Just Accepted by International Journal of Radiation Biology
The role of Beclin 1 in SDT induced apoptosis and autophagy in human leukemia cells Xiaomin Su, Xiaobing Wang, Quanhong Liu, Pan Wang , Chuanshan Xu, Albert Wingnang Leung Doi: 10.3109/09553002.2015.1021961
Int J Radiat Biol Downloaded from informahealthcare.com by Virginia Commonwealth University on 03/15/15 For personal use only.
Abstract Purpose: To prove the occurrence of autophagy after treatment by protoporphyrin IX (PpIX)- mediated sonodynamic therapy (SDT) of human chronic myelogenous leukemia K562 cells as well as its relationship with apoptosis. Materials and Methods: The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenylter-trazolium bromide tetrazolium (MTT) assay was adopted to examine cytotoxicity of different treatments. Nuclear morphology changes were observed under a fluorescence microscopy with 4’-6Diamidino-2-Phenylindole (DAPI) staining. Western blotting was used to analyze the expression of caspase-3, Beclin 1 (BECN 1) and the conversion of LC3-phosphatidylethanolamine conjugate / a cytosolic form of LC3 (LC3 II / I). Fluorescence microscope was used to identify the formation of autophagic vacuoles (AVO) during autophagy. Results: Under optimal conditions, SDT was shown to induce autophagy in K562 cells, which caused the up-regulation of Beclin-1 and the formation of AVO. In addition, pre-treatment of cancer cells with beclin 1-targeted short hairpin RNA (Beclin 1 shRNA) was shown to reduce the level of LC3-II accumulation and staining with punctate spots of monodansylcadaverine (MDC) staining. Besides, the cytotoxic effect of SDT was significantly increased by Beclin 1 shRNA. Furthermore, studies showed a marked effect on the apoptosis of cells by Beclin 1 shRNA to sonodamage with increased DAPI staining and caspase-3 cleavage. Conclusions: These results demonstrated that SDT significantly induced autophagy of K562 cells, probably to protect the K562 cells from sonodamage.
© 2015 Informa UK, Ltd. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted articles have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.
The role of Beclin 1 in SDT induced apoptosis and autophagy in human leukemia cells
Xiaomin Su1, Xiaobing Wang1, Quanhong Liu1, Pan Wang 1,2, Chuanshan Xu2, Albert Wingnang Leung2
Int J Radiat Biol Downloaded from informahealthcare.com by Virginia Commonwealth University on 03/15/15 For personal use only.
1
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, Shaanxi, China, 2School of Chinese Medicine, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong
Correspondence: Dr. Pan Wang and Prof. Albert Wingnang Leung, School of Chinese Medicine, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, China. E-mail: [email protected] or [email protected]
Short title: shRNA Beclin 1 enhanced SDT-induced apoptosis Abstract Purpose: To prove the occurrence of autophagy after treatment by protoporphyrin IX (PpIX)- mediated sonodynamic therapy (SDT) of human chronic myelogenous leukemia K562 cells as well as its relationship with apoptosis. Materials and Methods: The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltertrazolium bromide tetrazolium (MTT) assay was adopted to examine cytotoxicity of different treatments. Nuclear morphology changes were observed under a fluorescence microscopy with 4'-6-Diamidino-2-Phenylindole (DAPI) staining. Western blotting was used to analyze the expression of caspase-3, Beclin 1 (BECN 1) and the conversion of LC3-phosphatidylethanolamine conjugate / a cytosolic form of LC3 (LC3 II / I). Fluorescence microscope was used to identify the formation of autophagic vacuoles (AVO) during autophagy. Results: Under optimal conditions, SDT was shown to induce autophagy in K562 cells, which caused the up-regulation of Beclin-1 and the formation of AVO. In addition, pre-treatment of cancer cells with beclin 1-targeted short hairpin RNA (Beclin 1 shRNA) was shown to reduce the level of LC3-II accumulation and staining with punctate spots of monodansylcadaverine (MDC)
staining. Besides, the cytotoxic effect of SDT was significantly increased by Beclin 1 shRNA. Furthermore, studies showed a marked effect on the apoptosis of cells by Beclin 1 shRNA to sonodamage with increased DAPI staining and caspase-3 cleavage. Conclusions: These results demonstrated that SDT significantly induced autophagy of K562 cells, probably to protect the K562 cells from sonodamage.
Int J Radiat Biol Downloaded from informahealthcare.com by Virginia Commonwealth University on 03/15/15 For personal use only.
Keywords: Sonodynamic therapy, Beclin 1 shRNA, autophagy, apoptosis, Human leukemia K562 cells
Introduction Sonodynamic therapy (SDT), which involves the synergistic effect on cell killing by the combination of sonosensitizers and ultrasound, is a promising anticancer modality especially for cancer in deep-seated organs, terminal cancer and cancer resistant to chemotherapy (Rosenthal et al. 2004,
Int J Radiat Biol Downloaded from informahealthcare.com by Virginia Commonwealth University on 03/15/15 For personal use only.
Shibaguchi et al. 2011). Numerous studies have indicated that a specific sonosensitizer can be sonochemically activated and produce cytotoxic effects with ultrasound irradiation (Tachibana et al. 2008, El Maalouf et al. 2009). Currently, research on SDT mainly focuses on detecting tumor killing effects (He et al. 2010, Liu et al. 2008, Komori et al. 2009, Wang et al. 2011). In previous studies, some researchers also documented that protoporphyrin IX (PpIX) can accumulate specially in proliferative tumors and the ultrasound induced cytotoxicity could be significantly enhanced by the presence of PpIX (Mi et al. 2009). However, the mechanisms of PpIX mediated SDT have not been thoroughly understood. Recently, many mechanisms for SDT have been proposed by different research groups, including apoptosis (Wang et al. 2010a, Xiang et al. 2011) and autophagy (Wang et al. 2010b, Zhao et al. 2011). Apoptosis is defined as suicidal cell death which is one of the important mechanisms needed to maintain a stable internal environment (Lian et al. 2010). Autophagy is a reparative, life-sustaining process by which cytoplasmic components are sequestered in double-membrane vesicles and degraded on fusion with
lysosomes. It has been reported that autophagy may play an important role in the regulation of cancer development and progression (Ferraro and Cecconi 2007, Mizushima et al. 2011). In general, autophagy and apoptosis are two independent processes (Eisenberg-Lerner et al. 2009), but in other situations, possible molecular mechanisms for crosstalk between autophagy and
Int J Radiat Biol Downloaded from informahealthcare.com by Virginia Commonwealth University on 03/15/15 For personal use only.
apoptosis have been suggested (Marquez and Xu 2012). The crosstalk between the autophagy and apoptosis signaling pathways is critical to the overall fate of the cell depending on the nature of the stimulus and the actual metabolic status of the cell, thus autophagy can suppress or enable apoptosis (Maiuri et al. 2007). Therefore, an understanding of either pro-survival or pro-death functions of autophagy might shed light on enhancing the efficacy of anticancer therapy by the modulation of autophagy. Autophagy is regulated by specific genes known as autophagy-related genes (ATG). Many ATG proteins have been identified, including beclin 1 (BECN 1), which functions as a haplo-insufficient tumor suppressor gene and is a widely studied autophagy related gene (Qu et al. 2003). Beclin-1 levels appear to be one of the critical factors that affect the induction of autophagy. It assembles a multiprotein interactome which controls the initiation of autophagy and thus it has a critical role in cellular housekeeping and maintenance of homeostasis (Zhu et al. 2009, Kang et al. 2011, Liang et al. 1999). Moreover, Shrivastava et al. (2011) demonstrated that knockdown of Beclin 1 significantly inhibited autophagy. Beclin 1 plays a critical role in
the regulation of autophagy, apoptosis, differentiation, as well as in the development and progression of cancer. Therefore, the relationship between autophagy and apoptosis was further illustrated by short hairpin RNA (shRNA) studies with beclin 1-targeted shRNA (Beclin 1 shRNA) and explored the roles of beclin 1 in cell apoptosis and autophagy induction after
Int J Radiat Biol Downloaded from informahealthcare.com by Virginia Commonwealth University on 03/15/15 For personal use only.
SDT treatment. Materials and methods Chemicals Protoporphyrin IX disodium salt (PpIX), 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltertrazolium bromide tetrazolium (MTT), 4'-6-Diamidino-2Phenylindole (DAPI) and monodansylcadaverine (MDC), Antibody against microtubule-associated protein 1 light chain 3 (LC3) were purchased from Sigma Chemical Company (St Louis, MO, USA). Caspase-3 and β-actin antibody were acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody was supplied by EarthOX Company (San Francisco, CA, USA). All the other reagents were commercial products of analytical grade. Cell culture Human chronic myelogenous leukemia cell line K562 was obtained from Institute of Chinese Academy of Medical Sciences (Beijing, China). The cell line was cultured in Roswell Park Memorial Institute-1640 (RPMI-1640)
(Sigma, St. Louis, MO, USA) medium containing 10 % fetal bovine serum (FBS) (Life Technologies, Carlsbad, CA, USA), 1 % penicillin-streptomycin (100 U / ml penicillin and 100 μg / ml streptomycin) and 1 mM L- glutamine in cell culture flask under a humidified 5 % CO2 and 95 % air atmosphere at 37 ℃.
Int J Radiat Biol Downloaded from informahealthcare.com by Virginia Commonwealth University on 03/15/15 For personal use only.
Ultrasound exposure system The experimental set-up for insonation was the same as previously described (Su et al. 2014). Briefly, the focused ultrasound transducer (Institution of Applied Acoustics, Shaanxi Normal University (Xi’an, Shaanxi, China)) with a circular ceramic of 15 mm in diameter was submerged in degassed water in the tank facing directly upward. The focal length of the transducer is about 26 mm. The same transducer at a frequency of 1.1 MHz was used in this study. The continuous wave was generated and amplified by a multifunctional generator (AG1020, T&C Power Conversion, Inc, Rochester, NY, USA) before feeding the transducer. A sterile polystyrene tube containing a 0.5 ml cell suspension was placed into the focus center of ultrasound for 60 s duration, and was rotated at 20 rpm by a micro-motor to improve mixing and to provide a uniform exposure. Previous investigation indicated 1W/cm2 ultrasound intensity might be a threshold for cell damage (Wang et al. 2012), and was chosen to evaluate the sonodynamic responses when combined with PpIX throughout the present study.
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 (TP3001, Cheerman, Tianjin, China), and no significant variation of temperature was detected (± 1
Int J Radiat Biol Downloaded from informahealthcare.com by Virginia Commonwealth University on 03/15/15 For personal use only.
℃). Thus, any bio-effects observed in this study were considered to be nonthermal. SDT treatment protocols K562 cells in the exponential growth phase were collected and divided randomly into four groups: (1) control group, (2) PpIX alone group, (3) ultrasound alone group, and (4) ultrasound plus PpIX group. For PpIX alone group and ultrasound plus PpIX group, the cells were incubated with 2 μg / ml PpIX for 2 h in serum-free RPMI 1640 medium, allowing sufficient time for cell uptake of the sensitizer to reach a relatively high level. Instead of PpIX, an equivalent quantity of phosphate buffered saline (PBS) was used for the control group and ultrasound alone group. The cells in ultrasound group and ultrasound plus PpIX group were exposed to ultrasound at a frequency of 1.1 MHz and an intensity of 1 W/cm2 for 60 s duration. After various treatments, cells were re-suspended in fresh medium and cultured for an additional time as specified in the text and then subjected to different analysis. Cell toxicity assay
The cell toxicity was determined by MTT assay. At 24 h after ultrasound exposure, cells in 100 μl were added to 96 well culture plates, and cytotoxicity was determined by adding 10 μl MTT solution (5 mg/ml in PBS) to each well, and the mixture was incubated for 4 h at 37 ℃ in CO2 incubator. The formed formazan crystals were dissolved in 100 µl 10 %
Int J Radiat Biol Downloaded from informahealthcare.com by Virginia Commonwealth University on 03/15/15 For personal use only.
sodium dodecyl sulfate (SDS), 0.01 M HCl solution, and the absorbance at 570 nm was recorded using a microplate reader Bio-Tek ELX800 (Biotek Instruments, Winooski, VT, USA). Results were expressed as the percentage of MTT reduction, assuming that the absorbance of control was 100 %. MDC staining assay for autophagy detection MDC, a lysosomotropic fluorescent compound which selectively labels autophagic vacuoles (AVO), was used to analyze autophagic process (Yu et al. 2013). At 0.5 h post treatment, cells were stained with MDC at a final concentration of 20 mM for 20 minutes at 37 ℃. After labeling, cells were washed with PBS, and images were obtained with a Nikon E-600 fluorescence microscope (Niko, Tokyo, Japan). Excitation wave-length was 360-380 nm, and emission wavelength was 520-560 nm. shRNA transfection Recent developments indicate that beclin-1 is one of the most important mediators in the formation of the autophagosome because it is involved in the initial step of autophagosome formation (Pattingre et al. 2008). Beclin 1 shRNA and control shRNA were obtained from Sangon Biotech Co.
(Shanghai, China). Transfection was performed using turbofect transfection reagent according to the manufacturer’s protocol. DAPI staining DAPI is a fluorescence probe, which binds to natural double-stranded DNA and represents the change of the nuclei morphology to detect changes of
Int J Radiat Biol Downloaded from informahealthcare.com by Virginia Commonwealth University on 03/15/15 For personal use only.
nuclei morphology of K562 cells after different treatments. The cells were stained with 4 μg / ml DAPI for 30 min at 37 ℃. After washing with PBS, samples were viewed under a Nikon E-600 fluorescence microscopy with standard excitation filters (Nikon, Tokyo, Japan). Western blotting analysis SDS- polyacrylamide gelelectrophoresis (PAGE) and immunoblotting were performed according to standard procedures. Briefly, cells were lysed by Radio Immunoprecipitation Assay (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, Billerica, MA, USA) and blotted with primary antibodies at 4 ℃ overnight. The bound primary antibodies were then tagged with IRDye 680 Conjugated Immunoglobulin G (IgG) (Li-cor, Biosciences, Lincoln, NE, USA) at room temperature for 1 h. And the infrared fluorescence was detected with the Odyssey infrared imaging system (Li-Cor Bioscience, Lincoln, NE, USA). Statistical analysis
All values were expressed as means ± standard deviation (SD) of at least four independent experiments, the differences among the groups were analyzed of variance (one-way analysis of variance (ANOVA)), p
The role of Beclin 1 in SDT induced apoptosis and autophagy in human leukemia cells Xiaomin Su, Xiaobing Wang, Quanhong Liu, Pan Wang , Chuanshan Xu, Albert Wingnang Leung Doi: 10.3109/09553002.2015.1021961
Int J Radiat Biol Downloaded from informahealthcare.com by Virginia Commonwealth University on 03/15/15 For personal use only.
Abstract Purpose: To prove the occurrence of autophagy after treatment by protoporphyrin IX (PpIX)- mediated sonodynamic therapy (SDT) of human chronic myelogenous leukemia K562 cells as well as its relationship with apoptosis. Materials and Methods: The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenylter-trazolium bromide tetrazolium (MTT) assay was adopted to examine cytotoxicity of different treatments. Nuclear morphology changes were observed under a fluorescence microscopy with 4’-6Diamidino-2-Phenylindole (DAPI) staining. Western blotting was used to analyze the expression of caspase-3, Beclin 1 (BECN 1) and the conversion of LC3-phosphatidylethanolamine conjugate / a cytosolic form of LC3 (LC3 II / I). Fluorescence microscope was used to identify the formation of autophagic vacuoles (AVO) during autophagy. Results: Under optimal conditions, SDT was shown to induce autophagy in K562 cells, which caused the up-regulation of Beclin-1 and the formation of AVO. In addition, pre-treatment of cancer cells with beclin 1-targeted short hairpin RNA (Beclin 1 shRNA) was shown to reduce the level of LC3-II accumulation and staining with punctate spots of monodansylcadaverine (MDC) staining. Besides, the cytotoxic effect of SDT was significantly increased by Beclin 1 shRNA. Furthermore, studies showed a marked effect on the apoptosis of cells by Beclin 1 shRNA to sonodamage with increased DAPI staining and caspase-3 cleavage. Conclusions: These results demonstrated that SDT significantly induced autophagy of K562 cells, probably to protect the K562 cells from sonodamage.
© 2015 Informa UK, Ltd. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted articles have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.
The role of Beclin 1 in SDT induced apoptosis and autophagy in human leukemia cells
Xiaomin Su1, Xiaobing Wang1, Quanhong Liu1, Pan Wang 1,2, Chuanshan Xu2, Albert Wingnang Leung2
Int J Radiat Biol Downloaded from informahealthcare.com by Virginia Commonwealth University on 03/15/15 For personal use only.
1
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, Shaanxi, China, 2School of Chinese Medicine, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong
Correspondence: Dr. Pan Wang and Prof. Albert Wingnang Leung, School of Chinese Medicine, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, China. E-mail: [email protected] or [email protected]
Short title: shRNA Beclin 1 enhanced SDT-induced apoptosis Abstract Purpose: To prove the occurrence of autophagy after treatment by protoporphyrin IX (PpIX)- mediated sonodynamic therapy (SDT) of human chronic myelogenous leukemia K562 cells as well as its relationship with apoptosis. Materials and Methods: The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltertrazolium bromide tetrazolium (MTT) assay was adopted to examine cytotoxicity of different treatments. Nuclear morphology changes were observed under a fluorescence microscopy with 4'-6-Diamidino-2-Phenylindole (DAPI) staining. Western blotting was used to analyze the expression of caspase-3, Beclin 1 (BECN 1) and the conversion of LC3-phosphatidylethanolamine conjugate / a cytosolic form of LC3 (LC3 II / I). Fluorescence microscope was used to identify the formation of autophagic vacuoles (AVO) during autophagy. Results: Under optimal conditions, SDT was shown to induce autophagy in K562 cells, which caused the up-regulation of Beclin-1 and the formation of AVO. In addition, pre-treatment of cancer cells with beclin 1-targeted short hairpin RNA (Beclin 1 shRNA) was shown to reduce the level of LC3-II accumulation and staining with punctate spots of monodansylcadaverine (MDC)
staining. Besides, the cytotoxic effect of SDT was significantly increased by Beclin 1 shRNA. Furthermore, studies showed a marked effect on the apoptosis of cells by Beclin 1 shRNA to sonodamage with increased DAPI staining and caspase-3 cleavage. Conclusions: These results demonstrated that SDT significantly induced autophagy of K562 cells, probably to protect the K562 cells from sonodamage.
Int J Radiat Biol Downloaded from informahealthcare.com by Virginia Commonwealth University on 03/15/15 For personal use only.
Keywords: Sonodynamic therapy, Beclin 1 shRNA, autophagy, apoptosis, Human leukemia K562 cells
Introduction Sonodynamic therapy (SDT), which involves the synergistic effect on cell killing by the combination of sonosensitizers and ultrasound, is a promising anticancer modality especially for cancer in deep-seated organs, terminal cancer and cancer resistant to chemotherapy (Rosenthal et al. 2004,
Int J Radiat Biol Downloaded from informahealthcare.com by Virginia Commonwealth University on 03/15/15 For personal use only.
Shibaguchi et al. 2011). Numerous studies have indicated that a specific sonosensitizer can be sonochemically activated and produce cytotoxic effects with ultrasound irradiation (Tachibana et al. 2008, El Maalouf et al. 2009). Currently, research on SDT mainly focuses on detecting tumor killing effects (He et al. 2010, Liu et al. 2008, Komori et al. 2009, Wang et al. 2011). In previous studies, some researchers also documented that protoporphyrin IX (PpIX) can accumulate specially in proliferative tumors and the ultrasound induced cytotoxicity could be significantly enhanced by the presence of PpIX (Mi et al. 2009). However, the mechanisms of PpIX mediated SDT have not been thoroughly understood. Recently, many mechanisms for SDT have been proposed by different research groups, including apoptosis (Wang et al. 2010a, Xiang et al. 2011) and autophagy (Wang et al. 2010b, Zhao et al. 2011). Apoptosis is defined as suicidal cell death which is one of the important mechanisms needed to maintain a stable internal environment (Lian et al. 2010). Autophagy is a reparative, life-sustaining process by which cytoplasmic components are sequestered in double-membrane vesicles and degraded on fusion with
lysosomes. It has been reported that autophagy may play an important role in the regulation of cancer development and progression (Ferraro and Cecconi 2007, Mizushima et al. 2011). In general, autophagy and apoptosis are two independent processes (Eisenberg-Lerner et al. 2009), but in other situations, possible molecular mechanisms for crosstalk between autophagy and
Int J Radiat Biol Downloaded from informahealthcare.com by Virginia Commonwealth University on 03/15/15 For personal use only.
apoptosis have been suggested (Marquez and Xu 2012). The crosstalk between the autophagy and apoptosis signaling pathways is critical to the overall fate of the cell depending on the nature of the stimulus and the actual metabolic status of the cell, thus autophagy can suppress or enable apoptosis (Maiuri et al. 2007). Therefore, an understanding of either pro-survival or pro-death functions of autophagy might shed light on enhancing the efficacy of anticancer therapy by the modulation of autophagy. Autophagy is regulated by specific genes known as autophagy-related genes (ATG). Many ATG proteins have been identified, including beclin 1 (BECN 1), which functions as a haplo-insufficient tumor suppressor gene and is a widely studied autophagy related gene (Qu et al. 2003). Beclin-1 levels appear to be one of the critical factors that affect the induction of autophagy. It assembles a multiprotein interactome which controls the initiation of autophagy and thus it has a critical role in cellular housekeeping and maintenance of homeostasis (Zhu et al. 2009, Kang et al. 2011, Liang et al. 1999). Moreover, Shrivastava et al. (2011) demonstrated that knockdown of Beclin 1 significantly inhibited autophagy. Beclin 1 plays a critical role in
the regulation of autophagy, apoptosis, differentiation, as well as in the development and progression of cancer. Therefore, the relationship between autophagy and apoptosis was further illustrated by short hairpin RNA (shRNA) studies with beclin 1-targeted shRNA (Beclin 1 shRNA) and explored the roles of beclin 1 in cell apoptosis and autophagy induction after
Int J Radiat Biol Downloaded from informahealthcare.com by Virginia Commonwealth University on 03/15/15 For personal use only.
SDT treatment. Materials and methods Chemicals Protoporphyrin IX disodium salt (PpIX), 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltertrazolium bromide tetrazolium (MTT), 4'-6-Diamidino-2Phenylindole (DAPI) and monodansylcadaverine (MDC), Antibody against microtubule-associated protein 1 light chain 3 (LC3) were purchased from Sigma Chemical Company (St Louis, MO, USA). Caspase-3 and β-actin antibody were acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody was supplied by EarthOX Company (San Francisco, CA, USA). All the other reagents were commercial products of analytical grade. Cell culture Human chronic myelogenous leukemia cell line K562 was obtained from Institute of Chinese Academy of Medical Sciences (Beijing, China). The cell line was cultured in Roswell Park Memorial Institute-1640 (RPMI-1640)
(Sigma, St. Louis, MO, USA) medium containing 10 % fetal bovine serum (FBS) (Life Technologies, Carlsbad, CA, USA), 1 % penicillin-streptomycin (100 U / ml penicillin and 100 μg / ml streptomycin) and 1 mM L- glutamine in cell culture flask under a humidified 5 % CO2 and 95 % air atmosphere at 37 ℃.
Int J Radiat Biol Downloaded from informahealthcare.com by Virginia Commonwealth University on 03/15/15 For personal use only.
Ultrasound exposure system The experimental set-up for insonation was the same as previously described (Su et al. 2014). Briefly, the focused ultrasound transducer (Institution of Applied Acoustics, Shaanxi Normal University (Xi’an, Shaanxi, China)) with a circular ceramic of 15 mm in diameter was submerged in degassed water in the tank facing directly upward. The focal length of the transducer is about 26 mm. The same transducer at a frequency of 1.1 MHz was used in this study. The continuous wave was generated and amplified by a multifunctional generator (AG1020, T&C Power Conversion, Inc, Rochester, NY, USA) before feeding the transducer. A sterile polystyrene tube containing a 0.5 ml cell suspension was placed into the focus center of ultrasound for 60 s duration, and was rotated at 20 rpm by a micro-motor to improve mixing and to provide a uniform exposure. Previous investigation indicated 1W/cm2 ultrasound intensity might be a threshold for cell damage (Wang et al. 2012), and was chosen to evaluate the sonodynamic responses when combined with PpIX throughout the present study.
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 (TP3001, Cheerman, Tianjin, China), and no significant variation of temperature was detected (± 1
Int J Radiat Biol Downloaded from informahealthcare.com by Virginia Commonwealth University on 03/15/15 For personal use only.
℃). Thus, any bio-effects observed in this study were considered to be nonthermal. SDT treatment protocols K562 cells in the exponential growth phase were collected and divided randomly into four groups: (1) control group, (2) PpIX alone group, (3) ultrasound alone group, and (4) ultrasound plus PpIX group. For PpIX alone group and ultrasound plus PpIX group, the cells were incubated with 2 μg / ml PpIX for 2 h in serum-free RPMI 1640 medium, allowing sufficient time for cell uptake of the sensitizer to reach a relatively high level. Instead of PpIX, an equivalent quantity of phosphate buffered saline (PBS) was used for the control group and ultrasound alone group. The cells in ultrasound group and ultrasound plus PpIX group were exposed to ultrasound at a frequency of 1.1 MHz and an intensity of 1 W/cm2 for 60 s duration. After various treatments, cells were re-suspended in fresh medium and cultured for an additional time as specified in the text and then subjected to different analysis. Cell toxicity assay
The cell toxicity was determined by MTT assay. At 24 h after ultrasound exposure, cells in 100 μl were added to 96 well culture plates, and cytotoxicity was determined by adding 10 μl MTT solution (5 mg/ml in PBS) to each well, and the mixture was incubated for 4 h at 37 ℃ in CO2 incubator. The formed formazan crystals were dissolved in 100 µl 10 %
Int J Radiat Biol Downloaded from informahealthcare.com by Virginia Commonwealth University on 03/15/15 For personal use only.
sodium dodecyl sulfate (SDS), 0.01 M HCl solution, and the absorbance at 570 nm was recorded using a microplate reader Bio-Tek ELX800 (Biotek Instruments, Winooski, VT, USA). Results were expressed as the percentage of MTT reduction, assuming that the absorbance of control was 100 %. MDC staining assay for autophagy detection MDC, a lysosomotropic fluorescent compound which selectively labels autophagic vacuoles (AVO), was used to analyze autophagic process (Yu et al. 2013). At 0.5 h post treatment, cells were stained with MDC at a final concentration of 20 mM for 20 minutes at 37 ℃. After labeling, cells were washed with PBS, and images were obtained with a Nikon E-600 fluorescence microscope (Niko, Tokyo, Japan). Excitation wave-length was 360-380 nm, and emission wavelength was 520-560 nm. shRNA transfection Recent developments indicate that beclin-1 is one of the most important mediators in the formation of the autophagosome because it is involved in the initial step of autophagosome formation (Pattingre et al. 2008). Beclin 1 shRNA and control shRNA were obtained from Sangon Biotech Co.
(Shanghai, China). Transfection was performed using turbofect transfection reagent according to the manufacturer’s protocol. DAPI staining DAPI is a fluorescence probe, which binds to natural double-stranded DNA and represents the change of the nuclei morphology to detect changes of
Int J Radiat Biol Downloaded from informahealthcare.com by Virginia Commonwealth University on 03/15/15 For personal use only.
nuclei morphology of K562 cells after different treatments. The cells were stained with 4 μg / ml DAPI for 30 min at 37 ℃. After washing with PBS, samples were viewed under a Nikon E-600 fluorescence microscopy with standard excitation filters (Nikon, Tokyo, Japan). Western blotting analysis SDS- polyacrylamide gelelectrophoresis (PAGE) and immunoblotting were performed according to standard procedures. Briefly, cells were lysed by Radio Immunoprecipitation Assay (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, Billerica, MA, USA) and blotted with primary antibodies at 4 ℃ overnight. The bound primary antibodies were then tagged with IRDye 680 Conjugated Immunoglobulin G (IgG) (Li-cor, Biosciences, Lincoln, NE, USA) at room temperature for 1 h. And the infrared fluorescence was detected with the Odyssey infrared imaging system (Li-Cor Bioscience, Lincoln, NE, USA). Statistical analysis
All values were expressed as means ± standard deviation (SD) of at least four independent experiments, the differences among the groups were analyzed of variance (one-way analysis of variance (ANOVA)), p
