Functionalized selenium nanoparticles with nephroprotective activity, the important roles of ROS-mediated signaling pathways

Article abstract of DOI:10.1039/c3tb21168a

The most frequent adverse effect of cisplatin-based chemotherapy is nephrotoxicity. Oxidative stress has been implicated as an important mechanism in the pathogenesis of cisplatin-induced nephrotoxicity. In the present study, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) surface-functionalized selenium nanoparticles (Se@Trolox) with enhanced antioxidant activity have been prepared by self-assembly of trolox on the surface of the nanoparticles, and their nephroprotective effects have been investigated. Functionalization by trolox significantly enhanced cell uptake and in vitro antioxidant activities of the nanoparticles. In addition, pretreatment with Se@Trolox dose-dependently blocked cisplatin-induced cell growth inhibition against HK-2 cells. Mechanistic investigation suggested that Se@Trolox markedly prevented cisplatin-induced apoptosis in HK-2 cells, as evidenced by inhibition of chromatin condensation, DNA fragmentation, PARP cleavage and activation of caspase-3. Furthermore, Se@Trolox effectively blocked the cisplatin-induced reactive oxygen species (ROS) accumulation, activation of AKT and MAPK signaling and DNA damage-mediated p53 phosphorylation in HK-2 cells. Taken together, our findings suggest that Se@Trolox is a promising Se species with potential application in prevention of cisplatin-induced renal injury.

Full text of DOI:10.1039/c3tb21168a

As featured in:
Highlighting research from Prof Tianfeng Chen’s group
at the Department of Chemistry, Jinan University,
China.
Title: Functionalized selenium nanoparticles with
nephroprotective activity, the important roles of
ROS-mediated signaling pathways
Size controllable and highly stable SeNPs have been designed
and prepared by using trolox as a capping agent, which display
enhanced antioxidant activity and could suppress cisplatin-induced
nephrotoxicity by regulating ROS-mediated signaling pathways.
See Y. Li et al.,
J. Mater. Chem. B, 2013, 1, 6365.
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Functionalized selenium nanoparticles with
nephroprotective activity, the important roles of ROS-
mediated signaling pathways
Cite this: J. Mater. Chem. B, 2013, 1,
6365
*
Yinghua Li, Xiaoling Li, Wenjie Zheng, Cundong Fan, Yibo Zhang
*
and Tianfeng Chen
The most frequent adverse effect of cisplatin-based chemotherapy is nephrotoxicity. Oxidative stress has
been implicated as an important mechanism in the pathogenesis of cisplatin-induced nephrotoxicity. In
the present study, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) surface-functionalized
selenium nanoparticles (Se@Trolox) with enhanced antioxidant activity have been prepared by self-
assembly of trolox on the surface of the nanoparticles, and their nephroprotective effects have been
investigated. Functionalization by trolox significantly enhanced cell uptake and in vitro antioxidant
activities of the nanoparticles. In addition, pretreatment with Se@Trolox dose-dependently blocked
cisplatin-induced cell growth inhibition against HK-2 cells. Mechanistic investigation suggested that
Se@Trolox markedly prevented cisplatin-induced apoptosis in HK-2 cells, as evidenced by inhibition of
chromatin condensation, DNA fragmentation, PARP cleavage and activation of caspase-3. Furthermore,
Se@Trolox effectively blocked the cisplatin-induced reactive oxygen species (ROS) accumulation,
activation of AKT and MAPK signaling and DNA damage-mediated p53 phosphorylation in HK-2 cells.
Taken together, our findings suggest that Se@Trolox is a promising Se species with potential application
in prevention of cisplatin-induced renal injury.
Received 20th August 2013
Accepted 30th September 2013
DOI: 10.1039/c3tb21168a
www.rsc.org/MaterialsB
and non-small cell lung cancers since the 1960s.10–12 The
chemotherapeutic action of cisplatin is to cross-link with DNA,
1 Introduction
Trolox is a water-soluble analogue of a-tocopherol with a chro-
man ring but lacks a hydrophobic phytyl tail, where the side
chain in a-tocopherol is replaced by a more hydrophilic carboxyl
group.1 The antioxidant activity of trolox was proved to be better
than that of a-tocopherol under a wide range of conditions and
test systems compared with those of several food grade anti-
oxidants.2 Trolox has been investigated for its protective effects
against oxidative stress-related ailments as one of the major
phenolic compounds3 i.e. atherosclerosis,4 global cerebral
ischemia5 and neurodegenerative disorder.6 E. Soheili Majd
et al. showed that trolox signicantly reduced the cytotoxicity of
resin-based biomaterials but had no effect on GIC and RM-GIC
biomaterials.7 Although it has potent antioxidant function and
an important role in clinical treatment, it shows low utilization
due to its poor aqueous solubility and inferior stability when
exposed to an oxygen-containing environment owing to the
phenolic hydroxyl group of trolox.8,9
thereby interfering with transcription and/or DNA replication in
tumor cells.13 Although cisplatin has shown remarkable efficacy
at attacking various tumors, it has been found that 65–98%
of the platinum is bound to proteins in blood within 24 h of
intravenous injection of cisplatin, and the platination of
proteins may contribute to severe side effects, attributing to the
indiscriminate accumulation of the drug in both normal and
cancerous tissues, which leads to acute renal failure.14–16 In
renal proximal tubules, cisplatin causes intrastrand and inter-
strand crosslinking between N7 and O6 of the adjacent guanine
molecules, which results in local denaturation of the DNA.17
Although the molecular mechanism is not completely under-
stood, reactive oxygen species (ROS) have been suggested to be
as important mediators of cisplatin-induced nephrotoxicity.18
Lee and Ajith showed the protective effect of antioxidants
against cisplatin-induced nephrotoxicity.19,20 Therefore, the goal
of our work is to attempt to introduce nanotechnology into the
development of trolox to protect the kidney from cisplatin-
induced injury.
Cisplatin (cis-diamminedichloroplatinum(II)) is one of the
oldest available chemotherapeutic drugs that has been used for
the treatment of a wide spectrum of solid tumors, including
testicular, ovarian, esophageal, bladder, head and neck cancer
The essential micronutrient selenium (Se) plays a funda-
mental role in humans and animals and has been implicated to
have important health benets. In humans, selenium has one
of the narrowest ranges between dietary deciency (<40 mg per
day) and toxic levels (>400 mg per day). The dietary reference
Department of Chemistry, Jinan University, Guangzhou 510632, China. E-mail:
tchentf@jnu.edu.cn; fenling03@gmail.com
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value of Se intake has been set in the range of 30–55 mg per day diamidino-2-phenylindole (DAPI), bicinchoninic acid (BCA) kit
by international agencies.21 Selenium at supranutritional levels and Na2SeO3 were purchased from Sigma-Aldrich chemicals.
has been shown as a cancer preventive agent in reducing the Terminal transferase dUTP nick end labeling (TUNEL) assay kit
incidence of colon, prostate, and lung cancer.22 Studies showed was obtained from Roche Applied Science (Basel, Switzerland).
that the dose, chemical form and metabolic activity are the Caspase-3 substrate (Ac-DEVD-AMC) was purchased from Bio-
determinants of anticancer activities of Se compounds.23 Se mol (Germany). Caspase-9 substrate (Ac-LEHD-AFC) and cas-
displayed a narrow margin between the benecial and toxic pase-8 substrate (Ac-IETD-AFC) were purchased from Biomol
effects. As an anticancer agent, the effective dose of Se is close to (Germany). Dulbecco's modied Eagle's medium (DMEM) and
the toxic range, which greatly limits its clinical application. fetal bovine serum (FBS) were purchased from Gibco BRL
Although regarded as an essential trace element, Se is toxic if (Gaithersburg, MD). Vitamin C was purchased from Guangzhou
taken in excess.24 Selenocompounds act as an essential compo- chemical reagent factory. The water used in all experiments was
nent of several major metabolic pathways, including thyroid Milli-Q water.
hormone metabolism, antioxidant defense systems and in trolox
function.25 Se nanoparticles (SeNPs) attract increasing attention Preparation of Se@Trolox: synthesis of Trolox–SH
due to their excellent anticancer activities and low toxicity.26 We
Trolox (0.5 g) was mixed with cystamine dihydrochloride
have previously demonstrated that surface decoration of SeNPs
(0.11 g), benzotriazol-1-yloxytris (dimethylamino) phospho-
with polysaccharide and sialic acids signicantly enhanced the
nium hexauorophosphate (BOP), 0.39 g, 1-hydroxy-1H-benzo-
antiproliferative activities of SeNPs.27 Our previous work has
triazole, monohydrate (HOBt) 0.134 g, and 4-(dimethylamino)
pyridine (DMAP) 0.108 g in dry N,N-dimethylformamide (DMF)
(10 ml). Aer a brief activation period (10 min), N-methyl-
demonstrated that selenium nanoparticles by self-assembly of
11-mercapto-1-undecanol (Se@MUN) achieved enhanced anti-
oxidant activity and antagonism against cisplatin-induced
morpholine (0.9 g) was added to the reaction mixture and the
nephrotoxicity.18 But the stability of Se@MUN is poor, so we need
solution was stirred under N2 at room temperature for 18 h.
Solvent was removed under vacuum, and the residue was dis-
solved in ethyl acetate, then washed with 0.5 M H2SO4, 1 M
to nd more stable nanoparticles for future applications.
The combination of nanotechnology, biochemistry, biotech-
nology and molecular biology has developed into an emerging
Na2CO3, and brine, each for three times. The organic layer was
research area: nanobiotechnology, which offers exciting oppor-
dried over anhydrous MgSO4, and the solvent was evaporated in
tunities for developing new nanosized materials and providing
vacuo. The residue was separated by column chromatography
on silica gel to purify (Trolox–S)2. The reduction of (Trolox–S)2
by sodium borohydride in ethanol yielded the thiol ligand
Trolox–SH.
paradigm-breaking solutions in this area because these tech-
nologies provide extremely high sensitivity.28,29 The application of
nanotechnology to drug delivery is widely expected to change the
landscape of pharmaceutical and biotechnology industries for
the foreseeable future. These are a few of the many compelling
reasons that nanotechnology holds enormous promise for drug
delivery. For example, it may be possible to improve the delivery
Synthesis of Trolox–SH functionalized SeNPs (Se@Trolox)
Briey, 0.5 ml of 50 mM vitamin C solution was added dropwise
into 62.5 ml of 0.1 M Na2SeO3 solution under magnetic stirring,
of poorly water-soluble drugs and deliver drugs in a cell- or tissue-
and the mixture was reconstituted to a nal volume of 25 ml
specic manner. Additionally, the manufacturing complexity of
with Milli-Q water. Then the mixed solution was stirred for half
an hour at room temperature. When the colour of the solution
nanotechnology therapeutics may also create a signicant hurdle
for generic drug companies to develop equivalent therapeutics
completely became red, the pH was adjusted to 10 with 0.1 M
readily.30 The excellent performance of bionanomaterials opens
NaOH. Aer that, 3.86 mg of Trolox–SH powder dissolved in
1 ml absolute ethyl alcohol was added under magnetic stirring
new horizons for drug delivery and therapy of diseases that have
traditionally been recognized as incurable via basic therapies
overnight at room temperature. The color turned grey subse-
or surgical methods.31 In this study, we hypothesized that the
quently, and the nanoparticles were centrifuged at 10 000 ꢀ g
assembly of antioxidant ligands on SeNPs could exert the syner-
for 30 min and washed with Milli-Q water and absolute ethyl
gism of Se and the capping agents in scavenging free radicals,
alcohol each for three times. Finally, the solution was subjected
and the aim of the present study is to validate this hypothesis
to centrifugation at 8000 rpm for 15 min and freeze-dried.
Se@Trolox powder was stored at ꢁ20 ꢂC until use. To determine
and use trolox functionalized SeNPs (Se@Trolox) to antagonize
cisplatin-induced nephrotoxicity through scavenging free radi-
the cellular uptake of Se@Trolox, the nanoparticles containing
cals. Taken together, functionalization of SeNPs by trolox
a uorescent dye 6-coumarin were prepared using a similar
signicantly enhanced the ability of Se@Trolox antagonizing
procedure except that 100 mg of the dye was added to the
cisplatin-induced nephrotoxicity through scavenging ROS in HK-
reaction system aer the addition of trolox. The incorporated
dye acts as a probe for Se@Trolox and offers a sensitive method
2 cells and regulating the AKT and MAPK pathways.
to determine their intracellular uptake and localization.
2 Materials and methods
Materials
Characterization of Se@Trolox
Thiazolyl blue tetrazolium bromide (MTT), propidium The as-prepared products were characterized by using various
iodide (PI), 20,70-dichlorouorescein diacetate (DCF-DA), 40,6- methods. Briey, TEM samples were prepared by dispersing the
6366 | J. Mater. Chem. B, 2013, 1, 6365–6372
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powder particles onto a holey carbon lm on copper grids. The
micrographs were obtained on a Hitachi (H-7650) for TEM
operating at an accelerating voltage of 80 kV. EDX analysis was
carried out on an EX-250 System (Horiba) and employed to
examine the elemental composition of Se@Trolox. FT-IR
samples were recorded on an Equinox 55 IR spectrometer in the
range of 4000–500 cmꢁ1 using the KBr-disk method.
Determination of activities of caspase-3, -8 and -9
The caspase activities in the cells exposed to cisplatin and
Se@Trolox were determined by using specic caspase-3, -8, and
-9 substrates.38
Measurement of ROS generation
Intracellular ROS accumulation was evaluated by DCF uores-
cence assay. Briey, cells were harvested and suspended in PBS
(1 ꢀ 106 cells per ml) containing 10 mM of DCFH-DA. Aer
incubation at 37 ꢂC for 30 min, the stained cells were collected
and resuspended in PBS. Then, the ROS level was determined by
measuring the uorescence intensity using a microplate reader,
with the excitation and emission wavelengths set at 500 and
529 nm respectively. Experiments were performed in triplicate.
ABTS free radical scavenging assay
ABTS free radical scavenging activities of Se@Trolox were
measured as previously described.32
Cell culture and cell viability assay
HK-2 proximal tubular cell line derived from the normal human
kidney was purchased from American Type Culture Collection
(ATCC, Manassas, VA) and grown in DMEM medium, supple-
mented with 10% fetal bovine serum (FBS), 100 units per ml
penicillin and 50 units per ml streptomycin in a humidied
incubator at 37 ꢂC with a 5% CO2 atmosphere. Cell viability was
determined by measuring the ability of cells to transform MTT
to a purple formazan dye as previously described.33
Western blotting
The effects of cisplatin on the expression levels of various
intracellular proteins in HK-2 cells and the protection of
Se@Trolox were examined by Western blot analysis as previ-
ously described.23 The bands were visualized with enhanced
chemiluminescence reagent that detects the target proteins on
the X-ray lm.
In vitro cellular uptake of Se@Trolox
The cellular uptake of Se@Trolox was also monitored qualita-
tively by uorescence microscopy.31 Briey, treated cells
cultured on a cover glass in 6-well plates till 70% conuence
were incubated with different concentrations of 6-coumarin-
loaded Se@Trolox for various periods of time. The cells were
then washed three times with PBS and examined under a uo-
rescence microscope (AMG EVOS f1). For quantitative analysis
of cellular uptake, Se concentrations in the cells aer treat-
ments were determined by the ICP-MS method.34
Statistical analysis
All the data are presented as mean ꢃ SD. Differences between
two groups were evaluated using two-tailed Student's t test. One-
way analysis of variance39 was used in multiple group compar-
isons. These analyses were carried out using SPSS 13.0. Differ-
ence with P < 0.05 (*) or P < 0.01 (**) was considered statistically
signicant.
3 Results and discussion
Preparation and characterization of Se@Trolox
Localization and uptake pathways of Se@Trolox
The intracellular localization of coumarin-6-loaded Se@Trolox
in HK-2 cells was traced with the lysosomal marker Lyso Tracker
Red as previously described.35 Briey, the treated cells cultured
on a cover glass in 6-well plates to 70% conuence were incu-
bated with 6-coumarin-loaded Se@Trolox and Lyso Tracker Red
at 37 ꢂC for various periods of time. Then the stained cells were
rinsed three times with PBS and examined under a uorescence
microscope.
In this study, we demonstrated a simple method to synthesize
functionalized SeNPs through self-assembly of functional
ligands on the surface of nanoparticles. SeNPs were capped with
trolox molecules to form more compact and stable globular
Flow cytometric analysis
Cell cycle distribution was monitored by ow cytometric anal-
ysis as previously described.36 The proportion of cells in G0/G1,
S, G2/M phases was represented as a DNA histogram. Apoptotic
cells with hypodiploid DNA content were measured by quanti-
fying the sub-G1 peak in the cell cycle pattern. For each exper-
iment 10 000 events per sample were recorded.
TUNEL assay and DAPI staining
DNA fragmentation was examined by uorescence staining with
the TUNEL apoptosis detection kit according to the manu-
facturer's instructions.37
Scheme 1 Synthetic route towards Se@Trolox.
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To examine the effects of trolox on the surface properties and
stability of SeNPs, we measured the zeta potential and size
distribution of SeNPs and Se@Trolox. As shown in Fig. 2B, the
zeta potential of Se@Trolox was lower than SeNPs aer trolox
surface decoration, explaining the higher stability of Se@Trolox
than SeNPs. Fig. 2C and D show that the average particle size of
Se@Trolox was effectively decreased from 220 nm to about
100 nm. Furthermore, in Fig. 2E, we used a Zetasizer Nano-ZS
particle analyzer to test the changes in the size distribution of
Se@Trolox in aqueous solutions. The results revealed that
Se@Trolox remained stable at least for 30 days in aqueous
solutions.
Fig. 1 (A) TEM images of Se@Trolox (a and b) and SeNPS (c and d). (B) EDX
analysis of Se@Trolox.
Cellular uptake of Se@Trolox
Cellular uptake is an important factor that usually contributes
to nanomaterial-based drug cytotoxicity. In the present study, a
quantitative analysis of cellular uptake was conducted by
ICP-MS.
nanocomposites (Scheme 1). The morphology and chemical
composition of Se@Trolox were characterized using various
spectroscopic and microscopic methods. Fig. 1A shows the TEM
images of the SeNPs with the presence (a and b) and absence
(c and d) of the capping agent trolox, which clearly demon-
strated that Se@Trolox presented a monodisperse and homo-
geneous spherical structure with an average diameter of about
100 nm, whereas SeNPs in aqueous solutions easily aggregated
and precipitated. Fig. 1B, an elemental composition analysis
employing EDX showed the presence of a strong signal from the
Se atoms (36.04%), together with a S atom signal (5.43%), C
(53.38%) and O (5.15%) from trolox. The presence of an S atom
indicates that trolox was self-assembled on the surface of
Se@Trolox. Se@Trolox was further characterized by FTIR to
conrm the chemical binding of trolox to the surface of the
nanoparticles. As shown in Fig. 2A, the FTIR spectrum of
Se@Trolox resembles that of trolox, giving clear evidence that
this antioxidant ligand forms part of the nanocomposite. The
absence of an S–H peak at 2580 cmꢁ1 stretching mode in the IR
spectrum of Se@Trolox indicates the formation of the thiolate–
Se bond between the ligands and the SeNP surface. Stability is
an important issue for the nanoparticles' future applications.40
HK-2 cells were incubated with different concentrations of
nanoparticles and then subjected to acidic digestion and ICP-
MS analysis. As shown in Fig. 3A, HK-2 cells treated with 5, 10,
20 and 40 mg mlꢁ1 Se@Trolox displayed a dose-dependent
increase in Se cellular uptake, which was signicantly higher
than those of Se@MUN and SeNPs alone. The dramatic increase
in cellular uptake of SeNPs could be due to the enhancement of
endocytosis, one of the most important entry mechanisms for
extracellular nanomaterials.31 Taken together, these results
suggest that trolox surface decoration enhances the cellular
uptake of SeNPs.
Localization and uptake pathways of Se@Trolox
Endocytosis is an important uptake pathway for nano-
particles.41 In this study, the localization of Se@Trolox in HK-2
Fig. 2 (A) The FT-IR of Se@Trolox. (B) Zeta potential of Se@Trolox and SeNPs. (C)
and (D) Size distribution of Se@Trolox and SeNPs. (E) Stability of Se@Trolox in
aqueous solutions.
Fig. 3 (A) Quantitative analysis of Se concentrations by ICP-MS and (B) locali-
zation and uptake pathways of Se@Trolox.
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cells was investigated by using specic probes, Lyso Tracker Red control cells and cells treated with Se@Trolox alone appeared
and DAPI, for uorescence imaging of lysosomes and cell healthy, with regularity in shape and well developed cell-to-cell
nucleus, respectively. As shown in Fig. 3B, the combination of contact. These results indicated that Se@Trolox was able to
blue, red, and green uorescence clearly indicates the colocal- attenuate cisplatin-induced nephrotoxicity.
ization of Se@Trolox and lysosomes in HK-2 cells aer 5 min,
which increased in a time-dependent manner. Se@Trolox
escaped from lysosomes aer 15 min and then released into
Blocking of cisplatin-induced apoptosis by Se@Trolox
the cytosol and distributed in cells aer 60 min. During the
whole process, the uorescence from Se@Trolox could not be
observed in the nucleus. These results suggest that lysosomes
are the main target organelles of Se@Trolox.
Apoptosis or programmed cell death is an essential process of
cell death during embryonic and postnatal tissue remodeling
and some other pathological conditions, such as nephrotoxicity
caused by cisplatin. In the present study, ow cytometry was
used to evaluate the degree of apoptotic cell death caused by
cisplatin. As shown in Fig. 5A, the apoptotic cells with DNA
fragmentation show a typical sub-G1 peak in the DNA histo-
gram. For instance, 8 mg mlꢁ1 of cisplatin reduced the cell
viability of 30%. However, the sub-G1 apoptotic cell population
was signicantly decreased by co-treatment with Se@Trolox in a
Se@Trolox suppresses cisplatin-induced cytotoxicity
Several studies have demonstrated the protective effect of
antioxidants on kidneys against cisplatin-induced nephrotoxi-
city.42 Therefore, treatment with antioxidants could be an
effective way to ameliorate this damage. Our previous studies
have found that trolox is a thiol-containing functional ligand
with antioxidant activity.43 The present study aimed at evalu-
ating the nephrotoxicity protective effect of trolox-functional-
ized SeNPs against cisplatin-induced acute renal damage.
Firstly, we examined the cytotoxicity of cisplatin on HK-2 cells
and protective effects of Se@Trolox by using MTT reduction
assay. As shown in Fig. 4A, HK-2 cells treated with 8 mg mlꢁ1 of
cisplatin for 24 h showed a cell viability of 31.25% However, the
cytotoxicity was signicantly attenuated by Se@Trolox in a dose-
dependent manner. For instance, at concentrations 10 and
20 mg mlꢁ1, Se@Trolox increased the cell viability to 85.3 and
90.2% of control, which were signicantly higher than those of
free trolox and Se@MUN under the same concentrations. Cells
treated with 40 mg mlꢁ1 of Se@Trolox alone showed no differ-
ence in cell viability from those of control cells. These results
suggest that organized and ordered assembly of trolox on the
surface of nanoparticles effectively amplied the neph-
roprotective activity of SeNPs. The protective effects of
Se@Trolox were conrmed by phase-contrast observation. As
shown in Fig. 4B, cells treated with cisplatin showed cyto-
plasmic shrinkage, reduction in cell numbers and loss of cell-to-
cell contact. These cisplatin-induced morphological changes
were attenuated by co-treatment with Se@Trolox. By contrast,
dose-dependent manner. At concentrations of 10 and 20 mg mlꢁ1
,
Se@Trolox reduced the apoptotic cells to 12% and 12.8%,
respectively. Se@Trolox alone did not exhibit apoptosis-
inducing effect on HK-2 cells. The protection of Se@Trolox
against cell apoptosis was further conrmed by TUNEL enzy-
matic labeling and DAPI co-staining assay. DNA fragmentation
is an important biochemical hallmark of cell apoptosis. TUNEL
assay can be used to detect early stage of DNA fragmentation in
apoptotic cells prior to the changes in morphology. As shown in
Fig. 5B, aer treatment with 8 mg mlꢁ1 cisplatin, HK-2 cells
exhibited typical apoptotic features, such as DNA fragmentation
(green uorescence) and nuclear condensation (blue uores-
cence). A signicant increase in the number of TUNEL-positive
Fig. 5 Se@Trolox blocks cisplatin-induced apoptosis in HK-2 cells. (A) Apoptotic
cell population was determined by PI flow cytometric analysis. (B) Representative
photomicrographs of DNA fragmentation and nuclear condensation as detected
by TUNEL-DAPI co-staining assay. Cells were pretreated with Se@Trolox or SeNPs
for 12 h and then exposed to 8 mg mlꢁ1 cisplatin for 24 h. All results were
representative of three independent experiments. Cells were pretreated with
Se@Trolox for 12 h and then exposed to 8 mg mlꢁ1 cisplatin for 24 h. All results
were representative of three independent experiments.
Fig. 4 Se@Trolox rescues HK-2 cells from cisplatin-induced cytotoxicity. (A) Cells
were pretreated with Se@Trolox or SeNPs for 12 h and then exposed to 8 mg mlꢁ1
cisplatin for 24 h. Cell viability was measured by MTT assay. (B) Morphological
changes in HK-2 cells were observed by phase-contrast microscopy.
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cells and the uorescence intensity were observed aer 24 h situation of a marked imbalance between cellular defense
treatment with cisplatin. However, co-treatment with Se@Tro- mechanisms and ROS production. ROS is generated as a natural
lox effectively blocked the cisplatin-induced DNA fragmentation by product of normal cellular metabolism and has important
and changes in nuclear morphology. The number of TUNEL- roles in cell signaling. ROS has been proposed to involve in
positive cells was signicantly decreased in cisplatin-treated the initiation phase of apoptosis contributing to cell death
HK-2 cells. Moreover, cells exposed to Se@Trolox alone showed signaling.45 Several lines of evidence have demonstrated that
normal nuclei of oval shape, similar to those of the control cisplatin-induced renal failure was accompanied by ROS
cells. These results indicate that Se@Trolox rescues HK-2 cells production and peroxidative damage.46 In this study, we showed
from cisplatin-induced nephrotoxicity through blocking of cell that cisplatin signicantly increased the intracellular ROS
apoptosis.
generation in HK-2 cells to 390% of the control group (Fig. 7A).
Interestingly, we found that Se@Trolox signicantly inhibited
cisplatin-induced ROS generation in a dose-dependent manner.
For instance, at concentrations of 10 and 20 mg mlꢁ1, Se@Trolox
reduced the intracellular ROS to 350% and 310%, respectively.
Our results indicate that Se@Trolox is capable of scavenging
free radicals and protecting HK-2 cells against cisplatin-induced
oxidative stress.
Se@Trolox suppresses cisplatin-induced caspase activation
The caspase family of aspartate-specic cysteine proteases plays
important roles in the initiation and execution of apoptosis.44
Among them, caspase-3 has been regarded as the central
regulator of apoptosis, while caspase-8 and -9 act as initiators of
death receptor-mediated and mitochondria-mediated apoptotic
pathways, respectively. In this study, to determine whether
caspase family was activated in HK-2 cells exposed to cisplatin,
the enzymatic activities of caspase-3,-8 and -9 were measured by
using specic substrates. The results (Fig. 6A and B) show that
treatments of the cells with 20 mg mlꢁ1 of Se@Trolox signi-
cantly suppressed the activation of caspase-3,-8 and -9 induced
by cisplatin, demonstrating that Se@Trolox protects HK-2 cells
from cisplation-induced injury through death receptor-medi-
ated and mitochondria-mediated pathway inhibition of caspase
activation.
Antioxidant activity of Se@Trolox
The total antioxidant activity of Se@Trolox was determined by
using ABTS free radical scavenging assay. In this system, the
relatively long-lived ABTS+ generated by the direct oxidation of
ABTS with manganese dioxide is decolorized during the reac-
tion with hydrogen-donating antioxidants.47 This assay provides
a direct comparison of the antioxidant activities of tested
samples as expressed in terms of absorbance. As shown in
Fig. 7B, Se@Trolox signicantly inhibited the formation of
ABTS free radicals in a time-and dose-dependent manner,
which was higher than those of free trolox and Se@MUN. These
results support the enhancement of the antioxidant activity of
SeNPs by trolox surface decoration. Based on these results, the
strategy to assemble antioxidant ligands on the surface of SeNPs
Se@Trolox inhibits cisplatin-induced ROS generation
ROS consists of highly reactive molecules, including oxygen
ions, free radicals, and peroxides. Oxidative stress refers to a
Fig. 6 Inhibition of caspase activation by Se@Trolox. (A) Cells were pretreated
with or without Se@Trolox for 12 h and then cultured in the presence or absence
of 8 mg mlꢁ1 cisplatin for 24 h. Caspase activity was measured with whole cell
extracts by a fluorometric method. All data are expressed as means ꢃ SD from
three independent experiments, each performed in duplicate. Bars with different
characters are statistically different at P < 0.05 level. (B) Induction of caspase-3 and
PARP cleavage by cisplatin and the protective effects of Se@Trolox.
Fig. 7 Se@Trolox blocks cisplatin-induced ROS generation by scavenging free
radicals. (A) Cells were pretreated with Se@Trolox for 12 h and then cultured in the
presence of 8 mg mlꢁ1 cisplatin for 24 h. Intracellular ROS generation was deter-
mined by DCFH-DA assay and expressed as a percentage of control cells. All data
are expressed as means ꢃ SD from three independent experiments, each performed
in duplicate. Bars with different characters are statistically different at P < 0.05 level.
(B) Antioxidant activities of Se@Trolox as determined by ABTS assay.
6370 | J. Mater. Chem. B, 2013, 1, 6365–6372
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could be a highly efficient way to enhance the antioxidant suggest that MAPK pathways were involved in cancer cell
activity.
apoptosis induced by Se@Trolox. The role of the p53 signaling
pathway is one of the most common genetic alterations iden-
tied in human tumors and many chemotherapeutic drugs
result in either DNA damage or microtubule inhibition. The
action of p53 as a tumor suppressor protein has been elucidated
through the discovery and analysis of downstream target genes.
Therefore, the biochemical characterization of p53 could also
give us a glimpse of how p53 regulates cell physiology.51 As
shown in Fig. 8D, Se@Trolox treatment moderately decreased
the expression of total p53 protein.
Se@Trolox blocks various cisplatin-induced signaling
pathways
As shown in Fig. 8A, we proposed the signaling pathways
accounting for the nephroprotective effects of Se@Trolox. AKT
is a stress-sensitive kinase regulated by the intracellular redox
system.48 The status of ROS has been found to play an important
role in the regulation of the AKT signaling pathway.39 Once
activated, AKT can inhibit apoptosis by a number of actions,
including phosphorylation and inhibition of proapoptotic Bcl-2
family members, GSK3b, and forkhead family transcription
factors.49 In this study, Western blot analysis was used to
examine the effects of Se@Trolox on the expression and phos-
phorylation level of AKT in HK-2 cells. As shown in Fig. 8B,
Se@Trolox treatment led to an obvious decrease in total and
phosphorylated AKT in HK-2 cells in a dose-dependent manner,
suggesting that Se@Trolox induces cancer cell apoptosis
through inhibition of the AKT pathway. MAPK signaling path-
ways have also been found to play important roles in the action
of chemotherapeutics and are suggested to participate in
various cellular processes, including cell proliferation, differ-
entiation and apoptosis. MAPKs can be activated by a variety of
stimuli including growth factors, hormones, mitogens, and
stress factors. Oxidative stress is a key modulator of MAPK
signaling cascades.50 In this study, Western blot analysis was
used to examine the involvement of MAPK pathway Se@Trolox-
induced apoptosis.
4 Conclusions
In summary, we present a strategy to use trolox as a capping
agent for preparation of size controllable and highly stable
SeNPs with enhanced antioxidant activity and antagonism
against cisplatin-induced nephrotoxicity. The studies on the
underlying mechanisms revealed that Se@Trolox blocked the
caspase-mediated apoptosis induced by cisplatin through
inhibition of ROS mediated p53 phosphorylation. Furthermore,
Se@Trolox effectively protects HK-2 cells from cisplatin-
induced injury by regulating the AKT and MAPK pathways.
Taken together, our ndings suggest that Se@Trolox is a
promising Se species with potential application in prevention of
cisplatin-induced renal injury.
Acknowledgements
This work was supported by National Science and Technology
Support Program (2012BAC07B05), Science Foundation for
Distinguished Young Scholars of Guangdong Province, Natural
Science Foundation of China and Guangdong Province,
Program for New Century Excellent Talents in University,
Research Fund for the Doctoral Program of Higher Education of
China and China Postdoctoral Science Foundation.
It was found that Se@Trolox treatment triggered a differen-
tial response of JNK ERK and p38 MAPK in HK-2 cells (Fig. 8C).
A signicant decrease in total and phosphorylated ERK was
observed in cells exposed to Se@Trolox. A slight increase in
p-JNK and p38 was also detected in the treated cells, while
the expression of total JNK remained constant. These results
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