Chemico-Biological Interactions 232 (2015) 12–20

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Chemico-Biological Interactions

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Combination of chrysin and cisplatin promotes the apoptosis of Hep G2cells by up-regulating p53

http://dx.doi.org/10.1016/j.cbi.2015.03.0030009-2797/� 2015 Published by Elsevier Ireland Ltd.

Abbreviations: ERK1/2, extracellular signal-regulated knase 1 and 2; Bax, Bcl-2-associated X protein ; DR5, death receptor 5; Bcl-2, B-cell lymphoma 2.⇑ Corresponding authors. Tel.: +86 20 61648301; fax: +86 20 61648324 (F. Zou).

Tel./fax: +86 20 31051866 (X.-F. Yang).E-mail addresses: feizoudean@sina.com (F. Zou), xfenyang@163.com (X.-F. Yang).

1 These authors contributed equally to this study.

Xin Li a,b,1, Jun-Ming Huang b,1, Jian-Ning Wang c, Xi-Kun Xiong b, Xing-Fen Yang b,⇑, Fei Zou a,⇑a Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, 1838 Northern GuangzhouAvenue, Guangzhou, Guangdong Province 510515, PR Chinab Center for Disease Control and Prevention of Guangdong Province, 160 Qunxian Road, Dashi, Panyu District, Guangzhou, Guangdong Province 511430, PR Chinac Department of Oral and Maxillofacial Surgery, Guanghua School of Stomatology, Institute of Stomatological Research, Sun Yat-sen University, 56, Ling Yuan Xi Road, Guangzhou,Guangdong Province 510055, PR China

a r t i c l e i n f o

Article history:Received 23 September 2014Received in revised form 21 January 2015Accepted 4 March 2015Available online 11 March 2015

Keywords:ChrysinCisplatinApoptosisp53ERK1/2Cancer

a b s t r a c t

Cisplatin is a chemotherapy drug commonly used for the treatment of human cancers, however, drugresistance poses a major challenge to clinical application of cisplatin in cancer therapy. Recent studieshave shown that chrysin, a natural flavonoid widely found in various plants and foods, demonstratedeffective anti-cancer activity. In the present study, we found that the combination chrysin and cisplatinsignificantly enhanced the apoptosis of Hep G2 cancer cells. Combination of chrysin and cisplatinincreased the phosphorylation and accumulation of p53 through activating ERK1/2 in Hep G2 cells, whichled to the overexpression of the pro-apoptotic proteins Bax and DR5 and the inhibition of the anti-apoptotic protein Bcl-2. In addition, combination of chrysin and cisplatin promoted both extrinsicapoptosis by activating caspase-8 and intrinsic apoptosis by increasing the release of cytochrome cand activating caspase-9 in Hep G2 cells. Our results suggest that combination of chrysin and cisplatinis a promising strategy for chemotherapy of human cancers that are resistant to cisplatin.

� 2015 Published by Elsevier Ireland Ltd.

1. Introduction

Cis-Diammine-dichloro-platinumII (cisplatin) is a chemother-apy agent widely used for the treatment of numerous humantumors, such as head and neck, testicular, ovarian, cervical, lung,and colorectal cancers, as well as relapsed lymphoma [1].Cisplatin binds to DNA bases, leading to the activation of p53,P38 MAPKs, and/or JNKs, which finally induces tumor cell apopto-sis [2–4]. The role of p53 in cisplatin-induced cancer cell apoptosishas been extensively studied [5]. Typically, P53 mediates apoptosisin both transcription-dependent and transcription-independentmanners. The transcription-dependent pathway directly regulatesthe expression of p53 downstream genes that affect the sensitivityto apoptosis [6,7]. In addition, p53 can move out of nuclei andinteract with mitochondria or mitochondrial proteins such asBcl-2 and Bcl-xl to induce transcription-independent apoptosis

[8]. Under normal condition in unstressed cells, p53 is sequestratedin cytoplasm by MDM2, which promotes p53 degradation via theubiquitin/proteasome pathway [7]. In the case of DNA damagecaused by cisplatin, the function of MDM2 is impaired and phos-phorylated p53 is translocated to nuclei to cause apoptosis throughvarious p53 pathways.

Flavonoids are natural polyphenolic phytochemicals that areubiquitous in plants. Numerous epidemiological studies andanimal experiments have demonstrated the anti-cancer andchemopreventive properties of flavonoids [9,10]. Therefore, flavo-noids have been regarded as ideal candidates for chemotherapyof human cancers. It has been reported that chrysin (5,7-dihydrox-yflavone), a flavone commonly found in various plants and foods,had strong anti-inflammation [11] and antioxidative [12] proper-ties. In addition, chrysin exhibited anti-cancer activity in a diverserange of human [13–18] and animal derived cells [19]. However,the underlying mechanisms of the anti-cancer property of chrysinare not fully understood. Our previous studies have shown thatchrysin sensitized apoptotic cell death induced by tumor necrosisfactor (TNF) [20] or tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) in various human cancer cells [21].

X. Li et al. / Chemico-Biological Interactions 232 (2015) 12–20 13

Resistance to cisplatin is one of the major limitations ofcisplatin-mediated chemotherapy of human cancers. The presentstudy aimed to investigate whether the combination of chrysinand cisplatin can significantly improve the apoptosis of a numberof human cancers cells that are common in Southern China. Wealso studied the underline mechanism by evaluating the expres-sion of several proteins involved in the p53 pathway, which playsa central role in cancer cell apoptosis.

2. Materials and methods

2.1. Reagents and chemicals

Chrysin, cisplatin, and U0126 were purchased from Sigma (St.Louis, MO, USA). Pan-caspase inhibitor z-VAD-fmk was purchasedfrom Bio Mol (Minnesota, USA). Antibodies against caspase-3, cas-pase-8, caspase-9, Bcl2, Bax, p53, phosphorylated p53 (Ser15), DR5,a-tubulin, and GAPDH were purchased from Cell Signaling (Boston,MA, USA). Antibody against cytochrome c was from Epitomics(Burlingame, USA). Antibodies against MDM2 and phosphorylatedMDM2 were from Bioworld (Atlanta, USA). Antibody against PARPwas from BD Company (Franklin, USA). Goat anti-mouse IgG andFICT conjugated were from CW BIO (Beijing, China).Hoechst33342 was from GenMed (Boston, USA). The secondaryantibodies including horseradish peroxidase–conjugated goatanti-mouse IgG and goat anti-rabbit IgG, and the enhancedchemiluminescence substrate were from BIO-RAD (Berkeley,USA). All other common chemicals were from Sigma (St. Louis,MO, USA).

2.2. Cell culture and treatments

The human colorectal cancer cell line HCT-116 and the humanliver cancer cell lines HepG2 and Hep 3B were obtained from theAmerican Type Culture Collection (ATCC, Manassas, VA). Thehuman nasopharyngeal carcinoma cell line CNE1 was from SunYat-Sen University (Guangzhou, China). All cells were maintainedin DMEM medium (Gibco, USA), supplemented with penicillin(100 units/mL), streptomycin (100 lg/mL), 10% fetal bovine serum(Hyclone), and 5% CO2 at 37 �C. Regarding the combinationtreatment, cancer cells were pretreated with chrysin for 2 h, thenfollowed by cisplatin treatment for different periods of time.

2.3. Measurement of cell death and apoptosis

Cells in sub-G1 phase were measured to evaluate cell deathrates according to the method previously described [20]. Giventhat cells in sub-G1 phase can be either apoptotic or necrotic,apoptotic cancer cells was further confirmed by staining witheither Hoechst 33342 or DAPI. Typically, apoptotic cells exhibit acharacteristic chromatin condensation based on Hoechst 33342or DAPI staining. Briefly, cancer cells were stained with Hoechst33342 for 20 min or stained with DAPI after paraformaldehydefixation, then visualized under an inverted fluorescence micro-scope. The percentage of apoptotic cells were quantified by count-ing the cells with condensed nuclei among 200 randomly selectedcells.

2.4. Extraction of cytosolic proteins

Cytosolic proteins were extracted using the MitochondriaIsolation Kit (Thermo Scientific, USA) according to the manufac-turer’s instruction. Briefly, cancer cells were washed withReagent A at 4 �C, then resuspended in Reagent A for 2 min. AfterReagent B was added, the solution was incubated for 5 min before

Reagent C was added and mixed. Then, cells were centrifuged at700�g for 10 min to collect the supernatant. The cytosolic proteinswere obtained after centrifugation at 12,000g for 15 min. The levelof cytochrome c in the cytosolic protein samples was evaluatedusing Western blotting analysis.

2.5. Western blotting analysis

Cancer cells were harvested and washed in PBS once. Cellpellets were lysed in lysis buffer (20 mM Tris–HCl, pH 7.5, 1%NP-40, 1% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA,1 mM PMSF, 1 mM Na3VO4, 2.5 mM sodium pyrophosphate,1 mM b-glycerophosphate, 1 lg/mL leupeptin) with protease/phosphatase inhibitor cocktail (CST). Aliquots of the lysates weresubjected to SDS–PAGE and transferred to polyvinylidene-fluoridemembranes (Bio-Rad). After blocking with 5% nonfat milk in TBST[10 mmol/L Tris–HCl (pH7.5), 100 mmol/L NaCl, and 0.05%Tween20], the membrane was probed with various antibodiesand developed with enhanced chemiluminescence (Bio-Rad), usinga CHEMI-SMART 3000 image station (VILBER-LOURMAT).

2.6. Immunofluorescence

HepG2 cells were seeded in 24-well chamber slides 24 h beforetreatment. After treatment, cells were immediately washed withPBS and fixed in 3% paraformaldehyde for 1 h. They were thenpermeabilized for 2 min with 0.2% CHAPS in PBS, blocked in 2%BSA with 0.2% Tween-20 for 30 min, and then incubated withanti-p-p53 (ser15) antibody overnight at 4 �C. After washing withPBS (+0.2% Tween-20), cells were incubated with anti-mouseFITC secondary antibody for 1 h. Cells were visualized under aCellomics Toxinsight Reader (Thermo). The intensity of the p-p53(ser15) nuclear staining was measured in 49 fields.

2.7. RNA extraction and RT-PCR

Total RNA was isolated from HepG2 cells using the PureLinkRNA Mini Kit (Ambion). RT-PCR amplifications were performedusing OneStep RT-PCR Kit (QIAGEN) in a T100™ Thermal Cycler(Bio-rad). The RT-PCR program was 50 �C for 30 min, and 95 �Cfor 15 min., and then followed by 94 �C for 30 s, 55 �C for 30 sand 72 �C for 40 s, for a total of 36 cycles. The following primerswere used for RT-PCR assay of p53 mRNA: 50-TAACAGTTCCTGCATGGGCGGC-30 (forward) and 50-AGGACAGGCACAAACACGCACC-30 (reverse). The following primer sequences were used for RT-PCR assay of GAPDH mRNA 50-GAAGGTGAAGGTCGGAGTC-30

(forward), 50-GAAGATGGTGATGGGATTTC-30 (reverse).

2.8. Statistical analyses

All numerical data obtained from at least three independentexperiments were presented as mean ± standard deviation (SD).The differences among distinct groups were evaluated using aone-way ANOVA with LSD’s test (SPSS 13.0). A p value <0.05was considered statistically significant.

3. Results

3.1. The combination of chrysin and cisplatin promoted the death ofhuman cancer cells HepG2, CNE1, and HCT-116

A previous study showed that cisplatin-induced cell death wasa relatively slow process, normally requiring 2–3 days [3]. In thepresent study, no significant cell death was observed in the threetypes of cancer cells when they were treated with cisplatin alone

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(5 lg/mL for HepG2 cells, 10 lg/mL for CNE1, and HCT-116 cells)for 24 h, based on morphological examination using an invertedmicroscope (Fig. 1A). Similarly, no obvious cell death was observedwhen the cancer cells were treated with chrysin alone (40 lM forHepG2 and CNE1 cells, and 20 lM HCT-116 cells). We next testedthe effects of the combination of chrysin and cisplatin on cancercell death. The effects of the combination of chrysin and cisplatinon the death of Hep G2 cells was evaluated by measuring the per-centage of sub-G1 cells using flow cytometry. Our results showedthat chrysin alone (20 and 40 lM) and the combination of chrysin(20 or 40 lM) and cisplatin (5 lg/mL) significantly increased thedeath of Hep G2 cells compared with without any treatment(p < 0.05) (Fig. 1B). In addition, the rate of dead Hep G2 cells trea-ted with the combination of chrysin (40 lM) and cisplatin (5 lg/mL) was significantly higher than that of Hep G2 cells treated withchrysin (10-40 lM) or cisplatin (5 lg/mL) alone (p < 0.05) (Fig. 1B).Therefore, the combination of chrysin and cisplatin increased thedeath of Hep G2 cells at a dose-dependent manner. Similarly, thecombination of chrysin (40 lM) and cisplatin (5 lg/mL) signifi-cantly increased the death of Hep G2 cells at a time-dependentmanner (p < 0.05) (Fig. 1C). Significant increase of cell death wasobserved in Hep G2 cells after 6 h of co-treatment with chrysin(40 lM) and cisplatin (5 lg/mL) (Fig. 1C).

Fig. 1. Combination of chrysin and cisplatin promoted the death of human cancercells HepG2, CNE1, and HCT-116. (A) Cancer cells were treated with chrysin alone(40 lM for HepG2 and CNE1 cells, and 20 lM for HCT-116 cells) for 2 h, cisplatin

3.2. Caspase-dependent apoptosis was involved in increased death ofHep G2 cells induced by the combination of chrysin and cisplatin

Previous studies reported that cisplatin could induce both apop-tosis and necrosis [4,22]. Given that cells at sub-G1 phase can beeither apoptotic or necrotic [23], we then investigated whetherthe combination of chrysin and cisplatin induced cell deaththrough apoptosis based on DAPI staining. Hep G2 cells werestained with DAPI and examined under a fluorescent microscope.The treatment with the combination of chrysin and cisplatinresulted in evident shrinkage and chromatin condensation of HepG2 cells (Fig. 2A), which are the characteristic signs of apoptosis.To confirm the apoptosis, we evaluated the expression of PARPand caspase-3, two biochemical markers of apoptosis, in Hep G2cells. We found that the combination of chrysin and cisplatincaused a decrease of pro-proteins and an increase of PARP andcaspase-3 cleavages in a dose-dependent manner in Hep G2 cells(Fig. 2B). Consistently, pretreatment of Hep G2 cells with Z-VAD-fmk, a pan-caspase inhibitor, efficiently blocked PARP andcaspase-3 cleavage (Fig. 2B) and reversed the cell death (Fig. 2C).Taken together, our results suggest that the combination of chrysinand cisplatin promoted the apoptosis of Hep G2 cells, in whichcaspase was involved.

alone (5 lg/mL for HepG2 cells, and 10 lg/mL for HCT-116 and CNE1 cells) for 24 hor the combination of chrysin and cisplatin. Regarding the combination treatmentof chrysin and cisplatin, cells were first treated with chrysin (40 lM for HepG2 andCNE1 cells, and 20 lM for HCT-116 cells) for 2 h, and then treated with cisplatin(5 lg/mL for HepG2 cells, and 10 lg/mL for HCT-116 and CNE1 cells) for 24 h.Representative images of cells with different treatments were photographed using alight microscope. (B) The death rate of HepG2 cells based on flow cytometryanalysis of cells at sub-G1 phrase. HepG2 cells were treated with variousconcentrations of chrysin for 2 h, then treated with cisplatin (5 lg/mL) for 24 h.(C) The death rate of HepG2 cells based on flow cytometry analysis of cells at sub-G1 phrase. HepG2 cells were treated with chrysin (40 lM) for 2 h, then treated withcisplatin (5 lg/mL) for different periods of time (0, 6, 12, or 24 h). Data from threeindependent experiments were presented as means ± SD. ⁄p < 0.05 in comparison tocells without any treatment; #p < 0.05 in comparison to cisplatin alone and chrysinalone (one-way ANOVA with LSD’S test).

3.3. p53 was involved in the apoptosis of Hep G2 cells induced by thecombination of chrysin and cisplatin

Given that p53 is the key regulator involved in cisplatin-mediated apoptosis of cancer cells [24], we compared the apoptosisbetween wild type Hep G2 cells and Hep3B cells with p53 deletion(http://p53.free.fr/Database/Cancer_cell_lines/HCC.html) inducedby the combination of chrysin and cisplatin. Significantly moreapoptotic cells were observed in Hep G2 cells than in Hep3B cellsafter co-treatment with chrysin and cisplatin (Fig. 3A). Next, weexamined the expression of p53 in Hep G2 cells treated withchrysin, cisplatin, or a combination of both. The protein level ofp53 in Hep G2 cells was significantly increased as early as 6 h afterchrysin treatment. However, no significant change of p53 expres-sion was found in cisplatin-treated Hep G2 cells, which may beexplained by the low concentration of cisplatin and short treatmenttime employed in the present study (Fig. 3B). Interestingly, the

combination of chrysin and cisplatin further increased the proteinlevel of p53 in Hep G2 cells, especially after 12 h of co-treatment.

The accumulation of phosphorylated p53 (p-p53) was consis-tent with increased expression of p53 protein (Fig. 3B).Densitometric analysis of p53 and p-p53 protein levels showed

Fig. 2. The combination of chrysin and cisplatin significantly promoted caspase-dependent apoptosis of Hep G2 cells. (A) HepG2 cells were pretreated with chrysin (40 lM)for 2 h, then followed by incubation with cisplatin (5 lg/ mL) for 24 h. Then, Hep G2 cells were stained with DAPI and examined under an inverted fluorescence microscope.Apoptotic cells, which exhibited condensed nuclei, were counted. (B) Hep G2 cells were pretreated with different concentrations of chrysin for 2 h and followed by incubationwith cisplatin (5 lg/mL) for 24 h. Total proteins were extracted from Hep G2 cells and subjected to Western blotting analysis to evaluate the levels of caspase-3 and PARPproteins. GAPDH was used as a loading control. (C) Hep G2 cells were pretreated with pan-caspase inhibitor, Z-VAD-fmk (20 lM), for 30 min before cells were incubated withthe combination of cisplatin and chrysin as mentioned above. The cell death rate was measured by determining the sub-G1 cells using flow cytometry. Data in A and Cobtained from three independent experiments were expressed as mean ± SD. ⁄p < 0.05 in comparison to respective controls; #p < 0.05, in comparison to the combination ofcisplatin and chrysin.

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that the combination of cisplatin and chrysin significantlyincreased the expression of p53 and p-p53 (Ser15) after 12 h ofco-treatment, which led to the increase of the ratio of p-p53(Ser15) to p53. In addition, translocation of the phosphorylatedp53 (Ser15) protein to nuclei was also observed when Hep G2 cellswere treated with chrysin alone (40 lM), or the combination ofchrysin (40 lM) and cisplatin (5 lg/mL) for 12 h based onimmunofluorescence microscopy (Fig. 3C). Taken together, theseresults suggest that p53 was involved in the apoptosis of Hep G2cells induced by the combination of chrysin and cisplatin.

3.4. The combination of chrysin and cisplatin promoted the apoptosisof Hep G2 cells through p53 pathways

The p53 signaling pathways associated with apoptosis arecomplicated and involve a large number of molecules.Phosphorylation and activation of p53 by upstream kinases regu-late p53 downstream genes and the biological consequences [25].To further elucidate the mechanisms in which p53 is involved inapoptosis promoted by the combination of chrysin and cisplatin,we examined the downstream molecular targets and biologicalconsequences of p53 activation after Hep G2 cells were treatedwith the combination of chrysin and cisplatin. Bax and DR5 aretwo main pro-apoptotic genes targeted by p53 [26–28]. DR5 is amember of the tumor necrosis factor receptor superfamily andinitiates extrinsic apoptosis by participating in the formation ofdeath-inducing signaling complex (DISC) to activate caspase-8[27]. In the intrinsic apoptosis pathway, Bax is associated with

mitochondria and causes cytochrome c release, which activatespro-caspase-9 [29,30]. In contrast, the anti-apoptotic protein Bcl-2 can block intrinsic apoptosis by binding to Bax to form Bcl-2–Bax heterodimers [31]. While p53 is not the direct transcriptionalfactor for Bcl-2, it is an important negative regulator for Bcl-2[26,32]. Increased expressions of Bax and DR5 and reduced expres-sion of Bcl-2 were observed in Hep G2 cells treated with thecombination of chrysin and cisplatin. Based on densitometricanalysis, the ratio of Bax to Bcl-2 protein levels in Hep G2 cellswere significantly increased after 18 h of co-treatment of chrysinand cisplatin (Fig. 4A). In addition, we observed the release of cyto-chrome c to cytosol (Fig. 4B) and the activation of caspase-9 andcaspase-8 (Fig. 4C). The expression changes of proteins involvedin the p53 pathways suggest that the p53 pathways were involvedin the apoptosis of Hep G2 cells induced by the combination ofchrysin and cisplatin.

3.5. p53 was activated by ERK through phosphorylation at Ser15

While increased expression of p53 protein was observed in HepG2 cells treated with the combination of chrysin and cisplatin, nosignificant change of the mRNA level of P53 gene was detectedby RT-PCR assay (Fig. 5A). Therefore, we speculate that the changeof p53 protein is caused by post-translation modification of theprotein. We analyzed the oncoprotein MDM2, a p53-bindingprotein that promotes p53 degradation [7,33]. Nevertheless, nosignificant changes of MDM2 and phosphorylated MDM2 wereobserved at the early stage of chrysin treatment (Fig. 5B).

Fig. 3. P53 was involved in the apoptosis of Hep G2 cells induced by the combination of cisplatin and chrysin. (A) HepG2 and Hep3B cells were pretreated with chrysin for 2 h(40 lM), then incubated with cisplatin (5 lg/mL) for 24 h. Subsequently, cells were stained with Hochest 33342. Apoptotic cells exhibited condensed nuclei. The percentagesof apoptotic cells obtained from three independent experiments were presented as mean ± SD. p < 0.05 compared with the control group (Student’s t-test). (B) Hep G2 cellswere treated with cisplatin (5 lg/mL) and with or without chrysin pretreatment (40 lM for 2 h) for different periods of time (1, 3, 6, or 12 h). Cell lysates were collected forthe detection of phosphorylated p53 (Ser15) and total p53 protein levels by Western blotting. The content of GAPDH was used as a loading control. The expression levels oftotal p53 and p-p53 at 12 h were quantified by densitometry and normalized to the internal control GAPDH. The ratio of p-p53 to p53 was calculated by densitometry. (C)Hep G2 cells were treated with chrysin (40 lM), cisplatin (5 lg/mL), or a combination of the two for 12 h. The translocation of phosphorylated p53 (Ser15) to nuclei wasdetected by immunofluorescence. Images of nuclear localization of phosphorylated p53 (Ser15) were photographed under inverted fluorescence microscope. The intensity ofphosphorylated p53 (Ser15) fluorescence in nuclei was evaluated by Cellomics Toxinsight Reader. DAPI staining was used to visualize nuclei. Data obtained from threeindependent experiments in B and C are presented as means ± SD. ⁄p < 0.05 in comparison to chrysin or cisplatin alone (one-way ANOVA with LSD’S test).

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Phosphorylation-dependent post-translational modification playsan important role in p53 stabilization and activation [29], andthe antitumor effects of DNA damaging agents are dominantly gov-erned by phosphorylation events [34]. As shown in Fig 3B and C,dramatic p53 phosphorylation at the Ser15 site was detected afterchrysin pretreatment. Mitogen-activated protein kinases (MAPKs),including the stress-activated protein kinase/c-Jun-N-terminalkinase (SAPK/JNK), the p38 mitogen-activated protein kinase(MAPK), and the extracellular signal-regulated kinase (ERK), are

the key upstream kinases that phosphorylate and regulate p53[35]. Activation of ERK1/2 was observed in Hep G2 cells duringthe entire process of co-treatment with chrysin and cisplatin(Fig. 5C), as well as chrysin treatment alone. These observationwas concurrent with p53 activation in Hep G2 cells treated withchrysin alone as well as the combination of chrysin and cisplatin(Fig. 3B and C). Additionally, chrysin alone or the combination ofchrysin and cisplatin dramatically activated ERK1/2 at early stages(Fig. 5D). We showed that U0126, an inhibitor of ERK1/2, partially

Fig. 4. Chrysin pretreatment affected the expression of downstream molecules of the p53 pathways in Hep G2 cells. (A) Hep G2 cells were pretreated with or without chrysin(40 lM for 2 h), then followed by incubation with cisplatin (5 lg/mL) for 3, 6, 12, and 18 h. Cells were harvested and the extracted total proteins were subjected to Westernblotting analysis for detecting the expression levels of Bcl-2, Bax, and DR5 proteins. The expression levels of Bcl-2 and Bax proteins after 18 h of cisplatin treatment werequantified by densitometry, and normalized to the internal control tubulin. The ratio of Bax/Bcl-2 was calculated. The values were represented as means ± SD, ⁄p < 0.05 versusthe chrysin or cisplatin treatment alone (one-way ANOVA with LSD’S test). (B) Hep G2 cells were treated with chrysin (40 lM), cisplatin (5 lg/mL), or the combination of bothfor 12 h. Cytosolic proteins were extracted and subjected to Western blotting for the detection of cytochrome c. (C) Hep G2 cells were pretreated with various concentrationsof chrysin (10, 20, and 40 lM) for 2 h, followed by incubation with cisplatin (5 lg/mL) for 24 h. Cells were harvested and the extracted proteins were subjected to Westernblotting analysis for detecting the cleavages of caspase-8 and caspase-9. The expression of tubulin was used as a loading control.

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blocked p53 phosphorylation at Ser15 (Fig. 5E) and inhibited thetranslocation of phosphorylated p53 to nuclei induced by thecombination of chrysin and cisplatin (Fig. 5F).

4. Discussion

The anti-carcinogenic activity of chrysin has been reported bynumerous studies [16–18,20,21]. A number of molecular pathwayscontribute to the anticancer property of chrysin, such as sensitizingTNFa-mediated apoptosis by inhibiting NFjB activation [20], orreducing Nrf2 expression through down-regulating PI3K-Akt andthe ERK pathway to reverse doxorubicin resistance [36]. In thepresent study, we further addressed the anti-cancer mechanismof chrysin by combining it with cisplatin. Cisplatin has been usedas one of the most important chemotherapy agents for clinicalmanagement of human cancers over the last three decades.However, cisplatin resistance is a serious challenge to its wideapplication [37]. It has been reported that cisplatin resistancewas mainly caused by inhibited apoptosis of cancer cells [38].Therefore, combination of cisplatin with sensitizing agents mayallow us to overcome cisplatin resistance in chemotherapy ofhuman cancers [3,39]. Here, we provided experimental evidenceshowing that the combination of chrysin and cisplatin promotedapoptosis of Hep G2 cells in both dose- and time-dependent man-ners (Figs. 1 and 2), and p53 was involved in this process (Figs. 3and 4). In addition, activation of ERK partially contributed to theaccumulation and activation of p53 (Fig. 5). Our results suggest

that the combination of chrysin and cisplatin is a promising strat-egy for clinical treatment of human cancers that are resistant tocisplatin.

Loss of the function of p53 is a common event leading tochemotherapeutic resistance in tumors [24]. For instance,inappropriate regulation of p53 contribute to cisplatin-resistantovarian cancer cells [38]. In the present study, we found that thecombination of chrysin and cisplatin, which caused significantincrease of apoptosis of Hep G2 cells, had no significant effectson the apoptosis of Hep 3B cells in which p53 was deleted(Fig. 3A). Our results suggest that p53 was involved in increasedapoptosis of Hep G2 cells induced by the combination of chrysinand cisplatin, which is consistent with previous studies.

We also observed that the combination of chrysin and cisplatinenhanced the phosphorylation and activation of p53 in Hep G2cells, but no obvious activation of p53 was observed in Hep G2 cellstreated with cisplatin of the same concentration and duration oftime (Fig. 3B). We believe knock down of P53 gene in Hep G2 oroverexpression of p53 in Hep 3B cells in future studies would allowus to better understand the role of p53 in cancer cell apoptosisinduced by the combination of chrysin and cisplatin. Up-regulationof pro-apoptotic proteins DR5 and Bax was found due to p53activation (Fig. 4A). Both DR5 and Bax are transcriptional targetsof p53 [28]. Increasing the expression of DR5 by 5-allyl-7-gen-difluoromethoxychrysin (AFMC), a derivative of chrysin, has beenreported in A549 cells [40]. DR5 activation recruits Fas-associateddeath domain (FADD) and pro-caspase-8 to form DISC, which

Fig. 5. Chrysin activated ERK to elevate the phosphorylation and accumulation of p53. (A) Hep G2 cells were pretreated with chrysin (40 lM) for 2 h, then incubated withcisplatin (5 lg/mL) for 3, 6, or 12 h. The mRNA level of p53 was measured by reverse transcription-PCR. Glyceraldehyde-3-phosphate dehydrogenase was used as an internalcontrol. (B) Hep G2 cells were treated with chrysin (40 lM) for indicated periods. Cell lysates were collected for Western blot analysis to detect the total and phosphorylatedMDM2. (C) HepG2 cells were treated with or without chrysin (40 lM), followed by cisplatin incubation (5 lg/mL) for 3,6, 12, 18 h or for 5, 15, 30, or 60 min. (D) Cells lysateswere collected for Western blot analysis to detect ERK1/2 protein levels. (E) HepG2 cells were pretreated with U0126 (20 lM) for 1 h, then treated with chrysin (40 lM),cisplatin (5 lg/mL) or the combination of both for 12 h. Cell lysates were collected for Western blot analysis to determine the total p53, phosphorylated p53 (Ser15), andERK1/2 protein levels. (F) Cells were treated as mentioned in (E), then the translocation of phosphorylated p53 (Ser15) to nuclei was detected by immunofluorescence. Imagesof nucleus localization of phosphorylated p53 (Ser15) were photographed under inverted fluorescence microscope. The intensity of phosphorylated p53(Ser15) fluorescencein nuclei obtained from three independent experiments was presented as means ± SD, ⁄p < 0.05 in comparison to the combination of chrysin and cisplatin (one-way ANOVAwith LSD’S test). Nuclei were stained with DAPI. Tubulin was used as a loading control.

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activates the autocleavage of pro-caspase-8 to active caspase-8,then the downstream cleavage of caspase-3, leading to the execu-tion of extrinsic apoptosis [27,41]. Bax can perforate the outermitochondrial membrane, leading to rupture of the outermitochondrial membrane and release of cytochrome c to activatecaspase-9 [42]. Bcl-2 blocks apoptosis by binding to Bax to preventthe release of the pro-apoptotic proteins [30,31]. In the presentstudy, we observed cytochrome c release (Fig. 4B), and caspase-8and caspase-9 activation (Fig. 4C). These downstream molecularevents support the critical role of p53 in the apoptosis of Hep G2cells induced by the combination of chrysin and cisplatin. Theeffects of chrysin pretreatment in the apoptosis of Hep G2 cellsare consistent with previous reports in which chrysin derivates

induced cell apoptosis through increasing Bax expression anddecreasing Bcl-2 expression in both HCT-116 [43] and human gas-tric carcinoma SGC-7901 cells [13]. Activation of p53, caspase-3and -9, and the release of cytochrome c in HCT-116 cells were alsoreported in the previous study [43]. While chrysin or cisplatinalone can activate p53 and induce cancer cell apoptosis, thecombination of these two agents significantly promoted apoptosisof Hep G2 cell at an extremely low concentration and short time(Figs. 1 and 2). In addition, we also observed that the combinationof chrysin and cisplatin had significantly stronger effects on theratio of p-p53/p53 and Bax/Bcl-2 than any agent alone (Figs. 3Band 4A). Taken together, the combination of chrysin and cisplatindramatically promoted the apoptosis of Hep G2 cells.

X. Li et al. / Chemico-Biological Interactions 232 (2015) 12–20 19

The p53 protein is mainly regulated through post-translationalmodification by complex networks [29,35]. It is believed thatMAPK activation is an important upstream event causing p53phosphorylation in response to cisplatin [35,44]. ERK activationplays a pro-death role in promoting apoptosis or attenuatingcisplatin resistance [45–47]. It has also been shown that chrysinactivated JNK, p38, and ERK1/2, [17,18,36,48]. Our results demon-strated that no significant apoptosis of Hep G2 cells and ERK1/2activation were observed when chrysin was used alone (5 lg/mL)(Figs. 1, 5C and D), however, the combination of chrysin (40 lM)and cisplatin induced dramatic apoptosis of Hep G2 cells as wellas the activation of ERK1/2 (Fig. 5C and D). Especially, ERK1/2activation occurs prior to p53 phosphorylation, suggesting thatERK1/2 activation is an upstream event of p53 phosphorylation.Furthermore, U0126, an inhibitor of ERK1/2, could partially blockp53 phosphorylation at Ser15 and inhibited its translocation tonuclei (Fig. 5E and F). Therefore, ERK activated by chrysin con-tributes to some extent to the accumulation and activation ofp53 in Hep G2 cells. According to others studies, the phosphoryla-tion of p53 at Ser15 is the target of activated ERK1/2 [35,49].

In summary, the combination of chrysin and cisplatin showedsignificant anticancer effects in Hep G2 cells in vitro. The combina-tion of chrysin and cisplatin stabilized p53 protein through activat-ing ERK1/2, which promoted p53 phosphorylation in Hep G2 cells.Our results suggest that the combination of chrysin and cisplatinmay be a solution to the treatment of cisplatin-resistant cancersin clinic. In the future, the effects of the combination of chrysinand cisplatin on the apoptosis of cancer cells should be furtherinvestigated and confirmed in other human cancer cell lines as wellas animal model of cancers.

Conflict of interest

The authors declare that there are no conflicts of interest.

Transparency Document

The Transparency document associated with this article can befound in the online version.

Acknowledgements

This study was in part supported by a research grant from theNational Nature Science Foundation of China (No. 81202199), aresearch grant from the Nature Science Foundation of GuangdongProvince (No. 9151030003000004), a research grant from theScience and Technology Program of Guangzhou (2014J4100090),and a research grand from the Science and Technology plan projectof Guangdong Province (2013B021800044) to Xin Li, a researchgrant from the National Basic Research Program of China (No.2012CB518200) and a research grant from the National NaturalScience Foundation of China (No. 81272180) to Fei Zou.

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