Abstract
BackgroundWe isolated a novel monomeric peptide from antler plate polypeptide (APP) of sika deer and found that it inhibited rat breast cancer cell proliferation and telomerase activity.MethodsThe molecular mass and purity of this polypeptide was determined by ultra performance liquid chromatography (UPLC) and Bruker micOTOF OllQ TOF mass spectrometry, respectively. The full amino-acid sequence of the monomeric peptide was analyzed by sequential Edman degradation using a protein/peptide sequencer. The APP-1 markedly inhibited rat breast cancer cell proliferation as determined with an 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H- tetrazolium bromide (MTT) assay. Then, we used flow cytometry to detect the effects of the monomeric peptide on cell cycle. Relative quantitative fluorescence PCR was used to analyze the expression level telomerase reverse transcriptase (TERT).ResultsThe molecular mass and purity of this polypeptide was 10646 Da and 91.2 %. Amino acid sequence analyses indicated that the N-terminal amino-acid sequence of this monomeric peptide was: MTKLE DYLEG IVNIF HQYSV. The results showed that monomeric peptide halted most cancer cells stagnating in the G0/G1 phase. The percentage of cells in the G0/G1 is higher than control group after the monomeric peptide treatment. Relative quantitative fluorescence PCR results showed that TERT gene expression level obviously decreased after treatment with the monomeric peptide compared with control group.ConclusionsCollectively, the results suggest that this novel and monomeric APP has antitumor activities and imply that it is likely an important component of antitumor activities in antler plate polypeptide.
Highlights
We isolated a novel monomeric peptide from antler plate polypeptide (APP) of sika deer and found that it inhibited rat breast cancer cell proliferation and telomerase activity
Separation and purification of a new monomeric peptide from APP Crude total polypeptide extract was treated with acetic acid buffer and ethanol, analyzed by SDS-PAGE, and the Coomassie brilliant blue-stained gel suggested that it contained five polypeptides
APP was isolated as an amorphous white solid
Summary
Cell line MA782 cells are rat breast cancer cells, growing sticked a wall. Its cell form is epithelioid. Monomer peptide separation and purification As described above, the total peptide (300 mg dry mass) was applied to a Sephadex G-50 column (10 × 1000 mm) that had been pre-equilibrated with distilled water. The total peptide (S2) markedly inhibited MA782 cell proliferation after 72 h (in doses from 10–30 mg/L, as determined with MTT assay). MTT assay Prepare the protein solution of which the concentration is 20 μg/mL from the purified monomeric peptide freeze dried using RPMI-1640 culture solution that do not contain serum, and filtration sterilization. The monomeric peptide (20 μg/mL final concentration) was transfected into rat breast cancer cells that reached a growth density of 80 %, and total RNA was extracted from cell culture pellets using TRIzol reagent (Invitrogen) after 24, 48, and 72 h. SPSS version 12.0 software (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses, and p-values
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