Changes in the Serum Protein Composition in Mice with Transplanted ...

DOI 10.1007/s10517-015-2791-2 Bulletin of Experimental Biology and Medicine, Vol. 158, No. 4, February, 2015

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ONCOLOGY Changes in the Serum Protein Composition in Mice with Transplanted Ehrlich’s Carcinoma N. G. Kormosh, R. Kh. Ziganshin*, V. O. Shender*, K. E. Voyushin, and F. V. Donenko Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 158, No. 10, pp. 491-494, October, 2014 Original article submitted July 4, 2012 Injection of blood serum from mice with Ehrlich’s carcinoma stimulates the growth of transplanted tumor, which proves the presence of tumor-specific factors in the serum. Experiments on (CBA×C57Bl/6)F1 male mice with transplanted Ehrlich’s carcinoma demonstrated the appearance of new proteins in the serum, some of them are identified. The authors suggest continuing the search for tumor-associated factor by combining proteomic analytical methods and testing of identified candidate proteins for their effects on tumor growth. Key Words: blood serum proteins; Ehrlich’s carcinoma

Studies of tumor-specific factor and methods of its identification are in progress. The hypothesis on the presence of a factor regulating the tumor growth in the blood serum was proposed after analysis of the results of numerous experiments on mice [3]. Our studies indicate that partial elimination of the tumor stimulates mitotic activity of remaining tumor cells [2]. Presumably, this factor quantitatively and qualitatively differs in histological variants of tumors at different stages of the tumor process. Hence, the development of methods for laboratory identification of tumor-associated factor is expected to extend the potentialities of diagnosis and improve the results of therapy for cancer. However, despite the use of the most up to date proteomic methods for protein identification, the factor remains not identified. Search for potential protein markers of gastric cancer in human serum has detected tens of potential candidates [6]. It seems that search for this factor should be realized by means of a biological test N. N. Blokhin Russian Cancer Research Center, Russian Academy of Medical Sciences; *M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Medical Sciences, Moscow, Russia. Address for correspondence: nkormosh@hotmail. com. N. G. Kormosh

for analysis of specific activity of candidate proteins detected by proteomic analysis. This is expected to detect the differences in the serum protein composition in normal animals and animals with tumors and demonstrate the biological significance of the detected differences. We studied the serum protein composition in mice with transplanted Ehrlich’s carcinoma and the effect of this serum on primary tumor growth.

MATERIALS AND METHODS The study was carried out on male (CBA×C57Bl/6) F1 males hybrids. Ehrlich’s carcinoma (Tumor Strain Bank, N. N. Blokhin Russian Cancer Research Center) was transplanted intraperitoneally (106 cells in 0.2 ml RPMI-1640). Mice were decapitated on day 10 after tumor transplantation. The serum was separated by centrifugation and divided into two aliquots that were frozen at -18oC. One aliquot was then used for proteomic analysis, the other for studies of the biological activity of the sample. The experiments were repeated 3 times. The animals were divided into 3 groups, 10 per

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Bulletin of Experimental Biology and Medicine, Vol. 158, No. 4, February, 2015 ONCOLOGY

group. Group 1 were mice with transplanted carcinoma, group 2 were mice with transplanted carcinoma, injected with intact mouse serum, and group 3 were mice with transplanted carcinoma injected with the serum from mice with tumors. The data were statistically processed by Fisher– Student’s test. The results were considered significant at p≤0.05. Preparation of samples for proteomic analysis. Protease inhibitors and a mixture of nucleases were added to blood samples, after which the proteins were solubilized with lyzing buffer: 8 M urea, 2 M thiourea, 5% ampholines (pH 3-10), 80 mM dithiotreitol (DTT), 17% solution of 30% CHAPS, and 10% NP40. The samples were incubated at 25oC for 30 min and centrifuged at 13,000 rpm for 15 min. The supernatant was collected and protein concentrations in the samples were measured by the method Bradford with BSA as the reference sample. The samples were stained with CyDye DIGE Fluors (GE Healthcare) according to the instruction. A specimen of normal mouse serum (control) was stained with Cy3 stain, serum specimen from mouse with Ehrlich’s carcinoma (tumor) was stained with Cy5. Two-dimensional electrophoresis (2D-PAGE). Isoelectrofocussing was carried out in glass tubes on a Protean 2D Cell Components (Bio-Rad). The protein bands of the gel were then incubated in buffer (6 M urea, 30% glycerol, 20 g/liter SDS, 0.5 M Tris [pH 8.8], and 20 mM DTT) for 30 min, after which the preparations were transferred to vertical gradient (916%) PAAG prepared by the standard protocol [4,7]. Protein separation was carried out in a Protean XL Multi-Cell device (Bio-Rad). Protein spots were visualized by silver citrate staining. After staining, the gels were scanned on a PharosFX device (Bio-Rad). Trypsinolysis and mass spectrometry. Protein spots selected by the program were cut out of the gel by EXQuest Spot Cutter 165-7200 (Bio-Rad) and transferred into microtubes with a small volume of water for chromatography. Trypsinolysis and extraction of peptides were carried out according to the standard protocol [10]. Mass spectra were obtained on a MALDI Time-of-flight device (Ultraflex TOF/ TOF; Bruker Daltonik GmbH) in the reflection mode of registration of positively charged ions for 5002000 Da weights. The data were processed by Flex Analysis 3.3 software (Bruker Daltonics). The following parameters were used for analysis: weight evaluation precision (100 ppm), SwissProt database, Rodentia taxonomic category, one missed site of specific trypsin cleavage allowed. The method for estimation of the identification reliability was described in electron resource (www.matrixscience.com/help/scoring_help.html).

RESULTS Many new proteins are detected in the serum during tumor growth (Fig. 1); 16 of them are identified (Table 1). All the resultant proteins are classified in 2 groups – cellular and serum. Acute phase proteins are identified with high probability by Score test: serum amyloid A1 and A2 proteins and apolipoprotein A-I. Cellular proteins include apoptosis mediating proteins, transcription, DNA reparation and replication factors, structural and transport proteins, essential for mitosis. The increase in their serum content in mice with tumors can be attributed to the increase in the number of dividing cells. Importantly, 2D electrophoresis is a standard procedure and hence, the location of all protein spots and their mutual disposition is reproduced from one experiment to another and in comparison with the results of other scientists, except cases with analysis of partially destroyed protein molecule. The protein proportion is liable to change, and hence, we present the estimated weights of protein molecules and their isoelectric points (Table 1), essential for comparison of the results obtained by different research groups. The identified protein spots are not the major serum proteins, and the relative contribution of new spots in the total bulk of serum proteins is