Biologically synthesis of gold nanoparticles using Cirsium japonicum var. maackii extract and the study of anti-cancer properties on AGS gastric cancer cells

Plant extract-mediated synthesis of metal nanoparticles (NPs) is an eco-friendly and cost-effective biosynthesis method that is more suitable for biological applications than chemical ones. We prepared novel gold NPs (AuNPs), Cirsium japonicum mediated-AuNPs (CJ-AuNPs), using a biosynthetic process involving Cirsium japonicum (Herba Cirsii, CJ) ethanol extract. The physicochemical properties of CJ-AuNPs were characterized using spectrometric and microscopic analyses. The in vitro stability of CJ-AuNPs was studied for 3 months. Moreover, the selective human gastric adenocarcinoma (AGS) cell killing ability of CJ-AuNPs was verified in cancer and normal cells. An in vitro study revealed that CJ-AuNPs trigger oxidative stress and iron-dependent ferroptosis in AGS cells. Mechanistically, CJ-AuNPs induced mitochondrial reactive oxygen species (ROS), Fe2+, and lipid peroxidation accumulation, and mitochondrial damage by destroying the glutathione peroxidase-4 (GPX4)-dependent antioxidant capacity. Furthermore, in a xenograft mouse model implanted with AGS cells, treatment with 2.5, 5, and 10 mg/kg CJ-AuNPs for 16 days reduced tumor xenograft growth in a dose dependent manner in vivo without systemic toxicity. These results demonstrate that CJ-AuNPs exert anticancer effects in vitro and in vivo by inducing ferroptosis-mediated cancer cell death. This study, based on green-synthesized nanodrug-induced ferroptosis, provides new insight into potential developments in cancer therapies.

To detect the effect of CJ-AuNPs on cell viability, each cell suspension was seeded in a 96-well plate (1 × 10 4 cells/well) and stabilized for 24 h. After the cells were washed twice with phosphate-buffered saline (PBS), the fresh medium containing CJ-AuNPs at various concentrations (6.25, 12.5, 25, 50, 100, 150, 200 μg/mL) was added to the cells for 24 h incubation. The cell viability was measured using a conventional MTT solution according to the manufacturer 's instruction. The formed formazan crystal was dissolved in DMSO (100 μL/well) and detected at 570 nm using a microplate reader (SpectraMax® ABS plus).

Evaluation of intracellular uptake and localization of CJ-AuNPs
To observe the uptake capacity of CJ-AuNPs, the gastric adenocarcinoma (AGS) cells were treated with CJ-AuNPs (150 µg/mL) for 5 min and 3 h. Cells were washed with PBS and fixed in 4% paraformaldehyde for preparing slides. To monitor the distribution of the CJ-AuNPs within the cells, the slide was observed under a bright-field microscopy (Olympus Optical Co., Ltd., Tokyo, Japan) and enhanced dark-field (EDF) microscopy (CytoViva Inc., Auburn, AL, USA) to take the photographic results. Furthermore, the uptake and intracellular localization were detected using a Bio-TEM.
Briefly, after CJ-AuNPs (150 µg/mL) treatment for 5 min and 3 h, the cell pellets were collected and fixed in 2.5% glutaraldehyde at 4 °C. The 1% osmium tetroxide was used to post-fix. After gradually dehydrating with concentration gradient ethanol (50, 70, 90, and 100%), samples were embedded in Epon (Sigma-Aldrich, St. Louis, MO). an ultramicrotome (Leica EM UC7, Wetzlar, Germany) was used to make ultrathin sections (70 nm). The sections were then treated according to the standard protocol for Bio-TEM imaging. Finally, the sections were imaged using JEM-1010 TEM (JEOL, Tokyo, Japan) operated at 80 kV.

Colony formation assay and live/dead staining
For the colony-forming assay, AGS cells (2 × 10 3 cells/well) were plated in a 6-well plate (SPL Life Sciences, Pocheon, Korea) and stabilized for 24 h. After being washed twice with PBS, the cells were incubated in fresh RPMI 1640 culture medium containing samples (100, 150, and 200 μg/mL) for 36 h. After rinsing with PBS, AGS cells were dyed using 0.5% crystal violet solution (Sigma-Aldrich) for 3 min. The cell colony was observed under a Leica DM IRB inverted microscope. Furthermore, to compare the cytotoxic effect of CJ-AuNPs in AGS cells, a live/dead cell staining kit (Thermo Fisher Scientific) was employed according to the manufacturer's instruction.
Briefly, AGS cells (2 × 10 5 cells/well) were cultured in 6-well plates, stabilized for 24 h, and then incubated with CJ-AuNPs for 24 h. Cells were stained by Calcein (detect live cells) and Ethidium Homodimer-1 reagent (detect dead cells) for 30 min. The living cells (green) and dead cells (red) were subsequently visualized using fluorescence scanning microscope at Ex/Em 494/517 nm (Leica, Wetzlar, Germany).

Reactive oxygen species (ROS) quantification and lipid peroxidation assessment
The intracellular ROS release was detected by using a Cellular ROS/Superoxide Detection Assay Kit (Abcam, Cambridge, UK). AGS cells (2 × 10 5 cells/well) were seeded in 6-well plates (SPL Life Sciences) and stabilized for 24 h. After the cells were washed twice with PBS, they were treated respectively with fresh RPMI containing different samples for 4, 6, and 8 h. After rinsing twice with PBS, the cells were stained using a cellular ROS assay kit according to the manufacturer 's instructions. Following an incubation period of 30 min, the AGS cells-contained coverslips were inspected on the stage of a fluorescent microscope (Leica, Wetzlar, Germany) and quantified using the GraphPad software (Prism 8; San Diego, CA, USA). To detect the level of lipid peroxidation, the intracellular malondialdehyde (MDA) concentration was assessed at 4, 6, 8, and 24 h after samples treatment using a lipid peroxidation (MDA) colorimetric assay kit (Abcam) according to the manufacturer's protocol.

Iron assay
The iron assay kit obtained from Sigma-Aldrich was used to check the intracellular ferrous iron level in AGS cells. Briefly, cells (2 × 10 6 ) were rapidly homogenized in 5 volumes of iron assay buffer on ice and centrifuged at 16,000 × g for 10 min at 4 °C to remove insoluble material. The supernatant was collected for measuring ferrous iron according to the manufacturer's instructions.

Quantitative real-time PCR (qRT-PCR)
qRT-PCR was used to evaluate and quantify the mRNA expressions in the present study. Briefly, total cellular RNA was extracted using a Trizol reagent kit (Thermo Fisher Scientific) and quantified on a nanodrop plate using a microplate spectrophotometer (Epoch, BioTek Instruments, Winooski, VT, USA). Equal amounts (500 ng) of total RNA were reverse-transcribed using the AmfiRivert cDNA synthesis kit (GenDEPOT). qRT-PCR was performed using the AmfiSure qGreen Q-PCR master mix (GenDEPOT) following the manufacturer 's instructions on the CFX96TM

Real-Time RT-PCR System with SYBR®Premix Ex TaqII RT-PCR Kit (TaKaRa Bio
Inc., Kusatsu, Japan). All primers were designed and provided by Macrogen (Seoul, Korea). The gene-specific primer sequences used in this study are listed: HO-1 (F, R)

In vivo xenograft model and experimental schedule
Male nude mice (CAnN.Cg-Foxn1-nu, 5 weeks) weighing 20-22 g were provided by Orient Bio (Seongnam, South Korea). The mice were housed in a 12 h light-dark cycle chamber, at a temperature of 23 ± 2 °C in a humidity-controlled room (50% humidity) with food and water ad libitum. All experimental procedures for animal study were reviewed and approved by the Institutional Animal Care and Use Committee at Kyung Hee University (KHGASP-20-375, approval data: Oct 12th, 2020) and were conducted in accordance with the 2000 Helsinki Declaration. After acclimating the mice for one week, AGS cells (5 × 10 6 ) were injected subcutaneously into the right back of the mice. The mice were randomly divided into six groups (n=5) after tumor volume grew to about 100 mm 3 : control group, model group, CJ-AuNPs-administered groups with different concentrations (2.5, 5.0, and 10.0 mg/kg), and 5-Fu-administered group. The control and model groups (tumor injection group) received excipient (distilled water), and CJ-AuNPs-administered groups received different doses of CJ-AuNPs, respectively, once daily for 16 days. The 5-Fu-administered group received 5-Fu (5.0 mg/kg) once every 3 days for 16 days as a positive control. All mice were monitored daily for body weight throughout the study period. At the end of administration, all mice were euthanized by CO 2 inhalation at a flow rate of 6 L/min for 3 min, and blood, liver, kidney, and tumor tissues were surgically collected. The liver and kidney were fixed with 10% formalin buffer solution and embedded in paraffin for hematoxylin and eosin (H&E) staining using H&E solution (Abcam). The tumor tissues were performed by immunohistochemical (IHC) staining using Mouse and Rabbit-specific HRP/DAB (ABC) Detection IHC Kit (Abcam). The stained tissues were visualized using light microscopy (Leica Microsystems, Wetzlar, Germany), and brown-yellow color was used to determine the positive expression of specific proteins in the tumor tissues, which were quantified using ImageJ software.