Cheliensisin A Inhibits EGF-Induced Cell Transformation with Stabilization of p53 Protein Via a Hydrogen Peroxide/Chk1-Dependent Axis

The continuing high magnitude of the cancer incidence and the failure of treatments on cancers at advanced stage highlight the dire need for new approaches to control such diseases. Chemoprevention, which is a pharmacologic approach of intervention in an effort to arrest or reverse carcinogenesis at tumor promotion stage, has been increasingly appreciated as a new strategy in the fight against cancer (1–3). As diverse compounds isolated from plants were reported to interfere with a specific stage of the carcinogenic process or possess immense anti-oncologic value, numerous efforts have been devoted to identify such phytochemicals and phytochemical-derived agents with cancer prevention properties (4, 5).

Cheliensisin A (Chel A), a novel styryl-lactone isolated from Goniothalamus cheliensis Hu, has been shown to possess potent cytotoxicity against human promyelocytic leukemia HL-60 cells (6). Mechanically, Chel A was capable of inducing apoptosis of leukemia cells by downregulation of Bcl-2 expression (7). In addition, a recent study in our laboratory has shown that Chel A also displayed profound cytotoxic effects against HCT116 colon cancer cell line (data not shown). These results together suggest the potential of Chel A as an agent for cancer chemotherapy. Thus far, to the best of our knowledge, there has been no exploration of Chel A for chemopreventive intervention yet. Hence, the study here sought to evaluate the potential inhibitory effect of Chel A on EGF-induced cell transformation in JB6 Cl41 cell culture model.

p53 is a tumor suppressor that is inactivated or severely damaged during carcinogenesis of most human cancers (8). A multitude of studies have confirmed its pivotal role in anticancer functions, and there have been many studies of the multiple mechanisms that are involved, for example, activating DNA repair proteins, inducing cell-cycle arrest, and initiating apoptosis (9–11). Despite being abrogated in some cancer types, p53 can be activated in response to a myriad of stress types, which suggests that activating p53 by targeting p53-related pathways is a potential strategy for cancer prevention and therapy (12–14). Herein, we showed that Chel A exerted a chemopreventive effect on EGF-induced cell transformation with induction of apoptosis in Cl41 cells. Moreover, our results indicated that Chel A-induced apoptosis is mediated through stabilization and activation of p53 by hydrogen peroxide/Chk1-dependent axis.

Chel A [6(7,8-epoxy-styryl)-5-acetoxy-5,6-dihydro-2-pyrone; Fig. 1A] was isolated from Goniothalamus cheliensis by the Kunming Institute of Botany, Chinese Academy of Sciences (Kunming, China). Chel A is a white crystal with a purity more than 99.0%, as previously described (7). The chemicals MG132 and cycloheximide were purchased from Calbiochem. Dichlorofluorescein diacetate (DCFH-DA) and hydroethidine (HE) were purchased from Invitrogen.

Normal mouse epidermal Cl41 cells have been described previously (15). Cl41 cells and their stable transfectants were cultured in 5% FBS minimum essential medium (MEM) containing 1% penicillin/streptomycin and 2 mmol/L l-glutamine (Life Technologies) and were maintained at 37°C in 5% CO₂ incubator. Normal mouse embryonic fibroblasts (MEF) with wild-type p53 expression (p53^+/+) and p53-deficient MEFs (p53^−/−) have been described previously (16). Both p53^+/+ and p53^−/− MEFs were cultured in Dulbecco's modified Eagle medium (DMEM) with 10% FBS. The cultures were dissociated with trypsin and transferred to new 25 cm² culture flasks twice a week. No authentication of the cell lines was done by the authors. FBS was purchased from Life Technologies, Inc.and MEM and DMEM were from Calbiochem.

The pEGFP-Chk1 kinase-inactive mutant (GFP-Chk1 D130A) plasmids were kindly provided by Dr. Helen Piwnica-Worms (Department of Cell Biology and Physiology and Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO; ref. 17). Mitochondria Catalase expression plasmid (pZeo/mCat) and its parental control vector were kindly provided by Dr. J. Andres Melendez (Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY) as described in our previous publications (18, 19). The p53-promoter luciferase plasmid was kindly provided by Dr. Xianglin Shi (Graduate Center for Toxicology, University of Kentucky, Lexington, KY; ref. 20).

JB6 Cl41 cells were transfected with mCat and its corresponding control vector by using LipofectAMINE Transfection Kit (Gibco BRL) and stable transfectants were selected by Zeocin-resistant selection. Cl41 cells stably transfected with p53 promoter-driven luciferase reporter plasmid were established using FuGENE HD (Roche Applied Science), following the manufacturer's instructions. The basal level of luciferase activity in Cl41 p53 transfectant was verified to be significantly higher than that in Cl41 WT cells. DN-Chk1 plasmid and its corresponding control vector were transfected into Cl41 cells by using PolyJet DNA In Vitro Transfection Reagent (SignaGen Laboratories) following the manufacturer's instructions and stable transfectants were established by G418-resistant selection.

Soft agar colony formation assay was conducted as previously described (15). Briefly, 2.5 mL of 0.5% agar in basal modified Eagle's medium (BMEM) supplemented with 10% FBS and 20 ng/mL EGF, as well as Chel A at indicated concentrations, was layered onto each well of 6-well tissue culture plates. A total of 3 × 10⁴ Cl41 cells or their stable transfectants were mixed with 1 mL of 0.5% agar BMEM supplemented with 10% FBS with or without 20 ng/mL EGF, as well as with or without Chel A, and layered on top of the 0.5% agar layer. The plates were incubated at 37°C in 5% CO₂ for 3 weeks. The colonies were then counted under microscopy. Those with more than 32 cells were scored. The results were presented as colonies/10⁴ seeded cells.

Cl41 cells, MEFs, and their transfectants were cultured in each well of 6-well plates with normal medium until they reached 70% to 80% confluence. Cell culture medium was replaced to medium with 0.1% FBS for 24 hours. Then, the culture medium was changed to medium with 1% FBS and cells were treated with Chel A for 0.5 hour followed by treatment with Chel A and/or EGF with indicated concentrations and time periods. After exposure to EGF and Chel A, cells were washed with ice-cold PBS and then extracted with cell lysis buffer (10 mmol/L Tris-HCl, pH 7.4, 1% SDS, 1 mmol/L Na3VO4, and proteasome inhibitor). The cell extracts were subjected to Western blotting with each of the antibodies for determination of PARP, cleaved PARP, caspase-3, cleaved caspase-3, p53, p-p53 ser15, p-MDM2 Ser166, p-Chk1 Ser345, Chk1, p-Chk2 Thr68 (Cell Signaling Technology), Catalase (Calbiochem, EMD Biosciences, Inc.), MDM2, β-Actin (Sigma), as indicated. The protein bands specifically bound to the primary antibodies were detected using an alkaline phosphatase-linked secondary antibody and an ECF (enhanced chemifluorescence) Western blotting analysis system (Amersham Pharmacia Biotech), as previously described (15).

Cl41 cells were cultured in 6-well plates till they reached 70% to 80% confluence. Cell culture medium was changed to 0.1% FBS medium for 24 hours. Then, the cells were exposed to Chel A using the same method as was used for the cells that were treated for Western blotting assay. After treatment for the indicated time periods, total RNAs were extracted from cells using TRIzol reagent (Invitrogen). Total cDNAs were synthesized by using oligdT₍₂₀₎ primer by SuperScript First-Strand Synthesis system (Invitrogen). p53 and β-actin mRNA presented in the cells were determined by semiquantative reverse transcription (RT)-PCR assay. Mouse p53 (forward: 5′-CACGTACTCTCCTCCCCTCA-3′, reverse 5′-CTTCTGTACGGCGGTCT CTC-3′) and β-actin (forward: 5′-CCTGTGGCATCCATGAAACT-3′, reverse: 5′-GTGCTAGGAGCCAGAGCAGT-3′) primers (Invitrogen) were used to determine the mRNA amount of p53 and β-actin, respectively. The PCR products were separated onto 3% agarose gels, stained with ethidium bromide (EB), and the images scanned with a UV light as described previously (21).

Cl41 and its stable transfectant transfected with p53-promoter luciferase reporter plasmid were seeded into 96-well plates. The cell culture medium was replaced with 0.1% FBS MEM for 24 hours, and then the cells were exposed to Chel A at the indicated time periods and concentrations. The cells were then lysed for luciferase assay using luciferase substrate as previously described (21).

Cl41 cells were cultured in 6-well plates until they reached 70% to 80% confluence. Cell culture medium was replaced with 0.1% FBS medium for 36 hours. The cells were then treated with EGF (20 ng/mL) with or without Chel A at indicated concentrations in the medium containing 0.1% FBS. Cells were fixed in ice-cold 70% ethanol and stained with PI buffer (0.1% Triton X-100, 0.2 mg/mL RNase A, and 0.05 mg/mL PI) for 15 minutes. The samples were subjected to flow cytometry (Beckman Coulter) for apoptosis analysis.

Cl41 cells were seeded into 96-well plates until they reached 75% to 80% confluence. The cell culture medium was replaced with 0.1% FBS MEM for 36 hours. The cells were incubated with DCFH-DA or HE for 0.5 hour. Cells were then exposed to Chel A at indicated time periods followed by washing with PBS to remove the extracellular compounds and the fluorescence intensities were detected by SpectroMax M2 Microplate Reader (Molecular Devices Corp.). The cells incubated with DCFH-DA were used to monitor the production of hydrogen peroxide, with the excitation of 488 nm and emission of 530 nm, whereas cells treated with HE were used to the measure of superoxide, with excitation of 510 nm and emission of 600 nm (19, 22, 23).

The Student t test was used to determine the significance between Chel A-treated versus untreated group, or between specific gene transfectant versus vector transfectant. The results are expressed as mean ± SD from at least three independent experiments. P < 0.05 was considered as significant between the groups compared.

Although Chel A has been reported to possess the apoptosis-inducing effect on human promyelocytic leukemia HL-60 cells (6), its chemopreventive effect has not been explored yet. Thus, we used an EGF-induced Cl41 cell transformation model to evaluate the inhibitory effect of Chel A. As shown in Fig. 1B and C, coincubation of Cl41 cells with Chel A at various concentrations (0.5, 1.0, 2.0, and 4.0 μmol/L) for 3 weeks significantly inhibited EGF-induced anchorage-independent colony formation in mouse epidermal Cl41 cells, suggesting that Chel A might have a chemopreventive effect. To investigate whether the inhibitory effect of Chel A on EGF-induced cell transformation is due to its induction of apoptosis or cell-cycle arrest, the cell cycle distribution of Cl41 cells were first examined upon Chel A and/or EGF cotreatment. Cells were treated with Chel A at concentrations of 0, 2, and 4 μmol/L for 48 hours with or without exposure to EGF. High-resolution flow cytometry analysis of PI-stained nuclei displayed a substantial increase in apoptotic peak in a dose-dependent manner (Fig. 1D). Given that PARP cleavage and caspase-3 activation are well-characterized markers often associated with apoptosis (24), Western blot analysis was further carried out to check the induction of cleavage of caspase-3 and PARP. Consistent with flow cytometry analysis, Chel A treatment resulted in an increased cleavage of caspase-3 and PARP in both dose and time-dependent manners (Fig. 1E and F). Our results strongly showed that the Chel A treatment inhibited cell transformation with induction of apoptosis.

p53, a tumor suppressor, has been reported to participate directly in the mediation of caspase-dependent apoptosis (25). To explore the mechanism underlying Chel A-induced apoptosis, phosphorylation of p53 at ser15, which reflects its functional response to cellular stress and leads to apoptosis (26), and total p53 protein expression, were examined in Chel A-treated Cl41 cells, which express wild-type p53. Western blot analysis revealed that Chel A treatment induced both p53 phosphorylation at Ser15 and total p53 expression within as early as 1 hour and reaching maximum 3 hours (Fig. 2A), indicating that p53 participates in Chel A response. To further buttress this point, p53 phosphorylation and total expression were verified in MEFs, which express wild-type p53. Chel A treatment upregulated both Ser15 phosphorylated p53 and total p53 protein levels in MEFs (Fig. 2B), which is consistent with the results obtained from the Cl41cells. To determine whether p53 activation is necessary for Chel A-induced apoptosis, we used p53 knockout cells. Western blot analysis showed that p53 protein expression was depleted in p53^−/− cells in comparison with p53^+/+ cells (Fig. 2C). Moreover, p53 knockout significantly abrogated Chel A-induced cleavage of PARP and caspase-3, suggesting that p53 was critical in Chel A-induced apoptosis (Fig. 2D). In addition, we also examined the Chel A's effect on EGFR activation. As shown in Fig. 2A, Chel A treatment elicited neither increased EGFR phosphorylation at Tyr1068, which is crucial to the EGF-induced Ras/MAPK activation (27), nor elevated EGFR total protein expression, indicating that EGFR was not involved in Chel A-induced cell transformation inhibition and apoptotic induction.

To elucidate the molecular mechanisms for Chel A upregulation of p53 protein expression, we first examined the effect of Chel A on p53 transcription by using Cl41 p53 promoter-driven luciferase reporter stable transfectant. As shown in Fig. 3A, Chel A treatment did not significantly promote p53 promoter-driven luciferase reporter activity, whereas UVB radiation significantly induced p53 promoter-driven luciferase activity, revealing that Chel A did not upregulate p53 at transcription level. Furthermore, Chel A exerted no impact on p53 mRNA level within 6 hours along the time course by RT-PCR assay (Fig. 3B). Thus, we anticipated that p53 induction by Chel A might be regulated at the protein degradation or translational level.

In an attempt to clarify whether Chel A treatment is capable of preventing p53 protein from degradation, Cl41 cells were first pretreated with proteasome inhibitor MG132 to accumulate p53 protein. Then, MG132 was removed and the protein synthesis inhibitor cycloheximide was added to the cells, alone or in combination with Chel A. The effect of Chel A on the dynamics of p53 protein degradation was determined during the indicated time periods. As shown in Fig. 3C, the p53 protein level at 4 hours in cells cotreated with Chel A and cycloheximide is much higher than that observed in the cells treated with cycloheximide alone, showing that Chel A treatment significantly inhibited p53 protein degradation rate, further suggesting that Chel A treatment promoted p53 expression via inhibiting p53 protein degradation.

Several proteins, such as MDM2, Chk1, and Chk2, have been reported to be capable of regulating p53 protein degradation (26, 28). MDM2 serves as a p53-specific E3 ubiquitin ligase functioning to mediating p53 protein degradation, by which MDM2 limits p53-induced apoptosis (28). Posttranslational phosphorylation of the MDM2 protein at Ser166 promotes its mediation of p53 protein degradation (29). Thus, we examined whether Chel A had an effect on MDM2 phosphorylation at Ser166. The results showed that MDM2 total protein expression was not affected upon Chel A treatment, whereas its phosphorylation at Ser166 was increased in a time-dependent manner following Chel A treatment (Fig. 3D). These data suggest that Chel A did not show any inhibition of MDM2 phosphorylation at Ser166 and therefore did not account for accumulation of p53 protein upon Chel A treatment (Fig. 3D). Given the negative results received with MDM2, we then examined the kinases that can mediate p53 protein accumulation such as Chk1 and Chk2 (30). Chk1 and Chk2 are kinases capable of activating and stabilizing p53 protein by mediating phosphorylation of p53 protein, whereas the phosphorylation of Chk1 at Ser345 or of Chk2 at Thr68 is required for their activation (31). Our results showed that Chk1 phosphorylation at Ser345 was notably induced by Chel A treatment, whereas total Chk1 protein was slightly upregulated (Fig. 3D). In contrast, the phosphorylation of Chk2 at Thr68 did not show marked alteration following Chel A treatment (Fig. 3D). These results suggested that Chel A treatment might result in Chk1 phosphorylation and activation, subsequently mediating p53 stabilization.

To further substantiate this point, we analyzed protein levels of p53 protein phosphorylation and expression in the Chel A-treated Cl41 cells transfected with the plasmid-encoding kinase-inactive Chk1 mutant, GFP-Chk1 D130A, which can outcompete the endogenous Chk1 protein (17). Verification of GFP-Chk1 D130A expression in Cl41 stably transfectant cells was shown in Fig. 4A and ectopic expression of kinase inactive GFP-Chk1 D130A attenuated induction of p53 protein phosphorylation at Ser15 and expression by Chel A (Fig. 4B), strongly indicating that Chk1 mediated p53 stabilization. Furthermore, GFP-Chk1 D130A expression abrogated Chel A -induced cleavages of caspase-3 and PARP (Fig. 4C) as well as inhibition of EGF-induced Cl41 cell transformation (Fig. 4D and E).

As reactive oxygen species (ROS) has been well-characterized as a positive regulator in activating Chk1 and p53 (32), it is tempting to speculate whether ROS is also implicated in Chel A-induced Chk1 and p53 activation. Thus, two dyes, DCFH-DA and HE, were used to determine the intracellular hydrogen peroxide and superoxide anion following Cl41 treatment with Chel A, as illustrated in Fig. 5A. As a result, the amount of hydrogen peroxide significantly increased upon Chel A exposure in a time-dependent manner (Fig. 5B), whereas the generation of superoxide was not observed under the same experimental conditions (Fig. 5C). To further evaluate whether generation of hydrogen peroxide contributed to the induction of Chk1 and p53 by Chel A, Cl41 cells stably transfected with mitochondria catalase (Cl41 mCat) were used. The results showed that mCat overexpression attenuated the induction of Chk1 phosphorylation at Ser345 with consistent reduction of p53 protein phosphorylation at Ser15 and p53 protein accumulation following Chel A treatment (Fig. 6A), revealing that the increase in the intracellular level of hydrogen peroxide was required for Chk1 phosphorylation at Ser345, and subsequently leading to p53 protein accumulation by Chel A. Most importantly, Chel A-induced cleavages of caspase-3 and PARP were almost blocked in the Cl41 cells with mCat overexpressing (Fig. 6B). Consequently, the inhibition of cell transformation by Chel A was partially reversed by ectopic expression of mCat (Fig. 6C and D). Together, our results indicated that generation of hydrogen peroxide was required for Chel A inhibition of EGF-induced cell transformation in Cl41 cells.

Chemoprevention has become increasingly appreciated as a new strategy to fight against cancers. Discovering chemopreventive agents to reverse carcinogenesis is therefore of high significance (1–3). Chel A, a novel styryl-lactone isolated from Goniothalamus cheliensis Hu, has been shown to induce apoptosis in human promyelocytic leukemia HL-60 cells, and this process is mediated through the downregulation of Bcl-2 expression (6, 7). Nevertheless, the chemopreventive effect and mechanisms of Chel A on skin tumor promotion has not been explored. In this study, we have showed that Chel A is capable of inhibiting EGF-induced cell transformation in mouse epidermal JB6 Cl41 cells, suggesting the chemopreventive effect of Chel A on skin carcinogenesis. Our results indicated that Chel A exerted its inhibition of cell transformation via stabilization and activation of p53 with induction of apoptosis. Further studies showed that Chel A effect on p53 stabilization, apoptosis, and inhibition of cell transformation resulted from the generation of hydrogen peroxide and subsequent mediation of Chk1 phosphorylation at Ser345. Therefore, we identify a novel function of Chel A as a chemopreventive agent that is mediated by ROS/Chk1/p53 axis.

p53, a tumor suppressor, is inactivated or severely damaged during carcinogenesis of most human cancers (8). A multitude of studies have confirmed its pivotal role in anticancer functions, and multiple mechanisms have been involved, including activation of DNA repair proteins, induction of cell-cycle arrest, and initiation apoptosis (9–11). Inactivation of p53 by point mutations has been found to occur in more than 50% human cancers (33). Despite being abrogated in some cancer types, p53 can become activated in response to a myriad of stress types, suggesting that activating p53 by targeting p53-related pathways is a potential strategy for cancer prevention and therapy (12–14). In cancer harboring a WT p53, p53 often loses its function due to the degradation by overexpressed MDM2. Hence, targeting the p53-relavant pathway by reactivating its mutant or activating WT p53 could serve as an attractive strategy in both cancer prevention and therapy (34–36). Previous study showed that Chel A could trigger apoptosis in human promyelocytic leukemia HL-60 cells, which are deficient of p53 expression (37) and are unlikely to examine the effect of Chel A on p53-dependent pathway. In this study, Cl41 cells and MEFs with expression of wild-type p53 were used to explore whether Chel A could exert an impact on p53-associated anticancer effects. Our results strongly suggested that Chel A treatment could lead to both the accumulation and activation of p53 proteins. Additional studies indicated that Chel A regulated p53 by preventing p53 proteins from degradation.

Several factors may result in stress-induced p53 stabilization and activation (26, 28). Our results implied that activation of Chk1 by phosphorylation at Ser345 mediates Chel A-induced p53 stabilization and activation in Cl41 cells. However, the mechanism underlying p53 stabilization and activation regulated by Chk1 remains elusive. Building upon the previous study, that Chk1 is capable of phosphorylating p53 at Ser15 (38), it is likely that p53 phosphorylation at Ser15 is directly phosphorylated by Chk1 in our experimental system. If this is the case, then Chel A-induced p53 stabilization may result from p53 phosphorylation at Ser15 because p53 phosphorylation at Ser15 can lead to a reduced interaction of p53 with its negative regulator MDM2 (28). On the other hand, our data likewise suggest that MDM2 was activated due to its increased phosphorylation at Ser166. Although MDM2 activation would mediate p53 protein degradation, the overall p53 protein level is determined by multiple factors (26, 28). The results in this study presented the elevated total p53 protein levels in Chel A-treated cells, thus indicating that the force augmenting p53 protein stability, such as p53 phosphorylation at Ser15 by Chk1, outweighed that leading to p53 reduction.

ROS are products of cellular metabolism and have the potential to elicit DNA damage as well as play an important role in apoptosis induction under both physiologic and pathologic conditions (39–41). A multitude of stimulations has been identified thus far that can induce an elevated level of intracellular ROS, thereby leading to cell apoptosis (42–44). Similarly, our studies showed that Chel A treatment resulted in hydrogen peroxide generation, which played a critical role in Chel A-induced Chk1 phosphorylation at Ser345, p53 phosphorylation at Ser15, and apoptosis, as induction of these biologic effects by Chel A were almost completely impaired by ectopic expression of mCat in Cl41 cells. Consistently, overexpression of mCat could also reverse Chel A inhibition of EGF-induced cell transformation, suggesting that Chel A-induced apoptosis via H₂O₂/ChK1/p53 was crucial for the chemopreventive effect of Chel A. However, the mechanism underlying hydrogen peroxide generation by Chel A is still unclear and might be due to alteration of either oxidation–reduction metabolic pathways or interference with mitochondrial electron transport system (42). These possibilities are currently under investigation in our laboratory.

In summary, our studies showed that Chel A exerted a chemopreventive effect on EGF-induced cell transformation by inducing apoptosis in JB6 Cl41 cells. Mechanical insight into induction of apoptosis suggested that the signaling pathway is through hydrogen peroxide generation, which subsequently mediates Chk1 phosphorylation at Ser345 and p53 phosphorylation at Ser15. Taken together, these findings implicate the potential use of Chel A as a cancer chemopreventive agent.

No potential conflicts of interest were disclosed.

Conception and design: J. Zhang, Q. Zhao, C. Huang

Development of methodology: J. Zhang, X. Deng, J. Li, M. Zhang

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): G. Gao, F. Li

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Zhang, G. Gao, L. Chen, X. Deng, Y. Yu, Q. Zhao, C. Huang

Writing, review, and/or revision of the manuscript: J. Zhang, X. Deng, Y. Yu, Q. Zhao, C. Huang

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Zhang, X. Deng, D. Zhang, M. Zhang, Q. Zhao, C. Huang

Study supervision: Q. Zhao, C. Huang

The authors thank Dr. Helen Piwnica-Worms from Department of Cell Biology and Physiology and Howard Hughes Medical Institute, Washington University School of Medicine for the gift of GFP-Chk1 D130A construct, and Dr. J. Andres Melendez from The Center for Immunology and Microbial Disease, Albany Medical College for his generosity in providing us with the construct of mitochondria Catalase (pZeo/mCat).

This work was partially supported by grants from NIH/NCI RO1 CA112557, R01 CA177665 and NIH/NIEHS ES000260; NSFC81229002 and NSFC 90813004.

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