Zerumbone Induces Apoptosis in Breast Cancer Cells by Targeting αvβ3 Integrin upon Co-Administration with TP5-iRGD Peptide

Abstract

Cell-penetrating peptides (CPPs) are highly promising tools to deliver therapeutic molecules into tumours. αVβ3 integrins are cell-matrix adhesion receptors, and are considered as an attractive target for anticancer therapies owing to their roles in the process of metastasis and angiogenesis. Therefore, this study aims to assess the effect of co-administration of zerumbone (ZER) and ZERencapsulated in hydroxypropyl-β-cyclodextrin with TP5-iRGD peptide towards cell cytotoxicity, apoptosis induction, and proliferation of normal and cancerous breast cells utilizing in vitro assays, as well as to study the molecular docking of ZER in complex with TP5-iRGD peptide. Cell viability assay findings indicated that ZER and ZERencapsulated in hydroxypropyl-β-cyclodextrin (ZER-HPβCD) inhibited the growth of estrogen receptor positivebreast cancer cells (ER⁺ MCF-7) at 72 h treatment with an inhibitory concentration (IC)₅₀ of 7.51 ± 0.2 and 5.08 ± 0.2 µg/mL, respectively, and inhibited the growth of triple negative breast cancer cells (MDA-MB-231) with an IC₅₀ of 14.96 ± 1.52 µg/mL and 12.18 ± 0.7 µg/mL, respectively. On the other hand, TP5-iRGD peptide showed no significant cytotoxicity on both cancer and normal cells. Interestingly, co-administration of TP5-iRGD peptide in MCF-7 cells reduced the IC₅₀ of ZER from 7.51 ± 0.2 µg/mL to 3.13 ± 0.7 µg/mL and reduced the IC₅₀ of ZER-HPβCD from 5.08 ± 0.2 µg/mL to 0.49 ± 0.004 µg/mL, indicating that the co-administration enhances the potency and increases the efficacy of ZER and ZER-HPβCD compounds. Acridine orange (AO)/propidium iodide (PI) staining under fluorescence microscopy showed evidence of early apoptosis after 72 h from the co-administration of ZER or ZER-HPβCD with TP5-iRGD peptide in MCF-7 breast cancer cells. The findings of the computational modelling experiment provide novel insights into the ZER interaction with integrin αvβ3 in the presence of TP5-iRGD, and this could explain why ZER has better antitumor activities when co-administered with TP5-iRGD peptide.

Keywords: TP5-iRGD peptide; breast cancer; integrin; zerumbone.

Figure 1

Chemical structure of zerumbone (A) and zerumbone encapsulated in hydroxypropyl-β-cyclodextrin (ZER -HPβCD) (B).

Figure 2

Percentage of cell inhibition (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay) of MCF-7, MDA-MB-231, and Hs27 cells treated with zerumbone (ZER) (A), ZER-hydroxypropyl-β-cyclodextrin(HPβCD) (B), and TP5-iRGD peptide (C).

Figure 3

Of cell inhibition (MTT assay) of MCF-7 cells treated with ZER-TP5-iRGD for 72 h.

Figure 4

Of cell inhibition (MTT assay) of MCF-7 cells treated with ZER-HPβCD-TP5-iRGD for 72 h.

Figure 5

Acridine orange (AO)/propidium iodide (PI) assay for MCF-7 cells treated with the co-administration of zerumbone and TP5-iRGD peptide at three different time points of 24, 48, and 72 h. H: healthy, EA: early apoptosis, LA: late apoptosis, N: necrosis.

Figure 6

AO/PI assay for MCF-7 cells treated with the co-administration of ZER-HPβCD and TP5-iRGD peptide at three different time points of 24, 48 and 72h. H: healthy, EA: early apoptosis, LA: late apoptosis, N: necrosis.

Figure 7

Zerumbone in complex with integrin αvβ3 (Protein Data Bank (PDB) code: 1L5G).

Figure 8

The root mean square deviation (RMSD) values for protein are shown on the y-axis during the simulation time of 20 nsec for ZER-integrin αvβ3 protein complex in the presence of TP5-iRGD peptide.

Figure 9

The root meansquare fluctuation (RMSF) of ZER-integrin αvβ3 protein complex in the presence of TP5-iRGD peptide during 20 ns molecular dynamics simulation.

Figure 10

Per-residues analysis of the ZER in complex with integrin αvβ3 protein in the presence of TP5-iRGD peptide; the analysis was based throughout the 20 ns molecular dynamics simulations.

Figure 11

A representation of atomic interactions between ZER and integrin αvβ3 protein residues during 20 ns molecular dynamics simulation.