Activations of Both Extrinsic and Intrinsic Pathways in HCT 116 Human Colorectal Cancer Cells Contribute to Apoptosis through p53-Mediated ATM/Fas Signaling by Emilia sonchifolia Extract, a Folklore Medicinal Plant

Emilia sonchifolia (L.) DC (Compositae), an herbaceous plant found in Taiwan and India, is used as folk medicine. The clinical applications include inflammation, rheumatism, cough, cuts fever, dysentery, analgesic, and antibacteria. The activities of Emilia sonchifolia extract (ESE) on colorectal cancer cell death have not been fully investigated. The purpose of this study explored the induction of apoptosis and its molecular mechanisms in ESE-treated HCT 116 human colorectal cancer cells in vitro. The methanolic ESE was characterized, and γ-humulene was formed as the major constituent (63.86%). ESE induced cell growth inhibition in a concentration- and time-dependent response by MTT assay. Apoptotic cells (DNA fragmentation, an apoptotic catachrestic) were found after ESE treatment by TUNEL assay and DNA gel electrophoresis. Alternatively, ESE stimulated the activities of caspase-3, -8, and -9 and their specific caspase inhibitors protected against ESE-induced cytotoxicity. ESE promoted the mitochondria-dependent and death-receptor-associated protein levels. Also, ESE increased ROS production and upregulated the levels of ATM, p53, and Fas in HCT 116 cells. Strikingly, p53 siRNA reversed ESE-reduced viability involved in p53-mediated ATM/Fas signaling in HCT 116 cells. In summary, our result is the first report suggesting that ESE may be potentially efficacious in the treatment of colorectal cancer.


Introduction
Colorectal cancer is a major reason of death worldwide [1], and it is the third most frequent cause of cancer death in Taiwan. About 20.2 per 100,000 people died of colorectal cancer according to the reports of the Department of Health, R.O.C. (Taiwan) in 2010 (http://www.doh.gov.tw/EN2006/ index EN.aspx/). Recently, various studies have shown that traditional Chinese medicine (TCM) and folklore medicine possessed potential anticolorectal cancer activity and they are associated with a reduced risk of cancer [2][3][4]. It has been reported that the TCM and folklore medicine can induce reactive oxygen species (ROS) production and DNA damage, which leads to increased phosphorylation of ataxia-telangiectasia-mutated kinase (ATM) and p53 and then triggers apoptosis in human cancer cells [5]. The p53

GC-MS Analysis of Methanolic ESE. The compositions of methanolic ESE were analyzed by GC-MS (DSQ II
Single Quadrupole GC/MS, Thermofisher Scientific, USA), equipped with a 30 m × 0.25 mm × 0.25 μm DB-5MS (Agilent J&W Scientific). The GC oven temperature was programmed from 60 • C, held for 1 min and raised to 250 • C at 4 • C/min, held for 1 min, then increased by 10 • C/min to 300 • C, and held for 1 min to the end. The other parameters were as follows: injection temperature, 100 • C; ion source temperature, 250 • C; EI, 70 eV; carrier gas, He at 1.5 mL/min; injection volume, 5 μL; mass range, m/z 50-1050. The identification of the major compound, γ-humulene, was based on a comparison of MS spectra with an authentic standard purchased from Sigma-Aldrich Corp.

MTT Cell Viability Assay and Morphological
Observations. Cells seeded onto 96-well microplates at a density of 1 ×10 4 cells/100 μL per well were incubated with ESE at the concentrations of 0, 25, 50, 75, or 100 μg/mL for a 24-hour treatment. The medium was then removed, and the cells were incubated for 3 h with 100 μL of MTT solution (0.5 mg/mL MTT in PBS). The MTT-purple formazan productions were dissolved in 0.1 N isopropanol/hydrochloric acid (HCl) and optical densities of the solutions were measured by absorbance at 570 nm in an ELISA plate reader. Cell viability was expressed as the optical density ratio of the treatment to the control (% of control) as described previously [15,16]. For determining cell morphological experiment, cells were examined and photographed using a phase-contrast microscope as described elsewhere [16,17].

Assessments of Apoptosis by TUNEL Assay and DNA
Gel Electrophoresis. Terminal deoxynucleotidyltransferasemediated dUTP-biotin nick end labeling (TUNEL) assay was performed according to the manufacturer's protocols (In Situ Cell Death Detection Kit, Roche Diagnostics Corp., Indianapolis, IN, USA). Cells (1 × 10 6 /well) were plated onto six-well plates and exposed to 0, 25, 50, 75, or 100 μg/mL for 24 h. After treatment, cells were collected and determined as previously described [18,19]. TUNEL-positive cells were analyzed and quantitated using a FACSCalibur instrument (BD Biosciences, San Jose, CA, USA) equipped with BD Cell     Quest Pro software. Approximately 1 × 10 6 cells per well were incubated without (control) or with 50 μg/mL of ESE for 24-hour exposure. Cells from each sample were collected and the DNA was isolated for agarose gel electrophoresis as previously described [19,20]. After electrophoresis in a 1.5% agarose gel containing ethidium bromide (EtBr, Invitrogen) in 0.5x TBE buffer (AMRESCO Inc. Solon, OH, USA), the DNA in gel was resolved with UV light and photographed [19,20].

Determination of Levels of Proteins Associated with Apoptotic Death and p53/Fas Signaling by Western Blotting.
Cells at a density of 1 × 10 7 cells in 75 cm 2 flask were treated with 50 μg/mL of ESE for indicated intervals of time (0, 6, 12, and 24 h or 0, 2, and 4 h, resp.). Cells at the end of each treatment period were harvested, and isolated total proteins, mitochondrial and cytosolic proteins, and protein quantification were as described previously [22,23]. The lysates from each sample were centrifuged at 13000 ×g for 10 min and the protein concentration in the supernatant was determined with a PIERCE BCA protein assay kit (Thermo Fisher Scientific Inc. Rockford, IL, USA) as previously described [22,24]. Equal amounts (40 μg) from each sample of protein lysate were run on 10-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The iBotTM Dry Blotting System (Invitrogen) was used to electrotransferred to a PVDF membrane and thereafter the blot was blocked with 5% nonfat dry milk and 0.05% Tween 20 in PBS at pH 7.4 at room temperature for 1 h. After blocking, the membranes were incubated with anti-caspase-3, anti-caspase-8, anti-caspase-9, anticytochrome c (Cell Signaling Technology, Danvers, MA, USA), anti-Bcl-2, anti-Bax, anti-Bid, anti-PUMA, anti-Fas, anti-FasL, anti-DR4, anti-DR5, anti-ATM, anti-p-ATM Ser1981 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-p53 and p-p53 Ser15 (Abcam, Cambridge, U.K.) antibodies at 4 • C overnight. These membranes were then incubated with horseradish peroxidase-(HRP-) conjugated goat anti-mouse or anti-rabbit IgG secondary antibodies (Millipore, Billerica, MA, USA) for 2 h at room temperature with gentle shaking. After washing, bands were visualized by Immobilon Western chemiluminescent HRP substrate (ECL) kit (Millipore) according to the manufacturer's instructions followed by development on Kodak Bio-MAX MR film (Eastman Kodak, Rochester, NY, USA). The relative abundance of each band was quantified using ImageJ software (version 1.43, NIH, USA) for Windows [17,25]. Blots were reported with actin antibody as a loading control.

Measurement of Reactive Oxygen Species (ROS) Production and N-Acetylcysteine, Caffeine Pretreatment for Viability.
Cells at a density of 2 × 10 5 cells/well were plated onto 12well plates and treated with 50 μg/mL of ESE for 0, 2, and 4 h followed the determinations of the changes in ROS level. Cells were harvested from each treatment, resuspended in 500 μL of H 2 DCF-DA (5 μM) for ROS (hydrogen peroxide; H 2 O 2 ) at 37 • C for 30 min. Consequently, cells were immediately analyzed by flow cytometry as described elsewhere [28,29]. All fluorescence intensities were obtained from the mean intensity of the histogram constructed from approximately 10,000 cells using BD CellQuest Pro software. For viability assay, cells were pretreated with or without the 10 mM Nacetylcysteine (NAC, an antioxidant) or 1 mM caffeine (an ATM kinase inhibitor) for 1 h before exposure to 50 μg/mL of ESE for 24 h. Cells were harvested for determining the cell viability by MTT assay as described above [15,16].

Small
Interfering RNA Transfection. Cells at a density of 2 × 10 5 cells/well were seeded in 6-well plates and grown to 70% confluence. p53 siRNA (100 nM, Santa Cruz Biotechnology, Inc.) or control siRNA was transfected using Lipofectamine 2000 (Invitrogen) for 12 h according to the manufacturer's guideline [8]. After being transfected with p53 siRNA, cells were seeded and treated with 50 μg/mL of ESE for 24-h exposure. Cells were harvested for determining the protein abundance of p53, Fas, PUMA, caspase-8, and caspase-3 by Western blotting and analysis for cell viability using MTT assay and apoptosis by TUNEL as described above [15,16].
2.13. Statistical Analysis. All data were expressed as mean ± SD from at least three separate experiments. Statistical calculations of the data were obtained using Student's t-test Evidence-Based Complementary and Alternative Medicine 5 with significance value of * * * P < 0.001, which is considered significantly. (ESE). Results shown in Figure 1 indicated that major composition of methanolic Emilia sonchifolia extract (ESE) was "γ-humulene" (Figure 1(a)) and the content was 63.86% (Figure 1(b)) based on peak area integrated by Thermo Xcalibur TM data analysis program.  (Figure 2(c)). Moreover, DNA gel electrophoresis confirmed that ESE induced apoptosis and DNA ladders in HCT 116 cells after 50 μg/mL of ESE exposure (Figure 2(d)).

ESE Enhanced the Activities of Caspase-3, -8 and -9 in
HCT 116 Cells. The observation of apoptotic death induced by ESE raised the possibility that activations of caspase cascades were required for examining the treated HCT 116 cells in vitro. Thus, we next examined the proteolytic activation of caspase cascades in ESE-treated HCT 116 cells and our results indicated that ESE stimulated caspase-3, caspase-8, and caspase-9 activities in a time-dependent effect (Figure 3(a)). The protein levels of pro-caspase-3, pro-caspase-8, and pro-caspase-9 were downregulated in HCT 116 cells after ESE exposure (Figure 3(b)). In addition, the result in Figure 3(c) from confocal microscopy indicated ESE stimulated the translocation of caspase-3 trafficking to the nuclei when compared to the control sample. Importantly, pretreatment with specific inhibitors of caspase-3 inhibitor (Z-DEVD-FMK), caspase-8 inhibitor (Z-IETD-FMK), and caspase-9 inhibitor (Z-LEHD-FMK), respectively, significantly prevented against the ESE-induced cell growth inhibition (Figure 3(d)) and apoptosis in HCT 116 cells (Figure 3(e)).

ESE-Triggered Apoptosis in Mediated Mitochondria-and Death-Receptor-Dependent Signaling in HCT 116 Cells.
To assess the alterations in apoptosis-related protein levels in ESE-treated HCT 116 cells, we administered ESE at the concentration of 50 μg/mL for 0, 6, 12, and 24 h in HCT 116 cells and then evaluated the protein levels by Western blot analysis. Figure 4(a) shows that ESE promoted a decrease of Bcl-2 level (an antiapoptotic protein) and the increases of proapoptotic protein levels of Bax and PUMA in HCT 116 cells. Also, treatment of ESE showed that the level of Bid was downregulated in HCT 116 cells (Figure 4(a)). As shown in Figure 4(b), ESE enhanced the death receptor pathway-associated protein levels (Fas, DR4 and DR5) in HCT 116 cells. Alternatively, the level of cytochrome c from cytosolic fraction is upregulated and from mitochondrial fraction is downregulated in ESE-treated HCT 116 cells (Figure 4(c)). Collectively, these results suggest that both intrinsic (mitochondria) and extrinsic (death-receptor-) dependent pathways contributed to ESE-provoked apoptotic death in HCT 116 cells. Figure 5(a) revealed that ESE promoted the level of intracellular ROS production (2 h treatment: 81.26 ± 2.39%; 4 h treatment: 90.34±4.18%) in HCT 116 cells by flow cytometry and a specific fluorescent probe, H 2 DCFDA for determining ROS level. Cells pretreated with N-acetylcysteine (NAC, an antioxidant) and caffeine (an ATM kinase inhibitor) significantly reduced ESE-induced growth inhibition effect ( Figure 5(b)). Previous studies have stated that p53 gene and its phosphorylation at the Ser15 interacted Fas/CD95 activation when cell apoptosis occur [7,8,30]. To elucidate the crucial roles of ATM, p53 and Fas in HCT 116 cells after treatment with ESE, the protein levels of ATM, p-ATM Ser1981 , p53, and p-p53 Ser15 expression were investigated by Western blot analysis. Our results showed that ESE increased the protein levels of ATM, p-ATM Ser1981 , p53 and p-p53 Ser15 in HCT 116 cells as can be seen in Figure 5(b). Our further study investigated if p53 affects the Fas expression in ESEtreated HCT 116 cells. We hypothesized that ESE induces apoptosis through the increase of Fas/CD95 by p53-dependent transcriptional activation. real-time PCR analysis was performed to determine whether the induction of Fas/CD95 protein level by ESE was due to increased the level of mRNA. As shown in Figure 5(d), the 6 and 12 h treatment of HCT 116 cells with ESE (50 μg/mL) led to an increase in mRNA levels of Fas/CD95. Our results indicate that ESE increased the protein level of Fas/CD95 through the p53-dependent regulation of transcription levels. Therefore, our results showed that the induction of p53, Fas, PUMA, cleaved caspase-8, and cleaved caspase-3 due to ESE treatment was correlated with the decrease in p53, Fas, PUMA, cleaved caspase-8, and cleaved caspase-3 protein levels by the transfection with p53 siRNA in HCT 116 * * * * * * * * * * * * cells (Figure 6(a)). We found that ESE-reduced viability in HCT 116 cells was nearly enhanced after using p53 siRNA compared to the ESE alone sample as shown in Figure 6(b). We also found that ESE induced apoptosis in HCT 116 cells was nearly prevented after using p53 siRNA compared to the ESE alone sample as shown in Figure 6(c). Taken together, these results suggest that p53 activation is an important factor in ESE-induced apoptosis of HCT 116 cells, which is mediated through ROS productions (oxidative stress) and ATM/p53/Fas-dependent signaling pathways.

Discussion
Phytochemicals affect intracellular targets, and this characteristics often makes desirable on tumor cells as chemopreventive or chemotherapeutic agents against cancer [31].   Figure 4: ESE altered the protein abundance-associated with mitochondria-and death-receptor-dependent apoptotic signaling in HCT 116 cells. Cells were treated with ESE (50 μg/mL) for 0, 6, 12, 24 h, and total protein, cytosolic, and mitochondrial lysates were prepared and subjected to Western blotting analysis. The membranes were incubated with (a) anti-Bcl-2, anti-Bax, anti-Bid and anti-PUMA antibodies; (b) anti-Fas, anti-FasL, anti-DR4 and anti-DR5 antibodies; (c) anticytochrome c antibody. The blot was also probed with anti-Actin and anti-Complex V antibodies to confirm equal loading of samples. Each band was quantified using ImageJ software.
The previous study showed that ESE inhibited lymphoma, Ehrlich ascites carcinoma, and mouse lung L-929 fibroblast cells, and it is important that ESE is not toxic to normal cells in vitro [10]. In vivo study also indicated that oral administration of the ESE (100 mg/kg body weight) to mice increased the life span and reduced the solid tumor volume of tumor-bearing mice [10][11][12] [32][33][34][35].
The IC 50 values calculated from these results are reported in Table 1; SW480, HT29, and A549 cell lines, which carries a mutant form of the p53 gene, are significantly low cytotoxic action of ESE than the HCT116 cell line, carrying a wildtype p53 gene. However, H1299 cells are 2-fold IC 50 values to ESE than HCT116 cells (Table 1) effect was found in the wild-type p53 cell lines. The results suggested that ESE preferentially induced more cytotoxic effect in the wild-type p53 lines than in the mutant or null p53 colorectal cancer cells. The reasons for the differences in sensitivities in IC 50 of those cell lines may be due to the intrinsic different p53 gene in different types of cell lines. The p53 in SW480 and HT29 cells has been shown to be a mutated gene with a mutation at codon 273 and that in HCT 116 cells is future to be functional without mutation [36]. It is reported that p53 is a mediator of chemotherapyinduced cell death, resulting from ROS productions which is activated by chemotherapeutic agents [37]. Many studies reported that cisplatin did not significantly increase apoptosis in p53-mutant cells, but a significant increase in the apoptotic index was observed in wild type p53 cells which correlates with increased p53 protein level [38][39][40]  demonstrated that γ-humulene is the major constituent in Emilia sonchifolia by GC/MS analysis ( Figure 1). Recently, we first demonstrated that γ-humulene has anticancer activity by stimulating the clustering of DR4/DR5 and associated FADD protein levels, leading to caspase-8 and caspase-3 activation, and then induction of apoptosis in HT29 cells [11]. Correctively, our results suggest that γ-humulene is the major bioactive compound in ESE. Apoptosis is an intracellular suicide program possessing morphologic change and biochemical response. In the present study, we showed that ESE reduced the cell viability in HCT 116 cells in a concentration-dependent manner (Figure 2(a)) and triggered apoptotic morphological changes (Figure 2(b)). ESE induced DNA condensation and fragmentation by DNA gel electrophoresis and TUNEL staining (Figures 2(c) and 2(d)). Two major apoptotic pathways have been described the extrinsic (death receptor mediated) and the intrinsic pathway (mitochondria mediated) [41]. The intrinsic apoptotic pathway affected mitochondrial permeability, releases cytochrome c, Apaf-1, Endo G, and pro-caspase-9 proteins from mitochondria to cytosol, leading to activation of caspase-9. The extrinsic apoptotic   pathways originateing at membrane death receptors include Fas/CD95, DR4 and DR5 and then influence the intracellular apoptotic adaptor FADD protein and proximal caspase-8 as well as distal executioner caspases [41][42][43]. Our results demonstrated that ESE significantly increased activities of caspase-3, caspase-8, and caspase-9 (Figure 3(a)) after 6 to 24 h treatment, and pretreatment with specific inhibitors of caspase-3, -8, and -9, respectively, led to increased viable cells in ESE treatment (Figure 3(c)). On the other hand, pretreatment of cells with specific inhibitors to caspase-3, -8, and -9 significantly prevented the ESE-induced cell apoptosis (Figure 3(e)). Our results suggest that both of extrinsic and intrinsic apoptotic pathways may be involved in the ESEprovoked apoptotic death in HCT 116 cells.
In conclusion, the molecular signaling pathways are summarized in Figure 7. The present study revealed that ESE (i) decreased the percentage of viable cells; (ii) induced apoptotic morphological changes and DNA fragmentation; (iii) upregulated the protein levels and activated the levels of caspase-3, caspase-8, and caspase-9; (iv) increased the ROS production and upregulated the protein levels of ATM, p-ATM Ser1981 , p53 and p-p53 Ser15 ; (V) stimulated the p53 downstream protein levels of Fas/CD95 and PUMA. Our results suggest that ESE warrants further development as a colorectal cancer prevention or therapeutic agents in the future.