Exploring the Natural Piericidins as Anti-Renal Cell Carcinoma Agents Targeting Peroxiredoxin 1

Article abstract of DOI:10.1021/acs.jmedchem.9b00598

Anti-renal cell carcinoma (RCC) agents with new mechanisms of action are urgently needed. Twenty-seven natural products of the piericidin class, including 17 new ones, are obtained from a marine-derived Streptomyces strain, and several of them show strong

Full text of DOI:10.1021/acs.jmedchem.9b00598

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Article
Exploring the Natural Piericidins as Anti-Renal
Cell Carcinoma Agents Targeting Peroxiredoxin 1
Xuefeng Zhou, Zhi Liang, Kunlong Li, Wei Fang, Yuanxin Tian, Xiaowei Luo, Yulian Chen, Zhikun
Zhan, Tao Zhang, Shengrong Liao, Shuwen Liu, Yonghong Liu, William Fenical, and Lan Tang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00598 • Publication Date (Web): 12 Jul 2019
Downloaded from pubs.acs.org on July 12, 2019
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Exploring the Natural Piericidins as Anti-Renal Cell
Carcinoma Agents Targeting Peroxiredoxin 1
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Xuefeng Zhou,*,†,# Zhi Liang, Kunlong Li, Wei Fang, Yuanxin Tian , Xiaowei Luo, Yulian
‡,#
†
§
‡
†
‡
‡
‡
†
‡,⊥
†
Chen, Zhikun Zhan, Tao Zhang, Shengrong Liao, Shuwen Liu, Yonghong Liu, William
║
,‡,⊥
Fenical, and Lan Tang*
†CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key
Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese
Academy of Sciences, Guangzhou 510301, China
‡Biopharmaceutics, Guangdong Provincial Key Laboratory of New Drug Screening, School of
Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China
§Hubei Biopesticide Engineering Research Center, Hubei Academy of Agricultural Science,
Wuhan 430064, China
⊥State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou
5
10515, China
║Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography,
University of California at San Diego, La Jolla, CA 92093, USA
KEYWORDS: Piericidins; renal cell carcinoma; ACHN; peroxiredoxin 1
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ABSTRACT
Anti-renal cell carcinoma (RCC) agents with new mechanisms of action are urgently needed.
Twenty seven natural products of the piericidin class, including seventeen new ones, are
obtained from a marine-derived Streptomyces strain, and several of them show strong inhibitory
activities against ACHN renal carcinoma cells. By exploring the mechanisms of two
representative natural piericidin compounds, piericidin A (PA) and glucopiericidin A (GPA),
peroxiredoxin 1 (PRDX1) is detected as a potential target by transcriptome data of PA treated
ACHN cells, as well as the paired RCC tumor vs adjacent non-tumor tissues. PA and GPA
induce cell apoptosis through reducing ROS level caused by up-regulated PRDX1 mRNA and
protein level subsequently, and exhibit potent anti-tumor efficacy in nude mice bearing ACHN
xenografts, with increasing PRDX1 expression in tumor. The interaction between PA/GPA and
PRDX1 was supported by the docking analysis and SPR. Moreover, the translocation of PRDX1
into the nucleus forced by PA/GPA is proposed to be a key factor for the anti-RCC procedure.
Piericidins provide a novel scaffold for further development of potent anti-RCC agents, and the
new action mechanism of these agents targeting PRDX1, may improve upon the limitations of
existing targeted drugs for the treatment of renal cancer.
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1
. Introduction
Renal cell carcinoma (RCC) is the most common form of renal cancer, whose incidence rates
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have been gradually increasing by 2–4% every year over the past few decades. Chemotherapy
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and radiotherapy are not as successful in the case of RCC, although significant advances have
been derived from several target drugs approved by the FDA, such as sorafenib, sunitinib, and
2
pazopanib as tyrosine kinase inhibitors. In order to curb drug resistance, unwanted side effects,
and further improve efficacy, the discovery of more effective anti-RCC agents, especially those
3
with novel mechanisms of action, is warranted. The revolutionary technology employed in
medicine to interrogate human cancer is next-generation sequencing, which enabled a hitherto
4
unknown insight into the molecular and cellular machineries in RCC. Further studies to dissect
out the role of the mutated genes or key biomarkers in RCC tumorigenesis revealed by different
genomic platforms will hopefully provide the foundation for the development of effective forms
of therapy for this disease. 5
Microorganisms remain unrivalled in their potential to produce highly potent natural
products for drug development, and the actinomycetes are well-known as an inexhaustible source
for new chemical structures. The piericidin class metabolites, that feature a 4-pyridinol core
linked with a variable polyene side chain, are commonly produced by actinomycetes isolated
6
from soil, insects and marine samples. To date, there are about 40 natural piericidin derivatives
reported, including 11 piericidin glycosides. 6-8 Piericidin A (PA) is known as a potent inhibitor
of NADH−ubiquinone oxidoreductase (complex I). In addition to broad antimicrobial and
6
insecticidal activities, some piericidins are reported to have potential as antitumour agents. The
chemical diversity of the natural piericidins has not been reached, thus the pharmaceutical
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significance of this class will require further refinement and examination, especially regarding
their anticancer mechanisms of action.
Described herein is the isolation and structure determination of 27 natural piericidins from a
Streptomyces strain, with inhibitory activities against RCC ACHN cells. Driven by
transcriptomic analysis, a new action mechanism for the piericidins, targeting the anti-oxidative
protein peroxiredoxin 1 (PRDX1), has been observed and confirmed. Anti-RCC evaluation in
vitro and in vivo confirmed that the piericidins behave as potent anti-RCC agents.
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. Results
.1. Natural piericidins and their activities against renal cancer cells
In order to explore natural piericidins compounds, chemical dereplication by HPLC/MS was
performed to screen the strains with piericidins in marine Streptomyces strains in our lab. The
HPLC/MS analysis uncovered abundant and divers piericidin glycosides in the strain
Streptomyces psammoticus SCSIO NS126 (Supporting information, Figure S1), an actinomycete
strain isolated from a mangrove sediment sample collected from the Pearl River estuary to South
China Sea. Chemical study of the culture (60 L for liquid fermentation) extract of strain SCSIO
NS126 provided 27 natural piericidins (Figure 1).
Among the natural piericidins obtained, seventeen (2–5, 12, 13, 15, 17–26) were identified
as new natural products, by extensive NMR and HRMS analyses, including their configurations
as defined by NOESY data, coupling constant analysis, and ECD comparisons, along with their
biosynthesis taken into consideration.
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OH
9'
OH
O 3'
O
8
'
OH
5'
O
O
4
OH
10
7
13
7'
N 6'
1'
1
N
1
7
15
14
16
1
2
OH
OH
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O
OH
O
O
OH
OH
O
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O
N
1
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N
20
5
OH
3 13S,19R 4 13R,19S
OH
O
O
O
O
OH
OH
O
N
N
R
R1 R2
R = R = CH 9 R = R = H
6 R = CH3 7 R = H
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1
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OR4
OH
O
O
OR3
N
O
O
HO
OH
HO
HO
O
O
HO
OH
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O
OH
O
R1
R
N
R2
OH
10 R = H, R = CH , R = R = H
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21 R = H 22 R = OH
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OH
13 R = OH, R = H, R = R = H
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O
OH
N
14 R = H, R = CH , R = H, R = -D-Gal
O
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HO
HO
15 R = OH, R = CH , R = H, R = -D-Gal
O
O
1
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4
O
OH
16 R = H, R = CH , R = H, R = -D-Glu
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2
7 R = OH, R = CH , R = H, R = -D-Glu
1 2 3 3 4
8 R = H, R = CH , R = -D-Glu, R = H
1
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3
3
4
9 R = OH, R = CH , R = -D-Glu, R = H
23
1
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3
4
OH
0 R = H, R = CH , R = -D-Glu, R = -D-Gal
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4
O
OH
OH
OH
OH
OH
HO
HO
O
O
O
O
O
O
OH
R
HO
HO
HO
HO
HO
OH
OH
OH
N

-D-Gal

-D-Glu
-D-Glu
O
O
24 R =
25 R =
OH
OH
OH
O
OH
N
O
O
O
O
HO
HO
O
HO
HO
OH
OH
O
N
O
O
26
27
Figure 1. Structures of the piericidins obtained from the Streptomyces strain SCSIO NS126.
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Comparison of the NMR date of 2 with those of piericidin A (1) indicated that the only
difference was the absence of the 14-methyl group in 2, which was confirmed by the HMBC and
COSY correlations. Compound 2 was identified as 11-demethyl-piericidin A (2). Two hydroxyl
groups on C-13 and C-19 were confirmed in the structures of 3 and 4, and their absolute
configurations were determined by calculated and experimental ECD curves (Figure S9).
Compound 5 was identified as the same structure and same absolute configuration (9R) as
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synthetic C-10 ketone piericidin A, and obtained as a natural product firstly here. Compound 12
was suggested to be a glycoside of 7-demethylpiericidin A1 (9), and 10-O-β-D-glucoside linkage
was determined by the NMR analysis, as well as the acid hydrolysis. Compound 13 was
characterized as 7-demethyl-13-hydroxyglucopiericidin A (13). Compounds 14-19 were showed
to be diglycosides of piericidin. Compounds 14 and 16 were characterized as piericidin A 10-O-
α-D-galactose (1→6)-β-D-glucoside (14) and piericidin A 10-O-α-D-glucose (1→6)-β-D-
glucoside (16), respectively, by acid hydrolysis and NMR analysis. The additional β-D-glucose
group of 18 was linked on OH-4 of the pyridine ring, so the structure was characterized as 4-O-
β-D-glucose glucopiericidin A (18). Compounds 15, 17 and 19 were showed to be 13-hydroxy
derivatives of 14, 16 and 18, respectively. Compound 20 was indicated as a triglycoside of
piericidin by HR-ESIMS and NMR date, and characterized as 4-O-β-D-glucose piericidin A 10-
O-α-D-glucose (1→6)-β-D-glucoside (20). In the structure of 21, C-5/C-6 double bond was
replaced by 5-hydroxy-6-hydroxymethyl group, with their configurations determined by
coupling constant and NOESY correlation. Compound 22, the 13-hydroxy derivatives of 21, was
characterized as 5-hydroxy-6-hydroxymethyl-13-hydroxyglucopiericidin A (22). The
conventional C-2/C-3 double bond was moved to C-3/C-4 in glycoside 23, and an additional
hydroxymethyl was linked on C-2. Compound 23 was characterized as 2-hydroxymethyl-Δ3, 4-
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glucopiericidin A (23), with the absolute configuration of C-2 remained to be defined.
Glycosides 24 and 25 have the same planer structure, including the C-11/C-12 epoxy ring. The
configurations of the C-11/C-12 in 24 and 25 were determined to be 11S,12R and 11R,12S,
respectively, by NOESY and ECD analysis. The chemical shifts of CH-13 and HR-ESIMS data
suggested two methoxy groups linked on C-13 in 26, which were supported by HMBC
correlations. So, 26 was characterized as 13-dimethoxy glucopiericidin A (26). The other
reported compounds were identified by comparison of their spectroscopic data with those in the
literature. The details of the structure elucidation were supported in the Supporting
information.
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From the genome sequence of this actinomycete strain, we identified the biosynthetic gene
cluster of PA (1), which displays the same genetic organization as that in Streptomyces
piomogeues (Table S7), 10,11 together with several glycosyltransferases. Diverse oxygenated
piericidin derivatives, such as the seven 13-hydroxy piericidin glycosides and several derivatives
with 5,6-dihydroxy, 13,19-dihydroxy or those with the C-11/C-12 epoxide ring, suggest that
there are additional oxygenases taking part in the biosynthesis of these piericidin derivatives. The
1
3,19-dihydroxy functionality (in 3 and 4), 5,6-dihydroxy functionality (in 21 and 22), and also
the 2-hydroxymethyl group (in 23) are proposed to be formed by epoxidation and ring opening,
the latter of which is followed by dehydration. Eighteen glycosides (10–27), composed of eleven
monoglycosides, six diglycosides and one triglycoside, expand the diversity of natural piericidin
glycosides. Diglycosides of piericidin have been rarely reported, and the triglycoside (20) here is
the first piericidin triglycoside reported from nature.
Most of the piericidins were evaluated for their cytotoxicities against three renal carcinoma
cell lines, ACHN, OS-RC-2, and 786-O, as well as a normal renal cell line, HK-2 (Table 1).
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Most of the piericidins showed strong to moderate cytotoxicities toward ACHN, and several
analogs, such as PA (1), glucopiericidin A (GPA, 10) and 12, showed significant activities with
IC values less than 1 μM (Table 1). OS-RC-2, 786-O, and HK-2 cells were not as sensitive as
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ACHN to most of the piericidins.
Table 1. The cytotoxicity of the piericidins (IC , μM).
5
0
shPRDX1
ACHN
1.6
ACHN
OS-RC-2 786-O
HK-2
>100
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2
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1
1
1
1
1
0.40
4.1
5.2
30
11
>100
>100
>100
>100
>100
50
–
11
8.2
13
>100
>100
68
0.96
–
2.4
5.3
44
3.8
4.1
63
>100
>100
14
22
–
22
>100
45
17
0
1
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3
4
0.21
7.21
0.31
2.5
>100
>100
2.6
>100
>100
0.99
28
>100
>100
–
–
79
–
–
4.5
>100
>100
>100
>100
>100
>100
>100
60
>100
>100
>100
>100
>100
>100
>100
28
>100
>100
>100
>100
1.1
–
9.0
–
15
>100
21
1
1
1
6
7
8
–
23
58
–
28
21
2.4
–
2
2
2
3
60
–
–
1.7
–
–
24
2.8
30
19
–
–
2
7
3.8
14
15
–
–
Sora 3.3
14
13
2.6
–
Sora: Sorafenib; – Unrecorded.
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.2. PA/GPA up-regulates PRDX1 that is lower in ccRCC
To investigate the underlying molecular mechanisms of PA (1) on ACHN cells, we analyzed
transcriptome alterations using high-depth next generation sequencing with an Illumina HiSeq
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000. In total, more than 291 million stranded 150 basepairs (bp) paired-end reads were
sequenced from 3 groups of ACHN cells treated with two doses of PA (25 and 50 nM) for 24
hours along with untreated cells. All mRNA sequencing data has been submitted to the GEO
repository (GSE116158) (Table S1). Next, DEseq was used to identify the statistically
significant differentially expressed genes (DEGs) for the pairwise comparisons between these
three groups respectively. Among 17,847 detected unigenes, 6035 genes (2862 up regulated and
3173 down regulated) had significantly altered expression in cells treated with low dose PA,
while more DEGs (8,889 genes total, 4305 up regulated and 4584 down regulated) were
identified in high dose PA treatment (Figure 2A, Table S2). Given the significant effect of high
dose PA treatment, an intersection of DEGs in two experimental groups was performed to locate
the key genes affected. Then, KEGG pathway enrichment analysis, according to 1066 DEGs
from the intersection of three groups (Table S3), was employed to identify the biological
characterization of the significantly regulated genes. The results showed that the regulated genes
were involved in endoplasmic reticulum, pathways in cancer, HIF-1 signalling pathway, and
renal carcinoma pathways (Figure 2C, Table S4). Then supervised weighted correlation network
analysis (sWGCNA) was employed to define hub genes and the relationship among the selected
DEGs from those pathways (Figure S4).
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Figure 2. Transcriptome analysis of gene expression profile in ACHN cells treated with PA. A) Volcano
Plot: showed numbers of DEGs (FDR < 0.05 according to edgeR). B) The intersection of DEGs (the
statistical criteria using fold change > 1.5 and FDR < 0.05) effected by PA and those DEGs from pairs of
ccRCC tumours and adjacent non-tumour renal tissues from GSE40435 and GSE76207. C) KEGG
pathways enrichment analysis. The bars represent the enrichment scores, -log10 (P value). D) PA (25 and
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0 nM) and GPA (25 and 50 nM) reduced the activation of key genes in ACHN cells. E) mRNA level of
PRDX1 in ACHN cells after treatment of PA/GPA. F) Immunoblot analysis of PRDX1 protein of extracts
of paired tumour (T) versus adjacent non-tumour (N) samples from 6 human ccRCC patients.
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Consequently, multiple interaction genes (EGFR, SOS2, ETS1 and HK2, SLC2A1, LDHA,
MET, HIF1A, etc.,), among the above signalling pathways were chosen to be certificated treated
with PA and GPA (25 and 50 nM) in ACHN cells by RT-PCR (Figure 2D). GPA (10), with
similar inhibitory activity towards ACHN as PA, is the glycoside of PA (1). As expected, PA and
GPA could both reduce the activation of those key genes. Thus, there are reasons to believe that
PA and GPA exert their tumour-inhibition activity through the same pathway. To further clarify
the potential molecular targets, we examined the intersection of DEGs (the statistical criteria
using fold change  1.5 and FDR < 0.05) affected by PA and those DEGs between pairs of clear
cell renal cell carcinoma (ccRCC) tumour and the adjacent non-tumour renal tissues in GEO
database (GSE40435 and GSE76207) (Figure 2B). There were 129 common genes up-regulated
in the PA treatment group but down-regulated in the ccRCC tumour tissues compared to the
adjacent non-tumour renal tissues, including PRDX1 (Figure 2B). RT-PCR was employed to
certify the up-regulation of PRDX1 in ACHN cells after treatment of PA/GPA (Figure 2E).
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As a redox-regulating protein belongs to the ubiquitous PRDXs family, PRDX1 was
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implicated in regulating cell proliferation, differentiation, and apoptosis. All peroxiredoxins
have been shown to have altered expression in human cancer, being up-regulated in breast
cancer, oesophageal cancer, and lung cancer, but down-regulated in thyroid cancer as a tumour
suppressor.13,14 PRDX1 protein was decreased in ccRCC as compared to adjacent normal renal
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tissue. Remarkably, we make the same findings of the lower PRDX1 protein levels in six
ccRCC tumour samples (Figure 2F, Table S10). Along with the analysis of different GEO
datasets, it’s reasonable to believe that the mRNA and protein level of PRDX1 are lower in renal
tumour tissues, and PRDX1 could be considered as a potential target in RCC. However, the
mechanism of PRDX1 as an anti-RCC target still needs to be further explored.
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PRDX1 was firstly reported as an antioxidant enzyme because of its functions to eliminate
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peroxide in vivo and regulate reactive oxygen species (ROS). Aggressive cancer cells depend
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on elevated intracellular levels of ROS to proliferate, self-renew, metastasize, etc. These
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aggressive cancers maintain high basal levels of ROS compared to normal cells. With a high
ROS level and low PRDX1 expression, RCC becomes less sensitive to radiation and
chemotherapy. Thus, ROS elimination played a vital role in RCC treatment. To explore the
molecular mechanisms of piericidins (PA/GPA) on ACHN cells, clarifying the relationship
between PRDX1 and ROS levels in RCC is crucial.
To further illustrate the mechanism by which PRDX1 exerts its tumour-suppressing effect,
we created knockdown PRDX1 ACHN cells (PRDX1-Kd) using lentivirus PRDX1 shRNA
(shPRDX1) to speculate on the role of ROS (Figure 3B, Figure S5). As a result, the knockdown
of PRDX1 raised ROS levels compared to normal ACHN cells (Figure 3C1). ROS reduction in
normal ACHN cells was observed when treated with PA/GPA (25 and 50 nM) (Figure 3C2).
Notably, knockdown of PRDX1 eliminated these effects, which indicated that PRDX1 was the
target of PA/GPA (Figure 3C3). By decreasing ROS levels, PA showed significant proliferation
suppression as well as inducing apoptosis of ACHN cells (Figure 3A1/A2). In addition, the
cytotoxic IC values of PA/GPA against shPRDX1 ACHN cells were almost 4-fold higher than
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normal ACHN cells with slight toxicity towards HK2 cell (Table 1). These results indicated that
PA/GPA targets PRDX1 mainly increasing mRNA and protein levels rather than improving the
antioxidant activity (Figure S6).
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Figure 3. PA/GPA inhibits cell proliferation and induced cell apoptosis through increasing PRDX1 and
reducing ROS in ACHN. A1) Phase holographic imaging of ACHN cells treated with PA (50 nM) for
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2h. A2) Annexin V/PI staining and flow cytometry showed the percentages of apoptosis in ACHN cells
treated with PA (50 nM). B) Immunoblot analysis of PRDX1 protein in the normal ACHN cells and
PRDX1-Kd ACHN cells; C1) The intracellular ROS detection with flow cytometer in the normal ACHN
cells and PRDX1-Kd ACHN cells. C2) The intracellular ROS detection in the normal ACHN cell treated
by PA/GPA (25 and 50 nM) in 24h; C3) The intracellular ROS detection in the PRDX1-Kd ACHN cell
treated by PA/GPA (25 and 50 nM) in 24h. D1) Immunoblot analysis of PRDX1 protein in nuclear and
cytosol extracts of ACHN cell treated with PA/GPA (25 and 50 nM). D2) Fold change of protein
expression (PRDX1/β-Actin). E1) Representative images for immunofluorescent staining of ACHN
treated with PA/GPA (25 and 50 nM) after co-incubating for 24h. PRDX1 (green), nucleus (blue), n = 9.
Scale bar = 10 μm. E2) Mean fluorescence intensity of ACHN cell treated with PA/GPA (25 and 50 nM)
in 24h. All data represent mean ± SD. Statistical significance was determined using Student’s t test or
one-way ANOVA with Tukey’s post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001.
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Not only serving as antioxidant enzyme, PRDX1 can also act as a molecular chaperone with
the ability to modulate actions of numerous molecules or as a regulator of transcription. 17,18
PRDX1 could directly bind with nuclear transcriptional factors and modulate gene expression
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such as c-Myc and NF-κB. So, we further investigated the PRDX1 protein level in nuclear and
cytosol of ACHN cell. Along with increasing total PRDX1, obvious translocation of PRDX1
towards nucleus was also observed, indicated that PA/GPA not only raised the expression of
mRNA and protein of PRDX1 but also forced it into nucleus (Figure 3D/E). Some studies have
shown that PRDX1 in the nucleus leads to inhibition of NF-κB activation, a typical oncogene
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involved in most cancers. These data further suggested that the protein level of PRDX1, but
not antioxidant activity, is critical in regulating the ROS level and the inactivation of NF-κB.
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.4. Docking analysis of PA/GPA with PRDX1
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The interconversion between the dimeric and decameric forms of PRDX1 is influenced by redox
state or protein concentration, and the dimeric form is preferred for translocation into the nucleus.
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In order to further understand the interaction between PA/GPA and the potential target,
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especially regulatory mechanisms of dimer-oligomer interconversion of PRDX1, Induce-fit
module in the Schrödinger suite was employed to perform the in silico molecular docking analysis.
The chains a, b, c, and d, as a dimer-dimer interface unit, from the reduced decameric form of
PRDX1 (PDB entry 2Z9S, Figure S7) was used as the receptor structure (Figure 4A1). Since the
redox status CYS83 might influence the oligomeric structure and consequently the functions of
PRDX1 in regulating the peroxidase and chaperone activities, 22 it was selected as Trim residue
and the center of active pocket, which composed of the PHE48, TRP87 from the dimer-dimer
interface. 21 As the results, the pyridine ring of PA/GPA played a key role to form a π-π stacking
interaction with PHE48. Moreover, the π cation interaction was formed between pyridine ring and
LYS192, and hydrophobic interaction was formed between side chain and dimer-dimer interface
(Figure 4A2/A3). The hydroxyls at side chain and pyridine ring formed hydrogen bonds with the
residues GLN94, GLY95 in chain a and LYS192 in chain c, respectively. It is suggested that those
interactions inhibit the formation of the oligomeric state of PRDX1 and forced the active dimer
into the nucleus, which consistent with the experimental findings about the increase of PRDX1 in
nucleus (Figure 3D/E).
The docking study showed that the interactions between PA/GPA and single dimer (docking
scores about –6) of PRDX1 were much weaker than with dimer-dimer interface (docking scores
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about –10). The dissociation equilibrium constants (K ) of PRDX1 protein with PA (2.61 × 10
D
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M) and GPA (1.83 × 10 M), measured by a surface plasmon resonance (SPR) assay (Figure
4B), revealed the affinity interaction between PA/GPA and PRDX1 protein (Figure 4B1/B2).
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Therefore, it is suggested that, not only combined with the PRDX1, PA/GPA could force
PRDX1 translocate into nucleus is also proposed to be a key factor for the anti-RCC procedure
of PA/GPA.
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Figure 4. Molecular docking and SPR analysis of PA/GPA with PRDX1. A1) Dimer-dimer interface
region (PDB entry 2Z9S) was used as the receptor structure. Each chain of monomers is shown in
different colors. Chains a, b as a dimer and chains c, d as another dimer. CYS83-CYS83 disulfide bridge
is located between chains a and c. A2) Binding sites and the details of the predicted binding mode of PA
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in dimer-dimer interface of PRDX1 (2Z9S), 90° rotation from A1. A3) Binding sites and the details of the
predicted binding mode of GPA in dimer-dimer interface of PRDX1 (2Z9S) The contact residues are
shown and labelled by type and number. B) The affinity activities of PA (B1) and GPA (B2) to PRDX1
protein was analyzed using the SPR assay.
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.5. Anti-tumor efficacy of PA and GPA in nude mice bearing ACHN xenografts
Since PA and GPA showed significant inhibitory effects against ACHN in vitro, the in vivo their
anti-cancer therapeutic potential were further investigated. Nude mice bearing ACHN tumor
xenografts were divided into four groups (control group with only PBS injection, PA and GPA
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groups with both 0.8 mg/kg, sorafenib group with 50 mg/kg), and PA and GPA were
administered through intraperitoneal (ip) injection daily for three weeks. The treatment outcome
was assessed by monitoring relative tumor volumes and weights of the mice, and tumor tissue
ablation was also evaluated by HE (haematoxylin and eosin) staining and TUNEL (terminal
deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling) staining on tissue
sections.
As shown in Figure 5, administration of PA or GPA significantly reduced the final tumor
weight of the mice (mean 0.28 g to PA, and mean 0.23 g to GPA) as compared to the control
group (mean 0.55 g), basically matching the tumor volume curves (Figure 5A/B). All the mice
in the treatment groups survived more than three weeks, the mice body weights of PA and GPA
groups did not decrease as observed in the sorafenib group (Figure 5B). PA and GPA showed
significant anti-tumor efficacy in nude mice bearing ACHN xenografts with low effective dose
and without serious side effects.
After administration of PA and GPA for 21 days, the mRNA and protein expression of
PRDX1 in tumor tissues increased (Figure 5C), consistent with the results in vitro. In addition,
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TUNEL staining indicated by green colour staining and HE staining results in tumor tissues
treated with PA or GPA further demonstrated the inhibition of tumor growth (Figure 5D).
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Figure 5. PA and GPA show anti-tumor efficacy in nude mice bearing ACHN xenografts. A) Images of
mice bearing ACHN xenografts and injected ip once a day at day 21 post injection. B) Images of tumor
(B1) and progression of body weights (B2) and tumor volumes (B3) in mice during 21 days treatment; n
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4, ± SE. C) Immunoblot analysis of PRDX1 protein in tumor tissues after 21 days treatment with saline,
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PA and GPA. D) TUNEL and HE staining of tumor sections after treatment, respectively. Scale bar, 100
μm.
3. Discussion
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In this study, twenty seven natural piericidins were obtained from a Streptomyces strain isolated
from the mangrove sediments. Seventeen were identified as new natural products, including
thirteen new piericidin glycosides. This work has extended the chemical diversity of this family,
although several piericidin glycosides have been detected and structurally predicted in situ in the
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symbiotic Streptomyces strains of beewolf host species reported recently. The availability of
these diverse piericidin natural products creates an opportunity to fully understand the
importance of these compounds. Although the cytotoxicity of the piericidins are commonly
reported, the potential to treat renal cancer or related cancer cells was not realized. As one of the
high incidence cancers with poor prognosis, our findings highlight the inhibition activity of
piericidins against renal cancer in vitro and in vivo.
Cancer cells rely on the signaling capabilities of ROS for cell migration, proliferation, and
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survival. Studies have found that persistent high level of ROS can act as mitogenic or
genotoxic signals, inhibiting the sensitivity of radiotherapy and chemotherapy in cancer cells.
26,27
As a member of the ubiquitous family of redox-regulating proteins, PRDX1 is reported to
potentially eliminate various ROS. The dimeric form is considered to be the active and
functional form of PRDX1, and is preferred for translocation into the nucleus. 21 PRDX1 can
directly combine with some transcription factors in the nucleus such as c-Myc and NF-κB,
affecting their bioactivities upon gene regulation, which in turn induces or suppresses cell death.
Using bioinformatics analysis of clinical samples, ccRCC is thought to be a malignant tumor
with lower PRDX1 level. Remarkably, piericidins (PA/GPA) target PRDX1, increasing the
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mRNA and protein levels of PRDX1, which reduces ROS levels in ACHN cells along with
obvious cell apoptosis induction and proliferation inhibition. Besides, PA/GPA can interact
directly with PRDX1 protein and increase the nucleus co-localization, thereby inhibiting the
activation of key genes in the renal caner pathway. Moreover, our study revealed that ACHN
cells were less sensitive to piericidins when PRDX1 was down-regulated. Therefore, our direct
evidence shows that PA/GPA targets PRDX1 and reduces ROS in ACHN cells.
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In those twenty seven natural piericidins obtained in this study, PA and GPA were
investigated with their molecular mechanisms on renal carcinoma cells, as well as the in vivo
anti-cancer evaluation. PA and GPA were being chosen as representative molecules of piericidin
aglycones and glycosides, respectively, because of their relative high yield in the Streptomyces
strain and their significant inhibitory ability against ACHN cells in vitro. There were no obvious
differences between the piericidin glycosides and aglycones in the cytotoxicity against ACHN
cells. Moreover, the nearly indistinguishable anti-tumor effects in vivo between PA and GPA in
our study suggest that, the same metabolic substances might contribute to the efficacy after drug
metabolism in vivo. However, the up-regulation of PRDX1 protein expression by GPA in the
nucleus of ACHN cells appears stronger than that of PA (Figure 3D). And the affinity
interaction measured by SPR test also showed glycoside GPA bound more effectively than the
aglycone PA with PRDX1 (Figure 4B). In this study, we found that PRDX1 could be used as an
important new target for anti-RCC agents, and piericidins such as PA and GPA could be
considered as lead compounds targeting PRDX1.
4
. Conclusion
In conclusion, twenty seven natural piericidins were discovered by cultivation of a marine-
derived Streptomyces strain, and their potential in anti-RCC was demonstrated. The low effective
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dose and relative safety of PA and GPA in vivo suggests these maybe developed as new anti-
RCC agents. The site of action of these agents targeting PRDX1, may improve upon the
limitations of mechanisms of existing targeted drugs for the treatment of RCC. Although more
investigations are needed, our current studies reveal that piericidins could conceivably provide a
novel scaffold for further development of potent and mechanistically-novel anti-RCC agents.
1
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5
. Experimental Section
.1 Chemistry. IR spectra were measured on an IR Affinity-1 spectrometer (Shimadzu).
5
Optical rotations were performed on a PerkinElmer 341 polarimeter. ECD spectra were
measured with a Chirascan circular dichroism spectrometer (Applied Photophysics). The NMR
spectra including 1D and 2D NMR were recorded on Bruker AC 500/700 MHz spectrometers.
HR-ESIMS were determined with a Bruker maXis Q-TOF in positive/negative ion mode.
Column chromatography was performed on silica gel (200−300, 300−400 mesh) and Sephadex
LH-20 (Amersham Biosciences), respectively. All solvents used were of analytical grade
(Tianjin Fuyu Chemical and Industry Factory). The semipreparative HPLC was performed on an
HPLC (Hitachi-L2130, diode array detector, Hitachi L-2455, Tokyo, Japan) using a Phenomenex
ODS column (250 mm × 10.0 mm i.d., 5 μm; Phenomenex, USA).
5
.2 Strain Material. The strain SCSIO NS126 was isolated from a mangrove sediment
sample collected from the Pearl River estuary (E 113°33′11.15′′, N22°53′40.16′′) to South China
Sea in May 2015, by incubation at 28 °C for two weeks on ISP-2 medium (yeast extract 4 g, malt
extract 4 g, glucose 4 g, crude sea salt 30 g, agar powder 20 g, distilled water 1000 mL, pH, 7.2-
7
.4). The strain was identified as a Streptomyces psammoticus specie, by the 16S rRNA gene
sequence analysis (Table S8). It was deposited at the China Center for Type Culture Collection
(Wuhan) as CCTCC M2017099 (Streptomyces psammoticus SCSIO NS126).
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.3 Fermentation and Extraction. The strain SCSIO NS126 was fermented for 60 L. A
few loop of cells of the strains were inoculated into a 250 mL Erlenmeyer flask containing 50
mL of seed medium (mannitol 1 g, soya peptone 0.5 g, soya-bean oil 0.125 g, K HPO 0.02 g,
2
3
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pH 7.0, 50 mL distilled water), and then cultivated on a rotary shaker at 120 rpm, 28 °C for 48 h
as seed culture. Then, 2 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask
containing 100 mL media (cotton seed meal 2.5 g, soluble starch 1 g, glucose 1 g, yeast extract
0
.3 g, CaCO 0.5 g, sea salt, 0.2 g; in 100 mL distilled water, pH 7.2). After cultivation on a
3
rotary shaker at 180 rpm and 28 °C for 120 h, each bacterial culture broth was broken with
ultrasonic treatment apparatus for 10 min. Then culture broth was extracted with an equal
volume of ethyl acetate three times. The organic extract was then concentrated under vacuum to
afford the crude extract.
5
.4 Compounds Isolation. The extract of strain SCSIO NS126 (38.2 g) was
chromatographed on silica gel to give eight fractions (Frs.1–8). With the guide of HPLC
analysis, Frs.2 and Frs.3 were combined and further purified by silica gel to obtain pure
compound 1 (230 mg), 2 (13 mg), 5 (16 mg), 6 (12 mg), 8 (14 mg), and 9 (3.7 mg). Frs.4 was
further purified by semipreparative HPLC to get compound 3 (0.7 mg) and 4 (0.8 mg) and 7 (2.5
mg). Frs.5 was further purified by semipreparative HPLC to get 10 (78 mg), 12 (2.9 mg), 23 (1.9
mg), 26 (1.1 mg) and 27 (2.1 mg). The compound 10 (250 mg) with the highest content was
purified and obtained by semipreparative HPLC from Frs.6, together with 14 (6.5 mg), 16 (3.2
mg), 18 (2.5 mg), 21 (1.8 mg), 24 (1.6 mg) and 25 (0.6 mg). Frs.7 was also purified by semi-
preparative HPLC to get 11 (28 mg) 13 (1.8 mg), 15 (6.9 mg), 17 (4.1 mg), 19 (1.5 mg), 20 (1.5
mg), and 22 (1.9 mg). All obtained compounds were determined to have ≥95% purity by
analytical HPLC.
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1
1-Demethyl-piericidin A (2). Pale yellow oil; IR (film) νmax 3335, 2945, 1449, 1020, 667
−
1
1
13
+
cm ; H and C NMR data, Tables S2-1 and S2-2; HR-ESIMS m/z 402.2649 [M+H] (calcd for
+
C H NO , 402.2639).
2
4
36
4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
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2
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3
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1
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1
2
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0
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2
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2
3
4
5
6
7
8
9
0
(2E,5E,7E,11E,9R,10R,13S,19R)-13,19-Dihydroxyl-IT-143-A (3). Pale yellow oil; IR (film)
−
1
1
13
νmax 3335, 2945, 1449, 1020, 667 cm ; H and C NMR data, Tables S2-1 and S2-2; HR-
+
+
ESIMS m/z 504.3332 [M+H] (calcd for C H NO , 504.3320).
29 46
6
(2E,5E,7E,11E,9R,10R,13R,19S)-13,19-Dihydroxyl-IT-143-A (4). Pale yellow oil; IR (film)
−
1
1
13
νmax 3329, 2945, 1446, 1018, 667 cm ; H and C NMR data, Tables S2-1 and S2-2; HR-
+
+
ESIMS m/z 504.3320 [M+H] (calcd for C H NO , 504.3320).
29 46
6
10-Ketone piericidin A (5). Clear oil; [α]20 D – 5.5, c 0.2, CHCl ; IR (film) ν 3332,
3
max
−1 1
13
2
4
945, 1472, 1018, 667 cm ; H and C NMR data, Tables S2-1 and S2-2; HR-ESIMS m/z
+
+
14.2657 [M+H] (calcd for C H NO , 414.2639).
2
5
36
4
7-Demethylglucopiericidin A (12). Pale yellow oil; IR (film) νmax 3420, 2928, 1472, 1456,
−1
1
13
1
125, 1016, 652 cm ; H and C NMR data, Tables S2-3 and S2-5; HR-ESIMS m/z 564.3185
+
+
[
M+H] (calcd for C H NO , 564.3167).
30 46 9
7
-Demethyl-13-hydroxyglucopiericidin A (13). Pale yellow oil; IR (film) νmax 3302, 2930,
−
1
1
13
1
585, 1464, 1412, 1125, 1022, 650 cm ; H and C NMR data, Tables S2-3 and S2-5; HR-
+
+
ESIMS m/z 580.3126 [M+H] (calcd for C H NO , 580.3116).
3
0
46
10
1
3-Hydroxypiericidin A 10-O-α-D-galactose (1→6)-β-D-glucoside (15). Pale yellow oil; IR
−
1
1
13
(film) νmax 3319, 2930, 1650, 1412, 1126, 1022, 824, 764, 667 cm ; H and C NMR data,
–
–
Tables S2-3 and S2-5; HR-ESIMS m/z 754.3646 [M – H] (calcd for C H NO , 754.3655).
3
7
56
15
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3-Hydroxypiericidin A 10-O-α-D-glucose (1→6)-β-D-glucoside (17). Pale yellow oil; IR
−
1
1
13
(
film) νmax 3358, 2920, 1585, 1412, 1125, 1022, 824, 764, 667 cm ; H and C NMR data,
–
–
Tables S2-3 and S2-5; HR-ESIMS m/z 754.3660 [M – H] (calcd for C H NO , 754.3655).
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7
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15
0
1
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7
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9
0
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-O-β-D-Glucose glucopiericidin A (18). Pale yellow oil; IR (film) νmax 3366, 2920, 1582,
−
1
1
13
1
404, 1047, 669 cm ; H and C NMR data, Tables S2-3 and S2-5; HR-ESIMS m/z 738.3684
–
–
[M – H] (calcd for C H NO , 738.3706).
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7
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14
4
-O-β-D-Glucose 13-hydroxyglucopiericidin A (19). Pale yellow oil; IR (film) νmax 3292,
−
1
1
13
2
943, 1456, 1404, 1020, 660 cm ; H and C NMR data, Tables S2-3 and S2-5; HR-ESIMS m/z
+
+
756.3829 [M + H] (calcd for C H NO , 756.3801).
3
7
58
15
4
-O-β-D-Glucose piericidin A 10-O-α-D-glucose (1→6)-β-D-glucoside (20). Pale yellow
−
1
1
13
oil; IR (film) νmax 3342, 2930, 1568, 1456, 1018, 650 cm ; H and C NMR data, Tables S2-4
+
+
and S2-5; HR-ESIMS m/z 902.4390 [M + H] (calcd for C H NO , 902.4380).
4
3
68
19
5
-Hydroxy-6-hydroxymethyl glucopiericidin A (21). Pale yellow oil; IR (film) νmax 3356,
−
1
1
13
2
916, 1568, 1471, 1412, 1016, 667 cm ; H and C NMR data, Tables S2-4 and S2-5; HR-
+
+
ESIMS m/z 626.3543 [M + H] (calcd for C H NO , 626.3534).
3
2
52
11
5
-Hydroxy-6-hydroxymethyl-13-hydroxyglucopiericidin A (22). Pale yellow oil; IR (film)
−
1
1
13
νmax 3392, 1670, 1200, 1134, 723 cm ; H and C NMR data, Tables S2-4 and S2-5; HR-
+
+
ESIMS m/z 642.3481 [M + H] (calcd for C H NO , 642.3484).
3
2
52
12
2
-Hydroxymethyl-Δ3, 4-glucopiericidin A (23). Pale yellow oil; IR (film) νmax 3356, 2926,
−
1
1
13
1
471, 1411, 1124, 1016, 648 cm ; H and C NMR data, Tables S2-4 and S2-5; HR-ESIMS m/z
+
+
6
08.3444 [M + H] (calcd for C H NO , 608.3429).
32 50
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(
11S, 12R) Piericidin C1 10-O-β-D-glucoside (24). Pale yellow oil; IR (film) νmax 3354,
−
1
1
13
2
932, 1587, 1468, 1412, 1126, 1074 cm ; H and C NMR data, Tables S2-4 and S2-5; HR-
+
+
ESIMS m/z 594.3269 [M + H] (calcd for C H NO , 594.3273).
3
1
48
10
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
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3
3
3
4
4
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5
5
5
5
5
6
0
1
2
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9
0
1
2
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(11R, 12S) Piericidin C1 10-O-β-D-glucoside (25). Pale yellow oil; IR (film) νmax 3366,
−
1
1
13
2
926, 1585, 1464, 1412, 1125, 1076, 721 cm ; H and C NMR data, Tables S2-4 and S2-5;
+
+
HR-ESIMS m/z 594.3288 [M + H] (calcd for C H NO , 594.3273).
31 48
10
1
3-Dimethoxy glucopiericidin A (26). Pale yellow oil; IR (film) νmax 3358, 2926, 1670,
−
1
1
13
1
6
200, 1136, 802, 723 cm ; H and C NMR data, Tables S2-4 and S2-5; HR-ESIMS m/z
+
+
38.3535 [M + H] (calcd for C H NO , 638.3535).
33 52
11
5.5 Cell Culture and Cytotoxic Bioassay. ACHN (cat. TCHu199), OS-RC-2 (cat.
TCHu40) and 786-O (cat. TCHu186) cells, along with HK-2 (cat. SCSP-511) cells were
purchased from Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China.
ACHN cells were grown and maintained in MEM medium with 10% FBS, while OS-RC-2 and
7
86-O cells were grown in RPMI1640 medium with 10% FBS. HK-2 cells were grown in
DMEM medium with 10% FBS. Cell viability was determined by the CCK-8 (Dojindo) assay.28
Cells were seeded at a density of 400 to 800 cells/well in 384-well plates and then treated with
various concentrations of compounds or solvent control. Sorafenib was used as the positive
control. After 72h of incubation, CCK-8 reagent was added, and absorbance of the triplicate tests
were measured at 450 nm by an Envision 2104 multi-label reader (Perkin Elmer). Dose response
curves were plotted to determine IC using Prism 5.0 (GraphPad Software Inc.).
5
0
5
.6 Cell Cycle and Apoptosis Assay. Cell cycle arrest was analyzed by propidium iodide
2
8
(
PI) DNA staining using flow cytometry. Briefly, after treatment with piericidins for 24, 48
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and 72 h, respectively, cells were harvested, prepared, and then fixed overnight. The fixed cells
were harvested, washed, resuspended, and finally stained with PI (Sigma-Aldrich). Cell cycle
distribution was studied using an Accuri C6 (BD) flow cytometer. Cell apoptosis was analyzed
using a FITC Annexin V Apoptosis Detection Kit (BD), according to the manufacturer’s
protocol. Cells were treated with piericidins for 24, 48 and 72 h, stained with annexin V-FITC
and PI solution, examined and analyzed quantitatively using an Accuri C6 (BD) flow cytometer.
0
1
2
3
4
5
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0
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.7 Digital Holographic Microscopy. The Holomonitor™ M4 microscope (Phase
Holographic Imaging AB, Lund, Sweden) was used for imaging and tracking ACHN cell
movement and morphological changes induced by PA (1). This technique provides long-term
kinetic cellular analysis without any cell labelling. The principle behind this method is the
detection of the phase shift of the probing laser light transmitting or reflecting through a cell
2
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5
sample and comparison to the reference light. The ACHN cells were planted (2×10 cells) on a
6-well plate and adhered for 24h. A concentration of 50 nM PA (1) was added and then the field
of each sample was detected under the microscope automatically. For the morphometry analysis,
the color bar means the thickness of cells.
5
.8 Intracellular ROS detection. The intracellular ROS were measured by flow cytometry
in a BD Accuri™ C6 flow cytometer (BD Biosciences) using CellROX® Oxidative Stress
Reagents (Cat no. C10422; Life technologies, USA). ACHN cells were cultured in MEM
4
medium with 10% FBS by 24h. Then, the medium was replaced and the cells (2ꢀ×ꢀ10 cells/well)
were cultured in 6-well plates, together with PA/GPA (0, 25 nM, 50 nM) in medium and
incubated by 24ꢀh. Next, the cells were collected and incubated with CellROX® Oxidative Stress
Reagents 5ꢀμM for 30 min/37ꢀ°C in darkness. BD Accuri™ C6 flow cytometer was used to
analyze the cells with ROS.
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.9 Lentivirus-mediated RNA Interference. Lentivirus (Lv) expressing short hairpin RNA
(
shRNA) targeting PRDX1 was designed and chemically synthesized by Shanghai GeneChem
Co. Ltd. (Shanghai, China), and the non-target shRNA expressing green fluorescence protein
GFP) was only used as the RNA inference (RNAi) control. Target sequences are as followed: -
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
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4
5
6
7
8
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0
1
2
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7
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9
0
1
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0
(
TGAGACTTTGAGACTAGTT- for PRDX1; -TTCTCCGAACGTGTCACGT- for vector.
ACHN cell at 50-70% confluency was transfected and then selected in medium containing
puromycin (1 μg/mL). After confirming the stable transfection efficiency in 72h, cells were
treated with 25 nM and 50 nM PA or GPA for 24 hours.
5
.10 Immunofluorescent Staining and Confocal Microscopy. ACHN cells were co-
incubated with PA/GPA in 24h. After washing three times in PBS, ACHN cells were fixed in 4%
paraformaldehyde for 10ꢀmin, and permeabilized with 0.5% Triton X-100 in PBS. After washing,
cells were blocked with 10% FBS in 4℃ for 12h and incubated with anti-PRDX1 primary
antibody overnight. Cells were washed and then incubated with fluorescein isothiocyanate-
conjugated secondary antibody (green imaging) for 2ꢀh at room temperature. DAPI staining was
also performed for nucleus localization and shown in blue. Merge imaging was shown to indicate
the subcellular localization of PRDX1. Cell morphology was visualized under a Zeiss
fluorescence microscope (Carl Zeiss, LSM 880 with Airyscan, Germany).
5
.11 SPR Assay. The surface plasmon resonance (SPR) measurement was performed on
Biacore T100 system (GE Healthcare). Full-length human PRDX1 was immobilized on a CM5
chip (GE healthcare). Diluted in 10 mM Hepes (pH 7.4) for giving a concentration series,
PA/GPA samples were dispensed into single-used snap-capped vials and injected across the
protein and the protein-free (control) surfaces in a single step. Compound binding with PRDX1
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was analyzed in a single-cycle kinetic analysis range from 1–10 μM PA and 1–20 μM GPA
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respectively. The K values were calculated using dynamic fitting equation.
D
5.12 Molecular Docking. The Schrödinger 2017-1 suite (Schrödinger Inc., New York, NY)
was employed to perform dock analysis. The PRDX1 structure was retrieved from the available
crystal structure (PDB code: 2Z9S, chains a,b,c and d) and constructed following the Protein
Prepare Wizard workflow in Maestro11-1. The prepared ligands were then flexibly docked into
the receptor using Induce-fit module with default parameters.
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.13 Tumorigenesis Assays. Four-week old, male athymic nude (Balb/c nu/nu) mice were
purchased from Guangdong Medical Lab Animal Center (China) and allowed to acclimatize for
1 week. Ten million ACHN cells in logarithmic growth phase, suspended in 0.2mL PBS on ice,
were inoculated into the right flank of the nude mice. After the tumors became palpable (around
3
1
50 mm ), mice were randomly divided into four groups, when the average weights and tumors
volumes distributed equally in each group. Tumor-bearing mice were received PA (0.8 mg/kg) or
GPA (0.8 mg/kg) daily by intraperitoneal injection. Sorafenib tosylate (50 mg/kg) was dissolved
in Cremophor EL (Sigma) as a 10× stock solution, further diluted in PBS before use. During the
exposure period the animals were kept at a temperature of 21°C at a humidity of 55% under a 12
h/12 h light/dark cycle with free access to food and water. Tumor growth was measured through
calipers, and tumor volumes and body weight were in record. The calculation of tumor volumes
was according to the formula 0.5 × length × width2.
5.14 Histology. When nude mice were sacrificed at day 21, tumor tissues were rapidly fixed
in 4% paraformaldehyde (Sigma) at room temperature overnight. After fixation, tissues were
dehydrated in ethanol with increasing concentrations up to 100%. Dehydrated specimens were
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Journal of Medicinal Chemistry
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subsequently infiltrated with 100% xylene and then embedded in paraffin wax. Five μm-thick
sections were cut for immune-histochemical studies, which were performed, using standard
techniques.
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.15 Western Blot Analysis. ACHN cells, shPRDX1-ACHN cells and tumor tissues were
lysed in RIPA buffer (Beyotime), including 50 mM Tris (pH7.4), 150 mM NaCl, 1% Triton X-
00, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, EDTA and a protease inhibitor
1
cocktail (Sigma). The supernatants were collected after centrifugation at 13000 rpm at 4°C for 30
min, and the quantification of protein content was estimated by BCA Protein Assay kit
(Beyotime), detected with a spectrophotometer using absorption at 562 nm. Proteins (30–50 μg)
were separated on 10% or 12% polyacrylamide gels and transferred to PVDF membranes using a
Bio-Rad Trans-Blot wet transfer apparatus. Subsequently, the membranes were incubated with
primary antibody (1:1000 for PRDX1, 1:3000 for β-Actin, 1:3000 for GAPDH) overnight after
blocking with 5% nonfat milk for 2 h at room temperature. Then the appropriate HRP-
conjugated secondary antibody (1:3000) was used for detection by ECL substrates.
5
.16 Compliance with Ethical Standards. Animal used was permitted by the animal ethics
committee of Southern Medical University. The clinical samples were collected from the
Nanfang Hospital of Southern Medical University, Guangzhou, China. Informed consents were
obtained from all participants. Approval for tissue collection was obtained from the ethics
committee of Nanfang Hospital of Southern Medical University. Baseline clinical characteristics
of human kidney samples were listed in Table S10.
ASSOCIATED CONTENT
Supporting Information.
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