Dulbecco's modified Eagle's medium (DMEM), Roswell Park Memorial Institute (RPMI) medium, fetal bovine serum (FBS) and penicillin/streptomycin (P/S) were purchased from Welgene Inc. WST-1 and bovine serum albumin (BSA) were purchased from Sigma-Aldrich; Merck KGaA. A TUNEL assay kit was obtained from Promega Corporation. Hoechst dye, Mito-tracker^™ Red CMXRos and the Alexa-Fluor 488-conjugated donkey anti-Rabbit antibody were purchased from Thermo Fisher Scientific, Inc. The anti-Ki-67 antibody was purchased from Abcam (cat. no. ab15580). The aspirin conjugated DK143 [(E)-2-(4-(2,3-dimethoxyphenyl)acryloyl)-4-methoxyphenyl-2-acetoxybenzoate, AS-DK143] and AS-DK143-loaded methoxy poly(ethylene glycol)-poly(L-lactide) NPs (AS-DK143-NPs) were prepared and characterized according to our previous report (22). HeLa cell line, HepG2, which is a hepatoblastoma cell line, and AGS cell line were purchased from the Korean Cell Line Bank; Korean Cell Line Research Foundation. BXPC-3 cells were obtained from the Japanese Collection of Research Bioresources Cell Bank.

Cell lines were authenticated using short tandem repeat (STR) analysis as described in 2012 in the ANSI standard (ASN-0002) by the American Type Culture Collection Standards Development Organization and by Reid et al (23) The match criterion is based on an algorithm that compares the number of shared alleles between the reference and sample profile.

(E)-3-(2,3-dimethoxyphenyl)-1-(2-hydroxy-5-methoxyphenyl)prop-2-en-1-one (DK143), one of the chalcone derivatives, was used to synthesize AS-DK143 using the following procedures. 1-(3-Dimethylaminopropyl)-3-ethylcarbodimide hydrochloride (EDC·HCl, 0.641 g, 3.3 mmol) and 4-dimethylaminopyridine (DMAP, 0.816 g, 3.3 mmol) were added to chloroform (50 ml) containing aspirin (0.574 g, 3 mmol) and DK143 (1.0 g, 3 mmol). The solution was stirred at room temperature for 24 h. At the end of the reaction, ice water was added to the mixture solution and acidified with 6 N HCl. After extraction with ethyl acetate (100 ml, × 3), the organic layer was purified using silica gel flash column chromatography to yield 33.3% (0.48 g) as a pale-yellow solid.

AS-DK143 polymeric micelles (AS-DK143-NPs) were prepared using the thin-film hydration method. Briefly, methoxy poly(ethylene glycol)-b-poly(D,L-lactide) (mPEG-PLA, 60 mg) and AS-DK143 (10 mg) were dissolved in dichloromethane (50 ml) and then removed by rotary evaporator under reduced pressure at 40°C. The film obtained from this process contained AS-DK143 distributed in an amorphous form. The film was continuously hydrated in distilled water (50 ml) at room temperature. The solution was filtered through a syringe filter (0.2 µm) and lyophilized for 48 h.

HeLa (human cervical cancer cell line) and HepG2 cells (human liver cancer cell line) were cultured in DMEM supplemented with 10% FBS and 1% P/S and placed at 37°C in a humidified incubator containing 5% CO₂. AGS (human gastric adenocarcinoma hyperdiploid cell line) and BXPC-3 cells (human pancreatic cancer cell line) were cultured in RPMI supplemented with 10% FBS and 1% P/S and placed at 37°C in a humidified incubator containing 5% CO₂. Cells were seeded at the density of 1×10⁴ cells/well in 96-well plates, cultured for 24 h and treated with different concentration of AS-DK143-NPs (1, 3, 10, 30, 100 or 300 µM) for 24 h. To determine the cell viability, cells were incubated with 10 µl of WST-1 for 2 h at 37°C. Absorbance was read at 450 nm using an iMARK spectrophotometer (Bio-Rad Laboratories, Inc.). Cell-morphology images were captured using an inverted microscope (magnification, ×200; Nikon Corporation).

HeLa cells were plated in 8-chamber slides at a density of 0.5 × 10⁴ cells/well, and the cells were treated with AS-DK143-NPs for 24 h. Cells were fixed in 4% paraformaldehyde (PFA) and permeated in 0.1% TritonX-100 solution for 5 min at room temperature. Cells were incubated with equilibration buffer for 5 min and treated with recombinant terminal deoxynucleotidyl transferase (rTdT) reaction solution (consisting of equilibration buffer, a mixture of biotinylated nucleotides, and rTdT enzyme) for 1 h at 37°C. The cells were immersed in 2X SSC buffer (NaCl and sodium citrate) and washed with phosphate-buffered saline. The cells were incubated with streptavidin-conjugated horseradish peroxidase solution for 30 min. The cells were reacted with 3,3′-diaminobenzidine (DAB) solution (DAB chromogen and DAB substrate). Subsequently, the cells were mounted and observed under a light microscope.

HeLa cells (1×10⁵ cells/well) were seeded in 30-mm dishes and treated with AS-DK143-NPs for 24 h. Cells were then fixed with 4% PFA for 10 min at room temperature (RT) and permeabilized using 0.1% Triton-X 100 for 15 min at RT. The cells were blocked for 1 h in 5% BSA at room temperature and incubated with a primary antibody against Ki-67 (1:1,000) for 24 h at 4°C. Subsequently, cells were incubated for 1 h at RT with a fluorophore-conjugated secondary antibody (1:1,000). Cells were further incubated with Hoechst solution for 20 min at room temperature. Fluorescence was observed using a K1-fluo microscope (magnification, ×200; Nanoscope Systems, Inc.).

HeLa cells were seeded in 30-mm confocal dishes at the density of 1×10⁵ cells/well and were treated with 10 and 30 µM of AS-DK143-NPs for 24 h. Cells were loaded with 500 nM Mito-tracker^™ Red CMXRos (excitation: 579 nm, emission: 599 nm) for 30 min at 37°C. The medium was replaced and mitochondrial images were obtained using a Zeiss KSM 780 microscope (magnification, 400; Ziess GmbH).

Total RNA was isolated from HeLa cells using TRIzol reagent (Thermo Fisher Scientific, Inc.) and mixed with chloroform. The resulting supernatant was incubated with isopropanol for 20 h at 4°C and then centrifuged at 8,000 × g for 15 min at 4°C. The total RNA was quantified using a NanoDrop device (NanoDrop^™ 2000/2000c spectrophotometer; Thermo Fisher Scientific Inc.). cDNA was synthesized using 1 µg RNA and the Superscript III First Strand cDNA Synthesis Kit. qPCR was performed on an Exicyler^™ 96 instrument (Bioneer Corporation) using SYBR master mix (Bioneer Corporation). The following PCR thermocycling conditions were used: Initial denaturation at 95°C for 10 min, 40 cycles of denaturation for 10 sec at 95°C and annealing at 60°C for 30 sec, followed by a final extension step for 30 sec at 72°C. The sequences of the primers were as follows: CD36 forward, 5′-TGGAACAGAGGCTGACAACTT-3′, reverse, 5′-TTGATTTTGATAGATATGGGATGC-3′; BAX forward, 5′-GCGTCCACCAAGAAGCTGAG-3′, reverse, 5′-ACCACCCTGGTCTTGGATCC-3′; Bcl2 forward, 5′-TGTGGCCTTCTTTGAGTTCG-3′, reverse, 5′-TCACTTGTGGCCCAGATAGG-3′; p53 forward, 5′-TGTGGAGTATTTGGATGACA-3′, reverse, 5′-GAACATGAGTTTTTTATGGC-3′; and β-actin forward, 5′-GTGATGGTGGGCATGGGTC-3′ and reverse, 5′-ACGGCCAGAGGCGTACAGGG-3′. The relative expression levels were normalized to endogenous control β-actin and were expressed as 2^−ΔΔCq (24).

To detect the effects of AS-DK143-NPs on live cells, HeLa cells (1×10⁵ cells/well) were seeded in 60-mm Tomodishes and treated with 30 µM AS-DK143-NPs at 36°C for 30, 60 and 120 min. 3D holotomography images of live cells were obtained using an HT-1 microscope (magnification, ×600; Tomocube, Inc.). The cell structures were scanned using a digital micromirror device and reconstructed using a 3D refractive index tomogram. The masses of the cytoplasm and lipid droplets were analyzed based on densities of 0.135 g/ml and 0.178 g/ml, respectively.

Data are expressed as the means ± standard deviation of at least three independent experiments. Statistical analyses were performed using GraphPad Prism software version 4.0 (GraphPad Software, Inc.). Data were compared using Student's t-test and one-way ANOVA followed by with Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Fig. 1 shows the chemical structure of AS-DK143 and the formed NPs that were used as an efficient drug delivery system. Cell viability assay was performed to evaluate the anti-tumor effects of AS-DK143-NPs on the four cell lines HepG2, BXPC-3, AGS and HeLa. Cells were treated with increasing concentration of AS-DK143 for 24 h. The inhibitory rate was determined using WST-1 assay. As presented in Fig. 2, the IC₅₀ values of AS-DK143-NPs were 18.54, 35.74, 9.28 and 4.17 µM in HepG2, BXPC-3, AGS and HeLa cells, respectively. The AS-DK143-NPs revealed a reducing effect on cell viability in HeLa cells compared with other cell lines.

To determine the anticancer effects of AS-DK143-NPs, we performed cell morphology and immunocytochemistry assays. As presented in Fig. 2A, cells were treated with different concentrations of AS-DK143-NPs (10, 30, 100 and 300 µM) for 24 h. AS-DK143-NPs treatment induced changes in cell morphology and viability. Subsequently, the anticancer effects of AS-DK143-NPs were confirmed by studying cell proliferation arrest using Ki-67 antibody for immunocytochemical staining. As presented in Fig. 2B, AS-DK143-NPs treatment decreased Ki-67 expression in HeLa cells. In addition, AS-DK143-NPs induced mitochondrial fission (Fig. 2C).

To determine whether AS-DK143-NPs could induce apoptosis in HeLa cells, TUNEL assays, Hoechst staining and RT-qPCR were performed. HeLa cells were grown in the absence or presence of AS-DK143-NPs (10 and 30 µM) for 24 h. The results demonstrated that AS-DK143-NPs increased TUNEL-positive staining in a concentration dependent manner, whereas the vehicle control-treated cells were not stained with the TUNEL solution (Fig. 3A). In addition, AS-DK143-NPs induced nucleolus aggregation in HeLa cells (Fig. 3B). Furthermore, we confirmed that AS-DK143-NPs induced HeLa apoptosis by evaluating the mRNA expression levels of certain apoptosis-related genes. As presented in Fig. 3C, AS-DK143-NPs significantly increased the expression level of p53 and the BAX/Bcl-2 ratio.

To determine whether AS-DK143-NPs could induce lipid accumulation, we performed live cell imaging, 3-D holographic imaging using a holotomographic microscope and determined CD36 mRNA expression by RT-qPCR. Hela cells were treated with AS-DK143-NPs (10 µM) for 2 h and 2D images representing their cell morphology were taken (Fig. 4A). We observed the formation of apoptotic bodies over time. The 3D images revealed lipid droplet formation (red color) and lipid accumulation occurred in a time-dependent manner. The 3D holotomographic-imaging assay was performed to evaluate lipid masses in live cells. The results demonstrated that lipid accumulation was not altered in vehicle treated cells but was significantly increased (~6-fold) in HeLa cells treated with AS-DK143-NPs (Fig. 4B). As presented in Fig. 4C, AS-DK143-NPs significantly increased the mRNA level of CD36 compared with the vehicle.