The natural sesquiterpene lactones 
arglabin, grosheimin, agracin, 
parthenolide, and estafiatin inhibit T cell 
receptor (TCR) activation
Authors: Igor A. Schepetkin, Liliya N. Kirpotina, Pete T. Mitchell, 
Anarkul S. Kishkentaeva, Zhanar R. Shaimerdenova, Gayane A. 
Atazhanova, Sergazy M. Adekenov, and Mark T. Quinn
NOTICE: this is the author’s version of a work that was accepted for publication in 
Phytochemistry. Changes resulting from the publishing process, such as peer review, editing, 
corrections, structural formatting, and other quality control mechanisms may not be reflected in 
this document. Changes may have been made to this work since it was submitted for publication. 
A definitive version was subsequently published in Phytochemistry, VOL# 146, (February 2018), 
DOI# 10.1016/j.phytochem.2017.11.010.
Schepetkin, Igor A., Liliya N. Kirpotina, Pete T. Mitchell, Anarkul S. Kishkentaeva, Zhanar R. 
Shaimerdenova, Gayane A. Atazhanova, Sergazy M. Adekenov, and Mark T. Quinn. "The natural 
sesquiterpene lactones arglabin, grosheimin, agracin, parthenolide, and estafiatin inhibit T cell 
receptor (TCR) activation." Phytochemistry 146 (February 2018): 36-46. DOI:10.1016/
j.phytochem.2017.11.010.
Made available through Montana State University’s ScholarWorks 
scholarworks.montana.edu 
The natural sesquiterpene lactones arglabin, grosheimin, agracin,
parthenolide, and estafiatin inhibit T cell receptor (TCR) activation
Igor A. Schepetkin a, Liliya N. Kirpotina a, Pete T. Mitchell a, Аnarkul S. Kishkentaeva b,
Zhanar R. Shaimerdenova b, Gayane A. Atazhanova b, Sergazy M. Adekenov b,
Mark T. Quinn a, *
a Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, United States
b International Research and Production Holding “Phytochemistry”, Karaganda 100009, Kazakhstanc t
he T cel
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. Five o
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ith the
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ist WK
lactonea b s t r a
Inhibition of t
mediated infl
inflammatory
argolide, argra
sine, partheno
Jurkat T cells
inhibited anti
potent being p
extracellular
compounds, w
tively). These
intracellular g
lation in HL60
peptide agon
sesquiterpene
lation sites, as well a
site directed ligand
estafiatin also inhibit
Jurkat cells, but it d
grosheimin, agracin,
and may be natural c
Abbreviations used: DMSO, dimethyl sulfoxide; FB
formyl peptide receptor; HBSS, Hanks' balanced
triphosphate; ITAM, immune-receptor tyrosine-based
receptor; ERK, extracellular signal-regulated kinas
protein kinases; SERCA, sarco/endoplasmic reticulu
thione ethylene ester; GSH, glutathione; ELISA, enz
assay; PLC, phospholipase C.l receptor (TCR) pathway represents an effective strategy for the treatment of T cell-
ry and autoimmune diseases. To identify natural compounds that could inhibit
responses, we screened 13 sesquiterpene lactones, including achillin, arglabin,
-hydroxyarhalin, artesin, artemisinin, estafiatin, grosheimin, grossmisin, leucomi-
nd taurine, for their ability to modulate activation-induced Ca2þ mobilization in
f the compounds (arglabin, grosheimin, argracin, parthenolide, and estafiatin)
duced mobilization of intercellular Ca2þ ([Ca2⁺]i) in Jurkat cells, with the most
olide and argacin (IC50 ¼ 5.6 and 6.1 mM, respectively). Likewise, phosphorylation of
egulated kinase (ERK) 1/2 in activated Jurkat cells was inhibited by these five
most potent being parthenolide and estafiatin (IC50 ¼ 13.8 and 15.4 mM, respec-
nds also inhibited ERK1/2 phosphorylation in primary human T cells and depleted
ne. In contrast, none of the sesquiterpene lactones inhibited ERK1/2 phosphory-
ransfected with N-formyl peptide receptor 2 (FPR2) and stimulated with the FPR2
YMVM, indicating specificity for T cell activation. Estafiatin, a representative
, was also profiled in a cell-based phosphokinase array for 43 kinase phosphory-
s in a cell-free competition binding assay for its ability to compete with an active-
for 95 different protein kinases. Besides inhibition of ERK1/2 phosphorylation,
ed phosphorylation of p53, AMPKa1, CREB, and p27 elicited by TCR activation in
id not bind to any of 95 kinases evaluated. These results suggest that arglabin,
parthenolide, and estafiatin can selectively inhibit initial phases of TCR activation
ompounds with previously undescribed immunotherapeutic properties.1. Introduction
Sesquiterpene lactones are natural products that are abundantS, fetal bovine serum; FPR, N-
salt solution; IP3, inositol
activation motif; TCR, T cell
e; MAPK, mitogen-activated
m Ca2þ ATPase; GEE, gluta-
yme-linked immunosorbentin plants of the Asteraceae family and possess a broad spectrum of
biological activities, including anticancer, antibacterial, antifungal,
anti-inflammatory, and immunomodulatory activities [reviewed in
(Chadwick et al., 2013; Hou and Huang, 2016; Ren et al., 2016)].
Although the anticancer and anti-proliferative activities of various
sesquiterpene lactones and their synthetic derivatives are well
known, and some sesquiterpene lactones are currently under
clinical evaluation [reviewed in Ren et al. (2016)], the molecular
mechanisms involved in their anti-inflammatory and T cell
immunomodulatory activities have not been clearly elucidated. For
example, parthenolide has been found to inhibit p65 NF-kB binding
to DNA and IkB-kinase activity in Jurkat T cells (Garcia-Pineres et al.,
2001, 2004) and cytokine production in anti-CD3/CD28-stimulated
peripheral blood T cells from allergic and normal donors (Li-Weber
et al., 2002). However, it is still not clear if sesquiterpene lactones
can alter other pathways during T cell activation.
T cells are essential for inflammation and adaptive immune re-
sponses, and deregulation of T cell function can contribute to
autoimmune diseases [e.g., see (Sauer et al., 2015)]. The TCR rec-
ognizes and responds to antigenic peptides in the context of major
histocompatibility complex (MHC)-encoded molecules. Upon
MHC-peptide engagement, TCR components (associated with CD3
chains) initiate Ca2þ mobilization and mitogen-activated protein
kinase (MAPK) phosphorylation cascades, including extracellular
signal-regulated kinase (ERK) activation, which trigger multiple
signaling pathways (Koike et al., 2003). Thus, specific inhibitors of
TCR signaling represent potential therapeutics for treating auto-
immune diseases, and the identification of such compounds has
been of significant interest [e.g., see (Visperas et al., 2017)]. Analysis
of public microarray repositories predicts that TCR signalingmay be
involved in the effects of parthenolide and artemisinin on acute
myelogenous leukemia (Engreitz et al., 2010; Huang et al., 2013).
Indeed, SM905, a synthetic artemisinin derivative, was found to
inhibit TCR-mediated T cell proliferation and MAPK phosphoryla-
tion (Wang et al., 2007). Another artemisinin analogue, SM934, was
recently found to attenuate collagen-induced arthritis by sup-
pressing T follicular helper cells and T helper 17 (Th17) cells (Lin
et al., 2016). Similarly, parthenolide inhibited the initiation of
experimental autoimmune neuritis by decreasing Th1 and
Th17 cells (Zhang et al., 2017). However, molecular mechanisms
involved in the regulation of T cell activity by sesquiterpene lac-
tones are still not well defined. For example, the effects of sesqui-
terpene lactones on T cell Ca2þ mobilization and ERK
phosphorylation following TCR stimulation have not been previ-
ously assessed.
Here, we studied the capacity of 13 plant-derived sesquiterpene
lactones to inhibit initial phases of TCR activation and showed that
five of these natural sesquiterpene lactones (arglabin, parthenolide,
grosheimin, agracin, and estafiatin) inhibited Ca2þ mobilization
and ERK1/2 phosphorylation induced by TCR activation. Thus, our
studies demonstrate that these sesquiterpene lactones can selec-
tively inhibit initial phases of TCR activation.
2. Results
2.1. Effect of sesquiterpene lactones on lymphocyte Ca2þ
mobilization
TCR activation results in rapid mobilization of intracellular Ca2þ,
which represents one of the early signaling events that can be
monitored in lymphocyte activation (Ishikawa et al., 2003). Thus,
we evaluated the effect of a variety of natural sesquiterpene lac-
tones on Ca2þ mobilization induced by anti-CD3 antibodies in
Jurkat T cells (Table 1). The set of 13 natural sesquiterpene lactones
tested was assembled to maximize chemical diversity and included
compounds with germacranolide (compounds 3, 4, and 12),
eudesmane (compounds 5, 7, and 13), guaianolide (compounds 1, 2,
8, 9, 10, and 11), and cadinanolide (compound 6) structures. Six of
the germacranolide and eudesmane compounds contain an a-
methylene-g-lactone ring, two of guaianolide and germacranolide
compounds have an epoxide group, and one contains an endo-
peroxide bridge. As shown in Fig. 1A, the intracellular Ca2þ flux
rapidly induced after Jurkat cell activation was significantly
inhibited by pretreatment of these cells with estafiatin and par-
thenolide. Likewise, arglabin, agracin, and grosheimin also dose-
dependently inhibited Jurkat T cell activation-induced intracel-
lular Ca2þ flux, and a representative doseeresponse curve forinhibition of Jurkat cell Ca2þ mobilization by agracin is shown in
Fig.1B. In contrast, the other 8 sesquiterpene lactones tested had no
effect on this response (Table 2).
All five active sesquiterpene lactones contain an a-methylene-g-
lactone ring, which may react with nucleophiles, such as cysteine
sulfhydryl group(s) of glutathione (GSH), redox-sensitive kinases
and other enzymes, by a Michael-type addition (Butturini et al.,
2013; Visperas et al., 2015). To evaluate whether a redox-event is
important in modulation of TCR activation by these compounds,
Jurkat cells were pretreated for 3 h at 37 C with 5 mM glutathione
ethylene ester (GEE), a cell-permeable GSH (Butturini et al., 2013),
and thereafter for 20 min with different concentrations of agracin
and parthenolide, the most potent inhibitors of TCR activation-
induced Ca2þ flux (see Table 2). Interestingly, we found that pre-
treatment with GEE reversed much of the inhibitory effect of par-
thenolide (not shown) and agracin on intracellular Ca2þ
mobilization (Fig. 1B). Note, however, that the highest concentra-
tions of lactone could partially overcome these effects of GEE
(Fig. 1B).
Thapsigargin, a sesquiterpene lactone from Thapsia garganica, is
an inhibitor of the sarco/endoplasmic reticulum Ca2þ ATPase
(SERCA) and raises intracellular Ca2þ by blocking the ability of the
cell to pump Ca2þ into the sarcoplasmic and endoplasmic reticula
(Rogers et al., 1995). Thus, we evaluated the direct effect of our
selection of sesquiterpene lactones on Ca2þ flux in Jurkat T cells
compared to thapsigargin. While we confirmed that thapsigargin
could stimulate [Ca2þ]i accumulation in Jurkat cells, none of the 13
sesquiterpene lactones tested increased [Ca2þ]i in these cells. As
examples, the direct effects of thapsigargin, estafiatin, and arte-
misinin on Jurkat T cell Ca2þ levels are shown in Fig. 2.
To test cell specificity of the sesquiterpene lactones on Ca2þ flux,
we also evaluated if they modulated Ca2þmobilization in activated
human neutrophils and HL60 cells transfected with FPR1 and FPR2.
None of sesquiterpene lactones directly stimulated Ca2þ mobiliza-
tion in neutrophils or transfected HL60 cells (data not shown).
Likewise, treatment of these cells with various concentrations of
sesquiterpene lactones for 20 min had no effect on Ca2þ mobili-
zation induced by FPR1/FPR2 agonists (fMLF in neutrophils and
FPR1-HL60 cells or WKYMVM in FPR2-HL60 cells) (Table 2), indi-
cating that the inhibitory effect was specific for TCR activation.
Considering the apparent selectivity of sesquiterpene lactones
towards inhibition of Ca2þ flux in Jurkat T cells, we evaluated if N-
ethylmaleimide (NEM), a highly reactive but nonspecific cysteine
alkylating reagent, would also inhibit lymphocyte activation simi-
larly to the sesquiterpene lactones. However, we found that pre-
treatment with NEM led to a dose-dependent inhibition of Ca2þ
flux in all cells tested, with IC50 values in nanomolar range. Indeed,
NEM pretreatment inhibited Ca2þ flux in Jurkat cells stimulated
with anti-CD3/CD28 (IC50 ¼ 175 ± 70 nM) and in FPR1-HL60 cells
(IC50 ¼ 360 ± 34 nM) or human neutrophils (IC50 ¼ 860 ± 330 nM)
stimulated with 5 nM fMLF. Thus, comparison of the observed ef-
fects of NEM and sesquiterpene lactones in lymphoid and myeloid
cells supports the conclusion that these sesquiterpene lactones
modulate a specific target or targets in lymphocytes rather than
nonspecifically modifying cysteines in any cell type.
2.2. Effect of estafiatin on protein kinase activity
Stimulation of the TCR in Jurkat cells activates multiple signaling
pathways, including a variety of protein kinases (Kim and White,
2006; Koike et al., 2003). To evaluate the effect of sesquiterpene
lactones on protein kinase signaling, we selected estafiatin as a
representative compound, as it contains both a-methylene-g-
lactone and epoxide moieties (see Table 1). To simultaneously
evaluate effects of estafiatin on phosphorylation of various kinases
Table 1
Chemical structures and plant sources of the selected sesquiterpene lactones.and their substrates, we utilized a human phospho-kinase array
Proteome Profiler, which monitors the phosphorylation of 43 ki-
nase phosphorylation sites (see list of kinases in the Experimental
section). While estafiatin itself did not increase phosphorylation of
the arrayed kinases (Fig. 3A), treatment with anti-CD3/CD28
significantly increased phosphorylation of ERK1/2 [phosphoryla-
tion sites Thr202/Tyr204, Thr185/Tyr187; fold increase (FI) ¼ 6.7],
AMPKa1 (Thr183; FI ¼ 2.4), CREB (Ser133; FI ¼ 4.7), p53 (S392;FI ¼ 2.2), and p27 (Thr180/Tyr182; FI ¼ 7.3) in Jurkat cells (Fig. 3B,
grey bars). Importantly, pretreatment of Jurkat cells with estafiatin
(50 mM) for 20 min at 37 C completely inhibited the TCR
activation-induced phosphorylation of ERK1/2, p53, AMPKa1,
CREB, and p27 (Fig. 3C).
The suppression of ERK1/2 phosphorylation might result from
direct inhibition or inhibition of other upstream kinase(s). Thus, we
evaluated the direct binding activity of estafiatin against a panel of
Fig. 1. Effect of parthenolide, estafiatin, and agracin on activation-induced Ca2þ
mobilization in Jurkat T cells. Panel A. Jurkat cells were pretreated for 20 min with 1%
DMSO (negative control), estafiatin (50 mM), or parthenolide (50 mM), and the kinetics
of Ca2þ mobilization 15 s after activation with 5 mg/ml anti-CD3 monoclonal antibody
were measured (arrow indicates time of activation). The response of control cells
treated with DMSO alone is shown as a grey line. Panel B. Jurkat cells were pretreated
for 3 h with 5 mMGEE (þGEE) or medium (w/o GEE), followed by treatment for 20 min
with control 1% DMSO or increasing concentrations of argracin and activated with anti-
CD3. Activation-induced Ca2þ flux was measured as described, and the results are
shown as % of maximal activation measured in control cells. The results shown in both
panels are representative of three independent experiments. Statistically significant
differences between cells pretreated with argracin versus control (1% DMSO) are
indicated (ap<0.01). In addition, statistically significant differences between cells
incubated with GEE versus without GEE prior to the argracin treatment and activation
are indicated (bp < 0.01).95 protein kinases representing all known kinase families in a cell-
free competition binding assay for the ability of estafiatin to
compete with binding of an active-site directed ligand (DiscoveRxKINOMEscan). However, estafiatin did not bind directly to any of
the kinases tested (data not shown), including zeta-chain-
associated protein kinase 70 kDa (ZAP70), Fyn oncogene, spleen
tyrosine kinase (Syk), lymphocyte-specific protein tyrosine kinase
(Lck), liver kinase B1 (LKB1), ERK1, and ERK2. Thus, estafiatin likely
modulates kinase activity through alternative mechanisms. For
example, one possibility to be evaluated in future studies is that
estafiatin could prevent thiol-sensitive tandem-SH2 domains of
ZAP-70 and Syk from binding to phosphorylated ITAMs [see
(Visperas et al., 2015, 2017)].
ERK1/2 phosphorylation was one of the main TCR activation-
induced responses observed in our kinase array (Fig. 3B) [also see
(Kim and White, 2006)]. Thus we further characterized this
response and its modulation by the active sesquiterpene lactones.
Although none of compounds directly stimulated ERK1/2 phos-
phorylation (data not shown), pretreatment of Jurkat T cells with
various concentrations of these compounds, followed by activation
with anti-CD3/CD28 antibodies showed that the five compounds
that inhibited Ca2þ mobilization (arglabin, agracin, estafiatin,
grosheimin, and parthenolide) also significantly inhibited TCR
activation-induced ERK1/2 phosphorylation in a dose-dependent
manner, with IC50 values in the micromolar range (Table 2). As
examples, dose-dependent inhibition of ERK1/2 phosphorylation
by parthenolide and estafiatin is shown in Fig. 4. Likewise, pre-
treatment of human primary T cells with parthenolide or estafiatin
also suppressed ERK1/2 phosphorylation stimulated by anti-CD3/
CD28 antibodies (Fig. 5), verifying that this effect was relevant to
primary cells. Finally, pretreatment of Jurkat cells with GEE
reversed the inhibitory effect of parthenolide and estafiatin on
ERK1/2 phosphorylation (Fig. 4), indicating that restoring [GSH]i
could overcome at least some of the inhibitory effects of these
sesquiterpene lactones.
To evaluate target specificity of the effect on ERK1/2 phos-
phorylation activated through the TCR, we also evaluated if the
sesquiterpene lactones could inhibit ERK1/2 phosphorylation
stimulated by other receptors. For example, activation of FPR2 is
known to induce ERK1/2 phosphorylation (Schepetkin et al., 2014).
Thus, we pretreated FPR2-HL60 cells with various concentrations of
the sesquiterpene lactones (up to 50 mM), and the cells were acti-
vated with 5 nMWKYMVM, a peptide FPR2 agonist. However, none
of the compounds tested inhibited FPR2-dependent ERK1/2 phos-
phorylation (Table 2), suggesting again that sesquiterpene lactones
specifically inhibit TCR-dependent activation.
2.3. GSH reactivity of the sesquiterpene lactones
GSH is an important regulator of cellular redox equilibrium. The
TCR-mediated response is very sensitive to changes in intercellular
GSH levels ([GSH]i) (Gringhuis et al., 2002), and some sesquiter-
pene lactones can decrease [GSH]i in various human cells by
reacting with cysteine (Itoh et al., 2009; Scarponi et al., 2014). Thus,
we evaluated if the selected sesquiterpene lactones can react with
GSH in Jurkat T cells. Screening of sesquiterpene lactone reactivity
with cytosolic GSH showed that the compounds that inhibited Ca2þ
mobilization and ERK1/2 phosphorylation also depleted [GSH]i in
Jurkat cells, with the most potent being argracin and parthenolide
(Table 2), and a representative concentration-dependent decrease
of [GSH]i in Jurkat T cells after argracin treatment is shown in Fig. 6.
2.4. Effect of sesquiterpene lactones on anti-CD3 binding
As described above, sesquiterpene lactones can alkylate thiol
groups on biological macromolecules, including cysteine residues
of target proteins (Garcia-Pineres et al., 2001; Lagoutte et al., 2016;
Wagner et al., 2006; Zhang et al., 2015). Although the highly
Table 2
Effect of sesquiterpene lactones on Ca2þ mobilization, ERK1/2 phosphorylation, and GSH concentration.
Compd. Name Jurkat T cells FPR2-HL60 Jurkat T Cells FPR1-HL60 FPR2-HL60 PMN Jurkat T Cells
p-ERK1/2 Ca2þ mobilization [GSH]i
IC50 (mM)
1 achillin N.A. N.A. N.A. N.A. N.A. N.A. N.A.
2 arglabin 28.7 ± 6.3 N.A. 11.1 ± 2.7 N.A. N.A. N.A. 18.0 ± 1.8
3 argolide N.A. N.A. N.A. N.A. N.A. N.A. N.A.
4 argracin 16.8 ± 5.3 N.A. 6.1 ± 1.6 N.A. N.A. N.A. 8.9 ± 1.4
5 3b-hydroxyarhalin N.A. N.A. N.A. N.A. N.A. N.A. N.A.
6 artemisinin N.A. N.A. N.A. N.A. N.A. N.A. N.A.
7 artesin N.A. N.A. N.A. N.A. N.A. N.A. N.A.
8 estafiatin 15.4 ± 3.2 N.A. 29.3 ± 6.8 N.A. N.A. N.A. 46.9 ± 5.3
9 grosheimin 43.0 ± 7.5 N.A. 15.4 ± 4.3 N.A. N.A. N.A. 16.6 ± 4.8
10 grossmisin N.A. N.A. N.A. N.A. N.A. N.A. N.A.
11 leucomisine N.A. N.A. N.A. N.A. N.A. N.A. N.A.
12 parthenolide 13.8 ± 1.7 N.A. 2.4 ± 0.6 N.A. N.A. N.A. 9.9 ± 3.4
13 taurin N.A. N.A. N.A. N.A. N.A. N.A. N.A.
Inhibition was evaluated after 20 min pretreatment with test compounds at room temperature, followed by addition of 10 mg/ml anti-CD3/CD28 (p-ERK1/2 in Jurkat cells),
10 mg/ml anti-CD3 (Ca2þ flux in Jurkat cells), 5 nM fMLF [Ca2þ flux in FPR1-HL60 cells and human neutrophils (PMN)], or 5 nM WKYMVM (pERK1/2 and Ca2þ flux in FPR2-
HL60 cells). Changes in Jurkat T cell cytosolic [GSH]i were measured after treating cells with test compounds for 30 min at 37 C. N.A., no activity was observed at the highest
tested concentration (50 mM in the p-ERK1/2 assay and 100 mM in the GSH assay).
Fig. 2. Effect of thapsigargin, estafiatin, and artemisinin on Ca2þmobilization in Jurkat
T cells. Jurkat cells were treated with 1% DMSO (negative control), 200 nM thapsi-
gargin, 50 mM estafiatin, or 50 mM artemisinin, as indicated, and the kinetics of Ca2þ
mobilization were measured (arrow indicates time of compound addition). The results
shown are representative of three independent experiments.conserved membrane-proximal tetracysteine motif in the stalk
region of CD3 is not required for CD3 dimerization, modification of
these cysteine residues has been reported to reduce assembly of
CD3dε with TCRa (Xu et al., 2006). Thus, we evaluated if the active
sesquiterpene lactones could affect the ability of anti-CD3 mono-
clonal antibodies to bind the extracellular region of CD3. Although
pretreatment with anti-CD3 antibody (5 mg/ml) itself completely
blocked the binding of fluorescent anti-CD3 antibody to Jurkat cells,
neither arglabin, grosheimin, agracin, parthenolide, or estafiatin
(all tested at 50 mM for 20 min at 37 C) reduced binding of APC-
labeled anti-CD3 antibody to the extracellular region of CD3 (data
not shown).2.5. Effect of sesquiterpene lactones on cell toxicity
To ensure that the results were not influenced by possible
compound toxicity, cytotoxicity of the sesquiterpene lactones was
evaluated at various concentrations up to 50 mM in Jurkat and
isolated T cells during a 30 min incubation with the compounds.
None of the sesquiterpene lactones affected cell viability at the
highest tested concentrations (Table 2 for Jurkat T cells and data not
shown for primary human T cells), thereby verifying that these
compounds were not cytotoxic during the 30 min incubation
period of our assays.3. Discussion
Plant-derived sesquiterpene lactones exhibit a broad spectrum
of pharmacological properties, including immunomodulation and
anti-inflammatory activity (reviewed in (Chadwick et al., 2013; Hou
and Huang, 2016; Ren et al., 2016)). For example, the therapeutic
potential of arglabin and its chemical derivatives was recently
reviewed (Adekenov, 2016). Parthenolide was previously studied
regarding inhibition of NF-kB activation and cytokine production in
Jurkat and primary T cells (Garcia-Pineres et al., 2001, 2004; Li-
Weber et al., 2002). Although several sesquiterpenes have been
reported to inhibit ERK phosphorylation in different cell lines and
macrophages (D'Anneo et al., 2013; Saadane et al., 2011; Wang
et al., 2011; Ye et al., 2015), their effects on Ca2þ mobilization and
ERK phosphorylation following TCR stimulation have not been
previously assessed. In the present study, we evaluated the
immunomodulatory effects of 13 natural sesquiterpene lactones
and found that five of the tested compounds (arglabin, argracin,
estafiatin, grosheimin, and parthenolide) specifically inhibited
early phases of TCR activation but had no effect on phagocyte
activation through FPR1/2. Activation of Jurkat T and primary T cells
was suppressed by these sesquiterpene lactones, as demonstrated
by inhibition of Ca2þ mobilization and ERK1/2 phosphorylation.
These lactones also depleted [GSH]i in Jurkat T cells. Although
several naturally occurring compounds, including parthenolide,
have been previously reported to modulate T cell activity stimu-
lated through the TCR (Garcia-Pineres et al., 2001, 2004; Li-Weber
et al., 2002; Liu et al., 2011), this is the first report demonstrating
inhibition of TCR activation-induced Ca2þmobilization and ERK1/2
phosphorylation by natural sesquiterpene lactones.
Fig. 3. Effect of estafiatin on activation-induced kinase phosphorylation in Jurkat T cells. Jurkat T cells were pretreated for 20 minwith estafiatin (50 mM), followed by activationwith
anti-CD3/CD28 (10 mg/ml each) for 5 min, and the levels of protein phosphorylation in cell lysates were evaluated using a human phospho-kinase array. There are several kinases
with different phosphorylation sites, including Akt1/2/3a on Ser473 and Akt1/2/3b on Thr303; p70S6Ka on Thr389 and p70S6Kb on Thr421/S424; STAT3a on Tyr705 and STAT3b on
Ser727; p53a, p53b, and p53c, on Ser392, Ser46, and Ser15, respectively. The data are presented as mean ± SD of duplicate samples. Statistically significant differences (*p < 0.05)
between DMSO (control) and estafiatin-pretreated cells are indicated (also shown in shaded bars).Among of the sesquiterpene lactones evaluated, only parthe-
nolide and artemisinin have been studied previously for their ef-
fects on ERK phosphorylation, but in different cells (D'Anneo et al.,
2013; Saadane et al., 2011; Wang et al., 2011). For example, ERK1/2
phosphorylationwas inhibited by parthenolide in cystic fibrosis cell
cultures (Saadane et al., 2011) and murine osteoclast precursors
(Kim et al., 2014). However, parthenolide activated ERK1/2 phos-
phorylation in human osteosarcomaMG63 andmelanoma SK-MEL-
28 cells (D'Anneo et al., 2013). Although artemisinin can suppress
ERK1/2 phosphorylation in THP-1 macrophages (Wang et al., 2011),
we did not observe inhibition of ERK1/2 phosphorylation by this
compound in Jurkat T cells (Table 2). It should be noted that there
has been no reported correlation between [GSH]i depletion by
various natural compounds and ERK activity. For example, the
diterpenoid oridonin, which also contains an a-methylene-g-
lactone ring, reduced [GSH]i in hepatic stellate HSC-T6 cells but also
induced ERK1/2 phosphorylation (Kuo et al., 2014). Together, thesedata indicate the effects of sesquiterpene lactones are cell-specific
but may also depend on how various agonists engage down-
stream signaling pathways.
The precise target of the active sesquiterpene lactones identified
here is currently not known, although it is known that the a-
methylene-g-lactone group is important for their biological activ-
ity. For example, previous structureeactivity relationship analysis
showed that the a-methylene-g-lactone ring was the site of attack
of the cysteine residues on GSH, various receptors, protein kinases,
and transcription factor subunits (Garcia-Pineres et al., 2001;
Lagoutte et al., 2016; Wagner et al., 2006; Zhang et al., 2015).
Indeed, all five active sesquiterpene lactones that inhibited Ca2þ
flux, blocked ERK1/2 phosphorylation, and depleted [GSH]i in
Jurkat T cells contain an a-methylene-g-lactone ring, whereas only
one compound with an a-methylene-g-lactone ring (argolide) was
inactive (Tables 1 and 2). Note, however, that parthenolide, the
most potent compound in both the Ca2þ flux and ERK1/2 assays,
Fig. 4. Effect of parthenolide and estafiatin on activation-induced ERK1/2 phosphor-
ylation. Jurkat T cells were pretreated with 1% DMSO or increasing concentrations of
estafiatin () or parthenolide (☐) for 20 min, followed by activation with anti-CD3/
CD28 (10 mg/ml each) for 5 min, and the levels of ERK1/2 phosphorylation were
evaluated using ELISA. In some experiments, Jurkat cells were incubated overnight
with 2 mM GEE or medium (not shown), followed by treatment with DMSO or
increasing concentrations of estafiatin (C) or parthenolide (-) for 20 min, followed
by activation with anti-CD3/CD28 (10 mg/ml each) for 5 min, and ERK1/2 phophor-
ylation was evaluated using the same procedures described above. The data are pre-
sented as % of control levels and represent the mean ± SD of triplicate samples from
one experiment, which is representative of three independent experiments. Statisti-
cally significant differences between cells pretreated with sesquiterpene lactones
versus control (1% DMSO) are indicated (ap<0.01; bp < 0.05). In addition, statistically
significant differences between cells incubated with GEE versus without GEE prior to
the respective lactone treatment and activation are indicated (cp < 0.01).
Fig. 5. Effect of parthenolide and estafiatin on activation-induced ERK1/2 phosphor-
ylation in human primary T cells. Isolated human T cells were pretreated for 20 min
with 1% DMSO or the indicated concentrations of parthenolide and estafiatin, followed
by activation with human T-activator CD3/CD28 Dynabeads (bead-to-cell ratio 1:1) for
5 min, and the levels of ERK phosphorylation were evaluated using an ELISA for human
phospho-ERK1/2. The data are presented as mean ± SD of triplicate samples. Statis-
tically significant differences between cells treated with control DMSO versus
DMSO þ CD3/CD20 beads (ap<0.01) and between cells treated with DMSO þ CD3/
CD20 beads versus cells treated with the indicated lactones þ CD3/CD20 beads
(bp < 0.01) are indicated. The results shown are representative of three independent
experiments.
Fig. 6. Effect of agracin on Jurkat T cell cytosolic GSH. Jurkat cells (105 cells/ml) were
incubated with control 1% DMSO or increasing concentrations of agracin for 20 min at
room temperature, and the level of cytosolic [GSH]i in the cells was measured using a
GSHGlo assay. The results are presented as % of [GSH]i measured in control DMSO
treated cells (mean ± SD of triplicate samples) and are representative of three inde-
pendent experiments.also possesses an epoxide group. Thus, we cannot exclude the
possibility that inhibitory effect on TCR activation is greater when a
sesquiterpene lactone has two potentially reactive centers that
could react with biological nucleophiles, such as sulfhydryl groups.
Artemisinin also has a reactive endoperoxide bridge. However, this
compound was inactive in all of our assays, indicating that the
endoperoxide functionality and/or formation of free radicals via
cleavage of the endoperoxide bond does not play an important role
in modulation of TCR activity or that the geometry of this molecule
is not suitable for interaction with the relevant molecular targets.
Thus, based on the direct correlation between biological activity
and number of chemically reactive a-methylene-g-lactone/epoxide
groups in the compounds possessing very similar molecular
structure, we suggest that modulating influence of the other
structural factors on bioactivity is lower.
Recently, it was reported that GSH is dispensable for initial T cell
activation (Mak et al., 2017). However, sesquiterpene lactones likely
can act on several thiol-sensitive targets, and concurrent GSH
depletion would increase the aggregate effect of these compounds
on T cell activation. Following depletion of [GSH]i, the major anti-
oxidant molecule and redox-buffer in cells, the active sesquiter-
pene lactones could then induce oxidative stress and/or form
protein adducts (Carlisi et al., 2016). Indeed, regulation of protein
kinase activity can occur via several redox-cycles involving thio-
redoxin, GSH-dependent enzymes, and reactive oxygen species
(ROS). For example, apoptosis signal-regulating kinase 1 (ASK1) isphysically associated with thioredoxin, making thioredoxin a
redox-sensitive physiological regulator of ASK1 activity and ASK1-
associated MAPK (Davis et al., 2001). Likewise, glutathione S-
transferase P1-1 (GSTP) can interact with JNK, and this protein-
protein interaction can sequester JNK and act as a regulator of
this MAPK (Tew and Townsend, 2011). ROS-induced cysteine
oxidation in proteins involves initial formation of sulfenic acid,
which can subsequently react with GSH to form S-glutathionylated
proteins (Reddie and Carroll, 2008), and the importance of these
specific oxidative cysteine modifications in direct regulation of
tyrosine kinases was recently described (Heppner et al., 2016).
Because the inhibition of Ca2þ mobilization and ERK1/2 phos-
phorylation were significantly diminished when Jurkat cells were
incubated with GEE, S-glutathionylation of protein kinases engaged
in TCR activation is likely to be triggered by increased oxidative
stress induced by the decrease in cellular GSH.
It should be noted that the a-methylene-g-lactone ring of
sesquiterpene lactones plays a critical role inmediating cytotoxicity
via GSH depletion (Kupchan et al., 1971; Carlisi et al., 2016), as GSH
depletion can activate apoptotic caspases with subsequent cell
death (Lee et al., 2013; Khan et al., 2013). In the present studies, we
investigated very early events in T cell activation (first 30 min)
when no cytotoxicity was observed. Thus, assessment of the rela-
tionship between structure of the natural compounds and their
cytotoxicity profile during long-term treatment (hours and days)
and with a variety of immune cells will be important to evaluate in
future studies.
Sesquiterpene lactones can also directly react with proteins. For
example, parthenolide reacts with albumin cysteine residues
exclusively via its a-methylene-g-lactone moiety, while the
epoxide structure is not involved in the reaction (Ploger et al.,
2015). Thus, it is clearly feasible that our active sesquiterpene lac-
tones could form adducts with macromolecules engaged in TCR-
dependent activation. Upon TCR stimulation, tyrosine kinases Fyn
and Lck phosphorylate ITAMs in the TCR, which recruit tyrosine
kinases Syk and ZAP70 to phosphorylate the linker of activated T
cells (LAT), resulting in the formation of a complex consisting of
various components, including interleukin-2-inducible tyrosine
kinase (Itk) and phospholipase C (PLC) g1 (Fu et al., 2010; Zhang
et al., 1998). Activated PLCg1 hydrolyzes phosphatidylinositol 4,5-
bisphosphate to generate inositol 1,4,5-trisphosphate (IP3), which
stimulates the release of Ca2þ from intracellular stores. Ca2þ
mobilization leading to activation of multiple pathways, including
ERK1/2, which eventually activate specific nuclear factors (Winslow
et al., 2003). Using a high-throughput screen, Visperas et al. (2017,
2015) recently identified several small-molecule compounds that
covalently react with cysteine residues of the ZAP70 tandem SH2
module and inhibit its binding to a phosphorylated ITAM-derived
peptide. Thus, based on the reactivity of the active sesquiterpene
lactones for cysteine, it is possible that they can similarly induce
redox-dependent post-translational modification of cysteine resi-
dues in the ZAP70 SH2 module to regulate its function, and this is
currently being evaluated. Another possibility is that the active
sesquiterpene lactones interact with NEM-sensitive fusion protein
(NSF), soluble NEM-sensitive factor attachment protein receptor
(SNARE) proteins, or tubulin and/or dynein systems, which play
important roles in forming signaling clusters upon TCR activation
(Heller et al., 2001; Larghi et al., 2013; Philipsen et al., 2013).
Indeed, recent high-content screening identified parthenolide as an
inhibitor of cytoplasmic dynein-mediated transport and microtu-
bule formation and suggested it may react with detyrosinated
tubulin (Johnston et al., 2012; Miyata et al., 2008; Whipple et al.,
2013). Whether the other active sesquiterpene lactones identified
here can also inhibit these pathways in T cells will need to be
investigated in future studies.
Thapsigargin, a sesquiterpene lactone and potent inhibitor of
SERCA activity, induces Ca2þ release from the endoplasmic reticu-
lum and the subsequent opening of plasma membrane-specific
Ca2þ channels responsible for the store-operated calcium entry
(Cerveira et al., 2015). Consistent with these observations, we found
that thapsigargin stimulated Ca2þ accumulation in Jurkat T cells.
Although artemisinin inhibited SERCA activity in Plasmodiumfalciparum (Eckstein-Ludwig et al., 2003), none of the test com-
pounds, including artemisinin, induced Ca2þ flux in human neu-
trophils, Jurkat T cells, or FPR1/2 transfected HL60 cells. Based on
these data, we conclude that the active sesquiterpene lactones do
not inhibit SERCA in these cells.
Analysis of estafiatin, a representative active sesquiterpene
lactone with a-methylene-g-lactone and epoxide moieties, showed
that this compound inhibited AMPKa1 phosphorylation but did not
bind with LKB1, a main kinase that phosphorylates AMPKa1.
Because estafiatin also does not bind kinase modules of ZAP70, Fyn,
Syk, and Lck, which are the most proximal kinases to be activated
downstream of the TCR (Lovatt et al., 2006), it could be possible
that the active sesquiterpene lactones may directly interact with an
extracellular region of CD3 and prevent activation of the TCR
complex by anti-CD3. On the other hand, we also found that all of
the active sesquiterpene lactones did not affect anti-CD3 binding
with extracellular region of CD3 on Jurkat T cells, so such an
interactionwould have to involve a different region of the molecule
than that targeted by anti-CD3.
Our results show that the nonselective thiol-modifying reagent
NEM is a potent inhibitor of Ca2þ flux initiated via TCR activation in
Jurkat cells, as well as FPR1 stimulation in FPR1-HL60 cells and
neutrophils stimulated by fMLF, as reported previously (Hsu et al.,
2005). Given that the active sesquiterpene lactones did not
inhibit Ca2þ mobilization in activated FPR1-HL60 cells and neu-
trophils, we conclude that these compounds are clearly more se-
lective than NEM. Upon specific ligand binding and activation of
FPR, intracellular domains of FPR mediate signaling to G-proteins,
which trigger several agonist-dependent signal transduction
pathways, including Ca2þ mobilization and ERK1/2 phosphoryla-
tion (Cattaneo et al., 2013; Gripentrog and Miettinen, 2008).
Although intact thiol groups are important for FPR ligand binding
(Lane and Lamkin, 1982), the sesquiterpene lactones tested appar-
ently did not block binding of FPR1/2 with specific ligands since no
effect on activation was observed. Likewise, these compounds did
not appear to interfere with the PLC/IP3 pathway in these cells,
suggesting that their molecular target(s) could be located
upstream.
In conclusion, our experimental studies demonstrate that
arglabin, argracin, estafiatin, grosheimin, and parthenolide can
interact with pathways involved in initial phases of TCR activation,
resulting in inhibition of this response. All five active sesquiterpene
lactones that inhibited Ca2þ mobilization, blocked ERK1/2 phos-
phorylation, and depleted [GSH]i in Jurkat T cells contained an a-
methylene-g-lactone ring, indicating an important role for this
structure in compound bioactivity. Overall, these results suggest a
potential new strategy for developing novel medicines based on
natural sesquiterpene lactones that could effectively modulate TCR
responses. Further detailed studies are warranted to define the
molecular targets and define the therapeutic potential of these
natural sesquiterpene lactones as previously undescribed immu-
nomodulatory and anti-inflammatory agents.
4. Experimental
4.1. Natural compounds
The sesquiterpene lactones (achillin, arglabin, argolide, argracin,
3b-hydroxyarhalin, artesin, artemisinin, estafiatin, grosheimin,
grossmisin, leucomisine, parthenolide, and taurin) were isolated as
previously described from different plants of the Asteraceae family,
as shown in Table 1 (Adekenov, 2013, 2016; Adekenov et al., 2016,
1984, 2016, 2017; Akyev et al., 1972; Arystan et al., 2009; Rey
et al., 1992). The purity of each compound was determined to be
>98% by normalization of the peak areas detected on an automated
high-performance liquid chromatography (HPLC) system (Hew-
lettePackard Agilent 1100) with a Zorbax SB-C18 column
(4.6  150 mm) eluted with acetonitrile/water (50%/50%, v/v) or
methanol/water (50%/50%, v/v) at a flow rate of 0.5 ml/min at 25 C.
The elution was monitored at 204 nm.
4.2. Materials
Dimethyl sulfoxide (DMSO), N-formyl-Met-Leu-Phe (fMLF), and
Histopaque 1077 were purchased from Sigma-Aldrich Chemical Co.
(St. Louis, MO, USA). Fluo-4AM dye was from Invitrogen (Carlsbad,
CA, USA). NEM was from Chem-Impex International, Inc. (Wood
Dale, IL, USA). Anti-human CD3 and anti-human CD28 monoclonal
antibodies were purchased from eBioscience (San Diego, CA, USA).
Thapsigargin and Trp-Lys-Tyr-Met-Val-Met (WKYMVM) were from
Tocris Bioscience (Ellisville, MO, USA). GEE was from Cayman
Chemical Co. (Ann Arbor, MI, USA). Ficoll-Paque was from GE
Healthcare Bio-Science AB (Uppsala, Sweden). Pen-
icillinestreptomycin solution was purchased from Mediatech
(Herndon, VA, USA). Fetal bovine serum (FBS) was purchased from
Atlas Biologicals (Fort Collins, CO, USA). Hanks' balanced salt solu-
tion (HBSS; 0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM
KH2PO4, 4.2 mM NaHCO3, 5.56 mM glucose, and 10 mM HEPES, pH
7.4) was from Life Technologies (Grand Island, NY, USA). HBSS
without Ca2þ and Mg2þ is designated as HBSS; HBSS containing
1.3mMCaCl2 and 1.0mMMgSO4 is designated as HBSSþ. Phosphate
buffer solution (PBS, pH 7.2) supplemented with 0.1% sodium azide
and 1% bovine serum albumin is referred as flow cytometry buffer
(eBioscience).
4.3. Cell culture
Human Jurkat T acute lymphoblastic leukemia cells and human
promyelocytic leukemia cells (HL60 cells) stably transfected with
FPR1 (FPR1-HL60 cells) or FPR2 (FPR1-HL60 cells) (kind gifts from
Dr. Marie-Josephe Rabiet, INSERM, Grenoble, France) were cultured
in Roswell Park Memorial Institute (RPMI)-1640 supplemented
with 10% heat-inactivated FBS, 10 mM HEPES, 100 mg/mL strepto-
mycin, and 100 U/mL penicillin. Transfected HL60 cells were
cultured in the presence of G418 (1 mg/mL). All cell lines were
incubated in a humidified incubator at 37 C with an atmosphere of
5% CO2.
4.4. Isolation of human neutrophils
For isolation of human neutrophils, blood was collected from
healthy donors in accordance with a protocol approved by the
Institutional Review Board at Montana State University. Neutro-
phils were purified from the blood using dextran sedimentation,
followed by Ficoll-Paque 1077 gradient separation and hypotonic
lysis of red blood cells, as described previously (Schepetkin et al.,
2007). Isolated neutrophils were washed twice and resuspended
in HBSS. Neutrophil preparations were routinely >95% pure, as
determined by light microscopy, and >98% viable, as determined by
trypan blue exclusion. Neutrophils were obtained from multiple
donors (n¼ 8); however, the cells from different donors were never
pooled together during experiments.
4.5. Isolation of human T cells
For isolation of human T cells, blood was collected from healthy
donors in accordance with a protocol approved by the Institutional
Review Board at Montana State University. Peripheral blood
mononuclear cells (PBMCs) were isolated from the blood samples
by gradient centrifugation using Ficoll-Paque. CD3þ T cells wereisolated by negative selection using a pan T cell isolation kit, as
described by the manufacturer (Miltnyei Biotec Inc., Auburn, CA)
and cultured overnight in RPMI-1640 supplementedwith 10% heat-
inactivated FBS, 10 mMHEPES, 100 mg/mL streptomycin, and 100 U/
mL penicillin. The purified CD3þ T cells were used for experiments
the following day.
4.6. Protein kinase array
Analysis of the phosphorylation profiles of kinases and their
protein substrates was performed using a human phospho-kinase
array kit Proteome Profiler (R&D Systems, Minneapolis, MN). The
array simultaneously detects 43 kinase phosphorylation sites,
including MAPKs [ERK1/2, c-Jun N-terminal kinases (JNK 1e3), and
p38a], MSK1/2, mTOR, Akt 1e3, glycogen synthase kinases (GSK-3)
a/b, p90 ribosomal S6 kinases (RSK) 1e3, p70 S6 kinase (p70S6K),
AMP-activated protein kinase (AMPK) a1 and a2, WNK lysine
deficient protein kinase 1 (WNK1), checkpoint kinase 2 (Chk-2),
two receptor tyrosine-protein kinases [epidermal growth factor
receptor (EGF-R) and platelet-derived growth factor receptor
(PDGF-Rb)], non-receptor tyrosine-protein kinases (Src, Lyn, Lck,
Fyn, Yes, Fgr, Hck, PYK2, FAK/PTK2), cAMP response element-
binding protein (CREB), p53, STAT2, STAT3, STAT5a, STAT5b,
STAT6, chaperone heat shock protein (hsp) 27, endothelial nitric
oxide synthase (eNOS), phospholipase C-g1 (PLCg1), cyclin-
dependent kinase inhibitor p27 (Kip1), and proline-rich Akt1 sub-
strate 1 (PRAS). For the analysis, Jurkat T cells were incubated for
20 min with the selected compounds or negative control (1%
DMSO) at 37 C, followed by addition of anti-CD3/C28 (10 mg/ml of
each antibody) and a 5-min incubation at room temperature. The
cells were then lysed, and the arrays were incubated overnight at
4 Cwith lysates obtained from 107 cells for each sample. The arrays
were washed three times with 20 ml of the wash buffer and
incubated for 2 h with the detection antibody cocktail containing
phospho-site-specific biotinylated antibodies. The wash steps were
repeated, after which the arrays were exposed to chemilumines-
cent reagents, and the signal was captured with an Alpha Innotech
FluorChem FC2 imaging system.
4.7. Kinase profiling
Kinase profilingwas performed by KINOMEscan (DiscoveRx, San
Diego, CA, USA) using a panel of 95 protein kinases, as described
previously (Fabian et al., 2005). In brief, kinases were produced and
displayed on T7 phage or expressed in HEK-293 cells. Binding re-
actions were performed at room temperature for 1 h, and the
fraction of kinase not bound to test compound was determined by
capture with an immobilized affinity ligand and quantified by
quantitative polymerase chain reaction. Primary screening at fixed
concentration (10 mM) of a test compound was performed in
duplicate.
4.8. ERK1/2 enzyme-linked immunosorbent assay (ELISA)
Jurkat T cells or isolated human T cells were incubated for
20 min with the selected compounds or negative control (1%
DMSO) at 37 C, followed by addition of either anti-CD3/C28
monoclonal antibodies (10 mg/ml of each antibody) for Jurkat T
cells or Dynabead humanT-activator CD3/CD28 beads for primary T
cells (bead-to-cell ratio 1:1) and a 5-min incubation at room tem-
perature. The cells were lysed with a lysis buffer (R&D Systems),
and the levels of phosphorylated ERK1/2 were measured in the cell
lysates using an ELISA kit (R&D Systems) for human phospho-ERK1
(Thr202/Tyr204)/ERK2 (Thr185/Tyr187). The concentrations of
phospho-ERK1/2 in the cell lysates were determined using a
calibration curvewith recombinant human phosphor-ERK2 (Thr85/
Tyr187).
4.9. Ca2þ mobilization assay
Changes in intracellular Ca2þ concentrations ([Ca2þ]i) were
measured with a FlexStation 3 scanning fluorometer (Molecular
Devices, Sunnyvale, CA, USA). Briefly, cells (human neutrophils,
HL60 cells or Jurkat cells) were suspended in HBSS, loaded with
Fluo-4AM (Invitrogen, Carlsbad, CA, USA) at a final concentration of
1.25 mg/mL and incubated for 30 min in the dark at 37 C. After dye
loading, the cells were washed with HBSS, resuspended in HBSSþ,
separated into aliquots, and aliquoted into the wells of flat-bottom,
half-area well black microtiter plates (2  105 cells/well). Test
compounds diluted in DMSO were added to the wells (final con-
centration of DMSO was 1%), and changes in fluorescence were
monitored (lex¼ 485 nm, lem¼ 538 nm) every 5 s for 240 s at room
temperature after addition of test compounds to evaluate direct
agonist effects. To evaluate inhibitory effects, Jurkat T cells were
pretreated for 20 min with various concentrations of test com-
pound, followed by addition of 10 mg/ml anti-CD3 antibody. The
maximum change in fluorescence, expressed in arbitrary units over
baseline, was used to determine the agonist response. Responses
were normalized to the response induced by anti-CD3 for Jurkat
cells, 5 nM fMLF (neutrophils and FPR1 HL60 cells), or 5 nM
WKYMVM (FPR2 HL60 cells) which was assigned a value of 100%.
Curve fitting (at least five or six points) and calculation of median
effective concentration values (IC50) were performed by nonlinear
regression analysis of the doseeresponse curves generated using
Prism 7 (GraphPad Software, Inc., San Diego, CA, USA).
4.10. Flow cytometry
Jurkat T cells (1  106 cells) were incubated in 100 ml HBSSþ
containing different concentrations of test sesquiterpene lactones
or anti-human CD3 monoclonal antibody for 20 min at 37 C. The
cells were next stained with 1.25 mg/ml of allophycocyanin (APC)-
conjugated anti-human CD3 (Affymetrix eBioscience, San Diego,
CA, USA) or 5 mg/ml of APC-conjugated mouse IgG1 isotype control
(Affymetrix eBioscience) for 30 min on ice. The cells were then
washed, resuspended in flow cytometry buffer, and analyzed using
an LSR II flow cytometer (Becton Dickinson, Sunnyvale, CA, USA)
with FAXDiva Software, v.8.0.1.
4.11. Assessment of compound cytotoxicity
Cytotoxicity was analyzed with a CellTiter-Glo Luminescent Cell
Viability Assay Kit (Promega, Madison, WI, USA). Briefly, Jurkat T
cells or isolated human T cells were cultured at a density of
2 105 cells/well with different concentrations of compound under
investigation for 30 min at 37 C. Following treatment, substrate
was added, and the samples were analyzed with a Fluoroscan
Ascent FL microplate reader.
4.12. Glutathione (GSH) assay
Jurkat cells were plated at 10,000 cells per well in white 96-well
half-area plates. After addition of test sesquiterpene lactones or
vehicle (1% DMSO) negative control, the cells were incubated for
30 min at 37 C, and total GSH was measured using a GSHGlo™
assay (Promega Corp., Madison, WI, USA), according to manufac-
turer's instructions (Harling et al., 2013). Luminescence was
measured with a Fluroscan Ascent FL microplate reader (Thermo
Electron, Waltham, MA).Conflict of interest
The authors have declared that there is no conflict of interest.Acknowledgements
This research was supported in part by National Institutes of
Health IDeA Program COBRE Grant GM110732; USDA National
Institute of Food and Agriculture Hatch project 1009546; Montana
University System Research Initiative: 51040-MUSRI2015-03; and
the Montana State University Agricultural Experiment Station.References
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