Cell culture media and antibiotics were purchased from Thermo Fisher Scientific, Inc., and fetal bovine serum (FBS) was purchased from Hyclone; Cytiva. Celastrol was acquired from Sigma-Aldrich (Merck KGaA), and MEK162, from Selleck Chemicals. LY294002, SP600125, GM6001 and Bay11-7082 were purchased from Tocris Bioscience. The anti-matrix metalloproteinases (MMP) 1 antibody was purchased from Abcam (cat. no. ab137332; dilution, 1:1,000); anti-β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc. (cat. no. sc-47778; dilution, 1:1,000); and anti-phospho (p-)ERK and total (t-)ERK antibodies (cat. no. 9102 (t); and 4370 (p); dilution, 1:1,000) were purchased from Cell Signaling Technology, Inc. The human IL8 (cat. no. K0331216) IL1B (cat. no. K1331800) ELISA kits were purchased from Koma Biotech, Inc.

All breast cancer cells were obtained from the American Type Culture. T47D, ZR75-1, BT-474, SK-BR-3, HCC1143 and HCC1806 human breast cancer cells were cultured in RPMI1640 medium supplemented with 10% FBS, 2 mM glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin, and maintained at 37°C in a humidified atmosphere (95% air and 5% CO₂). MCF7, MDA-MB-157, MDA-MB-231, and Hs578T human breast cancer cells were maintained in DMEM under the same culture conditions.

As previously described (15), TNBC cells were trypsinized and counted using a Countess™ automated cell counter (Invitrogen; Thermo Fisher Scientific, Inc.). Each cell line was seeded into 96-well plates at a density of 1×10⁴ cells/well. After 24 h at 37°C in a cell culture incubator, fresh culture media with the indicated concentrations of celastrol were added, followed by further incubation for 24 h. Viable cells were photometrically quantified at 570 nm using a SpectraMax 190 microplate reader (Molecular Devices, LLC).

Hs578T TNBC cells were plated into 6-well culture plates (1×10³ cells/well). After 24 h at 37°C, the cells were treated with celastrol at the indicated concentrations, followed by an additional 7 days of incubation. The colonies were subsequently fixed with 10% ethanol for 5 min at RT and stained with 0.01% crystal violet for 30 min at RT (20).

As previously described (20), Hs578T TNBC cells (1×10⁶ cells/plate) were seeded into two separate 100-mm dishes, and treated with or without 0.5 µM celastrol in fresh serum-free media for 24 h. The conditioned culture media were collected 24 h later, and 300 µl was immediately used with The Proteome Profiler™ Human Cytokine Array Kit (R&D Systems, which was conducted according to manufacturer's instructions.

Total RNA was extracted from cells using TRIzol^® Reagent (Molecular Research Center, Inc.) according to the manufacturer's protocol. Isolated RNA samples (1 µg total RNA) were reverse-transcribed into cDNA in 20-µl reaction volumes using a first-strand cDNA synthesis kit (Fermentas; Thermo Fisher Scientific, Inc.) per the manufacturer's protocol. IL1B and IL8 gene expression were quantified by qPCR using the SensiMix SYBR Kit (Bioline) with 100 ng cDNA per reaction. The primer sets used for analysis were as follows: Human IL1B forward, 5′-GCCCTAAACAGATGAAGTGCTC-3′ and reverse, 5′-GAACCAGCATCTTCCTCAG-3′; human IL8 forward, 5′-AGGGTTGCCAGATGCAATAC-3′ and reverse, 5′-AAACCAAGGCACAGTGGAAC-3′; human MMP-1 forward, 5′-ATTCTACTGATATCGGGGCTTTGA-3′ and reverse, 5′-ATGTCCTTGGGGTATCCGTGTAG-3′; human MMP-2 forward, 5′-GGCCTCGTATACCGCATCAATC-3′ and reverse, 5′-GGCCTCTCCTGACATTGACCTT-3′; human MMP-9 forward, 5′-CCCGGACCAAGGATACAG-3′ and reverse, 5′-GGCTTTCTCTCGGTACTG-3′; and GAPDH forward, 5′-ATTGTTGCCATCAATGACCC-3′ and reverse, 5′-AGTAGAGGCAGGGATGATGT-3′. An annealing temperature of 60°C was used for all primers, and PCR were performed using a standard 384-well-plate format with an ABI 7900HT real-time PCR detection system. The following thermocycling conditions were used: 95°C for 10 min, 40 cycles of 95°C for 15 sec, 60°C for 15 sec and 72°C for 15 sec. For data analysis, the raw threshold cycle (C_T) value was normalized to the housekeeping gene for each sample to obtain ΔC_T. Normalized ΔC_T was calibrated to control cell samples to calculate ΔΔC_T (13,15).

IL8 and IL1B protein levels were determined using ELISA kits for human IL8 and IL1B (Koma Biotech, Inc.) according to the manufacturer's instructions. A microtiter plate reader was used to measure the absorbance at 450 nm (15).

Conditioned culture media (serum-free) and cell lysates were used for the western blot analysis of MMP-1, p-ERK, t-ERK and β-actin. The cells were lyzed using PRO-PREP™ Protein Extraction Solution (Intron Biotechnology, Inc.) and centrifuged (16,100 × g for 15 min). Total protein concentration was quantified by Bradford assay (Bio-Rad Laboratories, Inc.). The proteins were boiled for 5 min in Laemmli sample buffer and then electrophoresed onto 10% SDS-PAGE gels. The separated proteins (30 mg) were transferred to PVDF membranes, which were then blocked with 10% skim milk in 0.01% TBS-Tween (TBS/T) for 15 min at RT. The blots were incubated with anti-MMP-1, -p-ERK, -t-ERK and -β-actin antibodies in 1% TBS/T, at 4°C overnight. The blots were washed 3–4 times in TBS/T buffer, and then incubated with an anti-rabbit or anti-mouse HRP-conjugated antibody (1:2,000 dilution) in TBS/T. After a 1-h incubation at room temperature (RT), the blots were washed three times, and ECL prime reagents (Amersham; Cytiva) were used for development. The levels of protein expression (p/t-ERK ratio) were quantified using ImageJ version 2 (National Institutes of Health).

Conditioned culture media (serum-free) were mixed with loading buffer and run on a 10% SDS-PAGE gel containing 0.5 mg/ml gelatin without prior denaturation. After electrophoresis, the gels were washed to remove residual SDS, and incubated in renaturing buffer (50 mM Tris, 5 mM CaCl₂, 0.02% NaN₃ and 1% Triton X-100) for 30 min at RT. Next, the gels were incubated for 48 h at 37°C in developing buffer (50 mM Tris, 5 mM CaCl₂, 0.15 M NaCl and 1% Triton X-100), and subsequently stained with Coomassie brilliant blue G-250, destained in 30% methanol, and flooded with 10% acetic acid until bands can clearly visible (19).

Matrigel-precoated filter inserts (pore size, 8-µm); Becton, Dickinson and Company) were inserted into 24-well invasion chambers. Breast cancer cells were resuspended in culture medium (2×10⁵ cells/well) and added to the upper compartment of the invasion chamber in the presence or absence of 20 ng/ml IL-1β, and/or 0.5 µM celastrol or 10 µM GM6001. Fresh culture medium (700 µl) was added to the lower compartment of the chamber. The chambers were incubated at 37°C for 24 h. After incubation, the cells on the upper side of the filter were removed using cotton swabs, and the lower filters were fixed in 4% paraformaldehyde for 10 min and stained in 0.5% Toluidine blue for 1 h at RT (Sigma-Aldrick; Merck KGaA). The cells on the underside of the filter were photographed in four separate fields per condition using a CK40 inverted microscope (Olympus Corporation). The values obtained were calculated by averaging the total number of cells from four filters (15,21).

Data analysis was performed using Excel 2016 (Microsoft Corporation) and GraphPad Prism version 8 (GraphPad Software, Inc.). Statistical significances between two groups of data were calculated using unpaired two-tailed Student's t-test and Tukey's multiple comparison test was used for comparisons among multiple groups. Results are presented as the mean ± S.E.M. All quoted P-values are two-tailed, and P<0.05 was considered to indicate a statistically significant difference (20).

To investigate the effect of celastrol on TNBC cells, Hs578T cells that had been subjected to serum starvation for 24 h were treated with celastrol at the indicated concentrations for a further 24 h, after which cell viability was evaluated. The chemical structure of celastrol is shown in Fig. 1A. As shown in Fig. 1B, cell viability was significantly decreased following treatment with 2 and 4 µM celastrol. Moreover, the colony formation capacity of Hs578T TNBC cells treated with 2 and 4 µM celastrol was markedly reduced, compared with that of the untreated cells (Fig. 1C). Therefore, 0.25 and 0.5 µM celastrol were used for subsequent experimentation. Using the Proteome Profiler Human Cytokine Array Kit, the effect of celastrol on cytokine expression was evaluated using Hs578T TNBC cells. As shown in Fig. 1D, the amount of secreted IL1B protein was considerably decreased by celastrol treatment. Under similar conditions, the levels of IL1B mRNA and protein expression were examined in Hs578T TNBC cells. As predicted, treatment with celastrol significantly decreased the basal levels of IL1B mRNA and protein expression (Fig. 1E and F). Treatment with 0.5 µM celastrol decreased the levels of IL1B mRNA and protein expression 0.49±0.03- (Fig. 1E) and 0.58±0.04-fold (Fig. 1F), respectively, relative to the control. Celastrol also reduced the levels of IL1B mRNA expression (Fig. S1) in HCC1143, MDA231 and HCC1806 TNBC cells. Consequently, these results demonstrate that celastrol attenuated the inflammatory response in TNBC cells by suppressing IL1B expression.

In the present study, IL1B and IL8 mRNA expression were found to be significantly higher in TNBC cells (MDA-157, Hs578T, MDA-231, HCC1143 and HCC1806) compared with non-TNBC cells (MCF7, T47D, ZR75-1, BT474 and SK-BR-3) (Fig. 2A). The effect of IL1B on IL8 expression in Hs578T and HCC1143 TNBC cells was also determined. After serum starvation for 24 h, the two cell lines were treated with or without 20 ng/ml IL1B for 24 h in fresh serum-free media. An increase in IL8 expression was observed subsequent to IL1B treatment in Hs578T and HCC1143 TNBC cells (Fig. 2B and C). By contrast, IL8 treatment had no effect on IL1B expression (Fig. S2A and B). Compared with the control, treatment with 20 ng/ml IL1B led to a 14.9±2.8-fold and 585.6±50.6-fold increase in IL8 mRNA expression in Hs578T and in HCC1143 cells, respectively (Fig. 2B). In addition, the expression of secreted IL8 protein in the conditioned culture medium was assessed. IL1B treatment increased the level of secreted IL8 protein to 71.4±0.4 pg/ml in Hs578T cells and 72.9±1.2 pg/ml in HCC1143 cells, compared with the control (Fig. 2C). Under similar conditions, IL1B significantly increased the levels of MMPs, including MMP-1 and MMP-9, which play a pivotal role in the metastatic and invasion abilities of various cancer cell types (Fig. 2D and E). Moreover, a significant increase in TNBC cell invasiveness was observed in response to IL8 treatment (Fig. S2C). These results indicate that IL1B functions as a key mediator of inflammation and TNBC cell invasiveness through the induction of IL8, MMP-1 and MMP-9.

The mechanism underlying celastrol-regulated IL1B expression in TNBC cells was investigated. The cells that had been previously subjected to serum starvation for 24 h, both in the absence and presence of 5 µM specific inhibitors, were selected for analysis. As shown in Fig. 3A, MEK162, a MEK1/2-specific inhibitor, significantly decreased IL1B mRNA expression in Hs578T cells, though no effect was observed following treatment with Bay11-7082, LY294002 or SP600125. MEK162 decreased IL1B mRNA expression 0.53±0.02-fold (Hs578T cells) and 0.5±0.01-fold (HCC1143 cells), respectively (Fig. 3A). Under similar conditions, the amount of secreted IL1B protein in response to MEK162 treatment was analyzed in Hs578T and HCC1143 cells. As shown in Fig. 3B, basal levels of IL1B protein expression were decreased following treatment with 5 µM MEK162. These findings demonstrate that basal levels of IL1B expression are regulated through a MEK/ERK-dependent pathway in TNBC After serum starvation for 24 h, the cells were treated with celastrol at the indicated concentrations for a further 24 h. As shown in Fig. 3C, ERK phosphorylation was suppressed by celastrol treatment in both Hs578T and HCC1143 TNBC cells. In addition, the effect of celastrol was investigated at shorter time points, indicating that the levels of ERK phosphorylation began to decrease from 2 h in Hs578T cells (Fig. 3D). These results suggest that celastrol downregulated IL1B expression through the suppression of the MEK/ERK signaling pathway in TNBC cells.

Finally, the pharmacological effects of celastrol on IL1B-induced IL8 expression and cellular invasiveness were investigated. Cells that had been subjected to serum starvation for 24 h were pretreated with 0.5 µM celastrol for 3 h, followed by the addition of 20 ng/ml IL1B with incubation for a further 24 h. The effect of celastrol on MMP-1 and −9 expression was determined, as both proteins play an important role in cellular invasive and metastatic potential. The induction of IL1B-associated MMP-1 and −9 mRNA expression was decreased in response to celastrol treatment in Hs578T and HCC1143 TNBC cells (Fig. 4A). As anticipated, IL1B-induced MMP-1 and −9 protein expression was also decreased in the presence of celastrol (Fig. 4B). Furthermore, the effect of celastrol on IL1B-induced IL8 expression was investigated. As shown in Fig. 4C, celastrol decreased IL1B-induced IL8 mRNA expression. Under the control conditions (no celastrol), IL1B increased IL8 mRNA expression 133.5±6.9-fold in Hs578T TNBC cells and 765.4±127.6-fold in HCC1143 TNBC cells. Pretreatment with 0.5 µM celastrol decreased IL1B-induced IL8 expression by 64.8±4.3-fold in Hs578T TNBC cells and 7.6±1.9-fold in HCC1143 TNBC cells (Fig. 4C). In addition, celastrol suppressed IL1B-induced cell invasiveness (Fig. 4D). Furthermore, the effect of GM6001 (a reversible broad-spectrum MMP inhibitor) on IL1B-induced cell invasiveness was investigated, which was found to be suppressed (Fig. 4E). Therefore, it was demonstrated that celastrol suppressed cellular invasiveness by inhibiting IL1B-induced IL8, MMP-1 and MMP-9 expression in TNBC cells.