Sorafenib is the standard targeted drug used to treat hepatocellular
carcinoma (HCC), but the therapeutic response between individuals varies
markedly. Recently, cytokine-based immunotherapy has been a topic of intense
discussion in the fight against cancer. The aim of this study was to explore
whether cytokine IL-2 could augment the anti-tumour effects of sorafenib on
HCC.
Methods
HepG2 and Huh7 cells were co-treated with sorafenib and IL-2 in
vitro, and cellular viability and death were analysed through the MTT assay,
TUNEL staining, LDH release assay, and western blotting. Mitochondrial function
was measured via ELISA, immunofluorescence, and western blotting. Pathway
blockers were used to establish the role of the JNK-TAZ pathways in regulating
cancer cell phenotypes.
Results
Our data demonstrated that sorafenib treatment increased the HCC
apoptotic rate, repressed cell proliferation, and inhibited migratory responses,
and these effects were enhanced by IL-2 supplementation. Mechanistically, the
combination of IL-2 and sorafenib interrupted mitochondrial energy metabolism by
downregulating mitochondrial respiratory proteins. In addition, IL-2 and
sorafenib co-treatment promoted mitochondrial dysfunction, as evidenced by the
decreased mitochondrial potential, elevated mitochondrial ROS production,
increased leakage of mitochondrial pro-apoptotic factors, and activation of the
mitochondrial death pathway. A molecular investigation revealed that
mitochondrial fission was required for the IL-2/sorafenib-mediated mitochondrial
dysfunction. Mitochondrial fission was triggered by sorafenib and was largely
amplified by IL-2 supplementation. Finally, we found that IL-2/sorafenib
regulated mitochondrial fission via the JNK-TAZ pathways; blockade of the
JNK-TAZ pathways abrogated the inhibitory effects of L-2/sorafenib on cancer
survival, growth and mobility.
Conclusions
Altogether, these data strongly suggest that additional
supplementation with IL-2 enhances the anti-tumour activity of sorafenib by
promoting the JNK-TAZ-mitochondrial fission axis. This finding will pave the way
for new treatment modalities to control HCC progression by optimizing
sorafenib-based therapy.
The editor has retracted this article [1] because Figures 2
and 4 contain duplicated and modified figures from [2, 3, 4]. The data reported in
this article are therefore unreliable.
• Figure 2m in [1] contains figures that have been
duplicated and modified from Figure 2a in [2]
• Figure 2m in [1] contains figures that have been
duplicated and modified from Figure 6j in [3]
• Figure 4g in [1] is identical to Figure 4f in
[4]
• Figure 4i in [1] contains figures that are identical to
Figure 4h in [4]
None of the authors have responded to any correspondence
from the editor/publisher about this retraction.
1. Ding, X., Sun, W. & Chen, J. IL-2 augments the
sorafenib-induced apoptosis in liver cancer by promoting mitochondrial fission and
activating the JNK/TAZ pathway. Cancer Cell Int 18, 176 (2018) https://doi.org/10.1186/s12935-018-0671-3
3. Jieensinue, S., Zhu, H., Li, G. et al. Tanshinone IIA
reduces SW837 colorectal cancer cell viability via the promotion of mitochondrial
fission by activating JNK-Mff signaling pathways. BMC Cell Biol 19, 21 (2018) https://doi.org/10.1186/s12860-018-0174-z
4. Lijuan Zhang, Shuping Li, Rong Wang, Changyuan Chen, Wen
Ma, Hongyi Cai. Cytokine augments the sorafenib-induced apoptosis in Huh7 liver
cancer cell by inducing mitochondrial fragmentation and activating MAPK-JNK
signalling pathway. Biomedicine & Pharmacotherapy 110: 213-223 (2019), https://doi.org/10.1016/j.biopha.2018.11.037
transcriptional co-activator with PDZ-binding motif
Cyt-c
cytochrome c
mPTP
mitochondrial permeability transition pore
IL-2
interleukin-2
HCC
hepatocellular carcinoma
Background
Hepatocellular carcinoma (HCC), the sixth most common cancer worldwide,
accounts for ~ 5.7% of the overall incidence of cancer [1]. Several risk factors have been associated
with the development of HCC, including (but not limited to) hepatitis B infection,
alcohol consumption, diabetes mellitus and smoking [2]. Despite advancements in uncovering the molecular aetiology of
HCC, treatments for HCC are still unsatisfactory; the 5-year survival rate remains
approximately 26% in patients receiving standard chemotherapy and/or radiotherapy
[3].
Targeted therapy has been tested in several clinical trials and has
been proven to provide a survival advantage for patients with HCC. Sorafenib is the
first approved targeted therapy drug and is also the first-line FDA-approved
tyrosine kinase inhibitor, improving the median overall survival time from 7.9 to
10.7 months in patients with HCC [4]. At
the molecular level, sorafenib represses Raf kinase, a key protein mediating cancer
proliferation [5]. Sorafenib also
suppresses angiogenesis by modulating the Ras/Raf/MEK/ERK signalling pathway and
VEGFR [6]. Notably, the tolerance and
efficacy of sorafenib in Child–Pugh B patients have not been determined, and several
reports argue that sorafenib does not seem to be an option for these patients
[7]. Furthermore, the clinical
benefit of sorafenib treatment is limited to an overall increase in survival time of
3 months [8]. Thus, these data indicate
the therapeutic potential of sorafenib against the progression of HCC but suggest
that it is also clinically necessary to optimize sorafenib-based treatment by
combining it with other therapeutic strategies, such as immunotherapy.
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Immunotherapy has demonstrated great promise in specifically killing
cancer cells by multiple mechanisms [9].
Cytokine-based immunotherapy is currently a topic of intense discussion in the fight
against cancer [10]. For example,
supplementation with IL-7 has been found to repress the progression of acute
lymphoblastic leukaemia [11], and in
pancreatic cancer, the inhibition of IL-6 suppresses the metastatic invasion and
migration of tumours [12]. Moreover,
the regulation of CXCL13 modifies breast cancer cell viability via the CXCR5/ERK
pathway [13]. In animal studies and
cell experiments on liver cancer, the cytokine IL-2 has been documented to be a
potential therapeutic target to limit tumour growth [14, 15]. In a
clinical trial with small sample sizes, administration of IL-2 was found to play a
beneficial role in suppressing the development and progression of HCC [16]. This finding was also supported by a
previous study in which an IL-2 vaccine mediated the regression of HCC in mice
[15]. As there is strong evidence
supporting the suppressive effects of IL-2-based therapy against HCC progression, it
is worthwhile to explore whether IL-2, in combination with sorafenib, can further
reduce the proliferation of liver cancer cells.
Mitochondrial fission, which initiates the mitochondrial apoptosis
pathway, is an early hallmark of cancer cell death [17, 18]. Excessive
mitochondrial fission disrupts mitochondrial energy metabolism, evokes oxidative
stress, causes cellular calcium overload, and promotes the activation of
pro-apoptotic factors [19, 20]. Several attempts have been made to induce
the activation of mitochondrial fission in various tumours such as those in
pancreatic cancer [21], endometriosis
[22], and breast cancer
[23]. Based on the data gained from
these studies, we wanted to determine whether IL-2 could augment sorafenib-mediated
HCC apoptosis by activating mitochondrial fission. The JNK and TAZ pathways are the
primary upstream regulators for mitochondrial fission in liver cancer and in breast
cancer [24, 25]; however, whether IL-2 is capable of
modifying mitochondrial fission via the JNK-TAZ axis has remained unknown. Thus, the
aim of our study was to explore the efficacy of IL-2 in combination with sorafenib
on inducing HCC apoptosis, with a focus on mitochondrial fission and the JNK-TAZ
pathways.
Materials and methods
Cell culture and treatment
The HepG2 liver cancer cell line was purchased from the American
Type Culture Collection (ATCC® HB-8065™). The Huh7
liver cancer cell line and L02 normal liver cell line were purchased from the
Cell Bank of the Chinese Academy of Sciences. The HepG2 and Huh7 cells were
cultured in DMEM medium (#12800-017, Gibco) with 10% FBS (#10437-028, Gibco) at
37 °C/5% CO2. To induce damage, the cancer cells were
treated with sorafenib (5 μM) for approximately 12 h. Another group of cells was
treated with IL-2 (0–20 ng/ml) for 12 h according to a previous study
[16]. To inhibit the activity
of the JNK pathway, cells were treated with SP600125 (SP, 10 μM, Selleck
Chemicals) 2 h before sorafenib/IL-2 treatment [26].
Cellular viability and death evaluation
Cellular viability was measured with MTT and LDH release assays.
The MTT assay was performed according to the methods used in a previous study
[27]. Cells were plated onto a
96-well plate with the IL-2 and sorafenib treatment. MTT solution (Beyotime,
China, Cat. No. C0009) was then added into the medium, and the cells were
incubated for approximately 2 h at 37 °C/5% CO2. The
optical density (OD) of the MTT solution was recorded using a microplate reader
(490 nm absorbance; Epoch 2; BioTek Instruments, Inc., Winooski, VT, USA). An
LDH release assay was conducted using a commercial kit (Beyotime, China, Cat.
No. C0016) according to the manufacturer’s instructions [28].
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Cellular death was measured via a TUNEL assay and the measurement
of caspase-3 activity. TUNEL staining was performed using a One Step TUNEL
Apoptosis Assay Kit (Beyotime, China, Cat. No. C1086) according to the
manufacturer’s instructions. Caspase-3 activity was estimated using the Caspase
3 Activity Assay Kit (Beyotime, China, Cat. No. C1115), and the relative
caspase-3 activity was measured compared to that of the control group using a
microplate reader (430 nm absorbance; Epoch 2; BioTek Instruments, Inc.,
Winooski, VT, USA) [29].
Oxidative stress measurement
Cellular oxidative stress was determined via ELISA as described in
a previous study. Cells were washed with PBS and lysed using RIPA Lysis Buffer
(Beyotime, China, Cat. No. P0013C). Then, the proteins were collected through
high-speed centrifugation, and the concentrations of GSH (Beyotime, China, Cat.
No. S0073), SOD (Beyotime, China, Cat. No. S0086) and GPX (Beyotime, China, Cat.
No. S0058) were measured using commercial kits according to the manufacturers’
instructions [30].
EdU staining and transwell assay
To analyse the cellular proliferation, EdU staining was conducted
using the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 594 (Beyotime,
China, Cat. No. C00788L). Cells were first washed with PBS. Fresh DMEM was then
added, and 10 μM EdU was added into the medium. The cells were incubated for 2 h
at 37 °C/5% CO2. After the incubation, the cells were
again washed with PBS to remove the DMEM and the free EdU probe. The cells were
then fixed in 4% paraformaldehyde at room temperature for 30 min before being
stained with DAPI for 3 min. After an additional wash in PBS, the cells were
observed under an inverted microscope [31].
A transwell assay was carried out to observe the cell migration
response based on the methods of a previous study [32]. Cells at a density of
1 × 103 were added into the upper chamber. DMEM
with 2% FBS was loaded into the lower chamber. Subsequently, the cells were
cultured at 37 °C/5% CO2 for 12 h. After the culture
period, the non-migrated cells were removed, and the migrated cells were fixed
with 3.7% paraformaldehyde for 30 min at room temperature. The migrated cells
were then stained with 0.05% crystal violet for 15 min at room temperature in
the dark. The number of migrated cells was recorded, and images were captured
under an inverted microscope.
Mitochondrial function detection
Mitochondrial function was measured by analysing the mitochondrial
membrane potential, the mitochondrial permeability transition pore (mPTP)
opening rate and the mitochondrial ROS generation. The mitochondrial membrane
potential was determined by JC-1 staining [33]. Live cells were washed with PBS, and a JC-1 solution was
then added to the medium. The cells were incubated at 37 °C/5%
CO2 for 30 min, washed with PBS, loaded with DAPI,
and then observed under a fluorescence microscope. The mPTP opening rate was
recorded as described by a previous study. Cells were first washed with PBS and
incubated with calcein-AM/cobalt at 37 °C/5% CO2 for
30 min. The cells were then washed with PBS again to remove the free probe. The
optical density (OD) was recorded using a microplate reader (540 nm absorbance;
Epoch 2; BioTek Instruments, Inc., Winooski, VT, USA). The mPTP opening rate was
expressed relative to that of the control group [34]. Mitochondrial ROS production was measured via flow
cytometry as described by a previous study. Cells were washed three times in PBS
and incubated with MitoSOX Red Mitochondrial Superoxide Indicator (Molecular
Probes, USA) for 30 min at 37 °C/5% CO2 in the dark.
After incubation, the cells were washed three times in PBS at room temperature,
and the mitochondrial ROS production was measured via flow cytometry
[35].
Western blotting
Cells were lysed in RIPA Lysis Buffer (Beyotime, China, Cat. No.
P0013C). After high-speed centrifugation, the proteins were collected and
quantified with the Enhanced BCA Protein Assay Kit (Beyotime, China, Cat. No.
P0009). Subsequently, 40–60 μg of protein was loaded onto 10% SDS-PAGE gels and
transferred to PVDF membranes. The membranes were washed with TBST and then
blocked with 5% non-fat milk for 45 min at room temperature [36]. The membranes were then incubated at
4 °C overnight with the primary antibodies [CXCR4 (1:1000, Abcam, #ab1670),
CXCR7 (1:1000, Abcam, #ab38089), cyclin D1 (1:1000, Abcam, #ab134175), PCNA
(1:1000, Abcam, #ab18197), CDK4 (1:1000, Abcam, #ab137675), cadherin (1:1000,
Abcam, #ab133168), vimentin (1:1000, Abcam, #ab8978), TAZ (1:1000, Abcam,
#ab224239), complex III subunit core (CIII-core2, 1:1000, Invitrogen, #459220),
complex II (CII-30, 1:1000, Abcam, #ab110410), complex IV subunit II (CIV-II,
1:1000, Abcam, #ab110268), Drp1 (1:1000, Abcam, #ab56788), Fis1 (1:1000, Abcam,
#ab71498), Opa1 (1:1000, Abcam, #ab42364), Mfn1 (1:1000, Abcam, #ab57602), Mff
(1:1000, Cell Signaling Technology, #86668), Bcl2 (1:1000, Cell Signaling
Technology, #3498), Bax (1:1000, Cell Signaling Technology, #2772), caspase-9
(1:1000, Cell Signaling Technology, #9504), Bad (1:1000; Abcam; #ab90435), Tom20
(1:1000, Abcam, #ab186735), cyt-c (1:1000; Abcam; #ab90529), GAPDH (1:1000, Cell
Signaling Technology, #5174), JNK (1:1000; Cell Signaling Technology, #4672),
and p-JNK (1:1000; Cell Signaling Technology, #9251)]. After being washed with
TBST, the membranes were incubated with the secondary antibodies for 45 min at
room temperature. The bands were observed with an enhanced chemiluminescence
(ECL) substrate kit (Beyotime, China, Cat. No. P0018F). The mean densities of
the bands were represented as the optical density in
units/mm2 and normalized to that of loading
control (Quantity One, version 4.6.2; Bio-Rad Laboratories, Inc.)
Immunofluorescence
Cells were washed with PBS at room temperature to remove the DMEM.
Then, the cells were fixed in 3.7% paraformaldehyde for 30 min at room
temperature and permeabilized with 0.1% Triton X-100 for 10 min at 4 °C. The
cells were then washed with PBS, and 10% goat serum albumin was used to block
the samples for 45 min at room temperature. The samples were again washed with
PBS, and the primary antibodies [p-JNK (1:500; Cell Signaling Technology,
#9251), cyt-c (1:500; Abcam; #ab90529), Drp1 (1:500, Abcam, #ab56788), CDK4
(1:500, Abcam, #ab137675), cyclin D1 (1:500, Abcam, #ab134175), and Tom20
(1:500, Abcam, #ab186735)] were added. The samples were incubated overnight at
4 °C. After being washed with PBS three times to remove the primary antibodies,
the cells were incubated with the secondary antibodies for 45 min at room
temperature [37]. After the cells
were again washed with PBS to remove the free second antibodies and were loaded
with DAPI, they were observed under an inverted microscope. Mitochondrial
fission was observed via immunofluorescence using the Tom20 antibody. Images
were captured, and the average length of the mitochondria was used to quantify
the mitochondrial fission [38].
Statistical analysis
All statistical analyses in the present study were performed in
SPSS software (version 19.0). Our data are expressed as the mean ± SEM. Results
for more than two groups were evaluated by one-way analysis of variance followed
by Bonferroni’s multiple comparison test. A P value < 0.05 was considered
significant.
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Results
IL-2 promotes sorafenib-mediated apoptosis in HepG2 and Huh7
cells
First, sorafenib was added into the medium of liver cancer cell
lines (HepG2 cells and Huh7 cells) to repress the cancer cell viability.
Compared to the control group, the sorafenib treatment group displayed markedly
reduced cell viability, as assessed via MTT assay (Fig. 1a, b), suggesting that sorafenib is cytotoxic to
liver cancer cell lines. Similarly, the cell death rate, as evaluated by the LDH
release assay, also increased in response to sorafenib treatment in both the
HepG2 and the Huh7 cells (Fig. 1c, d).
To explore whether the tumour-suppressive effect of sorafenib could be enhanced
by combining sorafenib with IL-2-based therapy, different doses of IL-2 were
added to the medium. As shown in Fig. 1a, b, the cell viability of both HepG2 cells and Huh7 cells
progressively decreased with increasing IL-2 concentrations. IL-2 treatment also
dose-dependently elevated the cell death index, as determined by the LDH release
assay (Fig. 1c, d). Altogether, these
results indicated that IL-2 supplementation augmented the anti-tumour effect of
sorafenib in HepG2 and Huh7 cells. The minimum toxic concentration of IL-2 was
5 ng/ml; therefore, that dose was used in subsequent functional studies. To
exclude the influence of IL-2/sorafenib co-treatment on normal hepatocytes, L02
normal liver cells were treated with IL-2 and sorafenib. As shown in Additional
file 1: Figure S1, we found that
neither IL-2 nor sorafenib treatment affected the viability of L02 cells, as
assessed via MTT assay and LDH release assay. Subsequently, TUNEL staining was
used to detect cell apoptosis after IL-2 and sorafenib co-treatment in HepG2
cells. As shown in Fig. 1e–g, the number
of TUNEL-positive cells increased with sorafenib treatment and was further
elevated in response to IL-2 administration in both HepG2 cells and Huh7 cells.
Similarly, caspase-3 activity increased in response to sorafenib treatment, and
this effect was enhanced by IL-2 treatment (Fig. 1h, i). In all, our data indicated that IL-2 supplementation
augmented sorafenib-mediated cell apoptosis in both HepG2 cells and Huh7
cells.
Fig. 1
IL-2 treatment enhanced the pro-apoptotic effects of
sorafenib. a, b Cell viability was measured via MTT
assay in HepG2 cells and Huh7 cells. The different doses of IL-2
were added in the presence of 5 μM sorafenib. c, d
Cell death was evaluated via LDH release assay in HepG2 cells
and Huh7 cells. The different doses of IL-2 were added in the
presence of 5 μM sorafenib. e–g A TUNEL
assay was performed to observe the cell apoptotic rate. IL-2
(5 ng/ml) treatment was carried out in the presence of 5 μM
sorafenib. h, i Caspase-3 activity was measured in
HepG2 cells and Huh7 cells. IL-2 (5 ng/ml) treatment was carried
out in the presence of 5 μM sorafenib. *P < 0.05 vs. control
group; #P < 0.05 vs. sorafenib
group. Cont
control
×
IL-2 further repressed cell migration and proliferation in the presence of
sorafenib
Cancer proliferation was observed via EdU assay. The results shown
in Fig. 2a–c revealed that sorafenib
attenuated the percentage of EdU+ cells regardless of
whether they were HepG2 cells or Huh7 cells. Interestingly, the
anti-proliferative capacity of sorafenib was strengthened by IL-2 treatment
(Fig. 2a–c), suggesting that IL-2 in
combination with sorafenib further disrupted cancer growth. Similar results were
observed for the expression of proteins related to the cell cycle. Cyclin D1,
PCNA and CDK4 were abundant in the control group and were reduced in response to
sorafenib treatment (Fig. 2d–j). IL-2
administration caused a further decline in the expression of cyclin D1, PCNA and
CDK4 in both HepG2 cells and Huh7 cells (Fig. 2d–j). Taken together, our data support a synergistic role
for sorafenib and IL-2 in repressing the multiplication of cancer cells.
Fig. 2
IL-2 further repressed cell migration and proliferation
in the presence of sorafenib. a–c An EdU
assay was used to observe the proliferative cells. The number of
EdU-positive cells was recorded. d–j Western
blotting analysis for the proteins related to cell
proliferation. IL-2 (5 ng/ml) treatment was carried out in the
presence of 5 μM sorafenib. k–m A transwell
assay was conducted to determine the cell migration in response
to IL-2 and sorafenib co-treatment. n–r The
proteins related to cell migration were analysed via western
blotting. IL-2 (5 ng/ml) treatment was carried out in the
presence of 5 μM sorafenib. *P < 0.05 vs. control group;
#P < 0.05 vs. sorafenib group.Cont control
×
To examine cell migration, a transwell assay was performed. The
number of migrated cells was reduced by sorafenib treatment and was further
depressed with IL-2 treatment (Fig. 2k–m). In addition, proteins related to cancer migration, such as
cadherin and vimentin, were negatively regulated by sorafenib, and this effect
was enhanced by IL-2 treatment in both HepG2 and Huh7 cells (Fig. 2n–r). In summary, the sorafenib-induced
impairment of migration was strengthened by IL-2. Because no phenotypic
differences were noted between HepG2 and Huh7 cells with regards to apoptosis,
proliferation or migration, the HepG2 cell line was used for subsequent
molecular experiments.
IL-2 in combination with sorafenib interrupts mitochondrial
metabolism
Cellular proliferation, migration and survival are heavily
dependent on the production of sufficient energy by the mitochondria; thus,
mitochondrial metabolism was monitored. Cellular ATP production was repressed by
sorafenib in HepG2 cells, and this effect was reinforced by IL-2 supplementation
(Fig. 3a). Mitochondrial energy
production primarily relies on the activity of mitochondrial respiratory enzymes
[20, 39], which convert the mitochondrial
membrane potential into the chemical ATP. Interestingly, the expression levels
of the mitochondrial respiratory proteins were downregulated by sorafenib
(Fig. 3b–e); this tendency was
exacerbated by IL-2 treatment. In addition, the mitochondrial potential, as
assessed by JC-1 staining, was also negatively regulated by sorafenib
(Fig. 3f–g). IL-2 treatment further
repressed the mitochondrial potential, as evidenced by a lower ratio of
red/green fluorescence intensity.
Fig. 3
IL-2 and sorafenib co-treatment inhibited mitochondrial
energy metabolism. a ATP
production was measured in HepG2 cells subjected to IL-2 and
sorafenib co-treatment. b–e
Mitochondrial respiratory proteins were analysed via western
blotting in HepG2 cells. IL-2 (5 ng/ml) treatment was carried
out in the presence of 5 μM sorafenib. f, g
Mitochondrial potential was detected through JC-1 staining. Red
fluorescence, which indicated normal mitochondrial potential,
was converted into green fluorescence after a reduction in
mitochondrial potential. h,i The remaining glucose and
the produced LDH in the medium were analysed for HepG2 cells.
IL-2 (5 ng/ml) treatment was carried out in the presence of 5 μM
sorafenib. *P < 0.05 vs. control group;
#P < 0.05 vs. sorafenib group.Cont control
×
Anzeige
Finally, we measured the amount of glucose remaining in the medium
to directly evaluate the cellular mitochondrial metabolism. Compared to the
control group, the sorafenib treatment group showed reduced glucose uptake from
the medium (Fig. 3h). Lactate production
was also reduced in response to sorafenib treatment (Fig. 3j). IL-2 supplementation further repressed
glucose absorption and lactate generation (Fig. 3h–j), indicating the cessation of glucose absorption,
consumption and metabolism, possibly due to mitochondrial dysfunction.
Altogether, our data highlight a causal relationship between IL-2 administration
and mitochondrial dysfunction when sorafenib is present.
IL-2 induces mitochondrial apoptosis in sorafenib-treated cells
Given the links between IL-2 and mitochondrial dysfunction, we
tested whether IL-2 would amplify sorafenib-activated mitochondrial apoptosis in
HepG2 cells. As shown in Fig. 4a, b,
mitochondrial ROS production, an early molecular event in mitochondrial
apoptosis, increased significantly in response to sorafenib treatment in HepG2
cells, and ROS generation was further evoked by IL-2 (Fig. 4a, b). The sorafenib-mediated ROS production was
closely associated with a drop in the concentration of antioxidants such as GSH,
SOD and GPX (Fig. 4c–e). IL-2 treatment
contributed to a further loss of these antioxidants, suggesting a permissive
role for IL-2 in cancer oxidative stress.
Fig. 4
IL-2 activated the mitochondrial apoptotic pathway in
the presence of sorafenib. a,b Mitochondrial ROS
production was detected in HepG2 cells. IL-2 (5 ng/ml) treatment
was carried out in the presence of 5 μM sorafenib. c–e
The antioxidants in HepG2 cells under IL-2 and sorafenib
co-treatment were measured via ELISA. f The mPTP opening rate was analysed to
determine the mitochondrial damage. IL-2 (5 ng/ml) treatment was
carried out in the presence of 5 μM sorafenib. g, h
Cyt-c liberation was observed via immunofluorescence. i–o
Mitochondrial apoptotic proteins were analysed by western
blotting. The sorafenib-mediated upregulation of apoptotic
proteins was further augmented by IL-2 treatment. *P < 0.05
vs. control group; #P < 0.05 vs.
sorafenib group. Cont
control
×
A late molecular feature of mitochondrial damage is the opening of
the mitochondrial permeability transition pore (mPTP), a channel necessary to
enable the transmission of mitochondrial pro-apoptotic factors into the
cytoplasm/nucleus [40, 41]. Sorafenib-mediated mPTP opening was
enhanced by IL-2 in HepG2 cells (Fig. 4f). We also found through immunofluorescence assay that cyt-c, a
type of mitochondrial pro-apoptotic protein, was released into the nucleus upon
sorafenib treatment due to the prolonged open state of the mPTP
(Fig. 4g, h). IL-2 treatment
facilitated the cyt-c translocation, as determined by analysis of the
fluorescence intensity of cyt-c in the nucleus (Fig. 4g, h). This finding was also validated via western blotting.
The level of mitochondrial cyt-c declined in sorafenib-treated cells; this
decrease was accompanied by an increase in the expression of cytoplasmic cyt-c
(Fig. 4i, j), an effect that was
enhanced by IL-2. We also found that mitochondrial apoptotic proteins such as
Bad, Bax and caspase-9 were all upregulated by sorafenib treatment
(Fig. 4i–o). This upregulation was
followed by a fall in the content of anti-apoptotic factors (Fig. 4i–o). The sorafenib-initiated mitochondrial
apoptosis was amplified by IL-2 (Fig. 4i–o). Taken together, our data illustrate that IL-2 can promote
sorafenib-mediated mitochondrial apoptosis in HepG2 cells.
Mitochondrial fission is augmented by IL-2 in the presence of
sorafenib
To explain the additional action of IL-2 in activating
mitochondrial apoptosis in the presence of sorafenib, we focused on
mitochondrial fission, which is the upstream trigger of mitochondrial apoptosis
through multiple biological processes [19, 20].
Mitochondrial fission was first examined by western blotting. Mitochondrial
fission-related proteins such as Drp1, Fis1 and Mff [42] were slightly upregulated in
sorafenib-treated cells (Fig. 5a–f) and
were highly elevated in response to IL-2 supplementation. These data indicate
that mitochondrial fission seems to be initiated by sorafenib and is further
amplified by IL-2 supplementation. In addition, we examined the proteins related
to mitochondrial fusion, the defensive system used to correct excessive
mitochondrial division. Compared to those in the control group, the levels of
mitochondrial fusion-related proteins, such as Mfn1 and Opa1, were marginally
downregulated in the sorafenib-treated group (Fig. 5a–f), and this effect was exaggerated by IL-2. These data
suggest that IL-2 helps sorafenib to hinder the mitochondrial fusion system,
indirectly promoting mitochondrial fission.
Fig. 5
IL-2 enhanced sorafenib-initiated mitochondrial fission.a–f Western blotting was used to analyse the
proteins related to mitochondrial fusion and mitochondrial
fission. Drp1, Fis1 and Mff are factors involved in
mitochondrial fission. In contrast, mitochondrial fusion is
regulated by Mfn1 and Opa1. IL-2 and sorafenib co-treatment
elevated the mitochondrial fission proteins and repressed the
mitochondrial fusion factors. g, h Mitochondrial
fission was observed via immunofluorescence using the Tom20
antibody. Then, the average length of mitochondria was measured
in HepG2 cells. *P < 0.05 vs. control group;
#P < 0.05 vs. sorafenib group.Cont control
×
Anzeige
Subsequently, an immunofluorescence assay for mitochondria was
conducted to observe the mitochondrial fission. In sorafenib-treated cells, the
mitochondrial network divided into several fragmented mitochondria in response
to mitochondrial fission (Fig. 5g). This
alteration was more prominent in IL-2-challenged cells. We further measured the
average length of the mitochondria to quantify the mitochondrial fission. The
average length of the mitochondria was reduced to some extent under sorafenib
treatment (Fig. 5h), and this effect was
augmented by IL-2. Overall, we confirmed that IL-2 promotes sorafenib-triggered
mitochondrial fission in HepG2 cells.
IL-2 regulates mitochondrial fission via the JNK-TAZ pathways
The mechanism by which IL-2 boosts mitochondrial fission in the
presence of sorafenib was unclear. Since JNK and TAZ have been well documented
as activators of mitochondrial fission, we wondered whether JNK-TAZ pathways
were also involved in IL-2-exacerbated mitochondrial fission in the presence of
sorafenib. Western blotting analysis revealed that both JNK phosphorylation and
TAZ expression were slightly increased in response to sorafenib treatment
(Fig. 6a–c) and were considerably
upregulated with IL-2 supplementation. These findings suggest that the JNK-TAZ
pathways are regulated by IL-2 and sorafenib co-treatment.
Fig. 6
IL-2 and sorafenib co-treatment regulated mitochondrial
fission via the JNK-TAZ pathways. a–c JNK
phosphorylation and TAZ expression were measured via western
blotting. SP600125, an inhibitor of JNK, was used to inhibit the
activity of the JNK-TAZ pathways. d, e
Mitochondrial fission was observed via immunofluorescence, and
the average length of the mitochondria was recorded. f–h
The regulatory effects of IL-2 and sorafenib co-treatment on the
JNK-TAZ pathways and mitochondrial fission were monitored via
immunofluorescence. IL-2 and sorafenib co-treatment promoted the
upregulation of JNK phosphorylation, which was accompanied by an
increase in Drp1, a factor for mitochondrial fission.
*P < 0.05 vs. control group;
@P < 0.05 vs. IL-2+ sorafenib
group. Cont
control
×
To demonstrate whether the JNK-TAZ pathways were required to
initiate mitochondrial fission, we inhibited JNK activity with a pathway
blocker, SP600125. The inhibitory efficiency was validated via western blotting
as shown in Fig. 6a–c. After blockade of
JNK, the mitochondrial fission was monitored by immunofluorescence as described
previously. Compared to the fragmented mitochondria under IL-2 and sorafenib
co-treatment, the mitochondria of SP600125-treated cells maintained an
interconnected phenotype (Fig. 6d).
Similarly, the average length of the mitochondria was increased after SP600125
treatment when compared to the average length after IL-2 and sorafenib
co-treatment (Fig. 6e). We also measured
the alteration of mitochondrial fission-related proteins, such as Drp1, through
co-immunofluorescence. The fluorescence intensity of Drp1 closely paralleled the
content of p-JNK upon IL-2 and sorafenib co-treatment (Fig. 6f–h); higher p-JNK expression was accompanied by
increased Drp1 fluorescence intensity. However, inhibition of JNK abrogated the
stimulatory effect of IL-2/sorafenib on Drp1 expression (Fig. 6f–h). Collectively, the above data verify the
necessity of the JNK-TAZ pathways in IL-2/sorafenib-mediated mitochondrial
fission.
JNK-TAZ pathways are also involved in IL-2-mediated migration inhibition
and proliferation arrest
Finally, we wanted to know whether the JNK-TAZ pathways also
participate in the migration and proliferation of HepG2 cells. An
immunofluorescence assay for cell cycle proteins confirmed that IL-2/sorafenib
promoted the expression of CDK4 and cyclin D1 (Fig. 7a–c), and this effect was negated by blocking the JNK-TAZ
pathways. In addition, the EdU assay also illustrated that IL-2/sorafenib
co-treatment attenuated the ratio of EdU-positive cells by activating the
JNK-TAZ pathways (Fig. 7d, e). These
data indicate that IL-2/sorafenib-modulated cancer proliferation is dependent on
the activity of the JNK-TAZ pathways.
Fig. 7
Cell migration and proliferation were also regulated by
IL-2/sorafenib co-treatment through the JNK-TAZ pathways.a–c Immunofluorescence assay for cell
proliferation-related factors. IL-2/sorafenib co-treatment
elevated the expression of CDK4 and cyclin D1, which was
repressed by SP600125, an inhibitor of the JNK-TAZ pathways.d, e An EdU assay was performed to quantify the
cell proliferation. The number of EdU-positive cells was
recorded. f–h Cell migration factors such as
CXCR4 and CXCR7 were measured via western blotting. *P < 0.05
vs. control group; @P < 0.05 vs.
IL-2+ sorafenib group. Cont
control
×
With respect to cancer migration, molecular regulators, such as
CXCR4 and CXCR7, were reduced by IL-2/sorafenib co-treatment and were reversed
to near-normal levels after the inactivation of the JNK-TAZ pathways
(Fig. 7f–h). These data illustrate
the critical role played by the JNK-TAZ pathways in cancer migration.
Discussion
Despite advances in the molecular understanding of HCC, few effective
drugs are available in clinical practice to prevent its development. Sorafenib, a
first-line targeted therapy drug, has shown a significant survival benefit for
patients with HCC in global multiple-centre clinical trials [43, 44]. However, its efficacy is limited to a 3-month extension in
survival time [45, 46]. Although several attempts have been made to
elucidate the resistance mechanism of HCC against sorafenib, no solid conclusions
have been drawn [47]. Several studies
have suggested that the alteration of glucose metabolism and/or the downregulation
of the Raf-1 kinase inhibitory protein could be possible resistance mechanisms in
patients receiving sorafenib [48,
49]. In the present study, our data
suggest an option to enhance the therapeutic efficacy of sorafenib in killing liver
cancer cells. A combination of sorafenib and IL-2 reduced the viability of liver
cancer cell lines in vitro compared to the viability after sorafenib treatment
alone. Moreover, cancer cell migration and proliferation were also repressed by
sorafenib in conjunction with IL-2. At the molecular level, IL-2 supplementation
assisted sorafenib in inducing mitochondrial injury by activating fatal
mitochondrial fission. We also demonstrated that IL-2, in the presence of sorafenib,
modified mitochondrial fission via the JNK-TAZ pathways. This is the first
investigation to present a novel way to enhance the anti-tumour effect of sorafenib
on liver cancer in vitro. Our findings will pave the way for new treatment
modalities to control HCC progression by optimizing sorafenib-based therapy.
In the present study, we demonstrated that IL-2 facilitated the
pro-apoptotic effects of sorafenib by augmenting mitochondrial fission.
Mitochondrial fission is a physical process that modulates the quantity and quality
of mitochondrial mass [50]. Moderate
mitochondrial fission is necessary for cellular metabolism through the timely
production of daughter mitochondria [51]. Moreover, mitochondrial fission helps mitochondria to remove
damaged parts, thus enabling mitochondrial turnover and renewal [52]. However, excessive mitochondrial fission
converts the mitochondrial network into discontinuous debris, leading to
mitochondrial dysfunction. Previous studies on cardiac ischemia/reperfusion have
demonstrated that mitochondrial fission activates mitochondrial apoptosis via the
HK2-VDAC1-mPTP pathway and the mROS/cardiolipin/cyt-c axis [42]. More recent studies on pancreatic cancer
have also found that cancer cell proliferation, migration and survival are closely
regulated by mitochondrial fission [21]. Similar findings have been reported for colorectal cancer
[53], endometriosis [22], and liver cancer [25]. Consistent with these reports, our data
also identify mitochondrial fission as the critical upstream signal for
mitochondrial homeostasis in liver cancer cells.
We also demonstrated in this study that mitochondrial fission is
drastically activated by IL-2 in the presence of sorafenib, and this regulatory
mechanism is dependent on the JNK-TAZ pathways. Notably, no studies investigating
the detailed role of IL-2 in mitochondrial fission have yet been conducted. Thus,
our investigation provides the first evidence that the tumour-suppressive effects of
IL-2 on liver cancer may be attributable to the activation of mitochondrial fission.
Notably, the apoptotic rate of HepG2 cells was progressively increased with a rise
in the dose of IL-2. The minimum toxic concentration of IL-2 was 5 ng/ml, and
therefore, this dose was used to explore whether IL-2 could augment the efficiency
of sorafenib-based therapy. Subsequently, we demonstrated that IL-2 regulates
mitochondrial fission via the JNK-TAZ pathways. Previous studies have reported the
critical role of JNK and TAZ in activating mitochondrial fission in several disease
models. For example, in human rectal cancer cells, activation of the JNK pathway
promotes mitochondrial fission, thereby reducing cancer cell survival and migration
[53]. In primary hepatocytes, the
inhibition of mitochondrial fission through the modulation of JNK protects the cells
against senecionine-induced mitochondrial apoptosis [54]. In breast cancer cells, disruption of the JNK pathway
inhibits mitochondrial fission and represses cancer cell proliferation and survival
[55]. The above information lays a
foundation to help us understand the role of JNK in regulating mitochondrial
fission. With respect to TAZ, an early study revealed that mitochondrial fission
could be controlled by TAZ through the regulation of mitochondrial lipid synthesis
[56]. Subsequent experiments
verified that breast cancer migration is highly controlled by TAZ through
mitochondrial fission [57].
Furthermore, TAZ has been found to promote mitochondrial fission and induce stem
cell differentiation [58]. Such results
describe the causal relationship between TAZ and mitochondrial fission. Similar to
these findings, our study revealed that the JNK-TAZ pathways are activated by IL-2
in the presence of sorafenib and contribute to mitochondrial fission, ultimately
repressing liver cancer cell survival, migration and proliferation. These findings
inform us of the anti-tumour molecular mechanisms activated by IL-2 in combination
with sorafenib and suggest that strategies targeting mitochondrial fission and the
JNK-TAZ axis would yield additional clinical benefits for patients suffering from
HCC. To the end, we also found that the survival rate and proliferative index of
HepG2 cells were still high in response to IL-2/sorafenib co-treatment. Accordingly,
more attempts are required to further enhance the sensitivity of HCC to
sorafenib-based therapy. Although we observed the inhibitory effect of
IL-2/sorafenib co-treatment on HepG2 cell migration, the IL-2/sorafenib-mediated
cell apoptosis and proliferation arrest may also influence the HepG2 cell migration.
Further investigation of the direct role of IL-2/sorafenib co-treatment in HCC
migration is required.
Conclusions
Taken together, our data indicate that additional supplementation with
IL-2 can enhance the tumour-killing activity of sorafenib. IL-2 in combination with
sorafenib repressed liver cancer cell proliferation, migration and survival by
promoting mitochondrial dysfunction. The synergetic effects of IL-2 and sorafenib
were primarily dependent on mitochondrial fission through the activation of the
JNK-TAZ pathways. These findings provide new insights into the mechanisms of these
drugs and suggest novel strategies to induce cancer cell death with sorafenib
therapy.
Authors’ contributions
XYD and WS conceived the research; XYD and JLC performed the experiments;
all authors participated in discussing and revising the manuscript. All authors read
and approved the final manuscript.
Acknowledgements
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analysed during this study are included in
this published article.
Consent for publication
Not applicable.
Ethics approval and consent to participate
Not applicable.
Funding
This study was supported by Foundation of Clinical Research
Cooperation Capital Medical University (Project Number: 16JL77).
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