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Protoporphyrin IX-mediated sonodynamic action induces apoptosis of K562 cells
Ultrasonics 54 (2014) 275–284
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Protoporphyrin IX-mediated sonodynamic action induces apoptosis of K562 cells Xiaomin Su, Yixiang Li, Pan Wang, Xiaobing Wang ⇑, Quanhong Liu Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, Ministry of Education, National Engineering Laboratory for Resource Developing of Endangered Chinese Crude Drugs in Northwest of China, College of Life Sciences, Shaanxi Normal University, Xi’an, 710062 Shaanxi, China
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Article history: Received 18 December 2012 Received in revised form 14 July 2013 Accepted 21 July 2013 Available online 29 July 2013 Keywords: Sonodynamic therapy Protoporphyrin IX Human leukemia K562 cells ROS Apoptosis
a b s t r a c t Objectives: The present study aims to investigate apoptosis of human leukemia K562 cells induced by protoporphyrin IX (PpIX)-mediated sonodynamic therapy (PpIX-SDT). Methods: The uptakes of intracellular PpIX in K562 cells were detected by flow cytometry. The sub-cellular localization of PpIX was imaged by confocal microscope. The cytotoxic effect of PpIX-SDT was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenylter-trazolium bromide tetrazolium) assay. Apoptosis was evaluated by chromatin condensation with DAPI (40 -6-diamidino-2-phenylindole) staining, decrease of mitochondria membrane potential (MMP), re-distribution of Bax, and the expression changes of the key apoptosis-associated protein (Caspase-3 and polypeptide poly (ADP-robose) polymerase). The possible mechanism of SDT-induced apoptosis was investigated by detecting by intracellular ROS (reactive oxygen species) generation and effect of ROS scavenger-NAC (N-acetylcysteine) on SDT induced apoptosis. Results: The intracellular PpIX increased quickly within 2 h after PpIX administration and PpIX mainly localized in the mitochondria. Compared with PpIX alone and ultrasound alone groups, the synergistic cytotoxicity of PpIX plus ultrasound was significantly boosted. In addition, the ultrasound induced some extent of chromatin condensation and MMP loss was greatly enhanced by the presence of 2 lg/ml PpIX, where PpIX alone treatment showed no or only slight effect. Time-dependent Bax translocation, caspase3 activation and PARP cleavage were detected in SDT treatment groups. Besides, intracellular ROS production was significantly enhanced after SDT, and the general ROS scavenger NAC could obviously alleviate the SDT-caused cell viability loss, MMP loss, Bax redistribution and nuclear changes. Conclusions: These results indicated that PpIX-mediated sonodynamic action could induce apoptosis on K562 cells, and the intracellular ROS was involved in the PpIX-SDT induced apoptosis. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Leukemia is a malignant disorder of hematopoietic stem cell, which is harmful to human health. Presently, chemotherapy and bone marrow transplantation are still the common strategies for leukemia treatment, but with many side effects such as drug resistance, higher recurrence, and death by infection. Compared with these conventional cancer treatments, the contribution of sonodynamic therapy (SDT) to tumor treatment has got considerable attention in recent years [1–3]. SDT, which demonstrates a high level of tumor-specific cytotoxicity and minimal side-effects upon surrounding normal tissues, is based on photodynamic therapy (PDT), and it has been proven to be a promising new strategy for treatment of malignancies [4].
⇑ Corresponding author. Tel.: +86 29 85310275. E-mail address: [email protected] (X. Wang). 0041-624X/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ultras.2013.07.015
Sonosensitizer is a key component affecting the efficacy of SDT. Yumita et al. first reported that the photosensitive compounds activated by ultrasound can kill cancerous cells and suppress the growth of tumor [5]. PpIX is a most commonly used photosensitizer in PDT for treatment of skin premalignant and malignant lesions [6]. Kinoshita and Hynynen reported that PpIX could be considered as an effective sonosensitizer in SDT [7]. In previous studies, some researchers also documented that PpIX can accumulate specially in proliferative tumors and the ultrasound induced cytotoxicity could be significantly enhanced by the presence of PpIX [8,9]. These studies suggest that PpIX may be a good potential sonosensitizer for sonodynamic therapy in cancer treatment. Apoptosis is a programmed cell death in response to a variety of stimuli and usually characterized through distinct set of morphological and biochemical progresses [10]. Apoptosis induction is currently regarded as an important mechanism of chemotherapy and physical therapy which irreversibly induces tumor cell death [11,12]. Many recent reports concerning SDT have focused on
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apoptotic events after ultrasound irradiation with a sensitizer in vitro [4,13,14]. And, it was found that certain drugs, upon ultrasonic irradiation, created active oxygen species such as superoxide radicals, singlet oxygen, hydroxyl radical and other free radicals, and then effectively destroyed cancer cells/tissues [15]. Song et al. showed that the endogenous PpIX (synthesized by 5-aminolevulinic acid)-mediated SDT produced strong apoptotic effects on SAS cells, which were mainly related to the excessive intracellular ROS production followed by LPO (lipid pero-oxidation) increase and MMP (mitocondria membrane potential) decrease after exposure [16]. Li et al. suggested that SDT can induce C6 cell death through both necrosis and apoptosis, and the ROS generation during the process played a decisive role [17]. In the present study, we showed that the exogenous PpIX mediated SDT induced mitochondria–caspase dependent apoptosis in K562 cells, and ROS was involved in the process. 2. Materials and methods 2.1. Chemicals Protoporphyrin IX disodium salt (PpIX), 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltertrazolium bromide tetrazolium (MTT), rhodamine-123 (Rh123), 40 -6-Diamidino-2-Phenylindole (DAPI), and Nacetylcysteine (NAC) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Mito Tracker Green (MTG) and 20 ,70 dichlorodihydrofluorescein-diacetate (DCFH-DA) were supplied by Molecular Probes Inc. (Invitrogen, CA, USA). Antibodies against Bax and cleaved poly (ADP-ribose) polymerase (PARP) were obtained from Cell Signaling Technology (Beverly, USA). Caspase-3 antibody was acquired from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-GAPDH monoclonal antibody was supplied by EarthOX Company (San Francisco, CA, USA). All other reagents were analytical grade. 2.2. Cell culture Human chronic myelogenous leukemia cell line K562 was obtained from Institute of Chinese Academy of Medical Sciences, Beijing, China. Cells were grown in RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin, 100 lg/ml streptomycin, and 2 mM L-glutamine at 37 °C in a humidified 5% CO2 atmosphere. Each experiment utilized cells in the exponential phase (cell viability was above 98% using trypan blue exclusion test).
AG1020 apparatus indicated 3 W, which was used throughout the experiment. For all experiments, the coupling water was degassed before ultrasound treatment and was maintained at room temperature during irradiation. Temperature increase inside the test tube was measured before and after ultrasound treatment with a digital thermometer, and no significant variation of temperature was detected (±1 °C). Thus, any bio-effects observed in this study were considered to be non-thermal. 2.4. SDT treatment protocols K562 cells in the exponential phase were collected and divided randomly into four groups: (1) control, (2) PpIX alone, (3) ultrasound alone, and (4) ultrasound plus PpIX. For PpIX and ultrasound plus PpIX groups, cells were incubated with 2 lg/ml PpIX involved a 2 h drug-loading time in the serum-free RPMI 1640 medium, allowing sufficient time for cellular uptake of the sensitizer to reach a relatively high level. Instead of PpIX, an equivalent quantity of PBS was used for the control and ultrasound alone groups. The cells in ultrasound and ultrasound plus PpIX groups were exposed to ultrasound at a frequency of 1.1 MHz and an intensity of 1 W/ cm2 for 60 s duration. After the treatment procedure, cells were re-suspended in fresh medium and cultured for an additional time as specified in the text and then subjected to different analysis. 2.5. Cell uptake of PpIX detection by flow cytometry K562 cells were harvested in an exponential growth phase, washed once with serum-free medium and incubated with various concentrations of PpIX at 37 °C in a 6-well microplate for different time intervals. For determining PpIX content, cells were collected after different incubation time points and immediately detected with flow cytometry (Guava easyCyte 8HT, Millipore). The mean fluorescence intensity of PpIX was recorded at the same measurement conditions. 2.6. Sub-cellular localization of PpIX K562 cells were incubated with 2 lg/ml PpIX for 2 h, then coloaded with 2.5 nM Mito Tracker Green (MTG). After being washed twice with cold PBS (PBS, in g/L: NaCl 8.0 g, KCl 0.2 g, Na2HPO4 1.44 g, KH2PO4 0.24 g, pH 7.2), the cells were observed for the sub-cellular localization patterns of PpIX under a laser scanning confocal microscope (LSCM, Model TCS SP5, Leica, Germany). 2.7. Cell survival assay
2.3. Ultrasound exposure setup The experiment set-up for insonation was the same as previously described [18]. Briefly, the focused ultrasound transducer with a circular ceramic plate of 15 mm in diameter, manufactured by the Institution of Applied Acoustics, Shaanxi Normal University (Xi’an, Shaanxi, China), was submerged in degassed water in the tank facing directly upward. The electrical signal was generated and amplified by a multi-functional generator (AG1020, T&C Power Conversion, Inc, Rochester, NY, USA) before feeding the transducer. Ultrasound irradiation was conducted with a frequency of 1.1 MHz in a continuous wave mode for 60 s duration. 0.5 ml sample contained in a polypropylene test tube was placed into the center of the focal zone for irradiation. Samples were submerged entirely in degassed water and the test tube was rotated at 20 rpm by a micro-motor to improve mixing and to provide a uniform exposure. Suppose in the free field conditions, we previously evaluated the acoustic intensities in the tube were about 1 W/cm2 (ISATA, spatial average time average intensity) when the load power by the
The cytotoxicity of PpIX-mediated sonodynamic action on K562 cells was assessed using MTT assay. Briefly, 100 ll treated or untreated cells were incubated in a 96-well microplate at 37 °C for different time. 10 ll MTT solution (5 mg/ml in PBS) was added into each well, then incubated for 4 h at 37 °C. Finally, the formazan crystals were dissolved in the combined solution buffer (10% SDS, 5% isobutyl alcohol, 0.01 M HCl) [19], and the absorbance at 570 nm was recorded using a microplate reader (BIO-TEKELX800, USA) against the reference value at 630 nm. Results were expressed as the percentage of MTT reduction, assuming that the absorbance of control was 100%. 2.8. DAPI staining DAPI is a fluorescence probe, which binds to natural doublestranded DNAs, and represents the change of nuclei morphology. At 24 h after SDT treatment, K562 cells were collected and stained with 4 lg/ml DAPI for 30 min at 37 °C. The stained cells were
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The fluorescen intensity of intracellular PpIX (a.u.)
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Fig. 1. Uptakes and intracellular localization of PpIX in K562 cells. (a) K562 cells were incubated with different concentrations of PpIX for various incubation times. Data are presented as mean ± SD from three independent experiments (every sample has four replicates). (b) Sub-cellular localization of PpIX in K562 cells. After 2 h of incubation, K562 cells were co-loaded with mitochondria probe MTG. MTG and PpIX were visualized by confocal microscopy. Representative images are shown. PpIX displayed a red fluorescence pattern that corresponded well with that of the mitochondria probe MTG (green panel). (Scale bar: 7.5 lm). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
centrifuged at 2000 rpm for 5 min at 4 °C and washed three times in PBS, then observed using a fluorescence microscopy. The corresponding phase-contrast and fluorescence images were taken. 2.9. Mitochondria membrane potential detection
permeabilized with 0.1% Triton X-100 for 5 min at room temperature. After washing with PBS, cells were blocked with 1% bovine serum albumin (BSA) and incubated with anti-Bax antibody overnight at 4 °C, substitution of PBS for the primary antibody was used as negative controls. Then, all coverslips were incubated with a
Fluorescence probe Rh123 was used to evaluate perturbations in MMP (mitochondria membrane potential). Rh123 selectively enters mitochondria with an intact membrane potential and is retained in the mitochondria. Once the mitochondria membrane potential is lost, Rh123 is subsequently washed out of the cells. At 1 h post-treatment, cells were stained with 2 lg/ml Rh123 in an incubator for 20 min with gentle shaking. Then, the stained cells were centrifuged at 2000 rpm for 5 min at 4 °C and followed by washing with PBS, after that, the samples were immediately analyzed using flow cytometry (Guava easyCyte 8HT, Millipore). In addition, the stained cells were observed under a fluorescence microscopy (Nikon E-600, Japan). The excitation and emission waves of Rh123 were listed as 488 nm and 530 nm, respectively. 2.10. Bax translocation detection At the indicated times after SDT, cells were collected and incubated with 20 nM Mito Tracker Green for 20 min at 37 °C. Then cells were centrifuged at 2000 rpm for 5 min. The supernate was removed and the pellets were deposited on polylysine-coated coverslips, air dried, then the coverslips were immersed in 3.5% paraformaldehyde for 10 min for fixation. Cells were further
Fig. 2. The time course of the viability changes of K562 cells after different treatment. () Control group without any treatment; (j) 2 lg/ml PpIX alone; (N) 1 W/cm2 ultrasound alone; () 1 W/cm2 ultrasound plus 2 lg/ml PpIX. Cell viability was measured as MTT reduction and data were normalized as% control. The data indicate mean ± SD from four different experiments. Six replicates in each one experiment. p < 0.05 and p < 0.01 versus untreated control, ##p < 0.01 between groups.
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Fig. 3. Nuclear DAPI staining. The stained nuclei were visualized under a fluorescent microscope. Representative images are shown. Control, cells without any treatment; PpIX, cells were treated with 2 lg/ml PpIX alone; ultrosound, cells were irradiated with 1 W/cm2 ultrasound alone; SDT, cells were irradiated with 1 W/cm2 ultrasound in the presence of 2 lg/ml PpIX. Cells in the upper right corner of phase images were the zoom of representative cells of each group (indicated by the arrow). (Scale bar: 100 lm).
dilution (1:200) of the TRITC-labeled secondary antibody in 1% BSA in PBS for 1 h at room temperature in the dark; Coverslips were rinsed with PBS and mounted in glycerol-PBS (1:1) on slides. Cells were imaged with a Leica TCS SP5 inverted confocal laser scanning microscope associated with an Argon/HeNe laser light source.
To determine the role of ROS in SDT induced cell death and apoptosis, the pan ROS scavenger NAC (5 mM) was added to culture medium prior to loading PpIX by 1 h. The cell viability decrease, the MMP loss, the nuclear morphological changes and Bax translocations were specially analyzed as described above.
2.11. Western blotting analysis
2.13. Statistical analysis
SDS–PAGE and immunoblotting were performed according to standard procedures. Briefly, cells were lysed by RIPA buffer on ice. The protein samples were separated on a 10–15% SDS polyacrylamide gel, and then the gel was transferred to nitrocellulose membranes (Millipore, MA, USA) and blotted with primary antibodies (Caspase-3 and Cleaved-PARP) overnight at 4 °C. Caspase3 is a member of the apoptosis execution functional group of caspases, and is either partially or totally responsible for the proteolytic cleavage of many key proteins during apoptosis, such as poly (ADPribose) polymerase (PARP) [20]. Caspase-3 is a cytosolic protein found in cells as an inactive 32 kDa proenzyme. It is activated by proteolytic cleavage into active subunits (p17 and p20) only when cells undergo apoptosis. The used caspase-3 antibody detects endogenous levels of full length caspase-3 and the large fragment of caspase-3 resulting from cleavage. The used cleaved-PARP antibody specifically detects endogenous levels of the large fragment (89 kDa) of PARP produced by caspase cleavage, this antibody does not recognize full length PARP. The bound primary antibodies were then tagged with IRDye 680 Conjugated IgG (Li-cor, Biosciences) at room temperature for 1 h. And the infrared fluorescence was detected with the Odyssey infrared imaging system (Li-Cor Bioscience, Lincoln, NE).
All values were expressed as means ± SD of at least four independent experiments, the differences among the groups were analyzed of variance (one-way ANOVA), p < 0.05 were considered to be significant.
2.12. Intracellular ROS detection and its role in SDT caused cytotoxiciy Intracellular ROS was analyzed using a flow cytometer with DCFH-DA (2, 7-dichlorofluorescein-diacetate). DCFH-DA, a non fluorescent cell permeant compound, is cleaved by endogenous esterases within the cell and the de-esterified product can be converted into the fluorescent compound DCF upon oxidation by intracellular ROS. To estimate intracellular ROS, immediately after treatment, both of the control and treated cells were loaded with 100 nM DCFH-DA for 10 min at 37 °C, washed with PBS and immediately analyzed using flow cytometry.
3. Results 3.1. Uptakes and intracellular localization of PpIX in K562 cells The intracellular concentration changes of PpIX at different time points after addition to K562 cells were evaluated by the mean fluorescence intensity as determined by flow cytometry. As shown in Fig. 1a, immediately after PpIX administration, the intracellular PpIX increased quickly for the first 0.5 h, then slightly increased until 2 h after adding of PpIX, followed by a steady trend and almost sustained the same level at 3–5 h after adding of PpIX. While the intracellular PpIX accumulation curves at different concentrations showed similar trends. To assess the sub-cellular localization pattern of PpIX in K562 cells, we co-loaded cells with a mitochondrial specific dye-MTG at 2 h after incubation with PpIX. The fluorescence distributions of PpIX and MTG were captured using laser scanning confocal microscopy. In dual channel imaging, photomultiplier sensitivites and offsets were set to a level at which bleed through effects from one channel to another were negligible. The sub-cellular localization pattern of PpIX in K562 cells is shown in Fig. 1b. The red fluorescence pattern of PpIX corresponded well with that of MTG green fluorescence, indicating PpIX mainly localized in the mitochondria of K562 cells. 3.2. Cytotoxicity of PpIX-SDT on K562 cells The cytotoxic effect of PpIX-SDT on K562 cells was assessed by MTT assay. Compared with control, PpIX alone and ultrasound
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Fig. 4. Changes of mitochondrial membrane potential in K562 cells. At 1 h after treatment, cells were stained with 2 lg/ml Rh 123 and detected by flow cytometry. (a) Control group with untreated cell; (b) 2 lg/ml PpIX treatment alone; (c) 1 W/cm2 ultrasound treatment alone; (d) 1 W/cm2 ultrasound plus 2 lg/ml PpIX. The mean fluorescence intensity of Rh 123 was recorded and expressed as mean ± SD of three independent assessments.
alone caused some extent of cytotoxicity to K562 cells. Whereas the synergistic effect of PpIX plus ultrasound was significantly enhanced, which also displayed a time-dependent cell viability decrease after SDT treatment (Fig. 2). Immediately after exposure, PpIX alone and ultrasound alone decreased the cell viability to 91.1% (p > 0.05) and 85.9% (p > 0.05) of control, respectively, while SDT treatment significantly induced 39% cell viability loss (p < 0.01). After 24 h of incubation, the cell viability in PpIX group and ultrasound group was 76.8% (p < 0.05) and 84% (p > 0.05), and in SDT group was 45% (p < 0.01), suggesting the ultrasound induced K562 cytotoxicity was greatly enhanced by the presence of 2 lg/ml PpIX.
3.3. DAPI staining To investigate whether PpIX-mediated SDT could induce K562 cell apoptosis, the cells were stained with DAPI at 24 h post treatment at an ultrasonic intensity of 1 W/cm2 in the presence and absence of 2 lg/ml PpIX. As shown in Fig. 3, control cells displayed a weak fluorescence and had no visible nuclei changes, and the contrast phase indicated normal cell morphology with intact cell plasma membrane. Cells in PpIX alone and ultrasound alone groups
showed slight enhancing DAPI staining and few cells indicated damaged nuclei, and few cells with plasma membrane blebbing were observed under phase-contrast imaging. While in PpIX plus ultrasound group, typical apoptosis characteristics such as nuclear condensation with more enhancing DAPI staining and altered nuclei morphology were observed, and obvious cell lysis with broken cell membrane were observed under phase-contrast imaging. 3.4. MMP loss detection At early 1 h after treatment, the MMP loss as detected by flow cytometry with Rh123 staining significantly decreased in PpIXSDT treated cells (Fig. 4). The mean fluorescent intensity of Rh123 in SDT was calculated as 268.99 (p < 0.01), which was statistically much lower than that of PpIX (1009.13) and ultrasound (637.8) (p < 0.05) alone. 3.5. Confocal microscope observation Bax is a pro-apoptotic member of the Bcl-2 family which regulates programmed cell death. On the induction of apoptosis, Bax shifts from a soluble form to a membrane-bound form, which
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Fig. 5. Bax translocation of K562 cells after different incubation time following SDT treatment. (Scale bar: 25 lm).
Fig. 6. Western blots analysis of cleaved PARP, caspase-3 activation in K562 cells after different incubation time following SDT treatment. GAPDH was used as a loading control. The shown bolts are representative results of three independent experiments.
partially co-localizes with mitochondria. As shown in Fig. 5, in control cells, the MTG (mitochondria special dye) green fluorescence had little overlapping with the Bax red fluorescence. In contrast, SDT-treated cells exhibited colocalization between
mitochondria and Bax at 0.5 h post treatment, which was gradually enhanced by the incubation time and became more obvious at 2 h post treatment, which then restored to the similar distribution pattern of control at 4 h post SDT.
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Fig. 7. Measurement of intracellular ROS in K562 cells. The experiment was performed as specified in the methods, and the fluorescence intensity of DCF was recorded at the same measurement conditions. (a) Control group with untreated cell; (b) 2 lg/ml PpIX alone; (c) 1 W/cm2 ultrasound alone; (d) 1 W/cm2 ultrasound plus 2 lg/ml PpIX. The shown data indicate the percentage of cells with higher DCF fluorescence, which represent three independent assessments.
3.6. Changes of apoptosis related protein expression To further verify whether the apoptotic effect was involved in PpIX-SDT, we monitored the key apoptosis related protein (e.g. Caspase-3 and PARP) changes by using western blotting. As shown in Fig. 6, compare with control, SDT treatment caused slight increase of typical caspase-3 cleavage at 0.5 h post treatment, which was gradually enhanced by the incubation time and became more obvious at 4 h post treatment. Besides, we know, caspase-3 is either partially or totally responsible for the proteolytic cleavage of many key proteins such as the nuclear enzyme poly (ADP-ribose) polymerase (PARP). So, next, we examined PARP cleavage after treatment. The used cleaved PARP (Asp214) antibody detects endogenous levels of the large fragment (89 kDa) of human PARP produced by caspase cleavage. The result showed more obvious cleaved PARP fragment was detected at 4 h post SDT, which indirectly supported caspase activation and apoptotic response by SDT induction in K562 cells.
3.7. ROS involved in SDT-induced K562 cytotoxicity The intracellular ROS was analyzed by using flow cytometry with DCFH-DA staining immediately after different treatment. As
shown in Fig. 7, compared with control, 10.13% (p > 0.05) and 5.47% (p > 0.05) of cells in PpIX alone and ultrasound alone group showed higher DCF fluorescence, and which was enhanced to 23.87% in ultrasound plus PpIX group (p < 0.01). To further examine if intracellular ROS elevation was involved in SDT-induced cell death, we performed subsequent assays. Results in Fig. 8a showed that the decreased cell viability caused by SDT was partially rescued by NAC. In addition, the SDT caused MMP loss, Bax translocation and chromatin condensation were all visibly prevented by NAC (Fig. 8b–d). These results indicated that ROS was involved in SDT-induced damage in K562 cells.
4. Discussion Apoptosis is a complex process of cell death which involves various cellular factors and pathophysiologic pathways. The abnormal regulation of apoptosis may lead to numbers of diseases, including cancers. Due to the ability to specifically induce apoptosis on cancer cells, SDT represents one of the promising anticancer therapeutics [21,22]. Lagueaux et al. found that ultrasound exposure could induce apoptotic response in human leukemia cells, such as HL-60, Kgla and Nalm-60 [23]. Besides, our previous studies showed that the presence of PpIX could significantly enhance
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Fig. 8. ROS involved in SDT-induced death. (a) Effects of ROS inhibitor NAC on cell cytotoxicity of K562 cells. p < 0.01 versus SDT. (b) Effect of ROS inhibitor NAC on Rh123 labeling of K562 cells at 1 h after exposure. Control, cells without any treatment; NAC, cells with 5 mM NAC alone; SDT, cells were irradiated with 1 W/cm2 ultrasound in the presence of 2 lg/ml PpIX; SDT + NAC, cells were irradiated with 1 W/cm2 ultrasound in the presence of 2 lg/ml PpIX with NAC pre-treatment. Cells in the upper right corner of pictures were the magnification of representative cells of each group (indicated by the arrow). (Scale bar: 100 lm). (c) Bax translocation of K562 cells after 2 h incubation following SDT treatment with or without ROS inhibitor NAC. (Scale bar: 25 lm). (d) Nuclear DAPI staining in K562 cells at 24 h after SDT with or without ROS inhibitor NAC. Cells in the upper right corner of phase images were the zoom of representative cells of each group (indicated by the arrow). (Scale bar: 100 lm).
ultrasound-induced early and late apoptosis rate of murine hepatoma-22 cells and leukemia L1210 cells [24,25]. However, the cellular mechanisms of this process still remain unclear. In the present study, we aims to explore the underlining mechanisms of PpIX mediated SDT therapy in human leukemia K562 cells. As shown in Fig. 2, the cell survival rate was assessed by MTT assay after SDT treatment. The result showed that the combination of ultrasound and PpIX exerted more significant cytotoxic effect on K562 cells than ultrasound alone or PpIX alone under the same experimental conditions, implying PpIX might be a potential good sensitizer and PpIX-mediated SDT may provide an effective approach to combat with leukemia. The mitochondria–caspase pathway is widely considered as the main pathway in ultrasound-induced apoptosis [26]. Our previous results confirmed that porphyrins, such as hematoporphyrin (Hp) and PpIX, significantly enhanced the ultrasound-induced apoptosis
in S180 and Ehrlich ascites tumor cells, and mitochondrial damage was related [27,28]. In this study, we attempted to determine whether the mitochondria–caspase signal pathways were involved in SDT-induced apoptosis of K562 cells. So, firstly, the intracellular localization of PpIX in K562 cells was investigated. As shown in Fig. 1b, PpIX mainly localized in mitochondria. The sub-cellular localization of sonosensitizer is expected to be related to the probable primary sites of initial damage by SDT [8,29], so we speculated mitochondria played a central role in PpIX-ultrasound induced cell death, which was also in agreement with other reports [30]. Then, we monitored the mitochondrial membrane potential changes of K562 under the same experimental conditions. The MMP, as assessed by flow cytometry with Rh123, obviously decreased at early 1 h post SDT when compared with the other three groups. Moreover, the colocalization of mitochondria and Bax could be observed at 0.5 h post treatment, which turned to be more obvious with the
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Fig. 8 (continued)
prolonged incubation time, and could reach the peak at 2 h (Fig. 5). Also, DAPI staining evidenced typical apoptosis characteristics, such as nuclear condensation with enhancing DAPI staining. The altered nuclei morphology were observed in PpIX-SDT treated
cells, and the phase contrast image shows cell lysis and cytoplasma lost in the combined treatment group (Fig. 3). Besides, Western blot suggested after SDT, the apoptotic executioner protein-caspase-3 turned to its active sub-units, while the general substrate
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of caspase-3-PARP was cleaved to be 89 kDa fragment (Fig. 6). These data implied the mitochondria–caspase signaling pathway might be involved in PpIX-SDT induced apoptosis of K562 cells. Oxidative stress was thought to be one of the major initiators of apoptosis in SDT [27]. Recent studies found that except for a dramatic increase in apoptosis rate, ultrasound-activated sonosensitizer could also significantly enhance the intracellular ROS levels [31]. Xu et al. reported that the ROS generation during the synergistic effect of ultrasound and Photofrin decreased cell survival rates and enhanced apoptosis in GSCs [32]. Moreover, in 2011, Xiang et al. demonstrated that ROS from MB-mediated SDT may induce apoptosis in ovarian cancer cells [33]. In our paper, SDT could stimulate the generation of ROS in K562 cells obviously (Fig. 7). In order to confirm the role of ROS in PpIX-SDT induced effect, the general ROS scavenger-NAC was applied. Data indicated NAC significantly rescued SDT-induced K562 cytotoxicity, MMP loss, Bax redistribution and DNA damage, implying ROS may play a vital role in PpIX-SDT induced K562 cell apoptosis in our system (Fig. 8a–d). In conclusion, the presented results suggested that the synergy with PpIX and ultrasound led to more K562 cell death. ROS was involved in this process. Besides, mitochondria may be an important part in PpIX-SDT induced cell apoptosis. Further studies should focus on the relationship of ROS generation, mitochondria damage and cell apoptosis in this experiment system. Acknowledgments This work was supported by the Natural Science Foundation for Youth (No. 81000999, 10904087), the Fundamental Research Funds for the Central Universities (No. GK201102020) and the Innovation Funds of Graduate Programs, SNU (No. 2013CXB009). References [1] X. Wang, T.J. Lewis, D. Mitchell, et al., The tumoricidal effect of sonodynamic therapy (SDT) on S-180 sarcoma in mice, Integr. Cancer Ther. 7 (2) (2008) 96– 102. [2] K. Tomankova, H. Kolarova, P. Kolar, et al., Study of cytotoxic effect of photodynamically and sonodynamically activated sensitizers in vitro, Toxicol. In Vitro 23 (8) (2009) 1465–1471. [3] C. Komori, K. Okada, K. Kawamura, et al., The sonodynamic antitumor effect of methylene blue on sarcoma180 cells in vitro, Anticancer Res. 29 (6) (2009) 2411–2415. [4] Y. He, X. Xia, C. Xu, et al., 5-Aminolaevulinic acid enhances ultrasound-induced mitochondrial damage in K562 cells, Ultrasonics 50 (8) (2010) 777–781. [5] N. Yumita, R. Nishigaki, K. Umemura, et al., Hematoporphyrin as a sensitizer of cell damaging effect of ultrasound, Jpn. J. Cancer Res. 80 (3) (1989) 219–222. [6] N. Dögnitz, D. Salomon, M. Zellweger, et al., Comparison of ALA- and ALA hexyl-ester-induced PpIX depth distribution in human skin carcinoma, J. Photochem. Photobiol. B, Biol. 93 (3) (2008) 140–148. [7] M. Kinoshita, K. Hynynen, Mechanism of porphyrin-induced sonodynamic effect: possible role of hyperthermia, Radiat. Res. 165 (3) (2006) 299–306. [8] Z. Ji, G. Yang, V. Vasovic, et al., Subcellular localization pattern of protoporphyrin IX is an important determinant for its photodynamic efficiency of human carcinoma and normal cell lines, J. Photochem. Photobiol. B, Biol. 84 (3) (2006) 213–220. [9] N. Mi, Q. Liu, X. Wang, et al., Induction of sonodynamic effect with protoporphyrin IX on isolate Hepatoma-22 cells, Ultrasound Med. Biol. 35 (4) (2009) 680–686.
[10] Y.H. Kim, J.W. Park, J.Y. Lee, et al., Bcl-2 overexpression prevents daunorubicininduced apoptosis through inhibition of XIAP and Akt degradation, Biochem. Pharmacol. 66 (9) (2003) 1779–1786. [11] M.C. Jäckel, M.A. Dorudian, D. Marx, et al., Spontaneous apoptosis in laryngeal squamous cell carcinoma is independent of bcl-2 and bax protein expression, Cancer 85 (3) (1999) 591–599. [12] G.M. LaMuraglia, J. Schiereck, J. Heckenkamp, et al., Photodynamic therapy induces apoptosis in intimal hyperplastic arteries, Am. J. Pathol. 157 (3) (2000) 867–875. [13] J.B. Feril, T. Kondo, Q. Zhao, et al., Enhancement of hyperthermia-induced apoptosis by non-thermal effects of ultrasound, Cancer Lett. 178 (1) (2002) 63–70. [14] C. Scarponi, F. Nasorri, F. Pavani, et al., Low-frequency low-intensity ultrasounds do not influence the survival and immune functions of cultured keratinocytes and dendritic cells, J. Biomed. Biotechnol., 2009 (10.1155/2009/ 193260). [15] N. Miyoshi, V. Misik, P. Riesz, Sonodynamic toxicity of galliumporphyrin analogue ATX-70 in human leukemia cells, Radiat. Res. 148 (1) (1997) 43–47. [16] W. Song, H. Cui, R. Zhang, et al., Apoptosis of SAS cells induced by sonodynamic therapy using 5-aminolevulinic acid sonosensitizer, Anticancer Res. 31 (1) (2011) 39–45. [17] J. Li, W. Yue, Z. Huang, et al., Calcium overload induces C6 rat glioma cell apoptosis in sonodynamic therapy, Int. J. Radiat. Biol. 87 (10) (2011) 1061– 1066. [18] P. Wang, Y. Li, X. Wang, et al., Membrane damage effect of continuous wave ultrasound on K562 human leukemia cells, J. Ultrasound Med. 31 (12) (2012) 1977–1986. [19] J. Zhou, X. Yue, J. Han, et al., Improved MTT assay for activity of antitumor agents, Chin. J. Pharmaceut. 24 (10) (1993) 455–457. [20] G.M. Cohen, Caspases: the executioners of apoptosis, Biochem. J. 326 (Pt 1) (1997) 1–16. [21] K. Tachibana, L.B. Feril Jr., Y. Ikeda-Dantsuji, Sonodynamic therapy, Ultrasonics 48 (4) (2008) 253–259. [22] N. Yumita, K. Okudaira, Y. Momose, et al., Sonodynamically induced apoptosis and active oxygen generation by gallium–porphyrin complex, ATX-70, Cancer Chemother. Pharmacol. 66 (6) (2010) 1071–1078. [23] L. Lagneaux, E.C. de Meulenaer, A. Delforge, et al., Ultrasonic low-energy treatment: a novel approach to induce apoptosis in human leukemic cells, Exp. Hematol. 30 (11) (2002) 1293–1301. [24] X. Wang, Q. Liu, N. Mi, et al., Sonodynamically induced apoptosis by protoporphyrin IX on hepatoma-22 cells in vitro, Ultrasound Med. Biol. 36 (4) (2010) 667–676. [25] P. Zhao, Q. Liu, P. Wang, et al., Autophagic and apoptotic response to sonodynamic therapy induced cell damage in leukemia l1210 cells in vitro, Cancer Biother. Radiopharm. 26 (2) (2011) 209–218. [26] H. Honda, T. Kondo, Q. Zhao, et al., Role of intracellular calcium ions and reactive oxygen species in apoptosis induced by ultrasound, Ultrasound Med. Biol. 30 (5) (2004) 683–692. [27] W. Tang, Q. Liu, X. Wang, et al., Involvement of caspase 8 in apoptosis induced by ultrasound-activated hematoporphyrin in sarcoma 180 cells in vitro, J. Ultrasound Med. 27 (4) (2008) 645–656. [28] X. Zhao, Q. Liu, W. Tang, et al., Damage effects of protoporphyrin IXsonodynamic therapy on the cytoskeletal F-actin of Ehrlich ascites carcinoma cells, Ultrason. Sonochem. 16 (1) (2009) 50–56. [29] Q. Peng, J. Moan, J.M. Nesland, Correlation of subcellular localization and intratumoral photosensitizer localization with ultrastructural features after photodynamic therapy, Ultrastruct. Pathol. 20 (2) (1996) 109–129. [30] Y. Li, P. Wang, P. Zhao, et al., Apoptosis induced by sonodynamic treatment by protoporphyrin IX on MDA-MB-231 cells, Ultrasonics 52 (4) (2012) 490–496. [31] W. Tang, Q. Liu, X. Wang, et al., Potential mechanism in sonodynamic therapy and focused ultrasound induced apoptosis in sarcoma 180 cells in vitro, Ultrasonics 49 (8) (2009) 786–793. [32] Z. Xu, K. Wang, X. Li, et al., The ABCG2 transporter is a key molecular determinant of the efficacy of sonodynamic therapy with photofrin in glioma stem-like cells, Ultrasonics 53 (1) (2013) 232–238. [33] J. Xiang, X. Xia, Y. Jiang, et al., Apoptosis of ovarian cancer cells induced by methylene blue-mediated sonodynamic action, Ultrasonics 51 (3) (2010) 390– 395.
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Protoporphyrin IX-mediated sonodynamic action induces apoptosis of K562 cells Xiaomin Su, Yixiang Li, Pan Wang, Xiaobing Wang ⇑, Quanhong Liu Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, Ministry of Education, National Engineering Laboratory for Resource Developing of Endangered Chinese Crude Drugs in Northwest of China, College of Life Sciences, Shaanxi Normal University, Xi’an, 710062 Shaanxi, China
a r t i c l e
i n f o
Article history: Received 18 December 2012 Received in revised form 14 July 2013 Accepted 21 July 2013 Available online 29 July 2013 Keywords: Sonodynamic therapy Protoporphyrin IX Human leukemia K562 cells ROS Apoptosis
a b s t r a c t Objectives: The present study aims to investigate apoptosis of human leukemia K562 cells induced by protoporphyrin IX (PpIX)-mediated sonodynamic therapy (PpIX-SDT). Methods: The uptakes of intracellular PpIX in K562 cells were detected by flow cytometry. The sub-cellular localization of PpIX was imaged by confocal microscope. The cytotoxic effect of PpIX-SDT was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenylter-trazolium bromide tetrazolium) assay. Apoptosis was evaluated by chromatin condensation with DAPI (40 -6-diamidino-2-phenylindole) staining, decrease of mitochondria membrane potential (MMP), re-distribution of Bax, and the expression changes of the key apoptosis-associated protein (Caspase-3 and polypeptide poly (ADP-robose) polymerase). The possible mechanism of SDT-induced apoptosis was investigated by detecting by intracellular ROS (reactive oxygen species) generation and effect of ROS scavenger-NAC (N-acetylcysteine) on SDT induced apoptosis. Results: The intracellular PpIX increased quickly within 2 h after PpIX administration and PpIX mainly localized in the mitochondria. Compared with PpIX alone and ultrasound alone groups, the synergistic cytotoxicity of PpIX plus ultrasound was significantly boosted. In addition, the ultrasound induced some extent of chromatin condensation and MMP loss was greatly enhanced by the presence of 2 lg/ml PpIX, where PpIX alone treatment showed no or only slight effect. Time-dependent Bax translocation, caspase3 activation and PARP cleavage were detected in SDT treatment groups. Besides, intracellular ROS production was significantly enhanced after SDT, and the general ROS scavenger NAC could obviously alleviate the SDT-caused cell viability loss, MMP loss, Bax redistribution and nuclear changes. Conclusions: These results indicated that PpIX-mediated sonodynamic action could induce apoptosis on K562 cells, and the intracellular ROS was involved in the PpIX-SDT induced apoptosis. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Leukemia is a malignant disorder of hematopoietic stem cell, which is harmful to human health. Presently, chemotherapy and bone marrow transplantation are still the common strategies for leukemia treatment, but with many side effects such as drug resistance, higher recurrence, and death by infection. Compared with these conventional cancer treatments, the contribution of sonodynamic therapy (SDT) to tumor treatment has got considerable attention in recent years [1–3]. SDT, which demonstrates a high level of tumor-specific cytotoxicity and minimal side-effects upon surrounding normal tissues, is based on photodynamic therapy (PDT), and it has been proven to be a promising new strategy for treatment of malignancies [4].
⇑ Corresponding author. Tel.: +86 29 85310275. E-mail address: [email protected] (X. Wang). 0041-624X/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ultras.2013.07.015
Sonosensitizer is a key component affecting the efficacy of SDT. Yumita et al. first reported that the photosensitive compounds activated by ultrasound can kill cancerous cells and suppress the growth of tumor [5]. PpIX is a most commonly used photosensitizer in PDT for treatment of skin premalignant and malignant lesions [6]. Kinoshita and Hynynen reported that PpIX could be considered as an effective sonosensitizer in SDT [7]. In previous studies, some researchers also documented that PpIX can accumulate specially in proliferative tumors and the ultrasound induced cytotoxicity could be significantly enhanced by the presence of PpIX [8,9]. These studies suggest that PpIX may be a good potential sonosensitizer for sonodynamic therapy in cancer treatment. Apoptosis is a programmed cell death in response to a variety of stimuli and usually characterized through distinct set of morphological and biochemical progresses [10]. Apoptosis induction is currently regarded as an important mechanism of chemotherapy and physical therapy which irreversibly induces tumor cell death [11,12]. Many recent reports concerning SDT have focused on
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apoptotic events after ultrasound irradiation with a sensitizer in vitro [4,13,14]. And, it was found that certain drugs, upon ultrasonic irradiation, created active oxygen species such as superoxide radicals, singlet oxygen, hydroxyl radical and other free radicals, and then effectively destroyed cancer cells/tissues [15]. Song et al. showed that the endogenous PpIX (synthesized by 5-aminolevulinic acid)-mediated SDT produced strong apoptotic effects on SAS cells, which were mainly related to the excessive intracellular ROS production followed by LPO (lipid pero-oxidation) increase and MMP (mitocondria membrane potential) decrease after exposure [16]. Li et al. suggested that SDT can induce C6 cell death through both necrosis and apoptosis, and the ROS generation during the process played a decisive role [17]. In the present study, we showed that the exogenous PpIX mediated SDT induced mitochondria–caspase dependent apoptosis in K562 cells, and ROS was involved in the process. 2. Materials and methods 2.1. Chemicals Protoporphyrin IX disodium salt (PpIX), 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltertrazolium bromide tetrazolium (MTT), rhodamine-123 (Rh123), 40 -6-Diamidino-2-Phenylindole (DAPI), and Nacetylcysteine (NAC) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Mito Tracker Green (MTG) and 20 ,70 dichlorodihydrofluorescein-diacetate (DCFH-DA) were supplied by Molecular Probes Inc. (Invitrogen, CA, USA). Antibodies against Bax and cleaved poly (ADP-ribose) polymerase (PARP) were obtained from Cell Signaling Technology (Beverly, USA). Caspase-3 antibody was acquired from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-GAPDH monoclonal antibody was supplied by EarthOX Company (San Francisco, CA, USA). All other reagents were analytical grade. 2.2. Cell culture Human chronic myelogenous leukemia cell line K562 was obtained from Institute of Chinese Academy of Medical Sciences, Beijing, China. Cells were grown in RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin, 100 lg/ml streptomycin, and 2 mM L-glutamine at 37 °C in a humidified 5% CO2 atmosphere. Each experiment utilized cells in the exponential phase (cell viability was above 98% using trypan blue exclusion test).
AG1020 apparatus indicated 3 W, which was used throughout the experiment. For all experiments, the coupling water was degassed before ultrasound treatment and was maintained at room temperature during irradiation. Temperature increase inside the test tube was measured before and after ultrasound treatment with a digital thermometer, and no significant variation of temperature was detected (±1 °C). Thus, any bio-effects observed in this study were considered to be non-thermal. 2.4. SDT treatment protocols K562 cells in the exponential phase were collected and divided randomly into four groups: (1) control, (2) PpIX alone, (3) ultrasound alone, and (4) ultrasound plus PpIX. For PpIX and ultrasound plus PpIX groups, cells were incubated with 2 lg/ml PpIX involved a 2 h drug-loading time in the serum-free RPMI 1640 medium, allowing sufficient time for cellular uptake of the sensitizer to reach a relatively high level. Instead of PpIX, an equivalent quantity of PBS was used for the control and ultrasound alone groups. The cells in ultrasound and ultrasound plus PpIX groups were exposed to ultrasound at a frequency of 1.1 MHz and an intensity of 1 W/ cm2 for 60 s duration. After the treatment procedure, cells were re-suspended in fresh medium and cultured for an additional time as specified in the text and then subjected to different analysis. 2.5. Cell uptake of PpIX detection by flow cytometry K562 cells were harvested in an exponential growth phase, washed once with serum-free medium and incubated with various concentrations of PpIX at 37 °C in a 6-well microplate for different time intervals. For determining PpIX content, cells were collected after different incubation time points and immediately detected with flow cytometry (Guava easyCyte 8HT, Millipore). The mean fluorescence intensity of PpIX was recorded at the same measurement conditions. 2.6. Sub-cellular localization of PpIX K562 cells were incubated with 2 lg/ml PpIX for 2 h, then coloaded with 2.5 nM Mito Tracker Green (MTG). After being washed twice with cold PBS (PBS, in g/L: NaCl 8.0 g, KCl 0.2 g, Na2HPO4 1.44 g, KH2PO4 0.24 g, pH 7.2), the cells were observed for the sub-cellular localization patterns of PpIX under a laser scanning confocal microscope (LSCM, Model TCS SP5, Leica, Germany). 2.7. Cell survival assay
2.3. Ultrasound exposure setup The experiment set-up for insonation was the same as previously described [18]. Briefly, the focused ultrasound transducer with a circular ceramic plate of 15 mm in diameter, manufactured by the Institution of Applied Acoustics, Shaanxi Normal University (Xi’an, Shaanxi, China), was submerged in degassed water in the tank facing directly upward. The electrical signal was generated and amplified by a multi-functional generator (AG1020, T&C Power Conversion, Inc, Rochester, NY, USA) before feeding the transducer. Ultrasound irradiation was conducted with a frequency of 1.1 MHz in a continuous wave mode for 60 s duration. 0.5 ml sample contained in a polypropylene test tube was placed into the center of the focal zone for irradiation. Samples were submerged entirely in degassed water and the test tube was rotated at 20 rpm by a micro-motor to improve mixing and to provide a uniform exposure. Suppose in the free field conditions, we previously evaluated the acoustic intensities in the tube were about 1 W/cm2 (ISATA, spatial average time average intensity) when the load power by the
The cytotoxicity of PpIX-mediated sonodynamic action on K562 cells was assessed using MTT assay. Briefly, 100 ll treated or untreated cells were incubated in a 96-well microplate at 37 °C for different time. 10 ll MTT solution (5 mg/ml in PBS) was added into each well, then incubated for 4 h at 37 °C. Finally, the formazan crystals were dissolved in the combined solution buffer (10% SDS, 5% isobutyl alcohol, 0.01 M HCl) [19], and the absorbance at 570 nm was recorded using a microplate reader (BIO-TEKELX800, USA) against the reference value at 630 nm. Results were expressed as the percentage of MTT reduction, assuming that the absorbance of control was 100%. 2.8. DAPI staining DAPI is a fluorescence probe, which binds to natural doublestranded DNAs, and represents the change of nuclei morphology. At 24 h after SDT treatment, K562 cells were collected and stained with 4 lg/ml DAPI for 30 min at 37 °C. The stained cells were
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The fluorescen intensity of intracellular PpIX (a.u.)
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Fig. 1. Uptakes and intracellular localization of PpIX in K562 cells. (a) K562 cells were incubated with different concentrations of PpIX for various incubation times. Data are presented as mean ± SD from three independent experiments (every sample has four replicates). (b) Sub-cellular localization of PpIX in K562 cells. After 2 h of incubation, K562 cells were co-loaded with mitochondria probe MTG. MTG and PpIX were visualized by confocal microscopy. Representative images are shown. PpIX displayed a red fluorescence pattern that corresponded well with that of the mitochondria probe MTG (green panel). (Scale bar: 7.5 lm). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
centrifuged at 2000 rpm for 5 min at 4 °C and washed three times in PBS, then observed using a fluorescence microscopy. The corresponding phase-contrast and fluorescence images were taken. 2.9. Mitochondria membrane potential detection
permeabilized with 0.1% Triton X-100 for 5 min at room temperature. After washing with PBS, cells were blocked with 1% bovine serum albumin (BSA) and incubated with anti-Bax antibody overnight at 4 °C, substitution of PBS for the primary antibody was used as negative controls. Then, all coverslips were incubated with a
Fluorescence probe Rh123 was used to evaluate perturbations in MMP (mitochondria membrane potential). Rh123 selectively enters mitochondria with an intact membrane potential and is retained in the mitochondria. Once the mitochondria membrane potential is lost, Rh123 is subsequently washed out of the cells. At 1 h post-treatment, cells were stained with 2 lg/ml Rh123 in an incubator for 20 min with gentle shaking. Then, the stained cells were centrifuged at 2000 rpm for 5 min at 4 °C and followed by washing with PBS, after that, the samples were immediately analyzed using flow cytometry (Guava easyCyte 8HT, Millipore). In addition, the stained cells were observed under a fluorescence microscopy (Nikon E-600, Japan). The excitation and emission waves of Rh123 were listed as 488 nm and 530 nm, respectively. 2.10. Bax translocation detection At the indicated times after SDT, cells were collected and incubated with 20 nM Mito Tracker Green for 20 min at 37 °C. Then cells were centrifuged at 2000 rpm for 5 min. The supernate was removed and the pellets were deposited on polylysine-coated coverslips, air dried, then the coverslips were immersed in 3.5% paraformaldehyde for 10 min for fixation. Cells were further
Fig. 2. The time course of the viability changes of K562 cells after different treatment. () Control group without any treatment; (j) 2 lg/ml PpIX alone; (N) 1 W/cm2 ultrasound alone; () 1 W/cm2 ultrasound plus 2 lg/ml PpIX. Cell viability was measured as MTT reduction and data were normalized as% control. The data indicate mean ± SD from four different experiments. Six replicates in each one experiment. p < 0.05 and p < 0.01 versus untreated control, ##p < 0.01 between groups.
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Fig. 3. Nuclear DAPI staining. The stained nuclei were visualized under a fluorescent microscope. Representative images are shown. Control, cells without any treatment; PpIX, cells were treated with 2 lg/ml PpIX alone; ultrosound, cells were irradiated with 1 W/cm2 ultrasound alone; SDT, cells were irradiated with 1 W/cm2 ultrasound in the presence of 2 lg/ml PpIX. Cells in the upper right corner of phase images were the zoom of representative cells of each group (indicated by the arrow). (Scale bar: 100 lm).
dilution (1:200) of the TRITC-labeled secondary antibody in 1% BSA in PBS for 1 h at room temperature in the dark; Coverslips were rinsed with PBS and mounted in glycerol-PBS (1:1) on slides. Cells were imaged with a Leica TCS SP5 inverted confocal laser scanning microscope associated with an Argon/HeNe laser light source.
To determine the role of ROS in SDT induced cell death and apoptosis, the pan ROS scavenger NAC (5 mM) was added to culture medium prior to loading PpIX by 1 h. The cell viability decrease, the MMP loss, the nuclear morphological changes and Bax translocations were specially analyzed as described above.
2.11. Western blotting analysis
2.13. Statistical analysis
SDS–PAGE and immunoblotting were performed according to standard procedures. Briefly, cells were lysed by RIPA buffer on ice. The protein samples were separated on a 10–15% SDS polyacrylamide gel, and then the gel was transferred to nitrocellulose membranes (Millipore, MA, USA) and blotted with primary antibodies (Caspase-3 and Cleaved-PARP) overnight at 4 °C. Caspase3 is a member of the apoptosis execution functional group of caspases, and is either partially or totally responsible for the proteolytic cleavage of many key proteins during apoptosis, such as poly (ADPribose) polymerase (PARP) [20]. Caspase-3 is a cytosolic protein found in cells as an inactive 32 kDa proenzyme. It is activated by proteolytic cleavage into active subunits (p17 and p20) only when cells undergo apoptosis. The used caspase-3 antibody detects endogenous levels of full length caspase-3 and the large fragment of caspase-3 resulting from cleavage. The used cleaved-PARP antibody specifically detects endogenous levels of the large fragment (89 kDa) of PARP produced by caspase cleavage, this antibody does not recognize full length PARP. The bound primary antibodies were then tagged with IRDye 680 Conjugated IgG (Li-cor, Biosciences) at room temperature for 1 h. And the infrared fluorescence was detected with the Odyssey infrared imaging system (Li-Cor Bioscience, Lincoln, NE).
All values were expressed as means ± SD of at least four independent experiments, the differences among the groups were analyzed of variance (one-way ANOVA), p < 0.05 were considered to be significant.
2.12. Intracellular ROS detection and its role in SDT caused cytotoxiciy Intracellular ROS was analyzed using a flow cytometer with DCFH-DA (2, 7-dichlorofluorescein-diacetate). DCFH-DA, a non fluorescent cell permeant compound, is cleaved by endogenous esterases within the cell and the de-esterified product can be converted into the fluorescent compound DCF upon oxidation by intracellular ROS. To estimate intracellular ROS, immediately after treatment, both of the control and treated cells were loaded with 100 nM DCFH-DA for 10 min at 37 °C, washed with PBS and immediately analyzed using flow cytometry.
3. Results 3.1. Uptakes and intracellular localization of PpIX in K562 cells The intracellular concentration changes of PpIX at different time points after addition to K562 cells were evaluated by the mean fluorescence intensity as determined by flow cytometry. As shown in Fig. 1a, immediately after PpIX administration, the intracellular PpIX increased quickly for the first 0.5 h, then slightly increased until 2 h after adding of PpIX, followed by a steady trend and almost sustained the same level at 3–5 h after adding of PpIX. While the intracellular PpIX accumulation curves at different concentrations showed similar trends. To assess the sub-cellular localization pattern of PpIX in K562 cells, we co-loaded cells with a mitochondrial specific dye-MTG at 2 h after incubation with PpIX. The fluorescence distributions of PpIX and MTG were captured using laser scanning confocal microscopy. In dual channel imaging, photomultiplier sensitivites and offsets were set to a level at which bleed through effects from one channel to another were negligible. The sub-cellular localization pattern of PpIX in K562 cells is shown in Fig. 1b. The red fluorescence pattern of PpIX corresponded well with that of MTG green fluorescence, indicating PpIX mainly localized in the mitochondria of K562 cells. 3.2. Cytotoxicity of PpIX-SDT on K562 cells The cytotoxic effect of PpIX-SDT on K562 cells was assessed by MTT assay. Compared with control, PpIX alone and ultrasound
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Fig. 4. Changes of mitochondrial membrane potential in K562 cells. At 1 h after treatment, cells were stained with 2 lg/ml Rh 123 and detected by flow cytometry. (a) Control group with untreated cell; (b) 2 lg/ml PpIX treatment alone; (c) 1 W/cm2 ultrasound treatment alone; (d) 1 W/cm2 ultrasound plus 2 lg/ml PpIX. The mean fluorescence intensity of Rh 123 was recorded and expressed as mean ± SD of three independent assessments.
alone caused some extent of cytotoxicity to K562 cells. Whereas the synergistic effect of PpIX plus ultrasound was significantly enhanced, which also displayed a time-dependent cell viability decrease after SDT treatment (Fig. 2). Immediately after exposure, PpIX alone and ultrasound alone decreased the cell viability to 91.1% (p > 0.05) and 85.9% (p > 0.05) of control, respectively, while SDT treatment significantly induced 39% cell viability loss (p < 0.01). After 24 h of incubation, the cell viability in PpIX group and ultrasound group was 76.8% (p < 0.05) and 84% (p > 0.05), and in SDT group was 45% (p < 0.01), suggesting the ultrasound induced K562 cytotoxicity was greatly enhanced by the presence of 2 lg/ml PpIX.
3.3. DAPI staining To investigate whether PpIX-mediated SDT could induce K562 cell apoptosis, the cells were stained with DAPI at 24 h post treatment at an ultrasonic intensity of 1 W/cm2 in the presence and absence of 2 lg/ml PpIX. As shown in Fig. 3, control cells displayed a weak fluorescence and had no visible nuclei changes, and the contrast phase indicated normal cell morphology with intact cell plasma membrane. Cells in PpIX alone and ultrasound alone groups
showed slight enhancing DAPI staining and few cells indicated damaged nuclei, and few cells with plasma membrane blebbing were observed under phase-contrast imaging. While in PpIX plus ultrasound group, typical apoptosis characteristics such as nuclear condensation with more enhancing DAPI staining and altered nuclei morphology were observed, and obvious cell lysis with broken cell membrane were observed under phase-contrast imaging. 3.4. MMP loss detection At early 1 h after treatment, the MMP loss as detected by flow cytometry with Rh123 staining significantly decreased in PpIXSDT treated cells (Fig. 4). The mean fluorescent intensity of Rh123 in SDT was calculated as 268.99 (p < 0.01), which was statistically much lower than that of PpIX (1009.13) and ultrasound (637.8) (p < 0.05) alone. 3.5. Confocal microscope observation Bax is a pro-apoptotic member of the Bcl-2 family which regulates programmed cell death. On the induction of apoptosis, Bax shifts from a soluble form to a membrane-bound form, which
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Fig. 5. Bax translocation of K562 cells after different incubation time following SDT treatment. (Scale bar: 25 lm).
Fig. 6. Western blots analysis of cleaved PARP, caspase-3 activation in K562 cells after different incubation time following SDT treatment. GAPDH was used as a loading control. The shown bolts are representative results of three independent experiments.
partially co-localizes with mitochondria. As shown in Fig. 5, in control cells, the MTG (mitochondria special dye) green fluorescence had little overlapping with the Bax red fluorescence. In contrast, SDT-treated cells exhibited colocalization between
mitochondria and Bax at 0.5 h post treatment, which was gradually enhanced by the incubation time and became more obvious at 2 h post treatment, which then restored to the similar distribution pattern of control at 4 h post SDT.
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Fig. 7. Measurement of intracellular ROS in K562 cells. The experiment was performed as specified in the methods, and the fluorescence intensity of DCF was recorded at the same measurement conditions. (a) Control group with untreated cell; (b) 2 lg/ml PpIX alone; (c) 1 W/cm2 ultrasound alone; (d) 1 W/cm2 ultrasound plus 2 lg/ml PpIX. The shown data indicate the percentage of cells with higher DCF fluorescence, which represent three independent assessments.
3.6. Changes of apoptosis related protein expression To further verify whether the apoptotic effect was involved in PpIX-SDT, we monitored the key apoptosis related protein (e.g. Caspase-3 and PARP) changes by using western blotting. As shown in Fig. 6, compare with control, SDT treatment caused slight increase of typical caspase-3 cleavage at 0.5 h post treatment, which was gradually enhanced by the incubation time and became more obvious at 4 h post treatment. Besides, we know, caspase-3 is either partially or totally responsible for the proteolytic cleavage of many key proteins such as the nuclear enzyme poly (ADP-ribose) polymerase (PARP). So, next, we examined PARP cleavage after treatment. The used cleaved PARP (Asp214) antibody detects endogenous levels of the large fragment (89 kDa) of human PARP produced by caspase cleavage. The result showed more obvious cleaved PARP fragment was detected at 4 h post SDT, which indirectly supported caspase activation and apoptotic response by SDT induction in K562 cells.
3.7. ROS involved in SDT-induced K562 cytotoxicity The intracellular ROS was analyzed by using flow cytometry with DCFH-DA staining immediately after different treatment. As
shown in Fig. 7, compared with control, 10.13% (p > 0.05) and 5.47% (p > 0.05) of cells in PpIX alone and ultrasound alone group showed higher DCF fluorescence, and which was enhanced to 23.87% in ultrasound plus PpIX group (p < 0.01). To further examine if intracellular ROS elevation was involved in SDT-induced cell death, we performed subsequent assays. Results in Fig. 8a showed that the decreased cell viability caused by SDT was partially rescued by NAC. In addition, the SDT caused MMP loss, Bax translocation and chromatin condensation were all visibly prevented by NAC (Fig. 8b–d). These results indicated that ROS was involved in SDT-induced damage in K562 cells.
4. Discussion Apoptosis is a complex process of cell death which involves various cellular factors and pathophysiologic pathways. The abnormal regulation of apoptosis may lead to numbers of diseases, including cancers. Due to the ability to specifically induce apoptosis on cancer cells, SDT represents one of the promising anticancer therapeutics [21,22]. Lagueaux et al. found that ultrasound exposure could induce apoptotic response in human leukemia cells, such as HL-60, Kgla and Nalm-60 [23]. Besides, our previous studies showed that the presence of PpIX could significantly enhance
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Fig. 8. ROS involved in SDT-induced death. (a) Effects of ROS inhibitor NAC on cell cytotoxicity of K562 cells. p < 0.01 versus SDT. (b) Effect of ROS inhibitor NAC on Rh123 labeling of K562 cells at 1 h after exposure. Control, cells without any treatment; NAC, cells with 5 mM NAC alone; SDT, cells were irradiated with 1 W/cm2 ultrasound in the presence of 2 lg/ml PpIX; SDT + NAC, cells were irradiated with 1 W/cm2 ultrasound in the presence of 2 lg/ml PpIX with NAC pre-treatment. Cells in the upper right corner of pictures were the magnification of representative cells of each group (indicated by the arrow). (Scale bar: 100 lm). (c) Bax translocation of K562 cells after 2 h incubation following SDT treatment with or without ROS inhibitor NAC. (Scale bar: 25 lm). (d) Nuclear DAPI staining in K562 cells at 24 h after SDT with or without ROS inhibitor NAC. Cells in the upper right corner of phase images were the zoom of representative cells of each group (indicated by the arrow). (Scale bar: 100 lm).
ultrasound-induced early and late apoptosis rate of murine hepatoma-22 cells and leukemia L1210 cells [24,25]. However, the cellular mechanisms of this process still remain unclear. In the present study, we aims to explore the underlining mechanisms of PpIX mediated SDT therapy in human leukemia K562 cells. As shown in Fig. 2, the cell survival rate was assessed by MTT assay after SDT treatment. The result showed that the combination of ultrasound and PpIX exerted more significant cytotoxic effect on K562 cells than ultrasound alone or PpIX alone under the same experimental conditions, implying PpIX might be a potential good sensitizer and PpIX-mediated SDT may provide an effective approach to combat with leukemia. The mitochondria–caspase pathway is widely considered as the main pathway in ultrasound-induced apoptosis [26]. Our previous results confirmed that porphyrins, such as hematoporphyrin (Hp) and PpIX, significantly enhanced the ultrasound-induced apoptosis
in S180 and Ehrlich ascites tumor cells, and mitochondrial damage was related [27,28]. In this study, we attempted to determine whether the mitochondria–caspase signal pathways were involved in SDT-induced apoptosis of K562 cells. So, firstly, the intracellular localization of PpIX in K562 cells was investigated. As shown in Fig. 1b, PpIX mainly localized in mitochondria. The sub-cellular localization of sonosensitizer is expected to be related to the probable primary sites of initial damage by SDT [8,29], so we speculated mitochondria played a central role in PpIX-ultrasound induced cell death, which was also in agreement with other reports [30]. Then, we monitored the mitochondrial membrane potential changes of K562 under the same experimental conditions. The MMP, as assessed by flow cytometry with Rh123, obviously decreased at early 1 h post SDT when compared with the other three groups. Moreover, the colocalization of mitochondria and Bax could be observed at 0.5 h post treatment, which turned to be more obvious with the
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prolonged incubation time, and could reach the peak at 2 h (Fig. 5). Also, DAPI staining evidenced typical apoptosis characteristics, such as nuclear condensation with enhancing DAPI staining. The altered nuclei morphology were observed in PpIX-SDT treated
cells, and the phase contrast image shows cell lysis and cytoplasma lost in the combined treatment group (Fig. 3). Besides, Western blot suggested after SDT, the apoptotic executioner protein-caspase-3 turned to its active sub-units, while the general substrate
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of caspase-3-PARP was cleaved to be 89 kDa fragment (Fig. 6). These data implied the mitochondria–caspase signaling pathway might be involved in PpIX-SDT induced apoptosis of K562 cells. Oxidative stress was thought to be one of the major initiators of apoptosis in SDT [27]. Recent studies found that except for a dramatic increase in apoptosis rate, ultrasound-activated sonosensitizer could also significantly enhance the intracellular ROS levels [31]. Xu et al. reported that the ROS generation during the synergistic effect of ultrasound and Photofrin decreased cell survival rates and enhanced apoptosis in GSCs [32]. Moreover, in 2011, Xiang et al. demonstrated that ROS from MB-mediated SDT may induce apoptosis in ovarian cancer cells [33]. In our paper, SDT could stimulate the generation of ROS in K562 cells obviously (Fig. 7). In order to confirm the role of ROS in PpIX-SDT induced effect, the general ROS scavenger-NAC was applied. Data indicated NAC significantly rescued SDT-induced K562 cytotoxicity, MMP loss, Bax redistribution and DNA damage, implying ROS may play a vital role in PpIX-SDT induced K562 cell apoptosis in our system (Fig. 8a–d). In conclusion, the presented results suggested that the synergy with PpIX and ultrasound led to more K562 cell death. ROS was involved in this process. Besides, mitochondria may be an important part in PpIX-SDT induced cell apoptosis. Further studies should focus on the relationship of ROS generation, mitochondria damage and cell apoptosis in this experiment system. Acknowledgments This work was supported by the Natural Science Foundation for Youth (No. 81000999, 10904087), the Fundamental Research Funds for the Central Universities (No. GK201102020) and the Innovation Funds of Graduate Programs, SNU (No. 2013CXB009). References [1] X. Wang, T.J. Lewis, D. Mitchell, et al., The tumoricidal effect of sonodynamic therapy (SDT) on S-180 sarcoma in mice, Integr. Cancer Ther. 7 (2) (2008) 96– 102. [2] K. Tomankova, H. Kolarova, P. Kolar, et al., Study of cytotoxic effect of photodynamically and sonodynamically activated sensitizers in vitro, Toxicol. In Vitro 23 (8) (2009) 1465–1471. [3] C. Komori, K. Okada, K. Kawamura, et al., The sonodynamic antitumor effect of methylene blue on sarcoma180 cells in vitro, Anticancer Res. 29 (6) (2009) 2411–2415. [4] Y. He, X. Xia, C. Xu, et al., 5-Aminolaevulinic acid enhances ultrasound-induced mitochondrial damage in K562 cells, Ultrasonics 50 (8) (2010) 777–781. [5] N. Yumita, R. Nishigaki, K. Umemura, et al., Hematoporphyrin as a sensitizer of cell damaging effect of ultrasound, Jpn. J. Cancer Res. 80 (3) (1989) 219–222. [6] N. Dögnitz, D. Salomon, M. Zellweger, et al., Comparison of ALA- and ALA hexyl-ester-induced PpIX depth distribution in human skin carcinoma, J. Photochem. Photobiol. B, Biol. 93 (3) (2008) 140–148. [7] M. Kinoshita, K. Hynynen, Mechanism of porphyrin-induced sonodynamic effect: possible role of hyperthermia, Radiat. Res. 165 (3) (2006) 299–306. [8] Z. Ji, G. Yang, V. Vasovic, et al., Subcellular localization pattern of protoporphyrin IX is an important determinant for its photodynamic efficiency of human carcinoma and normal cell lines, J. Photochem. Photobiol. B, Biol. 84 (3) (2006) 213–220. [9] N. Mi, Q. Liu, X. Wang, et al., Induction of sonodynamic effect with protoporphyrin IX on isolate Hepatoma-22 cells, Ultrasound Med. Biol. 35 (4) (2009) 680–686.
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