High And Low Metastatic Potential Of Breast Cancer Cells Differentially Expressed Protein Research

Objective: To explore the feasibility of screening breast cancer cellline subgroup with different potentiality of metastasis through artificialmatrix, and to analyze the biological difference between these twosubgroups.Methods: Continuous penetrating of MDA-MB-435s cells throughartificial matrigel was conducted in Transwell chamber to obtain subgroupsof human breast cancer cell line with differently-metastatic potentiality.Transmission electron microscope was used to observe ultra-microstructureof cells showing different potentiality of metastasis. MTT method wasconducted to determine cell growth curve, doubling time and adhesion rate.Invasion test and angiopoiesis test were conducted in Transwell chamber.Immune histochemistry staining was employed to detect the expression ofmatrix metalloprotainese-2and-9, as well as epithelium cadherins.Results: Two subgroups of MDA-MB-435s cells, with highly-andlowly-metastatic potentiality, were obtained through continuous artificialmatrigel invasion test in Transwell chamber, showing significant difference in in-vitro growth velocity, invasive power, adhesive power, as well asinduction of human umbilical vascular endothelial cells.(1) It was shown that under the transmission electron microscope,MDA-MB-435s cells with highly-metastatic potentiality were mostly infusiform or round shape, with large nucleus, abundant euchromatin and fewallochromacy, as well as enlarged or multiple nucleoli. In some cells,nucleoli were located to the side of the nucleus, and nucleus abnormity wasalso observed. Moreover, caryocinesia was seen in a few cells. Fewkytoplasm and mitochondria, as well as a large amount of free ribosomewere observed in these cells. Besides, microvilli were observed on thesurface of these cells. Compared with cells showing highly-metastaticpotentiality, those cells with lowly-metastatic potentiality were constitutedwith more heterochromatin and mitochondria, as well as less nucleoli andfree ribosome. Moreover, caryocinesia was less than that in cells withhighly-metastatic potentiality.(2) It was seen from the cell growth curve and cell doubling time testthat, in-vitro growth velocity of cells with highly-metastatic potentialty wassignificantly faster than that of cells with lowly-metastatic potentiality.Doubling time of cells with highly-metastatic potentiality was39.797±2.018hours, significantly higher than that of cells withlowly-metastatic potentiality, which was34.166±1.405hours (P<0.05).(3) It was shown from the invasion test that, the number of cells passing through the artificial matrix membrane for cells withhighly-metastatic potentiality was61.46±7.08/per visual field, significantlyhigher than that for cells with lowly-metastatic potentiality, which was25.32±4.87/per visual field (P<0.05).(4) It was shown from the cell adhesion test that adhesion rate for cellswith highly-metastatic potentiality at30min,60min,90min and120minwas0.1696±0.0034,0.2779±0.0070,0.3913±0.01and0.5750±0.0202,respectively, significantly higher than that for cells with lowly-metastaticpotentiality at corresponding time point, which was0.1109±0.0068,0.1813±0.0144,0.2961±0.0375and0.4134±0.0996, respectively.(5) It was observed from angiopoiesis test that vascular lumensinduced by cells with highly-metastatic potentiality was12.44±1.32/pervisual field, significantly higher than that induced by cells withlowly-metastatic potentiality, which was5.41±0.82/per visual field (P＜0.01). Besides, length of lumens induced by cells with highly-metastaticpotentiality was243.54±44.31μm, significantly longer than that induced bycells with lowly-metastatic potentiality, which was117.47±19.78μm(P<0.05).(6) In the immune histochemistry staining test it was found that, incells with lowly-metastatic potentiality the average optical value formatrix metalloprotease-2and-9was0.16±0.02and0.13±0.03,respectively; while in cells with highly-metastatic potentiality, it was 0.24±0.04and0.23±0.05, respectively. The expression level of matrixmetalloprotease-2and-9was significantly higher in cells withhighly-metastatic potentiality (P<0.05). The average optical value forepithelium cadherins is0.16±0.04in cells with lowly-metastaticpotentiality, and0.11±0.01in cells with highly-metastatic potentiality. Theexpression level of epithelium cadherins was significantly lower in cellswith highly-metastatic potentiality, compared with that in cells withlowly-metastatic potentiality (P<0.05).Conclusions: Breast cancer cells with highly-and lowly-metastaticpotentiality could be screened by the difference in ability of cellspenetrating the artificial basal membrane. Cells with highly-andlowly-metastatic potentiality, originated from the same parental cell line,showed significantly different biological characterization. Objective: To screen metastasis-associated protein in breast cancercells, using2subgroups of cells with highly-and lowly-metastaticpotentiality established in part1.Methods: Two-phase gel electrophoresis was employed to separateproteins from2subgroups of breast cancer cells, follwed by argentationstaining of the gel. Subsequently, image analysis system ImageMaster2DV2002.1was used to analyze the gel graph, in order to find outdifferentially expressed proteins, i.e., more than2-fold in expression level.Enzymolysis was conducted using differentially expressed proteins, theresults analyzed with matrix assisted laser desorption ionization massspectrometry. Finally, the spectrum data of differentially expressed proteinspot were input into Mascot Database for data base querying.Results:36differentially expressed protein spots were identified bytwo-phase gel electrophoresis. Relative amount of11protein spots in cellswith highly-metastatic potentiality was higher than that in cells with lowly-metastatic potentiality; while relative amount of25protein spots incells with lowly-metastatic potentiality was higher than that in cells withhighly-metastatic potentiality.Twenty significantly different protein spots were found from massspectrographic analysis; then twenty differential expression protein weresuccessfully identified after matrix assisted laser desorption ionizationmass spectrometry, including CDK6,STAT1,PGAM1,PLD5,RAB7B,GSDM1and BRCA1.Detailed discussion and assessment of the biologicalsignificance of these differentially expressed proteins were conducted inthis part.Conclusion: Differentially expressed proteins existed in cells withhighly-and lowly-metastatic potentiality, separated from the same parentalbreast cancer cells MDA-MB-435s. Objective: To identify some of the candidate proteins obtained fromPart2.Methods: RT-PCR was applied to detect mRNA expression level ofANX A1,HSP70and MYLK2in breast cancer cells with highly-andlowly-metastatic potentiality; while Western-blot was conducted to detectprotein expression level of ANX A1,HSP70and MYLK2.Results: mRNA and protein expression level of ANX A1,HSP70andMYLK2were significantly different in cells with highly-andlowly-metastatic potentiality.(1) expression of Annexin A1: on the protein level, the averageoptical value was0.13±0.02in cells with highly-metastatic potentiality,significantly lower than that in cells with lowly-metastatic potentiality,which was0.42±0.06(P＜0.05); similarly, on the mRNA level, the averageoptical value was0.12±0.03in cells with highly-metastatic potentiality,significantly lower than that in cells with lowly-metastatic potentiality, which was0.31±0.06(P＜0.05);(2) expression of HSP70: on the protein level the average optical valuewas0.62±0.10in cells with highly-metastatic potentiality, significantlyhigher than that in cells with lowly-metastatic potentiality, which was0.21±0.04(P＜0.05); Besides, the same trend was found on the mRNAlevel, i.e., the average optical value was0.47±0.06in cells withhighly-metastatic potentiality, significantly higher than that in cells withlowly-metastatic potentiality, which was0.27±0.03(P＜0.05).(3) expression of MYLK2: on the protein level, the average opticalvalue was0.54±0.08in cells with highly-metastatic potentiality,significantly higher than that in cells with lowly-metastatic potentiality,which was0.18±0.03(P＜0.05); Moreover, on the mRNA level, theaverage optical value was0.49±0.07in cells with highly-metastaticpotentiality, significantly higher than that in cells with lowly-metastaticpotentiality, which was0.25±0.04(P＜0.05).Conclusion: Metastatic-associated proteins existed in breast cancercells MDA-MB-435s with highly-and lowly-metastatic potentiality. ANXA1,HSP70and MYLK2were related with breast cancer metastasis, itsexpression level of protein connected with transcription level of mRNA.