Rational Design of Cancer-Targeted Selenadiazole Derivative as Efficient Radiosensitizer for Precise Cancer Therapy

Article abstract of DOI:10.1021/acs.bioconjchem.8b00247

Chemical drug design based on the biochemical characteristics of cancer cells has become an important strategy for discovery of targeted therapies for personalized cancer medicine. Herein, cancer targeting RGD peptide has been covalently conjugated to selenadiazole derivative (RGD-SeD) to improve its cancer selectivity. The RGD decoration significantly enhances the anticancer efficacy of RGD-SeD in αVβ3 integrin-overexpressing HepG2 liver cancer cells but not in normal liver cells. Cellular uptake assay and fluorescent imaging confirmed the selectivity of RGD-SeD to integrin-overexpressing cancer cells. RGD-SeD strongly sensitizes HepG2 cells to clinically used X-ray radiotherapy through ROS overproduction, which triggers DNA damage-mediated apoptosis and G2/M cell cycle arrest. This X-ray-responsive DNA damage activates p53 signaling pathways by phosphorylation of ATM/ATR and γ-H2A.X. Furthermore, in a HepG2 nude mice xenograft model, the combined treatment of RGD-SeD and X-ray demonstrates potent in vivo antitumor efficacy via induction of apoptotic cell death but shows no toxicity on the functions of major organs. In summary, this study provides a strategy to design a selenium-based cancer targeting radiosensitizer for precise cancer therapy.

Full text of DOI:10.1021/acs.bioconjchem.8b00247

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Article
Rational Design of Cancer-targeted Selenadiazole Derivative
as Efficient Radiosensitizer for Precise Cancer Therapy
Delong Zeng, Shulin Deng, Chengcheng Sang, Jianfu Zhao Zhao, and Tianfeng Chen
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00247 • Publication Date (Web): 17 May 2018
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Rational Design of Cancerꢀtargeted Selenadiazole Derivative as Efficient
Radiosensitizer for Precise Cancer Therapy
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Delong Zeng#, Shulin Deng#, Chengcheng Sang, Jianfu Zhao*, Tianfeng Chen*
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The First Affiliated Hospital, and Department of Chemistry, Jinan University, Guangzhou 510632,
China.
E-mail addresses: tchentf@jnu.edu.cn, zhaojianfu@jnu.edu.cn.
# These authors contribute equally to this study.
* Corresponding author: Tel: +86 20 85223393.
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ABSTRACT:
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Chemical drug design based on the biochemical characteristics of cancer cells has become an
important strategy for discovery of targeted therapies for personalized cancer medicine. Herein,
cancer targeting RGD peptide has been covalently conjugated to selenadiazole derivative
(RGDꢀSeD) to improve its cancer selectivity. The RGD decoration significantly enhances the
anticancer efficacy of RGDꢀSeD in αVβ3 integrinꢀoverexpressing HepG2 liver cancer cells, but not
in normal liver cells. Cellular uptake assay and fluorescent imaging confirmed the selectivity of
RGDꢀSeD to integrin overexpressing cancer cells. RGDꢀSeD strongly sensitizes HepG2 cells to
clinicallyꢀused Xꢀray radiotherapy through ROS overproduction, which triggers DNA
damageꢀmediated apoptosis and G2/M cell cycle arrest. This Xꢀray responsive DNA damage
activates p53 signaling pathways by phosphorylation of ATM/ATR and γꢀH2A.X. Furthermore, in
HepG2 nude mice xenograft model, RGDꢀSeD combined with Xꢀray demonstrates potent in vivo
antitumor efficacy via induction of apoptotic cell death, but shows no toxicity on the functions of
major organs. In summary, this study provides a strategy to design seleniumꢀbased cancer targeting
radiosensitizer for precise cancer therapy.
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Key words:
Cancer targeting; Drug design; Selenadiazole derivative; Radiosensitizer
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INTRODUCTION
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Radiotherapy is one of the major therapeutic strategies to cure cancer, which is received by
approximately 50% of all cancer patients 1, 2. Using highꢀenergy ionizing radiation, such as Xꢀray or
γꢀray, to irradiate cancer tissue, radiotherapy causes DNA damages as well as lesions of plasma
membrane in cancer cells to kill them or slow down their growth 3. Radiotherapy is effective in
cancer treatment and has several advantages over surgery and chemotherapy, such as
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noninvasiveness and low systemic toxicity. However, a large proportion of cancer patients still
suffer from local recurrence of disease after radiotherapy 4. The effectiveness of radiotherapy is
limited by the maximum tolerated dose of the normal tissues around tumors. In addition, cancer
cells may resist to radiation inherently or acquire resistance during therapy 4. Therefore,
chemotherapy is widely used in combination with radiotherapy to gain additive of synergistic effect;
e.g. cisplatin, 5ꢀfluorouracil, mitomycin C are commonly used as radiosensitizers in head and neck
cancers, lung cancers and gastrointestinal cancers 5. Tirapazamine and nitroimidazoles can sensitize
hypoxic tumors to radiation. EGFR antibodies and inhibitors cetuximab, gefitinib, and erlotinib
show enhanced radiosensitivity in vitro and in vivo 6, 7. The inhibitor of PARPꢀ1, which is required
for the DNA base excision repair and single strand breaks (SSB) repair, has entered clinical trial 8.
Despite the progress in radiosensitizer research that has been made in recent years, the clinical
outcome of many of these radiosensitizers is not quite satisfactory. For example, although platinum
complexes, including cisplatin, oxaliplatin and carboplatin, are the most successful and bestꢀknown
metal complex radiosensitizers, their radiosensitization effects are not selective for tumor cells,
enhancing the damage to normal tissue during radiation 9. Therefore, it is of great significance to
develop new radiosensitizers with high efficacy and low toxicity.
Selenium compounds may be one kind of candidates that meet these criteria. Selenium (Se) is an
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essential trace element, which plays important roles in metabolism, inflammation and reproduction,
as well as in maintaining the functions of immune cells, thyroid and brain 10, 11. Selenium
compounds supplied in nutritional level can work as antioxidants and prevent the incidences of
cancer and cancer motility 12ꢀ14. Although higher level intake will shift selenium compounds into
proꢀoxidants and exhibit high toxicity, the fact that cancer cells are more vulnerable to selenium,15
due to their increased cellular reactive oxygen species (ROS) levels, endows the selenium
compounds with selective cancerꢀcell killing capacity. Various selenium compounds, both inorganic
and organic, has been reported to show anticancer activity against different types of cancer and to
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augment the efficacy of chemoꢀ and radiotherapy 12, 16
.
In our previous studies, we have designed and synthesized several selenadiazole derivatives and
seleniumꢀcontaining metal complexes, which showed high activities on tumor growth inhibition
17ꢀ20, antiꢀangiogenesis 21, 22 and antiꢀinvasion 23, as well as reversion of drug resistance 24, 25 and
synergism with chemoꢀ and radiotherapy 26, 27. However, cancer selectivity of these selenium
compound was limited. Therefore, to further improve the selectivity of selenadiazole derivatives to
cancer cells in radiotherapy, we decorated a novel selenadiazole derivative (SeD) with RGD peptide
that could bind to αVβ3 integrin frequently overexpressed on various types of cancer cells 28. The in
vitro and in vivo radiosensitization effects of RGDꢀSeD, and the underlying action mechanisms
were also elucidated (Figure 1A). Taken together, this study demonstrated an effective way to
improve the cancer selectivity of seleniumꢀbased radiosensitizer for personalized cancer medicine.
RESULTS
Rational design of cancerꢀtargeted RGDꢀSeD
We first examined the expression levels of integrin in HepG2 liver cancer cells and L02 normal
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liver cells. Western blot analysis showed about 1.7 folds higher of integrin expression in HepG2
cells than that in L02 cells (Figure 1B and C), which indicate that integrin may be an effective
target for anticancer drugs selectively recognizing liver cancer cells.
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In order to enhance the effect of radiotherapy, we designed and synthesis a new selenadiazole
derivative as a radiosensitizer, and further decorated it with a circular RGD peptide, which can
highly specifically bind to αVβ3 integrin. The synthesized selenadiazole derivatives were
characterized by UV/vis spectroscopy, ESI mass spectrometry, elemental analysis and HPLC (see
the Experimental Section for further details and Figures S1 and S2 in the Supporting Information).
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Figure 1. Scheme of the present work and cytotoxic and radiosensitization effects of SeD and RGDꢀSeD to
HepG2 and L02 cells expressing different levels of integrin. (A) Schematic illustration of the design SeD and
RGDꢀSeD and the synergistic mechanism of Xꢀray and RGD to selectively kill cancer cells. Expression of
Integrin in HepG2 and L02 cells were detected by Western blot (B), and the relative intensity of the bands were
analyzed by ImageJ software (C). (DꢀE) Cytotoxic and radiosensitization effect of SeD and RGDꢀSeD against
HepG2 (D) and L02 (E) cells. Bars with different characters are statistically significant (P < 0.05, Tukey’s test,
oneꢀway ANOVA).
In vitro antitumor and radiosensitization effects of RGDꢀSeD
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MTT assay was used to assess the in vitro antitumor activity and radiosensitization effects of SeD
and RGDꢀSeD. As shown in Figure 1 D and E, the IC50 values of SeD and RGDꢀSeD against
HepG2 cells were 50.18 ꢁM and 31.22 ꢁM, respectively; while in L02 cells the IC50 value were
93.69 ꢁM and 98.07 ꢁM, respectively. As a positive control, cisplatin showed the highest antitumor
activity with IC50 value at 3.9 ꢁM against HepG2. However, its cytotoxicity was not selective
toward tumor cells, as evidenced by the comparable IC50 of L02 cells (4.3 ꢁM) to that of HepG2
cells. Although, to some extent, SeD showed selectivity to cancer cells compared with normal cells,
the selectivity was much higher after decoration with RGD peptide. Similarly, the radiosensitization
effect of SeD on HepG2 cells was enhanced by RGD decoration; The IC50 of SeD in combination
with radiation (8 Gy) was 20.79 ꢁM, and it reduced to 10.26 ꢁM when decorated with RGD. In L02
cells, the radiosensitization effects of both SeD and RGDꢀSeD were remarkably weaker.
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Cellular uptake and localization of RGDꢀSeD
To evaluate whether the enhanced antitumor and radiosensitization effects of RGDꢀSeD were due
to its increase of cellular uptake by HepG2 cells mediated by binding of RGD group to integrin on
the cell surface, we measured the intracellular Se concentration in cells treated with SeD and
RGDꢀSeD for different time periods. We found that the uptake of RGDꢀSeD by HepG2 cells was
significantly higher than that of SeD at all the tested time points (Figure 2A and C). In L02 cells,
the uptake of SeD was comparable with RGDꢀSeD (Figure 2B and D). The different uptake
properties between RGDꢀSeD and SeD in HepG2 and L02 cells further confirmed the tumor
targeting ability of RGDꢀSeD.
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Figure 2. Cellular uptake of SeD and RGDꢀSeD by HepG2 and L02 cells. HepG2 (A) and L02 (B) cells were
treated with SeD (50 ꢁM) and RGDꢀSeD (50 ꢁM) for the indicated time before harvested. Then the cells were
digested and subjected to ICPꢀMS for Se measurement. HepG2 (C) and L02 (D) cells were treated as A and B and
the fluorescent images were taken at the indicated time points to visualize the intake of the compounds (green
fluorescence). DAPI (blue) was used to stain cell nucleus. Scale bar represents 100 ꢁm.
We next used fluorescence imaging technique to explore the intracellular trafficking of RGDꢀSeD
in HepG2 cells. As shown in Figure 3, green fluorescent RGDꢀSeD accumulated in HepG2 cells in
a timeꢀdependent manner. RGDꢀSeD appeared inside HepG2 cells as early as 2 h, and the
fluorescent intensity increased in 4 h and 8 h. We also used the LysoTracker® Red to label
lysosomes, and found that the intracellular RGDꢀSeD was coꢀlocalized with lysosome. The results
indicated that the uptake of RGDꢀSeD by HepG2 cells might be through endocytosis after binding
to integrin.
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Figure 3. Localization of RGDꢀSeD in HepG2 cells detected by fluorescent imaging. HepG2 cells were treated
with RGDꢀSeD (50 ꢁM) and labeled by DAPI for nucleus and LysoTracker® Red for lysosome. Images were
taken in different time points by fluorescent microscopy. Scale bar represents 100 ꢁm.
RGDꢀSeD synergizes with Xꢀray to inhibit colony formation of HepG2 cells
The decrease of IC50 when RGDꢀSeD combined with Xꢀray to treat HepG2 cells, implied the
radiosensitization effect of RGDꢀSeD. To further confirm the synergism between RGDꢀSeD and
Xꢀray, we measured the colony formation of HepG2 cells after treatment with RGDꢀSeD combined
with or without Xꢀray. As shown in Figure 4A, treatment of 2 Gy Xꢀray alone did not significantly
inhibit the colony formation ability of HepG2, compared with control, implying the capability of
HepG2 cell to repair DNA damage caused by low dose of Xꢀray. However, treatment with
RGDꢀSeD, even in a concentration much lower than IC50, greatly reduced the formation of colonies.
The inhibition were further enhanced by combining RGDꢀSeD with Xꢀray. Only several colonies
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were found after combination treatment; the survival fraction was less than 10% (Figure 4B). The
results demonstrated that colony inhibition effect on HepG2 cells of Xꢀray could be enhanced by
RGDꢀSeD.
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Figure 4. RGDꢀSeD synergizes with Xꢀray to attenuate the colony formation and cell cycle of HepG2 cells. (A
and B) HepG2 cells were seeded in low density (1000 cells/mL) and treated with RGDꢀSeD (8 ꢁM) and Xꢀray (2
Gy), and then continue to culture for 2 weeks. The colonies were visualized by staining with crystal violet and
imaged (A), colonies contained >50 cells in each well were counted and shown as percentage to untreated (control)
group (B). Bars with different characters are statistically significant (P < 0.05, Tukey’s test, oneꢀway ANOVA).
(C and D) HepG2 cells were treated with RGDꢀSeD (20 ꢁM) and Xꢀray (8 Gy). The cells were harvested, fixed
and stained with PI for cell cycle analysis by flow cytometry (C) and stacked bar chart of each cell cycle phase
were shown (D).
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RGDꢀSeD enhances Xꢀrayꢀcaused G2/M arrest and apoptosis of HepG2 cells
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Cell cycle arresting on G2/M phase and apoptosis are two main consequences of cells exposed to
radiation. We therefore examined the cell cycle distribution of HepG2 cells after treatment with
RGDꢀSeD and Xꢀray. Cell cycle analysis by flow cytometry showed that Xꢀray (8 Gy) significantly
caused G2/M arrest of HepG2 cells; the percentage increase from 15.27% to 30.51% (Figure 5).
G0/G1 phase was modestly increased, while apoptosis, which was indicated by subꢀG1 population,
was not changed. RGDꢀSeD treatment alone was able to arrest the cell cycle on G2/M phase and
caused apoptosis. Coꢀtreatment with RGDꢀSeD and Xꢀray caused more profound G2/M arrest and
apoptosis; nearly half of the cells were arrested on G2/M phase and subꢀG1 proportion reached
about 20% (Figure 4C and D). These data suggested that RGDꢀSeD inhibited HepG2 cell growth by
causing cell cycle arrest on G2/M phase and killed the cells by induction of apoptosis, and these
effects might contribute to the synergism with Xꢀray radiation.
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RGDꢀSeD enhances Xꢀrayꢀinduced cellular ROS overproduction
Reactive oxygen species (ROS) can be induced by organic selenium compounds 24 and radiation 29
which then play an important role in mediating cell death. In order to explore whether ROS is
produced in HepG2 cells treated with RGDꢀSeD and Xꢀray, we labeled the cells with
dihydroethidium (DHE), a fluorescent ROS probe, and recorded the fluorescent intensities in
different time points. As shown in Figure 5A, ROS level was increased to about 120% after Xꢀray
irradiation, compared with untreated control. RGDꢀSeD treatment alone did not changed the ROS
level significantly. When combined with Xꢀray, however, RGDꢀSeD strongly enhanced the ROS
produced by Xꢀray. The fluorescent images taken by fluorescent microscope also clearly showed
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the enhancement of ROS production in the combination treatment group (Figure 5B).
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Figure 5. RGDꢀSeD enhances ROS overproduction induced by Xꢀray. HepG2 cells were treated with RGDꢀSeD
(20 ꢁM) alone or in combination with Xꢀray (8 Gy). DHE fluorescent probe was added to the cells to labeled ROS,
fluorescent intensity (A) were measured by microplate reader in different time points. Fluorescent images (B)
were taken at the end of reading by a fluorescent microscope. Scale bar represents 100 ꢁm.
RGDꢀSeD enhanced Xꢀrayꢀcaused DNA damage in HepG2 cells
Excessive ROS in irradiated cells may react with DNA in the nuclei causing DNA damage, which is
the main cause of cell cycle arrest and apoptosis after radiation. Phosphorylation on serine 139 of
histone H2A.X, which is then referred as γH2AX, is an early step of DNA damage response and can
be served as an biomarker of DNA damage 30. As RGDꢀSeD enhanced the Xꢀrayꢀinduced
intracellular ROS production, we further investigated whether this effect was translated to more
DNA damage. Immunofluorescence analysis of RGDꢀSeD and Xꢀray treated HepG2 cells (Figure
6A) showed that RGDꢀSeD or Xꢀray treatment alone increased the γH2AX foci by a moderate
extent. However, coꢀtreatment of HepG2 cells with RGDꢀSeD and Xꢀray resulted in significant
increase of γH2AX focus and fluorescent intensity, indicating more DNA damage produced. We
then confirmed this result by examining the protein level of γH2AX using Western blot analysis
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(Figure 6B). Quantitative analysis of integrated density of the bands in Western blot showed that
RGDꢀSeD and Xꢀray treatment alone increased the γH2AX signal by about 2 folds, while the
combination treatment resulted in about 10ꢀfold increase (Figure 6C). These data indicated that
RGDꢀSeD enhanced Xꢀrayꢀcaused DNA damages in HepG2 cells, which promoted cell apoptosis.
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Figure 6. RGDꢀSeD increases γꢀH2A.X foci induced by Xꢀray in HepG2 cells. (A) HepG2 cells were treated with
RGDꢀSeD (20 ꢁM) and Xꢀray (8 Gy), and subjected to immunofluorescent analysis for γꢀH2A.X. DAPI was used
to label nuclei and images were taken by fluorescent microscopy. Scale bar represents 10 ꢁm. (B) HepG2 cells
were treated with RGDꢀSeD (20 ꢁM) and Xꢀray (8 Gy), and the cells were lysed and subjected to Western blot for
γꢀH2A.X. (C) The relative intensity of bands of γꢀH2A.X in (B) were measured using ImageJ software. Bars with
different characters are statistically significant (P < 0.05, Tukey’s test, oneꢀway ANOVA).
RGDꢀSeD and Xꢀray induce mitochondria fragmentation in HepG2 cells
Mitochondria play an important role in regulating apoptosis in addition to their energyꢀproducing
function. Extracellular and intracellular stresses may cause mitochondria dysfunction, which then
release several apoptosis promoting factors to activate apoptosis process. Mitochondria
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fragmentation is the morphology change reflecting the mitochondria dysfunction. We therefore
monitored the mitochondria status by labeling the cells with MitoTracker Red CMXRos. The
results showed that the mitochondria in untreated control group were filamentous, interconnected as
a network throughout the cytoplasm (Figure 7). Treatment with RGDꢀSeD or Xꢀray resulted in
mitochondria fragmentation, breaking the network. Coꢀtreatment with RGDꢀSeD and Xꢀray
enhanced this fragmentation. The results confirmed that mitochondria were involved in mediating
the apoptosis caused by RGDꢀSeD and Xꢀray.
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Figure 7. RGDꢀSeD and Xꢀray causes mitochondrial fragmentation of HepG2 cells. HepG2 cells were treated
with RGDꢀSeD (20 ꢁM) and Xꢀray (8 Gy), then the cells were labeled with MitoTracker® Red CMXRos and
DAPI, and images were by fluorescent microscopy. Scale bar represents 10 ꢁm. Arrows showed the fragmented
mitochondria.
DNA damage response and apoptosis signaling activated by RGDꢀSeD and Xꢀray
To further confirm the apoptosis induced by RGDꢀSeD and Xꢀray, we examined the expressions of
apoptosisꢀrelated proteins by Western blot. The hypothetic molecular mechanism of RGDꢀSeD and
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Xꢀray triggering apoptosis were depicted as Figure 8A, wherein RGDꢀSeD synergized with Xꢀray
to cause ROS overproduction, DNA damage and activation of ATM/ATR and p53, which then
induced cell cycle arrest and apoptosis. We first measured the phosphorylation level of ATM, ATR
and p53 in RGDꢀSeD and/or Xꢀray treated HepG2 cells. As shown in Figure 8B, treatment with
RGDꢀSeD or Xꢀray increased the phosphorylation of ATM, ATR and p53, which was further
enhanced by combination treatment. The results demonstrated the activation of DNA damage
response in the treated cells. The occurrence of apoptosis was then examined by detecting the
cleavages of caspaseꢀ8, ꢀ9 and ꢀ3, as well as their substrate PARP (Figure 8C). Strongest cleavage
of these proteins was demonstrated in the coꢀtreatment group, reflecting significant synergism of the
two treatments. We further confirmed the activation of caspase ꢀ9 and ꢀ3 by measurement of the
enzyme activities using specific fluorescent substrates. Figure 8D showed that the activities of
caspaseꢀ9 and ꢀ3 were significantly increased after treatment with Xꢀray or RGDꢀSeD, and the
combined treatment potently enhanced the activation.
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Figure 8. RGDꢀSeD synergizes with Xꢀray to activate DNA damage and apoptosis pathways in HepG2 cells. (A)
Proposed signaling pathways underlying RGDꢀSeD and Xꢀrayꢀmediated cancer cell killing. HepG2 cells were
treated with RGDꢀSeD (20 ꢁM) and/or Xꢀray (8 Gy). Cells were harvested, and cell lysate were subjected
Western blot to determine the activation of DNA damage pathway (B) and apoptosis pathway (C). (D) Caspaseꢀ3
and ꢀ9 activities of HepG2 cells treated with RGDꢀSeD (20 ꢁM) and/or Xꢀray (8 Gy) were determined by specific
fluorescent substrates. Bars with different characters are statistically significant (P < 0.05, Tukey’s test, oneꢀway
ANOVA).
In vivo radiosensitization activity of RGDꢀSeD
To further examine the in vivo radiosensitization activity of SeD and RGDꢀSeD, we tested their
tumor inhibition effects on nude mice bearing HepG2 xenograft. As shown in Figure 9A, treatment
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with Xꢀray alone significantly inhibited the growth of tumor. The mice receiving SeD or RGDꢀSeD
alone also showed substantial retardation of tumor growth, and better activity of RGDꢀSeD than
SeD can be observed. When combined SeD or RGDꢀSeD with Xꢀray treatment, the tumor growth
were further inhibited by factors of 1.60 and 2.11, respectively. Measurement of the tumor weights
shown similar results (Figure 9B and C). Treatment with SeD or RGDꢀSeD alone resulted in no
change of mice’s body weights, while Xꢀray treatment alone or in combination with SeD or
RGDꢀSeD caused considerable decrease of the mice’s body weights (Figure 9D), which might be
the results of the side effects of Xꢀray. No further loss of body weight in the mice of coꢀtreatment
groups comparing with Xꢀray group demonstrated no significant toxicity of SeD and RGDꢀSeD.
We next evaluated the apoptosis and proliferation status of cells in tumor tissues by
immunohistochemical staining of cleavedꢀcaspaseꢀ3 and Ki67. As shown in Figure 9E, lowest
expression of cleavedꢀcaspasꢀ3 was in control group, and the expression level increased in the
treatment groups, most strongly in RGDꢀSeD and Xꢀray combination group. Accordingly, the
control group showed highest expression of Ki67 and treatment with Xꢀray or SeD or RGDꢀSeD
resulted in decreased expression (Figure 9E).
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Finally, the side effects of SeD and RGDꢀSeD were evaluated by H&E staining of major organs
sections and examination of blood biochemical indexes. No obvious toxicity of SeD and RGDꢀSeD
to major organs was seen in H&E staining sections (Figure 10A). Blood biochemical indexes
analysis showed that RGDꢀSeD combined with Xꢀray most potently alleviated the
xenograftꢀinduced changes of blood glucose (GLU), blood urea nitrogen (BUN), creatine kinase
(CK), aspartate aminotransferase (AST) and highꢀdensity lipoprotein cholesterol (HDLꢀC) and
cholesterol (CHOL) (Figure 10B).
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Figure 9. In vivo radiosensitization effect of SeD and RGDꢀSeD. (A) Growth curves of tumors on mice receiving
the indicated treatments. (B, C) The weights (B) and image (C) of tumors dissected at the end of the experiment
from the mice in different treatment groups. Bars with different characters are statistically significant (P < 0.05,
Tukey’s test, oneꢀway ANOVA). (D) Body weight of the mice receiving the indicated treatments. (E)
Immunohistochemical analysis of the expressions of cleavedꢀcaspaseꢀ3 (CꢀCaspꢀ3) and Ki67 in tumor tissues of
mice. Scale bar represents 100 ꢁm.
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Figure 10. Histochemical and hematological analysis of in vivo radiosensitization effect and toxicity of SeD and
RGDꢀSeD. (A) H&E staining of sections from the mice’s major organs and tumor. The black arrows denoted the
sites of pulmonary hemorrhage in alveolus pulmonis. Scale bar represents 100 ꢁm. (B) Hematological analysis of
healthy (no xenograft) and treated nude mice. The biochemistry indexes included blood glucose (GLU), blood
urea nitrogen (BUN), highꢀdensity lipoprotein cholesterol (HDLꢀC), total cholesterol (CHOL), creatine kinase
(CK) and aspartate aminotransferase (AST). Bars with different characters are statistically significant (P < 0.05,
Tukey’s test, oneꢀway ANOVA).
DISCUSSION
In the past decade, promising anticancer activity had been found in Seꢀcontaining species, including
inorganic selenite and selenate, organic selenodiglutathione, selenoaminoacid derivatives,
methylseleninic acid, selenides and diselenides, selenocyanates, se containing heterocycles, as well
as selenium nanoparticles 12. Among these agents, we previously found that selenadiazole
derivatives had high anticancer activity with relative low toxicity, which trigged ROS
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overproduction and DNA damage to induce apoptosis in various cancer cell lines 17, 18, 20, 23ꢀ25
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Moreover, selenadiazole derivatives strongly sensitized cancer cells to radiotherapy by inhibition of
thioredoxin reductase (TrxR) 26. In order to find novel radiosensitizer, a selenadiazole derivative
SeD was synthesized in the present study. In vitro data showed high radiosensitization activity of
the newly synthesized selenadiazole derivative. However, like other traditional chemotherapy
agents, the selectivity of previous reported selenadiazole derivatives between cancer cells and
normal cells was limited.
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Decoration of cancer cellꢀtargeting groups is a useful strategy to improve the selectivity of both
small molecule drugs and nanoparticle delivery systems. RGD peptide, due to its selective binding
ability to ανβ3 integrin overexpressed in various types of cancer cells, has been considered to be an
ideal targeting molecule for modification of anticancer drugs and nanoparticle delivery systems 28.
RGD conjugates of several traditional chemotherapy drugs had been synthesized and evaluated,
such as paclitaxel 31., doxorubicin,32 platinum complexes 33 and camptothecin 34. Our previous study
also showed that surface decoration of nanoparticle with RGD peptide significantly increased their
selectivity between cancer and normal cells 35ꢀ37. Here, a c(RGDfk) group was conjugated to the
newly synthesized selenadiazole derivative, to improve its radiosensitization effect and lower its
toxicity in liver cancer, as we found that ανβ3 integrin was overexpressed in HepG2 cells but not in
L02 cells. After decoration, the radiosensitization effect of the conjugate was enhanced by increased
cellular uptake.
ROS is important in mediating the Xꢀrayꢀinduced cell death through damaging of DNA. Therefore,
anticancer agents which could induce ROS overproduction may synergize with Xꢀray to kill cancer
cells. Several of our previous reports had suggested that selenadiazole derivatives can induce ROS
overproduction in cancer cells 18, 19, 24, 26, which may be caused by the inhibition of ROSꢀeliminating
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enzyme, TrxR 26, 38. Consistent with this, RGDꢀSeD synthesized in the present study effectively
increased production of ROS in HepG2 cells, and showed strong synergism with Xꢀray. As a result
of ROS overproduction, the cell cycle of HepG2 cells were arrested in G2/M phase, and more DNA
damages were evidenced by increased γꢀH2A.X foci in the nuclei. Increased subꢀG1 fraction was
also observed in HepG2 cells treated with combination of RGDꢀSeD and Xꢀray, which implied the
apoptosis of HepG2 cells. DNA damage and apoptosis of HepG2 cells were further confirmed by
Western blot examining the phosphorylation of p53, ATM/ATR and cleavage of caspase. In
addition, mitochondrion fragmentation, which may happen in the early stage of apoptosis, was also
detected.
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Using HepG2 xenograft model we confirmed the in vivo antitumor activity of SeD and RGDꢀSeD,
which showed similar to in vitro results. The toxicities of SeD and RGDꢀSeD were also evaluated
by monitoring the mice body weight, blood biochemical indexes and histochemical analysis. SeD or
RGDꢀSeD showed no evident toxicity to the mice. However, Xꢀray treatment alone caused obvious
body weight loss of mice, which was consistent with the severe side effects of clinical radiotherapy.
But no further loss of mice body weight in the combination groups, suggesting that SeD or
RGDꢀSeD increased the therapeutic effects of Xꢀray without exaggerating its side effects.
CONCLUSIONS
In this study, cancer targeting RGD peptide has been covalently conjugated to selenadiazole
derivative to improve its cancer selectivity. The RGD decoration significantly enhances the
anticancer efficacy of RGDꢀSeD in αVβ3 integrinꢀoverexpressing HepG2 liver cancer cells, but not
in normal liver cells. Cellular uptake assay and fluorescent imaging confirmed the selectivity of
RGDꢀSeD to integrin overexpressing cancer cells. RGDꢀSeD strongly sensitizes HepG2 cells to
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Xꢀray radiotherapy through ROS overproduction, which triggers DNA damageꢀmediated apoptosis
and G2/M cell cycle arrest. This Xꢀray responsive DNA damage activates p53 signaling pathways
by phosphorylation of ATM/ATR and γꢀH2A.X. Furthermore, in HepG2 nude mice xenograft
model, RGDꢀSeD combined with Xꢀray demonstrates potent in vivo antitumor efficacy via
induction of apoptotic cell death, but shows no toxicity on the functions of major organs. Taken
together, this study demonstrates an effective way to improve the cancer selectivity of
seleniumꢀbased radiosensitizer for personalized cancer medicine.
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EXPERIMENTAL PROCEDURES
Synthesis and characterization
Synthesis of SeD: (1) (1,1’ꢀBiphenyl)ꢀ3,3’,4,4’ꢀtetraamine (30 mmol) was dissolved in 600 mL
hydrochloric acid solution (HCl/H2O=1:5) in a 1000 mL flask. Selenium dioxide (30 mmol) was
dissolved in 30 mL hot distilled water and transferred to constant pressure funnel, then the solution
was added drop wise to the flask. The mixture was stirred for 1.5 h at room temperature. The yellow
solid precipitated during the reaction were filtered and redissolved in water. The pH of the solution
was adjusted to about 7.0 with sodium hydroxide solution. The mixture was filtered again and
vacuum dried to afford 4ꢀ (benzo[c][1,2,5]selenadiazolꢀ5ꢀyl)benzeneꢀ1,2ꢀdiamine in a yield of 95%.
(2) The product of step (1) was dissolved (3 mmol) in 30 mL DMF in a 100 mL beaker. NꢀAcetic
acidꢀindoleꢀ3ꢀcarboxaldehyde was added with a catalytic amount of pꢀmethylbenzene sulfonic acid.
The mixture was stirred at 80 °C for 1 h. After cooling, the mixture was added into 200 mL
saturated Na2CO3 solution and stirred for 10 min at room temperature. The mixture was filtered and
the filtrate was transferred in to 500 mL lithium chloride (2 M) solution. A large amount of yellow
solid precipitated and the precipitate was filtered and vacuum dried to give crude product, which
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was then purified by column chromatography on silica gel (petroleum ether/EtOAc=5:1) to obtain
pure product [3ꢀ(5ꢀBenzo[1,2,5]selenadiazolꢀ5ꢀylꢀ1Hꢀbenzoimidazolꢀ2ꢀyl)ꢀindolꢀ1ꢀyl]ꢀacetic acid
(SeD); Yield: 60%.
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Synthesis of RGDꢀSeD: SeD (1 mmol) was dissolved in DMF then EDC and NHS (1.2 mmol each)
were added to activate the compound. After 2 h, 1 mmlo of c(RGDfK) (Gier biochemistry CO., Ltd,
Shanghai, China) and a catalytic amount of riethylamine were added and the mixture was stirred for
another 24 h. When the reaction completed, the mixture was centrifuged at 6000 rpm to obtain
supernatant, which were then added to diethyl ether to precipitate the produce. After filtration, the
cure produce was washed twice by diethyl ether then vacuum dried. The produce was further
purified by HPLC using acetonitrile ꢀ water as eluent. After freeze drying, the pure product
(RGDꢀSeD) was obtained; Yield: 80%.
SeD: Yellow solid (60%); elemental analysis calcd for C23H15N5O2Se (%): C, 58.48; H, 3.20; N,
14.83; found (%): C, 58.80; H, 3.28; N, 14.72; ESIꢀMS (in CH3OH): m/z calcd for C23H15N5O2Se
(M+H)+473.9; found 474.9.
RGDꢀSeD: Yellow solid (80%); elemental analysis calcd for C50H54N14O8Se (%): C, 56.76; H,
5.14; N, 18.53; found (%): C, 56.80; H, 5.28; N, 18.12; ESIꢀMS (in CH3OH): m/z calcd for
C50H54N14O8Se (M+H)+ 1059.9; found 1059.9.
Cell culture
HepG2 human liver cancer cell line was purchased from the American Type Culture Collection
(ATCC, Manassas, Virginia). L02 human normal liver cell line was obtained from Cell Source
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Center, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences
(Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum (FBS), penicillin (100 UmLꢀ1), and streptomycin (50 U
mLꢀ1) at 37 °C in a humidified incubator with 5% CO2 and 95% air. All the cell lines were tested
for mycoplasma by PCR and fluorescent DNA staining before used.
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Cell viability assay
Cells were plated in 96ꢀwell plates in a density of 2000 cells/well (HepG2) or 4000 cells/well (L02)
and cell viabilities after treatment with various concentrations of compounds were evaluated by
MTT assay 39, 40
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Determine of cellular uptake of compounds
Quantitative analysis of cellular uptake of SeD and RGDꢀSeD was determined by ICPꢀMS as
previously described 27, 41
.
Clonogenic assays
Colony formation ability of HepG2 cells were measured by clonogenic assay as previously
described 26, 42
.
Live cell imaging
Localization of RGDꢀSeD in HepG2 cells and fragmentation of mitochondrion induced by
RGDꢀSeD and Xꢀray were visualized by EVOS® FL Auto Imaging System (Life technologies).
Labeling of nucleus, lysosome and mitochondrion were done by incubation of DAPI (300 nM),
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LysoTracker® Red (50 nM) and MitoTrakcer® Red CMXRos (300 nM), respectively, according to
the manufactory’s protocol. The probes were all from Molecular Probes, Life technologies.
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Cell cycle analysis
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Cell cycle distribution of HepG2 cells were detected by flow cytometry after propidium iodide (PI)
staining as previously described 43ꢀ45
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Immunofluorescent analysis of γH2AX
HepG2 cells were plated on 2ꢀcm glass dishes and treated RGDꢀSeD (20 ꢁM) for 6 h then were
exposed to Xꢀray (8 Gy). After 2 h, the cells were fixed with 4% paraformaldehyde for 20 min at
room temperature, permeabilized with 1% Triton Xꢀ100 and 0.5% NPꢀ40 for 20 min at room
temperature. After washed by PBS for 3 times and blocked by 2% BSA for 1 h at room temperature,
cells were incubated with primary antibody against phosphoꢀHistone H2A.X (Ser139) (1:500, Cell
Signaling Technology) at 4 °C overnight. Cells were then washed 3 times with PBS and incubated
with Alexa fluorꢀ488 conjugated secondary antibody (1:500, Cell Signaling Technology) for 1 h at
room temperature. The cells were then washed with PBS again and immediately imaged by
EVOS® FL Auto Imaging System (Life technologies).
Measurement of intracellular reactive oxygen species (ROS)
The intracellular ROS level of HepG2 cells was measured by using DHE staining 46, 47
.
Western blotting
Expression level or phosphorylated form of proteins related to DNA damage and apoptosis were
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determined by Western blot analysis 48, 49. Rabbit antiꢀhuman phosphoꢀHistone H2A.X (Ser139)
antibody (#2577), rabbit antiꢀhuman Integrin β3 antibody (#13166), rabbit antiꢀHuman p53
antibody(#2527), mouse antiꢀhuman phosphoꢀp53 (Ser15) antibody (#9286), rabbit antiꢀhuman
phosphoꢀATM (Ser1981) antibody (#5883), rabbit antiꢀhuman phosphoꢀATR (Ser428) antibody
(#2853), rabbit antiꢀhuman PARP antibody (#9532), rabbit antiꢀhuman caspaseꢀ8 antibody (#4790),
rabbit antiꢀhuman caspaseꢀ9 antibody (#9520), rabbit antiꢀhuman caspaseꢀ3 antibody (#9662),
HRPꢀlinked horse antiꢀmouse IgG antibody (#7076) and HRPꢀlinked goat antiꢀrabbit IgG antibody
(#7074) were purchased from Cell Signaling Technology, Inc. Mouse antiꢀhuman βꢀActin (A1978)
antibody was from SigmaꢀAldrich.
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Caspase activity assay
Measurement of caspaseꢀ9 and ꢀ3 activities were conducted as previously described by using
specific substrates 44.
Nude mice xenograft model
All nude mice experiments were approved by the Laboratory Animal Ethics Committee of Jinan
University. Female Balb/c nude mice (3ꢀ4 weeks old and weighing 14–15 g) were purchased from
Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). The animals were
acclimated to the environment for 10 days before inoculation of cancer cells. Each mouse was
injected subcutaneously with 1.5 × 106 HepG2 cells in 100 ꢁL PBS. When the tumor volume
reached ~100 mm3, the mice were randomly divided into 6 groups, receiving Xꢀray, SeD,
RGDꢀSeD, Xꢀray + SeD and Xꢀray + RGDꢀSeD treatment, and saline as control group. Every other
day, the body weight and tumor volume (V = l × w2/2, l = tumor length, w = tumor width) of the
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mice were measured, and the compounds (SeD or RGDꢀSeD, 2 mg/kg) and saline were
administrated through intravenous injection at the same time. The mice that needed to be irradiated
were then exposed to Xꢀray (4 Gy). After 21 days, the mice were sacrificed to collect the blood,
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organs, and tumors for biochemical and immunohistochemical analyses 50ꢀ52
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Hematological and histological analyses
The blood samples of mice were centrifuged at 1500 rpm for 10 min, and the serum were obtained
and sent to the Blood Test Center of the First Affiliated Hospital of Jinan University for
hematological analysis. The tumor, heart, liver, spleen, lung and kidney were fixed in 4%
paraformaldehyde, embedded in paraffin and sectioned for hematoxylin and eosin (H&E) staining
and immunohistochemical analysis.
Statistical analysis
All experiments were carried out in triplicate and the data were expressed as mean ± standard
deviation (SD). Statistical analysis was carried out in GraphPad Prism 5 software (GraphPad
Software Inc.). Differences between two groups were analyzed by the twoꢀtailed Student’s t test.
Oneꢀway analysis of variance was used in multiple group comparisons. P < 0.05 was considered to
be significant differences between groups.
CONFLICTS OF INTEREST
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
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This work was supported by National Program for Support of Topꢀnotch Young Professionals
(W02070191), Yang Fan Innovative & Entepreneurial Research Team Project (201312H05),
Fundamental Research Funds for the Central Universities and China Postdoctoral Science
Foundation (20182018M633274).
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SUPPORTING INFORMATION
The Supporting Information is available free of charge on the ACS Publications website at DOI:
Detailed methods and ESIꢀMS, HPLC and UV/vis absorption spectra of SeD and RGDꢀSeD.
ABBREVIATIONS
RGD, Arginylglycylaspartic acid; ROS, reactive oxygen species; DHE, dihydroethidium; DAPI,
4',6ꢀdiamidinoꢀ2ꢀphenylindole; GLU, blood glucose; BUN, blood urea nitrogen; CK, creatine
kinase; AST, aspartate aminotransferase; HDLꢀC, highꢀdensity lipoprotein cholesterol; CHOL,
cholesterol.
REFERENCES
(1)
Begg, A. C., Stewart, F. A., and Vens, C. (2011) Strategies to improve radiotherapy with targeted drugs. Nat.
Rev. Cancer. 11, 239ꢀ253.
(2)
Delaney, G., Jacob, S., Featherstone, C., and Barton, M. (2005) The role of radiotherapy in cancer treatment:
estimating optimal utilization from a review of evidenceꢀbased clinical guidelines. Cancer 104, 1129ꢀ1137.
(3)
exposure. Lancet Oncol. 6, 520ꢀ528.
(4) Kim, B. M., Hong, Y., Lee, S., Liu, P., Lim, J. H., Lee, Y. H., Lee, T. H., Chang, K. T., and Hong, Y. (2015)
Therapeutic Implications for Overcoming Radiation Resistance in Cancer Therapy. Int. J. Mol. Sci. 16, 26880ꢀ26913.
(5) Seiwert, T. Y., Salama, J. K., and Vokes, E. E. (2007) The concurrent chemoradiation paradigmꢀꢀgeneral
principles. Nat. Clin. Pract. Oncol. 4, 86ꢀ100.
(6) Bonner, J. A., Harari, P. M., Giralt, J., Azarnia, N., Shin, D. M., Cohen, R. B., Jones, C. U., Sur, R., Raben, D.,
Prise, K. M., Schettino, G., Folkard, M., and Held, K. D. (2005) New insights on cell death from radiation
Jassem, J., et al. (2006) Radiotherapy plus cetuximab for squamousꢀcell carcinoma of the head and neck. N. Engl. J.
Med. 354, 567ꢀ578.
(7)
Chinnaiyan, P., Huang, S., Vallabhaneni, G., Armstrong, E., Varambally, S., Tomlins, S. A., Chinnaiyan, A. M.,
27
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 28 of 31
1
2
and Harari, P. M. (2005) Mechanisms of enhanced radiation response following epidermal growth factor receptor
signaling inhibition by erlotinib (Tarceva). Cancer. Res. 65, 3328ꢀ3335.
3
4
5
6
7
(8)
cancer treatment: rationale, mechanisms of action and clinical perspective. Drug Resist. Updat. 13, 29ꢀ43.
(9) Oronsky, B. T., Knox, S. J., and Scicinski, J. (2011) Six degrees of separation: the oxygen effect in the
Verheij, M., Vens, C., and van Triest, B. (2010) Novel therapeutics in combination with radiotherapy to improve
development of radiosensitizers. Transl. Oncol. 4, 189ꢀ198.
8
9
(10) Duntas, L. H., and Benvenga, S. (2015) Selenium: an element for life. Endocrine 48, 756ꢀ775.
(11) Rayman, M. P. (2012) Selenium and human health. The Lancet 379, 1256ꢀ1268.
(12) Fernandes, A. P., and Gandin, V. (2015) Selenium compounds as therapeutic agents in cancer. Biochim. Biophys.
Acta 1850, 1642ꢀ1660.
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
60
(13) Lu, J., Zhang, J., Jiang, C., Deng, Y., Ozten, N., and Bosland, M. C. (2016) Cancer chemoprevention research
with selenium in the postꢀSELECT era: Promises and challenges. Nutr. Cancer 68, 1ꢀ17.
(14) Clark, L. C., Combs, G. F., Jr., Turnbull, B. W., Slate, E. H., Chalker, D. K., Chow, J., Davis, L. S., Glover, R.
A., Graham, G. F., Gross, E. G., et al. (1996) Effects of selenium supplementation for cancer prevention in patients with
carcinoma of the skin. A randomized controlled trial. Nutritional Prevention of Cancer Study Group. J. Am. Med. Assoc.
276, 1957ꢀ1963.
(15) Wallenberg, M., Misra, S., Wasik, A. M., Marzano, C., Bjornstedt, M., Gandin, V., and Fernandes, A. P. (2014)
Selenium induces a multiꢀtargeted cell death process in addition to ROS formation. J. Cell Mol. Med. 18, 671ꢀ684.
(16) Evans, S. O., Khairuddin, P. F., and Jameson, M. B. (2017) Optimising Selenium for Modulation of Cancer
Treatments. Anticancer Res. 37, 6497ꢀ6509.
(17) Deng, S., Zeng, D., Luo, Y., Zhao, J., Li, X., Zhao, Z., and Chen, T. (2017) Enhancement of cell uptake and
antitumor activity of selenadiazole derivatives through interaction and delivery by serum albumin. RSC Adv. 7,
16721ꢀ16729.
(18) Liang, Y., Zhou, Y., Deng, S., and Chen, T. (2016) MicrowaveꢀAssisted Syntheses of BenzimidazoleꢀContaining
Selenadiazole Derivatives That Induce CellꢀCycle Arrest and Apoptosis in Human Breast Cancer Cells by Activation of
the ROS/AKT Pathway. ChemMedChem 11, 2339ꢀ2346.
(19) Zhou, M., Ji, S., Wu, Z., Li, Y., Zheng, W., Zhou, H., and Chen, T. (2015) Synthesis of selenazolopyridine
derivatives with capability to induce apoptosis in human breast carcinoma MCFꢀ7 cells through scavenge of
intracellular ROS. Eur. J. Med. Chem. 96, 92ꢀ97.
(20) Yang, L., Li, W., Huang, Y., Zhou, Y., and Chen, T. (2015) Rational design of cancerꢀtargeted
benzoselenadiazole by RGD peptide functionalization for cancer theranostics. Macromol. Rapid Commun. 36,
1559ꢀ1565.
(21) Xie, L., Luo, Z., Zhao, Z., and Chen, T. (2017) Anticancer and Antiangiogenic Iron(II) Complexes That Target
Thioredoxin Reductase to Trigger Cancer Cell Apoptosis. J. Med. Chem. 60, 202ꢀ214.
(22) Lai, H., Zhang, X., Feng, P., Xie, L., Chen, J., and Chen, T. (2017) Enhancement of Antiangiogenic Efficacy of
Iron(II) Complex by Selenium Substitution. Chem. Asian. J. 12, 982ꢀ987.
(23) Yang, Y., Deng, S., Zeng, Q., Hu, W., and Chen, T. (2016) Highly stable selenadiazole derivatives induce
bladder cancer cell apoptosis and inhibit cell migration and invasion through the activation of ROSꢀmediated signaling
pathways. Dalton T. 45, 18465ꢀ18475.
(24) Zhao, J., Zeng, D., Liu, Y., Luo, Y., Ji, S., Li, X., and Chen, T. (2017) Selenadiazole derivatives antagonize
hyperglycemiaꢀinduced drug resistance in breast cancer cells by activation of AMPK pathways. Metallomics 9,
535ꢀ545.
(25) Liu, Y., Luo, Y., Li, X., Zheng, W., and Chen, T. (2015) Rational design of selenadiazole derivatives to
antagonize hyperglycemiaꢀinduced drug resistance in cancer cells. Chem. Asian. J. 10, 642ꢀ652.
(26) He, L., Ji, S., Lai, H., and Chen, T. (2015) Selenadiazole derivatives as theranostic agents for simultaneous
28
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Page 29 of 31
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
cancer chemoꢀ/radiotherapy by targeting thioredoxin reductase. J. Mater. Chem. B 3, 8383ꢀ8393.
(27) Deng, Z., Yu, L., Cao, W., Zheng, W., and Chen, T. (2015) A seleniumꢀcontaining ruthenium complex as a
cancer radiosensitizer, rational design and the important role of ROSꢀmediated signalling. Chem. Commun. 51,
2637ꢀ2640.
(28) Katsamakas, S., Chatzisideri, T., Thysiadis, S., and Sarli, V. (2017) RGDꢀmediated delivery of smallꢀmolecule
drugs. Future Med. Chem. 9, 579ꢀ604.
9
(29) Baskar, R., Dai, J., Wenlong, N., Yeo, R., and Yeoh, K. W. (2014) Biological response of cancer cells to
radiation treatment. Front. Mol. Biosci. 1, 1ꢀ9.
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
60
(30) Rossetto, D., Avvakumov, N., and Cote, J. (2012) Histone phosphorylation: a chromatin modification involved
in diverse nuclear events. Epigenetics 7, 1098ꢀ1108.
(31) Bianchi, A., Arosio, D., Perego, P., De Cesare, M., Carenini, N., Zaffaroni, N., De Matteo, M., and Manzoni, L.
(2015) Design, synthesis and biological evaluation of novel dimeric and tetrameric cRGDꢀpaclitaxel conjugates for
integrinꢀassisted drug delivery. Org. Biomol. Chem. 13, 7530ꢀ7541.
(32) Ryppa, C., MannꢀSteinberg, H., Fichtner, I., Weber, H., SatchiꢀFainaro, R., Biniossek, M. L., and Kratz, F. (2008)
In vitro and in vivo evaluation of doxorubicin conjugates with the divalent peptide Eꢀ[c(RGDfK)2] that targets integrin
alphavbeta3. Bioconjug. Chem. 19, 1414ꢀ1422.
(33) Massaguer, A., GonzalezꢀCanto, A., Escribano, E., Barrabes, S., Artigas, G., Moreno, V., and Marchan, V. (2015)
Integrinꢀtargeted delivery into cancer cells of a Pt(IV) proꢀdrug through conjugation to RGDꢀcontaining peptides.
Dalton T. 44, 202ꢀ212.
(34) Dal Pozzo, A., Esposito, E., Ni, M., Muzi, L., Pisano, C., Bucci, F., Vesci, L., Castorina, M., and Penco, S.
(2010) Conjugates of a novel 7ꢀsubstituted camptothecin with RGDꢀpeptides as alpha(v)beta(3) integrin ligands: An
approach to tumorꢀtargeted therapy. Bioconjug. Chem. 21, 1956ꢀ1967.
(35) He, L., Chen, T., You, Y., Hu, H., Zheng, W., Kwong, W. L., Zou, T., and Che, C. M. (2014) A cancerꢀtargeted
nanosystem for delivery of gold(III) complexes: enhanced selectivity and apoptosisꢀinducing efficacy of a gold(III)
porphyrin complex. Angew. Chem. Int. Ed. 53, 12532ꢀ12536.
(36) He, L., Huang, Y., Zhu, H., Pang, G., Zheng, W., Wong, Y. S., and Chen, T. (2014) Cancerꢀtargeted
monodisperse mesoporous silica nanoparticles as carrier of ruthenium polypyridyl complexes to enhance theranostic
effects. Adv. Funct. Mater. 24, 2754ꢀ2763.
(37) Fu, X., Yang, Y., Li, X., Lai, H., Huang, Y., He, L., Zheng, W., and Chen, T. (2016) RGD peptideꢀconjugated
selenium nanoparticles: antiangiogenesis by suppressing VEGFꢀVEGFR2ꢀERK/AKT pathway. Nanomed.-Nanotechnol.
12, 1627ꢀ1639.
(38) Liang, Y. W., Zheng, J., Li, X., Zheng, W., and Chen, T. (2014) Selenadiazole derivatives as potent thioredoxin
reductase inhibitors that enhance the radiosensitivity of cancer cells. Eur. J. Med. Chem. 84, 335ꢀ342.
(39) Song, Z. H., Liu, T., and Chen, T. F. (2018) Overcoming bloodꢀbrain barrier by HER2ꢀtargeted nanosystem to
suppress glioblastoma cell migration, invasion and tumor growth. J. Mater. Chem. B 6, 568ꢀ579.
(40) He, L., Zeng, L., Mai, X., Shi, C., Luo, L., and Chen, T. (2017) Nucleolinꢀtargeted selenium nanocomposites
with enhanced theranostic efficacy to antagonize glioblastoma. J. Mater. Chem. B 5, 3024ꢀ3034.
(41) Deng, Z., Yu, L., Cao, W., Zheng, W., and Chen, T. (2015) Rational design of ruthenium complexes containing
2,6ꢀbis(benzimidazolyl)pyridine derivatives with radiosensitization activity by enhancing p53 activation.
ChemMedChem 10, 991ꢀ998.
(42) He, L., Lai, H., and Chen, T. (2015) Dualꢀfunction nanosystem for synergetic cancer chemoꢀ/radiotherapy
through ROSꢀmediated signaling pathways. Biomaterials 51, 30ꢀ42.
(43) Ma, B., He, L., You, Y., Mo, J., and Chen, T. (2018) Controlled synthesis and size effects of multifunctional
mesoporous silica nanosystem for precise cancer therapy. Drug Deliv. 25, 293ꢀ306.
(44) You, Y., He, L., Ma, B., and Chen, T. (2017) HighꢀDrugꢀLoading Mesoporous Silica Nanorods with Reduced
29
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