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Anti-oxidant and anti-cancer activities of Angelica dahurica extract via induction of apoptosis in colon cancer cells.

ABSTRACT

Introduction: Angelica dahurica Radix is the common herbal medicine with anti-cancer activities. However, details of its anti-cancer activities are lacking.

Materials and methods: We investigated the anti-cancer effects of Angelica dahurica extract in HT-29 colon cancer cell line. Cell viability, apoptotic and necrotic activities and the mechanism of actions of the active fraction were measured.

Results and discussion: The organic extract of Angelica dahurica Radi decreased significantly the gene expression of p53, Bd, Bax and induced apoptosis via caspase cascade and cell cycle arrest. The ethanol-ethyl acetate fraction showed anti-cancer activities in HT-29 cancer cells. A HPLC-DAD analysis of the fraction indicated the presence of Imperatorin and isoimperatorin, which are the major coumarins in the active fraction that contribute to the anti-cancer activities.

Conclusions: This study has evaluated the ant-cancer activity of the organic extract of Angelica dahurica Radix against colon cancer cells and provided a basis of further development of the herbal extract for treatment of colon cancer.

Keywords:

Angelica dahurica Radix

Colon cancer

Apoptosis

Cell cycle arrest

ROS scavenger

Introduction

Chemotherapy of cancer remains to be improved due to its side effects. Many anti-cancer drugs commonly used are developed from potent herbal phytochemicals. Thus, medicinal plants became the good source of anti-cancer agents. Herb-based drugs would be developed after systematic evaluation and the chemical modification.

Angelica dahurica Radix is the commonly used traditional herbal medicine in combination with other herbs for various antigens such as inflammation, liver dysfunction, infection and urinary disorders in China. A recent study reported the anti-oxidant effect of imperatorin, an active compound of Angelica dahurica Radix in hypertension by inhibiting NADPH oxidase and MAPK pathway (Cao et al., 2014; Li et al., 2015). Imperatorin and isoimperatorin were reported to be active components from roots of Angelica dahurica (Chen et al., 2012; Liang et al., 2015; Jeong et al., 2015). The anti-cancer effects of imperatorin were shown to induce apoptosis in HepG2 cell line (Luo et al., 2011; Zhao et al., 2014). The study suggested that imperatorin can inhibit cancer growth through cell death receptor. Imperatorin was also shown to be responsible for mediation of vasodilation (Wei & Ito, 2008; Nie et al., 2013; Zhu et al., 2013). The vasodilation effects of imperatorin were via inhibition of nitric oxide synthase. The protective activity of imperatorin in cultured neural cells was also reported (Wang et al., 2013). Though the medicinal properties of Angelica dahurica Radix is recorded in Chinese Pharmacopeia, details of its anti-cancer activities are lacking.

Materials and methods

Cells, chemicals and reagents

HT-29 cell line was obtained from ATCC (Manassas, VA, USA). Acetonitrile (ACN) (E. Merck, Germany), Methanol, trifluoroacetic acid and other reagents were of analytical grade purchased from Sigma Aldrich (Sigma Aldrich, St. Louis, MO, USA). All antibodies were purchased from Cell signaling (Danvers, MA, USA), except anti-caspase-8, Bax, Bcl-2, p21 and MDM2 which were obtained from Santa Cruz (Dallas, TX, USA).

Preparation of Extract of Angelica dahurica

Angelicae dahuricae Radix (Sichuan, China) used was purchased from a local vendor. Angelicae dahuricae Radix (100 g) was boiled in 1.5 1 of 95% ethanol for 1.5 h and the residue was boiled likewise again. The ethanol extract of Angelicae dahuricae Radix was collected and centrifuged at 10,000 x g for 15 min at 18 [degrees]C. The supernatant was condensed to 45 ml in a rotary evaporator system (R-210, BUCHI, Switzerland) at 120 mbar pressure at 60 [degrees]C. The condensed solution was partitioned with 50 ml ethyl acetate twice to yield the ethyl ac etate extract of Angelicae dahuricae Radix. The extract was dried by lyophilization overnight. The extract of Angelicae dahuricae Radix (abbreviated as EAD hereafter) was stored at -20 [degrees]C prior to use.

HPLC analysis of EAD

HPLC analysis of the EAD fraction was conducted according to the previous method (Park et al., 2009). The mixed standard solution for imperatorin and isoimperatorin (Sigma-Aldrich Inc., St. Louis, MO, USA) was prepared at a concentration of 0.1 mg/ml(ACN: DMSO = 1; 1, v/v). Likewise, 2.5 mg of extract was prepared at the same concentration. The standard solution and the extract were purified by 0.22 [micro]m polypropylene filter and injected into HP1100 series HPLC system equipped with a diode-array detector at 254 nm with an analytical HPLC column (ALLTIMA C18, 5 [micro]m, 250x4.6 mm i.d.). The flow rate of elution profile was set at 0.8 ml/min. The gradient mobile phase composed of solvent A (0.1% trifluoroacetic acid) and solvent B (acetonitrile). The gradient for separation was programmed: 0 min, 2% B; 10 min, 5% B; 15 min, 20% B; 35 min, 40% B; 40 min, 60% B; 70 min, 70% B; 80 min, 95% B; 90 min, 100% B and held for additional 5 min. Calibration curves were peak area versus concentration for each standard solution. Quantification was performed upon six levels of external standards. The limit of quantification (LOQ) was determined as the concentration with a signal-to-noise ration of ten.

M7T assay

The growth inhibitory effects of EAD were evaluated with HT-29 colon cancer cell line. All cells were seeded onto 96-well microtiter plates, at a density of 6 x [103.sup.] cells per well and allowed to incubate overnight. Then the cells were treated by either 0.5% DMSO (as solvent control; abbreviated as Ctrl) or various doses of EAD and incubated for 24, 48 and 72 h, respectively. After incubation, MTT assay was performed according to the manufacturer's protocols. The absorbance was measured at 540 nm by TECAN infinite M200. Each treatment or control was performed in replicates of 4 to 6 times. Mean absorbance readings from solvent control groups were defined as 100% viability and results from treatment groups were shown as % viability relative to the controls.

Lactate dehydrogenase activity (LDH) assay

The cells that undergo necrosis will lyse and release cytosolic LDH in the culture medium. CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega) was used to evaluate the cytotoxicity of EAD on HT-29 by assessing the activity of LDH presented in the culture medium. After 48-h treatment of EAD on HT-29, LDH assay was performed. The absorbance was measured at 490 nm by TECAN infinite M200. Maximum LDH activity was achieved by addition of lysis buffer and normalized by a corresponding volume correction control. Background values (medium only) were subtracted from the sample readings and percent necrotic cell death was calculated in relation to the maximum LDH release.

Flow cytometric analysis of cell cycle

HT-29 cells were seeded onto a 6-well plate, at a density of 1 x [10.sup.6] cells per well and allowed to incubate overnight. Then the cells were treated with EAD for 48 h. After 48-h treatment, the cells were washed with PBS once and harvested with trypsin. The harvested cells were washed with PBS (4 [degrees]C) twice fixed with 70% ethanol for 1 h. Then the cells were centrifuged at 500 g for 5 min and washed twice with PBS (4 [degrees]C), followed by addition of propidium iodide (PI; final cone; 40 [micro]g/ml). The cells were incubated away from light at 37 [degrees]C for 15 min. Fluorescence-activated cells in each sample were recorded with 10,000 events by using FACSVerse (BD Biosciences). The "event" here means a unit of data representing one fluorescence-activated cells or particles. The cell cycle distribution was analyzed with ModFit (Allen, 1990).

Flow cytometric analysis of apoptosis

HT-29 cells were cultured and treated with EAD in the same procedures performed for cell cycle analyses. The cells were harvested with trypsin and washed twice with PBS (4 [degrees]C). Then the cells were re-suspended in 1 x binding buffer at a density of 1 x [10.sup.6] cells/ml. One hundred microliter of ceil suspension per sample was transferred to a 5 ml culture tube and stained with 5 [micro]l FITC Annexin V and 5 [micro]l PI (FITC Annexin V Apoptosis Detection Kit I, BD Pharmingen) according to the manufacturer's protocols. Fluorescence-activated cell samples were recorded with 10,000 events by using FACSVerse System (BD Biosciences).

Caspase-3/7 assay

The Apo-ONE Homogeneous Caspase-3/7 Assay Kit was used to evaluate the activities of apoptosis by assessing the caspase-3/7 enzymatic activities in the EAD-treated HT-29 cells. HT-29 cells were seeded onto a 96-well black plate (with clear bottom), at a density of 6 x [10.sup.3] cells per well and allowed to incubate overnight. Then the cells were treated by either 0.5% DMSO (as negative control group; abbreviated as Ctrl) or various doses of EAD (as treatment group, abbreviated as EAD) and incubated for 48 h, respectively. After incubation, equal volume of Apo-ONE caspase-3/7 reagent was added to each well. The plate was incubated for 3 h at 20 [degrees]C prior to recording rate fluorescence (RFU) of each well at excitation 485 nm and emission 530 nm by TECAN infinite M200. The caspase-3/7 activity of each group was determined by their RFU, and TUNEL-based detection of DNA fragmentation was measured according to manufacturer's protocols (BD Biosciences).

Western blot analysis

Western blot analysis was carried out following the recommended protocol from Abeam. In brief, HT-29 cells were seeded onto 100 mm culture dishes, at a density of 2 x [10.sup.6] cells per dish and incubated for 24 h prior to 48-h treatment of various concentration of EAD. After 48-h treatment, cells were harvested and lysed by nonidet-P40 lysis buffer (150 mM sodium chloride, 1% Triton X-100, 50 mM Tris, pH 8.0, supplemented with protease inhibitor cocktails (Roche)). The protein concentration was determined by DC protein assay (Biorad) according to manufacturer's instruction using bovine serum albumin (BSA) as the standard, and subjected to 8-12% SDS-PAGE to separate proteins using 80-120 voltages for 2 h. Proteins were transferred from the gel to a 0.25 [micro]m PVDF membrane at 12 voltages for 30 min by Novex Semi-dry blotter (Invitrogen). Then the membrane was blocked by non-fat dry milk (5% w/v) in TBST for 1 h at 20 [degrees]C before incubating with specific primary antibody (at appropriate dilution suggested by product data sheet) in 4 ml of 5% non-fat milk at 4 [degrees]C overnight. After washing with TBST, the membrane was incubated with the species appropriate HRP-conjugated secondary antibody (at 1:2000 dilution) in 4 ml of 5% non-fat milk at 20 [degrees]C for 1 h. Signals were developed by using ECL chemiluminescence detection reagent (GE, Fairfield, CT) and blots were visualized after exposure to Fuji Super RX film. The membrane were re-probed with [beta]-tubulin as internal control. The blots were quantified by ImageJ image processing program.

Reactive oxygen species (ROS) assay

Image-iT LIVE Green Reactive Oxygen Species Detection Kit was used to detect the production of ROS (e.g. superoxide anion radical, hydrogen peroxide, hydroxyl radical or RNS) in live cells. HT29 cells were seeded on a coverslip in a 6-well plate, at a density of 1 x [10.sup.6] cells per well and allowed to incubate overnight. Then cells were administered with various concentrations of EAD and incubated for 24 h. Cells were then washed with Hank's balanced salt solution (HBSS, 37 [degrees]C) once before 25-min incubation with 25 [micro]M carboxy-H2DCFDA and additional 5-min incubation with 5 [micro]M Hoechst 33342. The coverslips were gently washed three times with HBSS (37 [degrees]C) and mounted for fluorescent imaging at excitation 485 nm and emission 530 nm by Nikon E80i (Nikon, Tokyo, Japan).

Data analysis

In all viability assays, the total viable cells was presented in percentage compared with the solvent control groups and expressed as means [+ or -] SD (n [greater than or equal to] 3). Nonlinear regression test was applied to the viability assays to obtain a fit curve ([R.sup.2] > 0.98). Statistical analysis of data was carried out by one-way ANOVA (coupled with post-test, Dunnett's test) to test for differences between groups, with * denotes p < 0.05; ** denotes p < 0.01; *** denotes p < 0.005.

Results

HPLC profiles of EAD

The main coumarins in the EAD were analyzed with HPLC-DAD (Fig. 1A-C). By referring to the standard compounds, the main components were imperatorin, isoimperatorin and phellopterin that was identified based on the established method (Park et al., 2009).

Effects of EAD on HT-29 cell line

HT-29 cell lines were treated with various concentrations of EAD for 24, 48 and 72 h (Fig. 2). The MTT assay showed that cell viability of HT-29 cells were decreased with increasing concentrations of EAD with [IC.sub.50] at 345,157, 73 [micro]g/ml after 24, 48 and 72 h incubation, respectively at 95% confidence level. EAD was more cytotoxic after 72 h of incubation.

Necrosis of HT-29 cell lines after treatment with EAD

HT-29 cell lines were treated with various concentrations of EAD for 48 h. The percentage of necrotic cells determined by LDH assay (Fig. 3). The results showed that EAD essentially did not induce necrosis of HT-29 cell with increasing concentrations of EAD. Solvent showed no toxic effects on the cell viability.

Effects of EAD on cell cycle of HT-29

After treatment of HT-29 cells line with EAD, cell cycle arrest at G1 phase was recorded (Fig. 4A-E). The percentage of cycle arrest was shown in Fig. 4A-E. The results suggested that EAD induced cell cycle arrest of HT-29. Fig. 5A-G showed EAD induce apoptosis of HT-29 cell in a concentration-dependent manner. The percentage of apoptotic cells was elevated with increasing concentration of EAD.

Effects of EAD on caspase cascade in HT-29 cell line

After treatment of HT-29 cell lines with EAD, apoptosis of HT-29 cells was observed. The over-expression of caspase 3/7 were recorded suggesting that EAD induced apoptosis via caspase 3/7 activities (Fig. 6). The expression Bax/Bcl-2 ratio was increased (Fig. 7A-0). The over-expression of p53, p21 and MDM2 provided supportive experimental evidences that EAD induced apoptosis via p53-dependent pathway (Fig. 7A-0) and DNA damage was evaluated by TUNEL-based detection of DNA fragmentation (Fig. 8).

Cytotoxicity of EAD in HT-29 cell line

After treatment of HT-29 cell lines with EAD, intracellular reactive oxygen species was measured (Fig. 9A-H). The cytotoxic effects of EAD on HT-29 cell lines were increased with the concentration of EAD suggesting the pharmacological effects of EAD on HT-29 cell lines.

Discussion

In this study, different concentrations of the EAD were used to incubate with HT-29 cell lines. The in vitro study of its effects on colon cancer cells revealed that EAD could induce apoptosis. Cell cycle analysis showed that EAD induced cell cycle arrest at G1 phase (Fig. 4A-E). The BCL-2 family members play a pivotal role in deciding cell viability. A major checkpoint in apoptosis is associated with the level of pro-apoptotic (BAX) and anti-apoptotic (BCL-2) members. Modulation of apoptotic pathway is associated with caspase 3, 6, 7, 8 and 9 while caspase 8 is important factor in external pathway. The results suggested Bax caused damage to the mitochondria membrane while Bcl-2 inhibited apoptosis, Cytochrome C release, and the function of p53 and p21. The results suggest that EAD exhibited anti-cancer activity in colon cancer cells. The apoptotic events suggest that EAD induces apoptosis via p53-dependent pathways (Ostrakhovitch & Cherian, 2005). The inhibitory activity of EAD on colon cancer damage was considerably attenuated. The gene expression of EAD-treated HT-29 colon cancer cells provided confirmative results on EAD-induced apoptosis. This result is reminiscent of analogous in vitro studies of imperatorin in HepG2 cells (Luo et al., 2011). Effects of EAD treatment of colon cancer cells suggest significant inhibition on cancer cell growth. The MTT assays reflect the inhibitory effects of EAD on cancer cell viability.

At the molecular level, an attempt was made to understand possible changes in gene expression associated with EAD-triggered apoptosis. The results revealed that remarkably changes in the level of p53 gene expression after incubation of HT-29 cells for 48 h. This change reflects the likelihood that EAD induces apoptosis via a p53-dependent pathway and caspases cascade. Levels of MDM2 were considerably higher than in the control group. This change in MDM2 expression was accompanied by mediation of gene expression of Bax, Bcl-2, p21 and PARP (Fig. 7A-P). In addition EAD triggered the change in gene expression of pro-caspase 8, cleaved caspase 8, pro-caspase 9, cleaved caspase 9, pro-caspase 7 and cleaved caspase 7 (Fig. 7A-P). Previous studies have revealed that activation of p53 in cancer cells could result in up-regulation of p21 and to increased expression of Bax and Bcl-2 activities (Ostrakhovitch & Cherian, 2005). Both p53 and p21 expression levels were increased. The over-expression of p53 is required for the execution of apoptosis in cancer cells. It is also known that p53 regulates the cell cycle and apoptosis by mediation of MDM2, p21, Bcl-2, PARP and Bax expression (Power et al., 1987).

These findings suggest that EAD exhibited anti-proliferative activity in HT-29 cancer cells, and caused cell cycle arrest and could ameliorate cell functions. The anti-proliferative effects of EAD was increased with increasing concentrations in HT-29 cell lines. The results demonstrate that EAD has remarkable potent medicinal properties that show effective anti-cancer activities in colon cancer cells. EAD induced a decrease of ROS which may be caused by the inhibition of ROS production of ROS scavenging capacity. Imperatorin and isoimperatorin, the active coumarins of Angelica dahurica Radix were shown to possess anti-cancer activities in HepG2 cancer cells in the previous study (Park et al., 2009). However, details of their anti-cancer activities were not available. The present results reveal that Bcl-2 protein expression was significantly lower in the EAD-treated HT-29 cancer cells compared with the control. Bax was overexpressed after incubation of cells for 48 h. These results showed that MDM2 was found to be over-expressed in association with changes in p53 level. EAD down-regulates the expression of MDM2 thus resulting in increase in p53 expression. The over-expression of p53 could mediate expression of Bax, Bcl-2, PARP and p21 leading to cell cycle arrest and apoptosis. The results suggest that colon cancer cell proliferation could be attenuated by EAD treatment. Treatment of colon cancer cells with EAD may not cause necrosis. The present findings suggest that colon cell damage by carcinogens can be reduced after EAD treatment. The histological changes observed between treated and un-treated HT-29 cancer cell lines reveal that EAD appears to attenuate the level of reactive oxygen species (Fig. 9A-H). EAD is believed to suppress the formation of toxic chemicals and solvents. These findings demonstrate that EAD exerts anti-cancer activities on concentration-dependent manner. The potential health benefits of EAD was demonstrated when a higher concentration of EAD was applied to the cancer cell lines. The results suggest that the anti-cancer activity of EAD is attributed to the radical scavenging capacity of EAD which is strongly believed to be able to play an important role in ameliorating colon cancer cell damage. The findings also suggest that EAD possesses anti-cytotoxic activities.

Conclusion

The present study demonstrates that EAD can inhibit colon cancer growth through a p53-dependent pathway. EAD can attenuate colon cancer cell functions and reduce the cancer burden. The changes in gene expression in colon cancer cells after incubation of EAD provide experimental evidences that EAD is a potent anticancer agent that can mediate cell cycle arrest through caspase cascade and p53-dependent pathway. EAD is an effective radical scavenger.

ARTICLE INFO

Article history:

Received 4 August 2015

Revised 10 November 2015

Accepted 14 November 2015

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgments

Zheng Yimei was supported by the Training Program of Fujian Excellent Talents in University in China and this work was supported in part by a grant no. 6903088 from the CUHK.

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Yi Mei Zheng (a), James Zheng Shen (b), Yan Wang (b), Amy Xiaoxu Lu (b), Wing Shing Ho (b), *

(a) Minnan Normal University, College of Biological Science and Technology, Zhangzhou 363000, PR China

(b) School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong

Abbreviations: (EAD), Ethyl acetate extract of Angelicae dahuricae Radix; (MTT), 3-(4,5-cimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide; (LDH), Lactate dehydrogenase activity; (ROS), reactive oxygen species; (HPLC-DAD), High-Performance Liquid Chromatography--Diode-Array Detection.

* Corresponding author. Tel.: +11 85239436114; fax: +11 852 2603 7732.

E-mail address: ws203ho@cuhk.edu.hk (Y.M. Zheng).

http://dx.doi.org/10.1016/j.phymed.2015.11.008

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Article Details
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Author:Zheng, Yi Mei; Shen, James Zheng; Wang, Yan; Lu, Amy Xiaoxu; Ho, Wing Shing
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUUK
Date:Oct 15, 2016
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