Structure based design and synthesis of peptide inhibitor of human LOX-12: in vitro and in vivo analysis of a novel therapeutic agent for breast cancer

Abstract

Human breast cancer cell proliferation involves a complex interaction between growth factors, steroid hormones and peptide hormones. The interaction of growth factors, such as epidermal growth factor (EGF), with their receptors on breast cancer cells can lead to the hydrolysis of phospholipids and release of fatty acid such as arachidonic acid, which can be further metabolized by cyclooxygenase (COX) and lipoxygenase (LOX) pathways to produce prostaglandins. The high concentration of prostaglandins has been associated with chronic inflammatory diseases and several types of human cancers. This is due to the over expression COX, LOX and other inflammatory enzymes. Ten peptides were designed and synthesized by solid phase peptide synthesis and analyzed in vitro for enzyme inhibition. Out of these peptides, YWCS had shown significant inhibitory effects. The dissociation constant (K(D)) was determined by surface plasmon resonance (SPR) analysis and was found to be 3.39 × 10(-8) M and 8.6 × 10(-8) M for YWCS and baicalein (positive control), respectively. The kinetic constant Ki was 72.45 × 10(-7) M as determined by kinetic assay. The peptide significantly reduced the cell viability of estrogen positive MCF-7 and estrogen negative MDA-MB-231 cell line with the half maximal concentration (IC(50)) of 75 µM and 400 µM, respectively. The peptide also induced 49.8% and 20.8% apoptosis in breast cancer cells MCF-7 and MDA-MB-231, respectively. The YWCS was also found to be least hemolytic at a concentration of 358 µM. In vivo studies had shown that the peptide significantly inhibits tumor growth in mice (p<0.017). This peptide can be used as a lead compound and complement for ongoing efforts to develop differentiation therapies for breast cancer.

Figure 1. Western blot showing LOX-12 expression:

(A) Molecular weight marker, supernatant of bacterial extract, inclusion bodies of bacterial cell extract, flow-through of the Ni-NTA column, purified refolded protein, (B) Activity profile of purified LOX-12. (C) Michaelis-Menten plot of LOX-12 activity versus arachidonic acid concentration showing decrease in activity at different concentrations (0, 25, 50, 75,100 µM) of YWCS. (D) Sensogram showing binding of different concentrations of YWCS (25, 50 and 75 µM) on immobilized with His-LOX-12 over the Ni²⁺-NTA chip.

Figure 2. Graph showing percentage of hemolysis with different concentrations (0–358 µM) of YWCS and of gentamycin as a control.

Figure 3. Dose-response curve:

(A) showing % viability of MCF-7 cells at 0–150 µM concentrations of YWCS (0.05% DMSO as a vehicle control), for three different time points (24, 48 and 72 h). IC₅₀ was found to be 75 µM at 72 h. (B) showing % viability of MDA-MB-231 cells at 0–500 µM concentration of YWCS (0.05% DMSO as a vehicle control), for three different time points (24,48 and 72 h). IC₅₀ was found to be 400 µM at 72 h.

Figure 4. Apoptotic analysis of cancer cells with YWCS by flow cytometry; MCF-7 cells:

(A) untreated cells (0.05% DMSO as a vehicle control), (B–D) treated with 50, 75 and 100 µM YWCS respectively, (E) treated with baicalein. MDA-MB231 cells :(F) untreated cells (0.05% DMSO as a vehicle control), (G–I) treated with 350,400 and 450 µM YWCS respectively,(J) treated with baicalein. (K) and (L) bar diagram showing mean percentage of apoptotic cells of the above cell lines treated and untreated with peptide and baicalein.[*** represent the comparison of bar of treated cells with the untreated cells].

Figure 5. Confocal microscopy pictures showing localization of FITC-labelled YWCS in MCF-7 and MDA-MB-231 cells.

White arrows in the merged image show that the peptide enters the cell cytoplasm. (A) MCF-7 cells treated with 75 µM YWCS. (B) MDA-MB-231 cells treated with 400 µM YWCS peptide.

Figure 6. In vivo experiments showing effect of YWCS on tumor initiation and progression:

(A)- Group I; untreated mice, Group II; treated mice with peptide after 20 days of treatment (treatment started after 10 days of tumor growth), Group III; peptide induce simultaneously with EAT tumor cells showing no tumor initiation and (B)The tumor size of Group I and Group II mice after sacrifice.