European Journal of Pharmacology 735 (2014) 52–58
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Molecular and cellular pharmacology
Dioscin induced activation of p38 MAPK and JNK via mitochondrial pathway in HL-60 cell line Ying Wang a,n, Qing-Yu He b, Jen-Fu Chiu c,d,nn a
State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes and Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou, China c Open Laboratory for Tumor Molecular Biology, Department of Biochemistry, Shantou University Medical College, Shantou, China d Department of Anatomy, The University of Hong Kong, Hong Kong SAR, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 24 November 2013 Received in revised form 9 April 2014 Accepted 10 April 2014 Available online 19 April 2014
Saponins have shown promise in cancer prevention and therapy; however, little is known about the detailed signaling pathways underlying their anticancer activities. In the present study, we examined the mechanisms of action of dioscin, a glucosides saponin isolated from Polygonatum zanlanscianense pump, in human myeloblast leukemia HL-60 cells. Dioscin suppressed HL-60 cell growth in a dose-dependent manner. This inhibition was due to the induction of apoptosis as revealed by the externalization of phosphatidylserine, and cleavages of lamin A/C and PARP-1. Treatment with dioscin induced apoptosis through activation of caspases 3, 7, 8, 9, and 10. Phosphorylation of p38 MAPK and JNK contributed to dioscin-induced apoptosis upstream of caspase activation. Using various inhibitors and antioxidant agents, we found that mitochondrial derived reactive oxygen species and depletion of mitochondrial transmembrane potential lead to the phosphorylation of p38 MAPK and JNK. Taken together, our results demonstrated that dioscin induces apoptosis by activation of p38 MAPK and JNK through the caspasedependent mitochondrial death pathway. This work suggests that dioscin may be used as a drug lead for the treatment of myeloblast leukemia. & 2014 Elsevier B.V. All rights reserved.
Keywords: Apoptosis Mitochondria Reactive oxygen species MAPK Saponin Dioscin
1. Introduction Saponins derived from plant sources have been investigated extensively for the treatment of various types of cancers (Koehn and Carter, 2005). Dioscin, a plant steroidal glycosides extracted from Polygonatum zanlanscianense pump, was first isolated and characterized in the mid-20th century (Tsukamoto et al., 1956). Dioscin exhibited cytotoxicity against a number of human malignant cell lines (Cai et al., 2002; Liu et al., 2004; Wang et al., 2001). Our previous proteomic study revealed that dioscin exerted cytotoxicity in human myeloblast leukemia HL-60 cell line through multiple pathways (Hu et al., 2013; Wang et al., 2013, 2007a, 2006); however, the detailed molecular basis of its mechanisms of action remained elusive.
Abbreviations: ASK1, signal-regulating kinase 1; FADD, Fas-associated death domain protein; JNK, c-Jun NH2-terminal protein kinase/stress-activated protein kinases; MAPK, mitogen-activated protein kinases; Δψm, mitochondrial transmembrane potential; Rho-123, Rhodamine 123 n Corresponding author at: Institute of Chinese Medicinal Sciences, University of Macau, Macao SAR, China. nn Corresponding author at: Department of Anatomy, The University of Hong Kong, Hong Kong SAR, China. E-mail addresses: [email protected] (Y. Wang), [email protected] (J.-F. Chiu). http://dx.doi.org/10.1016/j.ejphar.2014.04.018 0014-2999/& 2014 Elsevier B.V. All rights reserved.
Myeloid leukemia is highly curable with the treatment of all-trans retinoic acid in combination with anthracycline-based chemotherapy. Combined therapy achieved an approximately 90% complete remission rate newly diagnosed myeloid leukemia and approximately 20% relapse rate (Imagawa et al., 2010). However, long-term use of retinoic acid leads to development of differentiation syndrome with severe adverse effect (Leblebjian et al., 2013). Thus, development of new therapeutic agents with distinct mode of action could be expected to shorten the treatment or enhance remission rate when combined with all-trans retinoic acid. Apoptosis is a precisely controlled multi-step event that leads to cell death. Mitochondria play a central role in the intrinsic apoptotic pathway, in which the release of cytotoxic proteins from mitochondrial inter membrane to the cytoplasm allows subsequent activation of caspases 9 and 3 and/or DNA fragmentation (Wang et al., 2005). The extrinsic apoptotic pathway is characterized by the engagement of cellular death receptors (Gonzalvez and Ashkenazi, 2010). The biological effects of apoptotic signals are executed by a number of signal-responsive transcription factors and a broad range of substrates, including Bcl-2 family proteins and mitogen-activated protein kinases (MAPKs) (Azijli et al., 2013a; Gonzalvez and Ashkenazi, 2010). The precise role of p38 MAPK and JNK in cell death is cell type- and stimuli-dependent.
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Phosphorylation of p38 MAPK acted as a mediator for caspase 8 in manganese-induced mitochondria-dependent cell death (Azijli et al., 2013b). While transforming growth factor-β-activated p38 MAPK could induce cell death via Fas-associated death domain protein (FADD) independent of caspase activation in human Burkitt lymphoma B cells (Schrantz et al., 2001). Activation of JNK is necessary for apoptosis induced by tumor necrosis factor, osmotic stress, and chemotherapeutic drugs through oxidative stress (Yang et al., 2013). Additionally, p38 MAPK and JNK can initiate mitochondria-dependent intrinsic cell death pathway by direct phosphorylation of Bcl-2 family pro-apoptotic proteins BimEL (Mobasher et al., 2013) and Bax (Shi et al., 2013). In this study, we examined the molecular consequences by treating human myeloblast leukemia HL-60 with dioscin. We demonstrated that dioscin induced the intrinsic mitochondrial death pathway through p38 MAPK and JNK upstream of caspases. Our results illustrate the potential of dioscin as cancer chemotherapeutic agent for the treatment of myeloblast leukemia.
2. Materials and methods 2.1. Dioscin and other reagents Dioscin was provided by Shanghai Institute of Organic Chemistry (Wang et al., 2006) and kept as stock solution at 2 mM so that
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the final concentration of DMSO is less than 0.5% during treatment. The stock solution was diluted in medium prior to use. All other chemicals, except otherwise noted, were obtained from either Sigma-Aldrich Chemical Co. (St. Louis, MO) or GE Healthcare Life Sciences (Uppsala, Sweden).
2.2. Cell line and culture conditions Human myeloblast leukemia HL-60 cells were cultured in RPMI 1640 medium with 2.0 g/L sodium bicarbonate plus 10% fetal bovine serum, 1% L-glutamine, 1% penicillin and streptomycin (100 units/mL), in a humidified incubator with an atmosphere of 95% air and 5% CO2 at 37 1C.
2.3. Drug treatment The cells were grown to about 80% confluence and were then either subcultured or treated with the IC50 (half-maximal inhibitory concentration) value of dioscin (7.6 mM, or 6.6 μg/mL) to study the early direct effect (Fig. 1A). This dose is. In some experiments, cells were pretreated 1 h prior to the addition of dioscin with SB203580 (CalBioChem, La Jolla, CA), SP600125 (CalBioChem), N-acetyl-L-cysteine (NAC), DTT, aristolochic acid (ArA), or cyclosporine A (CyA).
Fig. 1. Dioscin induced apoptosis and activated caspases in HL-60 cells. (A) MTT assay showed that dioscin exhibited cytotoxicity in HL-60 cells in a dose-dependent manner after 24 h treatment, and the IC50 (half-maximal inhibition concentration) value is 7.6 7 0.12 mM. (B) Detection of dioscin-induced phosphatidylserine externalization in HL-60 cells by annexin-V analysis. (C) Western blot analysis of cleaved lamin A/C ( 50 kDa and 40 kDa) and PARP-1 ( 89 kDa) with dioscin treatment for different period of time. (D) Western blot analysis of cleaved caspases 3, 7, 8, 9, and 10 with dioscin treatment for different period of time. (A) Data are calculated from at least three separate experiments. (B–D) Data are representatives of three independent experiments; quantitative results of (C) and (D) are shown in Supplementary Fig. 1.
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2.4. Cytotoxicity assay The cytotoxicity of drug treatment was determined by MTT assay, in accordance to the procedure reported previously (Wang et al., 2007b). 2.5. Flow cytometric analysis of apoptosis Induction of apoptosis was determined by Annexin V staining (Molecular Probes, Eugene, OR). Cells were cultivated for 24 h and then either left untreated as control or treated with dioscin. At the end of each experiment, cells were harvested, resuspended in ice cold PBS, stained by Annexin V, and analyzed with a FACStar Plus flow cytometry. One million cells were analyzed for each sample, providing a solid statistical basis for the determination of the percentage of apoptotic cells in each treatment. 2.6. Measurement of mitochondrial transmembrane potential (Δψm) Changes in Δψm were assayed by Rhodamine 123 (Rho-123, Molecular Probes) staining in accordance with the procedure described previously (Wang et al., 2005). 2.7. Detection of oxidative stress Cells in HBSS were incubated for 15 min at 37 1C with 1 μM dichlorodihydrofluorescein diacetate (DCFH-DA, Molecular Probes), washed, re-suspended in complete growth media, and then treated with dioscin. Dye oxidation (increase in fluorescence) was measured using a FACStar Plus flow cytometer with excitation and emission settings of 488 nm and 530 nm, respectively (Wang et al., 2005). 2.8. Western blot analysis Western blot analysis was performed using primary antibodies against phospho-ERK (Tyr204, Santa Cruz Biotechnology), ERK (Santa Cruz Biotechnology), phospho-p38 MAPK (Thr180/Tyr182, Stressgen, Ann Arbor, MI), p38 MAPK (Cell Signaling Technology, Danvers, MA), phospho-JNK 1/2 (Thr183/Tyr185, Stressgen), JNK 1/2 (Cell Signaling Technology), caspase 3 (Lab Vision, Fremont, CA), caspase 7 (Lab Vision), caspase 8 (Lab Vision), caspase 9 (Lab Vision), caspase 10 (Lab Vision), lamin A/C (Santa Cruz Biotechnology), PARP-1 (Cell Signaling Technology), and alpha-tubulin (Sigma-Aldrich) at optimal dilution. 2.9. Statistical analysis Statistical analysis was performed using two-tailed Student's t-test (Microsoft Excel), and Po 0.05 was considered significant. Data were expressed as mean 7S.D. of triplicate samples, and reproducibility was confirmed in three separate experiments.
3. Results 3.1. Dioscin induces apoptosis and activation of caspases in HL-60 cells This study aims to analyze the mechanisms of action of dioscininduced apoptosis in HL-60 cells. The IC50 of dioscin for HL-60 cells was 7.6 mM detected by MTT assay (Fig. 1A). We then use the IC50 concentration all through the study to examine the early event with dioscin treatment. Microscopic results from previous study showed that HL-60 cells treated with 7.6 mM dioscin exhibited a shrunken morphology compared with untreated control cells (Wang et al., 2007a, 2007b), indicating that dioscin affected cell integrity
adversely. Significant increase in cell death was detected by annexinV staining after 24 h treatment with dioscin (Fig. 1B). The proteolytic cleavage of PARP-1 between Asp216 and Gly217 and the cleavage of 70 kDa lamin A/C to fragments of 40–45 kDa and 28 kDa served as early indicators of nuclear deregulation and apoptotic cell death (Takahashi et al., 1996). The treatment with dioscin induced fragmentation of lamin A/C and PARP-1 in a time-dependent manner (Fig. 1C and A). Together with the results from previous study, these findings confirmed that treatment with dioscin induced apoptosis in HL-60 cells (Wang et al., 2007a, 2007b). As major mediators of apoptotic cell death, caspases contribute to the overall morphological alterations by cleaving various cellular substrates in apoptotic cell death. Among them, caspases 8 and 10 are primarily activated by death receptors, caspase 9 is an initiator caspase downstream of the permeabilization of mitochondrial outer membrane, and caspases 3 and 7 serve as executioners that hence trigger apoptotic cell death (Green, 2005). Activation of caspases was measured by assessing the cleaved forms of these enzymes. Western blot results revealed that treatment with dioscin caused cleavage of caspases 8, 10, 7, and 3 as early as 6 h (Figs. 1D and S1B). The cleaved active forms were detectable after that throughout the period of the treatment in a time-dependent manner. Elevated expression of caspase 9 was detected after 6 h treatment, peaked at 9 to 18 h, and then tapered off. These results indicated that treatment with dioscin induced apoptotic cell death in a caspase-dependent manner. 3.2. Activation of p38 MAPK and JNK leads to cell death with the treatment of dioscin The MAPK signaling pathway plays important roles in regulating apoptosis induced by various cellular stresses and chemotherapeutic agents (Bruzzese et al., 2013). However, the detailed mechanisms of these pathways in cell death remain elusive. We examined the activation of the MAPK members with dioscin treatment. Western blot analysis showed that treatment with dioscin resulted in a significant activation of p38 MAPK and JNK after 1 h and 2 h, respectively (Figs. 2A and S2A). The phosphorylated form of p38 MAPK and JNK was detectable up to 8 h (Figs. 2A and S2A). In contrast, phosphorylated ERK remained unchanged all through the treatment (Figs. 2A and S2A). We next investigated the roles of p38 MAPK and JNK in dioscininduced apoptosis by using specific inhibitors. Western blot analysis showed that pretreatment with p38 MAPK inhibitor SB203580 and JNK inhibitor SP600125 diminished dioscininduced activation of p38 MAPK and JNK (Figs. 2B and S2B). Interestingly, MTT staining of viable cells suggested that inhibition of p38 MAPK or JNK activation only abrogated part of dioscininduced cell death (Fig. S2C). However, combined treatment of these inhibitors resulted in about 40% increases in cell viability compared with dioscin treatment alone (Fig. 2C). Fragmentations of lamin A/C and PARP-1 were also abrogated in the presence of SB203580 and SP600125 after 18 h treatment with dioscin (Figs. 2D and S2C). Inhibition of p38 MAPK reduced dioscinmediated cleavage of caspases 9, 3, and 7 (Figs. S2E and S2D); whereas co-treatment with SP600125 inhibited activation of caspase 8, 10, 3, and 7 (Figs. 2E and S2D). These results suggested that dioscin induced caspase activation and cell death mediated through phosphorylation of p38 MAPK and JNK. 3.3. Activation of p38 MAPK and JNK did not lead to mitochondrial dysfunction Mitochondria play a central role governing cell signaling pathways, caspases activation, and cell death (Gonzalvez and Ashkenazi, 2010; Wang et al., 2005). We next measured the mitochondrial
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Fig. 2. Inhibition of p38 MAPK and JNK abrogated dioscin-induced cell death. (A) Western blot analysis of phosphorylated ERK 1/2, p38 MAPK, and JNK 1/2 with dioscin treatment in HL-60 cells. The blots were stripped and reprobed with antibodies against ERK 1/2, p38 MAPK, and JNK 1/2 accordingly. (B) Western blot analysis showed the efficiency of SB203580 and SP600125 to inhibit p38 MAPK and JNK activation, respectively. (C) MTT assay showed cell viability with dioscin treatment alone or in combination with inhibitors of p38 MAPK and JNK after 18 h. The result is presented as mean 7 S.D. from at least three independent experiments (nPo 0.05 statistically significant difference between control, dioscin treatment, and combined treatment with inhibitors). (D) Western blot analysis of cleaved lamin A/C and PARP-1 in the presence of SB203580 and SP600125 with dioscin treatment for 18 h. (E) Western blot analysis of caspase activation in the presence of SB203580 and SP600125 with dioscin treatment for 18 h. (A, B, D, and E) Data are the representative from three independent experiments; quantitative results are shown in Supplementary Fig. 2.
membrane integrity using Rho-123 as a specific marker. Flow cytometric result revealed that dioscin induced attenuation of ΔΨm after 3 h treatment with dioscin (Fig. 3A). Accumulation of cytotoxic reactive oxygen species is secondary to mitochondrial dysfunction. We then conducted experiments to analyze dioscininduced oxidative stress. Increased fluorescent product of DCFH-DA in HL-60 cells reflected that treatment with dioscin elevated intracellular reactive oxygen species levels (Fig. 3B). The roles of mitochondria dysfunction and cellular reactive oxygen species in regulating p38 MAPK and JNK activation are controversial under different stimuli (Liu et al., 2013). The effect of p38 MAPK and JNK phosphorylation on ΔΨm and cellular reactive oxygen species generation was then studied by pre-treating cells with specific inhibitors. Pre-treatment with SB203580 and SP600125 did not abrogate dioscin-induced loss of ΔΨm (Fig. 3C) nor inhibited the elevated level of intracellular reactive oxygen species (Fig. 3D). These results suggested that phosphorylation of p38 MAPK and JNK did not affect mitochondrial function. 3.4. Dioscin-induced reactive oxygen species generation and mitochondria dysfunction is upstream of p38 MAPK and JNK activation Inhibition of p38 MAPK and JNK phosphorylation did not affect mitochondria permeabilization and cellular reactive oxygen species level, so we next tested whether activation of these kinases depends on mitochondrial function. Western blot analysis indicated that co-treatment with oxygen-free radical scavenger NAC (Figs. 4A and S4A) and thiol-reducing agent DTT (Figs. 4B and S4B) significantly suppressed dioscin-induced p38 MAPK and JNK
activation in a dose-dependent manner. Then we used CyA, an inhibitor of mitochondrial inner membrane permeability transition pore, and potent phospholipase A2 inhibitor ArA that can prevent ΔΨm depletion to determine the effect of mitochondrial permeability on phosphorylation of p38 MAPK and JNK. Increasing dose of CyA and ArA could suppress dioscin-induced JNK phosphorylation significantly (Figs. 4C–D and S4C–D). In contrast, the same dose of CyA and ArA did not alter p38 MAPK activation with dioscin treatment (Figs. 4C–D and S4C–D).
4. Discussion Our earlier proteomic study demonstrated that dioscin induced apoptosis through multiple pathways (Wang et al., 2006, 2007b). Here, we further elucidated the detailed molecular mechanisms of dioscin-induced cell death. Treatment with dioscin induced externalization of phosphatidylserine, activation of caspases, and cleavages of PARP-1 and lamin A/C in HL-60 cells (Figs. 1 and S1). These results support the notion that dioscin-induced apoptosis was caspase-dependent. Activation of caspase 9 is characteristic of the intrinsic mitochondrial death pathway. Mitochondria play a central role in cell death, and the mitochondrial outer membrane permeabilization serves as an integrator of upstream death signaling. Many death stimuli converge on the mitochondria to activate caspase 9, which then proceeds to cleave caspases 3, 7, PARP-1, and various target proteins resulting in the demise of the cell (Green, 2005). Death receptor-dependent recruitment of the FADD promotes dimerization and subsequent activation of caspases 8 and 10, and cleavage
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Fig. 3. Activation of p38 MAPK was downstream of mitochondrial permeabilization and reactive oxygen species generation. (A) Changes in ΔΨm after 3 h treatment with dioscin were measured by FACS. (B) Dioscin caused generation of reactive oxygen species after dioscin treatment for 3 h. (C) Effects of SB203580 and SP600125 on ΔΨm attenuation with dioscin treatment for 6 h. (D) Effects of SB203580 and SP600125 on reactive oxygen species generation after 6 h treatment with dioscin. (A and B) Results are the representative of three independent experiments. (C and D) Data are presented as mean 7 S.D. from three separate experiments (nPo 0.05 statistically significant difference between control, dioscin treatment, and combined treatment with inhibitors).
Fig. 4. Effects of antioxidants or inhibition of mitochondrial permeabilization on dioscin-induced p38 MAPK and JNK 1/2 phosphorylation. HL-60 cells were pretreated with NAC (A) and DTT (B) at the indicated concentration for 1 h before addition of dioscin. Phosphorylation of p38 MAPK and JNK 1/2 was determined by Western blot analysis. Effects of mitochondrial permeability transition inhibitors CsA (C) and ArA (D) on p38 MAPK and JNK 1/2 phosphorylation with dioscin-treatment. These results are representative from three independent experiments. (E) A proposed model for dioscin-induced apoptosis in HL-60 cells. Treatment with dioscin caused depletion of ΔΨm and oxidative stress soon after uptake, which lead to phosphorylation of p38 MAPK and JNK. Phosphorylated p38 MAPK activated caspase 9, and JNK initiated caspases 8 and 10, respectively. These signals then activated caspases 3 and 7, and cleaved PARP-1 and lamin A/C that ultimately led to apoptotic cell death. (A, B, D, and E) Data are the representative from three independent experiments; quantitative results are shown in Supplementary Fig. 3.
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of procaspases 3, 7, and lamin A/C (Inoue et al., 2009). Meanwhile, FADD-independent caspase 8 activation has been reported in other models under different apoptotic stimuli (Kavuri et al., 2011). The rapid phosphorylation of p38 MAPK and JNK with dioscin treatment suggests that these kinases play key roles in the early events of dioscin-induced apoptosis. Our results demonstrated that dioscin-induced activation of caspases 9, 3, and 7 was abrogated in the presence of SB203580 (Figs. 2E and S2D), suggesting that p38 MAPK acted at an early step prior to activation of caspase 9 (Zauli et al., 2003). De Chiara et al. (2006) reported that p38 MAPK activated caspases through conformational changes of Bcl-2 by phosphorylation at Ser87 and Thr56. It has also been proposed that p38 MAPK can regulate caspase activity at post-transcriptional level (Bachelor and Bowden, 2004). Caspases 8 and 10 involved in the signaling complex regulated by ligand binding-induced death receptor trimerization (Green, 2005). JNK acted upstream of caspase 8 through truncation of Bid (Deng et al., 2003). Phosphorylation of JNK can be regulated by transcription factors like c-Jun and NF-κB (Benedetti et al., 2013), and/or non-transcription factors such as induction of Fas ligand and proteasomal elimination of c-FLIPL (Chang et al., 2006). We demonstrated here that activation of caspases 8 and 10 was diminished by JNK inhibitor SP600125 (Fig. 2E and D), suggesting that JNK acted upstream of caspase 8 and 10 with dioscin treatment. A large body of evidence has been accumulated regarding the role of mitochondrial dysfunction and reactive oxygen species generation in the activation of p38 MAPK and JNK (Liu et al., 2012). For example, treatment with cyanidin-3-rutinoside induced p38 MAPK phosphorylation in a reactive oxygen species-dependent manner in HL-60 and CCRE-CEM cells (Feng et al., 2007). Hep3B cells overexpressing JNK are more susceptible to reactive oxygen species-induced cell death (Kim et al., 2004). It is generally believed that signal-regulating kinase 1 (ASK1) is an upstream activator of p38 MAPK and JNK upon mitochondria-initiated signaling pathways (Hatai et al., 2000). ASK1 activity is usually inactivated through binding to different kinds of cellular factors under normal conditions, including thioredoxin and 14-3-3 (Kuo et al., 2007). The critical step in ASK1 activation is the release of thioredoxin and 14-3-3 from the binding complex (Kuo et al., 2007). Free ASK1 is autophosphorylated at Thr 845, and then subsequently activates its downstream MAPK targets (Li et al., 2005). Consistent with these findings, we found that mitochondrial dysfunction is responsible for p38 MAPK activation with dioscin treatment. Co-treatment with antioxidants NAC or DTT abrogated dioscin-induced p38 MAPK activation (Figs. 4A–B and S4A–B); while inhibition of p38 MAPK by SB203580 did not affect ΔΨm attenuation nor the generation of reactive oxygen species (Fig. 3C). In addition, selective blockage of mitochondrial inner membrane permeability transition pore by CyA (Pucci et al., 2008) and potent mitochondrial outer membrane phospholipase A2 inhibitor ArA (Pastorino et al., 1998) decreased phosphorylation of and JNK with dioscin treatment (Figs. 4C–D and S4C–D). However, neither CyA nor ArA exhibited similar effect on p38 MAPK phosphorylation (Figs. 4C–D and S4C–D). These results suggest that the free radicals coordinating with mitochondrial permeability play a critical role in dioscin-induced activation of p38 MAPK and JNK in HL-60 cells. Taken together, the present stepwise analysis with various pharmacological inhibitors (SB203580, SP600125, NAC, DTT, CyA, and ArA) supports the notion that dioscin-induced apoptosis in HL-60 cells through the mitochondrial death pathway by activation of p38 MAPK and JNK (Fig. 4E). The sequential phosphorylation of p38 MAPK through reactive oxygen species activated caspase 9. Both the ΔΨm depletion and accumulated reactive oxygen species stimulated JNK activation, and subsequently lead to the cleavage of caspases 8 and 10. These signaling cascades
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synergistically activated caspases 3 and 7, and consequently cleaved lamin A/C and PARP-1 that ultimately induced cell death. Molecular dissection of the signaling pathways that activate the apoptotic cell death machinery is critical for both understanding of cell death events and the mechanisms of action of chemotherapeutic agents. These results suggest that dioscin has great potential as an alternative treatment strategy for myeloblast leukemia with demonstrated activity.
Acknowledgment This work was partially supported by University of Macau Startup Research Grant (SRG2013-00036-ICMS-QRCM) to YW.
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Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Dioscin induced activation of p38 MAPK and JNK via mitochondrial pathway in HL-60 cell line Ying Wang a,n, Qing-Yu He b, Jen-Fu Chiu c,d,nn a
State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes and Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou, China c Open Laboratory for Tumor Molecular Biology, Department of Biochemistry, Shantou University Medical College, Shantou, China d Department of Anatomy, The University of Hong Kong, Hong Kong SAR, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 24 November 2013 Received in revised form 9 April 2014 Accepted 10 April 2014 Available online 19 April 2014
Saponins have shown promise in cancer prevention and therapy; however, little is known about the detailed signaling pathways underlying their anticancer activities. In the present study, we examined the mechanisms of action of dioscin, a glucosides saponin isolated from Polygonatum zanlanscianense pump, in human myeloblast leukemia HL-60 cells. Dioscin suppressed HL-60 cell growth in a dose-dependent manner. This inhibition was due to the induction of apoptosis as revealed by the externalization of phosphatidylserine, and cleavages of lamin A/C and PARP-1. Treatment with dioscin induced apoptosis through activation of caspases 3, 7, 8, 9, and 10. Phosphorylation of p38 MAPK and JNK contributed to dioscin-induced apoptosis upstream of caspase activation. Using various inhibitors and antioxidant agents, we found that mitochondrial derived reactive oxygen species and depletion of mitochondrial transmembrane potential lead to the phosphorylation of p38 MAPK and JNK. Taken together, our results demonstrated that dioscin induces apoptosis by activation of p38 MAPK and JNK through the caspasedependent mitochondrial death pathway. This work suggests that dioscin may be used as a drug lead for the treatment of myeloblast leukemia. & 2014 Elsevier B.V. All rights reserved.
Keywords: Apoptosis Mitochondria Reactive oxygen species MAPK Saponin Dioscin
1. Introduction Saponins derived from plant sources have been investigated extensively for the treatment of various types of cancers (Koehn and Carter, 2005). Dioscin, a plant steroidal glycosides extracted from Polygonatum zanlanscianense pump, was first isolated and characterized in the mid-20th century (Tsukamoto et al., 1956). Dioscin exhibited cytotoxicity against a number of human malignant cell lines (Cai et al., 2002; Liu et al., 2004; Wang et al., 2001). Our previous proteomic study revealed that dioscin exerted cytotoxicity in human myeloblast leukemia HL-60 cell line through multiple pathways (Hu et al., 2013; Wang et al., 2013, 2007a, 2006); however, the detailed molecular basis of its mechanisms of action remained elusive.
Abbreviations: ASK1, signal-regulating kinase 1; FADD, Fas-associated death domain protein; JNK, c-Jun NH2-terminal protein kinase/stress-activated protein kinases; MAPK, mitogen-activated protein kinases; Δψm, mitochondrial transmembrane potential; Rho-123, Rhodamine 123 n Corresponding author at: Institute of Chinese Medicinal Sciences, University of Macau, Macao SAR, China. nn Corresponding author at: Department of Anatomy, The University of Hong Kong, Hong Kong SAR, China. E-mail addresses: [email protected] (Y. Wang), [email protected] (J.-F. Chiu). http://dx.doi.org/10.1016/j.ejphar.2014.04.018 0014-2999/& 2014 Elsevier B.V. All rights reserved.
Myeloid leukemia is highly curable with the treatment of all-trans retinoic acid in combination with anthracycline-based chemotherapy. Combined therapy achieved an approximately 90% complete remission rate newly diagnosed myeloid leukemia and approximately 20% relapse rate (Imagawa et al., 2010). However, long-term use of retinoic acid leads to development of differentiation syndrome with severe adverse effect (Leblebjian et al., 2013). Thus, development of new therapeutic agents with distinct mode of action could be expected to shorten the treatment or enhance remission rate when combined with all-trans retinoic acid. Apoptosis is a precisely controlled multi-step event that leads to cell death. Mitochondria play a central role in the intrinsic apoptotic pathway, in which the release of cytotoxic proteins from mitochondrial inter membrane to the cytoplasm allows subsequent activation of caspases 9 and 3 and/or DNA fragmentation (Wang et al., 2005). The extrinsic apoptotic pathway is characterized by the engagement of cellular death receptors (Gonzalvez and Ashkenazi, 2010). The biological effects of apoptotic signals are executed by a number of signal-responsive transcription factors and a broad range of substrates, including Bcl-2 family proteins and mitogen-activated protein kinases (MAPKs) (Azijli et al., 2013a; Gonzalvez and Ashkenazi, 2010). The precise role of p38 MAPK and JNK in cell death is cell type- and stimuli-dependent.
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Phosphorylation of p38 MAPK acted as a mediator for caspase 8 in manganese-induced mitochondria-dependent cell death (Azijli et al., 2013b). While transforming growth factor-β-activated p38 MAPK could induce cell death via Fas-associated death domain protein (FADD) independent of caspase activation in human Burkitt lymphoma B cells (Schrantz et al., 2001). Activation of JNK is necessary for apoptosis induced by tumor necrosis factor, osmotic stress, and chemotherapeutic drugs through oxidative stress (Yang et al., 2013). Additionally, p38 MAPK and JNK can initiate mitochondria-dependent intrinsic cell death pathway by direct phosphorylation of Bcl-2 family pro-apoptotic proteins BimEL (Mobasher et al., 2013) and Bax (Shi et al., 2013). In this study, we examined the molecular consequences by treating human myeloblast leukemia HL-60 with dioscin. We demonstrated that dioscin induced the intrinsic mitochondrial death pathway through p38 MAPK and JNK upstream of caspases. Our results illustrate the potential of dioscin as cancer chemotherapeutic agent for the treatment of myeloblast leukemia.
2. Materials and methods 2.1. Dioscin and other reagents Dioscin was provided by Shanghai Institute of Organic Chemistry (Wang et al., 2006) and kept as stock solution at 2 mM so that
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the final concentration of DMSO is less than 0.5% during treatment. The stock solution was diluted in medium prior to use. All other chemicals, except otherwise noted, were obtained from either Sigma-Aldrich Chemical Co. (St. Louis, MO) or GE Healthcare Life Sciences (Uppsala, Sweden).
2.2. Cell line and culture conditions Human myeloblast leukemia HL-60 cells were cultured in RPMI 1640 medium with 2.0 g/L sodium bicarbonate plus 10% fetal bovine serum, 1% L-glutamine, 1% penicillin and streptomycin (100 units/mL), in a humidified incubator with an atmosphere of 95% air and 5% CO2 at 37 1C.
2.3. Drug treatment The cells were grown to about 80% confluence and were then either subcultured or treated with the IC50 (half-maximal inhibitory concentration) value of dioscin (7.6 mM, or 6.6 μg/mL) to study the early direct effect (Fig. 1A). This dose is. In some experiments, cells were pretreated 1 h prior to the addition of dioscin with SB203580 (CalBioChem, La Jolla, CA), SP600125 (CalBioChem), N-acetyl-L-cysteine (NAC), DTT, aristolochic acid (ArA), or cyclosporine A (CyA).
Fig. 1. Dioscin induced apoptosis and activated caspases in HL-60 cells. (A) MTT assay showed that dioscin exhibited cytotoxicity in HL-60 cells in a dose-dependent manner after 24 h treatment, and the IC50 (half-maximal inhibition concentration) value is 7.6 7 0.12 mM. (B) Detection of dioscin-induced phosphatidylserine externalization in HL-60 cells by annexin-V analysis. (C) Western blot analysis of cleaved lamin A/C ( 50 kDa and 40 kDa) and PARP-1 ( 89 kDa) with dioscin treatment for different period of time. (D) Western blot analysis of cleaved caspases 3, 7, 8, 9, and 10 with dioscin treatment for different period of time. (A) Data are calculated from at least three separate experiments. (B–D) Data are representatives of three independent experiments; quantitative results of (C) and (D) are shown in Supplementary Fig. 1.
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2.4. Cytotoxicity assay The cytotoxicity of drug treatment was determined by MTT assay, in accordance to the procedure reported previously (Wang et al., 2007b). 2.5. Flow cytometric analysis of apoptosis Induction of apoptosis was determined by Annexin V staining (Molecular Probes, Eugene, OR). Cells were cultivated for 24 h and then either left untreated as control or treated with dioscin. At the end of each experiment, cells were harvested, resuspended in ice cold PBS, stained by Annexin V, and analyzed with a FACStar Plus flow cytometry. One million cells were analyzed for each sample, providing a solid statistical basis for the determination of the percentage of apoptotic cells in each treatment. 2.6. Measurement of mitochondrial transmembrane potential (Δψm) Changes in Δψm were assayed by Rhodamine 123 (Rho-123, Molecular Probes) staining in accordance with the procedure described previously (Wang et al., 2005). 2.7. Detection of oxidative stress Cells in HBSS were incubated for 15 min at 37 1C with 1 μM dichlorodihydrofluorescein diacetate (DCFH-DA, Molecular Probes), washed, re-suspended in complete growth media, and then treated with dioscin. Dye oxidation (increase in fluorescence) was measured using a FACStar Plus flow cytometer with excitation and emission settings of 488 nm and 530 nm, respectively (Wang et al., 2005). 2.8. Western blot analysis Western blot analysis was performed using primary antibodies against phospho-ERK (Tyr204, Santa Cruz Biotechnology), ERK (Santa Cruz Biotechnology), phospho-p38 MAPK (Thr180/Tyr182, Stressgen, Ann Arbor, MI), p38 MAPK (Cell Signaling Technology, Danvers, MA), phospho-JNK 1/2 (Thr183/Tyr185, Stressgen), JNK 1/2 (Cell Signaling Technology), caspase 3 (Lab Vision, Fremont, CA), caspase 7 (Lab Vision), caspase 8 (Lab Vision), caspase 9 (Lab Vision), caspase 10 (Lab Vision), lamin A/C (Santa Cruz Biotechnology), PARP-1 (Cell Signaling Technology), and alpha-tubulin (Sigma-Aldrich) at optimal dilution. 2.9. Statistical analysis Statistical analysis was performed using two-tailed Student's t-test (Microsoft Excel), and Po 0.05 was considered significant. Data were expressed as mean 7S.D. of triplicate samples, and reproducibility was confirmed in three separate experiments.
3. Results 3.1. Dioscin induces apoptosis and activation of caspases in HL-60 cells This study aims to analyze the mechanisms of action of dioscininduced apoptosis in HL-60 cells. The IC50 of dioscin for HL-60 cells was 7.6 mM detected by MTT assay (Fig. 1A). We then use the IC50 concentration all through the study to examine the early event with dioscin treatment. Microscopic results from previous study showed that HL-60 cells treated with 7.6 mM dioscin exhibited a shrunken morphology compared with untreated control cells (Wang et al., 2007a, 2007b), indicating that dioscin affected cell integrity
adversely. Significant increase in cell death was detected by annexinV staining after 24 h treatment with dioscin (Fig. 1B). The proteolytic cleavage of PARP-1 between Asp216 and Gly217 and the cleavage of 70 kDa lamin A/C to fragments of 40–45 kDa and 28 kDa served as early indicators of nuclear deregulation and apoptotic cell death (Takahashi et al., 1996). The treatment with dioscin induced fragmentation of lamin A/C and PARP-1 in a time-dependent manner (Fig. 1C and A). Together with the results from previous study, these findings confirmed that treatment with dioscin induced apoptosis in HL-60 cells (Wang et al., 2007a, 2007b). As major mediators of apoptotic cell death, caspases contribute to the overall morphological alterations by cleaving various cellular substrates in apoptotic cell death. Among them, caspases 8 and 10 are primarily activated by death receptors, caspase 9 is an initiator caspase downstream of the permeabilization of mitochondrial outer membrane, and caspases 3 and 7 serve as executioners that hence trigger apoptotic cell death (Green, 2005). Activation of caspases was measured by assessing the cleaved forms of these enzymes. Western blot results revealed that treatment with dioscin caused cleavage of caspases 8, 10, 7, and 3 as early as 6 h (Figs. 1D and S1B). The cleaved active forms were detectable after that throughout the period of the treatment in a time-dependent manner. Elevated expression of caspase 9 was detected after 6 h treatment, peaked at 9 to 18 h, and then tapered off. These results indicated that treatment with dioscin induced apoptotic cell death in a caspase-dependent manner. 3.2. Activation of p38 MAPK and JNK leads to cell death with the treatment of dioscin The MAPK signaling pathway plays important roles in regulating apoptosis induced by various cellular stresses and chemotherapeutic agents (Bruzzese et al., 2013). However, the detailed mechanisms of these pathways in cell death remain elusive. We examined the activation of the MAPK members with dioscin treatment. Western blot analysis showed that treatment with dioscin resulted in a significant activation of p38 MAPK and JNK after 1 h and 2 h, respectively (Figs. 2A and S2A). The phosphorylated form of p38 MAPK and JNK was detectable up to 8 h (Figs. 2A and S2A). In contrast, phosphorylated ERK remained unchanged all through the treatment (Figs. 2A and S2A). We next investigated the roles of p38 MAPK and JNK in dioscininduced apoptosis by using specific inhibitors. Western blot analysis showed that pretreatment with p38 MAPK inhibitor SB203580 and JNK inhibitor SP600125 diminished dioscininduced activation of p38 MAPK and JNK (Figs. 2B and S2B). Interestingly, MTT staining of viable cells suggested that inhibition of p38 MAPK or JNK activation only abrogated part of dioscininduced cell death (Fig. S2C). However, combined treatment of these inhibitors resulted in about 40% increases in cell viability compared with dioscin treatment alone (Fig. 2C). Fragmentations of lamin A/C and PARP-1 were also abrogated in the presence of SB203580 and SP600125 after 18 h treatment with dioscin (Figs. 2D and S2C). Inhibition of p38 MAPK reduced dioscinmediated cleavage of caspases 9, 3, and 7 (Figs. S2E and S2D); whereas co-treatment with SP600125 inhibited activation of caspase 8, 10, 3, and 7 (Figs. 2E and S2D). These results suggested that dioscin induced caspase activation and cell death mediated through phosphorylation of p38 MAPK and JNK. 3.3. Activation of p38 MAPK and JNK did not lead to mitochondrial dysfunction Mitochondria play a central role governing cell signaling pathways, caspases activation, and cell death (Gonzalvez and Ashkenazi, 2010; Wang et al., 2005). We next measured the mitochondrial
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Fig. 2. Inhibition of p38 MAPK and JNK abrogated dioscin-induced cell death. (A) Western blot analysis of phosphorylated ERK 1/2, p38 MAPK, and JNK 1/2 with dioscin treatment in HL-60 cells. The blots were stripped and reprobed with antibodies against ERK 1/2, p38 MAPK, and JNK 1/2 accordingly. (B) Western blot analysis showed the efficiency of SB203580 and SP600125 to inhibit p38 MAPK and JNK activation, respectively. (C) MTT assay showed cell viability with dioscin treatment alone or in combination with inhibitors of p38 MAPK and JNK after 18 h. The result is presented as mean 7 S.D. from at least three independent experiments (nPo 0.05 statistically significant difference between control, dioscin treatment, and combined treatment with inhibitors). (D) Western blot analysis of cleaved lamin A/C and PARP-1 in the presence of SB203580 and SP600125 with dioscin treatment for 18 h. (E) Western blot analysis of caspase activation in the presence of SB203580 and SP600125 with dioscin treatment for 18 h. (A, B, D, and E) Data are the representative from three independent experiments; quantitative results are shown in Supplementary Fig. 2.
membrane integrity using Rho-123 as a specific marker. Flow cytometric result revealed that dioscin induced attenuation of ΔΨm after 3 h treatment with dioscin (Fig. 3A). Accumulation of cytotoxic reactive oxygen species is secondary to mitochondrial dysfunction. We then conducted experiments to analyze dioscininduced oxidative stress. Increased fluorescent product of DCFH-DA in HL-60 cells reflected that treatment with dioscin elevated intracellular reactive oxygen species levels (Fig. 3B). The roles of mitochondria dysfunction and cellular reactive oxygen species in regulating p38 MAPK and JNK activation are controversial under different stimuli (Liu et al., 2013). The effect of p38 MAPK and JNK phosphorylation on ΔΨm and cellular reactive oxygen species generation was then studied by pre-treating cells with specific inhibitors. Pre-treatment with SB203580 and SP600125 did not abrogate dioscin-induced loss of ΔΨm (Fig. 3C) nor inhibited the elevated level of intracellular reactive oxygen species (Fig. 3D). These results suggested that phosphorylation of p38 MAPK and JNK did not affect mitochondrial function. 3.4. Dioscin-induced reactive oxygen species generation and mitochondria dysfunction is upstream of p38 MAPK and JNK activation Inhibition of p38 MAPK and JNK phosphorylation did not affect mitochondria permeabilization and cellular reactive oxygen species level, so we next tested whether activation of these kinases depends on mitochondrial function. Western blot analysis indicated that co-treatment with oxygen-free radical scavenger NAC (Figs. 4A and S4A) and thiol-reducing agent DTT (Figs. 4B and S4B) significantly suppressed dioscin-induced p38 MAPK and JNK
activation in a dose-dependent manner. Then we used CyA, an inhibitor of mitochondrial inner membrane permeability transition pore, and potent phospholipase A2 inhibitor ArA that can prevent ΔΨm depletion to determine the effect of mitochondrial permeability on phosphorylation of p38 MAPK and JNK. Increasing dose of CyA and ArA could suppress dioscin-induced JNK phosphorylation significantly (Figs. 4C–D and S4C–D). In contrast, the same dose of CyA and ArA did not alter p38 MAPK activation with dioscin treatment (Figs. 4C–D and S4C–D).
4. Discussion Our earlier proteomic study demonstrated that dioscin induced apoptosis through multiple pathways (Wang et al., 2006, 2007b). Here, we further elucidated the detailed molecular mechanisms of dioscin-induced cell death. Treatment with dioscin induced externalization of phosphatidylserine, activation of caspases, and cleavages of PARP-1 and lamin A/C in HL-60 cells (Figs. 1 and S1). These results support the notion that dioscin-induced apoptosis was caspase-dependent. Activation of caspase 9 is characteristic of the intrinsic mitochondrial death pathway. Mitochondria play a central role in cell death, and the mitochondrial outer membrane permeabilization serves as an integrator of upstream death signaling. Many death stimuli converge on the mitochondria to activate caspase 9, which then proceeds to cleave caspases 3, 7, PARP-1, and various target proteins resulting in the demise of the cell (Green, 2005). Death receptor-dependent recruitment of the FADD promotes dimerization and subsequent activation of caspases 8 and 10, and cleavage
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Fig. 3. Activation of p38 MAPK was downstream of mitochondrial permeabilization and reactive oxygen species generation. (A) Changes in ΔΨm after 3 h treatment with dioscin were measured by FACS. (B) Dioscin caused generation of reactive oxygen species after dioscin treatment for 3 h. (C) Effects of SB203580 and SP600125 on ΔΨm attenuation with dioscin treatment for 6 h. (D) Effects of SB203580 and SP600125 on reactive oxygen species generation after 6 h treatment with dioscin. (A and B) Results are the representative of three independent experiments. (C and D) Data are presented as mean 7 S.D. from three separate experiments (nPo 0.05 statistically significant difference between control, dioscin treatment, and combined treatment with inhibitors).
Fig. 4. Effects of antioxidants or inhibition of mitochondrial permeabilization on dioscin-induced p38 MAPK and JNK 1/2 phosphorylation. HL-60 cells were pretreated with NAC (A) and DTT (B) at the indicated concentration for 1 h before addition of dioscin. Phosphorylation of p38 MAPK and JNK 1/2 was determined by Western blot analysis. Effects of mitochondrial permeability transition inhibitors CsA (C) and ArA (D) on p38 MAPK and JNK 1/2 phosphorylation with dioscin-treatment. These results are representative from three independent experiments. (E) A proposed model for dioscin-induced apoptosis in HL-60 cells. Treatment with dioscin caused depletion of ΔΨm and oxidative stress soon after uptake, which lead to phosphorylation of p38 MAPK and JNK. Phosphorylated p38 MAPK activated caspase 9, and JNK initiated caspases 8 and 10, respectively. These signals then activated caspases 3 and 7, and cleaved PARP-1 and lamin A/C that ultimately led to apoptotic cell death. (A, B, D, and E) Data are the representative from three independent experiments; quantitative results are shown in Supplementary Fig. 3.
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of procaspases 3, 7, and lamin A/C (Inoue et al., 2009). Meanwhile, FADD-independent caspase 8 activation has been reported in other models under different apoptotic stimuli (Kavuri et al., 2011). The rapid phosphorylation of p38 MAPK and JNK with dioscin treatment suggests that these kinases play key roles in the early events of dioscin-induced apoptosis. Our results demonstrated that dioscin-induced activation of caspases 9, 3, and 7 was abrogated in the presence of SB203580 (Figs. 2E and S2D), suggesting that p38 MAPK acted at an early step prior to activation of caspase 9 (Zauli et al., 2003). De Chiara et al. (2006) reported that p38 MAPK activated caspases through conformational changes of Bcl-2 by phosphorylation at Ser87 and Thr56. It has also been proposed that p38 MAPK can regulate caspase activity at post-transcriptional level (Bachelor and Bowden, 2004). Caspases 8 and 10 involved in the signaling complex regulated by ligand binding-induced death receptor trimerization (Green, 2005). JNK acted upstream of caspase 8 through truncation of Bid (Deng et al., 2003). Phosphorylation of JNK can be regulated by transcription factors like c-Jun and NF-κB (Benedetti et al., 2013), and/or non-transcription factors such as induction of Fas ligand and proteasomal elimination of c-FLIPL (Chang et al., 2006). We demonstrated here that activation of caspases 8 and 10 was diminished by JNK inhibitor SP600125 (Fig. 2E and D), suggesting that JNK acted upstream of caspase 8 and 10 with dioscin treatment. A large body of evidence has been accumulated regarding the role of mitochondrial dysfunction and reactive oxygen species generation in the activation of p38 MAPK and JNK (Liu et al., 2012). For example, treatment with cyanidin-3-rutinoside induced p38 MAPK phosphorylation in a reactive oxygen species-dependent manner in HL-60 and CCRE-CEM cells (Feng et al., 2007). Hep3B cells overexpressing JNK are more susceptible to reactive oxygen species-induced cell death (Kim et al., 2004). It is generally believed that signal-regulating kinase 1 (ASK1) is an upstream activator of p38 MAPK and JNK upon mitochondria-initiated signaling pathways (Hatai et al., 2000). ASK1 activity is usually inactivated through binding to different kinds of cellular factors under normal conditions, including thioredoxin and 14-3-3 (Kuo et al., 2007). The critical step in ASK1 activation is the release of thioredoxin and 14-3-3 from the binding complex (Kuo et al., 2007). Free ASK1 is autophosphorylated at Thr 845, and then subsequently activates its downstream MAPK targets (Li et al., 2005). Consistent with these findings, we found that mitochondrial dysfunction is responsible for p38 MAPK activation with dioscin treatment. Co-treatment with antioxidants NAC or DTT abrogated dioscin-induced p38 MAPK activation (Figs. 4A–B and S4A–B); while inhibition of p38 MAPK by SB203580 did not affect ΔΨm attenuation nor the generation of reactive oxygen species (Fig. 3C). In addition, selective blockage of mitochondrial inner membrane permeability transition pore by CyA (Pucci et al., 2008) and potent mitochondrial outer membrane phospholipase A2 inhibitor ArA (Pastorino et al., 1998) decreased phosphorylation of and JNK with dioscin treatment (Figs. 4C–D and S4C–D). However, neither CyA nor ArA exhibited similar effect on p38 MAPK phosphorylation (Figs. 4C–D and S4C–D). These results suggest that the free radicals coordinating with mitochondrial permeability play a critical role in dioscin-induced activation of p38 MAPK and JNK in HL-60 cells. Taken together, the present stepwise analysis with various pharmacological inhibitors (SB203580, SP600125, NAC, DTT, CyA, and ArA) supports the notion that dioscin-induced apoptosis in HL-60 cells through the mitochondrial death pathway by activation of p38 MAPK and JNK (Fig. 4E). The sequential phosphorylation of p38 MAPK through reactive oxygen species activated caspase 9. Both the ΔΨm depletion and accumulated reactive oxygen species stimulated JNK activation, and subsequently lead to the cleavage of caspases 8 and 10. These signaling cascades
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synergistically activated caspases 3 and 7, and consequently cleaved lamin A/C and PARP-1 that ultimately induced cell death. Molecular dissection of the signaling pathways that activate the apoptotic cell death machinery is critical for both understanding of cell death events and the mechanisms of action of chemotherapeutic agents. These results suggest that dioscin has great potential as an alternative treatment strategy for myeloblast leukemia with demonstrated activity.
Acknowledgment This work was partially supported by University of Macau Startup Research Grant (SRG2013-00036-ICMS-QRCM) to YW.
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