TRAIL/S-layer/graphene quantum dot nanohybrid enhanced stability and anticancer activity of TRAIL on colon cancer cells

Abstract

Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL), known as a cytokine of the TNF superfamily, is considered a promising antitumor agent due to its ability to selectively induce apoptosis in a wide variety of cancer cells. However, failure of its successful translation into clinic has led to development of nano-based platforms aiming to improve TRAIL therapeutic efficacy. In this regard, we fabricated a novel TRAIL-S-layer fusion protein (S-TRAIL) conjugated with graphene quantum dots (GQDs) to benefit both the self-assembly of S-layer proteins, which leads to elevated TRAIL functional stability, and unique optical properties of GQDs. Noncovalent conjugation of biocompatible GQDs and soluble fusion protein was verified via UV-visible and fluorescence spectroscopy, size and ζ-potential measurements and transmission electron microscopy. The potential anticancer efficacy of the nanohybrid system on intrinsically resistant cells to TRAIL (HT-29 human colon carcinoma cells) was investigated by MTT assay and flow cytometry, which indicated about 80% apoptosis in cancer cells. These results highlight the potential of TRAIL as a therapeutic protein that can be extensively improved by taking advantage of nanotechnology and introduce S-TRAIL/GQD complex as a promising nanohybrid system in cancer treatment.

Figure 1

Schematic illustration of S-TRAIL and S-TRAIL/GQD preparation and biological function.

Figure 2

(a) Analysis of purified S-TRAIL using Comassie blue stained SDS-PAGE (12.5% polyacrylamide gel). Lane1. Molecular weight marker (62KDa), Lane 2–4. S-TRAIL (MW ~ 66 KDa). (b) Western blot analysis of S-layer and S-TRAIL using monoclonal anti-His tag antibody and goat anti-mouse AP conjugated antibody. Lane1. Molecular weight marker, Lane2. S-layer and Lane3. S-TRAIL.

Figure 3

(a) UV–Vis absorption spectrum of synthesized GQDs (Inset shows the standard curve drawn based on serial dilution results). (b) Photoluminescence spectra of GQDs under different excitation wavelengths of 300–420 nm. The insets are optical images of the solution of GQDs taken under visible light (left) and excited by 360 nm UV light (right). (c) FTIR spectrum of GQDs, arrows on the spectrum indicate characteristic bands of functional groups of GQDs.

Figure 4

(a) Schematic illustration of S-TRAIL/GQD preparation steps. (b) Analysis of S-TRAIL content in centrifugation products (both supernatant and pellet) using polyacrylamide gel electrophoresis (12.5%), protein marker is 62KDa. (c) UV–visible spectra of S-TRAIL, GQDs and prepared S-TRAIL/GQD after centrifugation. (d) Fluorescence spectra of S-TRAIL, GQDs and S-TRAIL/GQD (pellet) and the supernatant upon excitation at 360 nm. (e) Fluorescence spectra of GQDs (6 µg/ml) in the presence of different concentrations of S-TRAIL upon excitation at 360 nm. (f) ζ-potential of S-TRAIL and GQD before and after conjugation. (g) Intrinsic fluorescence of S-TRAIL (160 µg/ml) in the presence of different concentrations of GQD upon excitation at 280 nm.

Figure 5

TEM images of (a) S-TRAIL, (b) GQD and (c) S-TRAIL/GQD. (d) and (e) Size distribution of GQDs and S-TRAIL/GQDs, respectively. Analysis of TEM images was performed by ImageJ software version 1.52.

Figure 6

(a) In vitro cytotoxic activity of S-TRAIL and S-TRAIL/GQD with/without DXR pretreatment. (b) Cytotoxic effects of different concentrations of S-TRAIL and S-TRAIL/GQD following DXR pretreatment for 24 h. The statistical significance was determined using Student’s t-test. ${}^{\ast }p<0.05,{}^{\ast \ast }p<0.01,{}^{\ast \ast \ast }p<0.001.$

Figure 7

Evaluation of apoptosis induction in HT-29 cells. (a) Control cells without any treatment. (b) The cells treated with DXR (0.1 µM) for 24 h. (c) Treatment with S-TRAIL (160 µg/ml) for 12 h and (d) the cells pretreated with DXR (0.1 µM) for 24 h followed by treatment with S-TRAIL (160 µg/ml) for 12 h. (e) Quantitative comparison of the apoptosis rate of the cells.

Figure 8

Biological activity of TRAIL and S-TRAIL over 4 weeks storage at 4 °C. The molar concentration of 25 and 50 µg/ml of TRAIL protein are equal to the molar concentration of 80 and 160 µg/ml of S-TRAIL, respectively. Statistical significance was evaluated by repeated ANOVA test and student t-Test. ${}^{\ast }p<0.05,{}^{\ast \ast }p<0.01,{}^{\ast \ast \ast }p<0.001$.

Figure 9

Fluorescence microscopy images of HT-29 cells (a) untreated (b) treated by GQDs and (c) treated by S-TRAIL/GQD for 24 h. In each panel from the left, first image is the bright field, the second image is the fluorescence and the third one is the overlay image of the first and the second images.