Celastrol was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) as ≥98% powder. Stock solutions of celastrol were prepared with concentrations of 1, 2 and 4 mM in DMSO, and stored at −20°C. The final concentration of DMSO for all treatments was consistently <0.1%. MTT, U0126, SB203580 and DAPI reagents were obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

The human NPC cell lines HONE-1 and NPC-039 were purchased from American Type Culture Collection (Manassas, VA, USA), and cultured in Gibco RPMI-1640 Medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), 0.1 mM non-essential amino acids, 1 mM glutamine, 1% penicillin/streptomycin, 1.5 g/l sodium bicarbonate, and 1 mM sodium pyruvate (Sigma-Aldrich; Merck KGaA). The cell cultures were maintained at 37°C in a humidified atmosphere of 5% CO₂.

The influence of celastrol on cell growth was assayed by the MTT method. A total of 5×10⁴ HONE-1 and NPC-039 cells were seeded in each well of 24-well plates. Celastrol (0, 1, 2 or 4 µM) was then added to the culture media and incubated at 37°C for 24 h. Following celastrol treatment, MTT was added to each well to a 0.5 mg/ml final concentration, and the cells were incubated for a further 4 h. The viable cell number was directly proportional to the production of formazan following solubilization with isopropanol, as detected by the optical density at 595 nm.

To determine the effect of celastrol on the cell cycle, 1×10⁶ cells were cultured in 1 ml serum-free RPMI-1640 medium for 18 h to induce starvation, and then incubated with 1, 2 or 4 µM celastrol at 37°C for 24 h. Following the harvesting of cells by centrifugation (300 × g at room temperature for 5 min), washing in PBS and fixing with 70% ethanol overnight, they were incubated for 30 min in the dark at room temperature, with propidium iodide (PI) buffer (4 g/ml PI, 1% Triton X-100, 0.5 mg/ml RNase A (Affymetrix, Inc., Santa Clara, CA, USA) in PBS). The cells were filtered with a Falcon 40 µm nylon cell strainer (Corning Incorporated, Corning, NY, USA). The cell cycle distribution of 3,000 cells was analyzed by Muse Cell Analyzer flow cytometry (EMD Millipore, Billerica, MA, USA) and analysis data by Muse Cell Soft V1.4.0.0 Analyzer Assays (EMD Millipore).

To detect apoptosis in cells following celastrol exposure, a Muse Annexin V and Dead Cell Assay kit was used to quantify the number of cells at each stage of cell death (EMD Millipore). Briefly, 1×10⁵ cells were suspended in 100 µl Muse reagent. Following incubation for 20 min at room temperature, the cell suspension was gently mixed and analyzed by the Muse Cell Analyzer flow cytometer (EMD Millipore). The data was processed with Muse Cell Soft V1.4.0.0 Analyzer Assays (EMD Millipore).

Cells (3×10⁵) that had been incubated with 0, 1, 2 or 4 µM celastrol at 37°C for 24 h were fixed with 4% paraformaldehyde for 15 min at room temperature, then at 4°C overnight. The plates were washed twice with PBS and the cell nuclei from the plates were stained with 100 ng/ml DAPI for 15 min in the dark. Following 3 washes with tap water, the cells were examined under a fluorescence microscope. Cells that appeared to exhibit a rough surface and darkly-stained nuclei with fragmented chromosomes were considered to be apoptotic.

Cells were lysed in radioimmunoprecipitation assay buffer (EMD Millipore) containing protease inhibitor cocktail and phosphatase inhibitor cocktail (EMD Millipore). Cells were harvested by centrifugation (12,000 × g, 4°C, 10 min). Protein concentration was determined by the Pierce bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). Cell lysates (total 20 µg) were separated on a 10 or 15% polyacrylamide gel and transferred onto a PVDF membrane (EMD Millipore). The blot was subsequently incubated with 3–5% skimmed milk in PBS at room temperature for 1 h to block non-specific binding, and probed with the following antibodies: Cleaved caspase-3 (cat. no. 9664; dilution, 1:1,000); cleaved caspase-8 (cat. no. 9496; dilution, 1:1,000); cleaved caspase-9 (cat. no. 9505; dilution, 1:1,000); poly (ADP-ribose) polymerase (PARP) (cat. no. 556494; dilution, 1:1,000); cyclin A (cat. no. 4656; 1:1,000); cyclin B (cat. no. 12231; 1:1,000); cyclin-dependent kinase (CDK)1 (cat. no. 9116; dilution, 1:1,000); CDK2 (cat. no. 2546; dilution, 1:1,000); CDK inhibitor 1A (p21^Cip; cat. no, 2947; dilution, 1:1,000); CDK inhibitor 1B (p27^Kip; cat. no. 3686; dilution, 1:1,000); Bcl-2 (cat. no. 2870; dilution, 1:1,000); B-cell lymphoma-extra-large (Bcl-xL; cat. no. 2764; dilution, 1:1,000); Bcl-2 associated X, apoptosis regulator (Bax; cat. no. 5023; dilution, 1:1,000); Bcl-2 antagonist/killer 1 (Bak; cat. no. 12105; dilution, 1:1,000); Bcl-2-like 11 (Bim; cat. no. 2933; dilution, 1:1,000); BH3-interacting death agonist (Bid; cat. no. 2002; dilution, 1:1,000); Fas (cat. no. 4233; dilution, 1:1,000), Fas-associated via death domain (FADD; cat. no. 2782; dilution, 1:1,000); TNFRSF1A-associated via death domain (TRADD; cat. no. 3684; dilution, 1:1,000); TNF receptor superfamily member 1A (TNF-R1; cat. no. 3736; dilution, 1:1,000); TNF receptor superfamily member 10b (DR5; cat. no. 8074; dilution, 1:1,000); protein kinase B (AKT; cat. no. 4298; dilution, 1:1,000); phosphorylated (p-)AKT (cat. no. 4060; dilution, 1:1,000); mitogen-activated protein kinase 14 (p38; cat. no. 9212; dilution, 1:1,000); p-p38 (cat. no. 9211; dilution, 1:1,000); extracellular signal-regulated kinase 1/2 (ERK1/2; cat. no. 4695; dilution, 1:1,000); p-ERK1/2 (cat. no. 4370; dilution, 1:1,000); c-Jun N-terminal kinase 1/2 (JNK1/2; cat. no. 9258; dilution, 1:1,000); p-JNK1/2 (cat. no. 4668; dilution, 1:1,000); and β-actin (cat. no. NB600-501; dilution, 1:5,000). Membranes were incubated with primary antibodies overnight at 4°C, and then with an appropriate peroxidase-conjugated secondary antibody at room temperature for 1 h [anti-mouse Ig; dilution, 1:3,000 (cat. no. 7076); anti-rabbit IgG; dilution, 1:3,000 (cat. no. 7074)]. Following the final wash, the immunoreactive signal was detected with an enhanced chemiluminescence detection system (WBKLS0500; EMD Millipore), and the relative density was quantitated with gel documentation and analysis (AlphaImager 2000; Alpha Innotech Corporation, San Leandro, CA, USA).

The reduction in Δψm was detected with a Muse Mitopotential Assay kit (EMD Millipore). Briefly, 1×10⁵ cells which had been treated with celastrol were washed with PBS. The cells were suspended in Muse MitoPotential working solution and incubated at 37°C for 20 min. Following incubation, 5 µl Muse 7-AAD was added, and incubated at room temperature for 5 min. The reaction volume was thoroughly mixed, run on Muse Cell Analyzer flow cytometry (EMD Millipore), and analyzed by Muse Cell Soft V1.4.0.0 Analyzer Assays (EMD Millipore).

All values included represent the mean ± standard deviation of three repetitions per procedure. Statistical analyses of >3 groups were performed with a one-way analysis of variance test followed by Tukey's post hoc test. Comparisons between two groups were performed with a Student's t-test. Statistical tests were performed using SigmaStat 2.0 software (Systat Software, Inc., San Jose, CA, USA). P<0.05 was considered to represent a statistically significant difference.

To evaluate the effects of celastrol on cell viability, HONE-1 and NPC-039 cells were treated with celastrol for varying concentrations and durations, and viability was assessed with an MTT assay. The result demonstrated that celastrol decreased the viability of HONE-1 and NPC-039 cells, compared with untreated cells, in a dose-(Fig. 2A) and time-(Fig. 2B) dependent manner.

To determine whether the inhibitory effect on cell viability produced by celastrol was associated with the induction of apoptosis, HONE-1 and NPC-039 cells were treated with 0, 1, 2 or 4 µM of celastrol for 24 h and analyzed for cell cycle distribution analysis using flow cytometry. There was an accumulation of sub-G1 phase cells in HONE-1 cells (Fig. 3A). In NPC-039 cells, the treatment induced an increase of cells in the Sub-G1 and G2/M phases compared with the untreated control, in a dose dependent manner (Fig. 3A). The appearance of a sub-G1 population indicated apoptotic cells. These results suggested that the reduction in cell viability associated with celastrol may involve the induction of G2/M phase arrest and apoptosis. To further verify whether celastrol-induced cell death was associated with apoptosis, Annexin V/PI double staining was performed to quantify the level of apoptosis. A dose-dependent increase in apoptotic cells following 24 h of celastrol treatment was demonstrated for HONE-1 and NPC-039 cells (Fig. 3B). In addition, DAPI staining was applied to observe changes in nuclear morphology. In the two cell types, the condensed and fragmented nuclei in the treated cells was smaller compared with the control, which was observed following 24 h of 4 µM celastrol treatment (Fig. 3C).

To clarify whether caspase activation occurred in celastrol-induced apoptosis, apoptosis-associated molecules were examined with western blot analysis. Following treatment with celastrol at doses of 1–4 µM for 24 h, the levels of cleaved fragments of caspases-3, −8, and −9 and PARP increased in HONE-1 cells vs. untreated cells (Fig. 4A). A celastrol treatment of 4 µM for 24 h significantly increased the level of cleaved caspases-3, −8, −9 and PARP, by 52, 72, 30 and 42%, respectively, compared with the control (Fig. 4B). In addition, celastrol treatment significantly decreased the level of cyclin A and B and CDK1 and 2, and increased p21^Cip and p27^Kip levels, compared with the control (Fig. 4C and D).

Bcl-2 family proteins are associated with the regulation of apoptosis, therefore, western blot analysis was performed to determine whether Bcl-2 family members were involved in the process of apoptosis that was previously observed. The expression levels of the pro-apoptotic proteins Bax, Bak, t-Bid and Bim_S were increased, whereas the anti-apoptotic Bcl-2 and Bcl-xL decreased (Fig. 5A). Celastrol treatment at 4 µM for 24 h significantly increased the expression levels of Bax, Bak, t-Bid and Bim_S by 24, 135, 93 and 23% respectively, compared with the control (Fig. 5B). The 4 µM celastrol treatment also significantly decreased the expression levels of Bcl-2 and Bcl-xL by 30 and 35%, respectively, compared with the control (Fig. 5B).

To determine the molecular mechanism by which celastrol induced the apoptosis of HONE-1 cells, the mitochondrial membrane potential and the protein levels of death receptor proteins were assessed. The mitochondrial membrane potential was reduced in celastrol-treated HONE-1 cells (Fig. 6A). In addition, celastrol also increased Fas, FADD, TRADD, TNF-R1 and DR5 expression, as assessed with a western blot analysis (Fig. 6B). A 24 h, 4 µM celastrol treatment significantly increased the expression levels of Fas, FADD, TRADD, TNF-R1 and DR5 by 39, 51, 103, 126 and 88%, respectively, compared with the control (Fig. 6C).

The mitogen-activated protein kinase (MAPK) signaling pathway is involved in the induction of apoptosis by chemotherapeutic drugs (14). Western blot analysis was used to assess whether MAPKs were activated following celastrol treatment of HONE-1 cells. The relative phosphorylation level of JNK1/2 was increased and the phosphorylation of p38 and ERK1/2 was decreased, but AKT phosphorylation was not significantly altered (Fig. 7A). Celastrol treatment significantly increased the extent of JNK1/2 phosphorylation; phosphorylation was increased by 34% compared with the control (Fig. 7B). In addition, celastrol treatment significantly decreased the levels of ERK1/2 and p38 phosphorylation by 52 and 82%, respectively, compared with the control (Fig. 7B). Celastrol combined with an ERK1/2 inhibitor (U0126) or p38 inhibitor (SB203580) increased the rate of apoptosis in HONE-1 cells, compared with celastrol alone (Fig. 7C).