Demethoxycurcumin-Loaded Chitosan Nanoparticle Downregulates DNA Repair Pathway to Improve Cisplatin-Induced Apoptosis in Non-Small Cell Lung Cancer

Abstract

Demethoxycurcumin (DMC), through a self-assembled amphiphilic carbomethyl-hexanoyl chitosan (CHC) nanomatrix has been successfully developed and used as a therapeutic approach to inhibit cisplatin-induced drug resistance by suppressing excision repair cross-complementary 1 (ERCC1) in non-small cell lung carcinoma cells (NSCLC). Previously, DMC significantly inhibited on-target cisplatin resistance protein, ERCC1, via PI3K-Akt-snail pathways in NSCLC. However, low water solubility and bioavailability of DMC causes systemic elimination and prevents its clinical application. To increase its bioavailability and targeting capacity toward cancer cells, a DMC-polyvinylpyrrolidone core phase was prepared, followed by encapsulating in a CHC shell to form a DMC-loaded core-shell hydrogel nanoparticles (DMC-CHC NPs). We aimed to understand whether DMC-CHC NPs efficiently potentiate cisplatin-induced apoptosis through downregulation of ERCC1 in NSCLC. DMC-CHC NPs displayed good cellular uptake efficiency. Dissolved in water, DMC-CHC NPs showed comparable cytotoxic potency with free DMC (dissolved in DMSO). A sulforhodamine B (SRB) assay indicated that DMC-CHC NPs significantly increased cisplatin-induced cytotoxicity by highly efficient intracellular delivery of the encapsulated DMC. A combination of DMC-CHC NPs and cisplatin significantly inhibited on-target cisplatin resistance protein, ERCC1, via the PI3K-Akt pathway. Also, this combination treatment markedly increased the post-target cisplatin resistance pathway including bax, and cytochrome c expressions. Thymidine phosphorylase (TP), a main role of the pyrimidine salvage pathway, was also highly inhibited by the combination treatment. The results suggested that enhancement of the cytotoxicity to cisplatin via administration of DMC-CHC NPs was mediated by down-regulation of the expression of TP, and ERCC1, regulated via the PI3K-Akt pathway.

Keywords: ERCC1; NSCLC; chitosan; demethoxycurcumin.

Figure 1

Characteristics of CHC and DMC-CHC NPs. (A) TEM morphology of CHC and DMC-CHC NPs (scale bar: 200 nm). Left one is unloaded CHC, and right one is DMC-CHC nanoparticles. (B) Left panel: effect of DMC on the viability of A549 cells. Right panel: effect of free DMC-CHC NPs on the viability of A549 cells. (C) CHC nanomatrix (6.25, 12.5, 25, 50, and 100 μg/mL) were incubated with A549 for 24 h, 48 h, and 72 h. The results showed that the CHC nanomatrixes demonstrated no cytotoxicity on A549. Each data point is represented as the mean ± SD (n = 3). Error bars represent standard deviation. * Indicates the values significantly different from the control time points, 24 h, 48 h, and 72 h., respectively. (* p < 0.05; ** p < 0.01).

Figure 2

(A) Fluorescence images of A549 incubated with the DMC-CHC nanoparticles (5 μg/mL) for 0–4 h. The merged image was the overlapping image obtained using the DAPI channel (blue, nuclei), rhodamine channel (red, F-actin), and FITC channel (green, nanoparticles). (B) Nanoparticles uptake efficiency measured by flow cytometer (a) 20 μg/mL FITC-CHC and (b) 5 μg/mL FITC-DMC-CHC NPs in A549. A549 were administered with or without FITC-DMC-CHC NPs for various time intervals (0–4 h). (c) To compare the proportional amount of nanoparticles, and (d) relative fluorescence intensity between FITC-CHC and FITC-DMC-CHC NPs in A549 cancer cells (M1). 10,000 cells were randomly chosen for imaging analysis with flow cytometer. Each data point is represented as the mean ± SD (n = 3). Error bars represent the standard deviation.

Figure 3

Effects of DMC-CHC NPs on cytotoxicity of cisplatin in A549 cells.

Figure 4

An impact from DMC-CHC NPs on on- and post-target resistance of CDDP was examined. (A) The effects of DMC-CHC NPs on on-target resistance of cisplatin was determined. DMC-CHC NPs significantly decreased cisplatin-induced TP and PI3k/Akt/ERCC1 signaling in A549 cell. A549 cells were exposed to various concentrations of DMC-CHC NPs (7.5 µg/mL, 15 µg/mL, and 30 µg/mL) and cisplatin for 48 h. (B) The effects of DMC-CHC NPs (7.5 µg/mL, 15 µg/mL, and 30 µg/mL) on post-target resistance of cisplatin was examined. The protein expression of bax, and cytochrome c in A549 cells were determined using Western blotting analysis. The results represent the mean ± SD of three independent experiments. * Indicates the values significantly different from the control. (* p < 0.05; ** p < 0.01).