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Involvement of NF-[kappa]B and Bcl2/Bax signaling pathways in the apoptosis of MCF7 cells induced by a xanthone compound Pyranocycloartobiloxanthone A.


The plant Artoccirpus obtusus is a tropical plant that belongs to the tamily Moraceae. In the present study a xanthone compound Pyranocycloartobiloxanthone A (PA) was isolated from this plant and the apoptosis mechanism was investigated. PA induced cytotoxicity was observed using MU assay. High content screening (HCS) was used to observe the nuclear condensation, cell permeability, mitochondrial membrane potential (MMP) and cytochrorne c release. Reactive oxygen species formation was investigated on treated cells by using fluorescent analysis. Human apoptosis proteome profiler assays were performed to investigate the mechanism of cell death. In addition mRNA levels of Bax and Bcl2 were also checked using RT-PCR. Caspase 3/7,8 and 9 were measured for their induction while treatment. The involvement of NF-[kappa]B was analyzed using HCS assay. The results showed that PA possesses the characteristics of selectively inducing cell death of tumor cells as no inhibition was observed in non-tumorigenic cells even at 30 p.g/rnl. Treatment of MCF7 cells with PA induced apoptosis with cell death-transducing signaLs, that regulate the MMP by down-regulation of Bcl2 and up-regulation of Sax, triggering the cytochrorne c release from mitochondria to cytosol. The release of cytochrome c triggered the activation of caspases-9, then activates downstream executioner caspase-3/7 and consequently cleaved specific substrates leading to apoptotic changes. This form of apoptosis was found closely associated with the extrinsic pathway caspase (caspase-8) and inhibition of translocation of NF-[kappa]B from cytoplasm to nucleus. The results demonstrated that PA induced apoptosis of MCF7 cells through NF-[kappa]B and Bc12/Bax signaling pathways with the involvement of caspases.

[c] 2012 Elsevier GmbH. All rights reserved.


Keywords: Artocarpus obtusus Apoptosis Mitochondria Protein array Caspase 3/7 NF-[kappa]B


Traditional medicinal herbs are widely known to be effective in the treatment of many diseases, particularly those that could not be cured by modern medicine. In case of cancer, phytochemicals from these herbs has been proven to decrease the risk of cancer and increase the survival of patients (Issa et al. 2006; Yoon et al. 2011). Several phytochemicals from the nature have exhibited significant anticancer as well as apoptosis effects by targeting various molecular and cellular mechanisms towards breast cancer (Awad et al. 2007; Choi and Kim 2008).

Apoptosis is a vital physiological process essential for normal development and maintenance of tissue homeostasis (Desagher and Martinou 2000). This mode of cell death is widely studied, since the importance of regulation of apoptosis contributes to the key factor in the anticancer drug development. Among the various targets for cancer research, reactive oxygen species (ROS) is considered as an important one for anticancer drug research, since accumulation of excessive ROS will leads to oxidative DNA damage (Evans et al. 2004; Schumacker 2006) followed by disruption of the mitochondrial membrane potential (MMP) and release of cytochrome c into the cytosol, to triggers caspase-9 activation and initiates the executioner caspases which leads cell to apoptosis (Simon et al. 2000). In addition, the susceptibility of tumor cells to the induction of apoptosis by chemotherapeutic agents is controlled by the ratio of Bcl2/Bax proteins in the mitochondria (Mohan et al. 2010).

Subsequent to Bcl2 family proteins, heat shock proteins (HSP) also considered as promote tumorigenesis (Jaattela 1999). HSPs are also known to protect cells from stress by preventing the pro-tein aggregation and promote the refolding of denatured proteins (Bukau and Norwich 1998). Increased expression of HSP70 has been reported in high-grade malignant tumors (Knorr et al. 2010: Yang et al. 2011). As HSPs have the ability to prevent the drug induced apoptosis, inhibitors to I-1SP could be a target of right drug candidate identification. Not only HSPs, but nuclear factor-kappa B (NE-[kappa]B), a ubiquitous transcription factor also plays an important role in governing apoptosis and inflammation (Su et al. 1999).

The plant Artocarpus obtusus is tropical plant belongs to the family Moraceae. Recently Hashim et a]. have reported that a xanthone compound Pyranocycloartobiloxanthone A (PA) (Fig. 1) exert antiproliferative activity and apoptotic mode of cell death in MCF7 cells (Hashim et al. 2012). Now, we have further found that PA activates a complex signaling pathway required for cell death induction. In particular, an early downregulation of Bcl2, upregulation of Bax, release of cytochrome c from mitochondria into cytosol and the sequential activation of caspases were found to occur in PA-induced apoptosis. The production of ROS also was present in the cells after treatment. In addition, treatment with PA resulted in significant inhibition of NE-[kappa]B translocation from cytoplasm to nuclei activated by tumor necrosis factor alpha (TNF-[alpha]).

Materials and methods

Plant materials

The stem bark ofArtocarpus obtusus was collected from Sarawak, identified by Dr. Rusea Go, and a voucher specimen (S94402) has been deposited at the Herbarium, Department of Biology, Faculty of Science, Universiti Putra Malaysia.

Extraction and isolation of Pyranocycloartobiloxanthone A (PA)

Pyranocycloartobiloxanthone A as yellow needle-shaped crystals was purified from the dried and ground stem bark in our lab. Their chemical and physical data as obtained in our work were in agreement with those reported previously (Hashim et al. 2010).

Cell viability assay

All cells that are used in this study were obtained from American Type Cell Collection (ATCC) and were maintained in 37[degrees]C incubator with 5% CO2 saturation. MCF7 human breast adenocarcinoma cells, MCF 10A a non-tumorigenic epithelial cell line and WRL-68 normal hepatic cells were maintained in RPMI-1640 medium that is supplemented with 10% fetal bovine serum (FBS). Viability assay was done using MTT assay as previously described by Mosmann (1983). Briefly, cells (5 x 104 cells/m1) were treated with PA at different concentration in 96-well plate and incubated for 24 It The colorimetric assay is measured and recorded at absorbance of 570 nrn. Results were expressed as percentage of control giving percentage cell viability after 24 h exposure to test agent. The potency of cell growth inhibition for test agent was expressed as [IC.sub.50] value.

Measurement of reactive oxygen species generation

The production of intracellular ROS was measured using 2),7'-dichlorofluorescin diacetate (DCFH-DA). Briefly, 10 mM DCFH-DA stock solution (in methanol) was diluted 500-fold in Hank's bal-anced salt solution (HBSS) without serum or other additives to yield a 20 pLM working solution. After 24 h of exposure to PA the cells in the 96-well black plate was washed twice with HBSS and then incu-bated in 100 [mu]l working solution of DCFH-DA at 37 JC for 30 min. Fluorescence was then determined at 485-nm excitation and 520-nm emission using a fluorescence microplate reader (Tecan Infinite M 200 PRO, Mannedorf, Switzerland).

Multiple cytotoxicity assay

Cellomics Multiparameter Cytotoxicity 3 Kit was used as described in detail previously (Cheah et al. 2011), This kit enables simultaneous measurements in the same cell of six independent parameters that monitor cell health, including cell loss, nuclear size and morphological changes, mitochondrial membrane potential changes, cytochrome c release, and changes in cell permeability. Tamoxifen 0.04 [mu]g/m1 was used as positive control in this apopto-sis detection. Plates were analyzed using the ArrayScan HCS system (Cellomics, PA, USA).

Detection of NF-[kappa]B activity HCS was used to measure the inhibitory effects of PA on TNF-[alpha]-induced NE-[kappa]B activation, i.e. nuclear translocation of NE-[kappa]B. The experiments were performed according to manufacturer's instructions for the NE-[kappa]B activation kit (Cellomics). ArrayScan reader was used to quantify the difference between the intensity of nuclear and cytoplasmic NE-[kappa]B-associated fluorescence, reported as translocation parameter.

Image acquisition and cytometric analysis

Plates with stained cells were analyzed using the ArrayScan HCS system (Cellomics, PA, USA). This system is a computerized automated fluorescence imaging microscope that automatically identifies stained cells and reports the intensity and distribution of fluorescence in individual cells. The Array-Scan RCS system scans multiple fields in individual wells to acquire and analyze images of single cells according to defined algorithms. In each well, 1000 cells were analyzed. Automatic focusing was performed in the nuclear channel to ensure focusing regardless of staining intensities in the other channels. Images were acquired for each fluorescence channel, using suitable filters. Images and data regarding intensity and texture of the fluorescence within each cell, as well as the average fluorescence of the cell population within the well were stored in a Microsoft SQL database for easy retrieval. Data were captured, extracted and analyzed with ArrayScan II Data Acquisition and Data Viewer version 3.0 (Cellomics).

Human apoptosis proteome profiler array

To investigate the pathways by which PA induces apoptosis, we performed a determination of apoptosis-related proteins using the Proteome Profiler Array (RayBio[R] Human Apoptosis Antibody Array Kit, Raybiotech, USA), according to manufacturer's instruc-tions. In short, the cells where treated with 5 p.g/m1 PA. Three hundred micro gram proteins from each sample were incubated with the human apoptosis array overnight. The apoptosis array data were quantified by scanning the membrane on a Biospectrum AC ChemiHR 40 (UVP, Upland, CA) and analysis of the array image file was performed using image analysis software according to the manufacturer's instruction.

Analysis of mRNA expression by RT-PCR

Total RNA from MCF7 cells were extracted using RNeasy Mini Kit (Qiagen, Germany). 1 peg of total RNA was used to perform the reverse transcription with the QuantiTect Rev. Transcription Kit (Qiagen, Germany). The transcribed cDNA (1 [mu]l) was used for polymerase chain reaction (PCR) amplification with specific primers of Bax and Bcl2 genes, and the PCR reaction were carried out. [beta]-Actin mRNA served as the loading control.

The primers for Bax: sense, 5'-TTTGCTTCAGGGTTTCATCC-3', and antisense, 5'-GCCACTCGGAAAAAGACCTC-3'. The primers for Bcl2: sense, 5'-ATGMCTCTTCCGGGATGG-3', and antisense, 5'-TGGATCCAAGGCTCTAGGTG-3'. The primers for 13-actin: sense, 5'-CGGGMATCGTGCGTGAC-3', and antisen, 5'-GCCTAGAAGCATTTGCGGTG-3'. PCR amplification was performed with a thermal cycle, and the reaction was started with the initial denaturation at 95 'C for 5 min, followed by 30 cycles of denaturation, annealing and extension at 95 [degrees]C for 30 s, 60 [degrees]C for 40 s, and 72 [degrees]C for 1 min, respectively. The reaction was terminated after final extension at 72 "C for 10 min. The sizes of the amplification products of Bax, Bcl2 and P-actin were 213, 166 and 515 bp, respectively. The PCR products were subjected to 1.5% agarose gel electrophoresis and were stained with ethidium bromide. It was visualized under UV light using Gel Doc XR System (Bio-Rad, USA).

Western blot

MCF7 cells in 12-well plates were treated with different concentrations of PA. The total proteins of cells were extracted with cell lysis buffer (50 mM Tris-HCL pH 8.0, 120 mM NaC1, 0.5% NP-40, 1 mM PMSF), and 40 rig of protein extract was separated by 10% SDS-PAGE, then transferred to a polyvinylidenedifluo-ride (PVDF) membrane (Bio-Rad), blocked with 5% nonfat milk in TBS-Tween buffer 7 (0.12 M Tris-base, 1.5 M NaCI, 0.1% Tween20) for 1 h at room temperature, and incubated with the appropriate antibody overnight at 4 [degrees]C, then incubated with horseradish peroxidase conjugated secondary antibody for 30 min at room temperature. The bound antibody was detected with peroxidase-conjugated anti-rabbit antibody (1:10,000) or anti-mouse antibody (1:10,000) followed by chemiluminescence (ECL System) and exposed by autoradiography. The following primary antibodies [beta]-actin (1:10,000), Bcl2 (1:1000), Bax (1:1000), HSP70 (1:1000), were purchased from Santa Cruz Biotechnology, Inc., California, USA.

Statistical analysis

Results were reported as mean [+ or -] SEM for at least three analyses for each sample. Normality and homogeneity of variance assumptions were checked. Statistical analysis was performed according to the SPSS-16.0 package and GraphPad prism 3.0. Analyses of variance were performed using the ANOVA procedure.


PA inhibited the growth of MCF7 cells selectively in vitro

The cytotoxic effects of PA on MCF7 cells were assessed using the MTT assay. As shown in Table 1, PA inhibited the growth of MCF7 cells and exhibited significant inhibition at concentrations of 3.5 [+ or -] 0.50 and 2.4 [+ or -] 0.21 pig/m1 at 24 and 48 h respectively. Meanwhile, the normal cells (MCF10A and WRL 68) used in this study did not died significantly even at the highest concentrations (30 peg/m1) of PA.

Table 1

[IC.sub.50] concentration of PA.

Cell line     [IC.sub.50] [+
               or -] SD
               24h        48h

MCF7           3.5 [+ or   1.4 [+
                 -] 0.50    or -]

MCF10A               >30      >30

WRL-68               >30      >30

PA-induced apoptosis in MCF7 cells

To confirm the presence of apoptosis, we examined nuclear morphological changes of MCF7 cells by determining nuclear condensation and fragmentation hallmark for apoptosis (Fig. 2). Hoechst 33342 staining showed that a part of the cells displayed nuclear condensation at 24h after PA treatment. The nuclear intensity which is directly corresponding to apoptotic chromatin changes: blebbing, fragmentation and condensation where quantitated in Fig. 3A. Meanwhile, concurrent increase in the cell permeability also was observed (Fig. 3B).

PA-induced MMP disruption and release of cytochrome c

MMP was significantly reduced on cells treated with PA (55% at 51.1g/ml, p = 0.01) (Fig. 3C). Changes of mitochondrial membrane potential in MCF7 cells treated with PA 3 and 5 pig/m1 for 24 h showed a significant reduction of fluorescence intensity (Fig. 2), which reflected the collapse of MMP Meanwhile, PA triggered the MCF7 cells to translocate the cytochrome c from mitochondria into cytosol during apoptosis significantly (Fig. 2). At 5 [mu]g/ml PA triggered the cytochrome c release by 8-fold (p < 0.01) (Fig. 3D).

PA induced cell death includes increased ROS formation

The generation of intracellular ROS is always associated with MMP disruption and cell apoptosis (Castedo et al. 1996). Therefore, we examined the levels of ROS in MCF7 cells treated with PA. ROS was monitored by the oxidation-sensitive fluorescent dye DCFH-DA. A concentration depended increase in DCF fluorescence was detected in treated cells (Fig. 4). Rapid generation of ROS, up to 2-fold faster than the control, was detected at 5 pig/m1 treatment.

Effect of PA on apoptotic markers

After PA exposure for 24 h, MCF7 cells were lysed and apoptotic markers where screened using protein array. In Fig. 5 images are shown which are representative for the observed changes. All major markers which are involved in the apoptosis signaling pathway, such as bax, Bcl2, Bim, Caspase 3, 8, cytochrome c were induced in both models. HSP70, a significant chaperone involved in the apoptosis also was down regulated. In addition, cell proliferation repressor proteins, p27 and p21, also were induced in this in vitro model. Besides, various IGFBP also were induced while treatments.

RT-PCR analysis of Sax and Bcl2 mRNA

The expression levels of Bax and Bcl2 mRNA (Fig. 6) was evaluated by RT-PCR analysis. Expression of Bax was low in control group cells and was significantly increased in the PA treated group (p <0,05). Even though Bcl2 expression was down regulated compared to control, it was not significant (p> 0.05).

PA up-regulated Bax and suppressed the expression of Bcl2 and HSP70 protein

Although many proteins implicated with apoptosis were observed to be up or down regulated in the protein array, proteins such as bax, and FISP70 were significantly induced. Together with this, keeping in mind the changes occurred to the MMP and cytochrome c release, we were then confirmed the role of mitochondria in the apoptosis occurred by PA at protein level using western blot analysis. Exposure of MCF7 cells to PA increased the pro-apoptotic protein, Bax and decreased the expression of anti-apoptotic, Bcl2 protein. Further, the expression of HSP70 did down regulated in a concentration depended manner (Fig. 7).

PA induced caspases activation

All the caspases under investigation found to be induced while the treatment, and was found to be concentration-dependent. High level of caspase 3/7 was found at the highest treatment concentra-tion (5 [mu]g/ml) with a significance difference from control (p < 0.01). High levels of caspase-8 caspase 9 were also detected. But the activation of caspase was not significantly induced for caspase 3/7 at 3 pig/ml. These results further confirmed the activation of caspase3/7, -8 and -9 by PA in MCF7 cells (Fig. 8). PA inhibits TNE-[alpha]-induced NE-KB nuclear translocation

The blockage of apoptosis and cell proliferation is closely associated with activation of NF-[kappa]B. So we then examine the role of PA in the inhibition of activated NF-[kappa]B induced by the inflamma-tory cytokine, TNF-[alpha] using Alexa Fluor 488-conjugated anti-NF-[kappa]B antibody. In control cells (medium alone) (Fig. 9A), high NF-[kappa]B fluorescent intensity was found in cytoplasm but faintly in nuclei, which indicating that there was no NF-[kappa]B activation at the non-stimulated condition. Meanwhile, INF-[alpha] alone stimulated cell significantly increased the NF-[kappa]B fluorescent intensity in the nuclei. PA exhibited significant inhibitory effects on the activation of NF-[kappa]B (Fig. 9B). In the cells treated with curcumin, a known inhibitor of NF-[kappa]B activation, it was observed that significant inhibition of TNF-[alpha]-induced NF-[kappa]B nuclear translocation as evidenced by low nuclear NF-[kappa]B-related fluorescence intensity (Fig. 9B). In paral-lel, the morphological changes of NF-[kappa]B translocation indicated by immunofluorescence staining (Fig. 9B) showed an inhibitory effect of PA on TNE-[alpha]-induced NF-[kappa]B translocation in a dose-dependent manner with a strong inhibition at 5 [mu]g/ml concentration of PA.


Most approaches used in cancer treatment, such as chemotherapy and radiation therapy are likely to be affected by the apoptotic properties of cells; therefore, it has apparent therapeutic implications. Apoptosis is associated with many biochemical changes in the cells, which includes nuclear fragmentation, mitochondrial potential change, regulations in caspases, etc. (Hunter et al. 2007). In the present study, we analyzed the in vitro effect of PA against MCF7 cell line, and release of cytochrome c from mitochondria into cytosol and the sequential activation of caspases were found to occur in PA-induced apoptosis. Moreover, the production of ROS, significant inhibition of nuclear factor-kappa beta (NF-[kappa]B) translocation from cytoplasm to nuclei activated by tumor necrosis factor alpha (TNIF-[alpha]) and involvement of the up-regulation of pro-apoptotic Bax protein and the suppression of anti-apoptotic Bcl-2 protein expression also were observed. It is interesting to note that both the non tumorogenic cell lines (MCF 10A and WRL-68) were more resistant to the PA-mediated cytotoxic activity than the MCF7 cell line.

There is growing evidence that ROS and mitochondria play an important role in apoptosis induction (Simon et al. 2000). The apoptotic effect of PA on MCF7 cells was associated with a significant elevated level of intracellular ROS. After treatment with PA, rapid generation of ROS, up to 2-fold faster than the control was observed. ROS appear to be mitochondria derived and respon-sible for later mitochondria! events leading to full activation of the caspase cascade (Chen et al. 2009). There are sufficient evidences available that the oxidation of the mitochondria, pores by ROS may contribute to cytochrome c release due to disruption of the mitochondria! membrane potential (Tait and Green 2010; Zamzami et al. 1995). Treatment with PA significantly decreased nuclear area, cell morphology, cell membrane permeability as shown by the multiparameter apoptosis analysis. Due to the ability in activation of cellular apoptotic program directly, mitochondria have been described to play a central role in the apoptotic process (Wang and Youle 2009). So the complex role of mitochondria in MCF7 cell apo-ptosis was investigated by the detection of changes in MMP, as it is assumed that its disruption is the onset of mitochondrial membrane transition pores (MPTP) formation (Zamzami and Kroemer 2003). The high content analysis conducted in this research was revealed that PA may act on mitochondria, causing loss of MMP and subsequent apoptosis. The relocalization of apoptotic proteins such as cytochrome c shall be re localized due to this reduction MMP and subsequent MPTP (Ajenjo et al. 2004).

In the Intrinsic pathway, the release of cytochrome c from the mitochondria into the cytosol is fundamental to apoptosome formation and downstream caspase activation. The release of cytochrome c and the activation of caspase by PA clearly showed that the apoptosis happened are through this pathway. There are evidences that members of the Bcl2 protein family are key mediators of cytochrome c release in the context of apoptotic stimuli (Green 2006; Kluck et al. 1997). Moreover, the disastrous alteration of mitochondrial function which includes release of cytochrome c to cytosol, loss of MMP and induction of mitochondria! permeability transition events are occurs as the consequences of Bax movement in to the mitochondria (Xiang et al. 1996). The oncolysis induced by PA via apoptosis was exhibited clearly the involvement of Bax and Bcl2 both at protein level and gene level.

Many lines of evidence established that activation of caspase is a central mechanism of apoptosis (Vaux and Korsmeyer 1999). The treatment with PA on MCF7 cells activated the caspases 3/7, 8 and 9. Activation of caspase 9 and 8 occurred even at low concentration of PA, but caspase 3/7 activation occurred only at high concentration treatment. Caspase 9 is found in the intermembrane space of mitochondria, and released in a Bcl2-inhibitable fashion upon induction of permeability transition in isolated mitochondria and upon apoptosis induction in cells (Krajewski et al. 1999; Susin et al. 1999). The released caspase 9 is then activate post-mitochondrial caspases including caspase 3 and 7, the disassembly of the cell occurs in what is known as the execution phase of apoptosis (Li et al. 2010). Even though, caspase 8 activation was found both upstream and downstream of mitochondria, it is closely involved with apoptosis signaling through the extrinsic pathway (Jin et al. 2009). Besides, in many instance caspase 8 may interlinked to mitochondrial path-ways by cleavage of bcl2 family member Bid to tBid (Gu et al. 2005). Along with Bcl2 family members, NF-[kappa]B also have considered as apoptosis inhibitors and play a key role in the mechanism of antiapoptosis of tumors (Yang et al. 2004). If the activity of this factor is suppressed, tumor cells can undergo apoptosis (Dalen and Neuzil 2003). Hence we tested PA for its inhibitory effects against NE-KB translocation from cytoplasm to nucleus activated by TNF-[alpha]. The results obtained in this research support the fact that PA induced apoptosis may occur via mechanisms of NF-[kappa]B inhibition.

To sum up, PA possesses the characteristics of selectively inducing cell death of tumor cells. Treatment of MCF7 cells with PA induced apoptosis with cell death-transducing signals that regulate the MMP by down-regulation of 8c12 and up-regulation of Bax, triggering the cytochrome c release from mitochondria to cytosol. Upon entering the cytosol, cytochrome c triggers activation of caspases-9, then activates downstream executioner caspase-3/7 and consequently cleaves specific substrates leading to process apoptotic changes. This form of apoptosis was found closely associated with the extrinsic pathway caspase (caspase-8) and inhibition of translocation of NF-[kappa]B from cytoplasm to nucleus.

Conflict of interest

No conflict of interest.


The authors would like to express their utmost gratitude and appreciation to University of Malaya and Ministry of Higher Education (HIR grant F00009-21001) for providing grant to conduct this study.


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Syam Mohan (a), Siddig Ibrahim Abdelwahaba (a), (b), *, Behnam Kamalidehghan (a), Suvitha Syam (a), Koh Sue May (a), Nabil Saad Mohammed Harmal (a), Noor Shafifiyaz (a), A. Hamid A. Hadi (c), Najihah Mohd Hashim (a), Mawardi Rahmani (d), Manal Mohamed Elhassan Taha (a), Shiau-Chuen Cheah (e), Asdren Zajmi (a)

(a) Department of Pharmacy, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia

(b) Medical Research Center, Faculty of Medicine, Jazan University Jazan, Kingdom of Saudi Arabia

(c) Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia

(d) Department of Chemistry, Faculty of Science, 43400 UPM Serdang, University Putra Malaysia, Malaysia

(e) Centre for Natural Products and Drug Discovery (CENAR), Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia

Abbreviations: MMP, mitochondria! membrane potential; HSP, heat shock proteins; NE-[kappa]B, nuclear factor-kappa B; TNF-[alpha], tumor necrosis factor alpha; ROS, reactive oxygen species; DCFH-DA, 2',7'-dichlorofluorescin diacetate; MPTP, mitochondria! membrane transition pores; cFLIP, cellular FLICE inhibitory protein.

* Corresponding author. Tel.: +60 3 7967 4909; fax: +60 3 7967 4964.

E-mail address: (S.I. Abdelwahab).

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