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JNK-dependent mitochondrial pathway
Cancer Letters 298 (2010) 222–230
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Icariin, a natural flavonol glycoside, induces apoptosis in human hepatoma SMMC-7721 cells via a ROS/JNK-dependent mitochondrial pathway Songgang Li a,1, Ping Dong a,1, Jianwei Wang b, Jie Zhang c, Jun Gu a, Xiangsong Wu d, Wenguang Wu d, Xiaozhou Fei d, Zhiping Zhang d, Yong Wang d, Zhiwei Quan a, Yingbin Liu a,d,* a
Department of General Surgery, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, 1665 Kongjiang Road, Shanghai 200092, China Department of Oncological Surgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, 88 Jiefang Road, Hangzhou 310009, China c Department of General Surgery, Chongming Branch, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, 25 Nanmen Road, Chongming County, Shanghai 202150, China d Department of General Surgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, 88 Jiefang Road, Hangzhou 310009, China b
a r t i c l e
i n f o
Article history: Received 29 March 2010 Received in revised form 4 July 2010 Accepted 7 July 2010
Keywords: Hepatoma Icariin SMMC-7721 cells Apoptosis Reactive oxygen species c-Jun N-terminal kinase
a b s t r a c t In this study, the anticancer effect of icariin, a natural flavonol glycoside, against human hepatoma SMMC-7721 cells and the underlying mechanisms were investigated. Icariin triggered the mitochondrial/caspase apoptotic pathway indicated by enhanced Bax-toBcl-2 ratio, loss of mitochondrial membrane potential, cytochrome c release, and caspase cascade. Moreover, icariin induced a sustained activation of the phosphorylation of c-Jun N-terminal kinase (JNK) but not p38 and ERK1/2, and SP600125 (an inhibitor of JNK) almost reversed icariin-induced apoptosis in SMMC-7721 cells. In addition, icariin provoked the generation of reactive oxygen species (ROS) in SMMC-7721 cells, while the antioxidant N-acetyl cysteine almost completely blocked icariin-induced JNK activation and apoptosis. Taken together, these findings suggest that icariin induces apoptosis through a ROS/JNK-dependent mitochondrial pathway. Ó 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Hepatocellular carcinoma (HCC) is the third largest cause of cancer-related death behind only lung and colon cancers and is one of the commonest malignancies with >500,000 new tumors diagnosed annually [1,2]. It is one of the most aggressive human malignancies with extremely poor prognosis. A large retrospective populationbased study, including >7000 unselected USA patients, showed that the 5 year survival rate remained very poor
* Corresponding author at: Department of General Surgery, Xinhua Hospital, Affiliated to School of Medicine, Shanghai Jiao Tong University, 1665 Kongjiang Road, Shanghai 200092, China. Tel./fax: +86 21 65793206. E-mail address: [email protected] (Y. Liu). 1 These authors contributed equally to this work. This work was done in Shanghai Jiao Tong University. 0304-3835/$ - see front matter Ó 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2010.07.009
(2% in the period 1977–1981 and 5% in 1992–1996) [3]. The incidence of HCC is increasing in the United States and Europe [4]. Thus, HCC is becoming a major health problem worldwide. Usually, HCC is treated by surgical resection or liver transplantation, with curative options for the patients when the disease is diagnosed at an early stage. However, approximately 70% of patients are inoperable because of tumor metastasis or liver cirrhosis [5]. The current therapeutic options for HCC are not very effective because it is resistant to chemotherapy, thus novel therapeutic strategies are needed to decrease the incidence and severity associated with this cancer. Plant-derived natural products occupy a very important position in the area of cancer chemotherapy. Molecules such as vincristine, vinblastine, paclitaxel, camptothecin derivatives, epipodophyllotoxin, etc. are invaluable contributions of nature to modern medicine. However, the quest to find out novel therapeutic compounds for cancer
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treatment and management is a never-ending venture. Flavonoids, are plant polyphenols found in vegetables, fruit, and beverages of plant origin that is well known for their physiological antipyretic, analgesic, and anti-inflammatory activities [6]. The anti-tumor activity of flavonoid glycosides has recently attracted much attention [7,8]. In the present study, we found that icariin, a flavonol glycoside derived from Herba Epimedii, showed a strong growth inhibitory effect against three human hepatocellular carcinoma cell lines (SMMC-7721, Bel-7402, and HepG2). Therefore, we investigated both in vitro and in vivo anticancer activities and the mechanism of action of icariin in SMMC-7721 cells. Icariin is a prenylated flavonol glycoside derived from the medical plant Herba Epimedii, which exhibits a variety of pharmacological activities including tonic, aphrodisiac, antirheumatic, antidepressant, cardiovascular protective and immunomodulatory activities [9–11]. It has been previously demonstrated that icariin exerts potent osteogenic effect via activation of bone morphogenetic protein signaling [12]. Previous studies have demonstrated that icariin facilitates the directional differentiation of murine embryonic stem cells into cardiomyocytes in vitro [13,14]. Recently, Yang et al. have reported that icariin exerts antiproliferative efficacy on HepG2 bearing nude mice model [15], however, the exact mechanism of apoptosis inducing effect of icariin on human hepatoma cells remains unclear. In the present study, we demonstrate that icariin is a potent agent against human hepatoma cells both in vitro and in vivo, and reactive oxygen species (ROS)/c-Jun N-terminal kinase (JNK)-dependent mitochondrial pathway might be involved in the signaling of icariin-induced apoptosis. Our data provide the molecular theoretical basis for clinical application of icariin in patients with HCC.
2. Materials and methods 2.1. Cell culture SMMC-7721, Bel-7402 and L-02 cells were maintained in RPMI 1640 medium (2 g/l glucose; Invitrogen), and HepG2 cells were maintained in HG-Dulbecco’s modified Eagle’s medium (4.5 g/l glucose; Invitrogen); both media were supplemented with 10% fetal calf serum (Gibro, Invitrogen) plus 2 mM glutamine and 50 U/ml penicillin. All of the cell lines were purchased from the Shanghai Institute of Cell Biology (Shanghai, China), and they were grown at 37 °C in a 5% (v/v) CO2 atmosphere. Primary hepatocytes were isolated from mice by the modified two-step perfusion method [16]. In brief, the livers of the mice were first perfused in situ via the portal vein with Ca2+ and Mg2+ free Hank’s balanced salt solution (HBSS) supplemented with 0.5 mM EGTA and 25 mM HEPES at 37 °C until the blood in the organ was completely removed. Then, the buffer was replaced with 0.1% collagenase IV solution in HBSS. After a few minutes of perfusion, the liver was excised rapidly from the body cavity and dispersed into cold HBSS. The cell suspension generated was filtered through a 100 gauze mesh. After washing twice to remove dead cells and debris at 50g for 2 min, the hepa-
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tocytes were resuspended in RPMI 1640 medium containing 10% fetal calf serum and found to be 92% viable by trypan blue dye exclusion. The primary hepatocytes were used immediately for cytotoxic assay. 2.2. Mice Specific pathogen-free, eight- to ten-week-old female BALB/c and nude mice (BALB/c background) were purchased from National Rodent Laboratory Animal Resource (Shanghai, China). Animal welfare and experimental procedures were carried out strictly in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, the United States) and the related ethical regulations of our university. All efforts were made to minimize animals’ suffering and to reduce the number of animals used. 2.3. Drugs and reagents Icariin (2-(40 -methoxylphenyl)-3-rhamnosido-5-hydroxyl-7-glucosido-8-(30 -methyl-2-butylenyl)-4-chromanone, Fig. 1A, purity >99%, purchased from Xi’an Tongjiang Biotechnology Co., Ltd., Xi’an, China) was dissolved at a concentration of 20 mM in 100% DMSO as a stock solution, stored at 20 °C, and diluted with medium before each experiment. The final DMSO concentration did not exceed 0.1% throughout the study (all the control groups are composed of 0.1% DMSO). 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), N-acetyl cysteine (NAC) and collagenase IV were purchased from Sigma Chemical Co. (St. Louis, MO). The 5,50 ,6,60 -tetrachloro1,10 ,3,30 -tetraethylbenzimidazolcarbocyanine iodide (JC-1) and the 5-(and-6)-darboxy-20 -70 -dichlorofluorescin diacetate (carboxy-DCFDA) were purchased from Invitrogen (Carlsbad, CA). Annexin V-FITC (fluorescein isothiocyanate)/PI (propidium iodide) kit was purchased from BD Biosciences (San Jose, CA). The caspase-3 specific inhibitor Z-DEVD-FMK, JNK inhibitor SP600125, p38 inhibitor SB203580 and ERK1/2 inhibitor PD98059 were purchased from Calbiochem (San Diego, CA). Anti-caspase-3, anti-cleaved caspase-3, anti-caspase-8, anti-cleaved caspase-9, anti-poly(ADP-ribose) polymerase 1 (PARP1), anti-ERK1/2, anti-p-ERK1/2, anti-p38, anti-p-p38, antiJNK and anti-p-JNK were purchased from Cell Signaling Technology (Beverly, MA). Anti-Bax, anti-X-linked inhibitor of apoptosis protein (XIAP), anti-cytochrome c, anticytochrome c oxidase subunit IV (COX IV), and anti-a Tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Bcl-2 was purchased from BD Pharmingen (San Diego, CA). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). 2.4. HPLC analysis of the purity of icariin The test was applied on the Waters 600 pump, with a 2487 ultraviolet–vis detector, an online degasser, and a 5 lL injection loop. Icariin was applied to C18 column (Kromasil, 4.6 250 mm, 5 lm) and eluted with CH3OH/ H2O (45/55, v/v). The effluents were detected under 275 nm. Column temperature was set up at 25 °C and the
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Fig. 1. Icariin killed various human hepatoma cells in vitro. (A) HPLC analysis of the purity of icariin used in the present study. Insert: The chemical structure of icariin. (B) Icariin time- and dose-dependently inhibited the viability of human hepatoma SMMC-7721 cells. Cells were treated with different concentrations of icariin for 24, 48 and 72 h, respectively before cytotoxic analysis. (C) Two human hepatoma cell lines (Bel-7402 and HepG2), human normal hepatocyte line L-02 cells and primary murine hepatocytes were treated with 5–40 lM icariin for 24 h, then cytotoxicity was analyzed by MTT assay. Data represent the mean ± SEM of three different experiments with triplicate sets in each assay. P < 0.05, P < 0.01 vs. drug-untreated group.
flow rate was 1 ml/min. The peak areas and the relative contents were calculated using EmpowerTM workstation.
ProteoExtract Cytosol/Mitochondria Fractionation Kit (Merck Bioscience, Bad Soden, Germany) according to the procedures provided by the manufacturer.
2.5. Cytotoxicity assay 2.8. Western blot The cytotoxic activity of icariin was measured using the MTT assay. After cells were treated with icariin for indicated time in 96-well plates, MTT solution (5 mg/ml in RPMI 1640 medium; Sigma–Aldrich) was added (10 ll/ well), and plates were incubated for a further 4 h at 37 °C. The purple formazan crystals were dissolved in 100 ll of DMSO. After 5 min, the plates were read on an automated microplate spectrophotometer (Sunrise, Tecan, Austria) at 570 nm. Assays were performed in triplicate on three independent experiments. 2.6. Cell apoptosis assay The cells were stained with Annexin V-FITC/PI and measured by FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). Annexin V+/PI cells were considered as apoptotic cells. 2.7. Subcellular fractionation The proteins in the SMMC-7721 cells were separated into cytosolic and mitochondrial fractions using the
Proteins were extracted in lysis buffer (30 mmol/L Tris, pH 7.5, 150 mmol/L sodium chloride, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L sodium orthovanadate, 1% Nonidet P-40, 10% glycerol, and phosphatase and protease inhibitors). The proteins were then separated by SDS–PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes (Roche Applied Science). The membranes were probed with antibodies overnight at 4 °C, and then incubated with a horse radish peroxidase-coupled secondary antibody. Detection was performed using a LumiGLO chemiluminescent substrate system (KPL, Guildford, UK). Band intensity was quantified by BandScan software (Glyko, Novato, CA). 2.9. Cell mitochondrial membrane potential assay SMMC-7721 cells were treated with or without 10 lM icariin for 6–24 h, then the cells were harvested and the disruption of mitochondrial transmembrane potential was measured using fluorochrome dye JC-1 by flow cytometry as previously reported [17].
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2.10. Detection of intracellular ROS The production of intracellular ROS was measured in the SMMC-7721 cell line using the oxidation-sensitive fluorescent dye carboxy-DCFDA [18]. 2.11. Measurement of in vivo anti-tumor activity Tumors were established by injection of 5 106 SMMC7721 cells s.c. into the armpit of 4- to 5-week old BALB/c female athymic mice (National Rodent Laboratory Animal Resource, Shanghai, China). Treatments were initiated when tumors reached a mean group size of approximately 100 mm3. Tumor volume (cubic millimeters) was measured with calipers, and it was calculated as (W2 L)/2, where W is the width and L is the length of tumor. Icariin dissolved in ethanol/0.9% sterile sodium chloride solution (1:9, vol) was administered i.p. to athymic mice every day for 20 days. Tumor volume were recorded every 2 days until animals were sacrificed.
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biochem, USA) according to the manufacturer’s instructions [19]. The quantitation of DNA fragmentation was determined by photometric enzyme immunoassay using the Cell Death Detection ELISAplus kit (Roche, Penzberg, Germany) following the manufacturer’s instructions [20]. Activation of caspase-3 was determined using the caspase-3 colorimetric assay kit (MBL International, Woburn, MA) following the manufacturer’s instructions. [20]. 2.14. Statistical analysis Data are expressed as mean ± SEM. Student’s t test and one-way ANOVA test were used for statistical analyses of the data. All statistical analyses were conducted using SPSS 10.0 statistical software (SPSS, Chicago, IL, USA). Cases in which P values of <0.05 or <0.01 were considered statistically significant. 3. Results 3.1. Icariin kills human hepatoma cells in vitro
2.12. TUNEL assay Paraffin-embedded hepatoma sections were deparaffinized and rehydrated to PBS. Samples were treated with 10 lg/ml proteinase K (ICN) in 10 mM Tris–HCl (pH 7.4) for 15 min at room temperature. Sections were washed three times in PBS solution after proteinase treatment. TUNEL labeling was performed with the In Situ Cell Death detection kit with fluorescein (Roche, Mannheim, Germany) according to manufacturer recommendations. The slides were observed under the fluorescence microscope (Zeiss, Germany), and the images were captured by digital camera and analyzed by Axiovision software (Germany). 2.13. DNA fragmentation and caspase-3 activity assay The qualitation of DNA fragmentation was measured using the Suicide-TrackTM DNA Ladder Isolation Kit (Cal-
As shown in Fig. 1B, human hepatoma SMMC-7721 cells exhibited time- and dose-dependent sensitivity to icariin, with IC50 values at 24 h (the concentration of drug inhibiting 50% of cells) around 10 lM. In addition, icariin also killed other human hepatoma cell lines such as Bel-7402, and HepG2 cells in a dose-dependent manner (Fig. 1C). However, icariin at the concentration up to 40 lM did not affect the survival of normal human liver cell line L-02 cells as well as primary murine hepatocytes (Fig. 1C). The results suggest that icariin has promising antihepatoma activity with low cytotoxic effect on normal hepatocytes. 3.2. Icariin induces significant apoptosis in human hepatoma cells but not normal hepatocytes Compared with control group, icariin dramatically triggered apoptosis in human hepatoma SMMC-7721 cells (Fig. 2A) or Bel-7742 cells (Supplemental Fig. 1A) in a dose-dependent manner. To SMMC-7721 cells, approximately 40% of the cells were at early apoptosis in 10 lM icariin-treated group, compared with 1.2% of vehicle treated group. It was noted that 5–20 lM icariin did not induce significant apoptosis in human normal hepatocyte line L-02 cells (Supplemental Fig. 1B). To further confirm whether icariin-induced cell apoptotic death was indeed
Fig. 2. Icariin induced caspase-3-dependent apoptosis in human hepatoma SMMC-7721 cells. (A) Apoptosis in SMMC-7721 cells was assessed after 12 h of treatment with 5–20 lM icariin by Annexin V-FITC/PI binding and measured by flow cytometry analysis. Numbers indicate the percentage of cells in each quadrant. P < 0.01 vs. drug-untreated group. (B) SMMC-7721 cells were treated with 50 lM Z-DEVD-FMK, a caspase-3 specific inhibitor, for 1 h before treatment with 10 lM icariin for 12 h. Apoptosis was assessed by flow cytometry as mentioned above. P < 0.01.
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caspase-3-dependent, a caspase-3 specific inhibitor Z-DEVD-FMK was used. As indicated in Fig. 2B, the addition of Z-DEVD-FMK markedly reduced the apoptotic response from 40.5% to 8.4%. 3.3. Icariin triggers mitochondrial-related apoptosis in human hepatoma SMMC-7721 cells When SMMC-7721 cells were treated with 10 lM icariin for different time, significant proteolytic cleavage of procaspase-9 and procaspase-3 was detected using Western blot (Fig. 3A). By contrast, no cleavage of caspase-8 was found in the icariin-treated cells (Supplemental Fig. 2). Consistently, cytochrome c was released from mitochondria to cytosol (Fig. 3B), indicating activation of the intrinsic apoptosis pathway. Treatment of SMMC-7721 cells with 10 lM icariin for 24 h induced significant cleaved PARP1 protein expression (Fig. 3A). XIAP is able to inhibit activation of caspase-3 to protect cells from apoptosis. As shown in Fig. 3A, icariin decreased XIAP protein levels in a dose-dependent manner. To assess the role of mitochondria in icariin-induced cell death, we tested whether icariin caused a loss of mitochondrial membrane potential. Compared with the corresponding control, icariin caused an obvious decrease of mitochondrial membrane potential in SMMC-7721 cells in a time-dependent manner (Fig. 3C and D). The proto-oncoprotein Bcl-2 is a powerful antagonist of the mitochondrial pathway of apoptosis initiated by a variety of extra- and intracellular stresses. Therefore, we examined whether icariin could alter the balance between proapoptotic Bax and antiapoptotic Bcl-2 proteins at the mitochondrial membrane. Exposure to icariin resulted in decrease of Bcl-2 and increase of Bax (Fig. 3E), with an increase in Bax/Bcl-2 ratio (Fig. 3F). 3.4. JNK activation contributes to icariin-induced apoptosis in human hepatoma SMMC-7721 cells There is accumulating evidence that small compound-triggered apoptosis has been always associated with activation of mitogen-activated protein kinases (MAPK) [20–22], we next examined the effect of icariin
on MAPK signaling. As shown in Fig. 4A, the phosphorylation of JNK was gradually increased after the icariin treatment, but the phosphorylations of p38 and the extracellular signal-regulated protein kinase (ERK1/ 2) were hardly affected. To further analyze whether JNK activation plays a role in icariin-induced apoptosis, the effect of SP600125, a pharmacological inhibitor of JNK activation, were examined. Addition of 10 lM SP600125 remarkably suppressed JNK phosphorylation induced by 10 lM icariin, as observed after 24 h (Fig. 4D). As shown in Fig. 4B and C, SP600125 reversed the proapoptotic effect of icariin in SMMC-7721 cells assessed by Annexin V/PI binding assay. However, p38 inhibitor SB203580 or ERK1/2 inhibitor PD98059 did not blocked the icariin-induced apoptosis in SMMC-7721 cells (Supplemental Fig. 3). Moreover, SP600125 reduced Bax upregulation and increased Bcl-2 downregulation in response to icariin as well as resulted in reduction of caspase-3 activation and PARP cleavage (Fig. 4D and E). These results indicate the activation of JNK but not p38 or ERK1/2 plays a critical role in icariin-induced apoptosis in human hepatoma SMMC-7721 cells.
3.5. ROS is required for icariin-induced apoptosis in human hepatoma SMMC7721 cells Since a loss of mitochondrial membrane potential is associated with the generation of ROS [23], we detected the level of ROS in SMMC-7721 cells treated with icariin. As shown in Fig. 5A, the level of ROS in cells treated with icariin was increased in a time-dependent manner and pretreatment with antioxidant NAC significantly inhibited ROS generation. To illustrate the role of ROS in icariin-induced apoptosis, SMMC-7721 cells were treated with icariin in the presence or absence of antioxidant NAC. As shown in Fig. 5B, the proportion of apoptotic cells was changed from 38.4% to 12.2%, indicating that addition of antioxidant NAC almost completely reversed apoptotic cell death caused by icariin. Another antioxidant vitamin C also completely blocked the apoptosis induced by icariin in SMMC-7721 cells (Supplemental Fig. 3). In addition, NAC almost blocked icariin-induced decrease of Bcl-2, increase of Bax, activation of caspase-3, and cleavage of PARP1 (Fig. 5D and E). Moreover, the activation
Fig. 3. Icariin triggered mitochondrial-related apoptosis in human hepatoma SMMC-7721 cells. (A) SMMC-7721 cells were treated with or without 10 lM icariin for indicated time, and then cells were harvested and lysed. Caspase-9, caspase-3, PARP1 and XIAP were analyzed by Western blot. The data shown here are one of three different experiments. (B) SMMC-7721 cells were treated with or without 10 lM icariin for indicated time, then cells were harvested and separated into cytosolic and mitochondrial fractions using the commercial fractionation kit. The expressions of cytochrome c (Cyt c) in cytosol and mitochondria were analyzed by Western blot. The data shown here are one of three different experiments. (C) SMMC-7721 cells were treated with or without 10 lM icariin for indicated time, then cells were harvested and the disruption of mitochondrial transmembrane potential was measured using fluorochrome dye JC-1 by flow cytometry. The histogram shown here is one of three different experiments. (D) Quantification of cells with low mitochondrial transmembrane potential was depicted in the graph. n = 3, P < 0.01 vs. drug-untreated group. (E) Cells were treated with 10 lM icariin for indicated time, and whole-cell lysates were collected and immunoblotted with Bcl-2 and Bax. (F) The densitometric analyses of expression of Bcl-2 and Bax relative to the untreated control from three independent experiments. P < 0.05, P < 0.01 vs. drug-untreated group.
S. Li et al. / Cancer Letters 298 (2010) 222–230 of JNK in SMMC-7721 cells treated with icariin could be almost abolished by NAC (Fig. 5F). These observations suggest icariin-induced ROS generation is an early event that induces JNK activation and triggers mitochondrial apoptotic pathways in SMMC-7721 cells. 3.6. Icariin inhibits tumor growth in xenografted nude mice model by causing apoptotic cell death To further evaluate whether icariin had an effect to inhibit tumor growth in vivo, we measured the tumor volume in a xenograft tumor model in which SMMC-7721 cells were injected s.c. into nude mice. In
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the pre-experiment, we found that the drug concentration in the serum of mice with intraperitoneally administered 30 mg/kg of icariin daily for five days reached a peak at about 9.6 lM by a high performance liquid chromatography assay, which just fell within the range of the drug concentrations (5–20 lM) in vitro. So 15, 30 and 60 mg/kg of icariin were selected for in vivo experiments. When transplant tumors reached a mean group size of approximately 100 mm3, mice were treated every day for 20 days with various dose of icariin (i.p., 15, 30, and 60 mg/kg). Compared with the control group, icariin showed significant inhibitory effect on tumor volume, and the inhibitory rates on tumor volume at 20th day caused by 15, 30, and 60 mg/kg icariin were 38.7, 54.7, and 69.9%, respectively
Fig. 4. Involvement of JNK activation in icariin-induced apoptosis in human hepatoma SMMC-7721 cells. (A) Western blot of whole-cell extracts of SMMC7721 cells analyzed for total and phosphorylated MAPK members (JNK, p38, ERK) after treatment with 10 lM icariin for indicated time. P < 0.05, P < 0.01 vs. drug-untreated group. (B and C) SMMC-7721 cells were treated with 10 lM SP600125, a pharmacological inhibitor of JNK activation, for 1 h before treatment with 10 lM icariin for 12 h. Apoptosis was assessed by flow cytometry as mentioned above. P < 0.01. (D and E) SMMC-7721 cells were treated with 10 lM SP600125 for 1 h before treatment with 10 lM icariin for 24 h. The whole-cell extracts were assessed by Western blot. P < 0.01.
Fig. 5. Involvement of ROS in icariin-induced apoptosis in human hepatoma SMMC-7721 cells. (A) Icariin time-dependently increased ROS levels in SMMC7721 cells. Cells were pretreated with 5 mM NAC for 2 h before treatment with or without 10 lM icariin for indicated time, and then the intracellular levels of ROS were detected. P < 0.05, P < 0.01 vs. icariin-treated group. (B) Icariin-induced apoptosis could be almost reversed by antioxidant NAC. Cells were pretreated with 5 mM NAC for 2 h before treatment with or without 10 lM icariin for 12 h, then apoptosis was assessed by Annexin V-FITC/PI binding. (C) The statistical data of apoptosis from three independent experiments. P < 0.01. (D–F) SMMC-7721 cells, with or without pretreatment of 5 mM NAC for 2 h, were incubated with 10 lM icariin for 24 h, and whole-cell lysates were collected and immunoblotted with Bcl-2, Bax, caspase-3, cleaved caspase-3, and PARP1 (D); JNK and p-JNK (F). P < 0.05, P < 0.01.
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Fig. 6. Icariin suppressed tumor growth in mouse xenografted model through inducing apoptosis of tumor cells. Nude mice with SMMC-7721 transplant tumor were i.p. treated with 15, 30, or 60 mg/kg icariin every day for 20 days. (A) Transplant tumor volume of nude mice after the treatment of icariin. The number of mice in each group is eight. P < 0.05, P < 0.01 vs. control group (drug-untreated group) (B and C). At 12th day after the treatment of icariin, the transplant tumors were obtained from various groups of mice and subjected to apoptosis assay. Apoptosis was evaluated by enzyme-linked immunosorbent assay (B) and caspase-3 activation (C). n = 3, P < 0.05, P < 0.01 vs. control group. (D) DNA fragmentation of apoptotic cells in transplant tumor sections at 12th day after the treatment of icariin revealed by agarose gel electrophoresis. In icariin-treated group, the specific DNA cleavage became evident in electrophoresis analysis as a typical ladder pattern. The data shown here are one of three different experiments. (E) In situ detection of apoptotic cells in tumor sections at 12th day after the treatment of icariin by fluorescence microscopy using the TUNEL assay. Up: Bright-field image; Down: TUNEL reaction green fluorescence. Scale bar: 100 lm.
(Fig. 6A). To determine whether the reduced tumor growth rate following icariin treatment could be explained by the induction of apoptosis, we used various methods to characterize apoptosis in tumor sections. Icariin dose-dependently induced significant increase in DNA fragmentation (Fig. 6B) and caspase-3 activity (Fig. 6C) in tumor sections. Consistently, the formation of DNA ladder was evident in transplanted hepatoma cells of 60 mg/kg icariin-treated mice (Fig. 6D). As seen in Fig. 6E, TUNEL assay showed explicitly that apoptotic cells were induced by icariin in hepatoma tissue, whereas few TUNEL positive cells were observed in tumors treated vehicle alone. The results from different apoptosis assays reveal significant features of apoptosis, which strongly suggest that icariin-mediated inhibition of tumor growth in hepatoma-bearing mice is closely correlated with the enhanced apoptosis in cancer cells. In addition, there was no significant difference in the weight and cell numbers of lymphoid tissues (thymus, spleen and lymph nodes) between the mice intraperitoneally injected with icariin (30 mg/kg) daily for 7 days and the mice treated with normal saline (data not shown), suggesting icariin was not toxic for administration to mice in vivo.
4. Discussion HCC is a rapidly fatal disease, with a life expectancy of about 6 months from the time of the diagnosis. Therapeutic strategies employed to date have significantly improved the prognosis for patients with unresectable HCC [24]. This emphasizes the need for investigating the molecular mechanisms responsible for HCC development and seeking effective and non-cytotoxic chemical agents for chemoprevention and treatment. However, few synthetic antineoplastic compounds have been identified to be effective for the treatment of this disease. In this respect, more and more researchers paid much attention to natural active compounds for cancer chemoprevention and treatment [6]. In the present study, icariin, which was previously found to exert antineoplastic effect, was clearly demonstrated to kill all three human hepatoma cell lines,
such as SMMC-7721, Bel-7402, HepG2. However, it was interesting that icariin at the concentrations mentioned above hardly affected the viability of the benign non-tumor human hepatocyte L-02 cells as well as primary murine hepatocytes, suggesting a selective anti-tumor action of icariin to some degree. Similarly, Yang et al. had previously reported that gambogic acid could selectively induce apoptosis of human tumor cells while had relatively less effect on human normal embryon cells due to the higher distribution and longer retention time of gambogic acid in tumor cells compared to the normal cells [25]. The detailed mechanisms of icariin killing hepatoma cells but not benign cells need further investigation. Mitochondria play a pivotal role in the signal transduction of apoptosis [26–28]. The observation of icariin-mediated activation of caspase-9, caspase-3, and subsequent cleavage of PARP1, as well as the result that a caspase-3 specific inhibitor Z-DEVD-FMK almost completely blocked icariin-induced apoptosis in SMMC-7721 cells, suggesting that mitochondrial-mediated caspase cascade pathway plays a very important role in icariin-induced apoptosis. Furthermore, the icariin-caused downregulation of XIAP, one of the inhibitors of apoptosis proteins family [29,30] was detected in SMMC-7721 cells. Because XIAP had been reported to protect cells from apoptosis through inhibiting caspase-3 activation, the downregulation of XIAP provided an additional documentation that icariin-induced anti-tumor was related to activate caspase cascade. MAPKs are mediators of cellular responses to extracellular signals, including ERK1/2, JNK, and p38. The understanding of MAPK signaling may provide the explanation for chemical-triggered apoptotic processes. ERK1/2 is generally associated with proliferation and growth. In
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contrast, JNK and p38 MAPKs are induced by stress responses and closely associated with cell death [31]. There are a few reports about MAPKs related to the effects of icariin [32,33]. To elucidate the precise mechanism involved in the icariin-induced cell death, the effects of icariin on MAPK activation were examined. We found that icariin treatment induced a sustained activation of JNK in SMMC-7721 cells, and the phosphorylation was dependent on ROS generation since it was abrogated by addition of NAC. Furthermore, JNK activation was responsible for icariin-induced Bcl-2/Bax modulation, caspase-3 activation, PARP1 cleavage and consequent apoptosis, as shown by the reduction in these events when the JNK inhibitor SP600125 was present. To determine whether inhibition of JNK activity alone contributes to the reduced icariin-induced apoptosis, we assessed whether the other two MAPK members, p38 and ERK1/2 were involved. First, the activations of p38 and ERK1/2 were not significantly affected by the treatment of icariin from 6 h to 24 h. Second, unlike JNK inhibitor SP600125, p38 inhibitor SB203580 or ERK1/ 2 inhibitor PD98059 did not reversed the icariin-induced apoptosis in SMMC-7721 cells. These results suggest that JNK, but not p38 and ERK1/2, participates in icariin-induced apoptosis. ROS normally exist in all aerobic cells in balance with biochemical antioxidants. Oxidative stress occurs when this critical balance is disrupted because of excess ROS production and/or antioxidant depletion [34]. Evidence is accumulating which indicates that many chemotherapeutic agents may be selectively toxic to tumor cells because they increase oxidant stress and enhance these already stressed cells beyond their limit [35–37]. Cytotoxic ROS signaling appears to be triggered by the activation of the mitochondrial-dependent cell death pathway through activation of MAPK pathways and the proapoptotic protein Bax, with subsequent mitochondrial membrane permeabilization and cell death [38]. Our results showed that icariininduced apoptosis was initiated by the generation of ROS, which was followed by disruption of the mitochondrial membrane potential and release of cytochrome c into the cytosol leading to the activation of caspase-9/-3 cascade. The antioxidant NAC pretreatment not only blocked ROS generation but also offered significant protection against icariin-induced Bcl-2/Bax modulation, phosphorylation of JNK, caspase-3 activation, PARP1 cleavage and apoptosis in SMMC-7721 cells, suggesting ROS generation is an early event in the process of icariin-induced apoptosis. Apoptosis is a major mechanism to eliminate cancer cells. Many chemopreventive agents appear to target signaling intermediates in apoptosis-inducing pathways. For example, capsaicin, a cytotoxic alkaloid from various species of Capsicum, induced apoptosis of human pancreatic cancer cells through ROS generation and mitochondrial death pathway [39]. A recent report demonstrated that curcumin, a natural biologically active compound extracted from rhizomes of Curcuma species, initiated apoptosis of human cervical carcinoma cells through inhibiting human telomerase reverse transcriptase [40]. Thus, targeting apoptosis pathways in premalignant and malignant cells may be an effective strategy for cancer prevention and treatment [41]. In the present study, we conclude that icariin induces
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Fig. 7. Overview of pathways for icariin-induced apoptosis in human hepatoma cell line SMMC-7721. NAC, N-acetyl cysteine; JNK, c-Jun Nterminal kinase; SP600125, a pharmacological inhibitor of JNK activation; PARP, poly(ADP-ribose) polymerase.
human hepatoma SMMC-7721 cell apoptosis via ROS/JNKdependent mitochondrial pathway. Consistent with our results, Yang et al. had recently reported that icariin exerted anti-proliferative efficacy on HepG2 bearing nude mice through downregulating the expressions of both CD31 and Ki67 [15]. Moreover, hepatoma growth inhibition and induction of apoptosis in vivo were observed in the zenografted mice treated with 15–60 mg icariin/kg. These findings demonstrate that icariin is a potent compound against human hepatoma cells both in vitro and in vivo. Taken together, our results demonstrate that icariin-induced apoptosis in human hepatoma SMMC-7721 cells is mediated by the activation of mitochondrial death pathway that require ROS generation and JNK activation. Moreover, administration of icariin significantly suppressed the growth of hepatoma xenograft by inducing apoptosis in the tumor cells. Our studies thus provide a rationale for the development of icariin as chemotherapeutic agent against HCC in the clinical setting. Based on the results of the present study, the mechanisms by which icariin induces apoptosis in human hepatoma SMMC-7721 cells is summarized in Fig. 7. Conflict of interest The authors state no conflict of interest. Acknowledgments This study was supported by National Natural Science Foundation of China (Nos. 30872502, 30972918) and
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Icariin, a natural flavonol glycoside, induces apoptosis in human hepatoma SMMC-7721 cells via a ROS/JNK-dependent mitochondrial pathway Songgang Li a,1, Ping Dong a,1, Jianwei Wang b, Jie Zhang c, Jun Gu a, Xiangsong Wu d, Wenguang Wu d, Xiaozhou Fei d, Zhiping Zhang d, Yong Wang d, Zhiwei Quan a, Yingbin Liu a,d,* a
Department of General Surgery, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, 1665 Kongjiang Road, Shanghai 200092, China Department of Oncological Surgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, 88 Jiefang Road, Hangzhou 310009, China c Department of General Surgery, Chongming Branch, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, 25 Nanmen Road, Chongming County, Shanghai 202150, China d Department of General Surgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, 88 Jiefang Road, Hangzhou 310009, China b
a r t i c l e
i n f o
Article history: Received 29 March 2010 Received in revised form 4 July 2010 Accepted 7 July 2010
Keywords: Hepatoma Icariin SMMC-7721 cells Apoptosis Reactive oxygen species c-Jun N-terminal kinase
a b s t r a c t In this study, the anticancer effect of icariin, a natural flavonol glycoside, against human hepatoma SMMC-7721 cells and the underlying mechanisms were investigated. Icariin triggered the mitochondrial/caspase apoptotic pathway indicated by enhanced Bax-toBcl-2 ratio, loss of mitochondrial membrane potential, cytochrome c release, and caspase cascade. Moreover, icariin induced a sustained activation of the phosphorylation of c-Jun N-terminal kinase (JNK) but not p38 and ERK1/2, and SP600125 (an inhibitor of JNK) almost reversed icariin-induced apoptosis in SMMC-7721 cells. In addition, icariin provoked the generation of reactive oxygen species (ROS) in SMMC-7721 cells, while the antioxidant N-acetyl cysteine almost completely blocked icariin-induced JNK activation and apoptosis. Taken together, these findings suggest that icariin induces apoptosis through a ROS/JNK-dependent mitochondrial pathway. Ó 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Hepatocellular carcinoma (HCC) is the third largest cause of cancer-related death behind only lung and colon cancers and is one of the commonest malignancies with >500,000 new tumors diagnosed annually [1,2]. It is one of the most aggressive human malignancies with extremely poor prognosis. A large retrospective populationbased study, including >7000 unselected USA patients, showed that the 5 year survival rate remained very poor
* Corresponding author at: Department of General Surgery, Xinhua Hospital, Affiliated to School of Medicine, Shanghai Jiao Tong University, 1665 Kongjiang Road, Shanghai 200092, China. Tel./fax: +86 21 65793206. E-mail address: [email protected] (Y. Liu). 1 These authors contributed equally to this work. This work was done in Shanghai Jiao Tong University. 0304-3835/$ - see front matter Ó 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2010.07.009
(2% in the period 1977–1981 and 5% in 1992–1996) [3]. The incidence of HCC is increasing in the United States and Europe [4]. Thus, HCC is becoming a major health problem worldwide. Usually, HCC is treated by surgical resection or liver transplantation, with curative options for the patients when the disease is diagnosed at an early stage. However, approximately 70% of patients are inoperable because of tumor metastasis or liver cirrhosis [5]. The current therapeutic options for HCC are not very effective because it is resistant to chemotherapy, thus novel therapeutic strategies are needed to decrease the incidence and severity associated with this cancer. Plant-derived natural products occupy a very important position in the area of cancer chemotherapy. Molecules such as vincristine, vinblastine, paclitaxel, camptothecin derivatives, epipodophyllotoxin, etc. are invaluable contributions of nature to modern medicine. However, the quest to find out novel therapeutic compounds for cancer
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treatment and management is a never-ending venture. Flavonoids, are plant polyphenols found in vegetables, fruit, and beverages of plant origin that is well known for their physiological antipyretic, analgesic, and anti-inflammatory activities [6]. The anti-tumor activity of flavonoid glycosides has recently attracted much attention [7,8]. In the present study, we found that icariin, a flavonol glycoside derived from Herba Epimedii, showed a strong growth inhibitory effect against three human hepatocellular carcinoma cell lines (SMMC-7721, Bel-7402, and HepG2). Therefore, we investigated both in vitro and in vivo anticancer activities and the mechanism of action of icariin in SMMC-7721 cells. Icariin is a prenylated flavonol glycoside derived from the medical plant Herba Epimedii, which exhibits a variety of pharmacological activities including tonic, aphrodisiac, antirheumatic, antidepressant, cardiovascular protective and immunomodulatory activities [9–11]. It has been previously demonstrated that icariin exerts potent osteogenic effect via activation of bone morphogenetic protein signaling [12]. Previous studies have demonstrated that icariin facilitates the directional differentiation of murine embryonic stem cells into cardiomyocytes in vitro [13,14]. Recently, Yang et al. have reported that icariin exerts antiproliferative efficacy on HepG2 bearing nude mice model [15], however, the exact mechanism of apoptosis inducing effect of icariin on human hepatoma cells remains unclear. In the present study, we demonstrate that icariin is a potent agent against human hepatoma cells both in vitro and in vivo, and reactive oxygen species (ROS)/c-Jun N-terminal kinase (JNK)-dependent mitochondrial pathway might be involved in the signaling of icariin-induced apoptosis. Our data provide the molecular theoretical basis for clinical application of icariin in patients with HCC.
2. Materials and methods 2.1. Cell culture SMMC-7721, Bel-7402 and L-02 cells were maintained in RPMI 1640 medium (2 g/l glucose; Invitrogen), and HepG2 cells were maintained in HG-Dulbecco’s modified Eagle’s medium (4.5 g/l glucose; Invitrogen); both media were supplemented with 10% fetal calf serum (Gibro, Invitrogen) plus 2 mM glutamine and 50 U/ml penicillin. All of the cell lines were purchased from the Shanghai Institute of Cell Biology (Shanghai, China), and they were grown at 37 °C in a 5% (v/v) CO2 atmosphere. Primary hepatocytes were isolated from mice by the modified two-step perfusion method [16]. In brief, the livers of the mice were first perfused in situ via the portal vein with Ca2+ and Mg2+ free Hank’s balanced salt solution (HBSS) supplemented with 0.5 mM EGTA and 25 mM HEPES at 37 °C until the blood in the organ was completely removed. Then, the buffer was replaced with 0.1% collagenase IV solution in HBSS. After a few minutes of perfusion, the liver was excised rapidly from the body cavity and dispersed into cold HBSS. The cell suspension generated was filtered through a 100 gauze mesh. After washing twice to remove dead cells and debris at 50g for 2 min, the hepa-
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tocytes were resuspended in RPMI 1640 medium containing 10% fetal calf serum and found to be 92% viable by trypan blue dye exclusion. The primary hepatocytes were used immediately for cytotoxic assay. 2.2. Mice Specific pathogen-free, eight- to ten-week-old female BALB/c and nude mice (BALB/c background) were purchased from National Rodent Laboratory Animal Resource (Shanghai, China). Animal welfare and experimental procedures were carried out strictly in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, the United States) and the related ethical regulations of our university. All efforts were made to minimize animals’ suffering and to reduce the number of animals used. 2.3. Drugs and reagents Icariin (2-(40 -methoxylphenyl)-3-rhamnosido-5-hydroxyl-7-glucosido-8-(30 -methyl-2-butylenyl)-4-chromanone, Fig. 1A, purity >99%, purchased from Xi’an Tongjiang Biotechnology Co., Ltd., Xi’an, China) was dissolved at a concentration of 20 mM in 100% DMSO as a stock solution, stored at 20 °C, and diluted with medium before each experiment. The final DMSO concentration did not exceed 0.1% throughout the study (all the control groups are composed of 0.1% DMSO). 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), N-acetyl cysteine (NAC) and collagenase IV were purchased from Sigma Chemical Co. (St. Louis, MO). The 5,50 ,6,60 -tetrachloro1,10 ,3,30 -tetraethylbenzimidazolcarbocyanine iodide (JC-1) and the 5-(and-6)-darboxy-20 -70 -dichlorofluorescin diacetate (carboxy-DCFDA) were purchased from Invitrogen (Carlsbad, CA). Annexin V-FITC (fluorescein isothiocyanate)/PI (propidium iodide) kit was purchased from BD Biosciences (San Jose, CA). The caspase-3 specific inhibitor Z-DEVD-FMK, JNK inhibitor SP600125, p38 inhibitor SB203580 and ERK1/2 inhibitor PD98059 were purchased from Calbiochem (San Diego, CA). Anti-caspase-3, anti-cleaved caspase-3, anti-caspase-8, anti-cleaved caspase-9, anti-poly(ADP-ribose) polymerase 1 (PARP1), anti-ERK1/2, anti-p-ERK1/2, anti-p38, anti-p-p38, antiJNK and anti-p-JNK were purchased from Cell Signaling Technology (Beverly, MA). Anti-Bax, anti-X-linked inhibitor of apoptosis protein (XIAP), anti-cytochrome c, anticytochrome c oxidase subunit IV (COX IV), and anti-a Tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Bcl-2 was purchased from BD Pharmingen (San Diego, CA). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). 2.4. HPLC analysis of the purity of icariin The test was applied on the Waters 600 pump, with a 2487 ultraviolet–vis detector, an online degasser, and a 5 lL injection loop. Icariin was applied to C18 column (Kromasil, 4.6 250 mm, 5 lm) and eluted with CH3OH/ H2O (45/55, v/v). The effluents were detected under 275 nm. Column temperature was set up at 25 °C and the
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Fig. 1. Icariin killed various human hepatoma cells in vitro. (A) HPLC analysis of the purity of icariin used in the present study. Insert: The chemical structure of icariin. (B) Icariin time- and dose-dependently inhibited the viability of human hepatoma SMMC-7721 cells. Cells were treated with different concentrations of icariin for 24, 48 and 72 h, respectively before cytotoxic analysis. (C) Two human hepatoma cell lines (Bel-7402 and HepG2), human normal hepatocyte line L-02 cells and primary murine hepatocytes were treated with 5–40 lM icariin for 24 h, then cytotoxicity was analyzed by MTT assay. Data represent the mean ± SEM of three different experiments with triplicate sets in each assay. P < 0.05, P < 0.01 vs. drug-untreated group.
flow rate was 1 ml/min. The peak areas and the relative contents were calculated using EmpowerTM workstation.
ProteoExtract Cytosol/Mitochondria Fractionation Kit (Merck Bioscience, Bad Soden, Germany) according to the procedures provided by the manufacturer.
2.5. Cytotoxicity assay 2.8. Western blot The cytotoxic activity of icariin was measured using the MTT assay. After cells were treated with icariin for indicated time in 96-well plates, MTT solution (5 mg/ml in RPMI 1640 medium; Sigma–Aldrich) was added (10 ll/ well), and plates were incubated for a further 4 h at 37 °C. The purple formazan crystals were dissolved in 100 ll of DMSO. After 5 min, the plates were read on an automated microplate spectrophotometer (Sunrise, Tecan, Austria) at 570 nm. Assays were performed in triplicate on three independent experiments. 2.6. Cell apoptosis assay The cells were stained with Annexin V-FITC/PI and measured by FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). Annexin V+/PI cells were considered as apoptotic cells. 2.7. Subcellular fractionation The proteins in the SMMC-7721 cells were separated into cytosolic and mitochondrial fractions using the
Proteins were extracted in lysis buffer (30 mmol/L Tris, pH 7.5, 150 mmol/L sodium chloride, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L sodium orthovanadate, 1% Nonidet P-40, 10% glycerol, and phosphatase and protease inhibitors). The proteins were then separated by SDS–PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes (Roche Applied Science). The membranes were probed with antibodies overnight at 4 °C, and then incubated with a horse radish peroxidase-coupled secondary antibody. Detection was performed using a LumiGLO chemiluminescent substrate system (KPL, Guildford, UK). Band intensity was quantified by BandScan software (Glyko, Novato, CA). 2.9. Cell mitochondrial membrane potential assay SMMC-7721 cells were treated with or without 10 lM icariin for 6–24 h, then the cells were harvested and the disruption of mitochondrial transmembrane potential was measured using fluorochrome dye JC-1 by flow cytometry as previously reported [17].
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2.10. Detection of intracellular ROS The production of intracellular ROS was measured in the SMMC-7721 cell line using the oxidation-sensitive fluorescent dye carboxy-DCFDA [18]. 2.11. Measurement of in vivo anti-tumor activity Tumors were established by injection of 5 106 SMMC7721 cells s.c. into the armpit of 4- to 5-week old BALB/c female athymic mice (National Rodent Laboratory Animal Resource, Shanghai, China). Treatments were initiated when tumors reached a mean group size of approximately 100 mm3. Tumor volume (cubic millimeters) was measured with calipers, and it was calculated as (W2 L)/2, where W is the width and L is the length of tumor. Icariin dissolved in ethanol/0.9% sterile sodium chloride solution (1:9, vol) was administered i.p. to athymic mice every day for 20 days. Tumor volume were recorded every 2 days until animals were sacrificed.
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biochem, USA) according to the manufacturer’s instructions [19]. The quantitation of DNA fragmentation was determined by photometric enzyme immunoassay using the Cell Death Detection ELISAplus kit (Roche, Penzberg, Germany) following the manufacturer’s instructions [20]. Activation of caspase-3 was determined using the caspase-3 colorimetric assay kit (MBL International, Woburn, MA) following the manufacturer’s instructions. [20]. 2.14. Statistical analysis Data are expressed as mean ± SEM. Student’s t test and one-way ANOVA test were used for statistical analyses of the data. All statistical analyses were conducted using SPSS 10.0 statistical software (SPSS, Chicago, IL, USA). Cases in which P values of <0.05 or <0.01 were considered statistically significant. 3. Results 3.1. Icariin kills human hepatoma cells in vitro
2.12. TUNEL assay Paraffin-embedded hepatoma sections were deparaffinized and rehydrated to PBS. Samples were treated with 10 lg/ml proteinase K (ICN) in 10 mM Tris–HCl (pH 7.4) for 15 min at room temperature. Sections were washed three times in PBS solution after proteinase treatment. TUNEL labeling was performed with the In Situ Cell Death detection kit with fluorescein (Roche, Mannheim, Germany) according to manufacturer recommendations. The slides were observed under the fluorescence microscope (Zeiss, Germany), and the images were captured by digital camera and analyzed by Axiovision software (Germany). 2.13. DNA fragmentation and caspase-3 activity assay The qualitation of DNA fragmentation was measured using the Suicide-TrackTM DNA Ladder Isolation Kit (Cal-
As shown in Fig. 1B, human hepatoma SMMC-7721 cells exhibited time- and dose-dependent sensitivity to icariin, with IC50 values at 24 h (the concentration of drug inhibiting 50% of cells) around 10 lM. In addition, icariin also killed other human hepatoma cell lines such as Bel-7402, and HepG2 cells in a dose-dependent manner (Fig. 1C). However, icariin at the concentration up to 40 lM did not affect the survival of normal human liver cell line L-02 cells as well as primary murine hepatocytes (Fig. 1C). The results suggest that icariin has promising antihepatoma activity with low cytotoxic effect on normal hepatocytes. 3.2. Icariin induces significant apoptosis in human hepatoma cells but not normal hepatocytes Compared with control group, icariin dramatically triggered apoptosis in human hepatoma SMMC-7721 cells (Fig. 2A) or Bel-7742 cells (Supplemental Fig. 1A) in a dose-dependent manner. To SMMC-7721 cells, approximately 40% of the cells were at early apoptosis in 10 lM icariin-treated group, compared with 1.2% of vehicle treated group. It was noted that 5–20 lM icariin did not induce significant apoptosis in human normal hepatocyte line L-02 cells (Supplemental Fig. 1B). To further confirm whether icariin-induced cell apoptotic death was indeed
Fig. 2. Icariin induced caspase-3-dependent apoptosis in human hepatoma SMMC-7721 cells. (A) Apoptosis in SMMC-7721 cells was assessed after 12 h of treatment with 5–20 lM icariin by Annexin V-FITC/PI binding and measured by flow cytometry analysis. Numbers indicate the percentage of cells in each quadrant. P < 0.01 vs. drug-untreated group. (B) SMMC-7721 cells were treated with 50 lM Z-DEVD-FMK, a caspase-3 specific inhibitor, for 1 h before treatment with 10 lM icariin for 12 h. Apoptosis was assessed by flow cytometry as mentioned above. P < 0.01.
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caspase-3-dependent, a caspase-3 specific inhibitor Z-DEVD-FMK was used. As indicated in Fig. 2B, the addition of Z-DEVD-FMK markedly reduced the apoptotic response from 40.5% to 8.4%. 3.3. Icariin triggers mitochondrial-related apoptosis in human hepatoma SMMC-7721 cells When SMMC-7721 cells were treated with 10 lM icariin for different time, significant proteolytic cleavage of procaspase-9 and procaspase-3 was detected using Western blot (Fig. 3A). By contrast, no cleavage of caspase-8 was found in the icariin-treated cells (Supplemental Fig. 2). Consistently, cytochrome c was released from mitochondria to cytosol (Fig. 3B), indicating activation of the intrinsic apoptosis pathway. Treatment of SMMC-7721 cells with 10 lM icariin for 24 h induced significant cleaved PARP1 protein expression (Fig. 3A). XIAP is able to inhibit activation of caspase-3 to protect cells from apoptosis. As shown in Fig. 3A, icariin decreased XIAP protein levels in a dose-dependent manner. To assess the role of mitochondria in icariin-induced cell death, we tested whether icariin caused a loss of mitochondrial membrane potential. Compared with the corresponding control, icariin caused an obvious decrease of mitochondrial membrane potential in SMMC-7721 cells in a time-dependent manner (Fig. 3C and D). The proto-oncoprotein Bcl-2 is a powerful antagonist of the mitochondrial pathway of apoptosis initiated by a variety of extra- and intracellular stresses. Therefore, we examined whether icariin could alter the balance between proapoptotic Bax and antiapoptotic Bcl-2 proteins at the mitochondrial membrane. Exposure to icariin resulted in decrease of Bcl-2 and increase of Bax (Fig. 3E), with an increase in Bax/Bcl-2 ratio (Fig. 3F). 3.4. JNK activation contributes to icariin-induced apoptosis in human hepatoma SMMC-7721 cells There is accumulating evidence that small compound-triggered apoptosis has been always associated with activation of mitogen-activated protein kinases (MAPK) [20–22], we next examined the effect of icariin
on MAPK signaling. As shown in Fig. 4A, the phosphorylation of JNK was gradually increased after the icariin treatment, but the phosphorylations of p38 and the extracellular signal-regulated protein kinase (ERK1/ 2) were hardly affected. To further analyze whether JNK activation plays a role in icariin-induced apoptosis, the effect of SP600125, a pharmacological inhibitor of JNK activation, were examined. Addition of 10 lM SP600125 remarkably suppressed JNK phosphorylation induced by 10 lM icariin, as observed after 24 h (Fig. 4D). As shown in Fig. 4B and C, SP600125 reversed the proapoptotic effect of icariin in SMMC-7721 cells assessed by Annexin V/PI binding assay. However, p38 inhibitor SB203580 or ERK1/2 inhibitor PD98059 did not blocked the icariin-induced apoptosis in SMMC-7721 cells (Supplemental Fig. 3). Moreover, SP600125 reduced Bax upregulation and increased Bcl-2 downregulation in response to icariin as well as resulted in reduction of caspase-3 activation and PARP cleavage (Fig. 4D and E). These results indicate the activation of JNK but not p38 or ERK1/2 plays a critical role in icariin-induced apoptosis in human hepatoma SMMC-7721 cells.
3.5. ROS is required for icariin-induced apoptosis in human hepatoma SMMC7721 cells Since a loss of mitochondrial membrane potential is associated with the generation of ROS [23], we detected the level of ROS in SMMC-7721 cells treated with icariin. As shown in Fig. 5A, the level of ROS in cells treated with icariin was increased in a time-dependent manner and pretreatment with antioxidant NAC significantly inhibited ROS generation. To illustrate the role of ROS in icariin-induced apoptosis, SMMC-7721 cells were treated with icariin in the presence or absence of antioxidant NAC. As shown in Fig. 5B, the proportion of apoptotic cells was changed from 38.4% to 12.2%, indicating that addition of antioxidant NAC almost completely reversed apoptotic cell death caused by icariin. Another antioxidant vitamin C also completely blocked the apoptosis induced by icariin in SMMC-7721 cells (Supplemental Fig. 3). In addition, NAC almost blocked icariin-induced decrease of Bcl-2, increase of Bax, activation of caspase-3, and cleavage of PARP1 (Fig. 5D and E). Moreover, the activation
Fig. 3. Icariin triggered mitochondrial-related apoptosis in human hepatoma SMMC-7721 cells. (A) SMMC-7721 cells were treated with or without 10 lM icariin for indicated time, and then cells were harvested and lysed. Caspase-9, caspase-3, PARP1 and XIAP were analyzed by Western blot. The data shown here are one of three different experiments. (B) SMMC-7721 cells were treated with or without 10 lM icariin for indicated time, then cells were harvested and separated into cytosolic and mitochondrial fractions using the commercial fractionation kit. The expressions of cytochrome c (Cyt c) in cytosol and mitochondria were analyzed by Western blot. The data shown here are one of three different experiments. (C) SMMC-7721 cells were treated with or without 10 lM icariin for indicated time, then cells were harvested and the disruption of mitochondrial transmembrane potential was measured using fluorochrome dye JC-1 by flow cytometry. The histogram shown here is one of three different experiments. (D) Quantification of cells with low mitochondrial transmembrane potential was depicted in the graph. n = 3, P < 0.01 vs. drug-untreated group. (E) Cells were treated with 10 lM icariin for indicated time, and whole-cell lysates were collected and immunoblotted with Bcl-2 and Bax. (F) The densitometric analyses of expression of Bcl-2 and Bax relative to the untreated control from three independent experiments. P < 0.05, P < 0.01 vs. drug-untreated group.
S. Li et al. / Cancer Letters 298 (2010) 222–230 of JNK in SMMC-7721 cells treated with icariin could be almost abolished by NAC (Fig. 5F). These observations suggest icariin-induced ROS generation is an early event that induces JNK activation and triggers mitochondrial apoptotic pathways in SMMC-7721 cells. 3.6. Icariin inhibits tumor growth in xenografted nude mice model by causing apoptotic cell death To further evaluate whether icariin had an effect to inhibit tumor growth in vivo, we measured the tumor volume in a xenograft tumor model in which SMMC-7721 cells were injected s.c. into nude mice. In
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the pre-experiment, we found that the drug concentration in the serum of mice with intraperitoneally administered 30 mg/kg of icariin daily for five days reached a peak at about 9.6 lM by a high performance liquid chromatography assay, which just fell within the range of the drug concentrations (5–20 lM) in vitro. So 15, 30 and 60 mg/kg of icariin were selected for in vivo experiments. When transplant tumors reached a mean group size of approximately 100 mm3, mice were treated every day for 20 days with various dose of icariin (i.p., 15, 30, and 60 mg/kg). Compared with the control group, icariin showed significant inhibitory effect on tumor volume, and the inhibitory rates on tumor volume at 20th day caused by 15, 30, and 60 mg/kg icariin were 38.7, 54.7, and 69.9%, respectively
Fig. 4. Involvement of JNK activation in icariin-induced apoptosis in human hepatoma SMMC-7721 cells. (A) Western blot of whole-cell extracts of SMMC7721 cells analyzed for total and phosphorylated MAPK members (JNK, p38, ERK) after treatment with 10 lM icariin for indicated time. P < 0.05, P < 0.01 vs. drug-untreated group. (B and C) SMMC-7721 cells were treated with 10 lM SP600125, a pharmacological inhibitor of JNK activation, for 1 h before treatment with 10 lM icariin for 12 h. Apoptosis was assessed by flow cytometry as mentioned above. P < 0.01. (D and E) SMMC-7721 cells were treated with 10 lM SP600125 for 1 h before treatment with 10 lM icariin for 24 h. The whole-cell extracts were assessed by Western blot. P < 0.01.
Fig. 5. Involvement of ROS in icariin-induced apoptosis in human hepatoma SMMC-7721 cells. (A) Icariin time-dependently increased ROS levels in SMMC7721 cells. Cells were pretreated with 5 mM NAC for 2 h before treatment with or without 10 lM icariin for indicated time, and then the intracellular levels of ROS were detected. P < 0.05, P < 0.01 vs. icariin-treated group. (B) Icariin-induced apoptosis could be almost reversed by antioxidant NAC. Cells were pretreated with 5 mM NAC for 2 h before treatment with or without 10 lM icariin for 12 h, then apoptosis was assessed by Annexin V-FITC/PI binding. (C) The statistical data of apoptosis from three independent experiments. P < 0.01. (D–F) SMMC-7721 cells, with or without pretreatment of 5 mM NAC for 2 h, were incubated with 10 lM icariin for 24 h, and whole-cell lysates were collected and immunoblotted with Bcl-2, Bax, caspase-3, cleaved caspase-3, and PARP1 (D); JNK and p-JNK (F). P < 0.05, P < 0.01.
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Fig. 6. Icariin suppressed tumor growth in mouse xenografted model through inducing apoptosis of tumor cells. Nude mice with SMMC-7721 transplant tumor were i.p. treated with 15, 30, or 60 mg/kg icariin every day for 20 days. (A) Transplant tumor volume of nude mice after the treatment of icariin. The number of mice in each group is eight. P < 0.05, P < 0.01 vs. control group (drug-untreated group) (B and C). At 12th day after the treatment of icariin, the transplant tumors were obtained from various groups of mice and subjected to apoptosis assay. Apoptosis was evaluated by enzyme-linked immunosorbent assay (B) and caspase-3 activation (C). n = 3, P < 0.05, P < 0.01 vs. control group. (D) DNA fragmentation of apoptotic cells in transplant tumor sections at 12th day after the treatment of icariin revealed by agarose gel electrophoresis. In icariin-treated group, the specific DNA cleavage became evident in electrophoresis analysis as a typical ladder pattern. The data shown here are one of three different experiments. (E) In situ detection of apoptotic cells in tumor sections at 12th day after the treatment of icariin by fluorescence microscopy using the TUNEL assay. Up: Bright-field image; Down: TUNEL reaction green fluorescence. Scale bar: 100 lm.
(Fig. 6A). To determine whether the reduced tumor growth rate following icariin treatment could be explained by the induction of apoptosis, we used various methods to characterize apoptosis in tumor sections. Icariin dose-dependently induced significant increase in DNA fragmentation (Fig. 6B) and caspase-3 activity (Fig. 6C) in tumor sections. Consistently, the formation of DNA ladder was evident in transplanted hepatoma cells of 60 mg/kg icariin-treated mice (Fig. 6D). As seen in Fig. 6E, TUNEL assay showed explicitly that apoptotic cells were induced by icariin in hepatoma tissue, whereas few TUNEL positive cells were observed in tumors treated vehicle alone. The results from different apoptosis assays reveal significant features of apoptosis, which strongly suggest that icariin-mediated inhibition of tumor growth in hepatoma-bearing mice is closely correlated with the enhanced apoptosis in cancer cells. In addition, there was no significant difference in the weight and cell numbers of lymphoid tissues (thymus, spleen and lymph nodes) between the mice intraperitoneally injected with icariin (30 mg/kg) daily for 7 days and the mice treated with normal saline (data not shown), suggesting icariin was not toxic for administration to mice in vivo.
4. Discussion HCC is a rapidly fatal disease, with a life expectancy of about 6 months from the time of the diagnosis. Therapeutic strategies employed to date have significantly improved the prognosis for patients with unresectable HCC [24]. This emphasizes the need for investigating the molecular mechanisms responsible for HCC development and seeking effective and non-cytotoxic chemical agents for chemoprevention and treatment. However, few synthetic antineoplastic compounds have been identified to be effective for the treatment of this disease. In this respect, more and more researchers paid much attention to natural active compounds for cancer chemoprevention and treatment [6]. In the present study, icariin, which was previously found to exert antineoplastic effect, was clearly demonstrated to kill all three human hepatoma cell lines,
such as SMMC-7721, Bel-7402, HepG2. However, it was interesting that icariin at the concentrations mentioned above hardly affected the viability of the benign non-tumor human hepatocyte L-02 cells as well as primary murine hepatocytes, suggesting a selective anti-tumor action of icariin to some degree. Similarly, Yang et al. had previously reported that gambogic acid could selectively induce apoptosis of human tumor cells while had relatively less effect on human normal embryon cells due to the higher distribution and longer retention time of gambogic acid in tumor cells compared to the normal cells [25]. The detailed mechanisms of icariin killing hepatoma cells but not benign cells need further investigation. Mitochondria play a pivotal role in the signal transduction of apoptosis [26–28]. The observation of icariin-mediated activation of caspase-9, caspase-3, and subsequent cleavage of PARP1, as well as the result that a caspase-3 specific inhibitor Z-DEVD-FMK almost completely blocked icariin-induced apoptosis in SMMC-7721 cells, suggesting that mitochondrial-mediated caspase cascade pathway plays a very important role in icariin-induced apoptosis. Furthermore, the icariin-caused downregulation of XIAP, one of the inhibitors of apoptosis proteins family [29,30] was detected in SMMC-7721 cells. Because XIAP had been reported to protect cells from apoptosis through inhibiting caspase-3 activation, the downregulation of XIAP provided an additional documentation that icariin-induced anti-tumor was related to activate caspase cascade. MAPKs are mediators of cellular responses to extracellular signals, including ERK1/2, JNK, and p38. The understanding of MAPK signaling may provide the explanation for chemical-triggered apoptotic processes. ERK1/2 is generally associated with proliferation and growth. In
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contrast, JNK and p38 MAPKs are induced by stress responses and closely associated with cell death [31]. There are a few reports about MAPKs related to the effects of icariin [32,33]. To elucidate the precise mechanism involved in the icariin-induced cell death, the effects of icariin on MAPK activation were examined. We found that icariin treatment induced a sustained activation of JNK in SMMC-7721 cells, and the phosphorylation was dependent on ROS generation since it was abrogated by addition of NAC. Furthermore, JNK activation was responsible for icariin-induced Bcl-2/Bax modulation, caspase-3 activation, PARP1 cleavage and consequent apoptosis, as shown by the reduction in these events when the JNK inhibitor SP600125 was present. To determine whether inhibition of JNK activity alone contributes to the reduced icariin-induced apoptosis, we assessed whether the other two MAPK members, p38 and ERK1/2 were involved. First, the activations of p38 and ERK1/2 were not significantly affected by the treatment of icariin from 6 h to 24 h. Second, unlike JNK inhibitor SP600125, p38 inhibitor SB203580 or ERK1/ 2 inhibitor PD98059 did not reversed the icariin-induced apoptosis in SMMC-7721 cells. These results suggest that JNK, but not p38 and ERK1/2, participates in icariin-induced apoptosis. ROS normally exist in all aerobic cells in balance with biochemical antioxidants. Oxidative stress occurs when this critical balance is disrupted because of excess ROS production and/or antioxidant depletion [34]. Evidence is accumulating which indicates that many chemotherapeutic agents may be selectively toxic to tumor cells because they increase oxidant stress and enhance these already stressed cells beyond their limit [35–37]. Cytotoxic ROS signaling appears to be triggered by the activation of the mitochondrial-dependent cell death pathway through activation of MAPK pathways and the proapoptotic protein Bax, with subsequent mitochondrial membrane permeabilization and cell death [38]. Our results showed that icariininduced apoptosis was initiated by the generation of ROS, which was followed by disruption of the mitochondrial membrane potential and release of cytochrome c into the cytosol leading to the activation of caspase-9/-3 cascade. The antioxidant NAC pretreatment not only blocked ROS generation but also offered significant protection against icariin-induced Bcl-2/Bax modulation, phosphorylation of JNK, caspase-3 activation, PARP1 cleavage and apoptosis in SMMC-7721 cells, suggesting ROS generation is an early event in the process of icariin-induced apoptosis. Apoptosis is a major mechanism to eliminate cancer cells. Many chemopreventive agents appear to target signaling intermediates in apoptosis-inducing pathways. For example, capsaicin, a cytotoxic alkaloid from various species of Capsicum, induced apoptosis of human pancreatic cancer cells through ROS generation and mitochondrial death pathway [39]. A recent report demonstrated that curcumin, a natural biologically active compound extracted from rhizomes of Curcuma species, initiated apoptosis of human cervical carcinoma cells through inhibiting human telomerase reverse transcriptase [40]. Thus, targeting apoptosis pathways in premalignant and malignant cells may be an effective strategy for cancer prevention and treatment [41]. In the present study, we conclude that icariin induces
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Fig. 7. Overview of pathways for icariin-induced apoptosis in human hepatoma cell line SMMC-7721. NAC, N-acetyl cysteine; JNK, c-Jun Nterminal kinase; SP600125, a pharmacological inhibitor of JNK activation; PARP, poly(ADP-ribose) polymerase.
human hepatoma SMMC-7721 cell apoptosis via ROS/JNKdependent mitochondrial pathway. Consistent with our results, Yang et al. had recently reported that icariin exerted anti-proliferative efficacy on HepG2 bearing nude mice through downregulating the expressions of both CD31 and Ki67 [15]. Moreover, hepatoma growth inhibition and induction of apoptosis in vivo were observed in the zenografted mice treated with 15–60 mg icariin/kg. These findings demonstrate that icariin is a potent compound against human hepatoma cells both in vitro and in vivo. Taken together, our results demonstrate that icariin-induced apoptosis in human hepatoma SMMC-7721 cells is mediated by the activation of mitochondrial death pathway that require ROS generation and JNK activation. Moreover, administration of icariin significantly suppressed the growth of hepatoma xenograft by inducing apoptosis in the tumor cells. Our studies thus provide a rationale for the development of icariin as chemotherapeutic agent against HCC in the clinical setting. Based on the results of the present study, the mechanisms by which icariin induces apoptosis in human hepatoma SMMC-7721 cells is summarized in Fig. 7. Conflict of interest The authors state no conflict of interest. Acknowledgments This study was supported by National Natural Science Foundation of China (Nos. 30872502, 30972918) and
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