Quercetin and 5-Fu Loaded Chitosan Nanoparticles Trigger Cell-Cycle Arrest and Induce Apoptosis in HCT116 Cells via Modulation of the p53/p21 Axis

Abstract

Nanoparticles (NPs) are encapsulating agents that exist in the nanometer range. They can be classified into different classes based on their properties, shapes, or sizes. Metal NPs, fullerenes, polymeric NPs, ceramic NPs, and luminescent nanoporous hybrid materials are only a few examples. This study explored the anticancer potential of quercetin and 5-fluorouracil-encapsulated chitosan nanoparticles (CS-5-FU-QCT NPs). The nanoparticles were prepared by ionic gelation, and their efficacy and mechanism of action were examined. CS-5-FU-QCT NPs were characterized using dynamic light scattering (DLS), atomic force microscopy (AFM), UV-visible spectroscopy, and Fourier transform infrared spectroscopy (FTIR); cytotoxicity was analyzed using an MTT assay. Cells were treated with CS-5-FU-QCT NPs and incubated for 12, 24, and 36 h, and apoptosis analysis (using Annexin V/FITC), cell-cycle analysis, Western blotting, and confocal microscopic analysis were performed. Biophysical analysis revealed that the CS-5-FU-QCT NPs fall in the range of 300-400 nm with a near-spherical shape. The in vitro drug release profile indicates sustained release of drugs over a period of about 36 h. The cytotoxicity of CS-5-FU-QCT NPs was more prominent in HCT116 cells than in other cancer cells. This particular nanoformulation caused G0/G1 phase cell-cycle arrest in HCT116 cells and induced intracellular ROS generation, thereby causing apoptosis. It also downregulated Bcl2, cyclin D1, and Cdk4 and upregulated BAX, p53, and p21, causing cell-cycle arrest and apoptosis. In summary, CS-5-FU-QCT NPs hindered proliferation of HCT116 cells via ROS generation and altered the expression of key proteins in the p53/p21 axis and apoptotic machinery in a time-dependent manner.

Figure 1

Biophysical characteristics of quercetin and 5-Fu-loaded chitosan nanoparticles (CS-5-Fu-QCT NPs). (a) Nanoparticles were prepared by the ionic gelation method. (b) Dynamic light scattering (DLS) studies of CS-5-Fu-QCT nanoparticles-size distribution by intensity. (c) UV–visible spectroscopy of CS-5-Fu-QCT NPs. (d) Determination of surface topology of CS-5-Fu-QCT NPs using atomic force microscopy (AFM). (e) FTIR analysis of CS-5-Fu-QCT NPs and blank chitosan nanoparticles. (f) Transmission microscopic image (50 nm scale bar) of CS-5-Fu-QCT NPs.

Figure 2

Drug release kinetics and cytotoxicity of the prepared CS-5-Fu-QCT NPs. (a) Percentage of release of quercetin from CS-5-Fu-QCT NPs. (b) Percentage of release of 5-Fu from CS-5-Fu-QCT NPs. (c) Analysis of cytotoxicity of CS-5-Fu-QCT NPs using the MTT assay. (d) IC50 of CS-5-Fu-QCT NPs on different cancer cells and human embryonic kidney cells. Data are represented as the mean ± SEM (n = 3).

Figure 3

Induction of apoptosis in HCT116 cells by CS-5-Fu-QCT NPs. (a) Analysis of apoptosis by Annexin V/FITC followed by time-dependent administration of CS-5-Fu-QCT NPs. (b) Representative bar diagram indicating apoptosis at different time intervals. (c) Western blot of proapoptotic protein BAX and caspase 3 and antiapoptotic Bcl2. (d) Densitometric analysis of the expression of pro- and antiapoptotic proteins. Data are represented as the mean ± SEM (n = 3).

Figure 4

CS-5-Fu-QCT NPs altered the expression of proteins associated with apoptosis. (a) Confocal microscopic analysis of the proapoptotic protein BAX and the antiapoptotic protein Bcl2 in HCT116 cells followed by administration of CS-5-Fu-QCT NPs in a time-dependent manner. (b) Bar graph showing the comparative expression of BAX and Bcl2 proteins.

Figure 5

CS-5-Fu-QCT NPs caused ROS generation and arrested cell cycle in HCT116 cells. (a) Analysis of intracellular ROS using DCFDA fluorescence via flow cytometry. (b) Representative bar diagram of intracellular ROS generation. (c) Analysis of cell cycle using PI followed by treatment with CS-5-Fu-QCT NPs. (d) Representative bar diagram of percent distribution of cells across different phases of the cell cycle. Data are represented as the mean ± SEM (n = 3).

Figure 6

CS-5-Fu-QCT NPs modulated the expression of proteins associated with cell-cycle regulation. (a) Western blot analysis of p53, p21, cyclin D, and Cdk4. (b) Representative bar diagram of expression of proteins in Western blot analysis. (c) Confocal microscopic analysis of p53 and p21. Data are represented as the mean ± SEM (n = 3). (d) Bar diagram showing the comparative expression of P53 and P21.