Article pubs.acs.org/ac
Electrochemical Approach for the Development of a Simple Method for Detecting Cell Apoptosis Based on Caspase‑3 Activity Shinichiro Takano,† Shusaku Shiomoto,† Kumi Y. Inoue,*,†,‡ Kosuke Ino,† Hitoshi Shiku,†,§ and Tomokazu Matsue*,†,‡,§ †
Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki, Aoba, Sendai 980-8579, Japan Micro System Integration Center, Tohoku University, 519-1176 Aramaki, Aoba, Sendai 980-0845, Japan § Advanced Institute for Materials Research, Tohoku University, 2-1-1 Katahira, Aoba, Sendai 980-8577, Japan ‡
S Supporting Information *
ABSTRACT: This paper reports a novel approach for the simple detection of cell apoptosis using an electrochemical technique. This method uses caspase-3 activity as an indicator of apoptosis. Caspase-3 activity was detected with differential plus voltammetry (DPV) as an alternative to conventional spectrometry. In this method, p-nitroaniline (pNA) released from Asp-Glu-Val-Asp-pNA by caspase-3 enzyme reaction was measured with DPV by using a glassy carbon electrode. Using this method, we successfully detected cell apoptosis occurring inside living HepG2 cells without the need for a cell lysis step. This method provides an easy assay procedure and, more importantly, allows a live cell apoptosis detection format. This novel electrochemical apoptosis assay using living cells instead of typically used cell lysates will expand the applicable range of the apoptosis assay to include cell activity assays for drug discovery and cell transplantation medicine.
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sensitivity. Additionally, photometric assays are generally difficult to miniaturize into a compact device, which would be required for on-site analysis. A new technique for a photometrical caspase-3 assay has also been developed using fluorescence resonance energy transfer (FRET),15 a novel long-wave fluorescent probe,16 and surface plasmon resonance (SPR).17 However, they still have problems in terms of miniaturization and the cost of equipment. As an alternative to photometry, electrochemical detection potentially provides an easier method for apoptosis assays that could be presented in a compact device. However, only a limited number of papers have been published on the electrochemical method for the apoptosis assay, including impedance metric detection of morphology changes of the apoptotic cells,18 voltammetric detection of the translocation of the membrane phosphatidylserine using an annexin V-modified electrode,19 voltammetric analysis of intracellular redox activity changes,20 and a caspase-3 assay using a DEVD-peptidemodified electrode with voltammetry.21,22 We have previously developed simple electrochemical protease assay methods for endotoxin detection.23−25 In this method, we detected pNA released from Val-Pro-Arg-pNA or Leu-Gly-Arg-pNA with differential plus voltammetry (DPV). These works are based on
poptosis is a highly regulated process for eliminating unwanted cells from the body during normal cell turnover, development and function of the immune system, and tissue remodeling. Disorder of apoptosis regulation is associated with various diseases, including cancer, neurological disorders, cardiovascular disorders, and autoimmune diseases.1 Therefore, methods for assaying apoptosis are required, not only for biological science but also for use in many other areas such as medical treatment, pharmaceutical development, and food safety testing.2,3 There are huge numbers of established and developing assay methods for apoptosis,4,5 including methods based on the observation of cell morphology changes,6 the detection of DNA fragmentation,7 annexin V staining,8 and the detection of the release of cytochrome c from the mitochondria.9 Caspases make up a family of cysteine proteases that play key roles in apoptosis.10−12 Caspase-3 is especially important because it takes part in both internal and external apoptosis pathways13 and is therefore often targeted to detect apoptosis. Because caspase-3 specifically recognizes and cleaves the Cterminus of Asp-Glu-Val-Asp (DEVD), fluorometric and colorimetric methods are conventionally employed for apoptosis assays based on caspase-3, which use DEVD-4methyl-coumaryl-7-amide (DEVD-MCA) and DEVD-p-nitroaniline (DEVD-pNA) as substrates for active caspase-3.14 Commercially available kits are also provided for caspase-3based apoptosis assays; however, these photometric assays require laborious preparation of cell extracts to maintain © 2014 American Chemical Society
Received: October 18, 2013 Accepted: April 26, 2014 Published: April 26, 2014 4723
dx.doi.org/10.1021/ac403394z | Anal. Chem. 2014, 86, 4723−4728
Analytical Chemistry
Article
the results of the earlier studies done by Heiduschka et al.26 and Mir et al.,27 with respect to the DPV detection of protease activities using peptidyl substrates. As this method was considered potentially applicable to caspase-3 detection, we used it to provide a novel electrochemical apoptosis assay. In this study, we employed DPV to detect pNA released from DEVD-pNA by a caspase-3 enzyme reaction. We first confirmed DPV detection of pNA in a mixture of pNA and DEVD-pNA solutions. The activity of caspase-3 was then detected with DPV by using DEVD-pNA as a substrate for caspase-3. Finally, we detected the caspase-3 activity of a human liver carcinoma cell line (HepG2). In addition, we successfully eliminated the cell lysis, centrifugation, and debris removal steps included in the assay protocol used for conventional photometric detection, taking advantage of the electrochemical detection method. This novel protocol provides not only a simple assay procedure but also an assay that may be used for living cells. The availability of an apoptosis assay that uses living cells as opposed to the cell lysate will expand the applicable range of the apoptosis assay to include cell activity assays for drug discovery and cell transplantation medicine.
Apoptosis Assay Using Cell Samples. HepG2 cells (Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University) were used as the selected cell type for the apoptosis assay. To induce apoptosis, two types of carcinostatic agents, Dox and MMC, were used. HepG2 cells were seeded in 25 cm2 tissue culture flasks (Falcon, Becton, Dickinson and Co.) at a density of 1.5 × 106 cells/flask in DMEM supplemented with 10% fetal bovine serum and 50 μg mL−1 penicillin-streptomycin. Cells were cultured at 37 °C in a humidified atmosphere with 5% CO2. After cells had been cultured for 1 day, the medium was exchanged with apoptosis-inducing medium containing 5.0 μg mL−1 Dox or 5.0 μg mL−1 MMC.28 As a control, we use a medium without carcinostatic agent. Cell cultures were continued for 1 day and then used for the apoptosis assay. The apoptosis assay was performed as follows. The cells were transferred to a centrifuge tube after being detached from the flask with a 0.25% trypsin-EDTA solution. The cell solution was adjusted to a density of 1.5 × 106 cells/tube and centrifuged at 400g for 10 min at 4 °C. After the supernatant had been removed, the cell pellet was resuspended with 200 μL of icecold Cell Lysis Buffer. The cell suspension was placed on ice for 10 min and then centrifuged at 10000g for 5 min at 4 °C. The supernatant was then used as the cell extract for analysis. In a well of a 96-well plate, 100 μL of cell extract, 100 μL of 2× Reaction Buffer (10 mM DTT), and 10 μL of 10 mM DEVDpNA were mixed and incubated at 37 °C for 0−4 h. DPV or spectrophotometry was then performed to detect pNA.
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EXPERIMENTAL SECTION Chemicals and Apparatus. Active caspase-3 and DEVDfluoromethylketone (DEVD-FMK) were purchased from Medical & Biotechnological Laboratories Co., Ltd. Dulbecco’s phosphate-buffered saline (−) (PBS) and doxorubicin hydrochloride (Dox) were obtained from Wako Pure Chemical Industries, Ltd. Mitomycin-C (MMC) was purchased from Sigma-Aldrich Co. (St. Louis, MO). DMEM, fetal bovine serum, a 0.25% trypsin-EDTA solution, and penicillinstreptomycin were purchased from Gibco Life Technologies Co. Fetal bovine serum was used after being heat inactivated at 56 °C for 30 min. Electrochemical Measurements. Electrochemical measurements were performed using a potentiostat (CompactStat, Ivium Technologies B.V.) with an Ag/AgCl electrode used as a reference electrode, a Pt plate used as a counter electrode, and a glassy carbon disc electrode (BAS Inc., diameter of 1.0 mm) used as a working electrode. DPVs were performed with a pulse amplitude of 50 mV, a pulse width of 70 ms, a pulse period of 1 s, and a scan rate of 5 mV s−1. Spectrophotometry. Spectrophotometry was performed using a microplate reader (model 680, Bio-Rad Laboratories, Inc., Hercules, CA). The absorbance of the samples in a 96-well plate (model 9102, Corning Inc., Corning, NY) was measured at 405 nm. Caspase-3 Activity Assay Using Active Caspase-3. The caspase-3 activity assay was performed using the APOPCYTO Caspase-3 Colorimetric Assay Kit (Medical & Biotechnological Laboratories Co., Ltd.). This kit contains Cell Lysis Buffer, 2× Reaction Buffer, 1.0 M dithiothreitol (DTT), pNA, and DEVDpNA. DTT was diluted with 2× Reaction Buffer just before being used to yield a final DTT concentration of 10 mM. The active caspase-3 sample solution was prepared using Cell Lysis Buffer. Then, 100 μL of 2× Reaction Buffer containing 10 mM DTT, 100 μL of the sample solution, and 10 μL of 10 mM DEVD-pNA were mixed in a well of a 96-well plate. The mixed solution was incubated at 37 °C for 1 h to allow the hydrolysis of DEVD-pNA to proceed by the caspase-3 enzyme reaction. DPV or spectrophotometry was then performed to detect the liberated pNA.
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RESULTS AND DISCUSSION Detection of pNA in Mixed Solutions of pNA and DEVD-pNA with DPV. Preliminary evaluation of test samples consisting of mixed solutions of pNA and DEVD-pNA at various ratios was performed before measurements using caspase-3 or actual cell samples were tested. To prepare test samples, 10 μL of a 10 mM mixture solution of pNA and DEVD-pNA was diluted with 100 μL of Cell Lysis Buffer and 100 μL of 2× Reaction Buffer (containing 10 mM DTT) in the kit. Figure 1A shows differential pulse voltammograms of test samples. Both DEVD-pNA and pNA show a reduction peak current between −0.74 V versus Ag/AgCl and −0.75 V versus Ag/AgCl. However, the reduction peak current per unit concentration of pNA is 7.3 times higher than that of DEVD-pNA, based on background-subtracted data. This may due to the difference in the diffusion coefficients of DEVD-pNA and pNA. Therefore, the peak current increased with the concentration of pNA in the mixture solution with a constant total concentration of DEVD-pNA and pNA. Figure 1B shows the dependence of peak currents on pNA concentration. Each plot corresponds to a mean value of peak currents, and the error bars indicate the standard error obtained from three independent measurements. This graph exhibits the great linearity of the peak currents of DPV against the pNA concentrations with a correlation coefficient of 0.998. This result indicates the possibility of the detection of caspase-3 activity with DPV by using DEVD-pNA as a substrate. Detection of Caspase-3 Activity with DPV. As a model experiment, caspase-3 activity was detected by DPV. A mixture of 100 μL of a caspase-3 solution (from 0 to 10 units/well prepared with Cell Lysis Buffer), 100 μL of 2× Reaction Buffer (containing 10 mM DTT), and 10 μL of 10 mM DEVD-pNA were added to a 96-well plate and incubated for 1 h at 37 °C. Electrodes were then inserted into the wells to perform DPV. 4724
dx.doi.org/10.1021/ac403394z | Anal. Chem. 2014, 86, 4723−4728
Analytical Chemistry
Article
Figure 1. (A) Differential pulse voltammetric responses for mixtures of DEVD-pNA and pNA. The total concentration of DEVD-pNA and pNA was 476 μM. Ratios of DEVD-pNA to pNA were 0:1, 0.25:0.75, 0.5:0.5, 0.75:0.25, and 1:0. The background sample consisted of a mixture of Cell Lysis Buffer and 2× Reaction Buffer. The parameters for DPV were as follows: pulse amplitude of 50 mV, pulse width of 70 ms, pulse period of 1 s, and scan rate of 5 mV s−1. (B) Plots of reduction peak current of DPV vs pNA concentration. Error bars indicate the standard error (n = 3).
Typical voltammograms for high concentrations and low concentrations of caspase-3 are shown in panels A and B of Figure 2, respectively. The reduction peak current at −0.75 V versus Ag/AgCl increased with an increasing caspase-3 concentration. The peak shown in the voltammogram of 0 unit/well for caspase-3 originated from the reduction of DEVDpNA. The small peak at around −0.52 V versus Ag/AgCl was due to the reduction of dissolved oxygen. Figure 2C shows the dependence of peak currents on caspase-3 concentration. Each plot corresponds to the mean value of peak currents, and error bars indicate the standard error obtained from independent measurements. The peak current increased with an increase in caspase-3 concentration up to 4 units/well and became constant over 4 units/well. The same result was obtained in the spectrometric assay (Figure S-1 of the Supporting Information). All DEVD-pNA in a well (0.1 μmol) was hydrolyzed within 1 h with high caspase-3 concentrations of >4 units/well. Under low-caspase-3 conditions of
