Abstract
OBJECTIVE To identify metastasis-related biomarkers in human ovarian cancer cell lines and in serum.
METHODS We isolated total protein from cell lysis solutions and cultured supernatants from 2 human ovarian cancer cell lines and used SELDI-TOF-MS to detect the differential expression of the proteins in the 2 cell lines. The proteomic spectra were generated using weak cation exchange chips. The biomarkers were validated by analyzing serum proteins or peptides in ovarian cancer patients, relapsed ovarian cancer patients, patients with benign ovarian tumors, and healthy people.
RESULTS Four proteins in the culture supernatant from HO-8910PM cells were up-regulated, relative to the culture supernatant of HO-8910 cells. One protein (3,144 Da m/z value) was up-regulated in both the cell lysis solution and in the culture supernatant of HO-8910PM cells. In addition, expression of the 3,144 Da m/z protein differed significantly between serum from the 26 ovarian cancer patients, from the 22 relapsed ovarian patients and from the 37 healthy women (P < 0.01). However, there was no difference between patients with benign ovarian tumors and healthy people (P > 0.5).
CONCLUSION Ovarian cancer cell lines with high or low metastatic potential have distinct protein profiles. Protein 3,144 Da m/z could be a useful biomarker for diagnosing ovarian cancer metastasis.
keywords
Introduction
Ovarian cancer is one of the most common tumors of the female reproductive system. Unfortunately, the majority of epithelial ovarian cancers remain clinically undetected until progression to late stage disease, and in one study, only 25% of tumors were detected in stage I upon diagnosis[1]. Despite advancements in cytoreductive surgery and combination chemotherapy over the last 2 decades, long-term survival has remained stable[2]. In order to increase long-term survival, molecular therapeutics must be linked to molecular diagnostics in order to develop individualized therapy[3].
The profiling of low-molecular-weight serum proteins might help reflect the pathologic state of organs and aid in the early detection of cancer. Surface-enhanced laser desorption and ionization time-of-flight (SELDI-TOF) mass spectroscopy (MS) can detect proteins in this range, and several serum biomarkers identified by this method have been used to detect early ovarian cancer[4,5]. Our aim was to identify metastasis-related biomarkers by comparing the differential protein expression profiles of 2 human ovarian cancer cell lines with different metastasis potentials in vitro. We then aimed to validate these biomarkers by analyzing serum proteins or peptides in ovarian cancer patients, relapsed ovarian cancer patients, women with benign ovarian disorders, and healthy women.
Materials and Methods
Chemicals and apparatus
RPMI1640 was obtained from Gibco and the calf serum from Hangzhou Sijiqing Company. Acetonitrile, trifluoroacetic acid, urea, CHAPS, sinapic acid, DL-dithiothreitol, Tris-HCL, HCL, sodium acetate and water were purchased from Sigma-Aldrich. ProteinChip Biology System (PBSIIc) mass spectrograph, weak cation-exchange (WCX2) protein chips were purchased from Ciphergen Biosystems.
Cell lines
The ovarian cancer cell line HO-8910[6] and the highly metastatic ovarian cancer cell line HO-8910PM[7] were provided by the Zhejiang Cancer Institute. HO-8910 and HO-8910PM were cultured in RPMI1640 including 15% calf serum, at 37°C in 5% CO2. Calf serum was removed after 24 h. Samples were centrifuged at 15,000 rpm at 4°C for 30 min. The culture supernatant was immediately frozen in aliquots of 100 μl at -80°C. The cells were lysed using RIPA lysis buffer and centrifuged at 15,000 rpm at 4°C for 30 min. The supernatant was immediately frozen in aliquots of 100 μl at -80°C.
Cancer patients and the controls
We used a retrospective sample of 120 serum specimens. All samples were collected from May 2006 to May 2007 with the approval of Zhejiang Cancer Hospital. The study was performed after approval from the institutional Human Investigations Committee. The samples were collected from 26 patients with invasive epithelial ovarian cancer in stages III/IV (median age 53 years, range 37-74 years), and 22 patients with relapsed ovarian cancer (median age 48 years, range 26-67 years). All of the cancer patients had serous tumors. The remaining samples were collected from 35 patients with benign ovarian disorders (median age 42.5 years, range 21-71 years) and 37 healthy controls (median age 39 years, range 24-65 years). The malignant or benign diagnosis was confirmed by pathologists.
Serum samples
Blood samples were collected from the patients before treatment and stored at 2°C-8°C for a maximum of 2 h before centrifugation. They were processed according to standardized protocol. After collection, samples were centrifuged at 4,000 rpm for 6 min, and the serum was immediately frozen in aliquots of 100 μl at -80°C. For proteome fractionation, samples were thawed at room temperature for 5-10 min and processed immediately.
SELDI analysis
The weak cation-exchange (WCX2) ProteinChip was selected for our current study. WCX2 ProteinChip is a weak cation exchange array with a carboxylate surface to bind cationic proteins. The negatively charged carboxylate groups on the surface of the WCX2 chip interact with the positive charges exposed on the target proteins. Per protocol, 10 μl of serum were incubated with 20 μl of U9 (9 mol/L Urea, 2% CHAPS, 50 mmol/L Tris-HCl pH 9.0, 1% DTT) at room temperature for 30 min on a platform shaker. After the samples were ready, they were incubated at room temperature for 60 min on a platform shaker, and the array was washed twice with 200 μl of binding buffer for 5 min. Before SELDI analysis, 0.5 μl of a saturated EAM solution (sinapinic acid in 50% acetonitrile/0.5% trifluoroacetic acid) was applied to each chip array twice, allowing the array surface to air dry before each chip array was used. Chips were placed on the Protein Biological System II mass spectrometer reader, and time-of-flight spectra were generated by averaging 90 laser shots collected in the positive mode at a laser intensity of 185 and with a detector sensitivity of 8. Mass accuracy was calibrated on the day of measurement using the all-in-one peptide molecular mass standard (Ciphergen Biosystems).
Statistical analysis
Peak detection was performed using Ciphergen ProteinChip software 3.2 (Ciphergen Biosystems). A mass range of 1,000-50,000 Da was selected. Peak detection involved i) baseline subtraction, ii) mass accuracy calibration, and iii) automatic peak detection. Using Biomarker Wizard (Ciphergen Biosystems), biomarkers which were consistent with protein peaks across multiple spectra were identified. After biomarker identification, the quality and intensity of the readings of the selected peaks were manually reconfirmed from raw spectra. The Receiver Operating Characteristic (ROC) curve (AUC) was computed to identify the peaks with the highest potential to discriminate between ovarian cancers and benign tumors. For cell lines, different mass spectrometry peaks were defined as intensity ratios > 2[8].
Protein identification
Proteins were identified using TagIdent (http://expasy.org/tools/tagident.html). The peak sizes were determined by SELDI-TOF-MS analysis. The search criteria allowed for a 0.1% size error.
CA125 assay
CA125 assay was performed by chemiluminescent microparticle immunoassay using the Architect i2000 system (Abbott, USA). The cut-off value was set at 35 U/ml.
Results
Differential protein expression in HO-8910PM and HO-8910 cells
Four distinct protein peaks (3011, 3021, 3144 and 6635 Da) were detected in the culture supernatants of HO-8910PM and HO-8910 cells, all of which were relatively up-regulated in HO-8910PM cells (Table 1). The 3,021 m/z and 3,144 m/z peaks are shown in Fig. 1. In addition, 11 distinct protein peaks were detected in the lysis solution from HO-8910PM and HO-8910 cells. A distinct 3,144 m/z peak was found in both the lysis solutions and culture supernatants from HO-8910PM and HO-8910 cells.
The different concentrations of 3144m/z detected in sera
The average values of 3,144m/z in ovarian cancer patients in stage III-IV, relapsed ovarian cancer patients, patients with benign ovarian tumors and healthy women were 1.64 ± 1.01, 1.32 ± 0.72, 0.78 ± 0.50 and 0.76 ± 0.34, respectively (Table 2). In addition, 3,144 Da m/z expressions differed significantly among the 4 groups of ovarian cancer patients, ovarian cancer relapse patients, benign ovarian tumor patients and healthy people (P < 0.01). However, there were no differences between benign ovarian tumor patients and healthy people (P > 0.5).
Differences in concentrations of CA125 detected in sera between ovarian cancer and relapsed ovarian cancer patients
The average CA125 levels in 26 ovarian cancer patients in stage III-IV and 22 relapsed ovarian cancer patients were 2,473.36 ± 2758.99 U/ml and 590.89 ± 801.57 U/ml, respectively. CA125 levels were significantly higher in ovarian cancer patients than in relapsed ovarian cancer patients. The difference in CA125 levels between ovarian cancer and relapsed ovarian cancer patients was consistent with differences in the 3,144 m/z peak.
ROC curve of 3,144 m/z in stages III/IV invasive epithelial ovarian cancers and benign ovarian tumors
ROC analysis was performed by comparing samples from 26 patients with stages III/IV invasive epithelial ovarian cancer and 35 patients with benign disorders. The ROC AUC of the 3,144 m/z peak was 0.78. The sensitivity and specificity of the 3,144 m/z peak for discriminating between malignant and benign ovarian tumors were 65.4% and 91.4%, respectively, with a cut-off value of 1.15 (Fig. 2).
Proteins identified by TagIdent (http://expasy.org/tools/tagident.html)
We performed an online TagIdent (protein database) search using the size of 3,144 m/z determined from SELDI-TOF-MS analysis. Our search criteria allowed for a 0.1% size error. We identified a CD24 fragment (Chain: 27-59, pI: 5.72, MW: 3142) as a potential candidate protein for the 3144 Da peak.
Discussion
Complex serum proteomic patterns might reflect the underlying pathologic state of organs such as the ovary. SELDI-TOF-MS, an innovative proteomic technology, has overcome many of the limitations of two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry[9]. SELDI-TOF is widely used to investigate serum proteomic patterns[10]. The use of proteomics for identifying potential diagnostic biomarkers has been explored so far in several types of cancers, including prostate[11,12], colon[13], breast[14] and ovarian cancer[4].
We discovered a distinct 3,144 m/z protein peak that was consistently present in lysis solutions and culture supernatants from HO-8910PM and HO-8910 ovarian cancer cells. It differed significantly among ovarian cancer patients, relapsed ovarian cancer patients, benign ovarian tumor patients, and healthy women. Using the online TagIdent tool, we identified a CD24 fragment (Chain: 27-59, pI: 5.72, MW: 3142) as a potential candidate protein for the 3,144 Da peak. CD24 is a small, heavily glycosylated cell surface protein, which is expressed in a large variety of solid tumors. It is thought to play an important role in tumor progression and metastasis[15-17]. CD24 expression is associated with rapid cell spreading and high degrees of cell motility and invasion[18]. In addition, CD24 is an established marker for poor prognosis in ovarian and other carcinomas[19-21].
The origin and full identity of discriminatory proteins or peptides are still under investigation. They have been detected in the low-molecular-weight serum proteome but still remain unknown at present. These proteins or peptides could be derived from the host organ or the tumor, or could constitute metabolic fragments. By comparing the differential expression of proteins in 2 human ovarian cancer cell lines with different metastatic potential, and by comparing serum proteins or peptides in ovarian cancer patients, ovarian cancer relapse patients, benign ovarian tumor patients, and healthy women, we identified several metastasis-related proteins. These proteins or peptides might be proven to be useful biomarkers for the diagnosis of ovarian cancer metastasis.
Footnotes
This work was supported by grants from the Medical Scientific Research Foundation and Bureau of Health Care for Senior Officials of Zhejiang Province, China (No.2007B026) and the National Natural Science Foundation of China (No.0471819).
- Received May 14, 2009.
- Accepted September 15, 2009.
- Copyright © 2009 by Tianjin Medical University Cancer Institute & Hospital and Springer