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Keywords:

  • prostate cancer;
  • haptoglobin;
  • serum marker;
  • RM2

Abstract

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In our previous study, monoclonal antibody RM2, established toward the glycosyl epitope, reflected grade of malignancy of prostate cancer cells whereas RM2 reactivity to benign glands was negative or weak. RM2 reactivity was also detected in stroma, suggesting the glycoprotein RM2 recognizes could be released into the bloodstream. Then, we explored RM2 reactivity to sera of early prostate cancer. We compared RM2 reactivity to sera between 62 patients with early prostate cancer and 43 subjects with benign prostatic disease, and examined RM2 reactivity before and after radical prostatectomy in 15 patients by Western blotting. We also examined RM2 reactivity to sera of the other urogenital cancers. RM2 reactivity was significantly enhanced on a serum glycoprotein with molecular mass ∼40 kDa, hereby termed GPX, in the patients with early prostate cancer when compared with those with benign prostatic disease (p < 0.0001). Setting an appropriate cutoff level, RM2 reactivity to GPX for detection of prostate cancer had sensitivity of 87% and specificity of 84%, respectively. Furthermore, the level of RM2 reactivity significantly decreased after radical prostatectomy (p = 0.006). However, increased RM2 reactivity to GPX was also observed in the other urogenital cancers. The proteomics approach identified GPX as haptoglobin-β chain and RM2 showed preferential reactivity toward haptoglobin-β chain derived from prostate cancer when compared with polyclonal anti-haptoglobin antibody. Haptoglobin-β chain defined by RM2 is a novel serum marker that may be useful for detection of early prostate cancer when coupled with prostate-specific antigen because it is not specific to prostate cancer. © 2008 Wiley-Liss, Inc.

Prostate-specific antigen (PSA) has a problem especially with specificity, i.e., PSA is not only elevated in prostate cancer but also in benign prostatic disease (BPD). Thus, only 25% of men with PSA value of 4 to 10 ng/mL will have diagnosis of cancer after prostate biopsy.1–3 Furthermore, PSA alone cannot predict pathological stage of prostate cancer because PSA does not reflect the grade of malignancy.1–4 Recently, it was pointed out that the prevalence of prostate cancer was 15% among men with PSA 4 ng/mL or less.4 Therefore, a new serum marker to compensate for the problems of PSA is urgently required. However, there has been few serum markers reported, which have potential for clinical application.5, 6 We previously reported that monoclonal antibody (mAb) RM2 was established toward disialoganglioside and later found to recognize the glycosyl epitope (β1.4-GalNAcDSLc4).7, 8 In an attempt to find a new marker, we examined whether immunoreactivity of mAb RM2 was detected in radical prostatectomy specimens, and found that reactivity of mAb RM2 to prostate cancer cells was associated with grade of malignancy, whereas RM2 reactivity to benign glands was negative or weak.9 RM2 immunoreactivity was also detected in stroma,9 suggesting the glycoprotein RM2 recognizes may be shed from cancer cells into the surrounding stroma and then released into the bloodstream. In the current study, we explored RM2 reactivity to sera of early prostate cancer, and found that mAb RM2 also recognized haptoglobin-β chain and level of haptoglobin-β chain defined by RM2 significantly increased in sera of early prostate cancer.

Subjects and methods

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Serum samples

Of serum samples obtained from Department of Urology, Tohoku University Hospital between June 2004 and May 2006, serum samples of 62 patients with early prostate cancer and those of 43 with BPD were randomly selected. All these subjects had biopsy proven histological diagnosis and PSA less than 10 ng/mL. Fifteen serum samples of the patients with various PSA values who underwent radical prostatectomy, 6 of the subjects with renal cell carcinoma (RCC), 8 of those with urothelial carcinoma and 8 of those with testicular germ cell tumors were also randomly selected. The Ethics Committees of both Tohoku University Graduate School of Medicine and Pacific Northwest Research Institute approved the present study, and informed consent was obtained from each patient. Clinical tumor-node metastasis staging was assigned using the 1997 tumor-node metastasis staging system.10 Gleason scores of all slides were diagnosed by a single pathologist (M.E.).11

Cell lines

Prostate cancer cell lines PC3, LNCaP and DU145 were obtained from Human Science Research Resource Bank (Wako, Japan). Androgen-independent prostate cancer cell line AICaP1 was newly established from LNCaP in our laboratory (Taima et al., to be published elsewhere). PrEC, normal human prostatic epithelial cells were purchased from Cambrex Bioscience (Walkersville, MD). RCC cell line ACHN was purchased from Dainihonseiyaku. (Osaka, Japan) and TOS1 was previously established in our laboratory.7

Antibodies

mAb RM2 (isotype IgM) was established using RCC TOS1 cells as immunogen,7 based on our earlier studies that indicated that degree of RCC malignancy was correlated with disialoganglioside expression in RCC cells.12 RM2 is reactive with disialoganglioside but not with monosialoganglioside fraction, and the antigen was later identified as β1,4-GalNAc-disialyl-Lc4.8 Polyclonal anti-haptoglobin antibody was purchased from Dako (Dako Cytomation Kyoto, Japan).

Western blotting of serum

After removing albumin and IgG using Aurum™ Serum Protein Mini Kit (Bio-Rad), a 20 μl aliquot of serum was electrophoresed on an 10% SDS-PAGE and transferred to Hybond P PVDF membrane (Amersham Biosciences, Uppsala, Sweden). Immunoblotting was performed as described previously.9 Densitometric analysis of RM2 reactivity to a glycoprotein with molecular mass ∼40 kDa (GPX) in serum was performed using Scion image (Scion Corp., Frederick, MD) and each value of ∼40 kDa glycoprotein was normalized to that of a glycoprotein with molecular mass ∼75 kDa from the same lane.

Pretreatment of sera by Agilent column, followed by 2D sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE)

Agilent Multiple Affinity Removal System 1.6 × 100 mm2 column, designed to adsorb >98–99% of 6 abundant proteins (albumin, immunoglobulins IgG and IgA, transferrin, haptoglobin and antitrypsin) from human serum samples, was purchased from Agilent Technologies (Palo Alto, CA) together with solvents A and B used for adsorption and elution of the proteins.

Two-dimensional SDS gel electrophoresis, in situ alkylation, Western blotting and in gel digestion were performed as described previously.13

Identification of protein

The tryptic digest was analyzed using Agilent 110 capillary HPLC (Agilent Technologies) combined with LCQ ion trap mass spectrometer (Thermo Electron, Waltham, MA). Data were searched against NCBI human sequence database using TurboSEQUEST v.27, and in selected cases Mascot (Matrix Science).

mRNA levels of haptoglobin-β chain in prostate cancer cell lines

Total RNA was extracted from PC3, LNCaP, DU145 and PrEC using Trizol reagent (Gibco BRL, Grand Island, NY) following the manufacturer's instructions. Total RNA (2 μg) was reverse-transcribed into first-strand cDNA using ExScript reverse transcription kit (Takara Bio, Japan). PCR was performed using the primers of haptoglobin-β chain and those of β-actin.

Immunohistochemical staining

RM2 reactivity to prostate cancer was described in our previous study.9 Twelve cases of benign prostatic hyperplasia were immunostained by mAb RM2 in this study. To examine haptoglobin expression in prostate cancer, polyclonal anti-human haptoglobin antibody was used because antibody specific to haptoglobin-β chain was not available. Twenty cases of radical prostatectomy specimens as previously described9 were immunostained by polyclonal anti-haptoglobin antibody. Immunostaining intensity was graded as negative, weak and moderate to strong. Then, immunoreactivity of each slide was either classified as lower expression or higher expression based on staining intensity and percentage of cancer cells stained. When moderate to strong staining was observed in 10% or more of cancer cells, higher expression was assigned, and when moderate to strong staining was observed in less than 10% of cancer cells, lower expression was assigned.

Extraction of glycosphingolipids and thin-layer chromatography immunostaining

We investigated whether GalNAcDSLc4 as a ganglioside is also responsible for RM2 reactivity to prostate cancer cells. Briefly, glycosphingolipids were extracted from prostate cancer cell lines and separated from phospholipids by alkaline degradation of phospholipids, and glycosphingolipids were analyzed by thin-layer chromatography with immunostaining by mAb RM2 as described previously.14 To examine whether GalNAcDSLc4 as a ganglioside was released into the culture media, glycosphingolipids were extracted from 10 ml of supernatant of cell lines cultured in serum-free media for 3 days by solid phase extraction using C18 Sep-Pak cartridge (Waters, Milford, MA) as described previously.15 Monosialosyl globopentaosylceramide (MSGb5) from supernatant of ACHN cells was used as a positive control.

Treatment of protein extract from prostate cancer cells with the hemoglobin column and immunoblotting by mAb RM2

We examined whether RM2 reactivity to prostate cancer cells depends on haptoglobin-β chain because GalNAcDSLc4 as a ganglioside was not detected in prostate cancer cells as described in Results section. For this purpose, protein extract from prostate cancer cell line DU145 was treated with the hemoglobin column to adsorb haptoglobin. DEAE-purified human hemoglobin was purchased from Sigma and coupled to CNBr-activated Sepharose-4B (Sigma) according to the manufacturer's procedures. One hundred microliters of protein extract of DU145 was applied to the column and the eluate (termed fraction I) was immediately collected without incubation. Another 100 μl was reacted with the hemoglobin column beads at 4°C overnight, then the column was stood for 5 min, and the eluate (termed fraction II) passed through the column was collected. After collecting the fraction II, the column was washed with PBS and the washed fraction (termed wash I) was collected. RM2 reactivity to each fraction was examined by Western blotting.

Treatment of serum haptoglobin-β chain defined by mAb RM2 with glycosidase

Although our coworkers recently indicated that RM2 glycosyl epitope (β1.4-GalNAcDSLc4) was not found on haptoglobin-β chain,16 we examined changes of RM2 reactivity after β-hexosaminidase and/or sialidase treatment on the assumption that RM2 reactivity to haptoglobin-β chain is based on the RM2 glycosyl epitope. After transferring proteins from the gel to the membrane, the membrane was blocked with 1% bovine serum albumin solution at room temperature for 2 hr and washed with phosphate buffered saline/0.05% Tween-20. The membrane was placed into a vinyl bag and treated with 2.0 U of β-hexosaminidase from jack beans (Sigma) at 37°C overnight or with 25 mU of sialidase from Newcastle disease virus (NDV; Glyko) at 37°C overnight. The membrane was washed with phosphate buffered saline/0.05% Tween-20 and subjected to Western blotting by mAb RM2. The control membrane was treated in the same way without glycosidase.

Statistical analysis

Statistical analysis was performed using the software from the SAS Institute (SAS Institute, Cary, NC).

Results

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

RM2 reactivity to sera of prostate cancer and BPD

For comparison with RM2 reactivity to serum, examples of RM2 reactivity to prostate cancer cells and to benign prostatic hyperplasia were shown in Figures 1a and 1b, respectively. None of the 12 cases of benign prostatic hyperplasia examined was stained by mAb RM2. Level of RM2 reactivity to prostate cancer was associated with grade of malignancy as previously described.9

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Figure 1. Increased level of RM2 reactivity to GPX in sera of prostate cancer. (Panel a) Examples of immunohistochemical staining of prostate cancer cells (panels 1 and 2) and benign prostatic hyperplasia (panels 3 and 4). RM2 reactivity to stroma was also observed in prostate cancer tissues, but neither gland nor stroma in benign prostatic hyperplasia was stained by mAb RM2. (Panel b) Examples of serum Western blotting by mAb RM2. PCa: prostate cancer. BPD: benign prostatic disease. Arrow indicates position of size marker and GPX. In each panel, PSA level for each case was indicated at bottom. (Panel c) Comparison of RM2 reactivity to GPX between BPD and PCa. Large bars: standard deviation, Small bars: standard error of the mean. (Panel d) ROC curve of RM2 reactivity to GPX. The area under the ROC curve was 0.89. The difference between sensitivity and 1-specificity was highest at sensitivity of 87%.

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We found that RM2 reactivity was enhanced on a serum glycoprotein with molecular mass ∼40 kDa, hereby termed “GPX,” in the majority of 62 patients with early prostate cancer, when compared with 43 subjects with BPD (Figs. 1b and 4b right panel). No reactivity was observed for mouse IgM used as a negative control (data not shown). All these subjects had biopsy proven histological diagnosis and PSA less than 10 ng/mL (Tables I and II). There was no significant difference of age and PSA between the 2 groups. RM2 reactivity to GPX calculated by Scion image was normalized to the reactivity to ∼75 kDa protein, which was used as an internal control because RM2 reactivity to ∼75 kDa protein was relatively constant between prostate cancer and BPD. Level of RM2 reactivity to GPX in prostate cancer (0.96 ± 0.43) was significantly higher than that in BPD (0.35 ± 0.32) (p < 0.0001 by t test) (Fig. 1c). The receiver operating characteristic analysis was performed and the area under the receiver operating characteristic curve of RM2 reactivity to GPX was 0.89. Setting a cutoff level of RM2 reactivity to GPX as 0.59, RM2 reactivity to GPX for detection of prostate cancer had sensitivity of 87% and specificity of 84% (Fig. 1d). Then, we explored the variables predicting the level of RM2 reactivity to GPX by univariate analysis. Level of RM2 reactivity to GPX was not significantly associated with the pretreatment variables in 62 subjects with early prostate cancer (Table III). It was significantly associated with the origin of index (major) cancer among the postsurgical variables in 24 patients, who underwent radical prostatectomy (Table IV), i.e., the index cancer of transition zone origin predicted lower level of RM2 reactivity when compared with that of peripheral zone origin.

Table I. Clinical Characteristics of the Patients with Biopsy Proven Histological Diagnosis and PSA Value Less than 10 ng/ml
 PCaBPDp
  1. PCa, prostate cancer; BPD, benign prostatic disease; F/T, ratio of free to total PSA.

Age68.6 ± 6.466.8 ± 7.60.2
PSA5.3 ± 2.15.0 ± 2.10.4
F/T0.16 ± 0.10.22 ± 0.10.007
Table II. Clinicopathological Characteristics of the Prostate Cancer Patients with PSA Value Less than 10 ng/ml
ParametersNo. Patients
  1. cT, clinical T stage; bGS, biopsy Gleason score.

cT 
 lb1
 lc45
 2a11
 2b4
 3a1
bGS 
 68
 743
 85
 96
Table III. The Relationship Between the Pretreatment Variables and RM2 Reactivity
Variablesp
  1. cT, clinical T stage; bGS, biopsy Gleason score.

Age0.1769
PSA0.0922
Bgs0.4023
CT0.8196
No. positive biopsy core0.1429
Table IV. The Relationship Between the Postsurgical Variables and RM2 Reactivity
Variablesp
  1. RPGS, radical prostatectomy Gleason score; pT, pathological T stage.

Age0.0980
PSA0.9843
RPGS0.3723
Index cancer origin0.0117
Total cancer volume0.3433
pT0.6099

RM2 reactivity to GPX before and after radical prostatectomy was also examined in 15 patients with various preoperative PSA levels whose serum PSA level decreased to less than 0.1 ng/mL after radical prostatectomy, the value believed to be recurrence-free (Table V). Level of RM2 reactivity to GPX decreased in 13 of these 15 patients after radical prostatectomy, although the extent of decrease was varied (Figs. 2a and 2b). Level of RM2 reactivity significantly decreased after radical prostatectomy (pre- vs. postoperative value: 0.92 ± 0.52 vs. 0.60 ± 0.43; p = 0.006 by paired t test) (Fig. 2b). The profile of RM2 reactivity to sera of the other urogenital malignancies was almost the same as that to sera of prostate cancer, and level of RM2 reactivity to GPX 0.59 or more was observed in 5 of 6 subjects with RCC, 6 of 8 with urothelial carcinoma, and 2 of 8 with testicular germ cell tumor (data not shown), i.e., RM2 reactivity to GPX was not specific to prostate cancer.

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Figure 2. Changes of RM2 reactivity to GPX after radical prostatectomy. (Panel a) Examples of RM2 reactivity to sera before and after radical prostatectomy. Preoperative PSA level in each case was indicated at bottom. In all five cases radical prostatectomy Gleason scores were 7 and pT were 2b but No. 10 (3a). b, Before radical prostatectomy; a, after radical prostatectomy; M, size marker. (Panel b) Changes of RM2 reactivity to GPX after radical prostatectomy in 15 patients. Longitudinal axis: RM2 reactivity to GPX, RP: radical prostatectomy. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Table V. Clinicopathological Characteristics of the Patients Whose RM2 Reactivity was Compared Before and After Radical Prostatectomy
  1. Preop., preoperative; pT, pathological T stage; RPGS, radical prostatectomy Gleason score.

Age (median)55–75 yr (67)
Preop. PSA (median)3.07–24.29 ng/ml (5.41)
Pathological parametersNo. patients
pT 
 2a1
 2b9
 3a5
RPGS 
 51
 713
 81

Identification of GPX as haptoglobin-β chain

In view of the clinical significance of GPX, we analyzed its molecular parameters. GPX was separated by Agilent column, followed by application of proteomics approach with 2D gel electrophoresis, in gel digestion, HPLC and electrospray ionization mass spectrometry.13 The results showed clearly that GPX is haptoglobin-β chain (Fig. 3). In 2D SDS-PAGE, a clear difference was found in one fraction consisting of four contiguous spots (termed spot 1, 2, 3 and 4 from right to left), from malignant sera (specimen II-c: Fig. 3a-1, upper panel) vs. corresponding spots from nonmalignant sera (specimen II-b: Fig. 3a-1, lower panel). The difference between nonmalignant vs. malignant sera was more distinct when analysis was made by 2D SDS-PAGE with Western blotting by mAb RM2. RM2-blotted spots were strong for prostate cancer specimen II-c but absent for nonmalignant specimen II-b (Fig. 3a-2, upper and lower panels). The contiguous spots 1, 2, 3 and 4 as above from specimen II-c were excised from the gel, and subjected to in gel digestion by trypsin, followed by LC-MS/MS for protein identification. An example of a base peak chromatogram for tryptic peptides of spot 2 of GPX is shown in Figure 3b. The results of the TurboSEQUEST search on the data were displayed in Figure 3c, showing human haptoglobin-2 precursors (P00738). There were 9 tryptic peptides found, which corresponded to 25% coverage of the haptoglobin precursor. However, the protein precursor consists of signal peptide (1–18 residue), α-chain (19–160 residue) and β-chain (162–406 residue).17–20 All tryptic peptides identified were from β-chain. The corresponding peptide coverage for haptoglobin β-chain (40 kDa) was 38.8% for spot 2. The peptides found for the other 3 spots were similar, also identifying GPX as the haptoglobin β-chain protein. The coverage for spots 1, 3 and 4 was 35.5, 20.0 and 35.5%, respectively. Figure 3d shows the MS/MS spectrum of a doubly charged precursor ion at m/z 680 identifying SC(PAM)AVAEYGVYVK peptide with annotated amino acid sequence. Figure 3e shows the MS/MS spectrum of [M + 2H]2+ at m/z 710 of peptide DIAPTLTLYVGKK. The amino acid sequences in Figures 3d and 3e correspond to the residues 380–391 and 216–228 of the haptoglobin precursor.

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Figure 3. Identification of GPX as haptoglobin-β chain. (Panel a-1) Protein components separated by 2D SDS-PAGE: Staining by Coomassie Brilliant Blue (CBB). Agilent column-adsorbed fractions (fraction II) from nonmalignant serum and from prostate cancer serum were analyzed by 2D SDS-PAGE. Upper: The pattern from sample II-c (fraction II from serum of malignant subject c). Lower: The pattern from sample II-b (fraction II from serum of nonmalignant subject b). Horizontal direction: isoelectric focusing (IEF). Vertical direction: SDS-PAGE. Area circled by dotted line on both left and right: GPX. Note that sample II-c (malignant) has higher CBB staining than sample II-b (nonmalignant). For each sample, GPX is separated into four contiguous spots with different IEF, termed 1, 2, 3 and 4 as indicated. (Panel a-2) Upper and Lower. Same as in upper and lower in panel a-1, but stained by mAb RM2. Note that RM2-stained bands are clearly seen for sample II-c but not for sample II-b. (Panel b) LC-MS chromatogram of spot 2 on the 2D SDS-PAGE in Panel a after in gel trypsin digestion and extraction of the tryptic peptides. Abscissa: LC retention time. Ordinate: MS1 of total ion chromatogram. (Panel c) Identification of haptoglobin. (Panel d) MS/MS spectrum of m/z 680 at the [M + 2H]2+ ion. (Panel e) MS/MS spectrum of m/z 710 at the [M + 2H]2+ ion. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Preferential reactivity of mAb RM2 toward haptoglobin-β chain from prostate cancer when compared with polyclonal anti-haptoglobin antibody

The profile of reactivity to serum with mAb RM2 was very similar to that with polyclonal anti-haptoglobin antibody (Figs. 4a and 4b). This finding may support the results of the proteomics analysis described earlier, and also suggests that the 4 bands above haptoglobin-β chain may be various forms of haptoglobin-α and -β chain complex because haptoglobin occurs in vivo as polymers of an α and β chain complex.21 In addition, RM2 showed preferential reactivity toward haptoglobin-β chain from prostate cancer rather than that from BPD when compared with polyclonal anti-haptoglobin antibody (Figs. 4a and 4b).

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Figure 4. Preferential reactivity of mAb RM2 toward haptoglobin-β chain from prostate cancer. (Panel a and b) Examples of reactivity to sera of BPD and PCa by polyclonal anti-haptoglobin antibody and mAb RM2, respectively. Left and right panels are from the same patients. Left panel: Reactivity to sera by polyclonal anti-haptoglobin antibody. Right panel: Reactivity to sera by mAb RM2. PSA level in each case was indicated at bottom. Hpt: haptoglobin, Ab: antibody.

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Increased expression of haptoglobin in prostate cancer cells

Increased level of haptoglobin-β mRNA was observed in prostate cancer cell lines, LNCaP, PC3 and DU145, when compared with PrEC, normal human prostate epithelial cells (Fig. 5a). In immunohistochemical staining, negative to weak staining of polyclonal anti-haptoglobin antibody was observed in benign prostatic glands or stroma. Higher expression of polyclonal anti-haptoglobin antibody in prostate cancer cells was observed in 9 of 20 cases (Fig. 5b), whereas expression level was not clearly different between cancer cells and benign glands or stroma in 11 cases. There was no significant association between Gleason score and expression level of polyclonal anti-haptoglobin antibody in prostate cancer cells (data not shown).

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Figure 5. Increased expression of haptoglobin in prostate cancer cells. (Panel a) Level of haptoglobin-β mRNA in prostate cancer cell line. 1: LNCaP, 2: PC3, 3: DU145, 4: PrEC. (Panel b) Examples of immunohistochemical staining of prostate cancer by polyclonal anti-haptoglobin antibody. Left: Gleason pattern 3, Right: Gleason pattern 4.

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RM2 reactivity is largely due to haptoglobin-β chain in prostate cancer cells

Thin-layer chromatography immunostaining of glycolipids by mAb RM2 showed that expression of GalNAcDSLc4 was not observed in prostate cancer cell lines, whereas it was detected in RCC cell line TOS1 as a positive control (Fig. 6a). Expression of GalNAcDSLc4 was neither detected in culture media of prostate cancer cells nor in that of TOS1 cells, whereas MSGb5 as a positive control was detected in supernatant of ACHN cells (data not shown). Most of RM2 reactivity disappeared after treatment with the hemoglobin column (Fig. 6b, lane 3). These results indicate that RM2 reactivity in prostate cancer cells is largely due to haptoglobin-β chain.

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Figure 6. RM2 reactivity is largely due to haptoglobin-β chain in prostate cancer cells. (Panel a) TLC immunostaining by mAb RM2. Total glycolipid equivalent to 15 mg of wet weight cell pellet was applied per lane. Lane 1: PC3, lane 2: LNCaP, lane 3: AICaP1, lane 4: TOS1. Arrow: GalNAcDSLc4. (Panel b) Comparison of Western blotting of the protein extract of DU145 before and after treatment with the hemoglobin column. The major glycoprotein with approximate molecular mass of 50 kDa is thought to be a form of haptoglobin-α and -β chain complex. Lane 1: protein extract untreated, lane 2: fraction I (the eluate of protein extract passed through the column without incubation), lane 3: fraction II (the eluate of protein extract passed through the column 24 hr after incubation with the column beads), lane 4: wash I (the washed fraction after collecting fraction II).

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Involvement of glycosylation in RM2 reactivity to serum haptoglobin-β chain

RM2 reactivity to serum haptoglobin-β chain was little changed after treatment with β-hexosaminidase (Fig. 7b) or sialidase from NDV (data not shown). However, most of RM2 reactivity to serum haptoglobin-β chain disappeared after treatment with β-hexosaminidase followed by NDV sialidase (Fig. 7c).

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Figure 7. Involvement of glycosylation in RM2 reactivity to serum haptoglobin-β chain. (Panel a) No treatment, (Panel b) treatment with β-hexosaminidase and (Panel c) treatment with β-hexosaminidase followed by NDV sialidase. Arrow: haptoglobin-β chain. 1–4: sera from prostate cancer patients, 5–8: sera from BPD patients. PSA level for each case was indicated at bottom.

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Discussion

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In the current study, we found that haptoglobin-β chain defined by RM2 is a novel marker, which significantly increased in sera of early prostate cancer. Furthermore, RM2 showed preferential reactivity toward haptoglobin-β chain derived from sera of prostate cancer, suggesting haptoglobin-β chain is qualitatively different between prostate cancer and BPD, and haptoglobin-β chain defined by RM2 is more associated with prostate cancer than BPD. Qualitative change of haptoglobin was also suggested in head and neck cancer since haptoglobin derived from cancer sera was immunosuppressive whereas that from normal sera was not.22 Thus, both quantitative and qualitative changes of haptoglobin-β chain may be the basis of significant difference of RM2 reactivity to haptoglobin-β chain between prostate cancer and BPD. We also examined whether GalNAcDSLc4 as a ganglioside recognized by mAb RM2 was observed in prostate cancer cells. However, GalNAcDSLc4 was not detected in cell pellets or culture supernatants of prostate cancer cell lines. Furthermore, most of RM2 reactivity to protein extract from prostate cancer cells disappeared after incubation with the hemoglobin column. These results indicate RM2 reactivity to prostate cancer cells largely depends on haptoglobin-β chain.

Because haptoglobin-β chain defined by RM2 also increased in the other urogenital cancers, i.e., it is not specific to prostate cancer, it may be useful for detection of early prostate cancer when coupled with PSA as organ-specific marker. For clinical application, men with lower level of haptoglobin-β chain defined by RM2 may be a problem. Although the number of cases examined was small, transition zone cancer predicted lower level of RM2 reactivity when compared with peripheral zone cancer. Considering that transition zone cancer demonstrates more favorable pathologic features,23 the method that among men with increased PSA (4–10 ng/mL), those with increased level of haptoglobin-β chain defined by RM2 undergo prostate biopsy, and those without increase in haptoglobin-β chain undergo periodical PSA check and have prostate biopsy when rising PSA is observed may be one of the options for cancer screening. However, a large number of cases are necessary to confirm the relationship between haptoglobin-β chain defined by RM2 and the origin of index cancer.

mAb RM2 was originally established toward disialoganglioside and later found to recognize the glycosyl epitope (β1.4-GalNAcDSLc4).7, 8 In the present study, the proteomics approach showed that mAb RM2 also reacted with haptoglobin-β chain. Based on the findings of the current study, glycosylation status of serum haptoglobin-β chain was examined.16 Fujimura found that haptoglobin-β chain has minor O-glycosylation site in addition to 4 N-glycosylation sites,19, 20 and glycosylation status of serum haptoglobin-β chain was different between prostate cancer and BPD.16 However, the glycosyl epitope (β1,4-GalNAcDSLc4) recognized by mAb RM2 was not detected on haptoglobin-β chain.16 Nevertheless, large amount of serum is necessary for thorough analysis of glycosylation to conclude whether the RM2 glycosyl epitope exists or not on haptoglobin-β chain (personal communication with Dr. Fujimura). In the current study, RM2 reactivity to haptoglobin-β chain was little changed after treatment with β-hexosaminidase or NDV sialidase, but most of it disappeared after treatment with β-hexosaminidase followed by NDV sialidase. Because mAb RM2 showed reactivity to DSLc4 in addition to GalNAcDSLc4,8 changes of RM2 reactivity to haptoglobin-β chain after β-hexosaminidase/sialidase treatment may be compatible with RM2 reactivity to the glycosyl epitope (β1.4-GalNAcDSLc4). However, the existence of RM2 glycosyl epitope has not been verified on haptoglobin-β chain. Therefore, these results only indicate that glycosylation is involved in RM2 reactivity to haptoglobin-β chain, although it remains to be answered whether RM2 reacts directly with a glycosyl epitope or with possible conformational changes induced by glycosylation.

As to the production site, haptoglobin also known as an acute-phase protein has been reported to be mainly produced by liver and secreted into the bloodstream.24 In the patients with cancer, haptoglobin could be either produced by the tumor cells25 or the normal cells in response to the stimuli such as IL-6 produced from the tumor cells.26 Increased expression of haptoglobin in prostate cancer cells as suggested by RT-PCR and immunohistochemistry in the current study indicates that haptoglobin could be produced by cancer cells. In addition to quantitative increase, qualitative change of molecule also indicates haptoglobin is produced from cancer cells since modification of proteins such as aberrant glycosylation could be induced with carcinogenesis. Thus, the previous and the current studies indicate that haptoglobin could be produced from prostate cancer cells. Because haptoglobin occurs in vivo as polymers of an α and β chain complex,21 elevation of haptoglobin-β chain may be explained by dissociation of α and β chain from haptoglobin.27 However, although cleavage of haptoglobin by protease is assumed to be responsible for elevation of haptoglobin-α and -β chains, the exact mechanism of elevation of these chains remains to be clarified.27

There has been accumulating evidence of haptoglobin expression in sera of various cancers. Haptoglobin-α or -β chain is upregulated in sera of ovarian cancer, breast cancer, acute myeloid leukemia, hepatocellular carcinoma, small cell lung cancer and RCC.27–32 Furthermore, haptoglobin-β chain carrying α1 Fuc residue, blotted by Aleuria aurantia lectin, is significantly enhanced in sera of gastric cancer, colon cancer, hepatocellular carcinoma and pancreatic cancer.25 In the present study, increase in haptoglobin-β chain defined by RM2 was observed in sera of RCC, urothelial carcinoma and testicular germ cell tumors as well as prostate cancer. Because of this universal increase in haptoglobin-α or -β chain in various malignancies, haptoglobin-β chain defined by RM2 may have potential of exploiting a new approach to serum diagnosis of cancer other than the urogenital malignancies.

In conclusion, haptoglobin-β chain defined by RM2 is a novel serum marker that may be useful for detection of early prostate cancer when coupled with PSA as organ-specific marker because it is not specific to prostate cancer. However, larger trials are necessary to confirm the findings in the current study. It is also important to examine whether haptoglobin-β chain defined by RM2 can be detected in sera of various malignancies other than the urogenital cancers.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Dr. Sen-itiroh Hakomori and Dr. Taeko Miyagi for helpful advice to this study, Dr. Stephen Anderson for preparation and arrangement of the manuscript and figures and Ms. Emiko Idutsu for assisting serum sample collection.

References

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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