DrugRes/2015-01-0935/2.7.2015/MPS
Original Article
Anticancer Effects of Extracts from the Fruit of Morinda Citrifolia (Noni) in Breast Cancer Cell Lines
Authors
K. Sharma1, 3 * , S. D. Pachauri1 * , K. Khandelwal1, H. Ahmad1, A. Arya1, 3, P. Biala1, S. Agrawal1, 3, R. R. Pandey1, A. Srivastava1, A. Srivastav1, J. K. Saxena2, 3, A. K. Dwivedi1, 3
Affiliations
1
Key words ▶ cancer ● ▶ cell proliferation ● ▶ apoptosis ●
Abstract
received 08.01.2015 accepted 13.06.2015 Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1555804 Published online: 2015 Drug Res © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence A. K. Dwivedi Pharmaceutics Division CSIR-Central Drug Research Institute CDRI Communication No. 9017 B 10/1, Sector 10 Jankipuram Extension Sitapur Road Lucknow-226031 India Tel.: + 91/522/2623 405 Fax: + 91/522/2623 938 [email protected]
Division of Pharmaceutics, CSIR-Central Drug Research Institute, Lucknow, India Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow, India 3 Academy of Scientific and Innovative Research, New Delhi, India
▼
Morinda citrifolia L. (NONI) fruits have been used for thousands of years for the treatment of many health problems including cancer, cold, diabetes, flu, hypertension, and pain. Plant extracts have reported several therapeutic benefits, but extraction of individual compound from the extract often exhibits limited clinical utility as the synergistic effect of various natural ingredients gets lost. They generally constitute polyphenols and flavonoids. Studies have suggested that these phytochemicals, especially polyphenols, display high antioxidant properties, which help to reduce the risk of degenerative diseases, such as cancer and cardiovascular diseases. Several in-vitro and in-vivo studies have shown that Noni fruits have antioxidant, anti-inflammatory, anti-dementia, liver-protective, anticancer, analgesic, and immunomodulatory effects. Till date about 7 in vitro
Introduction
▼
Breast cancer continues to be one of the most common cancers and major cause of death among women worldwide. Breast cancer incidence in women in the United States is 1 in 8 (about 13 %). The American Cancer Society estimated 182 460 cases (which accounted for 26 % of all new cancer cases) for 2008 in the U.S.A alone, resulting in 40 480 deaths [1]. The existing therapeutic agents for breast cancer produce various side effects. Development of specifically breast cancer cell targeting drug without any side effect to normal is an ongoing effort in the field of cancer drug discovery. Apoptosis is well-regulated physiological process of cell death. Normal breast growth is controlled by a balance between cell proliferation and apoptosis, and breast tumor grows not just as a result of uncontrolled prolif * Authors contributed equally.
cancer studies have been done, but a detailed in vitro study including cell cycle and caspase activation assay on breast cancer cell line has not been done. In the present study different Noni fruit fractions have tested on cancer cell lines MCF-7, MDA-MB-231 (breast adenocarcinoma) and one non-cancer cell line HEK-293 (Human embryonic kidney). Out of which ethylacetate extract showed a higher order of in vitro anticancer activity profile. The ethylacetate extract strongly inhibited the proliferation of MCF-7, MDA-MB-231 and HEK-293 cell lines with IC50 values of 25, 35, 60 µg/ml respectively. The extract showed increase in apoptotic cells in MCF-7 and MDA-MB-231 cells and arrested the cell cycle in the G1/S phase in MCF-7 and G0/G1 phase in MDA-MB-231 cells. Noni extract also decreases the intracellular ROS generation and mitochondrial membrane potential.
eration but also due to reduced apoptosis. Consequently apoptosis inducing capability is considered as safer and better aspect of a therapeutic molecule. Fruits and vegetables have been shown to contain a diverse source of phytochemicals such as carotenoids, tocopherols and polyphenols which possess chemopreventive properties at multiple stages of cancer [2, 3]. Studies have suggested that these phytochemicals, especially polyphenols, display high antioxidant properties, which help to reduce the risk of degenerative diseases, such as cancer and cardiovascular diseases. The anticancer mechanisms of these polyphenols are well established [4], and the complex mechanisms of action of the phytochemicals implies that the cancer chemopreventive properties of fruits, vegetables and their products are likely to arise from the combinations of various chemicals present in them [5]. Approximately 41 % of women are utilizing complementary and alternative medicine (CAM) forms of medicine to Sharma K et al. Anticancer Activity of Noni Extract … Drug Res
Downloaded by: NYU. Copyrighted material.
2
manage their breast cancer, including products from the Morinda citrifolia (Noni) plant. Morinda citrifolia (Noni) an edible and medicinal tropical plant, Morinda citrifolia, has been used for over 2000 years by Polynesian cultures as an herbal remedy for infection, arthritis, diabetes, asthma, hypertension, and pain [6]. Several in-vitro and in-vivo studies have shown that Noni fruits have antioxidant, anti-inflammatory, anti-dementia, liver-protective, anticancer, analgesic, and immunomodulatory effects. Many authors reported that this Noni extract contains high levels of phenolic compounds, including rutin, scopoletin, quercetin, kaempferol and fruit also contain 6-O-(β-D-glucopyranosyl)-1O-octanoyl-β-D-glucopyranose, asperulosidic acid. Many of these are known to inhibit growth of breast cancer cells and so it is likely that at least some of the growth inhibitory effect of the extract can be ascribed to these compounds. Estimation of the concentration of these constituents in ethanol, chloroform, butanol and ethylacetate fraction of Noni fruits is already reported by us in our previous study [8]. Phytochemical investigation revealed presence of scopoletin, rutin, quercetin and kaempferol in ethyl acetate fraction while chloroform fraction showed presence of rutin and scopoletin with traces of quercetin. However, butanol fraction, which was ineffective, contained rutin with traces of scopoletin and quercetin. It may be that these individual phenolic phytochemicals act additively, synergistically, and/or antagonistically with other compounds to display the antiproliferative activity. Till date about 7 in vitro cancer studies have been done, but a detailed in vitro study including cell cycle and caspase activation assay on breast cancer cell line has not been done [7]. In the present study different Noni fruit fractions have tested on cancer cell lines MCF-7, MDA-MB-231 (breast adenocarcinoma) and one non-cancer cell line HEK-293 (Human embryonic kidney).
Materials and Methods
▼
General methods Plant material and preparation of extract
Noni fruits were collected during February to March 2011 in Bhubaneswar and were authenticated in the Central Horticulture Experiment Station [Voucher specimens of fruit (Noni: Lot Code. BAH-1)], Bhubaneswar, India. Noni fruits were shade dried at room temperature and dried powder (1.57 Kg) was first defatted with Hexane and extracted with ethanol (90 %) 3 times. Ethanolic extractive are separated and filtered. The filtered extractives were concentrated under reduced pressure by using rotavapour. The ethanolic extract (240 gm) was triturated with water and fractionated with chloroform, ethyl acetate and butanol. After drying at reduced pressure, Hexane (2.5 gm), chloroform (26.9 gm), ethyl acetate (25 gm) and butanol (50.3 gm) fractions were obtained. The identification and quantification of polyphenolic markers in ethyl acetate extract of Noni fruits by high-performance liquid chromatography HPLC [8] is well demonstrated in our previous work and we have used the same batch of ethylacetate extract for the present study [9].
Biological studies
The human cancer cell lines e. g., MCF-7, MDAMB-231 (breast adenocarcinoma), and normal non-transformed cell types human embryonic kidney (HEK-293) used in the present study were obtained from the ATCC (American Type Culture Collection, USA) and were maintained in RPMI 1 640 (Roswell Park Sharma K et al. Anticancer Activity of Noni Extract … Drug Res
DrugRes/2015-01-0935/2.7.2015/MPS
Memorial Institute, Merck) with 10 % FBS (Merck), supplemented with 1 % antibiotic and antimycotic solution (Gibco, USA) at 37 °C in a humidified incubator with 5 % CO2. All stock solution of the extracts were prepared in cell culture grade DMSO (dimethylsulphoxide) and stored in − 20 °C. Extracts were diluted in culture media prior to use in experiments. Annexin V FITC apoptosis detection kit was purchased from Calbiochem. MTT dye and caspase-3 activation assay kit was procured from Sigma-Aldrich. All the flow cytometry experiments were performed using a FACScan (Becton Dickinson, Mountain View, CA) flowcytometer, equipped with a single 488-nm argon laser.
MTT assay for cell viability
Cell viability was assessed by MTT assay [10], which is based on the reduction of MTT by mitochondrial dehydrogenases of viable cells to form a purple formazan product. Briefly MCF-7, MDA-MB-231 and HEK-293 cells (5 × 103/well) were plated in 96 well plates. After incubating for overnight, the cells were treated with different concentrations (200, 100, 50, 25, 12.5, 6.25 µg/ml, in triplicates) of extracts of Noni for 48 h. Subsequently 10 µL of MTT (10 mg/mL) was added to each well and incubated for 3 h. The MTT formazan formed by viable cells was dissolved in 100 µL of DMSO and shaken for 10 min. The absorbance was measured on an ELISA reader. Each test was repeated at least 3 times. The concentration of the compound which gives the 50 % growth inhibition value corresponds to IC50, calculated by using Graph Pad prism 5.
LDH release assay
The leakage into the media of LDH [11], as an indicator of cell membrane injury was detected with an LDH assay kit (Sigma Aldrich) according to the manufacturer instructions. Briefly, cells (4 × 103 cells/well) were cultured in 96 well tissue culture plates and treated with different concentrations (200, 100, 50, 25, 12.5, 6.25 µg/ml) of ethylacetate extract for 48 h. At the end of the incubation 20 µL of culture supernatants by different treatment was taken out for the activity analysis of extracellular LDH. Each sample was detected and the absorbance was read at wavelength 450 nm and the results were expressed as the percentage of LDH leakage from treated cells vs. control cells.
Apoptosis studies
Many of the cytotoxic drugs today, induces cell killing by the process known as apoptosis [12]. Quantitation of apoptotic cells by Annexin V staining was carried out according to the manufacturer’s instructions. Briefly, cells (5 × 105 cells/well) were seeded in 6 well plates and treated with ethylacetate extract at IC50 value for 24, 48 and 72 h. After the incubations, cells were washed with PBS stained with 1.25 μl of Annexin V-FITC and 10 μl of media binding reagent and incubated for 20 min. After that 10 μl of Propidium iodide (PI) was added and samples were analysed using flowcytometer. Annexin V-FITC was analyzed using excitation and emission settings of 488 nm and 535 nm (FL-1 channel); PI, 488 nm and 610 nm (FL-2 channel). Debris and clumps were gated out using forward and orthogonal light scatter. The experiment was repeated 2 times independently.
Cell cycle analysis
Cells at a density of 1 × 105 cells/ml were incubated with the extract at IC50 value for 24, 48 and 72 h. All adhering and floating cells were harvested and transferred to a sterile centrifuge tube (1 × 106 cells) [13]. The cells were then washed with cold phos-
Downloaded by: NYU. Copyrighted material.
Original Article
DrugRes/2015-01-0935/2.7.2015/MPS
Assay of caspase activity
ApoTarget caspase colorimetric protease assay [14] sample kit (Catalog No. KHZ10001, invitrogen) was used to determine the activity of caspase -2, -3, -6, -8 and -9. MCF-7 cells were treated with ethylacetate extract at IC50 value for 24, 48 and 72 h and lysed using the cell lysis buffer supplied. Samples (50 µl) of the lysate were aliquoted into wells of a 96-well microplate, to which 50 µl of reaction buffer containing 10 mM DTT were then added to the sample wells. Substrates selective for each of the caspase forms (5 µl; final concentration 4 mM) were added to the appropriate wells and the plate was then incubated at 37 °C for 2 h. Absorbance at 405 nm was then read in a microplate reader. The absorbance from treated samples was compared with untreated control to allow determination of the change in caspase activity. The selective substrates used were: VDVAD-pNA (substrate for caspase-2), DEVD-pNA (substrate for caspase-3), VEID-pNA (substrate for caspase-6), IETD-pNA (substrate for caspase-8), LEHDpNA (substrate for caspase-9).
Intracellular ROS measurement
For determination of the intracellular accumulation of ROS [15] (reactive oxygen species), the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) method was used. DCFH-DA crosses the cell membrane, subsequently undergoes deacetylation by intracellular esterases. The de acetylated 2′,7′-dichlorodihydrofluorescein (DCFH) reacts with intracellular hydrogen peroxide or other oxidizing ROS to give the fluorescent 2′,7′-dichlorofluorescein (DCF). After treatment of ethylacetate extract at IC50 value for 24, 48 and 72 h in MCF-7 cells, DCFH-DA (at a final concentration of 2 mM in the whole medium) was added to each well. After incubation for 20 min at 37 °C, cells were washed with PBS twice with 20 min each time. Intracellular ROS accumulation was measured by fluorimeter.
Results and Discussion
▼
Anti-proliferative activity
The MTT assay is a commonly used method to study the action of natural products on cell proliferation, viability and cytotoxicity. This assay is based on the reduction of a tetrazolium salt to a purple insoluble formazan by metabolically active cells. The absorbance of the solubilized formazan is taken as a measure of the number of living cells. All the extracts of Noni extracts (chloroform, ethylacetate, ethanol, and butanol) were evaluated for in vitro anticancer activity using a MTT assay across different concentrations (6.25–200 µg/ml) as triplicates. The growth-inhibitory effects were studied in 2 human cancer cell lines, MCF-7 (breast adenocarcinoma), MDA-MB-231 and one non-cancer cell line, HEK-293 (Human embryonic kidney). Paclitaxel was taken as positive control and its IC50 value is 15, 24 and 36 µM. Among all the extracts evaluated, the ethylacetate extract have IC50 of 25, 35 and 60 µg/ml in MCF-7, MDA-MB-231 and HEK-293. All other extracts have IC50 values more than 100 µg/ml. The IC50 values (amount of the extract to inhibit cell proliferation by 50 %) given in ● ▶ Table 1 were determined using Graph Pad Prism 5.0. From the above data it is obvious that ethy-
Table 1 IC50 values of different extracts of Morinda citrifolia L. (NONI) compared with commercially available anti-cancer drug Paclitaxel.
Ethylacetate extract Butanol extract Ethanol extract Chloroform extract Paclitaxel
MCF-7
MDA-MB-231
HEK-293
25 µg/ml > 100 µg/ml > 100 µg/ml > 100 µg/ml 15 µM
35 µg/ml > 100 µg/ml > 100 µg/ml > 100 µg/ml 24 µM
60 µg/ml > 100 µg/ml > 100 µg/ml > 100 µg/ml 36 µM
Detection of mitochondrial membrane potential (Δψm)
Mitochondrial Membrane Potential is a distinctive feature of the early stages of apoptosis which leads to mitochondrial disruption which includes changes in the membrane potential and alterations to the oxidation-reduction potential of the mitochondria. Changes in the membrane potential are presumed to be due to the opening of the mitochondrial permeability transition pore (MPTP), allowing passage of ions and small molecules. The resulting equilibration of ions leads in turn to the decoupling of the respiratory chain and the release of cytochrome c into the cytosol. The mitochondrial membrane potential (Δψm) was measured by flow cytometer using the cationic lipophilic green fluorochrome Rhodamine 123 [16]. Cells were harvested, washed twice with PBS, incubated with 1 mM Rhodamine 123 at 37 °C for 30 min, and washed twice with PBS. Fluorescence was determined by Flow Cytometry System.
Fig. 1 LDH release of MCF-7, MDA-MB-231 and HEK-293 cells exposed to Noni ethylacetate extract at different concentrations. (Color figure available online only).
Sharma K et al. Anticancer Activity of Noni Extract … Drug Res
Downloaded by: NYU. Copyrighted material.
phate buffer saline (PBS) and resuspended in 0.2 ml of cold PBS and 1.8 ml ice-cold 70 % ethanol was added to the cell suspension and incubated at 4 °C for overnight. The cells were then centrifuged at 1 000 rpm for 5 min, then suspended in 0.5 ml of cold PBS with ribonuclease A (100 μg/ml) and Triton-X (1 %) for 30 min at room temperature then added PI (50 μg/ml) and incubated in dark for 30 min and analyzed with flow cytometer and experiments were repeated 2 times independently.
Original Article
DrugRes/2015-01-0935/2.7.2015/MPS
Downloaded by: NYU. Copyrighted material.
Original Article
Fig. 2 Apoptosis study of a MCF-7, b MDA-MB-231, c HEK-293 cells exposed to Noni ethylacetate extract at IC50 value.
lacetate extract exhibits efficient anticancer activity in breast cancer cells compared to paclitaxel which showed only marginally higher activity compared to ethylacetate extract. One good thing is that ethylacetate extract is less toxic to non-cancerous cells as compared to standard drug Paclitaxel. As shown in ▶ Fig. 1 LDH leakage of MCF-7 and MDA-MB-231 cells was sig● nificantly increased and in HEK-293 there is no increase in LDH leakage with the presence of ethylacetate extract of Noni. The results imply that Noni ethylacetate extract with antiproliferative effects, which are consistent with the MTT results. In light of this finding, ethylacetate extract with high sensitivity to MCF-7 and MDA-MB-231 cancer cells may be amenable to be tested in its chemical characterization and the molecular mechanism of anticancer effects. For subsequent experiments to examine the cell cycle distribution and apoptosis-inducing effect of the ethySharma K et al. Anticancer Activity of Noni Extract … Drug Res
lacetate extract, the IC50 values were used (MCF-7: 25.0 µg/ml; MDA-MB-231: 35 µg/ml; HEK-293: 60 µg/ml).
Effects of Noni ethylacetate extract on early and late apoptosis
In this study we measured the apoptosis inducing ability of ethylacetate extract of Noni in MCF-7, MDA-MB-231 and HEK-293 cells by flow cytometry. Annexin V specially binds to phosphatidylserine and has been employed for determination of apoptotic cells. Annexin V/PI staining was performed to determine early (Annexin V positive, PI negative), late apoptotic (Annexin V positive, PI positive) and necrotic (Annexin V negative, PI positive) cells followed by 24, 48, 72 h treatment with 25, 35 and 60 µg/ml ▶ Fig. 2a), MDAof ethylacetate extract of Noni in MCF-7 ( ● ▶ Fig. 2b) and HEK-293 ( ▶ Fig. 2c) cells respectively. It MB-231 ( ● ●
DrugRes/2015-01-0935/2.7.2015/MPS
Original Article
is evident from the above data the ethylacetate extract induces apoptosis in MCF-7, MDA-MB-231 cells and does not induced apoptosis significantly in HEK-293 cells.
Effects of Noni ethylacetate extract on cell cycle distribution
Fig. 4 Activation of caspase-2, - 3, - 6, - 8, - 9 by Noni ethylacetate extract. MCF-7 cells and MDA-MB-231 cells Data are presented in mean ± SD (n = 3). Statistical difference was noticed at different time intervals comparing with normal control at significant level p
Original Article
Anticancer Effects of Extracts from the Fruit of Morinda Citrifolia (Noni) in Breast Cancer Cell Lines
Authors
K. Sharma1, 3 * , S. D. Pachauri1 * , K. Khandelwal1, H. Ahmad1, A. Arya1, 3, P. Biala1, S. Agrawal1, 3, R. R. Pandey1, A. Srivastava1, A. Srivastav1, J. K. Saxena2, 3, A. K. Dwivedi1, 3
Affiliations
1
Key words ▶ cancer ● ▶ cell proliferation ● ▶ apoptosis ●
Abstract
received 08.01.2015 accepted 13.06.2015 Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1555804 Published online: 2015 Drug Res © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence A. K. Dwivedi Pharmaceutics Division CSIR-Central Drug Research Institute CDRI Communication No. 9017 B 10/1, Sector 10 Jankipuram Extension Sitapur Road Lucknow-226031 India Tel.: + 91/522/2623 405 Fax: + 91/522/2623 938 [email protected]
Division of Pharmaceutics, CSIR-Central Drug Research Institute, Lucknow, India Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow, India 3 Academy of Scientific and Innovative Research, New Delhi, India
▼
Morinda citrifolia L. (NONI) fruits have been used for thousands of years for the treatment of many health problems including cancer, cold, diabetes, flu, hypertension, and pain. Plant extracts have reported several therapeutic benefits, but extraction of individual compound from the extract often exhibits limited clinical utility as the synergistic effect of various natural ingredients gets lost. They generally constitute polyphenols and flavonoids. Studies have suggested that these phytochemicals, especially polyphenols, display high antioxidant properties, which help to reduce the risk of degenerative diseases, such as cancer and cardiovascular diseases. Several in-vitro and in-vivo studies have shown that Noni fruits have antioxidant, anti-inflammatory, anti-dementia, liver-protective, anticancer, analgesic, and immunomodulatory effects. Till date about 7 in vitro
Introduction
▼
Breast cancer continues to be one of the most common cancers and major cause of death among women worldwide. Breast cancer incidence in women in the United States is 1 in 8 (about 13 %). The American Cancer Society estimated 182 460 cases (which accounted for 26 % of all new cancer cases) for 2008 in the U.S.A alone, resulting in 40 480 deaths [1]. The existing therapeutic agents for breast cancer produce various side effects. Development of specifically breast cancer cell targeting drug without any side effect to normal is an ongoing effort in the field of cancer drug discovery. Apoptosis is well-regulated physiological process of cell death. Normal breast growth is controlled by a balance between cell proliferation and apoptosis, and breast tumor grows not just as a result of uncontrolled prolif * Authors contributed equally.
cancer studies have been done, but a detailed in vitro study including cell cycle and caspase activation assay on breast cancer cell line has not been done. In the present study different Noni fruit fractions have tested on cancer cell lines MCF-7, MDA-MB-231 (breast adenocarcinoma) and one non-cancer cell line HEK-293 (Human embryonic kidney). Out of which ethylacetate extract showed a higher order of in vitro anticancer activity profile. The ethylacetate extract strongly inhibited the proliferation of MCF-7, MDA-MB-231 and HEK-293 cell lines with IC50 values of 25, 35, 60 µg/ml respectively. The extract showed increase in apoptotic cells in MCF-7 and MDA-MB-231 cells and arrested the cell cycle in the G1/S phase in MCF-7 and G0/G1 phase in MDA-MB-231 cells. Noni extract also decreases the intracellular ROS generation and mitochondrial membrane potential.
eration but also due to reduced apoptosis. Consequently apoptosis inducing capability is considered as safer and better aspect of a therapeutic molecule. Fruits and vegetables have been shown to contain a diverse source of phytochemicals such as carotenoids, tocopherols and polyphenols which possess chemopreventive properties at multiple stages of cancer [2, 3]. Studies have suggested that these phytochemicals, especially polyphenols, display high antioxidant properties, which help to reduce the risk of degenerative diseases, such as cancer and cardiovascular diseases. The anticancer mechanisms of these polyphenols are well established [4], and the complex mechanisms of action of the phytochemicals implies that the cancer chemopreventive properties of fruits, vegetables and their products are likely to arise from the combinations of various chemicals present in them [5]. Approximately 41 % of women are utilizing complementary and alternative medicine (CAM) forms of medicine to Sharma K et al. Anticancer Activity of Noni Extract … Drug Res
Downloaded by: NYU. Copyrighted material.
2
manage their breast cancer, including products from the Morinda citrifolia (Noni) plant. Morinda citrifolia (Noni) an edible and medicinal tropical plant, Morinda citrifolia, has been used for over 2000 years by Polynesian cultures as an herbal remedy for infection, arthritis, diabetes, asthma, hypertension, and pain [6]. Several in-vitro and in-vivo studies have shown that Noni fruits have antioxidant, anti-inflammatory, anti-dementia, liver-protective, anticancer, analgesic, and immunomodulatory effects. Many authors reported that this Noni extract contains high levels of phenolic compounds, including rutin, scopoletin, quercetin, kaempferol and fruit also contain 6-O-(β-D-glucopyranosyl)-1O-octanoyl-β-D-glucopyranose, asperulosidic acid. Many of these are known to inhibit growth of breast cancer cells and so it is likely that at least some of the growth inhibitory effect of the extract can be ascribed to these compounds. Estimation of the concentration of these constituents in ethanol, chloroform, butanol and ethylacetate fraction of Noni fruits is already reported by us in our previous study [8]. Phytochemical investigation revealed presence of scopoletin, rutin, quercetin and kaempferol in ethyl acetate fraction while chloroform fraction showed presence of rutin and scopoletin with traces of quercetin. However, butanol fraction, which was ineffective, contained rutin with traces of scopoletin and quercetin. It may be that these individual phenolic phytochemicals act additively, synergistically, and/or antagonistically with other compounds to display the antiproliferative activity. Till date about 7 in vitro cancer studies have been done, but a detailed in vitro study including cell cycle and caspase activation assay on breast cancer cell line has not been done [7]. In the present study different Noni fruit fractions have tested on cancer cell lines MCF-7, MDA-MB-231 (breast adenocarcinoma) and one non-cancer cell line HEK-293 (Human embryonic kidney).
Materials and Methods
▼
General methods Plant material and preparation of extract
Noni fruits were collected during February to March 2011 in Bhubaneswar and were authenticated in the Central Horticulture Experiment Station [Voucher specimens of fruit (Noni: Lot Code. BAH-1)], Bhubaneswar, India. Noni fruits were shade dried at room temperature and dried powder (1.57 Kg) was first defatted with Hexane and extracted with ethanol (90 %) 3 times. Ethanolic extractive are separated and filtered. The filtered extractives were concentrated under reduced pressure by using rotavapour. The ethanolic extract (240 gm) was triturated with water and fractionated with chloroform, ethyl acetate and butanol. After drying at reduced pressure, Hexane (2.5 gm), chloroform (26.9 gm), ethyl acetate (25 gm) and butanol (50.3 gm) fractions were obtained. The identification and quantification of polyphenolic markers in ethyl acetate extract of Noni fruits by high-performance liquid chromatography HPLC [8] is well demonstrated in our previous work and we have used the same batch of ethylacetate extract for the present study [9].
Biological studies
The human cancer cell lines e. g., MCF-7, MDAMB-231 (breast adenocarcinoma), and normal non-transformed cell types human embryonic kidney (HEK-293) used in the present study were obtained from the ATCC (American Type Culture Collection, USA) and were maintained in RPMI 1 640 (Roswell Park Sharma K et al. Anticancer Activity of Noni Extract … Drug Res
DrugRes/2015-01-0935/2.7.2015/MPS
Memorial Institute, Merck) with 10 % FBS (Merck), supplemented with 1 % antibiotic and antimycotic solution (Gibco, USA) at 37 °C in a humidified incubator with 5 % CO2. All stock solution of the extracts were prepared in cell culture grade DMSO (dimethylsulphoxide) and stored in − 20 °C. Extracts were diluted in culture media prior to use in experiments. Annexin V FITC apoptosis detection kit was purchased from Calbiochem. MTT dye and caspase-3 activation assay kit was procured from Sigma-Aldrich. All the flow cytometry experiments were performed using a FACScan (Becton Dickinson, Mountain View, CA) flowcytometer, equipped with a single 488-nm argon laser.
MTT assay for cell viability
Cell viability was assessed by MTT assay [10], which is based on the reduction of MTT by mitochondrial dehydrogenases of viable cells to form a purple formazan product. Briefly MCF-7, MDA-MB-231 and HEK-293 cells (5 × 103/well) were plated in 96 well plates. After incubating for overnight, the cells were treated with different concentrations (200, 100, 50, 25, 12.5, 6.25 µg/ml, in triplicates) of extracts of Noni for 48 h. Subsequently 10 µL of MTT (10 mg/mL) was added to each well and incubated for 3 h. The MTT formazan formed by viable cells was dissolved in 100 µL of DMSO and shaken for 10 min. The absorbance was measured on an ELISA reader. Each test was repeated at least 3 times. The concentration of the compound which gives the 50 % growth inhibition value corresponds to IC50, calculated by using Graph Pad prism 5.
LDH release assay
The leakage into the media of LDH [11], as an indicator of cell membrane injury was detected with an LDH assay kit (Sigma Aldrich) according to the manufacturer instructions. Briefly, cells (4 × 103 cells/well) were cultured in 96 well tissue culture plates and treated with different concentrations (200, 100, 50, 25, 12.5, 6.25 µg/ml) of ethylacetate extract for 48 h. At the end of the incubation 20 µL of culture supernatants by different treatment was taken out for the activity analysis of extracellular LDH. Each sample was detected and the absorbance was read at wavelength 450 nm and the results were expressed as the percentage of LDH leakage from treated cells vs. control cells.
Apoptosis studies
Many of the cytotoxic drugs today, induces cell killing by the process known as apoptosis [12]. Quantitation of apoptotic cells by Annexin V staining was carried out according to the manufacturer’s instructions. Briefly, cells (5 × 105 cells/well) were seeded in 6 well plates and treated with ethylacetate extract at IC50 value for 24, 48 and 72 h. After the incubations, cells were washed with PBS stained with 1.25 μl of Annexin V-FITC and 10 μl of media binding reagent and incubated for 20 min. After that 10 μl of Propidium iodide (PI) was added and samples were analysed using flowcytometer. Annexin V-FITC was analyzed using excitation and emission settings of 488 nm and 535 nm (FL-1 channel); PI, 488 nm and 610 nm (FL-2 channel). Debris and clumps were gated out using forward and orthogonal light scatter. The experiment was repeated 2 times independently.
Cell cycle analysis
Cells at a density of 1 × 105 cells/ml were incubated with the extract at IC50 value for 24, 48 and 72 h. All adhering and floating cells were harvested and transferred to a sterile centrifuge tube (1 × 106 cells) [13]. The cells were then washed with cold phos-
Downloaded by: NYU. Copyrighted material.
Original Article
DrugRes/2015-01-0935/2.7.2015/MPS
Assay of caspase activity
ApoTarget caspase colorimetric protease assay [14] sample kit (Catalog No. KHZ10001, invitrogen) was used to determine the activity of caspase -2, -3, -6, -8 and -9. MCF-7 cells were treated with ethylacetate extract at IC50 value for 24, 48 and 72 h and lysed using the cell lysis buffer supplied. Samples (50 µl) of the lysate were aliquoted into wells of a 96-well microplate, to which 50 µl of reaction buffer containing 10 mM DTT were then added to the sample wells. Substrates selective for each of the caspase forms (5 µl; final concentration 4 mM) were added to the appropriate wells and the plate was then incubated at 37 °C for 2 h. Absorbance at 405 nm was then read in a microplate reader. The absorbance from treated samples was compared with untreated control to allow determination of the change in caspase activity. The selective substrates used were: VDVAD-pNA (substrate for caspase-2), DEVD-pNA (substrate for caspase-3), VEID-pNA (substrate for caspase-6), IETD-pNA (substrate for caspase-8), LEHDpNA (substrate for caspase-9).
Intracellular ROS measurement
For determination of the intracellular accumulation of ROS [15] (reactive oxygen species), the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) method was used. DCFH-DA crosses the cell membrane, subsequently undergoes deacetylation by intracellular esterases. The de acetylated 2′,7′-dichlorodihydrofluorescein (DCFH) reacts with intracellular hydrogen peroxide or other oxidizing ROS to give the fluorescent 2′,7′-dichlorofluorescein (DCF). After treatment of ethylacetate extract at IC50 value for 24, 48 and 72 h in MCF-7 cells, DCFH-DA (at a final concentration of 2 mM in the whole medium) was added to each well. After incubation for 20 min at 37 °C, cells were washed with PBS twice with 20 min each time. Intracellular ROS accumulation was measured by fluorimeter.
Results and Discussion
▼
Anti-proliferative activity
The MTT assay is a commonly used method to study the action of natural products on cell proliferation, viability and cytotoxicity. This assay is based on the reduction of a tetrazolium salt to a purple insoluble formazan by metabolically active cells. The absorbance of the solubilized formazan is taken as a measure of the number of living cells. All the extracts of Noni extracts (chloroform, ethylacetate, ethanol, and butanol) were evaluated for in vitro anticancer activity using a MTT assay across different concentrations (6.25–200 µg/ml) as triplicates. The growth-inhibitory effects were studied in 2 human cancer cell lines, MCF-7 (breast adenocarcinoma), MDA-MB-231 and one non-cancer cell line, HEK-293 (Human embryonic kidney). Paclitaxel was taken as positive control and its IC50 value is 15, 24 and 36 µM. Among all the extracts evaluated, the ethylacetate extract have IC50 of 25, 35 and 60 µg/ml in MCF-7, MDA-MB-231 and HEK-293. All other extracts have IC50 values more than 100 µg/ml. The IC50 values (amount of the extract to inhibit cell proliferation by 50 %) given in ● ▶ Table 1 were determined using Graph Pad Prism 5.0. From the above data it is obvious that ethy-
Table 1 IC50 values of different extracts of Morinda citrifolia L. (NONI) compared with commercially available anti-cancer drug Paclitaxel.
Ethylacetate extract Butanol extract Ethanol extract Chloroform extract Paclitaxel
MCF-7
MDA-MB-231
HEK-293
25 µg/ml > 100 µg/ml > 100 µg/ml > 100 µg/ml 15 µM
35 µg/ml > 100 µg/ml > 100 µg/ml > 100 µg/ml 24 µM
60 µg/ml > 100 µg/ml > 100 µg/ml > 100 µg/ml 36 µM
Detection of mitochondrial membrane potential (Δψm)
Mitochondrial Membrane Potential is a distinctive feature of the early stages of apoptosis which leads to mitochondrial disruption which includes changes in the membrane potential and alterations to the oxidation-reduction potential of the mitochondria. Changes in the membrane potential are presumed to be due to the opening of the mitochondrial permeability transition pore (MPTP), allowing passage of ions and small molecules. The resulting equilibration of ions leads in turn to the decoupling of the respiratory chain and the release of cytochrome c into the cytosol. The mitochondrial membrane potential (Δψm) was measured by flow cytometer using the cationic lipophilic green fluorochrome Rhodamine 123 [16]. Cells were harvested, washed twice with PBS, incubated with 1 mM Rhodamine 123 at 37 °C for 30 min, and washed twice with PBS. Fluorescence was determined by Flow Cytometry System.
Fig. 1 LDH release of MCF-7, MDA-MB-231 and HEK-293 cells exposed to Noni ethylacetate extract at different concentrations. (Color figure available online only).
Sharma K et al. Anticancer Activity of Noni Extract … Drug Res
Downloaded by: NYU. Copyrighted material.
phate buffer saline (PBS) and resuspended in 0.2 ml of cold PBS and 1.8 ml ice-cold 70 % ethanol was added to the cell suspension and incubated at 4 °C for overnight. The cells were then centrifuged at 1 000 rpm for 5 min, then suspended in 0.5 ml of cold PBS with ribonuclease A (100 μg/ml) and Triton-X (1 %) for 30 min at room temperature then added PI (50 μg/ml) and incubated in dark for 30 min and analyzed with flow cytometer and experiments were repeated 2 times independently.
Original Article
DrugRes/2015-01-0935/2.7.2015/MPS
Downloaded by: NYU. Copyrighted material.
Original Article
Fig. 2 Apoptosis study of a MCF-7, b MDA-MB-231, c HEK-293 cells exposed to Noni ethylacetate extract at IC50 value.
lacetate extract exhibits efficient anticancer activity in breast cancer cells compared to paclitaxel which showed only marginally higher activity compared to ethylacetate extract. One good thing is that ethylacetate extract is less toxic to non-cancerous cells as compared to standard drug Paclitaxel. As shown in ▶ Fig. 1 LDH leakage of MCF-7 and MDA-MB-231 cells was sig● nificantly increased and in HEK-293 there is no increase in LDH leakage with the presence of ethylacetate extract of Noni. The results imply that Noni ethylacetate extract with antiproliferative effects, which are consistent with the MTT results. In light of this finding, ethylacetate extract with high sensitivity to MCF-7 and MDA-MB-231 cancer cells may be amenable to be tested in its chemical characterization and the molecular mechanism of anticancer effects. For subsequent experiments to examine the cell cycle distribution and apoptosis-inducing effect of the ethySharma K et al. Anticancer Activity of Noni Extract … Drug Res
lacetate extract, the IC50 values were used (MCF-7: 25.0 µg/ml; MDA-MB-231: 35 µg/ml; HEK-293: 60 µg/ml).
Effects of Noni ethylacetate extract on early and late apoptosis
In this study we measured the apoptosis inducing ability of ethylacetate extract of Noni in MCF-7, MDA-MB-231 and HEK-293 cells by flow cytometry. Annexin V specially binds to phosphatidylserine and has been employed for determination of apoptotic cells. Annexin V/PI staining was performed to determine early (Annexin V positive, PI negative), late apoptotic (Annexin V positive, PI positive) and necrotic (Annexin V negative, PI positive) cells followed by 24, 48, 72 h treatment with 25, 35 and 60 µg/ml ▶ Fig. 2a), MDAof ethylacetate extract of Noni in MCF-7 ( ● ▶ Fig. 2b) and HEK-293 ( ▶ Fig. 2c) cells respectively. It MB-231 ( ● ●
DrugRes/2015-01-0935/2.7.2015/MPS
Original Article
is evident from the above data the ethylacetate extract induces apoptosis in MCF-7, MDA-MB-231 cells and does not induced apoptosis significantly in HEK-293 cells.
Effects of Noni ethylacetate extract on cell cycle distribution
Fig. 4 Activation of caspase-2, - 3, - 6, - 8, - 9 by Noni ethylacetate extract. MCF-7 cells and MDA-MB-231 cells Data are presented in mean ± SD (n = 3). Statistical difference was noticed at different time intervals comparing with normal control at significant level p
