Induction of apoptosis by withaferin A in human leukemia U937 cells through down-regulation of Akt phosphorylation
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- Oh, J.H., Lee, TJ., Kim, S.H. et al. Apoptosis (2008) 13: 1494. doi:10.1007/s10495-008-0273-y
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Withaferin A, a major chemical constituent of Withania somnifera, has been reported for its tumor cell growth inhibitory activity, antitumor effects, and impairing metastasis and angiogenesis. The mechanism by which withaferin A initiates apoptosis remains poorly understood. In the present report, we investigated the effect of withaferin A on the apoptotic pathway in U937 human promonocytic cells. We show that withaferin A induces apoptosis in association with the activation of caspase-3. JNK and Akt signal pathways play crucial roles in withaferin A-induced apoptosis in U937 cells. Furthermore, we have shown that overexpression of Bcl-2 and active Akt (myr-Akt) in U937 cells inhibited the induction of apoptosis, activation of caspase-3, and PLC-γ1 cleavage by withaferin A. Taken together, our results indicated that the JNK and Akt pathways and inhibition of NF-κB activity were key regulators of apoptosis in response to withaferin A in human leukemia U937 cells.
KeywordsWithaferin A Apoptosis pAkt U937 JNK XIAP
The search for antitumor drugs from natural products represents an area of great interest worldwide. Natural products provide a rich source for developing novel anticancer agents since they show different mechanisms of action including cell growth suppression, modulation of cell differentiation and induction of apoptosis . It is suggested that there are several apoptosis pathways in cells responsive to apoptotic stimuli, such as the death receptor-mediated pathway, the mitochondrial apoptotic pathway, and the endoplasmic reticulum (ER) pathway [2, 3, 4]. Although each pathway is initially mediated by different mechanisms, they share a common final phase of apoptosis, consisting of the activation of the executioner caspases and dismantling of substrates critical for cell survival .
The mechanisms involved in induction of apoptosis by natural compounds are believed to be largely mediated by the mitochondrial apoptotic pathway, which in turn increase the permeability of outer mitochondrial membrane [6, 7, 8]. This involves release of mitochondrial apoptotic components such as cytochrome c, AIF, second mitochondrial-derived activator of caspase (Smac), endonuclease G and Omi1/HtrA2 [9, 10, 11, 12]. The released apoptotic proteins initiate caspase activation and trigger caspase-mediated apoptotic DNA fragmentation and eventually cell death .
PI3K/Akt signaling is activated in a wide variety of cancers including primary acute myeloid leukemia (AML) blast samples [14, 15, 16]. Therefore, the development of cytotoxic drugs that target the PI3K/Akt pathway has become an increasingly important strategy for targeting leukemia. Based on this concept, we have recently been interested in evaluating the anticancer activity of natural compounds that have a potential to induce apoptosis and target PI3K/Akt signaling pathway in cancer cells.
Withaferin A is a steroidal lactone purified from Withania somnifera . It exhibits a wide variety of activities, including antitumor, anti-inflammation, and immunomodulation properties. Recently, a bioactive properties of withaferin A have been reported; cytoskeletal architecture alteration by covalently binding annexin II , antitumor capacity by inhibition of the proteasomal chymotrypsin-like activity , and apoptosis induction through the inhibition of protein kinase C . However, the precise mechanism by which withaferin A induces cell death is not fully understood. The aim of this study was to determine the capacity of withaferin A to induce apoptosis and identify the biochemical mechanisms of this induction as well as novel target(s) including Akt signaling pathways in a human monocytic leukemic U937 cells.
Materials and methods
Cells and materials
Human leukemia U937 cells, Caki, AMC-HN-4 and HT-29 cells were obtained from the American Type Culture Collection (ATCC: Rockville, MD).The culture medium used throughout these experiments was Dulbecco’s modified Eagle’s medium, containing 10% fetal calf serum (FCS), 20 mM Hepes buffer, and 100 μg/ml gentamicin. Withaferin A was directly added to cell cultures at the indicated concentrations. Anti-cIAP2, anti-BclxL, anti-Bcl-2, anti-Mcl1, anti-Bax, anti-pro-caspase-3, anti-PLC-γ1 and anti-HSC70 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-XIAP and anti-cytochrome c antibodies were purchased from BD Biosciences PharMingen (San Jose, CA). Anti-OxPhos ComplexII subunit (QPs2) antibody was purchased from Molecular Probes (Eugene, OR). Anti-phospho-ERK, anti-phospho-JNK, anti-phospho-p38 MAPK, and anti-phospho-Akt were purchased from New England Biolabs. Caspase activity assay kits were obtained from R&D Systems (Minneapolis, MN). Withaferin A was purchased from Biomol (Plymouth Meeting, PA).
Analysis of cytochrome c release
2 × 106 cells were harvested, washed once with ice-cold PBS and gently lysed for 2 min in 80 μl ice-cold lysis buffer [250 mM sucrose, 1 mM EDTA, 20 mM Tris-HCl (pH 7.2), 1 mM DTT, 10 mM KCl, 1.5 mM MgCl2, 5 μg/ml pepstatin A, 10 μg/ml leupeptin and 2 μg/ml aprotinin]. Lysates were centrifuged at 12,000 × g at 4°C for 10 min to obtain the supernatants (cytosolic extracts free of mitochondria) and the pellets (fraction that contains mitochondria). The resulting cytosolic fractions were used for western blot analysis with an anti-cytochrome c antibody.
Cellular lysates were prepared by suspending 1 × 106 cells in 100 μl of lysis buffer (137 mM NaCl, 15 mM EGTA, 0.1 mM sodium orthovanadate, 15 mM MgCl2, 0.1% Triton X-100, 25 mM MOPS, 100 μM phenylmethylsulfonyl fluoride, and 20 μM leupeptin, adjusted to pH 7.2). The cells were disrupted by sonication and extracted at 4°C for 30 min. The proteins were electrotransferred to Immobilon-P membranes (Millipore Corporation, Bedford, MA, USA). Detection of specific proteins was carried out with an ECL Western blotting kit according to the manufacturer’s instructions.
Cell proliferation was detected by 2,3-Bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) (Wel Gene, Seoul, Korea) assay. When cells were cultured to the log phase, they were seeded on a 96-well plate (2 × 104 cells/100 μl/well) for 24 h. Cells were divided into a control (DMSO) group and a withaferin A-treated groups. Absorbance (A) was detected with an enzyme calibrator at 450 nm. Cell viability = (A of study group/A of control group) × 100%. The experiment was repeated twice. There were three wells for each concentration.
Cell count and flow cytometry analysis
Cell counts were performed using a hemocytometer. Approximately 1 × 106 U937 cells were suspended in 100 μl of PBS, and 200 μl of 95% ethanol were added while vortexing. The cells were incubated at 4°C for 1 h, washed with PBS, and resuspended in 250 μl of 1.12% sodium citrate buffer (pH 8.4) together with 12.5 μg of RNase. Incubation was continued at 37°C for 30 min. The cellular DNA was then stained by applying 250 μl of propidium iodide (50 μg/ml) for 30 min at room temperature. The stained cells were analyzed by fluorescent activated cell sorting (FACS) on a FACScan flow cytometer for relative DNA content based on red fluorescence.
Cell death assessment by DNA fragmentation assays
The cell death detection ELISAplus kit (Boerhringer Mannheim, Indianapolis, IN) was used for assessing apoptotic activity by detecting fragmented DNA within the nucleus in withafrin A-treated cells. Briefly, each culture plate was centrifuged for 10 min at 200 × g, the supernatant was removed, and the pellet was lysed for 30 min. After centrifuging the plate again at 200 × g for 10 min, the collected supernatant containing cytoplasmic histone-associated DNA fragments was incubated with an immobilized anti-histone antibody, and the reaction products were determined by spectrophotometry. Finally, absorbance at 405 nm and 490 nm (reference wavelength), upon incubating with a peroxidase substrate for 5 min, was determined with a microplate reader. Signals in the wells containing the substrate only were subtracted as background.
Determination of caspase activity
Caspase activities were determined by colorimetric assays using caspase-2, -3, -8, and -9 activity assay kits according to the manufacturer’s protocol. The kits utilize synthetic tetrapeptides labeled with p-nitroanilide. To evaluate caspase activity, cell lysates were prepared after their respective treatment with withaferin A. Assays were performed in 96-well microtiter plates by incubating 20 μg of cell lysates in 100 μl of reaction buffer (1% NP-40, 20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 10% glycerol) containing the caspases specific substrate at 5 μM. Lysates were incubated at 37°C for 2 h. Thereafter, the absorbance at 405 nm was measured with a spectrophotometer.
Expression of Bcl-2 and constitutively active Akt
Bcl-2 or Akt-overexpressing U937 cells were generated using a pMAX vector containing the human bcl-2 gene (provided by Dr Rakesh Srivastava, NIH/NIA) and Myc-His-tagged mouse Aktl (activated) under the control of the cytomegalovirus promoter (Upstate Biotechnology), respectively. U937 cells (2 × 106 cells/ml) in RPMI 1640 medium were transfected by preincubation with 15 μg Bcl-2 or Akt plasmid for 10 min at room temperature followed by electroporation at 500 V, 700 μF. The sample was immediately placed on ice for 10 min, and after 10 ml complete medium was added, the cells were incubated at 37°C for 24 h. The cells were selected in a medium containing 0.7 μg/ml geneticin (G418 sulfate) for 4 weeks. Single cell clones were obtained by limiting dilution and subsequently analyzed for an increase in Bcl-2 or Akt protein expression relative to the identically cloned empty vector control.
Determination of the mitochondrial membrane potential by DiOC6
DiOC6(3) (3,3-dihexyloxacarbocyanine iodide, Molecular Probes) uptake by mitochondria is directly proportional to its membrane potential. U937 cells subjected to 6 h after treatment were incubated with DiOC6 (1 μM) for 10 min in dark at 37°C. The cells were harvested and suspended in PBS. The mitochondrial membrane potential was subsequently analyzed using a Flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) with excitation and emission settings of 488 and 525 nm, respectively.
Three or more separate experiments were performed. Statistical analysis was done by Student’s t-test or ANOVA. A P value <0.05 was considered to have pronounced difference between experimental and control groups.
Withaferin A-induced anti-proliferation and apoptosis in U937 cells
Inhibition of withaferin A-induced apoptosis by caspase-3 inhibitor
Withaferin A exposure leads to the loss of MMP and release of cytochrome c
Ectopic expression of Bcl-2 reduced withaferin A-induced apoptosis
The JNK pathways play important roles in withaferin A-induced apoptosis
Ectopic expression of constitutive active Akt reduces withaferin A-induced apoptosis
To further evaluate the effect of Akt signaling on caspase-3 activation and PLC-γ1 degradation during withaferin A-induced apoptosis, we employed U937/vector and U937/DA-Akt cells generated by transfection of the constitutively active Myc-tagged form of Akt (myr-Akt). We isolated a clone stably expressing transfected myr-Akt (Fig. 6c). U937/vector and U937/myr-Akt cells were exposed to 1 μM withaferin A for various time points. Withaferin A treatment in U937/vector cells resulted in a markedly increased accumulation of sub-G1 phase. In contrast, expression of constitutively active Akt reduced withaferin A-induced accumulation of sub-Gl phase (Fig. 6c). We next analyzed the expression levels of pro-caspase-3, XIAP, and PLC-γ1 cleavage in U937/vector and U937/myr-Akt cells exposed to 1 μM withaferin A. As shown in Fig. 6c, degradation of pro-caspase-3, down-regulation of XIAP, Mcl-1 and Bcl-2, and cleavage of PLC-γ1 were inhibited by ectopic expression of constitutively active Akt. Taken together, these results indicate that Akt pathway plays a role in regulating withaferin A-induced apoptosis of U937 cells.
The PI3 K/Akt signal pathway plays role in cell survival through activation of anti-apoptotic proteins and can regulate NF-κB via activation of IKK, resulting in the increased phosphorylation of IκB and consequent release of NF-κB from the inhibitory complex. To determine whether withaferin A inhibits activation of NF-κB through the inhibition of DNA binding of NF-κB, we examined the effect of withaferin A on TNF-α-induced binding of NF-κB and constitutive NF-κB binding by EMSA. Treatment of U937 cells with 10 ng/ml TNF-α increased NF-κB DNA binding, but pretreatment with withaferin A prior to TNF-α reduced NF-κB DNA binding in a dose–dependent manner (Fig. 6d). To examine the effect of withaferin A on the constitutive DNA-binding activity of NF-κB, U937 cells were treated with various concentrations of withaferin A for 6 h. As shown in Fig. 6d, withaferin A treatment of U937 cells inhibited constitutive NF-κB binding activity in a dose–dependent manner. To confirm that higher-mobility bands were contained NF-κB DNA-protein complexes, we tested the binding of wild-type oligonucleotides against that of a mutant oligonucleotide lacking the NF-κB site. The wild-type competitor (50-fold excess) inhibited TNF-α-induced NF-κB binding activity, whereas a similar excess of the mutant-type competitor did not, showing that the band corresponded to a specific NF-κB DNA-protein complex. These findings suggest that withaferin A may inhibit TNF-α-induced NF-κB activation as well as constitutive NF-κB activation.
In the present study, we have demonstrated that withaferin A induces apoptosis in association with the activation of caspase-3 and the translocation of cytochrome c from the mitochondria to the cytosol, as well as the cleavage of PLC-γ1, resulting in the accumulation of cleavage products in a caspase-dependent manner, whereas ectopic expression of Bcl-2 significantly attenuates withaferin A-induced apoptosis. Furthermore, withaferin A altered the phosphorylation state of members of the MAPKs and Akt. Overexpression of active Akt (myr-Akt) in U937 cells inhibited induction of apoptosis, activation of caspase-3 and PLC-γ1 cleavage by withaferin A. We have also shown that JNK and Akt signal pathway plays a crucial role in withaferin A-induced apoptosis in U937 cells.
Withaferin A is a steroidal lactone isolated from medicinal plant “Indian Winter Cherry” and has anti-inflammation and anti-tumor . However, the molecular mechanism involved in withaferin A-induced apoptosis was poorly understood. In this study, we tested whether withaferin A treatment could be a new possibility for the treatment of human leukemia and examined the mechanism of withaferin A-induced apoptosis in human leukemia cells. We observed that withaferin A activated the mitochondrial caspase-dependent apoptotic pathway in dose–dependent manners in U937 cells, which were significantly prevented by pretreatment of a pancaspase inhibitor z-VAD-fmk. Previous reports have shown that withaferin A induced ROS generation, caspase-dependent apoptotic pathway and mitochondrial dysfunction in HL-60 cells trigger events responsible for mitochondrial-dependent and -independent apoptosis pathways . Members of the Bcl-2 family, including Bcl-2, Bcl-xL and Bax, have been shown to regulate apoptosis. In particular, Bcl-2 has been reported to directly inhibit members of the caspase family, including caspases-3 and -9 . In this study, withaferin A did not alter the expression levels of Bax in U937 cells but did selectively down-regulate the expression of Bcl-2. Furthermore, Bcl-2 overexpression significantly attenuated withaferin A-induced apoptosis in U937 cells by inhibiting the caspase-3 activity and PLC-γ1 cleavage.
Withaferin A modulated the activation of MAPKs. We next determined the functional relationship between MAPK activity and apoptosis in the presence of withaferin A, and thus determined that JNK, but not p38 MAPK, was associated with apoptosis. Surprisingly, we found that PD98059 (specific inhibitor of MEK1 which is an upstream kinase in the ERK pathway) enhanced withaferin A-induced apoptosis through JNK activation, suggesting that ERK is upstream of JNK-activated apoptosis in our experimental model (Fig. 5d). JNK is a key regulator of many cellular events, including apoptosis. Recent studies have shown that JNK phosphorylates and regulates the activity of transcription factors (c-Jun, ATF2, Elk-1, p53 and c-Myc) and non-transcription factors (Bcl-2, Bcl-xL, Bim and Bad), in response to a variety of extracellular stimuli . Prolonged JNK activation induced production of jBID, novel proteolytic fragment of BID. jBID induced the release of Smac from mitochondria and subsequently activated caspases cascade . However, the mechanisms associated with withaferin A-induced apoptosis are not well established. Thus, further studies are needed to determine whether JNK/ERK activation is sufficient to induce or only promote apoptosis.
Akt is activated in response to many extracellular stimuli including insulin-like growth factor, nerve growth factor, and lysophosphatidic acid [27, 28, 29]. We have shown that Akt is dephosphorylated after treatment with withaferin A. Ectopic expression of constitutively active Akt reduces withaferin A-induced apoptosis. These results suggest that withaferin A-induced apoptosis is associated with Akt inactivation. The ability of Akt promote cell survival is based on its ability to phosphorylate on residues necessary for their inactivation of several proapoptotic proteins, including Bad, caspase-9, transcription factors of the forkhead family, and IKK [30, 31, 32]. A possible candidate for mediating the effects of withaferin A-induced inhibition of Akt signaling is the prosurvival transcription factor NF-κB. Akt can regulate NF-κB via activation of IKK, resulting in the increased phosphorylation of IκB and consequent release of NF-κB from the inhibitory complex . NF-κB is crucial to cell survival as it regulates the transcription of anti-apoptotic genes, such as XIAP, which blocks caspases and neutralizes by interacting with cytosolic Smac [34, 35], thus down-regulates NF-κB and reduces XAIP expression in withaferin A treated U937 cells.
In conclusion, the results of our studies, for the first time, provide mechanistic evidence that withaferin A induces apoptosis by Akt dephosphorylation and down-regulation of XIAP. The apoptosis inducing ability of withaferin A, in conjunction with its nontoxic nature, could make it a potentially effective preventive and/or therapeutic agent against leukemia. However, additional in vivo studies are needed to establish the role of withaferin A as a chemopreventive and/or therapeutic agent for cancer.
This work was supported by the Korea Science and Engineering Foundation through the MRC at Keimyung University (R13-2002-028-03001-0).