarray Analysis—Single isolates of tumor samples and cancer cells were homogenized in the presence of TRIzol reagent (Invitrogen), and total cellular RNA was purified according to the manufacturer's procedures. RNA samples were further purified using RNeasy spin columns (Qiagen, Valencia, CA). RNA quality was assessed by 1% agarose gel electrophoresis in the presence of ethidium bromide. Samples that did not reveal intact and approximately equal 18 and 28 S ribosomal bands were excluded from further study. This study used commercially available high density microarrays (Affymetrix, Santa Clara, CA) that produced gene expression levels on 7129 known genes and expressed sequence tags (HuGeneFL Array). Preparation of cRNA, hybridization, and scanning of the arrays were performed according to the manufacturer's protocols. Briefly, 5 µg of the total RNA was used to generate double-stranded cDNA by reverse transcription using a cDNA synthesis kit (Superscript Choice system; Invitrogen) that uses an oligo(dT)₂₄ primer containing a T7 RNA polymerase promoter 3' to the poly(T) (Genset, La Jolla, CA), followed by second-strand synthesis. Labeled cRNA was prepared from the double-stranded cDNA by in vitro transcription by T7 RNA polymerase in the presence of biotin-11-CTP and biotin-16-UTP (Enzo, Farmingdale, NY). The labeled cRNA was purified over RNeasy columns. 15 µg of cRNA was fragmented at 94 °C for 35 min in 40 mM Tris acetate, pH 8.1, 100 mM potassium acetate, and 30 mM magnesium acetate. The cRNA was then used to prepare 300 µl of hybridization mixture (100 mM MES, 1 mM NaCl, 20 mM EDTA, 0.01% Tween 20) containing 0.1 mg/ml herring sperm DNA (Promega, Madison, WI) and 500 µg/ml acetylated bovine serum albumin (Invitrogen). Before hybridization, the cocktails were heated to 94 °C for 5 min, equilibrated at 45 °C for 5 min, and then clarified by centrifugation (16,000 x g) at room temperature for 5 min. Aliquots of this hybridization mixture containing 10 µg of fragmented cRNA were hybridized to HuGeneFL arrays at 45 °C for 16 h in a rotisserie oven set at 60 rpm. The arrays were washed using nonstringent buffer (6x SSPE) (sodium chloride/sodium phosphate/EDTA) at 25 °C, followed by stringent buffer (100 mM MES, pH 6.7, 0.1 M NaCl, 0.01% Tween 20) at 50 °C. The arrays were stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, OR), washed with 6x SSPE buffer, incubated with biotinylated anti-streptavidin IgG, stained again with streptavidinphycoerythrin, and washed again with 6x SSPE. The arrays were scanned using the GeneArray scanner (Affymetrix). Image analysis was performed with GeneChip software (Affymetrix).

Real-time Quantitative PCR—Total RNA was extracted from cells with TRIzol reagent (Invitrogen), following the protocol that was provided. For each sample, 2 µg of RNA were treated with DNase l (Roche Applied Science) at 37 °C for 30 min to remove contaminating DNA and then denatured in the presence of random hexamer primers (Promega). The samples were incubated with Superscript II (Invitrogen) reverse transcriptase in the presence of 1.0 mM dithiothreitol and 1.0 mM each of dTTP, dGTP, dCTP, and cATP at 42 °C for 40 min. The resulting cDNA was treated with RNase H (Roche Applied Science) and subjected to PCR amplification. We relied on the TaqMan assay (PerkinElmer model 7700) to quantitate the amount of defensin 6 mRNA. The forward and reverse primers and the FAM-tagged probe used for the defensin 6 gene in the assay were 5'-CAAGGCTGAGCCACTCCAA, 5'-CCTGGGCATCAGCCTCATA, 5'-6FAM-CTGAGGATGATCCACTGCAGGCAAAAG CTTTTGCCTGCAGTGGATCATCCTCAG-TAMRA, respectively.

The forward and reverse primers and FAM-tagged probe used for the -actin gene were 5'-AACTTGAGATGTATGAAGGCTTTTGG, 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTAAG, and 5'-6FAM-CAACTGGTCTCAAGTCAGTGTACAGGTAAGCCCT-TAMRA, respectively. To assay the initial concentration of the reactants (the sequence abundance), the number of cycles at which the reaction crosses a threshold value was measured. This number varies directly with the initial sequence abundance. To measure the relative abundance of the defensin 6 gene in any given RNA sample, the amplification value derived using the defensin 6 sequence was divided by the amplification value using the -actin sequence. Derivation of this fraction is independent of RNA sample concentration.

Two-dimensional Polyacrylamide Gel Electrophoresis—Cultured cells were solubilized in lysis buffer containing 9.5 M urea (Bio-Rad), 2% Nonidet P-40, 2% carrier ampholytes (pH 4–8; Gallard/Schlessinger, Carle Place, NY), 2% -mercaptoethanol, and 10 mM phenylmethylsulfonyl fluoride. Protein concentrations were measured by the Bradford assay (Bio-Rad). Proteins (175 µg) were applied to isoelectric focusing gels. Isoelectric focusing was conducted with pH 4–8 carrier ampholytes at 700 V for 16 h, followed by 1000 V for an additional 2 h. The first-dimension gel was loaded on the second-dimension gel after equilibration in second-dimension sample buffer (125 mM Tris (pH 6.8) containing 10% glycerol, 2% SDS, 1% dithiothreitol, and bromphenol blue). For the second-dimension separation, a gradient of 11–14% acrylamide (Serva/Crescent Chemical, Hauppauge, NY) was used. Proteins were transferred to an Immobilon-P polyvinylidene difluoride membrane (Millipore, Bedford, MA) or visualized by silver staining of the gels.

Western Blotting—After transfer, membranes were incubated for 2 h in blocking buffer containing 5% milk in 10 mM Tris-HCI (pH 7.5), 2.5 mM EDTA (pH 8), 50 mM NaCl. The membranes were incubated for 1 h at room temperature with defensin 6 antibody at a 1:1000 dilution. The membranes were then incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit (Amersham Biosciences) IgG antibodies at a dilution of 1:1000. Immunodetection was accomplished by enhanced chemiluminescence (Amersham Biosciences) followed by autoradiography on hyperfilm MP (Amersham Biosciences). Patterns visualized after hybridization following revelation with patients' sera were compared directly with Coomassie blue-stained blots from the same sample to determine correlations with proteins.

Immunofluorescence Microscopy—The cancer cells were grown in 2-well chamber slides for 48 h and then fixed in 2% formaldehyde, freshly prepared from paraformaldehyde. The fixed cells were washed briefly in Dulbecco's phosphate-buffered saline (D-PBS).¹ Aldehyde groups resulting from fixation were quenched in 50 mM L-lysine (in D-PBS), after which the fixed monolayers were washed three times in D-PBS (containing 1 mg/ml BSA). Each chamber was incubated with 0.5 ml of the defensin 6 antibody diluted as indicated in D-PBS (containing 2 mg/ml BSA) for 1 h at room temperature. The cells were washed in D-PBS (containing 2 mg/ml BSA), after which they were incubated in 0.5 ml of D-PBS (containing 2 mg/ml BSA) containing 10 µg/ml highly cross-adsorbed Alexa-594-conjugated goat anti-rabbit IgG. The stained monolayers were washed three times in D-PBS (containing 1 mg/ml BSA) and three times in D-PBS, after which a glass coverslip was mounted on the monolayers in GEL/MOUNT (Biomeda Corp., Foster City, CA). Fluorescence images were visualized through a Zeiss 510LSM confocal laser scanning microscope.

Competitive Enzyme-linked Immunosorbent Assay—A 96-well maxisorp microtiter plate (Nunc) was coated by incubation overnight at 4 °C with purified defensin 6 (10 µg/ml) in 0.1 M carbonate buffer, pH 9.6. After washing in phosphate-buffered saline (PBS, pH 7.4), any remaining protein binding sites in the microtiter plate were blocked by incubation with PBS/2% BSA overnight at 4 °C. Equal volumes of defensin antibody and serum competitor were mixed. The aliquots (100 µl) were added to the wells, in duplicate, after which the plate was incubated for 2 h at room temperature. After washing three times in PBS/2% BSA, 100 µl of horseradish peroxidase-conjugated goat anti-rabbit IgG (1: 1000 dilution) was added to each well and then incubated at room temperature for 1 h. After washing, bound peroxidase was determined using 3,3',5,5'-tetramethylbenzide as the substrate. The color reaction was developed for 30 min, stopped by the addition of 50 µl of 1 M HCl to each well, and then read at 450 nm using a microtiter plate reader.

Statistical Analysis—Serum levels of defensin 6 above 30 ng/ml were considered to be positive. Data were exported from a Microsoft Access data base, formatted, and then loaded into SAS 8.1v for analysis. An independent set of 67 serum samples had 95% power at the a = 0.05 level to reject an 80% sensitivity or specificity in favor of a true value of 95%, using an exact test for single proportions, with cutoff points for rejection based on the cumulative binomial distribution. Pearson's chisquare analysis was used to test the significance of the classification of colorectal cancer versus normal.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Expression of Defensin 6 mRNA Encoding Defensin 6 in Colon Cancer—We profiled the gene expression of 283 tumors and normal tissue, consisting of 6 normal brain, 73 brain tumors, 51 colon adenocarcinomas, 10 normal lung, 57 lung adenocarcinomas, 7 normal pancreas, 8 pancreatic adenocarcinomas, 63 ovarian cancers, and 8 cardia carcinoma by microarrays. To determine which genes were only expressed at high levels in colon cancer, we compared gene expression profiles from colon cancer to those from the other tissues analyzed. We found that defensin 6 expression was 5-fold higher in colon cancer than in any other tissue type analyzed (Fig. 1). Our microarray data were confirmed by NCI, National Institutes of Health Cancer Genome Anatomy Project data (cgap.nci.nih.gov/Tissues/VirtualNorthern) (Fig. 2). According to the SAGE data, defensin 6 is expressed at a frequency of 0/98089 in normal colon tissue and 23/325836 in colon cancer (p = 0.01). Interestingly, defensin 6 was not expressed in any other tissues except in the gastrointestinal tract (i.e. stomach and colon), thus validating the specificity of defensin 6 in colon cancers.

View larger version (8K): [in this window] [in a new window]   FIG. 1. DNA microarray analysis of defensin 6 in tissues. 283 tissues were analyzed using Affymetrix HuGeneFL GeneChip microarrays. A one-sided Wilcoxon signed-rank test of the individual oligonucleotide features for each probe set was used to determine whether a transcript was present or absent in a given sample. The mean value of each group was expressed. Note that defensin a6 was highly expressed in both microsatellite stable and microsatellite instability types of colon cancer.

View larger version (38K): [in this window] [in a new window]   FIG. 2. Virtual Northern of defensin 6. Spot images represent expression levels of the defensin 6 gene. For each combination of tissue and histology, expression is computed by dividing the number of expressed sequence tags (ESTs) or serial analysis of gene expression (SAGE) tags representing the gene divided by the total number of ESTs or SAGE tags in all libraries with the given tissue/histology. This ratio is then multiplied by 200,000, giving the number of ESTs or SAGE tags/200,000. This number is then placed on a logarithmic scale. Refer to cgap.nci.nih.gov/Tissues/VirtualNorthern.

View larger version (8K): [in this window] [in a new window]   FIG. 3. Real-time quantitative reverse transcription-PCR for the defensin 6 gene in different cancer cells. To measure the relative abundance of defensin 6 gene in a given RNA sample, the amplification value derived using the defensin 6 gene was divided by the amplification value using the -actin sequence.

View larger version (59K): [in this window] [in a new window]   FIG. 4. Two-dimensional PAGE and Western blot analysis of LoVo cell proteins. Left panels show LoVo cell two-dimensional protein pattern after silver staining. An arrow points to the location of defensin 6, recognized by a polyclonal rabbit anti-defensin 6 antiserum. Right panels show closeups of Western blots from colon tumor, SW480, SW620, and HCT15 cell proteins hybridized with anti-defensin 6 antiserum.

View larger version (9K): [in this window] [in a new window]   FIG. 5. Immunofluorescence staining of defensin 6 in SW480 cells. SW480 colon carcinoma cells were plated and grown for 48 h and then fixed and stained for immunofluorescence with rabbit anti-defensin 6 antibodies.

View this table: [in this window] [in a new window]   TABLE I Classification of serum samples from masked validation set by defensin 6 enzyme-linked immunosorbent assay

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

There are several explanations for a possible influence of the circulating levels of defensin 6 on cancer growth. For instance, circulating levels may be influenced by cancer growth as a result of altered expression of defensin 6 in cancer tissues. As the cytotoxic activity of the -defensins was noted (15), the protection mechanism would be increased with the cancer growth. Indeed, defensin 1, 2, and 3 expression were increased in renal cell carcinoma with a possible direct influence on tumor proliferation (16). Interestingly, cytolytic effects in tissues were occasionally correlated with the presence of the defensins in the study. In particular, groups of tumor cells in the center of primarily extended defensin patches tended to exhibit morphological signs of necrosis. By contrast, mitogenic effects of defensins have been described previously on mouse fibroblasts and epithelial cells (17). Thus, it is indeed plausible that defensins could contribute to cancer cell selection by promoting proliferation of a subset of malignant cells. At present it is unknown whether such selection mechanisms could favor colon cancer cell subpopulations that produce defensins. Considering the existing literature together with the data presented in this study, it is interesting to speculate that defensin 6 may contribute to host anti-tumor immunity or otherwise function as a tumor suppressor.

Screening based on a simple blood test that measures the concentrations of tumor-specific antigens would be far more likely than current diagnostic methods to gain general acceptance among patients and cancer specialists. The most widely used tumor marker for colon cancer is carcinoembryonic antigen (CEA), which is a glycoprotein (18). However, the clinical applications of CEA have been limited to the detection of recurrence after colorectal surgery. Because CEA is significantly less sensitive for earlier stage disease, it was abandoned as a screening marker for early cancer. Therefore, researchers are continually trying to develop new tumor markers with better diagnostic capabilities than CEA that provide a simple and accurate test for early detection.

The sensitivity and specificity for the masked validation set for defensin 6 was 69 and 83%, respectively. This result can be directly compared with a value of 40–45% and 88–90% for CEA, respectively (19–22). A sensitivity of 69% might be acceptable for high risk population screening. After proper validation, serum levels of defensin 6 might ultimately be applied in medical screening clinics as a diagnostic assessment. An important goal is confirmation of sensitivity and specificity for the prospective detection of colon cancer in trials of high and low risk populations.

In conclusion, defensin 6 mRNA was maximally expressed in colon cancer. Colon cancer cell lines showed significantly higher levels of defensin 6 expression than did non-colon cancer cell lines. The levels of defensin 6 in the serum of colon cancer patients were significantly higher than those found in normal subjects. Therefore, defensin 6 may have utility in colon cancer screening and diagnosis. Larger scale studies to establish the potential of these finding should be in progress.

	FOOTNOTES

 
^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel: 82-2-380-1512; Fax: 82-2-359-1397; E-mail: genetx{at}hanmail.net.

¹ The abbreviations used are: D-PBS, Dulbecco's phosphate-buffered saline; MES, 2-morpholinoethanesulfonic acid; BSA, bovine serum albumin: CEA, carcinoembryonic antigen.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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This Article Abstract Full Text (PDF) All Versions of this Article: 280/9/8260    most recent M410054200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nam, M. J. Articles by Hanash, S. M. Search for Related Content PubMed PubMed Citation Articles by Nam, M. J. Articles by Hanash, S. M. Social Bookmarking                      What's this?