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NF-κb facilitates oridonin-induced apoptosis and autophagy in HT1080 cells through a p53-mediated pathway
Archives of Biochemistry and Biophysics 489 (2009) 25–33
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NF-jb facilitates oridonin-induced apoptosis and autophagy in HT1080 cells through a p53-mediated pathway Yahong Zhang a,b, Yingliang Wu b, Di Wu a, Shin-ichi Tashiro c, Satoshi Onodera c, Takashi Ikejima a,* a
China-Japan Research Institute of Medical Pharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, PR China c Department of Clinical and Biomedical Sciences, Showa Pharmaceutical University, Tokyo 194-8543, Japan b
a r t i c l e
i n f o
Article history: Received 22 April 2009 and in revised form 15 July 2009 Available online 29 July 2009 Keywords: Oridonin Autophagy Apoptosis NF-jB p53
a b s t r a c t In this study, we investigated the molecular mechanisms involving in oridonin-induced apoptosis and autophagy. We found that apoptosis and autophagy were simultaneously induced by oridonin timedependently in HT1080 cells, and inhibition of autophagy by 3MA decreased oridonin-induced apoptosis, indicating that they act in synergy to mediate cell death. In addition, treatment with oridonin caused an increase in NF-jB and p53 activities in a time-dependent manner. Inhibition of NF-jB or p53 activation by its specific inhibitor PDTC or pifithrin-a respectively, significantly reduced both oridonin-induced apoptosis and autophagy accompanied by the decrease in Beclin 1 and LC3 levels. Further experiments confirmed that oridonin-induced p53 activation was reduced by the NF-jB inhibitor whereas the activation of NF-jB was not affected by p53 inhibition. Taken together, these results demonstrate that NF-jB promotes oridonin-induced apoptotic and autophagic cell death through regulating p53 activation in HT1080 cells. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Programmed cell death (PCD)1 is essential in multicellular organisms for development, tissue turnover and host defence [1]. Apoptosis, or type I PCD, is a genetically regulated cell suicide process, which plays an important role in maintaining homeostasis of higher organisms [2]. Autophagy is also an essential cellular homeostatic mechanism, whereby eukaryotic cells degrade unnecessary proteins and cytoplasmic organelles [3]. Although autophagy is considered to be a very important physiological process for cell survival during starvation, excessive autophagy can mediate cell death (type II PCD) under certain conditions [4,5]. Accumulating data suggest that there is a complex regulatory mechanism between apoptosis and autophagy [6,7]. So far, the molecular basis accounting for the mutual relationship between apoptotic and autophagic mechanisms remain largely unexplored. Thus, the signaling pathways regulating these two processes have achieved the great prominence.
* Corresponding author. Fax: +86 24 3844463. E-mail address: [email protected] (T. Ikejima). 1 Abbreviations used: PCD, Programmed cell death; NF-jB, nuclear factor jB; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; PI, propidium iodide; MDC, monodansylcadaverine; PDTC, pyrrolidine dithiocarbamate; ATCC, American type culture collection; PMSF, phenylmethyl-sulfonylfluoride; DAB, diamino-benzidine. 0003-9861/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2009.07.017
The nuclear factor jB (NF-jB) belongs to the transcription factors family and plays a critical role in many signal transduction pathways [8]. And it may act as pro- as well as anti-apoptotic transcription factor through regulation of different targets such as bcl2 family, JNK kinase and p53 [9–11]. Also, some recent studies have reported that NF-jB involves in autophagy, either promoting or inhibiting it, depending on the condition [12,13]. However, the mechanisms remain ambiguous. Oridonin (shown in Fig. 1), an active diterpenoid isolated from Rabdosia rubescens, has been reported to have various pharmacological and physiological effects such as anti-tumor, anti-bacterial and anti-inflammation effects [6,14,15]. And it was reported that the a-methylene cyclopentanone structure was the active site of oridonin for anti-tumor, changes in this structure (e.g. split ring or saturated methylene) could counteract its anti-tumor activity [16]. Our previous studies showed that oridonin could induce both apoptosis and autophagy in murine fibrosarcoma L929 cells, and autophagy antagonized apoptosis to play a protective role through activating p38-NF-jB signal pathway [17]. Here, HT1080, a human fibrosarcoma cell line which is highly aggressive and malignant was used to investigate the characteristics of cell death and their regulatory mechanisms by oridonin. In this study, we report for the first time that oridonin can simultaneously induce apoptotic and autophagic cell death in HT1080 cells, and in this process we further demonstrate that NF-jB plays both pro-apoptotic and pro-autophagic roles through activating p53.
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The analysis of monodansylcadaverine (MDC) staining
Fig. 1. Chemical structure of oridonin.
HT1080 cells (5 105/well) were cultured in 6-well culture plates and incubated with oridonin. After 24 h, the cells were incubated with 0.05 mM MDC at 37 °C for 1 h, and the changes of fluorescence were observed by OLYMPUS IX70 reverse fluorescence microscopy (Olympus, Tokyo, Japan) at excitation wave length 380 nm with emission filter 525 nm.
Materials and methods
Flowcytometric analysis of autophagy and apoptosis
Materials
HT1080 cells were dispensed in 25 ml culture bottle at a density of 1 106 per bottle. After 24 h incubation, they were treated with or without 3MA, PDTC and pifithrin-a at given concentrations 1 h prior to the administration of oridonin for the fixed time periods. The cells were harvested by trypsin and rinsed with PBS. For measuring autophagy, monodansylcadaverine (MDC) has been used as a marker for autophagic vacuoles [19]; collected cells were suspended with 0.05 mM MDC at 37 °C for 60 min. For measuring apoptosis, the cells were fixed with 500 lL PBS and 10 mL 70% ethanol at 4 °C for 18 h, then after washing twice with PBS, the cells were suspended with 1 mL PI solution (PI 50 mg/L and RNase A 1 g/L). Then the samples were analyzed by FACScan flowcytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
Oridonin was obtained from the Kunming Institute of Botany, The Chinese Academy of Sciences (Kunming, China). The purity of oridonin was confirmed by HPLC and determined to be 99.4%. Oridonin was dissolved in dimethyl sulfoxide (DMSO) to make a stock solution. The DMSO concentration was kept below 0.1% in cell culture and did not exert any detectable effect on cell growth or cell death. Fetal bovine serum (FBS) was obtained from TBD Biotechnology Development (Tianjin, China). 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), propidium iodide (PI), monodansylcadaverine (MDC), 3-methyladenine (3-MA) and NF-jB inhibitor pyrrolidine dithiocarbamate (PDTC) were purchased from Sigma Chemical (St. Louis, MO, USA). P53 inhibitor pifithrin-a was obtained from Calbiochem (La Jolla, CA, USA). Polyclonal antibodies to LC3, Beclin 1, NF-jB, phospho-NF-jB, IjBa, p53, phospho-p53 (serine 392), b-actin and horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cell culture The HT1080 cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in RPMI 1640 medium (GIBCO, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS), 0.03% L-glutamine (GIBIO, Grand Island, NY), 100 U/ml penicillin and 100 lg/ml streptomycin and maintained at 37 °C with 5% CO2 in a humidified atmosphere. Cell growth inhibition assay The inhibition of cell growth was measured by MTT assay as described previously [18]. The cells were dispensed in 96-well flat bottom microtiter plates (NUNC, Roskilde, Denmark) at a density of 1.2 104 cells per well. After 24 h incubation, they were treated with or without PDTC and pifithrin-a at given concentrations 1 h prior to the administration of oridonin for the indicated time periods. Then, MTT (5 mg/L) was added to each well for 3 h, and the resulting crystals were dissolved in DMSO. Optical density was measured by MTT assay using a plate microreader (TECAN SPECTRA, Wetzlar, Germany). The percentage of cell growth inhibition was calculated as follows:
Cell inhibitory ratio ð%Þ ¼ ðA492control A492sample Þ=ðA492control A492blank Þ 100
Western blot analysis HT1080 cells (2 106) were preincubated with or without specific inhibitors before treatment with oridonin. After the fixed time periods both adherent and floating cells were collected and frozen at 80 °C. Western blot analysis was performed as described before [20]. The cell pellets were resuspended in lysis buffer containing 50 mmol/L Hepes (pH 7.4), 1% Triton-X 100, 2 mmol/L sodium orthovanadate, 100 mmol/L sodium fluoride (NaF), 1 mmol/L edetic acid, 1 mmol/L egtazic acid (EGTA), 1 mmol/L phenylmethyl-sulfonylfluoride (PMSF), 0.1 g/L aprotinin, 0.01 g/L leupeptin and lysed in 4 °C for 1 h. Then the cells were centrifugated at 12,000g for 10 min, and the protein content of the supernatant was determined by Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA). The proteins were separated by 12% SDS polyacrylamide gel electrophoresis and blotted onto nitrocellulose membrane. The membranes were soaked in 5% skimmed milk, and then incubated with primary polyclonal antibodies overnight. The proteins were visualized by using anti-rabbit IgG conjugated with peroxidase and diamino-benzidine (DAB). Protein levels were quantified by densitometry (Fluochim v2.0 Alpha; Alpha Innotech, San Leandro, CA, USA). The relative density was calculated as follows:
Relative density ¼ ðband density of the sampleÞ= ðband density of controlÞ Statistical analysis All results and data were confirmed in at least three separate experiments. Data are expressed as means ± SD. Statistical comparisons were made by one way ANOVA. P < 0.05 was considered significant.
Observation of morphologic changes
Results
HT1080 cells (5 105/well) were seeded into 6-well culture plates and incubated with oridonin for 24 h. The cellular morphology was observed by a phase contrast microscope (Leica, Nussloch, Germany).
Oridonin induced apoptosis in HT1080 cells Oridonin caused remarkable inhibition of HT1080 cell growth in a time- and dose-dependent manner with an IC50
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Fig. 2. Oridonin-induced apoptosis in HT1080 cells. (A) The cells were treated with various doses of oridonin for 6, 12, 24, or 36 h. Cell growth inhibitory ratio was measured by MTT assay. n = 3, mean ± SD. (B) The cells were treated with 64 lM oridonin for 24 h, the cellular morphologic changes were observed by phase contrast microscopy (200 magnification, Bar = 20 lm). (C) The cells were treated with 64 lM oridonin for 12, 24, or 36 h, the apoptotic cells stained with PI (SubG1 fraction) were measured by flowcytometric analysis.
(at 24 h) of 63.7 lM (Fig. 2A). To characterize the cell growth inhibition of oridonin-treated HT1080 cells, the morphologic changes of the cells were observed. Compared to control cells, oridonin treatment induced marked apoptotic morphologic alterations, including cell shrinkage and granular apoptotic
bodies (Fig. 2B). The quantitative analysis of apoptosis by flowcytometry also showed that oridonin induced a significant time-dependent increase in the percentage of subG1 cells (Fig. 2C). These results indicate that oridonin induces apoptotic cell death in HT1080 cells.
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Fig. 3. Oridonin-induced autophagy in HT1080 cells. (A) The cells were treated with 64 lM oridonin for 24 h, the cellular morphologic changes were observed by fluorescence microscopy with MDC staining (400 magnification, Bar = 10 lm). (B–C) The cells were treated with 64 lM oridonin for 12, 24, or 36 h. The MDC-positive cells were measured by flowcytometric analysis (B), and the protein levels of Beclin 1 and MAP-LC3 were detected by Western blot analysis (C).
Y. Zhang et al. / Archives of Biochemistry and Biophysics 489 (2009) 25–33
Oridonin-induced autophagy in HT1080 cells Autophagy has been reported to co-exist with apoptosis in many experimental systems, therefore we examined the effect of oridonin on autophagy induction. The formation of autophagic vacuoles was assessed by staining cells with autophagosomal fluorescent dye MDC. As shown in Fig. 3A, treatment of oridonin induced marked increase in the number of MDC-labeled fluorescent particles in HT1080 cells compared to control cells. For quantitative analysis of autophagy, the MDC fluorescent intensity of oridonintreated cells for the indicated time periods was analyzed by flowcytometry. As shown in Fig. 3B, oridonin induced HT1080 cell autophagy in a time-dependent manner. Accordingly, the expression level of Beclin 1 and the conversion from LC3-I to LC3-II, as autophagic markers, were increased with time after administration of oridonin (Fig. 3C), suggesting that oridonin induces autophagy as well as apoptosis in HT1080 cells. Autophagy preceded apoptosis in oridonin-treated HT1080 cells We further investigate the role of autophagy in oridonin-induced apoptosis in HT1080 cells. Treatment with 3MA (a specific autophagy inhibitor), prior to the addition of oridonin, significantly decreased the percentage of MDC-positive cells as well as the apoptotic ratio compared with the group treated with oridonin alone (Fig. 4A and B), indicating that autophagy plays a death-promoting role in oridonin-treated HT1080 cells. All these results
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demonstrate that oridonin induces both apoptosis and autophagy in HT1080 cells, and inhibition of autophagy contributes to the down-regulation of apoptosis. Inhibition of NF-jB activation down-regulated apoptosis and autophagy in oridonin-treated HT1080 cells To investigate the role of NF-jB in oridonin-induced apoptosis and autophagy, we pretreated HT1080 cells with PDTC (a NF-jB inhibitor) before oridonin administration. As shown in Fig. 5A, preincubation with 10 or 20 lM PDTC decreased oridonin-induced cell growth inhibiton in a dose-dependant manner. Also pretreatment with 20 lM PDTC significantly decreased oridonin-induced apoptosis as well as autophagy (Fig. 5B and C). As to autophagy, the expression level of Beclin 1 and conversion from LC3-I to LC3-II revealed consistent results (Fig. 5D). To further ascertain the requirement of NF-jB activation for apoptosis and autophagy, expression levels of NF-jB, p-NF-jB and IjBa were examined in oridonintreated HT1080 cells for various time periods. As shown in Fig. 5E, oridonin administration enhanced both NF-jB and p-NFjB expression levels with accordingly attenuated levels of IjBa in a time-dependent manner. These results indicate that NF-jB can be activated by oridonin in HT1080 cells, and it promotes both apoptosis and autophagy in this circumstance. Inhibition of p53 activation decreased apoptosis and autophagy in oridonin-treated HT1080 cells Since the tumor suppressor protein p53 contributes to various types of cell death, and its expression is regulated by NF-jB, we examined the role of p53 in oridonin-induced apoptosis and autophagy in HT1080 cells. Pretreatment with 10 or 20 lM pifithrin-a (a p53 specific inhibitor) decreased the cell growth inhibitory ratio by oridonin (Fig. 6A). And both the apoptotic and the autophagic ratios of oridonin-treated cells were markedly decreased by addition of pifithrin-a (Fig. 6B and C). The down-regulations of Beclin 1 level and conversion from LC3-I to LC3-II further confirmed the negative regulatory effect of pifithrin-a on oridonin-induced autophagy (Fig. 6D). Then, we examined whether p53 was activated by oridonin. As shown in Fig. 6E, the levels of p53 and p-p53 were markedly elevated in a time-dependent manner after oridonin administration. Therefore, we conclude that activation of p53 also plays a promoting role in oridonin-induced apoptosis and autophagy in HT1080 cells. Inhibition of NF-jB suppressed the activation of p53 whereas inhibition of p53 had no effect on NF-jB activity Subsequently, to investigate the relationship between NF-jB and p53, we examined their expression levels respectively in oridonin-treated cells with or without indicated inhibitors. When the cells were treated with oridonin, inhibition of NF-jB by PDTC clearly decreased the expression levels of p53 and p-p53 (Fig. 7A). However, the expression levels of NF-jB, p-NF-jB and IjBa were not changed by pifithrin-a treatment compared with oridonin-treated alone (Fig. 7B). Thus, these results demonstrate that p53 is regulated by its upstream factor NF-jB to participate in oridonin-induced apoptosis and autophagy in HT1080 cells. Discussion
Fig. 4. Effects of 3MA on oridonin-induced autophagy and apoptosis in HT1080 cells. The cells were treated with 64 lM oridonin for 24 h in the presence or absence of 1 mM 3MA, the autophagic ratio (A) and the apoptotic ratio (B) were measured by flowcytometric analysis. n = 3, mean ± SD. *P < 0.05, **P < 0.01 vs oridonin group. Ori: oridonin.
Apoptosis and autophagy were reported to be co-existed and interconnected positively or negatively in some circumstances [6,17]. In the current study, our results from flowcytometric
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Fig. 5. Effects of NF-jB on oridonin-induced apoptosis and autophagy in HT1080 cells. (A) The cells were treated with 64 lM oridonin for 24 h, in the presence or absence of 10 or 20 lM PDTC, and the cell growth inhibitory ratio was measured by MTT assay. n = 3, mean ± SD. **P < 0.01 vs oridonin group. (B–D) The cells were treated with 64 lM oridonin for 24 h in the presence or absence of 20 lM PDTC, the apoptotic ratio (B) and the autophagic ratio (C) were measured by flowcytometric analysis. The protein levels of Beclin 1 and MAP-LC3 were detected by Western blot analysis, and the corresponding histograms quantified by densitometry were expressed at bottom (D). n = 3, mean ± SD. *P < 0.05, **P < 0.01 vs oridonin group. (E) The cells were treated with 64 lM oridonin for 12, 24, or 36 h, the protein levels of NF-jB, p-NF-jB and IjBa were detected by Western blot analysis, and the corresponding histograms quantified by densitometry were expressed at right.
analysis, morphologic observation and Western blot showed that oridonin could induce both apoptosis and autophagy in HT1080 cells, and autophagy exerted a synergistic function to apoptosis in this condition. Then, we investigated the possible mechanism which mediated oridonin-induced apoptosis and autophagy.
NF-jB is an important transcription factor involved in the regulation of cell death. It is well known that the nuclear localization of NF-jB is blocked by the binding of the NF-jB inhibitory proteins (IjB), and the activation of NF-jB results in the degradation of IjB proteins, followed by nuclear translocation and DNA binding of the
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Fig. 6. Effects of p53 on oridonin-induced apoptosis and autophagy in HT1080 cells. (A) The cells were treated with 64 lM oridonin for 24 h, in the presence or absence of 10 or 20 lM pifithrin-a, and the cell growth inhibitory ratio was measured by MTT assay. n = 3, mean ± SD. **P < 0.01 vs oridonin group. (B–D) The cells were treated with 64 lM oridonin for 24 h in the presence or absence of 20 lM pifithrin-a, the apoptotic ratio (B) and the autophagic ratio (C) were measured by flowcytometric analysis. The protein levels of Beclin 1 and MAP-LC3 were detected by Western blot analysis, and the corresponding histograms quantified by densitometry were expressed at bottom (D). n = 3, mean ± SD. *P < 0.05, **P < 0.01 vs oridonin group. (E) The cells were treated with 64 lM oridonin for 12, 24, or 36 h, and the protein levels of p53 and p-p53 were detected by Western blot analysis, and the corresponding histograms quantified by densitometry were expressed at right. PFT-a: pifithrin-a.
NF-jB [21]. Consistent with these observations, our results demonstrated that NF-jB was activated by oridonin in a time-dependent manner. Activation of NF-jB mediates cell survival signals in most tumor cells, but it may also act as pro-apoptotic factor in some conditions [22,11]. Here, we found that inhibition of NF-jB by PDTC
significantly decreased oridonin-induced cell death and apoptosis, indicating that NF-jB plays an apoptosis-promoting role in this research. And a few recent studies have been reported that NF-jB can be involved in the regulation of autophagy [12,13]. In our study, oridonin-induced autophagy was also markedly suppressed
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sis in p53-inducible Saos-2 cell line, and loss of p65 (a subunit of NF-jB) could also cause resistance to different agents that induced apoptosis through p53 [23,24]. Hence, we investigated the roles of p53 in apoptosis and autophagy in oridonin-treated HT1080 cells. The tumor suppressor protein p53 is activated in response to various cellular stresses to protect cells by inducing transcription of regulatory genes involved in cell cycle, apoptosis, and DNA repair [25]. Undoubtedly, oridonin has been shown to induce apoptosis through activating p53 in a various cells [26,27]. Here, we showed that oridonin induced the activation of p53, and inhibition of p53 by its inhibitor pifithrin-a led to the decrease in oridonininduced cell death and apoptosis. Furthermore, there are evidences that the activation of p53 also can involve in the regulation of autophagy. For instance, Zeng et al. reported that 6-thioguaninemediated DNA mismatch damage induced autophagy through p53 activation [28]. In another report, etoposide-induced p53 activation down-regulated mTOR activity resulting in increased autophagy levels in mouse embryonic fibroblasts [29]. In this study, oridonin-induced autophagy was significantly decreased by the disruption of p53 activation by pifithrin-a. As to the relationship between p53 and NF-jB, we found that inhibition of NFjB attenuated the expression levels of p53 and p-p53 whereas inhibition of p53 had no influence on the NF-jB, p-NF-jB and IjB expression levels, indicating that p53 activation is regulated by up-stream NF-jB. And this finding was supported by the fact that p53 activity which initiated pro-apoptotic signaling in response to doxycycline was NF-jB dependent in mouse embryonic fibroblasts [11]. Therefore, we conclude that activation of p53 mediates oridonin-induced apoptosis and autophagy in HT 1080 cells, and this process is regulated by NF-jB. In summery, NF-jB facilitates p53 activation to educe its proapoptotic and pro-autophagic functions in oridonin-treated HT1080 cells. Our results might provide new information about the role of NF-jB signal pathways existing between apoptosis and autophagy. Reference [1] [2] [3] [4] [5] [6]
Fig. 7. The relationship between NF-jB and p53 in oridonin-treated HT1080 cells. (A) The cells were treated with 64 lM oridonin for 24 h in the presence or absence of 20 lM PDTC, the protein levels of p53 and p-p53 were detected by Western blot analysis, and the corresponding histograms quantified by densitometry were expressed at bottom. n = 3, mean ± SD. *P < 0.05 vs oridonin group. (B) The cells were treated with 64 lM oridonin for 24 h in the presence or absence of 20 lM pifithrin-a, the protein levels of NF-jB, p-NF-jB and IjBa were detected by Western blot analysis, and the corresponding histograms quantified by densitometry were expressed at bottom.
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NF-jb facilitates oridonin-induced apoptosis and autophagy in HT1080 cells through a p53-mediated pathway Yahong Zhang a,b, Yingliang Wu b, Di Wu a, Shin-ichi Tashiro c, Satoshi Onodera c, Takashi Ikejima a,* a
China-Japan Research Institute of Medical Pharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, PR China c Department of Clinical and Biomedical Sciences, Showa Pharmaceutical University, Tokyo 194-8543, Japan b
a r t i c l e
i n f o
Article history: Received 22 April 2009 and in revised form 15 July 2009 Available online 29 July 2009 Keywords: Oridonin Autophagy Apoptosis NF-jB p53
a b s t r a c t In this study, we investigated the molecular mechanisms involving in oridonin-induced apoptosis and autophagy. We found that apoptosis and autophagy were simultaneously induced by oridonin timedependently in HT1080 cells, and inhibition of autophagy by 3MA decreased oridonin-induced apoptosis, indicating that they act in synergy to mediate cell death. In addition, treatment with oridonin caused an increase in NF-jB and p53 activities in a time-dependent manner. Inhibition of NF-jB or p53 activation by its specific inhibitor PDTC or pifithrin-a respectively, significantly reduced both oridonin-induced apoptosis and autophagy accompanied by the decrease in Beclin 1 and LC3 levels. Further experiments confirmed that oridonin-induced p53 activation was reduced by the NF-jB inhibitor whereas the activation of NF-jB was not affected by p53 inhibition. Taken together, these results demonstrate that NF-jB promotes oridonin-induced apoptotic and autophagic cell death through regulating p53 activation in HT1080 cells. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Programmed cell death (PCD)1 is essential in multicellular organisms for development, tissue turnover and host defence [1]. Apoptosis, or type I PCD, is a genetically regulated cell suicide process, which plays an important role in maintaining homeostasis of higher organisms [2]. Autophagy is also an essential cellular homeostatic mechanism, whereby eukaryotic cells degrade unnecessary proteins and cytoplasmic organelles [3]. Although autophagy is considered to be a very important physiological process for cell survival during starvation, excessive autophagy can mediate cell death (type II PCD) under certain conditions [4,5]. Accumulating data suggest that there is a complex regulatory mechanism between apoptosis and autophagy [6,7]. So far, the molecular basis accounting for the mutual relationship between apoptotic and autophagic mechanisms remain largely unexplored. Thus, the signaling pathways regulating these two processes have achieved the great prominence.
* Corresponding author. Fax: +86 24 3844463. E-mail address: [email protected] (T. Ikejima). 1 Abbreviations used: PCD, Programmed cell death; NF-jB, nuclear factor jB; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; PI, propidium iodide; MDC, monodansylcadaverine; PDTC, pyrrolidine dithiocarbamate; ATCC, American type culture collection; PMSF, phenylmethyl-sulfonylfluoride; DAB, diamino-benzidine. 0003-9861/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2009.07.017
The nuclear factor jB (NF-jB) belongs to the transcription factors family and plays a critical role in many signal transduction pathways [8]. And it may act as pro- as well as anti-apoptotic transcription factor through regulation of different targets such as bcl2 family, JNK kinase and p53 [9–11]. Also, some recent studies have reported that NF-jB involves in autophagy, either promoting or inhibiting it, depending on the condition [12,13]. However, the mechanisms remain ambiguous. Oridonin (shown in Fig. 1), an active diterpenoid isolated from Rabdosia rubescens, has been reported to have various pharmacological and physiological effects such as anti-tumor, anti-bacterial and anti-inflammation effects [6,14,15]. And it was reported that the a-methylene cyclopentanone structure was the active site of oridonin for anti-tumor, changes in this structure (e.g. split ring or saturated methylene) could counteract its anti-tumor activity [16]. Our previous studies showed that oridonin could induce both apoptosis and autophagy in murine fibrosarcoma L929 cells, and autophagy antagonized apoptosis to play a protective role through activating p38-NF-jB signal pathway [17]. Here, HT1080, a human fibrosarcoma cell line which is highly aggressive and malignant was used to investigate the characteristics of cell death and their regulatory mechanisms by oridonin. In this study, we report for the first time that oridonin can simultaneously induce apoptotic and autophagic cell death in HT1080 cells, and in this process we further demonstrate that NF-jB plays both pro-apoptotic and pro-autophagic roles through activating p53.
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The analysis of monodansylcadaverine (MDC) staining
Fig. 1. Chemical structure of oridonin.
HT1080 cells (5 105/well) were cultured in 6-well culture plates and incubated with oridonin. After 24 h, the cells were incubated with 0.05 mM MDC at 37 °C for 1 h, and the changes of fluorescence were observed by OLYMPUS IX70 reverse fluorescence microscopy (Olympus, Tokyo, Japan) at excitation wave length 380 nm with emission filter 525 nm.
Materials and methods
Flowcytometric analysis of autophagy and apoptosis
Materials
HT1080 cells were dispensed in 25 ml culture bottle at a density of 1 106 per bottle. After 24 h incubation, they were treated with or without 3MA, PDTC and pifithrin-a at given concentrations 1 h prior to the administration of oridonin for the fixed time periods. The cells were harvested by trypsin and rinsed with PBS. For measuring autophagy, monodansylcadaverine (MDC) has been used as a marker for autophagic vacuoles [19]; collected cells were suspended with 0.05 mM MDC at 37 °C for 60 min. For measuring apoptosis, the cells were fixed with 500 lL PBS and 10 mL 70% ethanol at 4 °C for 18 h, then after washing twice with PBS, the cells were suspended with 1 mL PI solution (PI 50 mg/L and RNase A 1 g/L). Then the samples were analyzed by FACScan flowcytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
Oridonin was obtained from the Kunming Institute of Botany, The Chinese Academy of Sciences (Kunming, China). The purity of oridonin was confirmed by HPLC and determined to be 99.4%. Oridonin was dissolved in dimethyl sulfoxide (DMSO) to make a stock solution. The DMSO concentration was kept below 0.1% in cell culture and did not exert any detectable effect on cell growth or cell death. Fetal bovine serum (FBS) was obtained from TBD Biotechnology Development (Tianjin, China). 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), propidium iodide (PI), monodansylcadaverine (MDC), 3-methyladenine (3-MA) and NF-jB inhibitor pyrrolidine dithiocarbamate (PDTC) were purchased from Sigma Chemical (St. Louis, MO, USA). P53 inhibitor pifithrin-a was obtained from Calbiochem (La Jolla, CA, USA). Polyclonal antibodies to LC3, Beclin 1, NF-jB, phospho-NF-jB, IjBa, p53, phospho-p53 (serine 392), b-actin and horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cell culture The HT1080 cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in RPMI 1640 medium (GIBCO, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS), 0.03% L-glutamine (GIBIO, Grand Island, NY), 100 U/ml penicillin and 100 lg/ml streptomycin and maintained at 37 °C with 5% CO2 in a humidified atmosphere. Cell growth inhibition assay The inhibition of cell growth was measured by MTT assay as described previously [18]. The cells were dispensed in 96-well flat bottom microtiter plates (NUNC, Roskilde, Denmark) at a density of 1.2 104 cells per well. After 24 h incubation, they were treated with or without PDTC and pifithrin-a at given concentrations 1 h prior to the administration of oridonin for the indicated time periods. Then, MTT (5 mg/L) was added to each well for 3 h, and the resulting crystals were dissolved in DMSO. Optical density was measured by MTT assay using a plate microreader (TECAN SPECTRA, Wetzlar, Germany). The percentage of cell growth inhibition was calculated as follows:
Cell inhibitory ratio ð%Þ ¼ ðA492control A492sample Þ=ðA492control A492blank Þ 100
Western blot analysis HT1080 cells (2 106) were preincubated with or without specific inhibitors before treatment with oridonin. After the fixed time periods both adherent and floating cells were collected and frozen at 80 °C. Western blot analysis was performed as described before [20]. The cell pellets were resuspended in lysis buffer containing 50 mmol/L Hepes (pH 7.4), 1% Triton-X 100, 2 mmol/L sodium orthovanadate, 100 mmol/L sodium fluoride (NaF), 1 mmol/L edetic acid, 1 mmol/L egtazic acid (EGTA), 1 mmol/L phenylmethyl-sulfonylfluoride (PMSF), 0.1 g/L aprotinin, 0.01 g/L leupeptin and lysed in 4 °C for 1 h. Then the cells were centrifugated at 12,000g for 10 min, and the protein content of the supernatant was determined by Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA). The proteins were separated by 12% SDS polyacrylamide gel electrophoresis and blotted onto nitrocellulose membrane. The membranes were soaked in 5% skimmed milk, and then incubated with primary polyclonal antibodies overnight. The proteins were visualized by using anti-rabbit IgG conjugated with peroxidase and diamino-benzidine (DAB). Protein levels were quantified by densitometry (Fluochim v2.0 Alpha; Alpha Innotech, San Leandro, CA, USA). The relative density was calculated as follows:
Relative density ¼ ðband density of the sampleÞ= ðband density of controlÞ Statistical analysis All results and data were confirmed in at least three separate experiments. Data are expressed as means ± SD. Statistical comparisons were made by one way ANOVA. P < 0.05 was considered significant.
Observation of morphologic changes
Results
HT1080 cells (5 105/well) were seeded into 6-well culture plates and incubated with oridonin for 24 h. The cellular morphology was observed by a phase contrast microscope (Leica, Nussloch, Germany).
Oridonin induced apoptosis in HT1080 cells Oridonin caused remarkable inhibition of HT1080 cell growth in a time- and dose-dependent manner with an IC50
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Fig. 2. Oridonin-induced apoptosis in HT1080 cells. (A) The cells were treated with various doses of oridonin for 6, 12, 24, or 36 h. Cell growth inhibitory ratio was measured by MTT assay. n = 3, mean ± SD. (B) The cells were treated with 64 lM oridonin for 24 h, the cellular morphologic changes were observed by phase contrast microscopy (200 magnification, Bar = 20 lm). (C) The cells were treated with 64 lM oridonin for 12, 24, or 36 h, the apoptotic cells stained with PI (SubG1 fraction) were measured by flowcytometric analysis.
(at 24 h) of 63.7 lM (Fig. 2A). To characterize the cell growth inhibition of oridonin-treated HT1080 cells, the morphologic changes of the cells were observed. Compared to control cells, oridonin treatment induced marked apoptotic morphologic alterations, including cell shrinkage and granular apoptotic
bodies (Fig. 2B). The quantitative analysis of apoptosis by flowcytometry also showed that oridonin induced a significant time-dependent increase in the percentage of subG1 cells (Fig. 2C). These results indicate that oridonin induces apoptotic cell death in HT1080 cells.
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Fig. 3. Oridonin-induced autophagy in HT1080 cells. (A) The cells were treated with 64 lM oridonin for 24 h, the cellular morphologic changes were observed by fluorescence microscopy with MDC staining (400 magnification, Bar = 10 lm). (B–C) The cells were treated with 64 lM oridonin for 12, 24, or 36 h. The MDC-positive cells were measured by flowcytometric analysis (B), and the protein levels of Beclin 1 and MAP-LC3 were detected by Western blot analysis (C).
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Oridonin-induced autophagy in HT1080 cells Autophagy has been reported to co-exist with apoptosis in many experimental systems, therefore we examined the effect of oridonin on autophagy induction. The formation of autophagic vacuoles was assessed by staining cells with autophagosomal fluorescent dye MDC. As shown in Fig. 3A, treatment of oridonin induced marked increase in the number of MDC-labeled fluorescent particles in HT1080 cells compared to control cells. For quantitative analysis of autophagy, the MDC fluorescent intensity of oridonintreated cells for the indicated time periods was analyzed by flowcytometry. As shown in Fig. 3B, oridonin induced HT1080 cell autophagy in a time-dependent manner. Accordingly, the expression level of Beclin 1 and the conversion from LC3-I to LC3-II, as autophagic markers, were increased with time after administration of oridonin (Fig. 3C), suggesting that oridonin induces autophagy as well as apoptosis in HT1080 cells. Autophagy preceded apoptosis in oridonin-treated HT1080 cells We further investigate the role of autophagy in oridonin-induced apoptosis in HT1080 cells. Treatment with 3MA (a specific autophagy inhibitor), prior to the addition of oridonin, significantly decreased the percentage of MDC-positive cells as well as the apoptotic ratio compared with the group treated with oridonin alone (Fig. 4A and B), indicating that autophagy plays a death-promoting role in oridonin-treated HT1080 cells. All these results
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demonstrate that oridonin induces both apoptosis and autophagy in HT1080 cells, and inhibition of autophagy contributes to the down-regulation of apoptosis. Inhibition of NF-jB activation down-regulated apoptosis and autophagy in oridonin-treated HT1080 cells To investigate the role of NF-jB in oridonin-induced apoptosis and autophagy, we pretreated HT1080 cells with PDTC (a NF-jB inhibitor) before oridonin administration. As shown in Fig. 5A, preincubation with 10 or 20 lM PDTC decreased oridonin-induced cell growth inhibiton in a dose-dependant manner. Also pretreatment with 20 lM PDTC significantly decreased oridonin-induced apoptosis as well as autophagy (Fig. 5B and C). As to autophagy, the expression level of Beclin 1 and conversion from LC3-I to LC3-II revealed consistent results (Fig. 5D). To further ascertain the requirement of NF-jB activation for apoptosis and autophagy, expression levels of NF-jB, p-NF-jB and IjBa were examined in oridonintreated HT1080 cells for various time periods. As shown in Fig. 5E, oridonin administration enhanced both NF-jB and p-NFjB expression levels with accordingly attenuated levels of IjBa in a time-dependent manner. These results indicate that NF-jB can be activated by oridonin in HT1080 cells, and it promotes both apoptosis and autophagy in this circumstance. Inhibition of p53 activation decreased apoptosis and autophagy in oridonin-treated HT1080 cells Since the tumor suppressor protein p53 contributes to various types of cell death, and its expression is regulated by NF-jB, we examined the role of p53 in oridonin-induced apoptosis and autophagy in HT1080 cells. Pretreatment with 10 or 20 lM pifithrin-a (a p53 specific inhibitor) decreased the cell growth inhibitory ratio by oridonin (Fig. 6A). And both the apoptotic and the autophagic ratios of oridonin-treated cells were markedly decreased by addition of pifithrin-a (Fig. 6B and C). The down-regulations of Beclin 1 level and conversion from LC3-I to LC3-II further confirmed the negative regulatory effect of pifithrin-a on oridonin-induced autophagy (Fig. 6D). Then, we examined whether p53 was activated by oridonin. As shown in Fig. 6E, the levels of p53 and p-p53 were markedly elevated in a time-dependent manner after oridonin administration. Therefore, we conclude that activation of p53 also plays a promoting role in oridonin-induced apoptosis and autophagy in HT1080 cells. Inhibition of NF-jB suppressed the activation of p53 whereas inhibition of p53 had no effect on NF-jB activity Subsequently, to investigate the relationship between NF-jB and p53, we examined their expression levels respectively in oridonin-treated cells with or without indicated inhibitors. When the cells were treated with oridonin, inhibition of NF-jB by PDTC clearly decreased the expression levels of p53 and p-p53 (Fig. 7A). However, the expression levels of NF-jB, p-NF-jB and IjBa were not changed by pifithrin-a treatment compared with oridonin-treated alone (Fig. 7B). Thus, these results demonstrate that p53 is regulated by its upstream factor NF-jB to participate in oridonin-induced apoptosis and autophagy in HT1080 cells. Discussion
Fig. 4. Effects of 3MA on oridonin-induced autophagy and apoptosis in HT1080 cells. The cells were treated with 64 lM oridonin for 24 h in the presence or absence of 1 mM 3MA, the autophagic ratio (A) and the apoptotic ratio (B) were measured by flowcytometric analysis. n = 3, mean ± SD. *P < 0.05, **P < 0.01 vs oridonin group. Ori: oridonin.
Apoptosis and autophagy were reported to be co-existed and interconnected positively or negatively in some circumstances [6,17]. In the current study, our results from flowcytometric
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Fig. 5. Effects of NF-jB on oridonin-induced apoptosis and autophagy in HT1080 cells. (A) The cells were treated with 64 lM oridonin for 24 h, in the presence or absence of 10 or 20 lM PDTC, and the cell growth inhibitory ratio was measured by MTT assay. n = 3, mean ± SD. **P < 0.01 vs oridonin group. (B–D) The cells were treated with 64 lM oridonin for 24 h in the presence or absence of 20 lM PDTC, the apoptotic ratio (B) and the autophagic ratio (C) were measured by flowcytometric analysis. The protein levels of Beclin 1 and MAP-LC3 were detected by Western blot analysis, and the corresponding histograms quantified by densitometry were expressed at bottom (D). n = 3, mean ± SD. *P < 0.05, **P < 0.01 vs oridonin group. (E) The cells were treated with 64 lM oridonin for 12, 24, or 36 h, the protein levels of NF-jB, p-NF-jB and IjBa were detected by Western blot analysis, and the corresponding histograms quantified by densitometry were expressed at right.
analysis, morphologic observation and Western blot showed that oridonin could induce both apoptosis and autophagy in HT1080 cells, and autophagy exerted a synergistic function to apoptosis in this condition. Then, we investigated the possible mechanism which mediated oridonin-induced apoptosis and autophagy.
NF-jB is an important transcription factor involved in the regulation of cell death. It is well known that the nuclear localization of NF-jB is blocked by the binding of the NF-jB inhibitory proteins (IjB), and the activation of NF-jB results in the degradation of IjB proteins, followed by nuclear translocation and DNA binding of the
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Fig. 6. Effects of p53 on oridonin-induced apoptosis and autophagy in HT1080 cells. (A) The cells were treated with 64 lM oridonin for 24 h, in the presence or absence of 10 or 20 lM pifithrin-a, and the cell growth inhibitory ratio was measured by MTT assay. n = 3, mean ± SD. **P < 0.01 vs oridonin group. (B–D) The cells were treated with 64 lM oridonin for 24 h in the presence or absence of 20 lM pifithrin-a, the apoptotic ratio (B) and the autophagic ratio (C) were measured by flowcytometric analysis. The protein levels of Beclin 1 and MAP-LC3 were detected by Western blot analysis, and the corresponding histograms quantified by densitometry were expressed at bottom (D). n = 3, mean ± SD. *P < 0.05, **P < 0.01 vs oridonin group. (E) The cells were treated with 64 lM oridonin for 12, 24, or 36 h, and the protein levels of p53 and p-p53 were detected by Western blot analysis, and the corresponding histograms quantified by densitometry were expressed at right. PFT-a: pifithrin-a.
NF-jB [21]. Consistent with these observations, our results demonstrated that NF-jB was activated by oridonin in a time-dependent manner. Activation of NF-jB mediates cell survival signals in most tumor cells, but it may also act as pro-apoptotic factor in some conditions [22,11]. Here, we found that inhibition of NF-jB by PDTC
significantly decreased oridonin-induced cell death and apoptosis, indicating that NF-jB plays an apoptosis-promoting role in this research. And a few recent studies have been reported that NF-jB can be involved in the regulation of autophagy [12,13]. In our study, oridonin-induced autophagy was also markedly suppressed
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sis in p53-inducible Saos-2 cell line, and loss of p65 (a subunit of NF-jB) could also cause resistance to different agents that induced apoptosis through p53 [23,24]. Hence, we investigated the roles of p53 in apoptosis and autophagy in oridonin-treated HT1080 cells. The tumor suppressor protein p53 is activated in response to various cellular stresses to protect cells by inducing transcription of regulatory genes involved in cell cycle, apoptosis, and DNA repair [25]. Undoubtedly, oridonin has been shown to induce apoptosis through activating p53 in a various cells [26,27]. Here, we showed that oridonin induced the activation of p53, and inhibition of p53 by its inhibitor pifithrin-a led to the decrease in oridonininduced cell death and apoptosis. Furthermore, there are evidences that the activation of p53 also can involve in the regulation of autophagy. For instance, Zeng et al. reported that 6-thioguaninemediated DNA mismatch damage induced autophagy through p53 activation [28]. In another report, etoposide-induced p53 activation down-regulated mTOR activity resulting in increased autophagy levels in mouse embryonic fibroblasts [29]. In this study, oridonin-induced autophagy was significantly decreased by the disruption of p53 activation by pifithrin-a. As to the relationship between p53 and NF-jB, we found that inhibition of NFjB attenuated the expression levels of p53 and p-p53 whereas inhibition of p53 had no influence on the NF-jB, p-NF-jB and IjB expression levels, indicating that p53 activation is regulated by up-stream NF-jB. And this finding was supported by the fact that p53 activity which initiated pro-apoptotic signaling in response to doxycycline was NF-jB dependent in mouse embryonic fibroblasts [11]. Therefore, we conclude that activation of p53 mediates oridonin-induced apoptosis and autophagy in HT 1080 cells, and this process is regulated by NF-jB. In summery, NF-jB facilitates p53 activation to educe its proapoptotic and pro-autophagic functions in oridonin-treated HT1080 cells. Our results might provide new information about the role of NF-jB signal pathways existing between apoptosis and autophagy. Reference [1] [2] [3] [4] [5] [6]
Fig. 7. The relationship between NF-jB and p53 in oridonin-treated HT1080 cells. (A) The cells were treated with 64 lM oridonin for 24 h in the presence or absence of 20 lM PDTC, the protein levels of p53 and p-p53 were detected by Western blot analysis, and the corresponding histograms quantified by densitometry were expressed at bottom. n = 3, mean ± SD. *P < 0.05 vs oridonin group. (B) The cells were treated with 64 lM oridonin for 24 h in the presence or absence of 20 lM pifithrin-a, the protein levels of NF-jB, p-NF-jB and IjBa were detected by Western blot analysis, and the corresponding histograms quantified by densitometry were expressed at bottom.
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