Gold nanoparticle resveratrol complex increases apoptosis in KRAS mutant pancreatic cancer cells

Abstract

The KRAS G12D mutation is the most prevalent type of pancreatic cancer and is found in about 35% of patients. Numerous natural chemicals are frequently investigated in cancer treatment to decrease side effects. Resveratrol (RVT) is a polyphenol that can promote cancer cell apoptosis and improve chemotherapy efficacy in cancers. To enhance delivery rate and efficacy, the size of about 30 nm gold nanoparticles (GNPs) was synthesized and conjugated to resveratrol via polyvinylpyrrolidone (GRs) for high bioavailability. Compared to RVT and GNPs, GRs had less inflammatory response and less toxicity on RAW 264.7 cells. This suggests that the toxicity of resveratrol can be alleviated by conjugation with gold nanoparticles. The viability of the human pancreatic cancer cell line (AsPC-1) decreased in sequence of GRs > RVT > GNPs, suggesting an enhanced anticancer effect of the GRs compared to resveratrol (RVT) alone. In addition, the extent of apoptosis was much bigger with GRs compared to RVT and GNPs. The apoptotic effects were confirmed with cell cycle arrest and expression of apoptosis-related genes and proteins. Thus, GRs had a better extent of anticancer effect than RVT, suggesting that GRs be considered as one of the prospective anti-cancer drugs for pancreatic cancer treatment.

Keywords: Apoptosis; Gold nanoparticles; KRAS mutation; Pancreatic cancer; Resveratrol.

Fig. 1

TEM images of GNPs and GRs. On the left below, the scale bar was presented. (A) and (B) are GNPs, while (C) and (D) are GRs.

Fig. 2

Nitric oxide production in RAW 264.7 cells after exposure to LPS, GNPs, RVT, and GRs for 24 h. Negative control (Con(-)) received no treatment; positive control (Con ( +)) received 1 μg/mL LPS. Various concentrations of GNPs, RVT, and GRs were added to the positivel control containing 1 μg/mL LPS. The data were measured in 3 independent experiments. The values were statistically different in the level of ***p < 0.001.

Fig. 3

Lactate dehydrogenase leakage level was conducted after exposure to LPS, GNPs, RVT, and GRs for 24 h. Negative control (Con(-)) received no treatment; positive control (Con ( +)) received 1 μg/mL LPS. Various concentrations of GNPs, RVT, and GRs were added to the positive control containing 1 μg/mL LPS. Data were expressed in 3 independent experiments. The values were statistically different in the level of ***p < 0.001.

Fig. 4

Raw 264.7 cells were treated with LPS to see which expressed more cytokines. Variation of expression levels of interleukin-6 (IL-6) (A) and tumor necrosis factor- α (TNF-α) (B) with treatments of various concentrations of GNPs, RVT, GRs to 1 μg/mL LPS-treated Raw 264.7 cells for 48 h. Negative control (Con(-)) received no treatment; positive control (Con ( +)) received 1 μg/mL LPS. The values were statistically different in the level of ***p < 0.001.

Fig. 5

Cell viability of AsPC-1 cells compared to the control (no treatment) after treatment of various concentrations of GNPs, RVT, and GRs for 72 h. The data were measured in 3 independent experiments. The values were statistically different in the level of *p < 0.05, ***p < 0.001.

Fig. 6

The percentages of apoptosis in AsPC-1 cells were shown for control (no treatment), GNPs, RVT, and GRs for 72 h. AsPC-1 cells were stained with Annexin V/PI (A) to measure the percentage of apoptotic cell death (B). The apoptotic cell death populations included early apoptotic cells (Annexin V + /PI-) and late apoptotic cells (Annexin V + /PI +). The values were statistically different in the level of *p < 0.05, ***p < 0.001 compared to the control (no treatment).

Fig. 7

AsPC-1 cells were treated for 72 h with GNPs, RVT, and GRs. mRNA expression levels of caspase-3 (A), caspase-7 (B), caspase-9 (C), BAK (D), and Bcl-2 (E) were determined by RT-qPCR. The values were obtained by normalization to the value of GAPDH for internal control (no treatment). Data are expressed as the mean ± SE (n = 3). The values were statistically different in the level of *p < 0.05, ***p < 0.001 compared to the internal control (no treatment).

Fig. 8

The treatment effects of GNPs, RVT, and GRs were analyzed by Western blotting in (A). Levels of n-fold expression of proteins in AsPC-1 cells were obtained by normalization to the value of β-actin for internal control and were presented in cleaved PARP (B), BAK (C), BAX (D), Cyclin D1 (E), Bcl-2 (F). Data are expressed as the mean ± SE (n = 3). The values were statistically different in the level of *p < 0.05, ***p < 0.001 compared to the internal control (no treatment).