Discovery of Turn-On Fluorescent Probes for Detecting PDEδProtein in Living Cells and Tumor Slices

Article abstract of DOI:10.1021/acs.analchem.0c00335

The first small-molecule fluorescent turn-on probes for detecting PDEδprotein were rationally designed, showing reasonable fluorescent properties and the fluorescent ability has been applied for visualization of the PDEδprotein in living cells and at tissue levels. The qPCR results showed that the mRNA expression of KRAS, PDEδ, AKT1, MAPK1, MEK7, RAF1, and mTOR were downregulated by probes 1-3 through PI3K/AKT/mTOR and MAPK signal pathways. The probes also can downregulate the protein level of pErk and tErk. Therefore, these small-molecule fluorescent probes are expected to be used in the screening of antipancreatic cancer drugs targeting the PDEδprotein, as well as in obtaining a better understanding of the pathological and physiological roles of PDEδprotein.

Full text of DOI:10.1021/acs.analchem.0c00335

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Article
Discovery of Novel Turn-On Fluorescent Probes for
Detecting PDE# Protein in Living Cells and Tumor Slices
Gaopan Dong, Long Chen, Jing Zhang, Tingting Liu, Lupei Du, Chunquan Sheng, and Minyong Li
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.0c00335 • Publication Date (Web): 22 Jun 2020
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Analytical Chemistry
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Discovery of Novel Turn-On Fluorescent Probes for Detecting PDEδ
Protein in Living Cells and Tumor Slices
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Gaopan Dong a,†, Long Chen b,†, Jing Zhang c,†, Tingting Liu a,d, Lupei Du a, Chunquan Sheng b,*
and Minyong Li a,*
a
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (MOE), School of Pharmaceutical
Sciences, Cheeloo College of Medicine, Shandong University, Jinan, Shandong 250012, China.
b Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical University, Shanghai 200433,
China.
c Department of Pathology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
d Institute of Pharmacology, School of Pharmaceutical Sciences, Shandong First Medical University & Shandong Academy
of Medical Sciences, Taian 271000, Shandong, China.
†Authors contributed equally to this work.
* Corresponding Author: Tel/Fax: +86-531-8838-2076. Email: mli@sdu.edu.cn; shengcq@smmu.edu.cn.
ABSTRACT: The first small molecule fluorescent turn-on probes for detecting PDEδ protein were rationally designed,
which showed reasonable fluorescent properties and can be applied into visualizing the PDEδ protein at the living cell and
tissue levels. The qPCR results showed that the mRNA expression of KRAS, PDEδ, AKT1, MAPK1, MEK7, RAF1 and mTOR
were down-regulated by probes 1-3 through PI3K/AKT/mTOR and MAPK signal pathways. The probes also can down-
regulate the protein level of pErk and tErk. Therefore, these small-molecule fluorescent probes are expected to be used into
screening anti-pancreatic cancer drugs targeting on the PDEδ protein, as well as in better understanding the pathological
and physiological roles of PDEδ protein.
prevalence in pancreatic (>90%), colorectal (about 50%),
INTRODUCTION
and lung (30%) cancers.5,7,8,10-13 KRAS protein played a
direct causal role in human cancer neoplastic,8 oncogenic
KRAS could induce metabolic disorders, while the aberrant
Pancreatic cancer is considered as the fourth lethal cancer
in the United States, and is highly possible raised to the
2nd place by the year 2030.1,2 Pancreatic cancer as a fatal
disease and one of the most leading cause of cancer-related
deaths among all malignances, for it with a horrible
mortality rate and the 5-year survival rate of pancreatic
cancer less than 5%.1-5 It's reported that during 2006 to
2010, the pancreatic cancer incidence rate had increased by
1.3% per year.3 Hence, there is an unquestionably need to
find effective systemic therapies to overcome this highly
lethal cancer.
metabolism is considered as a primary cause leading to
5,14,15
cancer.
Thus, intensive efforts were carried on the
KRAS protein research; the researchers take the oncogenic
KRAS as a prime therapeutic target for pancreatic cancer.
However, Ras proteins have not yielded to any types of
therapeutics, which was regarded as “undruggable” for
many years, even was compared to “the Everest” to climb.5,9
Phosphodiesterase 6 (PDE6) delta subunit (PDEδ), also
known as PDE6D, is the δ subunit of rod specific cyclic
GMP phosphodiesterase.7 PDEδ binds and solubilizes the
farnesylated Ras proteins, and then facilitate the KRAS
The oncogenic KRAS gene was first associated with
pancreatic cancer more than thirty years.2,6 Under
physiological conditions, the KRAS gene plays a crucial
role in regulating cell growth, while under abnormal
conditions, the mutant KRAS gene can lead to sustained
cell growth, finally drive pancreatic neoplasia.5,7 The RAS
proteins family include KRAS, NRAS and HRAS, and Ras
proteins are present with binary on-off switches: the GTP-
bound form is ON, or else the GDP-bound form is OFF,5,8
so the Ras proteins play a crucial role in cytoplasmic signal
transmission and participate in regulating diverse normal
cellular functions.9 The mutant KRAS protein is highly
protein diffusion in the cytoplasm.16-19 Bastiaens’s group
16
reported that the activity of PDEδ is mediated by the Ras-
dependent signaling, and then affect the Ras protein
dynamic distribution in the cell. So many researchers turn
to take the PDEδ as a therapeutic target for pancreatic
cancer.
Nowdays, there are two main pathways to therapeutic
target on the oncogenic KRAS: small interfering RNA
9,10,13
(siRNA)
mediated drug-sensitization on the
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oncogenic KRAS gene, and novel small-molecule inhibitors MeOH, r.t., overnight, 85% ; c) HBTU, TEA, DMF, r.t., 2 h,
Page 2 of 9
disturping KRAS-PDEδ interactions.9 siRNA only down-
regulate gene expression, while the small-molecule
inhibitors showed great advantages in regulating the
functions of gene.9 Based on an extensive biochemical
study, small molecule inhibitors could inhibit the KRAS-
PDEδ interactions, and impair the oncogenic KRAS
signaling was reported.18,20 Zimmermann et al. found
deltarasin could block RAS signaling, reduce the
proliferation of pancreatic cancer cell, and even lead to
cancer cell death.18 However, there are also many
disadvantages for deltarasin, such as poor selectivity and
16%-53%.
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EXPERIMENTAL SECTION
Materials and Instruments. The chemicals related to
synthesis probes were purchased from commercial sources.
NMR were assayed by the Bruker NMR spectrometer.
Photophysical properties were assayed by the Shimadzu
UV-2401PC UV-visible spectrometer and Bio Tek
microplate reader. The cell images were captured by the
Zeiss Axio Observer A1 fluorescence microscope. The
purity of probes were analyzed by Agilent Technologies
1260 Series HPLC with a C18 reversed phase column (250 x
4.60mm , Phenomenex). The Immunohistochemical and
tumor slice images were captured by OLYMPUS VS120
microscope, Beckman CytoFLEX flow cytometry was used
for assay the binding ability of probes to living cell, the
qPCR were assayed by LightCycler® 96 SW 1.1 qPCR
instrument (Roche, Switzerland).
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significant cytotoxicity. Subsequently,
a new PDEδ
inhibitor, named deltazinone was reported to be highly
selective, and less cytotoxic than deltarasin. It was
demonstrated to have a high correlation with the
phenotypic effect of PDEδ.19 Even though, the therapeutic
effects of these KRAS-PDEδ inhibitors on pancreatic
cancer were far from clinical application. Previously, our
group used fragment-based drug design (FBDD) to
discover and design novel chemotypes of KRAS−PDEδ
inhibitors, targeting on the Arg61 pocket and the Tyr149
pocket. Biochemical evaluations demonstrated our novel
PDEδ inhibitors were druggable and effective on the
inhibition of the KRAS-PDEδ protein-protein interactions
(PPI).7
Synthesis. The synthetic route for preparation of probes 1-
3 was shown in Scheme 1. NBD-Cl reacted with different
amino acid to produce compounds 4-6. Subsequently, the
reaction of intermediates 4-6 with compound 8, HBTU,
and TEA was conduct to afford the fluorescent probes 1-3.
Synthetic protocols and structure characteriztion can be
found in the Supporting Information.
Recently, small-molecule fluorescent probes are
prevalently used into imaging and detecting biotargets.
Compared with traditional methods such as bimolecular
Spectroscopy Assay of Probes 1-3. The Fluorescence
spectra of probes 1-3 are assayed at 5 μM and diluted into
1×PBS buffer and then scaned by UV-visible spectrometer,
BioTek microplate reader and HitachiF-2500 fluorescent
spectrometer. Further informations of the photophysical
properties of probes 1-3 are provided in Supporting
Information.
fluorescence
complementation
(BiFC)
assay
or
autofluorescent translocation biosensor techniques, small
meculor fluorescent probes have the advantages of
convenient and affordable detection. Most importantly,
they possess turn-on switch mechanism, which are highly
efficient and sensitive in imaging and detecting target
proteins21-24 even in the high signal-to-background ratios
environment.25,26 Inspired by our previous work,7 we
envisioned that novel turn-on small-molecule fluorescent
probes for PDEδ protein could be designed by
incoporating environmental sensitive fluorophores on
PDEδ inhibitors. Herein the first fluorescent probes for
PDEδ were identified on the basis of the binding mode of
quinazolinone inhibitors, which were able to selectively in
detecting and imaging PDEδ in the pancreatic cancer cell
lines and tumor slices. They are valuable chemical tools for
better understanding the pathological and physiological
functions of PDEδ protein and development of new drug
screening assay.
Fluorescence Anisotropy Assay. The fluorescence
anisotropy assay was referenced to our previous report.7,27
Determination of equilibrium dissociation constants KD2
for each compound was performed using the fluorescence
anisotropy assay with Atrovastatin-PEG3-FITC as ligand in
96-well flat-bottom black plates (Corning # 3650), further
information can be found in Supporting Information.
In Vitro Anti-proliferative Assay. We assay the
cytotoxicity of probes 1-3 and deltazinone in pancreatic cell
lines by the CCK-8 method. We selected two pancreatic
cell lines Capan-1 and MIA PaCa-2 to test the cytotoxicity
of probes 1-3. 5×103 cells each well were planted into the 96-
wells transparent plate and added 100 μL Dulbecco's
Modified Eagle Medium (DMEM) high glucose complete
medium with 1% Penicillin-Streptomycin, 10% FBS and
MIA PaCa-2 cell line also needed 5% house serum, then the
96-well plates were cultured in 37 °C with 5% CO2
humidified atmosphere for 12h. Then probes 1-
3/deltazinone were added to the wells with a series of
concentrations (3.125, 6.25, 12.5, 25, 50 and 100 μM),
respectively. Cells in the 96-well plates were treated with
probes 1-3 or deltazinone for another 48 h. Finally,
followed the manufacturer instruction of CCK-8 kit, all the
Cl
O
N
N
O
H
O
N
N
N
NH2
n
HO
O
HO
N
O
N
N
O
n
H
NO2
N
a
O
N
NO2
O
n
4-6
N
F
N
Bn
O2
O
NH
N
H
c
N
F
N
N
H
b
N
n=1-3
Probe 1-3
H
F
7
8
Scheme 1. Synthetic routes of fluorescent probes 1-3. a)
NaHCO3, MeOH, r.t., 24 h, 62%-77%; b) H2, Pd(OH)2,
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absorbance values at 450 nm of each well were recorded by
BMG multiscan spectrum. All the absorbance values were
analyzed by GraphPad Prism 7.0 software and got the IC50
value of each probe. Furture informations are provided in
supporting informations.
containing protease inhibitor (Sigma) and phosphatase
inhibitor I and II (Sigma) to each plate well. Scraped off on
ice and shake gently for 15 min. Collect the lysate, transfer
to a microcentrifuge tube and centrifugate at 4 oC, 12,000g
for 15 min. The extracted protein was denatured in boiling
water for 5 min. 30 μg protein samples from each lysate
were separated by SDS-PAGE, then transferred to the
PVDF membranes (Merck Millipore) and then blocked for
2 h in the 5% BSA blocking solution at room temperature.
The membranes were incubated with the primary antibody
overnight in dark at 4 °C. Then probed with the appropriate
secondary antibody for 2 h. After washed by TBST for three
times, the PVDF membranes were decteted by
chemiluminescence (Thermo Scientific Pierce ECL
Western Blotting) and scanned by Chemiluminescence
imaging system. More informations can be found in the
Supporting Informations.
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8
Fluorescence Imaging in Living Cells. MIA PaCa-2 and
Capan-1 cells are selected for the cell imaging assay. Cells
were transferred into confocal dish at the proliferation
period, and cultured for 12 h. Then, probes 1-3 were diluted
into DMEM medium (without FBS) and got a 5 μM probe
solution, all cells were co-staining with the nuclear dye
(500 nM Hoechst 33342). All the cells were cultured at 37 °C
atmosphere for 15 min, respectively. We also set MIA PaCa-
2 cell line and Capan-1 cell line co-incubated with KRAS-
PDEδ inhibitor deltazinone (100 µM) and 5 µM probe as
the positive control group. At the same time, we also
chosen the normal cell line HEK-293 cells at the negative
control incubated with 5µM probe. All the fluorescence
imagings in living cell lines were captured by the Zeiss Axio
Observer A1 fluorescent microscope. The background of
each image was adjusted by Image J software. Objective
lens: 63×.
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Immunohistochemical (IHC) Analysis of Capan-1,
HeLa Tumor Slices and Normal Mouse Skin Tissue
Slices. The Capan-1 cells and HeLa cells were grafted
subcutaneously under the forelimb armpit of BALB/c nude
mice, respectively. After 25 days, Capan-1 and HeLa
tumors were harvested, and fixed with 10% formalin for 24
h, and then embedded by paraffin. Subsequently, the
tumors were sectioned into 3 μm thickness layers.
According to the protocol of IHC, after antigen repaired,
the slices were incubated with primary anti-KRAS antibody
(1:150, Catalog Number: 12063-1-AP, Proteintech) at 4 °C for
12h, then followed the protocol of IHC as we previous
reported.25 Tumor sections were photoed by OLYMPUS
VS120 virtual slide microscope, with a 40× objective len.
Flow Cytometry Analysis the Binding Ability of Probes
1-3 to Living Cell Lines. The flow cytometry test was
performed on MIA PaCa-2 and Capan-1 cell lines. MIA
PaCa-2 and Capan-1 cells were collected and washed by
PBS for thrice. 5 µM probe was added together with
deltazinone (100 µM) or solely incubated with 1×105 cells in
300 μL PBS (pH=7.4), respectively, then all the cells were
added into the flow tubes. After incubation for about 0.5 h
at 37 °C in the dark environment, the fluorescence intensity
of cells were assayed by Beckman CytoFLEX Cell Counter.
Fluorescence Imaging Analysis of Capan-1, HeLa
Tumor Tissue Slices and Normal Mouse Tissue
Sections. 10 mM probes solutions diluted with Krebs
buffer (pH=7.4) at 1:1000, and get a final concentration of
10 µM. After antigen repaired, the tissue sections were
The Total RNA Isolation, cDNA Synthesized and qPCR
Test the Relative Expression Level of Related mRNA.
When the MIA PaCa-2 at the proliferation period, the cells
were collected and planted into the 6 well paltes. Total
RNA was extracted from MIA PaCa-2 cell and Capan-1,
using RNAiso Plus reagent (TaKaRa, Dalian, China). 1.5 µg
total RNA was reversed transcription to cDNA with
HiFiscript cDNA synthesis kit (CWBIO, CW2569M, Beijing,
China), following the manufacturer’s instructions. The
cDNA were stored at –20˚C until use. Quantitative real-
time PCR was performed by the FastStart Universal SYBR
Green Master (ROX) (Roche, Switzerland), and then
performed by the LightCycler® 96 SW 1.1 qPCR instrument
(Roche, Switzerland) at a final volume of 10 μL followed the
cycle parameters recommended by the manufacturer.
Relative expression levels of KRAS, PDEδ, AKT1, MAPK1,
MEK7, RAF1 and mTOR were calculated by the 2−△△CT
method,28,29 and GAPDH as the reference gene. The
primers of each gene for qPCR are shown in Table s3.
°
labeled by probes 1-3 at 4 C for 12h, Capan-1 tumor slice
incubated with probe and 200 µM deltazinone was set as
the positive group. Then tissue sections were photoed by
OLYMPUS VS120 virtual slide microscope, with a 40×
objective len.
RESULTS AND DISSCUSION
Design of Novel Small-molecule Fluorescent Probes.
Previously, we designed quinazoline inhibitors by
structural biology-inspired FBDD.7 The binding mode of
quinazoline inhibitor (Figure 1A) with PDEδ revealed that
the two quinazoline fragment was located into the Arg61
and Tyr149 pocket, respectively (PDB code: 5X73, Figure
1B). Considering the hydrophobic nature of the Tyr149
pocket, the quinazoline group was replaced by the
environmentally
sensitive
fluorophore
4-
nitrobenzoxadiazole (NBD). As a result, fluorescent
probes 1-3 with various linker length were designed
(Figure 1A). Molecular docking studies of probe 2
indicated that the it bound to the PDEδ hydrophobic
pocket and kept the key hydrogen bonding interactions
with Arg61 (Figure 1C). Furthermore, hydrogen bonding
Western Blot. Mia PaCa-2 cells were seeded at a density
of 5 × 105 each well in 6-well plates. After 24 h incubation,
cells were stimulated with 125 ng/ml EGF for 5 min and
then treated with various concentrations of compounds for
2 h. Briefly, add appropriate amount of ice-cold lysis buffer
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interactions with Gln78, π-π interaction with Trp32 were
observed, while the NBD moiety mainly form the
hydrophobic interaction with the Tyr149 pocket.
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Figure 2. Fluorescence-emission spectra of probes 1-3 (1
μM) before or after added PDEδ protein (0-2 μM). A: probe
1; B: probe 2; C: probe 3.
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PDEδ Binding Affinity Assay. The binding affinity to
PDEδ of probes 1-3 were presented in Table 2, we found
that with the fluorophore was introduced to the
pharmacophore, the binding affinity of probes to PDEδ
were decreased. Among the three novel probes, the KD2 of
probes 2-3 is 440 nM and 501 nM, respectively, which is
similar to the leading compound (KD2=465 ± 65 nM).7
Cytotoxicity Assay. The cytotoxicity of probes 1-3 and
deltazinone were tested in pancreatic cell lines: MIA PaCa-
2 and Capan-1 cell by CCK-8 method. From the Table 2, we
found that our probes can inhibit the tumor cell
proliferation, especially the probe 2 and 3 can inhibit the
pancreatic tumor cell proliferation better than deltazinone,
the IC50 values of probe 2 and 3 were 24.71 μM, 15.88 μM in
MIA PaCa-2 cell line and 30.89 μM, 33.39 μM in Capan-1
cell line, respectively. These antiproliferative results
proved that probes 1-3 may be used as the antitumor drugs.
Figure 1. Design of novel small-molecule fluorescent
probes of KRAS-PDEδ interaction. (A) Structure-based
design of fluorescent probes 1-3 by incorporating NBD
fluorophore into the quinazoline inhibitor. (B) Co-crystal
structure of quinazoline inhibitor with PDEδ protein (PDB
code: 5X74). (C) Proposed binding mode of Probe 2 with
PDEδ protein. Red dash lines represent hydrogen bonding
interactions. Green dash lines represent π-π interaction.
Spectroscopic Properties and Turn-On Response to
PDEδ Protein of Probes 1-3. The spectroscopic properties
of probes 1-3 were measured by the Bio Tek Instruments
microplate and the results were showed in Table 1. All
probes presented excellent fluorescent properties (Figure
S1). The fluorescence quantum yields of probes 1-3 is 2-5%
in the PBS (pH=7.4), while the fluorescence quantum
yields of probes 1-3 presented a huge increased in the
DMSO solution (27.61-31.42%). These results indicated that
probes 1-3 possessed the environment-sensitive turn-on
mechanism. We also test whether probes 1-3 own turn-on
mechanism to PDEδ protein, from the emission spectrum
of probes 1-3 (1 μM) before and after adding PDEδ protein,
we found that the fluorescence intensity of probes 1-3 were
obviously increased when incubated with PDEδ protein
than incubated probes 1-3 or PDEδ protein alone (Figure
2). And the fluorescence intensity gradually increased with
the increase of PDEδ protein concentration (0-2 μM). The
results shown that probes 1-3 own turn-on response to
PDEδ protein. Which provide many advantages in the
following tests.
Table 2. Biological Data of Probes 1-3
IC50 (μM)
compd
KD2 (nM)
MIA PaCa-2
37.7±4.18
24.7±4.48
15.9±1.28
Capan-1
43.4±8.83
30.9±7.88
33.4±8.25
35.3±1.54
Probe 1
Probe 2
Probe 3
682 ± 91
440 ± 73
501 ± 82
Deltazinone 1.06 ± 0.55
61.6±4.07
Fluorescence Imaging. Based on the excellent
fluorescent properties of probes 1-3, which were used into
detecting and imaging PDEδ protein in the living cell lines:
KRAS-dependent MIA PaCa-2 cells and Capan-1. The MIA
PaCa-2 living cell imaging results indicated that probes 1-3
possess strong fluorescence and could rapidly response to
the PDEδ protein in cells, when co-staining with the
nuclear dye 500 nM Hoechst 33342, we found that the our
probes 1-3 mainly labeled in the plasma membrane, which
demonstrated that PDEδ protein was mainly located in the
plasma membrane instead of the nuclear (Figure 3-5).
Furthermore, we also set MIA PaCa-2 and Capan-1 cell lines
co-incubated with 5 μM probes 1-3 and 100 μM deltazinone,
respectively. The results showed that the fluorescence
intensity of probes were decreased by deltazinone, which
indicated that deltazinone could competitively binding to
PDEδ with our probes. We also chosen the normal HEK
Table 1. Fluorescent Properties of Probe 1-3
λmax
(nm)
λex
(nm)
λem
Φ(%) Φ(%)
compd
(nm) PBS DMSO
Probe 1 337/476 340/470
Probe 2 342/476 345/475
Probe 3 342/476 345/480
555
555
555
4.99
3.21
27.74
27.61
31.42
2.46
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293 cell line, as the negative control, when HEK 293 cells
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incubated with 5 μM probes 1-3 in the same condition,
respectively. The results demonstrated that the
fluorescence in HEK 293 cell imagings are weaker than that
in the oncogenic KRAS-dependent cell lines, which is in
line with our hypothesis. The results provided that our
probes 1-3 displayed favorable selectivity for the oncogenic
KRAS-dependent cell lines. The Capan-1 cell imaging
results were in line with MIA PaCa-2 cell imaging results
(Figures S4-6).
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Figure 5. Fluorescence imagings of MIA PaCa-2 and HEK
293 cell lines incubated with probe 3 (5 μM; Bright field:
A1-C1; GFP channel: A2-C2; DAPI channel: A3-C3; merged
image: A4-C4). A: MIA PaCa-2 cells incubated with 5 μM
probe 3; B: MIA PaCa-2 cells co-incubated with 5 μM probe
3 and 100 μM deltazinone; C: HEK 293 cells incubated with
5 μM probe 3. Scale bar=67 μm.
Flow Cytometry (FCM) Assay. The binding ability to MIA
PaCa-2 living cell line of probes 1-3 were further validated
by flow cytometry (FCM). The FCM results exhibited the
fluorescence intensity of MIA PaCa-2 incubated with 5 μM
probes were stronger than of MIA PaCa-2 co-incubated
with 5 μM probes and 100 μM deltazinone, which indicated
that the binding capability of our probes 1-3 to the MIA
PaCa-2 cells was much higher than to MIA PaCa-2 cells co-
incubated with 5 μM probes and 100 μM deltazinone
(Figure 6), which is in line with the fluorescence
microscopic imaging results in MIA PaCa-2 cells. And the
FCM results of Capan-1 cell line were disclosed in Figure
S7. Further details can be found in the Supporting
Information.
Figure 3. Fluorescence imagings of MIA PaCa-2 and HEK
293 cell lines incubated with probe 1 (5 μM; Bright field: A1-
C1; GFP channel: A2-C2; DAPI channel: A3-C3; merged
image: A4-C4). A: MIA PaCa-2 cells incubated with 5 μM
probe 1; B: MIA PaCa-2 cells co-incubated with 5 μM probe
1 and 100 μM deltazinone; C: HEK 293 cells incubated with
5 μM probe 1. Scale bar=67 μm.
Figure 4. Fluorescence imagings of MIA PaCa-2 and HEK
293 cell lines incubated with probe 2 (5 μM; Bright field:
A1-C1; GFP channel: A2-C2; DAPI channel: A3-C3; merged
image: A4-C4). A: MIA PaCa-2 cells incubated with 5 μM
probe 2; B: MIA PaCa-2 cells co-incubated with 5 μM probe
2 and 100 μM deltazinone; C: HEK 293 cells incubated with
5 μM probe 2. Scale bar=67 μm.
Figure 6. Flow cytometry results of 5 μM probes 1-3 and
100 μM deltazinone binding to the living cells of MIA PaCa-
2. A: blank; B: 100 μM deltazinone; C: 5 μM probe 1; D: 5
μM probe 1+100 μM deltazinone; E: 5 μM probe 2; F: 5 μM
probe 2+100 μM deltazinone; G: 5 μM probe 3; H: 5 μM
probe 3+100 μM deltazinone.
Detecting the Relative Expression Level of mRNA by
qPCR. To well analyze the oncogenic KRAS-dependent
MIA PaCa-2 cells incubated with probes 1-3, whether can
change the transcriptional levels of related genes encoding
KRAS and PDEδ after treated by probe 1-3 The qPCR results
showed that the MIA PaCa-2 cells incubated with probes 1-
3 for 4 hours, the transcriptional levels of KRAS and PDEδ
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Figure 8. The protein expression level treated with probe
2 and detecting by Western Blot (MIA PaCa-2 cells). A: the
band images of protein; B: the quantitative results of
protein by the gray values of band from A with Image J
software, respectively.
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control group (with 0.1% DMSO) (Figure 7), the results
demonstrate that probes 1-3 could significantly reduce the
relative mRNA expression level of KRAS and PDEδ (Figure
7), and probes 1-3 could also mediate MAPK and
PI3K/AKT/mTOR signal pathways, and affect the mRNA
expression level of AKT1, MAPK1, MEK7, RAF1 and mTOR,
from the qPCR results we found these genes were also
down-regulated by probes 1-3, and interfered cell growth,
proliferation, differentiation and apoptosis.7,27,30
Hematoxylin-Eosin Staining(HE) and IHC Assay. The
HE staining results shown that the Capan-1 and HeLa
tumor slices showed high level of diseased. To furture
detecting the protein level of KRAS in tissue level, IHC
assay was carried out. The IHC relusts showed that the
anti-KRAS antibody could selectively label the oncogenic
KRAS tumor, Capan-1 tumor slice was staining the highest
immunostaining with anti-KRAS antibody (Proteintech,
Wuhan, China), while the normal mice skin tissue showed
the least staining (Figure 9). The results indicated the
Capan-1 tumor expression more KRAS protein than HeLa
tumor and normal mice skin tissue. The IHC results
provided the bases for the tissue slices imaging test.
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Figure 9. IHC of Capan-1 tumor, HeLa tumor and the
normal skin of BALB/c nude mice forelimb armpit slices.
A1, A2 and A3 are the HE staining of Capan-1 tumor, HeLa
tumor and the normal skin of BALB/c nude mice forelimb
armpit, respectively; B1, B2 and B3 are the Capan-1 tumor
slice, HeLa and the normal mice skin slices included with
primary antibodies Anti-KRAS antibody (1:150, Catalog
Number: 12063-1-AP, Proteintech), respectively. An
OLYMPUS VS120 virtual slide microscope was performed
the imaging with a 10× objective len.
Figure 7. The mRNA expression level dected by qPCR. A:
KRAS; B: PDEδ; C: AKT1; D: MAPK1; E: MEK7; F: RAF1; G:
mTOR and H: cluster analysis of genes in each groups. All
the histograms were present with mean ± SEM, all data
were analysed by one-way ANOVA followed by multiple
comparisons. The level of significance was defined as
*p<0.05, **p<0.01 and ***p<0.001 vs. control group.
Detecting the Protein Level by Western Blot. We also
used the western blot to detect the protein level of PDEδ,
tErk, and pErk, pAkt and tAkt in MIA PaCa-2 cell line. As
depicted in Figure 8, probe 2 could down-regulate the
level of tErk, and pErk, which is consistent with the qPCR
results, and the ratio of p-Erk/t-Erk showed a dose-
dependent manner. Moreover, probe 2 can mediate the
MAPK and PI3k/AKT/mTOR signal pathways.
Tissue Slices Imaging of Probes 1-3. We also evaluated
whether probes 1-3 could selectively detect PDEδ protein
in the tissue slices. When the tissue slices were antigen
repaired, and then incubated with 10 μM probes 1-3 for 12h.
The results (Figure 10) indicated that Capan-1 tumor slices
could be labelled with probe 1-3 and accompany with
stronger fluorescence; while the fluorescence intensity of
HeLa and normal mice skin tissue slice is relative weaker
than Capan-1 tumor slice incubated with probes 1-3. And
the fluorescence intensity of Capan-1 tumor slice incubated
with probe and 200 μM deltazinone decreased obviously
compared with Capan-1 tumor slice incubated probes 1-3
alone, the results are consist with the cell fluorescence
imaging results. The tissue sections imaging results
indicated that probes 1-3 could be used into tumor
diagnosis in the future.
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Analytical Chemistry
ZR2018ZC0233), the Taishan Scholar Program at Shandong
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Province, the Key Research and Development Project of
Shandong Province (No. 2017CXGC1401) and the
Innovation Program of Shanghai Municipal Education
Commission (No. 2019-01-07-00-07-E00073).
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CONCLUSION
In the current study, we designed and synthesized three
novel small molecule fluorescent probes 1-3 for PDEδ
protein, with reasonable fluorescent properties, and high
feasibility into detecting and imaging PDEδ protein. After
bioactivity evaluation, our probes can be applied into living
cell lines and tumor tissues slices imaging, they can down-
regulate the mRNA expression of KRAS, PDEδ, AKT1,
MAPK1, MEK7, RAF1 and mTOR, and decrease the protein
level of Erk and pErk. Compared with immunofluorescence
or fluorescent protein-based techniques, these novel non-
peptide small-molecule probes for PDEδ protein are more
affordable, rapid, convenient and with turn-on
mechanism, and thus resulting in the development of a
brand-new type of anticancer candidates. Furthermore,
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ASSOCIATED CONTENT
Supporting Information
More detail of synthetic experiment procedures, NMR and
MS spectra. The Supporting Information is available free of
charge on the ACS Publications website at DOI: 10.1021/.
AUTHOR INFORMATION
Author Contributions
(12) Malumbres, M.; Barbacid, M. RAS oncogenes: the first
30 years. Nat. Rev. Cancer. 2003, 3, 459-465.
The final manuscript was approved by all authors.
Conflict of Interest Disclosure
(13) Zorde Khvalevsky, E.; Gabai, R.; Rachmut, I. H.;
Horwitz, E.; Brunschwig, Z.; Orbach, A.; Shemi, A.; Golan,
T.; Domb, A. J.; Yavin, E.; Giladi, H.; Rivkin, L.; Simerzin,
A.; Eliakim, R.; Khalaileh, A.; Hubert, A.; Lahav, M.;
Kopelman, Y.; Goldin, E.; Dancour, A.; Hants, Y.; Arbel-
Alon, S.; Abramovitch, R.; Shemi, A.; Galun, E. Mutant
KRAS is a druggable target for pancreatic cancer. Proc.
Natl. Acad. Sci. U.S.A. 2013, 110, 20723-20728.
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The project is supported by the National Natural Science
Foundation of China (Nos. 21738002 and 81725020), the
Shandong
Natural
Science
Foundation
(No.
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