Carcinogenesis vol.34 no.2 pp.388–396, 2013
doi:10.1093/carcin/bgs352
Advance Access publication November 3, 2012
Oncrasin targets the JNK-NF-κB axis to sensitize glioma cells to TNFα-induced
apoptosis
Piyushi Gupta, Deobrat Dixit and Ellora Sen*
National Brain Research Centre, Manesar, Haryana 122051, India
*To whom correspondence should be addressed. Tel: +91-124-2845235;
Fax: +91-124-2338910/28;
Email: [email protected]
Resistance of glioblastoma multiforme (GBM) to tumor necrosis
factor (TNF) α-induced apoptosis have been attributed to increased
nuclear factor-kappaB (NF-κB) activation. As we have previously
reported that certain anticancer chemotherapeutics can sensitize
glioma cells to TNFα-induced apoptosis by abrogating NF-κB activation, we investigated the potential of oncrasin in sensitizing glioma
cells to TNFα-induced apoptosis. Oncrasin reduced glioma cell
viability, inhibited TNFα-mediated NF-κB activation and sensitized
cells to TNFα-induced apoptosis. Apoptosis was accompanied by elevated Fas and Fas-associated death domain (FADD) levels, increased
caspase-8 activation and formation of death-inducing signaling complex (DISC). Oncrasin also (i) affected expression of cell cycle regulators, (ii) triggered DNA damage response, (iii) induced G2/M cell
cycle arrest, (iv) decreased telomerase activity, (v) abrogated STAT3
activation and (v) mediated extracellular release of high mobility
group box 1 (HMGB1) along with its increased association with
nucleosomes. Oncrasin-induced apoptosis did not involve mitochondria. Importantly, oncrasin increased c-jun N-terminal kinase (JNK)
phosphorylation and pharmacological inhibition of JNK rescued
oncrasin-induced apoptosis. JNK inhibition prevented oncrasininduced decrease in TNFα-induced NF-κB activity and inhibition
of NF-κB increased JNK phosphorylation in TNFα-treated cells.
Oncrasin induced DISC formation and inhibited anchorage-independent growth of glioma cells in a JNK-dependent manner. By
elucidating the existence of JNK-NF-κB cross-talk that regulates
resistance to TNFα-induced apoptosis, this study has highlighted the
importance of JNK in regulating viability of glioma cells.
Introduction
Constitutive nuclear factor-kappaB (NF-κB) activation in glioblastoma
multiforme (GBM) tumors regulates expression of genes that promote
their growth and survival (1), and inhibition of NF-κB activity induces
glioma cell apoptosis (2). Being an important transcription factor
associated with tumor promotion (3), NF-κB is regarded as a potential antiglioma target (4). Importantly, the resistance of glioma cells to
tumor necrosis factor (TNF) α-mediated apoptosis (5) has been attributed to TNFα-induced NF-κB activation (6,7). We have reported that
certain chemotherapeutic drugs can sensitize glioma cells to TNFαinduced apoptosis by abrogating NF-κB activation (8,9).
Aberrant activation of Ras occurs in GBM (10), and Ras is known to
enhance NF-κB transcriptional activity (11,12). Besides Ras, STAT3
regulates constitutive NF-κB activation in tumors (13). STAT3 is
constitutively expressed in GBM, and STAT3 inhibitors have shown
promise as therapeutics for GBM (14). Importantly, STAT3 promotes
Ras-dependent oncogenic transformation (15). Oncrasin induces apoptosis by affecting cellular targets whose function is dependent on K-Ras
activation (16). NF-κB-mediated inhibition of c-jun N-terminal kinase
Abbreviations: DISC, death-inducing signaling complex; FACS, fluorescence-activated cell sorting; FADD, Fas-associated death domain; GBM,
glioblastoma multiforme; HMGB1, high mobility group box 1; JNK, c-jun
N-terminal kinase; NF-κB, nuclear factor-kappaB; TNF, tumor necrosis factor;
TUNEL, Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling;
SEM, standard error of the mean.
(JNK) activation contributes to resistance to TNFα-induced apoptosis
(17) and NF-κB negatively regulates TNFα-mediated JNK activation
(18). Besides, JNK is a negative regulator of Ras-induced tumorigenesis (19). We have shown that histone deacetylase inhibitor induces
glioma cell apoptosis in a Ras/JNK-dependent manner (20). Because
oncrasin analog mediates apoptosis through simultaneous inhibition of
STAT3 and increase of JNK activation (16), we evaluated the effect of
oncrasin on glioma cell viability in the presence and absence of TNFα.
Our results suggest that in addition to inducing glioma cell apoptosis through increased JNK activation and STAT3 inhibition,
oncrasin sensitizes glioma cells to TNFα-induced apoptosis in a JNKdependent manner. Oncrasin-mediated abrogation of TNFα-induced
NF-κB activation was concurrent with increased JNK activation.
Importantly, increased activation of JNK upon inhibition of NF-κB
activity and vice versa, highlighted the existence of a reciprocal interaction between JNK and NF-κB in glioma cells. Our findings suggest that TNFα might promote pro-survival advantage in glioma cells
through regulation of this JNK-NF-κB axis.
Materials and methods
Cell culture and treatment
Glioblastoma cell lines A172, LN18 and T98G obtained from American Type
Culture Collection were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Life Technologies-Invitrogen). On
attaining semi-confluence, cells were switched to serum-free media, and after
6 h, cells were treated with different combinations of 50 ng/ml TNFα (R&D
Systems) and oncrasin in the presence and absence of pan-caspase inhibitor
(Calbiochem), caspase-8 inhibitor Z-IETD-FMK (Tocris) or JNK (SP600125)
inhibitor. All reagents were purchased from Sigma unless otherwise stated.
Determination of cell viability
Viability of glioma cells treated with different combinations of TNFα
and oncrasin in the presence and absence of 50 µM pan-caspase inhibitor or 100 µM Z-IETD-FMK or 10 µM SP600125 was determined using
the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-­2H-tetrazolium) assay (Promega) as described earlier (21).
Western blot analysis and immunoprecipitation
Protein from whole cell lysates and nuclear extracts from cells treated with
different combinations of TNFα and oncrasin in the presence and absence of
10 μM JNK inhibitor, SP600125, were isolated, and western blot was performed as described (22). Following antibodies were used: pATM (S1981)
(Abcam), ATM (Abcam), high mobility group box 1 (HMGB1) (Abcam), cyclin D1 (Abcam), p27 (Abcam), caspase-8 (BD Pharmingen), Fas-associated
death domain (FADD) (Millipore), Fas (Santa Cruz), p65 (Santa Cruz), IκBα
(Santa Cruz), cyclin E (Santa Cruz), p21 (Santa Cruz), β-actin (Sigma),
γH2AX (Ser 139) (Upstate), pJNK (Thr183/Tyr185), JNK, histone H3 and
H4, pJAK2 (Tyr1007/1008), JAK2 pSTAT3 (Tyr705), STAT3 and pIκKα/β
(Ser176/177). Antibodies were purchased from Cell Signaling unless otherwise mentioned. Immunoprecipitation was performed with Fas antibody to
determine the association of Fas, FADD and caspase-8 in cells treated with
different combinations of TNFα, oncrasin and JNK inhibitor as described (23).
Transfections and luciferase assay
For determining JNK levels in presence of dominant-negative IκB, cells were
transfected with 0.3 µg of DN-IκB construct (Clontech and Takara) in the presence
and absence of TNFα as described (22). In experiments with DN-constructs,
control transfection using the appropriate empty vectors for each construct was
employed. Reporter assay of cells transfected with NF-κB luciferase constructs
and treated with different combinations of TNFα, oncrasin and JNK inhibitor
was performed using Lipofectamine 2000 (Life Technologies-Invitrogen) as
described (22). NF-κB luciferase construct was obtained from Clontech.
Flow cytometric analysis of DNA content
Fluorescence-activated cell sorting (FACS) analysis of DNA content was performed to determine the effect of TNFα and oncrasin on glioma cell cycle progression as described (8). 106 cells per condition were fixed with 70% ethanol
© The Author 2012. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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JNK-NF-κB axis in TNFα-induced apoptosis
Fig. 1. Oncrasin sensitizes glioma cells to TNFα-induced apoptosis and triggers release of extracellular HMGB1. (a) Viability of oncrasin-treated glioma
cells as determined by MTS assay. The graph represents the percentage viable cells of control observed when U87MG, T98G, LN18 and A172 cells were
treated with different concentrations of oncrasin for 24 h. (b) Viability of glioma cells treated with oncrasin in the presence and absence of 50 ng/ml TNFα
for 12 h as determined by MTS assay. The graphs (a and b) represent viable cells expressed as percentage of control. (c) The graph represent percentage of
TUNEL-positive glioma cells treated with oncrasin in the presence and absence of TNFα, as counted from multiple fields. (d) Oncrasin decreases
telomerase activity in glioma cells both in the presence and absence of TNFα. Glioma cells were treated with TNFα in the presence or absence of oncrasin;
and TeloTAGGG Telomerase PCR ELISA was performed. Values (a–d) represent the mean ± standard error of the mean (SEM) from three independent
experiments. *Significant change from control (P < 0.05). (e) Western blot analysis demonstrates oncrasin-mediated release of HMGB1 in the extracellular
milieu of TNFα-treated cells. Immunoprecipitation indicates increased interaction of HMGB1 with nucleosome in oncrasin-treated glioma cells in the
presence of TNFα. Culture media from cells treated with oncrasin or TNFα or both were immunoprecipitated with antibody to HMGB1 and nucleosome
components histone H3 and H4 were analyzed by western blot. A representative blot is shown from two independent experiments with identical results.
and stored at −20°C. The fixed cells were resuspended in propium iodide solution (BD Biosciences) for 20 min at room temperature, and flow cytometric
analysis was carried out using Cell Quest Program on FACS Calibur (Becton
Dickinson).
Determination of mitochondrial-mediated apoptosis
Mitochondrial-mediated apoptosis was detected in cells treated with different combinations of TNFα, oncrasin and JNK inhibitor using MitoLight®
mitochondrial apoptosis detection kit (Millipore) according to manufacturer’s
instruction, and cells were examined by fluorescence microscopy.
Telomerase activity
Telomerase activity of cells treated with TNFα in the presence and absence of
oncrasin was determined using the TeloTAGGG Telomerase PCR ELISA Plus
kit (Roche Diagnostics, Mannheim, Germany) as described (9).
Colony formation in soft agar
The soft agar colony formation ability of glioma cells treated with oncrasin in the presence and absence of TNFα and JNK inhibitor was performed
using CytoSelect™ 96-Well Cell Transformation Assay kit (Cell Biolabs), as
described previously (24).
Statistical analysis
A value of P < 0.05 was considered significant.
Results
Oncrasin sensitizes glioma cells to TNFα-induced apoptosis
Treatment with oncrasin decreased glioma cell viability in a dosedependent manner. Treatment with 20 µM oncrasin for 12 h resulted in
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~20–30% reduction in cell viability. In cells treated with 40 and 50 µM
concentration of oncrasin, the viability was similar to that observed at
20 µM of oncrasin. Although oncrasin had no significant effect on the
viability of U87MG cells at lower concentration, the viability was
significantly reduced at 50 µM concentration (Figure 1a). We, therefore, chose 20 µM dose of oncrasin for further characterization of its
effect on glioma cell death. Combination of some anticancer chemotherapeutic drugs and TNFα has been reported to synergistically kill
certain resistant tumor cells (25). As we have previously reported that
casein kinase 2 inhibitors sensitize glioma cells to TNFα-induced
apoptosis (9), we investigated whether oncrasin could induce apoptosis in TNFα-resistant glioma cells. Though glioma cell lines tested
were resistant to TNFα-induced apoptosis, oncrasin sensitized glioma
cells to TNFα-induced apoptosis. A ~35–40% decrease in cell viability was observed upon treatment of glioma cells with a combination
of TNFα and oncrasin (Figure 1b). To further confirm oncrasin-mediated apoptotic death, Terminal Deoxynucleotidyl Transferase dUTP
Nick End Labeling (TUNEL) assay was performed (Figure 1c). The
~20–30% increase in TUNEL-positive cells observed upon oncrasin
treatment was further elevated to ~35–50% when oncrasin was coadministered with TNFα (Figure 1c).
Oncrasin decreases hTERT activity
Telomerase inhibition is associated with apoptosis in cancers (26).
Because telomerase inhibition is considered a promising antiglioma
therapeutic approach (27), we investigated telomerase activity of
oncrasin-treated glioma cells in the presence and absence of TNFα.
Although TNFα had no significant effect on telomerase activity,
oncrasin abrogated telomerase activity by ~30–50%. However, in the
presence of both oncrasin and TNFα, there was a 50–60% decrease in
telomerase activity (Figure 1d).
Oncrasin mediates HMGB1 release
High mobility group box 1 (HMGB1), an architectural chromatin protein primarily associated with transcription regulation, is released into
extracellular milieu in apoptotic cells. Glioma cells release HMGB1
upon cell death induced by cytotoxic agents (28). Because HMGB1
release is associated with pro-apoptotic killing in glioma cells (28), we
determined the levels of secreted HMGB1 in glioma cells treated with
oncrasin. HMGB1 was not observed in the culture media of either
oncrasin or TNFα-treated cells. However, treatment with combination of oncrasin and TNFα resulted in a dramatic increase in HMGB1
release (Figure 1e). HMGB1 cofractionates with nucleosomes from
apoptotic cells (29). Immunoprecipitation indicated increased interaction of HMGB1 with histones H3 and H4 in the media of cells treated
with oncrasin and TNFα (Figure 1e).
Oncrasin increases DISC formation upon TNFα treatment by
enhancing association of Fas with FADD and elevating active caspase-8 expression
Fas activation is associated with caspase-dependent apoptotic cell
death as Fas stimulation results in the recruitment of adaptor molecule FADD (30). Interaction of Fas–FADD with procaspase-8 forms
the death-inducing signaling complex (DISC), which stimulates caspase-8 activation that subsequently results in apoptosis through caspase-3 activation. We performed western blot analysis to determine
the levels of FADD and Fas in cells treated with oncrasin in the presence and absence of TNFα. Although Fas and FADD levels remained
unaffected upon TNFα treatment, an increase in Fas and FADD
expression was observed in cells treated with oncrasin either alone or
in combination with TNFα (Figure 2a). Although the expression of
active caspase-8 was comparable between control, TNFα− and oncrasin-treated cells, a dramatic increase in active caspase-8 was observed
Fig. 2. Oncrasin induces formation of DISC in TNFα-treated glioma cells. (a) Fas, FADD and caspase-8 expressions in glioma cells treated with oncrasin in
the presence and absence of TNFα for 12 h, as demonstrated by western blot analysis. A representative blot is shown from two independent experiments with
identical results. Blots were reprobed for β-actin to establish equivalent loading. (b) Immunoprecipitation indicates increased DISC formation in oncrasin-treated
glioma cells both in the presence and absence of TNFα. Lysate from cells treated with oncrasin or TNFα or both were immunoprecipitated with antibody to Fas
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JNK-NF-κB axis in TNFα-induced apoptosis
Fig. 3. Oncrasin affects genes associated with cell cycle regulation, DNA damage response and induces cell cycle arrest. Altered expression of molecules
associated with (a) cell cycle progression and (b) DNA damage response in cells treated with oncrasin in the presence and absence of TNFα as demonstrated
by western blot analysis. Figures (a and b) are representative blots shown from three independent experiments with identical results. Blots were reprobed with
β-actin or C23 to establish equivalent loading. (c) Oncrasin induces G2/M phase cell cycle arrest in glioma cells. FACS analysis was performed on T98G cells
treated with different combinations of TNFα and oncrasin. Inset indicates percentage of cells in G1, S and G2/M phase of the cell cycle. C, T, and O denote
control, TNFα and oncrasin, respectively.
upon treatment with a combination of TNFα and oncrasin (Figure 2a).
Coimmunoprecipitation was performed to determine whether elevated Fas, FADD and caspase-8 levels in TNFα− and oncrasin-treated
cells were accompanied with increased DISC formation. Although
the interaction of Fas, FADD and caspase-8 in TNFα-treated cells
was comparable with the control, an increased interaction between
them was observed in cells treated with either oncrasin or a combination of TNFα and oncrasin (Figure 2b). Oncrasin-induced apoptosis
is accompanied by activation of caspase-8 in lung carcinoma cells
(16). To confirm the involvement of caspase-8 in oncrasin-induced
apoptosis, the viability of cells treated with TNFα and oncrasin in
the presence and absence of caspase-8 inhibitor was determined. The
cytotoxic effect of oncrasin on glioma cells was caspase dependent
as treatment with Pan-caspase inhibitor as well as caspase-8 inhibitor rescued glioma cells from oncrasin-induced apoptosis both in the
presence and absence of TNFα (Figure 2c).
←
and DISC components; Fas, FADD and cleaved caspase-8 were analyzed by western blot. IgG levels are shown to establish equivalent loading. Representative
blot is shown from two independent experiments with identical results. (c) Oncrasin-induced apoptosis is caspase dependent. The graph represents viable cells,
percentage of control, observed when glioma cells were treated with different combinations of TNFα, oncrasin, caspase-8 and pan-caspase inhibitor for 12 h,
as determined by MTS assay. Values represent the mean ± SEM from three independent experiments. *Significant change from control (P < 0.05). #Significant
change from oncrasin-treated cells (P < 0.05).
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P.Gupta, D.Dixit and E.Sen
Fig. 4. Oncrasin abrogates NF-κB activity and increases JNK phosphorylation in TNFα-treated glioma cells. (a) Western blot analysis demonstrates the levels of
p65, pIκKα/β and IκBα in glioma cells, treated with either TNFα or oncrasin or both for 12 h. (b) Decrease in TNFα-mediated NF-κB transcriptional activation
in oncrasin-treated glioma cells. Glioma cells transfected with NFκB reporter constructs were treated with oncrasin in the presence and absence of TNFα for
6 h, and luciferase reporter assay was performed to determine NF-κB activity. The graph represents fold change in NF-κB luciferase activity over control. Values
represent the mean ± SEM from three independent experiments. *Significant change from control (P < 0.05). #Significant change from TNFα-treated cells
(P < 0.05). (c) Oncrasin increases JNK phosphorylation both in the presence and absence of TNFα, as demonstrated by western blot analysis. Figures (a and c)
representative blot is shown from three independent experiments with identical results. Blots were reprobed for C23 or β-actin to establish equivalent loading. (d)
JNK inhibition rescues oncrasin-induced apoptosis both in the presence and absence of TNFα. The graph represents the percentage of viable cells of control as
determined by MTS assay, observed when glioma cells were treated with different combinations of oncrasin, TNFα and JNK inhibitor SP600125 for 12 h. Values
represent the mean ± SEM from three independent experiments. *Significant change from control (P < 0.05). #Significant change from TNFα and oncrasintreated cells (P < 0.05).
Oncrasin affects molecules associated with cell cycle progression
and DNA damage response
As oncrasin significantly inhibited the proliferation of glioma cells,
we determined the expression of molecules associated with cell cycle
progression in these cells. Although treatment with TNFα alone had
no effect on the expression of cell cycle regulators p21, p27, cyclin
D1 and E, oncrasin increased p21 and p27 levels and decreased cyclin
D1 and E levels in glioma cells both in the presence and absence of
TNFα (Figure 3a).
Oncrasin inhibits cell cycle progression
As oncrasin altered the expression of cell cycle regulators, FACS
analysis was performed to determine cell cycle profile in cells treated
with different combinations of oncrasin and TNFα. Although TNFα
had no significant effect on cell cycle progression, oncrasin increased
accumulation of cells at the G2/M phase (Figure 3c). A 8.21, 6.98,
15.23 and 19.13% cells at G2/M phase was observed in control, TNFα,
oncrasin, and TNFα− and oncrasin-treated T98G cells, respectively.
Similar trend was observed in A172 cells (data not shown).
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Oncrasin induces increased DNA damage signaling response
HMGB1 is involved in DNA damage response and enhances DNA
repair following damage (31). Because phosphorylation of histone
H2AX on Ser 139 is a sensitive reporter of DNA double-strand breaks
and as ATM is the main transducer of the double-strand break response
(32), we investigated γH2AX and pATM expression in oncrasintreated cells. Although treatment with TNFα or oncrasin alone had
no effect on pATM level, an increase in pATM was observed upon
treatment with a combination of both (Figure 3b). Although oncrasin
elevated γH2AX levels in the cell lines in combination with TNFα
(Figure 3b), it was capable of inducing γH2AX in T98G even in the
absence of TNFα.
Oncrasin abrogates TNFα-induced NF-κB activation in glioma
As we have reported that TNFα-induced NF-κB transcriptional activity
contributes to resistance to apoptosis in glioma cells (8), we determined
whether downregulation of NF-κB activity in oncrasin-treated cells could
have contributed to sensitivity to TNFα-induced death. IKBα, which is
the inhibitory subunit of NF-kB, is phosphorylated by IKKα/β, which
JNK-NF-κB axis in TNFα-induced apoptosis
Fig. 5. Oncrasin targets NF-κB-JNK axis in TNFα-treated glioma cells. (a) Oncrasin abrogates TNFα-induced NF-κB activation in a JNK-dependent manner.
Glioma cells transfected with NFκB reporter constructs were treated with different combinations of oncrasin and TNFα in the presence and absence of SP600125
for 6 h, and luciferase reporter assay was performed to determine NF-κB activity. The graph represents fold change in NF-κB luciferase activity over control.
Values represent the mean ± SEM from three independent experiments. *Significant change from control (P < 0.05), ΨSignificant change from TNFα-treated
cells, #Significant change from oncrasin-treated cells (P < 0.05). (b) TNFα regulates JNK activation in a NF-κB-dependent manner. Western blot demonstrates
increased JNK phosphorylation in TNFα-treated glioma cells transfected with DN-IκB as compared with mock-transfected control. The figure is representative of
three independent experiments. Blots were reprobed for β-actin to establish equivalent loading.
leads to IKBα degradation and increased nuclear import of NF-κB (p65)
(33). TNFα is known to increase phosphorylation of IKK, leading to
decreased IKBα levels and increased nuclear p65. Oncrasin mediated
decrease in TNFα-induced pIκKα/β level in glioma cells (Figure 4a)
was accompanied with increase in IκBα (nuclear factor of kappa light
polypeptide gene enhancer in B-cells inhibitor, alpha) and decrease in
NF-κB levels (Figure 4a). Moreover, TNFα mediated increase in NF-κB
luciferase activity was abrogated in the presence of oncrasin (Figure 4b).
Pro-apoptotic effects of oncrasin are mediated by JNK pathway
JNK is a member of the mitogen-activated protein kinase family that is
activated in response to a variety of chemical stresses. Activation of JNK
sensitizes glioma cells to apoptosis (34) by triggering apoptotic cascades
(35). Mitogen-activated protein kinase kinase is involved in JNK
activation, and the guanosine triphosphate-binding protein, Ras, plays
a crucial role in MAP kinase activation (36). Besides, we have reported
the involvement of JNK activation in glioma cell apoptosis (20). JNK
suppresses Ras-stimulated tumor formation (19). Oncrasin has been
shown to be effective against tumors bearing K-Ras mutations (16). As
the antitumor activity of oncrasin analog is dependent on JNK activation
(37), we investigated the phosphorylation status of JNK in oncrasintreated cells. Treatment with oncrasin increased JNK activation in a
time- and dose-dependent manner (Supplementary Figure 1, available
at Carcinogenesis Online). Because NF-κB negatively regulates TNFαmediated JNK activation (18) and as oncrasin abrogated TNFα-induced
NF-κB activation, the levels of pJNK in cells treated with oncrasin both
in the presence and absence of TNFα was determined. Although TNFα
had no effect on pJNK, increased JNK phosphorylation was observed
in oncrasin-treated cells both in the presence and absence of TNFα
(Figure 4c). The pJNK levels in oncrasin-treated cells in the presence
and absence of TNFα was comparable in LN18 cells. Despite pJNK
levels being less in A172 and T98G cells treated with combination of
oncrasin and TNFα as compared with that treated with oncrasin alone,
the pJNK levels was still much higher than in untreated or TNFα-treated
cells (Figure 4c). Because NF-κB antagonizes TNFα−mediated JNK
activation (18), residual NF-κB level could have possibly prevented
oncrasin from maximally activating JNK in the presence of TNFα in
A172 and T98G cells.
We next investigated the role of elevated pJNK in inducing apoptosis in oncrasin-treated cells, by determining the viability of glioma
cells treated with different combinations of TNFα and oncrasin in the
presence and absence of JNK inhibitor SP600125. JNK inhibition significantly rescued glioma cells from apoptosis induced by oncrasin
alone or in combination with TNFα (Figure 4d). Thus, increased JNK
activation triggers the antiproliferative effect of oncrasin in the presence of TNFα.
Non-involvement of mitochondria in oncrasin-triggered apoptosis
Sustained JNK activation that occurs in the absence of NF-κB initiates apoptosis through induction of mitochondrial death pathway
(38). We, therefore, investigated whether oncrasin-induced JNK activation triggers apoptosis through impairment of mitochondrial function. Accumulation of MitoLight red fluorescence, which is indicative
of intact mitochondrial function, was comparable between oncrasin-treated and control cells (Supplementary Figure 2a, available at
Carcinogenesis Online). Moreover, MitoLight accumulation was
unaffected by JNK inhibition (Supplementary Figure 2a, available
at Carcinogenesis Online). As JNK-mediated mitochondria-induced
apoptosis involves antiapoptotic proteins Bcl-2 (39) and cytochrome
c, we determined Bcl-2 and cytochrome c levels in oncrasin-treated
cells. No change in the levels of both Bcl-2 and cytochrome c was
observed upon oncrasin treatment both in the presence and absence of
JNK inhibitors (Supplementary Figure 2b, available at Carcinogenesis
Online).
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P.Gupta, D.Dixit and E.Sen
Fig. 6. Oncrasin induces DISC formation and inhibits anchorage-independent growth of glioma cells in a JNK-dependent manner. (a) Oncrasin-induced DISC
formation in TNFα-treated glioma cells is JNK dependent. Lysate from cells treated with different combinations of oncrasin, TNFα and JNK inhibitor for 12 h
were immunoprecipitated with antibody to Fas and DISC components; Fas, FADD and caspase-8 were analyzed by immunoblotting. A representative blot is
shown from two independent experiments with identical results. IgG levels are shown to establish equivalent loading. Inset shows pJNK levels in cells treated
with oncrasin and TNFα in the presence and absence of JNK inhibitor. (b) Oncrasin mediated decrease in pSTAT3 is JNK independent. Western blot analysis
demonstrates pSTAT3/ pJAK2 levels in glioma cells, treated with either TNFα or oncrasin or both in the presence of JNK inhibitor for 12 h. A representative blot
is shown from three independent experiments with identical results. Blots were reprobed for β-actin to establish equivalent loading. (c) The ability of oncrasin
to decrease the ability of glioma cells to form colonies in soft agar was reversed in the presence of JNK inhibitor. Soft agar assay was performed on cells treated
with different combinations of TNFα, oncrasin and JNK inhibitor SP600125 for 8 days. The graph indicates the percentage of colonies formed under different
conditions tested. Values represent the means ± SEM from three individual experiments. *Significant decrease from control and TNFα-treated cells. #Significant
increase from oncrasin-treated cells (P < 0.05). (d) Schematic depicting oncrasin-mediated sensitization of glioma cells to TNFα-induced apoptosis through
regulation of JNK-NF-κB axis.
Oncrasin mediated decrease in TNFα-induced NF-κB activation
is JNK dependent
NF-κB pathway negatively modulates TNFα-mediated JNK
activation (18). Besides, suppressed NF-κB and sustained JNK
activation sensitizes parthenolide to TNFα-induced apoptosis in
cancer cells (40). As oncrasin-mediated abrogation of TNFα-induced
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NF-κB activation was accompanied by increased JNK activation, we
questioned the involvement of JNK in NF-κB activation. The ability
of oncrasin to downregulate TNFα-induced NF-κB activation was
abolished in the presence of JNK inhibitor (Figure 5a). Importantly,
treatment with JNK inhibitor alone resulted in ~2–3-fold increase in
TNFα-induced NF-κB activity. This suggests that JNK downregulates
JNK-NF-κB axis in TNFα-induced apoptosis
NF-κB activity, and that oncrasin mediated decrease in TNFα-induced
NF-κB activation is JNK dependent.
TNFα-induced NF-κB prevent JNK activation
IKK negatively modulates JNK activity through induction of NF-κB
target genes (18). As oncrasin-induced abrogation of TNFα-mediated
NF-κB activation was concomitant with increased JNK activation, we
investigated whether low JNK phosphorylation level in TNFα-treated
cells was a consequence of heightened NF-κB activation. Interestingly,
an increase in JNK phosphorylation was observed in TNFαα-treated
cells transfected with DN-IκB (Figure 5b). This together with the
ability of JNK inhibitor to abrogate oncrasin-induced inhibition of
TNFα-induced NFκB activity suggests the existence of NFκB-JNK
axis that regulates the resistance of glioma cells to TNFα-induced
apoptosis.
JNK regulates DISC formation in glioma cells
Activation of JNK and its dependent Fas-mediated death signaling
sensitizes prostate cancer cells to etoposide-induced apoptosis
(41). Moreover, JNK activation regulates FADD phosphorylation
that subsequently sensitizes cancer cells to drug-induced apoptosis
(42). Importantly, the role of Fas activation in JNK-mediated cell
death in glioma cells is known (43). We, therefore, investigated
whether oncrasin-induced JNK activation affects DISC formation.
The increased interaction between DISC components—Fas,
FADD and caspase-8 observed in oncrasin-treated cells both in the
presence and absence of TNFα, was abrogated upon JNK inhibition
(Figure 6a). Thus, oncrasin-induced DISC formation in glioma
cells is JNK dependent (Figure 6a).
Oncrasin mediated decrease in STAT3 phosphorylation is JNK
independent
The antitumor activity of oncrasin analog is mediated by STAT3
inhibition in addition to JNK activation (37). Heightened STAT3
activation plays a critical role in glioblastoma, and STAT3 inhibitors have shown promise as therapeutics for GBM (14). Also, targeting STAT3/JAK2 sensitizes glioma cell to apoptosis (44). We,
therefore, investigated the effect of oncrasin on STAT3/JAK2 activation. Although treatment with TNFα had no effect on STAT3
or JAK2 activation, a decrease in pSTAT3 and pJAK2 levels was
observed in glioma cells treated with oncrasin both in the presence
and absence of TNFα. This decrease in STAT3 and JAK2 phosphorylation was independent of JNK as the decrease in STAT3 phosphorylation remained unaffected in the presence of JNK inhibitor
(Figure 6b).
Oncrasin mediated decrease in colony-forming ability of glioma
cells is JNK dependent
Although inhibition of JNK prevents epidermal growth factor-mediated anchorage-independent growth and protects glioma cells from
cell death, JNK attenuates anchorage-independent growth in insulinlike growth factor-treated breast cancer cells (45). Because this dual
function of JNK in terms of its ability to support anchorage-independent growth could be context dependent, we determined the ability of
oncrasin to form colonies in soft agar. Although TNFα had no significant effect on colony-forming ability of glioma cells in soft agar,
oncrasin reduced the anchorage-independent growth both in the presence and absence of TNFα. This decreased colony-forming ability of
glioma cells in the presence of oncrasin was reverted to control levels,
in the presence of JNK inhibitor (Figure 6c).
Discussion
We have reported the crucial involvement of TNFα-induced NF-κB
activation in mediating resistance of glioma cells to TNFα-induced
apoptosis (8,9). Here, we show that oncrasin sensitizes glioma cells
to TNFα-induced apoptosis by inhibiting TNFα-induced NF-κB
transcriptional activation. This decrease in NF-κB activation was
concomitant with increased JNK activation, and pharmacological
inhibition of JNK abrogated oncrasin-induced apoptotic cell death.
Oncrasin triggered release of HMGB1 in TNFα-treated cells, which
was detected in complex with nucleosomes. This ability of oncrasin to
impair resistance to TNFα-induced apoptosis was accompanied with
(i) arrest in G2/M phase of the cell cycle (ii) decreased STAT3/JAK2
activation and (iii) decreased telomerase activity (Figure 6d).
Although conflicting reports exist regarding the involvement of
JNK in apoptosis, evidences indicate that sustained JNK activation
triggers apoptosis (46). Although JNK activation in the absence of
NF-κB initiates apoptosis through mitochondrial death pathway (38),
oncrasin-mediated apoptosis did not involve mitochondria. Although
JNK activation protects glioma cells from stress or chemotherapeuticinduced apoptosis (47), other studies have indicated that attenuation
of TNFα-induced NF-κB activation leads to JNK-mediated glioma
cell death (43). NF-κB activation is known to suppress TNFα-induced
apoptosis through inhibition of caspase-8 and JNK activation (18).
Oncrasin-mediated suppression of NF-κB activation was concurrent
with increased JNK activation. This agrees with previous findings that
absence of NF-κB-mediated inhibition of JNK activation contributes to
TNFα-induced apoptosis (18). This negative effect of TNFα-induced
NF-κB on oncrasin-induced JNK activation should have, therefore,
reduced the apoptosis-inducing ability of oncrasin in the presence of
TNFα. However, it is probable that other mechanisms, which sensitize
cells to TNFα-induced apoptosis independent of JNK, contributed to
the increased apoptosis in oncrasin and TNFα-treated cells. Besides,
co-operativity between NF-κB and STAT3 regulates genes associated
with tumor progression (48). It is possible that simultaneous disruption
of both STAT3 (independent of JNK) and NF-κB activation could
trigger greater cell death upon treatment with both oncrasin and TNFα.
NF-κB activation is known to protect leukemic eosinophils from
Fas-mediated apoptosis (49). Also, JNK-mediated Fas (41) and FADD
(42)-dependent sensitization of cancer cells to drug-induced apoptosis
is known. Increased JNK activation in response to oncrasin promoted
DISC formation and caspase-8 activation leading to cell death
(Figure 6d). In addition, oncrasin inhibited anchorage-independent
growth of glioma cells in a JNK-dependent manner. Though
JNK-mediated regulation of STAT3 activation is associated with
doxorubicin resistance in cancer cell lines (50), oncrasin-mediated
decrease in STAT3 activation was JNK independent. Overall, this
study has highlighted the existence of an NF-κB-JNK axis in TNFαtreated glioma cells for the first time and has raised the possibility that
chemotherapeutic regimen, which modulates NF-κB-JNK balance,
might serve as an effective antiglioma therapeutic strategy.
Supplementary material
Supplementary Figures 1 and 2 can be found at http://carcin.oxfordjournals.org/
Funding
Department of Biotechnology (BT/PR12924/Med/30/235/09 to E.S.);
Council of Scientific and Industrial Research (CSIR, Government of
India) P.G. and D.D.
Conflict of Interest Statement: None declared.
References
1.Nozell,S. et al. (2008) The ING4 tumor suppressor attenuates NF-kappaB
activity at the promoters of target genes. Mol. Cell. Biol., 28, 6632–6645.
2.Robe,P.A. et al. (2004) In vitro and in vivo activity of the nuclear factorkappaB inhibitor sulfasalazine in human glioblastomas. Clin. Cancer Res.,
10, 5595–5603.
3.Aggarwal,B.B. (2004) Nuclear factor-kappaB: the enemy within. Cancer
Cell, 6, 203–208.
395
P.Gupta, D.Dixit and E.Sen
4.Sen,E. (2011) Targeting inflammation-induced transcription factor activation: an open frontier for glioma therapy. Drug Discov. Today, 16,
1044–1051.
5.Tsujimoto,M. et al. (1985) Tumor necrosis factor: specific binding and
internalization in sensitive and resistant cells. Proc. Natl. Acad. Sci. U.S.A.,
82, 7626–7630.
6.Beg,A.A. et al. (1996) An essential role for NF-kappaB in preventing TNFalpha-induced cell death. Science, 274, 782–784.
7.Wang,C.Y. et al. (1996) TNF- and cancer therapy-induced apoptosis:
potentiation by inhibition of NF-kappaB. Science, 274, 784–787.
8.Sharma,V. et al. (2008) Ebselen sensitizes glioblastoma cells to tumor
necrosis factor (TNFalpha)-induced apoptosis through two distinct pathways involving NF-kappaB downregulation and Fas-mediated formation
of death inducing signaling complex. Int. J. Cancer, 123, 2204–2212.
9.Dixit,D. et al. (2012) Inhibition of casein kinase-2 induces p53-dependent
cell cycle arrest and sensitizes glioblastoma cells to tumor necrosis factor
(TNFα)-induced apoptosis through SIRT1 inhibition. Cell Death Dis., 3,
e271.
10. Feldkamp,M.M. et al. (1999) Expression of activated epidermal growth
factor receptors, Ras-guanosine triphosphate, and mitogen-activated protein kinase in human glioblastoma multiforme specimens. Neurosurgery,
45, 1442–1453.
11. Finco,T.S. et al. (1997) Oncogenic Ha-Ras-induced signaling activates
NF-kappaB transcriptional activity, which is required for cellular transformation. J. Biol. Chem., 272, 24113–24116.
12. Norris,J.L. et al. (1999) Oncogenic Ras enhances NF-kappaB transcriptional activity through Raf-dependent and Raf-independent mitogen-activated protein kinase signaling pathways. J. Biol. Chem., 274, 13841–13846.
13. Lee,H. et al. (2009) Persistently activated Stat3 maintains constitutive
NF-kappaB activity in tumors. Cancer Cell, 15, 283–293.
14. Rahaman,S.O. et al. (2002) Inhibition of constitutively active Stat3 suppresses proliferation and induces apoptosis in glioblastoma multiforme
cells. Oncogene, 21, 8404–8413.
15. Gough,D.J. et al. (2009) Mitochondrial STAT3 supports Ras-dependent
oncogenic transformation. Science, 324, 1713–1716.
16. Guo,W. et al. (2008) Identification of a small molecule with synthetic
lethality for K-ras and protein kinase C iota. Cancer Res., 68, 7403–7408.
17. Tang,F. et al. (2002) The absence of NF-kappaB-mediated inhibition of
c-Jun N-terminal kinase activation contributes to tumor necrosis factor
alpha-induced apoptosis. Mol. Cell. Biol., 22, 8571–8579.
18. Tang,G. et al. (2001) Inhibition of JNK activation through NF-kappaB target genes. Nature, 414, 313–317.
19. Kennedy,N.J. et al. (2003) Suppression of Ras-stimulated transformation
by the JNK signal transduction pathway. Genes Dev., 17, 629–637.
20. Sharma,V. et al. (2010) HDAC inhibitor, scriptaid, induces glioma cell
apoptosis through JNK activation and inhibits telomerase activity. J. Cell
Mol. Med., 14, 2151–2161.
21. Koul,N. et al. (2010) Bicyclic triterpenoid Iripallidal induces apoptosis and
inhibits Akt/mTOR pathway in glioma cells. BMC Cancer, 10, 328.
22. Sharma,V. et al. (2011) Ras regulates interleukin-1β-induced HIF-1α transcriptional activity in glioblastoma. J. Mol. Med., 89, 123–136.
23. Tewari,R. et al. (2008) Involvement of miltefosine-mediated ERK activation in glioma cell apoptosis through Fas regulation. J. Neurochem., 107,
616–627.
24. Dixit,D. et al. (2009) Manumycin inhibits STAT3, telomerase activity, and
growth of glioma cells by elevating intracellular reactive oxygen species
generation. Free Radic. Biol. Med., 47, 364–374.
25. Safrit,J.T. et al. (1992) Sensitivity of resistant human tumor cell lines to
tumor necrosis factor and adriamycin used in combination: correlation
between down-regulation of tumor necrosis factor-messenger RNA induction and overcoming resistance. Cancer Res., 52, 6630–6637.
26. Zhang,X. et al. (1999) Telomere shortening and apoptosis in telomeraseinhibited human tumor cells. Genes Dev., 13, 2388–2399.
27. Mukai,S. et al. (2000) 2-5A antisense telomerase RNA therapy for intracranial malignant gliomas. Cancer Res., 60, 4461–4467.
396
28. Candolfi,M. et al. (2009) Release of HMGB1 in response to proapoptotic
glioma killing strategies: efficacy and neurotoxicity. Clin. Cancer Res., 15,
4401–4414.
29. Urbonaviciute,V. et al. (2008) Induction of inflammatory and immune
responses by HMGB1-nucleosome complexes: implications for the pathogenesis of SLE. J. Exp. Med., 205, 3007–3018.
30.Ashkenazi,A. (2002) Targeting death and decoy receptors of the tumournecrosis factor superfamily. Nat. Rev. Cancer, 2, 420–430.
31. Lange,S.S. et al. (2008) High mobility group protein B1 enhances DNA
repair and chromatin modification after DNA damage. Proc. Natl. Acad.
Sci. U.S.A., 105, 10320–10325.
32. Lavin,M.F. et al. (2007) ATM activation and DNA damage response. Cell
Cycle, 6, 931–942.
33. Häcker,H. et al. (2006) Regulation and function of IKK and IKK-related
kinases. Sci. STKE, 2006, re13.
34. Xia,S. et al. (2007) Ribotoxic stress sensitizes glioblastoma cells to death
receptor induced apoptosis: requirements for c-Jun NH2-terminal kinase
and Bim. Mol. Cancer Res., 5, 783–792.
35. Jin,H.O. et al. (2006) Up-regulation of Bak and Bim via JNK downstream
pathway in the response to nitric oxide in human glioblastoma cells. J. Cell.
Physiol., 206, 477–486.
36. Minden,A. et al. (1994) Differential activation of ERK and JNK
mitogen-activated protein kinases by Raf-1 and MEKK. Science, 266,
1719–1723.
37. Guo,W. et al. (2011) Antitumor activity of a novel oncrasin analogue
is mediated by JNK activation and STAT3 inhibition. PLoS ONE, 6,
e28487.
38. Liu,H. et al. (2002) NF-kappaB inhibition sensitizes hepatocytes to
TNF-induced apoptosis through a sustained activation of JNK and c-Jun.
Hepatology, 35, 772–778.
39.Schroeter,H. et al. (2003) c-Jun N-terminal kinase (JNK)-mediated modulation of brain mitochondria function: new target proteins for JNK signalling in
mitochondrion-dependent apoptosis. Biochem. J., 372(Pt 2), 359–369.
40. Zhang,S. et al. (2004) Suppressed NF-kappaB and sustained JNK activation contribute to the sensitization effect of parthenolide to TNFalpha-induced apoptosis in human cancer cells. Carcinogenesis, 25,
2191–2199.
41. Shimada,K. et al. (2003) c-Jun NH2-terminal kinase-dependent Fas activation contributes to etoposide-induced apoptosis in p53-mutated prostate
cancer cells. Prostate, 55, 265–280.
42. Shimada,K. et al. (2004) Phosphorylation of FADD is critical for sensitivity to anticancer drug-induced apoptosis. Carcinogenesis, 25, 1089–1097.
43. Kesanakurti,D. et al. (2011) Suppression of MMP-2 attenuates TNF-α
induced NF-κB activation and leads to JNK mediated cell death in glioma.
PLoS ONE, 6, e19341.
44. Lo,H.W. et al. (2008) Constitutively activated STAT3 frequently coexpresses with epidermal growth factor receptor in high-grade gliomas and
targeting STAT3 sensitizes them to Iressa and alkylators. Clin. Cancer Res.,
14, 6042–6054.
45. Mamay,C.L. et al. (2003) An inhibitory function for JNK in the regulation
of IGF-I signaling in breast cancer. Oncogene, 22, 602–614.
46. Deng,Y. et al. (2003) A JNK-dependent pathway is required for TNFalphainduced apoptosis. Cell, 115, 61–70.
47. Antonyak,M.A. et al. (2002) Elevated JNK activation contributes to the
pathogenesis of human brain tumors. Oncogene, 21, 5038–5046.
48. Atkinson,G.P. et al. (2010) NF-kappaB and STAT3 signaling in glioma:
targets for future therapies. Expert Rev. Neurother., 10, 575–586.
49. Qin,Y. et al. (2002) Fas resistance of leukemic eosinophils is due to activation of NF-kappa B by Fas ligation. J. Immunol., 169, 3536–3544.
50. Kim,J.H. et al. (2010) Jnk signaling pathway-mediated regulation of Stat3
activation is linked to the development of doxorubicin resistance in cancer
cell lines. Biochem. Pharmacol., 79, 373–380.
Received May 15, 2012; revised October 12, 2012; accepted October 30,
2012