Vinorelbine-induced oxidative injury in human endothelial cells
mediated by AMPK/PKC/NADPH/NF-
B pathways
Tsai et al: Vinorelbine-induced endothelial cell dysfunction
Kun-LingTsaia, Tsan-Hung Chiub, Mei-Hsueh Tsaic, Hsiao-Yun Chend, and Hsiu-Chung Ouc
a. Department of Physical Therapy and Graduate Institute of Rehabilitation Science,
China Medical University, Taichung, Taiwan; b. Department of Obstetrics and
Gynecology, China Medical University Hospital, Taichung, Taiwan; c. 3M Taiwan
LTD. Health Care Business; d. Institute of Clinical Medicine, National Yang-Ming
University, Taipei, Taiwan.
Correspondence to: Hsiu-Chung Ou, PhD, Department of Physical Therapy and
Graduate Institute of Rehabilitation Science, China Medical University,
Taichung,Taiwan No. 91, Shuch-Shih Road, Taichung 404, Taiwan
Fax: 886-4-22065051
Tel: 886-4-22053366 ext 7313
E-mail address: [email protected]
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Abstract Vinorelbine tartrate (VNR), a semi-synthetic vinca alkaloid acquired from
vinblastine, has extensively been used as an anticancer agent. However, VNR-induced
oxidative damage may cause several side-effects, such as venous irritation, vascular
pain and necrotizing vasculitis, thereby repressing clinical treatment efficiency. The
molecular mechanisms underlying the induced oxidative stress in endothelial cells are
still largely unknown. The present study was designed to test the hypothesis that VNR
induces oxidative injury via modulation of AMP-activated protein kinase (AMPK)
and possible mechanisms were then explored. Human umbilical vein endothelial cells
(HUVECs) were treated with VNR (5-0.625 M) to produce oxidative damage. The
VNR-mediated AMPK, PKC and NADPH oxidase expression were investigated by
Western blotting. Moreover, several oxidative stress-induced oxidative damage
markers as well as pro-inflammatory responses were also investigated.VNR treatment
resulted in dephosphorylation of AMPK, which in turn led to an activation of NADPH
oxidase by PKC, however, the phenomena were repressed by AICAR (an agonist of
AMPK) or AMPK siRNA. Moreover, VNR suppressed Akt/eNOS and enhanced p38
mitogen-activated protein kinase (MAPK), which in turn activated the NF-B
pathway. Furthermore, VNR facilitated several pro-inflammatory events, such as the
adherence of monocytic THP-1 cells to HUVECs, pro-inflammatory cytokines release
as well as over-expression of adhesion molecular. Our results highlight a possible
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molecular mechanism for VNR-mediated endothelial dysfunction.
Highlights:
1. VNR-mediated endothelial cells dysfunction by increasing oxidative stress.
2. VNR-facilitated oxidative stress by repressing AMPK- and activating PKC-
3. VNR-induced ROS production mainly go through NADPH oxidase activation.
4. Knockdown of AMPK impairs the VNR-induced PKC-and p47phox
activation.
5. VNR-induced endothelial cells inflammation by activating MAPKs and NF-B
pathway.
Key words: Vinorelbine tartrate, reactive oxygen species, endothelial cells, oxidative
stress, AMPK
Abbreviations: Vinorelbine tartrate (VNR), reactive oxygen species (ROS),
AMP-activated protein kinase (AMPK), protein kinase C (PKC), mitogen-activated
protein kinase (MAPK), diphenyleneiodonium (DPI), ethylene diaminotetraacetic
acid (EDTA), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion
molecules (ICAM-1), interleukin-8 (IL-8), nitric oxide (NO), endothelial NO synthase
(eNOS) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
Introduction
Vinca alkaloids remain among the most effective classes of anticancer drugs in
clinical use [1]. Vinorelbine tartrate (VNR) is a semi-synthetic vinca alkaloid acquired
from vinblastine. Vinorelbine has extensively been used as an anti-cancer or
anti-angiogenesis drug in clinical strategies [2, 3]. VNR represses the polymerization
of tubulin into microtubules and attenuates spindle formation by binding to tubulin [4,
5]. However, several side effects of VNR in clinical management have been reported,
such as venous irritation, vascular pain, phlebitis, and necrotizing vasculitis [6].
Moreover, Yamada et al. demonstrated VNR-mediated human endothelial cell
apoptosis through the induction of reactive oxygen species (ROS) production and
repression of antioxidant enzyme function [7].
In endothelial cells, AMP-activated protein kinase (AMPK) plays a central role
to maintain and detect intracellular homeostasis [8]. AMPK is also a well-known and
important actor in modulating cellular energy balance and metabolism and responses
to metabolic-related stress in endothelial cells [9], indicating that AMPK is functional
and effective in repressing oxidative stress-mediated injury in endothelial cells.
AMPK has several isoforms, including AMPK-1,β1, and1 in endothelial cells, that
manipulate multiple signal transduction pathways, with effects that include mitigating
intracellular ROS formation, attenuating NADPH oxidase activation, reinforcing the
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AKT pathway, and enhancing NO bioavailability [10, 11] . Moreover, Sag et al.
demonstrated that AMPK plays a role as a cardiovascular protector by counteracting
oxidative stress, inhibiting inflammatory responses, and activating eNOS expression
in endothelial cells [12].
Previous studies have shown that protein kinase C (PKC) is downstream of
AMPK, where inhibition of PKC expression contributes to the attenuation of NADPH
oxidase-derived ROS production [13]. NADPH oxidase is comprised of
membrane-bound gp91phox and p22hox, as well cytosolic subunits such as p47phox,
p67phox, and the small GTPase Rac. Endothelial NADPH oxidase-derived ROS
generation appears to be a driving force in the development of endothelial dysfunction
and cardiovascular disease, indicating that NADPH oxidase-activated ROS act as a
secondary messenger to initiate downstream signal transduction pathways [14, 15],
such as activation of p38 mitogen-activated protein kinase (MAPK), stimulation of
nitric oxide (NO) catabolism as a result of superoxide generation, and inhibition of
NO release via attenuated endothelial NO synthase (eNOS) [16], thereby activating
NF-B, which in turn triggers downstream pro-inflammatory responses[17, 18]
In clinical chemotherapy intervention, endothelial cells injury may contribute to
vascular dysfunction and decrease treatment efficiency [19]. Yamada et al. showed
that VNR-induced endothelial apoptosis was facilitated by ROS generation and
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eliminated antioxidant enzyme expression. However, the detailed mechanisms of
VNR-mediated injury in human endothelial cells are still unclear. In this study, we
hypothesize that VNR-mediated oxidative damage is modulated by AMPK, increasing
PKC and NADPH oxidase activation, thereby facilitating ROS generation, decreasing
AKT/eNOS expression and increasing the NF-B-mediated pro-inflammatory
response. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
Materials and Methods
Reagents. Fetal bovine serum, M199, and trypsin-EDTA were obtained from Gibco (Grand Island, NY, USA). Low serum growth supplement was obtained from Cascade (Portland, OR). Vinorelbine, diphenyleneiodonium (DPI), ethylene diaminotetraacetic acid (EDTA), pyrrolidine
dithiocarbamate (PDTC), metformin, penicillin, and streptomycin were obtained from Sigma (St. Louis, MO). SB203580 and compound C were purchased from Calbiochem (CA, USA). Anti-vascular cell adhesion molecule-1 (VCAM-1), anti-intercellular adhesion molecules (ICAM-1), anti-E-selectin, and Interleukin-8 (IL-8) ELISA kits were purchased from R&D Systems (Minneapolis, MN). Anti-p22phox and anti-gp91 were obtained from Santa Cruz (CA, USA), and anti-NF-κB/p65, anti-IκBα, anti-AMPK, anti-AMPK-, anti-AKT, anti-phospho AKT, anti-phospho eNOS, anti-eNOS, anti-PCNA, anti-phospho p38, anti-p38, anti-PKC, and anti-phospho PKC were obtained from Transduction Laboratories (CA, USA). Anti-Rac-1 and anti-p47phox were obtained from BD Biosciences (NJ, USA). Anti-cyclooxygenase-2 (COX-2) was obtained from Chemicon (MA, USA).
Cell cultures. These experiments were approved by the Research Ethics
Committee of the China Medical University Hospital. After receiving written
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informed consent from the parents, fresh human umbilical cords were obtained
from neonates after birth, collected and suspended in Hanks' Balanced Salt
Solution (HBSS) (Gibco, USA) at 4°C. Human umbilical vein endothelial cells
(HUVECs) were isolated with collagenase and used at passage 2-3 [20]. After
dissociation, the cells were collected and cultured on gelatin-coated culture
dishes in medium 199 with low serum growth supplement, 100 IU/mL penicillin,
and 0.1 mg/mL streptomycin. Subcultures were performed with trypsin-EDTA.
Media were refreshed every two days.
Total RNA isolation and real-time PCR reaction. To investigate the
effects of VNR-induced adhesion molecule gene expression in endothelial cells,
HUVECs were incubated with VNR for 24 hours. Total RNA was isolated using
TRIzol reagent. Reverse transcription was performed at 42°C for 60 min,
followed by incubation at 95°C for 5 min. The reaction 20 mixture (20 μl of total volume) consisted of 2 μg of isolated total RNA, 1 mM dNTP, 1 unit/μl of
recombinant RNasin ribonuclease inhibitor, 15 U/μg of avian myeloblastosis
22 virus (AMV) reverse transcriptase, 5× RT buffer, and 0.5 μg of oligo(dT)12
primer. The gene-specific primers used were as follows:
ICAM-1 sense: 5’-CCGAGCTCAAGTGTCTAAAG-3’; ICAM-1 antisense:
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5’-TGCCACCAATATGGGAAGGC-3’; VCAM-1 sense:
5’-AAGATGGTCGTGATCCTTGG-3’; VCAM-1 antisense:
5’-GGTGCTGCAAGTCAATGAGA-3’; E-selectin sense:
5’-AGCTTCCCATGGAACACAAC-3’; E-selectin antisense:
5’-CTGGGCTCCCATTAGTTCAA-3’; -actin sense:
5’-GGACTTCGAGCAAGAGATGG-3’; and -actin antisense:
5’-AGCACTGTGTTGGCGTACAG-3’. Real-time PCR reactions were
performed using the SYBR Green method in an ABI 7000 sequence detection
system (Applied Biosystems, Foster City, CA) following the manufacturer's
guidelines. Primers were designed using the computer software Primer Express
2.0 (Applied Biosystems, Foster City, CA). The reactions were set by mixing 12.5 μl of the SYBR Green Master Mix (Applied Biosystems, Foster City, CA)
with 1 μl of a solution containing 10 μM concentrations of both primers and 2 μl
of cDNA solution. The Ct value was defined as the number of PCR cycles
required for the fluorescence signal to exceed the detection threshold value. The
relative amounts of mRNA for each gene were normalized based on the amount
of the housekeeping gene -actin.
Measurement of ROS production. HUVECs (104 cells/well) in 96-well plates were pre-incubated with 10 M DCF-AM for 1 h; the fluorescence intensity was
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measured with a fluorescence microplate reader (Labsystems, CA, USA) calibrated for excitation at 485 nm and emission at 538 nm (before and after 24 hours of stimulation with various concentrations of VNR),The percentage increase in fluorescence per well was calculated by the formula [(Ft2-Ft0)/Ft0] X 100, where Ft2 is the fluorescence at at each time (15, 30, 60, 120 mins) of VNR exposure and Ft0 is the fluorescence at 0 min of VNR exposure.
Transfection with small interfering RNA (siRNA). ON-TARGETplus
SMARTpool siRNAs for non-targeting controls and NF-Bp65 siRNAs were
purchased from Dharmacon. Three days after transfection, cells were treated with the indicated reagent for further experiments.
Preparation of nuclear and cytosolic extracts. Nuclear and cytosolic extracts were isolated with a Nuclear and Cytoplasmic Extraction kit (Pierce Chemical, Rockford, IL). After the incubation period, HUVECs were collected by centrifugation at 600 g for 5 min at 4°C. The pellets were washed twice with ice-cold PBS, followed by the addition of 0.2 ml of cytoplasmic extraction buffer A and vigorous mixing for 15 s. Ice-cold cytoplasmic extraction buffer B (11l) was added to the solution. After vortex mixing, nuclei and cytosolic fractions were separated by centrifugation at 16,000 g for 5 min. The cytoplasmic extracts (supernatants) were stored at -80°C. Nuclear extraction buffer was added to the nuclear fractions (pellets), which were then mixed by vortex mixing on the highest setting for 15 s. The mixture was iced, and a 15-s vortex was performed every 10 min for a total of 40 min. Nuclei were centrifuged at 16,000 g for 10 min. The nuclear extracts (supernatants) were stored at -80°C until use.
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Preparation of membrane and cytosolic extracts. A cellular membrane fraction was prepared with Mem-PER (Pierce) according to the manufacturer’s instructions. The Mem-PER system consists of three reagents: reagent A is a cell lysis buffer, reagent B is a detergent dilution buffer, and reagent C is a membrane solubilization buffer. After the incubation period, HUVECs were collected by centrifugation at 600 g for 5 min at 4°C. Each cell pellet, containing 5x106 cells, was lysed at room temperature using Mem-PER reagent A. Membrane proteins were solubilized on ice with Mem-PER reagent C diluted 2:1 with Mem-PER reagent B. Reagents A and B/C were supplemented with Halt protease inhibitor cocktail (Pierce Biotechnology). The solubilized protein mixture was centrifuged at 10,000 g for 3 min at 4°C to remove cellular debris. The clarified supernatant was heated at 37°C for 10 min, followed by centrifugation at 10,000 g for 2 min to produce separated membrane and hydrophilic protein fractions. The hydrophobic fraction of the membrane proteins (bottom layer) was stored at -80°C until use.
Protein kinase C-assay. HUVECs were grown to confluence and then
stimulated with VNR for 1 hour. At the end of the incubation period, cells were rinsed
with ice-cold PBS and lysed by the addition of reaction buffer (50 mM HEPES, pH
7.2, 0.01% BSA, 10 mM MgCl2, 1 mM DTT, and 1x lipid activator, provided in the
kit). Protein kinase C- activity in wholecell lysate (10 μg) was measured with a
PKC- activity assay kit (nonradioactive) according to the manufacturer’s
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instructions (Upstate Biotechnology).
Immunoblotting. To determine how VNR-mediated signaling pathways are
altered, HUVECs were incubated with VNR for 24 hours. At the end of stimulation,
cells were washed, scraped from dishes, and lysed in RIPA buffer. Proteins were then
separated by electrophoresis on SDS-polyacrylamide gels. After the proteins had been
transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA), the
blots were incubated with blocking buffer for 1 h at room temperature and then probed with primary antibodies overnight at 4℃, followed by incubation with
horseradish peroxidase-conjugated secondary antibody for 1 h. To control for unequal
loading of total protein in all lanes, blots were stained with mouse anti-actin
antibody. The bound immunoproteins were detected via an enhancer
chemiluminescent assay (ECL; Amersham, Berkshire, UK). The intensities were
quantified by densitometric analysis (Digital Protein DNA Imagineware, Huntington
Station, NY).
Adhesion molecule expression. To determine whether VNR could enhance the
level of adhesion molecule expression, HUVECs were incubated with VNR for 24
hours. Following stimulation, HUVECs were harvested and incubated with
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fluorescence-conjugated anti-ICAM-1, anti-VCAM-1, and anti-E-selectin (R&D,
Minneapolis, MN) for 45 min at room temperature. After the HUVECs had been
washed three times, their immunofluorescence intensity was analyzed by flow
cytometry using a Becton Dickinson FACScan flow cytometer (Mountain View, CA).
Adhesion assay. HUVECs at 1X105 cells/mL were cultured in 96-well plates. HUVECs were incubated with VNR for 24 hours. The medium was then
removed, and 0.1 mL/well of THP-1 cells (prelabeled with 4 M BECF-AM for
30 min in RPMI at 1X106 cells/mL density) were added to fresh RPMI. The cells were allowed to adhere at 37◦C for 1 h in a 5% CO2 incubator. Plates were
washed three times with M199 to remove the non-adherent cells. The number of
adherent cells was estimated by microscopic examination, and the cells were
then lysed with 0.1 mL 0.25% Triton X-100. Fluorescence intensity was
measured with a fluorescence microplate reader (Labsystem, CA) calibrated for
excitation at 485 nm and for emission at 538 nm.
Assay for IL-8 secretion. HUVECs were seeded in 24-well plates at 0.5×105 cells. After 2 days, HUVECs were incubated with VNR for 24 hours. At the end
of the VNR incubation period, cell supernatants were removed and assayed for
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IL-8 concentration using an ELISA kit obtained from R&D Systems
(Minneapolis, MN). Data are expressed as ng/mL for duplicate samples.
Statistical analyses. Results are expressed as mean±SEM. Differences
between groups were analyzed using one-way ANOVA followed by Bonferroni's
post hoc test. A P-valueb0.05 was considered statistically significant.
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Results
VNR induced dephosphorylation of AMPK-, phosphorylation of
PKC-as well as PKC-activity. AMPK can reverse and alter many cellular
pathways to protect against oxidative injury [21]. We assumed that VNR-induced
endothelial cell dysfunction was caused by repression of AMPK phosphorylation. To
verify our hypothesis, the protein expression level of phosphorylated AMPK was
determined using a Western blotting assay. As shown in Fig. 1A and 1B, treatment of
HUVECs with VNR for 1 hour led to an attenuation of phosphorylated AMPK- in a
dose-dependent manner.
Moreover, previous studies have shown that PKC isoforms play a key role in the
regulation of NADPH subunit expression and, in particular, the translocation of
p47phox from the cytosol to the membrane [22, 23], and AMPK- can inhibit ROS production via suppression of protein kinase C (PKC), which in turn prevents the
activation of NADPH oxidase [13]. We therefore focused our attention on
determining whether VNR facilities PKC phosphorylation and activation in human
endothelial cells. As shown in Fig. 1C and 1E, VNR markedly increased
phosphorylation of PKC- and PKC- activity after a 1 h exposure. Pretreatment of
AICAR, one agonist of AMPK, significantly mitigated VNR-promoted
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phosphorylation of PKC-and PKC- activity indicating that PKC is implicated in
VNR-induced endothelial cell injury and mainly go through AMPK.
VNR induced membrane assembly of NADPH. A previous study revealed that
VNR induces ROS formation in endothelial cells, thereby facilitating endothelial cell
apoptosis[7]. We proposed that VNR facilitates ROS production mainly by promoting
PKC phosphorylation and activating NADPH oxidase. DPI, an inhibitor of NADPH
oxidase, was used to prove our hypothesis. In endothelial cells, the NOX family of
NADPH oxidases is an important source of ROS generation. NADPH oxidase is
composed of two membrane components, Nox2 (also called gp91phox) and p22phox,
and three cytoplasmic components, p47phox, p67phox, and the small GTPase Rac-1.
The process by which the NADPH oxidase enzyme complex is activated begins with
the phosphorylation of p47phox, which causes translocation of the p47 phox /p67phox
complex to the plasma membrane, where p47phox interacts with p22phox and
p67phox acts as a NOX activator through a direct protein-protein interaction.
Therefore, we verified the effects of NADPH oxidase activation after exposure to
VNR. The membrane translocation assay showed that the levels of p47phox and
Rac-1 in membrane fractions of HUVECs were higher in cells treated with VNR for 1
hour than control cells. In addition, we found that the protein levels of gp91 and p22
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phox were increased significantly in HUVECs exposed to VNR for 24 hours.
Moreover, pretreatment of VNR-exposed cells with AICAR led to a reduction in
membrane assembly of p47 phox and Rac-1, as well as suppression of gp91 and p22 phox protein expression (Fig. 2A-C).
VNR-induced intracellular ROS generation in HUVECs. Fluorescence
intensity were measured to clarify whether the VNR-promoted intracellular ROS
formation in human endothelial cells. As Fig.3, our data showed that exposure to
VNR for 2 hours resulted in an increasing of ROS in a dose-dependent manner. In
addition, pre-treatment with DPI and AICAR abrogated the VNR-elicited ROS
generation, suggesting that ROS generation were largely dependent on the repressing
AMPK function and the subsequent activation of NADPH oxidase.
VNR mediated oxidative injury involves Akt/eNOS deactivation. Akt serves
a major role in promoting cell survival in response to various death stimuli. Moreover,
Akt activates endothelial nitric oxide synthase (eNOS), which leads to nitric oxide
(NO) production. Studies have reported that oxidative stress decreases Akt and eNOS
phosphorylation in endothelial cells [24], while activation of Akt and eNOS are
known to repress apoptosis and promote cell survival [25]. To investigate whether
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AMPK/Akt/eNOS signaling is involved in the impaired effects of VNR, we
performed a Western blot analysis using phosphor-specific Akt (Ser473) and
phosphor-eNOS (Ser1177) antibodies. As expected, VNR significantly lessened the
phosphorylation of Akt and eNOS in a dose-dependent manner (Fig 4). Pretreatment
of AICAR manifestly restored the expression level of phosphorylated Akt and eNOS.
This finding suggested that AMPK/Akt/eNOS signaling is involved in the
VNR-induced oxidative injuries. Moreover, pretreatment DPI also reduced
VNR-repressed Akt and eNOS phosphorylation. This result suggesting that the
NADPH oxidase-derived ROS play an important role to repress Akt and eNOS
function, thereby inducing endothelial cells oxidative damage.
VNR induced ERK activation and decreased PPAR- expression. ERK
signaling plays an essential role in oxidative stress-mediated signaling. Peroxisome
proliferator-activated receptors (PPARs) comprise a superfamily of nuclear hormone
receptor proteins that function as transcription factors. Activation of PPAR-γ has been
displayed to repress expression of pro-inflammatory mediators such as
cyclooxygenase-2 (COX-2) and NF-κB [26, 27]. We next focused our attention on
determining whether VNR facilitates endothelial cell dysfunction by activating ERK
phosphorylation and attenuating PPAR- expression. As shown in Fig. 5, treatment of
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VNR markedly activated ERK phosphorylation and decreased PPAR- expression.
Nevertheless, pretreatment with AICAR suppressed the phenomenon. The results
were support out hypothesis. VNR increases ERK phosphorylation and decreases
PPAR- function by modulating AMPK expression. Pretreatment of DPI also
protected against VNR- activated ERK phosphorylation and decreased PPAR-
expression, revealing that the NADPH oxidase-derived ROS are key mediator
involved in this signaling.
VNR induced NF-B activation by modulation of p38MAPK. Oxidative
stress-mediated ROS can mediate p38MAPK and phosphoinositide 3-kinase (PI3K)
activation, and both of these mediators cause NFB activation, which facilitates
nuclear translocation and subsequent manipulation of pro-inflammatory events [2]. As
shown in Fig. 6A, VNR clearly induced p38MAPK phosphorylation as compared to
control cells. However, pre-treatment with SB203580, a specific inhibitor of
p38MAPK, decreases the phosphorylation level of p38MAPK. NF-B is an important
mediator of pro-inflammatory pathways. When pro-inflammatory responses are
activated, NF-B dissociates from the inhibitor factor I-B and subsequently
translocates to the nucleus, where it exists primarily as a p65/p50 heterodimer and
binds directly to its cognate DNA sequence. As shown in Fig. 6A, after exposure to
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VNR, I-B was degraded, thereby causing nuclear translocation of NF-p65. In
contrast, in cells pretreated with SB203580, VNR-induced NF-B activation was
markedly inhibited.
VNR increased the NF-B-related pro-inflammatory response in HUVECs.
NF-B is a vital player in regulation of the inflammatory response, apoptosis, and cell
survival [28]. Pro-inflammatory cytokines, cyclooxygenase II (COX-II), and adhesion
molecules are modulated by NF-B, with pro-inflammatory events subsequently
leading to the tethering and adherence of monocytic cells to endothelial cells. Our
results show that treatment with VNR facilitated the expression of COX-II (Fig.7A,
7B), increased the expression of adhesion molecules (ICAM-1, VCAM-1 and
E-selectin) based on protein levels (Fig. 7D) and the mRNA levels (Fig. 7E), and the
adhesion of monocytic THP-1 cells to HUVECs (Fig. 7F), and increased the secretion
of IL-8 (Fig. 7G). To further investigated whether NF-B plays a major role in
VNR-induced endothelial cells inflammation, we used a NF-Bp65 siRNA and
examined the changes of the pro-inflammatory responses. Our results showed that
NF-Bp65 siRNA and the NF-B inhibitor (PDTC) significantly antagonized the
VNR-facilitated pro-inflammatory events, indicating that VNR promoted endothelial
inflammation majorly by activating NF-B.
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Metformin, a clinical drug, is one of the most familiarly compound to promote
AMPK function [29], we used metformin and AICAR to investigate whether AMPK
agonist reduces VNR- derived inflammatory responses. As expect, both of metformin
and AICAR effectively mitigate VNR- derived inflammatory responses (Fig. 7A-7E).
Discussion 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
In clinical practice, vinorelbine has widely used as an anti-cancer drug. Several
side effects have been revealed, such as phlebitis [6]. A previous study showed that
VNR induces ROS generation, thereby facilitating downstream pro-apoptotic
responses, such as collapsed mitochondrial membrane potential and increased
phosphatidylserine translocation [7] . In this present study, we first demonstrated that
VNR induces ROS generation and oxidative injury by modulating
AMPK/PKC/NADPH oxidase. Our data shows that VNR elicited dephosphorylation
of AMPK, which led to PKC--mediated NADPH oxidase activation and
subsequent superoxide generation, as well as impaired Akt/eNOS signaling. VNR
increased ERK activation, which contributed to repression of PPAR- expression.
Moreover, VNR activated p38MAPK expression and NF-B- mediated
pro-inflammatory events.
AMPK acts as a detector of cellular homeostasis and also modulates oxidative
stress [8]. AMPK can also mediate several signaling cascades, leading to the
repression of free radical generation and the activation of angiogenic factors. Several
lines of evidence have demonstrated that PKC, which is negatively regulated by
AMPK, is required for the activation of NADPH oxidase, and the inhibition of PKC
contributes to the attenuation of NADPH oxidase-derived ROS production [13].
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Amassing research supports that AMPK negatively regulates PKC, which in turn
mediates diverse signaling pathways. For example, palmitic acid-activated endothelial
CRP expression involves PKC-facilitated oxidative injury by repressing AMPK
expression level [30]. Cellotto et al published the rosiglitazone enhances AMPK
function, in turn, protects against high glucose-induced hyperactivity of NADPH
oxidase by inhibiting PKC [13]. Moreover, AMPK has been considered as a protector
of the cardiovascular system by enhancing NO bioavailability [11]. Our data
confirmed that VNR induces de-activation of AMPK and activation of PKC (Fig.1).
Moreover, AMPK supports endothelial function by repressing NADPH
oxidase-derived superoxide production [31]. NADPH oxidase consists of
membrane-bound gp91phox and p22hox, as well cytosolic subunits such as p47phox,
p67phox, and the small GTPase Rac. Endothelial NADPH oxidase-derived ROS
generation appears to be a driving force in the development of endothelial dysfunction
and cardiovascular diseases, indicating that NADPH oxidase-activated ROS act as a
secondary messenger to turn on downstream signal transduction pathways leading to
endothelial cell dysfunction [14, 15]. Our results suggest that VNR enriches NADPH
oxidase activation (Fig. 2). These findings are in agreement with previous studies that
demonstrated that AMPK acts as a negative regulator of NADPH oxidase. For
example, AMPK negatively regulates NOX4-dependent activation of p53 and
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
epithelial cell apoptosis [32], in addition to preventing the serine phosphorylation and
membrane translocation of p47phox [33]. Those important finding revealed that VNR-mediated oxidative damage is regulated by the AMPK/PKC/NADPH oxidase
pathway.
The caspase pathway is an important modulator of apoptosis and a
well-identified downstream target for Akt/eNOS. One mechanism by which
Akt/eNOS regulates cell survival involves the S-nitrosylation of cysteine 163 in the
active center of the catalytic subunit p17 of caspase-3, resulting in inhibition of its
activity. Our data revealed that VNR repressed AKT and eNOS phosphorylation (Fig.
4). Moreover, a previous study reported that VNR induces endothelial cell apoptosis
by activating caspase 3 [7], suggesting that VNR activates caspase 3 expression by
decreasing AKT and eNOS phosphorylation.
Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors and
are able to modulate gene expression and activation via binding with the retinoid X
receptor as a heterodimeric partner to specific DNA sequence elements[34]. Moreover,
PPARs have been shown to affect lipid and glucose metabolisms. PPAR- is
expressed in endothelial cells and is inhibited by MAPK-mediated signaling [35].
PPAR- protects against TNF--mediated adhesion molecular expression [36, 37],
suggesting that PPAR- may play a role in suppressing the generation and progression
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
of oxidative injury by modulating metabolic disorders and modifying pro-
inflammatory reactions in human endothelial cells. Our data revealed that VNR
facilitated ERK activation and inhibited PPAR- expression (Fig. 5), thereby inducing
endothelial cell damage. Moreover, pretreatment with AICAR evidently reversed the
VNR-repressed PPAR- expression level, indicating that VNR-repressed PPAR-
expression mainly by regulating AMPK activity.
ROS can be a secondary signaling mediator to regulate signal transduction. Both
the MAPK and PI-3K pathways are involved in oxidative stress-activated NF-B
translocation [38], which may be a critical mechanism in endothelial inflammation.
NF-κB was one of the key mediator of adhesion molecules at the transcriptional level
in human vascular endothelial cells [39]. In this article, SB203580 observably
attenuates I-B degradation as well as NF-κB p65 translocation, indicating a
promotion of VNR-induced NF-κB p65 expression by enhancing p38MAPK
activation (Fig.6).
Inflammation mediated by overexpression of adhesion molecules and cytokines
is reported to participate to the pathogenesis of endothelial cells dysfunction. ICAM-1,
VCAM-1 as well as E-selectin are adhesion molecules of endothelial cells, they have
been validated to be up-regulated in the endothelial cells of oxidative damage, the
activation of the molecules might promote growth factor production and medial
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
smooth muscle cells migration [40]. Hattory et al reported the AMPK activator,
metformin, protects against TNF--induced NF-κB p65 activation an adhesion
molecules up-expression [41], supporting that oxidative stress-caused NF-κB p65
activation an adhesion molecules overexpression by impairing AMPK function. In our
study, NF-Bp65 siRNA, PDTC, AICAR and metformin manifestly antagonized the
VNR-activated adhesion molecules expression as well as pro-inflammation responses,
such as IL-8 secretion, adhesion molecule expression and monocyte attachment
(Fig.7), exhibiting the VNE-derived endothelial cells inflammation via weakening
AMPK and activating NF-B.
VNR is very lipophilic and can be rapidly distributed by peripheral tissue. After
intravenous injection of 30-30 mg/m2 VNR, a plasma concentration of 1 μM is quickly reached [42]. In the present study, the VNR concentrations we used (5-0.625
M) are very similar to those from other studies. For example, Yamada et al. reported
that 3 M VNR is able to induce ROS generation and apoptotic expression [7].
Moreover, our data shows that 1.25M VNR, which may be achieved under human
physiological conditions, can induce endothelial cell oxidative injury, increase
NADPH oxidase activation, and repress eNOS and PPARexpression, facilitated by
p38MAPK and NF-B activation.
In summary, in this study we demonstrate for the first time VNR-induced
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
oxidative injury caused by repression of AMPK function, increased PKC and NADPK
oxidase activation, attenuation of AKT/eNOS expression, enhanced ERK
phosphorylation, decreased PPAR- expression, and activation of NF-B expression,
all concomitantly triggering the pro-inflammatory response in endothelial cells
(Fig.8).
Acknowledgements
This study was supported by grants from The National Science Council, (NSC
98-2320-B-039-020-MY3, NSC 97-3111-B-075-001-MY3,
97-2320-B-075-003-MY3), China Medical University (CMU99-S-13), Taiwan, ROC.
This study was supported in part by Taiwan Department of Health Clinical Trial and
Research Center of Excellence (DOH100-TD-B-111-004)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
Figure legends
Figure 1. VNR repressed endothelial AMPK activation (A, B),induced PKC
activation (C-D). HUVECs were exposed to VNR (0.625-5 M) for 1 hours. At the
end of the incubation period, levels of phosphorylated AMPK and PKC were
determined by immunoblotting. The protein levels of p-AMPK- were normalized to
the level of AMPK-. The protein levels of p-PKC were normalized to the level of
PKC. (E) PKC- activity in whole-cell lysates was measured by a fluorescein green
assay kit. Data are the mean±SE of three different experiments. #P<0.05 compared
with untreated control HUVECs. *P<0.05 compared with 5M VNR-stimulated
HUVECs.
Figure 2. VNR increased the level of NADPH oxidase membrane assembly. HUVECs
were stimulated for 2 hours with the indicated concentrations of VNR. In one sample,
HUVECs were pretreated with AICAR for 1 hour before exposure to VNR.
Preparation of membrane and cytosolic proteins is described in the Materials and
Methods section. Representative Western blots (A) and summary data (B, C) showed
that VNR induced p47phox and Rac-1 translocation to the plasma membrane, as well as gp91 and p22phox expression. The levels of cytosolic protein and membrane protein
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
were normalized to the levels of -actin and flotillin-1, respectively. Data are the
mean±SE of three different experiments. #P<0.05 compared with untreated control
HUVECs. *P<0.05 compared with 5 M VNR-stimulated HUVECs.
Figure 3. Time course of VNR-induced ROS generation in HUVECs. After
pre-incubation for 2 hours with the DPI ( NADPH oxidase inhibitor) or
AICAR( AMPK agonist). After pre-incubation for Cells were treated with various
concentrations of VNR followed by 1 hour incubation with DCF-AM. Fluorescence
intensity of cells was measured with a fluorescence microplate reader. Data are
means±SE of 3 different experiments. #P<0.05 compared with untreated control
HUVECs. *P<0.05 compared with VNR-stimulated HUVECs.
Figure 4. VNR down-regulated Akt and eNOS activation (A-C). HUVECs were
stimulated for 2 hours with the indicated concentrations of VNR. In one sample,
HUVECs were pretreated with DPI or AICAR for 1 hour before exposure to VNR. At
the end of the incubation period, the levels of phosphorylated Akt and eNOS were
determined by immunoblotting. Data are the mean±SE of three different experiments.
#P<0.05 compared with untreated control HUVECs. *P<0.05 compared with 5M
VNR-stimulated HUVECs. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
Figure 5. VNR facilitated endothelial ERK activation and inhibition of PPAR-
expression (A-C). HUVECs were exposed to the indicated concentrations VNR for 1
hour (ERK) or 24 hours (PPAR-). In one sample, HUVECs were pretreated with DPI
or AICAR for 1 hour before exposure to VNR. At the end of the incubation period, the
levels of phosphorylated ERK and PPAR- were determined by immunoblotting. The
protein levels of p-ERK were normalized to the level of ERK. The protein levels of
PPAR-were normalized to the level of -actin. Data are the mean±SE of three
different experiments. #P<0.05 compared with untreated control HUVECs. *P<0.05
compared with 5M VNR-stimulated HUVECs.
Figure 6. VNR activated p38 MAPK phosphorylation and increased the translocation
of NF-B. HUVECs were stimulated for 2 hours with the indicated concentrations of
VNR. In some samples, HUVECs were pretreated with DPI or SB203580 for 1 hour
before exposure to VNR (A-D). Western blot analysis was used to evaluate the
expression of both phosphorylated and total p38 MAPK (B) and the activation of
NF-B. Anti--actin and anti-PCNA antibodies were used for normalization of
cytosolic and nuclear proteins (C, D), respectively. Data are the mean±SE of three
different experiments. #P<0.05 compared with untreated control HUVECs. *P<0.05
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
compared with 5M VNR-stimulated HUVECs.
Figure 7. VNR activated NF-kB-related pro-inflammation responses. VNR increased
COX-II expression (A,B). HUVECs were stimulated for 24 hours with the indicated
concentrations of VNR. HUVECs were pretreated with PDTC or AICAR or
metformin for 1 hour before exposure to VNR. In some sample, transfected with
NF-Bp65 siRNA or si-Control for 48 h followed by exposure to VNR. Western blot
analysis was used to evaluate the si-NF-Bp65 knockdown efficiency (C) and the
expression of COX-II, and Anti--actin antibodies were used for normalization of
protein expression level. VNR enhanced adhesion molecule expression at the mRNA
level (D) and the protein level (E) and increased IL-8 release (R). mRNA levels of
ICAM-1, VCAM-1 and E-selectin were determined by real-time PCR. Cell surface
expression of ICAM-1, VCAM-1, and E-selectin were determined by flow cytometry.
The protein level of VNR-induced IL-8 release was determined by ELISA. VNR
increased attachment of THP-1 monocytic cells to HUVECs (G). THP-1 cells
preloaded with BECEF were incubated with HUVECs for 1 h. The adhesiveness of
HUVECs to THP-1 was measured as described in the Materials and Methods.
#P<0.05 compared with untreated control HUVECs. *P<0.05 compared with 5M
VNR-stimulated HUVECs. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
Figure 8. Schematic diagram showing the signaling of VNR-induced endothelial
oxidative injury. As depicted, VNR induces AMPK- de-activation, PKC
phosphorylation, NADPH oxidase activation, and activated oxidative stress-related signal transduction pathways. The → indicates activation or induction, and ┤indicates
inhibition or blockade. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
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act iv ity ( U/mg pr otein) 6 8 10 12 14 16 18 # # # * Exp res s ion r ati o ( p- PKC- /PK C) 0.0 0.5 1.0 1.5 2.0 2.5 * # # VNR (M) 0 0.625 1.25 2.5 5 5 AICAR (500M) - - - - - + Expression ratio (p-AM PK- /AMPK) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 # # # VNR (M) 0 0.625 1.25 2.5 5
(A)
(C)
(B)
(D)
Fig.1
(E)
Ex pr ess ion r ation (tar ge t pr ote in/ -acti n) 0.5 1.0 1.5 2.0 gp91 p22phox # # # # # # * * # # Expre ssio n ra tio n (menb ren ce/cyto sol ) 0 2 4 6 8 p47phox rac-1 # # # # # # * * VNR (M) 0 0.625 1.25 2.5 5 5 AICAR (500 M) - - - - - +
(A)
(C)
Fig.2
Fig.3
min
0 100 200 300 400 VNR(2.5 M) VNR(5 M) VNR(5 M)+DPI (5 M) VNR(5 M)+AICARI (500 M)ROS(% of in
crea
se)
0 15 30 60 120
es s ion r ati o ( p- e-NOS/NO S) 0.4 0.6 0.8 1.0 1.2 1.4 1.6 # * # *
(C)
(B)
Fig.4
Ex pr es s ion r ati o ( p-AKT /AKT) 0.0 0.5 1.0 1.5 2.0 # # VNR (M) 0 0.625 1.25 2.5 5 5 5 DPI (5M) + -AICAR (500M) - - - - + * # *s ion r ati o ( PPAR- / -ac ti n) 1.0 1.5 2.0 2.5 3.0 3.5 # # # * *
(B)
(C)
Fig.5
Ex pr es s ion r ati o ( p-ERK/ERK) 0.0 0.5 1.0 1.5 2.0 2.5 # # # VNR (M) 0 0.625 1.25 2.5 5 5 5 DPI (5M) + -AICAR (500M) - - - - + * # *0 1 2 3 4 * * # # # VNR (M) 0 0.625 1.25 2.5 5 5 5 Ex pr es s ion r ati o (p -p38/p38) Ex pr es s ion r ati o (I-k B/ -ac ti n) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 * * # # # VNR (M) 0 0.625 1.25 2.5 5 5 5
(B)
Ex pr es s ion r ati o (NF -k Bp65/ -ac 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 * * # # VNR (M) 0 0.625 1.25 2.5 5 5 5 DPI (5M) + -SB203580 (10M) - - - - - - + #(D)
0 50 100 150 200 250 # * * * * si-Contr ol si-p65 PDTC ( 10 M) AICAR (500 M) met formi n (2 00 M) THP-1 adhes ion ( % of i nc reas 0 50 100 150 200 250 VNR (5M) # * * * * si-Contr ol si-p65 PDTC ( 10 M) AICAR (500 M) met formi n (2 00 M) IL-8 s ec reti on ( pg/m l)
(G)
Exp res s ion r ati o (CO X-2/ -ac ti n) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 VNR (5M) # * * * * si-Contr ol si-p65 PDTC ( 10 M) AICAR (500 M) met formi n (2 00 M)(B)
(E)
(C)
ICAM VCAM E-selectin 0 1 2 3 4 5 VNR 5 M+si-Control VNR 5 M+si-p65 VNR 5 M+PDTC 10 M VNR 5 M+AICAR 500 M VNR 5 M+metformin 200 M P rot ein ex pres sio n lev el (% of co nt rol) # # # * * * * * * * * * * * *
ICAM VCAM E-selectin
0 1 2 3 4 5 6 7 Control VNR 5 M VNR 5 M+si-Control VNR 5 M+si-p65 VNR 5 M+PDTC 10 M VNR 5 M+AICAR 500 M VNR 5 M+metformin 200 M Re lativ e m RN A / -ac tin ex pr es sio n # # # * ** * * * * * * * * *
