Pravin K.
Naoghare
a,
Hyeon A.
Ki
a,
Seung-Mann
Paek
a,
Yu Kyung
Tak
a,
Young-Ger
Suh
a,
Sang Geon
Kim
a,
Kyeong-Hee
Lee
b and
Joon Myong
Song
*a
aResearch Institute of Pharmaceutical Sciences and College of Pharmacy, Seoul National University, Seoul 151-742, South Korea. E-mail: jmsong@snu.ac.kr; Fax: +82 2 871 2238; Tel: +82 2 880 7841
bApplied Imaging Research Group, Korea Electrotechnology Research Institute, Gyeonggi-Technopark 1271-11, Ansan 426-901, Republic of Korea
First published on 8th December 2009
Caspases are the key mediators of apoptosis. The caspase cascade includes a series of events leading to the activation of initiator and downstream caspases in a cell. Analysis of the caspase cascade in intact cells, however, has generally been limited as the simultaneous monitoring of upstream and downstream caspases is not well executed. In an effort to monitor the activation of caspase cascades in an intact cell, high-content cellular imaging that allows simultaneous quantitative monitoring of caspase activation has been developed. This has great significance for the exploration of various cellular caspases involved in apoptotic pathways as possible therapeutic targets in the process of drug discovery. To explore the potential of simultaneous monitoring of caspase-mediated apoptotic pathways, human myeloid leukemia HL-60 cells were treated with SH-03 {(7S,7aR,13aS)-9,10-dimethoxy-3,3-dimethyl-7,7a,13,13a-tetrahydro-3H-chromeno [3,4-b]pyrano[2,3-h]chromen-7-ol} (a newly synthesized candidate), camptothecin or naringenin (agents known to induce apoptosis) with or without caspase inhibitors. SH-03 or naringenin treatment initiated the caspase cascade through an intrinsic apoptotic pathway, whereas camptothecin treatment triggered both intrinsic and extrinsic caspase cascades. We now report a new approach based on uniform threshold intensity distribution that facilitates rapid, quantitative monitoring of drug-induced caspase cascades through multi-spectral and multicolor imaging cytometry.
Insight, innovation, integrationCaspases have been recognized as emerging therapeutic targets in caspase-dependent diseases. The development of new methods to screen intracellular caspases as potential drug targets is difficult because the initiation of complex caspase cascade involves simultaneous activation of initiator and downstream caspases, leading to the distinct modes of cell death. We showed the application of high-content cellular imaging cytometry based on a new approach of uniform threshold intensity distribution that allows simultaneous quantitative monitoring of the caspase cascade in an intact cell. It facilitates rapid, quantitative, live cellular monitoring of drug-induced caspase cascades through multi-spectral and multicolor imaging cytometry. The present study provides important information for the design of new methodologies or the refinement of existing methodologies for efficient therapeutic drug target monitoring. |
The present work utilizes two commercially available drugs, camptothecin (CAM) and naringenin (NAR), and a newly synthesized drug, SH-03, to examine chemical-induced caspase cascades in human leukemia (HL-60) cells. SH-03 is a rotenoid-containing deguelin analog. Deguelin is an inhibitor of heat shock protein-90 (Hsp90) that was isolated from the African plant Mundulea sericea.9 It is known for its anti-angiogenic effect and its apoptotic effects in a variety of cell types.10 Despite the potential anticancer activity of deguelin in vivo and in vitro, this agent has toxic effects in rats.11 Moreover, long-term or high-dose deguelin treatment might cause Parkinson’s disease-like syndrome in rats.12 These constraints of high toxicity and low efficacy of deguelin prompted us to synthesize new deguelin derivatives with less toxicity and higher efficacy. Recently, we reported the isolation, synthesis and preliminary SAR study of SH-03.12 SH-03 activates intracellular hypoxia-inducible factor 1 subunit α (HIF-1α), Hsp90, the mammalian target of rapamycin (mTOR) and signal transducers and activators of transcription (STAT) proteins in malignant human bronchial epithelium (HBE) and non-small cell lung cancer (NSCLC) cell lines.13 SH-03 also exerts an antibacterial effect, specifically on microorganisms belonging to the genus Helicobacter (e.g., Helicobacter pylori).14 However, the anticancer activity and the dynamics of the caspase-mediated apoptotic cascade induced by SH-03 in myeloid leukemia cells are still unclear. Thus simultaneous monitoring of the drug-induced caspase cascade was studied in leukemia cells using a new drug-screening platform.
False-positive hits often interfere with discovery of aligned molecular targets and pathways after a new drug treatment in antagonistic cell-based screens.15 Thus, appropriate screening modalities are necessary to expand intense drug-development work. Developing new modalities against caspases as therapeutic targets has been limited because the activation of the caspase cascade is a highly complex process that includes a series of biochemical events. Biochemical assays for caspase activity demand comparatively large numbers of lysed cells for analysis. Consequently, the entire caspase activity in a cell population can be measured, but not the intercellular variation in the caspase activity within a population. Elucidation of intercellular variations in caspase activities may differentiate a defective cell (e.g., an oncogenic mutant) from its normal neighbors.16 To explore the mechanisms that underlie such variations, it is desirable to examine caspase activities in individual cells.
In addition to their role as executioners of apoptosis, some caspases are involved in inflammation, in which they act as pro-cytokine activators. Hence, initiation of a caspase cascade does not always lead to apoptosis. In this regard, activation of initiator caspases (caspase-2 and 8) involved in an inflammatory cellular response may be misinterpreted as an apoptotic event if the downstream caspases (e.g. caspases-3) are not monitored simultaneously. Here we demonstrate the simultaneous intracellular monitoring of caspase-mediated apoptotic cascades through high-content cellular imaging cytometry. The mechanism underlying the antineoplastic effects of a newly synthesized drug analog (SH-03) and two commercial drugs (CAM and NAR) were revealed. We report here for the first time that SH-03 triggers death signals in HL-60 cells via the caspase-9-mediated intrinsic apoptotic pathway by inhibiting the PI3K/AKT signal transduction pathway. Through this technology, we hope to mark cellular caspases as therapeutic targets in the process of drug discovery. This will strengthen the development of new methodologies necessary for high-content drug discovery screening.
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Fig. 1 (A) Effect of SH-03 on DNA fragmentation in HL-60 cells. Cells were incubated at 37 °C for 6 h in the presence of different SH-03 concentrations. (a) DNA was isolated from the control and treated samples and electrophorized on 0.8% agarose gel. M denotes the marker lane (1 kb DNA ladder), and lanes 1–5 depict results obtained using 0, 5, 10, 25, and 50 μM SH-03, respectively. (b) Line graph indicating the average gray scale intensities of each DNA sample. (B) Structure of SH-03 [(7S,7aR,13aS)-9,10-dimethoxy-3,3-dimethyl-7,7a,13,13a-tetrahydro-3H-chromeno [3,4-b] pyrano[2,3-h]chromen-7-ol], a rotenoid-containing deguelin analog, and a schematic of SH-03-mediated caspase-9 and caspase-3 activation. Hyperspectral fluorescent images (SH-03; 10 μM) were captured using cellular imaging cytometry. Here, 1. Sample, 2. Objective lens (60×), 3. Beam splitter, 4. Prism, 5. Focusing lens, 6. AOTF, 7. Un-diffracted beam, 8. Caspase-3 activation, 9. Caspase-3 and caspase-9 activation. θ. Bragg angle (the angle between diffracted and un-diffracted beam). (C) Representative hyperspectral fluorescent images of HL-60 cells displaying apoptosis triggered by SH-03 (10 μM) after 6 h of treatment (a). Images were taken over a spectral range from 463–688 nm with a total time interval of 60 s. The superimposed image refers to the image at 523 nm and 617 nm by collecting the fluorescence of Qdots and PI, respectively. (b) Scatter and dot plots of HL-60 cells obtained by flow cytometric analysis after 6 h of SH-03 treatment. The lower right quadrant of the dot plot represents early apoptotic cells with PS externalization (annexin V-FITC positive), whereas the upper left quadrant represents propidium iodide (PI)-positive dead cells (necrosis). The lower left quadrant represents control cells negative for both PI and annexin V-FITC. |
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Fig. 2 Focused, de-focused and fluorescent images of HL-60 cells acquired by cellular imaging cytometry. Fluorescent images refer to the image at 523 nm or 617 nm. Hyperspectral images depict caspase-9 and caspase-3 activation after SH-03 treatment (12 h) without (A) and with (B) caspase-2 inhibitor. (C and D) Caspase activation and inhibition assays after SH-03 treatment (5, 10 and 25 μM; 12 h). Cells were pre-incubated with fluorogenic (C) caspase-9 [(Ac-LEHD)2-Rh 110] and (D) caspase-3 [(z-DEVD)2-Magic Red] substrates in the presence or absence of caspase-2 inhibitor and were analyzed through cellular imaging cytometry. Error bars represents the percent deviations obtained in three individual measurements. |
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Fig. 3 Focused, de-focused and fluorescent images of HL-60 cells (caspase-8 and caspase-3 activation assay) acquired by cellular imaging cytometry after SH-03 treatment (12 h) without (A) and with (B) caspase-8 inhibitor. Fluorescent images refer to the images at 523 nm or 617 nm. Quantitative caspase-8 (C) and caspase-3 (D) activation measurements after SH-03 (5, 10 and 25 μM; 12 h) treatment in the presence or absence of caspase-8 inhibitor. Error bars represents the percent deviations obtained in three individual measurements. (E) Western blot analysis of HL-60 cells after 10 μM SH-03 treatment (12 h). |
Quantitative estimation of caspase activation in intrinsic apoptotic pathway | |||||
---|---|---|---|---|---|
Synthetic analog/drug | Concentration | Caspase-9 activation (%) | Caspase-3 activation (%) | ||
With caspase-2 inhibitor | Without caspase-2 inhibitor | With caspase-2 inhibitor | Without caspase-2 inhibitor | ||
SH-03 | Control | 9.3 | 5.39 | 7.5 | 6.86 |
5 μM | 10.9 | 72.00 | 11.51 | 76.00 | |
10 μM | 15.9 | 79.28 | 17.04 | 93.49 | |
25 μM | 13.33 | 79.90 | 15.83 | 85.78 | |
CAM | Control | 8.40 | 8.52 | 12.00 | 14.30 |
50 nM | 12.60 | 22.70 | 47.27 | 50.60 | |
100 nM | 19.37 | 40.27 | 52.30 | 68.97 | |
287 nM | 33.35 | 45.03 | 64.90 | 82.00 | |
NAR | Control | 5.90 | 6.35 | 5.90 | 4.65 |
0.1 mM | 8.27 | 14.27 | 6.73 | 13.83 | |
0.25 mM | 14.60 | 22.77 | 17.87 | 31.83 | |
0.5 mM | 20.87 | 31.50 | 21.10 | 44.23 | |
Quantitative estimation of caspase activation in extrinsic apoptotic pathway | |||||
Synthetic analog/drug | Concentration | Caspase-8 activation (%) | Caspase-3 activation (%) | ||
With caspase-8 inhibitor | Without caspase-8 inhibitor | With caspase-8 inhibitor | Without caspase-8 inhibitor | ||
SH-03 | Control | 9.6 | 1.44 | 6.25 | 5.25 |
5 μM | 8.02 | 6.83 | 19.49 | 35.93 | |
10 μM | 13.79 | 8.53 | 40.15 | 50.12 | |
25 μM | 20.90 | 10.10 | 56.41 | 65.61 | |
CAM | Control | 9.40 | 9.65 | 9.31 | 10.1 |
50 nM | 13.60 | 17.70 | 18.27 | 21.60 | |
100 nM | 21.37 | 45.27 | 28.30 | 49.97 | |
287 nM | 28.35 | 52.03 | 32.90 | 65.00 | |
NAR | Control | 5.90 | 6.20 | 5.91 | 6.50 |
0.1 mM | 6.27 | 9.27 | 6.73 | 9.83 | |
0.25 mM | 13.60 | 14.77 | 12.87 | 13.83 | |
0.5 mM | 11.87 | 15.50 | 15.10 | 16.23 |
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Fig. 4 Effect of SH-03 and LY294002 on PI3K/AKT activation and phosphorylation. HL-60 cells were incubated with 10 μM SH-03 or LY294002 for 12 h prior to quantitative imaging cytometry. Multicolor images were taken at 525 nm (AKT and pAKT) and 675 nm (PI3K and pPI3K). Superimposed images indicate the activation of either AKT/PI3K or pAKT/pPI3K. Inset in the fluorescent micrographs depicts de-focused and focused images of the representative cells in the respective samples. |
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Fig. 5 Effect of (A, B) CAM (50, 100 and 287 nM; 12 h) and (C, D) NAR (0.1, 0.25, 0.5 mM; 12 h) on intracellular caspase-9 and caspase-3 activation in HL-60 cells. The cells were pre-incubated with fluorogenic caspase-9 and caspase-3 substrates in the presence or absence of caspase-2 inhibitor and were analyzed using cellular imaging cytometry. (E, F) The effect of CAM (50, 100 and 287 nM; 12 h) and (G, H) NAR (0.1, 0.25, 0.5 mM; 12 h) on the extrinsic apoptotic pathway in HL-60 cells. Before each drug treatment, HL-60 cells were pre-incubated with fluorogenic caspase-8 and caspase-3 substrates in the presence or absence of caspase-8 inhibitor and were analyzed through cellular imaging cytometry. |
Among the key biochemical executioners of apoptosis, caspases are a group of proteolytic enzymes that sequentially disrupt normal cellular homeostasis by cleaving specific target proteins with structural, regulatory or housekeeping functions.19 Therefore, the cellular kinetics of the caspase cascade and its simultaneous monitoring can be an important way to assess drug efficacy. Insight into the SH-03-triggered caspase cascade can be achieved using quantitative high-content cellular imaging cytometry. Little is known about subsequent alterations in the extent and kinetics of effector caspase activation during apoptosis. Most of the available literature on apoptosis elucidates the mechanistic and regulatory changes in apoptosis after drug treatment. Relatively little is known about the kinetics of the hallmark apoptotic change that occurs during SH-03-induced apoptosis. In the present study, both the initiator and downstream caspases were examined to study the intracellular caspase activation. Our data provide important insight in terms of identifying the appropriate endpoints and the time points for drug-induced apoptosis. This study focused on exploring the cellular cell death pathways using two commercially available drugs (CAM and NAR) and SH-03. SH-03, being a newly synthesized drug analog, was tested to monitor its efficacy against leukemia cells through a DNA fragmentation assay (Fig. 1A). The hallmark of apoptosis is often coincident with internucleosomal DNA fragmentation. Thus, a DNA fragmentation assay was carried out to establish the appropriate drug concentration to be used for apoptosis and caspase activation/inhibition assays.20 SH-03 (Fig. 1B) is a rotenoid-containing deguelin analog. Apart from the application of SH-03 as an antibacterial,14 anti-angiogenic and apoptotic agent,10 its effect on the caspase-mediated apoptotic cascade in myeloid leukemia cells was not understood. Herein, we disclose its application as an anticancer agent and its mode of action against myeloid leukemia cells. The drug-induced cell death pathway was also revealed using the high-content cellular imaging cytometry. Moreover, cellular imaging cytometry may be partly utilized as an alternative analytical tool to commercial flow cytometry for rapid, automated drug screening. Our screening method based on cellular imaging cytometry provides simultaneous monitoring of cellular events. Moreover, cellular imaging cytometry should facilitate quantitative multivariate cellular analysis and eliminate false positive errors as it provides high spectral resolution among the fluorophores as shown in Fig. 1C.
Drugs can induce apoptosis in response to different stimuli. The proposed mechanisms for pro-apoptotic effects include activation of caspases,21 induction of cytochrome C release,22 regulation of protein kinase C isoform expression,23 and inhibition of NF-kB24 and AP-1.25 We elucidated the cell death-signaling mechanism triggered by SH-03 by examining the initial executioner of intrinsic and extrinsic apoptotic pathways. Our results suggest that SH-03 induces apoptosis in HL-60 cells via a caspase-9-mediated pathway (Fig. 2). Caspase 2 is known to play an important role in the induction of apoptosis in most of the cell systems and is most evolutionarily conserved.26 It is an initiator caspase working upstream of the mitochondria-dependent apoptotic cascade. It can be activated after a variety of cellular stresses, e.g. oxidative stress.26 The available literature demonstrates that caspase 2 is an upstream initiator of the JNK-mediated apoptotic signaling cascade in doxorubicin-induced apoptosis.26 Activation of caspase-9 is linked to mitochondrial dysfunction. Induction of stress signaling pathways mediated by Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) causes release of cytochrome C from mitochondria, followed by the activation of apaf-1 (apoptosome).27 This in turn cleaves the pro-enzyme of caspase-9 into the active form. Once activated, caspase-9 cleaves procaspase-3 and procaspase-7 and several cellular targets, including poly ADP ribose polymerase.27 The current data reveal drastic reductions in SH-03-induced apoptosis by the caspase-2 inhibitor, Z-VDVAD-FMK. Thus, SH-03-induced apoptosis operates through a caspase-9-mediated cell death pathway. As shown in Fig. 2 and Table 1, SH-03 treatment activates caspase-9 and caspase-3 in HL-60 cells. However, based on the data in Fig. 3 and Table 1, pretreatment with caspase-8 inhibitor (20 μM) did not change caspase-8 activation significantly. Cellular imaging cytometry revealed that SH-03 triggers apoptosis via a caspase-mediated intrinsic pathway. The additional information obtained by western blot analysis (Fig. 3E) showed elevated levels of cytosolic cytochrome C in SH-03-treated cells compared to the control. On the other hand, the activity of caspase-8 remained the same in the control as well as SH-03-treated cells. These observations suggest possible crosstalk between the caspase-8- and cytochrome C-mediated apoptotic pathways does not exist in SH-03-induced apoptosis in HL-60 cells. The time interval of caspase cascade activation is invariably short. Therefore, simultaneous monitoring of caspase-8, caspase-9 and caspases-3 has great potential advantages as it can differentiate between the inflammatory cellular response and the apoptotic stimulus induced by cellular caspases. Hyperspectral fluorescent images taken by cellular imaging cytometry at 15 s (caspase-3; 617 nm) and 45 s (caspase-8 and caspase-9; 523 nm) facilitate the rapid and efficient monitoring of initiator and downstream caspases after drug treatment.
The mechanism through which SH-03 activates caspase-9 is still not known. It has been reported that deguelin treatment arrests the cell cycle in the G2/M phase and induces apoptosis.28 In addition, deguelin is a phosphoinositide-3 kinase (PI3K) inhibitor; it also down-regulates pAKT levels but has minimal effect on the mitogen-activated protein kinase (MAPK) pathway.27 AKT phosphorylates a variety of intracellular substrates including insulin receptor susbtrate-1 (IRS-1), phosphodiesterase-3B (PDE-3B), BAD, and caspase-9. These findings suggest that deguelin activates pro-apoptotic caspases (e.g., caspase-9) by inhibiting the PI3K/AKT-mediated signaling pathway. Inhibition of the PI3K/AKT signaling pathway has significant importance with regard to the induction of apoptosis in several carcinoma cells.29 SH-03, being a deguelin analog, was further tested to elucidate the mechanistic insights of its effect on the PI3K/AKT signaling pathway. The results obtained (Fig. 4) clearly indicate that SH-03 inhibits the activation and phosphorylation of PI3K and AKT more efficiently compared to the known PI3K inhibitor (LY294002) in HL-60 cells. The existence of pAKT in control HL-60 cells followed its gradual inhibition over time in presence of drug has already been reported.30 The obtained results are in line with the above results. Fig. 6 shows the inhibition of PI3K/AKT signaling pathway by SH-03 in detail. Growth factors like VEGF, SCF, HGF, IGF1, and FGF1 trigger the PI3K/AKT signal transduction pathway in eukaryotic cells.31 This includes the growth factor receptor-mediated activation of PI3K followed by its phosphorylation. This in turn phosphorylates membrane phospholipids generating PIP3 from PIP2. PIP3 then activates PDK1 and PDK2 kinases resulting in the phosphorylation of AKT (pAKT). pAKT phosphorylates BAD and promotes cell survival. In this regard, SH-03 plays a crucial role in targeting the two important sites of the PI3K/AKT-mediated signal transduction pathway (i.e., activation and phosphorylation of PI3K and AKT). As a result, inactivated or non-phosphorylated AKT blocks BAD phosphorylation. This in turn blocks BCKXL and activates mitochondrial MMP. This results in the release of cytochrome C and the activation of a series of biomolecules (APAF1, caspase-9 and caspase-3) leading to apoptosis. Quantitative caspase activation and inhibition assays using cellular imaging cytometry and the validation of these assays with commercially available drugs (CAM and NAR) have strengthened the monitoring of cellular cascade events. NAR triggered the caspase cascade through an intrinsic apoptotic pathway, whereas CAM did so through both the intrinsic (caspase-9 activation) and extrinsic (caspase-8 activation) pathways (Fig. 5). These results were in agreement with the available literature.31
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Fig. 6 Effect of SH-03 on the AKT/PI3K signal transduction pathway in HL-60 cells. Abbreviations are VEGF: vascular endothelial growth factor; SCF: stem cell factor; HGF: hepatocyte growth factor; IGF: insulin-like growth factor; FGF: fibroblast growth factor; PI3K: phosphatidylinositol-3-kinase; PTEN: phosphatase and tensin homolog; PIP2: phosphatidylinositol-4,5-bisphosphate; PIP3: phosphatidylinositol-3,4,5-bisphosphate; PDK: PIP3-dependent protein kinase; AKT: protein kinase B; BAD: Bcl-2-associated death promoter; BCKXL: transmembrane molecule in the mitochondria; MMP: matrix metalloproteinase; APAF1: apoptotic peptidase activating factor 1; Cyto C: cytochrome C. |
Recent investigations are disentangling the complex apoptotic signaling pathways to develop new therapeutic approaches.32,33 Unfortunately, the conventional therapeutic approaches have focused on very few classes of drug target molecules such as regulatory enzymes and small-ligand receptors.34 To overcome this limitation, a more thorough understanding of new drug targets (e.g., initiator and effector caspases) that initiate and execute apoptosis is imperative in designing appropriate therapies. Considering the role of caspases as death effector molecules, a great deal of interest has focused on caspases as targets for various diseases (e.g., Alzheimer’s, Huntington and other neurodegenerative diseases).35 Recently, broad-spectrum caspase inhibitors have been evaluated in various clinical trials to determine their usefulness in delaying or preventing the progression of various diseases.36 However, a few studies point to the application of caspase inhibitors in leukemia therapy. In this regard, elucidation of caspase-mediated cell death dynamics through caspase activation and inhibition assays with quantitative cellular imaging (Fig. 2 and 3) could yield important insight regarding drug-induced cellular response. Here, simultaneous activation of caspase-3, caspase-8 and caspase-9 in the presence or absence of caspase-2 and caspase-8 inhibitors after drug treatment was efficiently monitored through cellular imaging cytometry. The developed protocol enabled us to accomplish rapid, quantitative and automated analysis to elucidate the possible mechanism of drug-induced cellular pathways (i.e., cascade of simultaneous cellular events). The results obtained via cellular imaging cytometry were in line with the data obtained through flow cytometric analysis and thus validated our approach.31 Using two marketed drugs as examples, we demonstrated the effectiveness of our method in detecting caspase cascades and cell death mechanisms. More importantly, the cell death mechanism induced by a newly developed drug (SH-03) was elucidated through cellular imaging cytometry. SH-03, a rotenoid-containing deguelin analog, displayed great potency as an anticancer drug against leukemia cells. Thus, SH-03 holds promise for future therapeutic modalities in leukemia. The developed concept may have fundamental significance in establishing image-based assays for elucidating simultaneous cellular events. It also offers great promise for exploring different targets involved in apoptotic pathways in order to design appropriate drug analogs necessary to strengthen the high-content drug discovery screening approaches.
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