br Ultraviolet and infrared spectrum analysis br APS was dis
2.2.2. Ultraviolet and infrared spectrum analysis
APS was dissolved in deionized water to final concentrations of 0.5–2 mg/mL. The samples were scanned with a UV–Vis spectro-photometer (Lambda 750 s, PerkinElmer, USA) in the range of 190 to 800 nm. The structural characteristics of APS were determined by Fourier transformed IR spectrophotometer (EQUINOX55, Bruker, Germany).
2.3. Macrophage activation
2.3.1. Nitrite determination
The generation of nitric oxide (NO) was determined by Griess re-action. Briefly, RAW 264.7 JSH23 (1 × 105 cells/mL) were seeded in 24-well plates and incubated for 24 h. Afterwards, cells were subjected to diﬀerent concentrations of APS. Here, LPS (1 μg/mL) and the complete medium was used as positive and negative control groups, respectively. Besides, cells were separately treated with TLR4 antibody (20 μg/mL) for 2 h following the addition of APS (1000 μg/mL) and LPS. After 24 h stimulation, 100 μL culture supernatant was mixed with an equal vo-lume of Griess reagent at room temperature for 10 min. The absorbance of the mixture was measured by a microplate reader at 540 nm. All of the experiments were performed in triplicates.
2.3.2. TNF-α expression by ELISA ELISA was performed to evaluate TNF-α expression in the presence
Fig. 1. Schematic diagram of the apoptosis of MCF-7 cells induced by APS-activated macrophages.
of APS. In brief, RAW 264.7 cells (1 × 105 cells/mL) were seeded into 24-well plates and incubated overnight, followed by the addition of serial dilutions of APS and LPS. Similarly, cells were subjected to TLR4 antibody as described above. After stimulation for 24 h, the level of TNF-α in cell-free supernatant (i.e. CM) was assessed by ELISA ac-cording to the manufacturer's instructions.
2.3.3. FITC-labeling of APS and interaction with macrophages
APS was labeled with fluorescein isothiocyanate (FITC) as pre-viously described . APS was dissolved in 50 mL formamide and 50 mL methylsulfoxide containing 0.05 g dibutyline-dilaurate. FITC (0.25 g) and NaHCO3 (0.1 g) were added and the mixture was heat-treated at 100 °C for 1 h. After several precipitations in ethanol, the FITC-labeled APS was dissolved in PBS and dialyzed against distilled water and lyophilized.
FITC-labeled polysaccharides were added to the macrophage cells and incubated for 20 min, 1 h, and 6 h, respectively. Similarly, un-labeled APS was treated with RAW264.7 cells for 1 h followed by ex-posure to FITC-labeled APS for 5 h. After being washed in PBS for three times, macrophages were imaged with a fluorescence microscopy at 488-nm.
2.4. Antineoplastic activities of APS
2.4.1. Cell culture and conditioned medium collection
MCF-7 cells were cultured in DMEM medium supplemented with 10% FBS at 37 °C and 5% CO2 in a humidified incubator. APS was dissolved in DMEM at diﬀerent concentrations (50, 100, 200, 500, and 1000 μg/mL, respectively) and filter-sterilized with a membrane filter (0.22-μm pore size). RAW264.7 cells at 1 × 105 cells/mL were seeded in 24-well culture plates, followed by the addition of diﬀerent con-centrations of APS. Besides, untreated RAW264.7 cells and cells treated by LPS (1 μg/mL) were used as the negative control and positive con-trol, respectively. At 24 h, the supernatants from the aforementioned groups were collected and centrifuged at 1000 rpm for 10 min. The supernatants with various concentrations of APS (hereafter simply re-ferred to as conditioned medium) were collected and stored at −20 °C for later use.
2.4.2. Inhibitory eﬀect of APS and conditioned medium
CCK-8 assay was conducted to investigate the eﬀect of APS and conditioned medium (CM) on the viability of MCF-7 cells. Briefly, 3 × 103 cells were seeded in each well of 96-well culture plates con-taining 100 μL complete medium. Following overnight incubation, se-rial concentrations of APS (50, 100, 200, 500 and 1000 μg/mL) and CM were added and maintained in a humidified incubator for 24, 48 and 72 h, respectively. MCF-7 cells incubated with complete medium and 5-FU (50 μg/mL) were used as negative and positive control, respectively. Subsequently, APS-containing medium and CM in each well were re-placed with 100 μL fresh medium and 10 μL CCK-8 solution, and the cells were then incubated at 37 °C for 3 h. Afterwards, the absorbance of each well was detected at 450 nm by a microplate reader (Varioskan Flash, Thermo Fisher Scientific, San Jose, CA, USA). All experiments were performed in triplicates. The absorbance of the negative control group was considered as 100% and the inhibitory rate was calculated according to the equation: ζ = (1 −A1/A0) × 100%, where A1 and A0 were the absorbance of the sample and negative control group, re-spectively.