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Colorimetric assay of apoptosis through in-situ biosynthesized gold nanoparticles inside living breast cancer cells
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Colorimetric assay of apoptosis through in-situ biosynthesized gold nanoparticles inside living breast cancer cells Yasaman-Sadat Borghei, Saman Hosseinkhani∗ Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
ARTICLE INFO
ABSTRACT
Keywords: Apoptosis assay In-situ synthesize Biosynthesized gold nanoparticles GSH/GSSG redox state Cytochrome c
Abnormalities in apoptosis (More or less than normal rate) is a central factor to detect many human disorders such as many types of cancer and to show therapeutic potential of drugs. During apoptosis, oxidation of reduced gluthation (GSH) to its oxidized form, GSSG, results in a decreased GSH to GSSG ratio. Here, we used altered GSH/GSSG redox state and the release of Cyt c from mitochondria to cytosol as an indicator for apoptosis assay. This study reports a visual method for cell apoptosis assay through in-situ biosynthesized gold nanoparticles (AuNPs) inside livingcells, only after adding a sufficient amount of gold ion. After incubation of apoptotic and non-apoptotic cells with chloroauric acid solution, high level of GSH act as reducing agent in formation of AuNPs using thiol inside living non-apoptotic cells. While in the apoptotic cells, what's happening is based on changes in plasmonic coupling between AuNPs embedded along the oxidized gluthation and Cyt c aggregates in cytosol which causes color changes from red to purple. In this study, we successfully reported cell-based (inside a living cells) and cell free (cell lysis) methods for apoptosis assay of breast cancer cells and we achieved very good results in comparison with a standard apoptosis assay procedure. The linear range for MCF-7 cells detection from 30 to 3 × 105 cells/ml was obtained with a detection limit of 30 cells. In addition, the proposed approach is applicable to detect other apoptotic cells.
1. Introduction The number of cells in a multicellular living organism is regulated not only by control in cell division but also by cell suicide. This suicide that occurs due to the activation of a number of intracellular pathways known as “programmed cell death” or “apoptosis” [1]. Initiation of Apoptosis can be occurred by various external “Extrinsic Apoptosis” and internal “Intrinsic Apoptosis” factors. The extrinsic (death receptor mediated) pathway can be initiated by attachment of death ligand to cell surface receptors. The intrinsic (Bcl-2 inhibit able or mitochondrial) pathway starts in response to different types of cellular stress such as DNA damage, endoplasmic reticulum (ER) stress, lysosomal stress, reactive oxygen species (ROS), and calcium (Ca2+) overload. Following the reception of stress signals, Bcl-2 family members translocated into the mitochondria, which can be changed in mitochondrial permeability and consequent release of cytochrome c (Cyt c) as an essential mitochondrial electron transport releases from the intermembrane space of the mitochondria to the cytosol [1–6]. During apoptosis process, oxidation of reduced gluthation (GSH) to its oxidized form, GSSG, results in a decreased GSH to GSSG ratio. In ∗
fact, reducing thiol proteins is a defense of the cell against oxidative conditions such as apoptosis process [2–4]. While typically, reduced glutathione (GSH, comprised of three amino acids; cysteine-glutamic acid-glycine), with millimolar concentrations in various cell types, due to free thiol groups preserves a cellular redox system (NAD+/NADH, NADP+/NADPH, and GSH/GSSG) for native function of cytosolic proteins [5,6]. Apoptosis is a vital process for organism development and cellular/ tissue homeostasis. So, there is a direct relationship between changes in the rate of apoptosis and various forms of disease [7]. For example, degenerative disorders such as Alzheimers, Parkinsons and autoimmune and infectious diseases caused by too much apoptosis. Conversely, too little apoptosis is often associated with cancer and leukemia. Hence, various methods have been developed for measuring apoptosis, including annexin V staining which is due to a change in cellular membrane asymmetry and flip-flop of phosphatidylserine (PS) residues in plasma membrane, propidium iodide (PI) staining for analysis of DNA Fragmentation, Western blot for detection of cytochrome c release from damaged mitochondria, and mechanism based assays such as caspases activity assay [7,8]. However, each of these traditional methods has its own individual limitations. western blotting is complex
Corresponding author. E-mail address: [email protected] (S. Hosseinkhani).
https://doi.org/10.1016/j.talanta.2019.120463 Received 8 August 2019; Received in revised form 8 October 2019; Accepted 9 October 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Yasaman-Sadat Borghei and Saman Hosseinkhani, Talanta, https://doi.org/10.1016/j.talanta.2019.120463
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Scheme 1. Schematic illustration of in-situ synthesis of gold nanoparticles on the basis of alterations in different redox state of GSSG/2GSH ratio and the release of Cyt c from mitochondria in apoptotic MCF-7 cells. And microscopy images of MCF-7 cells under Visible light from Non-Apoptotic (left) and Apoptotic (right) MCF7 cells.
and needs labeling procedures, which can lead to low efficiency; while, annexin V staining has good detection limits, but the required equipment and materials are costly. Although propidium iodide (PI) staining can be used for dead cells, but it is only applied in late stage of apoptosis. Due to these limitations, developing advanced techniques for apoptosis detection by using novel material and nanomaterial has attracted great attention. Nanomaterials have encouraged and enabled researchers to develop new detection methods for biomedical applications. Among the different types of nanomaterials, gold nanoparticles have attracted tremendous interests because of their optical and electronic properties that are tunable by changing the size, shape, surface chemistry, or aggregation state. For example, the different colorimetric detection method of analytes by using the aggregation of AuNPs have been developed [9–14]. Here, for the first time, we successfully show a detection procedure for apoptosis in living MCF-7 cells (cell-based system) in addition to the cell lysate (cell free system) using spontaneously biosynthesis of gold nanoparticles by living cells or in the presence of cell lysates. In this method, after incubation of non-apoptotic MCF-7 cells with a sufficient
amount of chloroauric acid solution, the high level of gluthations could form and stabilize large gold nanoparticles (AuNPs) based on typically strong gold–sulfur bonds (GSH-gold ion). While in apoptotic MCF7 cells, on one side alteration in GSH/GSSG redox state and low level of GSH causes small AuNPs and on the other side release of cytochromc protein in cytosol causes color changes that result from electronic dipole–dipole coupling between neighboring gold nanoparticles (Scheme 1). 2. Experimental section 2.1. Materials and reagents Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were purchased from Invitrogen, and penicillin/streptomycin was purchased from Gibco (USA). All chemicals used of analytical grade or of the highest purity available. Chloroauric acid (HAuCl4 99.9%) was obtained from Sigma Aldrich. Caspase 3/7 activity was measured by a luminescent assay kit from promega. Used phosphate buffer (PB) contains 20 mmol L−1 NaH2PO4. Hypotonic lysis buffer is 10 mmol L−1 2
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Fig. 1. (A) Microscopy images of MCF-7 cells under Visible light from Non-Apoptotic (left) and Apoptotic (right) MCF-7 cells. (B) the biosynthesized red color AuNPs inside the non-apoptotic cells and the aggregated purple color AuNPs indicating non-apoptotic cells that are dying (showed with the arrow) after incubation with trace amount of Dox (20 nmol L−1 for 15 h).
HEPES (pH:7.5) including 1.5 mmol L−1 MgCl2, 1 mmol L−1 Na-EDTA, 68 mmol L−1 sucrose and 1 mmol L−1 PMSF.
2.4. Cell free apoptosis assay To measure apoptosis in cell free system, Dox-treated cells were collected by centrifugation at 1000 rpm for 5 min after trypsinization and washing with cold PBS. Then the cell pellet (about 3 × 105 cells/ ml) was suspended in 30 μl of cold hypotonic lysis buffer and kept on ice for 10 min. In the final stage after 30 s vortex, it is also centrifuged for 30 s in 12,000 rpm whichresulted in the cytoplasmic content in the supernatant, and the cell debris was pelleted [16]. The supernatant cell lysate was used for proposed colorimetric assay. In this way, 10 μl of cold HAuCl4 (1.0 mmol L−1) were added to 30 μl of supernatant at room temperature for 50 min.
2.2. Cell culture and apoptosis induction The human breast carcinoma MCF-7 cells were cultured in DMEM complemented with 10% FBS and 1% penicillin/streptomycin at 37 °C with 5% CO2 in a humid atmosphere. To induce apoptosis, we usedDoxorubicin (Dox), a well-known Anthracycline-derived chemotherapeutics in apoptosis [15], at different concentration and different time. For this purpose, MCF-7 cells (3 × 105 cell/ml) were seeded in each well of a 48-well plate for overnight, then the cells incubated with Dox at different concentration (0–700 nmol L−1 in DMEM). After 15 h incubation, the cells were washed with PBS buffer. Three replicates were done for each treatment.
2.5. Caspase 3/7 assay Caspase 3/7 assay was done to measure doxorubicin-induced apoptosis in MCF-7 cells. Hence, cells were incubated with various concentrations of doxorubicin (0–700 nmol L−1) for 15 h and then hypotonic cell lysis was used for caspase 3/7 activity assay.
2.3. Cell-based apoptosis assay In order to measure apoptosis in cell-based system, the cells were incubated with Doxorubicin at different concentrations and different times. Then after washing the cells with PBS, they were treated dropwise with various concentration of cold HAuCl4 solution (PB buffer (pH:7.5)) and incubated at 4 °C for 24–48 h. 3
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Fig. 2. (A) UV–Vis of Non-Apoptotic (red line) and Apoptotic (after incubation with Dox (4000 nmol L−1 for 15 h) (purple line) MCF-7 cells after incubation with HAuCl4 solution (0.5 mmol L−1). Inset: photographs of them under Visible light. And (B) DLS analysis on the basis of number distribution (a) and intensity distribution (b) of Non-Apoptotic (black line) and Apoptotic (green line) MCF-7 cells. (C) TEM images are taken from biosynthesized AuNPs by non-apoptotic cells (a–c) and apoptotic cells (d–f); at different scales (40, 80 and 150 nm). 4
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Fig. 3. Optimization of HAuCl4 solution in PB buffer (pH:7.5): Microscopy images of them at different concentration of HAuCl4 solution (0.05, 0.1, 0.5, 1, 1.5 and 2 mmol L−1).
3. Results and discussion
aggregated purple color AuNPs are also visible in the apoptotic cells after 15 h of incubation with Dox (400 nmol L−1) (Fig. 1A (right)). Also, it can be seen in Fig. 1B that the aggregated purple color of AuNPs indicating non-apoptotic cells that goes towards apoptosis after incubation with trace amount of Dox (20 nmol L−1 for 15 h) (showed with the arrow). Also Fig. 2A showed the UV–Vis absorption spectra of non-apoptotic/apoptotic cell@AuNPs over a wavelength range of 400–800 nm. In the non-apoptotic MCF-7 cells the red color solution was observed with an absorption peak at around 535 nm. While in apoptotic MCF7 cells, it produced a color change (purple, Fig. 2A, inset) with an absorption peak around 560 nm. In addition, as shown in Fig. 2B, DLS results give the size distribution by intensity and number. As indicated in Fig. 2B, a, the size distribution of gold nanoparticles in non-apoptotic MCF-7 cells were about 25.0 nm while in apoptotic MCF-7 cells, it was about 24.0 nm which did not show significant difference. In fact, these results are not consistent with our suggestion, because in apoptotic cells, the amount of reducing agents (NADH, NADPH, and GSH) are lower than non-apoptotic cells [2–4,17–19], and therefore, the number of reduced gold ions is less than non-apoptotic. As shown in Fig. 2B, b, relative intensity and size distribution of the biosynthesized AuNPs in non-apoptotic and apoptotic MCF-7 cells were about 58 and 100 nm; respectively. This increase in the light scattering (intensity distribution) in apoptotic cells also shows the more aggregation of biosynthesized AuNPs compared with non-apoptotic cells. We also suggest that these AuNPs aggregations were due to the accumulation of oxidized glutathione and the release of cytochrome c in the cytosol [24]. In order to confirm more, the transmission electron microscopy (TEM) images of the biosynthesized AuNPs in non-apoptotic/apoptotic MCF-7 cells were taken after 15 h of incubation with Dox
3.1. Cell-based assay and characterization Scheme 1 shows the mechanism of proposed colorimetric apoptosis sensor which is based on the difference in redox state of the glutathione/disulfide-glutathione couple (GSSG/2GSH) in non apoptotic/ apoptotic cells and also the release of cytochrome c from mitochondria in apoptotic cells. There are numerous reports confirming that in apoptotic cells, the amount of oxidized glutathion (GSSG) is more than reduced glutathione (GSH) [2–4,17–19]. On the other hand, by induction of apoptosis and activation of the apoptotic pathway, cytochrome c releases from mitochondria and binds to Apaf-1 molecules thereby bring about with formation of apoptosome complex within cell death and differentiation [16,20–23]. So in the presence of HAuCl4, the cells can synthesis gold nanoparticles (AuNPs) inside themselves through reducting conditions such as high content of thiol group of GSH. But in apoptotic cells due to the overcoming of the oxidation conditions and the aggregation of oxidized glutathion (GSSG with a larger molecular structure than GSH) and release of cytochrome c, AuNPs aggregated [24]. The presence of in-situ synthesized AuNPs by MCF-7 cells and their aggregation in apoptotic MCF-7 cells was characterized by invert microscopy in the whole cells under visible light, UV–Visible spectroscopy and Dynamic light scattering (DLS) analysis. For cellular imaging (Fig. 1), the cells were treated with HAuCl4 solutions (0.5 mmol L−1) and incubated at 4 °C for 24 h or 48 h before imaging on an Inverted Microscopy (BX51, Olympus) under visible light using 20x and 40x objective. As shown in Fig. 1A, the biosynthesized red color AuNPs inside the non-apoptotic cells (Fig. 1A (left)) and the 5
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Fig. 4. (A) UV–Vis of Non-Apoptotic (red line) and Apoptotic (purple line) MCF-7 cell lysate after addition of HAuCl4 for 50 min at room temperature. Inset: photographs of them under Visible light. (B) the Scheme depicts the principle mechanism of the proposed sensing system by addition of GSH in apoptotic cell lysate and the addition of Cyt c in non-apoptotic cell lysate. (C) UV–Vis of Non-Apoptotic cell lysate (a), Non-Apoptotic cell lysate after addition of Cyt c (b), Apoptotic cell lysate after addition of GSH (c), And Apoptotic MCF-7 cell lysate (d) Inset: photographs of them under Visible light.
(400 nmol L−1) (Fig. 2C). These images are as good as DLS results; Images (a-c)/(d-f) are taken from AuNPs synthesized by non-apoptotic/ apoptotic cells; respectively. It is well clear that the biosynthesized AuNPs in non-apoptotic cells are bigger (Fig. 2C, a-c) than apoptotic cells, and also the aggregation of AuNPs in apoptotic cells is well visible
(Fig. 2C, d-f). In order to optimize the performance of the assay, concentration of HAuCL4 was optimized. For this purpose, the cells were treated with various concentrations of cold HAuCl4 solution (final concentration in PB buffer (pH:7.5): 0.05, 0.1, 0.5, 1, 1.5 and 2 mmol L−1) and 6
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Fig. 5. (A) Monitoring of apoptosis progress by the activity of caspase 3/7 by a luminescent assay kit as a gold standard method at different concentrations of Dox (0, sample signal at [x ] × 100 . 10, 25, 50, 75, 100, 200, 300, 400, 500, 600 and 700 nmol L−1, for 15 h) and it was compared with the proposed method based on: signal (%) = signal at [Y ]
([Y] is the concentration of Dox in which the maximum amount of signal is obtained and [x] is the other concentrations of Dox.) (B) photographs of them under Visible light. (C)The linear calibration curve of different numbers of MCF-7 cells (3 × 101, 3 × 102, 3 × 103, 3 × 104 and 3 × 105 cells/ml) after incubation with Dox (400 nmol L−1 for 15hr). (D) photographs of them under Visible light.
Fig. 6. The absorption signal at 650 nm for the Dox-treated non-apoptotic MCF-7 cell lysate (e), MCF-7 cells with H2O2–induced apoptosis (d), Dox-treated HEK293 cells (c) and Dox-treated MCF-7 cell ((b) as a positive control) and non treated MCF-7 cells ((a) as a negative control).
incubated at 4 °C (Fig. 3). As shown in Fig. 3, when the HAuCl4 concentration was 0.5 mmol L−1, the cells synthesize more red gold nanoparticles. It appears that at high concentrations of gold salt, death is induced into the cell.
3.2. Cell free optical assay To further confirm the in-situ synthesis of intracellular AuNPs, the non-apoptotic/apoptotic MCF-7 cells were lysed with hypotonic buffer and after addition of HAuCl4 for 50 min at room temperature it was 7
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investigated by UV–vis absorption spectrum. As shown in Fig. 4A, the non-apoptotic/apoptotic cell lysis is represented by red/purple-colored solution with absorption peak at around 550/580 nm, respectively. To investigate the principle mechanism of the proposed sensing system (Fig. 4B), we evaluated the effect of GSH on biosynthesized AuNPs by spiking GSH (1 mg/ml) in apoptotic cell lysate, and also Cyt c (1 mmol L−1) in non-apoptotic cell lysate to understand the effect of accumulation of the release of cytochrome c on aggregation of AuNPs. As can be seen, after addition of cytochrom c in non-apoptotic cell lysate the absorption peak shifted from 550 nm toward the long wavelength region to 580 nm (Fig. 4C (a,b)) which means aggregation of AuNPs and obtaining their larger size by addition of Cyt c. Moreover, size of AuNPs is increased after adding GSH to apoptotic cell lysate and therefore the absorption peak is observed in the range of 740 nm, while the absorption peak was in the range of 580 nm before it was added (Fig. 4C (c,d)) which means larger size of biosynthesized AuNPs by addition of GSH and aggregation of them due to the presence of GSSG and Cyt c.
treated non-apoptotic MCF-7 cell lysate (e), MCF-7 cells with H2O2–induced apoptosis (d), Dox-treated HEK-293 cells (c) and Doxtreated MCF-7 cell ((b) as a positive control) and non treated MCF7 cells ((a) as a negative control). Fig. 6 e, d shows that the Dox itself is not effective, and this difference in the signal is due to activation of the pathway of apoptosis, and Fig. 6 c indicates that proposed apoptotic assay method is not specific to MCF-7 cells, but can also be extended to other cells. 4. Conclusion In this study, we report an efficient and low-cost visual detection method for apoptosis assay of cancer cells and achieved good results in comparison with a standard apoptosis assay procedure. The linear range for MCF-7 cells in a different cell number from 30 to 3 × 105 cells was obtained with a detection limit of 30 cells. In addition, the proposed approach is applicable to detect other apoptotic cells. This method developed on the basis of: (1) spontaneous biosynthesis of gold nanoparticles inside a living cells (cell-based), (2) spontaneous biosynthesis of gold nanoparticles in the presence of cell lysate (cell free system), (3) alterations in redox state of the glutathione/disulfide-glutathione couple (GSSG/2GSH) and (4) the release of cytochrome c from mitochondria in apoptotic MCF-7 cells. This method offers a simple assay after addition of a sufficient amount of gold ion and, more importantly, enable us to determine apoptosis in a living cell format.
3.3. Analytical performance Under the optimal condition, the performance of proposed method for apoptosis assay is shown in Fig. 5. For this purpose, we obtained the optimal concentration of doxorubicin to induce apoptosis in breast cancer MCF-7 cells by using this colorimetric method and caspase 3/7 apoptosis detection assay. So the cells were incubated with different concentrations of doxorubicin (0, 10, 25, 50, 75, 100, 200, 300, 400, 500, 600 and 700 nmol L−1) for 15 h and then the absorbance of samples was measured. As shown in Fig. 5A, the absorbance at 650 nm increased gradually for samples containing increasing concentrations of Dox from 0 to 400 nmol L−1 and at concentrations higher than 400 nmol L−1, this amount of absorption decreases. This decrease in absorption at higher concentration of Dox can be attributed to a decrease in the number of cells (Fig. 5C). In addition, the results achieved by the suggested strategy were in good agreement with caspase 3/7 apoptosis detection kit. As shown in Fig. 5C, plotting the absorption at 650 nm versus the number of cells (30–3 × 105), a linear slope was obtained with a regression coefficient of 0.9. The limit of detection (LOD) was determined to be 30 cells for MCF-7 cells. Progress of apoptosis is attributed to the appearance of some specific features. Early stage apoptosis is represented by changes to, and ultimate loss of, the mitochondrial membrane potential which can be detected by JC-1, a dye that selectively enters the mitochondria [25]. Upon mitochondrial loss of integrity, mid-term signs are appeared. Amongst them, translocation of phosphatidylserin (PS), a protein located in the cellular membrane which can be detected by Annexin V upon its translocation to the cell surface. Late stage apoptosis is accompanied with defragmentation of DNA which can be detected by a two color stain using fluorescein-deoxyuridine triphosphate (FITC-dUTP) for DNA Break detection and Propidium Iodide for total DNA counterstain. However, the current method uses of release of cytochrome c upon loss of mitochondrial membrane integrity and can be considered as a mid-term sign of apoptosis. However, release of cytochrome c is accumulative and in a conservative way, it might be suggested that signal of sensor up to 400 nM of doxorubicin (Fig. 5A) is corresponded to apoptotic cells in early-mid term stages and those treated with more than 400 nM with loss of intensity are late apoptotic cells.
Acknowledgements Financial support of this work was provided by National Institute for Medical Research Development (NIMAD, Grant No: 957982). The authors are grateful to the Iran National Elites Foundation for support of Yasaman-Sadat Borghei as a post-doc fellow in this work. References [1] S. Elmore, Apoptosis: a review of programmed cell death, Toxicol. Pathol. 35 (2007) 495–516. [2] M.L. Circu, T.Y. Aw, Glutathione and apoptosis, Free Radic. Res. 42 (2008) 689–706. [3] M.L. Circu, T.Y. Aw, Glutathione and modulation of cell apoptosis, Biochim. Biophys. Acta Mol. Cell Res. 1823 (2012) 1767–1777. [4] A.L. Ortega, S. Mena, J.M. Estrela, Glutathione in cancer cell death, Cancers 3 (2011) 1285–1310. [5] M.L. Circu, T.Y. Aw, Reactive oxygen species, cellular redox systems, and apoptosis, Free Radic. Biol. Med. 48 (2010) 749–762. [6] D. Chandra, D.G. Tang, Detection of apoptosis in cell-free systems, Methods Mol. Biol. 559 (2009) 65–75. [7] M.M. Martinez, R.D. Reif, D. Pappas, Detection of apoptosis: a review of conventional and novel techniques, Analytical methods 2 (2010) 996–1004. [8] G. Banfalvi, Methods to detect apoptotic cell death, Apoptosis 22 (2017) 306–323. [9] J. Wang, G. Zhang, Q. Li, H. Jiang, C. Liu, C. Amatore, X. Wang, In vivo self-bioimaging of tumors through in situ biosynthesized fluorescent gold nanoclusters, Sci. Rep. 3 (2013) 1157. [10] A.L. West, N.M. Schaeublin, M.H. Griep, E.I. Maurer-Gardner, D.P. Cole, A.M. Fakner, S.M. Hussain, S.P. Karna, In situ synthesis of fluorescent gold nanoclusters by nontumorigenic microglial cells, ACS Appl. Mater. Interfaces 8 (2016) 21221–21227. [11] F. Dong, E. Feng, T. Zheng, Y. Tian, In situ synthesized silver nanoclusters for tracking the role of telomerase activity in the differentiation of mesenchymal stem cells to neural stem cells, ACS Appl. Mater. Interfaces 10 (2018) 2051–2057. [12] S. Chattoraj, K. Bhattacharyya, Fluorescent gold nanocluster inside a livingbreast cell: etching and higher uptake in cancer cell, J. Phys. Chem. C 118 (2014) 22339–22346. [13] D. Drescher, H. Traub, T. Büchner, N. Jakubowski, J. Kneipp, Properties of in situ generated gold nanoparticles in the cellular context, Nanoscale 9 (2017) 11647–11656. [14] Y.S. Borghei, M. Hosseini, M. Dadmehr, S. Hosseinkhani, M.R. Ganjali, R. Sheikhnejad, Visual detection of cancer cells by colorimetric aptasensor based on aggregation of gold nanoparticles induced by DNA hybridization, Anal. Chim. Acta 904 (2016) 92–97. [15] I. Müller, D. Niethammer, G. Bruchelt, Anthracycline-derived chemotherapeutics in apoptosis and free radical cytotoxicity, Int. J. Mol. Med. 1 (1998) 491–495. [16] M. Torkzadeh-Mahani, F. Ataei, M. Nikkhah, S. Hosseinkhani, Design and development of a whole-cell luminescent biosensor for detection of early-stage of
3.4. Specificity To confirm that the increase in the absorption at 650 nm was derived from the activation of apoptosis pathway not a dox presence, on the one hand, we added 400 nmol L−1 of Dox to the non-apoptotic cell lysate and on the other hand, we used hydrogen peroxide (H2O2) as an apoptosis induction. Fig. 6 shows the absorption recorded for the Dox8
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Y.-S. Borghei and S. Hosseinkhani apoptosis, Biosens. Bioelectron. 38 (2012) 362–368. [17] T. Xia, M. Kovochich, J. Brant, M. Hotze, J. Sempf, T. Oberley, C. Sioutas, J.I. Yeh, M.R. Wiesner, A.E. Nel, Comparison of the abilities of ambient and manufactured nanoparticles to induce cellular toxicity according to an oxidative stress paradigm, Nano Lett. 6 (2006) 1794–1807. [18] D. Shen, T.P. Dalton, D.W. Nebert, H.G. Shertzer, Glutathione redox state regulates mitochondrial reactive oxygen production, J. Biol. Chem. 280 (2005) 25305–25312. [19] D.W. Voehringer, BCL-2 and glutathione: alterations in cellular redox state that regulate apoptosis sensitivity, Free Radic. Biol. Med. 27 (1999) 945–950. [20] A.R. Noori, E.S. Hosseini, M. Nikkhah, S. Hosseinkhani, Apoptosome formation upon overexpression of native and truncated Apaf-1 in cell-free and cell-based systems, Arch. Biochem. Biophys. 642 (2018) 46–51. [21] S. Karimzadeh, S. Hosseinkhani, A. Fathi, F. Ataei, H. Baharvand, Insufficient Apaf1 expression in early stages of neural differentiation of human embryonic stem cells
might protect them from apoptosis, Eur. J. Cell Biol. 97 (2018) 126–135. [22] S. Akbari-Birgani, S. Hosseinkhani, S. Mollamohamadi, H. Baharvand, Delay in apoptosome formation attenuates apoptosis in mouse embryonic stem cell differentiation, J. Biol. Chem. 289 (2014) 16905–16913. [23] M. Shamsipur, F. Molaabasi, S. Hosseinkhani, F. Rahmati, Detection of early stage apoptotic cells based on label-free cytochrome c assay using bioconjugated metal nanoclusters as fluorescent probes, Anal. Chem. 88 (2016) 2188–2197. [24] I. Choi, Y.I. Yang, E. Jeong, K. Kim, S. Hong, T. Kang, J. Yi, Colorimetric tracking of protein structural evolution based on the distance-dependent light scattering of embedded gold nanoparticles, Chem. Commun. 48 (2012) 2286–2288. [25] P. Ghiasi, S. Hosseinkhani, H. Ansari, N. Aghdami, S. Balalaei, S. Pahlavan, H. Baharvand, Reversible permeabilization of the mitochondrial membrane promotes human cardiomyocyte differentiation from embryonic stem cells, J. Cell. Physiol. 234 (2019) 521–536.
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Talanta journal homepage: www.elsevier.com/locate/talanta
Colorimetric assay of apoptosis through in-situ biosynthesized gold nanoparticles inside living breast cancer cells Yasaman-Sadat Borghei, Saman Hosseinkhani∗ Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
ARTICLE INFO
ABSTRACT
Keywords: Apoptosis assay In-situ synthesize Biosynthesized gold nanoparticles GSH/GSSG redox state Cytochrome c
Abnormalities in apoptosis (More or less than normal rate) is a central factor to detect many human disorders such as many types of cancer and to show therapeutic potential of drugs. During apoptosis, oxidation of reduced gluthation (GSH) to its oxidized form, GSSG, results in a decreased GSH to GSSG ratio. Here, we used altered GSH/GSSG redox state and the release of Cyt c from mitochondria to cytosol as an indicator for apoptosis assay. This study reports a visual method for cell apoptosis assay through in-situ biosynthesized gold nanoparticles (AuNPs) inside livingcells, only after adding a sufficient amount of gold ion. After incubation of apoptotic and non-apoptotic cells with chloroauric acid solution, high level of GSH act as reducing agent in formation of AuNPs using thiol inside living non-apoptotic cells. While in the apoptotic cells, what's happening is based on changes in plasmonic coupling between AuNPs embedded along the oxidized gluthation and Cyt c aggregates in cytosol which causes color changes from red to purple. In this study, we successfully reported cell-based (inside a living cells) and cell free (cell lysis) methods for apoptosis assay of breast cancer cells and we achieved very good results in comparison with a standard apoptosis assay procedure. The linear range for MCF-7 cells detection from 30 to 3 × 105 cells/ml was obtained with a detection limit of 30 cells. In addition, the proposed approach is applicable to detect other apoptotic cells.
1. Introduction The number of cells in a multicellular living organism is regulated not only by control in cell division but also by cell suicide. This suicide that occurs due to the activation of a number of intracellular pathways known as “programmed cell death” or “apoptosis” [1]. Initiation of Apoptosis can be occurred by various external “Extrinsic Apoptosis” and internal “Intrinsic Apoptosis” factors. The extrinsic (death receptor mediated) pathway can be initiated by attachment of death ligand to cell surface receptors. The intrinsic (Bcl-2 inhibit able or mitochondrial) pathway starts in response to different types of cellular stress such as DNA damage, endoplasmic reticulum (ER) stress, lysosomal stress, reactive oxygen species (ROS), and calcium (Ca2+) overload. Following the reception of stress signals, Bcl-2 family members translocated into the mitochondria, which can be changed in mitochondrial permeability and consequent release of cytochrome c (Cyt c) as an essential mitochondrial electron transport releases from the intermembrane space of the mitochondria to the cytosol [1–6]. During apoptosis process, oxidation of reduced gluthation (GSH) to its oxidized form, GSSG, results in a decreased GSH to GSSG ratio. In ∗
fact, reducing thiol proteins is a defense of the cell against oxidative conditions such as apoptosis process [2–4]. While typically, reduced glutathione (GSH, comprised of three amino acids; cysteine-glutamic acid-glycine), with millimolar concentrations in various cell types, due to free thiol groups preserves a cellular redox system (NAD+/NADH, NADP+/NADPH, and GSH/GSSG) for native function of cytosolic proteins [5,6]. Apoptosis is a vital process for organism development and cellular/ tissue homeostasis. So, there is a direct relationship between changes in the rate of apoptosis and various forms of disease [7]. For example, degenerative disorders such as Alzheimers, Parkinsons and autoimmune and infectious diseases caused by too much apoptosis. Conversely, too little apoptosis is often associated with cancer and leukemia. Hence, various methods have been developed for measuring apoptosis, including annexin V staining which is due to a change in cellular membrane asymmetry and flip-flop of phosphatidylserine (PS) residues in plasma membrane, propidium iodide (PI) staining for analysis of DNA Fragmentation, Western blot for detection of cytochrome c release from damaged mitochondria, and mechanism based assays such as caspases activity assay [7,8]. However, each of these traditional methods has its own individual limitations. western blotting is complex
Corresponding author. E-mail address: [email protected] (S. Hosseinkhani).
https://doi.org/10.1016/j.talanta.2019.120463 Received 8 August 2019; Received in revised form 8 October 2019; Accepted 9 October 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Yasaman-Sadat Borghei and Saman Hosseinkhani, Talanta, https://doi.org/10.1016/j.talanta.2019.120463
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Scheme 1. Schematic illustration of in-situ synthesis of gold nanoparticles on the basis of alterations in different redox state of GSSG/2GSH ratio and the release of Cyt c from mitochondria in apoptotic MCF-7 cells. And microscopy images of MCF-7 cells under Visible light from Non-Apoptotic (left) and Apoptotic (right) MCF7 cells.
and needs labeling procedures, which can lead to low efficiency; while, annexin V staining has good detection limits, but the required equipment and materials are costly. Although propidium iodide (PI) staining can be used for dead cells, but it is only applied in late stage of apoptosis. Due to these limitations, developing advanced techniques for apoptosis detection by using novel material and nanomaterial has attracted great attention. Nanomaterials have encouraged and enabled researchers to develop new detection methods for biomedical applications. Among the different types of nanomaterials, gold nanoparticles have attracted tremendous interests because of their optical and electronic properties that are tunable by changing the size, shape, surface chemistry, or aggregation state. For example, the different colorimetric detection method of analytes by using the aggregation of AuNPs have been developed [9–14]. Here, for the first time, we successfully show a detection procedure for apoptosis in living MCF-7 cells (cell-based system) in addition to the cell lysate (cell free system) using spontaneously biosynthesis of gold nanoparticles by living cells or in the presence of cell lysates. In this method, after incubation of non-apoptotic MCF-7 cells with a sufficient
amount of chloroauric acid solution, the high level of gluthations could form and stabilize large gold nanoparticles (AuNPs) based on typically strong gold–sulfur bonds (GSH-gold ion). While in apoptotic MCF7 cells, on one side alteration in GSH/GSSG redox state and low level of GSH causes small AuNPs and on the other side release of cytochromc protein in cytosol causes color changes that result from electronic dipole–dipole coupling between neighboring gold nanoparticles (Scheme 1). 2. Experimental section 2.1. Materials and reagents Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were purchased from Invitrogen, and penicillin/streptomycin was purchased from Gibco (USA). All chemicals used of analytical grade or of the highest purity available. Chloroauric acid (HAuCl4 99.9%) was obtained from Sigma Aldrich. Caspase 3/7 activity was measured by a luminescent assay kit from promega. Used phosphate buffer (PB) contains 20 mmol L−1 NaH2PO4. Hypotonic lysis buffer is 10 mmol L−1 2
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Fig. 1. (A) Microscopy images of MCF-7 cells under Visible light from Non-Apoptotic (left) and Apoptotic (right) MCF-7 cells. (B) the biosynthesized red color AuNPs inside the non-apoptotic cells and the aggregated purple color AuNPs indicating non-apoptotic cells that are dying (showed with the arrow) after incubation with trace amount of Dox (20 nmol L−1 for 15 h).
HEPES (pH:7.5) including 1.5 mmol L−1 MgCl2, 1 mmol L−1 Na-EDTA, 68 mmol L−1 sucrose and 1 mmol L−1 PMSF.
2.4. Cell free apoptosis assay To measure apoptosis in cell free system, Dox-treated cells were collected by centrifugation at 1000 rpm for 5 min after trypsinization and washing with cold PBS. Then the cell pellet (about 3 × 105 cells/ ml) was suspended in 30 μl of cold hypotonic lysis buffer and kept on ice for 10 min. In the final stage after 30 s vortex, it is also centrifuged for 30 s in 12,000 rpm whichresulted in the cytoplasmic content in the supernatant, and the cell debris was pelleted [16]. The supernatant cell lysate was used for proposed colorimetric assay. In this way, 10 μl of cold HAuCl4 (1.0 mmol L−1) were added to 30 μl of supernatant at room temperature for 50 min.
2.2. Cell culture and apoptosis induction The human breast carcinoma MCF-7 cells were cultured in DMEM complemented with 10% FBS and 1% penicillin/streptomycin at 37 °C with 5% CO2 in a humid atmosphere. To induce apoptosis, we usedDoxorubicin (Dox), a well-known Anthracycline-derived chemotherapeutics in apoptosis [15], at different concentration and different time. For this purpose, MCF-7 cells (3 × 105 cell/ml) were seeded in each well of a 48-well plate for overnight, then the cells incubated with Dox at different concentration (0–700 nmol L−1 in DMEM). After 15 h incubation, the cells were washed with PBS buffer. Three replicates were done for each treatment.
2.5. Caspase 3/7 assay Caspase 3/7 assay was done to measure doxorubicin-induced apoptosis in MCF-7 cells. Hence, cells were incubated with various concentrations of doxorubicin (0–700 nmol L−1) for 15 h and then hypotonic cell lysis was used for caspase 3/7 activity assay.
2.3. Cell-based apoptosis assay In order to measure apoptosis in cell-based system, the cells were incubated with Doxorubicin at different concentrations and different times. Then after washing the cells with PBS, they were treated dropwise with various concentration of cold HAuCl4 solution (PB buffer (pH:7.5)) and incubated at 4 °C for 24–48 h. 3
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Fig. 2. (A) UV–Vis of Non-Apoptotic (red line) and Apoptotic (after incubation with Dox (4000 nmol L−1 for 15 h) (purple line) MCF-7 cells after incubation with HAuCl4 solution (0.5 mmol L−1). Inset: photographs of them under Visible light. And (B) DLS analysis on the basis of number distribution (a) and intensity distribution (b) of Non-Apoptotic (black line) and Apoptotic (green line) MCF-7 cells. (C) TEM images are taken from biosynthesized AuNPs by non-apoptotic cells (a–c) and apoptotic cells (d–f); at different scales (40, 80 and 150 nm). 4
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Fig. 3. Optimization of HAuCl4 solution in PB buffer (pH:7.5): Microscopy images of them at different concentration of HAuCl4 solution (0.05, 0.1, 0.5, 1, 1.5 and 2 mmol L−1).
3. Results and discussion
aggregated purple color AuNPs are also visible in the apoptotic cells after 15 h of incubation with Dox (400 nmol L−1) (Fig. 1A (right)). Also, it can be seen in Fig. 1B that the aggregated purple color of AuNPs indicating non-apoptotic cells that goes towards apoptosis after incubation with trace amount of Dox (20 nmol L−1 for 15 h) (showed with the arrow). Also Fig. 2A showed the UV–Vis absorption spectra of non-apoptotic/apoptotic cell@AuNPs over a wavelength range of 400–800 nm. In the non-apoptotic MCF-7 cells the red color solution was observed with an absorption peak at around 535 nm. While in apoptotic MCF7 cells, it produced a color change (purple, Fig. 2A, inset) with an absorption peak around 560 nm. In addition, as shown in Fig. 2B, DLS results give the size distribution by intensity and number. As indicated in Fig. 2B, a, the size distribution of gold nanoparticles in non-apoptotic MCF-7 cells were about 25.0 nm while in apoptotic MCF-7 cells, it was about 24.0 nm which did not show significant difference. In fact, these results are not consistent with our suggestion, because in apoptotic cells, the amount of reducing agents (NADH, NADPH, and GSH) are lower than non-apoptotic cells [2–4,17–19], and therefore, the number of reduced gold ions is less than non-apoptotic. As shown in Fig. 2B, b, relative intensity and size distribution of the biosynthesized AuNPs in non-apoptotic and apoptotic MCF-7 cells were about 58 and 100 nm; respectively. This increase in the light scattering (intensity distribution) in apoptotic cells also shows the more aggregation of biosynthesized AuNPs compared with non-apoptotic cells. We also suggest that these AuNPs aggregations were due to the accumulation of oxidized glutathione and the release of cytochrome c in the cytosol [24]. In order to confirm more, the transmission electron microscopy (TEM) images of the biosynthesized AuNPs in non-apoptotic/apoptotic MCF-7 cells were taken after 15 h of incubation with Dox
3.1. Cell-based assay and characterization Scheme 1 shows the mechanism of proposed colorimetric apoptosis sensor which is based on the difference in redox state of the glutathione/disulfide-glutathione couple (GSSG/2GSH) in non apoptotic/ apoptotic cells and also the release of cytochrome c from mitochondria in apoptotic cells. There are numerous reports confirming that in apoptotic cells, the amount of oxidized glutathion (GSSG) is more than reduced glutathione (GSH) [2–4,17–19]. On the other hand, by induction of apoptosis and activation of the apoptotic pathway, cytochrome c releases from mitochondria and binds to Apaf-1 molecules thereby bring about with formation of apoptosome complex within cell death and differentiation [16,20–23]. So in the presence of HAuCl4, the cells can synthesis gold nanoparticles (AuNPs) inside themselves through reducting conditions such as high content of thiol group of GSH. But in apoptotic cells due to the overcoming of the oxidation conditions and the aggregation of oxidized glutathion (GSSG with a larger molecular structure than GSH) and release of cytochrome c, AuNPs aggregated [24]. The presence of in-situ synthesized AuNPs by MCF-7 cells and their aggregation in apoptotic MCF-7 cells was characterized by invert microscopy in the whole cells under visible light, UV–Visible spectroscopy and Dynamic light scattering (DLS) analysis. For cellular imaging (Fig. 1), the cells were treated with HAuCl4 solutions (0.5 mmol L−1) and incubated at 4 °C for 24 h or 48 h before imaging on an Inverted Microscopy (BX51, Olympus) under visible light using 20x and 40x objective. As shown in Fig. 1A, the biosynthesized red color AuNPs inside the non-apoptotic cells (Fig. 1A (left)) and the 5
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Fig. 4. (A) UV–Vis of Non-Apoptotic (red line) and Apoptotic (purple line) MCF-7 cell lysate after addition of HAuCl4 for 50 min at room temperature. Inset: photographs of them under Visible light. (B) the Scheme depicts the principle mechanism of the proposed sensing system by addition of GSH in apoptotic cell lysate and the addition of Cyt c in non-apoptotic cell lysate. (C) UV–Vis of Non-Apoptotic cell lysate (a), Non-Apoptotic cell lysate after addition of Cyt c (b), Apoptotic cell lysate after addition of GSH (c), And Apoptotic MCF-7 cell lysate (d) Inset: photographs of them under Visible light.
(400 nmol L−1) (Fig. 2C). These images are as good as DLS results; Images (a-c)/(d-f) are taken from AuNPs synthesized by non-apoptotic/ apoptotic cells; respectively. It is well clear that the biosynthesized AuNPs in non-apoptotic cells are bigger (Fig. 2C, a-c) than apoptotic cells, and also the aggregation of AuNPs in apoptotic cells is well visible
(Fig. 2C, d-f). In order to optimize the performance of the assay, concentration of HAuCL4 was optimized. For this purpose, the cells were treated with various concentrations of cold HAuCl4 solution (final concentration in PB buffer (pH:7.5): 0.05, 0.1, 0.5, 1, 1.5 and 2 mmol L−1) and 6
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Fig. 5. (A) Monitoring of apoptosis progress by the activity of caspase 3/7 by a luminescent assay kit as a gold standard method at different concentrations of Dox (0, sample signal at [x ] × 100 . 10, 25, 50, 75, 100, 200, 300, 400, 500, 600 and 700 nmol L−1, for 15 h) and it was compared with the proposed method based on: signal (%) = signal at [Y ]
([Y] is the concentration of Dox in which the maximum amount of signal is obtained and [x] is the other concentrations of Dox.) (B) photographs of them under Visible light. (C)The linear calibration curve of different numbers of MCF-7 cells (3 × 101, 3 × 102, 3 × 103, 3 × 104 and 3 × 105 cells/ml) after incubation with Dox (400 nmol L−1 for 15hr). (D) photographs of them under Visible light.
Fig. 6. The absorption signal at 650 nm for the Dox-treated non-apoptotic MCF-7 cell lysate (e), MCF-7 cells with H2O2–induced apoptosis (d), Dox-treated HEK293 cells (c) and Dox-treated MCF-7 cell ((b) as a positive control) and non treated MCF-7 cells ((a) as a negative control).
incubated at 4 °C (Fig. 3). As shown in Fig. 3, when the HAuCl4 concentration was 0.5 mmol L−1, the cells synthesize more red gold nanoparticles. It appears that at high concentrations of gold salt, death is induced into the cell.
3.2. Cell free optical assay To further confirm the in-situ synthesis of intracellular AuNPs, the non-apoptotic/apoptotic MCF-7 cells were lysed with hypotonic buffer and after addition of HAuCl4 for 50 min at room temperature it was 7
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investigated by UV–vis absorption spectrum. As shown in Fig. 4A, the non-apoptotic/apoptotic cell lysis is represented by red/purple-colored solution with absorption peak at around 550/580 nm, respectively. To investigate the principle mechanism of the proposed sensing system (Fig. 4B), we evaluated the effect of GSH on biosynthesized AuNPs by spiking GSH (1 mg/ml) in apoptotic cell lysate, and also Cyt c (1 mmol L−1) in non-apoptotic cell lysate to understand the effect of accumulation of the release of cytochrome c on aggregation of AuNPs. As can be seen, after addition of cytochrom c in non-apoptotic cell lysate the absorption peak shifted from 550 nm toward the long wavelength region to 580 nm (Fig. 4C (a,b)) which means aggregation of AuNPs and obtaining their larger size by addition of Cyt c. Moreover, size of AuNPs is increased after adding GSH to apoptotic cell lysate and therefore the absorption peak is observed in the range of 740 nm, while the absorption peak was in the range of 580 nm before it was added (Fig. 4C (c,d)) which means larger size of biosynthesized AuNPs by addition of GSH and aggregation of them due to the presence of GSSG and Cyt c.
treated non-apoptotic MCF-7 cell lysate (e), MCF-7 cells with H2O2–induced apoptosis (d), Dox-treated HEK-293 cells (c) and Doxtreated MCF-7 cell ((b) as a positive control) and non treated MCF7 cells ((a) as a negative control). Fig. 6 e, d shows that the Dox itself is not effective, and this difference in the signal is due to activation of the pathway of apoptosis, and Fig. 6 c indicates that proposed apoptotic assay method is not specific to MCF-7 cells, but can also be extended to other cells. 4. Conclusion In this study, we report an efficient and low-cost visual detection method for apoptosis assay of cancer cells and achieved good results in comparison with a standard apoptosis assay procedure. The linear range for MCF-7 cells in a different cell number from 30 to 3 × 105 cells was obtained with a detection limit of 30 cells. In addition, the proposed approach is applicable to detect other apoptotic cells. This method developed on the basis of: (1) spontaneous biosynthesis of gold nanoparticles inside a living cells (cell-based), (2) spontaneous biosynthesis of gold nanoparticles in the presence of cell lysate (cell free system), (3) alterations in redox state of the glutathione/disulfide-glutathione couple (GSSG/2GSH) and (4) the release of cytochrome c from mitochondria in apoptotic MCF-7 cells. This method offers a simple assay after addition of a sufficient amount of gold ion and, more importantly, enable us to determine apoptosis in a living cell format.
3.3. Analytical performance Under the optimal condition, the performance of proposed method for apoptosis assay is shown in Fig. 5. For this purpose, we obtained the optimal concentration of doxorubicin to induce apoptosis in breast cancer MCF-7 cells by using this colorimetric method and caspase 3/7 apoptosis detection assay. So the cells were incubated with different concentrations of doxorubicin (0, 10, 25, 50, 75, 100, 200, 300, 400, 500, 600 and 700 nmol L−1) for 15 h and then the absorbance of samples was measured. As shown in Fig. 5A, the absorbance at 650 nm increased gradually for samples containing increasing concentrations of Dox from 0 to 400 nmol L−1 and at concentrations higher than 400 nmol L−1, this amount of absorption decreases. This decrease in absorption at higher concentration of Dox can be attributed to a decrease in the number of cells (Fig. 5C). In addition, the results achieved by the suggested strategy were in good agreement with caspase 3/7 apoptosis detection kit. As shown in Fig. 5C, plotting the absorption at 650 nm versus the number of cells (30–3 × 105), a linear slope was obtained with a regression coefficient of 0.9. The limit of detection (LOD) was determined to be 30 cells for MCF-7 cells. Progress of apoptosis is attributed to the appearance of some specific features. Early stage apoptosis is represented by changes to, and ultimate loss of, the mitochondrial membrane potential which can be detected by JC-1, a dye that selectively enters the mitochondria [25]. Upon mitochondrial loss of integrity, mid-term signs are appeared. Amongst them, translocation of phosphatidylserin (PS), a protein located in the cellular membrane which can be detected by Annexin V upon its translocation to the cell surface. Late stage apoptosis is accompanied with defragmentation of DNA which can be detected by a two color stain using fluorescein-deoxyuridine triphosphate (FITC-dUTP) for DNA Break detection and Propidium Iodide for total DNA counterstain. However, the current method uses of release of cytochrome c upon loss of mitochondrial membrane integrity and can be considered as a mid-term sign of apoptosis. However, release of cytochrome c is accumulative and in a conservative way, it might be suggested that signal of sensor up to 400 nM of doxorubicin (Fig. 5A) is corresponded to apoptotic cells in early-mid term stages and those treated with more than 400 nM with loss of intensity are late apoptotic cells.
Acknowledgements Financial support of this work was provided by National Institute for Medical Research Development (NIMAD, Grant No: 957982). The authors are grateful to the Iran National Elites Foundation for support of Yasaman-Sadat Borghei as a post-doc fellow in this work. References [1] S. Elmore, Apoptosis: a review of programmed cell death, Toxicol. Pathol. 35 (2007) 495–516. [2] M.L. Circu, T.Y. Aw, Glutathione and apoptosis, Free Radic. Res. 42 (2008) 689–706. [3] M.L. Circu, T.Y. Aw, Glutathione and modulation of cell apoptosis, Biochim. Biophys. Acta Mol. Cell Res. 1823 (2012) 1767–1777. [4] A.L. Ortega, S. Mena, J.M. Estrela, Glutathione in cancer cell death, Cancers 3 (2011) 1285–1310. [5] M.L. Circu, T.Y. Aw, Reactive oxygen species, cellular redox systems, and apoptosis, Free Radic. Biol. Med. 48 (2010) 749–762. [6] D. Chandra, D.G. Tang, Detection of apoptosis in cell-free systems, Methods Mol. Biol. 559 (2009) 65–75. [7] M.M. Martinez, R.D. Reif, D. Pappas, Detection of apoptosis: a review of conventional and novel techniques, Analytical methods 2 (2010) 996–1004. [8] G. Banfalvi, Methods to detect apoptotic cell death, Apoptosis 22 (2017) 306–323. [9] J. Wang, G. Zhang, Q. Li, H. Jiang, C. Liu, C. Amatore, X. Wang, In vivo self-bioimaging of tumors through in situ biosynthesized fluorescent gold nanoclusters, Sci. Rep. 3 (2013) 1157. [10] A.L. West, N.M. Schaeublin, M.H. Griep, E.I. Maurer-Gardner, D.P. Cole, A.M. Fakner, S.M. Hussain, S.P. Karna, In situ synthesis of fluorescent gold nanoclusters by nontumorigenic microglial cells, ACS Appl. Mater. Interfaces 8 (2016) 21221–21227. [11] F. Dong, E. Feng, T. Zheng, Y. Tian, In situ synthesized silver nanoclusters for tracking the role of telomerase activity in the differentiation of mesenchymal stem cells to neural stem cells, ACS Appl. Mater. Interfaces 10 (2018) 2051–2057. [12] S. Chattoraj, K. Bhattacharyya, Fluorescent gold nanocluster inside a livingbreast cell: etching and higher uptake in cancer cell, J. Phys. Chem. C 118 (2014) 22339–22346. [13] D. Drescher, H. Traub, T. Büchner, N. Jakubowski, J. Kneipp, Properties of in situ generated gold nanoparticles in the cellular context, Nanoscale 9 (2017) 11647–11656. [14] Y.S. Borghei, M. Hosseini, M. Dadmehr, S. Hosseinkhani, M.R. Ganjali, R. Sheikhnejad, Visual detection of cancer cells by colorimetric aptasensor based on aggregation of gold nanoparticles induced by DNA hybridization, Anal. Chim. Acta 904 (2016) 92–97. [15] I. Müller, D. Niethammer, G. Bruchelt, Anthracycline-derived chemotherapeutics in apoptosis and free radical cytotoxicity, Int. J. Mol. Med. 1 (1998) 491–495. [16] M. Torkzadeh-Mahani, F. Ataei, M. Nikkhah, S. Hosseinkhani, Design and development of a whole-cell luminescent biosensor for detection of early-stage of
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