Akt signaling pathway

Journal Pre-proofs Cocoa tea (Camellia ptilophylla) induces mitochondria-dependent apoptosis in HCT116 cells via ROS generation and PI3K/Akt signaling pathway Xiong Gao, Xiaofei Li, Chi-Tang Ho, Xiaorong Lin, Yuanyuan Zhang, Bin Li, Zhongzheng Chen PII: DOI: Reference:

S0963-9969(19)30740-9 https://doi.org/10.1016/j.foodres.2019.108854 FRIN 108854

To appear in:

Food Research International

Received Date: Revised Date: Accepted Date:

16 June 2019 17 November 2019 20 November 2019

Please cite this article as: Gao, X., Li, X., Ho, C-T., Lin, X., Zhang, Y., Li, B., Chen, Z., Cocoa tea (Camellia ptilophylla) induces mitochondria-dependent apoptosis in HCT116 cells via ROS generation and PI3K/Akt signaling pathway, Food Research International (2019), doi: https://doi.org/10.1016/j.foodres.2019.108854

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Cocoa tea (Camellia ptilophylla) induces mitochondria-dependent apoptosis in HCT116 cells via ROS generation and PI3K/Akt signaling pathway

Xiong Gaoa,b, Xiaofei Lib, Chi-Tang Hoc, Xiaorong Linb, Yuanyuan Zhangb, Bin Lib,*, Zhongzheng Chenb,*

a

State Key Laboratory of Applied Microbiology Southern China, Guangdong

Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, Guangdong Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou 510070, China b

College of Food Science, South China Agricultural University, 483 Wushan Street,

Tianhe District, Guangzhou 510642, China c

Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick,

NJ 08901, USA

* Corresponding author: Prof. Bin Li, Tel/Fax: +86 20 85281029 E-mail: [email protected] * Corresponding author: Dr. Zhongzheng Chen, Tel/Fax: +86 20 85281029 E-mail: [email protected]

ABSTRACT Cocoa tea (Camellia ptilophylla), a natural gallocatechin gallate (GCG)-rich and low caffeine-containing tea species, has been recently reported to possess various bioactivities. However, the anti-colon cancer effects of Cocoa tea and its underlying mechanisms remain virtually unknown. This study aimed to assess the anti-proliferative and pro-apoptotic effects of water extract of Cocoa tea (CWE) on human colon cancer HCT116 cells compared with Yunnan Daye tea (YWE). Primarily, CWE showed stronger anti-proliferation and apoptosis induction than YWE. Moreover, reduction of mitochondrial membrane potential (MMP), up-regulation of Bax/Bcl-2 ratio, release of cytochrome c, activation of caspase-9 and -3, and cleavage of poly (ADP-ribose) polymerase (PARP) were observed, suggesting that mitochondrial apoptotic pathway was activated by CWE. Furthermore, CWE-induced apoptosis in HCT116 cells was dependent on the generation of intracellular reactive oxygen species (ROS) and down-regulation of phosphatidylinositol-3-kinase (PI3K)/Akt pathway. Pretreatment with ROS scavenger N-acetyl cysteine (NAC) attenuated the impact of CWE on mitochondria-related apoptosis proteins, and partially recovered the inhibition of Akt phosphorylation. These results indicated that ROS generation mediated mitochondrial dysfunction and inactivation of PI3K/Akt pathway in CWE-induced HCT116 cell apoptosis. Additionally, CWE significantly inhibited tumor growth in HCT116 tumor-bearing mice, suggesting that Cocoa tea could act as a potential functional beverage to prevent or treat colorectal cancer.

Keywords: Cocoa tea; GCG-rich; low-caffeine; colon cancer; HCT116 cells; apoptosis; ROS; PI3K/Akt

1. Introduction Colorectal cancer (CRC) is the second most commonly diagnosed cancer in women and the third in men with over 1.84 million new cases and 881,000 deaths reported worldwide in 2018 (Bray et al., 2018). In the United States, despite considerable advancement in CRC treatment, mortality rates remain relatively high (Siegel et al., 2017). In China, the incidence and mortality rates of CRC showed an increasing in the past decades (Chen et al., 2019). Except for environmental and genetic factors, dietary patterns play an important role in the prevention of CRC (Tabung, Brown, & Fung, 2017). Healthy dietary patterns have been associated with decreased risk of CRC (Petimar et al., 2018). During the past few decades, increasing attention has been focused on the role of diet in human health. Phenolic compounds, which are widely distributed in food, are regarded as an important source of antioxidants in the daily diet (Zhang et al., 2019). Various studies have demonstrated that dietary intake of phenolic compounds can reduce the risk of chronic diseases, including cancer, diabetes, cardiovascular and neurodegenerative diseases (Shahidi & Yeo, 2018). Tea (Camellia sinensis), the second most consumed beverage worldwide, is well known for its potential health benefits. Green tea and its major constituent tea polyphenols, particularly epigallocatechin gallate (EGCG), have been shown to exert colon cancer prevention activity in mouse models (Zhang et al., 2019). However, high intake of tea increases caffeine level in the human body that may cause some detrimental effects, e.g. palpitations, anxiety, tremor, insomnia, osteoporosis,

gastrointestinal disturbances, and increased blood pressure (Mohanpuria, Kumar, & Yadav, 2010). Therefore, many techniques such as hot water, supercritical carbon dioxide, and organic solvents have been applied to produce decaffeinated tea. However, these techniques are not widely accepted by consumers because tea catechins, aromas, or flavors would be lost during the decaffeination process (Dong, Ye, Lu, Zheng, & Liang, 2011; Shiono et al., 2017). Furthermore, caffeine shows no inhibitory effects on colonic carcinogenesis (Yang & Hong, 2013). Cocoa tea (Camellia ptilophylla Chang), a unique and naturally occurring low caffeine-containing tea species, was discovered in Southern China in 1981 (Peng et al., 2011). Locally, Cocoa tea has been consumed as a daily beverage for a long time. This rare species comprised 4−7% of theobromine instead of 3−5% of caffeine in regular tea (Camellia sinensis). Moreover, different from Camellia sinensis, dominant catechin monomer in Cocoa tea is gallocatechin gallate (GCG) (Lin et al., 2014). Cocoa tea has been confirmed to possess various beneficial effects such as anti-obesity (Peng et al., 2019), anti-angiogenesis (Li et al., 2014), and antioxidant activity (Peng et al., 2011). Additionally, previous studies have suggested that Cocoa tea also exerts inhibitory effects on human prostate cancer and liver cancer both in vitro and in vivo (Peng, Khan, Afaq, Ye, & Mukhtar, 2010; Yang et al., 2012). However, the anti-cancer effects of Cocoa tea on CRC and its underlying mechanisms remain virtually unknown. In the present study, we focused on the inhibitory effects of water extract of green tea from Cocoa tea on HCT116 cells, using YD tea (Yunnan Daye tea, Camellia sinensis) as a control. The underlying mechanism

of apoptosis induced by Cocoa tea in HCT116 cells was investigated. With our current knowledge, this is the first study to report the anti-cancer activity of Cocoa tea on CRC. The results presented in this study may be helpful for understanding cancer chemopreventive properties of Cocoa tea and provide scientific evidence for its future application in colorectal cancer therapy. 2. Materials and methods 2.1. Chemicals and reagents Epicatechin (EC, ≥98%), (−)-catechin (C, ≥98%), epicatechin gallate (ECG, ≥98%), catechin gallate (CG, ≥98%), epigallocatechin (EGC, ≥98%), gallocatechin (GC, ≥98%), EGCG (≥95%), GCG (≥98%), theobromine (TB, ≥99%), gallic acid (GA, 97.5−102.5% by titration), 2',7'-dichlorofluorescein diacetate (DCFH-DA), 5-fluorouracil (5-FU), and methyl thiazolyl tetrazolium (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Caffeine (CAF) was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS, Australia Origin), Dulbecco’s phosphate-buffered saline (DPBS, pH 7.4), and 0.25% trypsin-ethylene diamine tetraacetic acid (EDTA) were purchased from Gibco (Life Technologies, Carlsbad, CA, USA). Pierce bicinchoninic acid (BCA) protein assay kit and enhanced chemiluminescent substrate were purchased from Thermo Scientific (Rockford, IL, USA). Penicillin/streptomycin solution was purchased from HyClone (General Electric Healthcare, Uppsala, Sweden). Primary antibodies against β-actin, phosphatidylinositol-3-kinase (PI3K) (p110β), PI3K (p85), Akt, p-Akt (Ser 473), cytochrome c, Bcl-2, Bax, cleaved

caspase-3, apoptosis antibody sampler kit, and horseradish peroxidase-conjugated secondary antibody were purchased from Cell Signaling Technology (Boston, MA, USA). Mitochondrial membrane potential assay kit, cell mitochondria isolation kit, colorimetric terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) apoptosis assay kit, N-acetyl-L-cysteine (NAC), and LY294002 were purchased from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). Milli-Q water (18.2 MΩ) was prepared by Milli-Q Integral 3 from Millipore (Molsheim, Alsace, France). All other reagents and chemicals were of analytical grade. 2.2. Materials and extraction Cocoa tea was plucked with an apical bud and two or three adjoining leaves in 2014 spring at Nankun Mountain (Longmen, Guangdong, China), and then processed into green tea as described in our previous study. Briefly, fresh tea leaves were roasted at 260−300 °C for fixation followed by a 30 min-rolling. Then, rolled tea leaves were dried at 110−120 °C for 10−15 min and re-dried at 85−95 °C after a 40 min-cooling period. YD green tea harvested during 2014 spring was purchased from Huahai Sugar Development Co., Ltd. (Xuwen, Guangdong, China). Green tea leaves (4.0 g), ground and sieved ranging from 20 to 30 mesh, were extracted with 100 mL of Milli-Q water in a boiling water bath (100 °C) for 45 min (Erlenmeyer flask was shaken every 10 min). Extract infusions were filtered and lyophilized with a freeze drier (Christ Alpha 1-2 LD plus, Germany), the freeze-dried powder of water extracts of Cocoa green tea and YD green tea were weighed and

designated as CWE and YWE, respectively. The extraction yields of CWE and YWE were about 43.0% and 40.7% of dry weight, respectively. The extracts were stored at −20 °C until tested. For cellular experiments, samples were dissolved in culture medium and filtered through 0.22 µm pore size syringe-driven filters and then diluted to certain concentrations. 2.3. Cell culture Human colon cancer cell line HCT116, obtained from the Chinese Academy of Sciences (Shanghai, China), was cultured in DMEM with 10% FBS and 1% penicillin/streptomycin. Cells were incubated in a humidified atmosphere at 37 °C with 5% CO2. 2.4. Chemical compositions of tea extracts As described in our previous study (Lin et al., 2014), analyses for catechins and purine alkaloids were conducted using an Agilent 1200 HPLC (G1322A Degasser, G1311A Quat Pump, G1329A ALS, G1315D DAD, Agilent Technologies, Inc., Santa Clara, CA, USA). An Eclipse XDB-C18 column (250 mm × 4.6 mm × 5 μm, Agilent) was used. All the contents of chemical components were expressed as the percentage of freeze-dried powder of water extract. The total phenolics content of tea extracts was determined using the Folin−Ciocalteu method described by Ramos et al. (2019) with some modifications. Briefly, 20 µL of tea sample or standard were added into each well of the microplate, followed by 100 µL of 10% Folin−Ciocalteu reagent and mixed. After 8 min of reaction, 80 µL of 7.5% Na2CO3 (w/v) was added. After gentle mixing, the microplate

was incubated in the dark for another 1 h and the absorbance at 765 nm was then measured with a VersaMax ELISA Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). Gallic acid was used as a standard and total phenolics content was expressed as mg gallic acid equivalents (GAE) per 100 mg dry powder. 2.5. Cell viability assay The MTT reduction assay was used to determine relative cell viability as described by Mosmann (1983) with some modification. Briefly, HCT116 cells were seeded at 5 × 103 cells/well on a 96-well plate and allowed to adhere for 24 h at 37 °C in 5% CO2. The cells were then treated with tea extracts (100−500 µg/mL), GCG or EGCG (100−300 µM) in fresh medium for 24 h, 48 h or 72 h. Subsequently, the medium was carefully removed and cells were incubated with MTT solution (0.25 mg/mL) for 2 h, and the formazan crystals were dissolved with dimethyl sulphoxide (DMSO). The plate was then incubated at 37 °C under gentle shaking for 15 min and the absorbance at 550 nm was measured with a VersaMax ELISA Microplate Reader (Molecular Devices). The cell viability was expressed as the percentage of the control group, which was set as 100%. The half inhibitory concentration (IC50) values were calculated using Origin 8.0 software. 2.6. Apoptosis assay Detection of apoptosis in HCT116 cells was carried out using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (eBioscience, San Diego, CA, USA). Briefly, cells were seeded at 5 × 104 cells/mL in a 60-mm culture dish for 24 h at 37 °C in 5% CO2. After treatment with tea extracts

(100, 200, and 300 µg/mL), GCG or EGCG (50, 100, and 200 µM) for 48 h, cells were harvested, centrifuged at 500 g for 5 min and washed twice with ice-cold DPBS. For inhibitor treatment, cells were pretreated with NAC (5 mM) and LY294002 (30 µM) for 1 h, respectively, and followed by treatment with or without CWE (300 µg/mL) for another 48 h. Subsequently, cells were resuspended in binding buffer with 1 × 106 cells/mL and stained with Annexin V-FITC and PI for 15 min at room temperature in the dark. For each sample, 10,000 events were analyzed using a FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA). 2.7. Measurement of mitochondrial membrane potential (MMP) Mitochondrial membrane potential assay kit with JC-1 was used to detect the loss of MMP in HCT116 cells. After treatment with CWE (100, 200, and 300 µg/mL) for 48 h, the cells were collected, washed twice with DPBS and stained with JC-1 according to the manufacturer’s protocols. Stained cells were analyzed using a FACSVerse flow cytometer (BD Biosciences). 2.8. Measurement of intracellular reactive oxygen species (ROS) The intracellular ROS of HCT116 cells was determined with DCFH-DA fluorescence probe. After treatment with CWE (100, 200, and 300 µg/mL) for 24 h, the cells were collected, washed twice with DPBS and stained with 10 µM DCFH-DA in DMEM for 30 min at 37 °C in the dark. Then the medium was removed and cells were washed twice with DPBS again. Finally, the DCF fluorescence intensity was measured using a FACSVerse flow cytometer (BD Biosciences). 2.9. Western blot analysis

Cells were harvested and the whole protein was extracted with RIPA buffer containing protease and phosphatase inhibitor. Cytosolic and mitochondrial proteins were extracted using the cell mitochondria isolation kit. After centrifugation at 14000 g for 15 min at 4 °C, the protein concentration was quantified using Pierce BCA protein assay kit. Equal amounts of proteins (25 µg) were separated on SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Each membrane was blocked with 5% skim milk in TBST (Sangon Biotech Co., Ltd., Shanghai, China) at room temperature for 1.5 h, and incubated with different primary antibodies overnight at 4 °C. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. The protein bands were visualized by using enhanced chemiluminescent substrate and performed with OmegaLum G imaging software (Aplegen, San Francisco, CA, USA). 2.10. HCT116 xenograft tumor study Five-weeks-old male BALB/c nude mice were purchased from the Laboratory Animal Center of Southern Medical University (Guangzhou, Guangdong, China). All experiments were conducted following procedures approved by the Animal Ethics Committee of Guangdong Institute of Microbiology (No. SYXK(Yue)2016-0156). The mice were maintained under specific pathogen-free conditions, fed with standard laboratory food and water, and housed in an air-conditioned room with 12 h light/dark cycle. HCT116 cells (1 × 107 cells/mL, 100 µL) were injected subcutaneously into the flank of each mouse to form a tumor. When the diameter of the tumor reached to

approximately 5 mm, the mice were randomly divided into four groups (n=6), and daily administered intragastrically with water as a control, 80 mg/kg and 160 mg/kg of CWE as treatment groups, and 20 mg/kg of 5-FU as a positive control, respectively. The tumor volume was measured every 4 days and calculated from the formula: tumor volume (mm3) = (A ×B2)/2, where A indicates the longest diameter and B indicates the shortest diameter of the tumor as measured using calipers. The mice were sacrificed on day 28, and the tumors were excised and weighed. Then tumors were photographed, dissected, fixed with 4% (V/V) paraformaldehyde solution, and embedded in paraffin. After deparaffinization and rehydration, the sections of tumor tissues were stained with hematoxylin and eosin (H&E) for histological evaluation. Apoptosis in vivo was visualized by colorimetric TUNEL apoptosis

assay

kit

according

to

the

manufacturer’s

protocols.

For

immunohistochemical analysis, the sections of tumor tissues were stained using primary antibody against cleaved caspase-3. Finally, the sections were observed under an optical microscope equipped with an Olympus digital camera (Olympus, Tokyo, Japan). Images were captured and analyzed using Image-Pro Plus 6.0 software. 2.11. Statistical analysis Experimental results were presented as means ± standard deviation (S.D.). The statistical significance of the differences was assessed by Fisher's least significant difference (LSD) test using SAS 9.2 software for Windows. Origin 8.6 software was used for illustration. 3. Results

3.1. Distinctive chemical compositions of Cocoa tea To compare chemical basis of Cocoa tea and YD tea, total phenolics content, monomeric catechins and purine alkaloids in the water extract of Cocoa tea (CWE) and YD tea (YWE) were determined with Folin−Ciocalteu and HPLC method. As shown in Table 1, CWE contained small amount of caffeine (0.70 mg/100 mg dry powder) and its major alkaloid was theobromine (10.78 mg/100 mg dry powder), while the main alkaloid in YWE was caffeine (6.62 mg/100 mg dry powder). Moreover, the total contents of catechins and phenolics in CWE were significantly higher than those in YWE. Additionally, CWE contained lower 2,3-cis-catechins, like EGCG, EGC, ECG,

and EC

than YWE, however,

it

contained more

2,3-trans-catechins including GCG and C than the latter. Furthermore, the predominant catechin in CWE was GCG (18.21 mg/100 mg dry powder), which accounted for about 52% of total catechins, while the main catechin in YWE was EGCG (6.85 mg/100 mg dry powder). These results revealed that Cocoa tea contained more total phenolics, total catechins, GCG, and theobromine than YD tea, which is in accordance with previous study (Lin et al., 2014). 3.2. Anti-proliferative effects of CWE and GCG on HCT116 cells The cell viability of HCT116 cells treated with various concentrations of CWE, YWE, GCG, and EGCG was evaluated using the MTT assay. As shown in Fig. 1A and C, CWE exerted anti-proliferative activity in a time- and dose-dependent manner in HCT116 cells, and the consistent results were also observed in GCG. After treatment with various concentrations of CWE for 24 h, 48 h, and 72 h, the IC50

values of CWE for HCT116 cells were 413.16, 339.30, and 236.97 µg/mL, respectively, which were significantly lower than those of YWE (Fig. 1B). Additionally, compared with EGCG, GCG showed stronger anti-proliferative activity in HCT116 cells. For 24 h, 48 h, and 72 h treatment, the IC50 values of GCG were 220.41, 194.00, and 174.18 µM, respectively (Fig. 1D). These results suggested that CWE and GCG could inhibit the proliferation of HCT116 cells. 3.3. Pro-apoptotic effects of CWE and GCG on HCT116 cells The induction of apoptosis in cancer cells is an appealing approach to intervene cancer progression (Vukovic, Obradovic, Vukic, Jovanovic, & Djurdjevic, 2018). To investigate whether the anti-proliferation in HCT116 cells was associated with apoptosis, the cell apoptosis induced by CWE, YWE, GCG, and EGCG was analyzed by flow cytometry using Annexin V-FITC and PI double staining. As shown in Fig. 2A and B, after 48 h of treatment, both CWE and GCG displayed a dose-dependent increase in apoptosis induction in HCT116 cells. Compared with the control group, the apoptotic rates of HCT116 cells treated with CWE at 300 µg/mL and GCG at 200 µM were increased by 19.86 and 25.91%, respectively. In addition, CWE and GCG showed significantly better pro-apoptotic effect on HCT116 cells in comparison to YWE and EGCG, respectively, which was consistent with MTT assay. These results indicated that CWE and GCG could induce apoptosis in HCT116 cells. 3.4. CWE induced mitochondrial dysfunction in HCT116 cells To examine whether CWE-induced apoptosis in HCT116 cells was involved in the mitochondria-mediated pathway, the change of mitochondrial membrane potential

(MMP) was measured using the JC-1 fluorescence probe. It is well known that red fluorescent JC-1 dimers in normal cells and loss of MMP caused the increase of green fluorescent JC-1 monomers. As shown in Fig. 3A, green fluorescence in CWE-treated cells was increased in a dose-dependent manner, suggesting that CWE treatment triggered mitochondrial damage. In addition, the protein levels of Bcl-2, Bax, cytochrome c, caspase-9 and -3, and poly (ADP-ribose) polymerase (PARP) were detected. As shown in Fig. 3B, CWE treatment markedly upregulated the expression of pro-apoptotic Bax, while it downregulated the expression of anti-apoptotic Bcl-2, leading to a significant dose-dependent increase in Bax/Bcl-2 ratio. Moreover, CWE effectively increased the protein level of cytochrome c in cytosol. We also demonstrated that treatment of HCT116 cells with CWE significantly activated caspase-9 and -3, and increased the cleaved form of PARP (Fig. 3C). These results implied that CWE could induce apoptosis in HCT116 cells through the mitochondrial apoptotic pathway. 3.5. PI3K/Akt signaling pathway involved in CWE-induced apoptosis in HCT116 cells The PI3K/Akt signaling pathway plays an important role in various biological consequences critical for human cancer progression, including proliferation, growth, and motility, whereby inhibition of this pathway is crucial to both decreased cell proliferation and promotion of cell death (Bauer, Patel, & Infante, 2015). To explore the potential role of PI3K/Akt pathway in CWE-induced apoptosis in HCT116 cells, the protein levels of PI3K (p110β), PI3K (p85), p-Akt (Ser 473), and Akt were examined. As shown in Fig. 4A, treatment of HCT116 cells with 300 µg/mL of CWE

appreciably decreased the levels of the catalytic subunit (p110β) and regulatory subunit (p85) of PI3K protein. At the same time, 300 µg/mL of CWE remarkably reduced the expression of p-Akt (Ser 473) without significant change in total Akt level. To further determine whether CWE-induced apoptosis was modulated by inhibiting PI3K/Akt pathway, HCT116 cells were treated with CWE in the presence or absence of LY294002 (PI3K inhibitor). As expected, compared with CWE treatment alone, pretreatment with 30 µM LY294002 significantly enhanced CWE-induced apoptosis and the inhibitory effect of CWE on Akt phosphorylation (Fig. 4B and C). These results suggested that CWE-induced apoptosis in HCT116 cells was modulated, at least partially, by the inactivation of PI3K/Akt signaling pathway. 3.6. ROS generation triggered CWE-induced apoptosis in HCT116 cells ROS generation plays a vital role in the process of apoptosis (Liang, Cheng, Dong, & Ju, 2019). To investigate whether CWE treatment could provoke ROS accumulation in HCT116 cells, the intracellular ROS level was measured using DCFH-DA fluorescence probe. As shown in Fig. 5A, the DCF fluorescence intensity was increased in a dose-dependent manner in CWE-treated cells, which suggested CWE treatment triggered intracellular ROS accumulation in HCT116 cells compared with the control group. Next, to elucidate whether ROS generation was associated with CWE-induced apoptosis, HCT116 cells were treated with CWE in combination with NAC (ROS scavenger). Flow cytometric assay showed that pretreatment with 5 mM NAC significantly ameliorated CWE-induced apoptosis (Fig. 5B). Consistently,

NAC pretreatment effectively attenuated CWE-induced up-regulation of Bax/Bcl-2 ratio, release of cytochrome c, activation of caspase-9 and -3, and cleavage of PARP (Fig. 6A and B). These results indicated that ROS generation mediated mitochondria-dependent apoptosis elicited by CWE. Furthermore, the relationship between ROS production and inactivation of PI3K/Akt pathway in CWE-induced apoptosis was confirmed. As shown in Fig. 6C, compared with CWE treatment alone, the protein level of p-Akt (Ser 473) was observed to be markedly reversed by pretreatment with NAC prior to CWE treatment, indicating that CWE-mediated ROS accumulation might be involved in the inhibition of PI3K/Akt pathway in HCT116 cells. 3.7. CWE inhibited tumor growth in vivo To evaluate the anti-tumor effects of CWE in vivo, the HCT116 colon cancer xenograft nude mice model was used. Compared with the control group, CWE at the doses of 80 mg/kg and 160 mg/kg decreased tumor volumes in tumor-bearing mice during the 4-week treatment (Fig. 7A). At the end of the experiment, significant reductions of tumor weights were observed in the CWE-treated groups. Moreover, the efficacy of CWE at 160 mg/kg was comparable to that of 5-FU at 20 mg/kg (positive control), and tumor weight were reduced by 45.12% and 51.22%, respectively (Fig. 7B). Additionally, a slight decrease in body weight was observed in the 160 mg/kg CWE-treated group, while 5-FU treatment rapidly decreased the body weight of mice (Table 2). Furthermore, histology analysis by H&E staining showed the shrinkage of tumor cells, numerous gaps between tumor cells, and karyopyknosis, indicating that

CWE treatment could induce necrosis (Fig. 7C). Therefore, CWE exhibited significant anti-tumor activity in vivo. To investigate whether tumor growth inhibitory effects of CWE in vivo was related to the apoptosis induction, TUNEL analysis and immunohistochemistry for cleaved caspase-3 were used to examine the apoptotic cells in the tumor. Similar to the 5-FU treatment group, CWE treatment increased the percentage of TUNEL positive cells and cleaved caspase-3 positive cells compared with the control group, indicating that CWE exerted an anti-tumor activity in vivo was correlated with apoptosis (Fig. 7C). To elucidate whether PI3K/Akt pathway and mitochondrial apoptotic pathway were involved in CWE-induced apoptosis in vivo, we further determined the expression levels of p-Akt (Ser 473), Bcl-2, and Bax in tumor tissues. As shown in Fig. 7D, similar to the results in vitro, CWE treatment significantly decreased the protein levels of p-Akt (Ser 473) and Bcl-2, and increased the expression level of Bax. These results revealed that the anti-tumor effects of CWE on HCT116 xenograft tumor was connected with the mitochondria-mediated apoptotic pathway and PI3K/Akt pathway. 4. Discussion Colorectal cancer is one of the leading causes of cancer death threatening human health worldwide (Bray et al., 2018). Green tea polyphenols have been demonstrated to be effective in inhibiting colon tumorigenesis in mouse models, but caffeine shows no inhibitory effects (Yang & Hong, 2013). In addition, many attempts have been made to remove caffeine from tea for its occasional adverse effects on human health

in recent years (Shiono et al., 2017). Cocoa tea (Camellia ptilophylla) is a natural low caffeine-containing tea species, and its caffeine content is about 0.3% by dry weight in our study. Previous studies have shown that N-methyltransferases from Cocoa tea mainly participates in the synthesis of theobromine, and it lacks adequate enzymatic activity to catalyze theobromine into caffeine (Ashihara, Kato, & Ye, 1998; Jin, Yao, Ma, Ma, & Chen, 2016). Moreover, compared with regular green tea (Camellia sinensis), Cocoa tea contains more polyphenols and is characterized by abundant 2,3-trans-catechins, especially GCG (Lin et al., 2014), which is consistent with our results. Hence, we are interested to investigate the anti-proliferative and pro-apoptotic effects of Cocoa tea on HCT116 cells and its underlying mechanisms. Previous studies have demonstrated that tea polyphenols show inhibitory effects on colon cancer both in vitro and in vivo. Cerezo-Guisado et al. (2015) reported that EGCG induced human colon cancer HT-29 cell apoptosis through mitogen-activated protein kinase (MAPK) and Akt signaling pathways. Hao et al. (2017) demonstrated that dietary green tea polyphenols (0.24%) for 34 weeks significantly inhibited colorectal tumorigenesis in azoxymethane-treated F344 rats. Additionally, several studies have demonstrated that the molecular structure of catechins including galloyl group at the 3-position on the C-ring (gallated catechins) and hydroxyl group at the 5'-position on the B-ring are vital for their anti-cancer activity in both HCT116 and LoVo colon cancer cell lines (Du et al., 2012; Tan et al., 2000). As can be seen from Table 1, CWE contained higher total phenolics content. Moreover, the content of gallated catechins (EGCG, GCG, ECG, and CG) in CWE (24.93 mg/100 mg dry

powder) was significantly higher than that in YWE (14.79 mg/100 mg dry powder). Also, CWE (25.71 mg/100 mg dry powder) contained more catechins with hydroxyl group at the 5'-position (EGCG, GCG, EGC, and GC) than YWE (14.95 mg/100 mg dry powder). Interestingly, compared with EGCG, better anti-proliferation and apoptosis induction were observed in HCT116 cells treated with GCG, which was probably attributed to differences between their stereochemical structures or cellular uptake. In this study, 300 µg/ml of CWE is equivalent to 119 µM of GCG since the content of GCG in CWE was 18.21%. Our result showed that 100 µM of GCG could already induce significant apoptosis in HCT116 cells. Therefore, CWE exhibited relatively stronger anti-proliferative and pro-apoptotic effects than YWE in HCT116 cells at least attributing to its higher total phenolics and catechins content, especially GCG. Apoptosis is mainly initiated through two pathways: mitochondria-mediated intrinsic pathway and death receptor-mediated extrinsic pathway (Pfeffer & Singh, 2018). Many studies have confirmed that mitochondrial damage displays a pivotal role in apoptosis (Cui et al., 2016; Wang et al., 2018). The interaction of Bcl-2 family members can trigger the loss of MMP and mitochondrial outer membrane permeabilization, and in turn cause mitochondrial damage. Thus, the balance between pro-apoptotic and anti-apoptotic Bcl-2 family members plays an important role in the regulation and execution of intrinsic apoptosis (Singh, Letai, & Sarosiek, 2019). The pro-apoptotic Bax and anti-apoptotic Bcl-2 protein are two crucial members of the Bcl-2 family (Kalkavan & Green, 2018). Our results indicated that CWE treatment

prompted disruption of MMP, enhanced the Bax/Bcl-2 ratio, suggesting that Bcl-2 family proteins were involved in CWE-induced mitochondrial damage. The disruption of MMP can lead to the release of cytochrome c from mitochondria into cytosol and the activation of caspase cascade in the mitochondrial pathway. Caspases are a family of cysteine proteases responsible for apoptosis in mammalian cells. Caspase-9 is an initiator caspase of the mitochondria-dependent pathway. Caspase-3, an important executor caspase of apoptosis, can be directly activated by caspase-9 and in turn cleaves PARP, which participates in DNA fragmentation (Singh, Letai, & Sarosiek, 2019). Our results demonstrated that CWE treatment increased the level of cytochrome c, activated caspase-9 and -3, and promoted the cleavage of PARP. Moreover, in our in vivo studies, CWE significantly reduced both tumor weight and volume in HCT116 xenograft tumor model. Immunohistochemical staining and western blot analysis showed a significant increase in DNA fragmentation, active caspase-3 expression, and Bax/Bcl2 protein ratio, which are in accordance with our findings in vitro. These results indicated that the mitochondria-mediated apoptotic pathway participated in apoptosis of HCT116 cells induced by CWE both in vitro and in vivo. The PI3K/Akt pathway represents one of the most important signaling cascades crucial for cell survival, proliferation, and growth. Class IA PI3K kinase, a heterodimeric enzymatic complex consisted of one catalytic subunit (p110) and regulatory subunit

(p85),

phosphorylate

PI

4,5-bisphosphate

to

yield

PI

3,4,5-triphosphate (Bauer, Patel, & Infante, 2015; Danielsen et al., 2015). This

process triggers the phosphorylation of Akt and in turn inhibits apoptosis through regulating its downstream targets including the Bcl-2 family proteins (Jafari, Ghadami, Dadkhah, & Akhavan-Niaki, 2019). The aberration of PI3K/Akt signaling pathway is involved in about 40% of CRCs has been reported (Danielsen et al., 2015). Extensive studies have demonstrated that inhibiting PI3K/Akt signaling pathway promotes apoptosis in colorectal cancer cells (Song, Chang, & Li, 2016; Wani et al., 2016). In the current study, it was found that CWE treatment inhibited PI3K/Akt signaling pathway in HCT116 cells both in vitro and in vivo. Moreover, pretreatment with LY294002 significantly exacerbated the apoptosis induction of CWE and reduced the phosphorylation of Akt at Ser 473, indicating that CWE-induced apoptosis in HCT116 cells was partly attributed to the inhibition of PI3K/Akt signaling pathway. ROS are products of normal cellular metabolism and play essential roles in intracellular signaling regulation at moderate concentrations. However, excessive production of ROS can attack cellular macromolecules such as lipid, DNA, or protein, and consequently affect the normal functions of cells (Liang, Cheng, Dong, & Ju, 2019). Among apoptotic stimuli, ROS accumulation can alter the expression of the Bcl-2 family proteins, which in turn trigger mitochondria-mediated apoptosis (Durand et al., 2017). Several studies have indicated that tea catechins can induce apoptosis by modulating the generation of ROS in human hepatocellular carcinoma HepG2 and endometrial adenocarcinoma cells (Khiewkamrop, Phunsomboon, Richert, Pekthong, & Srisawang, 2018; Manohar et al., 2013). Manohar et al. (2013) found that EGCG could induce apoptosis in endometrial carcinoma cells via ROS generation and p38

MAPK activation, while NAC pretreatment blocked the phosphorylation of p38 MAPK and reduced the apoptosis induction of EGCG. The present results showed that CWE treatment caused an elevation of intracellular ROS levels in HCT116 cells. Moreover, NAC pretreatment appreciably downregulated Bax/Bcl-2 ratio, reduced release of cytochrome c, suppressed activation of caspase-9 and -3, and attenuated apoptosis induction in CWE-treated cells. These findings implied that ROS generation contributed to mitochondria-dependent apoptosis induced by CWE. Various studies have found that the activation of PI3K/Akt pathway could elevate intracellular ROS production in apoptosis induction (Zhou et al., 2018). Also, ROS accumulation could suppress the phosphorylation of Akt (Guo et al., 2018; Posadino et al., 2017). Our present results showed that NAC pretreatment significantly recovered the inactivation of Akt phosphorylation in CWE-treated cells, which was supported by previous studies (Guo et al., 2018; Lai et al., 2017). Therefore, these results suggested that the inhibition of PI3K/Akt pathway was mediated, at least partially, by ROS accumulation in CWE-induced apoptosis in HCT116 cells. In conclusion, the present study demonstrates that CWE can inhibit proliferation and induce apoptosis in human colon cancer HCT116 cells. Further, its pro-apoptotic effect may be mainly attributed to the mitochondrial dysfunction modulated by ROS generation and PI3K/Akt signaling pathway. Also, CWE significantly suppresses tumor growth in HCT116 tumor bearing-mice. With higher content of polyphenols and GCG, our research suggests that Cocoa tea could be a promising functional beverage to control colorectal cancer.

Conflict of Interest The authors have declared no conflict of interest. Acknowledgements This work was supported by the Earmarked Fund for China Agriculture Research System (CARS-19), Science and Technology Planning Project of Guangdong Province, China (2015A030302065), Foundation for Distinguished Young Talents in Higher Education of Guangdong (2017KQNCX019), and Natural Science Foundation of Guangdong Province, P. R. China (2018A030313917). Thanks are given to Bingming Chen for editing this manuscript. Acknowledgment is also given to senior agronomist Songlin Qin (College of Horticulture in South China Agricultural University) for tea processing. This work was also supported by Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University.

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Table 1 Chemical compositions of water extract of Cocoa tea and YD tea Content (mg/100 mg dry powder)

Δ

Components

CWEΔ

YWE

GA

0.89 ± 0.01a

0.30 ± 0.01b

GC

1.53 ± 0.01b

1.96 ± 0.11a

TB

10.78 ± 0.04a

0.21 ± 0.01b

EGC

1.18 ± 0.14b

2.52 ± 0.11a

C

6.28 ± 0.04a

2.33 ± 0.05b

EGCG

4.79 ± 0.12b

6.85 ± 0.49a

CAF

0.70 ± 0.05b

6.62 ± 0.43a

EC

1.11 ± 0.06b

3.12 ± 0.10a

GCG

18.21 ± 0.76a

3.62 ± 0.16b

ECG

1.62 ± 0.11b

3.50 ± 0.02a

CG

0.31 ± 0.02b

0.82 ± 0.04a

Total catechins

35.03 ± 0.95a

24.71 ± 0.78b

Total phenolics

52.38 ± 0.96a

37.86 ± 1.24b

Abbreviations: CWE, water extract of Cocoa tea; YWE, water extract of YD tea; GA, gallic acid; GC,

gallocatechin; TB, theobromine; EGC, epigallocatechin; C, (−)-catechin; EGCG, epigallocatechin gallate; CAF, caffeine; EC, epicatechin; GCG, gallocatechin gallate; ECG, epicatechin gallate; CG, catechin gallate; Total catechins content was expressed as the sum of eight monomeric catechins (EGCG, ECG, EGC, EC, GCG, CG, GC, and C); Total phenolics content was expressed as mg GAE per 100 mg dry powder. Data from both CWE and YWE were converted to freeze-dried powder weight basis. All data were presented as means ± S.D. from two separate experiments with two replicates. Within each row, values with different letters denote the significant differences (p < 0.05).

Table 2 The body weight of tumor-bearing mice under different treatment condition Body weight (g) Components

Day 0

Day 28

Control

18.51 ± 0.52a

22.66 ± 0.58a

CWE (80 mg/kg)

18.91 ± 0.54a

22.10 ± 0.85a

CWE (160 mg/kg) 18.88 ± 0.59a

20.10 ± 0.92b

18.30 ± 0.37a

17.89 ± 0.73c

5-FU (20 mg/kg)

All data were presented as means ± S.D. (n=6). Within each column, values with different letters denote the significant differences (p < 0.05).

Figure captions Fig. 1. Cytotoxic effects of CWE, YWE, GCG, and EGCG on HCT116 cells were determined by MTT assay. Cell viability of HCT116 cells treated with different concentrations of CWE (A) and GCG (C) for 24 h, 48 h, and 72 h, assuming that viability of the control group was 100%. The bar diagram showed the IC50 values of CWE, YWE (B), GCG and EGCG (D) obtained from cell viability assay. The data in (B) and (D) were presented as mean values ± S.D. (n=3). Different letters represent significant difference (p < 0.05) between CWE and YWE, or GCG and EGCG. Fig. 2. Apoptosis of HCT116 cells was analyzed by flow cytometry. HCT116 cells were treated with different concentrations of CWE, YWE (A), GCG, and EGCG (B) for 48 h, and cell apoptosis was measured using Annexin V-FITC/PI double-staining. The data were presented as mean values ± S.D. (n=3). *p < 0.05 and **p < 0.01 denote significant difference from the control group; #p < 0.05 and ##p < 0.01 denote significant difference between CWE and YWE, or GCG and EGCG. Fig. 3. Activation of the mitochondrial apoptotic pathway in CWE-treated HCT116 cells. Cells were treated with different concentrations of CWE for 48 h and then stained with JC-1 for 20 min. Cells in the lower right represent low MMP (green fluorescent JC-1 monomers), upper right represents high MMP (red fluorescent JC-1 dimers) (A). Western blot analysis of the expression levels of Bcl-2, Bax, cytochrome c (B), caspase-9 and -3, and PARP (C) in HCT116 cells treated with different concentrations of CWE for 48 h. The relative density of the bands was normalized with β-actin. The data were presented as means ± S.D. (n=3). *p < 0.05 and **p < 0.01 denote significant difference from the control group. Fig. 4. Involvement of PI3K/Akt signaling pathway in CWE-induced HCT116 cell apoptosis. Western blot analysis of the expression levels of PI3K (p110β), PI3K (p85), p-Akt (Ser 473), and Akt in

HCT116 cells treated with different concentrations of CWE for 48 h (A). Cells were pretreated with LY294002 (30 µM) for 1 h and followed by treatment with or without CWE (300 µg/mL) for another 48 h. Cell apoptosis was measured using Annexin V-FITC/PI double-staining (B), and the expression levels of p-Akt (Ser 473) and Akt were detected by western blot analysis (C). The relative density of the bands was normalized with β-actin. The data were presented as mean values ± S.D. (n=3). *p < 0.05 and **p < 0.01 denote significant difference from the control group; ##p < 0.01 denote significant difference between combination treatment group (CWE + LY294002) and CWE-treated group. Fig. 5. Involvement of intracellular ROS generation in CWE-induced HCT116 cell apoptosis. Cells were treated with different concentrations of CWE for 24 h and then stained with 10 µM DCFH-DA for 30 min. Dotted line represents the control group, and solid line represents the CWE-treated group, assuming that the fluorescence intensity of the control group was 100% (A). Cells were pretreated with NAC (5 mM) for 1 h and followed by treatment with or without CWE (300 µg/mL) for another 48 h, and cell apoptosis was measured using Annexin V-FITC/PI double-staining (B). The data were presented as mean values ± S.D. (n=3). **p < 0.01 denote significant difference from the control group; ##

p < 0.01 denote significant difference between combination treatment group (CWE + NAC) and

CWE-treated group. Fig. 6. ROS generation modulates mitochondrial apoptotic pathway and PI3K/Akt pathway in CWE-induced HCT116 cell apoptosis. Cells were pretreated with NAC (5 mM) for 1 h and followed by treatment with or without CWE (300 µg/mL) for another 48 h, and the expression levels of Bcl-2, Bax, cytochrome c (A), caspase-9 and -3, PARP (B), p-Akt (Ser 473), and Akt (C) were detected by western blot analysis. The relative density of the bands was normalized with β-actin. The data were presented as means ± S.D. (n=3). *p < 0.05 and **p < 0.01 denote significant difference from the

control group; ##p < 0.01 denote significant difference between combination treatment group (CWE + NAC) and CWE-treated group. Fig. 7. Anti-tumor effects of CWE on HCT116 xenograft tumor model. The tumor-bearing mice were daily administered intragastrically with 80 mg/kg and 160 mg/kg of CWE, or 20 mg/kg of 5-FU (positive control), or water (control) for 4 weeks. Tumor volumes were measured every 4 days (n=6) (A). Tumor weights at the end of the study (n=6) (B). Tumors were excised at the end point and photographs of three representative tumors in each group were displayed. Representative photographs of H&E staining, TUNEL assay, and cleaved caspase-3 immunostaining in tumor tissue of different groups were shown (scale bar: 20 µm) (C). The expression levels of Bcl-2, Bax, p-Akt (Ser 473), and Akt in tumor tissue were detected by western blot analysis. The relative density of the bands was normalized with β-actin (n=3) (D). The data were presented as mean values ± S.D., *p < 0.05 and **p < 0.01 denote significant difference from the control group.

Figures

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

 Cocoa tea is a naturally GCG-rich and low caffeine-containing tea cultivar.  Cocoa tea water extract (CWE) induced apoptosis in HCT116 cells.  CWE triggered reactive oxygen species generation and mitochondrial dysfunction.  Inhibition of PI3K/Akt signaling pathway was involved in CWE-induced apoptosis.  CWE significantly inhibited tumor growth in HCT116 tumor-bearing mice.

Authors' Contributions Xiong Gao performed experiments, interpreted the results, and drafted the manuscript. Xiaofei Li offered extensive help in experimental performance and data collection. Bin Li and Zhongzheng Chen designed the study. In addition, Chi-Tang Ho, Xiaorong Lin, and Yuanyuan Zhang assisted with the manuscript editing.