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Nitric oxide induces apoptosis and autophagy; autophagy down-regulates NO synthesis in physalin A-treated A375-S2 human melanoma cells
Food and Chemical Toxicology 71 (2014) 128–135
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Nitric oxide induces apoptosis and autophagy; autophagy down-regulates NO synthesis in physalin A-treated A375-S2 human melanoma cells Hao He a,b, Yong-Sheng Feng b, Ling-He Zang b, Wei-Wei Liu b, Li-Qin Ding c, Li-Xia Chen a, Ning Kang c, Toshihiko Hayashi b, Shin-ichi Tashiro d, Satoshi Onodera e, Feng Qiu a,c,⇑, Takashi Ikejima b,⇑ a Department of Natural Products Chemistry, School of Traditional Chinese Materia Medica, Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, People’s Republic of China b China-Japan Research Institute of Medical Pharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, People’s Republic of China c Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, People’s Republic of China d Institute for Clinical and Biomedical Sciences, Kyoto 603-8072, Japan e Department of Clinical and Biomedical Sciences, Showa Pharmaceutical University, Tokyo 194-8543, Japan
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Article history: Received 11 January 2014 Accepted 10 June 2014 Available online 19 June 2014 Keywords: Physalin A A375-S2 cells Nitric oxide mTOR Apoptosis Autophagy
a b s t r a c t Physalin A is an active withanolide isolated from Physalis alkekengi var. franchetii, a traditional Chinese herbal medicine named Jindenglong, which has been used for the treatment of sore throat, hepatitis, eczema and tumors in China. Our previous study demonstrated that physalin A induced apoptosis and cyto-protective autophagy in A375-S2 human melanoma cells. Induction of reactive oxygen species (ROS) with physalin A triggered apoptosis. In this study, NO generated by physalin A induced apoptosis and autophagy in A375-S2 cells, since physalin A induced the expression of inducible nitric oxide synthase (iNOS) in the cells. Generation of NO partially promoted both apoptosis and autophagy in A375-S2 cells. NO suppressed mTOR expression, which led to autophagy induction. An autophagic inhibitor, 3-methyladenine (3MA) promoted NO production, while acceleration of autophagy with an autophagic agonist rapamycin repressed NO production, suggesting that autophagy and NO production form a negative feedback loop that eventually protects the cells from apoptosis. The results together with the previous study indicate apoptosis and autophagy induced by physalin A in A375-S2 cells; the autophagy, repressing production of reactive nitrogen species (RNS) and ROS, protects the cells from apoptosis. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Abbreviations: 1400W, N-(3-(aminomethyl) benzyl) acetamidine; 3MA, 3methyladenine; ATCC, American Type Culture Collection; DAF2-DA, 4,5-diaminofluorescein diacetate; DAF-2T, diaminofluoresceine-2 triazole; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; ICAD, inhibitor of caspase-activated DNase; iNOS, inducible nitric oxide synthase; MDC, monodansylcadaverine; MEM, Minimum Essential Medium; mTOR, the mammalian target of rapamycin; MTT, 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NO, nitric oxide; NOS, nitric oxide synthase; PARP, poly ADP-ribose polymerase; PI, propidium iodide; RNS, reactive nitrogen species; ROS, reactive oxygen species. ⇑ Corresponding authors. Address: China-Japan Research Institute of Medical Pharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, People’s Republic of China. Tel./fax: +86 22 59596163 (F. Qiu). Tel./fax: +86 24 23844463 (T. Ikejima). E-mail addresses: [email protected] (F. Qiu), [email protected] (T. Ikejima). http://dx.doi.org/10.1016/j.fct.2014.06.007 0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.
Physalin A is an active withanolide isolated from Physalis alkekengi var. franchetii (Chinese name: ‘‘Jindenglong’’). We previously demonstrated that physalin A induced both apoptosis and autophagy in A375-S2 human melanoma cells, and autophagy played a protective role against apoptosis. However, the mechanism of the protective autophagy was not fully understood (He et al., 2013). NO, a highly reactive free radical, is a signaling molecule of major importance. Synthesised by NOS from L-arginine, it is involved in inflammation, immunological function, apoptosis and autophagy (Murad, 2006). Previous studies reported that NO induced cytotoxicity for several tumor cells, suggesting that NO could induce cancer cell apoptosis and autophagy (Fan et al., 2011; Kim, 2012; Zang et al., 2012). Our study showed that
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Fig. 1. Physalin A induced NO generation in A375-S2 cells. (A) The chemical structure of physalin A. (B) A375-S2 cells were treated with 15 lM physalin A for 0, 12, 24 and 36 h, and DAF-2T, the fluorescent dye product of DAF-2 in reaction with NO, was measured by flow cytometric analysis. (C) The cells were lysed after treatment with 15 lM physalin A for 6, 12, 18 and 24 h, and the protein levels of iNOS were determined by western blot analysis. b-Actin was used as a loading reference. Band density of the specific protein was analyzed with Quantity One image software and the results were expressed as average density to b-actin. (D–E) A375-S2 cells were pre-treated with 1400W (20 lM) or Carboxy-PTIO (200 lM) for 1 h prior to 15 lM physalin A treatment for 24 h. DAF-2T positive ratio was measured by flow cytometric analysis (D), and the cell viability was measured by MTT assay (E). Mean ± S.D. of three independent experiments. ⁄p < 0.05, ⁄⁄p < 0.01 vs. control group, #p < 0.05, ##p < 0.01 vs. physalin A group.
physalin A treatment was correlated with the induction of reactive oxygen species (He et al., 2013), and NO was found to promote autophagy under severe oxidative stress (Fan et al., 2011; Yang et al., 2010). It is also widely accepted that iNOS, which produces NO, is expressed in response to several stresses (Kang et al., 2013; Nomura and Kitamura, 1993).
Apoptosis is a process of cellular suicide followed by many physiological process, including nuclear fragmentation, cell shrinkage, chromatin condensation and membrane blebbing (Gerl and Vaux, 2005). The regulation of apoptosis is not an intrinsic function of drugs and genes. If the goal of a drug therapy or gene regulation is to induce the death of cancer cells, its ability to induce indirect
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Fig. 2. NO was involved in physalin A induced apoptosis in A375-S2 cells. (A) A375-S2 cells were incubated with 15 lM physalin A in the presence or absence of 1400W (20 lM) or Carboxy-PTIO (200 lM) for 24 h, and cell morphologies were observed by phase contrast microscopy (200 magnification, bar = 20 lm). Arrows indicate the apoptotic bodies. (B–C) A375-S2 cells were pre-treated with 20 lM 1400W or 200 lM Carboxy-PTIO for 1 h prior to the addition of 15 lM physalin A and then incubated for 24 h. The apoptotic rate was determined by flow cytometric analysis with PI staining (B), and the protein levels of caspase-3, PARP and ICAD were detected by western blot analysis. b-Actin was used as a loading reference. Band density of the specific protein was analyzed with Quantity One image software and the results were expressed as average density to b-actin (C). n = 3, mean ± S.D. ⁄p < 0.05, ⁄⁄p < 0.01 vs. control group, #p < 0.05, ##p < 0.01 vs. physalin A group.
cell suicide might be just as important as its direct cytotoxic activity. Extreme level of autophagy is also a process of cellular suicide, as well as lysosome-dependent protein degradation, and organelle turnover and removal (Finkel et al., 2007). Sometimes, autophagy is a survival mechanism under starvation, differentiation or severe oxidative stress. NO is also involved in apoptosis and autophagy under several stresses, including some kind of drug therapy, severe oxidative stress, differentiation and hormonal stimulation. The mechanisms of NO involvement in relation to apoptosis and autophagy are complex (Lee et al., 2012); both inhibitory role and accelerating role of NO in autophagy can be found in the literature (Barsoum et al., 2006; Sarkar et al., 2011). The mammalian target of rapamycin (mTOR) is a protein kinase at the nexus between oncogenic phosphoinositide 3-kinase (PI3K) signaling and downstream pathways. Two complexes, called mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), are known
to play crucial roles in cell growth, proliferation, and cellular metabolism (Guertin and Sabatini, 2009). The activity of mTORC1 can be inhibited by rapamycin or starvation, well-established inducers of autophagy (Noda and Ohsumi, 1998). Autophagy is controlled by many diverse signals, such as growth factors, amino acids, and energy status, and in these pathways, phosphatidylinositol 3-kinases and the protein kinase mTOR play important roles (Meijer and Codogno, 2006). In this study, we examined the effect of physalin A on the mTOR expression. We have recently demonstrated that physalin A induced apoptosis and cyto-protective autophagy in A375-S2 human melanoma cells. Reactive oxygen species (ROS) triggered apoptotic cell death under such circumstance (He et al., 2013). However, the relation between apoptosis and autophagy were unclear. In this study, we attempted to elucidate the mechanism of physalin A-induced NO production involved in the death of A375-S2 cells. Elucidation of
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Fig. 3. NO was involved in physalin A induced autophagy in A375-S2 cells. (A) A375-S2 cells were incubated with 15 lM physalin A for 6, 12, 18 and 24 h, followed by western blot analysis for detection of mTOR, p-mTOR and ATG5. (B) The cells were pre-treated with 1400W (20 lM) or Carboxy-PTIO (200 lM) for 1 h prior to 15 lM physalin A treatment for 24 h. The MDC positive ratio was determined by flow cytometric analysis with MDC staining. (C) Cells were transfected with GFP-LC3 and then treated with physalin A in the presence or absence of 1400W and Carboxy-PTIO for 24 h. Scale bar 10 lm. GFP-LC3 puncta per cell were calculated in 30 cells and expressed as the mean ± S.D. (D) Effects of 1400W and Carboxy-PTIO on the expression of iNOS, mTOR and p-mTOR, and the conversion from LC3 I to LC3 II were detected by western blot analysis. (E) The cells were treated with physalin A for 24 h in the absence or presence of chloroquine (CQ). At the end of treatment, the levels of LC3 and p62 were examined by western blot analysis. b-Actin was used as a loading reference. Band density of the specific protein was analyzed with Quantity One image software and the results were expressed as average density to b-actin. n = 3, mean ± S.D. ⁄p < 0.05, ⁄⁄p < 0.01 vs. control group, #p < 0.05, ##p < 0.01 vs. physalin A group.
detailed mechanisms might provide cues for understanding the relationship between physalin A-induced apoptosis and autophagy in A375-S2 cells.
2. Material and methods 2.1. Chemicals and reagents Physalin A was prepared from P. alkekengi var. franchetii by our laboratory (Qiu et al., 2008) and identified by comparing its chemical and spectroscopic (1H NMR, 13 C NMR) data with those reported in the literature (Kawai et al., 1992). The purity was measured by HPLC and determined to be 98.0% [column: Aglient ZORBAX SBC18, 4.6 150 mm, 5 lm; solvent phase: methanol–H2O (60:40)]. Physalin A was
dissolved in dimethyl sulfoxide (DMSO) as a stock solution, which was diluted in Minimum Essential Medium (MEM) before the experiments. The DMSO concentration was kept below 0.05% when used in all cell cultures that DMSO did not exert any detectable effect on cell growth or viability. Fetal bovine serum (FBS) was obtained from TBD Biotechnology Development (Tianjin, People’s Republic of China); MEM was obtained from Gibco (Grand Island, NY, USA). 4,5-Diaminofluorescein diacetate (DAF2-DA) was obtained from Cayman Chemical (Ann Arbor, MI, USA), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), monodansylcadaverine (MDC), chloroquine (CQ), N-(3-(aminomethyl) benzyl) acetamidine (1400W), 3MA, and rapamycin were purchased from Sigma (St. Louis, MO, USA). Carboxy-PTIO was from Beyotime Institute of Biotechnology (Haimen, China). Nonspecific-siRNA and Beclin 1-siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). TranSmarter was from Abmart (Shanghai, People’s Republic of China). Polyclonal antibodies against iNOS, caspase-3, PARP (poly ADP-ribose polymerase), ICAD (inhibitor of caspase-activated
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Fig. 3 (continued)
DNase), mTOR, p-mTOR, b-actin, LC3, Beclin 1, ATG5, p62 and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Electrochemiluminescence (ECL) reagent was from Thermo Scientific (Rockford, IL, USA).
2.2. Cell culture The A375-S2 human melanoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). They were cultured in MEM medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 lg/mL streptomycin and maintained at 37 °C in a humidified atmosphere with 5% CO2. The cells in the exponential phase of growth were used in the experiments.
2.3. Cell viability assay A375-S2 cells were incubated in 96-well flat bottom cell culture clusters (Corning, NY, USA) at a density of 8 103 cells per well and cultured for 24 h. Thereafter, the cells were treated with or without an inhibitor of nitric oxide synthase 1400W or NO scavenger Carboxy-PTIO at the given concentrations for 1 h prior to physalin A treatment for 24 h. After that, the cells were rinsed with ice-cold PBS twice and incubated with 5.0 mg/mL MTT solution at 37 °C for 2 h. The resulting crystals were dissolved in 150 lL DMSO and the optical density was measured by MTT assay using a mirco-plate reader (Thermo Multiskan MK3, Thermo scientific, Helsinki, Finland). The percentage of cell viability was calculated as follows:
Cell viability ð%Þ ¼ ðA492sample A492blank Þ=ðA492control A492blank Þ 100
2.6. Flow cytometric analysis of apoptosis and autophagy A375-S2 cells were incubated in 25 mL culture bottle at a density of 2 105 per bottle for 24 h. Then the cells were treated with physalin A in the presence or absence of 1400W and Carboxy-PTIO for indicated time periods. The cells were harvested and rinsed with PBS. For measuring apoptosis, the collected cells were then fixed with 500 lL PBS and 10 mL 70% ethanol at 4 °C for 18 h, after washing twice with PBS, the cells were suspended with 1 mL PI solution (PI 50 lg/mL and RNase A 1 mg/mL) and placed in an ice bath for 1 h. For measuring autophagy, the collected cells were suspended in 0.05 mM MDC at 37 °C for 1 h. Then the samples were analyzed by FACScan flow cytometry. 2.7. Western blot analysis A375-S2 cells were treated with 15 lM physalin A for indicated time periods, or treated with 15 lM physalin A for 24 h in the presence or absence of chloroquine, 1400W, Carboxy-PTIO, or with 3MA or rapamycin at given concentrations. All the cells were harvested and lysed in lysis buffer (50 mM Hepes pH 7.4, 1% Triton X100, 2 mM sodium orthovanadate, 100 mM sodium fluoride, 1 mM edetic acid, 1 mM egtazic acid, 1 mM PMSF, 10 lg/mL aprotinin and 10 lg/mL leupeptin) at 4 °C for 60 min. Lysates were centrifuged at 12,000g for 15 min, and the protein content of the supernatant was determined with the Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of the total protein were separated by 12% SDS–polyacrylamide gel electrophoresis and electrophoretically transferred to ImmobilonÒ-P Transfer Membrane (Millipore Corporation, Billerica, MA, USA). The membranes were soaked in blocking buffer (5% skimmed milk). Proteins were detected with the primary antibodies against iNOS, mTOR, p-mTOR, caspase-3, PARP, ICAD, b-actin, LC3 or Beclin 1, followed by horseradish perosidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology) and visualized by using ECL as the HRP substrate.
2.4. Observation of morphologic changes A375-S2 cells (2 104 cells/well) were inoculated into 24-well culture plates (Corning, NY, USA). After 24 h incubation, the cells were treated with or without 1400W or Carboxy-PTIO at the given concentration for 1 h prior to physalin A treatment for 24 h. The cellular morphology was observed by using a phase contrast microscope (Leica, Nussloch, Germany).
2.5. Flow cytometric analysis of nitric oxide production After treatment with 15 lM physalin A in the presence or absence of 1400W, Carboxy-PTIO, 3MA or rapamycin for indicated time periods, A375-S2 cells were harvested and rinsed with PBS, then mixed with 10 lM NO specific dye DAF-2DA and incubated at 37 °C for 30 min. The samples were analyzed by FACScan flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA) with the excitation wavelength at 480 nm and the emission wavelength at 525 nm (Cheng et al., 2009).
2.8. Study of autophagy by the quantification of GFP-LC3 puncta in cells Cells cultured in 24-well cell culture clusters were transfected with 2 lg of GFPLC3 plasmid (kindly provided by Y. Chen, Peking University Center for Human Disease Genomics) using 1 lL of TranSmarter per well according to instructions of the manufacturer. The fluorescence of GFP-LC3 was observed under the fluorescence microscope. In each treatment group, 30 cells were selected randomly for the quantification of GFP-LC3 puncta per cell. 2.9. siRNA Transfection One day before transfection, 5 105 A375-S2 cells were inoculated in a 75 mL culture bottle and incubated overnight. Then, 48 lL siRNA duplex was diluted with 800 lL diluent, and 16 lL TranSmarter was diluted with 800 lL the same diluent. The siRNA solution was added to the TranSmarter solution dropwise and mixed
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Fig. 4. NO was also regulated by autophagy in physalin A-treated A375-S2 cells. (A) The cells were incubated with 15 lM physalin A in the presence or absence of 3MA (200 lM) or rapamycin (1 nM) for 24 h, quantitative analysis detected a positive ratio of DAF-2T by flow cytometric analysis. (B) A375-S2 cells were transfected with either nonspecific-siRNA (NS-siRNA) or Beclin 1-siRNA, and Beclin 1 protein was detected 24 h after transfection by western blot analysis (a), the transfected cells were treated with physalin A for 24 h and DAF-2T positive ratio was measured by flow cytometric analysis (b). (C) Effects of 3MA and rapamycin on the expression of mTOR, p-mTOR, Beclin 1 and the conversion from LC3 I to LC3 II were determined by western blot analysis. b-Actin was used as a loading reference. n = 3, mean ± S.D. ⁄p < 0.05, ⁄⁄p < 0.01 vs. (NSsiRNA) control group, #p < 0.05, ##p < 0.01 vs. physalin A group, $p < 0.05 vs. NS-siRNA + physalin A group.
by pipet, then incubated at room temperature for 45 min. The cells were washed once with the same diluent, and the mixture was added dropwise into the culture and mixed by gentle shaking of the flask. The cells were incubated at 37 °C, 5% CO2 for 7 h, then the transfection mixture was removed and replaced with normal growth medium. The transfected cells were maintained for 24 h before harvesting. 2.10. Statistical analysis All the presented data and results were confirmed in at least three independent experiments and were expressed as mean ± SD. Statistical comparisons were made by Student’s t-test and One-way ANOVA followed by Tukey’s post hoc test. p < 0.05 was considered to represent a statistically significant difference.
3. Results
15 lM physalin A resulted in increased expression of iNOS in cell lysates (Fig. 1C). As shown in Fig. 1D, the inhibitor of nitric oxide synthase 1400W and NO scavenger Carboxy-PTIO significantly suppressed the stimulation of NO expression induced by physalin A treatment. Moreover, decrease in viable cells with physalin A was partially reversed by 1400W and Carboxy-PTIO. No change was observed in 1400W and Carboxy-PTIO treatment alone (Fig. 1E). These results showed that physalin A induced NO generation in A375-S2 cells, and there was a reciprocal relationship between NO and cell viability in physalin A treated A375-S2 cells. 3.2. NO was involved in physalin A induction of apoptosis in A375-S2 cells
3.1. Physalin A induced nitric oxide (NO) generation in A375-S2 cells A375-S2 cells were cultured with 15 lM physalin A for 0, 12, 24 and 36 h, and an increase in the levels of NO was observed in a time-dependent manner (Fig. 1B). Meanwhile, treatment with
The morphologic changes were observed by phase contrast microscopy. Physalin A treatment caused membrane blebbing and granular apoptotic bodies. However, the changes were less significant in the presence of 1400W and Carboxy-PTIO (Fig. 2A). Flow
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cytometric analysis of PI staining was carried out and showed that physalin A treatment induced a significant increase in the percentage of subG1 cells, while 1400W and Carboxy-PTIO pretreatment reversed the ratio of subG1 cells to a certain extent in physalin A treated A375-S2 cells (Fig. 2B). Western blot analysis also showed that physalin A treatment induced the cleavage of procaspase-3 and PARP, down-regulated ICAD expression, leading to DNA fragmentation in nuclei. However, the affects were diminished by 1400W and Carboxy-PTIO pretreatment (Fig. 2C). All the results indicated that NO induced by physalin A treatment promoted apoptosis in A375-S2 cells. 3.3. NO was also involved in physalin A induction of autophagy in A375-S2 cells Previous study showed that interconnections between apoptosis and autophagy existed in physalin A-treated A375-S2 cells (He et al., 2013). To further clarify the relationship between NO and cell viability in physalin A-treated A375-S2 cells, the relevance of NO level to autophagy was examined. Western blot analysis showed that mTOR and p-mTOR were down-regulated while ATG5 was up-regulated in A375-S2 cells after physalin A treatment (Fig. 3A). Quantitative analysis of physalin A-induced autophagy by flow cytometric analysis showed that the MDC-positive cells decreased after pretreatment with 1400W and Carboxy-PTIO, indicating that autophagy was reduced after suppression of NO generation in A375-S2 cells (Fig. 3B). The same results were came out by the marked increase of GFP-LC3 puncta in A375-S2 cells (Fig. 3C). Western blot analysis revealed that iNOS expression augmented with physalin A was down-regulated after 1400W pretreatment, while Carboxy-PTIO showed no effect on iNOS expression. Moreover, decreased in mTOR and p-mTOR levels by physalin A treatment was reversed by 1400W or Carboxy-PTIO treatment with concomitant suppression of LC3 I to LC3 II conversion (Fig. 3D). Since an increased level of LC3 II can be due to either stimulation of the LC3 conversion or the inhibition of the fusion of autophagosome with lysosome, in order to validate the effects of physalin A on autophagy, we measured autophagic flux in the presence of chloroquine, an inhibitor of autophagosomelysosome fusion (Klionsky et al., 2012). It illustrates that LC3 II and p62 significantly accumulated in the presence of chloroquine in the cells treated with physalin A, indicating that autophagic flux was enhanced (Fig. 3E). It is concluded that NO induced by physalin A treatment promoted autophagy in A375-S2 cells. 3.4. NO production was regulated by autophagy in physalin A-treated A375-S2 cells Physalin A-induced NO generation is responsible for induction of apoptosis and autophagy in A375-S2 cells as shown above. To elucidate the role of autophagy in NO generation, the specific autophagic inhibitor 3MA and the autophagic agonist rapamycin were used. NO generation in the cells treated with physalin A was markedly increased as analyzed by flow cytometry in the presence of 3MA, while it was decreased by pretreatment with rapamycin. No change was observed in the group treated by 3MA and rapamycin alone (Fig. 4A). To further elucidate the results, the expression of the autophagy associated marker protein Beclin 1 in A375-S2 cells was knocked down by application of short interfering RNA (siRNA) (Fig. 4B(a)), and Beclin 1 knockdown led to an increase of NO generation (Fig. 4B(b)). Western blot analysis showed that mTOR and p-mTOR were down-regulated by rapamycin pretreatment. Furthermore, the expression of autophagic marker Beclin 1 as well as the conversion from LC3 I to LC3 II was up-regulated (Fig. 4C). These findings demonstrate that physalin A suppressed the expression of mTOR and finally led to autophagy. Meanwhile,
autophagy, diminishing the NO production, forms a negative feedback loop to protect cell apoptosis. 4. Discussion We previously demonstrated that physalin A induced both apoptosis and autophagy in A375-S2 cells, and that autophagy played a protective role against apoptosis (He et al., 2013). However, the relationship between apoptosis and autophagy was unclarified. This study demonstrated the involvement of NO in the effect of physalin A: (1) physalin A induced the expression of iNOS and NO generation, (2) NO then promoted apoptosis and autophagy in A375-S2 cells, and (3) autophagy diminished the NO production, reducing the apoptotic ratio and thereby protecting the cells from death. NO, a diffusible short-lived highly reactive free radical, is also an important signaling molecule that acts in many tissues as a regulating factor of a diverse range of physiological processes, including cell growth, differentiation and apoptosis (Moncada et al., 1991; Murad, 2006; Schmidt and Walter, 1994). It was reported that NO induced cytotoxicity against several tumor cells. On the other hand, NO promoted cancer growth and progression (Mocellin et al., 2007). High concentrations of NO induce cell death in several cell types (Mocellin et al., 2007). The present study demonstrates that physalin A induced apoptosis in A375-S2 cells through NO generation. This is consistent with the previous studies by other investigators showing that NO induced apoptosis in mammalian cells (Kim, 2012; Moriya et al., 2000). This study also showed that NO induced autophagy in A375-S2 cells. Previous studies reported that NO has a variety of effects on autophagy depending upon the cell type (Barsoum et al., 2006; Chen and Gibson, 2008; Rabkin, 2007). In A375-S2 cells, our data showed that NO was released after physalin A treatment. Inhibition of NO production caused a decrease in autophagic ratio. However, suppression of autophagy caused an increase in NO releasing. These results indicated that NO induced autophagy, and simultaneously, autophagy formed a negative feedback loop to diminish the NO generation that contributes to apoptosis, thereby leading to protection from cell apoptosis. This finding was consistent with our previous observations that autophagy plays an protective role in physalin A-treated A375-S2 cells (He et al., 2013). The regulation of autophagy is highly complex. The mTOR pathway is widely studied in autophagic pathways (Rouschop and Wouters, 2009). TOR kinase, mTOR, is a negative control element in autophagy and plays an important role in the regulation of cell growth (Schmelzle and Hall, 2000). This study demonstrated (1) physalin A inhibited the mTOR expression, (2) inhibition of NO production by 1400W and Carboxy-PTIO partially reversed the down-regulation of mTOR. These findings suggest that NO induced autophagy through decreasing mTOR expression in physalin A treated A375-S2 cells. Application of rapamycin further decreased mTOR expression and up-regulated the autophagic rate, resulting in repression of NO production. On the contrary, reduction of Beclin 1 expression by 3MA down-regulates the autophagic rate, resulting in decrease of NO clearance. Taken together with our previous study (He et al., 2013), we can draw a conclusion that NOinduced autophagy formes a negative feedback loop to repression of NO production, which eventually protects the physalin A-treated A375-S2 cells from apoptosis. 5. Conclusions This study provides the evidence that NO is involved in physalin A-induced apoptosis and autophagy in A375-S2 cells. Furthermore, autophagy diminishes NO production and thereby reduces
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apoptotic rate or contributes to protection of the cells from apoptosis. The study may add to understanding of the mechanism of physalin A-induced apoptosis and autophagy in A375-S2 human melanoma cells. Conflict of Interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version.
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Nitric oxide induces apoptosis and autophagy; autophagy down-regulates NO synthesis in physalin A-treated A375-S2 human melanoma cells Hao He a,b, Yong-Sheng Feng b, Ling-He Zang b, Wei-Wei Liu b, Li-Qin Ding c, Li-Xia Chen a, Ning Kang c, Toshihiko Hayashi b, Shin-ichi Tashiro d, Satoshi Onodera e, Feng Qiu a,c,⇑, Takashi Ikejima b,⇑ a Department of Natural Products Chemistry, School of Traditional Chinese Materia Medica, Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, People’s Republic of China b China-Japan Research Institute of Medical Pharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, People’s Republic of China c Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, People’s Republic of China d Institute for Clinical and Biomedical Sciences, Kyoto 603-8072, Japan e Department of Clinical and Biomedical Sciences, Showa Pharmaceutical University, Tokyo 194-8543, Japan
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Article history: Received 11 January 2014 Accepted 10 June 2014 Available online 19 June 2014 Keywords: Physalin A A375-S2 cells Nitric oxide mTOR Apoptosis Autophagy
a b s t r a c t Physalin A is an active withanolide isolated from Physalis alkekengi var. franchetii, a traditional Chinese herbal medicine named Jindenglong, which has been used for the treatment of sore throat, hepatitis, eczema and tumors in China. Our previous study demonstrated that physalin A induced apoptosis and cyto-protective autophagy in A375-S2 human melanoma cells. Induction of reactive oxygen species (ROS) with physalin A triggered apoptosis. In this study, NO generated by physalin A induced apoptosis and autophagy in A375-S2 cells, since physalin A induced the expression of inducible nitric oxide synthase (iNOS) in the cells. Generation of NO partially promoted both apoptosis and autophagy in A375-S2 cells. NO suppressed mTOR expression, which led to autophagy induction. An autophagic inhibitor, 3-methyladenine (3MA) promoted NO production, while acceleration of autophagy with an autophagic agonist rapamycin repressed NO production, suggesting that autophagy and NO production form a negative feedback loop that eventually protects the cells from apoptosis. The results together with the previous study indicate apoptosis and autophagy induced by physalin A in A375-S2 cells; the autophagy, repressing production of reactive nitrogen species (RNS) and ROS, protects the cells from apoptosis. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Abbreviations: 1400W, N-(3-(aminomethyl) benzyl) acetamidine; 3MA, 3methyladenine; ATCC, American Type Culture Collection; DAF2-DA, 4,5-diaminofluorescein diacetate; DAF-2T, diaminofluoresceine-2 triazole; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; ICAD, inhibitor of caspase-activated DNase; iNOS, inducible nitric oxide synthase; MDC, monodansylcadaverine; MEM, Minimum Essential Medium; mTOR, the mammalian target of rapamycin; MTT, 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NO, nitric oxide; NOS, nitric oxide synthase; PARP, poly ADP-ribose polymerase; PI, propidium iodide; RNS, reactive nitrogen species; ROS, reactive oxygen species. ⇑ Corresponding authors. Address: China-Japan Research Institute of Medical Pharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, People’s Republic of China. Tel./fax: +86 22 59596163 (F. Qiu). Tel./fax: +86 24 23844463 (T. Ikejima). E-mail addresses: [email protected] (F. Qiu), [email protected] (T. Ikejima). http://dx.doi.org/10.1016/j.fct.2014.06.007 0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.
Physalin A is an active withanolide isolated from Physalis alkekengi var. franchetii (Chinese name: ‘‘Jindenglong’’). We previously demonstrated that physalin A induced both apoptosis and autophagy in A375-S2 human melanoma cells, and autophagy played a protective role against apoptosis. However, the mechanism of the protective autophagy was not fully understood (He et al., 2013). NO, a highly reactive free radical, is a signaling molecule of major importance. Synthesised by NOS from L-arginine, it is involved in inflammation, immunological function, apoptosis and autophagy (Murad, 2006). Previous studies reported that NO induced cytotoxicity for several tumor cells, suggesting that NO could induce cancer cell apoptosis and autophagy (Fan et al., 2011; Kim, 2012; Zang et al., 2012). Our study showed that
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Fig. 1. Physalin A induced NO generation in A375-S2 cells. (A) The chemical structure of physalin A. (B) A375-S2 cells were treated with 15 lM physalin A for 0, 12, 24 and 36 h, and DAF-2T, the fluorescent dye product of DAF-2 in reaction with NO, was measured by flow cytometric analysis. (C) The cells were lysed after treatment with 15 lM physalin A for 6, 12, 18 and 24 h, and the protein levels of iNOS were determined by western blot analysis. b-Actin was used as a loading reference. Band density of the specific protein was analyzed with Quantity One image software and the results were expressed as average density to b-actin. (D–E) A375-S2 cells were pre-treated with 1400W (20 lM) or Carboxy-PTIO (200 lM) for 1 h prior to 15 lM physalin A treatment for 24 h. DAF-2T positive ratio was measured by flow cytometric analysis (D), and the cell viability was measured by MTT assay (E). Mean ± S.D. of three independent experiments. ⁄p < 0.05, ⁄⁄p < 0.01 vs. control group, #p < 0.05, ##p < 0.01 vs. physalin A group.
physalin A treatment was correlated with the induction of reactive oxygen species (He et al., 2013), and NO was found to promote autophagy under severe oxidative stress (Fan et al., 2011; Yang et al., 2010). It is also widely accepted that iNOS, which produces NO, is expressed in response to several stresses (Kang et al., 2013; Nomura and Kitamura, 1993).
Apoptosis is a process of cellular suicide followed by many physiological process, including nuclear fragmentation, cell shrinkage, chromatin condensation and membrane blebbing (Gerl and Vaux, 2005). The regulation of apoptosis is not an intrinsic function of drugs and genes. If the goal of a drug therapy or gene regulation is to induce the death of cancer cells, its ability to induce indirect
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Fig. 2. NO was involved in physalin A induced apoptosis in A375-S2 cells. (A) A375-S2 cells were incubated with 15 lM physalin A in the presence or absence of 1400W (20 lM) or Carboxy-PTIO (200 lM) for 24 h, and cell morphologies were observed by phase contrast microscopy (200 magnification, bar = 20 lm). Arrows indicate the apoptotic bodies. (B–C) A375-S2 cells were pre-treated with 20 lM 1400W or 200 lM Carboxy-PTIO for 1 h prior to the addition of 15 lM physalin A and then incubated for 24 h. The apoptotic rate was determined by flow cytometric analysis with PI staining (B), and the protein levels of caspase-3, PARP and ICAD were detected by western blot analysis. b-Actin was used as a loading reference. Band density of the specific protein was analyzed with Quantity One image software and the results were expressed as average density to b-actin (C). n = 3, mean ± S.D. ⁄p < 0.05, ⁄⁄p < 0.01 vs. control group, #p < 0.05, ##p < 0.01 vs. physalin A group.
cell suicide might be just as important as its direct cytotoxic activity. Extreme level of autophagy is also a process of cellular suicide, as well as lysosome-dependent protein degradation, and organelle turnover and removal (Finkel et al., 2007). Sometimes, autophagy is a survival mechanism under starvation, differentiation or severe oxidative stress. NO is also involved in apoptosis and autophagy under several stresses, including some kind of drug therapy, severe oxidative stress, differentiation and hormonal stimulation. The mechanisms of NO involvement in relation to apoptosis and autophagy are complex (Lee et al., 2012); both inhibitory role and accelerating role of NO in autophagy can be found in the literature (Barsoum et al., 2006; Sarkar et al., 2011). The mammalian target of rapamycin (mTOR) is a protein kinase at the nexus between oncogenic phosphoinositide 3-kinase (PI3K) signaling and downstream pathways. Two complexes, called mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), are known
to play crucial roles in cell growth, proliferation, and cellular metabolism (Guertin and Sabatini, 2009). The activity of mTORC1 can be inhibited by rapamycin or starvation, well-established inducers of autophagy (Noda and Ohsumi, 1998). Autophagy is controlled by many diverse signals, such as growth factors, amino acids, and energy status, and in these pathways, phosphatidylinositol 3-kinases and the protein kinase mTOR play important roles (Meijer and Codogno, 2006). In this study, we examined the effect of physalin A on the mTOR expression. We have recently demonstrated that physalin A induced apoptosis and cyto-protective autophagy in A375-S2 human melanoma cells. Reactive oxygen species (ROS) triggered apoptotic cell death under such circumstance (He et al., 2013). However, the relation between apoptosis and autophagy were unclear. In this study, we attempted to elucidate the mechanism of physalin A-induced NO production involved in the death of A375-S2 cells. Elucidation of
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Fig. 3. NO was involved in physalin A induced autophagy in A375-S2 cells. (A) A375-S2 cells were incubated with 15 lM physalin A for 6, 12, 18 and 24 h, followed by western blot analysis for detection of mTOR, p-mTOR and ATG5. (B) The cells were pre-treated with 1400W (20 lM) or Carboxy-PTIO (200 lM) for 1 h prior to 15 lM physalin A treatment for 24 h. The MDC positive ratio was determined by flow cytometric analysis with MDC staining. (C) Cells were transfected with GFP-LC3 and then treated with physalin A in the presence or absence of 1400W and Carboxy-PTIO for 24 h. Scale bar 10 lm. GFP-LC3 puncta per cell were calculated in 30 cells and expressed as the mean ± S.D. (D) Effects of 1400W and Carboxy-PTIO on the expression of iNOS, mTOR and p-mTOR, and the conversion from LC3 I to LC3 II were detected by western blot analysis. (E) The cells were treated with physalin A for 24 h in the absence or presence of chloroquine (CQ). At the end of treatment, the levels of LC3 and p62 were examined by western blot analysis. b-Actin was used as a loading reference. Band density of the specific protein was analyzed with Quantity One image software and the results were expressed as average density to b-actin. n = 3, mean ± S.D. ⁄p < 0.05, ⁄⁄p < 0.01 vs. control group, #p < 0.05, ##p < 0.01 vs. physalin A group.
detailed mechanisms might provide cues for understanding the relationship between physalin A-induced apoptosis and autophagy in A375-S2 cells.
2. Material and methods 2.1. Chemicals and reagents Physalin A was prepared from P. alkekengi var. franchetii by our laboratory (Qiu et al., 2008) and identified by comparing its chemical and spectroscopic (1H NMR, 13 C NMR) data with those reported in the literature (Kawai et al., 1992). The purity was measured by HPLC and determined to be 98.0% [column: Aglient ZORBAX SBC18, 4.6 150 mm, 5 lm; solvent phase: methanol–H2O (60:40)]. Physalin A was
dissolved in dimethyl sulfoxide (DMSO) as a stock solution, which was diluted in Minimum Essential Medium (MEM) before the experiments. The DMSO concentration was kept below 0.05% when used in all cell cultures that DMSO did not exert any detectable effect on cell growth or viability. Fetal bovine serum (FBS) was obtained from TBD Biotechnology Development (Tianjin, People’s Republic of China); MEM was obtained from Gibco (Grand Island, NY, USA). 4,5-Diaminofluorescein diacetate (DAF2-DA) was obtained from Cayman Chemical (Ann Arbor, MI, USA), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), monodansylcadaverine (MDC), chloroquine (CQ), N-(3-(aminomethyl) benzyl) acetamidine (1400W), 3MA, and rapamycin were purchased from Sigma (St. Louis, MO, USA). Carboxy-PTIO was from Beyotime Institute of Biotechnology (Haimen, China). Nonspecific-siRNA and Beclin 1-siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). TranSmarter was from Abmart (Shanghai, People’s Republic of China). Polyclonal antibodies against iNOS, caspase-3, PARP (poly ADP-ribose polymerase), ICAD (inhibitor of caspase-activated
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Fig. 3 (continued)
DNase), mTOR, p-mTOR, b-actin, LC3, Beclin 1, ATG5, p62 and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Electrochemiluminescence (ECL) reagent was from Thermo Scientific (Rockford, IL, USA).
2.2. Cell culture The A375-S2 human melanoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). They were cultured in MEM medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 lg/mL streptomycin and maintained at 37 °C in a humidified atmosphere with 5% CO2. The cells in the exponential phase of growth were used in the experiments.
2.3. Cell viability assay A375-S2 cells were incubated in 96-well flat bottom cell culture clusters (Corning, NY, USA) at a density of 8 103 cells per well and cultured for 24 h. Thereafter, the cells were treated with or without an inhibitor of nitric oxide synthase 1400W or NO scavenger Carboxy-PTIO at the given concentrations for 1 h prior to physalin A treatment for 24 h. After that, the cells were rinsed with ice-cold PBS twice and incubated with 5.0 mg/mL MTT solution at 37 °C for 2 h. The resulting crystals were dissolved in 150 lL DMSO and the optical density was measured by MTT assay using a mirco-plate reader (Thermo Multiskan MK3, Thermo scientific, Helsinki, Finland). The percentage of cell viability was calculated as follows:
Cell viability ð%Þ ¼ ðA492sample A492blank Þ=ðA492control A492blank Þ 100
2.6. Flow cytometric analysis of apoptosis and autophagy A375-S2 cells were incubated in 25 mL culture bottle at a density of 2 105 per bottle for 24 h. Then the cells were treated with physalin A in the presence or absence of 1400W and Carboxy-PTIO for indicated time periods. The cells were harvested and rinsed with PBS. For measuring apoptosis, the collected cells were then fixed with 500 lL PBS and 10 mL 70% ethanol at 4 °C for 18 h, after washing twice with PBS, the cells were suspended with 1 mL PI solution (PI 50 lg/mL and RNase A 1 mg/mL) and placed in an ice bath for 1 h. For measuring autophagy, the collected cells were suspended in 0.05 mM MDC at 37 °C for 1 h. Then the samples were analyzed by FACScan flow cytometry. 2.7. Western blot analysis A375-S2 cells were treated with 15 lM physalin A for indicated time periods, or treated with 15 lM physalin A for 24 h in the presence or absence of chloroquine, 1400W, Carboxy-PTIO, or with 3MA or rapamycin at given concentrations. All the cells were harvested and lysed in lysis buffer (50 mM Hepes pH 7.4, 1% Triton X100, 2 mM sodium orthovanadate, 100 mM sodium fluoride, 1 mM edetic acid, 1 mM egtazic acid, 1 mM PMSF, 10 lg/mL aprotinin and 10 lg/mL leupeptin) at 4 °C for 60 min. Lysates were centrifuged at 12,000g for 15 min, and the protein content of the supernatant was determined with the Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of the total protein were separated by 12% SDS–polyacrylamide gel electrophoresis and electrophoretically transferred to ImmobilonÒ-P Transfer Membrane (Millipore Corporation, Billerica, MA, USA). The membranes were soaked in blocking buffer (5% skimmed milk). Proteins were detected with the primary antibodies against iNOS, mTOR, p-mTOR, caspase-3, PARP, ICAD, b-actin, LC3 or Beclin 1, followed by horseradish perosidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology) and visualized by using ECL as the HRP substrate.
2.4. Observation of morphologic changes A375-S2 cells (2 104 cells/well) were inoculated into 24-well culture plates (Corning, NY, USA). After 24 h incubation, the cells were treated with or without 1400W or Carboxy-PTIO at the given concentration for 1 h prior to physalin A treatment for 24 h. The cellular morphology was observed by using a phase contrast microscope (Leica, Nussloch, Germany).
2.5. Flow cytometric analysis of nitric oxide production After treatment with 15 lM physalin A in the presence or absence of 1400W, Carboxy-PTIO, 3MA or rapamycin for indicated time periods, A375-S2 cells were harvested and rinsed with PBS, then mixed with 10 lM NO specific dye DAF-2DA and incubated at 37 °C for 30 min. The samples were analyzed by FACScan flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA) with the excitation wavelength at 480 nm and the emission wavelength at 525 nm (Cheng et al., 2009).
2.8. Study of autophagy by the quantification of GFP-LC3 puncta in cells Cells cultured in 24-well cell culture clusters were transfected with 2 lg of GFPLC3 plasmid (kindly provided by Y. Chen, Peking University Center for Human Disease Genomics) using 1 lL of TranSmarter per well according to instructions of the manufacturer. The fluorescence of GFP-LC3 was observed under the fluorescence microscope. In each treatment group, 30 cells were selected randomly for the quantification of GFP-LC3 puncta per cell. 2.9. siRNA Transfection One day before transfection, 5 105 A375-S2 cells were inoculated in a 75 mL culture bottle and incubated overnight. Then, 48 lL siRNA duplex was diluted with 800 lL diluent, and 16 lL TranSmarter was diluted with 800 lL the same diluent. The siRNA solution was added to the TranSmarter solution dropwise and mixed
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Fig. 4. NO was also regulated by autophagy in physalin A-treated A375-S2 cells. (A) The cells were incubated with 15 lM physalin A in the presence or absence of 3MA (200 lM) or rapamycin (1 nM) for 24 h, quantitative analysis detected a positive ratio of DAF-2T by flow cytometric analysis. (B) A375-S2 cells were transfected with either nonspecific-siRNA (NS-siRNA) or Beclin 1-siRNA, and Beclin 1 protein was detected 24 h after transfection by western blot analysis (a), the transfected cells were treated with physalin A for 24 h and DAF-2T positive ratio was measured by flow cytometric analysis (b). (C) Effects of 3MA and rapamycin on the expression of mTOR, p-mTOR, Beclin 1 and the conversion from LC3 I to LC3 II were determined by western blot analysis. b-Actin was used as a loading reference. n = 3, mean ± S.D. ⁄p < 0.05, ⁄⁄p < 0.01 vs. (NSsiRNA) control group, #p < 0.05, ##p < 0.01 vs. physalin A group, $p < 0.05 vs. NS-siRNA + physalin A group.
by pipet, then incubated at room temperature for 45 min. The cells were washed once with the same diluent, and the mixture was added dropwise into the culture and mixed by gentle shaking of the flask. The cells were incubated at 37 °C, 5% CO2 for 7 h, then the transfection mixture was removed and replaced with normal growth medium. The transfected cells were maintained for 24 h before harvesting. 2.10. Statistical analysis All the presented data and results were confirmed in at least three independent experiments and were expressed as mean ± SD. Statistical comparisons were made by Student’s t-test and One-way ANOVA followed by Tukey’s post hoc test. p < 0.05 was considered to represent a statistically significant difference.
3. Results
15 lM physalin A resulted in increased expression of iNOS in cell lysates (Fig. 1C). As shown in Fig. 1D, the inhibitor of nitric oxide synthase 1400W and NO scavenger Carboxy-PTIO significantly suppressed the stimulation of NO expression induced by physalin A treatment. Moreover, decrease in viable cells with physalin A was partially reversed by 1400W and Carboxy-PTIO. No change was observed in 1400W and Carboxy-PTIO treatment alone (Fig. 1E). These results showed that physalin A induced NO generation in A375-S2 cells, and there was a reciprocal relationship between NO and cell viability in physalin A treated A375-S2 cells. 3.2. NO was involved in physalin A induction of apoptosis in A375-S2 cells
3.1. Physalin A induced nitric oxide (NO) generation in A375-S2 cells A375-S2 cells were cultured with 15 lM physalin A for 0, 12, 24 and 36 h, and an increase in the levels of NO was observed in a time-dependent manner (Fig. 1B). Meanwhile, treatment with
The morphologic changes were observed by phase contrast microscopy. Physalin A treatment caused membrane blebbing and granular apoptotic bodies. However, the changes were less significant in the presence of 1400W and Carboxy-PTIO (Fig. 2A). Flow
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cytometric analysis of PI staining was carried out and showed that physalin A treatment induced a significant increase in the percentage of subG1 cells, while 1400W and Carboxy-PTIO pretreatment reversed the ratio of subG1 cells to a certain extent in physalin A treated A375-S2 cells (Fig. 2B). Western blot analysis also showed that physalin A treatment induced the cleavage of procaspase-3 and PARP, down-regulated ICAD expression, leading to DNA fragmentation in nuclei. However, the affects were diminished by 1400W and Carboxy-PTIO pretreatment (Fig. 2C). All the results indicated that NO induced by physalin A treatment promoted apoptosis in A375-S2 cells. 3.3. NO was also involved in physalin A induction of autophagy in A375-S2 cells Previous study showed that interconnections between apoptosis and autophagy existed in physalin A-treated A375-S2 cells (He et al., 2013). To further clarify the relationship between NO and cell viability in physalin A-treated A375-S2 cells, the relevance of NO level to autophagy was examined. Western blot analysis showed that mTOR and p-mTOR were down-regulated while ATG5 was up-regulated in A375-S2 cells after physalin A treatment (Fig. 3A). Quantitative analysis of physalin A-induced autophagy by flow cytometric analysis showed that the MDC-positive cells decreased after pretreatment with 1400W and Carboxy-PTIO, indicating that autophagy was reduced after suppression of NO generation in A375-S2 cells (Fig. 3B). The same results were came out by the marked increase of GFP-LC3 puncta in A375-S2 cells (Fig. 3C). Western blot analysis revealed that iNOS expression augmented with physalin A was down-regulated after 1400W pretreatment, while Carboxy-PTIO showed no effect on iNOS expression. Moreover, decreased in mTOR and p-mTOR levels by physalin A treatment was reversed by 1400W or Carboxy-PTIO treatment with concomitant suppression of LC3 I to LC3 II conversion (Fig. 3D). Since an increased level of LC3 II can be due to either stimulation of the LC3 conversion or the inhibition of the fusion of autophagosome with lysosome, in order to validate the effects of physalin A on autophagy, we measured autophagic flux in the presence of chloroquine, an inhibitor of autophagosomelysosome fusion (Klionsky et al., 2012). It illustrates that LC3 II and p62 significantly accumulated in the presence of chloroquine in the cells treated with physalin A, indicating that autophagic flux was enhanced (Fig. 3E). It is concluded that NO induced by physalin A treatment promoted autophagy in A375-S2 cells. 3.4. NO production was regulated by autophagy in physalin A-treated A375-S2 cells Physalin A-induced NO generation is responsible for induction of apoptosis and autophagy in A375-S2 cells as shown above. To elucidate the role of autophagy in NO generation, the specific autophagic inhibitor 3MA and the autophagic agonist rapamycin were used. NO generation in the cells treated with physalin A was markedly increased as analyzed by flow cytometry in the presence of 3MA, while it was decreased by pretreatment with rapamycin. No change was observed in the group treated by 3MA and rapamycin alone (Fig. 4A). To further elucidate the results, the expression of the autophagy associated marker protein Beclin 1 in A375-S2 cells was knocked down by application of short interfering RNA (siRNA) (Fig. 4B(a)), and Beclin 1 knockdown led to an increase of NO generation (Fig. 4B(b)). Western blot analysis showed that mTOR and p-mTOR were down-regulated by rapamycin pretreatment. Furthermore, the expression of autophagic marker Beclin 1 as well as the conversion from LC3 I to LC3 II was up-regulated (Fig. 4C). These findings demonstrate that physalin A suppressed the expression of mTOR and finally led to autophagy. Meanwhile,
autophagy, diminishing the NO production, forms a negative feedback loop to protect cell apoptosis. 4. Discussion We previously demonstrated that physalin A induced both apoptosis and autophagy in A375-S2 cells, and that autophagy played a protective role against apoptosis (He et al., 2013). However, the relationship between apoptosis and autophagy was unclarified. This study demonstrated the involvement of NO in the effect of physalin A: (1) physalin A induced the expression of iNOS and NO generation, (2) NO then promoted apoptosis and autophagy in A375-S2 cells, and (3) autophagy diminished the NO production, reducing the apoptotic ratio and thereby protecting the cells from death. NO, a diffusible short-lived highly reactive free radical, is also an important signaling molecule that acts in many tissues as a regulating factor of a diverse range of physiological processes, including cell growth, differentiation and apoptosis (Moncada et al., 1991; Murad, 2006; Schmidt and Walter, 1994). It was reported that NO induced cytotoxicity against several tumor cells. On the other hand, NO promoted cancer growth and progression (Mocellin et al., 2007). High concentrations of NO induce cell death in several cell types (Mocellin et al., 2007). The present study demonstrates that physalin A induced apoptosis in A375-S2 cells through NO generation. This is consistent with the previous studies by other investigators showing that NO induced apoptosis in mammalian cells (Kim, 2012; Moriya et al., 2000). This study also showed that NO induced autophagy in A375-S2 cells. Previous studies reported that NO has a variety of effects on autophagy depending upon the cell type (Barsoum et al., 2006; Chen and Gibson, 2008; Rabkin, 2007). In A375-S2 cells, our data showed that NO was released after physalin A treatment. Inhibition of NO production caused a decrease in autophagic ratio. However, suppression of autophagy caused an increase in NO releasing. These results indicated that NO induced autophagy, and simultaneously, autophagy formed a negative feedback loop to diminish the NO generation that contributes to apoptosis, thereby leading to protection from cell apoptosis. This finding was consistent with our previous observations that autophagy plays an protective role in physalin A-treated A375-S2 cells (He et al., 2013). The regulation of autophagy is highly complex. The mTOR pathway is widely studied in autophagic pathways (Rouschop and Wouters, 2009). TOR kinase, mTOR, is a negative control element in autophagy and plays an important role in the regulation of cell growth (Schmelzle and Hall, 2000). This study demonstrated (1) physalin A inhibited the mTOR expression, (2) inhibition of NO production by 1400W and Carboxy-PTIO partially reversed the down-regulation of mTOR. These findings suggest that NO induced autophagy through decreasing mTOR expression in physalin A treated A375-S2 cells. Application of rapamycin further decreased mTOR expression and up-regulated the autophagic rate, resulting in repression of NO production. On the contrary, reduction of Beclin 1 expression by 3MA down-regulates the autophagic rate, resulting in decrease of NO clearance. Taken together with our previous study (He et al., 2013), we can draw a conclusion that NOinduced autophagy formes a negative feedback loop to repression of NO production, which eventually protects the physalin A-treated A375-S2 cells from apoptosis. 5. Conclusions This study provides the evidence that NO is involved in physalin A-induced apoptosis and autophagy in A375-S2 cells. Furthermore, autophagy diminishes NO production and thereby reduces
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apoptotic rate or contributes to protection of the cells from apoptosis. The study may add to understanding of the mechanism of physalin A-induced apoptosis and autophagy in A375-S2 human melanoma cells. Conflict of Interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version.
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