Apoptosis (2015) 20:399–409 DOI 10.1007/s10495-014-1071-3
ORIGINAL PAPER
FOXO3-mediated up-regulation of Bim contributes to rhein-induced cancer cell apoptosis Jiao Wang • Shu Liu • Yancun Yin • Mingjin Li • Bo Wang • Li Yang • Yangfu Jiang
Published online: 12 December 2014 Ó Springer Science+Business Media New York 2014
Abstract The anthraquinone compound rhein is a natural agent in the traditional Chinese medicine rhubarb. Preclinical studies demonstrate that rhein has anticancer activity. Treatment of a variety of cancer cells with rhein may induce apoptosis. Here, we report that rhein induces atypical unfolded protein response in breast cancer MCF-7 cells and hepatoma HepG2 cells. Rhein induces CHOP expression, eIF2a phosphorylation and caspase cleavage, while it does not induce glucose-regulated protein 78 (GRP78) expression in both MCF-7 and HepG2 cells. Meanwhile, rhein inhibits thapsigargin-induced GRP78 expression and X box-binding protein 1 splicing. In addition, rhein inhibits Akt phosphorylation and stimulates FOXO transactivation activity. Rhein induces Bim expression in MCF-7 and HepG2 cells, which can be abrogated by FOXO3a knockdown. Knockdown of FOXO3a or Bim abrogates rhein-induced caspase cleavage and apoptosis. The chemical chaperone 4-phenylbutyrate acid antagonizes the induction of FOXO activation, Bim expression and caspase cleavage by rhein, indicating that protein misfolding may be involved in triggering these deleterious effects. We conclude that FOXO3a-mediated upregulation of Bim is a key mechanism underlying rheininduced cancer cells apoptosis.
J. Wang School of Basic Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, China S. Liu Y. Yin B. Wang L. Yang Y. Jiang (&) State Key Laboratory of Biotherapy, Section of Oncogene, West China Hospital, Sichuan University, Chengdu, China e-mail: [email protected] M. Li Medicine & Pharmacy Research Center, Binzhou Medical University, Yantai, Shandong, China
Keywords The unfolded protein response FOXO Bim Cancer Apoptosis
Introduction Previous studies have demonstrated that there are many natural compounds that have anti-cancer effects. Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) is one of the anthraquinone compounds in the root of rhubarb (R. palmatum L. or R. tanguticum Maxim). Previous studies have shown that rhein can induce apoptosis in cervical cancer cells [1], human nasopharyngeal carcinoma cells [2], breast cancer cells and tongue squamous cancer cells [3, 4]. Moreover, rhein and its derivatives reportedly inhibit tumor promotion or progression in multiple animal models [5–7]. Mechanistically, rhein may induce apoptosis through the generation of nitric oxide, reactive oxygen species (ROS) and Ca2? release, which may lead to endoplasmic reticulum (ER) stress [8, 9]. Induction of ER stress is a mechanism underlying the induction of apoptosis by many anti-cancer agents [10, 11]. Perturbations in the endoplasmic reticulum (ER) homeostasis would elicit the unfolded protein response (UPR). Although the UPR is basically a cytoprotective response to ER stress, persistent or unalleviated ER stress will cause cell death. A major UPR regulator is the ER chaperone glucose-regulated protein 78 (GRP78). As a multifunctional protein, GRP78 can interact with transmembrane ER stress sensors such as IRE1, PERK, and ATF6 and control their activation; maintain ER Ca2? homeostasis; and target misfolded proteins for proteasomal degradation [12]. While GRP78 represents a pro-survival factor during ER stress, CHOP is one of the mediators of ER-stress-induced apoptosis [13]. In addition, the BCL-2 family member Bim
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is an ER-stress-induced gene that is involved in mitochondrial-dependent apoptosis pathway [14]. Bax and Bak are ‘executioner’ proapoptotic proteins, which require activation via BH3-only proteins such as Bim and Bid. Previous studies have demonstrated that Bim is essential in some anti-cancer drugs-induced apoptosis, such as taxol, imatinib and sorafenib [15–17]. Here, we report that rhein induces atypical UPR in MCF-7 and HepG2 cells. Treatment of MCF-7 and HepG2 cells leads to an increase in the transactivation activity of FOXO, which leads to Bim overexpression. Knockdown of FOXO3a or Bim abrogates rhein-induced apoptosis.
Materials and methods Reagents and antibodies Rhein was purchased from MUST Biotech. (Chengdu, China). Thapsigargin, tunicamycin and 4-phenylbutyrate acid were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA).PI3K inhibitor LY294002 was purchased from Beyotime (Haimen,China). AKT inhibitor was purchased from Merck Millipore (Billerica, MA, USA). GRP78, CHOP, FOXO3a, cleaved caspase-7, cleaved caspase-3, cleaved caspase-8, eIF2a and phospho-eIF2a antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). XBP-1 and actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Bim antibody was purchased from Epitomics (Burlingame, CA, USA). Bid antibody was purchased from Proteintech (Wuhan, China). Phospho-FOXO3a antibody was purchased from Abzoom (Dallas, TX, USA). Cell culture The normal human mammary gland epithelial cell line MCF-10A was purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA) and grown in Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F-12 (DMEM/F12) supplemented with 5 % horse serum, 1 ng/mL cholera toxin, 10 lg/mL human insulin, 10 ng/mL epidermal growth factor and 0.5 lg/mL hydrocortisone. Breast cancer cell line MCF7 and hepatoma cell line HepG2 were obtained from Cell Lines Bank, Chinese Academy of Science (Shanghai, China), and grown in DMEM supplemented with 10 % fetal bovine serum. The cells were incubated at 37 °C in a humidified atmosphere of 5 % CO2. CCK-8 assay The cells were seeded in 96-well plates and allowed to attach and grow for 24 h. Then, the cells were exposed to various
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concentrations of rhein for 24–72 h. Cell viability was assessed by incubating cells with CCK-8 (Cell Counting Kit8) reagents (Dojindo Laboratory Co., Ltd., Kumamoto, Japan) for 1–2 h and measuring the absorbance at 450 nm with a microplate reader (Bio-Rad, Hercules, CA, USA).The half maximal inhibitory concentration (IC50) was calculated by probit regression analysis with SPSS statistics software package. Plasmids and transfection The plasmids contained the cDNAs encoding wild-type Akt1 and constitutive active Akt1 were purchased from Addgene (Cambridge, MA, USA). Transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). MCF-7 and HepG2 cells were plated in 6-well plates and grown to 60–70 % confluence and then transfected with 2.5–3 lg of plasmids. The maximal levels of protein expression were observed between 48 and 72 h. RNA interference All siRNA are synthesized products of Ribobio Co., Ltd. (Guangzhou, China). The cells grown in 6-well plates were transfected with siRNA using Lipofectamine 2000 reagent according to the manufacturer’s instructions. siRNA target sequences were as follows: Bim-siRNA (50 -CAACCACT ATCTCAGTGCA-30 ), FOXO3a-siRNA (50 -CAACCTGTC ACTGCATAGT-30 ). As nonspecific control, a scramble siRNA was used. Western blotting Cells were disrupted in lysis buffer containing protease inhibitors and phosphatase inhibitors. Protein contents in the supernatants were determined by BCA (bicinchoninic acid) protein assay (Pierce, Rockford, IL, USA). Aliquots of 30 lg of proteins were loaded into 10 % Tris–HCl polyacrylamide gels, and transferred to PVDF membrane. The membranes were incubated with the appropriate primary antibody overnight at 4 °C followed by incubation with secondary antibodies for 1 h. Protein bands were detected by chemiluminescent agents after hybridization with appropriate HRP-secondary antibodies. Hoechst 33342 staining The cells were plated in 24-well plate at a density of 3 9 105 cells per well, and transfected with siRNA and/or treated with rhein. After a change of fresh medium 24 h later, the cells were incubated with Hoechst 33342 at 37 °C for 10 min. Apoptotic cells with condensed or fragmented nuclei were detected by fluorescence microscopy.
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Fig. 1 The effects of rhein on MCF-7, HepG2 and MCF-10A cells growth. a The growth curves for HepG2, MCF-7 and MCF-10A cells treated with or without rhein. The percentage of cell growth compared
with control (0 h) was shown. Bars SD;# p \ 0.05; * p \ 0.01. b The IC50 values of rhein for HepG2, MCF-7 and MCF-10A cells
Quantification of apoptotic cells was performed by counting cells in four random fields in each well.
rhein for 24–72 h, followed by measuring cell viability. Rhein inhibited MCF-7 and HepG2 cells growth in timeand dose-dependent manner. Treatment of MCF-7 and HepG2 cells with 40 or 60 lM rhein for 48 h significantly inhibited cell growth, while treatment with 20 lM rhein for 72 h also significantly inhibited cell growth (Fig. 1a). The IC50 values of rhein were listed in (Fig. 1b). Upon treatment with rhein for 72 h, the mean IC50 values were 37.8 and 34.5 lM for MCF-7 and HepG2 cells, respectively. Also, we detected the effect of rhein on MCF-10A, an immortalized mammary epithelial cell line. After treatment with rhein for 48 or 72 h, the IC50 for MCF-10A cells was almost two-fold of that for MCF-7 and HepG2 cells (Fig. 1).
Luciferase assay The cells were plated in 24-well plate, and cotransfected with FOXO-responsive luciferase construct (Addgene) and Renilla luciferase reporter plasmid (pRL-TK, Promega) per well mixed with the LipoFectamine reagent (Invitrogen). 48 h after transfection, the cells were treated with rhein or not. 24 h after rhein treatment, cells were collected and FOXO transactivation activities were analyzed using DualGlo luciferase assay system (Promega) according to the manufacturer’s instructions. The luciferase activities were normalized against the Renilla luciferase activity. All of the treatments were performed in triplicate. Statistical analysis Data are presented as mean ± SD. For statistical comparison, one-way analysis of variance (ANOVA) was used. P \ 0.05 were considered statistically significant.
Results Rhein inhibits MCF-7 and HepG2 cells growth To detect the effect of rhein on cancer cells growth, we treated MCF-7 and HepG2 cells with increasing doses of
Rhein induces atypical UPR in MCF-7 and HepG2 cells Typically, the UPR is characteristic of GRP78 up-regulation, CHOP overexpression and eIF2a phosphorylation. To determine the effects of rhein on the UPR, MCF-7 and HepG2 cells were treated with 75–150 lM rhein for 24 h, followed by western blot analysis of GRP78 and CHOP expression, and eIF2a phosphorylation.. While rhein induced eIF2a phosphorylation and CHOP expression in dose-dependent manner, it did not up-regulate GRP78 expression in both MCF-7 and HepG2 cells (Fig. 2a). Treatment of MCF-10A cells with 75-150 lM rhein did not induce eIF2a phosphorylation and CHOP expression, while treatment with 250 lM rhein induce eIF2a phosphorylation and CHOP expression (Fig. 2b). Treatment of MCF-7
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Fig. 3 The effects of rhein on TG-induced GRP78, CHOP expression, eIF2a phosphorylation and XBP-1 splicing. a MCF7 and HepG2 cells were treated with or without 100 lM rhein or 300 nM TG for 24 h, followed by western blot analysis of GRP78, CHOP expression and eIF2a phosphorylation. b MCF7 and HepG2 cells were treated with or without 100 lM rhein or 300 nM TG for 6 h, followed by western blot analysis of XBP-1 splicing. XBP-1s, spliced XBP-1. c MCF7 and HepG2 cells were treated with or without 100 lM rhein for 24 h, followed by western blot analysis of ERK1/2 phosphorylation. A representative of three experiments was shown Fig. 2 The effects of rhein on GRP78, CHOP expression and eIF2a phosphorylation. a MCF7 and HepG2 cells were treated with or without 75-150 lM rhein for 24 h, followed by western blot analysis of GRP78, CHOP expression and eIF2a phosphorylation. b MCF10A cells were treated with or without 75-250 lM rhein for 24 h, followed by western blot analysis of GRP78, CHOP expression and eIF2a phosphorylation. c MCF7 and HepG2 cells were treated with or without 30 lM rhein for 24–72 h, followed by western blot analysis of GRP78, CHOP expression and eIF2a phosphorylation. A representative of three experiments was shown
and HepG2 cells with 30 lM rhein for 48 or 72 h also induced eIF2a phosphorylation and CHOP expression (Fig. 2c). These data indicate that rhein induce atypical UPR in MCF-7 and HepG2 cells.
than single agent (Fig. 3a). While treatment with TG alone up-regulated GRP78 expression, combination of rhein and TG did not induce GRP78 expression in both MCF-7 and HepG2 cells (Fig. 3a). Moreover, rhein did not induce XBP-1 splicing, another hallmark of the UPR, in both MCF-7 and HepG2 cells (Fig. 3b). Instead, the induction of XBP-1 splicing by TG was inhibited by rhein (Fig. 3b). Inhibition of Erk usually blocks ER stress-induced GRP78 up-regulation [18, 19]. Indeed, rhein inhibited Erk phosphorylation in both MCF-7 and HepG2 cells (Fig. 3c). These data indicate that rhein selectively induces some branches of the UPR but compromises other branches. Bim mediates the induction of apoptosis by rhein
Rhein suppresses ER-stress-induced GRP78 expresison and XBP-1 splicing Next, we detect the effects of rhein on other ER stressinduced UPR. To this end, MCF-7 and HepG2 cells were treated with or without rhein and classical ER stress inducer, thapsigargin (TG). Both rhein and TG induced eIF2a phosphorylation and CHOP expression in MCF-7 and HepG2 cells. Combination of rhein and TG led to more abundant CHOP expression and eIF2a phosphorylation
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Bim and caspases are critical mediators in the apoptosis induced by ER stress and some anticancer drugs [14, 20– 22]. We then detected the effects of rhein on Bim expression, caspase-3, caspase-7 and caspase-8 cleavage, and the levels of truncated Bid (t-Bid). Treatment of MCF7 and HepG2 cells with 75–150 lM rhein for 24 h led to increased Bim expression, caspase-3, caspase-7 and caspase-8 cleavage in dose-dependent manner (Fig. 4a). Also, rhein increased the levels of t-Bid in both MCF-7 and
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HepG2 cells (Fig. 4a). By contrast, treatment of MCF-10A cells with 75–150 lM rhein did not increase Bim expression, caspase-3, caspase-7, caspase-8 and Bid cleavage, while treatment with 250 lM rhein induce these events (Fig. 4b).Treatment of MCF-7 and HepG2 cells with 30 lM rhein for 48 or 72 h also led to increased Bim
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expression, caspase-3 and caspase-7 cleavage (Fig. 4c). Combination of rhein and TG led to more abundant Bim expression and caspase-3, -7 cleavage than single agent (Fig. 5). To determine whether Bim has a critical role in rheininduced apoptosis, we detected the effects of Bim knockdown on rhein-induced caspase cleavage. Bim knockdown abrogated rhein-induced caspase-3 and -7 cleavage in both MCF-7 and HepG2 cells, while it had no effect on CHOP expression and eIF2a phosphorylation (Fig. 6a, b). In addition, we detected the effects of Bim knockdown on rhein-induced apoptosis by Hoechst 33342 staining. Treatment of MCF-7 cells with rhein significantly induced apoptosis. Bim knockdown led to dramatic decrease in rhein-induced apoptosis (Fig. 6c). Similar effects were observed in HepG2 cells (Fig. 6d). FOXO3a mediates the induction of Bim by rhein
Fig. 4 The effects of rhein on Bim expression and caspase cleavage. a MCF7 and HepG2 cells were treated with or without 75–150 lM rhein for 24 h, followed by western blot analysis of Bim expression, caspase-3, -7, -8 cleavage and truncated Bid (T-Bid). b MCF10A cells were treated with or without 75–250 lM rhein for 24 h, followed by western blot analysis of Bim expression, caspase-3, -7, -8 cleavage and truncated Bid (T-Bid). c MCF7 and HepG2 cells were treated with or without 30 lM rhein for 24–72 h, followed by western blot analysis of Bim expression, caspase-3 and -7 cleavage. A representative of three experiments was shown
Given that Bim is regulated by FOXO transcription factors [15, 23], we detected the effects of rhein on the transcriptional activity of FOXO. Treatment with rhein led to increased FOXO activity in both MCF-7 and HepG2 cells (Fig. 7a). It is wellknown that Akt-mediated phosphorylation of FOXO negatively regulates FOXO activity [24, 25]. Indeed, treatment with rhein inhibited Akt and FOXO3a phosphorylation in MCF-7 and HepG2 cells (Fig. 7b). While rhein induced CHOP expression and eIF2a phosphorylation, neither PI3K inhibitor LY294002 nor Akt inhibitor IX induced CHOP expression and eIF2a phosphorylation, suggesting that the induction of UPR by rhein is not due to the inhibition of Akt (Fig. 7c). Consistent with the inactivation of FOXO3a by Akt, overexpression of constitutively active Akt in MCF-7 and HepG2 cells rescued the decrease in Akt and FOXO3a phosphorylation (Fig. 7d). Meanwhile, the induction of Bim expression and caspase-3, -7 cleavage by rhein was attenuated by constitutively active Akt (Fig. 7d). These data indicate that the inhibition of Akt by rhein may contribute, in part, to the induction of Bim expression and caspase cleavage.
Fig. 5 The effects of rhein on thapsigargin (TG)-induced Bim expression and caspase cleavage. MCF7 and HepG2 cells were treated with or without 100 lM rhein or 300 nM TG for 24 h, followed by western blot analysis of Bim expression and caspase-3, -7 cleavage. A representative of three experiments was shown
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Fig. 6 The effects of Bim knockdown on rhein-induced caspase cleavage and apoptosis. a MCF7 cells were transfected with siCtrl or siBim, followed by treating with or without 100 lM rhein for 24 h, Cell lysates were subjected to western blot analysis of Bim expression, caspase-3, -7 cleavage, CHOP expression and eIF2a phosphorylation. b HepG2 cells were transfected with siCtrl or siBim, followed by treating with or without 100 lM rhein for 24 h, Cell lysates were subjected to western blot analysis of Bim expression, caspase-3, -7 cleavage, CHOP expression and eIF2a phosphorylation. c MCF7 cells were transfected with siCtrl or siBim, followed by treating with or without 100 lM rhein for 24 h. Apoptosis were detected by Hoechst 33342 staining. Apoptotic cells were counted and plotted. Bars SD. d HepG2 cells were transfected with siCtrl or siBim, followed by treating with or without 100 lM rhein for 24 h. Apoptosis were detected by Hoechst 33342 staining. Apoptotic cells were counted and plotted. Bars SD. A representative of two or more experiments was shown
Next, we detected the effects of FOXO3a knockdown on rhein-induced Bim expression and caspase cleavage. FOXO3a knockdown reduced the induction of Bim expression, caspase-3 and caspase-7 cleavage by rhein (Fig. 8a, b). In addition, FOXO3a knockdown blunted rhein-induced apoptosis in both MCF-7 and HepG2 cells (Fig. 8c, d). These data demonstrate that FOXO3a contributes to rhein-induced Bim expression and apoptosis.
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Chemical chaperone relieves rhein-induced Bim expression and caspase cleavage Finally, we determined if ER stress was responsible for the stimulation of FOXO activity by rhein. To this end, we treated HepG2 cells with or without rhein and 4-phenylbutyrate acid (4-PBA), a chemical chaperone that could relieve ER stress [26–28]. 4-PBA significantly compromised the stimulation of
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Fig. 7 The effects of rhein on Akt phosphorylation and FOXO transactivation activity. a MCF7 and HepG2 cells were transfected with FOXO-responsive luciferase construct and Renilla luciferase reporter plasmid (pRL-TK), followed by treating with or without 100 lM rhein for 24 h and detecting the dual-luciferase activity. The FOXO transactivation activity was normalized against the renilla luciferase activity. The relative luciferase activity was plotted. Bars SD. b MCF7 and HepG2 cells were treated with or without 100 lM rhein for 24 h, followed by western blot analysis of Akt
phosphorylation. c MCF7 and HepG2 cells were treated with or without 20 lM LY294002 and 20 lM Akt inhibitor IX for 24 h, followed by western blot analysis of CHOP expression, Akt and eIF2a phosphorylation. d MCF7 and HepG2 cells were transfected with wild type (WT) Akt1 or constitutively active (CA) Akt1, followed by treatment with or without 100 lM rhein for 24 h. Cell lysates were subjected to western blot analysis of indicated proteins. A representative of three experiments was shown
FOXO activity by rhein (Fig. 9a). Also, treatment with 4-PBA suppressed rhein-induced Bim expression, CHOP expression and caspase cleavage, while it did not affect the inhibition of Akt phosphorylation by rhein (Fig. 9b).
ER stress can be induced by multiple stimuli such as low pH, glucose starvation, protein misfolding and disruption of Ca2? homeostasis [12]. Rhein reportedly induces ER stress through depletion of ER Ca2? store [2, 9]. The typical ER stress response or unfolded protein response includes up-regulation of GRP78, activation of IRE1,ATF6 and PERK [12, 32]. However, our study demonstrates that rhein does not up-regulate GRP78, while it induces eIF2a phosphorylation and CHOP expression. These data suggest that rhein may induce atypical ER stress response. Given that GRP78 is an ER-resident chaperone that has multiple protective roles, the failure to up-regulate GRP78 may lead to a disadvantage in adaptation to ER stress and then more cell death [12, 33]. Rhein also inhibits ER stress-induced GRP78 expression. Inhibition of Erk usually blocks ER stress-induced GRP78 up-regulation in melanoma cells and gastric cancer cells [18, 19]. Similar to rhein, the raf inhibitor vemurafenib inhibits ER stress-induced up-regulation of GRP78 expression [34]. Rhein also inhibits Erk phosphorylation. Inhibition of ERK may contribute, at least in part, to the inhibition of ER stress-induced GRP78 expression by rhein. Notably, rhein not only antagonizes
Discussion Rhein is a natural compound that has anticancer effects in vitro and in vivo. Based on our current study and other reports, rhein can induce massive cell death in a variety of cancer cells [1–4, 29–31]. The mechanisms underlying rhein-induced cell death including activation of p53, enhancement of CD95 and its ligands [29], generation of ROS and initiation subsequent mitochondria-mediated apoptosis [30]. In addition, rhein has been found to affect the aerobic and anaerobic glycolysis in Ehrlich ascites tumor cells [31]. Our current study demonstrates that rhein induces atypical unfolded protein response, FOXO activation, Bim overexpression, caspase cleavage and apoptosis. These findings uncover some molecular events in MCF7 and HepG2 cells after rhein treatment.
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Fig. 8 The effects of FOXO3a knockdown on rhein-induced Bim expression, caspase cleavage and apoptosis. a MCF7 cells were transfected with siCtrl or siFOXO3a, followed by treating with or without 100 lM rhein for 24 h. Cell lysates were subjected to western blot analysis of FOXO3a, Bim expression and caspase-3, -7 cleavage. b HepG2 cells were transfected with siCtrl or siFOXO3a, followed by treating with or without 100 lM rhein for 24 h. Cell lysates were subjected to western blot analysis of FOXO3a, Bim expression and caspase-3, -7 cleavage. c MCF7 cells were transfected with siCtrl or siFOXO3a, followed by treating with or without 100 lM rhein for 24 h. Apoptosis were detected by Hoechst 33342 staining. Apoptotic cells were counted and plotted. Bars SD. d HepG2 cells were transfected with siCtrl or siFOXO3a, followed by treating with or without 100 lM rhein for 24 h. Apoptosis were detected by Hoechst 33342 staining. Apoptotic cells were counted and plotted. Bars SD. A representative of two experiments was shown
ER stress-induced GRP78 expression, but also inhibits ER stress-induced XBP-1 splicing, an event downstream of IRE1 activation. These data suggest that rhein may inhibit selective branches of the ER stress response. Although rhein effectively induces cell apoptosis, the key mediators of rhein-induced apoptosis remains elusive. Here, we show that the B cell lymphoma-2 (Bcl-2)-family member Bim is a critical mediator of rhein-induced apoptosis. There are mainly two distinct apoptosis signaling pathways, the death receptor pathway and the
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mitochondrial pathway [35]. Previous studies have shown that the induction of cancer cell apoptosis by rhein is mitochondria-dependent [4]. The Bcl-2 family of proteins has either antiapoptotic role or proapoptotic role in the mitochondrial pathway [36]. Bim is one of the proapoptotic BH3-only proteins in Bcl-2 family. Bim up-regulation triggers cytochrome c release from mitochondria, thereby induces the formation of the apoptosome and the activation of its effector caspase [37]. Previous studies have demonstrated that Bim plays important roles in paclitaxol therapy,
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Fig. 9 The effects of chemical chaperone on rhein-induced FOXO activation, CHOP, Bim expression and caspase cleavage. a MCF7 cells were transfected with FOXO-responsive luciferase construct and Renilla luciferase reporter plasmid (pRL-TK), followed by treating with or without 5 mM 4-PBA and 100 lM rhein for 24 h and detecting the dual-luciferase activity. The FOXO tranactivation
activity was normalized against the renilla luciferase activity. The relative FOXO transactivation activity was plotted. Bars SD. b MCF7 cells were treated with or without 100 lM rhein or 5 mM 4-PBA for 24 h, followed by western blot analysis of GRP78, CHOP, Bim expression, Akt phosphorylation and caspase cleavage. A representative of three experiments was shown
but not in cisplatin treatment. In addition, Bim-targeted drugs include Bcr/Abl inhibitors and EGFR inhibitors [38]. Our current study demonstrates that Bim may contribute, at least in part, to the induction of cancer cell apoptosis by rhein. Hence, Bim down-regulation may leads to resistance to this agent. Both CHOP and FOXO3a may regulate Bim expression [39]. Indeed, we find that rhein up-regulates FOXO transactivation activity. The protein kinase Akt can phosphorylate and inactivate FOXO [24, 25]. We also find that rhein inhibits Akt and FOXO3a phosphorylation. Overexpression of constitutively active Akt can rescue the decrease in both Akt and FOXO3a phosphorylation, and attenuate rheininduced Bim expression and caspase cleavage. These data suggest that inhibition of Akt-FOXO axis may contribute, in part, to up-regulation of pro-apoptotic Bim. Other factors such as CHOP may also be involved in the induction of Bim. FOXO3a knockdown significantly compromised rhein-induced Bim expression and apoptosis. The ability of chemical chaperone to relieve rhein-induced FOXO activation and Bim expression suggests that ER stress may be attributable, at least in part, to these pro-apoptotic events induced by rhein. Meanwhile, the chemical chaperone does not relieve the inhibition of Akt phosphorylation by rhein. Thus, the induction of FOXO activity, Bim expression and apoptosis by rhein may involve, but not limited to, two mechanisms including inhibition of Akt and induction of ER stress. Previous studies demonstrate that ER stress may lead to Akt phosphorylation, which is dependent on GRP78
[40, 41]. The failure to induce Akt phosphorylation by rhein may be due to the inhibition of ER stress-induced GRP78 expression. It is less likely that the induction of ER stress is a consequence of Akt inhibition, since neither Akt inhibitor nor PI3K inhibitor induces CHOP expression and eIF2a phosphorylation. Bim may be one of key nodes for mediation of rhein-induced apoptosis. Preclinical studies demonstrate that rhein and its derivatives can induce apoptosis, suppress proliferation and angiogenesis in vitro and in vivo [1–7]. A wide range of concentrations of rhein (30–180 lM) have been used in previous studies with various efficacy [1–4, 29–31]. Our current study shows that, upon treatment with rhein for 72 h, the mean IC50 values are 34.5 and 37.8 lM for HepG2 and MCF-7 cells, respectively. The bioavailability of rhein may be a key determinant of its effectiveness in vivo. Phamacokinetic study in rat model demonstrates that a peak value of 149.7 lM in plasma can be reached after single oral administration of 70 mg/kg rhein [42]. In human, the maximal plasma concentration of approximately 35.9 lM was detected after single oral administration of 200 mg rhein [43]. Thus, the concentrations of rhein in the current study may have in vivo relevance. After treatment with rhein for 48 or 72 h, the IC50 for MCF-10A, an untransformed breast cell line, was almost two-fold of that for MCF-7 and HepG2 cells. These data suggest a narrow treatment window between the treatment dose and toxicity dose, which may impede the clinical application of rhein. It warrants further research to determine if chemical modification, enhancement of solubility and bioavailability, and targeted or intravenous delivery can lower the
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treatment dose of rhein thereby increasing its anticancer effect and avoiding potential adverse effects in vivo. Acknowledgments We thank for the support from National Natural Science Foundation of China (No. 81001587). Conflict of interest
The authors declare no conflicts of interest.
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ORIGINAL PAPER
FOXO3-mediated up-regulation of Bim contributes to rhein-induced cancer cell apoptosis Jiao Wang • Shu Liu • Yancun Yin • Mingjin Li • Bo Wang • Li Yang • Yangfu Jiang
Published online: 12 December 2014 Ó Springer Science+Business Media New York 2014
Abstract The anthraquinone compound rhein is a natural agent in the traditional Chinese medicine rhubarb. Preclinical studies demonstrate that rhein has anticancer activity. Treatment of a variety of cancer cells with rhein may induce apoptosis. Here, we report that rhein induces atypical unfolded protein response in breast cancer MCF-7 cells and hepatoma HepG2 cells. Rhein induces CHOP expression, eIF2a phosphorylation and caspase cleavage, while it does not induce glucose-regulated protein 78 (GRP78) expression in both MCF-7 and HepG2 cells. Meanwhile, rhein inhibits thapsigargin-induced GRP78 expression and X box-binding protein 1 splicing. In addition, rhein inhibits Akt phosphorylation and stimulates FOXO transactivation activity. Rhein induces Bim expression in MCF-7 and HepG2 cells, which can be abrogated by FOXO3a knockdown. Knockdown of FOXO3a or Bim abrogates rhein-induced caspase cleavage and apoptosis. The chemical chaperone 4-phenylbutyrate acid antagonizes the induction of FOXO activation, Bim expression and caspase cleavage by rhein, indicating that protein misfolding may be involved in triggering these deleterious effects. We conclude that FOXO3a-mediated upregulation of Bim is a key mechanism underlying rheininduced cancer cells apoptosis.
J. Wang School of Basic Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, China S. Liu Y. Yin B. Wang L. Yang Y. Jiang (&) State Key Laboratory of Biotherapy, Section of Oncogene, West China Hospital, Sichuan University, Chengdu, China e-mail: [email protected] M. Li Medicine & Pharmacy Research Center, Binzhou Medical University, Yantai, Shandong, China
Keywords The unfolded protein response FOXO Bim Cancer Apoptosis
Introduction Previous studies have demonstrated that there are many natural compounds that have anti-cancer effects. Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) is one of the anthraquinone compounds in the root of rhubarb (R. palmatum L. or R. tanguticum Maxim). Previous studies have shown that rhein can induce apoptosis in cervical cancer cells [1], human nasopharyngeal carcinoma cells [2], breast cancer cells and tongue squamous cancer cells [3, 4]. Moreover, rhein and its derivatives reportedly inhibit tumor promotion or progression in multiple animal models [5–7]. Mechanistically, rhein may induce apoptosis through the generation of nitric oxide, reactive oxygen species (ROS) and Ca2? release, which may lead to endoplasmic reticulum (ER) stress [8, 9]. Induction of ER stress is a mechanism underlying the induction of apoptosis by many anti-cancer agents [10, 11]. Perturbations in the endoplasmic reticulum (ER) homeostasis would elicit the unfolded protein response (UPR). Although the UPR is basically a cytoprotective response to ER stress, persistent or unalleviated ER stress will cause cell death. A major UPR regulator is the ER chaperone glucose-regulated protein 78 (GRP78). As a multifunctional protein, GRP78 can interact with transmembrane ER stress sensors such as IRE1, PERK, and ATF6 and control their activation; maintain ER Ca2? homeostasis; and target misfolded proteins for proteasomal degradation [12]. While GRP78 represents a pro-survival factor during ER stress, CHOP is one of the mediators of ER-stress-induced apoptosis [13]. In addition, the BCL-2 family member Bim
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is an ER-stress-induced gene that is involved in mitochondrial-dependent apoptosis pathway [14]. Bax and Bak are ‘executioner’ proapoptotic proteins, which require activation via BH3-only proteins such as Bim and Bid. Previous studies have demonstrated that Bim is essential in some anti-cancer drugs-induced apoptosis, such as taxol, imatinib and sorafenib [15–17]. Here, we report that rhein induces atypical UPR in MCF-7 and HepG2 cells. Treatment of MCF-7 and HepG2 cells leads to an increase in the transactivation activity of FOXO, which leads to Bim overexpression. Knockdown of FOXO3a or Bim abrogates rhein-induced apoptosis.
Materials and methods Reagents and antibodies Rhein was purchased from MUST Biotech. (Chengdu, China). Thapsigargin, tunicamycin and 4-phenylbutyrate acid were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA).PI3K inhibitor LY294002 was purchased from Beyotime (Haimen,China). AKT inhibitor was purchased from Merck Millipore (Billerica, MA, USA). GRP78, CHOP, FOXO3a, cleaved caspase-7, cleaved caspase-3, cleaved caspase-8, eIF2a and phospho-eIF2a antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). XBP-1 and actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Bim antibody was purchased from Epitomics (Burlingame, CA, USA). Bid antibody was purchased from Proteintech (Wuhan, China). Phospho-FOXO3a antibody was purchased from Abzoom (Dallas, TX, USA). Cell culture The normal human mammary gland epithelial cell line MCF-10A was purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA) and grown in Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F-12 (DMEM/F12) supplemented with 5 % horse serum, 1 ng/mL cholera toxin, 10 lg/mL human insulin, 10 ng/mL epidermal growth factor and 0.5 lg/mL hydrocortisone. Breast cancer cell line MCF7 and hepatoma cell line HepG2 were obtained from Cell Lines Bank, Chinese Academy of Science (Shanghai, China), and grown in DMEM supplemented with 10 % fetal bovine serum. The cells were incubated at 37 °C in a humidified atmosphere of 5 % CO2. CCK-8 assay The cells were seeded in 96-well plates and allowed to attach and grow for 24 h. Then, the cells were exposed to various
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concentrations of rhein for 24–72 h. Cell viability was assessed by incubating cells with CCK-8 (Cell Counting Kit8) reagents (Dojindo Laboratory Co., Ltd., Kumamoto, Japan) for 1–2 h and measuring the absorbance at 450 nm with a microplate reader (Bio-Rad, Hercules, CA, USA).The half maximal inhibitory concentration (IC50) was calculated by probit regression analysis with SPSS statistics software package. Plasmids and transfection The plasmids contained the cDNAs encoding wild-type Akt1 and constitutive active Akt1 were purchased from Addgene (Cambridge, MA, USA). Transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). MCF-7 and HepG2 cells were plated in 6-well plates and grown to 60–70 % confluence and then transfected with 2.5–3 lg of plasmids. The maximal levels of protein expression were observed between 48 and 72 h. RNA interference All siRNA are synthesized products of Ribobio Co., Ltd. (Guangzhou, China). The cells grown in 6-well plates were transfected with siRNA using Lipofectamine 2000 reagent according to the manufacturer’s instructions. siRNA target sequences were as follows: Bim-siRNA (50 -CAACCACT ATCTCAGTGCA-30 ), FOXO3a-siRNA (50 -CAACCTGTC ACTGCATAGT-30 ). As nonspecific control, a scramble siRNA was used. Western blotting Cells were disrupted in lysis buffer containing protease inhibitors and phosphatase inhibitors. Protein contents in the supernatants were determined by BCA (bicinchoninic acid) protein assay (Pierce, Rockford, IL, USA). Aliquots of 30 lg of proteins were loaded into 10 % Tris–HCl polyacrylamide gels, and transferred to PVDF membrane. The membranes were incubated with the appropriate primary antibody overnight at 4 °C followed by incubation with secondary antibodies for 1 h. Protein bands were detected by chemiluminescent agents after hybridization with appropriate HRP-secondary antibodies. Hoechst 33342 staining The cells were plated in 24-well plate at a density of 3 9 105 cells per well, and transfected with siRNA and/or treated with rhein. After a change of fresh medium 24 h later, the cells were incubated with Hoechst 33342 at 37 °C for 10 min. Apoptotic cells with condensed or fragmented nuclei were detected by fluorescence microscopy.
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Fig. 1 The effects of rhein on MCF-7, HepG2 and MCF-10A cells growth. a The growth curves for HepG2, MCF-7 and MCF-10A cells treated with or without rhein. The percentage of cell growth compared
with control (0 h) was shown. Bars SD;# p \ 0.05; * p \ 0.01. b The IC50 values of rhein for HepG2, MCF-7 and MCF-10A cells
Quantification of apoptotic cells was performed by counting cells in four random fields in each well.
rhein for 24–72 h, followed by measuring cell viability. Rhein inhibited MCF-7 and HepG2 cells growth in timeand dose-dependent manner. Treatment of MCF-7 and HepG2 cells with 40 or 60 lM rhein for 48 h significantly inhibited cell growth, while treatment with 20 lM rhein for 72 h also significantly inhibited cell growth (Fig. 1a). The IC50 values of rhein were listed in (Fig. 1b). Upon treatment with rhein for 72 h, the mean IC50 values were 37.8 and 34.5 lM for MCF-7 and HepG2 cells, respectively. Also, we detected the effect of rhein on MCF-10A, an immortalized mammary epithelial cell line. After treatment with rhein for 48 or 72 h, the IC50 for MCF-10A cells was almost two-fold of that for MCF-7 and HepG2 cells (Fig. 1).
Luciferase assay The cells were plated in 24-well plate, and cotransfected with FOXO-responsive luciferase construct (Addgene) and Renilla luciferase reporter plasmid (pRL-TK, Promega) per well mixed with the LipoFectamine reagent (Invitrogen). 48 h after transfection, the cells were treated with rhein or not. 24 h after rhein treatment, cells were collected and FOXO transactivation activities were analyzed using DualGlo luciferase assay system (Promega) according to the manufacturer’s instructions. The luciferase activities were normalized against the Renilla luciferase activity. All of the treatments were performed in triplicate. Statistical analysis Data are presented as mean ± SD. For statistical comparison, one-way analysis of variance (ANOVA) was used. P \ 0.05 were considered statistically significant.
Results Rhein inhibits MCF-7 and HepG2 cells growth To detect the effect of rhein on cancer cells growth, we treated MCF-7 and HepG2 cells with increasing doses of
Rhein induces atypical UPR in MCF-7 and HepG2 cells Typically, the UPR is characteristic of GRP78 up-regulation, CHOP overexpression and eIF2a phosphorylation. To determine the effects of rhein on the UPR, MCF-7 and HepG2 cells were treated with 75–150 lM rhein for 24 h, followed by western blot analysis of GRP78 and CHOP expression, and eIF2a phosphorylation.. While rhein induced eIF2a phosphorylation and CHOP expression in dose-dependent manner, it did not up-regulate GRP78 expression in both MCF-7 and HepG2 cells (Fig. 2a). Treatment of MCF-10A cells with 75-150 lM rhein did not induce eIF2a phosphorylation and CHOP expression, while treatment with 250 lM rhein induce eIF2a phosphorylation and CHOP expression (Fig. 2b). Treatment of MCF-7
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Fig. 3 The effects of rhein on TG-induced GRP78, CHOP expression, eIF2a phosphorylation and XBP-1 splicing. a MCF7 and HepG2 cells were treated with or without 100 lM rhein or 300 nM TG for 24 h, followed by western blot analysis of GRP78, CHOP expression and eIF2a phosphorylation. b MCF7 and HepG2 cells were treated with or without 100 lM rhein or 300 nM TG for 6 h, followed by western blot analysis of XBP-1 splicing. XBP-1s, spliced XBP-1. c MCF7 and HepG2 cells were treated with or without 100 lM rhein for 24 h, followed by western blot analysis of ERK1/2 phosphorylation. A representative of three experiments was shown Fig. 2 The effects of rhein on GRP78, CHOP expression and eIF2a phosphorylation. a MCF7 and HepG2 cells were treated with or without 75-150 lM rhein for 24 h, followed by western blot analysis of GRP78, CHOP expression and eIF2a phosphorylation. b MCF10A cells were treated with or without 75-250 lM rhein for 24 h, followed by western blot analysis of GRP78, CHOP expression and eIF2a phosphorylation. c MCF7 and HepG2 cells were treated with or without 30 lM rhein for 24–72 h, followed by western blot analysis of GRP78, CHOP expression and eIF2a phosphorylation. A representative of three experiments was shown
and HepG2 cells with 30 lM rhein for 48 or 72 h also induced eIF2a phosphorylation and CHOP expression (Fig. 2c). These data indicate that rhein induce atypical UPR in MCF-7 and HepG2 cells.
than single agent (Fig. 3a). While treatment with TG alone up-regulated GRP78 expression, combination of rhein and TG did not induce GRP78 expression in both MCF-7 and HepG2 cells (Fig. 3a). Moreover, rhein did not induce XBP-1 splicing, another hallmark of the UPR, in both MCF-7 and HepG2 cells (Fig. 3b). Instead, the induction of XBP-1 splicing by TG was inhibited by rhein (Fig. 3b). Inhibition of Erk usually blocks ER stress-induced GRP78 up-regulation [18, 19]. Indeed, rhein inhibited Erk phosphorylation in both MCF-7 and HepG2 cells (Fig. 3c). These data indicate that rhein selectively induces some branches of the UPR but compromises other branches. Bim mediates the induction of apoptosis by rhein
Rhein suppresses ER-stress-induced GRP78 expresison and XBP-1 splicing Next, we detect the effects of rhein on other ER stressinduced UPR. To this end, MCF-7 and HepG2 cells were treated with or without rhein and classical ER stress inducer, thapsigargin (TG). Both rhein and TG induced eIF2a phosphorylation and CHOP expression in MCF-7 and HepG2 cells. Combination of rhein and TG led to more abundant CHOP expression and eIF2a phosphorylation
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Bim and caspases are critical mediators in the apoptosis induced by ER stress and some anticancer drugs [14, 20– 22]. We then detected the effects of rhein on Bim expression, caspase-3, caspase-7 and caspase-8 cleavage, and the levels of truncated Bid (t-Bid). Treatment of MCF7 and HepG2 cells with 75–150 lM rhein for 24 h led to increased Bim expression, caspase-3, caspase-7 and caspase-8 cleavage in dose-dependent manner (Fig. 4a). Also, rhein increased the levels of t-Bid in both MCF-7 and
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HepG2 cells (Fig. 4a). By contrast, treatment of MCF-10A cells with 75–150 lM rhein did not increase Bim expression, caspase-3, caspase-7, caspase-8 and Bid cleavage, while treatment with 250 lM rhein induce these events (Fig. 4b).Treatment of MCF-7 and HepG2 cells with 30 lM rhein for 48 or 72 h also led to increased Bim
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expression, caspase-3 and caspase-7 cleavage (Fig. 4c). Combination of rhein and TG led to more abundant Bim expression and caspase-3, -7 cleavage than single agent (Fig. 5). To determine whether Bim has a critical role in rheininduced apoptosis, we detected the effects of Bim knockdown on rhein-induced caspase cleavage. Bim knockdown abrogated rhein-induced caspase-3 and -7 cleavage in both MCF-7 and HepG2 cells, while it had no effect on CHOP expression and eIF2a phosphorylation (Fig. 6a, b). In addition, we detected the effects of Bim knockdown on rhein-induced apoptosis by Hoechst 33342 staining. Treatment of MCF-7 cells with rhein significantly induced apoptosis. Bim knockdown led to dramatic decrease in rhein-induced apoptosis (Fig. 6c). Similar effects were observed in HepG2 cells (Fig. 6d). FOXO3a mediates the induction of Bim by rhein
Fig. 4 The effects of rhein on Bim expression and caspase cleavage. a MCF7 and HepG2 cells were treated with or without 75–150 lM rhein for 24 h, followed by western blot analysis of Bim expression, caspase-3, -7, -8 cleavage and truncated Bid (T-Bid). b MCF10A cells were treated with or without 75–250 lM rhein for 24 h, followed by western blot analysis of Bim expression, caspase-3, -7, -8 cleavage and truncated Bid (T-Bid). c MCF7 and HepG2 cells were treated with or without 30 lM rhein for 24–72 h, followed by western blot analysis of Bim expression, caspase-3 and -7 cleavage. A representative of three experiments was shown
Given that Bim is regulated by FOXO transcription factors [15, 23], we detected the effects of rhein on the transcriptional activity of FOXO. Treatment with rhein led to increased FOXO activity in both MCF-7 and HepG2 cells (Fig. 7a). It is wellknown that Akt-mediated phosphorylation of FOXO negatively regulates FOXO activity [24, 25]. Indeed, treatment with rhein inhibited Akt and FOXO3a phosphorylation in MCF-7 and HepG2 cells (Fig. 7b). While rhein induced CHOP expression and eIF2a phosphorylation, neither PI3K inhibitor LY294002 nor Akt inhibitor IX induced CHOP expression and eIF2a phosphorylation, suggesting that the induction of UPR by rhein is not due to the inhibition of Akt (Fig. 7c). Consistent with the inactivation of FOXO3a by Akt, overexpression of constitutively active Akt in MCF-7 and HepG2 cells rescued the decrease in Akt and FOXO3a phosphorylation (Fig. 7d). Meanwhile, the induction of Bim expression and caspase-3, -7 cleavage by rhein was attenuated by constitutively active Akt (Fig. 7d). These data indicate that the inhibition of Akt by rhein may contribute, in part, to the induction of Bim expression and caspase cleavage.
Fig. 5 The effects of rhein on thapsigargin (TG)-induced Bim expression and caspase cleavage. MCF7 and HepG2 cells were treated with or without 100 lM rhein or 300 nM TG for 24 h, followed by western blot analysis of Bim expression and caspase-3, -7 cleavage. A representative of three experiments was shown
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Fig. 6 The effects of Bim knockdown on rhein-induced caspase cleavage and apoptosis. a MCF7 cells were transfected with siCtrl or siBim, followed by treating with or without 100 lM rhein for 24 h, Cell lysates were subjected to western blot analysis of Bim expression, caspase-3, -7 cleavage, CHOP expression and eIF2a phosphorylation. b HepG2 cells were transfected with siCtrl or siBim, followed by treating with or without 100 lM rhein for 24 h, Cell lysates were subjected to western blot analysis of Bim expression, caspase-3, -7 cleavage, CHOP expression and eIF2a phosphorylation. c MCF7 cells were transfected with siCtrl or siBim, followed by treating with or without 100 lM rhein for 24 h. Apoptosis were detected by Hoechst 33342 staining. Apoptotic cells were counted and plotted. Bars SD. d HepG2 cells were transfected with siCtrl or siBim, followed by treating with or without 100 lM rhein for 24 h. Apoptosis were detected by Hoechst 33342 staining. Apoptotic cells were counted and plotted. Bars SD. A representative of two or more experiments was shown
Next, we detected the effects of FOXO3a knockdown on rhein-induced Bim expression and caspase cleavage. FOXO3a knockdown reduced the induction of Bim expression, caspase-3 and caspase-7 cleavage by rhein (Fig. 8a, b). In addition, FOXO3a knockdown blunted rhein-induced apoptosis in both MCF-7 and HepG2 cells (Fig. 8c, d). These data demonstrate that FOXO3a contributes to rhein-induced Bim expression and apoptosis.
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Chemical chaperone relieves rhein-induced Bim expression and caspase cleavage Finally, we determined if ER stress was responsible for the stimulation of FOXO activity by rhein. To this end, we treated HepG2 cells with or without rhein and 4-phenylbutyrate acid (4-PBA), a chemical chaperone that could relieve ER stress [26–28]. 4-PBA significantly compromised the stimulation of
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Fig. 7 The effects of rhein on Akt phosphorylation and FOXO transactivation activity. a MCF7 and HepG2 cells were transfected with FOXO-responsive luciferase construct and Renilla luciferase reporter plasmid (pRL-TK), followed by treating with or without 100 lM rhein for 24 h and detecting the dual-luciferase activity. The FOXO transactivation activity was normalized against the renilla luciferase activity. The relative luciferase activity was plotted. Bars SD. b MCF7 and HepG2 cells were treated with or without 100 lM rhein for 24 h, followed by western blot analysis of Akt
phosphorylation. c MCF7 and HepG2 cells were treated with or without 20 lM LY294002 and 20 lM Akt inhibitor IX for 24 h, followed by western blot analysis of CHOP expression, Akt and eIF2a phosphorylation. d MCF7 and HepG2 cells were transfected with wild type (WT) Akt1 or constitutively active (CA) Akt1, followed by treatment with or without 100 lM rhein for 24 h. Cell lysates were subjected to western blot analysis of indicated proteins. A representative of three experiments was shown
FOXO activity by rhein (Fig. 9a). Also, treatment with 4-PBA suppressed rhein-induced Bim expression, CHOP expression and caspase cleavage, while it did not affect the inhibition of Akt phosphorylation by rhein (Fig. 9b).
ER stress can be induced by multiple stimuli such as low pH, glucose starvation, protein misfolding and disruption of Ca2? homeostasis [12]. Rhein reportedly induces ER stress through depletion of ER Ca2? store [2, 9]. The typical ER stress response or unfolded protein response includes up-regulation of GRP78, activation of IRE1,ATF6 and PERK [12, 32]. However, our study demonstrates that rhein does not up-regulate GRP78, while it induces eIF2a phosphorylation and CHOP expression. These data suggest that rhein may induce atypical ER stress response. Given that GRP78 is an ER-resident chaperone that has multiple protective roles, the failure to up-regulate GRP78 may lead to a disadvantage in adaptation to ER stress and then more cell death [12, 33]. Rhein also inhibits ER stress-induced GRP78 expression. Inhibition of Erk usually blocks ER stress-induced GRP78 up-regulation in melanoma cells and gastric cancer cells [18, 19]. Similar to rhein, the raf inhibitor vemurafenib inhibits ER stress-induced up-regulation of GRP78 expression [34]. Rhein also inhibits Erk phosphorylation. Inhibition of ERK may contribute, at least in part, to the inhibition of ER stress-induced GRP78 expression by rhein. Notably, rhein not only antagonizes
Discussion Rhein is a natural compound that has anticancer effects in vitro and in vivo. Based on our current study and other reports, rhein can induce massive cell death in a variety of cancer cells [1–4, 29–31]. The mechanisms underlying rhein-induced cell death including activation of p53, enhancement of CD95 and its ligands [29], generation of ROS and initiation subsequent mitochondria-mediated apoptosis [30]. In addition, rhein has been found to affect the aerobic and anaerobic glycolysis in Ehrlich ascites tumor cells [31]. Our current study demonstrates that rhein induces atypical unfolded protein response, FOXO activation, Bim overexpression, caspase cleavage and apoptosis. These findings uncover some molecular events in MCF7 and HepG2 cells after rhein treatment.
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Fig. 8 The effects of FOXO3a knockdown on rhein-induced Bim expression, caspase cleavage and apoptosis. a MCF7 cells were transfected with siCtrl or siFOXO3a, followed by treating with or without 100 lM rhein for 24 h. Cell lysates were subjected to western blot analysis of FOXO3a, Bim expression and caspase-3, -7 cleavage. b HepG2 cells were transfected with siCtrl or siFOXO3a, followed by treating with or without 100 lM rhein for 24 h. Cell lysates were subjected to western blot analysis of FOXO3a, Bim expression and caspase-3, -7 cleavage. c MCF7 cells were transfected with siCtrl or siFOXO3a, followed by treating with or without 100 lM rhein for 24 h. Apoptosis were detected by Hoechst 33342 staining. Apoptotic cells were counted and plotted. Bars SD. d HepG2 cells were transfected with siCtrl or siFOXO3a, followed by treating with or without 100 lM rhein for 24 h. Apoptosis were detected by Hoechst 33342 staining. Apoptotic cells were counted and plotted. Bars SD. A representative of two experiments was shown
ER stress-induced GRP78 expression, but also inhibits ER stress-induced XBP-1 splicing, an event downstream of IRE1 activation. These data suggest that rhein may inhibit selective branches of the ER stress response. Although rhein effectively induces cell apoptosis, the key mediators of rhein-induced apoptosis remains elusive. Here, we show that the B cell lymphoma-2 (Bcl-2)-family member Bim is a critical mediator of rhein-induced apoptosis. There are mainly two distinct apoptosis signaling pathways, the death receptor pathway and the
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mitochondrial pathway [35]. Previous studies have shown that the induction of cancer cell apoptosis by rhein is mitochondria-dependent [4]. The Bcl-2 family of proteins has either antiapoptotic role or proapoptotic role in the mitochondrial pathway [36]. Bim is one of the proapoptotic BH3-only proteins in Bcl-2 family. Bim up-regulation triggers cytochrome c release from mitochondria, thereby induces the formation of the apoptosome and the activation of its effector caspase [37]. Previous studies have demonstrated that Bim plays important roles in paclitaxol therapy,
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Fig. 9 The effects of chemical chaperone on rhein-induced FOXO activation, CHOP, Bim expression and caspase cleavage. a MCF7 cells were transfected with FOXO-responsive luciferase construct and Renilla luciferase reporter plasmid (pRL-TK), followed by treating with or without 5 mM 4-PBA and 100 lM rhein for 24 h and detecting the dual-luciferase activity. The FOXO tranactivation
activity was normalized against the renilla luciferase activity. The relative FOXO transactivation activity was plotted. Bars SD. b MCF7 cells were treated with or without 100 lM rhein or 5 mM 4-PBA for 24 h, followed by western blot analysis of GRP78, CHOP, Bim expression, Akt phosphorylation and caspase cleavage. A representative of three experiments was shown
but not in cisplatin treatment. In addition, Bim-targeted drugs include Bcr/Abl inhibitors and EGFR inhibitors [38]. Our current study demonstrates that Bim may contribute, at least in part, to the induction of cancer cell apoptosis by rhein. Hence, Bim down-regulation may leads to resistance to this agent. Both CHOP and FOXO3a may regulate Bim expression [39]. Indeed, we find that rhein up-regulates FOXO transactivation activity. The protein kinase Akt can phosphorylate and inactivate FOXO [24, 25]. We also find that rhein inhibits Akt and FOXO3a phosphorylation. Overexpression of constitutively active Akt can rescue the decrease in both Akt and FOXO3a phosphorylation, and attenuate rheininduced Bim expression and caspase cleavage. These data suggest that inhibition of Akt-FOXO axis may contribute, in part, to up-regulation of pro-apoptotic Bim. Other factors such as CHOP may also be involved in the induction of Bim. FOXO3a knockdown significantly compromised rhein-induced Bim expression and apoptosis. The ability of chemical chaperone to relieve rhein-induced FOXO activation and Bim expression suggests that ER stress may be attributable, at least in part, to these pro-apoptotic events induced by rhein. Meanwhile, the chemical chaperone does not relieve the inhibition of Akt phosphorylation by rhein. Thus, the induction of FOXO activity, Bim expression and apoptosis by rhein may involve, but not limited to, two mechanisms including inhibition of Akt and induction of ER stress. Previous studies demonstrate that ER stress may lead to Akt phosphorylation, which is dependent on GRP78
[40, 41]. The failure to induce Akt phosphorylation by rhein may be due to the inhibition of ER stress-induced GRP78 expression. It is less likely that the induction of ER stress is a consequence of Akt inhibition, since neither Akt inhibitor nor PI3K inhibitor induces CHOP expression and eIF2a phosphorylation. Bim may be one of key nodes for mediation of rhein-induced apoptosis. Preclinical studies demonstrate that rhein and its derivatives can induce apoptosis, suppress proliferation and angiogenesis in vitro and in vivo [1–7]. A wide range of concentrations of rhein (30–180 lM) have been used in previous studies with various efficacy [1–4, 29–31]. Our current study shows that, upon treatment with rhein for 72 h, the mean IC50 values are 34.5 and 37.8 lM for HepG2 and MCF-7 cells, respectively. The bioavailability of rhein may be a key determinant of its effectiveness in vivo. Phamacokinetic study in rat model demonstrates that a peak value of 149.7 lM in plasma can be reached after single oral administration of 70 mg/kg rhein [42]. In human, the maximal plasma concentration of approximately 35.9 lM was detected after single oral administration of 200 mg rhein [43]. Thus, the concentrations of rhein in the current study may have in vivo relevance. After treatment with rhein for 48 or 72 h, the IC50 for MCF-10A, an untransformed breast cell line, was almost two-fold of that for MCF-7 and HepG2 cells. These data suggest a narrow treatment window between the treatment dose and toxicity dose, which may impede the clinical application of rhein. It warrants further research to determine if chemical modification, enhancement of solubility and bioavailability, and targeted or intravenous delivery can lower the
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treatment dose of rhein thereby increasing its anticancer effect and avoiding potential adverse effects in vivo. Acknowledgments We thank for the support from National Natural Science Foundation of China (No. 81001587). Conflict of interest
The authors declare no conflicts of interest.
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