Postoperative recurrence of epithelial ovarian cancer patients and chemoresistance related protein analyses

This study was approved by the Ethics Committee of Hebei Medical University and signed consent was obtained from each patient. Forty patients from September 2013 to September 2014 at Gynecology Department in The Fourth Hospital of Hebei Medical University were enrolled in the study. All patients were diagnosed to be epithelial ovarian cancer and underwent complete ovarian cancer excision or ovarian tumor cell subtraction surgery. The diagnoses were further verified at postoperative pathological confirmation. After surgery, all patients received 6–8 cycles of the standard of platinum-based chemotherapy, and were followed up to at least 6 months. The 6–8 cycles of treatment consisted of platinum-based combination chemotherapy with paclitaxel of 135-175 mg/m2, carboplatin AUC 5-7 mg/ml/min, oxaliplatin of 130 mg/m2, and nedaplatin of 80-100 mg/m2. Inclusion criteria are as following: Pathological types were epithelial ovarian cancer; there was no history of other malignant tumors; the initial treatment was paclitaxel and carboplatin-based chemotherapy of 6 to 8 courses with follow-up satisfactory cytoreductive surgery or comprehensive staging surgery for early-staged ovarian cancer. Exclusion criteria are as following: Non-epithelial ovarian cancer; in the absence of a comprehensive staging of early ovarian cancer, or satisfactory cytoreductive surgery or without completion of 6–8 courses of chemotherapy; with other malignancies or history other tumors.

According to the National Comprehensive Cancer Network (NCCN) guidelines, after the initial treatment of platinum-based chemotherapy, the cancer recurrence within 6 month was defined as ovarian cancer chemotherapy-resistant, while the recurrence with more than 6-month were defined as ovarian cancer chemotherapy- sensitive. Ovarian cancer clinic outcome can be classified into following types: 1) chemotherapy-sensitive: after the initial administration of platinum-based chemotherapy, the patient has reached clinical remission but undergoes recurrence after more than 6-month following the termination of chemotherapy; 2) chemotherapy resistant: after the initial administration of platinum-based chemotherapy, the patient has reached clinical remission but undergoes recurrence within 6-months following termination of chemotherapy; 3) persistent ovarian malignancy: after the initial administration of platinum-based chemotherapy, the patient has demonstrated reaction or response but shows tumors after further examination; 4) refractory ovarian malignancy: platinum-based chemotherapy is ineffective and the tumor undergoes stabilization or progression. The chemotherapy-resistant, persistent ovarian malignancy and refractory ovarian malignancies are classified in the ovarian cancer chemotherapy-resistant group.

Urea, thiourea, dithiothreitol (DTT), 3-[3-(cholamidopropyl) dimethylamino] propanesulfonic acid salt (CHAPS), iodoacetamide, tricarboxylaminomethane (Tris), glycine and low melting point agar (10 cm) were from GE Corporation (Shanghai, China); sugar, trichloroacetic acid (TCA), ammonium persulfate (APS), sodium carbonate, N, N, N ‘, N’-tetramethyldiethylamine (TEMED), silver nitrate sodium thiosulfate and EDTA were from Amresco (Washington DC, USA); DC protein assay kit was from Bio-Rad (CA, USA). Bovine serum albumin (BSA) was purchased from Pierce; albumin and IgG protein removal and desalting kits were Merck (NJ, USA); trypsin was from Promega (Madison, WI, USA); formic acid, acetonitrile, tris-HCl (pH 6.8, pH 8.5, pH 8.8), coomassie Brilliant blue R-250, coomassie brilliant blue G-250, bromophenol blue, acetone, methanol, phosphoric acid, n-butanol, ethanol, glacial acetic acid, ammonium sulfate were obtained from Sigma (St. Louis, MI, USA). Low temperature high speed centrifuger (TTICH) was from Heraeus (Hanau, Germany); ultraviolet spectrophotometer was from Beckman (Bernard, FL, USA), eppendorf refrigerated centrifuge was from Eppendorf (German); IPGhor isoelectric focusing instrument, DALT-SIX SDS (TTICH type) and ImageScanner Scanner were from Shanghai GE Healthcare; PDquest Analysis Software was from Bio-Rad; ABI 5800 MALDI-TOF/TOF Tandem Mass Spectrometer was from ABI (USA).

The morning fasting peripheral venous blood of 2 ml from each patient was placed into EDTA anticoagulation vacuum blood collection tube (EP tube) and centrifuged at 3000 rpm for 15 min. The plasma was aspirated and divided into 5 EP tubes with 150 μl per tube. The plasma was stored in a refrigerator at − 80 °C.

The frozen samples were removed from − 80 °C and thawed. Plasma samples in the same group were mixed and the high abundant proteins in the samples were removed using the albumin and IgG protein removal kit. The salt ion was removed by a desalting kit and then the sample was added with protein lysis buffer containing 9 M of urea, 1% IPG buffer (GE Healthcare), 1% Dithiothreitol (DTT), 4% of 3- [3- (cholamidopropyl) dimethylamino] propanesulfonic acid salt (CHAPS). After incubation in 30 °C water bath for 1 h, the sample was centrifuged at 15000 g for 15 min at room temperature. The supernatant was aspirated and centrifuged again at 15000 g for 15 min at room temperature. The supernatant from the second centrifugation was taken and the protein concentration was determined with DC Protein Assay kit.

A total of 300 μg protein sample was adjusted to a total volume of 450 μL with sample hydrating solution and loaded onto the slot of IPG strip (GE Healthcare, 24 cm, pH = 3–10). Isoelectrophoresis was performed at 20 °C with the maximum current of 50 μA/strip. The isoelectric focusing procedure was: 50 V for 12 h, 500 V for 1 h, 1000 V for 1 h, gradient from1000 to 10,000 V for 1 h, and finally 10,000 V for 10 h. After soaking in SDS-PAGE electrophoresis buffer for 10 min, the gel was transferred to an electrophoresis tank of Ettan-DALT-Six system and electrophoresed at 100 V for 45 min and then 200 V for bromine to run out of the gel. The gel was then subjected to silver nitrate staining as described [7].

The images were scanned with the Image Scanner with the optical density of 300dpi. The images were analyzed by PDquest 8.0 software. The main operations included gel protein spot detection, image background subtraction, protein point gray value standardization, and different gel protein spots matching. The 2-D electrophoretic images were compared and the protein spots with 2 times or less than 0.5 times were selected as the differential protein spots. The differential protein spots were selected as the follow-up mass spectrometry.

The protein was digested and extracted with 50 μL extraction buffer. The protein extract was lyophilized and the dry powder was redissolved with 5 μL of 0.1% TFA solution. The solution was mixed with 5 μL saturated α-cyano-4-hydroxycinnamic acid containing 50% ACN and 1% TFA. 1 μL of the sample was subjected to mass spectrometry (MALDI-TOF/TOF) using ABI5800 Series Time-of-Flight Mass Spectrometer. The data were automatically acquired using positive ion mode. The primary and secondary mass spectrometry data were integrated using GPS 3.6 (Applied Biosystems) and Mascot2.3 (Matrix Science) to analyze and identify proteins. The search parameters are as follows: the database is human database; the enzyme is Trypsin; the maximum allowable breakpoint is 1; the fixed modification is carbamidomethyl (C); the variable modification is Acetyl (Protein N-term), deamidated (NQ), dioxidation (W) and oxidation (M); MS tolerance is 100 ppm and MS/MS tolerance is 0.5 Da. Protein score C.I. % of greater than 95% is defined as successful identification of a protein.

All data were analyzed using SPSS19.0 statistical software. Comparison was made by t test between two groups. A P value of < 0.05 was considered statistically significant.