Introduction
Anaplastic thyroid carcinoma (ATC) is a highly aggressive malignancy of the thyroid gland characterized as extrathyroidal invasion, distant metastasis, and resistance to conventional treatment.1 In ATC, multimodal therapeutic options including surgery, chemotherapy, and external beam radiotherapy do not have potential effectiveness, and thus new therapeutic strategies to improve efficacy against cancer cells are under consideration.1
Celastrol is a quinine methide triterpenoid purified from Tripterygium wilfordii Hook.f, also known as Thunder God Vine, used for treatment of inflammatory diseases such as rheumatoid arthritis.2,3 Intriguingly, the nuclear factor-kappaB (NF-κB) inhibitor triptolide, another extract from T. wilfordii Hook.f, shares common properties to celastrol despite different molecular structures.2,3 Celastrol represents a novel class of heat shock protein (hsp) 90 inhibitors, and modulates hsp90 client proteins including Akt.4–6 In cancer cells, celastrol exerts antitumor activities by involving hsp90 client proteins, Bcl2 family proteins, death receptor (DR), and caspase and poly (ADP-ribose) polymerase (PARP).4–10 Celastrol leads to cell death via regulation of NF-κB, Akt, and mitogen-activated protein kinase (MAPK) such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, and induction of endoplasmic reticulum (ER) stress and reactive oxidative species (ROS) in cancer cells.8,11–20 Although celastrol alone or in combination with chemotherapeutic agents has a cytotoxic effect on various cancer cells,4,21,22 the influence of celastrol on ATC cells has not been investigated.
In the American Thyroid Association guideline, it is recommended that the regimen of chemotherapeutic agents such as paclitaxel in combination with external radiation is considered in ATC patients.23 Furthermore, there is accumulating evidence that paclitaxel is a more effective cytotoxic agent than those traditionally used in ATC patients.24 With regard to combination of chemotherapeutic agents with paclitaxel, it was shown that celastrol augmented paclitaxel-induced cytotoxicity in HeLa cells.25 In contrast, we recently reported that the hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) antagonized with paclitaxel in induction of cytotoxicity in ATC cells.26 However, the impact of celastrol in combination with paclitaxel on survival of ATC cells has not been identified.
The aim of this study was to evaluate the effect of celastrol alone or in combination with paclitaxel on survival of ATC cells. Our results demonstrate that celastrol results in cell death through involvement of Bcl2 family proteins and DR, and modulation of NF-κB, Akt, and MAPK accompanied by ER stress and ROS production in ATC cells. Moreover, celastrol synergizes with paclitaxel in induction of death of ATC cells.
Materials and methods
Materials
Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI1640, fetal bovine serum (FBS), l-glutamine, and streptomycin/penicillin were purchased from Life Technologies (Carlsbad, CA, USA). Celastrol, paclitaxel, and 17-AAG were obtained from Sigma (St Louis, MO, USA). These were dissolved in dimethylsulfoxide (DMSO), which was provided to the control within permissible concentrations. The final concentration of the vehicle DMSO in the control did not exceed 0.1% in all treatments. The primary antibodies raised against hsp90, hsp70, ErbB2, Raf-1, Bcl-xL, Bcl2, Bax, Bid, DR5, cleaved caspase-3, cleaved PARP, total and phospho-NF-κB (Ser536), total and phospho-ERK1/2 (Thr402/Tyr404), total and phospho-JNK (Thr183/Tyr185), total and phospho-p38 (Thr180/Tyr182), Bip, CCAAT/enhancer-binding protein-homologous protein (CHOP), and cyclooxygenase 2 (COX2) were purchased from Cell Signaling Biotechnology (Danvers, MA, USA). The primary antibodies raised against total and phospho-Akt (Ser473), and β-actin from Sigma were obtained. All other reagents were purchased from Sigma unless otherwise stated.
Cell culture
For experiments, 8505C and SW1736 human ATC cells were used. The 8505C cells were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ GmbH, Braunschweig, Germany), and grown in DMEM supplemented with 10% heat-inactivated FBS and 1% streptomycin/penicillin. SW1736 cells were obtained from Cell Lines Service (CLS GmbH, Eppelheim, Germany) and grown in RPMI1640 supplemented with 2 mM l-glutamine, 10% heat-inactivated FBS, and 1% streptomycin/penicillin. Cells received fresh medium at regular intervals. Treatments and experiments were performed using cells that were 70% confluent.
CCK-8 assay
Cell viability was determined by the CCK-8 Assay Kit (Dojindo laboratories, Kumamoto, Japan). Cells (5 × 103/100 µL) in each well on 96-well plates were incubated overnight and treated with celastrol and paclitaxel for an additional 4 h at 37°C. Absorbance was measured using Glomax™ Discover System GM3000 (Promega, Madison, WI, USA). All experiments were performed in triplicate.
Cytotoxicity assay
Cytotoxic activity was measured by the LDH Cytotoxicity Assay Kit (BioVision, Linda, CA, USA). Cells (5 × 103/100 µL) in each well on 96-well plates were incubated and centrifuged at 250g for 10 min. Supernatant of 100 µL was transferred in clear 96-well plates. After addition of reaction mixture (2.5 µL catalyst solution in 112.5 µL dye solution), cells were incubated for 30 min at room temperature. Absorbance was measured using GloMaxTM Discover System GM3000 (Promega). All experiments were performed in triplicate.
Multiplexed cytotoxicity assay
Cells (5 × 103/100 µL) were seeded in 96-well plates, and reagents of the MultiTox-Glo Multiplex Cytotoxicity Assay Kit (Promega) were added to cells after treatments as indicated by manufacturer’s protocol.27 Fluorescent and luminescent values were measured using GloMaxTM Discover System GM3000 (Promega). Viability was calculated as a ratio of live/dead cells and expressed as percentage of untreated cells. All experiments were performed in triplicate.
Measurement of ROS production
ROS production was measured by the ROS-Glo H2O2 Assay Kit (Promega). Cells (1 × 104/mL) in each well on 96-well plates were incubated and treated with H2O2 substrate solution (25 µM/well), and reincubated at 37°C. After addition of ROS-Glo Detection solution (100 µL/well), cells were incubated for 20 min at room temperature. Absorbance was measured using GloMaxTM Discover System GM3000 (Promega). All experiments were performed in triplicate.
Western blotting
The total protein was extracted by radioimmunoprecipitation assay (RIPA) buffer (Sigma) containing 1 × protease inhibitor cocktail and 1 × phophatase inhibitor cocktail set V (Calbiochem, La Jolla, CA, USA). Western blotting was performed using specific primary antibodies and horseradish peroxidase–conjugated anti-rabbit and anti-mouse secondary antibodies. Bands were detected using ECL Plus Western Blotting Detection System (Thermo Fisher Scientific, Rockford, IL, USA). The protein levels were quantified by densitometry using ImageJ software (NIH) and normalized to β-actin levels. The relative levels of protein to β-actin were calculated. All experiments were performed in triplicate.
Reverse transcription–polymerase chain reaction (RT-PCR)
Total RNA was isolated using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA) according to manufacturer’s protocol. Complementary DNA (cDNA) was synthesized using AmpiRevert cDNA Synthesis Platinum Master Mix (GenDEPOT, Barker, TX, USA). Amplification of the resulting cDNA was conducted. The following primers were used: Bip, 5′-ATGAGGACCTGCAAGAG-3’ and 5′-TCCTCCTCAGTCAGCC-3′; CHOP, 5′-GCACCTCCCAGAGCCCTCACTCTCC-3′ and 5′-GTCTACTCCAAGCCTTCCCCCTGCG-3′; β-actin, 5′-CAAGAGTGGCCACGGCTGCT-3′ and 5′-TCCTTCGCATCCTGTCGGCA-3′. Reactions were successively incubated at 95°C for 15 s, at 60°C for 15 s, and at 72°C for 15 s using a GeneAmp PCR System 9700 thermal cycler. The number of cycle (optimized in a preliminary study which was to determine exponential range of amplification for each gene) was 22 for Bip, CHOP, and β-actin. The amplified products were analyzed by 2% agarose gel electrophoresis. The messenger RNA (mRNA) expression of Bip and CHOP was compared with the mRNA expression of β-actin. All experiments were performed in triplicate.
Drug combination analysis
Combination Index (CI) and isobologram were calculated by CalcuSyn program version 2.11 (Biosoft, Great Shelford, Cambridge, UK), and the effect of drug interactions was quantitatively assessed. CI values less than 1.0, 1.0, and greater than 1.0 demonstrate synergism, additivity, and antagonism, respectively. The isobologram is formed by plotting the doses of each drug required for 50% inhibition (ED50) on the x- and y-axis, and connecting them to draw a line segment, which is ED50 isobologram. Combination data points that below, fall on, and above the line segment reveal synergism, additivity, and antagonism, respectively. All experiments were performed in triplicate.
Statistical analysis
All data are expressed as mean ± standard error (SE). Data were analyzed by unpaired Student’s t-test or analysis of variance (ANOVA) as appropriate. A p value <0.05 was considered to be statistically significant. All analyses were performed using SPSS program version 23.0 (SPSS, Chicago, IL, USA).
Results
Celastrol induces death of ATC cells
In this study, the effect of celastrol on cell survival was evaluated in 8505C and SW1736 ATC cells. First, to compare the influence between two hsp90 inhibitors, celastrol and 17-AAG, on cell survival, cells were treated with celastrol and 17-AAG at 0.25, 0.5, 0.75, 1, 1.5, and 2 µM for 48 h, respectively. Cell viability was measured using CCK-8 assay, and 50% inhibitory concentration (IC50) values were calculated by nonlinear regression using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA; Figure S1(a)). As a result of treatment, the IC50 values of celastrol were 0.94 µM in 8505C cells and 1.08 µM in SW1736 cells, whereas those of 17-AAG were 4.48 µM in 8505C cells and 4.41 µM in SW1736 cells. When cells were treated with celastrol at 1 µM and 17-AAG at 10 µM for 48 h, cleaved caspase-3 protein was detected after treatment of celastrol, but not 17-AAG (Figure S1(b)).
Next, to clarify the impact of celastrol on cell survival, cells were treated with celastrol at 0.25, 0.5, 0.75, 1, 1.5, and 2 µM for 24 and 48 h, and cell viability was measured (Figure 1(a)). As a result of treatment, cell viability was reduced in a dose- and time-dependent manner. When cells were treated with celastrol at same doses for 48 h, cytotoxic activity measured using cytotoxicity assay was elevated in a dose-dependent manner (Figure 1(b)).
Figure 1.
The effect of celastrol on survival of ATC cells. (a) 8505C and SW1736 cells were treated with celastrol at 0.25, 0.5, 0.75, 1, 1.5, and 2 µM for 24 and 48 h, and cell viability was measured using CCK-8 assay. (b) 8505C and SW1736 cells were treated with celastrol at 0.25, 0.5, 0.75, 1, 1.5, and 2 µM for 48 h, and cytotoxic activity was measured using cytotoxicity assay. All experiments were performed in triplicate. Data are expressed as mean ± SE.
[Figure omitted. See PDF]
Hsp90 client proteins, Bcl2 family proteins, and DR are involved in celastrol-induced death of ATC cells. To identify the involvement of hsp90 client proteins, Bcl2 family proteins, and DR in celastrol-induced cell death, cells were treated with celastrol at 0.1, 0.5, 1, and 2 µM for 48 h, and at 1 µM for 6, 12, 24, and 48 h, and then the protein levels of hsp90, hsp70, ErbB2, Raf-1, Bcl-xL, Bcl2, Bax, Bid, DR5, cleaved caspase-3, and cleaved PARP were measured (Figure 2(a) and (b)). After treatment, the protein levels of hsp90, hsp70, Bax, DR5, cleaved caspase-3, and cleaved PARP increased, while those of ErbB2, Raf-1, and Bcl2 decreased. The protein levels of Bcl-xL and Bid were unchanged.
Figure 2.
The involvement of hsp90 client proteins, Bcl2 family proteins, and DR in celastrol-induced death of ATC cells. (a) 8505C and (b) SW1736 cells were treated with celastrol at 0.1, 0.5, 1, and 2 µM for 48 h, and at 1 µM for 6, 12, 24, and 48 h, and then the protein levels of hsp90, hsp70, ErbB2, Raf-1, Bcl-xL, Bcl2, Bax, Bid, DR5, cleaved caspase-3, and cleaved PARP were measured. All experiments were performed in triplicate. The blots are representative of independent experiments.
[Figure omitted. See PDF]
Celastrol induces cell death through modulation of NF-κB, Akt, and MAPK in ATC cells
To examine the influence of celastrol on signal proteins, cells were treated with celastrol at 0.1, 0.5, 1, and 2 µM for 48 h, and at 1 µM for 6, 12, 24, and 48 h, and then the total and phospho-protein levels of NF-κB, Akt, ERK1/2, JNK, and p38 were measured (Figure 3(a) and (b)). After treatment, the protein levels of phospho-ERK1/2 and phospho-JNK were enhanced, whereas those of phospho-NF-κB, and total and phospho-Akt were diminished without alteration in those of total NF-κB, total ERK1/2, total JNK, and total and phospho-p38. When cells were treated with celastrol at 1 µM for 1, 3, and 6 h shortly, the protein levels of phospho-NF-κB and phospho-Akt were diminished without change in those of total NF-κB and total Akt (Figure S2). Overall, in cells treated with celastrol at 1 µM from 1 h to 48 h, the protein levels of NF-κB and Akt were sequentially diminished in order of phospho-NF-κB, phospho-Akt, and total Akt without alteration in total NF-κB.
Figure 3.
The influence of celastrol on signal proteins in ATC cells. (a) 8505C and (b) SW1736 cells were treated with celastrol at 0.1, 0.5, 1, and 2 µM for 48 h, and at 1 µM for 6, 12, 24, and 48 h, and then the total and phospho-protein levels of NF-κB, Akt, ERK1/2, JNK, and p38 were measured. All experiments were performed in triplicate. The blots are representative of independent experiments.
[Figure omitted. See PDF]
To establish the role of NF-κB in cell survival, cells were transfected with NF-κB p65 siRNA, and cell viability and cytotoxic activity were measured (Figure S3). In NF-κB p65 siRNA-transfected cells, compared with control, cell viability was diminished, while cytotoxic activity was enhanced.
Celastrol-induced cell death is related to induction of ER stress in ATC cells
To explore the impact of celastrol on ER stress markers, cells were treated with celastrol at 0.1, 0.5, 1, and 2 µM for 48 h, and at 1 µM for 6, 12, 24, and 48 h, and then the mRNA and protein levels of Bip and CHOP were measured (Figure 4(a)–(d)). After treatment, the mRNA and protein levels of Bip and CHOP were elevated.
Figure 4.
The impact of celastrol on ER stress markers in ATC cells. (a and b) 8505C and (c and d) SW1736 cells were treated with celastrol at 0.1, 0.5, 1, and 2 µM for 48 h, and at 1 µM for 6, 12, 24, and 48 h, and then the mRNA and protein levels of Bip and CHOP were measured, and quantified by densitometry, and normalized to β-actin levels. All experiments were performed in triplicate. The blots are representative of independent experiments. Data are expressed as mean ± SE. *p < 0.05 versus each matched control. WB, Western blotting.
[Figure omitted. See PDF]
Celastrol-induced cell death is relevant to ROS production in ATC cells
In this study, whether celastrol-induced cell death is associated with ROS production was assessed. First, cells were treated with celastrol at 0.5, 1, 1.5, and 2 µM for 48 h, and ROS production was measured (Figure 5(a)). After treatment, ROS production increased in a dose-dependent manner.
Figure 5.
The association of ROS production with celastrol-induced death of ATC cells. (a) 8505C and SW1736 cells were treated with celastrol at 0.5, 1, 1.5, and 2 µM for 48 h, and ROS production was measured. (b–e) 8505C and SW1736 cells were pretreated with NAC at 20 mM for 1 h before treatment of celastrol at 1 µM for 48 h, after which (b) ROS production, (c) cell viability, (d) cytotoxic activity, and (e) the protein levels of COX2, cleaved PARP, hsp90, hsp70, total and phospho-NF-κB, total and phospho-ERK1/2, total and phospho-JNK, and Bip were measured. All experiments were performed in triplicate. The blots are representative of independent experiments. Data are expressed as mean ± SE. *p < 0.05 versus control. †p < 0.05 versus cells treated with celastrol alone.
[Figure omitted. See PDF]
Next, cells were pretreated with N-acetylcysteine (NAC) at 20 mM for 1 h before treatment of celastrol at 1 µM for 48 h, after which ROS production (Figure 5(b)), cell viability (Figure 5(c)), cytotoxic activity (Figure 5(d)), and the protein levels of COX2, cleaved PARP, hsp90, hsp70, total and phospho-NF-κB, total and phospho-ERK1/2, total and phospho-JNK, and Bip (Figures 5(e) and S4) were measured. In celastrol-treated cells, NAC increased cell viability, and decreased cytotoxic activity with concomitant decrement of ROS production. Furthermore, NAC increased phospho-NF-κB protein levels, whereas it decreased the protein levels of COX2, cleaved PARP, hsp90, hsp70, phospho-ERK1/2, phospho-JNK, and Bip without change in those of total NF-κB, total ERK1/2, and total JNK in celastrol-treated cells.
Celastrol synergizes with paclitaxel in induction of death of ATC cells
We recently reported that 17-AAG antagonized with paclitaxel in induction of cytotoxicity in ATC cells,26 and thus the effect of celastrol in combination with paclitaxel on survival of ATC cells was evaluated.
To investigate the influence of combination with two agents, cells were simultaneously treated with both celastrol and paclitaxel, and the interactions were estimated by calculating CI using Chou–Talalay equation, where CI < 1.0 indicates synergism, and CI = 1.0 indicates additivity, and CI > 1.0 indicates antagonism (Figure 6(a) and (b), Table 1). After cotreatment, cell viability was measured using CCK-8 assay, and death rate was calculated as 100-cell viability (%). All of the CI values were lower than 1.0 in combination of celastrol with paclitaxel. In the isobologram analysis, the combination data points were all located below the isobologram line at ED50, suggesting the synergism between celastrol and paclitaxel inducing cytotoxicity in ATC cells.
Figure 6.
The effect of celastrol in combination with paclitaxel on survival of ATC cells. (a) 8505C and (b) SW1736 cells were simultaneously treated with both celastrol at 0.25, 0.5, 0.75, and 1 µM and paclitaxel at 2, 4, 6, and 8 nM for 48 h. Cell viability was measured using CCK-8 assay, and death rate was calculated as 100-cell viability (%). Combination Index (CI) and isobologram were calculated. The horizontal dash lines at CI = 1.0 are drawn. (c–g) 8505C and SW1736 cells were simultaneously treated with both celastrol at 1 µM and paclitaxel at 8 nM for 48 h. (c) The percentage of viable cells was measured using multiplexed cytotoxicity assay, and (d) cytotoxic activity was measured. (e) The protein levels of cleaved PARP, total and phospho-NF-κB, total and phospho-ERK1/2, total and phospho-JNK, Bip, and COX2 were measured, quantified by densitometry, and normalized to β-actin levels. (f) The relative levels of protein to β-actin were calculated. (g) ROS production was measured. All experiments were performed in triplicate. The blots are representative of independent experiments. Data are expressed as mean ± SE. *p < 0.05 versus control. †p < 0.05 versus cells treated with paclitaxel alone. Cel: celastrol; Pac: paclitaxel; C. PARP: cleaved PARP.
[Figure omitted. See PDF]
Table 1.Combination Index (CI) values at combined doses determined by the median effect analysis method in ATC cells simultaneously treated with both celastrol and paclitaxel.
| Celastrol (µM) | Paclitaxel (nM) | Celastrol + Paclitaxel |
|
|---|---|---|---|
| 8505C cells | SW1736 cells | ||
| 0.25 | 2 | 0.848 | 0.792 |
| 0.5 | 4 | 0.958 | 0.867 |
| 0.75 | 6 | 0.897 | 0.880 |
| 1 | 8 | 0.928 | 0.838 |
ATC: anaplastic thyroid carcinoma.
CI values less than 1.0, 1.0, and greater than 1.0 indicate synergism, additivity, and antagonism, respectively.
To confirm synergistic activity of celastrol with paclitaxel in induction of cell death, cells were simultaneously treated with both celastrol at 1 µM and paclitaxel at 8 nM for 48 h, and the percentage of viable cells was measured using multiplexed cytotoxicity assay (Figure 6(c)), and cytotoxic activity was measured (Figure 6(d)). The percentage of viable cells was diminished, while cytotoxic activity was enhanced as a result of cotreatment of celastrol and paclitaxel, compared with treatment of paclitaxel alone.
When cells were simultaneously treated with both celastrol at 1 µM and paclitaxel at 8 nM for 48 h, compared with paclitaxel alone, the protein levels of cleaved PARP, phospho-ERK1/2, phospho-JNK, Bip, and COX2 were enhanced, whereas phospho-NF-κB protein levels were diminished without alteration in those of total NF-κB, total ERK1/2, and total JNK (Figures 6(e) and (f) and Figure S4). Moreover, ROS production was enhanced in cells treated with both celastrol and paclitaxel, compared with paclitaxel alone (Figure 6(g)).
Discussion
This study demonstrates for the first time that celastrol leads to cell death by involving Bcl2 family proteins and DR, and regulating NF-κB, Akt, and MAPK in conjunction with ER stress and ROS production in ATC cells. Moreover, our results indicate that celastrol has a synergistic activity with paclitaxel in induction of death of ATC cells.
Celastrol exerts antitumor activities in various cancer cells.4,21,22 However, in regard to the influence of celastrol on thyroid cancer cells, there is only one study reporting the result that celastrol suppresses TGFβ1-induced epithelial–mesenchymal transition in thyroid cancer cells derived from BRAFV600E mice.28 Meanwhile, celastrol results in cell death through modulation of hsp90 client proteins including Akt, upregulation of anti-survival proteins including Bax, downregulation of pro-survival proteins including Bcl2, overexpression of DR, and activation of caspase and PARP in cancer cells.4–10 In this study, celastrol, compared with 17-AAG, caused cell death with detection of cleaved caspase-3 protein and low IC50 values, suggesting relatively potent cytotoxicity. In addition, cell death was accompanied by alterations in hsp90 client proteins; elevation of Bax, DR5, cleaved caspase-3, and cleaved PARP; and reduction of Bcl2 in celastrol-treated cells. Taken together, these results imply that celastrol has a cytotoxic effect on ATC cells with concomitant modulation of hsp90 client proteins, Bcl2 family proteins, and DR.
NF-κB is activated by phosphorylation, and translocated to the nucleus.29 After posttranscriptional modification in the nucleus, NF-κB controls target genes involved in multiple cellular processes.29 In this regard, celastrol leads to cell death via repression of NF-κB in in vitro and in vivo models of cancers.11–13 With regard to the impact of celastrol on Akt, celastrol attenuates vascular endothelial growth factor-triggered activation of Akt/mTOR/p70S6K signaling and has cytostatic and cytotoxic effects in prostate cancer cells.14 In this study, celastrol decreased expression of phospho-NF-κB, phospho-Akt, and total Akt in sequential order without change in total NF-κB. Furthermore, inhibition of NF-κB by siRNA also resulted in cell death. All taken together, these results connote that celastrol induces cytotoxicity by chronologically regulating NF-κB and Akt in ATC cells. However, further study for alterations in nuclear NF-κB of ATC cells will be needed.
MAPK is activated by a diversity of environmental stimuli including oxidative and genotoxic stresses.30,31 Upon stimulation, activation of MAPK signifies burden of stress itself or resistance to stress.30,31 As a result, MAPK signaling either promotes cell proliferation or induces cell death depending on molecular kinetics and downstream signaling.30,31 In view of the influence of celastrol on MAPK, celastrol modulates ERK without change in p38 in human hepatoma cells.15 Moreover, celastrol activates JNK by suppressing transcriptional activity of ATF2 in melanoma cells and causes cell death through ROS/JNK signaling in cancer cells.16–18 In this study, celastrol stimulated phosphorylation of ERK1/2 and JNK without alteration in p38. In celastrol-treated cells, supplementation of NAC abrogated cell death as well as expression of phospho-ERK1/2 and phospho-JNK. In addition, combination of celastrol with paclitaxel, compared with paclitaxel alone, augmented cell death with overexpression of phospho-ERK1/2 and phospho-JNK. Taken collectively, these results denote that celastrol-induced cytotoxicity is associated with MAPK in ATC cells. Considering that MAPK kinase (MKK) 4 and MKK7 activate JNK, whereas MKK3 and MKK6 activate p38,30,31 it is a possible explanation that celastrol may have a different effect on MKKs. However, dynamics of MAPK under exposure of celastrol in ATC cells should be further scrutinized.
Celastrol leads to cell death in conjunction with induction of ER stress in cancer cells.8 In this regard, induction of DR requires induction of CHOP, and silencing of CHOP diminishes TNF-related apoptosis-inducing ligand (TRAIL)-induced death of cancer cells exposed to celastrol.8 Furthermore, celastrol represses proteosomal activity, and thereby results in induction of ER stress and overexpression of Bax in cancer cells.32,33 In this study, celastrol enhanced the mRNA and protein expression of Bip and CHOP, suggesting that celastrol-induced cytotoxicity is relevant to induction of ER stress in ATC cells. In addition, time lag between induction of mRNA and protein of CHOP in celastrol-treated ATC cells may be attributable to impediment of mRNA translation and/or increment of protein degradation.
In thyroid cancer, crosstalk between NF-κB and hypoxia-inducible factor-1α (HIF-1α) systems plays a role in tumor progression.34 In both benign and malignant cells, hsp90 inhibitors cause RACK1-dependent, VHL-independent ubiquitination and proteasomal degradation of HIF-1α.35 Meanwhile, celastrol inhibits HIF-1α and accumulates ROS followed by suppression of hsp90, activation of JNK, and cell death in cancer cells.15,17,19,20 In this study, celastrol elevated ROS production, and pretreatment of the ROS scavenger NAC mitigated decrement of cell viability and increment of cytotoxic activity in celastrol-treated cells. Moreover, pretreatment of NAC elevated phospho-NF-κB, and reduced COX2, cleaved PARP, hsp90, hsp70, phospho-ERK1/2, phospho-JNK, and Bip in celastrol-treated cells. Taken together, these results imply that celastrol-induced cytotoxicity is related to ROS production in ATC cells. In regard to the impact of celastrol on NF-κB-HIF-1α network, it is presumed that celastrol inactivates NF-κB-HIF-1α network via repression of hsp90 and release of ROS in ATC cells.
The various cytotoxic agents sensitize ATC cells to paclitaxel.24 With regard to combination of celastrol with paclitaxel, it was shown that celastrol potentiated death of paclitaxel-resisted HeLa cells.25 By contrast, we recently reported that 17-AAG antagonized with paclitaxel in induction of cytotoxicity in ATC cells,26 and thus the effect of celastrol in combination with paclitaxel on survival of ATC cells was investigated. In this study, when celastrol was combined with paclitaxel, all of the CI values in drug combination analysis were lower than 1.0, connoting that celastrol synergizes with paclitaxel in induction of cytotoxicity in ATC cells. In addition, celastrol in combination with paclitaxel, compared with paclitaxel alone, augmented cytotoxicity, based on the data of the percentage of viable cells (multiplexed cytotoxicity assay) and cytotoxic activity (cytotoxicity assay), providing additional evidences for the synergism. Furthermore, cotreatment of celastrol and paclitaxel, compared with treatment of paclitaxel alone, increased cleaved PARP, phospho-ERK1/2, phospho-JNK, Bip, COX2, and ROS production, and decreased phospho-NF-κB without change in total NF-κB. In regard to the influence of paclitaxel on NF-κB in ATC cells, it was shown that paclitaxel-induced activation of NF-κB led to overexpression of anti-apoptotic proteins in FRO ATC cells.36 However, in contrast with our study, paclitaxel weakly increased total NF-κB, but not phospho-NF-κB, protein levels in one ATC cell line. Moreover, the data of NF-κB siRNA transfection in our study strongly support the positive role of NF-κB in survival of ATC cells. All taken together, these results denote that celastrol has a synergistic activity with paclitaxel in induction of cytotoxicity in ATC cells, and combination of celastrol with paclitaxel may be an excellent therapeutic regimen in human ATC.
In conclusion, our results suggest that celastrol induces cytotoxicity through involvement of Bcl2 family proteins and DR, and regulation of NF-κB, Akt, and MAPK in association with ER stress and ROS production in ATC cells. In addition, celastrol synergizes with paclitaxel in induction of cytotoxicity in ATC cells. This study will provide the clinical implications of celastrol alone or in combination with paclitaxel as an attractive therapeutic option in ATC patients refractory to conventional chemotherapeutic agents.
Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
FundingThe author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2015R1A2A2A01003589) to S.J. Lee, Republic of Korea.
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Abstract
The influence of celastrol alone or in combination with paclitaxel on survival of anaplastic thyroid carcinoma cells was investigated. In 8505C and SW1736 cells, after treatment of celastrol, cell viability decreased, and cytotoxic activity increased. The protein levels of heat shock protein (hsp) 90, hsp70, Bax, death receptor 5, cleaved caspase-3, cleaved poly (ADP-ribose) polymerase, phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), and phospho-c-Jun N-terminal kinase (JNK) were elevated, and those of Bcl2, phospho-nuclear factor-kappaB (NF-κB), and total and phospho-Akt were reduced. The endoplasmic reticulum stress markers expression and reactive oxygen species production were enhanced. In celastrol-treated cells, N-acetylcysteine increased cell viability and phospho-NF-κB protein levels, and decreased reactive oxygen species production and cytotoxic activity. The protein levels of cyclooxygenase 2, phospho-ERK1/2, phospho-JNK and Bip were diminished. After treatment of both celastrol and paclitaxel, compared with paclitaxel alone, cell viability and the percentage of viable cells were reduced, and death rate and cytotoxic activity were elevated. The protein levels of phospho-ERK1/2, phospho-JNK, Bip, and cyclooxygenase 2, and reactive oxygen species production were enhanced. All of the Combination Index values calculated by Chou–Talalay equation were lower than 1.0, implying the synergism between celastrol and paclitaxel in induction of cell death. In conclusion, our results suggest that celastrol induces cytotoxicity through involvement of Bcl2 family proteins and death receptor, and modulation of phospho-NF-κB, Akt, and mitogen-activated protein kinase in association with endoplasmic reticulum stress and reactive oxygen species production in anaplastic thyroid carcinoma cells. Moreover, celastrol synergizes with paclitaxel in induction of cytotoxicity in anaplastic thyroid carcinoma cells.
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1 Division of Endocrinology and Metabolism, Department of Internal Medicine, College of Medicine, Hallym University, Chuncheon, Republic of Korea