Plumbagin induces ROS-mediated apoptosis in human .pdf

Document Sample
Plumbagin induces ROS-mediated apoptosis in human .pdf Powered By Docstoc
					                                                                         Leukemia Research 34 (2010) 658–665

                                                                    Contents lists available at ScienceDirect

                                                                        Leukemia Research
                                                   journal homepage:

Plumbagin induces ROS-mediated apoptosis in human promyelocytic
leukemia cells in vivo
Kai-Hong Xu a , Dao-Pei Lu a,b,∗
    Peking University People’s Hospital, Institute of Hematology, Beijing, China
    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.
                                                             The incidence of formation, growth characteristics, body weight and volume of tumors were observed.
                                                             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 [1].                      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 [2], antiatherosclerotic [3] and anticancer                         brane potential loss, cytochrome c release and caspase-9 activation
[4]. 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 [11].                            [21]. 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 (D.-P. Lu).                                       confirmed the validity of plumbagin in APL mouse model.

0145-2126/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
                                                            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

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 [24].
                                                                                      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 [21]. 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-                         [23]. 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
[37].                                                                                      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.
                                                                                          [14] 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.
                                                                                          [15] Scandalios JG. The rise of ROS. Trends Biochem Sci 2002;27:483–6.
                                                                                          [16] Schumacker PT. Reactive oxygen species in cancer cells: live by the sword, die
Acknowledgements                                                                               by the sword. Cancer Cell 2006;10:175–6.
                                                                                          [17] 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                         [18] 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
executant.                                                                                [19] Kim WH, Park WB, Gao B, et al. Critical role of reactive oxygen species and mito-
                                                                                               chondrial membrane potential in Korean mistletoe lectin-induced apoptosis in
                                                                                               human hepatocarcinoma cells. Mol Pharmacol 2004;66:1383–96.
References                                                                                [20] Kuo PL, Chen CY, Hsu YL. Isoobtusilactone A induces cell cycle arrest and
                                                                                               apoptosis through reactive oxygen species/apoptosis signal-regulating kinase
 [1] Tallman MS. Treatment of relapsed or refractory acute promyelocytic leukemia.             1 signaling pathway in human breast cancer cells. Cancer Res 2007;67:7406–
     Best Pract Res Clin Haematol 2007;20:57–65.                                               20.
 [2] Durga R, Sridhar P, Polasa H. Effects of plumbagin on antibiotic resistance in       [21] Zhao YL, Lu DP. Effects of plumbagin on the human acute promyelocytic
     bacteria. Indian J Med Res 1990;91:18–20.                                                 leukemia cells in vitro. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2006;14:208–
 [3] Sharma I, Gusain D, Dixit VP. Hypolipidaemic and antiatherosclerotic effects of           11.
     plumbagin in rabbits. Indian J Physiol Pharmacol 1991;35:10–4.                       [22] Green DR, Reed JC. Mitochondria and apoptosis. Science 1998;281:1309–
 [4] Hazra B, Sarkar R, Bhattacharyya S, et al. Synthesis of plumbagin deriva-                 12.
     tives and their inhibitory activities against Ehrlich ascites carcinoma in vivo      [23] Gross A, McDonnell JM, Korsmeyer SJ. BCL-2 family members and the mito-
     and Leishmania donovani Promastigotes in vitro. Phytother Res 2002;16:                    chondria in apoptosis. Genes Dev 1999;13:1899–911.
     133–7.                                                                               [24] Jacobson MD. Apoptosis: Bcl-2-related proteins get connected. Curr Biol
 [5] Powolny AA, Singh SV. Plumbagin-induced apoptosis in human prostate cancer                1997;7:R277–281.
     cells is associated with modulation of cellular redox status and generation of       [25] Kosugi H, Ito M, Yamamoto Y, et al. In vivo effects of a histone deacetylase
     reactive oxygen species. Pharm Res 2008;25:2171–80.                                       inhibitor, FK228, on human acute promyelocytic leukemia in NOD/Shi-scid/scid
 [6] Ahmad A, Banerjee S, Wang Z, et al. Plumbagin-induced apoptosis of human                  mice. Jpn J Cancer Res 2001;92:529–36.
     breast cancer cells is mediated by inactivation of NF-kappaB and Bcl-2. J Cell       [26] Zhang SY, Zhu J, Chen GQ, et al. Establishment of a human acute promyelocytic
     Biochem 2008;105:1461–71.                                                                 leukemia-ascites model in SCID mice. Blood 1996;87:3404–9.
 [7] Hsu YL, Cho CY, Kuo PL, et al. Plumbagin (5-hydroxy-2-methyl-1,4-                    [27] Srinivas G, Annab LA, Gopinath G, et al. Antisense blocking of BRCA1 enhances
     naphthoquinone) induces apoptosis and cell cycle arrest in A549 cells through             sensitivity to plumbagin but not tamoxifen in BG-1 ovarian cancer cells. Mol
     p53 accumulation via c-Jun NH2-terminal kinase-mediated phosphoryla-                      Carcinog 2004;39:15–25.
     tion at serine 15 in vitro and in vivo. J Pharmacol Exp Ther 2006;318:               [28] Srinivas P, Gopinath G, Banerji A, et al. Plumbagin induces reactive oxygen
     484–94.                                                                                   species, which mediate apoptosis in human cervical cancer cells. Mol Carcinog
 [8] Wang CC, Chiang YM, Sung SC, et al. Plumbagin induces cell cycle                          2004;40:201–11.
     arrest and apoptosis through reactive oxygen species/c-Jun N-terminal                [29] Kuo PL, Hsu YL, Cho CY. Plumbagin induces G2-M arrest and autophagy by
     kinase pathways in human melanoma A375.S2 cells. Cancer Lett 2008;259:                    inhibiting the AKT/mammalian target of rapamycin pathway in breast cancer
     82–98.                                                                                    cells. Mol Cancer Ther 2006:3209–21.
 [9] Thasni KA, Rakesh S, Rojini G, et al. Estrogen-dependent cell signaling and apop-    [30] Sun XM, MacFarlane M, Zhuang J, et al. Distinct caspase cascades are ini-
     tosis in BRCA1-blocked BG1 ovarian cancer cells in response to plumbagin and              tiated in receptor-mediated and chemical-induced apoptosis. J Biol Chem
     other chemotherapeutic agents. Ann Oncol 2008;19:696–705.                                 1999;274:5053–60.
[10] Aziz MH, Dreckschmidt NE, Verma AK. Plumbagin, a medicinal plant-derived             [31] Budihardjo I, Oliver H, Lutter M, et al. Biochemical pathways of caspase activa-
     naphthoquinone, is a novel inhibitor of the growth and invasion of hormone-               tion during apoptosis. Annu Rev Cell Dev Biol 1999;15:269–90.
     refractory prostate cancer. Cancer Res 2008;68:9024–32.                              [32] Wieder T, Essmann F, Prokop A, et al. Activation of caspase-8 in drug-induced
[11] Nair S, Nair RR, Srinivas P, et al. Radiosensitizing effects of plumbagin in cer-         apoptosis of B-lymphoid cells is independent of CD95/Fas receptor–ligand
     vical cancer cells is through modulation of apoptotic pathway. Mol Carcinog               interaction and occurs downstream of caspase-3. Blood 2001;97:
     2008;47:22–33.                                                                            1378–87.
[12] Acharya BR, Bhattacharyya B, Chakrabarti G. The natural naphthoquinone               [33] Akay C, Gazitt Y. Arsenic trioxide selectively induces early and extensive apop-
     plumbagin exhibits antiproliferative activity and disrupts the microtubule net-           tosis via the APO2/caspase-8 pathway engaging the mitochondrial pathway in
     work through tubulin binding. Biochemistry 2008;47:7838–45.                               myeloma cells with mutant p53. Cell Cycle 2003;2:358–68.
[13] Sandur SK, Ichikawa H, Sethi G, et al. Plumbagin (5-hydroxy-2-methyl-1,4-            [34] Ramos AM, Aller P. Quercetin decreases intracellular GSH content and poten-
     naphthoquinone) suppresses NF-kappaB activation and NF-kappaB-regulated                   tiates the apoptotic action of the antileukemic drug arsenic trioxide in human
     gene products through modulation of p65 and IkappaBalpha kinase activation,               leukemia cell lines. Biochem Pharmacol 2008;75:1912–23.
                                                        K.-H. Xu, D.-P. Lu / Leukemia Research 34 (2010) 658–665                                                       665

[35] Jing Y, Dai J, Chalmers-Redman RM, et al. Arsenic trioxide selectively           [37] Kawiak A, Piosik J, Stasilojc G, et al. Induction of apoptosis by plumbagin
     induces acute promyelocytic leukemia cell apoptosis via a hydrogen peroxide-          through reactive oxygen species-mediated inhibition of topoisomerase II. Tox-
     dependent pathway. Blood 1999;94:2102–11.                                             icol Appl Pharmacol 2007;223:267–76.
[36] Dai J, Weinberg RS, Waxman S, et al. Malignant cells can be sensitized to        [38] Zhu ZF, Chen LJ, Lu R, et al. Tripeptide tyroserleutide plus doxorubicin: thera-
     undergo growth inhibition and apoptosis by arsenic trioxide through modu-             peutic synergy and side effect attenuation. BMC Cancer 2008;8:342.
     lation of the glutathione redox system. Blood 1999;93:268–77.

Shared By:
liningnvp liningnvp http://