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Enhancement of radiotherapy efficacy by silver

nanoparticles in hypoxic glioma cells

Zhujun Liu, Hongye Tan, Xiaohong Zhang, Feng Chen, Zhuo Zhou, Xiaodan
Hu, Shuquan Chang, Peidang Liu & Haiqian Zhang

To cite this article: Zhujun Liu, Hongye Tan, Xiaohong Zhang, Feng Chen, Zhuo Zhou, Xiaodan
Hu, Shuquan Chang, Peidang Liu & Haiqian Zhang (2018) Enhancement of radiotherapy efficacy
by silver nanoparticles in hypoxic glioma cells, Artificial Cells, Nanomedicine, and Biotechnology,
46:sup3, S922-S930, DOI: 10.1080/21691401.2018.1518912

To link to this article: https://doi.org/10.1080/21691401.2018.1518912

Published online: 11 Oct 2018.

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ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY
2018, VOL. 46, NO. S3, S922–S930
https://doi.org/10.1080/21691401.2018.1518912

Enhancement of radiotherapy efficacy by silver nanoparticles in hypoxic

glioma cells
Zhujun Liua, Hongye Tana, Xiaohong Zhanga,b,c, Feng Chena, Zhuo Zhoua, Xiaodan Hua, Shuquan Changa,
Peidang Liub and Haiqian Zhanga,b,c
a
Department of Nuclear Science and Engineering, Nanjing University of Aeronautics and Astronautics, Nanjing P.R. China; bJiangsu Key
Laboratory for Biomaterials and Devices, Southeast University, Nanjing, P.R. China; cCollaborative Innovation Center of Radiation Medicine of
Jiangsu Higher Education Institutions, Suzhou University, Suzhou, P.R. China

ABSTRACT ARTICLE HISTORY

Radiotherapy is one of the most widely used treatments for therapy of malignant tumors, but resist- Received 11 July 2018
ance to radiation of hypoxic cells in tumor tissues is still a serious concern. Previous studies have dem- Revised 26 August 2018
onstrated that silver nanoparticles (AgNPs) enhance the radiosensitivity of human glioma cells in vitro, Accepted 27 August 2018
but the effect of AgNPs on hypoxic glioma cells has not been investigated in detail. The main purpose
KEYWORDS
of this study is to evaluate the radiosensitizing efficacy of AgNPs on hypoxic glioma cells. The half max- Radiotherapy; radiosensitiv-
imal inhibitory concentration (IC50) values of AgNPs for the hypoxic U251 cells and C6 cells were ity; silver nanoparticles;
30.32 lg/mL and 27.53 lg/mL, respectively. The sensitization enhancement ratio (SER) demonstrated hypoxia; autoph-
that AgNPs exhibit higher capacity in radiosensitization in hypoxic cells (U251: 1.78; C6: 1.84) than that agy; apoptosis
in normoxic cells (U251: 1.34; C6: 1.45). The underlying mechanism of AgNPs’ radiosensitization in hyp-
oxic cells is through the promotion of apoptosis and enhanced destructive autophagy. There is evi-
dence of crosstalk between apoptosis and autophagy in AgNPs-radiosensitized hypoxic cells where
inhibition of autophagy results in decreased apoptosis. These findings suggest that AgNPs can be used
as a highly effective nano-radiosensitizer for the treatment of hypoxic glioma.

Introduction combination [10–12]; however, further investigation is

Glioma is the most common primary intracranial tumor in
the central nervous system, and it is caused by canceration
of the brain and spinal cord glial cells [1]. Radiotherapy is the
standard therapeutic modality for patients with brainstem gli-
oma [2]. However, the current median survival of patients
with glioma is only 7 to 15 months from therapy. Brainstem
glioma contains many hypoxic cells [3], which may be the
cause of radioresistance due to oxygen effects in radiobiol-
ogy in glioma. Therefore, there is an urgent need to identify
a radiosensitizer that can increase the radiosensitivity of hyp-
oxic glioma to improve radiotherapy efficacy.
Traditionally, radiosensitizers are pharmacologic or chem-
ical agents; however, in recent years, nanoparticles with a
high atomic number have provided new opportunities for
radiosensitization of tumors because they have higher prob-
ability of emitting secondary radiation [4,5]. Silver nanopar-
ticles (AgNPs) are most commonly used in several fields, such
as industry, agriculture, medicine and biomedical, and are
well known for their broad-spectrum antibacterial, antiviral
and anticancer effects as well as their surface-enhanced
Raman scattering activity [6–9]. Previous studies have
reported significantly enhanced anti-glioma effects in vitro
and in vivo when AgNPs and radiotherapy are used in
required to test the efficacy of this combination in hyp-
oxic cells.
Ionizing radiation-induced cell death includes necrosis,
apoptosis and autophagy [13]. The synergistic effect of
nano-radiosensitizers under irradiation is a fundamental
cause of cancer cell death during radiosensitivity enhance-
ment [14]. Although this combined induction of cell death
is not the same as cell death caused by radiation alone,
the former is also classified as ionizing radiation-induced
cell death [15]. Recent studies suggest that apoptosis and
autophagy can interconvert into each other [16,17], which
makes the mechanisms of the ionizing radiation-induced
cell death significantly more complicated. Therefore, this
study aimed to evaluate the radiosensitizing effect of
AgNPs on hypoxic glioma cell line U251 and C6, and
investigate the possible underlying mechanisms of radio-
sensitization. Firstly, the toxicity of AgNPs on hypoxic cells
was assessed. Secondly, the radiosensitizing effect of
AgNPs was evaluated using colony formation assay. Thirdly,
the apoptosis and mitochondrial membrane potential
(MMP) were detected and analysed. Finally, destructive
autophagy was measured and the crosstalk between
autophagy and apoptosis was investigated.

CONTACT Haiqian Zhang zhanghq@nuaa.edu.cn Department of Nuclear Science and Engineering, Nanjing University of Aeronautics and Astronautics,
Nanjing 210016, P.R. China
Supplemental data for this article can be accessed here.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

Materials and methods
Synthesis and characterization of AgNPs
AgNPs were synthesized with a green continuous flow elec-
trochemical process, as previously reported [18–20]. More
specifically, we designed a silver electrolytic reactor in a con-
tinuous flow system. The production process is described
below. Firstly, two pure silver rods were fixed on the cover of
the reactor to serve as the anode and cathode, respectively.
Secondly, 5 mg/mL of PVP was added to the container to
avoid a thick oxidation deposition coating on the anode sur-
face. Finally, the collected colloidal silver was purified using a
.22-lm filter membrane. The morphology of AgNPs was esti-
mated using a transmission electron microscope (TEM; JEOL
Ltd., Tokyo, Japan). The optical property of synthesized nano-
particles was measured with UV–visible spectroscopy (UV-
3600, Shimadzu, Japan).
Cell culture and hypoxic exposure
U251 and C6 glioma cells were obtained from the Biological
Science and Medical Engineering College of Southeast
University (Nanjing, People’s Republic of China). The cells
were nourished with Dulbecco’s modified Eagle’s medium
(DMEM) containing 10% foetal bovine serum (FBS), 100 unit/
mL penicillin and 100 lg/mL streptomycin, and sustained at
37
C in a humidified incubator with 5% CO2 and 95% air. To
achieve a hypoxic condition, a chamber with an air intake
and outlet was used to maintain the mixed gas (94% N2, 5%
CO2 and 1% O2) for 24 h. The oxygen concentration was
measured using an oxygen meter to ensure that the chamber
contained 1% O2.
Cell viability assay
The cytotoxicity effect of AgNPs on U251 and C6 cells was
evaluated by using the cell viability assay. The cells were
treated with different concentrations of AgNPs (0, 10, 15, 20,
25, 30, 40 and 60 lg/mL). The cell viability was estimated
using CCK-8 assay according to the manufacturer’s
instructions.
Radiation treatment
The cells were placed in a glass chamber and were irradiated
with X-rays (6 MeV, 200 cGy/min) from a linear accelerator
(Primus-M, Siemens, Germany). In particular, in order to main-
tain hypoxic environment, the cells ready to be irradiated
were placed in the glass chamber and flushed with the
gas mixture.
Uptake of AgNPs
The normoxic and hypoxic U251 cells were treated with
AgNPs and collected after different times’ incubation (10 min,
4, 8, 16 and 24 h). The uptake of AgNPs into the treated cells
was evaluated with forward scatter (FSC) and side scatter
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY S923
(SSC), two parameters of the flow cytometry (FACS Calibur,
BD, USA).
Colony formation assay
The cells were treated with AgNPs for 24 h before being
exposed to X-rays at doses of 0, 2, 4, 6 or 8 Gy. Following
another 10 days of incubation, the colony forming ability of
the cells was evaluated using a clonogenic formation assay.
The sensitization enhancement ratio (SER) was used as a
quantitative index of radiosensitivity. SER was determined by
a classical multi-target single-hit model using the following
equation [21].
n
S ¼ 1ð1eD=D0 Þ (1)
SER ¼ D0 ðcontrol groupÞ=D0 ðtreated groupÞ (2)
where S is the survival fraction, D is the radiation dose, D0 is
the mean lethal dose and n is the extrapolation number.
Apoptosis assay
To detect apoptosis rate and cell viability, the Apoptosis
Detection Kit (KeyGEN) was used according to the man-
ufacturer’s protocol. The normoxic and hypoxic U251 cells
were treated with AgNPs and then exposed to 6 Gy dose of
X-rays. The cells were stained with the Annexin V-FITC/propi-
dium iodide (PI) and analysed using the flow cytome-
ter (FCM).
Mitochondrial membrane potential analysis
MMP was detected using the fluorescent lipophilic cationic
dye JC-1 (Beyotime, China). This dye reagent easily enters the
mitochondria, aggregates and emits red fluorescence. When
MMP decreases, the dye reagent no longer gathers in the
mitochondria and emits green fluorescence. Briefly, the
treated U251 cells were stained with the dye reagent in the
dark at 37
C for 20 min. The cells were then assayed
using FCM.
Quantitative assay of autophagy
To quantify the autophagic potency, the treated U251 cells
were stained with Cyto-ID green autophagy detection
reagent (Enzo Life Sciences) and analysed using FCM accord-
ing to the manufacturer’s instructions.
Inhibition of autophagy to detect cell viability and
apoptosis rate
The AgNPs-treated U251 cells were incubated for the desig-
nated times with 3-MA (3-methyladenine, an autophagy
inhibitor, 5 mM), then exposed to 6 Gy dose of X-rays and
finally subjected to apoptosis assay.
S924 Z. LIU ET AL.
Statistical analysis
The data were presented as mean ± SD and processed with
OriginPro 8.5 (Origin Lab, Northampton, MA, USA). One-way
analysis of variance and a Duncan test were used to analyse
the difference between the untreated and treated groups.
A p-value less than .05 was regarded as statistically
significant.
Results
Synthesis and characterization of AgNPs
The morphology and the optical absorption of the AgNPs
were characterized by TEM and spectrophotometry, respect-
ively. The AgNPs synthesized in colloidal solution are pre-
dominantly spherical in morphology with good
monodispersity (Figure 1(A)), and the average size of AgNPs
is 26.87 ± 3.68 nm, determined by analysing the recorded TEM
image with ImageJ software (Figure 1(B)). The absorption
peak of UV–Visible spectroscopy for the AgNPs is at 422 nm,
which was in accordance with the characteristic surface plas-
mon resonance absorption bands of the corresponding
nanoparticles.
Cytotoxicity of AgNPs in the hypoxic U251 and C6 cells
The cytotoxicity of the AgNPs in the hypoxic U251 and C6
cells was evaluated using the CCK-8 assay. This result shows
that the AgNPs cause a concentration-dependent reduction
in the hypoxic and normoxic U251 (Figure 2(A)) and C6
(Figure 2(B)) cells. What’s more, the cytotoxicity of the AgNPs
Figure 1. Characterization of the AgNPs. (A) TEM images of AgNPs (scale bar, 50 nm). (B) Size distribution measurements of the AgNPs. (C) Ultraviolet-visible spectra
of the AgNPs. TEM: transmission electron microscope; AgNPs: silver nanoparticles.
to the hypoxic cells is higher than that to the normoxic cells.
The half maximal inhibitory concentration (IC50) values of
the AgNPs for the hypoxic and normoxic U251 cells are
30.32 lg/mL and 34.73 lg/mL, and 27.53 lg/mL and
32.47 lg/mL for hypoxic and normoxic C6 cells. The concen-
tration of the AgNPs for inducing the mild cytotoxicity
(20%) in the hypoxic cells is 15 lg/mL, which was selected
for subsequent experiments of radiosensitivity enhancement.
Cellular uptake of AgNPs in hypoxic cells
The cellular uptake is a very important process for the bio-
medical application of nanomaterials, since it plays an
important role in the realization of their functions [22]. The
FSC and SSC are two parameters relating to the scattered
light of the FCM. The former reflects the size of the cell and
increases with the cross-sectional area of the same cell popu-
lation. The latter refers to the grain properties inside the cell
and gives a susceptive response to larger particles in the
cytoplasm [23,24]. The FSC and SSC were used to evaluate
the capacity of the hypoxic U251 cells in AgNPs uptake. As
shown in Figure 3(A), both the normoxic and hypoxic U251
cells increase in number in their clusters and the clusters
tend to move to the upper right during 24 h’s incubation,
suggesting that the AgNPs are absorbed by the cells. The
quantitative analyses of the SSC value are shown in Figure
3(B), which increase from 483.26 ± 5.16 to 622.44 ± 3.96 for
the hypoxic U251 cells and from 501.17 ± 5.66 to
605.92 ± 3.98 for the normoxic U251 cells. The SSC values
indicate that the capacity of the hypoxic U251 cells in AgNPs
uptake is higher than that of the normoxic U251 cells.
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY S925

Figure 2. Cytotoxicity of the hypoxic U251 (A) and C6 (B) cells treated with AgNPs at various concentrations. IC50: half maximal inhibitory concentration.

Figure 3. Uptake of AgNPs was assessed by the change of SSC. (A) FCM result of the hypoxic U251 cells incubated with AgNPs (10 min, 4, 8, 12 and 24 h). (B)
Quantitative analysis of SSC-H. SSC: side scatter; FSC: forward scatter; FCM: flow cytometry.

Radiosensitizing effect of AgNPs on the hypoxic U251 that of the normoxic glioma cells (Figure 4(A,C)). Figure
and C6 cells 4(B,D) presents survival fraction curves, determined by the
photographs of clonogenic formation of the U251 or C6 cells
Figure 4 shows radiosensitizing data of AgNPs in the hypoxic
treated with AgNPs and X-rays. The corresponding SER based
U251 and C6 cells. Reduction in survival of the hypoxic U251
on the survival fractions is 1.78 and 1.34 in the hypoxic and
and C6 cells treated with AgNPs and X-rays is much greater
normoxic U251 cells, and 1.84 and 1.45 in the hypoxic and
than that treated with X-rays alone, and this reduction
normoxic C6 cells.
becomes more significant as the doses increase. This result
indicates that the AgNPs can radiosensitize the hypoxic U251
and C6 cells. Similarly, reduction in survival of the hypoxic
AgNPs combined with X-rays inducing apoptosis in the
U251 and C6 cells treated with AgNPs and X-rays is also
hypoxic U251 cells
much greater than the normoxic cells treated with AgNPs
and X-rays. This result suggests that the radiasensitizing effi- The apoptotic response of the normoxic and hypoxic U251
cacy of AgNPs in the hypoxic glioma cells is greater than cells to AgNPs with or without radiation was evaluated with
S926 Z. LIU ET AL.

Figure 4. Cloning formations of hypoxic cells treated with AgNPs and X-rays. (A and C) Photography of the hypoxic U251 and C6 cell’s colony formed by combined
treatment of AgNPs and X-rays. (B and D) Survival fraction of the hypoxic U251 and C6 cells induced by AgNPs and X-rays.

Annexin V-FITC/PI assay. Figure 5(A) shows the apoptotic

image of FCM for the hypoxic U251 cells treated with AgNPs
and X-rays. Figure 5(B) presents the apoptosis quantitative
data based on the Figure 5(A). As shown in Figure 5(A,B), the
apoptotic rate (30.39 ± 0.67%) in the hypoxic U251 cells
treated with AgNPs and X-rays is higher than that in the hyp-
oxic U251 cells treated with X-rays only (15.65 ± 0.87%). At
the same time, it is also higher than that in the normoxic
U251 cells treated with AgNPs and X-rays (13.62 ± 0.69%).

AgNPs combined with X-rays decreasing MMP in the

hypoxic U251 cells
The decrease of MMP is the earliest event in the apoptosis
cascade [25,26]. The fluorescent lipophilic cationic dye, JC-1,
was used to investigate the MMP in the hypoxic U251 cells
treated with the AgNPs and X-rays. Figure 6 shows the MMP
data detected with FCM for the hypoxic U251 cells treated
with the AgNPs and X-rays. It is evident from Figure 6 that
MMP is significantly lower in the hypoxic U251 cells treated
with the AgNPs and X-rays, compared with the hypoxic U251
cells treated with X-rays only.

AgNPs combined with X-rays enhancing autophagy in
the hypoxic U251 cells
The autophagy level of the hypoxic U251 cells treated with
AgNPs and X-rays was evaluated using the green fluorescent
probe Cyto-ID, which labels vacuolar components of the
autophagy pathway. Figure 7(A) shows the autophagy levels
of hypoxic U251 cells treated with AgNPs and X-rays deter-
mined by FCM, and Figure 7(B) shows the autophagy
Figure 5. Apoptosis rates of the hypoxic U251 cells treated with AgNPs and X-
rays. (A) FCM images of the hypoxic U251 cells treated with AgNPs (15 lg/mL)
and X-rays (6 Gy). (B) Quantitative apoptotic data of the hypoxic U251 cells
treated with AgNPs and X-rays. FITC: fluorescein isothiocyanate; PI: propidium
iodide; FCM: flow cytometry. (p < .001).
fluorescence intensity on account of Figure 7(A). As shown in
Figure 7(A,B), the autophagy fluorescence intensity
(56.58 ± 2.37%) in the hypoxic U251 cells treated with AgNPs
and X-rays is stronger than that in the hypoxic U251 cells

Figure 6. Changes in the mitochondrial membrane potential detected with
FCM. The mitochondrial membrane potential of the hypoxic U251 cells treated
with AgNPs and X-rays by FCM using the JC-1 apoptosis detection kit. FCM:
flow cytometry. (p < .001).
Figure 7. Elevated autophagy in the hypoxic U251 cells treated with AgNPs
and X-rays. (A) FCM results of the hypoxic U251 cells treated with AgNPs and
X-rays using Cyto-ID as the probe. (B) Quantitative analysis of fluorescence
intensity based on FCM. FCM: flow cytometry. (p < .001).
treated with X-rays (28.15 ± 2.09%). At the same time, it is
also stronger than that in the normoxic U251 cells treated
with AgNPs and X-rays (31.04 ± 1.89%).
Many studies have indicated that autophagy appears to
have dual and conflicting functions in oncogenesis. One is
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY S927
facilitating the cell survival, and the other is delay of the for-
mation of tumour cells. To conduct further investigation on
the function of autophagy, 3-MA was used to evaluate the
survival rate in the hypoxic U251 cells treated with AgNPs
and X-rays. Figure 8(A) shows apoptotic graphs of the hyp-
oxic U251 cells treated with AgNPs and 3-MA under X-rays.
The lower left area of the apoptotic graphs presents the sur-
vival rate. The quantitative data of cell viability is acquired
from the apoptotic graphs (Figure 8(B)). Three-MA can signifi-
cantly increase the cell viability in the hypoxic U251 cells
treated with AgNPs and X-rays (from 66.26 ± 1.06% to
80.38 ± 1.56%), indicating that autophagy induced by AgNPs
and X-rays is destructive in the hypoxic U251 cells. By con-
trast, 3-MA reduced the cell viability in the normoxic U251
cells treated with AgNPs and X-rays (from 83.61 ± 1.86 to
75.16 ± 2.56%), demonstrating that autophagy induced by
AgNPs and X-rays is protective in the normoxic U251 cells.
The autophagy-related cell death and apoptosis have
been recognized as two modes of cell death with independ-
ent mechanisms, and there is a crosstalk linking these two
types of cell death. In this study, both apoptosis and autoph-
agy participate in the death of hypoxic U251 cells treated
with AgNPs and X-rays. Whether the crosstalk exists between
apoptosis and autophagy should be elucidated. As can be
seen in Figure 8(C), 3-MA can significantly decrease the rate
of apoptosis in hypoxic cells treated with AgNPs and X-rays
(from 32.96 ± 1.27% to 20.89 ± 0.97%). This result indicates
that inhibition of autophagy results in decreased apoptosis
rate in the hypoxic U251 cells treated with AgNPs and X-rays,
suggesting autophagy and apoptosis may have a crosstalk.
Discussion
The ability of radiosensitivity enhancement of AgNPs in nor-
moxic cells has been previously reported. However, there was
little concern about the radiosensitivity enhancement of
AgNPs in hypoxic cells. Our findings demonstrate that AgNPs
exhibits greater radiosensitivity in the hypoxic U251 and C6
cells than that in normoxia. The underlying mechanism of
AgNPs’ radiosensitivity enhancement is through promotion of
apoptosis and destructive autophagy.
We first evaluated the toxic effects of AgNPs in hypoxic
cells. Liu et al. [27] reported that smaller nanoparticles are
more likely to enter cells than larger ones, which may be the
cause of higher toxic effect of smaller nanoparticles. AgNPs
with the size of about 20 nm has better radiosensitizing
effects [28]. Considering these factors, AgNPs with the aver-
age size of 26.87 nm (Figure 1) were prepared using the
green continuous flow electrochemical method. AgNPs at the
same concentrations show higher cytotoxicity in hypoxic cells
compared with that in normoxic cells (Figure 2). AgNPs have
the strong ability to enhance the radiosensitivity of hypoxic
cells compared with normoxic cells (Figure 4). These results
may be explained by the phenomenon of higher capacity in
AgNPs uptake of hypoxic cells (Figure 3).
Increasing research has evidenced that apoptosis contrib-
utes significantly to radiotherapy-induced tumour cell death
[29,30]. The hypoxic U251 cells treated with AgNPs and
S928 Z. LIU ET AL.

Figure 8. The effect of autophagy in the hypoxic U251 cells treated with AgNPs and X-rays. (A) FCM results of the hypoxic U251 cells treated with AgNPs and X-
rays respectively with 3-MA (5 mM) using annexin-V/PI staining. In the four regions shown in the figure, the lower left portion represents living cells, and the sum of
the lower right portion and upper right portion signals apoptotic cells. (B and C) The quantitative analysis for cell viability and apoptotic rates of the hypoxic U251
cells treated with AgNPs and X-rays. 3-MA: 3-methyladenine; (p < .01 and p < .001).

X-rays presented significant apoptosis response (Figure 5).
Our result is in alignment with results reported in previous
studies. The decrease of MMP is considered to be the earliest
event in the apoptosis cascade of events [31]. A variety of
mitochondrial apoptotic stimulators can enter the cytoplasm
and induce apoptosis following the reduction of MMP
[32,33]. The decrease of MMP in hypoxic U251 cells treated
with AgNPs and X-rays further supports the increase of apop-
tosis in the hypoxic U251 cells treated with AgNPs and X-rays
(Figure 6). It is plausible that the significant increase in apop-
tosis is correlated with the change of the cell cycle distribu-
tion. Jin et al. [34] reported that DNA damage will be
repaired in the arrested G2 phase, and apoptosis will occur if
the repair fails. In this study, G2/M phase increases in the
hypoxic U251 cells treated with AgNPs and X-rays (Figure S1).
Cells are most sensitive to the effects of radiation in the G2/
M phase [35]. Therefore, the apoptosis in the hypoxic U251
cells induced by AgNPs and X-rays may be related to the
arrest of the G2/M phase.
Autophagy has been shown to play an important role in
cancer cell survival and death [36]. AgNPs can induce signifi-
cantly the destructive autophagy in the hypoxic U251 cells
treated with X-rays (Figure 8(B)). It has been demonstrated
that the enhancement of autophagy is accompanied by the
increase of reactive oxygen species (ROS) levels [37]. We
found that there were higher ROS levels in the hypoxic U251
cells treated with AgNPs and X-rays in comparison with the
hypoxic U251 cells treated with X-rays only (Figure S2). It is
therefore possible that the increased autophagy detected in
the hypoxic U251 cells treated with AgNPs and X-rays may
be due to the higher ROS levels.
Inhibition of autophagy can decrease the apoptosis in
AgNPs-radiosensitized hypoxic cells (Figure 8(C)), which indi-
cates that increase in the levels of destructive autophagy can
lead to increased apoptosis. This suggests that there is a
crosstalk between the autophagy and apoptosis in the hyp-
oxic U251 treated with AgNPs and X-rays. A possible explan-
ation for this may be that the destruction of cytoplasm and
organelles by autophagy leads to the collapse of important
cell functions, causing apoptosis. Another possibility is that
autophagy develops as a primary response to stress stimuli
and then triggers apoptosis through signalling pathways
such as JNK (c-Jun N-terminal kinase) and ERK (extracellular
signal-regulated kinase) [38] or regulatory proteins such as
Bcl-2, caspase, ATG and P53 [39–42]. The exact mechanism
remains to be investigated.
Conclusions
In summary, the purpose of the current study was to evalu-
ate the radiosensitizing efficacy of AgNPs in hypoxic glioma
cells. The combination of AgNPs and radiotherapy showed
significantly enhanced anti-glioma effects in hypoxia, which
may be due to its higher apoptosis activity and strongly
destructive autophagy. Collectively, the findings reported
here may be useful to lay the foundation for potential

treatment strategies of hypoxic glioma. Further investigation
of AgNPs’ radiosensitization should be performed on animal
glioma hypoxia model to explore this promising
radiotherapy.
Acknowledgements
We thank the College of Materials Science and Technology, Nanjing
University of Aeronautics and Astronautics, and Jiangsu Laboratory for
Biomaterials and Devices, Southeast University, for technical assistance
for this research.
Disclosure statement
The authors declare that they have no competing interests.
Funding
This work was supported by the National Natural Science Foundation of
the People’s Republic China [grant number: 31400721, 11575086 and
81571805], the Fundamental Research Funds for the Central Universities
[grant number: NP2015207], the Postdoctoral Science Foundation of
China [2017M611660], and the Priority Academic Program Development
of Jiangsu Higher Education Institutions and Collaborative Innovation
Center of Radiation Medicine of Jiangsu Higher Education Institutions.
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