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Research Article
Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α Hui Gaoa,1, Jing Xiea,1, Jianjun Pengb,n, Yantao Hana,n, Qixiao Jianga, Mei Hana, Chunbo Wanga a
Medical College, Qingdao University, Qingdao, Shandong 266071, China College of Life Sciences, Chongqing Normal University, Chongqing 401331, China
b
article information
abstract
Article Chronology:
Gallbladder cancer (GBC) is an aggressive malignancy of the bile duct, which is associated with
Received 16 October 2014
a low (5-year) survival and poor prognosis. The transcription factor HIF-1α is implicated in the
Received in revised form
angiogenesis, cell survival, epithelial mesenchymal transition (EMT) and invasiveness of GBC. In
24 November 2014
this study, we have investigated the role of HIF-1α in the pathobilogy of GBC and effect of
Accepted 27 November 2014
hispidulin on the molecular events controlled by this transcription factor. We observed that hispidulin caused induction of apoptosis, blockade of growth and cell cycle progression in GBC
Keywords:
cells. Our results have demonstrated for the first time that hispidulin-exerted anti-tumor effect
Gallbladder cancer
involved the suppression of HIF-1α signaling. Hispidulin was found to repress the expression of
Hispidulin
HIF-1α protein dose-dependently without affecting the HIF-1α mRNA expression. In addition, the
HIF-1α
inhibition of HIF-1α protein synthesis was revealed to be mediated through the activation of
P-gp
AMPK signaling. Hispidulin also sensitized the tumor cells to Gemcitabine and 5-Fluoroucil by
Chemosensitivity
down-regulating HIF-1α/P-gp signaling. Given the low cost and exceedingly safe profile, hispidulin appears to be a promising and novel chemosensitizer for GBC treatment. & 2014 Elsevier Inc. All rights reserved.
Introduction
for patients with unresectable GBC include radiotherapy and
Gallbladder cancer (GBC) is the most widespread malignancy of biliary tract and the fifth common gastrointestinal cancer that is associated with high mortality rate worldwide [1,2]. Although GBC can be cured by complete surgical resection at an initial stage, less than 30% patients are diagnosed early enough to benefit from the radical operation [3]. The treatment options
systemic chemotherapy. However, the response to chemotherapeutics, such as, gemcitabine (Gem) and 5-fluorouracil (5-FU) has not been shown to be satisfactory. Hence, despite the treatment, recurrence and/or metastasis occur in a large proportion of the patients and the prognosis remains poor [3]. The tumor related hypoxia has been recognized to play a crucial role in rendering the tumor cells insensitive to drugs and radiations. The main
n
Corresponding authors. E-mail addresses: [email protected] (J. Peng), [email protected] (Y. Han). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.yexcr.2014.11.021 0014-4827/& 2014 Elsevier Inc. All rights reserved.
Please cite this article as: H. Gao, et al., Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.11.021
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reason for the insensitivity is poor vascularization of the hypoxic tissue that hinders the transport of drugs to the tumor cells. Moreover, these drugs are known to target the rapidly multiplying cells, whereas, the poorly vacularized cells exhibit retarded multiplication due to the deprivation of sufficient nutrients. As a consequence, they are not the right candidates to be targeted by the anti-proliferative drugs. Clinical efforts aimed at inhibiting hypoxia have been reported to improve therapeutic outcome [4–6]. Therefore, finding novel therapeutic agents are expected to impede the hypoxia related mechanisms and therefore offer the most rational therapeutic strategy against GBC. Hypoxia inducible factor 1 (HIF1), a heterodimeric transcription factor is comprised of two subunits α and β which are sensitive and insensitive to oxygen respectively. The proline residue on alpha subunit is hydroxylated in the normoxic atmosphere and it leads to degradation of this molecule. As a result, the cells growing in normal environment of rich oxygen are deficient in this protein. However, under the hypoxic condition, such as that of a tumor, the posttranslational hydroxylation of the α subunit on its proline residue is restricted and that prevents its degradation. The stabilized α unit binds with its counterpart, the β subunit to form the activated HIF1 which is translocated to the nucleus to bind with the hypoxia-response element of the target gene promoter and enhances the transcription [4,5]. HIF-1α has been known to regulate the expression of a multitude of genes that are critically involved in tumor growth, angiogenesis, metastasis, venous invasion and chemoresistance [6,7]. A poor prognosis of GBC has also been associated with tumor neovascularisation promoted by the HIF-1α over expression [7]. Therefore, targeting HIF-1α appears to be a promising therapeutic approach for GBC. Hispidulin (40 ,5,7-trihydroxy-6-methoxyflavone), a naturally occurring flavonoid, has been isolated from a traditional Chinese medicinal herb, Salvia involucrata [8,9]. The compound has been shown to have antifungal, anti-inflammatory, antioxidant, antithrombosis, antiepileptic, neuroprotective and antiosteoporotic activities [10–17]. The anti-proliferative effect of hispidulin has been observed in pancreatic, gastric, ovarian and glioblastoma cancer cells in vitro [18–21]. Previously, we have evidenced the hispidulin-induced apoptosis in hepatocellular carcinoma cells [22]. In the present study, we sought to investigate the effects of hispidulin on AMPK/HIF-1α signaling in gallbladder carcinoma cell line, GBC-SD. Since the cells growing in normal environment exhibit negligible quantities of HIF-1a (as discussed in the earlier paragreaph) we over expressed the GBC cells with HIF-1α protein so as to establish a phenotype akin to that of the hypoxic tumor cells. Our results also showed that hispidulin enhanced the effect of Gem and 5-FU by down regulating P-gp (P-glycoproteins) through the repression of HIF-1α.
Materials and methods Cell culture Human gallbladder carcinoma cell line GBC-SD was obtained from Shanghai Cell Institute Country Cell Bank (Shanghai, China). All cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Life Sciences, Carlesbad, CA) containing 10% fetal bovine serum, 100 units/ml penicillin and 100 mg/ml streptomycin in a humidified incubator at 37 1C and 5% CO2.
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Cell viability assay Cell proliferation was determined by the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) (Sigma, St. Louis, MO). Briefly, the cells were plated at a density of 5 103 cells/well in 96-well culture plates. Following the drug treatment, the cells in each well were incubated for two hours with 20 mL of 5 mg/ml MTT solution in phosphate-buffered saline (PBS)). The end-product, MTT formazan was dissolved in 150 mL of isopropanol and the absorbance was measured at 570 nm with an ELISA reader (Bio-Rad Laboratories Ltd., Shanghai, China).
Western blot analysis Cells were washed with ice-cold PBS and treated with 100 mL of cell lysis buffer (Cell Signaling, Danvers, MA) containing protease inhibitors (Sigma, St. Louis, MO). Extracted proteins were separated on SDS-PAGE and then transferred electrophoretically onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA). Proteins were probed with specific antibodies as previously described [23]. The antibodies against HIF-1α, cyclin D1 were from Santa Cruz Biotechnology (Santa Cruz, CA), antibodies against Bcl-2 and Bax were from Boster Ltd. (Wuhan, China). Antibodies against 4EBP-1, phospho-4EBP-1 (Thr70), AMPKα, phospho-AMPKα (Thr172), p70 S6 kinase, phospho-p70 S6 kinase (Thr389) and P-gp were purchased from Cell Signaling Technology (Beverly,MA). The rabbit polyclonal antibody to β-actin was used as a gel loading control (Santa Cruz, Santa Cruz, CA, USA, 1:1000 dilution).
Analysis of apoptosis by flow cytometry Flow cytometry analysis was performed using FITC Annexin V apoptosis kit (BD Pharmingen, Franklin Lakes, NJ) according to manufacturer's instructions. Following treatment, the cells were washed with ice-cold phosphate buffered saline and then resuspended in binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2) at a concentration of 1 106 cells/ml. Cells were stained with annexin V-FITC and propidium (PI) for 15 min in dark before analyzing with flow cytometer (Beckman Coulter Inc., Miami, Florida, USA).
Caspase-3 activity assay Following the treatment, the cytosolic proteins of GBC-SD cells were extracted in hypotonic cell lysis buffer (25 mm HEPES, pH 7.2, 5 mM MgCl2, 5 mm EDTA, 5 mM dithiothreitol, 0.05% phenylmethylsulfonyl fluoride). The cytosolic extracts corresponding to 30 mg total protein were added to the reaction mixture containing 312.5 mm HEPES buffer, pH 7.5, 31.25% sucrose, 0.3125% CHAPS and the enzyme substrate, benzyloxycarbonyl-DEVD-7-amido-4-(trifluoromethyl) coumarin (Calbiochem, Darmstadt, Germany). After 1 h of incubation at 37 1C, the reaction product, 7-amido-4-(trifluoromethyl)coumarin (AFC) was detected with a fluorescence reader, set to the excitation and emission wavelengths of 355 nm and 525 nm respectively.
Analysis of cell cycle Cell cycle distribution was determined by DNA staining with propidium iodide (PI). Following the treatment, the cells were
Please cite this article as: H. Gao, et al., Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.11.021
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Fig. 1 – Effect of hispidulin on the proliferation (A), apoptosis (B), caspas-3 activity (C) and Bcl2 and Bax expression (D) in GBC-SD cells. The cells were incubated with hispidulin at the indicated concentration for 48 h before conducting the analyses. n and nn indicate that P values were o0.05 and o0.01 respectively and the effect was significant compared to the control. fixed with 70% cold ethanol at 4 1C overnight. DNA was stained with propidium iodide (0.05 mg/ml) and RNase (2 mg/ml) for 30 min at room temperature. Cell cycle was analyzed by flow cytometry (FCM, Becton-Dickinson, San Jose, CA) and the percentages of cells in the different phases of cell cycle were analyzed by FlowJo 7.6 software.
Overexpression of HIF-1α The GBC-SD cells were seeded at the sub-confluent densities and transfected with pcDNA3-EGFP empty vector or scrambled vector (SV) or pcDNA3-HA-HIF-1α expression construct TransIT-LT-1 following the manufacturer's instructions (Mirus Bio Corp., Madison, WI). Overexpression of HIF-1α was confirmed by Western blotting and real-time PCR analyses.
HIF-1α reporter activity assay Cells (1 105) were plated in 6-well culture plates and then transfected with HIF-1α reporter vector (Panomics, Redwood, CA) using lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Briefly, the cells were incubated with 0.5 μg HIF-1α reporter vector (Panomics, Redwood, CA) and 0.5 μg pEGFP-C2 per well in the presence and absence of hispidulin. After transfection, the luciferase activity assay was measured using a commercial kit (Promega Corp., Madison, MI) according the manufacturer's manual. pEGFP-C2 vector was used to normalize for transfection efficiency, and relative luciferase activity (defined as reporter activity) was calculated as the ratio of luciferase/EGFP activity.
Quantitative RT-PCR (qRT-PCR) Total RNA was extracted from the cells using the Total RNA Kit (TIANGEN Co., Beijing, China) and 3 mg of RNA was converted into cDNA using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). The primers were synthesized based on the published sequence [24]. The master mix of each PCR reaction included SYBR GREEN master mix (Solarbio Co., Beijing, China), forward and reverse primers, and 10 ng of template cDNA. The PCR reaction was carried out at 95 1C for 5 min followed by 40 cycles of 95 1C for 30 s, 60 1C for 30 s, and 72 1C for 30 s. Data were analyzed using the comparative ΔCt method (ABPrism software,
Please cite this article as: H. Gao, et al., Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.11.021
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Fig. 2 – Effect of hispidulin on the distribution of cells in different phases of cell cycle (A) and cyclin-D1 expression (B). The GBC-SD cells were incubated with hispidulin at the indicated concentrations for 48 h before the flow cyometric (A) and western blot analyses (B). * indicate that the effect was significant (Po0.05). Applied Biosystems, Foster City, CA) using GAPDH as the internal normalization control.
AMPKα1 shRNA transfection The transfection and colony selection was conducted as previously described [25]. The AMPKα1 short hairpin RNA (shRNA)containing lentiviral particles (Santa Cruz Biotech, Santa Cruz, CA, sc-29673-SH) or the scramble shRNA control lentiviral particles (Santa Cruz Biotech, Santa Cruz, CA, sc-108080) (20 μL/ml medium each) were added to GBC-SD cells and the incubation was continued for 48 h. The culture medium (containing 1 μg/ml of puromycin) was replaced every 48 h, and incubation of the cells continued until puromycin resistant colonies were formed. The single colony of the cells expressing shRNA was sub-cultured. The expression of AMPKα1 was confirmed by western blotting.
In vivo xenograft experiments The animal experiments were performed in accordance with CAPN (China Animal Protection Law), and the protocols were approved by the Animal Care and Use Committee of Qingdao University. Female BALB/c (nu/nu) mice, 6 weeks old, purchased from the Animal Centre of China (Beijing, China), were housed with a light/dark cycle of 12/12 h and allowed free access to rodent chow and water. GBC-SD cells were harvested from subconfluent cultures and washed in serum-free medium before being resuspended in PBS. The mice were anesthetized using ethyl ether, and 105 cells were injected subcutaneously into the right and left abdominal regions with a gauge needle. Tumor appearance was inspected every 3 days. Once tumor volume
reached 100 mm3, animals were randomized into 4 groups (n¼ 6 per group) to receive intraperitoneal (IP) injections as follows: (A) vehicle (0.9% sodium chloride plus 1% DMSO), (B) hispidulin (40 mg/kg/day, dissolved in vehicle), (C) hispidulin (20 mg/kg/ day, dissolved in vehicle) and (D) hispidulin (10 mg/kg/day, dissolved in vehicle) on a daily basis. Tumor volumes and body weight were measured twice per week. Tumor volume was measured along the longest orthogonal axes and calculated as volume¼ (length width2)/2, where width was the shortest measurement. All mice were sacrificed 30 days after the first day of treatment.
Statistical analysis The data are expressed as Mean7SD and represent the results of three separate experiments each performed in quadruplicate unless otherwise stated. The Student's test was used to evaluate if the difference between two groups was significant and One Way ANOVA with post hoc test was used for comparison between three or more groups. Po0.05 was considered as statistically significant.
Results Hispidulin inhibited the growth of GBC cells GBC-SD cells at logarithmic growth phase were seeded in 6-well tissue culture plates at a density of 1 104/ml and incubated overnight at 37 C with 5% CO2. The following day, cells were exposed to various concentrations (0, 12.5, 25, 50 μM) of hispidulin (Shanghai Tauto Biotech Co., Ltd., Shanghai, China, purity498%) and incubation
Please cite this article as: H. Gao, et al., Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.11.021
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Fig. 3 – Effect of hispidulin on HIF-1α mRNA and protein expressions measured by qRT-PCR and Western blotting respectively (A), cell viability and apoptosis (B) expression of Bcl-2 and Bax and caspase-3 activity (C) in the cells stably transfected with HIF-1α. The cells were incubated with 50 μM of hispidulin for 48 h before conducting the analyses. HIF-1α OE and His denote HIF-1α over expressed and Hispidulin respectively. ** and b represents Po0.01 vs. control, and Po0.05 vs. HIF-1αOE respectively.
was continued for 24, 48 and 72 h, then the number of viable cells was determined. As shown in Fig. 1A, hispidulin exhibited dose- and time-dependent anti-proliferative effects. Cells treated with hispidulin for 24 h at concentrations of 12.5, 25 and 50 μM exhibited the viability that was reduced by 10.373.2%, 17.575.2% and 33.676.7%, respectively. After 48 h incubation with hispidulin, the cell viability was decreased by 14.373.2%, 31.276.3% and 44.374.6%, respectively. When the incubation was prolonged to 72 h, further decrease in cell viability was observed. The inhibition of cells incubated for 48 h with 25 μM hispidulin was statistically significant as compared with the cells treated with vehicle.
Hispidulin promoted the apoptosis in GBC cells As shown in Fig. 1B, hispidulin increased the number of apoptotic cells in a dose-dependent manner. Thus, compared to the vehicletreated control cells, those incubated with 25 and 50 μM hispidulin, showed a significantly increased percentage of apoptotic cells (18.073.6% and 33.474.3% respectively). We also examined the effect of hispidulin on the activity of caspase-3, an enzyme that promotes apoptosis. As shown in Fig. 1C, the hispidulin-treated cells showed an elevated activity of caspase-3, indicating that hispidulin promoted apoptosis involved activation of this enzyme.
Please cite this article as: H. Gao, et al., Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.11.021
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The dose-dependent effect of hispidulin on the expression of apoptosis modulating molecules, Bcl-2 and Bax was also evidenced. As shown in Fig. 1D, the expression of Bcl-2 was decreased while that of the Bax was upregulated with the increasing doses of hispidulin. Collectively, these results suggest that hispidulin mediated the inhibition of cancer cells growth by promoting apoptosis.
Hispidulin-induced the cell arrest in G0/G1 phase of the cell cycle. The effect of hispidulin on cell cycle regulation was determined by flow cytometry. As shown in Fig. 2A, compared to the controls, the cells treated with hispidulin were accumulated in an increased proportion in G0/G1 phase with a concomitant decrease in their number in S and G2 phases. Since cyclin D1 plays an important role in the regulation of G1 progression and G1-S transition, we examined the effect of hispidulin on the expression of cyclin D1. As shown in Fig. 2B, hispidulin was found to down-regulate the cyclin D1 expression in a dose-dependent manner, suggesting the involvement of cyclin D1 in hispidulin-mediated blockade of the cell cycle progression in G1 phase.
Hispidulin-exerted anti-tumor effect was mediated through HIF-1α inhibition To investigate the effect of hispidulin on HIF-1α, we over expressed this protein by transfecting its cDNA into the GBC-SD cells. Cells transfected with an empty vector or non-transfected cells were used as controls. As depicted in Fig. 3A, cells transfected with HIF-1α showed increased expressions of HIF-1α protein and mRNA. The treatment of HIF-1α-transfected cells with hispidulin resulted in a reduced expression of HIF-1α protein. In addition, compared to the non-transfected cells or those transfected with an empty vector, the cells overexpressing HIF-1α exhibited a significantly increased viability and decreased apoptosis (Fig. 3B). Upon treatment with hispidulin, a marked reduction in proliferation and increased apoptosis of the cells was observed. The cells with HIF-1α overexpression also exhibited an upregulation of Bcl-2, and downregulation of Bax expressions. Hispidulin suppressed this effect of HIF-1α, and it also decreased the expression of Bcl-2 while increased that of Bax in control cells. The HIF-1α-overexpressing cells also showed a decreased activity of caspase-3 (Fig. 3C). In contrast, the cells treated with 50 μM of hispidulin exhibited a several fold increase in the activity of caspase-3 and suppressed the HIF-1α-mediated inhibition of this enzyme. Taken together, these results suggest that hispidulin blocked the HIF-1α-promoted cell proliferation and inhibition of apoptosis.
Hispidulin repressed the transactivation activity and expression of HIF-1α in GBC cells In order to examine the effect of hispidulin on the transfection of HIF-1α, we treated the GBC cells with a series of hispidulin concentrations before carrying out the transfection procedures. As shown by the reporter luciferase assay in Fig. 4B, the transactivation activity of HIF-1α was decreased with hispidulin in a dose-dependent way. The expression of HIF-1α was confirmed by determining mRNA and protein levels. Thus, in GBC cells the expression of HIF-1α protein
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determined by Western blotting was reduced with hispidulin dosedependently (Fig. 4A). However, the expression of HIF-1α mRNA remained unchanged even with the highest concentration of hispidulin (Fig. 4C). These results clearly indicate that hispidulin-mediated decrease in HIF-1α content occurred at the level subsequent to mRNA transcription (Fig. 4C).
Hispidulin-mediated down-regulated of HIF-1α protein expression involved the modulation of p70S6K and 4E-BP1 In order to further dissect the site of hispidulin's action, we examined its effect on the expression of phosphorylated 70S6K and 4E-BP (p70S6K and p4E-BPs), the key regulators of protein translation (see Section Discussion). Fig. 4D shows that hispidulin caused a decrease in the expression of phosphorylated p70S6K (on Thr389) and p4E-BP-1 (on Thr70) in a dose-dependent manner. This observation suggests that hispidulin-mediated reduction in HIF-1α expression was executed through blockade of protein translational activity involving p70S6K (on Thr389) and p4E-BP-1.
Hispidulin suppressed the HIF-1α expression via AMPK signaling The reduced phosphorylation of p70(S6K) and 4E-BP-1 has been shown to be associated with repressed HIF-1α expression and mitochondrial dysfunction. Moreover, the modulation of AMPK (AMP kinase) signaling has been proposed to play a major role in the expression of HIF-1α and cancer progression in HepG2 cells [27]. Therefore, we explored whether AMPK was involved in the hispidulinmediated suppression of HIF-1α synthesis. As shown in Fig. 5A, hispidulin produced a dramatic elevation of phosphorylated AMPK (on Thr172) expression in a dose-dependent manner. In the cells pretreated with compound C (C24H25N5O, Pyrazolo[1,5-a]pyrimidine,6[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl), a pharmacological AMPK inhibitor) or short hairpin RNA (shRNA) for 4 h, the expression of p-AMPK was found to be significantly reduced (Fig. 5B). This effect of the inhibitors on p-AMPK expression was not revealed to be reversed by hispidulin. However, the substantially increased HIF-1α expression caused by the inhibition of p-AMPK (both with compound C and shRNA) was significantly reduced by hispidulin (Fig. 5C). These results suggest that hispidulin reversed the p-AMPK-mediated inhibition of HIF-1α through a mechanism other than blocking the expression of AMPK-α. For instance, promoting the activation of the existing p-AMPK that is not bound with inhibitors or by releasing the bound inhibitor from pAMPK. Further work is required to find out if hispidulin accelerates the acivity of AMPK. Also, treatment of the cells with AMPK activator AICAR was found to significantly increase the pAMPK but reduce the HIF-1α protein expression (Fig. 5D). Corresponding to these observations, the cell viability was significantly reduced with hispidulin (Fig. 5E). Collectively, these findings suggest that hispidulin mediated the activation of AMPK which in turn caused the repression of HIF-1α protein synthesis resulting in reduced cell viability.
Hispidulin enhanced the chemosensitivity of GBC-SD cells by down regulating P-gp (P glycoproteins) P-gp are known to play an important role in imparting chemoresistance to gallbladder cancer and their downregulation has
Please cite this article as: H. Gao, et al., Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.11.021
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Fig. 4 – Effect of hispidulin on HIF-1α expression analyzed by Western blotting (A), the HIF-1α-transactivation measured by reporter activity (B), mRNA expression measured by qRT-PCR (C) and expression of phosphorylated p70S6K and 4EBP1 examined by Western blotting (D) in GBC-SD cells stably transfected with HIF-1α. The cells were incubated with various concentrations of hispidulin for 48 h before the analyses. * indicates that compared to the control, the effect was significant (Po0.05). been shown to increase the chemosensitivity of GBC cells [26]. We tested the effect of hispidulin on P-gp expression in the presence and absence of Gem or 5-FU, the chemotherapeutic agents frequently-used for the gallbladder cancer [27]. As depicted in Fig. 6A, Gem (0.5 μM) or 5-FU (1 μM) showed a moderate decrease in the viability and increase in the apoptosis of GBC-SD cells. However, the cells treated with a relatively low dose of hispidulin (12.5 μM), exhibited a significantly enhanced sensitivity to Gem or 5-FU. Hispidulin alone at this dose produced a minor effect. In order to explore the mechanism of hispidulin-enhanced cell sensitization, we determined its effect on P-gp expression. As shown in Fig. 6B, hispidulin markedly suppressed the P-gp expression in a dose-dependent manner. Given the fact that HIF-1α overexpression is associated with chemoresistance, we investigated whether it involved a role of P-gp. As shown in Fig. 6C, HIF-1α overexpression was associated with a significant increase in P-gp expression which was significantly reduced with hispidulin, indicating that the effect was, in part, mediated through blocking HIF-1α signaling.
Hispidulin suppressed tumor growth in vivo Based on the aforementioned results, we evaluated the in vivo therapeutic effect of hispidulin using an in vivo xenograft mouse model. Hispidulin was administered at 40 mg/kg/day, 20 mg/kg/ day, and 10 mg/kg/day in different treating groups, respectively. Treatment started when the tumor size reached 100 mm3. The
efficacy of the treatment was evaluated by measuring the tumor volume during treatment. As shown in Fig. 7, treatment with hispidulin dose-dependently inhibited the growth of tumor and dose at 20 mg/kg/day was found be able to exert significant antitumor effect (Po0.05 vs. vehicle).
Discussion Epidemiological studies have suggested that high consumption of flavonoids decreases the risk of a variety of cancers [28]. While investigating the anti-cancer activities of flavonoids, a few of the recent studies have focused on a small flavonoid, hispidulin [18–22]. Previously, we showed that hispidulin suppressed the cell growth and induced apoptosis in heptocellular carcinoma cells [22]. This study was designed to evaluate the therapeutic effect of hispidulin in GBC and to investigate the underlying mechanism(s) of its action. Our results revealed that hispidulin exhibited a dose- and timedependent anti-proliferation effect on GBC-SD cells by promoting apoptosis and cell cycle arrest in G0/G1 phase. In the previous study, we found that hispidulin treatment resulted in mitochondrial dysfunction in heptocellular carcinoma cells [22]. Interestingly, a recent study has reported that mitochondrial dysfunction induced by mitochondrial inhibitor or uncoupler under normoxia led to the repression of HIF-1α expression [24]. Given that HIF-1α plays a critical role in the tumor cell physiology, we postulated
Please cite this article as: H. Gao, et al., Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.11.021
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Fig. 5 – Hispidulin-mediated inhibition of HIF-1α expression via AMPK activation. Effect of hispidulin on phosphorylated to unphosphorylated AMPK ratio (A), p-AMPK/AMPK in the presence of Com C or shAMPK (B), HIF-1α expression in the presence of Com C or shAMPK (C), on p-AMPK/AMPK ratio in the presence of ALCAR (D), on cell viability (E). GBC-SD cells were incubated with hispidulin at the indicated concentrations for 48 h before the analyses. His and Com C denote hispidulin and compound C respectively. * and ** represent Po0.05 and Po0.01 respectively, significant effect vs. control, b represents Po0.05, significant effect of hispidulin vs. compound C or shAMPKα treatment. that hispidulin might suppress tumor cell growth by targeting HIF1α. In experimental models, expression/activation of HIF-1α can be achieved by transfection or by hypoxia. In this study, we stably transfected the GBC-SD cells with HIF-1α gene to over express this transcription factor so as to rule out the interference from non-HIF1α-mediated cellular response to hypoxia. Our results showed that over-expression of HIF-1α resulted in promoted cell proliferation and inhibited apoptosis. Hispidulin blocked the expression of HIF1α and the consequent actions of increased cell viability and attenuated apoptosis. Our results are in complete agreement with
other studies reporting that downregulation of HIF-1α resulted in reduced tumor cell survival [29]. A number of studies have identified HIF-1α as the target molecule of flavonoids action in tumor cells. For instance, Mylonis et al. [33] reported that the dietary flavonoid kaempferol effectively inhibited the viability of hepatoma cancer cells by impeding HIF-1α expression and activity under hypoxic conditions. Another flavonoid, apigenin has been found to inhibit expressions of both HIF-1α mRNA and protein in pancreatic cancer cells under normoxic and hypoxic conditions [30]. Silibinin and epigallocatechin-3-gallate (EGCG) have been shown
Please cite this article as: H. Gao, et al., Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.11.021
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Fig. 6 – Hispidulin enhanced the chemosensitivity of GBC-SD cells to Gem and 5-FU, by downregulating P-gp expression. (A) Hispidulin-mediated sensitization of GBC-SD cells to Gem (0.5 μM) and 5-FU (1 μM), (B) Hispidulin repressed the P-gp expression, (C) effect of 50 μM hispidulin on HIF-1α-induced increased P-gp expression. The analyses were performed after incubating the cells with the reactants for 48 h. His and HIF-1α-OP denote hispidulin and HIF-1α over expressed respectively. * and b represent Po0.05 and significant effect vs. control, and vs. HIF-1α OE respectively.
Fig. 7 – Measurements of gallbladder tumor volume at the indicated time points, depicting the in vivo therapeutic efficacy of hispidulin with a representative picture of mouse model at the end of the experiment. * represent Po0.05, significant effect vs. vehicle.
to inhibit the HIF-1α protein expression without affecting that of the mRNA in prostate cancer and pancreatic carcinoma cells respectively [31]. In this study, we found that GBC-SD cells treated with hispidulin displayed a dose-dependent decrease in the expression and activity of HIF-1α. Whereas, the protein expression of HIF-1α was markedly inhibited, the mRNA expression was not changed with hispidulin, indicating that the effect was exerted at the post transcriptional level. The hispidulin-induced significant reduction of p-4E-BP1 and p-p70 S6 kinase expression suggests that the compound repressed the initiation of HIF-1α translation. Based on the aforementioned studies and our results, we suggest that different flavonoids modulate HIF-1α synthesis and/or activity by modulating distinct signaling pathways.
The key regulators of protein translation, p70S6K and 4E-BP are known to be the targets of mTOR signaling [32]. In addition, AMPK has been identified to play an important regulatory role in the activation of mTOR signaling that involves protein synthesis, cell growth and viability. During the energy stress, AMPK represses mTOR signaling by phosphorylating tuberous sclerosis complex 2 (TSC2), and thereby enhancing the formation of TSC complex (TSC1/TSC2), a key modulator of mTOR functioning [33]. AMPK is also known to inhibit mTOR signaling independent of TSC1/TSC2 pathway by directly phosphorylating raptor, a component of the mTOR complex 1 (mTORC1) [34]. Recent studies have revealed a correlation between pharmacological activation of AMPK and decreased HIF-1α expression [25,35]. Our results showed that hispidulin promoted the increased
Please cite this article as: H. Gao, et al., Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.11.021
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the expression of phosphorylated AMPK, the active form of AMPK. Furthermore, our results showed that pharmacological inhibition (using compound C) or gene knock-down (using shRNA) led to an enhanced expression of HIF-1α, suggesting a negative correlation between the two molecules. We observed that hispidulin reversed the effect of AMPK inhibition and that caused an up-regulation of HIF-1α, at least in part. Since in this study, hispidulin did not reverse the effect of inhibitors on the expression of p-AMPK, we propose that hispidulin-retrieved inhibition of HIF-1α was mediated through a mechanism other than protein expression. Further work is needed to find out if hispiduline accelerates the activity of pAMPK. In the presence of compound C and shAMPKα, the hispidulin-induced decrease in cell viability was partially prevented, further supporting the notion that the anti-tumor activity of hispidulin involved the activation of AMPK and consequent inhibition of HIF-1α. Because of the complexity of AMPK functions in the tumor cell physiology, role of other molecule(s) than HIF-1α in hispidulin-stimulated loss of cell viability cannot be ruled out. However, our study has evidenced that hispidulin exerts its anti-tumor effect through modulation of AMPK/ HIF-1α signaling at least in part. In future, we intend to investigate the role of TSC1/TSC2 in AMPK-regulated HIF-1α expression and function to further understand the mechanism(s) involved in this process. Because of the hypoxic conditions, cancer cells have been shown to be resistant to anti-neoplastic agents [36]. The multidrug resistance gene (MDR1) that encodes P-glycoproteins has been found to be up-regulated in hypoxia in an HIF-1α dependent manner. MDR1/P-gp, a member of the ABC (adenosine triphosphate binding cassette) transporter family, promotes the excretion of xenobiotics and drugs including chemotherapeutics and results in their decreased intracellular concentrations [37]. Moreover, the presence of HIF-1α binding domain and auxiliary sequence similar to hypoxia-responsive element (HRE) detected in the MDR1 gene promoter has led to the suggestion that MDR1 might actually be a hypoxia-responsive gene [38]. Recently, a number of studies have shown the contribution of HIF-1αmediated up-regulation of P-gp expression to hypoxia-induced drug resistance in a variety of human tumors, such as gastric, colon, gliomas, and breast carcinoma [38–41]. Clearly inferring that HIF-1α/MDR1 signaling may serve as a promising target of anti-neoplastic drugs. Consistent with these reports, our results showed that pharmacological suppression of HIF-1α resulting in down-regulation of P-gp enhanced the chemosensitivity of GBCSD cells to Gem and 5-FU. Taken together, our results suggest that hispidulin mediated the downregulation of MDR1/P-gp expression by interfering with the translation of HIF-1α in GBC-SD. In summary, we observed that hispidulin inhibited cell growth, induced apoptosis and blocked cell cycle progression. Our study has demonstrated for the first time that the anti-neoplastic effects of hispidulin were mediated at least in part, by down-regulating HIF-1α expression through the activation of AMPK. The hispidulin-mediated repression of HIF-1α expression was furthered to P-gp downregulation which in turn potentiated the anti-tumor efficacy of Gem and 5-FU. Indeed, it is a novel mechanism that explains the hispidulin-elicited sensitization of the cancer cells to chemotherapeutic agents.
Conflict of interest We declare that we have no conflict of interest.
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Acknowledgment This work was supported by the National Natural Science Foundation (Nos. 81473384 and 31470570) and funds from the Qingdao University (Nos. 14-2-3-50-nsh, 13-1-3-74 and 600201304).
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Research Article
Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α Hui Gaoa,1, Jing Xiea,1, Jianjun Pengb,n, Yantao Hana,n, Qixiao Jianga, Mei Hana, Chunbo Wanga a
Medical College, Qingdao University, Qingdao, Shandong 266071, China College of Life Sciences, Chongqing Normal University, Chongqing 401331, China
b
article information
abstract
Article Chronology:
Gallbladder cancer (GBC) is an aggressive malignancy of the bile duct, which is associated with
Received 16 October 2014
a low (5-year) survival and poor prognosis. The transcription factor HIF-1α is implicated in the
Received in revised form
angiogenesis, cell survival, epithelial mesenchymal transition (EMT) and invasiveness of GBC. In
24 November 2014
this study, we have investigated the role of HIF-1α in the pathobilogy of GBC and effect of
Accepted 27 November 2014
hispidulin on the molecular events controlled by this transcription factor. We observed that hispidulin caused induction of apoptosis, blockade of growth and cell cycle progression in GBC
Keywords:
cells. Our results have demonstrated for the first time that hispidulin-exerted anti-tumor effect
Gallbladder cancer
involved the suppression of HIF-1α signaling. Hispidulin was found to repress the expression of
Hispidulin
HIF-1α protein dose-dependently without affecting the HIF-1α mRNA expression. In addition, the
HIF-1α
inhibition of HIF-1α protein synthesis was revealed to be mediated through the activation of
P-gp
AMPK signaling. Hispidulin also sensitized the tumor cells to Gemcitabine and 5-Fluoroucil by
Chemosensitivity
down-regulating HIF-1α/P-gp signaling. Given the low cost and exceedingly safe profile, hispidulin appears to be a promising and novel chemosensitizer for GBC treatment. & 2014 Elsevier Inc. All rights reserved.
Introduction
for patients with unresectable GBC include radiotherapy and
Gallbladder cancer (GBC) is the most widespread malignancy of biliary tract and the fifth common gastrointestinal cancer that is associated with high mortality rate worldwide [1,2]. Although GBC can be cured by complete surgical resection at an initial stage, less than 30% patients are diagnosed early enough to benefit from the radical operation [3]. The treatment options
systemic chemotherapy. However, the response to chemotherapeutics, such as, gemcitabine (Gem) and 5-fluorouracil (5-FU) has not been shown to be satisfactory. Hence, despite the treatment, recurrence and/or metastasis occur in a large proportion of the patients and the prognosis remains poor [3]. The tumor related hypoxia has been recognized to play a crucial role in rendering the tumor cells insensitive to drugs and radiations. The main
n
Corresponding authors. E-mail addresses: [email protected] (J. Peng), [email protected] (Y. Han). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.yexcr.2014.11.021 0014-4827/& 2014 Elsevier Inc. All rights reserved.
Please cite this article as: H. Gao, et al., Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.11.021
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reason for the insensitivity is poor vascularization of the hypoxic tissue that hinders the transport of drugs to the tumor cells. Moreover, these drugs are known to target the rapidly multiplying cells, whereas, the poorly vacularized cells exhibit retarded multiplication due to the deprivation of sufficient nutrients. As a consequence, they are not the right candidates to be targeted by the anti-proliferative drugs. Clinical efforts aimed at inhibiting hypoxia have been reported to improve therapeutic outcome [4–6]. Therefore, finding novel therapeutic agents are expected to impede the hypoxia related mechanisms and therefore offer the most rational therapeutic strategy against GBC. Hypoxia inducible factor 1 (HIF1), a heterodimeric transcription factor is comprised of two subunits α and β which are sensitive and insensitive to oxygen respectively. The proline residue on alpha subunit is hydroxylated in the normoxic atmosphere and it leads to degradation of this molecule. As a result, the cells growing in normal environment of rich oxygen are deficient in this protein. However, under the hypoxic condition, such as that of a tumor, the posttranslational hydroxylation of the α subunit on its proline residue is restricted and that prevents its degradation. The stabilized α unit binds with its counterpart, the β subunit to form the activated HIF1 which is translocated to the nucleus to bind with the hypoxia-response element of the target gene promoter and enhances the transcription [4,5]. HIF-1α has been known to regulate the expression of a multitude of genes that are critically involved in tumor growth, angiogenesis, metastasis, venous invasion and chemoresistance [6,7]. A poor prognosis of GBC has also been associated with tumor neovascularisation promoted by the HIF-1α over expression [7]. Therefore, targeting HIF-1α appears to be a promising therapeutic approach for GBC. Hispidulin (40 ,5,7-trihydroxy-6-methoxyflavone), a naturally occurring flavonoid, has been isolated from a traditional Chinese medicinal herb, Salvia involucrata [8,9]. The compound has been shown to have antifungal, anti-inflammatory, antioxidant, antithrombosis, antiepileptic, neuroprotective and antiosteoporotic activities [10–17]. The anti-proliferative effect of hispidulin has been observed in pancreatic, gastric, ovarian and glioblastoma cancer cells in vitro [18–21]. Previously, we have evidenced the hispidulin-induced apoptosis in hepatocellular carcinoma cells [22]. In the present study, we sought to investigate the effects of hispidulin on AMPK/HIF-1α signaling in gallbladder carcinoma cell line, GBC-SD. Since the cells growing in normal environment exhibit negligible quantities of HIF-1a (as discussed in the earlier paragreaph) we over expressed the GBC cells with HIF-1α protein so as to establish a phenotype akin to that of the hypoxic tumor cells. Our results also showed that hispidulin enhanced the effect of Gem and 5-FU by down regulating P-gp (P-glycoproteins) through the repression of HIF-1α.
Materials and methods Cell culture Human gallbladder carcinoma cell line GBC-SD was obtained from Shanghai Cell Institute Country Cell Bank (Shanghai, China). All cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Life Sciences, Carlesbad, CA) containing 10% fetal bovine serum, 100 units/ml penicillin and 100 mg/ml streptomycin in a humidified incubator at 37 1C and 5% CO2.
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Cell viability assay Cell proliferation was determined by the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) (Sigma, St. Louis, MO). Briefly, the cells were plated at a density of 5 103 cells/well in 96-well culture plates. Following the drug treatment, the cells in each well were incubated for two hours with 20 mL of 5 mg/ml MTT solution in phosphate-buffered saline (PBS)). The end-product, MTT formazan was dissolved in 150 mL of isopropanol and the absorbance was measured at 570 nm with an ELISA reader (Bio-Rad Laboratories Ltd., Shanghai, China).
Western blot analysis Cells were washed with ice-cold PBS and treated with 100 mL of cell lysis buffer (Cell Signaling, Danvers, MA) containing protease inhibitors (Sigma, St. Louis, MO). Extracted proteins were separated on SDS-PAGE and then transferred electrophoretically onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA). Proteins were probed with specific antibodies as previously described [23]. The antibodies against HIF-1α, cyclin D1 were from Santa Cruz Biotechnology (Santa Cruz, CA), antibodies against Bcl-2 and Bax were from Boster Ltd. (Wuhan, China). Antibodies against 4EBP-1, phospho-4EBP-1 (Thr70), AMPKα, phospho-AMPKα (Thr172), p70 S6 kinase, phospho-p70 S6 kinase (Thr389) and P-gp were purchased from Cell Signaling Technology (Beverly,MA). The rabbit polyclonal antibody to β-actin was used as a gel loading control (Santa Cruz, Santa Cruz, CA, USA, 1:1000 dilution).
Analysis of apoptosis by flow cytometry Flow cytometry analysis was performed using FITC Annexin V apoptosis kit (BD Pharmingen, Franklin Lakes, NJ) according to manufacturer's instructions. Following treatment, the cells were washed with ice-cold phosphate buffered saline and then resuspended in binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2) at a concentration of 1 106 cells/ml. Cells were stained with annexin V-FITC and propidium (PI) for 15 min in dark before analyzing with flow cytometer (Beckman Coulter Inc., Miami, Florida, USA).
Caspase-3 activity assay Following the treatment, the cytosolic proteins of GBC-SD cells were extracted in hypotonic cell lysis buffer (25 mm HEPES, pH 7.2, 5 mM MgCl2, 5 mm EDTA, 5 mM dithiothreitol, 0.05% phenylmethylsulfonyl fluoride). The cytosolic extracts corresponding to 30 mg total protein were added to the reaction mixture containing 312.5 mm HEPES buffer, pH 7.5, 31.25% sucrose, 0.3125% CHAPS and the enzyme substrate, benzyloxycarbonyl-DEVD-7-amido-4-(trifluoromethyl) coumarin (Calbiochem, Darmstadt, Germany). After 1 h of incubation at 37 1C, the reaction product, 7-amido-4-(trifluoromethyl)coumarin (AFC) was detected with a fluorescence reader, set to the excitation and emission wavelengths of 355 nm and 525 nm respectively.
Analysis of cell cycle Cell cycle distribution was determined by DNA staining with propidium iodide (PI). Following the treatment, the cells were
Please cite this article as: H. Gao, et al., Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.11.021
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Fig. 1 – Effect of hispidulin on the proliferation (A), apoptosis (B), caspas-3 activity (C) and Bcl2 and Bax expression (D) in GBC-SD cells. The cells were incubated with hispidulin at the indicated concentration for 48 h before conducting the analyses. n and nn indicate that P values were o0.05 and o0.01 respectively and the effect was significant compared to the control. fixed with 70% cold ethanol at 4 1C overnight. DNA was stained with propidium iodide (0.05 mg/ml) and RNase (2 mg/ml) for 30 min at room temperature. Cell cycle was analyzed by flow cytometry (FCM, Becton-Dickinson, San Jose, CA) and the percentages of cells in the different phases of cell cycle were analyzed by FlowJo 7.6 software.
Overexpression of HIF-1α The GBC-SD cells were seeded at the sub-confluent densities and transfected with pcDNA3-EGFP empty vector or scrambled vector (SV) or pcDNA3-HA-HIF-1α expression construct TransIT-LT-1 following the manufacturer's instructions (Mirus Bio Corp., Madison, WI). Overexpression of HIF-1α was confirmed by Western blotting and real-time PCR analyses.
HIF-1α reporter activity assay Cells (1 105) were plated in 6-well culture plates and then transfected with HIF-1α reporter vector (Panomics, Redwood, CA) using lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Briefly, the cells were incubated with 0.5 μg HIF-1α reporter vector (Panomics, Redwood, CA) and 0.5 μg pEGFP-C2 per well in the presence and absence of hispidulin. After transfection, the luciferase activity assay was measured using a commercial kit (Promega Corp., Madison, MI) according the manufacturer's manual. pEGFP-C2 vector was used to normalize for transfection efficiency, and relative luciferase activity (defined as reporter activity) was calculated as the ratio of luciferase/EGFP activity.
Quantitative RT-PCR (qRT-PCR) Total RNA was extracted from the cells using the Total RNA Kit (TIANGEN Co., Beijing, China) and 3 mg of RNA was converted into cDNA using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). The primers were synthesized based on the published sequence [24]. The master mix of each PCR reaction included SYBR GREEN master mix (Solarbio Co., Beijing, China), forward and reverse primers, and 10 ng of template cDNA. The PCR reaction was carried out at 95 1C for 5 min followed by 40 cycles of 95 1C for 30 s, 60 1C for 30 s, and 72 1C for 30 s. Data were analyzed using the comparative ΔCt method (ABPrism software,
Please cite this article as: H. Gao, et al., Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.11.021
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Fig. 2 – Effect of hispidulin on the distribution of cells in different phases of cell cycle (A) and cyclin-D1 expression (B). The GBC-SD cells were incubated with hispidulin at the indicated concentrations for 48 h before the flow cyometric (A) and western blot analyses (B). * indicate that the effect was significant (Po0.05). Applied Biosystems, Foster City, CA) using GAPDH as the internal normalization control.
AMPKα1 shRNA transfection The transfection and colony selection was conducted as previously described [25]. The AMPKα1 short hairpin RNA (shRNA)containing lentiviral particles (Santa Cruz Biotech, Santa Cruz, CA, sc-29673-SH) or the scramble shRNA control lentiviral particles (Santa Cruz Biotech, Santa Cruz, CA, sc-108080) (20 μL/ml medium each) were added to GBC-SD cells and the incubation was continued for 48 h. The culture medium (containing 1 μg/ml of puromycin) was replaced every 48 h, and incubation of the cells continued until puromycin resistant colonies were formed. The single colony of the cells expressing shRNA was sub-cultured. The expression of AMPKα1 was confirmed by western blotting.
In vivo xenograft experiments The animal experiments were performed in accordance with CAPN (China Animal Protection Law), and the protocols were approved by the Animal Care and Use Committee of Qingdao University. Female BALB/c (nu/nu) mice, 6 weeks old, purchased from the Animal Centre of China (Beijing, China), were housed with a light/dark cycle of 12/12 h and allowed free access to rodent chow and water. GBC-SD cells were harvested from subconfluent cultures and washed in serum-free medium before being resuspended in PBS. The mice were anesthetized using ethyl ether, and 105 cells were injected subcutaneously into the right and left abdominal regions with a gauge needle. Tumor appearance was inspected every 3 days. Once tumor volume
reached 100 mm3, animals were randomized into 4 groups (n¼ 6 per group) to receive intraperitoneal (IP) injections as follows: (A) vehicle (0.9% sodium chloride plus 1% DMSO), (B) hispidulin (40 mg/kg/day, dissolved in vehicle), (C) hispidulin (20 mg/kg/ day, dissolved in vehicle) and (D) hispidulin (10 mg/kg/day, dissolved in vehicle) on a daily basis. Tumor volumes and body weight were measured twice per week. Tumor volume was measured along the longest orthogonal axes and calculated as volume¼ (length width2)/2, where width was the shortest measurement. All mice were sacrificed 30 days after the first day of treatment.
Statistical analysis The data are expressed as Mean7SD and represent the results of three separate experiments each performed in quadruplicate unless otherwise stated. The Student's test was used to evaluate if the difference between two groups was significant and One Way ANOVA with post hoc test was used for comparison between three or more groups. Po0.05 was considered as statistically significant.
Results Hispidulin inhibited the growth of GBC cells GBC-SD cells at logarithmic growth phase were seeded in 6-well tissue culture plates at a density of 1 104/ml and incubated overnight at 37 C with 5% CO2. The following day, cells were exposed to various concentrations (0, 12.5, 25, 50 μM) of hispidulin (Shanghai Tauto Biotech Co., Ltd., Shanghai, China, purity498%) and incubation
Please cite this article as: H. Gao, et al., Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.11.021
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Fig. 3 – Effect of hispidulin on HIF-1α mRNA and protein expressions measured by qRT-PCR and Western blotting respectively (A), cell viability and apoptosis (B) expression of Bcl-2 and Bax and caspase-3 activity (C) in the cells stably transfected with HIF-1α. The cells were incubated with 50 μM of hispidulin for 48 h before conducting the analyses. HIF-1α OE and His denote HIF-1α over expressed and Hispidulin respectively. ** and b represents Po0.01 vs. control, and Po0.05 vs. HIF-1αOE respectively.
was continued for 24, 48 and 72 h, then the number of viable cells was determined. As shown in Fig. 1A, hispidulin exhibited dose- and time-dependent anti-proliferative effects. Cells treated with hispidulin for 24 h at concentrations of 12.5, 25 and 50 μM exhibited the viability that was reduced by 10.373.2%, 17.575.2% and 33.676.7%, respectively. After 48 h incubation with hispidulin, the cell viability was decreased by 14.373.2%, 31.276.3% and 44.374.6%, respectively. When the incubation was prolonged to 72 h, further decrease in cell viability was observed. The inhibition of cells incubated for 48 h with 25 μM hispidulin was statistically significant as compared with the cells treated with vehicle.
Hispidulin promoted the apoptosis in GBC cells As shown in Fig. 1B, hispidulin increased the number of apoptotic cells in a dose-dependent manner. Thus, compared to the vehicletreated control cells, those incubated with 25 and 50 μM hispidulin, showed a significantly increased percentage of apoptotic cells (18.073.6% and 33.474.3% respectively). We also examined the effect of hispidulin on the activity of caspase-3, an enzyme that promotes apoptosis. As shown in Fig. 1C, the hispidulin-treated cells showed an elevated activity of caspase-3, indicating that hispidulin promoted apoptosis involved activation of this enzyme.
Please cite this article as: H. Gao, et al., Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.11.021
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The dose-dependent effect of hispidulin on the expression of apoptosis modulating molecules, Bcl-2 and Bax was also evidenced. As shown in Fig. 1D, the expression of Bcl-2 was decreased while that of the Bax was upregulated with the increasing doses of hispidulin. Collectively, these results suggest that hispidulin mediated the inhibition of cancer cells growth by promoting apoptosis.
Hispidulin-induced the cell arrest in G0/G1 phase of the cell cycle. The effect of hispidulin on cell cycle regulation was determined by flow cytometry. As shown in Fig. 2A, compared to the controls, the cells treated with hispidulin were accumulated in an increased proportion in G0/G1 phase with a concomitant decrease in their number in S and G2 phases. Since cyclin D1 plays an important role in the regulation of G1 progression and G1-S transition, we examined the effect of hispidulin on the expression of cyclin D1. As shown in Fig. 2B, hispidulin was found to down-regulate the cyclin D1 expression in a dose-dependent manner, suggesting the involvement of cyclin D1 in hispidulin-mediated blockade of the cell cycle progression in G1 phase.
Hispidulin-exerted anti-tumor effect was mediated through HIF-1α inhibition To investigate the effect of hispidulin on HIF-1α, we over expressed this protein by transfecting its cDNA into the GBC-SD cells. Cells transfected with an empty vector or non-transfected cells were used as controls. As depicted in Fig. 3A, cells transfected with HIF-1α showed increased expressions of HIF-1α protein and mRNA. The treatment of HIF-1α-transfected cells with hispidulin resulted in a reduced expression of HIF-1α protein. In addition, compared to the non-transfected cells or those transfected with an empty vector, the cells overexpressing HIF-1α exhibited a significantly increased viability and decreased apoptosis (Fig. 3B). Upon treatment with hispidulin, a marked reduction in proliferation and increased apoptosis of the cells was observed. The cells with HIF-1α overexpression also exhibited an upregulation of Bcl-2, and downregulation of Bax expressions. Hispidulin suppressed this effect of HIF-1α, and it also decreased the expression of Bcl-2 while increased that of Bax in control cells. The HIF-1α-overexpressing cells also showed a decreased activity of caspase-3 (Fig. 3C). In contrast, the cells treated with 50 μM of hispidulin exhibited a several fold increase in the activity of caspase-3 and suppressed the HIF-1α-mediated inhibition of this enzyme. Taken together, these results suggest that hispidulin blocked the HIF-1α-promoted cell proliferation and inhibition of apoptosis.
Hispidulin repressed the transactivation activity and expression of HIF-1α in GBC cells In order to examine the effect of hispidulin on the transfection of HIF-1α, we treated the GBC cells with a series of hispidulin concentrations before carrying out the transfection procedures. As shown by the reporter luciferase assay in Fig. 4B, the transactivation activity of HIF-1α was decreased with hispidulin in a dose-dependent way. The expression of HIF-1α was confirmed by determining mRNA and protein levels. Thus, in GBC cells the expression of HIF-1α protein
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determined by Western blotting was reduced with hispidulin dosedependently (Fig. 4A). However, the expression of HIF-1α mRNA remained unchanged even with the highest concentration of hispidulin (Fig. 4C). These results clearly indicate that hispidulin-mediated decrease in HIF-1α content occurred at the level subsequent to mRNA transcription (Fig. 4C).
Hispidulin-mediated down-regulated of HIF-1α protein expression involved the modulation of p70S6K and 4E-BP1 In order to further dissect the site of hispidulin's action, we examined its effect on the expression of phosphorylated 70S6K and 4E-BP (p70S6K and p4E-BPs), the key regulators of protein translation (see Section Discussion). Fig. 4D shows that hispidulin caused a decrease in the expression of phosphorylated p70S6K (on Thr389) and p4E-BP-1 (on Thr70) in a dose-dependent manner. This observation suggests that hispidulin-mediated reduction in HIF-1α expression was executed through blockade of protein translational activity involving p70S6K (on Thr389) and p4E-BP-1.
Hispidulin suppressed the HIF-1α expression via AMPK signaling The reduced phosphorylation of p70(S6K) and 4E-BP-1 has been shown to be associated with repressed HIF-1α expression and mitochondrial dysfunction. Moreover, the modulation of AMPK (AMP kinase) signaling has been proposed to play a major role in the expression of HIF-1α and cancer progression in HepG2 cells [27]. Therefore, we explored whether AMPK was involved in the hispidulinmediated suppression of HIF-1α synthesis. As shown in Fig. 5A, hispidulin produced a dramatic elevation of phosphorylated AMPK (on Thr172) expression in a dose-dependent manner. In the cells pretreated with compound C (C24H25N5O, Pyrazolo[1,5-a]pyrimidine,6[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl), a pharmacological AMPK inhibitor) or short hairpin RNA (shRNA) for 4 h, the expression of p-AMPK was found to be significantly reduced (Fig. 5B). This effect of the inhibitors on p-AMPK expression was not revealed to be reversed by hispidulin. However, the substantially increased HIF-1α expression caused by the inhibition of p-AMPK (both with compound C and shRNA) was significantly reduced by hispidulin (Fig. 5C). These results suggest that hispidulin reversed the p-AMPK-mediated inhibition of HIF-1α through a mechanism other than blocking the expression of AMPK-α. For instance, promoting the activation of the existing p-AMPK that is not bound with inhibitors or by releasing the bound inhibitor from pAMPK. Further work is required to find out if hispidulin accelerates the acivity of AMPK. Also, treatment of the cells with AMPK activator AICAR was found to significantly increase the pAMPK but reduce the HIF-1α protein expression (Fig. 5D). Corresponding to these observations, the cell viability was significantly reduced with hispidulin (Fig. 5E). Collectively, these findings suggest that hispidulin mediated the activation of AMPK which in turn caused the repression of HIF-1α protein synthesis resulting in reduced cell viability.
Hispidulin enhanced the chemosensitivity of GBC-SD cells by down regulating P-gp (P glycoproteins) P-gp are known to play an important role in imparting chemoresistance to gallbladder cancer and their downregulation has
Please cite this article as: H. Gao, et al., Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.11.021
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Fig. 4 – Effect of hispidulin on HIF-1α expression analyzed by Western blotting (A), the HIF-1α-transactivation measured by reporter activity (B), mRNA expression measured by qRT-PCR (C) and expression of phosphorylated p70S6K and 4EBP1 examined by Western blotting (D) in GBC-SD cells stably transfected with HIF-1α. The cells were incubated with various concentrations of hispidulin for 48 h before the analyses. * indicates that compared to the control, the effect was significant (Po0.05). been shown to increase the chemosensitivity of GBC cells [26]. We tested the effect of hispidulin on P-gp expression in the presence and absence of Gem or 5-FU, the chemotherapeutic agents frequently-used for the gallbladder cancer [27]. As depicted in Fig. 6A, Gem (0.5 μM) or 5-FU (1 μM) showed a moderate decrease in the viability and increase in the apoptosis of GBC-SD cells. However, the cells treated with a relatively low dose of hispidulin (12.5 μM), exhibited a significantly enhanced sensitivity to Gem or 5-FU. Hispidulin alone at this dose produced a minor effect. In order to explore the mechanism of hispidulin-enhanced cell sensitization, we determined its effect on P-gp expression. As shown in Fig. 6B, hispidulin markedly suppressed the P-gp expression in a dose-dependent manner. Given the fact that HIF-1α overexpression is associated with chemoresistance, we investigated whether it involved a role of P-gp. As shown in Fig. 6C, HIF-1α overexpression was associated with a significant increase in P-gp expression which was significantly reduced with hispidulin, indicating that the effect was, in part, mediated through blocking HIF-1α signaling.
Hispidulin suppressed tumor growth in vivo Based on the aforementioned results, we evaluated the in vivo therapeutic effect of hispidulin using an in vivo xenograft mouse model. Hispidulin was administered at 40 mg/kg/day, 20 mg/kg/ day, and 10 mg/kg/day in different treating groups, respectively. Treatment started when the tumor size reached 100 mm3. The
efficacy of the treatment was evaluated by measuring the tumor volume during treatment. As shown in Fig. 7, treatment with hispidulin dose-dependently inhibited the growth of tumor and dose at 20 mg/kg/day was found be able to exert significant antitumor effect (Po0.05 vs. vehicle).
Discussion Epidemiological studies have suggested that high consumption of flavonoids decreases the risk of a variety of cancers [28]. While investigating the anti-cancer activities of flavonoids, a few of the recent studies have focused on a small flavonoid, hispidulin [18–22]. Previously, we showed that hispidulin suppressed the cell growth and induced apoptosis in heptocellular carcinoma cells [22]. This study was designed to evaluate the therapeutic effect of hispidulin in GBC and to investigate the underlying mechanism(s) of its action. Our results revealed that hispidulin exhibited a dose- and timedependent anti-proliferation effect on GBC-SD cells by promoting apoptosis and cell cycle arrest in G0/G1 phase. In the previous study, we found that hispidulin treatment resulted in mitochondrial dysfunction in heptocellular carcinoma cells [22]. Interestingly, a recent study has reported that mitochondrial dysfunction induced by mitochondrial inhibitor or uncoupler under normoxia led to the repression of HIF-1α expression [24]. Given that HIF-1α plays a critical role in the tumor cell physiology, we postulated
Please cite this article as: H. Gao, et al., Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.11.021
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Fig. 5 – Hispidulin-mediated inhibition of HIF-1α expression via AMPK activation. Effect of hispidulin on phosphorylated to unphosphorylated AMPK ratio (A), p-AMPK/AMPK in the presence of Com C or shAMPK (B), HIF-1α expression in the presence of Com C or shAMPK (C), on p-AMPK/AMPK ratio in the presence of ALCAR (D), on cell viability (E). GBC-SD cells were incubated with hispidulin at the indicated concentrations for 48 h before the analyses. His and Com C denote hispidulin and compound C respectively. * and ** represent Po0.05 and Po0.01 respectively, significant effect vs. control, b represents Po0.05, significant effect of hispidulin vs. compound C or shAMPKα treatment. that hispidulin might suppress tumor cell growth by targeting HIF1α. In experimental models, expression/activation of HIF-1α can be achieved by transfection or by hypoxia. In this study, we stably transfected the GBC-SD cells with HIF-1α gene to over express this transcription factor so as to rule out the interference from non-HIF1α-mediated cellular response to hypoxia. Our results showed that over-expression of HIF-1α resulted in promoted cell proliferation and inhibited apoptosis. Hispidulin blocked the expression of HIF1α and the consequent actions of increased cell viability and attenuated apoptosis. Our results are in complete agreement with
other studies reporting that downregulation of HIF-1α resulted in reduced tumor cell survival [29]. A number of studies have identified HIF-1α as the target molecule of flavonoids action in tumor cells. For instance, Mylonis et al. [33] reported that the dietary flavonoid kaempferol effectively inhibited the viability of hepatoma cancer cells by impeding HIF-1α expression and activity under hypoxic conditions. Another flavonoid, apigenin has been found to inhibit expressions of both HIF-1α mRNA and protein in pancreatic cancer cells under normoxic and hypoxic conditions [30]. Silibinin and epigallocatechin-3-gallate (EGCG) have been shown
Please cite this article as: H. Gao, et al., Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.11.021
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Fig. 6 – Hispidulin enhanced the chemosensitivity of GBC-SD cells to Gem and 5-FU, by downregulating P-gp expression. (A) Hispidulin-mediated sensitization of GBC-SD cells to Gem (0.5 μM) and 5-FU (1 μM), (B) Hispidulin repressed the P-gp expression, (C) effect of 50 μM hispidulin on HIF-1α-induced increased P-gp expression. The analyses were performed after incubating the cells with the reactants for 48 h. His and HIF-1α-OP denote hispidulin and HIF-1α over expressed respectively. * and b represent Po0.05 and significant effect vs. control, and vs. HIF-1α OE respectively.
Fig. 7 – Measurements of gallbladder tumor volume at the indicated time points, depicting the in vivo therapeutic efficacy of hispidulin with a representative picture of mouse model at the end of the experiment. * represent Po0.05, significant effect vs. vehicle.
to inhibit the HIF-1α protein expression without affecting that of the mRNA in prostate cancer and pancreatic carcinoma cells respectively [31]. In this study, we found that GBC-SD cells treated with hispidulin displayed a dose-dependent decrease in the expression and activity of HIF-1α. Whereas, the protein expression of HIF-1α was markedly inhibited, the mRNA expression was not changed with hispidulin, indicating that the effect was exerted at the post transcriptional level. The hispidulin-induced significant reduction of p-4E-BP1 and p-p70 S6 kinase expression suggests that the compound repressed the initiation of HIF-1α translation. Based on the aforementioned studies and our results, we suggest that different flavonoids modulate HIF-1α synthesis and/or activity by modulating distinct signaling pathways.
The key regulators of protein translation, p70S6K and 4E-BP are known to be the targets of mTOR signaling [32]. In addition, AMPK has been identified to play an important regulatory role in the activation of mTOR signaling that involves protein synthesis, cell growth and viability. During the energy stress, AMPK represses mTOR signaling by phosphorylating tuberous sclerosis complex 2 (TSC2), and thereby enhancing the formation of TSC complex (TSC1/TSC2), a key modulator of mTOR functioning [33]. AMPK is also known to inhibit mTOR signaling independent of TSC1/TSC2 pathway by directly phosphorylating raptor, a component of the mTOR complex 1 (mTORC1) [34]. Recent studies have revealed a correlation between pharmacological activation of AMPK and decreased HIF-1α expression [25,35]. Our results showed that hispidulin promoted the increased
Please cite this article as: H. Gao, et al., Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.11.021
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the expression of phosphorylated AMPK, the active form of AMPK. Furthermore, our results showed that pharmacological inhibition (using compound C) or gene knock-down (using shRNA) led to an enhanced expression of HIF-1α, suggesting a negative correlation between the two molecules. We observed that hispidulin reversed the effect of AMPK inhibition and that caused an up-regulation of HIF-1α, at least in part. Since in this study, hispidulin did not reverse the effect of inhibitors on the expression of p-AMPK, we propose that hispidulin-retrieved inhibition of HIF-1α was mediated through a mechanism other than protein expression. Further work is needed to find out if hispiduline accelerates the activity of pAMPK. In the presence of compound C and shAMPKα, the hispidulin-induced decrease in cell viability was partially prevented, further supporting the notion that the anti-tumor activity of hispidulin involved the activation of AMPK and consequent inhibition of HIF-1α. Because of the complexity of AMPK functions in the tumor cell physiology, role of other molecule(s) than HIF-1α in hispidulin-stimulated loss of cell viability cannot be ruled out. However, our study has evidenced that hispidulin exerts its anti-tumor effect through modulation of AMPK/ HIF-1α signaling at least in part. In future, we intend to investigate the role of TSC1/TSC2 in AMPK-regulated HIF-1α expression and function to further understand the mechanism(s) involved in this process. Because of the hypoxic conditions, cancer cells have been shown to be resistant to anti-neoplastic agents [36]. The multidrug resistance gene (MDR1) that encodes P-glycoproteins has been found to be up-regulated in hypoxia in an HIF-1α dependent manner. MDR1/P-gp, a member of the ABC (adenosine triphosphate binding cassette) transporter family, promotes the excretion of xenobiotics and drugs including chemotherapeutics and results in their decreased intracellular concentrations [37]. Moreover, the presence of HIF-1α binding domain and auxiliary sequence similar to hypoxia-responsive element (HRE) detected in the MDR1 gene promoter has led to the suggestion that MDR1 might actually be a hypoxia-responsive gene [38]. Recently, a number of studies have shown the contribution of HIF-1αmediated up-regulation of P-gp expression to hypoxia-induced drug resistance in a variety of human tumors, such as gastric, colon, gliomas, and breast carcinoma [38–41]. Clearly inferring that HIF-1α/MDR1 signaling may serve as a promising target of anti-neoplastic drugs. Consistent with these reports, our results showed that pharmacological suppression of HIF-1α resulting in down-regulation of P-gp enhanced the chemosensitivity of GBCSD cells to Gem and 5-FU. Taken together, our results suggest that hispidulin mediated the downregulation of MDR1/P-gp expression by interfering with the translation of HIF-1α in GBC-SD. In summary, we observed that hispidulin inhibited cell growth, induced apoptosis and blocked cell cycle progression. Our study has demonstrated for the first time that the anti-neoplastic effects of hispidulin were mediated at least in part, by down-regulating HIF-1α expression through the activation of AMPK. The hispidulin-mediated repression of HIF-1α expression was furthered to P-gp downregulation which in turn potentiated the anti-tumor efficacy of Gem and 5-FU. Indeed, it is a novel mechanism that explains the hispidulin-elicited sensitization of the cancer cells to chemotherapeutic agents.
Conflict of interest We declare that we have no conflict of interest.
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Acknowledgment This work was supported by the National Natural Science Foundation (Nos. 81473384 and 31470570) and funds from the Qingdao University (Nos. 14-2-3-50-nsh, 13-1-3-74 and 600201304).
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Please cite this article as: H. Gao, et al., Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.11.021
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Please cite this article as: H. Gao, et al., Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.11.021
