| BackgroundBreast cancer is one of the most frequent types of female malignant tumors, in the past 10 years, the morbidity of breast cancer is gradually increasing. Despite the fact that the morbidity rises with age and the postmenopausal woman are at a higher risk of suffering from breast cancer there are yet 15%~26% of the females who were diagnosed with breast cancer in premenopausal, 7% of which were below 40 years old. A considerable number of young women of childbearing age were diagnosed with breast cancer and this affected their physical and mental health as well as the quality of life. The prognosis of breast cancer is associated with multiple factors, such as the histomorphological types and clinical staging(whether distant metastasis exists). The breast cancer patients will be at high risk of metastasis to other organs such as the liver in all life. Metastasis and relapse have yet been the main obstacles of survival time of breast cancer patients. Recently, the main reason of high mortality rate of breast cancer is the lack of biomarkers that could be used to screen and diagnose cancer in the early stage and the lack of knowledge about the disease among women. If we can find a panel of biomarkers which can be used to detect the breast cancer in the subclinical stage, and then can help to prevent or reverse thedevelopment of breast cancer by early interventions, the prognosis of the patients will be significantly improved. Although the serum biomarkers have not yet played a major role in breast cancer diagnostic or prognostic practice, an effective biomarker panel in an easily accessible biological fluid would be a valuable and minimally invasive adjunct to other clinical and pathological approaches. Hence it is important to find new protein markers which can help to make an early diagnosis, a correct prognosis and evaluate the curative effect.Proteomics can contact with and reflect the changes of the oncogene and cellular physiology, thus people place high hopes on finding tumor biomarkers via protein analysis technology. Matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry(MALDI-TOF-MS) and the derived surface-enhanced laser desorption/ionization mass spectrometry(SELDI-TOF-MS) enable the development of high-throughput proteome analysis based on comprehensive reliable biomarkers,and it also provide very large development space in the researches of new panel of biomarkers in the serum of breast cancer. The protein profiles between tumor tissue and normal tissue, or the body fluid of the tumor sufferer and normal person can be compared via SELDI-TOF-MS, proteins of differential expression can be found such as tumor associated proteins. Many researchers think that the accuracy can be improved if we can predict and diagnose disease according to the proteome changes.It is more accurate and effective to detect the proteome changes than the gene changes.In this research, we analyzed the serum of breast cancer and healthy females by surface-enhanced laser desorption ionization time-of-flight mass spectrometry(SELDI-TOF-MS), investigated the proteome profiles between the breast cancer women and normal women, pre-operative and post-operative patients, and the triple-negative breast cancer and non-triple negative patients, to find the related serum biomarkers. We also used other peoteomics technologies, such as MALDI-TOF-MS,Tricine-SDS-PAGE, and Western blot to identify and verify the potential protein biomarkers of the breast cancer. We performed the in vitro study in breast cancer cell lines, and observed its effect on tumor growth in the tumor-bearing mice, and compared the difference of immunohistochemical results between the experimentalgroup and control group so as to explore its biological characteristics and effect on breast cancer.ObjectiveThe objectives of this study were to:(a) identify the proteome profiles between the breast cancer patients and normal individuals,(b) identify the proteome profiles between the pre-operative and post-operative patients,(c) identify the proteome profiles between triple-negative and non-triple negative patients,(d) find the differential expressing proteins and screen out a panel of biomarkers which can reflect the existence of tumor, evaluate the curative effect and identify different molecular types of tumor, and build a perfect protein fingerprint pattern model for early diagnosis, curative evaluation and prognosis analyzing. Breast cancer cell lines and zoology experiments were conducted for the screened and identified target protein to know more about its biological characteristics and explore its role in the treatment of breast cancer, and new method will be provided for clinical diagnosis and treatment of breast cancer and the discovery of new drug targets.Materials and methods1 MaterialsSixty(60) cases of female breast cancer patients, aged 23~70 years old with the mean age of 46.7±0.4, without any treatment between June 2011 to June 2014 were enrolled. All of them underwent operation, and all diagnoses were confirmed by postoperative pathological examination. The patients received regular radiotherapy or chemotherapy after operation, while these factors were not considered in the research.22 out of 60 cases were triple-negative breast cancer patients. Twenty(20)participants who had no disease were enrolled as controls. Written informed consent was obtained from the participants by the researchers. Serum was collected from all the breast cancer patients before operation, 2 weeks after operation in all the 60 cases.All the blood specimens were collected on an empty stomach in the morning, andkept at room temperature for 30-60 minutes. Specimens were then run on a centrifuge at 3000 r/min for about 20-30 min.The serums were extracted and preserved under-80℃ for 1 hour.2 Instruments and reagentsSELDI-TOF-MS, 96 holes protein chip(Bioprocessor), SELDI chip, WCX2 chip, CHAPS, Acetonitrile, DTT, TEMED were all purchased from Ciphergen Company in USA. MALDI-TOF-MS was purchased from Kratos Analytical Company in UK. SPD Speed Vac was purchased from Thermo Electron Inc Company in USA, Trypsase were purchased from Promega Inc Company in USA. The Analogous Apolipoprotein C-I peptides were synthesised by the Ze River Source Company in China.3 Methods3.1 Selection of specific proteins in breast cancer serumFirstly, the serum specimens were unfrozen, then dealt with the samples by weak positive factor(WCX) and mixed the matrix with the samples. The handled samples were loaded into protein chip board. The reading operating procedures were set as follows: the molecular weight range was 2000Da-20000 Da, and the largest molecular weight was 30000 Da. The operating status and the sensitivity of the instruments were tested. After that, every protein chip board which was loaded with samples was put into the mass spectrograph. The data were collected using MELDI-TOF/TOF and were processed using the corresponding software in order to get m/z peaks in each sample Protein samples with less than 0.3% difference between their m/z peaks were considered to be the same kind. The groups with different m/z peaks were analyzed by Wilcoxon rank sum test. The less the P value was, the more significant between the different groups were, which was of more significance to find out the protein with clear difference.3.2 Identification and confirmation of target proteinAfter screening the samples, the preparation of the gel was started. We collected2 ml of serum samples from the breast cancer patients and dilute them to 5ml by adding ultrapure water. Then we started Tricine-SDS-PAGE. After that, rubber cutting operation was performed. The samples were put into different EP and marked to get the target protein by enzymatic hydrolysis. We added the enzymatic hydrolysis to the samples and extracted the liquid supernatant and mixed with the matrix, then made the sample application in the protein chip board. After laser bombardment in the MALDI-TOF-MS, We collected the sequences of peptides by Mascot software, which were then compared and matched with that in the Swiss Prot database to get the targeted protein. Finally, we used Western blot to verify the target protein biomarkers of the breast cancer.3.3 The effect and biological characteristics of Analogous Apolipoprotein C-I peptides in breast cancer cell lines in vivo and zoology experimentsThen the effect of targeted protein in breast cancer cell lines and the influence on breast cancer cell lines tumor-burdened bone tumors in mice were studied.Meanwhile immunohistochemical detection of tumors was in process to further understand the biological characteristics and clinical significance of the target protein in breast cancer.Results1 Detection of different breast cancer protein peak1.1 The breast cancer group and normal control groupIn order to get the peaks in each group, the protein map data of breast cancer group and normal control group was processed by denoising, baseline deduction,standardized treatment and clustering analysis. Through Wilcoxon rank sum test, we obtained 14 specific peaks(P<0.01), of which 11 peaks had high expression and 3peaks had low expression in breast cancer group. Through SVM, the models with the maximum Youden index were screened out, and the protein marker with an m/z of6447.9 was obtained. This obtained protein had a low expression in preoperative group with the intensity of 38.0187±34.2194, while in the control group it had a high intensity of 337.5261±207.6438. The results between the two groups were contracted obviously, with remarkable statistical significance(P<0.01).1.2 Preoperative group and postoperative groupMass spectrometry experiments were performed using the original data, 6specific peaks(P<0.01) were obtained, of which 4 peaks had high expression and 2peaks had low expression in preoperative group. SVM was used to find out the models with the maximum Youden index, and the protein with the m/z 6447.9 was also obtained. It had a low expression in preoperative group with the intensity of38.0187±34.2194 which it had a high expression in postoperative group with the intensity of was 294.1245±102.8634. The results have great statistical significance(P<0.01).1.3 Triple-negative group and non-triple negative groupSELDI-TOF-MS was used to obtain specific peaks between Triple-negative group and non-triple negative group, we got approximate peaks ranging about +0.3%.Among the 9 specific peaks(P<0.01), 4 peaks with low expression in triple-negative group, and 5 peaks with high expression in the non-triple negative group. We used SVM to find out models with the maximum Youden index, and the protein with the m/z 6447.9 was also obtained. The intensity in triple-negative was 5.1351±3.6437.while the intensity in the non-triple negative group was 43.6162±18.8542. The results have great statistical significance(P<0.01).2 Identification of confirmation of the specific protein peaksAfter separation, purification and enzymolysis to specific protein in tumour serum, we used the MALDI-TOF/TOF to detect the peptide fragment mixture and m/z 6447.9 was identified as Analogous Apo C-I. Finally the Western blot method was used and verified that the target protein biomarkers with the m/z 6447.9 was Analogous Apo C-I.3 The effect and biological characteristics of Analogous Apolipoprotein C-I peptides in breast cancer3.1 Analotides have lethal effect on breast cancer gous Apo C-I pepcell linesWith the increased concentration of peptides, damage to the breast cancer cell line MCF-7, MDA-MB-231 increased after the injection 24 h, 48 h, 72 h, 96 h, 120 h of the injection.3.2 Analogous Apo C-I peptides may inhibit the growth of breast cancer tumorburdened bone tumors in miceThe nude mice inoculated MCF-7 tumor cells were divided into two groups, the control group(not to) and the experimental group(peptides injection). By continuous caudal vein, the dosing tumors of the nude mice in the experimental group grew slower than that in the control group, the volume and weight of the tumors also had differences. At the same time, vital signs of the mice in the experimental group were stable, adverse reactions such as death did not occur. We can conclude that the adverse of the peptides is small but the effect is remarkable.3.3 The control group and experimental group tumors immunohistochemical resultsIn the healthy control group, the expression quantity of PCNA, Ki67, Bcl-2increased obviously, while the Bax expression quantity decreased. In the experimental group, the expression quantity of PCNA, Ki67, Bcl-2 decreased while the Bax expression quantity increased, visible tumor necrosis area can be found at the same time.ConclusionIn summary, we have identified a set of protein peaks that could discriminate breast cancer from normal controls. The 6447.9 peak was identified as Analogous Apo C-I. It can be considered as a potential proteomic biomarker of breast cancer, and can help to identify triple-negative breast cancer, which can be combined with CA-153 or CEA to improve the accuracy of early diagnosis, curative effect evaluation and observation of prognosis. We also verified the lethal effects of the Analogous Apo C-I peptides to breast cancer cell lines, which provides the theoretical foundation to further study the internal relations. Analogous Apo C-I peptides is a new type of anti-tumor protein drug, may become a new target of the breast cancer treatment, and it may play an important role in other types of tumors. |
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