ObjectiveTo investigate the inhibitory effect of antimicrobial peptide WK-13-3D on the proliferation of triple negative breast cancer MDA-MB-231 cells and its potential mechanism.p-eIF2α, and Bax proteins was significantly increased (P<0.05), with no significant change in eIF2α protein expression (P>0.05); Compared with si-BiP-592 group, si-BiP-592+WK-13-3D group showed a decrease in BiP protein expression (P<0.05) and an increase in PERK, p-eIF2α, and Bax protein expression (P<0.05 or P<0.01), with no significant change in eIF2α protein expression (P>0.05). Tumor volumes in mice treated with antimicrobial peptide WK-13-3D and TAX were significantly smaller than those in PBS groupMethodsThe effect of antimicrobial peptide WK-13-3D at concentrations of 0, 10, 15, 20, 25, 30, 35, and 40 μmol/L on MDA-MB-231 cells proliferation was assessed using the CCK-8 assay. A pull-down assay was conducted to identify interacting proteins of antimicrobial peptide WK-13-3D with MDA-MB-231 cells. MDA-MB-231 cells were obtained and divided into the following groups: (1) control group and treatment groups with 10 and 20 μmol/L antimicrobial peptide WK-13-3D. Apoptosis was evaluated using flow cytometry and Western blotting was conducted to detect the expression change of heavy chain binding protein (BiP), protein kinase R-like endoplasmic reticulum kinase (PERK), eukaryotic translation initiation factor 2α (eIF2α), phosphorylated eIF2α (p-eIF2α), and Bax proteins within the cells. (2) Control group (transfected with no-load plasmid), si-BiP-592 group (transfected with si-BiP-592 interference plasmid) and si-BiP-592+WK-13-3D group (co-treated with si-BiP-592 interference plasmid and 10 μmol/L antimicrobial peptide WK-13-3D). Western blotting was used to detect the expression changes of BiP, PERK, eIF2α, p-eIF2α and Bax proteins. Twelve BALB/c mice were randomly divided into PBS group (n=4), taxol (TAX) group (n=4) and WK-13-3D group (n=4). All mice were subcutaneously injected with MDA-MB-231 cells to establish a triple-negative breast cancer transplant tumor model. WK-13-3D group received local injections of antimicrobial peptide WK-13-3D [200 mg/(kg·d)], TAX group was administered TAX intraperitoneally at the same dose [200 mg/(kg·d)], and PBS group was injected with an equivalent volume of PBS. Two weeks post-injection, the mice were killed, and the tumor weight and volume were measured and photographed. Immunohistochemistry staining was performed to evaluate the expressions of BiP and Ki-67 proteins in the tumor tissues.ResultsCCK-8 assay showed a gradual decrease in MDA-MB-231 cell survival rates with increasing concentrations of WK-13-3D, with an inhibitory concentration 50 (IC₅₀) of 19.82 μmol/L. The pull-down assay identified 268 interacting proteins of antimicrobial peptides and MDA-MB-231 cells, mainly including heavy-chain binding protein (BiP), heat shock protein 90 beta family member 1 (HSP90B1), valerin-containing protein (VCP), heat shock cognate 71 kD protein (HSPA8). Compared with control group, treatment with 10 and 20 μmol/L antimicrobial peptide WK-13-3D significantly increased the apoptosis rate of MDA-MB-231 cells (P<0.05 or P<0.01), decreased BiP protein expression (P<0.05), and increased the expression levels of PERK, p-eIF2α, and Bax (P<0.05 or P<0.01), with no significant change in eIF2α protein expression (P>0.05). Compared with control group, si-BiP-592 group showed BiP protein expression significantly decreased (P<0.05), and the expression of PERK,(P<0.05), and the immunohistochemical staining showed that the proportion of Ki-67 and BiP positive cells in tumor tissues of WK-13-3D treated mice was significantly lower than that in PBS group (P<0.01).ConclusionAntimicrobial peptide WK-13-3D could inhibit the proliferation of MDA-MB-231 cells and its mechanism may involve the activation of endoplasmic reticulum stress and the induction of cell apoptosis.

antimicrobial peptides;triple negative breast cancer;endoplasmic reticulum stress;apoptosis