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RETRACTED: In vitro cytotoxic and apoptotic effect of Mimusops elengi Linn. methanolic bark extract
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Original article
In vitro cytotoxic and apoptotic effect of Mimusops elengi Linn. methanolic bark extract Harish Kumar a,∗ , K. Sreedhara Ranganath Pai a , Naseer Maliyakkal a , Savaliya Mihir a , Charanjeet Singh b a b
Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal 576104, India Department of Pharmacology, Arya College of Pharmacy, Jaipur, India
a r t i c l e
i n f o
Article history: Received 12 June 2014 Accepted 10 July 2014 Keywords: Cytotoxic Mimusops elengi Linn. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay Sulphorhodamine B assay Hoechst33343 dye Comet
a b s t r a c t The ayurvedic system of medicine recommends Mimusops elengi Linn. (Sapotaceae) for the treatment of tumors. The study is needed as cancer is the second most common cause for death in the world; most of the traditional drugs have shown resistance, low-cost approaches, and higher toxicity of the existing compound. The present study aims to determine the mode of cell death induced by the methanol extract of ME in human cancer cell lines. Among M. elengi (ME) crude extract and fractions screened for potential anticancer activity, MED and MEE fractions (dichloromethane and ethyl acetate) were found to have promising cytotoxic activities in all cell lines studied by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay and Suforhodamine B (SRB). MEE found to have more potent activity in cervical cancer (HeLa), colon cancer (HCT15), lung (A549), breast cancer (BT549), glioblastoma (U343 cells). Interestingly, ME was less cytotoxic in normal cell line (HBL-100) indicate the specific activity towards cancer cells. AO/EB staining and Hoechst-33342 staining indicate membrane blebbing, condensed and fragmented nuclei upon treatment with MEE and MED in HeLa, A549 and U343 cells. Fragmented DNA ladder and genomic DNA fragmentation were observed with DNA fragmentation assay based on gel electrophoresis and COMET assay by fluorescent microscopy. Similarly, cell cycle analysis by flow cytometer indicates distortion of normal cell cycle and increased subG0 phase. ME was found genotoxic by micronuclei formation. These results indicate that both MED and MEE fractions induces apoptosis but not necrosis cells in the cancer cells. © 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction Apoptosis is cellular suicide or programmed cell death that is mediated by activation of an evolutionary conserved intracellular pathway. Recently the relation of apoptosis and cancer has been emphasized and increasing evidence suggests that the processes of neoplastic transformation, progression and metastasis involve alterations of normal apoptotic pathway [1]. Apoptotic also gives some clues about effective anticancer therapy and many chemotherapeutic agents were reported to event their anti-tumor effect including apoptosis of cancer cells. Medicinal plants have been used as remedies for human diseases for centuries. The reason for using them as medicine lies
∗ Corresponding author. Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal 576104, India. Mobile: +9587775613. E-mail addresses: [email protected], [email protected] (H. Kumar).
in the fact that they contain chemical components of therapeutic value [2]. The medicinal value of plants lies in some chemical substances (usually secondary metabolites) that produce a definite physiological action on the human body. Mimusops elengi Linn. (Family – Sapotaceae) (“Syn.”: Bakul, Maulsari) is commonly known as Spanish cherry or bullet wood is a small to large tree found in all parts of India. It is an evergreen tree whose distribution extends to India, Burma, Pakistan and Thailand. The plant has been used as febrifuges, astringents, headache, cardiotonic, alexipharmic and stomachic, anthelmintic, teeth cleaner and purgatives and stimulants [3]. The various extracts of the plant have been reported (bark, fruit, leaves, seed, flowers) to have antimicrobial, anti-ulcer, anti-hyperglycemic, diuretic, wound healing, anti-inflammatory, analgesic and antipyretic, antigastric ulcers, hypotensive, anti-HIV and spasmolytic activities. Recent reports suggest that bark of the plant contains presence of steroids, alkaloids, taraxerol, taraxerone, urosolic acid, betulinic acid, ␣-spinosterol, -sitosterol glycoside, quercitol, lupeol, -amyrin, farnane type triterpenoid and mixture of
http://dx.doi.org/10.1016/j.bionut.2014.07.004 2210-5239/© 2014 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Kumar H, et al. In vitro cytotoxic and apoptotic effect of Mimusops elengi Linn. methanolic bark extract. Biomed Prev Nutr (2014), http://dx.doi.org/10.1016/j.bionut.2014.07.004
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triterpenoidsaponins in the bark of M. elengi Linn. have been isolated from the bark such as mimusopgenone and mimugenone in the seeds [4], triterpenoid saponins, such as mimusopsides A and B, mimusopin, mimusopsin, mimusin, Mi-saponin A and 16␣-hydroxyMi-saponin A from the seeds [5,6]. Members of the lupane, ursane and oleanane triterpenoids have demonstrated antiproliferative activity on various cancer cell lines. 2. Materials and methods 2.1. Collection of plant material The fresh plant bark was collected from parkala (Udupi), Karnataka in September month and authenticated by Dr GopalakrishnaBhat, Professor of Botany, PoornaPrajna College, Udupi, Karnataka. An authenticated specimen was submitted in herbarium of Manipal college of Pharmaceutical sciences, Manipal.
The cytotoxicity assay was performed by using the Sulforhodamine B (SRB) colorimetric method to assess growth inhibition according to Vanicha and Kirtikara [10]. Briefly, till 48 h treatment same as that of MTT method. At the end of the exposed time, cells in each well were fixed by addition of 100 mL of cold [(4 ◦ C) 10% (w/v)] trichloroacetic acid (TCA) into the growth medium. Each plate was incubated at4 ◦ C for 1 h before gently washed five times with Mili-Q water to remove TCA, the growth medium and dead cells. Plates were allowed to dry in air and to each well were added 50 mL of 0.057% (v/v) SRB dye in 1% acetic acid in deionized water and allowed to stand for 30 min at room temperature. At the end of the staining period, unbound SRB was removed by washing four times with 1% of an acetic acid solution. The plate was air-dried and
2.2. Preparation of extract The fresh plant bark was harvested, rinsed under tap water and oven dried. The coarsely powder material was extracted with methanol by using soxhlet apparatus and solvent was removed by distillation and concentrated using a rotavapor. 2.3. Fractionation of crude extract Crude methanolic extract of M. elengi Linn. was suspended in water and then fractionated with organic solvents in order of increasing polarity to get petroleum ether, dichloromethane, n-butanol, ethyl acetate and methanol-water (hydro-alcoholic mixture) [7]. 2.4. Preparation of different concentrations Different concentrations of crude extract and fractions were prepared by dissolving the extract in DMSO and then diluting it with DMEM medium under sterile conditions.
Fig. 1. Cytotoxicity of most active fractions in selected human cancer cells (MTT Method). 3A: human cervical carcinoma (HeLa), 3B: human glioblastoma cells (LN229), 3C: human glioblastoma cells (U343), 3D: human adenocarcinoma alveolar basal epithelial cells (A549), 3E: rat glioma cells (C6), 3F: human breast adenocarcinoma epithelial cells (MCF7).
2.5. Cell culture conditions A cell line was procured from NCCS, Pune. Cancer cells were maintained in Dulbecco’s modified eagle medium (DMEM) with 1000 mg/mL of glucose, supplemented with 10% FBSFBS (fetal bovine serum) and penicillin/streptomycin-L-glutamine and cultured in a humified atmosphere of 5% CO2 and 95% air at 37 ◦ C in incubator [8]. 2.5.1. Screening of cytotoxicity/anticancer potential Cancer cell line was used for the determination of cytotoxic activity. Cells were seeded in 96-well plates at the density of 6000 cells/well (HeLa cells) in 100 L medium. Then various concentrations of the crude extract were added to the cells in 100 L medium. Cells were incubated for 24 h with test extract concentrations. Each concentration was tested in triplicate. The MTT assay is a laboratory test that measures changes in color for measuring the activity of enzyme that reduces MTT to formazan, giving a purple color. Yellow MTT (3-(4, 5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide, a tetrazole) reduce to purple formazan in living cells [9]. After 48 h incubation, 20 L (5 mg/mL) MTT reagent was added to each well and incubated for additional 4 h. Then 200 L of DMSO solution was added to each well to solubilize the formazan crystals. The plates were read for optical density at 540 nm with reference 630 nm, using a plate reader. By using optical density, percentage inhibition of was calculated.
Fig. 2. Cytotoxicity of most active fractions in selected human cancer cells (SRB Method). A: human cervical carcinoma (HeLa), B: human glioblastoma cells (U343), C: human adenocarcinoma alveolar basal epithelial cells (A549), D: human breast ductal carcinoma cells (BT-549), E: human colorectal adenocarcinoma (HCT-15), F: human breast adenocarcinoma epithelial cells (MCF7).
Please cite this article in press as: Kumar H, et al. In vitro cytotoxic and apoptotic effect of Mimusops elengi Linn. methanolic bark extract. Biomed Prev Nutr (2014), http://dx.doi.org/10.1016/j.bionut.2014.07.004
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150 mL of 10 mM aqueous Tris base buffer of pH 10.5 was added to each well to dissolve the cell-bound dye. The plate was then shaken for 15 to 30 min on a gyratory shaker and the optical density (OD) was read at 540 nm with reference 630 nm in a microplate reader; control wells were used as blanks. Inhibition concentration at 50% of cell population or IC50 was calculated prism graph pad [11]. 2.5.2. Apoptosis studies 2.5.2.1. AOEB dual staining. AO/EB staining identifies live, early apoptotic, late apoptotic, and necrotic cells. 5 × 105 cells/well was seeded in a 6-well plate the night before the treatment. Cells were treated with selected fraction, vehicle control and positive control at their IC50 for 48 hrs at 37 ◦ C in CO2 incubator. After treatment, media containing floating cells and attached cells in 6 well plate were collected into centrifuge tubes and centrifuged. Supernatant discarded and cell pellets were resuspended in 1 mL of HBSS. Ten microlitre of EtBr solutions were added and kept in incubator for 10 min and cells were centrifuged at 1200 rpm for 4 min at 4 ◦ C. Supernatant was drained and pellets were dislodged. Twenty microlitre of the cell suspensions was placed on a slide and observed under the fluorescent microscope equipped with 450–490 nm excitation and 520 nm emission wavelength (blue filter) [12]. Fig. 3. Cytotoxicity of selected fractions in human normal breast epithelial cells.
2.5.2.2. Nuclear staining with Hoechst-33342. The nuclear morphology of cells was studied by using cell permeable DNA dye
Fig. 4. Acridine orange assay for the discrimination of apoptosis vs necrotic cells after treatment with selected fractions. A. Human cervical carcinoma (HeLa). B. Human neuronal glioblastoma (U343). C. Lung adenocarcinoma (A549). D. Human breast carcinoma (BT549).
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Fig. 5. Hoechst-33342 DNA staining assay. A. Human cervical carcinoma (HeLa). B. Human neuronal glioblastoma (U343). C. Normal epithelial breast cells (HBL100).
Hoechst33342. Cells with homogeneously stained nuclei were considered to be viable, whereas the presence of chromatin condensation and/or fragmentation was indicative of apoptosis. The cells were treated with IC50 of the selected fractions and further incubated for 48 h. Cells were centrifuged and supernatant discarded. Cell pellets were resuspended in 1 mL of HBSS. Five microlitre Hoechst-33342-dye solutions were added and kept in incubator for 10 min and centrifuged at 1200 rpm for 4 min at 4 ◦ C. Supernatant was drained and pellets were resuspended. Twenty microlitre of the cell suspensions was placed on a slide and observed under the fluorescent microscope equipped with excitation source of 350 nm and emission at 450 nm (UV filter) [13]. 2.5.2.3. Determination of DNA fragmentation. The characteristic ladder pattern of DNA breakage was analyzed by gel electrophoresis. Cancer cells were placed in a 6-well plate at a concentration of 5 × 105 cell/mL. The cells were treated with IC50 concentration of selected fraction with positive control and were further incubated for 48 h. The DNA was isolated and electrophoretically analyzed on 1.5% agerose gel containing 10 L/mL ethidium bromide [14]. 2.5.2.4. Alkaline comet assay. Twenty-four hours after culture initiation, the cells were treated for 48 h with IC50 values of drugs. The cells were washed with HBSS and harvested. The slides were covered with 1.5% normal melting point agarose, allowed to solidify at 25 ◦ C and stored at 4 ◦ C for 1 h.
An aliquot of the cell suspension was diluted of 0.75% low melting point agarose. This mixture was rapidly pipetted on to the agarose layer on the slide, gently spread by placing a coverslip on top and allowed to solidify at 4 ◦ C for 5 min. After removal of the coverslip, the slides were immersed in freshly prepared lysis solution (2.5 M NaCl, 100 mM EDTA, 10mMTris, pH 10, with 1% Triton X-100 and 10% DMSO) for overnight at4 ◦ C. Then, the slides were left in the electrophoresis solution (1 mMEDTA and 300mMNaOH, pH 13 at 4 ◦ C) for 1 h to allow for DNA unwinding and expression of alkali-labile damage before electrophoresis. Electrophoresis was done at 22 V and 300 mA for 20 min at 4 ◦ C. Then, the slides were washed in neutralizing buffer (0.4 M Tris/HCl, pH 7.5) and stained with 80 L of an aqueous solution containing20 mg/mL ethidium bromide. Nucleoids were evaluated visually [15]. 2.5.2.5. Cell cycle analysis. In order to study the relationship between cell proliferation inhibition and the induction of apoptosis, we decided to study the subdiploid DNA contends as indicative of DNA fragmentation by apoptosis. DNA content and cell cycle distribution were assessed using PI staining. After sample treatment, both adherent and floating cells were harvested, washed with phosphate buffered saline (PBS) and fixed with ice-cold absolute ethanol at –20 ◦ C overnight. Fixed cells were washed and resuspended in a buffer containing 50 g/mL of RNAse A for 3 hrs, added 25 g/mL of Propidium iodide and analysed by flow cytometry. Propidium
Please cite this article in press as: Kumar H, et al. In vitro cytotoxic and apoptotic effect of Mimusops elengi Linn. methanolic bark extract. Biomed Prev Nutr (2014), http://dx.doi.org/10.1016/j.bionut.2014.07.004
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Fig. 6. DNA fragmentation assay. A. HeLa (Human cervical carcinoma). B. A549 (human lung adenocarcinoma). C. U343 (human glioblastoma).
Fig. 7. Single cell gel electrophoresis (COMET) assay. A. Human cervical carcinoma (HeLa). B. Human neuronal glioblastoma (U343). C. Lung adenocarcinoma (A549). D. Human breast carcinoma (BT549).
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Fig. 8. Cell cycle analysis. Human cervical carcinoma (HeLa).
iodide was excited by a 488 nm laser and emission was captured at FL-2 (570/20 BP filter). DNA content in different phases of the cell cycle were analysed for both the controls and the treated cells. Data was analysed in Summit v4.3 software [16,17].
as mean ± SEM. and were analyzed by one-way analysis of variance (ANOVA) followed by Tukey test and P < 0.05 was considered statistically significant. 4. Results and Discussion
2.5.3. Genotoxic assessment 2.5.3.1. Micronucleus assay. The micronucleus assay was performed according to Matsuoka et al. [18]. About 1 × 106 cells/mL medium were exposed and incubated. Complete media were treated as positive and negative controls respectively. At the end of incubation the cells were harvested by low centrifugation, treated with a hypotonic solution of KCl (0.075 M) and fixed in methanol: acetic acid (3:1) for 3–4 h. Two to three drops of the fixed cell suspension were dispensed on to the surface of cold micro slides, air-dried and stained with 3% Giemsa solution in Sorenson phosphate buffer (pH 6.8) for 5–7 min. The bi, tri or multinuclei cell was observed in slides [19]. 3. Statistical analysis All the experiments were independently preformed thrice with three replicates for each treatment. The results were expressed
Recently, the use of some herbs has attracted a great deal of attention as one of the alternative cancer therapies from the point of less toxicity and cost benefits. Therefore, an attempt has been made to evaluate the anticancer activity of the bark extract of M. elengi L., which is commonly used in ayurvedic system of medicine for various purposes. 4.1. Cytotoxic effect of the extracts and fractions on human cancer cells In preliminary screening HeLa cells were the most susceptible to the treatment, while U343 were the least. Assay is based upon reduction of yellow tetrazolium salt (MTT) by the reductase enzyme in metabolically active cells to a darkblue formazan [20]. These activities were supported by Sulphorhodamine B assay. The inhibitory effect on cell viability was evident after 48 h of incubation and most cytotoxic fraction (DCM and ethyl acetate) was selected
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for further evaluated at various cells by determining the IC50 value. In ethyl acetate fraction potent cytotoxic IC50 values were found against human cervical carcinoma184.2 g/mL and 11.68 g/mL by MTT and SRB method respectively. But significant cytotoxic activity was found only by SRB method (144.1 g/mL IC50 value) in DCM fraction. IC50 values in Human adenocarcinoma alveolar basal epithelial cells 43.89 g/mL, Human breast ductal carcinoma cells (BT-549) 49.33 g/mL, Human colorectal adenocarcinoma (HCT15) 43.04 g/mL by SRB method were found potent cytotoxic activity on ethyl acetate fraction. MTT showed significant cytotoxic activity in Rat glioma cells (C6) with IC50 value82.39 g/mL. Results showed that ethyl acetate fraction significantly reduced viability of cancer cells in dose-dependent manner (Figs. 1–3, Table 1). 4.2. Induction of apoptosis in cancer cells by the selected active fraction
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Table 1 IC50 value of selected extracts/fractions in different cells by MTT and SRB method. Type of cells
IC50 (g/mL) MTT method
HeLa U343 A549 MCF 7 BT549 HCT15 LN 229 C6
SRB method
MED
MEE
MED
MEE
773.6 917.7 256.4 461.6 – – 234.8 367.8
184.2 3230 334.1 313.7 – – 384.7 82.39
144.1 366.0 237.3 200.7 168.1 358.2 – –
11.68 268.8 43.89 251.9 49.33 43.04 – –
MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SRB: Suforhodamine B; MED: Mimusops elengi Dichloromethane fraction; MEE: Mimusops elengi Ethyl acetate fraction.
Induction of apoptosis in cancer cells is one useful strategy for anticancer drug development [12]. In this respect, many
Fig. 9. Cell cycle analysis. Lung adenocarcinoma (A549).
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8 Table 2 COMET parameters. Drugs
Types of cell lines HeLa
A549
% Tail DNA ± ± ± ± ±
Untreated Doxo Mtx MED MEE
4.26 29.48 48.82 79.49 27.54
1.08 4.01 6.41 1.87 3.49
Drugs
Types of cell lines
Olive tail moment 5.37 65.04 141.65 328.92 64.33
± ± ± ± ±
1.53 10.89 15.11 11.00 6.31
3.65 45.15 48.01 8.90 67.12
U343
11.2 3.1 53.1 50.1 44.5
± ± ± ± ±
1.58 9.99 6.02 3.42 2.23
Olive tail moment 2.35 95.92 84.33 15.60 139.13
± ± ± ± ±
1.03 9.98 9.03 7.17 8.21
BT549
% Tail DNA Untreated Doxo Mtx MED MEE
% Tail DNA
± ± ± ± ±
3.8 1.5 6.6 7.4 7.0
Olive tail moment 19.6 3.4 190.2 100.6 126.7
± ± ± ± ±
6.9 1.9 8.4 13.0 12.4
compounds from plant origin have been tested for their apoptotic inducing capacity [12,21–23]. After 48 hours incubation of cancer calls at IC50 value, resulted in the appearance of a large number of cells in the sub-G1 phase (apoptotic cells). Moreover, a reduction of cells in the G0 /G1 phase and cell arrest in the S phase was observed after 48 treatments. Exposure of cancer cells to the selected fraction at IC50 values for 48 h led to typical morphological hallmarks
% Tail DNA 8.71 49.81 50.85 64.84 69.07
± ± ± ± ±
2.31 6.49 2.81 5.07 5.07
Olive tail moment 9.05 84.93 94.72 125.38 140.02
± ± ± ± ±
2.21 6.72 8.25 8.55 9.92
of apoptosis. Those changes included cells shrinkage, membrane blebbing, fragmented cell nuclei forming apoptotic bodies, nuclear fragmentation, cytoplasmatic membrane shrinkage and loss of contact with neighboring cells. Similar observations were made on cells treated with doxorubicin, whereas the exposure to the vehicle did not caused nuclear alterations as confirmed by the absence of staining.
Fig. 10. Micronuclei formation assay. A. Human cervical carcinoma (HeLa). B. Human neuronal Glioblastoma (U343). C. Lung adenocarcinoma (A549), D. Human breast carcinoma (BT549).
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Fig. 11. Effect on normal epithelial breast cells (HBL100). A. AOEB staining. B. Comet assay. C. Micronuclei assay.
4.3. AO/EB dual staining indicate that apoptosis induction Acridine orange permeates all cells and makes the nuclei to appear green, while EtBr is taken up only by cells that have lost cytoplasmic membrane integrity, and stains the nuclei red. Cells treated with positive control (methotrexate and doxorubicin), MED and MEE with IC50 values. Untreated cells showed green stained unfragmented (normal DNA distribution) nuclei, indicating non-apoptotic live cells; while cells treated with positive control and selected fraction (MED and MEE) resulted in a significant increase in the number of green fragmented (condensed DNA) nuclei indicating early apoptotic cells and green-red fragmented nuclei (condensed DNA) shows late apoptotic cells. Treated cells showed highly condensed chromosome with green fragment nuclei (Fig. 4).
with a typical apoptotic nuclear morphology (nuclear shrinkage, DNA condensation and fragmentation), was present in the most active fraction treated cells, but not in the untreated controls (Fig. 5). 4.5. DNA Fragmentation DNA fragmentation is a characteristic feature of apoptosis. Increased DNA fragmentation (DNA ladders) was apparent in HeLa, A549 and U343 cells after treatment with IC50 values of MED, MEE and methotrexate (positive control) for 48 h whereas treatment with DMSO (0.5%) (vehicle control) did not produce DNA fragment ladders (Fig. 6). 4.6. Comet assay for apoptosis
4.4. Hoechst-33342 staining reveals induction of apoptosis To test whether, the decrease in cell viability observed after treatment with most active selected fractions (DCM and ethyl acetate) was due to apoptosis, the cells (HeLa, U343, HBL100) were stained with Hoechst-33342-dye. This dye stains the condensed chromatin of apoptotic cells more brightly than it stain the chromatin of normal cells. Which correlates with the presence of cells
Data suggest that an increase in comet parameters, such as percentage DNA in tail and olive tail moment (OTM) upon treatment with selected fractions as compared to untreated control or solvent control (DMSO). MEE showed highest increase in percentage tail DNA content and OTM in A549, BT549 and U343. Treatment with positive control (methotrexate) MED and MEE show significant increase in comet parameters as compared to untreated cells.
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In HeLa and BT-549 cells, MED showed highest increase in comet parameters (Fig. 7, Table 2). 4.7. Cell cycle analysis shows G1 arrest etc. The effect of the selected fractions on cell cycle progression on HeLa and A549 was determined by flow cytometry. MED and MEE treatment showed a significant increase in the percentage of G1 phase from72% (untreated) of cells at the concentration of IC50 suggesting that MED arrested the HeLa cells at G0 /G1 phase (82%) and MEE treatment showed 18% G2 /M phase which is significant as compare to untreated cells (12%). in A549 cells same as HeLa MED significantly increases the percentage of G0 /G1 phase (89%) comparative to untreated cells (75%) and MEE increases the concentration in sub G0 (7%) and G2 /M (21%) than untreated cells sub G0 (0%) and G2 /M (14%). Doxorubicin was taken as positive control and DMSO (0.5%) as vehicle control. Doxorubicin showed completely G2 arrest (Figs. 8 and 9). 4.8. Active fractions induces genotoxicity in cancer cells Micronuclei formation is a hallmark of property of drug-induced genotoxicity. Results show that MED and MEE were found to form micronuclei in all tested cell lines. This indicates ability of fractions to cause chromosomal damage and genome instability in cancer cells (Figs. 10 and 11). 5. Conclusion Alcoholic extracts of M. elengi Linn. bark was analyzed for their anticancer activity. In which two fractions (DCM and ethyl acetate) was found most active in cytotoxic activity. We would like to conclude that, the present study highlights the anticancer and cytotoxic potential of bark extract of M. elengi Linn. These results suggest that the cytotoxic activity of this plant has been due to its apoptosis inducing properties. This was evidenced by disruption of mitochondrial membrane potential, DNA fragmentation, externalized phosphatidyl serine and accumulation of sub-G0 and G1 population. Acknowledgements My science thanks to Dr. K. Gopala Krishna Bhat, Professor of Botany (Rtd.) for his help in authentication of plant, Dr. Annapoorni rangarajan, MRDG lab, IISc Bangalore for helps in flow cytometry based assay.
References [1] Bold RJ, Temuhlen PM, McConkey DJ. Apoptosis, cancer and cancer therapy. Surg Oncol 1997;6:113–42. [2] Nostro A, Germano MP, Dángelo V, Cannatelli MA. Extraction methods and bioautography for evaluation of medicinal plant antimicrobial activity. Lett Appl Microbiol 2000;30:379–84. [3] Jahan N, Ahmed W, Malik A. New steroidal glycosides from Mimusops elengi. J Nat Prod 1995;8(8):1244–7. [4] Sen K. Novel migrated Oleanane Triterpenoid Sapogenins from Mimusops elengi. Tetrahydron 1993;49. [5] Sahu NP, Koike K, Jia Z, Nikaido T. Triterpenoid saponins from Mimusopselengi. Phytochemistry 1997;44:1145–9. [6] Sahu A. Pentacyclictriterpenoids from mimusopselengi. Phytochemistry 1995:38. [7] Bibi Y, Nisa S, Waheed A, Zia M, Sarwar S, Ahmed S, Chaudhary MF. Evaluation of Viburnum foetens for anticancer and antibacterial potential and phytochemical analysis. Afr J Biotechnol 2010;9(34):5611–5. [8] Freshney I. Culture of animal cells: a manual of basic techniques. J Pharm Res 2011:4. [9] Mossman BT. In vitro approaches for determining mechanisms of toxicity and carcinogenicity by asbestos in the gastrointestinal and respiratory tracts. Environ Health Perspect 1983;53:155–61. [10] Vanicha V, Kirtikara K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc 2006;1(3):1112–6. [11] Sakuma T. A counterexample to the bold conjecture. J Graph Theory 1997;25(2):165–8. [12] Hu W, Kavanagh JJ. Anticancer therapy targeting the apoptotic pathway. Lancet Oncol 2003;4:721–9. [13] Kil-Nam K, Young-Min H, Min-Seok Y. Molecular mechanism of apoptosis induced by Scytosiphon gracilis Kogame in HL-60 cells. Int J Pharmacol 2010;6(3):249–56. [14] Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988;175:184–91. [15] Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, et al. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology tetsing. Environ Molec Mutagen 2000;35:206–21. [16] Nunez R. DNA measurement and cell cycle analysis by flow cytometry. Curr Issues Mol Biol 2001;3(3):67–70. [17] Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 1991;139:271–9. [18] Matsuoka A, Yamazaki N, Suzuki T. Evaluation of micronucleus test using a chinese hamster cell lines as a alternative to the conventional to invitro chromosomal aberration test. Mutat Res 1993;272:223–36. [19] Fenech M, Morley AA. Measurement of micronuclei in lymphocytes. Mutat Res 1985;147:29–36. [20] Van De Loosdrecht AA, Beelen RH, Ossenkoppele GJ, Broekhoven MG, Langenhuijsen MM. A tetrazolium-based colorimetric MTT assay to quantitate human monocyte mediated cytotoxicity against leukemic cells from cell lines and patients with acute myeloid leukemia. J Immunol Methods 1994;174:311–20. [21] Kwon HJ, Hong YK, Kim KH, Han CH, Cho SH, Choi JS, Kim BW. Methanolic extract of Pterocarpussantalinus induces apoptosis in HeLa cells. J Ethnopharmacol 2006;105:229–34. [22] Gonc¸alves S, Xavier C, Costa P, Alberício F, Romano A. Antioxidant, cytotoxic and apoptotic activity of Drosophyllum lusitanicum extracts. J Med Plant Res 2010;4:1601–8. [23] Lee SB, Park HR. Anticancer activity of guava (Psidiumguajava L.) branch extracts against HT-29 human colon cancer cells. J Med Plant Res 2010;4:891–6.
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Original article
In vitro cytotoxic and apoptotic effect of Mimusops elengi Linn. methanolic bark extract Harish Kumar a,∗ , K. Sreedhara Ranganath Pai a , Naseer Maliyakkal a , Savaliya Mihir a , Charanjeet Singh b a b
Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal 576104, India Department of Pharmacology, Arya College of Pharmacy, Jaipur, India
a r t i c l e
i n f o
Article history: Received 12 June 2014 Accepted 10 July 2014 Keywords: Cytotoxic Mimusops elengi Linn. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay Sulphorhodamine B assay Hoechst33343 dye Comet
a b s t r a c t The ayurvedic system of medicine recommends Mimusops elengi Linn. (Sapotaceae) for the treatment of tumors. The study is needed as cancer is the second most common cause for death in the world; most of the traditional drugs have shown resistance, low-cost approaches, and higher toxicity of the existing compound. The present study aims to determine the mode of cell death induced by the methanol extract of ME in human cancer cell lines. Among M. elengi (ME) crude extract and fractions screened for potential anticancer activity, MED and MEE fractions (dichloromethane and ethyl acetate) were found to have promising cytotoxic activities in all cell lines studied by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay and Suforhodamine B (SRB). MEE found to have more potent activity in cervical cancer (HeLa), colon cancer (HCT15), lung (A549), breast cancer (BT549), glioblastoma (U343 cells). Interestingly, ME was less cytotoxic in normal cell line (HBL-100) indicate the specific activity towards cancer cells. AO/EB staining and Hoechst-33342 staining indicate membrane blebbing, condensed and fragmented nuclei upon treatment with MEE and MED in HeLa, A549 and U343 cells. Fragmented DNA ladder and genomic DNA fragmentation were observed with DNA fragmentation assay based on gel electrophoresis and COMET assay by fluorescent microscopy. Similarly, cell cycle analysis by flow cytometer indicates distortion of normal cell cycle and increased subG0 phase. ME was found genotoxic by micronuclei formation. These results indicate that both MED and MEE fractions induces apoptosis but not necrosis cells in the cancer cells. © 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction Apoptosis is cellular suicide or programmed cell death that is mediated by activation of an evolutionary conserved intracellular pathway. Recently the relation of apoptosis and cancer has been emphasized and increasing evidence suggests that the processes of neoplastic transformation, progression and metastasis involve alterations of normal apoptotic pathway [1]. Apoptotic also gives some clues about effective anticancer therapy and many chemotherapeutic agents were reported to event their anti-tumor effect including apoptosis of cancer cells. Medicinal plants have been used as remedies for human diseases for centuries. The reason for using them as medicine lies
∗ Corresponding author. Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal 576104, India. Mobile: +9587775613. E-mail addresses: [email protected], [email protected] (H. Kumar).
in the fact that they contain chemical components of therapeutic value [2]. The medicinal value of plants lies in some chemical substances (usually secondary metabolites) that produce a definite physiological action on the human body. Mimusops elengi Linn. (Family – Sapotaceae) (“Syn.”: Bakul, Maulsari) is commonly known as Spanish cherry or bullet wood is a small to large tree found in all parts of India. It is an evergreen tree whose distribution extends to India, Burma, Pakistan and Thailand. The plant has been used as febrifuges, astringents, headache, cardiotonic, alexipharmic and stomachic, anthelmintic, teeth cleaner and purgatives and stimulants [3]. The various extracts of the plant have been reported (bark, fruit, leaves, seed, flowers) to have antimicrobial, anti-ulcer, anti-hyperglycemic, diuretic, wound healing, anti-inflammatory, analgesic and antipyretic, antigastric ulcers, hypotensive, anti-HIV and spasmolytic activities. Recent reports suggest that bark of the plant contains presence of steroids, alkaloids, taraxerol, taraxerone, urosolic acid, betulinic acid, ␣-spinosterol, -sitosterol glycoside, quercitol, lupeol, -amyrin, farnane type triterpenoid and mixture of
http://dx.doi.org/10.1016/j.bionut.2014.07.004 2210-5239/© 2014 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Kumar H, et al. In vitro cytotoxic and apoptotic effect of Mimusops elengi Linn. methanolic bark extract. Biomed Prev Nutr (2014), http://dx.doi.org/10.1016/j.bionut.2014.07.004
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triterpenoidsaponins in the bark of M. elengi Linn. have been isolated from the bark such as mimusopgenone and mimugenone in the seeds [4], triterpenoid saponins, such as mimusopsides A and B, mimusopin, mimusopsin, mimusin, Mi-saponin A and 16␣-hydroxyMi-saponin A from the seeds [5,6]. Members of the lupane, ursane and oleanane triterpenoids have demonstrated antiproliferative activity on various cancer cell lines. 2. Materials and methods 2.1. Collection of plant material The fresh plant bark was collected from parkala (Udupi), Karnataka in September month and authenticated by Dr GopalakrishnaBhat, Professor of Botany, PoornaPrajna College, Udupi, Karnataka. An authenticated specimen was submitted in herbarium of Manipal college of Pharmaceutical sciences, Manipal.
The cytotoxicity assay was performed by using the Sulforhodamine B (SRB) colorimetric method to assess growth inhibition according to Vanicha and Kirtikara [10]. Briefly, till 48 h treatment same as that of MTT method. At the end of the exposed time, cells in each well were fixed by addition of 100 mL of cold [(4 ◦ C) 10% (w/v)] trichloroacetic acid (TCA) into the growth medium. Each plate was incubated at4 ◦ C for 1 h before gently washed five times with Mili-Q water to remove TCA, the growth medium and dead cells. Plates were allowed to dry in air and to each well were added 50 mL of 0.057% (v/v) SRB dye in 1% acetic acid in deionized water and allowed to stand for 30 min at room temperature. At the end of the staining period, unbound SRB was removed by washing four times with 1% of an acetic acid solution. The plate was air-dried and
2.2. Preparation of extract The fresh plant bark was harvested, rinsed under tap water and oven dried. The coarsely powder material was extracted with methanol by using soxhlet apparatus and solvent was removed by distillation and concentrated using a rotavapor. 2.3. Fractionation of crude extract Crude methanolic extract of M. elengi Linn. was suspended in water and then fractionated with organic solvents in order of increasing polarity to get petroleum ether, dichloromethane, n-butanol, ethyl acetate and methanol-water (hydro-alcoholic mixture) [7]. 2.4. Preparation of different concentrations Different concentrations of crude extract and fractions were prepared by dissolving the extract in DMSO and then diluting it with DMEM medium under sterile conditions.
Fig. 1. Cytotoxicity of most active fractions in selected human cancer cells (MTT Method). 3A: human cervical carcinoma (HeLa), 3B: human glioblastoma cells (LN229), 3C: human glioblastoma cells (U343), 3D: human adenocarcinoma alveolar basal epithelial cells (A549), 3E: rat glioma cells (C6), 3F: human breast adenocarcinoma epithelial cells (MCF7).
2.5. Cell culture conditions A cell line was procured from NCCS, Pune. Cancer cells were maintained in Dulbecco’s modified eagle medium (DMEM) with 1000 mg/mL of glucose, supplemented with 10% FBSFBS (fetal bovine serum) and penicillin/streptomycin-L-glutamine and cultured in a humified atmosphere of 5% CO2 and 95% air at 37 ◦ C in incubator [8]. 2.5.1. Screening of cytotoxicity/anticancer potential Cancer cell line was used for the determination of cytotoxic activity. Cells were seeded in 96-well plates at the density of 6000 cells/well (HeLa cells) in 100 L medium. Then various concentrations of the crude extract were added to the cells in 100 L medium. Cells were incubated for 24 h with test extract concentrations. Each concentration was tested in triplicate. The MTT assay is a laboratory test that measures changes in color for measuring the activity of enzyme that reduces MTT to formazan, giving a purple color. Yellow MTT (3-(4, 5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide, a tetrazole) reduce to purple formazan in living cells [9]. After 48 h incubation, 20 L (5 mg/mL) MTT reagent was added to each well and incubated for additional 4 h. Then 200 L of DMSO solution was added to each well to solubilize the formazan crystals. The plates were read for optical density at 540 nm with reference 630 nm, using a plate reader. By using optical density, percentage inhibition of was calculated.
Fig. 2. Cytotoxicity of most active fractions in selected human cancer cells (SRB Method). A: human cervical carcinoma (HeLa), B: human glioblastoma cells (U343), C: human adenocarcinoma alveolar basal epithelial cells (A549), D: human breast ductal carcinoma cells (BT-549), E: human colorectal adenocarcinoma (HCT-15), F: human breast adenocarcinoma epithelial cells (MCF7).
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150 mL of 10 mM aqueous Tris base buffer of pH 10.5 was added to each well to dissolve the cell-bound dye. The plate was then shaken for 15 to 30 min on a gyratory shaker and the optical density (OD) was read at 540 nm with reference 630 nm in a microplate reader; control wells were used as blanks. Inhibition concentration at 50% of cell population or IC50 was calculated prism graph pad [11]. 2.5.2. Apoptosis studies 2.5.2.1. AOEB dual staining. AO/EB staining identifies live, early apoptotic, late apoptotic, and necrotic cells. 5 × 105 cells/well was seeded in a 6-well plate the night before the treatment. Cells were treated with selected fraction, vehicle control and positive control at their IC50 for 48 hrs at 37 ◦ C in CO2 incubator. After treatment, media containing floating cells and attached cells in 6 well plate were collected into centrifuge tubes and centrifuged. Supernatant discarded and cell pellets were resuspended in 1 mL of HBSS. Ten microlitre of EtBr solutions were added and kept in incubator for 10 min and cells were centrifuged at 1200 rpm for 4 min at 4 ◦ C. Supernatant was drained and pellets were dislodged. Twenty microlitre of the cell suspensions was placed on a slide and observed under the fluorescent microscope equipped with 450–490 nm excitation and 520 nm emission wavelength (blue filter) [12]. Fig. 3. Cytotoxicity of selected fractions in human normal breast epithelial cells.
2.5.2.2. Nuclear staining with Hoechst-33342. The nuclear morphology of cells was studied by using cell permeable DNA dye
Fig. 4. Acridine orange assay for the discrimination of apoptosis vs necrotic cells after treatment with selected fractions. A. Human cervical carcinoma (HeLa). B. Human neuronal glioblastoma (U343). C. Lung adenocarcinoma (A549). D. Human breast carcinoma (BT549).
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Fig. 5. Hoechst-33342 DNA staining assay. A. Human cervical carcinoma (HeLa). B. Human neuronal glioblastoma (U343). C. Normal epithelial breast cells (HBL100).
Hoechst33342. Cells with homogeneously stained nuclei were considered to be viable, whereas the presence of chromatin condensation and/or fragmentation was indicative of apoptosis. The cells were treated with IC50 of the selected fractions and further incubated for 48 h. Cells were centrifuged and supernatant discarded. Cell pellets were resuspended in 1 mL of HBSS. Five microlitre Hoechst-33342-dye solutions were added and kept in incubator for 10 min and centrifuged at 1200 rpm for 4 min at 4 ◦ C. Supernatant was drained and pellets were resuspended. Twenty microlitre of the cell suspensions was placed on a slide and observed under the fluorescent microscope equipped with excitation source of 350 nm and emission at 450 nm (UV filter) [13]. 2.5.2.3. Determination of DNA fragmentation. The characteristic ladder pattern of DNA breakage was analyzed by gel electrophoresis. Cancer cells were placed in a 6-well plate at a concentration of 5 × 105 cell/mL. The cells were treated with IC50 concentration of selected fraction with positive control and were further incubated for 48 h. The DNA was isolated and electrophoretically analyzed on 1.5% agerose gel containing 10 L/mL ethidium bromide [14]. 2.5.2.4. Alkaline comet assay. Twenty-four hours after culture initiation, the cells were treated for 48 h with IC50 values of drugs. The cells were washed with HBSS and harvested. The slides were covered with 1.5% normal melting point agarose, allowed to solidify at 25 ◦ C and stored at 4 ◦ C for 1 h.
An aliquot of the cell suspension was diluted of 0.75% low melting point agarose. This mixture was rapidly pipetted on to the agarose layer on the slide, gently spread by placing a coverslip on top and allowed to solidify at 4 ◦ C for 5 min. After removal of the coverslip, the slides were immersed in freshly prepared lysis solution (2.5 M NaCl, 100 mM EDTA, 10mMTris, pH 10, with 1% Triton X-100 and 10% DMSO) for overnight at4 ◦ C. Then, the slides were left in the electrophoresis solution (1 mMEDTA and 300mMNaOH, pH 13 at 4 ◦ C) for 1 h to allow for DNA unwinding and expression of alkali-labile damage before electrophoresis. Electrophoresis was done at 22 V and 300 mA for 20 min at 4 ◦ C. Then, the slides were washed in neutralizing buffer (0.4 M Tris/HCl, pH 7.5) and stained with 80 L of an aqueous solution containing20 mg/mL ethidium bromide. Nucleoids were evaluated visually [15]. 2.5.2.5. Cell cycle analysis. In order to study the relationship between cell proliferation inhibition and the induction of apoptosis, we decided to study the subdiploid DNA contends as indicative of DNA fragmentation by apoptosis. DNA content and cell cycle distribution were assessed using PI staining. After sample treatment, both adherent and floating cells were harvested, washed with phosphate buffered saline (PBS) and fixed with ice-cold absolute ethanol at –20 ◦ C overnight. Fixed cells were washed and resuspended in a buffer containing 50 g/mL of RNAse A for 3 hrs, added 25 g/mL of Propidium iodide and analysed by flow cytometry. Propidium
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Fig. 6. DNA fragmentation assay. A. HeLa (Human cervical carcinoma). B. A549 (human lung adenocarcinoma). C. U343 (human glioblastoma).
Fig. 7. Single cell gel electrophoresis (COMET) assay. A. Human cervical carcinoma (HeLa). B. Human neuronal glioblastoma (U343). C. Lung adenocarcinoma (A549). D. Human breast carcinoma (BT549).
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Fig. 8. Cell cycle analysis. Human cervical carcinoma (HeLa).
iodide was excited by a 488 nm laser and emission was captured at FL-2 (570/20 BP filter). DNA content in different phases of the cell cycle were analysed for both the controls and the treated cells. Data was analysed in Summit v4.3 software [16,17].
as mean ± SEM. and were analyzed by one-way analysis of variance (ANOVA) followed by Tukey test and P < 0.05 was considered statistically significant. 4. Results and Discussion
2.5.3. Genotoxic assessment 2.5.3.1. Micronucleus assay. The micronucleus assay was performed according to Matsuoka et al. [18]. About 1 × 106 cells/mL medium were exposed and incubated. Complete media were treated as positive and negative controls respectively. At the end of incubation the cells were harvested by low centrifugation, treated with a hypotonic solution of KCl (0.075 M) and fixed in methanol: acetic acid (3:1) for 3–4 h. Two to three drops of the fixed cell suspension were dispensed on to the surface of cold micro slides, air-dried and stained with 3% Giemsa solution in Sorenson phosphate buffer (pH 6.8) for 5–7 min. The bi, tri or multinuclei cell was observed in slides [19]. 3. Statistical analysis All the experiments were independently preformed thrice with three replicates for each treatment. The results were expressed
Recently, the use of some herbs has attracted a great deal of attention as one of the alternative cancer therapies from the point of less toxicity and cost benefits. Therefore, an attempt has been made to evaluate the anticancer activity of the bark extract of M. elengi L., which is commonly used in ayurvedic system of medicine for various purposes. 4.1. Cytotoxic effect of the extracts and fractions on human cancer cells In preliminary screening HeLa cells were the most susceptible to the treatment, while U343 were the least. Assay is based upon reduction of yellow tetrazolium salt (MTT) by the reductase enzyme in metabolically active cells to a darkblue formazan [20]. These activities were supported by Sulphorhodamine B assay. The inhibitory effect on cell viability was evident after 48 h of incubation and most cytotoxic fraction (DCM and ethyl acetate) was selected
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for further evaluated at various cells by determining the IC50 value. In ethyl acetate fraction potent cytotoxic IC50 values were found against human cervical carcinoma184.2 g/mL and 11.68 g/mL by MTT and SRB method respectively. But significant cytotoxic activity was found only by SRB method (144.1 g/mL IC50 value) in DCM fraction. IC50 values in Human adenocarcinoma alveolar basal epithelial cells 43.89 g/mL, Human breast ductal carcinoma cells (BT-549) 49.33 g/mL, Human colorectal adenocarcinoma (HCT15) 43.04 g/mL by SRB method were found potent cytotoxic activity on ethyl acetate fraction. MTT showed significant cytotoxic activity in Rat glioma cells (C6) with IC50 value82.39 g/mL. Results showed that ethyl acetate fraction significantly reduced viability of cancer cells in dose-dependent manner (Figs. 1–3, Table 1). 4.2. Induction of apoptosis in cancer cells by the selected active fraction
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Table 1 IC50 value of selected extracts/fractions in different cells by MTT and SRB method. Type of cells
IC50 (g/mL) MTT method
HeLa U343 A549 MCF 7 BT549 HCT15 LN 229 C6
SRB method
MED
MEE
MED
MEE
773.6 917.7 256.4 461.6 – – 234.8 367.8
184.2 3230 334.1 313.7 – – 384.7 82.39
144.1 366.0 237.3 200.7 168.1 358.2 – –
11.68 268.8 43.89 251.9 49.33 43.04 – –
MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SRB: Suforhodamine B; MED: Mimusops elengi Dichloromethane fraction; MEE: Mimusops elengi Ethyl acetate fraction.
Induction of apoptosis in cancer cells is one useful strategy for anticancer drug development [12]. In this respect, many
Fig. 9. Cell cycle analysis. Lung adenocarcinoma (A549).
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8 Table 2 COMET parameters. Drugs
Types of cell lines HeLa
A549
% Tail DNA ± ± ± ± ±
Untreated Doxo Mtx MED MEE
4.26 29.48 48.82 79.49 27.54
1.08 4.01 6.41 1.87 3.49
Drugs
Types of cell lines
Olive tail moment 5.37 65.04 141.65 328.92 64.33
± ± ± ± ±
1.53 10.89 15.11 11.00 6.31
3.65 45.15 48.01 8.90 67.12
U343
11.2 3.1 53.1 50.1 44.5
± ± ± ± ±
1.58 9.99 6.02 3.42 2.23
Olive tail moment 2.35 95.92 84.33 15.60 139.13
± ± ± ± ±
1.03 9.98 9.03 7.17 8.21
BT549
% Tail DNA Untreated Doxo Mtx MED MEE
% Tail DNA
± ± ± ± ±
3.8 1.5 6.6 7.4 7.0
Olive tail moment 19.6 3.4 190.2 100.6 126.7
± ± ± ± ±
6.9 1.9 8.4 13.0 12.4
compounds from plant origin have been tested for their apoptotic inducing capacity [12,21–23]. After 48 hours incubation of cancer calls at IC50 value, resulted in the appearance of a large number of cells in the sub-G1 phase (apoptotic cells). Moreover, a reduction of cells in the G0 /G1 phase and cell arrest in the S phase was observed after 48 treatments. Exposure of cancer cells to the selected fraction at IC50 values for 48 h led to typical morphological hallmarks
% Tail DNA 8.71 49.81 50.85 64.84 69.07
± ± ± ± ±
2.31 6.49 2.81 5.07 5.07
Olive tail moment 9.05 84.93 94.72 125.38 140.02
± ± ± ± ±
2.21 6.72 8.25 8.55 9.92
of apoptosis. Those changes included cells shrinkage, membrane blebbing, fragmented cell nuclei forming apoptotic bodies, nuclear fragmentation, cytoplasmatic membrane shrinkage and loss of contact with neighboring cells. Similar observations were made on cells treated with doxorubicin, whereas the exposure to the vehicle did not caused nuclear alterations as confirmed by the absence of staining.
Fig. 10. Micronuclei formation assay. A. Human cervical carcinoma (HeLa). B. Human neuronal Glioblastoma (U343). C. Lung adenocarcinoma (A549), D. Human breast carcinoma (BT549).
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Fig. 11. Effect on normal epithelial breast cells (HBL100). A. AOEB staining. B. Comet assay. C. Micronuclei assay.
4.3. AO/EB dual staining indicate that apoptosis induction Acridine orange permeates all cells and makes the nuclei to appear green, while EtBr is taken up only by cells that have lost cytoplasmic membrane integrity, and stains the nuclei red. Cells treated with positive control (methotrexate and doxorubicin), MED and MEE with IC50 values. Untreated cells showed green stained unfragmented (normal DNA distribution) nuclei, indicating non-apoptotic live cells; while cells treated with positive control and selected fraction (MED and MEE) resulted in a significant increase in the number of green fragmented (condensed DNA) nuclei indicating early apoptotic cells and green-red fragmented nuclei (condensed DNA) shows late apoptotic cells. Treated cells showed highly condensed chromosome with green fragment nuclei (Fig. 4).
with a typical apoptotic nuclear morphology (nuclear shrinkage, DNA condensation and fragmentation), was present in the most active fraction treated cells, but not in the untreated controls (Fig. 5). 4.5. DNA Fragmentation DNA fragmentation is a characteristic feature of apoptosis. Increased DNA fragmentation (DNA ladders) was apparent in HeLa, A549 and U343 cells after treatment with IC50 values of MED, MEE and methotrexate (positive control) for 48 h whereas treatment with DMSO (0.5%) (vehicle control) did not produce DNA fragment ladders (Fig. 6). 4.6. Comet assay for apoptosis
4.4. Hoechst-33342 staining reveals induction of apoptosis To test whether, the decrease in cell viability observed after treatment with most active selected fractions (DCM and ethyl acetate) was due to apoptosis, the cells (HeLa, U343, HBL100) were stained with Hoechst-33342-dye. This dye stains the condensed chromatin of apoptotic cells more brightly than it stain the chromatin of normal cells. Which correlates with the presence of cells
Data suggest that an increase in comet parameters, such as percentage DNA in tail and olive tail moment (OTM) upon treatment with selected fractions as compared to untreated control or solvent control (DMSO). MEE showed highest increase in percentage tail DNA content and OTM in A549, BT549 and U343. Treatment with positive control (methotrexate) MED and MEE show significant increase in comet parameters as compared to untreated cells.
Please cite this article in press as: Kumar H, et al. In vitro cytotoxic and apoptotic effect of Mimusops elengi Linn. methanolic bark extract. Biomed Prev Nutr (2014), http://dx.doi.org/10.1016/j.bionut.2014.07.004
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In HeLa and BT-549 cells, MED showed highest increase in comet parameters (Fig. 7, Table 2). 4.7. Cell cycle analysis shows G1 arrest etc. The effect of the selected fractions on cell cycle progression on HeLa and A549 was determined by flow cytometry. MED and MEE treatment showed a significant increase in the percentage of G1 phase from72% (untreated) of cells at the concentration of IC50 suggesting that MED arrested the HeLa cells at G0 /G1 phase (82%) and MEE treatment showed 18% G2 /M phase which is significant as compare to untreated cells (12%). in A549 cells same as HeLa MED significantly increases the percentage of G0 /G1 phase (89%) comparative to untreated cells (75%) and MEE increases the concentration in sub G0 (7%) and G2 /M (21%) than untreated cells sub G0 (0%) and G2 /M (14%). Doxorubicin was taken as positive control and DMSO (0.5%) as vehicle control. Doxorubicin showed completely G2 arrest (Figs. 8 and 9). 4.8. Active fractions induces genotoxicity in cancer cells Micronuclei formation is a hallmark of property of drug-induced genotoxicity. Results show that MED and MEE were found to form micronuclei in all tested cell lines. This indicates ability of fractions to cause chromosomal damage and genome instability in cancer cells (Figs. 10 and 11). 5. Conclusion Alcoholic extracts of M. elengi Linn. bark was analyzed for their anticancer activity. In which two fractions (DCM and ethyl acetate) was found most active in cytotoxic activity. We would like to conclude that, the present study highlights the anticancer and cytotoxic potential of bark extract of M. elengi Linn. These results suggest that the cytotoxic activity of this plant has been due to its apoptosis inducing properties. This was evidenced by disruption of mitochondrial membrane potential, DNA fragmentation, externalized phosphatidyl serine and accumulation of sub-G0 and G1 population. Acknowledgements My science thanks to Dr. K. Gopala Krishna Bhat, Professor of Botany (Rtd.) for his help in authentication of plant, Dr. Annapoorni rangarajan, MRDG lab, IISc Bangalore for helps in flow cytometry based assay.
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Please cite this article in press as: Kumar H, et al. In vitro cytotoxic and apoptotic effect of Mimusops elengi Linn. methanolic bark extract. Biomed Prev Nutr (2014), http://dx.doi.org/10.1016/j.bionut.2014.07.004