Post on 14-Aug-2020
1
Itraconazole-induced inhibition on human
esophageal cancer cell growth requires AMPK
activation
Min-Bin Chen 1#, Yuan-Yuan Liu 2#, Zhao-yu Xing 3#, Zhi-qing Zhang 4, Qin Jiang 5*, Pei-Hua
Lu 6* and Cong Cao 4, 5, 7*
1 Department of Radiotherapy & Oncology, Kunshan First People’s Hospital Affiliated to
Jiangsu University, Kunshan, China. 2 Department of Laboratory Center, Kunshan First People’s Hospital Affiliated to Jiangsu
University, Kunshan, China. 3 The Department of Urology, the Third Affiliated Hospital of Soochow University,
Changzhou, China. 4 Institute of Neuroscience, Soochow University, Suzhou, China. 5 The Affiliated Eye Hospital of Nanjing Medical University, Nanjing, China 6 Department of Medical Oncology, Wuxi People’s Hospital Affiliated to Nanjing Medical
University, Wuxi, China. 7 North District, The Municipal Hospital of Suzhou, Suzhou, China
# Co-first authors.
Correspondence to:
* Prof. Qin Jiang M.D. Ph.D., the Affiliated Eye Hospital, Nanjing Medical University. 138
Han-zhong Road, Nanjing, 210029, China. Tel/Fax:+86-025-86677699. E-mail:
Jqin710@vip.sina.com.
* Prof. Pei-Hua Lu, M.D. Ph.D., Department of Medical Oncology, Wuxi People’s Hospital
Affiliated to Nanjing Medical University, No. 299, Qingyang Road, Wuxi, 214023, China
Tel/Fax: +86-510-85350070. Email: lphty1_1@163.com.
* Prof. Cong Cao, M.D. Ph.D., Institute of Neuroscience, Soochow University, 199 Ren’ai
Road, SIP, Suzhou, 215021, China. Tel/Fax: +86-512-65883602. E-mail:
caocong@suda.edu.cn
Running head. Itraconazole activates AMPK to inhibit cancer cells.
The authors declare no potential conflicts of interest.
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Abstract.
We here evaluated the anti-esophageal cancer cell activity by the antifungal drug
itraconazole. Our results show that μg/mL concentrations of itraconazole potently inhibited
survival and proliferation of established (TE-1 and Eca-109) and primary human esophageal
cancer cells. Itraconazole activated AMP-activated protein kinase (AMPK) signaling, which
was required for subsequent esophageal cancer cell death. Pharmacological AMPK
inhibition, AMPKα1 shRNA or dominant negative mutation (T172A) almost completely
abolished itraconazole-induced cytotoxicity against esophageal cancer cells. Significantly,
itraconazole induced AMPK-dependent autophagic cell death (but not apoptosis) in
esophageal cancer cells. Further, AMPK activation by itraconazole induced multiple receptor
tyrosine kinases (RTKs: EGFR, PDGFRα and PDGFRβ) lysosomal translocation and
degradation to inhibit downstream Akt activation. In vivo, itraconazole oral gavage potently
inhibited Eca-109 tumor growth in severe combined immunodeficient (SCID) mice. It was yet
ineffective against AMPKα1 shRNA-expressing Eca-109 tumors. The in vivo growth of the
primary human esophageal cancer cells was also significantly inhibited by itraconazole
administration. AMPK activation, RTKs degradation and Akt inhibition were observed in
itraconazole-treated tumors. Together, itraconazole inhibits esophageal cancer cell growth
via activating AMPK signaling.
Keywords. Esophageal cancer; Itraconazole; AMPK signaling; Autophagy; Receptor
tyrosine kinase (RTK).
Introduction
Esophageal cancer is one important cause of cancer-related human mortalities (1).
Treatment of this devastating disease in the past decades has achieved significant
achievements (1). The application of conventional cytotoxic drugs and recently-developed
molecular-targeted agents are however discouraging against cancer cells with pre-existing
and/or acquired resistance (1). Therefore, there is an urgent need to explore novel, more
efficient, but less toxic anti-esophageal cancer agents (1).
Itraconazole blocks the synthesis of ergosterol in the fungal cell membrane, and it
functions as a systematic antifungal drug (2). Studies have tested itraconazole as a novel
anti-cancer drug (2-5). Preclinical studies have demonstrated its superior anti-cancer activity
against a number of different cancer cells (3-5). It is currently under phase II clinical trials of
non-small-cell lung cancer, basal cell carcinoma, and prostate cancer (5-7). The potential
effect of this antifungal drug in human esophageal cancer cells has not been studied. The
underlying signaling mechanism of itraconazole-mediated anti-cancer activity is still largely
unclear (5).
AMP-activated protein kinase (AMPK) activation maintains cellular energy homeostasis
under many stress conditions (8, 9). It is also critical in controlling cell survival and death
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(see review (8-10)). Our group and others have been focusing on the anti-cancer ability by
AMPK. AMPK activation could promote cancer cell death via regulating its downstream
signaling effectors, including p53 activation, mammalian target of rapamycin (mTOR)
complex 1 (mTORC1) inhibition as well as autophagy induction and oncogenic protein
degradation (11). Here, we show that itraconazole activates AMPK signaling to promote
esophageal cancer cell death.
Materials and methods
Ethics approval and consent to participate. The animal procedures in this study
were approved by Soochow University Ethics Review Board and IACUC. All studies
requiring human tissue samples were conducted according to the principles of Declaration of
Helsinki, and protocols were approved by the Soochow University Ethics Review Board.
Each of the participant provided written-informed consent.
Chemicals, reagents and antibodies. Itraconazole,
5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR, catalog no. A9978) (12),
ammonium chloride (NH4Cl) and 3-methyaldenine (3-MA) were purchased from Sigma
(Shanghai, St. Louis, MO). Cell permeable short-chain C6 ceramide was described
previously (13). The applied caspase inhibitors, Z-DEVD-fmk, Z-LEHD-fmk and Z-VAD-fmk
were purchased from Merck-Sigma (Catalog nos. C0605, C1355, V116). The molecular
structures can be found from the supplier’s website (https://www.sigmaaldrich.com/) and
from https://pubchem.ncbi.nlm.nih.gov (SID: 329774802, 329774878 and 347914303). All
the antibodies were obtained from Cell Signaling Technology (Beverly, MA). Necrostatin-1
and ferrostatin-1 were purchased from Sigma.
Culture of established cell lines. The human esophageal cancer lines, Eca-109 and
TE-1, as well as the human esophageal epithelial cell (HEEC) line (14), were purchased
from the Cell Bank of Shanghai Institute of Biological Science (Shanghai, China, 08-2015).
Cells were maintained in the DMEM plus 10% FBS (Sigma) medium. All cell lines were
routinely subjected to mycoplasma and microbial contamination examination. STR profiling,
population doubling time, colony forming efficiency, and morphology were checked every
three months to confirm the genotype.
Primary culture of human esophageal cancer cells. One patient (47 year old, female)
with primary esophageal cancer (Stage-III) at Wuxi People’s Hospital of Nanjing Medical
University (Wuxi, China) was enrolled in the study. Written-informed consent was obtained
from the patient, who received no therapy before surgery. As described early (15), the
surgery-dissected esophageal cancer tissue specimen was washed and minced. Cancer
tissues were then mechanically dissociated and filtered through a 70 µm strainer. Single-cell
suspensions of primary cancer cells were achieved by incubation cells in collagenase I
(Sigma)-containing DMEM. Afterwards, individual cells were pelleted and rinsed, which were
re-suspended in the cell culture medium as described (15).
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Methyl thiazol tetrazolium (MTT) assay of cell viability. Cell viability was examined
by the routine MTT (Sigma) assay as described (15-18).
BrdU incorporation assay of cell proliferation. Cell proliferation was tested by the
incorporation of 5-bromo-2’-deoxyuridine (BrdU) assay via an ELISA format, the detailed
protocol was described previously (19).
Colony formation assay. Cells with applied treatment were suspended in 1 mL of
DMEM with 0.5% agar (Sigma). The suspension was then added on the pre-solidified cell
culture dish. After 10-day incubation, the remaining colonies were stained and counted.
Trypan blue staining assay of cell death. As described (15), the “dead” cell
percentage was determined via counting cells in an automatic hemocytometer with trypan
blue dye, which stains dead cells.
Apoptosis assays, including Histone-DNA ELISA assay, Annexin V
fluorescence-activated cell sorting (FACS) assay and TUNEL assay were described in detail
in our studies elsewhere (16-18).
Western blot assay. Western blot assay was described in detail in our previous reports
(15-18). Blot intensity was quantified via the ImageJ software (NIH). Note that the exact
same set of lysate samples were run in sister gels to examine different proteins, and the blot
was stripped and re-probed. For each lane, the exact amount (30 μg) of total cell/tissue
lysates were loaded. The intensity of each band was quantified via Image J software (NIH).
LC3B immunochemistry. After applied treatment, Eca-109 cells were fixed, washed
and blocked. The cells were then incubated with the primary antibody (anti-LC3B-Alexa
Fluor 555 Conjugate, Cell Signaling Tech, 1:25). Afterwards, LC3B fluorescence puncta was
visualized under the Leica microscope. The percentage LC3B puncta positive cells (green
fluorescence) was recorded. For each treatment, total 100 cells (DAPI stained) in 5 random
views were counted (20).
Subcellular fractionation. Isolation of lysosomes: Following the treatment, 20 millions
cells per treatment were re-suspended in 250 mM sucrose/10 mM Tris/HCl buffer solution,
which were then disrupted by sonication with two 15s pulses (Sonics and Materials VCX 600
Watt, Danbury, CT) (21). The disrupted cells were then subjected to rotation (1,200 g for 15
min) to remove nuclei and cell debris, and the supernatants were submitted to centrifugation
at 30,000 g for 30 min to achieve the enriched lysosome fraction. Lysosome-enriched
protein LAMP1 (Lysosome-associated membrane protein 1) was tested in the fraction.
Isolation of cell plasma membrane was described exactly in detail in other studies (22). Na,
K-ATPase was tested as a plasma membrane marker protein.
Short hairpin RNA (shRNA) knockdown. As described (13), lentiviral AMPKα1
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shRNA (Santa Cruz, sc-29673-V, 10 μL/mL per well) was added to cultured esophageal
cancer cells for 24 hours. Afterwards, puromycin (2.0 μg/mL, Sigma) was added to the
culture medium for 2 passages. shRNA knockdown of EGFR, PDGFRα and PDGFRβ was
performed similarly.
AMPKα1 mutation. The dominant-negative (dn) mutant of AMPKα1
(dn-AMPKα1,T172A, Flag-tagged) construct, the constrictively-active AMPKα1 (ca-AMPKα1,
T172D, Flag-tagged) and the empty vector were described previously (13). The construct
(0.25 μg/mL per well) was transfected to cultured esophageal cancer cells using the
Lipofectamine 2000 reagent (13), and stable cells were selected for 8 days. Transfection
efficiency was determined again by Western blot assay.
Tumor xenograft assay. Briefly, Eca-109 cells at logarithmic growing phase (5×106
cells per mouse), expressing either scramble control shRNA or AMPKα1 shRNA, were
inoculated (via s.c. injection) to the female severe combined immuno-deficient (SCID) mice
(4-5 week old, 17-18 g weight). Within 3-4 weeks, the tumors reached the average volume of
150 mm3, the mice were treated with/without itraconazole through oral gavage. Tumor
volumes were recorded weekly, calculated via the following formula: π/6 × larger diameter ×
(smaller diameter)2. Estimated daily tumor growth (in mm3 per day) was also calculated as
described (19). The establishment of xenograft tumors using the primary human cancer cells
was performed similarly.
Statistical analysis. Data were presented as mean ± standard deviation (SD).
Statistics were analyzed by one-way ANOVA followed by the Scheffe' and Tukey Test using
SPSS software. Significance was chosen as P < 0.05. To determine significance between
only 2 treatment groups, a 2-tailed unpaired t test was used (Excel 2007 for Windows).
Results
Itraconazole is cytotoxic and anti-proliferative to human esophageal cancer cells
First, Eca-109 esophageal cancer cells were treated with gradually-increasing
concentrations of itraconazole. The MTT viability assay results in Figure 1A demonstrate
that itraconazole dose-dependently inhibited Eca-109 cell survival. Itraconazole also
displayed a time-dependent response in inhibiting Eca-109 cells, it would require at least 24
to 48 hours for itraconazole (0.3-3.0 μg/mL) to exert a significant anti-survival activity (Figure
1A). Clonogenicity assay results in Figure 1B show that treatment with itraconazole (0.3-3.0
μg/mL) dramatically decreased the number of viable Eca-109 colonies. Meanwhile, with the
itraconazole (0.3-3.0 μg/mL) treatment, the number of trypan blue-stained cells (“dead” cells)
was significantly increased (Figure 1C).
The potential effect of itraconazole on other esophageal cancer cells was also studied.
MTT assay results in Figure 1D show that itraconazole (0.3-3.0 μg/mL) was cytotoxic to
TE-1 cells (another established esophageal cancer cell line) and patient-derived primary
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esophageal cancer cells. The same itraconazole treatment failed to inhibit survival of HEEC
esophageal epithelial cells (14) (Figure 1D). These results demonstrate that itraconazole is
only cytotoxic to human esophageal cancer cells.
Next, we tested the potential effect of itraconazole on esophageal cancer cell
proliferation. BrdU incorporation assay results in Figure 1E show that itraconazole (0.3-3.0
μg/mL, 24 hours) significantly inhibited Eca-109 cell proliferation (BrdU ELISA OD reduction).
In TE-1 cells and primary esophageal cancer cells, BrdU incorporation was also inhibited by
itraconazole (Figure 1F). However, proliferation of HEEC esophageal epithelial cells was
unchanged (Figure 1F). These results imply that itraconazole inhibits esophageal cancer cell
proliferation.
Itraconazole fails to induce apoptosis in esophageal cancer cells
The potential effect of itraconazole on cell apoptosis was tested. Several different
apoptosis assays were performed, including the Annexin V FACS assay (Figure 2A),
Histone DNA apoptosis ELISA assay (Figure 2B) and TUNEL staining assay (Figure 2C).
Results from all the assays show that itraconazole (48 hours treatment) failed to induce
significant apoptosis in Eca-109 cells (Figure 2A-C). On the other hand, short-chain C6
ceramide (13), tested as a positive control, induced significant apoptosis activation in
Eca-109 cells (Figure 2A-C).
Next, the caspase-3 specific inhibitor Z-DEVD-fmk, the caspase-8 specific inhibitor
Z-IETD-fmk and the pan caspase inhibitor Z-VAD-fmk were applied. Results show that the
caspase inhibitors failed to inhibit itraconazole (3.0 μg/mL)-induced Eca-109 cell viability
reduction (Figure 2D) and cell death (Figure 2E). Similarly, in TE-1 cells and primary
esophageal cancer cells, treatment with itraconazole (3.0 μg/mL) failed to induce apparent
cell apoptosis (TUNEL staining assay, Figure 2F). No apoptosis was induced in itraconazole
(3.0 μg/mL)-treated HEEC epithelial cells (Figure 2F).
Necrostatin-1 is a specific inhibitor of necroptosis, acting by directly blocking
receptor-interacting serine/threonine-protein kinase 1/3 (RIP1) (23). Ferrostatin-1 is the
known ferroptosis inhibitor (24). We show that pre-treatment with necrostatin-1 or
ferrostatin-1 failed to inhibit itraconazole-induced cytotoxicity against Eca-109 cells (Figure
S1A and B). The results suggest that necroptosis and ferroptosis are unlikely involved in
itraconazole-induced cancer cell death.
AMPK activation mediates itraconazole-induced esophageal cancer autophagic cell
death
The tumor-suppressing function of AMPK has been well-established. AMPK signaling in
itraconazole-treated cells was then tested. Western blot results in Figure 3A demonstrate
that itraconazole (3.0 μg/mL) treatment in Eca-109 cells induced increased phosphorylation
(“p-“) of AMPKα1 (Thr-172) and its major downstream substrate acetyl-CoA carboxylase
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(ACC, Ser-79), indicating AMPK signaling activation. AMPK activation lasted for at least 12
hours (Figure 3A). In order to study the role of AMPK activation in itraconazole-induced
activity, the AMPK inhibitor Compound C (“CC”) was applied. Compound C completely
blocked itraconazole-induced AMPK activation (AMPKα1-ACC phosphorylation) (Figure 3B),
which also largely attenuated itraconazole-induced Eca-109 cell viability reduction (Figure
3C).
Next, the lentiviral shRNA strategy was applied to stably knockdown AMPKα1 in
Eca-109 cells. AMPKα1 protein expression was almost completely silenced in AMPKα1
shRNA-expressing stable cells (Figure 3B). Consequently, itraconazole-induced AMPK
activation was blocked (Figure 3B). Itraconazole-induced cytotoxicity in Eca-109 cells was
also largely ameliorated by AMPKα1-shRNA (Figure 3C). In TE-1 cells, treatment with
itraconazole also induced AMPK activation (AMPKα/ACC phosphorylation, Figure S2A),
which was completely blocked by AMPKα1 shRNA (Figure S2B). Significantly, AMPKα1
shRNA almost nullified itraconazole-induced TE-1 cell viability reduction (Figure S2C) and
cell death (Figure S2D). The results further support the requirement of AMPK activation in
itraconazole-mediated esophageal cancer cell death.
The dominant negative mutant AMPKα1 (“dn-AMPKα1”, T172A, Flag-tagged) (13, 19)
was transfected to Eca-109 cells. As shown in Figure 3D, dn-AMPKα1 almost abolished
itraconazole-induced AMPK activation, and also largely protected cells from itraconazole
(Figure 3E). Conversely, the constitutively-active AMPKα1 (“ca-AMPKα1”, T172D) (25) was
introduced to Eca-109 cells. Results in Figure 3F confirmed expression of the exogenous
ca-AMPKα1 (Flag-tagged) in Eca-109 cells. AMPKα1-ACC phosphorylation was significantly
elevated in the ca-AMPKα1-expressing cells (Figure 3F). As compared to the vector-control
cells, expression of ca-AMPKα1 reduced cell viability in Eca-109 cells (Figure 3G).
AMPK activation was able to trigger autophagic cell death, via phosphorylating and
activating Ulk1 (the direct mechanism) (26) or inhibiting mTORC1 (the indirect mechanism)
(26). Results in Figure 3H show that itraconazole (3.0 μg/mL) induced Ulk1 phosphorylation
(at Ser-317 (26)), light chain 3B-I (LC3B-I) to LC3B-II convention, Beclin-1 and ATG-5
upregulation as well as p62 downregulation in Eca-109 cells. Further, the percentage of
Eca-109 cells with LC3B-GFP puncta was significantly increased following itraconazole
treatment (Figure 3I), indicating autophagy activation. Significantly, AMPKα1 shRNA or
dominant negative mutation almost completely blocked itraconazole-induced LC3B-GFP
puncta formation (Figure 3I). The results suggest that itraconazole-provoked autophagy is
dependent on AMPK. To study the role of autophagy in itraconazole-induced cytotoxicity, two
known autophagy inhibitors were applied: ammonium chloride (NH4Cl) and 3-methyaldenine
(3-MA). Both largely attenuated itraconazole-induced Eca-109 cell death (Figure 3J).
Collectively, the results suggest that itraconazole induces AMPK-dependent autophagic
death of esophageal cancer cells.
Itraconazole in-activates Akt-mTORC1 signaling and induces multiple receptor
tyrosine kinases (RTKs) degradation
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mTORC1 is important for esophageal cancer cell progression (27, 28). As shown in
Figure 4A, treatment with itraconazole (3.0 μg/mL, 12 hours) in Eca-109 cells inhibited
phosphorylation of the mTORC1 substrates 4E-binding protein 1 (4E-BP1, Ser-65) and S6
(Ser-235/236), suggesting mTORC1 inhibition (29, 30). Compound C or AMPKα1 shRNA
(see Figure 3) almost reversed itraconazole-induced mTORC1 inhibition (Figure 4A).
Conversely, AICAR or ca-AMPKα1 inhibited mTORC1 activation (p-4E-BP1/p-S6) in
Eca-109 cells (Figure 4B). Akt activation, tested by p-Akt at both Ser-473 and Thr-308, was
also largely inhibited by itraconazole (Figure 4C), which was almost completely blocked by
Compound C or AMPKα1 shRNA (Figure 4C). Further, AICAR or ca-AMPKα1 similarly
inhibited Akt activation in Eca-109 cells (Figure 4D). The results suggest that AMPK
activation by itraconazole also inhibits Akt-mTORC1 signaling.
Co-current activation of multiple receptor tyrosine kinases (RTKs) is responsible for
downstream Akt-mTORC1 activation (31, 32). Expression of several RTKs in
itraconazole-treated cells were then tested. As demonstrated, treatment with itraconazole in
Eca-109 cells induced downregulation of several RTKs, including epidermal growth factor
receptor (EGFR), platelet-derived growth factor receptor α (PDGFRα) and PDGFRβ (Figure
4E). Notably, such effect by itraconazole was also dependent on AMPK, and was abolished
by Compound C or AMPKα1 shRNA (Figure 4E). AICAR or ca-AMPKα1 similarly induced
downregulation of RTKs (EGFR, PDGFRα and PDGFRβ) (Figure 4F). In the patient-derived
primary esophageal cancer cells, itraconazole treatment also induced AMPK activation
(Figure S2E), RTKs degradation (Figure S2F) and Akt inhibition (Figure S2G). Notably,
RTKs expression and pAkt were quite low in the non-cancerous HEEC cells (Figure S2F-G),
which could explain why the epithelial cells were not killed by itraconazole (Figure 1D), even
though AMPK was activated in the cells (Figure S2E). The results indicate that AMPK
activation by itraconazole induces degradation of several RTKs, causing downstream Akt
inhibition.
AMPK activation by itraconazole dictates RTKs lysosomal enrichment and
degradation
Next, the underlying mechanism of itraconazole-induced RTKs degradation was
assessed. As shown in Figure 5A, itraconazole (3.0 μg/mL, 1 hour) treatment in Eca-109
cells induced RTKs (EGFR, PDGFRα and PDGFRβ) translocation from plasma membrane
to lysosome. The plasma membrane-located RTKs were decreased, and the
lysosome-enriched RTKs were increased after itraconazole treatment (Figure 5A).
Na-K-ATPase was tested as the plasma membrane marker protein, and LAMP1
(Lysosome-associated membrane protein 1) is the lysosomal maker (Figure 5A). Such
activity by itraconazole was almost abolished by AMPKα1 shRNA (Figure 5A). ca-AMPKα1
also promoted RTKs translocation to lysosome (Figure 5B). The results suggest that
itraconazole dictates RTKs (EGFR, PDGFRα and PDGFRβ) lysosomal translocation in a
AMPK-dependent manner.
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NH4Cl increases the intra-lysosomal pH and prevents autophagic protein degradations
(33). Further, 3-MA is known to block conversion of LC3B-I to LC3B-II, and to inhibit
autophagosome formation (33). Remarkably, the autophagy/lysosomal inhibitors almost
completely blocked RTKs degradation in Eca-109 cells (Figure 5C). The results suggest that
itraconazole promotes RTKs translocation to lysosome for possible degradation.
Considering that the two autophagy inhibitors also largely attenuated
itraconazole-induced cytotoxicity (Figure 3), we speculate that RTKs degradation might be
the key mechanism of itraconazole-induced cytotoxicity. Therefore, shRNA strategy was
applied to knockdown the RTKs (EGFR, PDGFRα and PDGFRβ) in Eca-109 cells. Western
blot assay results (Figure 5D) confirmed knockdown of all three RTKs by the designated
shRNAs, and activation of Akt (p-Akt at Thr-308) was also largely inhibited (Figure 5D).
Compared to the control cells, shRNA knockdown of all three RTKs inhibited Eca-109 cell
survival (Figure 5E). Significantly, itraconazole was unable to further exert cytotoxicity to the
cells with depleted-RTKs (Figure 5E). The results imply that degradation of RTKs (EGFR,
PDGFRα and PDGFRβ) by itraconazole could be the main cause of Akt inhibition and
subsequent Eca-109 cell death.
Itraconazole oral administration inhibits esophageal cancer cell growth in mice
The potential in vivo anti-esophageal cancer activity by itraconazole was also tested.
Eca-109 cells were inoculated (s.c. injection) to SCID mice, and xenograft tumors were
established. Tumor growth curve results show that daily administration of itraconazole (50
mg/kg, gavage) efficiently inhibited Eca-109 tumor growth (Figure 6A). Estimated daily
tumor growth (in mm3 per day) was also significantly lower in itraconazole-treated mice
(Figure 6B). The Eca-109 tumors were lighter after itraconazole treatment (Figure 6C).
Significantly, same itraconazole (50 mg/kg, daily, gavage) treatment was almost in-effective
against tumors of AMPKα1-shRNA-expressing Eca-109 cells (Figure 6A-C). Thus,
itraconazole failed to inhibit Eca-109 tumor growth when AMPKα1 was silenced. Notably,
mice body weight was not different between the groups (Figure 6D). We also failed to
observe any apparent toxicities in the experimental mice. Thus, the mice were well-tolerated
to the itraconazole administration, as reported by other studies (3, 4).
The signalings were also tested in itraconazole-treated tumor tissues. Western blot
assay was employed. Results show that itraconazole administration in vivo activated AMPK
signaling in Eca-109 tumor tissues, and p-AMPKα1 and p-ACC were significantly increased
at Day-1 after the itraconazole administration (Figure 6E). Further, RTKs (EGFR, PDGFRα
and PDGFRβ) were downregulated in tumor tissues with itraconazole treatment (Figure 6F).
Remarkably, AMPKα1-shRNA-expressing tumors show depleted AMPKα1 (Figure 6E and F).
Itraconazole-induced AMPK activation (p-AMPKα1/p-ACC) and RTKs degradation were
almost completely reversed in AMPKα1-shRNA-expressing tumors (Figure 6E and F). The in
vivo growth of sh-AMPKα1-expresssing Eca-109 tumors was not significantly different from
the control tumors with shRNA-C (Figure S3A-C). The mice body weight was indifferent
between the two groups as well (Figure S3D). One possibility is that the basal AMPK
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activation (AMPK/ACC phosphorylation) was quite low in the control Eca-109 tumors (Figure
S3E). The results indicate that AMPK activation likely mediates itraconazole-induced
anti-tumor activity in vivo.
In order to further confirm the anti-esophageal cancer cell activity in vivo, we next
inoculated primary human esophageal cancer cells to SCID mice. Within three weeks,
tumors of primary cancer cells (“primary tumors”) were established. As shown in Figure 6G
and H, growth of the primary tumors in SCID mice was largely inhibited with the itraconazole
treatment (50 mg/kg, oral administration, daily). Tumor weights (at the terminal) were also
significantly lower after itraconazole administration (Figure 6I). The mice body weight was
not affected (Figure 6J). Therefore, itraconazole efficiently inhibited in vivo growth of the
primary human cancer cells.
Discussion
The results of this study suggest that AMPK activation is the key signaling mechanism
responsible for itraconazole-mediated anti-esophageal cancer cell activity. Pharmacological
AMPK inhibition, AMPKα1 shRNA or dominant negative mutation (T172A) almost
completely nullified itraconazole-induced cytotoxicity in esophageal cancer cells. Conversely,
AMPK activator AICAR or exogenous expression of ca-AMPKα1 mimicked itraconazole
actions. Further mechanistic studies show that itraconazole provoked AMPK-dependent
autophagic cell death (but not apoptosis) in esophageal cancer cells. In vivo, itraconazole
gavage potently inhibited esophageal cancer cell growth in SCID mice. It was however
ineffective against AMPKα1-shRNA tumors.
There are several mechanisms responsible for AMPK-mediated cancer suppressing
actions. For example, activated AMPK directly (by phosphorylating Raptor (34)) or indirectly
(by phosphorylating TSC2 (35)) inhibits mTORC1 signaling (26). Meanwhile, AMPK
activation could provoke pro-apoptotic p53 (36) and JNK (37) cascades. AMPK could also
induce autophagic cancer cell death, directly (vs. phosphorylating Ulk1 (38)) or indirectly (via
inhibiting mTORC1 (26)). In this study, mTORC1 inhibition and autophagy induction were
observed in itraconazole-treated cancer cells. Remarkably, we here propose another
AMPK-dependent mechanism to inhibit cancer cells: by inducing degradation of multiple
RTKs (EGFR, PDGFRα and PDGFRβ).
Concurrent activation of multiple RTKs in human esophageal cancer cells leads to
sustained activation of oncogenic signaling cascades (i.e. PI3K-Akt-mTOR) to promote
cancer progression (39). Tyrosine kinase inhibitor (TKI) against single RTKs could only
result in part or even no inhibition of the downstream cascades, it is therefore less effective
against human cancer cells. The novel finding of this study is that AMPK activation by
itraconazole induces lysosomal enrichment and subsequent degradation of multiple RTKs
(EGFR, PDGFRα and PDGFRβ), causing downstream Akt inhibition and esophageal cancer
cell autophagic death. Such activity by itraconazole was almost reversed with AMPK
blockage, silence or mutation. Notably, the anti-cancer cell activity by itraconazole was
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11
compromised in Eca-109 cells with depleted-RTKs (EGFR, PDGFRα and PDGFRβ).
Additionally, expression of RTKs as well as downstream Akt activation were quite low in the
HEEC epithelial cells (Figure S2), this might explain why these cells were not killed by
itraconazole (Figure 1D). Therefore, itraconazole-induced AMPK-dependent lysosomal
degradation of RTKs (EGFR, PDGFRα and PDGFRβ) could be the primary reason of its
superior and specific anti-cancer cell activity.
The clinical studies have demonstrated that patients with prostate cancer, lung cancer,
and basal cell carcinoma shall benefit from the itraconazole treatment (5-7). Further studies
also proposed the superior anti-cancer activity of itraconazole again leukemia, ovarian,
breast, and pancreatic cancers (5-7). Itraconazole has safe pharmacokinetics as well as a
defined toxicity profile (5-7). Given these information, it would be certainly interesting to
further test itraconazole as a promising anti-esophageal cancer agent in clinical settings.
Declarations
Fundings. This work is supported by the National Natural Science Foundation
(81771457/81302195/31371139/81571282 to C. Cao, 81472786/81773192 to M. B. Chen,
81472305 to P. H. Lu, 81502162 to Z.Q. Zhang), the Six Talents Peak Project of Jiangsu
Province (2014-WSN-012 to M. B. Chen, 2014-WSN-061 to P. H. Lu), Kunshan Science and
Technology Program (KS1418 to M. B. Chen), and by grants from Natural Science
Foundation of Jiangsu Province (BK20130301/BK20170060 to C. Cao, and BK20171248 to
M. B. Chen). The funders had no role in study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Competing interests. The authors declare that they have no competing interests.
Consent for publication. Not applicable
Availability of data and materials. All data generated or analyzed during this study are
included in this published article [and its supplementary information files].
Acknowledgements. Not applicable.
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Figure legends
Figure 1. Itraconazole is cytotoxic and anti-proliferative to human esophageal cancer
cells. Established esophageal cancer cells (Eca-109 and TE-1 lines), primary human
esophageal cancer cells (“Primary cancer cells”) or HEEC esophageal epithelial cells were
treated with/without applied concentrations (0.1-3.0 μg/mL, 0.14-4.25 μM) of itraconazole
(“Itra”), cells were then cultured in conditional medium for designated time, and were
subjected to MTT assay (A and D), clonogenicity assay (B) and trypan blue staining assay
(C). Cell proliferation was evaluated by the BrdU ELISA assay (E and F). n=5 for each assay.
“Ctrl” stands for untreated control group (Same for all figures). * P < 0.05 vs. group “Ctrl”.
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14
Experiments in this and all following figures of in vitro experiments were repeated three
times, with similar results obtained.
Figure 2. Itraconazole fails to induce apoptosis in esophageal cancer cells. Listed cells
were treated with/without applied concentrations (0.3-3.0 μg/mL) of itraconazole (“Itra”) or
C6 ceramide (“C6 Cer”), cells were then cultured in conditional medium for designated time,
and were tested by designated apoptosis assays (A-C and F, data were quantified). Eca-109
cells were pre-treated for 1 hour with 50 μM of Z-DEVD-fmk (“DEVD”), Z-IETD-fmk (“IETD”)
or Z-VAD-fmk (“VAD”), followed by itraconazole (“Itra”, 3.0 μg/mL) treatment for 72 hours,
cell viability (MTT assay, D) and cell death (trypan blue staining assay, E) were tested. * P <
0.05 vs. group “Ctrl”.
Figure 3. AMPK activation mediates itraconazole-induced esophageal cancer
autophagic cell death. Eca-109 cells were treated with itraconazole (“Itra”, 3.0 μg/mL) for
applied time, expression of listed proteins were shown (A and H). Puromycin-selected stable
Eca-109 cells, with scramble control shRNA (“shRNA-Ctrl”) or AMPKα1 shRNA
(“shRNA-AMPKα1”), were treated with itraconazole, or plus AMPK inhibitor Compound C
(“CC”, 10 μM); Expression of listed proteins were shown (B); Cell viability (C) was tested.
Eca-109 cells with dominant negative mutant AMPKα1 (T172A, “dn-AMPKα1”, Flag-tagged),
constitutively-active AMPKα1 (T172D, “ca-AMPKα1”, Flag-tagged) or empty vector
(“Vector”), were treated with/without itraconazole (3.0 μg/mL); Expression of listed proteins
were shown (D and F); Cell viability was also tested (E and G). Stable Eca-109 cells, with
shRNA-Ctrl, shRNA-AMPKα or dn-AMPKα1, were treated with itraconazole (24 hours),
LC3B-GFP positive cells were counted (I); Eca-109 cells, pretreated with 3-methyladenine
(“3-MA”, 2.5 mM) or ammonium chloride (“NH4Cl”, 1 mM) for 1 hour, were treated
with/without itraconazole (3.0 μg/mL) for 72 hours, cell viability was tested (J). Note that the
exact same set of lysate samples were run in sister gels to examine different proteins, and
the blot was stripped and re-probed (Same for all Figures). “DMSO” stands for 0.1% of
DMSO. “C” stands for parental Eca-109 cells (D, E and G). * P < 0.05 vs. group “Ctrl”. # P <
0.05 vs. group “shRNA-Ctrl” or “Vector” (E and G). & P < 0.05 (I and J).
Figure 4. Itraconazole in-activates Akt-mTORC1 signaling and induces multiple
receptor tyrosine kinases (RTKs) degradation. Puromycin-selected stable Eca-109 cells,
expressing scramble control shRNA (“shRNA-Ctrl”) or AMPKα1 shRNA (“shRNA-AMPKα1”),
were treated with itraconazole (3.0 μg/mL) or plus Compound C (“CC”, 10 μM) for applied
time; Expression of listed proteins were tested (A, C and E); Eca-109 cells with
constitutively-active AMPKα1 (T172D, “ca-AMPKα1”) or empty vector (“Vector”,
p-Super-puro), were treated with/without AICAR (1 mM, 12 hours), expression of listed
proteins were shown (B, D and F); Expression of indicated proteins was quantified.
Figure 5. AMPK activation by itraconazole dictates RTKs lysosomal enrichment and
degradation. Eca-109 cells, expressing scramble control shRNA (“shRNA-C”), AMPKα1
shRNA (“sh-AMPKα1”) or constitutively-active AMPKα1 (T172D, “ca-AMPKα1”), were
treated with/without itraconazole (“Itra”, 3.0 μg/mL) for 1 hour; Cell plasma fraction and
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15
lysosomal fraction were isolated, expression of listed proteins were shown (A and B);
Eca-109 cells, pretreated with 3-methyladenine (“3-MA”, 2.5 mM) or ammonium chloride
(“NH4Cl”, 1 mM) for 1 hour, were treated with/without itraconazole (“Itra”,3.0 μg/mL) for 24
hours, RTKs expression were tested (C). Expression of the listed RTKs in Eca-109 cells
infected with EGFR-shRNA plus PDGFRα/β-shRNA or scramble control shRNA (“sc-sh”)
were shown, p-Akt and regular Akt1 were also tested (D). Above cells were also treated with
itraconazole (“Itra”, 3.0 μg/mL) for 72 hours, cell viability (E) was tested. * P < 0.05.
Figure 6. Itraconazole-induced the anti-esophageal cancer cell activity in vivo. Exact
same amount of stable Eca-109 cells, expressing scramble control shRNA (“shRNA-C”) or
AMPKα1 shRNA (“sh-AMPKα1”) (A-F), as well as the primary human esophageal cancer
cells (G-J), were inoculated to the SCID mice. When the tumor volumes were around 150
mm3, mice were randomly assigned to the listed groups, with 10 mice per group.
Itraconazole (50 mg/kg, gavage, daily) or vehicle (“Veh”) administration was performed, the
tumor volume (A and G) and the mice body weight (D and J) were recorded every 7 days;
Daily tumor growth (in mm3 per day) was also calculated (B and H); At day-42, all animals
were subjected to surgery, tumors were separated and weighted (C and I). At experimental
Day-1, 16 hours after the itraconazole administration, one Eca-109 tumor per group was
isolated, expression of listed proteins in tumor lysates were shown (E and F). * P < 0.05 vs.
group “Veh” (A and G). # P < 0.05 vs. group “Itra+shRNA-C” (A).* P < 0.05 (B-C).
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0
20
40
60
80
100
120
Ctrl 0.1 0.3 1.0 3.0
24h48h72h96h
** *
** *
** **
Itra (μg/mL)
MTT
OD
(% v
s. “
Ctr
l”)
0
20
40
60
80
100
120
Ctrl 0.1 0.3 1.0 3.0
TE-1Primary cancer cellsHEEC
Itra (μg/mL), 72 h
MTT
OD
(% v
s. “
Ctr
l”)
* **
*
**
*
Eca-109
0
10
20
30
40
50
Ctrl 0.1 0.3 1.0 3.0
*
**N
umbe
r of c
olon
ies
per d
ish
Itra (μg/mL), 10 days
Eca-109
0
10
20
30
Ctrl 0.1 0.3 1.0 3.0
*
* *
Tryp
an b
lue
(%)
Itra (μg/mL), 72 h
Eca-109
Figure 1.
A. B.
C. D.
0
20
40
60
80
100
120
Ctrl Itra Ctrl Itra Ctrl Itra (3.0 μg/mL), 24 h
TE-1Primary cancer cells HEEC
* *
Brd
U O
D (%
vs.
“C
trl”
)
E. F.
20
40
60
80
100
120
Ctrl 0.1 0.3 1.0 3.0Itra (μg/mL), 24 h
Brd
U O
D (%
vs.
“C
trl”
)
Eca-109
** *
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0
0.3
0.6
0.9
Ctrl 0.1 0.3 1.0 3.0 C6 Cer
Apo
ptos
is E
LISA
OD
*
P > 0.05
Itra (μg/mL), 48 h 10 μg/mL
0
4
8
12
16
Ctrl 0.1 0.3 1.0 3.0 C6 Cer
P > 0.05
*
Itra (μg/mL), 48 h 10 μg/mL
Ann
exin
V (%
)
0
5
10
15
20
25
TUN
EL (%
)
Ctrl 0.1 0.3 1.0 3.0 C6 Cer
Itra (μg/mL), 48 h 10 μg/mL
*
Figure 2.
Eca-109 Eca-109 Eca-109A. B. C.
0
20
40
60
80
100
120 DMSODEVDIETDVAD
MTT
OD
(% v
s. “
Ctr
l”)
Ctrl Itra (3.0 μg/mL), 72 h
P > 0.05
Eca-109 Eca-109
0
10
20
30DMSODEVDIETDVAD
Tryp
an b
lue
(%)
Ctrl Itra (3.0 μg/mL), 72 h
P > 0.05
0
1
2
3
4
5
6
Ctrl Itra Ctrl Itra Ctrl Itra (3.0 μg/mL), 48 h
TUN
EL (%
)
TE-1
Primary cancer cells
HEEC
D. E. F.
P > 0.05
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0
20
40
60
80
100
120DMSOshRNA-CtrlCCshRNA-AMPKα1
MTT
OD
(% v
s. “
Ctr
l”)
*
* *
#
Ctrl Itra (3.0 μg/mL), 72 h
Ctrl Itra Ctrl Itra, 3.0 μg/mL, 12hp-AMPKα1 T172
p-ACCS79
AMPKα1
ACC
DMSO CC DMSOshRNA-Ctrl shRNA-AMPKα1
Ctrl Itra
A. B. C.
*#
0
20
40
60
80
100
120CVectordn-AMPKα1
MTT
OD
(% v
s. “
Ctr
l”)
*#
* *
Ctrl Itra (3.0 μg/mL), 72 h
Figure 3
Vector
ca-A
MPKα1
0
20
40
60
80
100
120
CVec
tor
ca-A
MPKα1
#
MTT
OD
(% v
s. “
C”)
At day-4
p-AMPKα1 T172
p-ACCS79
AMPKα1
ACC
Akt1
ca-AMPKα1
Itra, 3.0 μg/mL, 12hD. E. F. G.
p-AMPKα1 T172
62kD-
p-ACCS79
280kD-
AMPKα1
ACC280kD-
62kD-
1h 3h 6h 12h
Itra, 3.0 μg/ml
Ctrl
Akt160kD-
Eca-109
C Vector
dn-A
MPKα1
p-AMPKα1 T172
p-ACCS79
AMPKα1
ACC
Akt1
dn-AMPKα1
62kD-
280kD-
280kD-
62kD-
60kD-
-LC3B-II
Beclin-1
β-actin
p-Ulk1
p62
55kD-
60kD-
14kD-
150kD-
45kD-
62kD-
Ulk1
-LC3B-I
ATG-5
150kD-
16kD-
Ctrl 12h 24h
Itra (3.0 μg/mL)
0
20
40
60
80
100
120
MTT
OD
(% v
s. “
Ctr
l”)
Itra (3.0 μg/mL), 72 hCtrl
DMSO3-MANH4Cl
*
&&
H. I. J.
0
10
20
30shRNA-Ctrlsh-AMPKα1 dn-AMPKα1
Itra (3.0 μg/mL), 24 hCtrl
LC3B
pun
cta
(%)
&&
*
62kD-
280kD-
280kD-
62kD-
62kD-
280kD-
280kD-
62kD-
60kD-
Akt160kD-
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A. B.
C.
Figure 4
p-4E-BP1Ser65
S6
4E-BP1
pS6Ser235/236
20kD-
20kD-
32kD-
32kD-
1.00 0.14 1.09 0.92 0.99 0.86
1.00 0.12 0.93 0.97 1.31 1.12
Ctrl Itra Ctrl Itra, 3.0 μg/mL, 12hDMSO CC DMSOshRNA-Ctrl shRNA-AMPKα1
Ctrl Itra
1.00 0.01 0.89 0.85 0.77 1.06
1.00 0.01 0.93 1.07 1.15 0.89
p-Akt S473
Akt1
p-Akt T308
60kD-
60kD-
60kD-
Ctrl Itra Ctrl Itra, 3.0 μg/mL, 24hDMSO CC DMSOshRNA-Ctrl shRNA-AMPKα
Ctrl Itra
1.00 0.16 0.15
1.00 0.11 0.04
Ctrl
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AR
ca-A
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α1p-Akt S473
Akt1
p-Akt T308
D.
E. F.
p-4E-BP1
p-S6
S6
4E-BP1
Ctrl
AIC
AR
ca-A
MPK
α1
1.00 0.06
1.00 0.16 0.24
0.22
PDGFRα
EGFR
PDGFRβ
β-actin
Ctrl Itra Ctrl Itra, 12hDMSO CC DMSOshRNA-Ctrl shRNA-AMPKα1
Ctrl Itra
1.00 0.00 1.04 1.01 1.28 1.12
1.01 0.11 0.89 0.91 0.93 0.87
1.00 0.06 0.91 0.88 0.90 0.68
190kD-
190kD-
42kD-
170kD-EGFR
β-actin
Ctrl
AIC
AR
ca-A
MPK
α1
PDGFRβ
PDGFRα
1.00 0.26 0.02
1.00 0.11 0.07
1.00 0.04 0.10
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175kD-
190kD-
190kD-
100kD-
110kD-
Ctrl+shRNA-C
Itra+shRNA-C
Itra+sh-AMPKα1
1.00 0.26 0.78
1.00 0.27 0.99
1.00 0.31 1.00
175kD-
190kD-
190kD-
100kD-
110kD-
PDGFRα
EGFR
PDGFRβ
LAMP1
Na,K-ATPase
PDGFRα
EGFR
PDGFRβ
LAMP1
Na,K-ATPase
175kD-
190kD-
190kD-
100kD-
110kD-
Plasma membrane Plasma membrane
1.00
1.00
1.00
PDGFRα
EGFR
PDGFRβ
LAMP1
Na,K-ATPase
0.19
0.14
0.07
Lysosome Lysosome
0
20
40
60
80
100
120
Ctrl Itra Ctrl Itra
P > 0.05
MTT
OD
(% v
s. “
Ctr
l” o
f “sc
-sh”
)
shRNA-C
*
sh-PDGFRα/β+sh-EGFR
A. B.
Figure 5
C. D. E.
EGFR
PDGFRα
PDGFRβ
Tubulin
Itra
+NH4C
l
+3-M
ACtrl
175kD-
190kD-
190kD-
55kD-
175kD-
190kD-
190kD-
60kD-
60kD-
PDGFRα
EGFR
PDGFRβ
Akt1
pAkt
shRNA-C sh-PDGFRα/β+sh-EGFR
Ctrl+shRNA-C
Itra+shRNA-C
Itra+sh-AMPKα1
Vectorca-AMPKα1
Vectorca-AMPKα1
175kD-
190kD-
190kD-
100kD-
110kD-
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A. B. C.Figure 6
D.E. F.
0
5
10
15
20
25
30
35
Estim
ated
tum
or g
row
th
(mm
per
day
)3
* *
n=10
Veh+shRNA-C
Itra+shRNA-C
Itra+sh-AMPKα1
0
400
800
1200
Tum
or w
eigh
ts (m
g)
* *
n=10
Veh+shRNA-C
Itra+shRNA-C
Itra+sh-AMPKα1
15
17
19
21
23
D0 D7 D14 D21 D28 D35 D42
Ani
mal
wei
ght (
g)
n=10
Veh+shRNA-CItra+shRNA-CItra+sh-AMPKα1
0
300
600
900
Tum
or w
eigh
ts (m
g)
*
n=10
Veh Itra0
400
800
1200
D0 D7 D14 D21 D28 D35 D42
Tum
or v
olum
es (m
m )3 Veh
Itra
n=10
*
G. H. I. J.
0
5
10
15
20
25
Estim
ated
tum
or g
row
th
(mm
per
day
)
*
n=10
Veh Itra
30
400
800
1200
1600
D0 D7 D14 D21 D28 D35 D42
Veh+shRNA-CItra+shRNA-CItra+sh-AMPKα1
n=10
Tum
or v
olum
es (m
m )3
*
#
Eca-109 tumors
Primary tumors
16
18
20
22
24
D0 D7 D14 D21 D28 D35 D42
VehItra
n=10
Ani
mal
wei
ght (
g)
PDGFRα
EGFR
PDGFRβ
β-actin
Veh+shRNA-C
Itra+shRNA-C
Itra+sh-AMPKα1
175kD-
190kD-
190kD-
45kD-
D1
p-AMPKα1 T172
p-ACCS79
AMPKα
ACC
Tubulin
Veh+shRNA-C
Itra+shRNA-C
Itra+sh-AMPKα1
62kD-
280kD-
280kD-
62kD-
55kD-
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Published OnlineFirst March 28, 2018.Mol Cancer Ther Min-Bin Chen, Yuan-Yuan Liu, Zhao-yu Xing, et al. cell growth requires AMPK activationItraconazole-induced inhibition on human esophageal cancer
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