Ultrasensitive Fluorescent Probes Reveal an Adverse Action of

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Ultrasensitive Fluorescent Probes Reveal an Adverse Action of Dipeptide Peptidase IV and Fibroblast Activation Protein during Proliferation of Cancer Cells Qiuyu Gong, Wen Shi, Lihong Li, Xiaofeng Wu, and Huimin Ma Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02231 • Publication Date (Web): 22 Jul 2016 Downloaded from http://pubs.acs.org on July 27, 2016

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Analytical Chemistry

Ultrasensitive Fluorescent Probes Reveal an Adverse Action of Dipeptide Peptidase IV and Fibroblast Activation Protein during Proliferation of Cancer Cells Qiuyu Gong,†,‡ Wen Shi,† Lihong Li,† Xiaofeng Wu† and Huimin Ma*,†,‡ †

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for

Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China ‡

University of Chinese Academy of Sciences, Beijing 100049, China

Corresponding Author *E-mail: [email protected]; phone: +86-10-62554673; fax: +86-10-62559373

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Abstract Dipeptide peptidase IV (DPPIV) and fibroblast activation protein (FAP) are isoenzymes. Evidence shows that DPPIV is related to anti-tumor immunity, and FAP may be a drug target in the cancer therapy, seeming that the two enzymes might have a synergistic role during the proliferation of cancer cells. Surprisingly, herein we find an adverse action of DPPIV and FAP in the proliferation process by analyzing their changes with two tailor-made ultrasensitive fluorescent probes. First, the up-regulation of DPPIV and down-regulation of FAP in cancer cells under the stimulation of genistein are detected. Then, we find that MGC803 cells with a higher FAP but lower DPPIV level than SGC7901 cells exhibit a faster proliferation rate. Importantly, inhibiting the DPPIV expression with siRNA increases the proliferation rate of MGC803 cells, whereas the FAP inhibition decreases the rate. These findings suggest that the two enzymes play an adverse role during the proliferation of cancer cells, which provides us a new viewpoint for cancer studies.


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INTRODUCTION Dipeptide peptidase IV (DPPIV), also known as CD26, is found in many cells and tissues,1-4 and plays a role in several physiological processes.5-11 Current evidence shows that DPPIV is related to anti-tumor immunity,12,13 which provides us a new thought for cancer therapy; moreover, fibroblast activation protein (FAP), belonging to the isoenzyme of DPPIV, may be a new target in cancer therapeutics.14-16 Thus, it seems that DPPIV and FAP might have a synergistic role during the proliferation of cancer cells. Surprisingly, herein we find an adverse action of DPPIV and FAP in the proliferation process by analyzing their changes with two tailor-made ultrasensitive fluorescent probes. As is known, the purpose of developing a new spectroscopic probe is for analysis and detection of a target of interest,17-19 therefore the most important evaluation standard is whether the developed probe exhibits excellent analytical performance (e.g., high sensitivity, selectivity, and applicability) or not. On the above basis, the simpler both the design and synthesis of the probe the better. This rule means that a probe even with a long and complicated synthetic route but poor performance would not be a good choice. In contrast, the idea of obtaining a superior probe by utilizing either new synthetic chemistry or the smart modification of the existing fluorochromes should be encouraged. Regretfully, an irrational phenomenon in this field seems appearing that excessive attention is paid to the longer and more complicated synthetic route to a probe instead of its analytical performance and validated bioapplication. According to the rule discussed above, the previous DPPIV and FAP fluorescent probes are unsuited to image the changes of DPPIV and FAP in live cells due to their insufficient sensitivity and/or short analytical wavelengths.20-22 It is also noted that nile blue-based fluorescent probes have long analytical wavelengths,23,24 but they suffer from several drawbacks such as rather low fluorescence quantum yields (Φ ≈ 0.1), high background fluorescence and low sensitivity (signal/background ≈ 7 only), implying their incapacity to monitor the subtle change of the two enzymes in cells. In this work, therefore, we develop two new probes 1 and 2 (Figure 1A), which are constructed by using cresyl violet as a superior fluorochrome (Φ ≈ 0.5), Boc-protected Gly-L-Pro and de-protected Gly-L-Pro as a recognition unit for 3 Environment ACS Paragon Plus


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FAP and DPPIV, respectively. The probes show high sensitivity (detection limits, 0.35 ng/mL for DPPIV; 2.7 ng/mL for FAP). Using the probes combined with confocal fluorescence microscopy, we detect the up-regulation of DPPIV and down-regulation of FAP in cancer cells (MGC803 or HO8910PM cells), respectively, under the treatment of a drug (genistein), as further confirmed by western blot and immunohistochemical (IHC) assays. Moreover, we find that MGC803 cells with a higher level of FAP but lower level of DPPIV than SGC7901 cells display a faster proliferation rate. Most notably, inhibiting the expression of DPPIV with siRNA increases the proliferation rate of MGC803 cells, whereas the FAP inhibition decreases the rate. These observations reveal that DPPIV and FAP play an adverse role during the proliferation of cancer cells, which provides us a new viewpoint for the study of cancer therapy (for instance, inhibiting FAP and simultaneously up-regulating DPPIV with suitable drugs might be a useful route to cancer studies).

EXPERIMENTAL SECTION Reagents and Materials. (S)-1-(2-((tert-Butoxycarbonyl)amino)acetyl)pyrrolidine-2-carboxylic acid (N-Boc-Gly-L-Pro)

and

linagliptin

were

obtained

from

Ark

Pharm,

Inc.

(USA).

N,N-

Diisopropylethylamine (DIPEA) and cresyl violet acetate were purchased from Acros Organics. Trifluoroacetic acid (HPLC grade) and O-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) were purchased from Alfa Aesar Chemicals. Talabostat mesylate (PT-100) was obtained from MedChem Express Inc. (USA). Genistein, dimethyl sulfoxide (DMSO), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), vildagliptin, reduced glutathione (GSH), leucine aminopeptidase (LAP), esterase, V8 protease, prolidase, trypsin, thrombin, DPPIV, FAP, matrix metalloproteinase-2 (MMP-2) and commercial DPPIV probe (Gly-L-Pro-7-amido-4-methylcoumarin hydrobromide) were purchased from Sigma-Aldrich. Pyroglutamate aminopeptidase 1 (PGP-1) was obtained from State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai (China). Commercial FAP probe (Z-Gly-L-Pro-7-amido-4-methylcoumarin) was obtained from ATT Bioquest, Inc. (USA). RIPA (radio immunoprecipitation assay) lysis buffer (CW2333) was purchased from 4 Environment ACS Paragon Plus

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CWbiotech. Co. Ltd, Beijing (China). Proteins were pure, as judged by Coomassie-stained SDSpolyacrylamide

gel

electrophoresis

(SDS-PAGE).

DPPIV

siRNA

(sequences:

I

=

CACGGCAACACAUUGAAAUTTAUUUCAAUGUG-UUGCCGUGTT, II = GGGUCAUAAAUUGGCAUAUTTAUAUGCCAAUUUAUGACCCTT, III = GAGGGUACGUAACCUCAAUTTAUUGAGGUUACGUACCCUCTT, IV = GCACAGCACACCAACAUAUTTAUAUGUUGGUGUGCUGUGCTT), FAP siRNA (sequences: I’ = CGCCCUUCAAGAGUUCAUATTUAUGAACUCUUGAAGGGCGTT, II’ = CCUGAUCGGCAAUUUGUAUTTAUACAAAUUGCCGAUCAGGTT, III’ = GAUGGCAAGAGCAGAAUAUTTAUAUUCUGCUCUUGCCAUCTT, IV’ = GGUGGAUUCUUUGUUUCAATTUUGAAACAAAGAAUCCACCTT),

poly(vinylidene

fluoride)

membranes

(PVDF

membranes), gel electrophoresis kits, western blot kits, enhanced chemiluminescence kits (ECL), Braford protein assay kits, Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute-1640 medium (RPMI-1640), calf serum, MGC803, MKN28, SGC7901 and HO8910PM cell lines were purchased from KeyGEN BioTECH Co. LTD, Nanjing (China). Lipofectamine 2000 and fetal bovine serum were purchased from Invitrogen (USA). A phosphate buffered saline (PBS: 9 g/L NaCl, 795 mg/L Na2HPO4 and 144 mg/L KH2PO4) of pH 7.4 was obtained from Invitrogen. AntiDPPIV antibody was purchased from ProteintechTM, USA. Anti-FAP antibody was purchased from Signalway Antibody, Inc. (USA). Ultrapure water (over 18 MΩ·cm) from a Milli-Q reference system (Millipore) was used throughout. The stock solution (1.0 mM) of probe 1 or 2 was prepared by dissolving requisite amount of it in DMSO. Stock solutions of other substances were prepared by dissolving in PBS or water. Apparatus. Unless otherwise noted, the instruments used for absorption, fluorescence and pH measurements were the same as described previously.25-27 1H NMR and

13

C NMR spectra were

measured with a Bruker Avance 400 or 600 spectrometer in CD3OD. Electrospray ionization mass spectra (ESI-MS) were measured with an LC-MS 2010A (Shimadzu) instrument. High resolution electrospray ionization mass spectra (HR-ESI-MS) were recorded on an APEX IV FTMS instrument 5 Environment ACS Paragon Plus


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(Bruker, Daltonics). The fluorescence quantum yield (Φ) was determined by using fluorescein (Φ = 0.85 in 0.1 M NaOH) as a standard. The western blot signal was detected using an ECL kit. The SDS-PAGE was operated with an electrophoresis apparatus (Jun Yi, Beijing, China). Cell imaging experiments were operated on a FV 1000-IX81 confocal laser scanning microscope (Olympus, Japan). Image processing was made with Olympus ImageJ software (National Institutes of Health, USA). The cytotoxicity assay was operated in microtiter plate assay system (Molecular Devices, USA). Synthesis of Probes 1 and 2. N-Boc-Gly-Pro (272 mg, 1.0 mmol), HATU (570 mg, 1.5 mmol) and DIPEA (2.0 mmol, 435 µL) were added to 30 mL of CH2Cl2 at 0 °C. After stirring for 40 min, cresyl violet acetate (321 mg 1.0 mmol) was added slowly, and the reaction mixture was further stirred at room temperature for 3 h. Then, the mixture was diluted with ethyl acetate, and washed three times with water (100 mL×3). The collected organic solvent was removed by evaporation under reduced pressure, and the residue as crude product was purified by silica gel chromatography eluted with CH2Cl2/methanol (v/v, 15/1), affording probe 1 as dark red solid (92 mg, yield 18%). The 1H and 13C NMR spectra and the HRESI-MS of probe 1 are shown below in Figures. S1 and S2, respectively. 1H NMR (400 MHz, CD3OD): δ 8.49-8.51 (d, 1H, J = 8 Hz), 8.11 (s, 1H), 7.69-7.70 (s, 3H), 7.49 (s, 1H), 7.21-7.23 (d, 1H, J = 8 Hz), 6.39 (s, 1H), 4.65-4.68 (m, 1H), 3.97-4.10 (m, 2H), 3.67-3.77 (m, 2H), 2.02-2.37 (m, 6H), 1.46 (s, 9H). 13

C NMR (100 MHz, CD3OD): δ 179.2, 173.1, 170.5, 158.4, 133.1, 132.5, 132.0, 130.7, 126.0, 125.2,

118.3, 80.5, 71.3, 62.5, 43.7, 36.4, 30.7, 30.6, 30.2, 28.6, 27.9, 26.8, 25.8, 23.6, 19.3, 14.3. HR-ESI-MS, calcd for C28H30N5O5 [M]+: m/z 516.2247; found: m/z 516.2241. Trifluoroacetic acid (2.5 mL) in CH2Cl2 (2.5 mL) was added dropwise to the solution of probe 1 in 5 mL of CH2Cl2 at 0 °C, and then the reaction mixture was stirred at room temperature for 3 h. The solvent was removed by evaporation under reduced pressure, and the crude product was purified by flash silica gel chromatography eluted with CH2Cl2/methanol (v/v, 5/1), affording probe 2 as crimson solid (52 mg, yield 70%). The 1H and

13

C NMR spectra and the HR-ESI-MS of probe 2 are shown in

Figure S3 and Figure S4, respectively. 1H NMR (600 MHz, CD3OD): δ 8.87-8.88 (d, 1H, J = 4 Hz), 8.31-8.32 (d, 1H, J = 4 Hz), 8.09 (s, 1H), 7.85-7.94 (m, 3H), 7.53 (s, 1H), 6.81 (s, 1H), 3.81 (s, 2H), 6 Environment ACS Paragon Plus

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3.47-3.62 (m, 2H), 2.25-2.27 (m, 1H), 1.99-2.10 (m, 3H), 1.88 (s, 2H). 13C NMR (100 MHz, CD3OD): δ 172.9, 166.6, 166.0, 153.8, 146.2, 145.4, 143.6, 135.1, 130.6, 126.8, 125.6, 125.3, 119.8, 106.7, 100.0, 62.3, 41.3, 32.8, 27.9, 26.7, 25.5, 23.5, 14.2. HR-ESI-MS, calcd for C23H22N5O3 [M]+: m/z 416.1717; found: m/z 416.1718. General Procedures for DPPIV and FAP Detection. Unless otherwise stated, all the fluorescence measurements were made according to the following procedure. In a test tube, 5 µL of stock solution of probe 1 (or 10 µL of probe 2) and appropriate volume of PBS were mixed, followed by adding an appropriate volume of the solution of DPPIV, FAP or other substances. The mixed solution was adjusted to 1 mL with PBS. After incubation at 37 °C for 30 min (for DPPIV detection) or 180 min (for FAP detection), the reaction solution was transferred to a quartz cell of 1-cm optical length to measure fluorescence with λex/em = 585/625 nm (both excitation and emission slit widths were set to 5 nm for DPPIV, and 10 nm for FAP, respectively). For absorbance measurements, 2 mL of the reaction solution was prepared and used. At the same time, a blank solution without DPPIV or FAP was prepared and measured under the same conditions for comparison. Data are expressed as mean ± standard deviation (SD) of three separate measurements. Cell Imaging. Cells (MGC803, HO8910PM or MKN28) were treated at 37 °C for 24 h with various concentrations of genistein in Petri dishes. Before cell imaging, the culture media were removed, and the cells were washed using RPMI-1640 for three times. Then, the cells were incubated with probe 1 (5 µM) or probe 2 (10 µM) at 37 °C for 30 min (DPPIV detection) or 60 min (FAP detection) in RPMI-1640, washed with RPMI-1640 to remove the free probe, and subjected to fluorescence imaging experiments. SGC7901 cells were incubated directly with probe 1 (5 µM) or probe 2 (10 µM) for imaging experiments. Unless otherwise noted, data are expressed as mean ± standard deviation (SD) of three separate measurements. Immunohistochemical (IHC) Assay. Similar to the reported method,28 IHC images were obtained in KeyGEN BioTECH Co. LTD, Nanjing (China). The integral optical density values in the images represent the relative concentrations of intracellular DPPIV or FAP. 7 Environment ACS Paragon Plus


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Transfection. The siRNA-transfected MGC803 cells were obtained according to the following procedure. MGC803 cells were cultured at 37 °C for one day before transfection. The corresponding siRNA at a concentration of 0.4 µM was prepared by dissolving in DMEM (Dulbecco’s modified Eagle’s medium), and lipofectamine 2000 was diluted 50 times by DMEM. The siRNA and lipofectamine 2000 solutions were then mixed well. The mixture was added to Petri dishes, and the cells were cultured at 37 °C for 6 h. Then the culture medium was discarded, and the fresh RPMI-1640 medium was added. The cells were incubated at 37 °C for 72 h for further use. Western Blot. The western blot analyses were made by using glyceraldehyde-3-phosphate dehydrogenase, GAPDH, as a protein standard.29 Viabilities of MGC803 and SGC7901 Cells. The viabilities of MGC803 and SGC7901 cells were measured as follows. Cells were seeded in 96-well U-bottom plates at a density of 7000 cells/well, and incubated at 37 °C for 1-5 days. Then, the absorbance at 490 nm was recorded using a standard MTT assay with microtiter plate assay system, and the cell viability was calculated according to the previous method.30 Cytotoxicity Assay. The cytotoxicity of probe 1 or 2 was tested on MGC803 cells using a standard MTT assay, as described previously.30 Statistical Tests. The t analysis was made according to our previous method.30

RESULTS AND DISCUSSION Synthesis of Probes and Their Spectroscopic Properties. As shown in Figure 1, probes 1 and 2 were

readily

prepared

by

treating

cresyl

violet

with

(S)-1-(2-((tert-

butoxycarbonyl)amino)acetyl)pyrrolidine-2-carboxylic acid, followed by removing the protecting group in the presence of trifluoroacetic acid (Figures S1-S4). Here, cresyl violet is chosen as a fluorescent skeleton, because substitution of its amino group usually causes complete fluorescence quenching,30 which would favor low background signal and thus sensitive detection.


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A OH N O C

N

O

O O

NH

HATU, DIPEA

N H

N

cresyl violet acetate

O

NH 2

O N H

O

O

FAP

cresyl violet

CH 3 COO -

O C

1 (18% yield) CF 3COOH

N

O N

N H

DPPIV

O

NH2 CF 3COO -

NH2

O

1 2

B 1

2

0.05

C

0.2

450 D ∆F

600 400 200 0 0.0 0.3 0.6 0.9 C (µg/mL)

0 630 675 720 Wavelength (nm)

450 600 750 Wavelength (nm) Intensity (a.u.)

450 600 750 Wavelength (nm)

150

1

0.0

0.00

300

12 2

150 E

120 ∆F

0.10

0.4 Absorbance

Absorbance

2 (70% yield)

Intensity (a.u.)

1 2 3 4 5 6 7 8 9 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


100

80 40 0

0

20

40

60

C (ng/mL)

50 0

630 675 720 Wavelength (nm)

Figure 1. Synthesis of probes and their spectroscopic properties. (A) Synthesis of probes and their reactions with enzymes. (B) Absorption spectra of 1 (5 µM) before (curve 1) and after (curve 2) reaction with FAP (1.5 µg/mL). The inset shows the corresponding color change. (C) Absorption spectra of 2 (10 µM) before (curve 1) and after (curve 2) reaction with DPPIV (0.2 µg/mL). The inset shows the corresponding color change. (D) Fluorescence response of 1 (5 µM) to FAP at different concentrations (from bottom to top: 0 - 1.5 µg/mL). The inset shows the plot of ∆F versus the FAP concentration. (E) Fluorescence response of 2 (10 µM) to DPPIV at different concentrations (from bottom to top: 0 - 0.8 µg/mL). The inset shows the plot of ∆F versus the DPPIV concentration. λex/em = 585/625 nm. 9 Environment ACS Paragon Plus


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Then, spectroscopic properties and analytical performances of probes 1 and 2 were studied. As shown in Figure 1, both of the probes exhibit an absorption peak at about 494 nm, but reaction of 1 with FAP or 2 with DPPIV produces a distinct color change, accompanied by a red-shifted absorption peak to about 585 nm (Figures 1B and 1C) and a large fluorescence off-on response at 625 nm (Figures 1D and 1E; the quantum yields of both the probes themselves, Φ < 0.01). It is worth noting that the absorption and fluorescence spectra from the reaction systems accord well with those from cresyl violet,30-32 suggesting the release of cresyl violet (Φ = 0.51), which is further verified by mass spectral analysis (m/z = 262.1 [M]+; Figure S5). The inhibitor experiments also support that the fluorescence off-on responses result from the action of the corresponding enzyme (Figure S6), because the presence of the inhibitors can largely decrease the enzymatic activity (i.e., diminishing fluorescence). Under the optimized conditions (reaction with FAP and DPPIV for 3 h and 30 min, respectively, at 37 °C in pH 7.4 PBS; Figures S7 and S8), probe 1 exhibits a good linear fluorescence response to FAP in the concentration range of 20 - 900 ng/mL (see also inset of Figure 1D), with an equation of ∆F = 5.44 × C (ng/mL) + 0.46 (R = 0.996), where ∆F is the difference of fluorescence intensity of the probe after and before reaction with the enzyme; probe 2 displays a linear fluorescence response to DPPIV in the concentration range of 2 – 60 ng/mL (Figure 1E), with an equation of ∆F = 10.18 × C (ng/mL) + 1.63 (R = 0.999). The detection limits33,34 of probes 1 and 2 were determined to be 2.7 ng/mL FAP and 0.35 ng/mL DPPIV, respectively, which are about 4 times more sensitive than those from the previous FAP and DPPIV probes (Figure S9). The present ultrasensitive fluorescent probes make the detection and imaging of intracellular DPPIV and FAP possible. Furthermore, probe 1 shows high selectivity for FAP over various potential interfering substances tested (the relative error

Ultrasensitive Fluorescent Probes Reveal an Adverse Action of

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