Strategy to Enhance Anticancer Activity and Induced Immunogenic Cell Death of Antimicrobial Peptides by Using Non-Nature Amino Acid Substitutions

Abstract

There is an urgent and imminent need to develop new agents to fight against cancer. In addition to the antimicrobial and anti-inflammatory activities, many antimicrobial peptides can bind to and lyse cancer cells. P-113, a 12-amino acid clinically active histatin-rich peptide, was found to possess anti-Candida activities but showed poor anticancer activity. Herein, anticancer activities and induced immunogenic cancer cell death of phenylalanine-(Phe-P-113), β-naphthylalanine-(Nal-P-113), β-diphenylalanine-(Dip-P-113), and β-(4,4'-biphenyl)alanine-(Bip-P-113) substituted P-113 were studied. Among these peptides, Nal-P-113 demonstrated the best anticancer activity and caused cancer cells to release potent danger-associated molecular patterns (DAMPs), such as reactive oxygen species (ROS), cytochrome c, ATP, and high-mobility group box 1 (HMGB1). These results could help in developing antimicrobial peptides with better anticancer activity and induced immunogenic cell death in therapeutic applications.

Keywords: DAMPs; antimicrobial peptides; bulky non-nature amino acid; cancer; membrane integrity; oncolytic peptides.

Figure 1

Anticancer activities of P-113, Phe-P-113, Nal-P-113, Bip-P-113, and Dip-P-113 against various cancer cell lines by MTT cell viability assay. Data represent mean ± SD of three independent experiments.

Figure 2

Time killing analysis of Nal-P-113, Bip-P-113, and Dip-P-113 against PC 9 cell line. Data represent mean ± SD of three independent experiments. ** p < 0.01 (Nal-P-113 vs. Dip-P-113; Bip-P-113 vs. Dip-P-113), ns = no significant differences (Nal-P-113 vs. Bip-P-113).

Figure 3

Fluorescence images of PC 9 cells treated with or without Nal-P-113, Bip-P-113, and Dip-P-113 at 2× IC₅₀ for 120 min and stained with PI (red) and nuclear dye Hoechst 33342 (blue) using fluorescence microscopy. Scale bar represents 50 μm. The experiments were repeated three times independently.

Figure 4

Fluorescence images of PC 9 cells treated with 2× IC₅₀ Nal-P-113, Bip-P-113, and Dip-P-113 for 120 min and stained with JC-1. Samples treated with CCCP served as a positive control. Scale bar represents 50 μm. The experiments were repeated three times independently.

Figure 5

ROS release after the treatment with 2× IC₅₀ of Nal-P-113, Bip-P-113, and Dip-P-113 detected by DCFDA cellular ROS detection kit. The untreated cells were used as control. Results are presented as mean ± SD of three independent experiments, ** p < 0.01 compared with control.

Figure 6

The cytochrome c secretion was quantified using ELISA assay. Results are presented as mean ± SD of three independent experiments. The cells were treated with Nal-P-113, Bip-P-113, and Dip-P-113 peptides at 2× IC₅₀ concentration for 0.5, 1, 1.5, 2, and 4 h, respectively. Untreated cells were used as a control. * p < 0.05 compared with control.

Figure 7

ATP release of PC 9 cells after treatment with Nal-P-113, Bip-P-113, and Dip-P-113. Untreated cells were served as the control group. Results are presented as mean ± SD of three independent experiments, * p < 0.05; ** p < 0.01; *** p < 0.001 compared with control.

Figure 8

HMGB1 protein secreted from cell lysate to supernatant after incubation with Nal-P-113, Bip-P-113, and Dip-P-113. (A) The Western blot images of the PC 9 cells were treated with 2× IC₅₀ Nal-P-113, Bip-P-113, and Dip-P-113 peptides for 1, 2, and 4 h, respectively. Untreated cells were served as a control group. L and S represented cell lysate and supernatant, respectively. The relative band intensities of HMGB1 for each group from cell lysate (B) and supernatant (C).

Figure 9

Proposed mechanism of anticancer action for the designed peptides. The intratumoral administration of Nal-P-113 () induced tumor cellular lysis through membrane destabilization, caused the release of danger-associated molecular patterns (DAMPs), including ATP (), HMGB1 (), and ROS (), as well as tumor antigens into the tumor microenvironment. Thereafter, these DAMPs recruited the immature dendritic cells (DCs) and further induced the maturation of dendritic cells. The mature DCs were then priming for antigen presentation to T cells and subsequent antitumor immune responses.