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Elephantopus scaber induces apoptosis through ROS-dependent mitochondrial signaling pathway in HCT116 Human colorectal carcinoma cells Chim Kei Chan, Hadi Supriady, Bey Hing Goh, Habsah Abdul Kadir

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S0378-8741(15)00231-7 http://dx.doi.org/10.1016/j.jep.2015.03.072 JEP9432

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Journal of Ethnopharmacology

Received date: 30 November 2014 Revised date: 10 March 2015 Accepted date: 13 March 2015 Cite this article as: Chim Kei Chan, Hadi Supriady, Bey Hing Goh, Habsah Abdul Kadir, Elephantopus scaber induces apoptosis through ROS-dependent mitochondrial signaling pathway in HCT116 Human colorectal carcinoma cells, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2015.03.072 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Elephantopus scaber induces apoptosis through ROS-dependent mitochondrial signaling pathway in HCT116 human colorectal carcinoma cells. Chim Kei Chan1, Hadi Supriady1, Bey Hing Goh2 and Habsah Abdul Kadir1* 1

Biomolecular Research Group, Biochemistry Program, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia 2

Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Jalan Lagoon Selatan, Bandar Sunway, 46150 Selangor, Malaysia. * Author to whom correspondence should be addressed; E-Mail: [email protected] ; Tel.: +603-79674363; Fax: +603-79614178. Abstract Ethnopharmacological relevance

Elephantopus scaber also known as Elephant’s foot (Asteraceae family) has a plethora of traditional applications including dysuria, diarrhea, dysentery, leukemia and cancer. This study aimed to investigate the apoptosis inducing effects of E. scaber and the underlying mechanisms in HCT116 colorectal cell line. Methods The MTT assay was used to determine the IC50 values on cancer cell lines by the ethanol, hexane, ethyl acetate and water fractions. Apoptosis was detected by cell morphologic observation through Hoechst 33342/PI dual staining, phosphatidylserine externalization by annexin V/PI staining and DNA fragmentation by TUNEL assay. The caspase activity, Bcl-2 family and p53 proteins was determined by flow cytometric analysis. The cleaved PARP protein expression was assessed by western blot analysis Results The ethanol extract of E. scaber and its fractions significantly inhibited the growth of HCT116 and HT-29 cells and induced apoptosis. The E. scaber ethyl acetate fraction (ESEAF) was the most potent on HCT116 cell line with the IC50 value of 1.42 ± 0.10 µg/mL. The induction of apoptosis was marked by nuclear shrinkage accompanied with chromatin condensation, DNA fragmentation and phosphatidylserine externalization. The results showed that ESEAF-induced apoptosis was associated with an upregulation of proapoptotic Bax, elevation of Bax/Bcl-2 ratio, dissipation of mitochondrial membrane potential, activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP). In addition, a compromised mitochondrial membrane potential and overproduction of ROS demonstrated the involvement of the mitochondrial signaling pathway. Mechanistic studies further revealed that ESEAF caused the augmentation of the intracellular ROS, subsequently incited the increase in p53 protein expression and led to oligomerization of 1

Bax, depolarization of mitochondrial membrane potential and caspases cascade (caspase3/7 and -9) in a time-dependent manner. The attenuation of intracellular ROS level by Nacetyl-L-cysteine (NAC) preserved the integrity of mitochondrial membrane and rescued the cells from cell death. Furthermore, caspase cascade results in the cleavage of PARP which ultimately activated DNA fragmentation and eventually apoptosis. Conclusion Taken together, cumulative evidences in this study suggest that ESEAF induces apoptosis through ROS-dependent mitochondrial signaling pathway and holds potential therapeutic effect for colorectal cancer. Keywords: Anticancer, Elephantopus scaber, Apoptosis, ROS and Mitochondrial pathway

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1. Introduction Cancer is one of the most life-threatening diseases which is ranked second leading cause of lethality. Among the various cancers, colorectal cancer (CRC) is the third most prevalent malignancy in the world, after lung and prostate/breast cancer (Society, 2012). Over decades, vast array of cancer therapy is available and tailored to improve patient overall survival including chemotherapy, radiotherapy or surgery. However, the progress made in cancer therapy has not been sufficient to significantly lower annual death rates and therefore there is an urgent need to develop new approaches based on natural products as chemotherapeutic/chemopreventive agents, and to endow with more effective and safer therapies. ROS are a family of active molecules containing superoxide anion radical, hydrogen peroxide, singlet oxygen and hydroxyl radical which mainly produced by mitochondria. Low levels of ROS serve as a physiological regulator for normal cell proliferation and differentiation. In contrast, excess intracellular ROS level can cause oxidative damage to lipids, DNA and proteins via apoptosis (Mieyal et al., 2008). Recently, copious evidence showed that cancer cells could be regulated by redox system and more susceptible to oxidative stress which is triggered by ROS-inducing agents (Circu and Aw, 2010). These ROS-inducing agents which induce oxidative stress eventually lead to cell death including apoptosis act as potential anticancer agents. In response to ROS stress stimuli, mitochondria-mediated apoptosis pathway is activated (Li-Weber, 2013). Activation of intrinsic pathway (mitochondria-apoptotic signaling pathway) involves mitochondrial membrane potential loss, the activation of p53, the release of a set of apoptotic substrates including cytochrome c, endonuclease G and Smac/Diablo which is followed by the 3

activation of caspase cascade and release of cleaved PARP eventually lead to apoptosis (Li-Weber, 2013). Numerous substances present in fresh fruits, vegetables and medicinal plants are associated with the prevention of diseases such as cancer, diabetes mellitus and cardiovascular diseases (Chan et al., 2012; Chan et al., 2011; Hashim et al., 2013; Li et al., 2010; Zhang et al., 2014). E. Scaber, commonly known as Prickly-leaved elephant’s foot and can be found in various countries such as Asia including Malaysia, Japan, Hong Kong, Taiwan, Thailand, Vietnam, Indonesia, India as well as China, Europe, Africa and America. E. scaber is anecdotally used in folk medicines for hepatoprotection, antipyretic, hepatitis, arthritis, diarrhea, anticancer, leukemia, diabetes and eczema (Hammer and Johns, 1993; Lin et al., 1995; Rasoanaivo et al., 1992). With the diverse traditional application of E. scaber, the potential of this herb raises great attention among the scientists to ascertain these claims and discover more new therapeutic properties. Therefore, numerous studies showed that E. scaber possesses various medicinal properties including hepatoprotective, anticancer, antidiabetic, anti-inflammatory, analgesic, anti-asthmatic, antibacterial and wound healing ability (Anitha et al., 2012; Daisy et al., 2009; Ho et al., 2012; Huang et al., 2010; Kabeer et al., 2013; Sagar and Sahoo, 2012; Su et al., 2011; Tsai and Lin, 1999). E. scaber can be considered as ‘cancer killer’ due to its remarkable cytotoxic activity against various cancer cell lines. In India, it was traditionally used as ayuverdic medicine for the treatment of cancer and leukemia (Balachandran and Govindarajan, 2005). Cumulative scientific reports have shown that E. scaber possesses anticancer effect against various human cancer cell lines including leukemia, lung cancer, nasopharyngeal cancer, mammary adenocarcinoma and cervical 4

carcinoma cells (Huang et al., 2010; Ichikawa et al., 2006; Kabeer et al., 2013; Su et al., 2011). To date, the effect of E. scaber on human colorectal carcinoma cells has not yet been reported and its mechanisms remain an enigma. In this study, we demonstrated that ESEAF induced apoptosis in HCT116 cells through mitochondria-dependent pathway. We further identified the potential mechanisms of ESEAF-induced ROS-mediated apoptosis in HCT116 cells. 2.

Materials and methods

2.1 Cell Culture HT-29, HCT116 cancer cells and CCD841-CoN normal cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cells were cultured in RPMI-1640 medium (Sigma, St. Louis, MO, USA) supplemented with 10% v/v heatinactivated fetal bovine serum (FBS) (PAA Lab, Pasching, Austria), 100 µg/mL penicillin streptomycin (PAA Lab) and 50µg/mL amphotericin B (PAA Lab). The media was filtered by using a sterilized 0.22 µm filter membrane (Minisart, Sartorius Stedim, Goettingen, Germany) and stored at 4°C. The cells were maintained at 37°C in a humidified atmosphere with 5% CO2. The cells were sub cultured every two to three days and checked every day under an inverted microscope for any contamination. The cells were detached and harvested from culture flasks by Accutase (Innovative Cell Technologies, San Diego, CA, USA). The viable cell count was done by using tryphan blue exclusion assay with haemocytometer.

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2.2 Preparation of crude Elephantopus scaber extract and fractions The leaves of E. scaber were purchased from a local supplier and upon authentication, a voucher specimen (No. KLU47976) was deposited at the herbarium in the Institute of Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia. Grounded leaves of E. scaber (1.5 kg) were soaked with 70% ethanol (three times) at room temperature for 3 days. The extract obtained was filtered from the residue through Whatman No.1 filter paper. The filtrate was concentrated using a rotary evaporator (Buchi) under reduced pressure at 40 °C. The dark green crude extract (50 g) was obtained and further partitioned with hexane (room temperature). The hexane insoluble fraction was partitioned with ethyl acetate and water in 1:1 ratio (room temperature) to yield an ethyl acetate-soluble fraction. The ethyl acetate-soluble fraction was evaporated under reduced pressure at 45 °C, while the aqueous filtrate was lyophilized to yield the aqueous fraction. The E. scaber leaves ethanol extract (ESEE), E. scaber leaves hexane fraction (ESHF), E. scaber leaves ethyl acetate fraction (ESEAF) and E. scaber leaves aqueous fraction (ESAF) were dissolved in DMSO prior to each assay. The final concentration of DMSO in all the experiments did not exceed 0.5% v/v. All samples were filter sterilized with 0.22 µm filters before use. 2.3 In vitro MTT Cytotoxicity Assay To investigate the cell viability, viable cells were seeded to 96-well plates and allowed to adhere overnight prior to the treatment with different concentrations (0.20 - 200.00 µg/mL) of E. scaber ethanol extract, hexane, ethyl acetate and water fraction and 56

Fluorouracil (5-FU) as positive control. Untreated cells (negative control) were treated with vehicle dimethyl sulfoxide (DMSO) instead of the sample. After 72 h incubation, MTT (5 mg/mL, Sigma) was then added to each well and incubated for another 4 h at 37 °C. After 4 h incubation, the culture medium was discarded by gentle aspiration and replaced by 150 µL of DMSO to dissolve the formazan crystals. The amount of formazon product was measured at 570 nm and 650 nm as a background using a microplate reader (Asys UVM340, Eugendorf, Austria). The percentage of cell viability was calculated according to the following formula: Percentage of cell viability (%) = (Absorbance of treated cells/absorbance of untreated cells) × 100%. 2.4 Phytochemical profiling by GC-MS analysis GC-MS analysis was performed using Agilent Technologies 7890A equipped with 7000 Mass Selective Detector, HP-5MS (5% phenyl methyl siloxane) capillary column of dimensions 30.0m x 250µm x 0.25 µm and used helium as carrier gas at 1 ml min-1. The column temperature was programmed initially at 100 ºC for 10 min, followed by an increase of 5 ºC min-1 to 300 ºC and was kept isothermally for 45 min. The MS was operating at 70 eV. The constituents were identified by comparison of their mass spectral data with those from NIST 08 Spectral Library. Only mass spectral fragmentation pattern that gave greater than 90% match were accepted. The compounds known as deoxyelephantophin and isodeoxyelephantopin were validated by using purified marker compounds.

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2.5 Nuclear Morphology Detection using Hoechst 33342/PI HCT116 cells (1×106 cells) were plated in 60 mm2 culture dishes and incubated in 5% CO2 incubator at 37 °C for 24 h. After incubation, the cells were treated with different incubation period (6, 12 and 24 h) with 10 µg/mL of ESEAF while negative control was treated with vehicle DMSO. After the indicated period, the cells were harvested and washed with PBS. The cells were subsequently stained with Hoechst 33342 (40 µg/mL) and Propidium Iodide solution (10 µg/mL) at room temperature in the dark for 30 mins. Then, the cells were observed under inverted fluorescence microscope (Leica DM1600B, Wetzlar, Germany). 2.6 Detection of Phosphatidylserine Externalization by Annexin V and PI Staining HCT116 cells (1 × 106 cells) were seeded in 60 mm2 culture dishes and incubated in 5% CO2 incubator at 37°C for 24 h. Then, the cells were treated with different concentrations (2.5 -10.0 µg/mL) of ESEAF while negative control was treated with vehicle DMSO. After the cells were treated for 24 h, both adherent and suspension cells were harvested, washed twice with PBS and resuspended in 1× Annexin V binding buffer (BD). Subsequently, the treated cells were stained with Annexin V-fluorescein-isothiocyanate (FITC) (BD) and propidium iodide (PI) (50 µg/mL). The cells were then vortex and incubated in dark at room temperature for 15 min. After that, 1× Annexin V binding buffer (BD) was added into each tube. The cells were then analyzed by flow cytometry using quadrant statistics for apoptotic and necrotic cell populations. The fluorescence intensity was detected in FL1-A (x-axis) and FL2-A channel (y-axis). The discrimination 8

between viable, early apoptotic, late apoptotic and necrotic cells was attained by quantitatively estimating the relative amounts of the Annexin V/PI-stained cells in the cell population. 2.7 Terminal Deoxynucleotidyl Transferased UTP Nick End Labeling (TUNEL) Assay For detection of DNA breakage, a TUNEL assay was performed following the protocol provided by the manufacturer (Sigma). In brief, HCT116 cells (1 × 106 cells) were seeded in 60 mm2 culture dish and treated with ESEAF or vehicle DMSO (control). ESEAFtreated cells were harvested, washed with PBS and fixed with 1% (w/v) paraformaldehyde in PBS on ice for 15 min. After fixation, the cells were washed and then incubated in DNA labeling solution containing terminal deoxynucleotidyl transferase enzyme, bromodeoxyuridine (BrdU), and TdT reaction buffer for 60 min at 37 °C. The cells were then rinsed and incubated with FITC-conjugated anti-BrdU antibody for 30 min at room temperature in the dark. Subsequently, the propidium iodide/RNase A solution was added to the cells and further incubated for another 30 min in the dark. The cells were then analysed by using Accuri C6 flow cytometry and the fluorescence intensity in X-axis and Y-axis were detected in FL1-A and FL2-A channel respectively.

2.8 Measurement of Intracellular Reactive Oxygen Species (ROS) The fluorescent probe 2'-7'-dichlorofluorescein diacetate (DCFH-DA) used to monitor intracellular accumulation of ROS. HCT116 cells (1 × 106 cells) were seeded in 60 mm2 culture dish and treated with ESEAF or vehicle DMSO (control) while tert-butyl

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hydroperoxide (TBHP) was served as positive control. After 4 h treatment with various concentrations (1.25 µg/mL to 10 µg/mL) of ESEAF, the cells were washed and incubated with medium containing 50 µM DCFH-DA for 1 h. Cells were then harvested and washed again with PBS. The cell suspension was resuspended in PBS and the fluorescence intensity was measured by flow cytometry and detected in FL1-A channel. 2.9 Measurement of Mitochondrial Membrane Potential The alteration in mitochondrial membrane potential was assessed by using the fluorescent cationic dye, JC-1, which is cell-permeable. HCT116 cells (1 × 106 cells) were cultured in 60 mm2 culture dishes and incubated for 24 h. Then, the cells were treated with different concentrations (2.5 - 10.0 µg/mL) of ESEAF. For the untreated cells (control), the sample was replaced with vehicle DMSO. After 24 h treatment with ESEAF, the cells were harvested, washed and stained with medium containing JC-1. The cells were then incubated at 37 °C in 5% CO2 incubator for 15 min. Subsequently, the cell suspension was centrifuged, washed and re-suspended in the medium. The cells were subjected to flow cytometry analysis by detecting the green and red fluorescence signals in FL1-A and FL2-A channels respectively. 2.10 Determination of p53 Tumor Suppressor Protein Expression Level The expression level of p53 protein was determined by immunofluorescence staining using flow cytometry. HCT116 cells (1 x 106 cells) were seeded into 60mm2 culture dishes and allowed to adhere for 24 h. The cells were then incubated with or without 10 µg/mL of ESEAF. After the incubation period, cells were then washed twice with PBS, fixed and subsequently permeabilized using Cytofix/Cytoperm. kit (BD Biosciences). A 10

total of 1 x 106 cells were resuspended in 500 µL of fixation/permeabilization solution and incubated at 4 °C for 20 min. The cells were then washed twice with Perm/Wash buffer and incubated for another 15 min in 1 mL of Perm/Wash buffer. The fixed and permeabilized cells were incubated with 100 µL of Perm/Wash buffer containing corresponding antibodies. The cells were either stained directly with 10 µL FITCconjugated mouse anti-human p53 monoclonal antibody or IgG2b isotype control (BD Biosciences) at 4 °C in the dark for 30 min. Finally, the cells were then washed with Perm/Wash buffer before analyzed by using BD AccuriC6 flow cytometer.

2.11 Determination of Bax and Bcl-2 protein expression level The protein expression level of Bax and Bcl-2 was assessed by immunofluorescence staining using flow cytometry. This method was based on Roussi et al. (Roussi et al., 2007) with some modifications. After HCT116 cells (1 × 106 cells/mL) were treated with ESEAF or DMSO (control), both adherent and suspension cells were harvested, washed twice with PBS and then fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA, USA). ESEAF-treated cells (1 × 106 cells) were resuspended in fixation/permeabilization solution (500 µL) and incubated for 20 min at 4 °C. The cells were washed twice with Perm/Wash buffer and incubated for 15 min in this buffer (1 mL). To detect Bax or Bcl-2, the fixed and permeabilized cells were incubated with Perm/Wash buffer (100 µL) containing the antibodies. For Bcl-2 protein, the cells were stained directly for 30 min with FITC-conjugated mouse anti-human Bcl-2 monoclonal antibody or IgG1 isotype control (10 µL, BD Biosciences) at 4 °C. For indirect Bax

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staining, the cells were incubated for 30 min with either rabbit anti-human Bax polyclonal antibody or IgG1 isotype control (BD Biosciences) at 4 °C. After washing, the cells were further incubated for 30 min with FITC-conjugated goat anti-rabbit F(ab')2 polyclonal secondary antibody (Abcam) at 4 °C. The cells were then washed with Perm/Wash buffer and analyzed using C6 Accuri flow cytometer and detected in FL1-H channel. 2.12 Caspase activity HCT116 cells (1 × 106 cells) were seeded in 60 mm2 culture dish and treated with ESEAF or vehicle DMSO (control). After treatment, cells were harvested and cell suspensions were stained with 30× FLICA solution (caspase 3/7-FAM-DEVD-FMK; caspase 9-FAMLEHD-FMK) for 1 h at 37 °C under 5% CO2 in darkness. Cells were then washed twice with wash buffer followed by resuspending of cell pellet in wash buffer. FAM-DEVDFMK and FAM-LEHD-FMK will bind to caspase-3/7, caspase-9, , respectively that are present in the cells and appear as green fluorescence. Increase in fluorescence intensity indicating caspase-3/7 and caspase-9 activities were detected by flow cytometry and detected in FL1-A channel.

2.13 Western blot analysis HCT116 cells (1×106 cells) were seeded in 60 mm2 culture dishes and treated with different concentration 2.5, 5.0 and 10.0 µg/mL of ESEAF for 24 h. Negative control was treated with vehicle DMSO. Then the cells were harvested, washed with cold PBS and resuspended in cold RIPA buffer containing protease and phosphatase inhibitors. The 12

cells were then kept on ice for 5 min and then centrifuged at 14,000 × g for 15 min at 4°C to pellet the cell debris. The total content of protein was determined by using Bradford assay. 25 µg of total protein of each lysates were loaded and separated by electrophoresis on the 12 % SDS-PAGE gel. After electrophoresis, the proteins were transferred onto a nitrocellulose membrane and followed by the blocking using skim milk for 1 h and was incubated with primary antibodies (cleaved PARP (Asp214)(D64E10) and GAPDH, Cell signaling) at 4°C overnight. Subsequently, the membrane was washed with TBST (0.05% Tween 20 in TBS) and incubated with corresponding anti-mouse/rabbit immunoglobulin G-horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Then, the membrane was washed again with TBST. For detection, the membrane was incubated using enhanced chemiluminescence (ECL) detection kit and visualized using gel documentation system. Protein bands were analyzed quantitatively and qualitatively with Vilber Lourmart. 2.14 Statistical analysis In all the experiments, data were expressed as means ± standard error. A significant difference from the respective control for each experiment was assessed using ANOVA followed by Dunnett’s test or Student’s t-test, with p values < 0.05 being regarded as statistically significant. 3. Results 3.1 Inhibition of cancer cell growth by the extract and fractions of Elephantopus scaber The cytotoxic activities of E.scaber crude and fractions against human colon adenocarcinoma cell line (HT-29), human colon carcinoma cell line (HCT116) and 13

normal colon cells (CCD841-CoN) were assessed by MTT (3-4,5-Dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay. All the IC50 value were summarized and encapsulated in Table 1. Both cell lines were exposed to increasing concentrations of E. scaber’s ethanol extract, hexane, ethyl acetate and aqueous fractions ranging from 0.1953 – 200 µg/mL. Among the fractions, ESEAF exhibited the greatest inhibitory effect against both cancer cell lines HCT116 and HT-29 in a dose-dependent manner, followed by ESHF, ESEE and ESAF. The cell viability for both cell lines was significantly decreased dose-dependently after 72 h of exposure and IC50 values were summarized in Table 1. ESAF showed the highest IC50 value on HCT116 cells with >200 µg/mL, followed by the crude extract in ethanol and hexane fractions with the IC50 values of 71.90 ± 1.09 µg/mL and 6.48 ± 0.15 µg/mL respectively (Table 1). However, ESEAF displayed remarkable cytotoxic effect on HCT116 cells with the IC50 value of 1.42 ± 0.10 µg/mL as shown in Fig. 1. CCD-841CoN cells as representatives of normal colon cells was observed to survive at the highest concentration of 25 µg/mL. Thus, ESEAF was selected and subjected for further investigation on apoptosis induction against HCT116 cells.

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Fig. 1. The effect of crude extract and fractions of Elephantopus scaber leave against different cancer cell lines and normal cell line. (A) Bar chart showed the percentage of cell viability after treatment with crude extract and fractions of Elephantopus scaber against HCT116 cells. (B)) Bar chart showed the percentage of cell viability after treatment with crude extract and fractions of Elephantopus scaber against HT-29 HT cells. (C)) Bar chart showed the percentage of cell viability after treatment with different concentration of ESEAF against two different colon cancer cell lines (HCT116 and HTHT 29) and normal colon cells (CCD841CoN) for 72 h. The data exp expressed ressed as mean ± S.E. of three independent experiments (n = 9). Asterisks indicate significantly different value from control (* p < 0.05). Table 1. IC50 values of ethyl acetate fraction of Elephantopus scaber leaves and 5fluorouracil against HCT116 and H HT-29 cancer cell lines ines and normal cell line. line IC50 (µg/mL) Cell lines

HCT116 HT-29 CCD-841CoN

Ethyl acetate fraction 1.42 ± 0.08

5-fluorouracil* 0.73 ± 0.02

2.41 ± 0.26

NA

>25

>25

The data represent mean ± S.E. of three independent experiments (n = 9). NA: Not available. * 5-Fluorouracil Fluorouracil served as positive control. 15

3.2 Phytochemical profile le of ESEAF using GC–MS analysis As depicted in Fig. 2, chemical ical profiling of ESEAF was investi investigated by using us GC-MSTOF. The chromatogram showed the presence of eight different nt compounds (Table 2.). Some of these compounds are known for anticancer activity.

Fig. 2. GC-MS MS analysis of ESEAF Table 2. Identified ESEAF chemical constituents by using GC GC-MS No

Compounds

M.W.

M.F.

1. 2. 3. 4. 5. 6. 7. 8.

Deoxyelephantophin Isodeoxyelephantopin Stigmasterol Olean-12-en en-3-one Lup-20(29)--en-3-one Lupeol Betulin Lup-20(29)-en-3-ol, ol, acetate, (3 (3β)-

344 344 412 424 424 426 442 468

C19H20O6 C19H20O6 C29H48O C30H48O C30H48O C30H50O9 C30H50O2 C32H52O2

Retention Time (s) 43.196 45.436 50.494 51.403 52.060 52.457 53.324 54.083

M.W.: molecular weight; M.F.: molecular formula 16

3.2 Morphological alterations induced by ESEAF In order to determine the nuclear morphological alterations in response to ESEAF treatment, HCT116 cells were stained by Hoescht 33342 and Propidium Iodide (PI) and visualized by fluorescence microscope. Cells which displayed dull blue colour represent healthy and viable cells. Apoptotic cells were characterized into early and late apoptosis. Early apoptotic cells stained with bright blue colour whereas the late apoptotic cells double-stained with bright blue and red colour. Dead cells were only displayed in red colour. After treatment with ESEAF, the occurrence of apoptosis was observed as shown in Fig. 3. As the incubation period increased, the appearance of apoptotic cell morphological characteristics such as nuclear shrinkage and chromatin condensation as well as DNA fragmentation became more apparent as indicated by the arrows. We found that the abundance of late apoptotic cells which exhibited DNA fragmentation characteristic increased with longer exposure to ESEAF (Fig. 3).

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Fig. 3. Effect of ESEAF oon n nuclear morphological changes of HCT116 cells. (A) untreated cells as control. After exposure of 6 h ((B), 12 h (C) and 24 h (D)) of 10 µg/mL ESEAF, cells were stained with Hoechst 333342 and PI. Magnification: 400X Arrow 1 chromatin condensation, 2 late apoptosis/ necrosis, 3 cell shrinkage, 4 DNA fragmentation 3.3 Induction of DNA Fragmentation by ESEAF DNA fragmentation is one of the apoptosis hallmark hallmarkss and one of the late apoptotic characteristics which can be detected by TdT-mediated mediated dUTP Nick End Labelling 18

(TUNEL) TUNEL) assay. HCT116 cells were exposed with or without 10 µg/mL of ESEAF for 6, 12 and 24 h respectively. The percentage of TUNEL-positive positive cells increased to 1.13 ± 0.09, 1.33 ± 0.07 and 3.09 ± 0.03% with the increasi increasing ng incubation times of 6, 12 and 24 h, respectively (Fig. 4). These data indicated that ESEAF induced DNA fragmentation in a time time-dependent manner.

Fig. 4. Effect of ESEAF on DNA fragmentation of HCT116 cells. HCT116 cells were exposed for different incubation period (6, 12 and 24 h) with 10 µg/mL of ESEAF. (A) Positive TUNEL staining was shown by the M2 region in which cells were stained with FITC-conjugated anti- BrdU antibody. (B) Bar chart showed the percentage age of TUNEL positive cells. Histograms are representatives of three separate experiments (n=3). Asterisks indicate significantly different value from control (* p < 0.05).

3.4 Externalization of Phosphatidylserine by ESEAF The externalization of phosphat phosphatidylserine as one of the hallmarks of apoptosis was detected by using AnnexinV AnnexinV-FITC/ FITC/ Propidium Iodide. The HCT116 cells were exposed to increasing concentrationss of ESEAF for 6, 12 and 24 h.. The dual parametric dot plots showed the significant increase in externalization tion of phosphatidylserine in dose-

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dependent and time-dependent manner (Fig. 5(A) & (D)). Fig. 5 ((B) & (E)) shows the proportion of the viable, early apoptotic, late apoptotic and dead cells. The untreated control cells showed low or negative staining with both Annexin V and PI (Annexin V./PI.) indicating viable cells. When treatment with different concentrations of ESEAF, the results showed the progression from early to late apoptosis in HCT116 cells. The total Annexin V-positive cells (%) which consist of early and late apoptotic cells significantly increased to 4.64 ± 0.12, 8.14 ± 0.47, 16.39 ± 1.28 with the increasing ESEAF concentrations of 2.5, 5.0 and 10.0 µg/mL, respectively (Fig. 5 (C)). Similarly, the percentages of total Annexin V-positive cells (Annexin+/PI- and Annexin+/PI+) increased with the values of 1.77 ± 0.03, 2.29 ± 0.57, 3.62 ± 0.30 and 16.39 ± 1.28 with the increase in incubation periods of 6, 12 and 24 h, respectively as shown in Fig. 5(F).

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Fig. 5. Induction of early and late apoptosis by ESEAF in HCT116 cells. (A) (A showed the flow cytometric fluorescence patterns of Annexin V V-FITC/PI FITC/PI staining in different concentrations of ESEAF (2.5 (2.5-10.0 µg/mL) at 24 h. (B) The bar chart indicated the percentage of viable, early apoptotic, late apoptotic and necrotic cells in different concentrations of ESEAF. (C (C) The bar chart depicted the percentage of annexin V positive cells. (D)) showed the flow cytometric fluorescence patterns of Annexin VV FITC/PI staining after treatment with 10.0 µg/mL of ESEAF in different time incubation periods (6, 12 and 24 h).. (E (E) The bar chart indicated the percentage of viable, early apoptotic, late apoptotic optotic and necrotic cells in time incubation periods. (F) The bar chart depicted the percentage of annexin V positive cells in a time course study. The data 21

expressed as mean ± S.E. from three individual experiments. Asterisks indicate significantly different value from control (*p

Elephantopus scaber induces apoptosis through ROS-dependent mitochondrial signaling pathway in HCT116 human colorectal carcinoma cells.

Elephantopus scaber also known as Elephant's foot (Asteraceae family) has a plethora of traditional applications including dysuria, diarrhea, dysenter...
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