2.1 Cell culture and treatment
The MCF-7 cells were obtained from the ATCC cell bank. MCF-7 cells were cultured in DMEM medium (Gibco,1853128) supplemented with 10% fetal bovine serum (Biological Industries,1707254) 1% penicillin/streptomycin in a humidified 5% CO2 at 37 °C incubator. Cells were passaged every 3 days at desired confluence. Curcumin was purchased from Sigma Aldrich (CAS No.458-37-7, assay≥94%) and dissolved in DMSO to make a stock solution of 50mM. MCF-7 cells were pretreated with 0.2μg/ml lipopolysaccharide (LPS, Sigma Aldrich, L4391) for 4h and then expose to curcumin (8μM) for 48h.This experiment involved pharmacological inhibitors. MCF-7 cells were pretreated with the addition of autophagy inhibitor 3-methyladenine (3-MA,5mM, Sigma, assay: 98%), CTSB inhibitor CA-074 Me (10μM, AdooQ, A13256,assay>98%), NLRP3 inhibitor MCC950 (5Mm,MCE, HY-12815A,assay>98%) and 0.2μg/ml LPS for 4h, then to expose to curcumin (8μM) for 48h.
2.2 Animal experiment
Ten six-year-old female BALB/c-Nude mice (weight:20g) were purchased from Beijing Vital River, raised in the Institute of Genome Engineered Animal Models for Human Disease of Dalian Medical University (SPF level). The mice were kept under sterile conditions and fed a sterilized mouse diet and water. All mice were anaesthetized via inhalation of isoflurance and a tumor cell suspension of 107 MCF-7 cells in 0.2 ml DMEM was injected subcutaneously into the inguinal of each mouse. When tumors reached about 30-60mm3 at 1 week, the mice were randomly seperated into two groups (n = 5/group). The mice were treated with curcumin 200 ug/kg or saline (control) by intraperitoneal injections every day for 4 weeks. Tumor size was measured every week in two perpendicular dimensions with vernier calipers and converted to tumor volume using the formula: a*b*b (a:longer, b:shorter). At the end of the experimental period, all mice were euthanized and tumors were segregated and weighed.
2.3 Cell viability assays
The cytotoxicity of curcumin was measured by using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. MCF-7 cells (5*104/ml) were seeded in 96-well plates and treated with 0,2,4,8,16,32,64,128μM for 24h or 48h.After treatment, MTT (5 mg/ml in sterile PBS, Solarbio, M8180) was added and the 96-well plates were incubated for 4h at 37℃.Thereafter the supernatant was removed and 100μl DMSO was added into each well for 30 min at 37℃.After that, the plates were shaken carefully until the blue formazan crystals were fully dissolved. Then measured the absorbance at 570 nm using a Bio-Rad Microplate Reader, and the cell viability (%) was calculated using the formula: (A570 of treated group/A570 of control) × 100%.
2.4 Western blot analysis
Proteins were exracted from the MCF-7 cells or from the tumors, completely lysed in the lysis buffer by using a nuclear and cytoplasmic protein extraction kit (Keygen Biotech, China). Then quantified with the BCA Protein Assay kit (Thermo, MK164230). An equal amount (30μg) of total proteins were loaded into different lanes and separated on 10%-15% SDS-Page gel, then blotted and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked for 1h with 10% skimmed milk by gentle shaking in a water bath shaker at 37 °C and then incubated with primary antibody LC3 (Abcam, ab192890, 1:1000), p62 (Protein tach, 18420-1-AP, 1:1000), CTSB (Wanleibio, WL01089, 1:500), ASC (Wanleibio, WL02462, 1:500), pro-Caspase-1 (Wanleibio, WL02996,1:500), GSDMD (Cell Signaling Technology, #93709, 1:500), NLRP3 (Wanleibio, WL02635, 1:1000), Caspase-1 (Affinity, AF4005, 1:500), GSDMD-N(Abcam, ab215203), IL-1β (Wanleibio, WL00891, 1:500), IL-18 (Wanleibio, WL01127, 1:1000), β-actin (Cell Signaling Technology, #3700, 1:1000) at 4℃ overnight. After washing 3 times with PBS, membranes were incubated with the Secondary Goat anti-mouse IgG and Goat anti-rabbit-IgG at room temperature for 1.5h. The bound antibody was visualized using the Super Signal West Pico Kit (Thermo Scientific) and Bio-Rad ChemiDoc™ MP imaging system (Bio-Rad Laboraturies), then analyzed by ImageJ software. The experiments above were independently repeated three times.
2.5 Immunohistochemical assays
The tissue samples of the tumors, preserved in 2.5% glutaraldehyde-polyoxymethylene solution, were dehydrated and embedded in paraffin following routine methods. The paraffin sections were dewaxed and hydrated following routine methods. Rinsed the paraffin sections in PBS-T (3×5 min before each following steps), and then blocked with 3% peroxide-methanol at room temperature for endogenous peroxidase ablation. Afterwards, the sections were immersed in a boiling sodium citrate buffer for 15min, cooled down to room temperature. After that, incubated with blocking buffer (normal goat serum at room temperature for 20 min. Then incubated with primary antibody Caspase1(Affinity, AF4005, 1:100), IL-1β (Wanleibio, WL00891, 1:100), IL-18 (Wanleibio, WL01127, 1:100) at 4℃ overnight. After that, the sections were incubated with the Secondary Goat anti-rabbit-IgG at 37℃ for 30min.Then incubated with the S-A/ HRP at 37°C for 30 min. Colored with 3,3-diaminobenzidin (DAB), and kept at room temperature without light for 10 min. After rinsing adequately with water, the sections were stained with hematoxylin for 5s, then dehydrated and sealed with neutral resins. We observed the sections under an upright microscope.
2.6 Immunofluorescence staining
In order to observe distribution of CTSB in cytoplasm, MCF-7 cells were incubated on slides and treated with primary rabbit monoclonal antibodies against CTSB (Abcam, ab125067, 1:200) after fixation and blocking. Lysosomes were labeled with Lysotracker Red (Beyotime) and Alexa Fluor 488 secondary antibodies (Proteintech, SA00006-2,1:400) was added in the dark. We analyzed the 4,6-diamidino-2-phenylindole (DAPI) counterstained slides under a fluorescence microscope (40 × 10).
2.7 Lactate dehydrogenase (LDH) release assays
To access the toxic effect of curcumin, the LDH release of MCF-7 cells were measured using an LDH Cytotoxicity Assay Kit (Beyotime, C0016). MCF-7 cells were seeded in 96 well plates to desired confluence and treated with inhibitor for 4h then treated with curcumin for 48h. One hour before the end of the treatment, we added 10% of the LDH release reagent and the original culture volume when the sample showed maximum enzyme activity. The culture supernatants were collected, and the absorbance was read at 490 nm with a microplate reader (Thermo Fisher Scientific). The percentage of LDH released was calculated as the percentage of the total release amount and considered to be the sum of the enzyme activity in the cell lysate and the enzyme activity in the medium.
2.8 Enzyme-linked immunosorbent assays (ELISA)
IL-1β and IL-18 levels were measured by using ELISA kits (Lengton, BPE10083, BPE10092) according to the manufacturer's instructions. MCF-7 cells were seeded in 96-well plates to desired confluence and treated with inhibitor for 4h then treated with curcumin for 48h. The 96-well plates were centrifuged at 3000rpm for 20 min at 4 °C. Finally, the supernatants were collected. A total of 50μl of serially diluted samples and standard were added to the ELISA plate wells and incubated with horseradish peroxidase-conjugated specific antibodies for IL-1β and IL-18 for 60 min at 37 °C. The OD values were detected at 450nm by using microplate reader. The linear regression equation of the standard curve was calculated according to the concentration of the standard corresponding OD value. Finally, the sample concentration was calculated on the linear regression equation according to its OD value.
2.9 Cell migration assays
For the transwell migration assays, MCF-7 cells were treated with various treatments. After indicated treatment, cells were resuspended at serum-free medium then seeded onto the upper chambers with non-coated membrane (24-well insert; 8-mm pore size). After that, we filled the lower chambers with 600ml DMEM containing 10% FBS. After 24 hours of incubation, non-invading cells on the upper surface of the upper chamber were utterly removed by using cotton swabs. Other cells on the lower surface of filters were fixed with methanol for 30 min then stained with 0.1% crystal violet for 30 min. The number of invaded cells was counted under an upright microscope.
2.10 Matrigel tube formation assay
Via a precooled pipette, 50 µL of freeze thawing liquid Matrigel (Corning, 354248, USA) was embedded into a 96-well plate at 4 °C. HUVECs (2 × 104 cells per well) were seeded onto the solidified matrigel 96-well plate and cultured for 12 h at 37°C in 5% CO2. After the HUVECs were overlaid, we replaced the culture medium of HUVECs with MCF-7 cell culture medium treated by curcumin and inhibitor. Capillary-like structures were evident and counted using a phase-contrast microscope and the networks formed by HUVECs were quantified with ImageJ.
2.11 Plate clone formation assay
MCF-7 cells were cultured and seeded onto 6-well plates (1000 cells per well) to prepare the cells for the plate clone formation assay. Different wells were treated variously. When the cells in 6-well plates growth to a density of 50% per cluster, which were washed with the phosphate-buffered saline(PBS) and fixed by 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA). Giemsa stain (Sigma-Aldrich) was used to stain the cells. Eventually, cell clones were counted and analyzed.
2.12 Statistical analysis
All Data were expressed as means ± standard deviation (SD) from at least three independent experiments performed in triplicates and analyzed by using the SPSS 20.0. Significance was determined using one-way analysis of variance (ANOVA) or t-test, and P value < 0.05 was considered statistically significant.