Compound K, a metabolite of ginseng saponin, induces apoptosis via caspase-8-dependent pathway in HL-60 human leukemia cells
- Sung-Hee Cho†1,
- Kyung-Sook Chung†1, 3,
- Jung-Hye Choi2,
- Dong-Hyun Kim1 and
- Kyung-Tae Lee1, 3Email author
© Cho et al; licensee BioMed Central Ltd. 2009
Received: 5 August 2009
Accepted: 18 December 2009
Published: 18 December 2009
Compound K [20-O-β-(D-glucopyranosyl)-20(S)-protopanaxadiol], a metabolite of the protopanaxadiol-type saponins of Panax ginseng C.A. Meyer, has been reported to possess anti-tumor properties to inhibit angiogenesis and to induce tumor apoptosis. In the present study, we investigated the effect of Compound K on apoptosis and explored the underlying mechanisms involved in HL-60 human leukemia cells.
We examined the effect of Compound K on the viabilities of various cancer cell lines using MTT assays. DAPI assay, Annexin V and PI double staining, Western blot assay and immunoprecipitation were used to determine the effect of Compound K on the induction of apoptosis.
Compound K was found to inhibit the viability of HL-60 cells in a dose- and time-dependent manner with an IC50 of 14 μM. Moreover, this cell death had typical features of apoptosis, that is, DNA fragmentation, DNA ladder formation, and the externalization of Annexin V targeted phosphatidylserine residues in HL-60 cells. In addition, compound-K induced a series of intracellular events associated with both the mitochondrial- and death receptor-dependent apoptotic pathways, namely, (1) the activation of caspases-3, -8, and -9; (2) the loss of mitochondrial membrane potential; (3) the release of cytochrome c and Smac/DIABLO to the cytosol; (4) the translocation of Bid and Bax to mitochondria; and (5) the downregulations of Bcl-2 and Bcl-xL. Furthermore, a caspase-8 inhibitor completely abolished caspase-3 activation, Bid cleavage, and subsequent DNA fragmentation by Compound K. Interestingly, the activation of caspase-3 and -8 and DNA fragmentation were significantly prevented in the presence of cycloheximide, suggesting that Compound K-induced apoptosis is dependent on de novo protein synthesis.
The results indicate that caspase-8 plays a key role in Compound K-stimulated apoptosis via the activation of caspase-3 directly or indirectly through Bid cleavage, cytochrome c release, and caspase-9 activation.
Apoptosis is a selective process of physiological cell deletion that plays an important role in the balance between cellular replication and death. Furthermore, it has been suggested that some cancer chemotherapeutics and chemopreventives exert their effects by triggering either apoptotic cell death or cell cycle transition, and accordingly, the induction of tumor cell apoptosis is used to predict tumor treatment response [9, 10]. Apoptotic signaling can proceed via two pathways, i.e., via death receptors expressed on the plasma membranes of cells or alternatively via mitochondria, which contain several proteins that regulate apoptosis. The death receptor pathway is initiated by the ligation of membrane bound tumor necrosis factor (TNF) or Fas receptors, which result in a caspase-8-dependent cascade and subsequent cell death . During this cascade, caspase-8 cleaves Bid and induces cytochrome c release and/or directly activates caspase-3 . On the other hand, the mitochondrial pathway involves cytochrome c release, which leads to caspase-9 activation and a proteolytic caspase cascade . Thus, caspase cascades appear to be a central component in the apoptotic process. However, accumulating evidence indicates that apoptosis is also induced by caspase-independent pathways , which has been suggested to involve mitochondrial release of apoptosis-inducing factor (AIF) and/or endonuclease G [15, 16].
Human leukemia results from multiple mutations that lead to abnormalities in the expressions and functions of genes that maintain the delicate balance between proliferation, differentiation, and apoptosis. Continued research on the molecular aspects of leukemia cells has resulted in the developments of several potentially useful therapeutic agents. In this context, we investigated that Compound K induced apoptosis and the mechanism involved the activation of caspase-8 system.
Cells and reagents
HL-60 human promyelocytic leukemia, U-937 human histocytic lymphoma, HeLa human negroid cervix epitheloid carcinoma, A549 human lung adenocarcinoma, HepG2 human hepatoblastoma, P388 mouse lymphoblast, and A431 human epidermoid carcinoma cells were obtained from the Korean cell line bank (KCLB, Seoul, Korea) and were cultured according to the KCLB recommendations.
Compound K used in this study was isolated from Panax ginseng and structural identities were determined spectroscopically (1H and 13NMR, IR, MS) as described previously . The identity of isolated compound was confirmed by LC-MS and was found to be >98% pure. RPMI 1640 medium, fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Life Technologies Inc. (Grand Island, NY). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tertazolium bromide (MTT), dimethyl sulfoxide (DMSO), RNase A, leupeptin, aprotinin, phenylmethylsulfonylfluoride (PMSF), 4',6-diamidino-2-phenylindole-dihydrochloride (DAPI), Triton X-100, Nonidet P-40, protein A/G-Sepharose beads, and propidium iodide (PI) were purchased from Sigma Chemical Co. (St. Louis, MO). The following antibodies for caspase-3, poly(ADP-ribose) polymerase (PARP), Bid, tBid, Bax, Bcl-2, Bcl-xL, anti-Smac/DIABLO, Fas, FasL, α-tubulin and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies for Fas-associated death domain protein (FADD), X-linked inhibitor of apoptosis protein (XIAP), caspase-8, caspsae-9, and cytochrome c were purchased from BD Biosciences, Pharmingen (San Diego, CA). The antibody for COX IV was purchased from Cell Signaling Technology (Beverly, MA), and Bax 6A7 antibody was obtained from Trevigen, Inc. (Gaithersburg, MD). z-VAD-fmk and z-DEVD-fmk, z-IETD-fmk, and z-LEHD-fmk were purchased from Calbiochem (Bad Soden, Germany).
The cytotoxicity was assessed using a MTT assay . Briefly, the cells (5 × 104) were seeded in each well containing 100 μL of the RPMI medium supplemented with 3% FBS in a 96-well plate. After 24 h, various concentrations of Compound K were added. After 48 h, 50 μL of MTT (5 mg/mL stock solution) was added and the plates were incubated for an additional 4 h. The medium was discarded and the formazan blue, which was formed in the cells, was dissolved with 100 μLDMSO. The optical density was measured at 540 nm.
Detection and quantification of DNA fragmentation
DNA fragmentation was quantified using DAPI staining and analysis of DNA fragmentation by agarose gel electrophoresis was performed as described previously . In brief, cells were lysed in a solution of 5 mM Tris-HCl (pH 7.4) and 1 mM EDTA containing 0.5% (w/v) Triton X-100 for 20 min on ice. After centrifugation at 25,000 g for 20 min, the lysate and supernatant were sonicated for 15 sec and the level of DNA in each fraction was measured by a fluorometric method using DAPI. The amount of the fragmented DNA was calculated as the ratio of the amount of DNA in the supernatant to that in the lysate. Genomic DNA was prepared and was performed in a 1.5% (w/v) agarose gel in 40 mM Tris-acetate buffer (pH 7.4) at 50 V for 1 h. The fragmented DNA was visualized by staining with ethidium bromide after electrophoresis.
PI and Annexin V double staining
For PI and Annexin V double staining, cells were suspended with 100 μl of binding buffer (10 mM HEPES/NaOH, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) and stained with 5 μL of FITC-conjugated Annexin V and 10 μL of PI (50 μg/mL) for 15 min at room temperature in dark place and then added 400 μl binding buffer, and analyzed by the fluorescence-activated cell sorting (FACS) cater-plus flow cytometry (Becton Dickinson Co, Heidelberg, Germany)
Analysis of mitochondrial membrane potential (MMP, ΔΨ m )
Changes in mitochondrial transmembrane potential were monitored by flow cytometric analysis. Cells were incubated with 50 nM 3,3'-dihexyloxacarbocyanine iodide (DiOC6) for 30 min, washed twice with PBS, and analyzed by flow cytometric analysis (Becton Dickinson Co, Heidelberg, Germany) with excitation and emission settings of 484 and 500 nm, respectively. To ensure that DiOC6 uptake was specific for ΔΨ m , we also treated cells with 50 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) or 5 μM cyclosporine A (CsA). CCCP was used as a reference depolarizing agent and CsA was used as an inhibitor of mitochondrial permeability transition.
Protein extraction and Western blot analysis
Cells were collected by centrifugation at 200 g for 10 min at 4°C. The cells were then washed twice with ice-cold PBS, and centrifuged at 200 g for 5 min. The cell pellet obtained was then resuspended in 1× protein lysis buffer (Intron, Seoul, Korea). Equal amounts of cell lysates were separated by SDS- polyacrylamide gel and transferred to nitrocellulose membranes for Western blot analysis using the indicated primary antibodies. HRP-conjugated secondary antibodies were detected using an ECL (Amersham, Buckingham-shire, England) detection system.
Preparation of mitochondrial proteins
Cells were collected by centrifugation at 200 g for 10 min at 4°C. The cells were then washed twice with ice-cold PBS, and centrifuged at 200 g for 5 min. The cell pellet obtained was then resuspended in ice-cold cell extraction buffer (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 100 μM PMSF, and protease inhibitor cocktail) for 30 min on ice. The cells were then homogenized with a glass dounce and a B-type pestle (80 strokes), homogenates were spun at 15,000 g for 15 min at 4°C, and the supernatant (cytosolic fraction) was removed whilst taking care to avoid the pellet. The resulting pellet (mitochondrial fraction) was resuspended in extraction buffer. Cell lysates were fractionated in SDS- polyacrylamide gels and transferred to nitrocellulose membranes fot immunoblot analysis using the indicated primary antibodies. Immuno-positive bands were visualized by ECL kit (Amersham, Buckingham-shire, England).
After harvesting and washing, pellets were lysed in EBC buffer (30 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 2.5 mM EGTA, 5 mM NaF, 0.1 mM Na3VO4, 1% Triton X-100 and protease inhibitor) for 15 min on ice. After centrifugation (10,000 g, 5 min), protein concentrations were determined. Equal amount of protein (100 μg) was incubated with anti-Smac/DIABLO, anti-tBid, anti-Bax, and anti-Fas antibodies for 12 h at 4°C, followed by incubation with 40 μL protein A-Sepharose beads for 4 h. The protein complex was washed 4 times with EBC buffer and released from the beads by boiling in 6× sample buffer (350 mM Tris, pH 6.8, 10% SDS, 30% β-mercaptoethanol, 6% glycerol, 0.12% bromophenolblue) for 5 min. The reaction mixture was then resolved by a 10 - 12% SDS- polyacrylamide gels, transferred to nitrocellulose membrane and probed with anti-XIAP, anti-Bax 6A7 (Trevigen Inc., Gaithersburg, MD), anti-Bcl-xL, anti-Bcl-2, anti-tBid, anti-Bax, anti-caspase-8, anti-Fas, anti-FasL, and anti-FADD antibodies. Immuno-positive bands were visualized by ECL kit (Amersham, Buckingham-shire, England).
Data presented are the means ± S.D. of results from three independent experiments with similar patterns. *P < 0.05, ** P < 0.01, *** P < 0.001 vs control group, † P < 0.05, †† P < 0.01, ††† P < 0.001 vs Compound K-treated group; significance of difference between treated groups by Student's t-test.
Compound K inhibited HL-60 cell growth and induced apoptosis
Cell viabilities of Compound K in various cancer cell lines in vitro
Caspase involvement in Compound K-induced apoptosis
Compound K induced apoptosis involving the loss of ΔΨ m and the release of cytochrome c and Smac/DIABLO
Compound K induced the translocations of tBid and Bax and downregulated Bcl-2 and Bcl-xL
Compound K induced apoptosis via the caspase-8- dependent pathway
Cycloheximide prevented Compound K-induced caspase-8 and -3 activation and DNA fragmentation
Compound K is a novel ginseng saponin metabolite that is formed by the action of intestinal bacteria on ginseng extract in man and rats . The intestinal bacteria Prevotella oris, which is responsible for the hydrolysis of ginsenoside Rb1 to Compound K, has been found in 79% of human fecal specimens . Since Compound K was detected in blood after oral administration of ginsenoside Rb1 to mice, it has been speculated that it is probably the major form of protopanaxadiol saponin absorbed by the intestine. Furthermore, evidence that Compound K is the active metabolite responsible for the anti-carcinogenic effects of ginseng saponins has prompted several groups to investigate its effects in detail [22–26].
Compound K has been reported to have potent anti-tumorigenic activity, and in particular, to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion in mouse skin, and COX-2 expression in mouse skin and in cultured human mammary epithelial cells . In addition, Compound K has been reported to inhibit glucose uptake by tumor cells and to reverse multidrug resistance in bacterial and tumor cells . Compound K was also found to have antiangiogenic activities in a spontaneous metastasis model [24, 27], and to inhibit the expression of matrix metalloproteinase (MMP)-9, which regulates the growth and invasiveness of brain tumors . It has also been reported to induce apoptosis in activated rat hepatic stellate  and hepatoma [7, 26] cancer cells. However, the effects of Compound K on leukemia have not been studied in detail, although Lee et al found that Compound K induced apoptosis via the cytochrome c-mediated activation of caspase-3 in HL-60 cells . Our present results confirm those of Lee et al., in as much as Compound K showed cytotoxic and apoptosis-inducing activities in HL-60 cells.
Apoptosis is a fundamental cellular activity and provides protection against cancer development by eliminating genetically altered cells and hyper-proliferative cells. Thus, defects in apoptosis signaling pathways contribute to carcinogenesis and chemoresistance . As mentioned above, there are two major apoptotic pathways; the extrinsic death receptor-mediated pathway and the intrinsic mitochondria-mediated pathway, and truncated Bid protein provides cross-talk between the two . Both of these pathways are regulated by caspases, which are responsible, either directly or indirectly, for the cleavages of cellular proteins, a characteristic of apoptosis . In the present study, Compound K increased the levels of cleaved caspase-8, -9, and -3 and PARP in a time-dependent manner and pretreatment with various caspase inhibitors markedly prevented Compound K-induced DNA fragmentation (Figure 2). These findings indicate that caspase-3 and -8 play fundamental roles in Compound K-induced apoptosis in HL-60 cells. It is noteworthy that the specific inhibition of caspase-9 by z-LEHD-fmk had only a mild inhibitory effect on Compound K-stimulated apoptosis. In this regard, it appears that activation of the intrinsic mitochondria-mediated pathway alone is not sufficient to explain the apoptotic effects of Compound K in HL-60 leukemia cells. In addition, caspase-9 was activated about 2 h after treatment with Compound-K while activation of caspase-3 was observed at 0.5 h. Recent studies have suggested that caspase-3 has feedback action on caspase-9 . For example, caspase-3-mediated activation of caspase-9 was involved in cisplatin-induced apoptosis . This data suggested a possibility that tBid/caspase 9 activation is a consequence of active caspase 3 in the Compound-K-induced apoptosis.
Activation of the intrinsic mitochondrial pathway leads to the release of Smac/DIABLO, which removes the IAP blockage of caspase activation . The IAP family, which includes XIAP and survivin, functions by binding to and inhibiting several caspases . In the present study, we observed Smac/DIABLO increased in cytoplasm and correspondingly decreased in mitochondria after treating cells with Compound K (data not shown), which suggests that the translocation of Smac/DIABLO from mitochondria to cytoplasm contributes to Compound K-induced apoptosis in HL-60 leukemia cells. Furthermore, Compound K-induced releases of cytochrome c and Smac/DIABLO from mitochondria were seemingly associated with mitochondrial depolarization (Figure 3A). Bid, a BH3-only proapoptotic member of the Bcl-2 family, undergoes proteolysis by caspase-8 activated by cell surface death receptors, such as, Fas or TNF [36, 37], and the tBid so produced, translocates to mitochondria and binds to anti-apoptotic proteins, such as, Bcl-2 and Bcl-xL, which leads to a conformational change in Bax, mitochondrial depolarization, and cytochrome c release from mitochondria . Moreover, conformationally altered Bax stimulates the release of Smac/DIABLO from mitochondria , and the cytochrome c-releasing activity of Bid by cell surface death receptors such as Fas and TNF is antagonized by Bcl-2 .
In the present study, Compound K-induced releases of cytochrome c and Smac/DIABLO from mitochondria were seemingly associated with mitochondrial depolarization (Figure 3) and Compound K increased the translocations of Bid and Bax to mitochondria and their bindings to Bcl-2 and Bcl-xL, which is presumed to induce a conformational change in Bax and to reduce the mitochondrial levels of Bcl-2 and Bcl-xL in HL-60 cells (Figure 4). These findings suggest that the tBid triggered-imbalance between pro- and anti-apoptotic Bcl-2 family members is associated with the mitochondrial membrane depolarization that leads to the releases of cytochrome c and Smac/DIABLO in Compound K-treated cells.
The bindings of FasL, TRAIL, and TNF to their cell surface receptors are known to lead to caspase-8 activation, which then amplifies the apoptotic signal either by directly activating downstream caspases or by cleaving Bid, a BH3-only proapoptotic member of the Bcl-2 family . In the present study, caspase-8 inhibition prevented the cleavages of Bid and caspase-3 and subsequent apoptosis (Figure 5A), which suggests that Bid is a substrate of caspase-8, and that caspase-8 is required for Compound K-induced apoptosis. Furthermore, we found that Compound K stimulates DISC formation with the FasL, Fas, FADD, and procaspase-8, which indicates that Compound K-induced caspase-8 activation is probably due to the stimulation of the death receptors. The mechanism by which Compound K promotes DISC formation appears to be dependent on de novo protein synthesis since the Compound K-induced activation of caspases-8 and -3 and DNA fragmentation were prevented by cycloheximide. During our earlier studies, we found FasL is abundantly expressed in HL-60 cells, and that Compound K increases Fas protein expression in a time-dependent manner, but does not affect FasL (data not shown). In this regard, we speculate that Compound K stimulates apoptosis by upregulating Fas in HL-60 cells. Indeed, in a previous study, the triterpene saponin platycodin D was demonstrated to induce DNA fragmentation and caspase-3/-8 activation within 3 h in immortalized keratinocyte HaCa cells . Recent studied have demonstrated that the general translation rate is about 2-2.8 amino acid/sec suggestting that it would take only 15 min to make a 50% change in protein concentration [40, 41]. Furthermore, Fas and FasL protein upregulations were observed at 15 and 30 min, respectively, after platycodin D treatment, and NF-κB activation (mediated by the degradation of IκB) was found to be involved in these upregulations. Other studies have also demonstrated the involvement of NF-κB in the regulation of Fas and FasL expression in a variety of cell lines [42, 43]. However, whether Compound K-induced apoptosis is mediated by NF-κB-regulated Fas expression in HL-60 cells remains to be determined. Taken together, we hypothesize Compound K-induced apoptosis in HL-60 cells is associated with the upregulations of cleaved caspases-8 and -3, tBid, and mitochondrial membrane depolarization, and that crosstalk between the caspase-8/Bid and mitochondrial pathways appears to contribute to Compound K-induced apoptosis.
Our results demonstrate that Compound K inhibit the viability of HL-60 cells and these effects were mediated through the induction of apoptosis. Compound K induced the activation of caspase-3, -8, and -9, and modulation of Bcl-2 families. In addition, a caspase-8 inhibitor completely abolished caspase-3 activation, Bid cleavage, and subsequent DNA fragmentation by Compound K. Therefore caspase-8 plays a key role in Compound K-stimulated apoptosis. Based on these finding, we suggest that Compound K may be used as a potential therapeutic and chemopreventive agent for leukemia via the potent apoptotic activity.
cystein arspartyl-specific protease
second mitochondria-derived activator of caspase/direct inhibitor of apoptosis protein binding protein with low pI
X-linked inhibitor of apoptosis protein
Fas-associated death domain protein.
This work was supported partly by a grant from the Korean Food Research Institute (2003) and partly by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (No. R13-2002-020-03002-0(2007).
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