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Antiproliferative activity of ionic liquid-graviola fruit extract against human breast cancer (MCF-7) cell lines using flow cytometry techniques
Journal of Ethnopharmacology 236 (2019) 466–473
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Antiproliferative activity of ionic liquid-graviola fruit extract against human breast cancer (MCF-7) cell lines using flow cytometry techniques
T
Djabir Daddiouaissaa, Azura Amidb,∗, Nassereldeen A. Kabbashia, Fazia A.A. Fuada, AhmedA.M. Elnoura, Mohamad A.K.M. S. Epandyc a
Biotechnology Engineering, Kulliyyah of Engineering, International Islamic University, Malaysia (IIUM), P. O. Box 10, Gombak, 50728, Kuala Lumpur, Malaysia International Institute for Halal Research and Training (INHART), Level 3, KICT Building, International Islamic University Malaysia (IIUM), Jalan Gombak, 53100, Kuala Lumpur, Malaysia c Adikafirdaus Resources, Lot 24, Jalan Klebang Selatan, 2/5 Kampung Tersusun, Batu 6 Klebang Selatan, 31200, Ipoh, Perak, Malaysia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Breast cancer Graviola Ionic liquid Flow cytometry Apoptosis
Ethnopharmacological relevance: Medicinal plants have been used for ages by indigenous communities around the world to help humankind sustain its health. Graviola (Annona muricata), also called soursop, is a member of the Annonaceae family and is an evergreen plant that is generally distributed in tropical and subtropical areas of the world. Graviola tree has a long history of traditional use due to its therapeutic potential including anti-inflammatory, antimicrobial, antioxidant, insecticide and cytotoxic to tumor cells. Aim of the study: This study aimed to investigate the in vitro antiproliferative effects and apoptotic events of the ionic liquid extract of Graviola fruit (IL-GFE) on MCF-7 breast cancer cells and their cytokinetics behaviour to observe their potential as a therapeutic alternative in cancer treatment. Materials and methods: The cell viability assay of the extract was measured using tetrazolium bromide (MTT assay) to observe the effects of Graviola fruit extract. Then the cytokinetics behaviour of MCF-7 cells treated with IL-GFE is observed by plotting the growth curve of the cells. Additionally, the cell cycle distribution and apoptosis mechanism of IL-GFE action on MCF-7 cancer cells were observed by flow cytometry. Results: IL-GFE exhibited anti-proliferative activity on MCF-7 with the IC50 value of 4.75 μg/mL, compared to Taxol with an IC50 value of 0.99 μg/mL. IL- GFE also reduced the number of cell generations from 3.71 to 1.67 generations compared to 2.18 generations when treated with Taxol. Furthermore, the anti-proliferative activities were verified when the growth rate was decreased dynamically from 0.0077 h to 1 to 0.0035 h-1. Observation of the IL-GFE-treated MCF-7 under microscope demonstrated detachment of cells and loss of density. The growth inhibition of the cells by extracts was associated with cell cycle arrest at the G0/G1 phase, and phosphatidylserine externalisation confirms the anti-proliferation through apoptosis. Conclusions: ionic liquid Graviola fruit extract affect the cytokinetics behaviour of MCF-7 cells by reducing cell viability, induce apoptosis and cell cycle arrest at the G0/G1 phase.
1. Introduction Fruit consumption is highly recommended to maintain human health (Halliwell, 2012). Graviola (Annona muricata L.) is also called soursop because of the sour and sweet flavour of its great fruit (Patel and Patel, 2016). Graviola is native to the tropical zones, South America and Africa but is currently widely cultivated in the tropical and subtropical zones around the world. Recently, Graviola fruit is widely used to make candies, syrups and beverages (Ioannis et al., 2015). Ethnobotanical studies reported that all parts of Graviola tree are used in an alternative medicine, especially the fruit which is taken to treat fever ∗
(Taylor, 2002), eliminate worms and parasites (Yajid et al., 2018), increase mother's milk (Badrie and Schauss, 2010), as well as hypertension (Samuel et al., 2010), liver and renal infections (Coe, 2008). The popular use of Graviola as an anticancer treatment reported ethnobotanically may be related to the reports of its selective cytotoxicity (George et al., 2012; Monigatti et al., 2013). This activity is considered selective as some reported in vitro studies have shown that the Graviola extract was more toxic to cancer cells than normal cells (BetancurGalvis et al., 1999; Dai et al., 2011; Gavamukulya et al., 2014). The major benefit of this fruit is the active compounds known as annonaceous acetogenins which are thought to be potent anticancer
Corresponding author. E-mail address: [email protected] (A. Amid).
https://doi.org/10.1016/j.jep.2019.03.003 Received 14 December 2018; Received in revised form 25 February 2019; Accepted 2 March 2019 Available online 07 March 2019 0378-8741/ © 2019 Elsevier B.V. All rights reserved.
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identified from the Adikafirdaus Resources Farm in Kampong Bawong, Perak, Malaysia. After that, the fruits were washed with distilled water to remove all traces of dust and insects. The fresh pulp of each fruit separated from the pericarp and seeds, then the pulp was frozen at −20 °C and lyophilised using a Christ Alpha freeze drier for 72 h according to Hoeing (2001), with slight modification. After that, the powder was weighed and placed in an airtight bottle and stored until further extraction procedure (Gyamfi et al., 2011). A plant sample deposited in the herbarium at the Kulliyyah of Architecture and Environmental Design (KAED), International Islamic University Malaysia with voucher number KAED/HBL/S1A047/2018/ 707. The name of the plant was checked on www.theplantlist.org.
toward several cancer cell lines including breast cancer as highlighted recently by Magadi et al. (2015) and Daddiouaissa & Amid (2018). At present, the attention is being focused on anticancer properties of Graviola acetogenins which are the primary bioactive compounds found in the fruit (Sun et al., 2017). Breast adenocarcinoma cell lines (MCF-7) is one of the most frequent incident cancers among women, with an estimated 266,120 new cases, and 40,920 breast cancer (BC) deaths will occur in the United States' women in 2018 (Siegel et al., 2017). Therefore, BC is a challenge among research communities around the world; this is because of the BC incidence rate keep increasing by 0.4% annually worldwide according to the final report conducted by Jemal et al. (2017). Current protocols of treatment include radiation therapy, surgical intervention, and chemotherapy which induce numerous side effects including nausea, fatigue, vomiting, weak of the immune system and hair loss (Griffin et al., 1996). Thus, the search for alternative treatment is necessary. Apoptosis also called programmed cell death, is a mechanism that allows self-destruction of the cells when stimulated by an appropriate trigger. Distinct morphological changes such as cell shrinkage, nuclear condensation, and pyknosis, complemented with biochemical phenomena (BP) such as the cleavage of DNA between the nucleosomes (Elmore, 2007; Peter, 2011). New insights into the process of apoptosis lead to different parameters which can be used to detect and measure apoptosis. One of these parameters is the appearance of phosphatidylserine (PS) on the surface of the cell membrane and loss of the asymmetry which is a specific signal for macrophages to recognise and remove the apoptotic cells (Fadok et al., 1992; Verhoven et al., 1995). Apoptosis is mainly created by either death receptors (extrinsic pathway) involving caspases 8 and 10 or the mitochondrial pathway (intrinsic pathway) via caspase 9 (Elmore, 2007). The alteration of the mitochondrial membrane potential translocates the pro-apoptotic protein Bax which leads to the release of cytochrome C, into the cytoplasm, and subsequent activation of caspases and lead to apoptosis (Wyllie, 1997). The constant breast cell number is maintained and regulated via an equilibrium between cell proliferation and apoptosis. After that, any changes in this balance lead to the loss of homeostasis which can cause the growth of cancer cells (Hao et al., 1998). Thus, the arrestation of cell cycle machinery and induction of apoptosis are the key mechanism to prevent and suppress the breast cancer cells (BCs). Hence, there is a massive interest in the development of new apoptosis-inducing agents with efficacy and more specificity with minimal side effects. Recently, our group has successfully extracted the Graviola fruit using ionic liquid method resulted in high productivity. Thus, this study aimed to investigate the possible inhibitions induced in breast cancer cell lines the MCF-7using the ionic liquid extract of Graviola fruit.
2.3. Fruit extraction One gram of the dried sample was mixed with 30 mL of 0.5 mol/L of 1-butyl-3-methylimidazolium chloride [C4MIM] [Cle] solution. Then the suspension was heated with microwave oven under irradiation power of 700 W for 3 min. After each irradiation, the obtained extracts (IL-GFE) were cooled down to 25 °C and then filtered through Whatman 3 mm filter paper and transferred into a test tube. The tubes were frozen at −20 °C then lyophilised using a Christ Alpha freeze drier for 72 h. Then, the powder was stored in 4 °C freezer until use (Bhan et al., 2017; Zhang et al., 2014).
2.4. Cell culture This study used breast cancer cell lines the MCF-7 as the experimental cancer cells (ATCC No: HTB-22™), and VERO cell line (ATCC® No: CCL-81™) as a control cell line. These cells were obtained from American type Collection Culture. Frozen cells were thawed and inoculated into 5 mL of Dulbecco's Modification of Eagle's Medium (DMEM) supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS). The cells were cultured in T-25 flasks and incubated in 95% humidified incubator with 5% CO2 at 37 °C. The cell cultures were used when reached approximately 70% confluence for experimental treatments.
2.5. Cell viability assay (cytotoxicity assay) The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to quantify the percentage of cell viability. The cultured cells were detached with accutase, suspended in media and counted using a hemocytometer. Approximately 50,000 cells were plated in 100 μL of medium per well in 96-well plate then incubated for 24 h in 5% CO2 incubator. The cells were then treated (triplicate wells per condition) by adding 20 μL of serial dilutions of the sample extract in DMEM free-fetal bovine serum (the control samples received only DMSO). Then incubate the cells for another 24 h before adding 20 μL (5 mg/mL) solution of MTT reagent into each well. Then the incubation was continued for another 3 h before the media was pipetted out carefully. 100 μL of solubilization solution (DMSO) was added to each well and mixed by gentle shaking for 15 min (Liu et al., 2016). Finally, the amount of formazan was determined by measuring the absorbance at 570 nm using a microplate reader. The obtained data were used to calculate the percentage of viable/dead cells, and fifty percent of inhibitory concentration (IC50) of each extract was determined, using equation (1):
2. Materials and methods 2.1. Chemicals and reagents All the chemicals and reagents used in this project are laboratory grade. 1-Butyl-3-Methylimidazolium Chloride 96% [C4MIM] [Cle] reagent was purchased from (Alfa, USA), cell culture media, including Dulbecco's modified Eagle's medium (DMEM), penicillin/streptomycin and fetal bovine serum (FBS) were obtained from GIBCO® (Invitrogen, USA). The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium Bromide (MTT) was purchased from Sigma-Aldrich, (USA). The accutase cell detachment solution purchased from (ICT, USA). RNase A/PI cell cycle assay kit and Annexin V/PI apoptosis assay kit was purchased from Beckman Coulter, (USA). These chemicals were used to investigate the anticancer effect of IL-GFE on cancer cell lines.
% cell viability =
Abs of sample x 100 Abs of control
(1)
2.2. Sample collection and preparation where Abs of the sample is the absorbance of treated cells, and Abs of control is the absorbance of untreated cells.
Graviola fruit (DB3) samples were randomly collected and 467
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Coulter, USA). The cells in S and G2M phases are defined as proliferative cells and as apoptotic cells in the sub-G1 phase (Agu et al., 2018).
2.6. Estimation of the inhibition concentration at 50% (IC50) The IC50 values were graphically obtained by plotting the percentage of cell viability against the corresponding of the IL-GFE sample used. Data were reported as the mean ± standard error (SE) and statically analyzed. The significance was accepted when P ≤ 0.05 level.
2.10. Annexin-V-FITC assay The Annexin -V-FITC methods were conducted using the early, and late apoptotic cells after treatment with IL-GFE was determined using Annexin-V/PI staining assay (Verhoven et al., 1995). The MCF-7 have treated with GFE at IC50 value for 24, 48, and 72 h. After that, media removed, washed with PBS and centrifuged at 1500 rpm for 5 min. Then, cells were resuspended in the binding buffer and stained with Annexin-V-FITC and PI according to the procedure from the manufacturer's manual. Finally, the fluorescence intensity of the stained MCF7 cells was determined using CytoFLEX S flow cytometry (Moghadamtousi et al., 2014).
2.7. Preparation of ionic liquid-graviola fruit extract (IL-GFE) and taxol About 1 mg of IL-GFE was dissolved in 1 mL of PBS (phosphate buffered saline). Taxol was used as a positive control (+ve), and 50 μg of Taxol was dissolved in DMSO. 2.8. Growth kinetics study The growth kinetics of treated and non-treated MCF-7 breast cancer cells with IL-GFE and Taxol as a positive control (+ve) was studied and compared to observe the antiproliferative effects of IL-GFE on the growth of MCF-7 cell lines. Firstly, MCF-7 cells were harvested and seeded into T-25 flasks at 2 × 105 cells/mL. There were three replicates of flasks in this study: untreated MCF-7; IL-GFE-treated MCF-7 at the IC50 value of 4.75 μg/mL and taxol-treated MCF-7 at the IC50 value of 0.99 μg/mL (Chik et al., 2010). Each group has 20 flasks to count samples every 8 h interval from 0 to 144 h. Each sample was performed in three independent experiments. After incubation for 24 h, media was removed, and fresh media containing samples as described before with the IC50 value concentrations were added into separate flasks. Then, cells were incubated from 0 to 144 h. Every 8 h, cell images were observed and documented using phase contrast microscope at 100 magnification then harvested from monolayer and proceed to cell quantification using trypan blue dye exclusion assay (Fouz, Amid, and Hashim, 2014). The number of viable cells was calculated as the equation below:
C = nx 2x
2.11. Statistical analysis The cell viability experiments were carried out in triplicates, and each data point represents the overall mean of at least 3 independent experiments. The findings were expressed as mean ± standard deviation (SD). The results were analyzed using GraphPad Prism (Version 7.00) and Microsoft Office, Excel software to carry out the statistical analysis. One-way analysis of variance technique followed by Tukey's test was applied to observe the significance between the groups. The entire statistical analysis was carried out at (p ≤ 0.05). 3. Results 3.1. The IC50 of IL-GFE against MCF-7 and VERO cell lines To investigate the half maximal inhibitory concentration (IC50) of IL-GFE against MCF-7 cell lines, the data of MTT assay were interpreted by plotting graph of the percentage of cell viability (%) versus different concentrations (μg/mL). After that, the IC50 values for IL-GFE (Fig. 1) and Taxol (Fig. 2), were observed as 4.759 μg/mL and 0.99 μg/mL, respectively. This result shows that the proliferation of MCF-7 cells has dramatically and significantly inhibited after the addition of IL-GFE in a dose-dependent manner. Moreover, MTT assays of IL-GFE against the normal VERO cell lines (Fig. 3) did not show any IC50 value compared to the tumor cell lines. This signifies that the IL-GFE is more cytotoxic against cancer cells than the normal cell lines and will not affect the growth of healthy cells, even with a high concentration of 100 μg/mL. This result confirms those obtained in the previous study (Dai et al., 2011). Thus, the Graviola fruit serves as is a promising alternative or complementary supplement towards reducing breast cancer growth generally and MCF-7
(2)
10 4
4
where n is the average cell number, 2 is the dilution factor, and 10 is the conversion of volume 0.1 mm3 to mL. The data were used to plot a growth kinetics graph and calculate the cell generation number using the following equations:
X=
Log10 N − Log10 N0 Log10 2
Log10 N = Log10 N0 + μt td =
Log 102 μ
=
0.301 μ
(3) (4)
(5)
where X is the number of generations; N is the final cells number; N0 is the initial cells number; μ is the specific growth rate (slope); t is the duration of treatment; td is the doubling time. 2.9. Cell cycle assay Firstly, the flow cytometry analysis was applied to detect changes in cell cycle distribution induced by IL-GFE treatment at an IC50 value in a time-dependent manner. In brief, MCF-7 cells (5 × 104 cells/mm) were treated with IL-GFE at 4.75 μg/mL then incubated for 24, 48 and 72 h, respectively. The cells were harvested and washed with 1x phosphate buffered saline (PBS), then collected and centrifuged at 1500 rpm for 5 min. After that, cells were fixed in 70% ethanol at 4 °C for 1 h then centrifuged at 4000 rpm for 10 min, at ambient temperature, removing the supernatant and wash twice with PBS. Later, cells were stained with 500 μL of RNase A/PI solution containing 0.05 mg/mL of propidium iodide (PI) and 0.05 mg/mL of RNase A in PBS (Magadi et al., 2015). The percentages of cell cycle distribution of the treated and untreated cells were determined using CytoFLEX S flow cytometry (Beckman
Fig. 1. The percentage of cell viability vs. concentrations of IL-GFE treated on MCF-7 cells. 468
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Fig. 2. The percentage of cell viability vs. concentrations of Taxol treated on MCF-7 cells. Fig. 4. MCF-7 Growth profiles of Untreated (control) and Treated with IL-GFE (IC50 = 4.75 μg/mL) and Taxol (IC50 = 0.99 μg/mL).
Fig. 3. Graph of the percentage cell viability vs. concentrations of IL-GFE against VERO cell lines.
specifically. 3.2. Effect of IL-GFE on MCF-7 cell growth kinetics The growth kinetics of untreated (control) and treated MCF-7 with IL-GFE (4.75 μg/mL) and Taxol (0.99 μg/mL) was studied and compared. Fig. 4 shows the growth behaviour and the changes of MCF7 cells after treatment. The number of cells was initially inoculated at 2 × 105 cells/mL. The lag phase was similar for the untreated and the treated MCF-7 cells at first 16 h. Then the cells started proliferation and showed clear log phase after 24 h. However, the treated cells did not show a clear log phase and took 88 h to reach the late log phase. The taxol showed clear log phase but produced less total cell number. It can be observed that the IL-GFE and Taxol treated MCF-7 shows a great reduction in the number of cell proliferation compared to the control. The highest number of cells obtained at the stationary phase for the ILGFE and Taxol-treated cells were only 6.4 × 105 and 9.1 × 105 cells/ mL, respectively, as compared to the control 26.3 × 105. Based on the fourth equation that was obtained from the growth curve (Fig. 4), cell growth at exponential phase and death phase (Fig. 5) and the cell generation number (Table 1) were obtained for all treatments. The number of cell generation was reduced from 3.71 generations in the untreated cells to 1.67 generations in IL-GFE treated cells. While Taxol reduced cell generation from 3.71 to 2.18 generations, this output
Fig. 5. MCF-7 Cells Growth at A: Exponential Phase and B: Death Phase, for untreated MCF-7 and the treated MCF-7 with IL-GFE and Taxol.
is significantly (p ≤ 0.05) deferent when the cell generations number were compared between different treatments and the control. The exemplary photographs of the untreated and the IL-GFE-treated MCF-7 at 24, 72 and 120 h were presented in Fig. 6. The longer the treatment time, the less density of MCF-7 observed. This probably due to the antiproliferative effect of IL-GFE toward MCF-7 cells growth. Cancer growth model in some types of cancers can give a good approximation of the volume of cancer cells using specific growth rate (Mehrara et al., 2007). Therefore, to evaluate the effect of IL-GFE on the specific growth of MCF-7, the graph of the log of viable cells number vs. time was plotted, and the regression slope was taken as a specific growth rate (μ). The growth rate of IL-GFE treated cells and taxol were observed to reduce significantly 0.0035 h−1 and 0.012 h−1, respectively compared to 0.0077 h−1 in control.
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Table 1 Number of Generations (X), specific growth rate (μ) and doubling time (td) for Untreated MCF-7 Cells (control), MCF-7 Cells Treated with IL-GFE and Taxol. Cell growth Untreated MCF-7 cells IL-GFE treated MCF-7 cells (IC50 = 4.75 μg/mL) Taxol treated MCF-7 cells (IC50 = 0.99 μg/mL)
Maximum cells volume (N) (cells/mL) 5
26.3 × 10 + 1.193 6.4 × 105+0.709* 9.1 × 105+0.482*
Number of generations (X)
Specific growth rate μ (h−1)
Doubling time td (h)
3.71 1.67 2.18
0.0077 0.0035 0.012
39.09 86.00 25.62
Initial cells number (N0) was constant at 2 × 105 cells/mL for all experiments. * Identical superscripts in the same column indicate significant difference (p ≤ 0.05).
4. Discussion A healthy diet (HD) rich in fruits and vegetables is associated with maintenance of health and reduced risk of diseases including cancers. This is due to the various phytochemicals such as phenols, alkaloids, flavonoids, carotenoids, and vitamins that play an essential role in boosting immunity (Wang et al., 2012). Cancer is treated nowadays using various anticancer drugs such as Taxol, cisplatin, topotecan, Doxorubicin, and etoposide. However, these drugs are known to give severe side effects to the cancer patients due to their ability to kill healthy cells (Magadi et al., 2015). Hence, the search for new anticancer drugs with minimal side effects is vital. According to the U.S. National Cancer Institute (NCI), drugs with IC50 values under 20 μg/mL after an incubation time of 48–72 h are considered as a potent cytotoxic substance (Boik, 2001). Graviola fruit is rich in secondary metabolites such as alkaloids, flavonoids, saponins, and importunately acetogenins which have proved to be a promising anticancer drug. In the present study, the anticancer activity (AC) of ionic liquid-Graviola fruit extract was investigated on adherent breast cancer MCF-7 and standard VERO cell lines. The IL-GFE could induce anti-proliferative effects as determined by MTT assay. However, IL-GFE was found to have slight anti-proliferation effect on the normal VERO cells at markedly higher doses of 100 μg/mL compared with cancer cells. Previous studies have reported that Graviola fruit extract induced cytotoxicity toward human prostate cancer PC-3 cells, pancreatic tumor PACA-2, lung carcinoma A-549 and human hepatoma HepG2 cell lines with comparable results to our study (Consolacion et al., 2012; Sun et al., 2014, 2016, 2017). Moreover, the representative photographs of the untreated and IL-GFE treated MCF7 cells at different times showed a reduction in cells density. The specific morphological and biochemical changes such as cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation are indicating the increase of apoptotic cells (Pieme et al., 2014). To understand the anti-proliferative and cytotoxicity effect of ILGFE, the present experimental study focused on a general kinetics model that would predict the inhibitory effects of IL-GFE on MCF-7 cells growth. From the growth kinetics curve, the number of generations was significantly reduced for the IL-GFE-treated MCF-7 compared with the control. A general model for breast adenocarcinoma MCF-7 cell line in response to IL-GFE treatment was developed. It is expected that an effective treatment may reduce the rate of cell proliferation (cytostatic effect) and raise the rate of cell death (cytotoxic effect). Most chemotherapeutic agents interfere with cell division processes and disturb cell cycle regulation, either by damaging DNA or interfering with DNA synthesis (O'Reilly et al., 1997). This is proved by the doubling time when expanded from 39.09 h for the control to 86.00 h for the IL-GFEtreated MCF-7, indicating a cytotoxic effect. On the other hand, Taxol had reduced the doubling time of MCF-7 cell growth to 25.62 h compared to the control. The exponential phase demonstrates the cytostatic effects (Fig. 5A) while cytotoxic effects were inferred at the death phase (Fig. 5B). According to the American Cancer Society, a biopsy is usually used to achieve certain tests to determine the speed of cancer growth and find the successful treatment for that, and therefore, the kinetics model with simple formulas and logical precision may have determined the size of a tumor in a function of time. For the untreated breast cancer
Fig. 6. Representative photographs of A; Untreated MCF-7 cells, and B; MCF7 cells treated with IL-GFE at a designated time point.
3.3. Cell cycle distribution and apoptosis Cell cycle progression is well-correlated to the proliferation of the cell. The untreated and the treated MCF-7 cells with IL-GFE were analyzed using RNase A/PI staining in combination with flow cytometry. The results show that IL-GFE treatment at IC50 for 24, 48 and 72 h increased the cells in the G0/G1 phase from 59.07% in control into 72.82% in GFE-treated cells for 72 h, while decreased in the S and G2M phase compared with the control. Moreover, cell number at the sub G0 phase of the treated MCF-7 cells increased in a time-dependent manner compared to the control, taking cells at the sub G0 phase as apoptotic cells (Agu et al., 2018); there was a remarkable increase in the number of apoptotic cells starting from 24 h of IL-GFE treatment (Fig. 7). Next, Annexin-V-FITC assay has been performed to confirm the apoptotic effect of the IL-GFE on MCF-7 cells. Figure (8) shows that the extract has significantly increased the percentage of cells positive to the Annexin-V-FITC staining in range of 0.32% for the control to 14.09% after 72 h of IL-GFE-treatment which indicates apoptosis.
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Fig. 7. Cell cycle distribution of the MCF-7 cells treated with IL-GFE at corresponding IC50 in a time-dependent manner.
MCF-7 cells growth, the first order linear equation is presented as follows: Log10 N = Log10 N0 + 0.0077t
tumor growth characteristic can be neglected (Mehrara et al., 2007). Thus, the final kinetic growth model in response to IL-GFE treatment gives the following equation:
(6)
Log10 Nf = (Log10 N0 +0.0077t)-(Log10 N0+0.0035t)
After treating MCF7 cells with IL-GFE, the first order linear equation become: Log10 N = Log10 N0 + 0.0035t
Moreover, the kinetics’ final growth model after treating with Taxol gives the equation as:
(7)
Log10 Nf =(Log10 N0 +0.0077t)-(Log10 N0+0.012t)
Additionally, when treating MCF-7 cells with Taxol, the first-order linear equation can be expressed as: Log10 N = Log10 N0 + 0.012t
(9)
(10)
Where, log10Nf = is the final volume of a tumor after treatment, log10N0 = is the initial volume of a tumor and t = is the duration of treatment (tf-to). The specific growth rate (μ) was then used to achieve growth
(8)
However, to underestimate the effectiveness of the treatment, the 471
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Fig. 8. The percentage of apoptotic MCF7 cells treated with IL-GFE at the corresponding IC50 for 24, 48 and 72 h.
et al., 2012; Moghadamtousi et al., 2014). Hence, these studies revealed that most acetogenins act as a DNA topoisomerase I poison, induced apoptotic cell death at a Bax and caspase 3 related pathways, arrested cancer cells at the G1 phase and inhibited NADH-ubiquinone oxidoreductase of the complex I mitochondrial (Rieser et al., 1991). It can be proposed that the synergistic effects of phytochemicals present in the extracts of Graviola fruit including acetogenins may be responsible for the potential chemotherapy agents of Graviola. These acetogenins found in a large quantity in Graviola extracts may act in synergy and might be responsible for their antiproliferation activity. Thus, further studies on the pure isolated compounds from Graviola fruit through bioassay-guided approach are still required to undoubtedly explore the potent active compounds responsible for the antiproliferative activity of the fruit.
kinetics model based on the fourth equation. This model allows to identify the tumor size at an early stage (less than 1 cc) and predict interpolation and extrapolation of sizes at any time points. It appears from the kinetics model constants that IL-GFE treatment has inhibited MCF-7 cells growth. Cancer is known as a disease of the cell cycle dysfunction. The efficient anticancer drug can block the cell cycle progression in cancer cells (Mantena et al., 2006). To investigate whether IL-GFE induced growth inhibition by the cell cycle arrest, MCF7 cell cycle distribution was examined after IL-GFE-treatment using flow cytometry analysis. The current study demonstrated that IL-GFE arrests the MCF-7-cell cycle at the growth-static G1 phase which explains the anti-proliferation of MCF7 by IL-GFE. Furthermore, the results of the flow cytometry analysis using Annexin V/PI staining showed that GFE has remarkedly induced apoptosis in breast cancer cells. This finding was confirmed by Moghadamtousi et al. (2014) and Chamcheu et al. (2018) when treated human HCT-116 and HT-29 colon cancer, and non-melanoma skin cancer NMSC cell lines with ethyl acetate extract of Annona muricata leaves. Moreover, Fig. 8 indicates the appearance of the necrotic cells in a time-dependent manner which is in close agreement with the results of Torres et al. (2012), after treating pancreatic cancer PC cells with Graviola extract. They observed that Graviola extract inhibited cellular metabolism by inhibiting multiple signaling pathways that regulate metabolism, cell cycle and metastatic properties in pancreatic cancer cells. Thus, both mechanisms either apoptosis and necrosis are induced by Graviola fruit ionic liquid extract. Intensive phytochemical investigations of the fruit and leaves of Graviola have isolated and identified a great number of acetogenins with a potent biological and pharmacological activities, such as anticancer, cytotoxicity, and apoptotic on human cancer cells (Gajalakshmi
5. Conclusion In summary, IL-GFE exhibit antiproliferative effects against MCF-7 breast cancer cell lines by inducing loss of cell viability via apoptosis, morphology changes, and cell cycle arrest at the G0/G1 phase. This inhibition was selective to the growth of MCF-7 cells without any effect on nontumorigenic VERO cells, suggesting that IL-GFE possesses selective antitumor properties toward cancer cells. It also showed their potentiality as an anticancer agent when compared with Taxol as a positive control. Moreover, IL-GFE treatment has inhibited breast cancer MCF-7 cell growth by reducing the number of cell generations and increasing the doubling time compared with the control. These data suggest that the IL-GFE can be developed as a novel mechanism-based supplement agent for the cure and prevention of breast cancer. Further investigations are necessary to elucidate the mechanisms underlying 472
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the therapeutic effects of the fruit extract as well as the determination of the active compounds responsible for cytotoxicity.
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Functional proteomic analysis revels that the ethanol extract of Annona muricata L. induces liver cancer cell apoptosis through endoplasmic reticulum stress pathway. J. Ethnopharmacol. 189, 210–217. Magadi, V.P., Ravi, V., Arpitha, A., 2015. Evaluation of cytotoxicity of aqueous extract of Graviola leaves on squamous cell carcinoma cell-25 cell lines by 3-(4, 5-dimethylthiazol-2-Yl)-2, 5-diphenyltetrazolium bromide assay and determination of percentage of cell inhibition at G2M phase of cell cycle by flow cytometry: an in vitro study. Contemp. Clin. Dent. 6 (4), 529. Mantena, S.K., Sharma, S.D., Katiyar, S.K., 2006. Berberine, a natural product, induces G1-phase cell cycle arrest and caspase-3-dependent apoptosis in human prostate carcinoma cells. Mol. Canc. Therapeut. 5 (2), 296–308. Mehrara, E., Forssell-Aronsson, E., Ahlman, H., Bernhardt, P., 2007. Specific growth rate versus doubling time for quantitative characterization of tumor growth rate. Cancer Res. 67 (8), 3970–3975. Moghadamtousi, S.Z., Karimian, H., Rouhollahi, E., Paydar, M., Fadaeinasab, M., Kadir, H.A., 2014. Annona muricata leaves induce G1 cell cycle arrest and apoptosis through mitochondria-mediated pathway in human HCT-116 and HT-29 colon cancer cells. J. Ethnopharmacol. 156, 277–289. Monigatti, M., Bussmann, R.W., Weckerle, C.S., 2013. Medicinal plant use in two Andean communities located at different altitudes in the Bolívar Province, Peru. J. Ethnopharmacol. 145 (2), 450–464. O'Reilly, M.S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W.S., Flynn, E., Birkhead, J.R., Olsen, B.R., Folkman, J., 1997. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88 (2), 277–285. Patel, M.S., Patel, J.K., 2016. A review on a miracle fruits of Annona muricata. J. Pharmacogn. Phytochem. 5 (1), 137. Peter, M.E., 2011. Programmed cell death: apoptosis meets necrosis. Nature 471 (7338), 310. Pieme, C.A., Kumar, S.G., Dongmo, M.S., Moukette, B.M., Boyoum, F.F., Ngogang, J.Y., Saxena, A.K., 2014. Antiproliferative activity and induction of apoptosis by Annona muricata (Annonaceae) extract on human cancer cells. BMC Complement Altern. Med. 14 (1), 516. Rieser, M.J., Kozlowski, J.F., Wood, K.V., McLaughlin, J.L., 1991. Muricatacin: a simple biologically active acetogenin derivative from the seeds of Annona muricata (Annonaceae). Tetrahedron Lett. 32 (9), 1137–1140. Samuel, A.J.S.J., Kalusalingam, A., Chellappan, D.K., Gopinath, R., Radhamani, S., Husain, H.A., Muruganandham, V., Promwichit, P., 2010. Ethnomedical survey of plants used by the orang asli in kampung Bawong, Perak, west Malaysia. J. Ethnobiol. Ethnomed. 6 (1), 5. Siegel, R., Miller, K., Jemal, A., 2017. Cancer statistics, 2018 CA: a cancer. J Clin 68, 7–30. Sun, S., Liu, J., Kadouh, H., Sun, X., Zhou, K., 2014. Three new anti-proliferative Annonaceous acetogenins with mono-tetrahydrofuran ring from graviola fruit (Annona muricata). Bioorg. Med. Chem. Lett 24 (12), 2773–2776. Sun, S., Liu, J., Sun, X., Zhu, W., Yang, F., Felczak, L., Dou, Q.P., Zhou, K., 2017. Novel Annonaceous acetogenins from Graviola (Annona muricata) fruits with strong antiproliferative activity. Tetrahedron Lett. 58 (19), 1895–1899. Sun, S., Liu, J., Zhou, N., Zhu, W., Dou, Q.P., Zhou, K., 2016. Isolation of three new annonaceous acetogenins from Graviola fruit (Annona muricata) and their anti-proliferation on human prostate cancer cell PC-3. Bioorg. Med. Chem. Lett 26 (17), 4382–4385. Taylor, L., 2002. Graviola (Annona Muricata). Herbal Secrets of the Rainforest. Torres, M.P., Rachagani, S., Purohit, V., Pandey, P., Joshi, S., Moore, E.D., Johansson, S.L., Singh, P.K., Ganti, A.K., Batra, S.K., 2012. Graviola: a novel promising naturalderived drug that inhibits tumorigenicity and metastasis of pancreatic cancer cells in vitro and in vivo through altering cell metabolism. Cancer Lett. 323 (1), 29–40. Verhoven, B., Schlegel, R., Williamson, P., 1995. Mechanisms of phosphatidylserine exposure, a phagocyte recognition signal, on apoptotic T lymphocytes. J. Exp. Med. 182 (5), 1597–1601. Wang, H., Oo Khor, T., Shu, L., Su, Z.-Y., Fuentes, F., Lee, J.-H., Tony Kong, A.-N., 2012. Plants vs. cancer: a review on natural phytochemicals in preventing and treating cancers and their druggability. Anti Cancer Agents Med. Chem. 12 (10), 1281–1305. Wyllie, A.H., 1997. Apoptosis: an overview. Br. Med. Bull. 53 (3), 451–465. Yajid, A.I., Ab Rahman, H.S., Wong, M.P.K., Zain, W.Z.W., 2018. Potential benefits of Annona muricata in combating cancer: a review. Malays. J. Med. Sci.: MJMS 25 (1), 5. Zhang, Y., Liu, Z., Li, Y., Chi, R., 2014. Optimization of ionic liquid-based microwaveassisted extraction of isoflavones from Radix puerariae by response surface methodology. Separ. Purif. Technol. 129, 71–79.
Conflicts of interest There is no conflict of interest in this work. Acknowledgment The authors are grateful to the International Islamic University Malaysia, for providing the P-RIGS grant and laboratory facilities to carry out the research. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jep.2019.03.003. References Agu, K.C., Okolie, N.P., Falodun, A., Engel-Lutz, N., 2018. In vitro anticancer assessments of Annona muricata fractions and in vitro antioxidant profile of fractions and isolated acetogenin (15-acetyl guanacone). Journal of Cancer Research and Practice 5 (2), 53–66. Badrie, N., Schauss, A.G., 2010. Soursop (Annona Muricata L.): Composition, Nutritional Value, Medicinal Uses, and Toxicology, Bioactive Foods in Promoting Health. Elsevier, pp. 621–643. Betancur-Galvis, L.A., Saez, J., Granados, H., Salazar, A., Ossa, J.E., 1999. Antitumor and antiviral activity of Colombian medicinal plant extracts. Memórias do Inst. Oswaldo Cruz 94 (4), 531–535. Bhan, M., Satija, S., Garg, C., Dureja, H., Garg, M., 2017. A novel approach towards green extraction for glycyrrhitinic acid by ionic liquid based microwave assisted extraction and optimization through response surface methodology. Phcog. J. 9 (6). Boik, J., 2001. Natural Compounds in Cancer Therapy. Chamcheu, J., Rady, I., Chamcheu, R.-C., Siddique, A., Bloch, M., Banang Mbeumi, S., Babatunde, A., Uddin, M., Noubissi, F., Jurutka, P., 2018. Graviola (Annona muricata) exerts anti-proliferative, anti-clonogenic and pro-apoptotic effects in human nonmelanoma skin cancer UW-BCC1 and A431 cells in vitro: involvement of hedgehog signaling. Int. J. Mol. Sci. 19 (6), 1791. Chik, W.W., Amid, A., Jamal, P., 2010. Purification and cytotoxicity assay of tomato (Lycopersicon esculentum) leaves methanol extract as potential anti-cancer agent. J. Appl. Sci. 10 (24), 3283–3288. Coe, F.G., 2008. Rama midwifery in eastern Nicaragua. J. Ethnopharmacol. 117 (1), 136–157. Consolacion, Y., Geneveve, S., Ming-Jaw, D., Chien-Chang, S., 2012. Acetogenins from Annona muricata. Phcog. J. 4 (32). Daddiouaissa, D., Amid, A., 2018. Anticancer activity of acetogenins from Annona muricata fruit. The international medical journal malaysia 17 (3), 103–112. Dai, Y., Hogan, S., Schmelz, E.M., Ju, Y.H., Canning, C., Zhou, K., 2011. Selective growth inhibition of human breast cancer cells by graviola fruit extract in vitro and in vivo involving downregulation of EGFR expression. Nutr. Canc. 63 (5), 795–801. Elmore, S., 2007. Apoptosis: a review of programmed cell death. Toxicol. Pathol. 35 (4), 495–516. Fadok, V., Savill, J., Haslett, C., Bratton, D., Doherty, D., Campbell, P., Henson, P., 1992. Different populations of macrophages use either the vitronectin receptor or the phosphatidylserine receptor to recognize and remove apoptotic cells. J. Immunol. 149 (12), 4029–4035. Fouz, N., Amid, A., 2014. Hashim YZ., Cytokinetic study of MCF-7 cells treated with commercial and recombinant bromelain, Asian Pac. J. Cancer Prev. APJCP 14 (11), 6709–6714. Gajalakshmi, S., Vijayalakshmi, S., Devi Rajeswari, V., 2012. Phytochemical and pharmacological properties of Annona muricata: a review. Int. J. Pharm. Pharm. Sci. 4 (2), 3–6. Gavamukulya, Y., Abou-Elella, F., Wamunyokoli, F., AEl-Shemy, H., 2014. Phytochemical screening, anti-oxidant activity and in vitro anticancer potential of ethanolic and water leaves extracts of Annona muricata (Graviola). Asian Pacific journal of tropical medicine 7, S355–S363. George, V.C., Kumar, D., Rajkumar, V., Suresh, P., Kumar, R.A., 2012. Quantitative assessment of the relative antineoplastic potential of the n-butanolic leaf extract of Annona muricata Linn. in normal and immortalized human cell lines. Asian Pac. J. Cancer Prev. APJCP 13 (2), 699–704. Griffin, A., Butow, P., Coates, A., Childs, A., Ellis, P., Dunn, S., Tattersall, M., 1996. On the receiving end V: patient perceptions of the side effects of cancer chemotherapy in 1993. Ann. Oncol. 7 (2), 189–195. Gyamfi, K., Sarfo, D., Nyarko, B., Akaho, E., Serfor-Armah, Y., Ampomah-Amoako, E., 2011. Assessment of elemental content in the fruit of graviola plant, Annona muricata,
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Antiproliferative activity of ionic liquid-graviola fruit extract against human breast cancer (MCF-7) cell lines using flow cytometry techniques
T
Djabir Daddiouaissaa, Azura Amidb,∗, Nassereldeen A. Kabbashia, Fazia A.A. Fuada, AhmedA.M. Elnoura, Mohamad A.K.M. S. Epandyc a
Biotechnology Engineering, Kulliyyah of Engineering, International Islamic University, Malaysia (IIUM), P. O. Box 10, Gombak, 50728, Kuala Lumpur, Malaysia International Institute for Halal Research and Training (INHART), Level 3, KICT Building, International Islamic University Malaysia (IIUM), Jalan Gombak, 53100, Kuala Lumpur, Malaysia c Adikafirdaus Resources, Lot 24, Jalan Klebang Selatan, 2/5 Kampung Tersusun, Batu 6 Klebang Selatan, 31200, Ipoh, Perak, Malaysia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Breast cancer Graviola Ionic liquid Flow cytometry Apoptosis
Ethnopharmacological relevance: Medicinal plants have been used for ages by indigenous communities around the world to help humankind sustain its health. Graviola (Annona muricata), also called soursop, is a member of the Annonaceae family and is an evergreen plant that is generally distributed in tropical and subtropical areas of the world. Graviola tree has a long history of traditional use due to its therapeutic potential including anti-inflammatory, antimicrobial, antioxidant, insecticide and cytotoxic to tumor cells. Aim of the study: This study aimed to investigate the in vitro antiproliferative effects and apoptotic events of the ionic liquid extract of Graviola fruit (IL-GFE) on MCF-7 breast cancer cells and their cytokinetics behaviour to observe their potential as a therapeutic alternative in cancer treatment. Materials and methods: The cell viability assay of the extract was measured using tetrazolium bromide (MTT assay) to observe the effects of Graviola fruit extract. Then the cytokinetics behaviour of MCF-7 cells treated with IL-GFE is observed by plotting the growth curve of the cells. Additionally, the cell cycle distribution and apoptosis mechanism of IL-GFE action on MCF-7 cancer cells were observed by flow cytometry. Results: IL-GFE exhibited anti-proliferative activity on MCF-7 with the IC50 value of 4.75 μg/mL, compared to Taxol with an IC50 value of 0.99 μg/mL. IL- GFE also reduced the number of cell generations from 3.71 to 1.67 generations compared to 2.18 generations when treated with Taxol. Furthermore, the anti-proliferative activities were verified when the growth rate was decreased dynamically from 0.0077 h to 1 to 0.0035 h-1. Observation of the IL-GFE-treated MCF-7 under microscope demonstrated detachment of cells and loss of density. The growth inhibition of the cells by extracts was associated with cell cycle arrest at the G0/G1 phase, and phosphatidylserine externalisation confirms the anti-proliferation through apoptosis. Conclusions: ionic liquid Graviola fruit extract affect the cytokinetics behaviour of MCF-7 cells by reducing cell viability, induce apoptosis and cell cycle arrest at the G0/G1 phase.
1. Introduction Fruit consumption is highly recommended to maintain human health (Halliwell, 2012). Graviola (Annona muricata L.) is also called soursop because of the sour and sweet flavour of its great fruit (Patel and Patel, 2016). Graviola is native to the tropical zones, South America and Africa but is currently widely cultivated in the tropical and subtropical zones around the world. Recently, Graviola fruit is widely used to make candies, syrups and beverages (Ioannis et al., 2015). Ethnobotanical studies reported that all parts of Graviola tree are used in an alternative medicine, especially the fruit which is taken to treat fever ∗
(Taylor, 2002), eliminate worms and parasites (Yajid et al., 2018), increase mother's milk (Badrie and Schauss, 2010), as well as hypertension (Samuel et al., 2010), liver and renal infections (Coe, 2008). The popular use of Graviola as an anticancer treatment reported ethnobotanically may be related to the reports of its selective cytotoxicity (George et al., 2012; Monigatti et al., 2013). This activity is considered selective as some reported in vitro studies have shown that the Graviola extract was more toxic to cancer cells than normal cells (BetancurGalvis et al., 1999; Dai et al., 2011; Gavamukulya et al., 2014). The major benefit of this fruit is the active compounds known as annonaceous acetogenins which are thought to be potent anticancer
Corresponding author. E-mail address: [email protected] (A. Amid).
https://doi.org/10.1016/j.jep.2019.03.003 Received 14 December 2018; Received in revised form 25 February 2019; Accepted 2 March 2019 Available online 07 March 2019 0378-8741/ © 2019 Elsevier B.V. All rights reserved.
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identified from the Adikafirdaus Resources Farm in Kampong Bawong, Perak, Malaysia. After that, the fruits were washed with distilled water to remove all traces of dust and insects. The fresh pulp of each fruit separated from the pericarp and seeds, then the pulp was frozen at −20 °C and lyophilised using a Christ Alpha freeze drier for 72 h according to Hoeing (2001), with slight modification. After that, the powder was weighed and placed in an airtight bottle and stored until further extraction procedure (Gyamfi et al., 2011). A plant sample deposited in the herbarium at the Kulliyyah of Architecture and Environmental Design (KAED), International Islamic University Malaysia with voucher number KAED/HBL/S1A047/2018/ 707. The name of the plant was checked on www.theplantlist.org.
toward several cancer cell lines including breast cancer as highlighted recently by Magadi et al. (2015) and Daddiouaissa & Amid (2018). At present, the attention is being focused on anticancer properties of Graviola acetogenins which are the primary bioactive compounds found in the fruit (Sun et al., 2017). Breast adenocarcinoma cell lines (MCF-7) is one of the most frequent incident cancers among women, with an estimated 266,120 new cases, and 40,920 breast cancer (BC) deaths will occur in the United States' women in 2018 (Siegel et al., 2017). Therefore, BC is a challenge among research communities around the world; this is because of the BC incidence rate keep increasing by 0.4% annually worldwide according to the final report conducted by Jemal et al. (2017). Current protocols of treatment include radiation therapy, surgical intervention, and chemotherapy which induce numerous side effects including nausea, fatigue, vomiting, weak of the immune system and hair loss (Griffin et al., 1996). Thus, the search for alternative treatment is necessary. Apoptosis also called programmed cell death, is a mechanism that allows self-destruction of the cells when stimulated by an appropriate trigger. Distinct morphological changes such as cell shrinkage, nuclear condensation, and pyknosis, complemented with biochemical phenomena (BP) such as the cleavage of DNA between the nucleosomes (Elmore, 2007; Peter, 2011). New insights into the process of apoptosis lead to different parameters which can be used to detect and measure apoptosis. One of these parameters is the appearance of phosphatidylserine (PS) on the surface of the cell membrane and loss of the asymmetry which is a specific signal for macrophages to recognise and remove the apoptotic cells (Fadok et al., 1992; Verhoven et al., 1995). Apoptosis is mainly created by either death receptors (extrinsic pathway) involving caspases 8 and 10 or the mitochondrial pathway (intrinsic pathway) via caspase 9 (Elmore, 2007). The alteration of the mitochondrial membrane potential translocates the pro-apoptotic protein Bax which leads to the release of cytochrome C, into the cytoplasm, and subsequent activation of caspases and lead to apoptosis (Wyllie, 1997). The constant breast cell number is maintained and regulated via an equilibrium between cell proliferation and apoptosis. After that, any changes in this balance lead to the loss of homeostasis which can cause the growth of cancer cells (Hao et al., 1998). Thus, the arrestation of cell cycle machinery and induction of apoptosis are the key mechanism to prevent and suppress the breast cancer cells (BCs). Hence, there is a massive interest in the development of new apoptosis-inducing agents with efficacy and more specificity with minimal side effects. Recently, our group has successfully extracted the Graviola fruit using ionic liquid method resulted in high productivity. Thus, this study aimed to investigate the possible inhibitions induced in breast cancer cell lines the MCF-7using the ionic liquid extract of Graviola fruit.
2.3. Fruit extraction One gram of the dried sample was mixed with 30 mL of 0.5 mol/L of 1-butyl-3-methylimidazolium chloride [C4MIM] [Cle] solution. Then the suspension was heated with microwave oven under irradiation power of 700 W for 3 min. After each irradiation, the obtained extracts (IL-GFE) were cooled down to 25 °C and then filtered through Whatman 3 mm filter paper and transferred into a test tube. The tubes were frozen at −20 °C then lyophilised using a Christ Alpha freeze drier for 72 h. Then, the powder was stored in 4 °C freezer until use (Bhan et al., 2017; Zhang et al., 2014).
2.4. Cell culture This study used breast cancer cell lines the MCF-7 as the experimental cancer cells (ATCC No: HTB-22™), and VERO cell line (ATCC® No: CCL-81™) as a control cell line. These cells were obtained from American type Collection Culture. Frozen cells were thawed and inoculated into 5 mL of Dulbecco's Modification of Eagle's Medium (DMEM) supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS). The cells were cultured in T-25 flasks and incubated in 95% humidified incubator with 5% CO2 at 37 °C. The cell cultures were used when reached approximately 70% confluence for experimental treatments.
2.5. Cell viability assay (cytotoxicity assay) The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to quantify the percentage of cell viability. The cultured cells were detached with accutase, suspended in media and counted using a hemocytometer. Approximately 50,000 cells were plated in 100 μL of medium per well in 96-well plate then incubated for 24 h in 5% CO2 incubator. The cells were then treated (triplicate wells per condition) by adding 20 μL of serial dilutions of the sample extract in DMEM free-fetal bovine serum (the control samples received only DMSO). Then incubate the cells for another 24 h before adding 20 μL (5 mg/mL) solution of MTT reagent into each well. Then the incubation was continued for another 3 h before the media was pipetted out carefully. 100 μL of solubilization solution (DMSO) was added to each well and mixed by gentle shaking for 15 min (Liu et al., 2016). Finally, the amount of formazan was determined by measuring the absorbance at 570 nm using a microplate reader. The obtained data were used to calculate the percentage of viable/dead cells, and fifty percent of inhibitory concentration (IC50) of each extract was determined, using equation (1):
2. Materials and methods 2.1. Chemicals and reagents All the chemicals and reagents used in this project are laboratory grade. 1-Butyl-3-Methylimidazolium Chloride 96% [C4MIM] [Cle] reagent was purchased from (Alfa, USA), cell culture media, including Dulbecco's modified Eagle's medium (DMEM), penicillin/streptomycin and fetal bovine serum (FBS) were obtained from GIBCO® (Invitrogen, USA). The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium Bromide (MTT) was purchased from Sigma-Aldrich, (USA). The accutase cell detachment solution purchased from (ICT, USA). RNase A/PI cell cycle assay kit and Annexin V/PI apoptosis assay kit was purchased from Beckman Coulter, (USA). These chemicals were used to investigate the anticancer effect of IL-GFE on cancer cell lines.
% cell viability =
Abs of sample x 100 Abs of control
(1)
2.2. Sample collection and preparation where Abs of the sample is the absorbance of treated cells, and Abs of control is the absorbance of untreated cells.
Graviola fruit (DB3) samples were randomly collected and 467
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Coulter, USA). The cells in S and G2M phases are defined as proliferative cells and as apoptotic cells in the sub-G1 phase (Agu et al., 2018).
2.6. Estimation of the inhibition concentration at 50% (IC50) The IC50 values were graphically obtained by plotting the percentage of cell viability against the corresponding of the IL-GFE sample used. Data were reported as the mean ± standard error (SE) and statically analyzed. The significance was accepted when P ≤ 0.05 level.
2.10. Annexin-V-FITC assay The Annexin -V-FITC methods were conducted using the early, and late apoptotic cells after treatment with IL-GFE was determined using Annexin-V/PI staining assay (Verhoven et al., 1995). The MCF-7 have treated with GFE at IC50 value for 24, 48, and 72 h. After that, media removed, washed with PBS and centrifuged at 1500 rpm for 5 min. Then, cells were resuspended in the binding buffer and stained with Annexin-V-FITC and PI according to the procedure from the manufacturer's manual. Finally, the fluorescence intensity of the stained MCF7 cells was determined using CytoFLEX S flow cytometry (Moghadamtousi et al., 2014).
2.7. Preparation of ionic liquid-graviola fruit extract (IL-GFE) and taxol About 1 mg of IL-GFE was dissolved in 1 mL of PBS (phosphate buffered saline). Taxol was used as a positive control (+ve), and 50 μg of Taxol was dissolved in DMSO. 2.8. Growth kinetics study The growth kinetics of treated and non-treated MCF-7 breast cancer cells with IL-GFE and Taxol as a positive control (+ve) was studied and compared to observe the antiproliferative effects of IL-GFE on the growth of MCF-7 cell lines. Firstly, MCF-7 cells were harvested and seeded into T-25 flasks at 2 × 105 cells/mL. There were three replicates of flasks in this study: untreated MCF-7; IL-GFE-treated MCF-7 at the IC50 value of 4.75 μg/mL and taxol-treated MCF-7 at the IC50 value of 0.99 μg/mL (Chik et al., 2010). Each group has 20 flasks to count samples every 8 h interval from 0 to 144 h. Each sample was performed in three independent experiments. After incubation for 24 h, media was removed, and fresh media containing samples as described before with the IC50 value concentrations were added into separate flasks. Then, cells were incubated from 0 to 144 h. Every 8 h, cell images were observed and documented using phase contrast microscope at 100 magnification then harvested from monolayer and proceed to cell quantification using trypan blue dye exclusion assay (Fouz, Amid, and Hashim, 2014). The number of viable cells was calculated as the equation below:
C = nx 2x
2.11. Statistical analysis The cell viability experiments were carried out in triplicates, and each data point represents the overall mean of at least 3 independent experiments. The findings were expressed as mean ± standard deviation (SD). The results were analyzed using GraphPad Prism (Version 7.00) and Microsoft Office, Excel software to carry out the statistical analysis. One-way analysis of variance technique followed by Tukey's test was applied to observe the significance between the groups. The entire statistical analysis was carried out at (p ≤ 0.05). 3. Results 3.1. The IC50 of IL-GFE against MCF-7 and VERO cell lines To investigate the half maximal inhibitory concentration (IC50) of IL-GFE against MCF-7 cell lines, the data of MTT assay were interpreted by plotting graph of the percentage of cell viability (%) versus different concentrations (μg/mL). After that, the IC50 values for IL-GFE (Fig. 1) and Taxol (Fig. 2), were observed as 4.759 μg/mL and 0.99 μg/mL, respectively. This result shows that the proliferation of MCF-7 cells has dramatically and significantly inhibited after the addition of IL-GFE in a dose-dependent manner. Moreover, MTT assays of IL-GFE against the normal VERO cell lines (Fig. 3) did not show any IC50 value compared to the tumor cell lines. This signifies that the IL-GFE is more cytotoxic against cancer cells than the normal cell lines and will not affect the growth of healthy cells, even with a high concentration of 100 μg/mL. This result confirms those obtained in the previous study (Dai et al., 2011). Thus, the Graviola fruit serves as is a promising alternative or complementary supplement towards reducing breast cancer growth generally and MCF-7
(2)
10 4
4
where n is the average cell number, 2 is the dilution factor, and 10 is the conversion of volume 0.1 mm3 to mL. The data were used to plot a growth kinetics graph and calculate the cell generation number using the following equations:
X=
Log10 N − Log10 N0 Log10 2
Log10 N = Log10 N0 + μt td =
Log 102 μ
=
0.301 μ
(3) (4)
(5)
where X is the number of generations; N is the final cells number; N0 is the initial cells number; μ is the specific growth rate (slope); t is the duration of treatment; td is the doubling time. 2.9. Cell cycle assay Firstly, the flow cytometry analysis was applied to detect changes in cell cycle distribution induced by IL-GFE treatment at an IC50 value in a time-dependent manner. In brief, MCF-7 cells (5 × 104 cells/mm) were treated with IL-GFE at 4.75 μg/mL then incubated for 24, 48 and 72 h, respectively. The cells were harvested and washed with 1x phosphate buffered saline (PBS), then collected and centrifuged at 1500 rpm for 5 min. After that, cells were fixed in 70% ethanol at 4 °C for 1 h then centrifuged at 4000 rpm for 10 min, at ambient temperature, removing the supernatant and wash twice with PBS. Later, cells were stained with 500 μL of RNase A/PI solution containing 0.05 mg/mL of propidium iodide (PI) and 0.05 mg/mL of RNase A in PBS (Magadi et al., 2015). The percentages of cell cycle distribution of the treated and untreated cells were determined using CytoFLEX S flow cytometry (Beckman
Fig. 1. The percentage of cell viability vs. concentrations of IL-GFE treated on MCF-7 cells. 468
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Fig. 2. The percentage of cell viability vs. concentrations of Taxol treated on MCF-7 cells. Fig. 4. MCF-7 Growth profiles of Untreated (control) and Treated with IL-GFE (IC50 = 4.75 μg/mL) and Taxol (IC50 = 0.99 μg/mL).
Fig. 3. Graph of the percentage cell viability vs. concentrations of IL-GFE against VERO cell lines.
specifically. 3.2. Effect of IL-GFE on MCF-7 cell growth kinetics The growth kinetics of untreated (control) and treated MCF-7 with IL-GFE (4.75 μg/mL) and Taxol (0.99 μg/mL) was studied and compared. Fig. 4 shows the growth behaviour and the changes of MCF7 cells after treatment. The number of cells was initially inoculated at 2 × 105 cells/mL. The lag phase was similar for the untreated and the treated MCF-7 cells at first 16 h. Then the cells started proliferation and showed clear log phase after 24 h. However, the treated cells did not show a clear log phase and took 88 h to reach the late log phase. The taxol showed clear log phase but produced less total cell number. It can be observed that the IL-GFE and Taxol treated MCF-7 shows a great reduction in the number of cell proliferation compared to the control. The highest number of cells obtained at the stationary phase for the ILGFE and Taxol-treated cells were only 6.4 × 105 and 9.1 × 105 cells/ mL, respectively, as compared to the control 26.3 × 105. Based on the fourth equation that was obtained from the growth curve (Fig. 4), cell growth at exponential phase and death phase (Fig. 5) and the cell generation number (Table 1) were obtained for all treatments. The number of cell generation was reduced from 3.71 generations in the untreated cells to 1.67 generations in IL-GFE treated cells. While Taxol reduced cell generation from 3.71 to 2.18 generations, this output
Fig. 5. MCF-7 Cells Growth at A: Exponential Phase and B: Death Phase, for untreated MCF-7 and the treated MCF-7 with IL-GFE and Taxol.
is significantly (p ≤ 0.05) deferent when the cell generations number were compared between different treatments and the control. The exemplary photographs of the untreated and the IL-GFE-treated MCF-7 at 24, 72 and 120 h were presented in Fig. 6. The longer the treatment time, the less density of MCF-7 observed. This probably due to the antiproliferative effect of IL-GFE toward MCF-7 cells growth. Cancer growth model in some types of cancers can give a good approximation of the volume of cancer cells using specific growth rate (Mehrara et al., 2007). Therefore, to evaluate the effect of IL-GFE on the specific growth of MCF-7, the graph of the log of viable cells number vs. time was plotted, and the regression slope was taken as a specific growth rate (μ). The growth rate of IL-GFE treated cells and taxol were observed to reduce significantly 0.0035 h−1 and 0.012 h−1, respectively compared to 0.0077 h−1 in control.
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Table 1 Number of Generations (X), specific growth rate (μ) and doubling time (td) for Untreated MCF-7 Cells (control), MCF-7 Cells Treated with IL-GFE and Taxol. Cell growth Untreated MCF-7 cells IL-GFE treated MCF-7 cells (IC50 = 4.75 μg/mL) Taxol treated MCF-7 cells (IC50 = 0.99 μg/mL)
Maximum cells volume (N) (cells/mL) 5
26.3 × 10 + 1.193 6.4 × 105+0.709* 9.1 × 105+0.482*
Number of generations (X)
Specific growth rate μ (h−1)
Doubling time td (h)
3.71 1.67 2.18
0.0077 0.0035 0.012
39.09 86.00 25.62
Initial cells number (N0) was constant at 2 × 105 cells/mL for all experiments. * Identical superscripts in the same column indicate significant difference (p ≤ 0.05).
4. Discussion A healthy diet (HD) rich in fruits and vegetables is associated with maintenance of health and reduced risk of diseases including cancers. This is due to the various phytochemicals such as phenols, alkaloids, flavonoids, carotenoids, and vitamins that play an essential role in boosting immunity (Wang et al., 2012). Cancer is treated nowadays using various anticancer drugs such as Taxol, cisplatin, topotecan, Doxorubicin, and etoposide. However, these drugs are known to give severe side effects to the cancer patients due to their ability to kill healthy cells (Magadi et al., 2015). Hence, the search for new anticancer drugs with minimal side effects is vital. According to the U.S. National Cancer Institute (NCI), drugs with IC50 values under 20 μg/mL after an incubation time of 48–72 h are considered as a potent cytotoxic substance (Boik, 2001). Graviola fruit is rich in secondary metabolites such as alkaloids, flavonoids, saponins, and importunately acetogenins which have proved to be a promising anticancer drug. In the present study, the anticancer activity (AC) of ionic liquid-Graviola fruit extract was investigated on adherent breast cancer MCF-7 and standard VERO cell lines. The IL-GFE could induce anti-proliferative effects as determined by MTT assay. However, IL-GFE was found to have slight anti-proliferation effect on the normal VERO cells at markedly higher doses of 100 μg/mL compared with cancer cells. Previous studies have reported that Graviola fruit extract induced cytotoxicity toward human prostate cancer PC-3 cells, pancreatic tumor PACA-2, lung carcinoma A-549 and human hepatoma HepG2 cell lines with comparable results to our study (Consolacion et al., 2012; Sun et al., 2014, 2016, 2017). Moreover, the representative photographs of the untreated and IL-GFE treated MCF7 cells at different times showed a reduction in cells density. The specific morphological and biochemical changes such as cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation are indicating the increase of apoptotic cells (Pieme et al., 2014). To understand the anti-proliferative and cytotoxicity effect of ILGFE, the present experimental study focused on a general kinetics model that would predict the inhibitory effects of IL-GFE on MCF-7 cells growth. From the growth kinetics curve, the number of generations was significantly reduced for the IL-GFE-treated MCF-7 compared with the control. A general model for breast adenocarcinoma MCF-7 cell line in response to IL-GFE treatment was developed. It is expected that an effective treatment may reduce the rate of cell proliferation (cytostatic effect) and raise the rate of cell death (cytotoxic effect). Most chemotherapeutic agents interfere with cell division processes and disturb cell cycle regulation, either by damaging DNA or interfering with DNA synthesis (O'Reilly et al., 1997). This is proved by the doubling time when expanded from 39.09 h for the control to 86.00 h for the IL-GFEtreated MCF-7, indicating a cytotoxic effect. On the other hand, Taxol had reduced the doubling time of MCF-7 cell growth to 25.62 h compared to the control. The exponential phase demonstrates the cytostatic effects (Fig. 5A) while cytotoxic effects were inferred at the death phase (Fig. 5B). According to the American Cancer Society, a biopsy is usually used to achieve certain tests to determine the speed of cancer growth and find the successful treatment for that, and therefore, the kinetics model with simple formulas and logical precision may have determined the size of a tumor in a function of time. For the untreated breast cancer
Fig. 6. Representative photographs of A; Untreated MCF-7 cells, and B; MCF7 cells treated with IL-GFE at a designated time point.
3.3. Cell cycle distribution and apoptosis Cell cycle progression is well-correlated to the proliferation of the cell. The untreated and the treated MCF-7 cells with IL-GFE were analyzed using RNase A/PI staining in combination with flow cytometry. The results show that IL-GFE treatment at IC50 for 24, 48 and 72 h increased the cells in the G0/G1 phase from 59.07% in control into 72.82% in GFE-treated cells for 72 h, while decreased in the S and G2M phase compared with the control. Moreover, cell number at the sub G0 phase of the treated MCF-7 cells increased in a time-dependent manner compared to the control, taking cells at the sub G0 phase as apoptotic cells (Agu et al., 2018); there was a remarkable increase in the number of apoptotic cells starting from 24 h of IL-GFE treatment (Fig. 7). Next, Annexin-V-FITC assay has been performed to confirm the apoptotic effect of the IL-GFE on MCF-7 cells. Figure (8) shows that the extract has significantly increased the percentage of cells positive to the Annexin-V-FITC staining in range of 0.32% for the control to 14.09% after 72 h of IL-GFE-treatment which indicates apoptosis.
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Fig. 7. Cell cycle distribution of the MCF-7 cells treated with IL-GFE at corresponding IC50 in a time-dependent manner.
MCF-7 cells growth, the first order linear equation is presented as follows: Log10 N = Log10 N0 + 0.0077t
tumor growth characteristic can be neglected (Mehrara et al., 2007). Thus, the final kinetic growth model in response to IL-GFE treatment gives the following equation:
(6)
Log10 Nf = (Log10 N0 +0.0077t)-(Log10 N0+0.0035t)
After treating MCF7 cells with IL-GFE, the first order linear equation become: Log10 N = Log10 N0 + 0.0035t
Moreover, the kinetics’ final growth model after treating with Taxol gives the equation as:
(7)
Log10 Nf =(Log10 N0 +0.0077t)-(Log10 N0+0.012t)
Additionally, when treating MCF-7 cells with Taxol, the first-order linear equation can be expressed as: Log10 N = Log10 N0 + 0.012t
(9)
(10)
Where, log10Nf = is the final volume of a tumor after treatment, log10N0 = is the initial volume of a tumor and t = is the duration of treatment (tf-to). The specific growth rate (μ) was then used to achieve growth
(8)
However, to underestimate the effectiveness of the treatment, the 471
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Fig. 8. The percentage of apoptotic MCF7 cells treated with IL-GFE at the corresponding IC50 for 24, 48 and 72 h.
et al., 2012; Moghadamtousi et al., 2014). Hence, these studies revealed that most acetogenins act as a DNA topoisomerase I poison, induced apoptotic cell death at a Bax and caspase 3 related pathways, arrested cancer cells at the G1 phase and inhibited NADH-ubiquinone oxidoreductase of the complex I mitochondrial (Rieser et al., 1991). It can be proposed that the synergistic effects of phytochemicals present in the extracts of Graviola fruit including acetogenins may be responsible for the potential chemotherapy agents of Graviola. These acetogenins found in a large quantity in Graviola extracts may act in synergy and might be responsible for their antiproliferation activity. Thus, further studies on the pure isolated compounds from Graviola fruit through bioassay-guided approach are still required to undoubtedly explore the potent active compounds responsible for the antiproliferative activity of the fruit.
kinetics model based on the fourth equation. This model allows to identify the tumor size at an early stage (less than 1 cc) and predict interpolation and extrapolation of sizes at any time points. It appears from the kinetics model constants that IL-GFE treatment has inhibited MCF-7 cells growth. Cancer is known as a disease of the cell cycle dysfunction. The efficient anticancer drug can block the cell cycle progression in cancer cells (Mantena et al., 2006). To investigate whether IL-GFE induced growth inhibition by the cell cycle arrest, MCF7 cell cycle distribution was examined after IL-GFE-treatment using flow cytometry analysis. The current study demonstrated that IL-GFE arrests the MCF-7-cell cycle at the growth-static G1 phase which explains the anti-proliferation of MCF7 by IL-GFE. Furthermore, the results of the flow cytometry analysis using Annexin V/PI staining showed that GFE has remarkedly induced apoptosis in breast cancer cells. This finding was confirmed by Moghadamtousi et al. (2014) and Chamcheu et al. (2018) when treated human HCT-116 and HT-29 colon cancer, and non-melanoma skin cancer NMSC cell lines with ethyl acetate extract of Annona muricata leaves. Moreover, Fig. 8 indicates the appearance of the necrotic cells in a time-dependent manner which is in close agreement with the results of Torres et al. (2012), after treating pancreatic cancer PC cells with Graviola extract. They observed that Graviola extract inhibited cellular metabolism by inhibiting multiple signaling pathways that regulate metabolism, cell cycle and metastatic properties in pancreatic cancer cells. Thus, both mechanisms either apoptosis and necrosis are induced by Graviola fruit ionic liquid extract. Intensive phytochemical investigations of the fruit and leaves of Graviola have isolated and identified a great number of acetogenins with a potent biological and pharmacological activities, such as anticancer, cytotoxicity, and apoptotic on human cancer cells (Gajalakshmi
5. Conclusion In summary, IL-GFE exhibit antiproliferative effects against MCF-7 breast cancer cell lines by inducing loss of cell viability via apoptosis, morphology changes, and cell cycle arrest at the G0/G1 phase. This inhibition was selective to the growth of MCF-7 cells without any effect on nontumorigenic VERO cells, suggesting that IL-GFE possesses selective antitumor properties toward cancer cells. It also showed their potentiality as an anticancer agent when compared with Taxol as a positive control. Moreover, IL-GFE treatment has inhibited breast cancer MCF-7 cell growth by reducing the number of cell generations and increasing the doubling time compared with the control. These data suggest that the IL-GFE can be developed as a novel mechanism-based supplement agent for the cure and prevention of breast cancer. Further investigations are necessary to elucidate the mechanisms underlying 472
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the therapeutic effects of the fruit extract as well as the determination of the active compounds responsible for cytotoxicity.
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