Enhanced induction of cell cycle arrest and apoptosis ...

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Enhanced induction of cell cycle arrest and apoptosis via the mitochondrial membrane potential disruption in human U87 malignant glioma cells by aloe emodin a

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Samhani Ismail , Khalilah Haris , Abdul Rahman Izaini Abdul a

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Ghani , Jafri Malin Abdullah , Muhammad Farid Johan & Abdul Aziz Mohamed Yusoff

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Department of Neurosciences, Universiti Sains Malaysia, Kubang KerianKelantan16150, Malaysia b

Department of Haematology, Universiti Sains Malaysia, Kubang KerianKelantan16150, Malaysia Published online: 22 Jul 2013.

To cite this article: Samhani Ismail, Khalilah Haris, Abdul Rahman Izaini Abdul Ghani, Jafri Malin Abdullah, Muhammad Farid Johan & Abdul Aziz Mohamed Yusoff (2013) Enhanced induction of cell cycle arrest and apoptosis via the mitochondrial membrane potential disruption in human U87 malignant glioma cells by aloe emodin, Journal of Asian Natural Products Research, 15:9, 1003-1012, DOI: 10.1080/10286020.2013.818982 To link to this article: http://dx.doi.org/10.1080/10286020.2013.818982

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Journal of Asian Natural Products Research, 2013 Vol. 15, No. 9, 1003–1012, http://dx.doi.org/10.1080/10286020.2013.818982

Enhanced induction of cell cycle arrest and apoptosis via the mitochondrial membrane potential disruption in human U87 malignant glioma cells by aloe emodin Samhani Ismaila1, Khalilah Harisa1, Abdul Rahman Izaini Abdul Ghania, Jafri Malin Abdullaha, Muhammad Farid Johanb and Abdul Aziz Mohamed Yusoffa* a


Department of Neurosciences, Universiti Sains Malaysia, Kubang Kerian, Kelantan 16150, Malaysia; bDepartment of Haematology, Universiti Sains Malaysia, Kubang Kerian, Kelantan 16150, Malaysia (Received 13 January 2013; final version received 20 June 2013) Aloe emodin, one of the active compounds found in Aloe vera leaves, plays an important role in the regulation of cell growth and death. It has been reported to promote the anti-cancer effects in various cancer cells by inducing apoptosis. However, the mechanism of inducing apoptosis by this agent is poorly understood in glioma cells. This research is to investigate the apoptosis and cell cycle arrest inducing by aloe emodin on U87 human malignant glioma cells. Aloe emodin showed a time- and dosedependent inhibition of U87 cells proliferation and decreased the percentage of viable U87 cells via the induction of apoptosis. Characteristic morphological changes, such as the formation of apoptotic bodies, were observed with confocal microscope by Annexin V-FITC/PI staining, supporting our viability study and flow cytometry analysis results. Our data also demonstrated that aloe emodin arrested the cell cycle in the S phase and promoted the loss of mitochondrial membrane potential in U87 cells that indicated the early event of the mitochondria-induced apoptotic pathway. Keywords: aloe emodin; apoptosis; mitochondrial membrane potential; U87 glioma cells

1. Introduction Malignant gliomas are the most common fatal of all human brain tumors. Despite advanced multiple modalities of the treatment, malignant gliomas remain incurable in most cases [1]. Patients with malignant gliomas have poor prognosis because it is highly resistant to current chemotherapeutic drugs, and there is no single effective chemical against it. Two or three agents are often combined to enhance the efficacy of chemical agents. Chemotherapy drugs can cause serious toxic effects. Thus, it is crucial to develop new therapeutic

approaches with new mechanisms of action. One of the best approaches for destroying cancer cells is via induction of apoptosis in cancer cells. Apoptosis can be induced by two major pathways. One is the best known as the intrinsic apoptosis pathway that involves the collapse of mitochondrial membrane potential [2] and the release of mitochondrial proteins such as cytochrome c into the cytoplasm [3]. For many years now, researchers have been focusing on the natural products from plant to kill cancer cells growth through the induction of apoptosis.

*Corresponding author. Email: [email protected] q 2013 Taylor & Francis


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Aloe emodin (1,8-dihydroxy-3-hydroxymethyl-anthraquinone) is a natural active compound found in the leaves of A. vera, well known for its wide spectrum of pharmacological effects [4,5]. The chemical structure of aloe emodin is shown in Figure 1, and it has a molecular weight of 270.24 g/mol. Recently, researchers have found that aloe emodin can inhibit growth of several cancer cell lines [6–8]. Moreover, aloe emodin anti-cancer activity involves induction of apoptosis through mitochondrial pathways, as demonstrated in various cancers [9–11]. So far, there is no available information about the anti-cancer effects of aloe emodin on human glioma cells involving the mitochondria-induced apoptotic pathway. In this study, we determined the anti-proliferative activity and cell cycle arrest induction of aloe emodin and investigated whether aloe emodin might induce apoptosis in human U87 malignant glioma cells by the loss of mitochondrial membrane potential. 2. Results and discussion 2.1 Aloe emodin inhibits proliferation of U87 cells The anti-proliferative effect of aloe emodin on exponentially grown U87 cells was evaluated using MTS. The MTS assay showed that aloe emodin significantly inhibited the viability of U87 cells (Figure 2 (a)). The cells were incubated in the absence or presence of increasing concentrations of aloe emodin (0, 20, 40, 60, 80 mg/ml) for specified time periods (6, 24, 48, 72 h), and

OH

O

OH

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Figure 1. Chemical structure of aloe emodin.

the 50% inhibitory concentration, IC50, values were 58.6, 25.0, and 24.4 mg/ml for 24, 48, and 72 h, respectively. This IC50 value for 6 h treatment cannot be achieved as the cell proliferation was not reaching even 50% of cell viability. The MTS assay showed that aloe emodin decreased the viability of U87 cells in a time- and dosedependent manner. Observations of cell death under phase contrast microscope using unstained cells further confirmed the cytotoxic effects of aloe emodin on U87 cells (Figure 2(b)).

2.2 Aloe emodin induced DNA fragmentation A time-dependent occurrence of cellular DNA fragmentation was examined by gel agarose electrophoresis and a ladder-like pattern, typical of DNA cleavage between nucleosomes, was observed during 24 – 48 h after incubation with aloe emodin (58.6 mg/ml) (Figure 3), whereas control cells did not provide ladders. Thereby, it is possible that aloe emodin causes apoptosis of U87 cells.

2.3 Aloe emodin induces apoptosis in U87 glioma cell lines To quantify the induction of apoptosis, we treated U87 cells for 24 and 48 h with (58.6 mg/ml) or without aloe emodin and assayed by flow cytometry after staining with Annexin V-FITC/PI. The percentage of early apoptotic U87 cells was significantly increased (63.88 ^ 35.05%) after 24 h of treatment with aloe emodin (58.6 mg/ml) (Figure 4). After treatment with aloe emodin for 48 h, early apoptotic cells gradually decreased to 51.77 ^ 16.12%, whereas late apoptotic cells increased to 30.04 ^ 11.03%. Meanwhile, the necrosis cells remained at minimal count indicating that the inhibition of U87 cells growth by aloe emodin was due to the induction of apoptosis.

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Effect of various concentration of aloe emodin on U87

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Figure 2. Effects of aloe emodin on cell viability in U87 cells. (a) Time- and dose-dependent effect of aloe emodin was performed when the U87 cells were treated with various concentrations of aloe emodin for various time. Results are mean values ^ SD of three independent experiments performed in triplicate (n ¼ 3). (b) U87 cells were treated with DMSO (control) or with aloe emodin for 48 h. Cells were observed under phase contrast microscopy (10 £ magnification). Phase contrast microscopy shows a reduction in U87 viability induced by aloe emodin treatment as evaluated by the MTS assay (5 £ 104 cells/mm2 vs 2 £ 104 cells/mm2). Scale bar ¼ 200 mm.

2.4 Aloe emodin-treated U87 glioma cells showed apoptotic morphological changes

untreated cells did not show evident apoptotic morphological changes.

To obtain further evidence for the induction of apoptosis, the morphological changes of cells such as apoptotic body formation in aloe emodin-treated U87 cells were also observed in cells stained with Annexin VFITC/PI using confocal microscope. As shown in Figure 5, typical morphological changes, such as the formation of apoptotic bodies with condensed chromatin appeared after the cells, were treated for 24 h with 58.6 mg/ml aloe emodin, whereas the

2.5 Aloe emodin induces S phase cell cycle arrest In addition to apoptosis, we also examined the distribution of cell cycle after the U87 cells were exposed to aloe emodin. This experiment was performed by flow cytometry, in order to determine its inhibitory effect via the induction of cell cycle arrest. As shown in Figure 6, our study revealed that aloe emodin induced S phase arrest in U87 cells. After treatment with


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S. Ismail et al. initiating cellular apoptosis, we evaluated whether the treatment with aloe emodin on U87 cells had any effect on the mitochondrial membrane potential. To examine mitochondrial membrane dysfunction induced by aloe emodin, JC-1 staining was performed followed by flow cytometry analysis. As shown in Figure 7, exposure to aloe emodin for 24 – 48 h resulted in slightly significant decrease in the ratio of red/green fluorescence intensity. Flow cytometry analysis also showed that high integrity of mitochondrial membranes was maintained in control cells (Figure 7). The data suggested that aloe emodin indeed depolarized the mitochondrial membrane potential.

Figure 3. Assessment of apoptosis in U87 cells by the DNA fragmentation assay. U87 cells were under untreated or treated with aloe emodin for 6 h (lane 6), 24 h (lane 24), and 48 h (lane 48). Molecular weight standards are shown on the same gel at left (lane M).

58.6 mg/ml aloe emodin, the accumulations of cells in S phase were observed in the increasing rate, which reached 22.5% at 24 h and 39.8% at 48 h compared with the control (16.9%). A peak of the sub-G0/G1 cell population, an indicative of apoptosis, was seen following 48 h exposure. 2.6 Aloe emodin disrupts mitochondrial membrane potential in U87 cells As the disruption of the mitochondrial membrane potential is a crucial event

2.7 Examination of aloe emodininduced mitochondrial membrane potential disruption by confocal microscope Confocal microscope was used to validate the results obtained from flow cytometry after staining cells with JC-1 dye. JC-1, a cationic dye, produces red fluorescent J-aggregates in healthy cells with high mitochondrial membrane potential, whereas exhibits green fluorescence in apoptotic cells with low mitochondrial membrane potential (Figure 8). As expected after 3 h of aloe emodin treatment, U87 cells exhibited green fluorescence, indicating a collapse of mitochondrial membrane potential,

Figure 4. Scatter plot of Annexin V/PI-stained U87 cells untreated (A), treated by aloe emodin for 24 h (B) and 48 h (C). They are under four situations quadrant analysis; they are living cells (Q3), early apoptotic cells (Q4), late apoptosis cells (Q2), and necrotic cells (Q1).


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Figure 5. Confocal images after AnnexinV/PI/DAPI-staining showing features of apoptosis induced by aloe emodin on U87 malignant glioblastoma cell lines. Cells after treated 24 h and with 40 £ resolution reveal cell shrinkage, blebs, apoptotic bodies, and condensed chromatin. Cells untreated (control); most of the cells are healthy and have fibroblast-like shape.

whereas most control cells displayed in red J-aggregation corresponding to a high potential. The results suggested that the

disruption of mitochondrial membrane potential in U87 cells could be induced by aloe emodin.

Figure 6. Cell cycle analysis of U87 cells during aloe emodin treatment. Cells were treated or not (control) with 58.6 mg/ml for 24 and 48 h. The distribution of cell cycle was assessed by flow cytometry. The cell cycle distribution was represented as and compared in the 100% stacked column chart. Aloe emodin led to an arrest of cell cycle progression at the S phase, and the appearance of sub-G0/G1 population represented the apoptotic cells.


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Figure 7. There is a slightly significant increase in the number of cells with lowered red fluorescence time-dependent manner, indicating change in the mitochondrial membrane potential (DCm) in the populations induced by aloe emodin to undergo apoptosis.

Figure 8. JC-1 analysis of mitochondrial membrane potential in U87 cells by confocal microscopy. U87 cells were exposed to 58.6 mg/ml aloe emodin for 3 h. Red fluorescence in control U87 cells indicated high membrane potential (A), and green fluorescence in aloe emodin-treated U87 cells indicated loss of mitochondrial membrane potential (B).

3. Discussion Phytochemicals and herbal extracts have recently been examined for their induction ability of apoptosis in cancer cells [12]. These compounds are believed to be the candidates of novel chemotherapeutic agents that can improve the anti-cancer properties for the treatment of cancer. Aloe emodin is one of the active components present in A. vera leaves and has been determined to have significant anticancer effect on human lung cancer cells [13], cervical cancer cells [14], nasopharyngeal cancer cells [15], and leukemia cells [16,17] without any appreciable toxic effects. Despite that its effect on human

malignant glioma represented by U87 cell lines has not yet been addressed. In order to gain a better understanding of the anti-cancer effect of aloe emodin in glioma cells, we studied the anti-proliferation and early apoptosis induction by aloe emodin on human U87 malignant glioma cells. In our study, we found that aloe emodin inhibits U87 cells proliferation in a time- and dose-dependent manner with the IC50 of 58.6 mg/ml at 24 h, 25.0 mg/ml at 48 h, and 24.4 mg/ml at 72 h. The IC50 value observed in our study is much higher than the value of previously reported in human lung squamous cell carcinoma CH27 [7], human liver cancer cells HepG2 and Hep3B [8], human gastric


Journal of Asian Natural Products Research carcinoma AGS [18], and human nasopharyngeal carcinoma NPC cells [15], showing that U87 cells are less sensitive to aloe emodin than other cells. This study also suggested that aloe emodin has differential cytotoxic effects against the sensitivity of different human cells. In addition, the same dose of aloe emodin does not affect normal human astrocytes cells proliferation (data not shown). The term ‘apoptosis’ was first defined in 1972 by Kerr et al., to describe a fundamental biological process of programed cell death, which is essential for the development and survival of all multicellular organisms [19]. The characteristic morphological changes observed during apoptosis include membrane blebbing, cell shrinkage, chromatin condensation, DNA fragmentation, and apoptotic body formation [19]. To determine whether apoptosis is involved in the cell death caused by aloe emodin on U87 cells, we used multiple approaches to assess apoptosis, including flow cytometry analysis of Annexin VFITC/PI staining, morphological changes, and DNA laddering pattern on agarose gel electrophoresis. This study revealed that aloe emodin resulted in apoptosis (cell stained positive with Annexin V), but not necrosis of treated U87 cells in a timedependent manner (Figure 4). The percentage of late apoptotic increased with the incubation time. Our flow cytometry results were supported by morphological changes experiment where it showed the hallmark features of apoptotic cells (Figure 5). From these data, we can conclude that aloe emodin induced apoptosis in U87 cells. DNA fragmentation analysis by agarose gel electrophoresis also showed a typical apoptotic oligonucleosomal fragmentation of the DNA. In addition, we subsequently examined whether aloe emodin could induce cell cycle arrest, in order to determine another mechanisms, besides apoptosis, involved in aloe emodin-induced inhibition of U87

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cell proliferation. Indeed, in our experiment, it was found that aloe emodin induced S phase arrest of the cell cycle in U87 cells. The S phase cell cycle arrest ability of aloe emodin was in consistence with other reports in different types of cancer cells. For instances, the S phase arrest was reported in human gastric cancer cells [14] and tongue squamous cancer cells [11]. Other studies have also reported that aloe emodin caused G2/M phase arrest in human cervical cancer [14] and nasopharyngeal cancer cells [15], whereas the G1 phase arrest was identified in hepatoma cells [8]. Our results suggested that the ability of aloe emodin in arresting multiple phase of cell cycle may be responsible for the anticancer effects on different types of cancers. Mitochondria are deeply believed as a regulatory role in initiating apoptosis [20]. The disruption of the mitochondrial membrane potential is an established indicator of mitochondrial dysfunction where it triggers the first step in the intrinsic caspase/proapoptotic activation [21]. Activation of these proapoptotic proteins leads to cytochrome c release into the cytoplasm [11,15]. In the current study, we used a fluorescent dye, JC-1, which has advantages over the detection of the mitochondrial membrane potential changes in cells. We report that aloe emodin-treated cells resulted in a higher green/red fluorescence ratio and conclude that aloe emodin induced the disruption of mitochondrial membrane potential in U87 cells. In conclusion, aloe emodin has a powerful anti-proliferative effect by inducing early apoptotic cell death against U87 malignant glioma cells, suggesting as a potential candidate for preventive and therapeutic approach for brain cancer treatment. However, further investigations are needed in order to assess the activation of proapoptotic/caspase cascade and confirm the finding on the molecular mechanisms

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involved in aloe emodin-induced apoptosis in U87 cells.


4.

Experimental

4.1 Chemical and reagents Aloe emodin and dimethyl sulfoxide (DMSO) were purchased from SigmaAldrich Co. (St. Louis, MO, USA). Dulbecco’s-modified eagle’s medium (DMEM), fetal bovine serum (FBS), L glutamine, penicillin, streptomycin, and trypsin were purchased from Gibco Invitrogen Corp. (Carlsbad, CA, USA). The CellTiter 96w AQueous Non-Radioactive Cell Proliferation Assay (MTS) was bought from Promega (Madison, WI, USA). The FITC Annexin V Apoptosis Detection Kit I and CycleTESTe PLUS DNA Reagent Kit were purchased from BD Biosciences (San Jose, CA, USA). The JC-1 (5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolcarbocyanine iodide) was product of Molecular Probes, Inc. (Carlsbad, CA, USA). 4.2

Cell culture and treatment

The human glioma cell line U87 was purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM supplemented with 10% FBS, 2 mM L -glutamine, 100 units/ ml penicillin, and 100 mg/ml streptomycin and maintained at 378C with 5% CO2 in a humidified atmosphere. The medium was changed every 2 – 3 days. When the cultures were about 90% of confluence, the cultured cells were washed with phosphate-buffered saline (PBS, pH 7.4), detached with 0.25% trypsin-EDTA, and sub-cultured. Aloe emodin was dissolved in less than 1% DMSO to a stock concentration at 10 mg/ml. It was further diluted in fresh growth medium to the desired working concentration before use. The cells were treated with different concentrations of aloe emodin for various lengths of time.

4.3 Cell viability assay The viability of U87 cells treated with and without aloe emodin was analyzed by CellTiter 96w AQueous Non-Radioactive Cell Proliferation Assay, also known as MTS assay. MTS is a tetrazolium salt [3(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salt] that undergoes a color change caused by its bioreduction of MTS into a blue formazan product. Briefly, cells were seeded on 96-well culture plates at density of 5 £ 104 cells/ well. Twenty h post plating, the medium was removed and cells were incubated with aloe emodin at concentrations of 0, 20, 40, 60, and 80 mg/ml in fresh media. At the end of various treatments, 10 ml of MTS solution was added to each well and further incubated for 1 h. The OD samples at 490 nm were read using a microplate reader. The inhibition percentage of cell growth was calculated as (OD of drug-treated sample/OD of control) £ 100%. All experimental data were derived from at least three independent experiments, and the sigmoidal inhibition concentration dose curves were plotted as concentration of aloe emodin (mg/ml) versus percentage of viable cells.

4.4

DNA fragmentation assay

U87 cells (1 £ 105cells/well) were seeded in 6-well plates and treated with 58.6 mg/ ml of aloe emodin for 6, 24, and 48 h. Both attached and floating cells were collected by trypsinization and centrifugation. Total DNA was purified using a rapid preparation of genomic DNA kit (Genomic DNA Isolation Kit; Norgen Biotek Corporation, Ontario, Canada) according to the manufacturer’s instructions. The DNA was separated on 1% agarose gel electrophoresis and visualized under UV light after staining with ethidium bromide.


Journal of Asian Natural Products Research 4.5 Flow cytometry analysis of Annexin V/ propidium iodide (PI) staining U87 cells were plated 5 £ 105 on 6-well culture plates and allowed to adhere; 58.6 mg/ml aloe emodin was used to treat the cells for 24 and 48 h. Mode of cell death induced by aloe emodin was measured by staining the cells with Annexin V conjugated with fluorescein isothiocyanate (FITC) combined with (PI), according to FITC Annexin V Apoptosis Detection Kit I protocol outlined by manufacturer. Percentages of cells in viable, early apoptosis, late apoptosis, and necrosis states were detected by flow cytometry (BD FACS CantoTM II analyzer) (BD Biosciences, San Jose, CA, USA) and analyzed by BD FACSDiva software version 6.1.2 (Becton, Dickinson and Company, San Jose, CA, USA). 4.6 Microscopic analysis of cell morphology To validate our flow cytometry assay, characteristic morphological changes of cells, such as the formation of apoptotic bodies, were observed with confocal microscope after Annexin V-FITC/PI staining. Cells were cultured in 8-chamber slides in the presence of 58.6 mg/ml aloe emodin for 24 and 48 h. Cells were then washed with PBS twice and stained with FITC Annexin V Apoptosis Detection Kit I according to the manufacturer’s instructions. In brief, 5 ml Annexin V-FITC and 5 ml PI were diluted with 400 ml binding buffer, and then the mixture was added to the cells for 15 min at room temperature in the dark. Stained cells were examined under Laser Scanning Confocal Microscope LSM 700 (Carl Zeiss, Jena, Germany). 4.7 Cell cycle arrest study Cell cycle arrest analysis was performed using the CycleTESTe PLUS DNA Reagent Kit, which is based on the

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measurement of the DNA content of nuclei labeled with PI. Briefly, cells were seeded at 5 £ 105 cells/well in 6-well culture plates and incubated with or without aloe emodin for 24 and 48 h. Cells were harvested, washed with PBS, treated with trypsin and Rnase, and then stained with PI in accordance with the manufacturers’ instructions. Subsequently, the cell cycle phases (G0/G1, S, and G2/M) were determined with flow cytometry FACS Canto II (BD Biosciences) and analyzed using BD FACSDiva software version 6.1.2 (Becton, Dickinson and Company).

4.8 Measurement of mitochondrial membrane potential U87 cells at 1 £ 105 cells/ml were treated with 58.6 mg/ml of aloe emodin for 24 and 48 h, then incubated with 10 mg/ml JC-1 at room temperature for 15 – 20 min. Stained cells were washed with PBS, followed by flow cytometric analysis using green (FL-1) and red (FL-2) channels according to manufacturer protocol with some modification. Red aggregates indicated high mitochondrial membrane potential (590 nm), whereas green monomer was measured as depolarization of DC m (523 nm).

4.9 Mitochondrial membrane potential imaging using confocal microscope To support our flow cytometry results that JC-1 stained mitochondria in a potential dependent manner, the changes of mitochondrial membrane potential were visualized by confocal microscope. Cells were seeded 5 £ 105 on square cover glass in 6well plates. Cells were then treated with aloe emodin and stained with JC-1 in the same way as for the flow cytometry. Cells were rinsed once with warmed dye-free culture media followed by PBS and viewed under confocal microscope.

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4.10 Statistical analysis All data presented were obtained from three independent experiments and are presented as mean ^ standard deviation (SD). Statistical significance was assessed by the Student’s t-test, performed using the statistical software package SPSS 19.0 (SPSS).


Acknowledgments We are very grateful to Mr Jamaruddin Mat Asan from Department of Immunology, Universiti Sains Malaysia, for his advice and help regarding flow cytometry. This study was financially supported by Fundamental Research Grant Scheme (FRGS) (203/PPSP/6171125) from the Ministry of Higher Education of Malaysia.

Note 1.

These authors contributed equally to this work.

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