Chitosan nanoparticles loaded with aspirin and 5-fluororacil enable synergistic antitumour activity through the modulation of NF-κB/COX-2 signalling pathway

Abstract

Based on the enhancement of synergistic antitumour activity to treat cancer and the correlation between inflammation and carcinogenesis, the authors designed chitosan nanoparticles for co-delivery of 5-fluororacil (5-Fu: an as anti-cancer drug) and aspirin (a non-steroidal anti-inflammatory drug) and induced synergistic antitumour activity through the modulation of the nuclear factor kappa B (NF-κB)/cyclooxygenase-2 (COX-2) signalling pathways. The results showed that aspirin at non-cytotoxic concentrations synergistically sensitised hepatocellular carcinoma cells to 5-Fu in vitro. It demonstrated that aspirin inhibited NF-κB activation and suppressed NF-κB regulated COX-2 expression and prostaglandin E2 (PGE2) synthesis. Furthermore, the proposed results clearly indicated that the combination of 5-Fu and aspirin by chitosan nanoparticles enhanced the intracellular concentration of drugs and exerted synergistic growth inhibition and apoptosis induction on hepatocellular carcinoma cells by suppressing NF-κB activation and inhibition of expression of COX-2.

Fig. 1

Characterisation of 5‐FACN (a) TEM images of CN and 5‐FACN, (b) DLS analysis of NPs, (c) Accumulative release of 5‐FACN and free drugs in PBS (pH 7.4 and 5.8), mean ± SD (n  = 3). Scale bar: 100 nm

Fig. 2

Confocal images of RhB‐labelled NPs at 6 h and uptake ratio of free RhB and RhB‐labelled NPs in SMMC‐7721 and HepG2 cells. Results are expressed as means ± standard deviation (n = 3). *P < 0.05

Fig. 3

Viability of SMMC‐7721 and HepG2 cells treated by different formulations (a) Viability of CNs treated SMMC‐7721 and HepG2 cells, (b) Viability of SMMC‐7721 and HepG2 cells treated with 5‐FCN, 5‐Fu and free aspirin, (c) Viability of SMMC‐7721 and HepG2 cells treated with 5‐FCN and the combination of 5‐FCN and aspirin. Results are expressed as means ± standard deviation (n  = 3), *P  < 0.05, (d) Viability of SMMC‐7721 and HepG2 cells after incubation with 5‐FACN and the combination of 5‐FCN and aspirin with the same concentration of aspirin at 0.1 and 0.5 mM. Results are expressed as means ± standard deviation (n  = 3). *P  < 0.05, ^# P  < 0.05, comparing with control. ^& P  < 0.05

Fig. 4

Cell cycle progression in SMMC‐7721 cell lines treated with free aspirin, 5‐FCN, 5‐FACN and the combination of 5‐FCN and free aspirin for 24 h by flow cytometry

Fig. 5

Western blot analysis and ELISA analysis (a) Determination of NF‐κB p65 levels in SMMC‐7721 cells treated with free aspirin. The results are expressed as the mean ± standard deviation (n  = 3), ^# P  < 0.05, (b) Determination of COX‐2 level in SMMC‐7721 cells treated with free aspirin. The results are expressed as the mean ± standard deviation (n  = 3), ^# P  < 0.05, (c) Determination of PGE2 levels in the supernatant of SMMC‐7721 cells treated with free aspirin by ELISA. The results are expressed as the mean ± standard deviation (n  = 3), ^# P  < 0.05

Fig. 6

Western blot analysis and ELISA analysis (a) Determination of caspase‐3 and Bax levels treated with aspirin, 5‐FCN, 5‐FACN and the combination of 5‐FCN and free aspirin. The results are expressed as the mean ± standard deviation (n  = 3), ^# P  < 0.05. The determination of NF‐κB p65 levels, (b) COX‐2 levels, (c) PEG2 levels, (d) Treated with 5‐FCN, 5‐FACN and the combination of 5‐FCN and free aspirin. The results are expressed as the mean ± standard deviation (n  = 3), ^# P  < 0.05