Leukemia Research 34 (2010) 658–665 Contents lists available at ScienceDirect Leukemia Research journal homepage: www.elsevier.com/locate/leukres Plumbagin induces ROS-mediated apoptosis in human promyelocytic leukemia cells in vivo Kai-Hong Xu a , Dao-Pei Lu a,b,∗ a Peking University People’s Hospital, Institute of Hematology, Beijing, China b Beijing Daopei Hospital, Beijing, China a r t i c l e i n f o a b s t r a c t Article history: Plumbagin, a naphtoquinone from the roots of Plumbago zeylanica is known to possess anticancer and Received 15 June 2009 anti-bacterial activity. Based on the former ﬁnding of our group in vitro demonstrating its effectiveness in Received in revised form 15 August 2009 human promyelocytic leukemia cells, NB4, in this study we further revealed the mitochondrial pathway Accepted 15 August 2009 involved in plumbagin-induced apoptosis. We also found that the generation of ROS was a critical medi- Available online 12 September 2009 ator in plumbagin-induced apoptosis, which would be abrogated completely by antioxidant, NAC. The anticancer effect of plumbagin was investigated in vivo using NB4 tumor xenograft in NOD/SCID mice. Keywords: The incidence of formation, growth characteristics, body weight and volume of tumors were observed. Plumbagin Apoptosis The histopathologic examination of tumors and organs were made. The results showed that intraperi- Leukemia toneal injection of plumbagin (2 mg/kg body weight) daily for 3 weeks resulted to a 64.49% reduction Reactive oxygen species of tumor volume compared with the control. Furthermore, there was no overt manifestation of toxicity Xenograft such as weight loss, tissue damage and behavior change which appeared in Doxorubicin-treated mice (1 mg/kg thrice a week). These results indicate that plumbagin has potential as a novel therapeutic agent for myeloid leukemia. © 2009 Elsevier Ltd. All rights reserved. 1. Introduction true by disrupting microtubule polymerization through tubulin binding and inducing apoptosis [12,13]. In addition, plumbagin Acute promyelocytic leukemia (APL) was once a very fulminant does not exert an apoptotic effect on normal cells and therefore disease with a severe bleeding tendency and a fatal course of only may have potential as a chemotherapeutic agent [7,10]. weeks. The introduction of ATRA and arsenic trioxide (ATO) has ROS are derived from the metabolism of molecular oxygen. increased the cure rate to 70–85%. However, approximately 20–30% ROS normally exist in balance with biochemical antioxidants in of patients are still relapsing, especially extramedullary relapse . all aerobic cells. Oxidative stress occurs when this critical bal- APL relapse attributes to repetitive or prolonged exposure to ATRA ance is disrupted due to excess of ROS, antioxidant depletion, or and ATO, which are more likely to develop the retinoic acid syn- both [14,15]. Evidence is accumulating to indicate that chemother- drome (RAS), cardiac toxicity and drug resistance. Therefore, other apeutic agents may be selectively toxic to tumor cells because highly effective and safe drugs still need to be explored. they increase oxidant stress and enhance these already stressed Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a cells beyond their limit [16–18]. Cytotoxic ROS signaling seems quinonoid constituent isolated from the root of Plumbago zey- to trigger the mitochondrial apoptotic pathway, as indicated by lanica L. has been shown to have diverse pharmacological effects a change in Bax/Bcl-2 ratios, resulting in mitochondrial mem- including anti-bacterial , antiatherosclerotic  and anticancer brane potential loss, cytochrome c release and caspase-9 activation . The research in cancer is most promising, plumbagin can exert [19,20]. anticancer and antiproliferative properties in a variety of cell lines Previous study carried out in vitro by our group has demon- and animal models [5–10]. Plumbagin can also as a radiosensitizer strated that plumbagin can induce apoptosis of APL cell line, NB4 modulate the effects of radiation in the treatment of tumor . . In the present study, we explored the apoptosis pathway The anticancer effect of plumbagin has been postulated to come involved in plumbagin-treated NB4 cells by measuring the activ- ity of caspase-3, -8 and -9, the change of mitochondrial membrane potential as well as the expression of the Bcl-2 family. We showed for the ﬁrst time that the natural product, plumbagin, induces apop- ∗ Corresponding author at: Peking University People’s Hospital, Institute of Hema- tosis of NB4 through the increase of ROS, which then serves as a tology, 11 Xizhimen South Street, Beijing 100044, China. Tel.: +86 10 88324618; fax: +86 10 68333439. signal triggering the mitochondrial apoptotic pathway, and also E-mail address: lu email@example.com (D.-P. Lu). conﬁrmed the validity of plumbagin in APL mouse model. 0145-2126/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2009.08.017 K.-H. Xu, D.-P. Lu / Leukemia Research 34 (2010) 658–665 659 2. Materials and methods parafﬁn and cut into 3–5 m sections. The apoptotic cells were identiﬁed by TUNEL assay using In situ Cell Death Detection Kit (Roche Applied Science, Indianapolis, IN), 2.1. Materials according to the manufacturer’s protocol. The apoptotic index (AI) was calculated as number of apoptotic cells × 100/total number of cells. All animal experimental RPMI 1640 media and fetal bovine serum (FBS) were from GIBCO-BRL procedures were approved by the Ethics Committee of Peking University People’s (Grand Island, NY, USA). N-acetyl-l-cysteine (NAC), DCFH-DA (2 ,7 -dichloro ﬂuo- Hospital. roscein diacetate), dimethyl sulphoxide (DMSO) and Rhodamine 123 were from Sigma–Aldrich Inc. (St. Louis, MO, USA). Plumbagin (Sigma–Aldrich, USA) solved in DMSO to a stock concentration of 50 mM, was stored at −20 ◦ C. The ﬁnal DMSO con- 2.8. Toxicity assessment centrations in the medium were not greater than 0.1%. Annexin V-FITC Apoptosis Detection Kit was from BD Bioscience (BD Pharmingen, CA, USA). All other chemicals To evaluate the potential side effects on treated mice, they were continuously were of analytical grade. observed for relevant indexes such as weight loss, diarrhea, behavior and toxic death. Tissues of heart, liver and kidney were ﬁxed in 4% paraformaldehyde and embed- ded in parafﬁn when treatment terminated. Parafﬁn sections were stained with 2.2. Cell hematoxylin–eosin (H–E) for microscopic examination. NB4 cells were cultured in RPMI 1640 with 10% FBS, 100 U/ml penicillin and 100 g/ml streptomycin in a humidiﬁed atmosphere with 5% CO2 . 2.9. Statistical analysis 2.3. Assay for caspase activity Data were expressed as means ± SD of three determinations. Statistical com- parisons of the results were made using analysis of variance (ANOVA). Signiﬁcant Caspase-related protease activity was determined by using commercially avail- differences between the means of test groups were analyzed by Dunnett’s test. able kits (BioVision, Mountain View, CA, USA), according to the manufacturer’s Statistical signiﬁcance was set at P < 0.05. instructions. The assay was based on spectrophotometric detection of the chro- mophore p-nitroanilide (pNA) at 405 nm after cleavage from the labeled substrate, LEHD-pNA (for caspase-9), DEVD-pNA (for caspase-3) and IETD-pNA (for caspase- 3. Results 8). An equal amount of protein (∼200 g) from each sample was used to determine caspase activity using a standard microplate reader (Thermo Varioskan. Flash, Van- taa, Finland) and the relative caspase activity was absorbance at 405 nm subtracting 3.1. The activation of caspase blank one. Experiments were performed in triplicate. At multiple time points after treatment of 10 M plumbagin, 2.4. Mitochondrial membrane potential ( « ) assay caspase activity was monitored by colorimetric assay. Caspase- 9, as an initiator caspase, whose activity increased distinctly and The « was determined by ﬂow cytometry using Rhodamine 123. Brieﬂy, treated cells were washed twice with PBS and incubated with 5 g/ml Rhodamine was at the peak after 4 h, whereas, caspase-3 and -8 were acti- 123 at 37 ◦ C for 30 min. Rhodamine 123 intensity was determined by ﬂow cytometry vated later than caspase-9, getting to the peak after 8 h (Fig. 1a). (Becton Dickinson). « determinations were based on mean ﬂuorescence intensity Activation of caspase-3, -8 and -9 induced by plumbagin was (MFI). also conﬁrmed by cleavage of proforms into the active cleaved forms using polyclonal antihuman casapase-3, -8, and -9 antibodies 2.5. Western blot analysis (Fig. 1b). After 24 h of incubation, the cells (8 × 106 ) were treated with plumbagin 10 M for various time periods. Total cell extracts were prepared by extracting proteins with lysis buffer [40 mmol/l Tris–Cl (pH 8.0), 120 mmol/l NaCl and 0.1% NP40] sup- plemented with protease inhibitors. The cell lysate was cleared by centrifugation at 12,000 × g for 15 min, the protein content of the supernatant was determined using Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA). Equal amounts of pro- tein were separated by 12% SDS-PAGE and transferred to Immobilon-P membrane (Millipore, Bedford, MA), according to a standard protocol. The protein expres- sion was detected by immunoblotting with the corresponding primary antibodies. The following antibodies were used in this study: anti-caspase-3, -9 and anti-Bcl- XL (Cell Signaling Technology, Inc., Beverly, MA); anti-Bcl-2, anti-Bax, anti- Bak and anti- -actin (Santa Cruz Biotechnology, Santa Cruz, CA); anti-caspase-8 (Lab Vision Corporation, Fremont, CA), followed by incubation with the IRDye® 800CW Conjugated Goat (polyclonal) Anti-Rabbit IgG (LI-COR Biosciences, Lincoln, NE). The signals were visualized using the Odyssey infrared imaging system (LI-COR Biosciences). 2.6. ROS measurement To assess the generation of ROS, control- and plumbagin-treated cells (1 × 106 ) were incubated with 10 M DCFH-DA. Within the cells, DCFH-DA is converted to DCFH, which can be oxidized to the ﬂuorescent compound DCF in the presence of ROS. After incubation for 30 min at 37 ◦ C, the cells were washed twice with PBS and analyzed by ﬂow cytometry for DCF. 2.7. Xenograft assay 4–6-week-old male NOD/SCID mice (National Science Council Animal Center, Beijing, China) were bred and maintained under a speciﬁc pathogen-free condition in the Department of Laboratory Animal Science, Peking University Health Science Center. NB4 cells (5 × 106 /0.2 ml) were injected subcutaneously (s.c.) into NOD/SCID mice. Treatment initiated on day 10 when tumors reached 100–200 mm3 . Mice were randomly assigned to one of the following treatment groups: (i) mice (n = 8) had intraperitoneal injection (i.p.) daily with 2 mg/kg plumbagin in vehicle (25% PEG in PBS); (ii) mice (n = 12) treated daily with an equal volume of the same vehicle; (iii) mice (n = 8) treated i.p. with 1 mg/kg of Doxorubicin (DOX) (Pharmacia Italia S.P.A, Fig. 1. Effects of plumbagin on caspase activation. (a) NB4 cells were incubated with Italy) three times a week. Tumor volumes and body weights were measured twice a 10 M plumbagin for the indicated times and analyzed for activities of caspase- week. Tumor size was based on the formula: tumor volume (mm3 ) = 1/2 (long diam- 3, -8 and -9 by colorimetric assay. Values are means (±SD) of three independent eter) × (short diameter)2 , measured by calipers. After 3 weeks of treatment, mice experiments. (b) NB4 cells were cultured with 10 M plumbagin for the indicated were sacriﬁced and tumor tissues were ﬁxed in 4% paraformaldehyde, embedded in times and analyzed by immunoblotting with anti-caspase-3, -8, and -9 antibodies. 660 K.-H. Xu, D.-P. Lu / Leukemia Research 34 (2010) 658–665 signal was predominant over anti-apoptotic signal in this course (Fig. 3). 3.4. Plumbagin increased intracellular superoxide and apoptosis was inhibited by NAC in NB4 cells ROS have been suggested as a possible mediator of apoptosis induced by plumbagin [5,8]. We examined ROS production in NB4 cells with 10 M plumbagin using the ﬂuorescent probe DCFH-DA. Within 0.5 h, the treated NB4 cells showed an increase in intra- cellular ROS compared with the control, reaching the maximum at 1.5 h and declining after 2 h (Fig. 4a). NAC, an excellent sup- plier of GSH, can remove ROS from cells. Pretreated with10 mM Fig. 2. Time course analysis of mitochondrial membrane potential in Plumbagin- NAC for 2 h, and then exposed to 10 M plumbagin for 24 h, treated cells. NB4 cells were exposed to 10 M plumbagin for the indicated times. plumbagin-induced apoptosis was completely inhibited (Fig. 4b). « determinations were based on mean ﬂuorescence intensity (MFI) of Rhodamine These results indicated that ROS generation may be an initiating- 123 by ﬂow cytometry. Each value is the mean ± SD of three determinations. event in plumbagin-induced apoptosis. 3.2. Change of mitochondrial membrane potential 3.5. The anti-tumor effect of plumbagin in vivo Mitochondria play a key role in the regulation of apoptotic cell Our in vitro data prompted us to examine whether the effect of death and change in « m is known to be one of the important plumbagin was valid in vivo. NB4 cells were injected s.c. into the factors for mitochondrial dysfunction [22,23]. Therefore, we exam- ﬂanks of NOD/SCID mice, because the anti-tumor effect could be ined « m by ﬂow cytometry using Rhodamine 123, a lipophilic quantitatively and pathologically assessed. Subcutaneous tumors ﬂuorochrome taken up by the mitochondria in proportion to the appeared with 100% after 1 week and all treatment initiated on « m . The results showed that NB4 cells treated with 10 M day 10 by i.p. route. Plumbagin showed markedly suppression of plumbagin for 0–24 h, led to the levels of « collapsed, which tumor growth after only 1-week treatment (day 17 after implanta- was remarkable after 4 h and these effects were time-dependent tion). The tumor volume was 193 ± 40 mm3 in vehicle control and (Fig. 2). 153 ± 37 mm3 in plumbagin-treated group (P < 0.05). Moreover, the difference increased with time; at the termination of the experi- 3.3. Expression of the Bcl-2 family protein in plumbagin-treated ment, the average volume of the tumors in plumbagin-treated mice NB4 cells was reduced by 64.49% when compared with the mice treated with vehicle (P < 0.01) (Fig. 5a and b). The Bcl-2 family includes a number of pro-apoptotic and For APL mouse models developed with implantation of NB4 cell anti-apoptotic proteins which are known to regulate apoptosis do not respond to ATRA impressively as showed in vitro [25,26], we at the level of mitochondria by changing its relative levels . initially chose ATO to further evaluate the efﬁcacy of plumbagin In the study, levels of the major anti-apoptotic protein, Bcl-2 in NB4 xenograft, but ﬁnally ﬁxed on DOX due to dismal inhibi- and Bcl-XL; the major pro-apoptotic protein, Bax and Bak, were tion of ATO on tumor growth (data not shown). Compared with visualized by western blot. After incubated with 10 M plumbagin plumbagin, DOX treatment had a superior suppression of NB4 for 0, 2, 4 and 8 h, the protein levels of Bax and Bak protein were xenograft (P < 0.05) (Fig. 5a and b), whereas, without exception, the increased; in contrast, plumbagin decreased Bcl-XL protein level mice experienced weight loss, diarrhea and decreased activity after and had no obvious effect on Bcl-2 expression, implying that the treatment with DOX, and one of the mice died of cachexia at the ratio between pro-apoptotic and anti-apoptotic members was day 11 after treatment; Fig. 6a showed the change of body weight increased by plumbagin treatment. It is likely that pro-apoptotic in treated mice. Furthermore, DOX treatment group showed toxic pathologic change in tissue of heart and liver. Contrarily, mice in plumbagin group and vehicle group were in good physical condi- tion and pathologic analysis revealed no obvious tissue changes in plumbagin-treated mice (Fig. 6b). An increase of TUNEL-positive cells was observed in tumors of the plumbagin-treated mice compared with tumors taken from vehicle-treated mice (Fig. 7). There was signiﬁcant difference in AI (9.63 ± 1.69% for the plumbagin-treated group and 2.44 ± 1.23% for the vehicle group, P < 0.01). These data indicated that the admin- istration of plumbagin induces tumor regressions associated with apoptosis in vivo. 4. Discussion Plumbagin has been approved to effectively inhibit the prolifer- ation of various cancer cells including breast cancer, cervical cancer, lung cancer, melanoma, ovarian cancer and prostate cancer. There are different sensitivity to plumbagin: breast cancer, cervical can- Fig. 3. Effects of plumbagin on the expression of Bcl-2 family protein. NB4 cells cer, ovarian cancer and prostate cancer are more susceptible to were exposed to 10 M plumbagin for the indicated time intervals, and total cell lysates were subjected to western blot with the corresponding antibodies. The - plumbagin with the IC50 less than 5 M, while lung cancer and actin protein was used as protein loading control as shown for each blot. melanoma cells have the IC50 more than 14 M [5,7,8,27–29]. Our K.-H. Xu, D.-P. Lu / Leukemia Research 34 (2010) 658–665 661 Fig. 4. ROS and apoptosis in plumbagin-treated cells. (a) Generation of intracellular ROS in NB4 cells treated with plumbagin. ROS levels were detected by ﬂow cytometry using H2 DCFDA probes at different time points after exposure to 10 M plumbagin. Abscissa: representing DCF ﬂuorescence intensities as logarithmic scale (increases from left to right); ordinate: number of events (cells). Open peaks represent control cells; shaded peaks represent cells treated with plumbagin. Results shown are representative of three independent experiments. (b) Abrogation of plumbagin-induced apoptosis by NAC. NB4 cells were pretreated with 10 mM NAC for 2 h, and then exposed to 10 M plumbagin for 24 h. The apoptosis was determined by annexin V/propidium iodide double staining. 662 K.-H. Xu, D.-P. Lu / Leukemia Research 34 (2010) 658–665 Fig. 5. Anti-tumor efﬁcacy of plumbagin or DOX on NB4 xenograft in NOD/SCID mice. From day 10 to day 31, mice with established tumors (100–200 mm3 ) were dosed i.p. with plumbagin (2 mg/kg) daily or DOX (1 mg/kg) thrice a week, and the control mice were treated with vehicle as detailed in Section 2. Data were presented as means ± SD. (a) Representative tumor-bearing mice and tumors from the control and drug-treated groups. (b) Tumor volume measured at the indicated number of days after implantation. Inhibition on tumor growth by plumbagin had been signiﬁcant since day 17 and enforced to the end. *P < 0.05 and **P < 0.01, indicated plumbagin vs. vehicle-treated mice, as analyzed by Dunnett’s test. DOX had higher efﬁcacy against tumor growth compared with plumbagin. group ﬁrst approved that plumbagin can inhibit the proliferation reached the maximum at 4 h, while, caspase-3 and -8 delayed to of NB4 cells with an IC50 value of ∼9 M, suggesting APL cells are 8 h, implying that caspase-9 was the herald in caspase pathway more susceptible to plumbagin than lung cancer and melanoma and caspase-8 activation maybe a secondary event derived from cells. The other ﬁndings included that plumbagin-treated cells mitochondrial activation which has been put forward in different accumulated in the G2/M phase of the cell cycle underwent apop- leukemia cell models [32–34]. As the upstream event of activation, tosis in a dose- and time-dependent manner . In this study, mitochondrial function was evaluated by mitochondrial membrane we generated myeloid sarcoma in NOD/SCID mice with implan- potential, which collapsed remarkably after 4 h, conﬁrming that tation of NB4 cells to further evaluate the effect of plumbagin apoptosis did bloom at 4–8 h in plumbagin-induced apoptosis. It in vivo. As proved in vitro, plumbagin can signiﬁcantly inhibit has been accepted that the ratio of pro-apoptotic to anti-apoptotic the growth of NB4 xenograft in NOD/SCID mice, along with cell members is the major checkpoint of the downstream execution apoptosis. programs: mitochondria dysfunction and the caspase pathway Current available data on the molecular mechanism underly- . Our ﬁndings also showed a corresponding increase of Bax ing the sequential activation of caspase has led to a model in which and Bak expression as well as the decrease of Bcl-XL within 8 h caspase-9 is activated through chemotherapy and caspase-8 is acti- of plumbagin treatment. Even without the change of Bcl-2 expres- vated by death receptor signaling. The caspase pathway headed by sion, the increased pro-apoptotic proteins over anti-apoptotic ones caspase-9 mediates apoptosis, which follows a prescribed sequence might contribute to the apoptosis-promotion activity of plumba- of events that center on the mitochondria, known as the intrinsic or gin. These occurrences of mitochondrial apoptotic events suggest mitochondria-dependent pathway [30,31]. We evaluated the mito- that in promyelocytic leukemia NB4 cells, mitochondria-dependent chondria pathway involved by measuring the activity of caspase-3, caspase activation plays an important role in plumbagin-induced -8 and -9, the change of mitochondrial membrane potential as well apoptosis. as the expression of the Bcl-2 family in plumbagin-induced apopto- We detected that plumbagin-induced apoptosis in NB4 cell was sis. In the course of plumbagin treatment, the activity of caspase-9 associated with a rapid increase in the level of intracellular ROS K.-H. Xu, D.-P. Lu / Leukemia Research 34 (2010) 658–665 663 Fig. 6. Toxicity assessment of plumbagin and DOX in NOD/SCID mice bearing NB4 xenograft. (a) Body weight of NOD/SCID mice in course of treatment. Data were presented as means ± SD and the percentage at the right was average weight before sacriﬁced to that at initiation of the test with corresponding treatment. (b) Tissue sections of livers, hearts, and kidneys from experimental mice discerned by H&E stain. The histopathologic examination of organs was made after mice were sacriﬁced. DOX-treated mice, heart: vacuole formation; liver: focal cellular swelling and an eosinophilic “councilman body” (arrow); kidney: no detectable abnormalities. Plumbagin-treated mice: no sign of toxic pathologic change; original magniﬁcation, 200×. less than 2 h, and then declined. NB4 cells have relative low levels of chemotherapy is another major clinical concern besides the efﬁ- antioxidant defense system and constitutively higher H2 O2 content cacy of treatment. To assess the efﬁcacy as well as the side effect comparing with other tumor cell [35,36], chances are that apoptosis of plumbagin, DOX, one of the most useful anti-tumor drugs ser- will be onset even with slightly extra increase in ROS. Pretreatment viced as a control, and the dose was effective according to informed with the antioxidant NAC, completely blocked plumbagin-induced research [26,38]. We discerned that plumbagin was less effective apoptosis in NB4 cells and this fact was also found in other cancer against tumor xenograft than DOX, but it did have an advantage of cells, such as human cervical cancer cells (ME-180), human prostate side effect over DOX. cancer cells (LNCaP) and promyelocytic leukemia (HL-60) [5,28,37], In conclusion, this present study ﬁrst demonstrates that conﬁrming that plumbagin induces the death pathway by genera- plumbagin effectively induces apoptotic cell death and inhibits tion of ROS. ROS has been reported to mediate inhibition of Topo tumor growth in acute promyelocytic leukemia mouse model with- II contributing to the apoptosis-inducing properties of plumbagin out obvious toxicity. The mechanism involved is triggering the . mitochondria-dependent apoptosis of tumor cells by increasing In vivo, we demonstrated that plumbagin efﬁciently inhib- ROS. It can potentially serve as a new anticancer agent for certain ited the growth of NB4 xenograft. Nevertheless, toxicity following speciﬁc malignancies such as APL. 664 K.-H. Xu, D.-P. Lu / Leukemia Research 34 (2010) 658–665 Fig. 7. Plumbagin-induced apoptosis in NB4 xenograft as determined by TUNEL assay. The histopathologic examination of tumors was made after mice were sacriﬁced. TUNEL+ cells appear blue/black in this assay. Representative ﬁeld from control and plumbagin groups; original magniﬁcation, 200×. Conﬂict of interest leading to potentiation of apoptosis induced by cytokine and chemotherapeutic agents. J Biol Chem 2006;281:17023–33.  Waris G, Ahsan H. Reactive oxygen species: role in the development of cancer The authors report no potential conﬂicts of interest and various chronic conditions. J Carcinog 2006;5:14.  Scandalios JG. The rise of ROS. Trends Biochem Sci 2002;27:483–6.  Schumacker PT. Reactive oxygen species in cancer cells: live by the sword, die Acknowledgements by the sword. Cancer Cell 2006;10:175–6.  Moungjaroen J, Nimmannit U, Callery PS, et al. Reactive oxygen species medi- We would like to thank Dr Rui Han (Institute of Materia Med- ate caspase activation and apoptosis induced by lipoic acid in human lung epithelial cancer cells through Bcl-2 down-regulation. J Pharmacol Exp Ther ica, Chinese Academy of Medical Sciences) for providing NB4 cells, 2006;319:1062–9. and Dr Fei Pei (Department of Pathology, Peking University Health  Kim BC, Kim HG, Lee SA, et al. Genipin-induced apoptosis in hepatoma cells is Science Center) for her excellent technical assistance. mediated by reactive oxygen species/c-Jun NH2-terminal kinase-dependent activation of mitochondrial pathway. Biochem Pharmacol 2005;70:1398– Contributions: Dao-Pei Lu as a deviser and Kai-Hong Xu as an 407. executant.  Kim WH, Park WB, Gao B, et al. 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