- Email: [email protected]
Combination of chrysin and cisplatin promotes the apoptosis of Hep G2 cells by up-regulating p53
Chemico-Biological Interactions 232 (2015) 12–20
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Combination of chrysin and cisplatin promotes the apoptosis of Hep G2 cells by up-regulating p53 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 Guangzhou Avenue, Guangzhou, Guangdong Province 510515, PR China b Center for Disease Control and Prevention of Guangdong Province, 160 Qunxian Road, Dashi, Panyu District, Guangzhou, Guangdong Province 511430, PR China c 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 2014 Received in revised form 21 January 2015 Accepted 4 March 2015 Available online 11 March 2015 Keywords: Chrysin Cisplatin Apoptosis p53 ERK1/2 Cancer
a b s t r a c t Cisplatin is a chemotherapy drug commonly used for the treatment of human cancers, however, drug resistance poses a major challenge to clinical application of cisplatin in cancer therapy. Recent studies have shown that chrysin, a natural flavonoid widely found in various plants and foods, demonstrated effective anti-cancer activity. In the present study, we found that the combination chrysin and cisplatin significantly enhanced the apoptosis of Hep G2 cancer cells. Combination of chrysin and cisplatin increased the phosphorylation and accumulation of p53 through activating ERK1/2 in Hep G2 cells, which led to the overexpression of the pro-apoptotic proteins Bax and DR5 and the inhibition of the antiapoptotic protein Bcl-2. In addition, combination of chrysin and cisplatin promoted both extrinsic apoptosis by activating caspase-8 and intrinsic apoptosis by increasing the release of cytochrome c and activating caspase-9 in Hep G2 cells. Our results suggest that combination of chrysin and cisplatin is a promising strategy for chemotherapy of human cancers that are resistant to cisplatin. Ó 2015 Published by Elsevier Ireland Ltd.
1. Introduction Cis-Diammine-dichloro-platinumII (cisplatin) is a chemotherapy agent widely used for the treatment of numerous human tumors, 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 apoptosis [2–4]. The role of p53 in cisplatin-induced cancer cell apoptosis has been extensively studied [5]. Typically, P53 mediates apoptosis in both transcription-dependent and transcription-independent manners. The transcription-dependent pathway directly regulates the expression of p53 downstream genes that affect the sensitivity to apoptosis [6,7]. In addition, p53 can move out of nuclei and interact with mitochondria or mitochondrial proteins such as Bcl-2 and Bcl-xl to induce transcription-independent apoptosis Abbreviations: ERK1/2, extracellular signal-regulated knase 1 and 2; Bax, Bcl-2associated 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: [email protected] (F. Zou), [email protected] (X.-F. Yang). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.cbi.2015.03.003 0009-2797/Ó 2015 Published by Elsevier Ireland Ltd.
[8]. Under normal condition in unstressed cells, p53 is sequestrated in cytoplasm by MDM2, which promotes p53 degradation via the ubiquitin/proteasome pathway [7]. In the case of DNA damage caused by cisplatin, the function of MDM2 is impaired and phosphorylated p53 is translocated to nuclei to cause apoptosis through various p53 pathways. Flavonoids are natural polyphenolic phytochemicals that are ubiquitous in plants. Numerous epidemiological studies and animal experiments have demonstrated the anti-cancer and chemopreventive properties of flavonoids [9,10]. Therefore, flavonoids have been regarded as ideal candidates for chemotherapy of human cancers. It has been reported that chrysin (5,7-dihydroxyflavone), a flavone commonly found in various plants and foods, had strong anti-inflammation [11] and antioxidative [12] properties. In addition, chrysin exhibited anti-cancer activity in a diverse range of human [13–18] and animal derived cells [19]. However, the underlying mechanisms of the anti-cancer property of chrysin are not fully understood. Our previous studies have shown that chrysin sensitized apoptotic cell death induced by tumor necrosis factor (TNF) [20] or tumor necrosis factor–related apoptosisinducing ligand (TRAIL) in various human cancer cells [21].
X. Li et al. / Chemico-Biological Interactions 232 (2015) 12–20
Resistance to cisplatin is one of the major limitations of cisplatin-mediated chemotherapy of human cancers. The present study aimed to investigate whether the combination of chrysin and cisplatin can significantly improve the apoptosis of a number of human cancers cells that are common in Southern China. We also studied the underline mechanism by evaluating the expression of several proteins involved in the p53 pathway, which plays a 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 purchased from Bio Mol (Minnesota, USA). Antibodies against caspase-3, caspase-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 phosphorylated MDM2 were from Bioworld (Atlanta, USA). Antibody against PARP was from BD Company (Franklin, USA). Goat anti-mouse IgG and FICT conjugated were from CW BIO (Beijing, China). Hoechst33342 was from GenMed (Boston, USA). The secondary antibodies including horseradish peroxidase–conjugated goat anti-mouse IgG and goat anti-rabbit IgG, and the enhanced chemiluminescence 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 human liver cancer cell lines HepG2 and Hep 3B were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The human nasopharyngeal carcinoma cell line CNE1 was from Sun Yat-Sen University (Guangzhou, China). All cells were maintained in 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 combination treatment, cancer cells were pretreated with chrysin for 2 h, then followed 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 death rates according to the method previously described [20]. Given that cells in sub-G1 phase can be either apoptotic or necrotic, apoptotic cancer cells was further confirmed by staining with either Hoechst 33342 or DAPI. Typically, apoptotic cells exhibit a characteristic chromatin condensation based on Hoechst 33342 or DAPI staining. Briefly, cancer cells were stained with Hoechst 33342 for 20 min or stained with DAPI after paraformaldehyde fixation, then visualized under an inverted fluorescence microscope. The percentage of apoptotic cells were quantified by counting the cells with condensed nuclei among 200 randomly selected cells. 2.4. Extraction of cytosolic proteins Cytosolic proteins were extracted using the Mitochondria Isolation Kit (Thermo Scientific, USA) according to the manufacturer’s instruction. Briefly, cancer cells were washed with Reagent A at 4 °C, then resuspended in Reagent A for 2 min. After Reagent B was added, the solution was incubated for 5 min before
13
Reagent C was added and mixed. Then, cells were centrifuged at 700g for 10 min to collect the supernatant. The cytosolic proteins were obtained after centrifugation at 12,000g for 15 min. The level of cytochrome c in the cytosolic protein samples was evaluated using Western blotting analysis. 2.5. Western blotting analysis Cancer cells were harvested and washed in PBS once. Cell pellets 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 were subjected to SDS–PAGE and transferred to polyvinylidene-fluoride membranes (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 antibodies and developed with enhanced chemiluminescence (Bio-Rad), using a CHEMI-SMART 3000 image station (VILBER-LOURMAT). 2.6. Immunofluorescence HepG2 cells were seeded in 24-well chamber slides 24 h before treatment. After treatment, cells were immediately washed with PBS and fixed in 3% paraformaldehyde for 1 h. They were then permeabilized for 2 min with 0.2% CHAPS in PBS, blocked in 2% BSA with 0.2% Tween-20 for 30 min, and then incubated with anti-p-p53 (ser15) antibody overnight at 4 °C. After washing with PBS (+0.2% Tween-20), cells were incubated with anti-mouse FITC secondary antibody for 1 h. Cells were visualized under a Cellomics 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 PureLink RNA Mini Kit (Ambion). RT-PCR amplifications were performed using OneStep RT-PCR Kit (QIAGEN) in a T100™ Thermal Cycler (Bio-rad). The RT-PCR program was 50 °C for 30 min, and 95 °C for 15 min., and then followed by 94 °C for 30 s, 55 °C for 30 s and 72 °C for 40 s, for a total of 36 cycles. The following primers were used for RT-PCR assay of p53 mRNA: 50 -TAACAGTTCC TGCATGGGCGGC-30 (forward) and 50 -AGGACAGGCACAAACACGCACC30 (reverse). The following primer sequences were used for RTPCR assay of GAPDH mRNA 50 -GAAGGTGAAGGTCGGAGTC-30 (forward), 50 -GAAGATGGTGATGGGATTTC-30 (reverse). 2.8. Statistical analyses All numerical data obtained from at least three independent experiments were presented as mean ± standard deviation (SD). The differences among distinct groups were evaluated using a one-way ANOVA with LSD’s test (SPSS 13.0). A p value <0.05 was considered statistically significant. 3. Results 3.1. The combination of chrysin and cisplatin promoted the death of human cancer cells HepG2, CNE1, and HCT-116 A previous study showed that cisplatin-induced cell death was a relatively slow process, normally requiring 2–3 days [3]. In the present study, no significant cell death was observed in the three types of cancer cells when they were treated with cisplatin alone
14
X. Li et al. / Chemico-Biological Interactions 232 (2015) 12–20
(5 lg/mL for HepG2 cells, 10 lg/mL for CNE1, and HCT-116 cells) for 24 h, based on morphological examination using an inverted microscope (Fig. 1A). Similarly, no obvious cell death was observed when the cancer cells were treated with chrysin alone (40 lM for HepG2 and CNE1 cells, and 20 lM HCT-116 cells). We next tested the effects of the combination of chrysin and cisplatin on cancer cell death. The effects of the combination of chrysin and cisplatin on the death of Hep G2 cells was evaluated by measuring the percentage of sub-G1 cells using flow cytometry. Our results showed that chrysin alone (20 and 40 lM) and the combination of chrysin (20 or 40 lM) and cisplatin (5 lg/mL) significantly increased the death of Hep G2 cells compared with without any treatment (p < 0.05) (Fig. 1B). In addition, the rate of dead Hep G2 cells treated with the combination of chrysin (40 lM) and cisplatin (5 lg/ mL) was significantly higher than that of Hep G2 cells treated with chrysin (10-40 lM) or cisplatin (5 lg/mL) alone (p < 0.05) (Fig. 1B). Therefore, the combination of chrysin and cisplatin increased the death of Hep G2 cells at a dose-dependent manner. Similarly, the combination of chrysin (40 lM) and cisplatin (5 lg/mL) significantly increased the death of Hep G2 cells at a time-dependent manner (p < 0.05) (Fig. 1C). Significant increase of cell death was observed in Hep G2 cells after 6 h of co-treatment with chrysin (40 lM) and cisplatin (5 lg/mL) (Fig. 1C). 3.2. Caspase-dependent apoptosis was involved in increased death of Hep G2 cells induced by the combination of chrysin and cisplatin Previous studies reported that cisplatin could induce both apoptosis and necrosis [4,22]. Given that cells at sub-G1 phase can be either apoptotic or necrotic [23], we then investigated whether the combination of chrysin and cisplatin induced cell death through apoptosis based on DAPI staining. Hep G2 cells were stained with DAPI and examined under a fluorescent microscope. The treatment with the combination of chrysin and cisplatin resulted in evident shrinkage and chromatin condensation of Hep G2 cells (Fig. 2A), which are the characteristic signs of apoptosis. To confirm the apoptosis, we evaluated the expression of PARP and caspase-3, two biochemical markers of apoptosis, in Hep G2 cells. We found that the combination of chrysin and cisplatin caused a decrease of pro-proteins and an increase of PARP and caspase-3 cleavages in a dose-dependent manner in Hep G2 cells (Fig. 2B). Consistently, pretreatment of Hep G2 cells with Z-VADfmk, a pan-caspase inhibitor, efficiently blocked PARP and caspase-3 cleavage (Fig. 2B) and reversed the cell death (Fig. 2C). Taken together, our results suggest that the combination of chrysin and cisplatin promoted the apoptosis of Hep G2 cells, in which caspase was involved. 3.3. p53 was involved in the apoptosis of Hep G2 cells induced by the combination of chrysin and cisplatin Given that p53 is the key regulator involved in cisplatinmediated apoptosis of cancer cells [24], we compared the apoptosis between wild type Hep G2 cells and Hep3B cells with p53 deletion (http://p53.free.fr/Database/Cancer_cell_lines/HCC.html) induced by the combination of chrysin and cisplatin. Significantly more apoptotic cells were observed in Hep G2 cells than in Hep3B cells after co-treatment with chrysin and cisplatin (Fig. 3A). Next, we examined the expression of p53 in Hep G2 cells treated with chrysin, cisplatin, or a combination of both. The protein level of p53 in Hep G2 cells was significantly increased as early as 6 h after chrysin treatment. However, no significant change of p53 expression was found in cisplatin-treated Hep G2 cells, which may be explained by the low concentration of cisplatin and short treatment time employed in the present study (Fig. 3B). Interestingly, the
Fig. 1. Combination of chrysin and cisplatin promoted the death of human cancer cells 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 alone (5 lg/mL for HepG2 cells, and 10 lg/mL for HCT-116 and CNE1 cells) for 24 h or the combination of chrysin and cisplatin. Regarding the combination treatment of chrysin and cisplatin, cells were first treated with chrysin (40 lM for HepG2 and CNE1 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 a light microscope. (B) The death rate of HepG2 cells based on flow cytometry analysis of cells at sub-G1 phrase. HepG2 cells were treated with various concentrations 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 subG1 phrase. HepG2 cells were treated with chrysin (40 lM) for 2 h, then treated with cisplatin (5 lg/mL) for different periods of time (0, 6, 12, or 24 h). Data from three independent experiments were presented as means ± SD. ⁄p < 0.05 in comparison to cells without any treatment; #p < 0.05 in comparison to cisplatin alone and chrysin alone (one-way ANOVA with LSD’S test).
combination of chrysin and cisplatin further increased the protein level of p53 in Hep G2 cells, especially after 12 h of co-treatment. The accumulation of phosphorylated p53 (p-p53) was consistent with increased expression of p53 protein (Fig. 3B). Densitometric analysis of p53 and p-p53 protein levels showed
X. Li et al. / Chemico-Biological Interactions 232 (2015) 12–20
15
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 incubation with 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 PARP proteins. 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 with the 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 C obtained 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 of cisplatin and chrysin.
that the combination of cisplatin and chrysin significantly increased the expression of p53 and p-p53 (Ser15) after 12 h of co-treatment, which led to the increase of the ratio of p-p53 (Ser15) to p53. In addition, translocation of the phosphorylated p53 (Ser15) protein to nuclei was also observed when Hep G2 cells were treated with chrysin alone (40 lM), or the combination of chrysin (40 lM) and cisplatin (5 lg/mL) for 12 h based on immunofluorescence microscopy (Fig. 3C). Taken together, these results suggest that p53 was involved in the apoptosis of Hep G2 cells induced by the combination of chrysin and cisplatin. 3.4. The combination of chrysin and cisplatin promoted the apoptosis of Hep G2 cells through p53 pathways The p53 signaling pathways associated with apoptosis are complicated and involve a large number of molecules. Phosphorylation and activation of p53 by upstream kinases regulate p53 downstream genes and the biological consequences [25]. To further elucidate the mechanisms in which p53 is involved in apoptosis promoted by the combination of chrysin and cisplatin, we examined the downstream molecular targets and biological consequences of p53 activation after Hep G2 cells were treated with the combination of chrysin and cisplatin. Bax and DR5 are two main pro-apoptotic genes targeted by p53 [26–28]. DR5 is a member of the tumor necrosis factor receptor superfamily and initiates extrinsic apoptosis by participating in the formation of death-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 activates pro-caspase-9 [29,30]. In contrast, the anti-apoptotic protein Bcl2 can block intrinsic apoptosis by binding to Bax to form Bcl-2– Bax heterodimers [31]. While p53 is not the direct transcriptional factor for Bcl-2, it is an important negative regulator for Bcl-2 [26,32]. Increased expressions of Bax and DR5 and reduced expression of Bcl-2 were observed in Hep G2 cells treated with the combination of chrysin and cisplatin. Based on densitometric analysis, the ratio of Bax to Bcl-2 protein levels in Hep G2 cells were significantly increased after 18 h of co-treatment of chrysin and cisplatin (Fig. 4A). In addition, we observed the release of cytochrome c to cytosol (Fig. 4B) and the activation of caspase-9 and caspase-8 (Fig. 4C). The expression changes of proteins involved in the p53 pathways suggest that the p53 pathways were involved in the apoptosis of Hep G2 cells induced by the combination of chrysin and cisplatin. 3.5. p53 was activated by ERK through phosphorylation at Ser15 While increased expression of p53 protein was observed in Hep G2 cells treated with the combination of chrysin and cisplatin, no significant change of the mRNA level of P53 gene was detected by RT-PCR assay (Fig. 5A). Therefore, we speculate that the change of p53 protein is caused by post-translation modification of the protein. We analyzed the oncoprotein MDM2, a p53-binding protein that promotes p53 degradation [7,33]. Nevertheless, no significant changes of MDM2 and phosphorylated MDM2 were observed at the early stage of chrysin treatment (Fig. 5B).
16
X. Li et al. / Chemico-Biological Interactions 232 (2015) 12–20
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 percentages of 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 cells were 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 for the 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 of total 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 was detected by immunofluorescence. Images of nuclear localization of phosphorylated p53 (Ser15) were photographed under inverted fluorescence microscope. The intensity of phosphorylated p53 (Ser15) fluorescence in nuclei was evaluated by Cellomics Toxinsight Reader. DAPI staining was used to visualize nuclei. Data obtained from three independent 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).
Phosphorylation-dependent post-translational modification plays an important role in p53 stabilization and activation [29], and the antitumor effects of DNA damaging agents are dominantly governed by phosphorylation events [34]. As shown in Fig 3B and C, dramatic p53 phosphorylation at the Ser15 site was detected after chrysin pretreatment. Mitogen-activated protein kinases (MAPKs), including the stress-activated protein kinase/c-Jun-N-terminal kinase (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 during the entire process of co-treatment with chrysin and cisplatin (Fig. 5C), as well as chrysin treatment alone. These observation was concurrent with p53 activation in Hep G2 cells treated with chrysin alone as well as the combination of chrysin and cisplatin (Fig. 3B and C). Additionally, chrysin alone or the combination of chrysin and cisplatin dramatically activated ERK1/2 at early stages (Fig. 5D). We showed that U0126, an inhibitor of ERK1/2, partially
X. Li et al. / Chemico-Biological Interactions 232 (2015) 12–20
17
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 Western blotting 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 were quantified 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 versus the 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 both for 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 concentrations of 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 Western blotting analysis for detecting the cleavages of caspase-8 and caspase-9. The expression of tubulin was used as a loading control.
blocked p53 phosphorylation at Ser15 (Fig. 5E) and inhibited the translocation of phosphorylated p53 to nuclei induced by the combination of chrysin and cisplatin (Fig. 5F). 4. Discussion The anti-carcinogenic activity of chrysin has been reported by numerous studies [16–18,20,21]. A number of molecular pathways contribute to the anticancer property of chrysin, such as sensitizing TNFa-mediated apoptosis by inhibiting NFjB activation [20], or reducing Nrf2 expression through down-regulating PI3K-Akt and the ERK pathway to reverse doxorubicin resistance [36]. In the present study, we further addressed the anti-cancer mechanism of chrysin by combining it with cisplatin. Cisplatin has been used as one of the most important chemotherapy agents for clinical management of human cancers over the last three decades. However, cisplatin resistance is a serious challenge to its wide application [37]. It has been reported that cisplatin resistance was mainly caused by inhibited apoptosis of cancer cells [38]. Therefore, combination of cisplatin with sensitizing agents may allow us to overcome cisplatin resistance in chemotherapy of human cancers [3,39]. Here, we provided experimental evidence showing that the combination of chrysin and cisplatin promoted apoptosis of Hep G2 cells in both dose- and time-dependent manners (Figs. 1 and 2), and p53 was involved in this process (Figs. 3 and 4). In addition, activation of ERK partially contributed to the accumulation and activation of p53 (Fig. 5). Our results suggest
that the combination of chrysin and cisplatin is a promising strategy for clinical treatment of human cancers that are resistant to cisplatin. Loss of the function of p53 is a common event leading to chemotherapeutic resistance in tumors [24]. For instance, inappropriate regulation of p53 contribute to cisplatin-resistant ovarian cancer cells [38]. In the present study, we found that the combination of chrysin and cisplatin, which caused significant increase of apoptosis of Hep G2 cells, had no significant effects on the apoptosis of Hep 3B cells in which p53 was deleted (Fig. 3A). Our results suggest that p53 was involved in increased apoptosis of Hep G2 cells induced by the combination of chrysin and cisplatin, which is consistent with previous studies. We also observed that the combination of chrysin and cisplatin enhanced the phosphorylation and activation of p53 in Hep G2 cells, but no obvious activation of p53 was observed in Hep G2 cells treated with cisplatin of the same concentration and duration of time (Fig. 3B). We believe knock down of P53 gene in Hep G2 or overexpression of p53 in Hep 3B cells in future studies would allow us to better understand the role of p53 in cancer cell apoptosis induced by the combination of chrysin and cisplatin. Up-regulation of pro-apoptotic proteins DR5 and Bax was found due to p53 activation (Fig. 4A). Both DR5 and Bax are transcriptional targets of p53 [28]. Increasing the expression of DR5 by 5-allyl-7-gendifluoromethoxychrysin (AFMC), a derivative of chrysin, has been reported in A549 cells [40]. DR5 activation recruits Fas-associated death domain (FADD) and pro-caspase-8 to form DISC, which
18
X. Li et al. / Chemico-Biological Interactions 232 (2015) 12–20
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 with cisplatin (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 internal control. (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 phosphorylated MDM2. (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 lysates were 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), and ERK1/2 protein levels. (F) Cells were treated as mentioned in (E), then the translocation of phosphorylated p53 (Ser15) to nuclei was detected by immunofluorescence. Images of nucleus localization of phosphorylated p53 (Ser15) were photographed under inverted fluorescence microscope. The intensity of phosphorylated p53(Ser15) fluorescence in 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 ANOVA with LSD’S test). Nuclei were stained with DAPI. Tubulin was used as a loading control.
activates the autocleavage of pro-caspase-8 to active caspase-8, then the downstream cleavage of caspase-3, leading to the execution of extrinsic apoptosis [27,41]. Bax can perforate the outer mitochondrial membrane, leading to rupture of the outer mitochondrial membrane and release of cytochrome c to activate caspase-9 [42]. Bcl-2 blocks apoptosis by binding to Bax to prevent the release of the pro-apoptotic proteins [30,31]. In the present study, we observed cytochrome c release (Fig. 4B), and caspase-8 and caspase-9 activation (Fig. 4C). These downstream molecular events support the critical role of p53 in the apoptosis of Hep G2 cells induced by the combination of chrysin and cisplatin. The effects of chrysin pretreatment in the apoptosis of Hep G2 cells are consistent with previous reports in which chrysin derivates
induced cell apoptosis through increasing Bax expression and decreasing Bcl-2 expression in both HCT-116 [43] and human gastric carcinoma SGC-7901 cells [13]. Activation of p53, caspase-3 and -9, and the release of cytochrome c in HCT-116 cells were also reported in the previous study [43]. While chrysin or cisplatin alone can activate p53 and induce cancer cell apoptosis, the combination of these two agents significantly promoted apoptosis of Hep G2 cell at an extremely low concentration and short time (Figs. 1 and 2). In addition, we also observed that the combination of chrysin and cisplatin had significantly stronger effects on the ratio of p-p53/p53 and Bax/Bcl-2 than any agent alone (Figs. 3B and 4A). Taken together, the combination of chrysin and cisplatin dramatically promoted the apoptosis of Hep G2 cells.
X. Li et al. / Chemico-Biological Interactions 232 (2015) 12–20
The p53 protein is mainly regulated through post-translational modification by complex networks [29,35]. It is believed that MAPK activation is an important upstream event causing p53 phosphorylation in response to cisplatin [35,44]. ERK activation plays a pro-death role in promoting apoptosis or attenuating cisplatin resistance [45–47]. It has also been shown that chrysin activated JNK, p38, and ERK1/2, [17,18,36,48]. Our results demonstrated that no significant apoptosis of Hep G2 cells and ERK1/2 activation 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 well as the activation of ERK1/2 (Fig. 5C and D). Especially, ERK1/2 activation occurs prior to p53 phosphorylation, suggesting that ERK1/2 activation is an upstream event of p53 phosphorylation. Furthermore, U0126, an inhibitor of ERK1/2, could partially block p53 phosphorylation at Ser15 and inhibited its translocation to nuclei (Fig. 5E and F). Therefore, ERK activated by chrysin contributes to some extent to the accumulation and activation of p53 in Hep G2 cells. According to others studies, the phosphorylation of p53 at Ser15 is the target of activated ERK1/2 [35,49]. In summary, the combination of chrysin and cisplatin showed significant anticancer effects in Hep G2 cells in vitro. The combination of chrysin and cisplatin stabilized p53 protein through activating ERK1/2, which promoted p53 phosphorylation in Hep G2 cells. Our results suggest that the combination of chrysin and cisplatin may be a solution to the treatment of cisplatin-resistant cancers in clinic. In the future, the effects of the combination of chrysin and cisplatin on the apoptosis of cancer cells should be further investigated and confirmed in other human cancer cell lines as well as 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 be found in the online version.
Acknowledgements This study was in part supported by a research grant from the National Nature Science Foundation of China (No. 81202199), a research grant from the Nature Science Foundation of Guangdong Province (No. 9151030003000004), a research grant from the Science and Technology Program of Guangzhou (2014J4100090), and a research grand from the Science and Technology plan project of Guangdong Province (2013B021800044) to Xin Li, a research grant from the National Basic Research Program of China (No. 2012CB518200) and a research grant from the National Natural Science Foundation of China (No. 81272180) to Fei Zou. References [1] D.W. Shen, L.M. Pouliot, M.D. Hall, M.M. Gottesman, Cisplatin resistance: a cellular self-defense mechanism resulting from multiple epigenetic and genetic changes, Pharmacol. Rev. 64 (2012) 706–721. [2] P. Bragado, A. Armesilla, A. Silva, A. Porras, Apoptosis by cisplatin requires p53 mediated p38alpha MAPK activation through ROS generation, Apoptosis 12 (2007) 1733–1742. [3] R. Shi, Q. Huang, X. Zhu, Y.B. Ong, B. Zhao, J. Lu, C.N. Ong, H.M. Shen, Luteolin sensitizes the anticancer effect of cisplatin via c-Jun NH2-terminal kinasemediated p53 phosphorylation and stabilization, Mol. Cancer Ther. 6 (2007) 1338–1347.
19
[4] V.M. Gonzalez, M.A. Fuertes, C. Alonso, J.M. Perez, Is cisplatin-induced cell death always produced by apoptosis?, Mol Pharmacol. 59 (2001) 657–663. [5] Z.H. Siddik, Cisplatin: mode of cytotoxic action and molecular basis of resistance, Oncogene 22 (2003) 7265–7279. [6] Y. Aylon, M. Oren, Living with p53, dying of p53, Cell 130 (2007) 597–600. [7] S. Nag, J. Qin, K.S. Srivenugopal, M. Wang, R. Zhang, The MDM2-p53 pathway revisited, J. Biomed. Res. 27 (2013) 254–271. [8] U.M. Moll, S. Wolff, D. Speidel, W. Deppert, Transcription-independent proapoptotic functions of p53, Curr. Opin. Cell Biol. 17 (2005) 631–636. [9] S. Ramos, Effects of dietary flavonoids on apoptotic pathways related to cancer chemoprevention, J. Nutr. Biochem. 18 (2007) 427–442. [10] I.C. Arts, A review of the epidemiological evidence on tea, flavonoids, and lung cancer, J. Nutr. 138 (2008) 1561S–1566S. [11] A. During, Y. Larondelle, The O-methylation of chrysin markedly improves its intestinal anti-inflammatory properties: structure–activity relationships of flavones, Biochem. Pharmacol. 86 (2013) 1739–1746. [12] N. Ali, S. Rashid, S. Nafees, S.K. Hasan, S. Sultana, Beneficial effects of Chrysin against Methotrexate-induced hepatotoxicity via attenuation of oxidative stress and apoptosis, Mol. Cell. Biochem. 385 (2014) 215–223. [13] X.H. Ai, X. Zheng, X.Q. Tang, L. Sun, Y.Q. Zhang, Y. Qin, H.Q. Liu, H. Xia, J.G. Cao, Induction of apoptosis of human gastric carcinoma SGC-7901 cell line by 5,7dihydroxy-8-nitrochrysin in vitro, World J. Gastroenterol. 13 (2007) 3824– 3828. [14] M.S. Weng, Y.S. Ho, J.K. Lin, Chrysin induces G1 phase cell cycle arrest in C6 glioma cells through inducing p21Waf1/Cip1 expression: involvement of p38 mitogen-activated protein kinase, Biochem. Pharmacol. 69 (2005) 1815–1827. [15] X.M. Yu, T. Phan, P.N. Patel, R. Jaskula-Sztul, H. Chen, Chrysin activates Notch1 signaling and suppresses tumor growth of anaplastic thyroid carcinoma in vitro and in vivo, Cancer 119 (2013) 774–781. [16] F. Yang, H. Jin, J. Pi, J.H. Jiang, L. Liu, H.H. Bai, P.H. Yang, J.Y. Cai, Anti-tumor activity evaluation of novel chrysin–organogermanium(IV) complex in MCF-7 cells, Bioorg. Med. Chem. Lett. 23 (2013) 5544–5551. [17] S.K. Ha, E. Moon, S.Y. Kim, Chrysin suppresses LPS-stimulated proinflammatory responses by blocking NF-kappaB and JNK activations in microglia cells, Neurosci. Lett. 485 (2010) 143–147. [18] E. Pichichero, R. Cicconi, M. Mattei, A. Canini, Chrysin-induced apoptosis is mediated through p38 and Bax activation in B16–F1 and A375 melanoma cells, Int. J. Oncol. 38 (2011) 473–483. [19] S. Miyamoto, H. Kohno, R. Suzuki, S. Sugie, A. Murakami, H. Ohigashi, T. Tanaka, Preventive effects of chrysin on the development of azoxymethaneinduced colonic aberrant crypt foci in rats, Oncol. Rep. 15 (2006) 4. [20] X. Li, Q. Huang, C.N. Ong, X.F. Yang, H.M. Shen, Chrysin sensitizes tumor necrosis factor-alpha-induced apoptosis in human tumor cells via suppression of nuclear factor-kappaB, Cancer Lett. 293 (2010) 109–116. [21] X. Li, J.N. Wang, J.M. Huang, X.K. Xiong, M.F. Chen, C.N. Ong, H.M. Shen, X.F. Yang, Chrysin promotes tumor necrosis factor (TNF)-related apoptosisinducing ligand (TRAIL) induced apoptosis in human cancer cell lines, Toxicol. In Vitro 25 (2011) 630–635. [22] D. Wang, S.J. Lippard, Cellular processing of platinum anticancer drugs, Nat. Rev. Drug Discovery 4 (2005) 307–320. [23] Z. Darzynkiewicz, E. Bedner, F. Traganos, T. Murakami, Critical aspects in the analysis of apoptosis and necrosis, Hum. Cell 11 (1998) 3–12. [24] S. Fujioka, C. Schmidt, G.M. Sclabas, Z. Li, H. Pelicano, B. Peng, A. Yao, J. Niu, W. Zhang, D.B. Evans, J.L. Abbruzzese, P. Huang, P.J. Chiao, Stabilization of p53 is a novel mechanism for proapoptotic function of NF-kappaB, J. Biol. Chem. 279 (2004) 27549–27559. [25] A.J. Levine, W. Hu, Z. Feng, The P53 pathway: what questions remain to be explored?, Cell Death Differ 13 (2006) 1027–1036. [26] F. Esposito, M. Tornincasa, P. Chieffi, I. De Martino, G.M. Pierantoni, A. Fusco, High-mobility group A1 proteins regulate p53-mediated transcription of Bcl-2 gene, Cancer Res. 70 (2010) 5379–5388. [27] N. Finnberg, J.J. Gruber, P. Fei, D. Rudolph, A. Bric, S.H. Kim, T.F. Burns, H. Ajuha, R. Page, G.S. Wu, Y. Chen, W.G. McKenna, E. Bernhard, S. Lowe, T. Mak, W.S. ElDeiry, DR5 knockout mice are compromised in radiation-induced apoptosis, Mol. Cell. Biol. 25 (2005) 2000–2013. [28] J.K. Sax, W.S. El-Deiry, P53 downstream targets and chemosensitivity, Cell Death Differ. 10 (2003) 413–417. [29] M. Martinez-Rivera, Z.H. Siddik, Resistance and gain-of-resistance phenotypes in cancers harboring wild-type p53, Biochem. Pharmacol. 83 (2012) 1049–1062. [30] M.L. Tan, J.P. Ooi, N. Ismail, A.I. Moad, T.S. Muhammad, Programmed cell death pathways and current antitumor targets, Pharm. Res. 26 (2009) 1547–1560. [31] X. Yi, X.M. Yin, Z. Dong, Inhibition of Bid-induced apoptosis by Bcl-2. tBid insertion, Bax translocation, and Bax/Bak oligomerization suppressed, J. Biol. Chem. 278 (2003) 16992–16999. [32] J.S. Fridman, S.W. Lowe, Control of apoptosis by p53, Oncogene 22 (2003) 9030–9040. [33] D. Thomasova, S.R. Mulay, H. Bruns, H.J. Anders, P53-independent roles of MDM2 in NF-kappaB signaling: implications for cancer therapy, wound healing, and autoimmune diseases, Neoplasia 14 (2012) 1097–1101. [34] E. Appella, C.W. Anderson, Post-translational modifications and activation of p53 by genotoxic stresses, Eur. J. Biochem./FEBS 268 (2001) 2764–2772. [35] G.S. Wu, The functional interactions between the p53 and MAPK signaling pathways, Cancer Biol. Ther. 3 (2004) 156–161. [36] A.M. Gao, Z.P. Ke, F. Shi, G.C. Sun, H. Chen, Chrysin enhances sensitivity of BEL7402/ADM cells to doxorubicin by suppressing PI3K/Akt/Nrf2 and ERK/Nrf2 pathway, Chem. Biol. Interact. 206 (2013) 100–108.
20
X. Li et al. / Chemico-Biological Interactions 232 (2015) 12–20
[37] I. Kudo, M. Esumi, A. Kida, M. Ikeda, P53 mutation, but not in vitro predictor genes of therapeutic efficacy of cisplatin, is clinically relevant in comparing partial and complete responder cases of maxillary squamous cell carcinoma, Oncol. Rep. 24 (2010) 851–856. [38] E.L. Leung, M. Fraser, R.R. Fiscus, B.K. Tsang, Cisplatin alters nitric oxide synthase levels in human ovarian cancer cells: involvement in p53 regulation and cisplatin resistance, Br. J. Cancer 98 (2008) 1803–1809. [39] T.M. de Kok, S.G. van Breda, M.M. Manson, Mechanisms of combined action of different chemopreventive dietary compounds: a review, Eur. J. Nutr. 47 (Suppl. 2) (2008) 51–59. [40] Z.H. Xie, M.F. Quan, F. Liu, J.G. Cao, J.S. Zhang, 5-Allyl-7-gendifluoromethoxychrysin enhances TRAIL-induced apoptosis in human lung carcinoma A549 cells, BMC Cancer 11 (2011) 322. [41] Z. Kang, S.Y. Sun, L. Cao, Activating death receptor DR5 as a therapeutic strategy for rhabdomyosarcoma, ISRN Oncol. 2012 (2012) 395952. [42] R. Eskes, S. Desagher, B. Antonsson, J.C. Martinou, Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane, Mol. Cell. Biol. 20 (2000) 929–935. [43] J. Ren, H. Cheng, W.Q. Xin, X. Chen, K. Hu, Induction of apoptosis by 7piperazinethylchrysin in HCT-116 human colon cancer cells, Oncol. Rep. 28 (2012) 1719–1726.
[44] A. Brozovic, M. Osmak, Activation of mitogen-activated protein kinases by cisplatin and their role in cisplatin-resistance, Cancer Lett. 251 (2007) 1–16. [45] I. Arany, J.K. Megyesi, H. Kaneto, P.M. Price, R.L. Safirstein, Cisplatin-induced cell death is EGFR/src/ERK signaling dependent in mouse proximal tubule cells, Am. J. Physiol. Renal Physiol. 287 (2004) F543–F549. [46] S. Schweyer, A. Soruri, O. Meschter, A. Heintze, F. Zschunke, N. Miosge, P. Thelen, T. Schlott, H.J. Radzun, A. Fayyazi, Cisplatin-induced apoptosis in human malignant testicular germ cell lines depends on MEK/ERK activation, Br. J. Cancer 91 (2004) 589–598. [47] P.Y. Yeh, S.E. Chuang, K.H. Yeh, Y.C. Song, C.K. Ea, A.L. Cheng, Increase of the resistance of human cervical carcinoma cells to cisplatin by inhibition of the MEK to ERK signaling pathway partly via enhancement of anticancer druginduced NF kappa B activation, Biochem. Pharmacol. 63 (2002) 1423–1430. [48] W. Zeng, Y. Yan, F. Zhang, C. Zhang, W. Liang, Chrysin promotes osteogenic differentiation via ERK/MAPK activation, Protein Cell 4 (2013) 539–547. [49] Q.B. She, A.M. Bode, W.Y. Ma, N.Y. Chen, Z. Dong, Resveratrol-induced activation of p53 and apoptosis is mediated by extracellular-signal-regulated protein kinases and p38 kinase, Cancer Res. 61 (2001) 1604–1610.
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Combination of chrysin and cisplatin promotes the apoptosis of Hep G2 cells by up-regulating p53 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 Guangzhou Avenue, Guangzhou, Guangdong Province 510515, PR China b Center for Disease Control and Prevention of Guangdong Province, 160 Qunxian Road, Dashi, Panyu District, Guangzhou, Guangdong Province 511430, PR China c 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 2014 Received in revised form 21 January 2015 Accepted 4 March 2015 Available online 11 March 2015 Keywords: Chrysin Cisplatin Apoptosis p53 ERK1/2 Cancer
a b s t r a c t Cisplatin is a chemotherapy drug commonly used for the treatment of human cancers, however, drug resistance poses a major challenge to clinical application of cisplatin in cancer therapy. Recent studies have shown that chrysin, a natural flavonoid widely found in various plants and foods, demonstrated effective anti-cancer activity. In the present study, we found that the combination chrysin and cisplatin significantly enhanced the apoptosis of Hep G2 cancer cells. Combination of chrysin and cisplatin increased the phosphorylation and accumulation of p53 through activating ERK1/2 in Hep G2 cells, which led to the overexpression of the pro-apoptotic proteins Bax and DR5 and the inhibition of the antiapoptotic protein Bcl-2. In addition, combination of chrysin and cisplatin promoted both extrinsic apoptosis by activating caspase-8 and intrinsic apoptosis by increasing the release of cytochrome c and activating caspase-9 in Hep G2 cells. Our results suggest that combination of chrysin and cisplatin is a promising strategy for chemotherapy of human cancers that are resistant to cisplatin. Ó 2015 Published by Elsevier Ireland Ltd.
1. Introduction Cis-Diammine-dichloro-platinumII (cisplatin) is a chemotherapy agent widely used for the treatment of numerous human tumors, 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 apoptosis [2–4]. The role of p53 in cisplatin-induced cancer cell apoptosis has been extensively studied [5]. Typically, P53 mediates apoptosis in both transcription-dependent and transcription-independent manners. The transcription-dependent pathway directly regulates the expression of p53 downstream genes that affect the sensitivity to apoptosis [6,7]. In addition, p53 can move out of nuclei and interact with mitochondria or mitochondrial proteins such as Bcl-2 and Bcl-xl to induce transcription-independent apoptosis Abbreviations: ERK1/2, extracellular signal-regulated knase 1 and 2; Bax, Bcl-2associated 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: [email protected] (F. Zou), [email protected] (X.-F. Yang). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.cbi.2015.03.003 0009-2797/Ó 2015 Published by Elsevier Ireland Ltd.
[8]. Under normal condition in unstressed cells, p53 is sequestrated in cytoplasm by MDM2, which promotes p53 degradation via the ubiquitin/proteasome pathway [7]. In the case of DNA damage caused by cisplatin, the function of MDM2 is impaired and phosphorylated p53 is translocated to nuclei to cause apoptosis through various p53 pathways. Flavonoids are natural polyphenolic phytochemicals that are ubiquitous in plants. Numerous epidemiological studies and animal experiments have demonstrated the anti-cancer and chemopreventive properties of flavonoids [9,10]. Therefore, flavonoids have been regarded as ideal candidates for chemotherapy of human cancers. It has been reported that chrysin (5,7-dihydroxyflavone), a flavone commonly found in various plants and foods, had strong anti-inflammation [11] and antioxidative [12] properties. In addition, chrysin exhibited anti-cancer activity in a diverse range of human [13–18] and animal derived cells [19]. However, the underlying mechanisms of the anti-cancer property of chrysin are not fully understood. Our previous studies have shown that chrysin sensitized apoptotic cell death induced by tumor necrosis factor (TNF) [20] or tumor necrosis factor–related apoptosisinducing ligand (TRAIL) in various human cancer cells [21].
X. Li et al. / Chemico-Biological Interactions 232 (2015) 12–20
Resistance to cisplatin is one of the major limitations of cisplatin-mediated chemotherapy of human cancers. The present study aimed to investigate whether the combination of chrysin and cisplatin can significantly improve the apoptosis of a number of human cancers cells that are common in Southern China. We also studied the underline mechanism by evaluating the expression of several proteins involved in the p53 pathway, which plays a 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 purchased from Bio Mol (Minnesota, USA). Antibodies against caspase-3, caspase-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 phosphorylated MDM2 were from Bioworld (Atlanta, USA). Antibody against PARP was from BD Company (Franklin, USA). Goat anti-mouse IgG and FICT conjugated were from CW BIO (Beijing, China). Hoechst33342 was from GenMed (Boston, USA). The secondary antibodies including horseradish peroxidase–conjugated goat anti-mouse IgG and goat anti-rabbit IgG, and the enhanced chemiluminescence 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 human liver cancer cell lines HepG2 and Hep 3B were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The human nasopharyngeal carcinoma cell line CNE1 was from Sun Yat-Sen University (Guangzhou, China). All cells were maintained in 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 combination treatment, cancer cells were pretreated with chrysin for 2 h, then followed 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 death rates according to the method previously described [20]. Given that cells in sub-G1 phase can be either apoptotic or necrotic, apoptotic cancer cells was further confirmed by staining with either Hoechst 33342 or DAPI. Typically, apoptotic cells exhibit a characteristic chromatin condensation based on Hoechst 33342 or DAPI staining. Briefly, cancer cells were stained with Hoechst 33342 for 20 min or stained with DAPI after paraformaldehyde fixation, then visualized under an inverted fluorescence microscope. The percentage of apoptotic cells were quantified by counting the cells with condensed nuclei among 200 randomly selected cells. 2.4. Extraction of cytosolic proteins Cytosolic proteins were extracted using the Mitochondria Isolation Kit (Thermo Scientific, USA) according to the manufacturer’s instruction. Briefly, cancer cells were washed with Reagent A at 4 °C, then resuspended in Reagent A for 2 min. After Reagent B was added, the solution was incubated for 5 min before
13
Reagent C was added and mixed. Then, cells were centrifuged at 700g for 10 min to collect the supernatant. The cytosolic proteins were obtained after centrifugation at 12,000g for 15 min. The level of cytochrome c in the cytosolic protein samples was evaluated using Western blotting analysis. 2.5. Western blotting analysis Cancer cells were harvested and washed in PBS once. Cell pellets 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 were subjected to SDS–PAGE and transferred to polyvinylidene-fluoride membranes (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 antibodies and developed with enhanced chemiluminescence (Bio-Rad), using a CHEMI-SMART 3000 image station (VILBER-LOURMAT). 2.6. Immunofluorescence HepG2 cells were seeded in 24-well chamber slides 24 h before treatment. After treatment, cells were immediately washed with PBS and fixed in 3% paraformaldehyde for 1 h. They were then permeabilized for 2 min with 0.2% CHAPS in PBS, blocked in 2% BSA with 0.2% Tween-20 for 30 min, and then incubated with anti-p-p53 (ser15) antibody overnight at 4 °C. After washing with PBS (+0.2% Tween-20), cells were incubated with anti-mouse FITC secondary antibody for 1 h. Cells were visualized under a Cellomics 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 PureLink RNA Mini Kit (Ambion). RT-PCR amplifications were performed using OneStep RT-PCR Kit (QIAGEN) in a T100™ Thermal Cycler (Bio-rad). The RT-PCR program was 50 °C for 30 min, and 95 °C for 15 min., and then followed by 94 °C for 30 s, 55 °C for 30 s and 72 °C for 40 s, for a total of 36 cycles. The following primers were used for RT-PCR assay of p53 mRNA: 50 -TAACAGTTCC TGCATGGGCGGC-30 (forward) and 50 -AGGACAGGCACAAACACGCACC30 (reverse). The following primer sequences were used for RTPCR assay of GAPDH mRNA 50 -GAAGGTGAAGGTCGGAGTC-30 (forward), 50 -GAAGATGGTGATGGGATTTC-30 (reverse). 2.8. Statistical analyses All numerical data obtained from at least three independent experiments were presented as mean ± standard deviation (SD). The differences among distinct groups were evaluated using a one-way ANOVA with LSD’s test (SPSS 13.0). A p value <0.05 was considered statistically significant. 3. Results 3.1. The combination of chrysin and cisplatin promoted the death of human cancer cells HepG2, CNE1, and HCT-116 A previous study showed that cisplatin-induced cell death was a relatively slow process, normally requiring 2–3 days [3]. In the present study, no significant cell death was observed in the three types of cancer cells when they were treated with cisplatin alone
14
X. Li et al. / Chemico-Biological Interactions 232 (2015) 12–20
(5 lg/mL for HepG2 cells, 10 lg/mL for CNE1, and HCT-116 cells) for 24 h, based on morphological examination using an inverted microscope (Fig. 1A). Similarly, no obvious cell death was observed when the cancer cells were treated with chrysin alone (40 lM for HepG2 and CNE1 cells, and 20 lM HCT-116 cells). We next tested the effects of the combination of chrysin and cisplatin on cancer cell death. The effects of the combination of chrysin and cisplatin on the death of Hep G2 cells was evaluated by measuring the percentage of sub-G1 cells using flow cytometry. Our results showed that chrysin alone (20 and 40 lM) and the combination of chrysin (20 or 40 lM) and cisplatin (5 lg/mL) significantly increased the death of Hep G2 cells compared with without any treatment (p < 0.05) (Fig. 1B). In addition, the rate of dead Hep G2 cells treated with the combination of chrysin (40 lM) and cisplatin (5 lg/ mL) was significantly higher than that of Hep G2 cells treated with chrysin (10-40 lM) or cisplatin (5 lg/mL) alone (p < 0.05) (Fig. 1B). Therefore, the combination of chrysin and cisplatin increased the death of Hep G2 cells at a dose-dependent manner. Similarly, the combination of chrysin (40 lM) and cisplatin (5 lg/mL) significantly increased the death of Hep G2 cells at a time-dependent manner (p < 0.05) (Fig. 1C). Significant increase of cell death was observed in Hep G2 cells after 6 h of co-treatment with chrysin (40 lM) and cisplatin (5 lg/mL) (Fig. 1C). 3.2. Caspase-dependent apoptosis was involved in increased death of Hep G2 cells induced by the combination of chrysin and cisplatin Previous studies reported that cisplatin could induce both apoptosis and necrosis [4,22]. Given that cells at sub-G1 phase can be either apoptotic or necrotic [23], we then investigated whether the combination of chrysin and cisplatin induced cell death through apoptosis based on DAPI staining. Hep G2 cells were stained with DAPI and examined under a fluorescent microscope. The treatment with the combination of chrysin and cisplatin resulted in evident shrinkage and chromatin condensation of Hep G2 cells (Fig. 2A), which are the characteristic signs of apoptosis. To confirm the apoptosis, we evaluated the expression of PARP and caspase-3, two biochemical markers of apoptosis, in Hep G2 cells. We found that the combination of chrysin and cisplatin caused a decrease of pro-proteins and an increase of PARP and caspase-3 cleavages in a dose-dependent manner in Hep G2 cells (Fig. 2B). Consistently, pretreatment of Hep G2 cells with Z-VADfmk, a pan-caspase inhibitor, efficiently blocked PARP and caspase-3 cleavage (Fig. 2B) and reversed the cell death (Fig. 2C). Taken together, our results suggest that the combination of chrysin and cisplatin promoted the apoptosis of Hep G2 cells, in which caspase was involved. 3.3. p53 was involved in the apoptosis of Hep G2 cells induced by the combination of chrysin and cisplatin Given that p53 is the key regulator involved in cisplatinmediated apoptosis of cancer cells [24], we compared the apoptosis between wild type Hep G2 cells and Hep3B cells with p53 deletion (http://p53.free.fr/Database/Cancer_cell_lines/HCC.html) induced by the combination of chrysin and cisplatin. Significantly more apoptotic cells were observed in Hep G2 cells than in Hep3B cells after co-treatment with chrysin and cisplatin (Fig. 3A). Next, we examined the expression of p53 in Hep G2 cells treated with chrysin, cisplatin, or a combination of both. The protein level of p53 in Hep G2 cells was significantly increased as early as 6 h after chrysin treatment. However, no significant change of p53 expression was found in cisplatin-treated Hep G2 cells, which may be explained by the low concentration of cisplatin and short treatment time employed in the present study (Fig. 3B). Interestingly, the
Fig. 1. Combination of chrysin and cisplatin promoted the death of human cancer cells 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 alone (5 lg/mL for HepG2 cells, and 10 lg/mL for HCT-116 and CNE1 cells) for 24 h or the combination of chrysin and cisplatin. Regarding the combination treatment of chrysin and cisplatin, cells were first treated with chrysin (40 lM for HepG2 and CNE1 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 a light microscope. (B) The death rate of HepG2 cells based on flow cytometry analysis of cells at sub-G1 phrase. HepG2 cells were treated with various concentrations 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 subG1 phrase. HepG2 cells were treated with chrysin (40 lM) for 2 h, then treated with cisplatin (5 lg/mL) for different periods of time (0, 6, 12, or 24 h). Data from three independent experiments were presented as means ± SD. ⁄p < 0.05 in comparison to cells without any treatment; #p < 0.05 in comparison to cisplatin alone and chrysin alone (one-way ANOVA with LSD’S test).
combination of chrysin and cisplatin further increased the protein level of p53 in Hep G2 cells, especially after 12 h of co-treatment. The accumulation of phosphorylated p53 (p-p53) was consistent with increased expression of p53 protein (Fig. 3B). Densitometric analysis of p53 and p-p53 protein levels showed
X. Li et al. / Chemico-Biological Interactions 232 (2015) 12–20
15
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 incubation with 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 PARP proteins. 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 with the 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 C obtained 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 of cisplatin and chrysin.
that the combination of cisplatin and chrysin significantly increased the expression of p53 and p-p53 (Ser15) after 12 h of co-treatment, which led to the increase of the ratio of p-p53 (Ser15) to p53. In addition, translocation of the phosphorylated p53 (Ser15) protein to nuclei was also observed when Hep G2 cells were treated with chrysin alone (40 lM), or the combination of chrysin (40 lM) and cisplatin (5 lg/mL) for 12 h based on immunofluorescence microscopy (Fig. 3C). Taken together, these results suggest that p53 was involved in the apoptosis of Hep G2 cells induced by the combination of chrysin and cisplatin. 3.4. The combination of chrysin and cisplatin promoted the apoptosis of Hep G2 cells through p53 pathways The p53 signaling pathways associated with apoptosis are complicated and involve a large number of molecules. Phosphorylation and activation of p53 by upstream kinases regulate p53 downstream genes and the biological consequences [25]. To further elucidate the mechanisms in which p53 is involved in apoptosis promoted by the combination of chrysin and cisplatin, we examined the downstream molecular targets and biological consequences of p53 activation after Hep G2 cells were treated with the combination of chrysin and cisplatin. Bax and DR5 are two main pro-apoptotic genes targeted by p53 [26–28]. DR5 is a member of the tumor necrosis factor receptor superfamily and initiates extrinsic apoptosis by participating in the formation of death-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 activates pro-caspase-9 [29,30]. In contrast, the anti-apoptotic protein Bcl2 can block intrinsic apoptosis by binding to Bax to form Bcl-2– Bax heterodimers [31]. While p53 is not the direct transcriptional factor for Bcl-2, it is an important negative regulator for Bcl-2 [26,32]. Increased expressions of Bax and DR5 and reduced expression of Bcl-2 were observed in Hep G2 cells treated with the combination of chrysin and cisplatin. Based on densitometric analysis, the ratio of Bax to Bcl-2 protein levels in Hep G2 cells were significantly increased after 18 h of co-treatment of chrysin and cisplatin (Fig. 4A). In addition, we observed the release of cytochrome c to cytosol (Fig. 4B) and the activation of caspase-9 and caspase-8 (Fig. 4C). The expression changes of proteins involved in the p53 pathways suggest that the p53 pathways were involved in the apoptosis of Hep G2 cells induced by the combination of chrysin and cisplatin. 3.5. p53 was activated by ERK through phosphorylation at Ser15 While increased expression of p53 protein was observed in Hep G2 cells treated with the combination of chrysin and cisplatin, no significant change of the mRNA level of P53 gene was detected by RT-PCR assay (Fig. 5A). Therefore, we speculate that the change of p53 protein is caused by post-translation modification of the protein. We analyzed the oncoprotein MDM2, a p53-binding protein that promotes p53 degradation [7,33]. Nevertheless, no significant changes of MDM2 and phosphorylated MDM2 were observed at the early stage of chrysin treatment (Fig. 5B).
16
X. Li et al. / Chemico-Biological Interactions 232 (2015) 12–20
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 percentages of 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 cells were 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 for the 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 of total 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 was detected by immunofluorescence. Images of nuclear localization of phosphorylated p53 (Ser15) were photographed under inverted fluorescence microscope. The intensity of phosphorylated p53 (Ser15) fluorescence in nuclei was evaluated by Cellomics Toxinsight Reader. DAPI staining was used to visualize nuclei. Data obtained from three independent 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).
Phosphorylation-dependent post-translational modification plays an important role in p53 stabilization and activation [29], and the antitumor effects of DNA damaging agents are dominantly governed by phosphorylation events [34]. As shown in Fig 3B and C, dramatic p53 phosphorylation at the Ser15 site was detected after chrysin pretreatment. Mitogen-activated protein kinases (MAPKs), including the stress-activated protein kinase/c-Jun-N-terminal kinase (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 during the entire process of co-treatment with chrysin and cisplatin (Fig. 5C), as well as chrysin treatment alone. These observation was concurrent with p53 activation in Hep G2 cells treated with chrysin alone as well as the combination of chrysin and cisplatin (Fig. 3B and C). Additionally, chrysin alone or the combination of chrysin and cisplatin dramatically activated ERK1/2 at early stages (Fig. 5D). We showed that U0126, an inhibitor of ERK1/2, partially
X. Li et al. / Chemico-Biological Interactions 232 (2015) 12–20
17
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 Western blotting 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 were quantified 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 versus the 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 both for 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 concentrations of 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 Western blotting analysis for detecting the cleavages of caspase-8 and caspase-9. The expression of tubulin was used as a loading control.
blocked p53 phosphorylation at Ser15 (Fig. 5E) and inhibited the translocation of phosphorylated p53 to nuclei induced by the combination of chrysin and cisplatin (Fig. 5F). 4. Discussion The anti-carcinogenic activity of chrysin has been reported by numerous studies [16–18,20,21]. A number of molecular pathways contribute to the anticancer property of chrysin, such as sensitizing TNFa-mediated apoptosis by inhibiting NFjB activation [20], or reducing Nrf2 expression through down-regulating PI3K-Akt and the ERK pathway to reverse doxorubicin resistance [36]. In the present study, we further addressed the anti-cancer mechanism of chrysin by combining it with cisplatin. Cisplatin has been used as one of the most important chemotherapy agents for clinical management of human cancers over the last three decades. However, cisplatin resistance is a serious challenge to its wide application [37]. It has been reported that cisplatin resistance was mainly caused by inhibited apoptosis of cancer cells [38]. Therefore, combination of cisplatin with sensitizing agents may allow us to overcome cisplatin resistance in chemotherapy of human cancers [3,39]. Here, we provided experimental evidence showing that the combination of chrysin and cisplatin promoted apoptosis of Hep G2 cells in both dose- and time-dependent manners (Figs. 1 and 2), and p53 was involved in this process (Figs. 3 and 4). In addition, activation of ERK partially contributed to the accumulation and activation of p53 (Fig. 5). Our results suggest
that the combination of chrysin and cisplatin is a promising strategy for clinical treatment of human cancers that are resistant to cisplatin. Loss of the function of p53 is a common event leading to chemotherapeutic resistance in tumors [24]. For instance, inappropriate regulation of p53 contribute to cisplatin-resistant ovarian cancer cells [38]. In the present study, we found that the combination of chrysin and cisplatin, which caused significant increase of apoptosis of Hep G2 cells, had no significant effects on the apoptosis of Hep 3B cells in which p53 was deleted (Fig. 3A). Our results suggest that p53 was involved in increased apoptosis of Hep G2 cells induced by the combination of chrysin and cisplatin, which is consistent with previous studies. We also observed that the combination of chrysin and cisplatin enhanced the phosphorylation and activation of p53 in Hep G2 cells, but no obvious activation of p53 was observed in Hep G2 cells treated with cisplatin of the same concentration and duration of time (Fig. 3B). We believe knock down of P53 gene in Hep G2 or overexpression of p53 in Hep 3B cells in future studies would allow us to better understand the role of p53 in cancer cell apoptosis induced by the combination of chrysin and cisplatin. Up-regulation of pro-apoptotic proteins DR5 and Bax was found due to p53 activation (Fig. 4A). Both DR5 and Bax are transcriptional targets of p53 [28]. Increasing the expression of DR5 by 5-allyl-7-gendifluoromethoxychrysin (AFMC), a derivative of chrysin, has been reported in A549 cells [40]. DR5 activation recruits Fas-associated death domain (FADD) and pro-caspase-8 to form DISC, which
18
X. Li et al. / Chemico-Biological Interactions 232 (2015) 12–20
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 with cisplatin (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 internal control. (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 phosphorylated MDM2. (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 lysates were 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), and ERK1/2 protein levels. (F) Cells were treated as mentioned in (E), then the translocation of phosphorylated p53 (Ser15) to nuclei was detected by immunofluorescence. Images of nucleus localization of phosphorylated p53 (Ser15) were photographed under inverted fluorescence microscope. The intensity of phosphorylated p53(Ser15) fluorescence in 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 ANOVA with LSD’S test). Nuclei were stained with DAPI. Tubulin was used as a loading control.
activates the autocleavage of pro-caspase-8 to active caspase-8, then the downstream cleavage of caspase-3, leading to the execution of extrinsic apoptosis [27,41]. Bax can perforate the outer mitochondrial membrane, leading to rupture of the outer mitochondrial membrane and release of cytochrome c to activate caspase-9 [42]. Bcl-2 blocks apoptosis by binding to Bax to prevent the release of the pro-apoptotic proteins [30,31]. In the present study, we observed cytochrome c release (Fig. 4B), and caspase-8 and caspase-9 activation (Fig. 4C). These downstream molecular events support the critical role of p53 in the apoptosis of Hep G2 cells induced by the combination of chrysin and cisplatin. The effects of chrysin pretreatment in the apoptosis of Hep G2 cells are consistent with previous reports in which chrysin derivates
induced cell apoptosis through increasing Bax expression and decreasing Bcl-2 expression in both HCT-116 [43] and human gastric carcinoma SGC-7901 cells [13]. Activation of p53, caspase-3 and -9, and the release of cytochrome c in HCT-116 cells were also reported in the previous study [43]. While chrysin or cisplatin alone can activate p53 and induce cancer cell apoptosis, the combination of these two agents significantly promoted apoptosis of Hep G2 cell at an extremely low concentration and short time (Figs. 1 and 2). In addition, we also observed that the combination of chrysin and cisplatin had significantly stronger effects on the ratio of p-p53/p53 and Bax/Bcl-2 than any agent alone (Figs. 3B and 4A). Taken together, the combination of chrysin and cisplatin dramatically promoted the apoptosis of Hep G2 cells.
X. Li et al. / Chemico-Biological Interactions 232 (2015) 12–20
The p53 protein is mainly regulated through post-translational modification by complex networks [29,35]. It is believed that MAPK activation is an important upstream event causing p53 phosphorylation in response to cisplatin [35,44]. ERK activation plays a pro-death role in promoting apoptosis or attenuating cisplatin resistance [45–47]. It has also been shown that chrysin activated JNK, p38, and ERK1/2, [17,18,36,48]. Our results demonstrated that no significant apoptosis of Hep G2 cells and ERK1/2 activation 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 well as the activation of ERK1/2 (Fig. 5C and D). Especially, ERK1/2 activation occurs prior to p53 phosphorylation, suggesting that ERK1/2 activation is an upstream event of p53 phosphorylation. Furthermore, U0126, an inhibitor of ERK1/2, could partially block p53 phosphorylation at Ser15 and inhibited its translocation to nuclei (Fig. 5E and F). Therefore, ERK activated by chrysin contributes to some extent to the accumulation and activation of p53 in Hep G2 cells. According to others studies, the phosphorylation of p53 at Ser15 is the target of activated ERK1/2 [35,49]. In summary, the combination of chrysin and cisplatin showed significant anticancer effects in Hep G2 cells in vitro. The combination of chrysin and cisplatin stabilized p53 protein through activating ERK1/2, which promoted p53 phosphorylation in Hep G2 cells. Our results suggest that the combination of chrysin and cisplatin may be a solution to the treatment of cisplatin-resistant cancers in clinic. In the future, the effects of the combination of chrysin and cisplatin on the apoptosis of cancer cells should be further investigated and confirmed in other human cancer cell lines as well as 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 be found in the online version.
Acknowledgements This study was in part supported by a research grant from the National Nature Science Foundation of China (No. 81202199), a research grant from the Nature Science Foundation of Guangdong Province (No. 9151030003000004), a research grant from the Science and Technology Program of Guangzhou (2014J4100090), and a research grand from the Science and Technology plan project of Guangdong Province (2013B021800044) to Xin Li, a research grant from the National Basic Research Program of China (No. 2012CB518200) and a research grant from the National Natural Science Foundation of China (No. 81272180) to Fei Zou. References [1] D.W. Shen, L.M. Pouliot, M.D. Hall, M.M. Gottesman, Cisplatin resistance: a cellular self-defense mechanism resulting from multiple epigenetic and genetic changes, Pharmacol. Rev. 64 (2012) 706–721. [2] P. Bragado, A. Armesilla, A. Silva, A. Porras, Apoptosis by cisplatin requires p53 mediated p38alpha MAPK activation through ROS generation, Apoptosis 12 (2007) 1733–1742. [3] R. Shi, Q. Huang, X. Zhu, Y.B. Ong, B. Zhao, J. Lu, C.N. Ong, H.M. Shen, Luteolin sensitizes the anticancer effect of cisplatin via c-Jun NH2-terminal kinasemediated p53 phosphorylation and stabilization, Mol. Cancer Ther. 6 (2007) 1338–1347.
19
[4] V.M. Gonzalez, M.A. Fuertes, C. Alonso, J.M. Perez, Is cisplatin-induced cell death always produced by apoptosis?, Mol Pharmacol. 59 (2001) 657–663. [5] Z.H. Siddik, Cisplatin: mode of cytotoxic action and molecular basis of resistance, Oncogene 22 (2003) 7265–7279. [6] Y. Aylon, M. Oren, Living with p53, dying of p53, Cell 130 (2007) 597–600. [7] S. Nag, J. Qin, K.S. Srivenugopal, M. Wang, R. Zhang, The MDM2-p53 pathway revisited, J. Biomed. Res. 27 (2013) 254–271. [8] U.M. Moll, S. Wolff, D. Speidel, W. Deppert, Transcription-independent proapoptotic functions of p53, Curr. Opin. Cell Biol. 17 (2005) 631–636. [9] S. Ramos, Effects of dietary flavonoids on apoptotic pathways related to cancer chemoprevention, J. Nutr. Biochem. 18 (2007) 427–442. [10] I.C. Arts, A review of the epidemiological evidence on tea, flavonoids, and lung cancer, J. Nutr. 138 (2008) 1561S–1566S. [11] A. During, Y. Larondelle, The O-methylation of chrysin markedly improves its intestinal anti-inflammatory properties: structure–activity relationships of flavones, Biochem. Pharmacol. 86 (2013) 1739–1746. [12] N. Ali, S. Rashid, S. Nafees, S.K. Hasan, S. Sultana, Beneficial effects of Chrysin against Methotrexate-induced hepatotoxicity via attenuation of oxidative stress and apoptosis, Mol. Cell. Biochem. 385 (2014) 215–223. [13] X.H. Ai, X. Zheng, X.Q. Tang, L. Sun, Y.Q. Zhang, Y. Qin, H.Q. Liu, H. Xia, J.G. Cao, Induction of apoptosis of human gastric carcinoma SGC-7901 cell line by 5,7dihydroxy-8-nitrochrysin in vitro, World J. Gastroenterol. 13 (2007) 3824– 3828. [14] M.S. Weng, Y.S. Ho, J.K. Lin, Chrysin induces G1 phase cell cycle arrest in C6 glioma cells through inducing p21Waf1/Cip1 expression: involvement of p38 mitogen-activated protein kinase, Biochem. Pharmacol. 69 (2005) 1815–1827. [15] X.M. Yu, T. Phan, P.N. Patel, R. Jaskula-Sztul, H. Chen, Chrysin activates Notch1 signaling and suppresses tumor growth of anaplastic thyroid carcinoma in vitro and in vivo, Cancer 119 (2013) 774–781. [16] F. Yang, H. Jin, J. Pi, J.H. Jiang, L. Liu, H.H. Bai, P.H. Yang, J.Y. Cai, Anti-tumor activity evaluation of novel chrysin–organogermanium(IV) complex in MCF-7 cells, Bioorg. Med. Chem. Lett. 23 (2013) 5544–5551. [17] S.K. Ha, E. Moon, S.Y. Kim, Chrysin suppresses LPS-stimulated proinflammatory responses by blocking NF-kappaB and JNK activations in microglia cells, Neurosci. Lett. 485 (2010) 143–147. [18] E. Pichichero, R. Cicconi, M. Mattei, A. Canini, Chrysin-induced apoptosis is mediated through p38 and Bax activation in B16–F1 and A375 melanoma cells, Int. J. Oncol. 38 (2011) 473–483. [19] S. Miyamoto, H. Kohno, R. Suzuki, S. Sugie, A. Murakami, H. Ohigashi, T. Tanaka, Preventive effects of chrysin on the development of azoxymethaneinduced colonic aberrant crypt foci in rats, Oncol. Rep. 15 (2006) 4. [20] X. Li, Q. Huang, C.N. Ong, X.F. Yang, H.M. Shen, Chrysin sensitizes tumor necrosis factor-alpha-induced apoptosis in human tumor cells via suppression of nuclear factor-kappaB, Cancer Lett. 293 (2010) 109–116. [21] X. Li, J.N. Wang, J.M. Huang, X.K. Xiong, M.F. Chen, C.N. Ong, H.M. Shen, X.F. Yang, Chrysin promotes tumor necrosis factor (TNF)-related apoptosisinducing ligand (TRAIL) induced apoptosis in human cancer cell lines, Toxicol. In Vitro 25 (2011) 630–635. [22] D. Wang, S.J. Lippard, Cellular processing of platinum anticancer drugs, Nat. Rev. Drug Discovery 4 (2005) 307–320. [23] Z. Darzynkiewicz, E. Bedner, F. Traganos, T. Murakami, Critical aspects in the analysis of apoptosis and necrosis, Hum. Cell 11 (1998) 3–12. [24] S. Fujioka, C. Schmidt, G.M. Sclabas, Z. Li, H. Pelicano, B. Peng, A. Yao, J. Niu, W. Zhang, D.B. Evans, J.L. Abbruzzese, P. Huang, P.J. Chiao, Stabilization of p53 is a novel mechanism for proapoptotic function of NF-kappaB, J. Biol. Chem. 279 (2004) 27549–27559. [25] A.J. Levine, W. Hu, Z. Feng, The P53 pathway: what questions remain to be explored?, Cell Death Differ 13 (2006) 1027–1036. [26] F. Esposito, M. Tornincasa, P. Chieffi, I. De Martino, G.M. Pierantoni, A. Fusco, High-mobility group A1 proteins regulate p53-mediated transcription of Bcl-2 gene, Cancer Res. 70 (2010) 5379–5388. [27] N. Finnberg, J.J. Gruber, P. Fei, D. Rudolph, A. Bric, S.H. Kim, T.F. Burns, H. Ajuha, R. Page, G.S. Wu, Y. Chen, W.G. McKenna, E. Bernhard, S. Lowe, T. Mak, W.S. ElDeiry, DR5 knockout mice are compromised in radiation-induced apoptosis, Mol. Cell. Biol. 25 (2005) 2000–2013. [28] J.K. Sax, W.S. El-Deiry, P53 downstream targets and chemosensitivity, Cell Death Differ. 10 (2003) 413–417. [29] M. Martinez-Rivera, Z.H. Siddik, Resistance and gain-of-resistance phenotypes in cancers harboring wild-type p53, Biochem. Pharmacol. 83 (2012) 1049–1062. [30] M.L. Tan, J.P. Ooi, N. Ismail, A.I. Moad, T.S. Muhammad, Programmed cell death pathways and current antitumor targets, Pharm. Res. 26 (2009) 1547–1560. [31] X. Yi, X.M. Yin, Z. Dong, Inhibition of Bid-induced apoptosis by Bcl-2. tBid insertion, Bax translocation, and Bax/Bak oligomerization suppressed, J. Biol. Chem. 278 (2003) 16992–16999. [32] J.S. Fridman, S.W. Lowe, Control of apoptosis by p53, Oncogene 22 (2003) 9030–9040. [33] D. Thomasova, S.R. Mulay, H. Bruns, H.J. Anders, P53-independent roles of MDM2 in NF-kappaB signaling: implications for cancer therapy, wound healing, and autoimmune diseases, Neoplasia 14 (2012) 1097–1101. [34] E. Appella, C.W. Anderson, Post-translational modifications and activation of p53 by genotoxic stresses, Eur. J. Biochem./FEBS 268 (2001) 2764–2772. [35] G.S. Wu, The functional interactions between the p53 and MAPK signaling pathways, Cancer Biol. Ther. 3 (2004) 156–161. [36] A.M. Gao, Z.P. Ke, F. Shi, G.C. Sun, H. Chen, Chrysin enhances sensitivity of BEL7402/ADM cells to doxorubicin by suppressing PI3K/Akt/Nrf2 and ERK/Nrf2 pathway, Chem. Biol. Interact. 206 (2013) 100–108.
20
X. Li et al. / Chemico-Biological Interactions 232 (2015) 12–20
[37] I. Kudo, M. Esumi, A. Kida, M. Ikeda, P53 mutation, but not in vitro predictor genes of therapeutic efficacy of cisplatin, is clinically relevant in comparing partial and complete responder cases of maxillary squamous cell carcinoma, Oncol. Rep. 24 (2010) 851–856. [38] E.L. Leung, M. Fraser, R.R. Fiscus, B.K. Tsang, Cisplatin alters nitric oxide synthase levels in human ovarian cancer cells: involvement in p53 regulation and cisplatin resistance, Br. J. Cancer 98 (2008) 1803–1809. [39] T.M. de Kok, S.G. van Breda, M.M. Manson, Mechanisms of combined action of different chemopreventive dietary compounds: a review, Eur. J. Nutr. 47 (Suppl. 2) (2008) 51–59. [40] Z.H. Xie, M.F. Quan, F. Liu, J.G. Cao, J.S. Zhang, 5-Allyl-7-gendifluoromethoxychrysin enhances TRAIL-induced apoptosis in human lung carcinoma A549 cells, BMC Cancer 11 (2011) 322. [41] Z. Kang, S.Y. Sun, L. Cao, Activating death receptor DR5 as a therapeutic strategy for rhabdomyosarcoma, ISRN Oncol. 2012 (2012) 395952. [42] R. Eskes, S. Desagher, B. Antonsson, J.C. Martinou, Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane, Mol. Cell. Biol. 20 (2000) 929–935. [43] J. Ren, H. Cheng, W.Q. Xin, X. Chen, K. Hu, Induction of apoptosis by 7piperazinethylchrysin in HCT-116 human colon cancer cells, Oncol. Rep. 28 (2012) 1719–1726.
[44] A. Brozovic, M. Osmak, Activation of mitogen-activated protein kinases by cisplatin and their role in cisplatin-resistance, Cancer Lett. 251 (2007) 1–16. [45] I. Arany, J.K. Megyesi, H. Kaneto, P.M. Price, R.L. Safirstein, Cisplatin-induced cell death is EGFR/src/ERK signaling dependent in mouse proximal tubule cells, Am. J. Physiol. Renal Physiol. 287 (2004) F543–F549. [46] S. Schweyer, A. Soruri, O. Meschter, A. Heintze, F. Zschunke, N. Miosge, P. Thelen, T. Schlott, H.J. Radzun, A. Fayyazi, Cisplatin-induced apoptosis in human malignant testicular germ cell lines depends on MEK/ERK activation, Br. J. Cancer 91 (2004) 589–598. [47] P.Y. Yeh, S.E. Chuang, K.H. Yeh, Y.C. Song, C.K. Ea, A.L. Cheng, Increase of the resistance of human cervical carcinoma cells to cisplatin by inhibition of the MEK to ERK signaling pathway partly via enhancement of anticancer druginduced NF kappa B activation, Biochem. Pharmacol. 63 (2002) 1423–1430. [48] W. Zeng, Y. Yan, F. Zhang, C. Zhang, W. Liang, Chrysin promotes osteogenic differentiation via ERK/MAPK activation, Protein Cell 4 (2013) 539–547. [49] Q.B. She, A.M. Bode, W.Y. Ma, N.Y. Chen, Z. Dong, Resveratrol-induced activation of p53 and apoptosis is mediated by extracellular-signal-regulated protein kinases and p38 kinase, Cancer Res. 61 (2001) 1604–1610.