| Objectives: The aim of this study is to screen differentially expressed proteins in serum of breast cancer rats by TMT-labeled proteomics and compare them with differentially expressed proteins in premenopausal breast cancer women screened in previous clinical studies and selected common proteins;thereafter verify the common proteins and explore the relationship between abnormal expression of candidate proteins and breast carcinogenesis at premenopausal stages.The inhibitory effect of allicin on breast cancer and the expression level of candidate proteins were studied.The anti-breast cancer mechanism of allicin was discussed.Methods: 1)Twenty five sprague-dawley(SD)female rats of?27 days were randomly divided into normal control(NC,n=10)group and breast tumor(BT,n=15)group after 14 days(?42 days)SPF acclimation.At 49-57 days of age,the rats in the BT group were administered 50 mg/kg DMBA in corn oil twice(day49 and 56)at an interval of one week.The SD rats in the NC group were fed normal diet and were given no treatment.Seven weeks after DMBA administration,the rats in the BT group were palpated twice weekly to monitor tumor development,and the palpable tumor multiplicity,location,and latency were recorded.After 17 weeks,all the rats were fasted overnight and sacrificed.The blood and tissue samples were collected for further analysis.For proteomic analysis,two groups of pooled samples were prepared by mixing serum samples from each of the ten rats within each group.After depilation of high abundance proteins,digestion,labeled and purification,the peptides form pooled samples were analysed by nano LC-tandom Q-exactive MS spectrometry.The candidate proteins provided by proteomics analysis were selected and multi reaction monitoring mass spectrometry(MRMS)was used to validate the protein expression.The relationship between the breast carcinogenesis at premenopausal stage and the protein expression was explored;2)Using IPA @ software(ingenuity pathway analysis)and its online data base,the function and interaction of candidate proteins were studied by bioinformatics;3)quantitative reverse transcription polymerase chain reaction(q RT-PCR)and immunohistochemistry(IHC)were used to analyze the expression levels of differentially expressed proteins in normal breast tissues and tumor tissues of NC and BT.The results of proteomics and bioinformatics analysis were further verified,and the relationship between breast tumorigenesis and protein expression was discussed;4)MCF-7(ER positive)cells and MDA-MB-231 cells(ER negative)were treated with different concentrations of allicin.MTT and CCK8 cell viability test,apoptosis,cell cycle and cell scratch methods were used to analysis the effect of allicin on proliferation,cell cycle and cell migration.q RT-PCR and Western bolt(WB)were employed to analyze the effect of allicin on the expression of candidate proteins and apoptosis related proteins in MCF-7 and MDA-MB-231.Results: 1)The first breast tumor was observed ten weeks after the first DMBA administration.Multiple tumors of different sizes were frequently found in the same rat.Among the fifteen rats in BT,ten developed tumors with 66.7% tumor incidence12.50±2.63 weeks of tumor latency,5.51±2.02 g of tumor weight,1.76±0.78 cm of tumor size,and 1.74±0.95 of tumor multiplicity were observed by morphological analysis.There was no any weight loss of all rats in experimental period.Histopathological analysis showed that all tumors in the BT group were invasive ductal carcinoma,nine of ten cases were grade I,and one case was grade II;Proteomic analysis revealed that the expression level profiles of 106 proteins between the BT and NC groups were significantly altered(fold change?1.2 refers to upregulated,fold change ? 0.83 refers downregulated,P<0.05).Among them,seven candidate proteins,namely TPM4,KRT1,SAA,PF4,THBS1,A1 BG,and APOC1 were found to be identical to our clinical proteomics study for pre-menopausal breast cancer.The APOC1,KRT1,THBS1,PF4 and SAA1 were up-regulated(P<0.05),while A1 BG and TPM4 were down regulated(P<0.05)in BT group rats serum compared to NC;2)Gene ontology(GO)results showed that the proteins were localized in the extracellular space and cytoplasmand involved in heparin binding,glycosaminoglycan binding,sulfur compound binding,and lipid binding functions,and different biological processes such as inflammatory response,defense response,regulation of body fluid levels,and negative regulation of lipid transport.Pathway analysis showed that the identified proteins participate in many signaling pathways such as inhibition of angiogenesis by THBS1,bladder cancer signaling,p53 signaling,LXR/RXR activation,FXR/RXR activation,and corticotrophin-releasing hormone signaling.the network contained all seven candidate proteins and were directly or indirectly related to tumor factors such as HDL,TP53,AQP3,TINCR,IL-1 ?,DMTF1,p14 ARF and CD1 glycoprotein family proteins which is closely related to tumorigenesis;3)q RT-PCR results showed that the m RNA expression of AIBG and THBS1 in BT group were significantly higher than those in NC group.The m RNA expression of APOC1,KRT1,PF4,TPM4 and SAA1 were lower than those in NC group(P<0.05).IHC analysis demonistrated that KRT1?TPM4?THBS1?PF4 and SAA1 expression leveles in BT group were up-regulated compared to NC group,suggesting that these indicators may have a relationship with the occurrence of breast cancer;4)The results of MTT and CCK8 showed that allicin(8?23?g/ml)significantly decreased the cell viability of breast cancer MCF-7 and MDA-MB-231 cells(P<0.01);the results of cell apoptosis,cell cycle and scratch test showed that allicin could increase the early apoptosis rate of breast cancer MCF-7 and MDA-MB-231 cells,arrest the cells in M2/G0 phase.Results of q RT-PCR and western blot showed that compared with NC group,the expression levels of A1 BG and THBS1 decreased in MCF-7 cells,but increased in MDA-MB-231 cells(P<0.05);the expression levels of SAA1,caspase 3 and p53 were increased(P<0.05),while the expression levels of TPM4 were decreased(P<0.05);the m RNA expression levels of APOC1 in MCF-7 cells were increased(P<0.05),but not in MDA-MB-231 cells(P>0.05).The protein expression of KRT1 protein increased in MDA-MB-231 cells(P<0.05),but not in MCF-7 cells(P>0.05).Conclusion: 1)In this paper,we successfully induced the breast cancer rat model by using DMBA and simulated premenopausal ER positive breast cancer according to the ER positivity of breast tumors and ovariectomized properties.We screened seven differentially expressed proteins common to premenopausal breast cancer patients and breast cancer rats by proteomics and comparision analysis.The relationship between the regulation of candidate protein expression and breast tumorigenesis is further clarified by bioinformatics and tissue level analysis,thereby overcome the limitations of clinical research;2)Allicin inhibits the proliferation and migration;induced apoptosis and cell cycle arrest of breast cancer cells through p53 apoptosis pathway.MDA-MB-231 cells are more sensitive to allicin compared to MCF-7;allicin regulates the expression of breast cancer specific candidate proteins in the MCF-7 and MDA-MB-231 cells through p53 dependent and independent pathways.In conclution,this study will provide a basis for early diagnosis and treatment of breast cancer. |
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