Effect Of Synthetic Peptides Derived From HKa D5 On Proliferation And Apoptosis Of MDA-MB-231 Cells

High molecular weight kininogen (HK) is a multifunctional protein that plays important roles in many pathophysiological processes, such as fibrinolysis, thrombosis, and inflammation. HK is comprised of heavy and light chains that contain domains 1 through 3, and 5 and 6, respectively, and are linked by domain 4, which contains the vasoactive nonapeptide bradykinin. Cleavage of HK between Lys362-Arg363 and Arg371-Ser372 by kallikrein, an event that may occur on the cell surface, results in the release of bradykinin and generation of cleaved high molecular weight kininogen (HKa). HKa undergoes dramatic conformational changes as detected by electron microscopy. As a result of domain rearrangement, HKa acquires new properties, such as the ability to bind to an anionic charged surface. In comparison with HK, HKa has an increased antiadhesive effect, perhaps due to the exposure of D5 on the surface of the molecule. Recombinant D5 (GST-D5) mimicked the antiangiogenic activity of HKa, indicating that D5 is most likely to be the active region responsible for the antiangiogenic activity of HKa. Therefore, D5 was named kininostatin by Colman et al.The first evidence showing that HKa is an angiogenic inhibitor came from a study which showed that HKa or its recombinant D5 inhibited endothelial cell proliferation and induced apoptosis, two important steps required for angiogenesis. Domain 5 (D5) of kininogen inhibits endothelial cell adhesion, migration, proliferation and angiogenesis by inducing apoptosis and disrupting a signaling pathway initiated by binding to the urokinase receptor (uPAR). Recent studies indicate that tumor cells frequently overexpress uPAR, however, Cancer cells have not been used in most previous experiments on HK, and it is not clear that the core motif for inhibitory activity of D5 or its derived peptides. We queried whether tumor cells, such as the estrogen and progesterone receptor-negative human breast cancer cell line (MDA-MB-231), which overexpresses uPAR, would behave similarly to endothelial cells. We investigated the responsible mechanisms and the core motif for inhibitory activity.Materials1. MDA-MB-231 Cells.2. Recombinant histidine-rich domain of HK and peptides derived from HKa D5.3. Reagents for WST colorimetric assay.4. Reagents for Annexin V -FITC / PI double labeled assay.Methods1. Synthetic PepeidesAll peptides, P-5 (His479-Lys493), P-5n (Lys480-Lys487), P-5m (Gly484-Lys491), P-5c (His488-Asn495), were synthesized based on the amino acid sequence of HK domain 5 (amino acid residues 402-502 of HKa), and they were purified to more than 90% purity by high performance liquid chromatography.2. Preparation of Recombinant HK Domain 5A human HK cDNA fragment (from the codon for His 411 to the codon for Trp 501) was amplified by PCR using forward and reverse primers tagged with Nde I and Xba I sites, respectively. The sense primers was5'-CCGGCATATGTCTAGACCCAACCATTGTGTGCTTTCC-3', and the antisense primer was 5'-CCGGCATATGCATGACTGGGGCCATG-3'.3. Cell CultureMDA-MB-231 cells were cultured in RPMI 1640 medium with 8% fetal bovine serum, 100units/ml penicillin, and 100ug/ml streptomycin. They were maintained in a 37℃, 5% CO₂, folly humidified incubator.4. Cell Proliferation AssayEffects of HK r-HRD and peptides derived from HKa D5 on MDA-MB-231 cellsproliferation was assessed using WST kit-8. Cells in logarithm growth stage were transferred in 96-well plates. HK r-HRD and peptides derived from HKa D5 was added at various concentrations, and 10ul WST kit-8 was added after 24 hours. Two hour later, cell survival rate were assayed by a Spectra max miroplate at 450 nm. Complete medium was used as blank control. Repeated three wells were set up for each concentration in each time.5. Observation of Cell MorphologyThe morphologic change and adherence condition of MDA-MB-231 cells were observed in the inverted phase contrast microscope with or without r-HRD and peptides derived from HKa D5.6. Cell Apoptosis AnalysisMDA-MB-231 cells, treated by 2umol/L r-HRD and 200 400 800umol/L peptides derived from HKa D5 for 24 hours, were incubated with FITC-conjugated Annexin V and counterstained with propidium iodide (PI) in order to allow exclusion of necrotic cells. The cells were subsequently analyzed using a flow cytometer. 10 000 cells were analyzed in every sample.Results1. Compared with control group, the cell numbers were decreasing significantly with the increase r-HRD, P-5, P-5n, and P-5m, P-5c did not show inhibitory activity.2. After treated with r-HRD, P-5, P-5n, and P-5m for 24 hours, the morphological changes of MDA-MB-231 cells that shrank and turned round, cytoplasm condensed were observed through the inverted phase contrast microscope.3. HK r-HRD, P-5, P-5n, and P-5m induced apoptosis in MDA-MB-231 cells, P-5c have no function of induced apoptosis.ConclusionHK r-HRD and peptides derived from HKa D5 could inhibit MDA-MB-231 cells proliferation through inducing apoptosis, the acid sequence His-Gly-Lys (HGK) in D5 is the core motif for inhibitory activity.