Lysine-Rich Polypeptide Modulates Forkhead Box O3 and Phosphoinositide 3-Kinase-Protein Kinase B Pathway To Induce Apoptosis in Breast Cancer

Abstract

The PI3K/AKT/FOXO3 pathway is one of the most frequently involved signaling pathways in cancer, including breast cancer. Therefore, we synthesized a novel lysine-rich polypeptide (Lys-PP) using de novo assembly method and evaluated its anticancer effect. We characterized the structural and physicochemical properties of Lys-PP using various techniques. Later, we used integrated approaches such as in silico, in vitro, and in vivo analysis to confirm the anticancer and therapeutic effect of Lys-PP. First, RNA sequencing suggests Lys-PP disrupted the central carbon metabolic pathway through the modulation of prolactin signaling. Additionally, docking analysis also confirmed the significant association of PI3K/AKT and FOXO3 pathway to induce an apoptotic effect on cancer. Second, Lys-PP exhibited a significant cytotoxicity effect against MDA-MB-231 but no cytotoxic effects on RAW 264.7 and HEK-293, respectively. The cytotoxic effect of Lys-PP-induced apoptosis by an increase in FOXO3a protein expression and a decrease in PI3K/AKT pathway was confirmed by quantitative real-time polymerase chain reaction, immunoblotting, and fluorescent microscopy. Later, immunohistochemistry and hematoxylin and eosin staining on MDA-MD-231 showed increased FOXO3a expression and cell death in the xenograft mice model. Further, liver function, metabolic health, or lipid profile upon Lys-PP showed the absence of significant modulation in the biomarkers except for kidney-related biomarkers. Overall, our comprehensive study provides the first evidence of Lys-PP antibreast cancer action, which could serve as a potential treatment in an alternative or complementary medicine practice.

Figure 1

Identification and refinement of Lys-PP 3D structure. (A) Crude, refined, and superimposed Lys-PP structure. (B) The Ramachandran plot analysis of Lys-PP was performed using the MolProbity server to examine the main-chain conformational preferences of the amino acid residues. (C) The z-score value was employed to assess the overall quality of the Lys-PP structure.

Figure 2

Structure and physicochemical properties of Lys-PP. (A) Physiochemical characteristics of Lys-PP. (B) Average molecular mass (Da) of Lys-PP was obtained using MALDI-TOF. (C) Determination of major peaks of Lys-PP through FTIR analysis; (D) Lys-PP purity determined by HPLC analysis.

Figure 3

Anticancer activity of Lys-PP in MDA-MB-231 cells. (A) Cell viability of Lys-PP in neoplastic cell lines (RAW264.7 and HEK293 cells) and breast cancer cell lines (MDA-MB-231); (B) fluorescence microscopic images of live (green)/dead (red) MDA-MB-231 cells stained using live/dead assay kit; (C) colony formation of MDA-MB-231 cells treated with Lys-PP.

Figure 4

Transcriptomic analysis of DEGs. (A) Volcano plot of Lys-PP-treated cells compared with control cells. Red and blue indicate the upregulated and downregulated genes, respectively [log2 (fold change) ≥ |1.0|, p ≤ 0.05]. (B,C) KEGG pathway enrichment analysis of DEGs (p < 0.01). (D) Gene ontology (GO) enrichment analysis (cellular component, molecular function, and biological process) of DEGs (p < 0.01). Color and node size indicate the p-value and gene count, respectively.

Figure 5

Visualization of the DEGs involved in the PI3K-AKT signaling pathway. (A) Network of DEGs in the PI3K-AKT signaling (hsa04151) KEGG pathway regulated by prolactin and the central carbon metabolic signaling pathway. Color and node size indicate the fold change value and p-value, respectively. Heatmap of DEGs in the PI3K-AKT signaling (hsa04151) KEGG pathway. Red and blue indicate the upregulated and downregulated genes, respectively. Clustering was analyzed based on the similarity of fold change and intensity by Euclidean distance metric. (B) Distribution of query genes on chromosomes, Chi-squared test P = 1. (C) Gene–disease association of targeted genes. (D) KEGG pathway and PPI network.

Figure 6

Molecular docking of the significantly up- and downregulated proteins with Lys-PP. (A–F) 3D visualization of the Lys-PP docked pose of proteins such as AKT1, Protein kinase B; BCL2L11, BCL2-like 11 (apoptosis facilitator); FOXO3, Forkhead Box O3; ITGA1, Integrin Subunit Alpha 1; PIK3CA, Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha; PTEN, Phosphatase and Tensin Homologue.

Figure 7

Lys-PP triggers apoptotic cell death of MDA-MB-231 cells. (A, B) Fluorescein isothiocyanate-labeled annexin V (Annexin V-FITC)/propidium iodide (PI) staining analysis of MDA-MB-231 cells; (C, D) Effect of Lys-PP on the protein expressions during apoptosis in MDA-MB-231 cells by Immunoblotting as quantified by ImageJ. Cis was used as the positive control. Data are presented as representative results of three independent experiments. All values are expressed as the mean ± standard deviation (S.D). *p < 0.05, **p < 0.01, and ***p < 0.001 vs control group.

Figure 8

Lys-PP regulated PI3K-AKT/FOXO3a signaling pathway of MDA-MB-231 cells. (A) Effect of Lys-PP on gene expression; the six most significantly regulated genes such as AKT1, protein kinase B; BCL2L11, BCL2-like 11 (apoptosis facilitator); FOXO3, Forkhead Box O3; ITGA1, Integrin Subunit Alpha 1; PIK3CA, Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha; PTEN, Phosphatase and Tensin Homologue were presented in the PI3K-AKT signaling (hsa04151) KEGG pathway in MDA-MB-231 cells. (B) Protein expression of the PI3K-AKT signaling including PTEN, p-FOXO3a/FOXO3a, p-PI3K/PI3K, and p-AKT/AKT by immunoblotting was quantified by ImageJ. Cis was used as the positive control. Data are presented as representatives of the results of three independent experiments. All values are expressed as the mean ± standard deviation (S.D). *p < 0.05, **p < 0.01, and ***p < 0.001 vs control group.

Figure 9

Anticancer effect of Lys-PP in a xenograft mouse model. (A) Workflow of treatment planning a xenograft mouse model. (B,C) tumor volume (mm³) and weight (g); (D) hematoxylin and Eosin (H&E) staining. (E) IHC staining of FOXO3a protein in tumor tissues isolated from breast-cancer xenograft mice. Results are presented as the mean ± SD. Significance is indicated by *p < 0.05, **p < 0.01, and ***p < 0.001 vs Con-T.

Figure 10

Toxicity evaluation of Lys-PP in a xenograft mouse model. (A) Body weight (g). (B) Organ index of the liver and kidney. (C) Serum biochemical analysis, including ALT, alanine aminotransferase (U/L); AST, aspartate aminotransferase (U/L); albumin; A/G, albumin to globulin ratio; Crea—creatinine (mg/dL); BUN, blood urea nitrogen (mg/dL); total protein (g/dL); GLU, glucose (mg/dL); TG, total triglycerides (mg/dL); T-Cho, total cholesterol (mg/dL); (D) Heatmap visualization of the serum biochemical analysis.