Research Mitochondrial-Mediated Apoptosis in Lymphoma ...grapholide or andrographolide + BSO where indicated. Fifty micromoles in DMSO or 100 mmol/L in water stock solution were prepared

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Published OnlineFirst August 26, 2010; DOI: 10.1158/1078-0432.CCR-10-0883

Clinical
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cer Therapy: Preclinical

chondrial-Mediated Apoptosis in Lymphoma Cells byDiterpenoid Lactone Andrographolide, the Active

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Yang, Andrew M. Evens, Sheila Prachand, Amareshwar T.K. Singh, Savita Bhalla,
David, and Leo I. Gordon
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pose: Andrographolide is a diterpenoid lactone isolated from Andrographis paniculata (King of), an herbal medicine used in Asia. It has been reported to have anti-inflammatory, antihyperten-ntiviral, and immune-stimulant properties. Furthermore, it has been shown to inhibit cancer cellration and induce apoptosis in leukemia and solid tumor cell lines.erimental Design: We studied the Burkitt p53-mutated Ramos cell line, the mantle cell lymphoma) line Granta, the follicular lymphoma (FL) cell line HF-1, and the diffuse large B-cell lymphomaL) cell line SUDHL4, as well as primary cells from patients with FL, DLBCL, and MCL.ults: We found that andrographolide resulted in dose- and time-dependent cell death as measuredT. Andrographolide significantly increased reactive oxygen species (ROS) production in all cell lines.termine mechanism of cell death, we measured apoptosis by Annexin V/propidium iodide in thece and absence of the antioxidantN-acetyl-L-cysteine (NAC), the glutathione (GSH)–depleting agentnine sulfoxamine (BSO), or caspase inhibitors. We found that apoptosis was greatly enhanced byblocked by NAC, and accompanied by poly(ADP-ribose) polymerase cleavage and activation ofe-3, caspase-8, and caspase-9.Wemeasured BAX conformational change andmitochondrialmembranetial, and using mouse embryonic fibroblast (MEF) Bax/Bak double knockouts (MEFBax−/−/Bak−/−), wethat apoptosis was mediated through mitochondrial pathways, but dependent on caspases in bothes and patient samples.clusions: Andrographolide caused ROS-dependent apoptosis in lymphoma cell lines and in pri-tumor samples, which was enhanced by depletion of GSH and inhibited by NAC or the pan-caspase

inhibitor Z-VAD-FMK. Further studies of diterpenoid lactones in lymphoma are warranted. Clin Cancer Res;

16(19); 4755–68. ©2010 AACR.

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have bceed t

rographolide is a diterpenoid lactone isolated fromgraphis paniculata (King of Bitters; refs. 1–3), an im-t herbal medicine used in Asia to treat a range ofes, such as respiratory infection, fever, bacterialtery, and diarrhea (4–6). It also has been studied

HIV (7). The major bioactive componentA. paniculata is andrographolide, and the

fore hdeathrelategraphas inlarge(MCLdrogracaspasin primand t(GSH(NACmore,pathw

n: Division of Hematology/Oncology, Department ofoma Program, Northwestern University Feinberge and the Robert H. Lurie Comprehensive Cancerstern University, Chicago, Illinois

ry data for this article are available at Clinical Cancerttp://clincancerres.aacrjournals.org/).

uthor: Leo I. Gordon, Northwestern University, 676eet, Suite 850, Chicago, IL 60611. Phone: 312-695--6189; E-mail: [email protected].

0432.CCR-10-0883

ssociation for Cancer Research.

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Research. on February 13, clincancerres.aacrjournals.org d from

hydroxyls at C-3, C-19, and C-14 are responsiblebiological activity (8).ently, the anticancer properties of andrographolideeen recognized, and some of its effects seem to pro-hrough redox-mediated pathways (9–12). We there-ypothesized that andrographolide would lead to cellin lymphoma cell lines and that the effect may bed to altered cellular redox state. We studied andro-olide in non–Hodgkin's lymphoma cell lines as wellprimary malignant B cells from patients with diffuseB-cell lymphoma (DLBCL), mantle cell lymphoma), and follicular lymphoma (FL). We found that an-pholide induced reactive oxygen species (ROS) ande-dependent apoptosis in lymphoma cell lines andary tumor samples but not in normal lymphocytes,

hat this was enhanced by depletion of glutathione) and inhibited by the antioxidant N-acetyl-L-cysteine) or the pan-caspase inhibitor Z-VAD-FMK. Further-
these effects seemed to proceed through BAX/BAKays.
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2020. © 2010 American Association for Cancer

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Translational Relevance

The anticancer properties of the diterpenoid lactoneandrographolide have recently been recognized, andthe biological effects of this naturally occurring prod-uct derived from Andrographis paniculata are likely relat-ed to reactive oxygen species (ROS) signaling. Basedon these observations, we hypothesized that androgra-pholide would be cytotoxic to lymphoma cells. Inthese studies, we examined lymphoma cell lines, in-cluding Ramos, Granta, SUDHL4, and HF-1, as wellas primary lymphoma cells derived from patients. Weshow that andrographolide results in apoptosis in lym-phoma cell lines and in primary lymphoma cells, thatthis is mediated through ROS-mediated caspase activa-tion, and that these effects proceed through BAX/BAKmitochondrial pathways. These studies will providethe preclinical rationale to bring this novel natural com-pound to clinical trials for the treatment of lymphoma.

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rials and Methods

ntsrographolide (Supplementary Fig. S1), buthionineamine (BSO), and NAC were purchased from Sigmaical Co. Z-VAD-FMK, Ac-DEVD-CHO, Ac-IETD-and Ac-LEHD-CHO were obtained from BioMol.dies to caspase-3, caspase-9, and caspase-8 were pur-fromCell Signaling Technology, and glyceraldehyde-sphate dehydrogenase (GAPDH)was purchased fromicon.

ultureos (Burkitt lymphoma) cell line was obtained frommerican Type Culture Collection, HF-1 (FL) wasDr. Richard Miller (Stanford University, Palo Alto,SUDHL4 (DLBCL) was from Dr. Ron Gartenhausersity of Maryland, Baltimore, MD), and Granta) was a kind gift from Dr. Steven Bernstein (Univer-f Rochester, Rochester, NY). Malignant cells fromts with FL, DLBCL, and MCL were cultured in RPMIcontaining 10% fetal bovine serum (FBS) and 1%m pyruvate (Granta only) and in the presence ofllin/streptomycin/glutamine at 37°C in a humidi-% CO2 incubator. Cell viability was measured usingpan blue or propidium iodide (PI) exclusionmethodT assay (see below). Cells were treated with andro-olide or andrographolide + BSO where indicated.micromoles in DMSO or 100 mmol/L in watersolution were prepared for both substances. Beforeent, PBS was used to dilute andrographolide stockon, and PBS/andrographolide solution was addedmedium to achieve the desired working concen-

n. The control groups used the same amount ofand PBS in the medium as the treatment groups.

tion ocence

ancer Res; 16(19) October 1, 2010

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ationsls were incubated with the following drugs: 0 tomol/L andrographolide (10), 100 μmol/L BSO10 mmol/L NAC (14), and 50 μmol/L caspase inhi-(Z-VAD-FMK, Ac-DEVD-CHO, Ac-IETD-CHO, andHD-CHO; ref. 10).

ry MCL, DLBCL, and FL cellslowing written consent approved by the Northwest-niversity Institutional Review Board, peripheralwas drawn from three patients with leukemic-FL, one with MCL, and two with transformed

L. The three FL patients had bulky abdominal ade-hy (>12 cm) and a rapidly rising lymphocyte countlute lymphocyte count, 238.5 K/μL) with fluores-in situ hybridization (FISH) confirmation of8) in 95% of nuclei. The patient with MCL had new-gnosed MCL with a WBC of 28,000/μL, with 96%nant cells, and had t(11;14) by FISH in the bloodone marrow. The two patients with DLBCL hadormed lymphoma with >100,000/μL circulatingcells. The peripheral blood was diluted 1:1 withCa2+- and Mg2+-free) and layered over Ficoll-PaqueSigma). Samples were then centrifuged at 150 × gminutes at room temperature; the buffy coat layer

emoved and centrifuged again. Isolated peripheralmononuclear cells (all malignant cells in the six

les) were then resuspended in RPMI 1640 + 10%1 × 106 cells/mL.

ssayeffects of andrographolide on cell viability werered by MTT assay in Ramos, Granta, HF-1, andL4 cells according to the instructions of an im-

d detection kit provided by the manufacturer (Cell-6 AQueous One Solution Cell Proliferation Assay,ega). Briefly, 2.5 × 104 cells/90 μL were seeded inll microtiter plates. After incubating with differentntrations of andrographolide (10 μL) for theated times, 20 μL MTT solution was added to eachnd the plates were incubated for an additional 1 tors at 37°C. The absorbance was read at 490 nma microplate reader (MRX Revelation; DYNEXologies). The absorbance values were expressed asr the control group. Because reduction of MTT canccur in metabolically active cells, the level of activityeasure of the viability of the cells.

measurementS accumulation in treated and untreated cells wasred by fluorescence-activated cell sorting (FACS;

15, 16). Cells were incubated in 5 μmol/L 2′7′rofluorescein diacetate (H2DCFDA) for 30 minutesC in the dark. After washing, cells were suspendedmL of cold PI (200 ng/mL)/PBS for 5 minutes toOS in living cells. ROS were measured by oxida-
f H2DCFDA to dichlorofluorescein (DCF). Fluores-intensity was read by flow cytometry using the
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Andrographolide-Induced Apoptosis in Lymphoma

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an Coulter EPICS XL-MCL Cytometer on the FL1el. Results were analyzed and calculated by FCSss V3 software (De Novo Software) and Excelsoft).

titation of apoptosisr incubations and washing, 1 × 106 cells wered with Annexin V–FITC and PI reagent in the bind-uffer according to the Annexin V–FITC apoptosision kit instruction provided by the manufacturerrogen). The fluorescent signals of FITC and PI wereed at 518 and 620 nm, respectively, on a Beckmaner FACS machine. For each analysis, 30,000 eventsrecorded. Results were analyzed and calculated byxpress V3 software and Excel. The % apoptosis
he sum of (Annexin V–FITC+/PI−) and (AnnexinC+/PI+) cells.
Folwith

os, 40 μmol/L for Granta, 15 μmol/L for HF-1, and 30 μmol/L for SUDHL4. The darapholide concentration at P > 0.05 by Student's t test as described in Materials

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nofluorescent stainingr washing and cytospin, cells were fixed for 20 min-y formaldehyde (4% in PBS). Then, cells wereed in blocking buffer (1% bovine serum albumin.02% Triton X-100) for 1 hour and further incubatednti-Bax 6A7monoclonal antibody (Sigma) overnight. AfterwashingwithPBS (+0.02%TritonX-100 inPBS)blocking with blocking buffer for 1 hour, cells wereated with anti-mouse Alexa Fluor 633 or Cy3 second-tibody for another 1 hour. After washing, coverslipsounted onto slides using ProLong antifade mount-

agent (Invitrogen). Cells were visualized under Nikononfocal or UV LSM510 Meta confocal microscopes.

rn blotting
lowing the various incubations, cells were washedPBS and centrifuged, and cell pellets were treated
MTT cell viability assay. Cells were treated with andrographolide (Andro) at the indicated concentration for the indicated period of time. Theagent (Promega) as described in Materials and Methods was added, and colored solution was quantified by measuring at 490-nm wavelength.rapholide-induced inhibition of cell viability was dose and time dependent in Ramos, Granta, HF-1, and SUDHL4 cell lines. IC50 at 48 h was 20 μmol/L

ta are expressed as % of control and are significant for eachand Methods.

Clin Cancer Res; 16(19) October 1, 2010 4757



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ysis buffer containing protease and phosphatase in-rs (Roche; Sigma). Protein concentrations were de-ed with the Bio-Rad protein assay kit. An aliquoth cell lysate was used for protein assay (17). Totaln samples (25-50 μg) were subjected to 12% SDS-electrophoresis. Proteins were electrophoretically

erred to a nitrocellulose membrane. After incubatingblocking buffer containing 5% nonfat milk for 1the membranes were incubated with the primary an-y overnight at 4°C, washed with TBS–Tween 20) thrice, incubated with secondary antibody forr at room temperature, and washed thrice with TBST.ne complexes were visualized by enhanced chemilu-cence kit and film (Amersham Biosciences, Milli-or Denville Scientific, Inc.).

ration of cytosolic and mitochondrial fractions
ls were treated with andrographolide for 0, 3, 18,4 hours. Mitochondrial and cytosolic fractions were
viabilsion m

Fig. 2.in lympandroghistogrGranta,producrepresethree eandrogrepreseexperimandrographolide



red using a mitochondria isolation kit for culturedrom Pierce according to the manufacturer's instruc-. The mitochondrial pellet was resuspended inle buffer for SDS-gel electrophoresis and analyzedestern blotting for BAX antibody (Cell Signalingology). COX IV (Cell Signaling Technology) is usedinternal control for the mitochondrial fraction,APDH (Chemicon) for the cytosolic fraction. Cyto-fractions were also subjected to Western blottingX.

t and MEFBax−/−/Bak−/− experimentsuse embryonic fibroblasts (MEF) were a kind giftDr. Craig Thompson (University of Pennsylvania,elphia, PA). Cells were cultured inDMEM containingBS and in the presence of penicillin/streptomycin/ine at 37°C in a humidified 5% CO2 incubator. Cell

ity was measured using the trypan blue or PI exclu-ethod. Cells were treated with andrographolide at

Andrographolide-induced ROS accumulation and Δψm changehoma cell lines were dose dependent. Cells were treated withrapholide at the indicated dose and time period. A, representativeams show that andrographolide induced ROS production in Ramos,HF-1, and SUDHL4 cell lines. A shift to the right (red) indicates ROStion. B, quantification of ROS accumulation in four cell lines. The datant percentage increase compared with control. Columns, mean ofxperiments completed in triplicate; bars, SD. *, P < 0.05, control versusrapholide. C, quantification of Δψm in three cell lines. The datant percentage decrease compared with control. Points, mean of threeents completed in triplicate; bars, SD. *, P ≤ 0.05, control versus

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Fig. 3.inhibitoandroganalyzelines (Pdose an(P ≤ 0.0with 50 μmol/L of the indicated caspase inhibitors (Ac-DEVD-CHO, Ac-IETD-CHO, Ac-LEHD-CHO, or Z-VAD-FMK) for 1 h, and then andrographolide wasadded antly i


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60 μmol/L for 24 or 30 hours. Cell morphology wased by microscope. Further, Annexin V–FITC/PI apo-assay was performed as described above.

hondrialmembranepotential (Δψm)measurementamethylrhodamine (Invitrogen) was added to thee medium to a final concentration of 250 nmol/Lefore the end of the incubation time. Cells were fur-cubated for 30 minutes at 37°C. Cells were washed

at the indicated concentrations for 48 h. All of the caspase inhibitors signific

S twice and resuspended in FACS buffer containingol/L tetramethylrhodamine and 2% FBS. Fluores-

minedefine

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intensity was measured on the FL-2 channel of aytometer. Results were analyzed and calculated byxpress V3 software and Excel.

ticsa are expressed as the mean ± SD. Comparisonseen two values were performed by unpairednt's t test. For multiple comparisons among differ-oups of data, the significant differences were deter-

nhibited andrographolide-induced apoptosis (P ≤ 0.05).

Dependence of apoptosis on ROS and caspases. Andrographolide-induced apoptosis was inhibited by the antioxidant NAC, or caspasers, but enhanced by BSO in Ramos, Granta, HF-1, and SUDHL4 cell lines. Cells were treated with andrographolide, andrographolide + BSO,rapholide + NAC, or andrographolide + caspase inhibitors at the indicated dose and time period. Annexin V–FITC and PI reagent were added andd by FACS. A, andrographolide-induced apoptosis is dose dependent. NAC completely inhibited andrographolide-induced apoptosis in all cell≤ 0.05). B, cells were pretreated with BSO (100 μmol/L) overnight and then treated with andrographolide or andrographolide + NAC at the indicatedd time period. BSO significantly increased andrographolide-induced apoptosis (P ≤ 0.05), and apoptosis was completely inhibited by NAC5). Lower-dose andrographolide (72 h, 10 μmol/L in Ramos; 48 h, 5 μmol/L in HF-1) with BSOdramatically increased apoptosis. C, cells were pretreated

d by the Bonferroni method. Significance wasd at P ≤ 0.05.




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lts

grapholide inhibits cell viabilityevaluate the effect of andrographolide on cell via-, we treated Ramos, Granta, HF-1, and SUDHL4ith 0 to 100 μmol/L andrographolide for 24 orurs. Andrographolide resulted in loss of cell viabil-y MTT assay) in all four lymphoma cell lines in aand time-dependent manner (Fig. 1). IC50 (definedas that concentration that achieved 50% inhibi-f cell viability) at 48 hours was 20 μmol/L fors, 40 μmol/L for Granta, 15 μmol/L for HF-1,0 μmol/L for SUDHL4. These data indicate thatgrapholide inhibits cell viability in Ramos, Granta,and SUDHL4 lymphoma cell lines in a dose- andependent manner.

grapholide induces ROS accumulation inoma cell lines

examine whether andrographolide affects the oxida-
nction of the cell, we quantified ROS at differentoints by measuring the fluorescent signal of DCF
whethlines,

ase-9 in Ramos. E and F, andrographolide-induced cleavage of PARP, caspase-4) and completely inhibited by NAC and by the pan-caspase inhibitor Z-VAD-FM



FACS. Cells were treated with andrographolide atdicated dose and time period (Fig. 2). We showedicant (P ≤ 0.05) ROS accumulation at 48 hours fors, 1 to 6 hours for Granta, 0.5 to 3 hours for HF-1,hours for SUDHL4 (Fig. 2A). A shift to the rightindicates ROS accumulation. Figure 2B showsification of ROS accumulation in all four cell lines.ata shown represent percentage increase comparedontrol and represent the mean ± SD. Taken together,data show that andrographolide-induced ROSulation is dose and time dependent in lymphomanes.

grapholide induces disruption of mitochondrialrane potentialruption of the mitochondrial membrane potentialis one of the earliest intracellular events that occur

optosis. It has been shown that andrographolides in loss of matrix metalloproteinase (MMP) in aocellular carcinoma cell line (12). To investigate
er andrographolide affects Δψm in lymphoma cellwe measured Δψm in Ramos (19 hours), HF-1
Mechanism of andrographolide-induced apoptosis. Cells were treated with andrographolide in the presence or absence of NAC (10 mmol/L)D-FMK (50 μmol/L) at the indicated dose and time period, and Western blot was performed. A, andrographolide resulted in cleavage of PARPtivation of caspase-3 and caspase-8 in Granta. B, andrographolide-induced PARP cleavage started at 16-h incubation. NAC and Z-VAD-FMKd andrographolide-induced PARP cleavage in Granta. C and D, andrographolide resulted in cleavage of PARP and activation of caspase-3, caspase-8,

3, caspase-8, and caspase-9 was seen at 18 h (HF-1) or 16 hK.




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Fig. 5.and the(three Fwith FLAc-IETDapoptohowevegroup.malignaof PARmalignant cells from a patient MCL, with some baseline PARP cleavage increasing by 17 h after exposure to low doses of andrographolide (5 μmol/L).E, andr AC an


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urs), and SUDHL4 (16 hours; Fig. 2C). For Ramos, atconcentrations of andrographolide (20-40 μmol/L),rt to see the change of Δψm at 19 hours (Fig. 2C).r results were seen in HF-1 and SUDHL4 cells. Thesehow that andrographolide results in dose-dependentf Δψm.

grapholide induces NAC-reversible,se-dependent apoptosis in Ramos, Granta,and SUDHL4 cell lines

ographolide-induced cleavage of PARP and caspase-9 was inhibited by N

determine whether andrographolide-induced loss ofability was related to apoptosis, we quantified apo-

NAC cptosis

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by FACS after staining with Annexin V–FITC and PI). We found that andrographolide resulted in signif-(P < 0.05) dose-dependent apoptosis in Ramos aturs and in Granta, HF-1, and SUDHL4 at 48 hours.C50 (defined herein as that concentration thated 50% apoptosis) was 40 μmol/L for Ramos aturs, 40 μmol/L for Granta or HF-1 at 48 hours,0 μmol/L for SUDHL4 at 48 hours.determine if apoptosis was related to ROS, we coin-ed cells with the antioxidant NAC. In all cell lines,

d Z-VAD-FMK in malignant cells from a patient with MCL.

Effects of andrographolide on malignant cells from patients with FL, DLBCL, and MCL. Cells were pretreated with caspase inhibitors for 1 h,n andrographolide was added at the indicated time period. These data are representative. Similar data were seen for all patient samples studiedL, one MCL, and two DLBCL). Annexin V–FITC and PI reagent were added and analyzed by FACS. A, apoptosis in malignant cells from patients, DLBCL, and MCL after the indicated time exposure to 1 to 20 μmol/L andrographolide with or without the caspase inhibitors Z-VAD-FMK,-CHO, or Ac-LEHD-CHO, or antioxidant NAC, showing significant inhibition with Z-VAD-FMK or NAC (P ≤ 0.05). Andrographolide-induced

sis in patient samples was dose dependent, and it was also significantly inhibited by Ac-IETD-CHO and Ac-LEHD-CHO in FL (P < 0.05). Right,r, at the same andrographolide concentrations, we saw no significant apoptosis in normal human lymphocytes compared with the controlB, andrographolide-induced cleavage of PARP, BID, caspase-3, caspase-8, and caspase-9 was seen at concentrations as low as 5 μmol/L innt cells from a patient with FL, and all are inhibited by NAC. C, in malignant cells from a patient with DLBCL, andrographolide-induced cleavageP, caspase-8, and caspase-9 was dose dependent and completely inhibited by NAC. D, cleavage of PARP and BID was time dependent in

ompletely abrogated andrographolide-induced apo-(Fig. 3A; P < 0.05). These data suggest that apoptosis




Fig. 6.mitocho(C) HF-or HF-1were seas intertime de

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Andrographolide resulted in BAX conformational changes in cell lines (HF-1 and SUDHL4) and patient samples (FL and DLBCL), and in BAXndrial translocation. Andrographolide induced BAX conformational changes in (A) SUDHL4 cell line, (B) primary DLBCL cells (n = 1 patient sample),1 cell line, and (D) primary FL cells (n = 1 patient sample). All BAX conformational changes were blocked by NAC or Z-VAD-FMK. SUDHL4 (E)(F) cells were treated with 30 and 40 μmol/L andrographolide, respectively, for the indicated period of time. Mitochondrial and cytosolic fractionsparated as described in Materials and Methods and analyzed by Western blotting for proapoptotic BAX protein. COX IV and GAPDH were used
nal controls for mitochondrial and cytosolic extracts, respectively. Andrographolide-induced BAX translocation from cytoplasm to mitochondria waspendent. Mito BAX, mitochondrial-related BAX; Cyto BAX, cytosolic-related BAX.
ancer Res; 16(19) October 1, 2010 Clinical Cancer Research

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ndrographolide-induced cell death was dependent on the BAX/BAK pathway. Apoptosis in wild-type (WT) MEFs (MEFwt) and in MEFs obtained fromk DKO mice (MEFBax−/−/Bak−/−) after treatment with andrographolide was examined. Andrographolide significantly increased cell death in MEFwt

5), but in MEFBax−/−/Bak−/−, there was minimal cell death. A, the progressive detachment and rounding up of cells after 24-h andrographolidere was dose dependent in MEFwt, and that was not seen in MEFBax−/−/Bak−/− under the microscope. B, apoptosis (Annexin V–FITC and PI) wased after 30-h andrographolide treatment. The bar graph shows that there was a significant difference (*, P < 0.05) in apoptosis between MEFwt and−/−/Bak−/− at 60 μmol/L andrographolide, but no change from baseline in MEFBax−/−/Bak−/− cells compared with MEFwt. C, similarly, there was a

wt Bax−/−/Bak−/−
ant decrease in MMP (Δψm) after 4-h andrographolide exposure in MEF (*, P ≤ 0.05) but not in MEF cells. D, Western blots confirmedFBax−/−/Bak−/− had no BAX and BAK proteins.
Clin Cancer Res; 16(19) October 1, 2010acrjournals.org 4763

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lated to the cellular redox state, perhaps as a conse-e of depletion of the endogenous antioxidant GSH,se NAC restores intracellular GSH.further investigated the involvement of ROS ingrapholide-induced apoptosis by pretreating cells withduced GSH-depleting agent BSO (100 μmol/L) anddded andrographolide or andrographolide + NAC atdicated dose and time periods (Fig. 3B). We foundSO greatly enhanced andrographolide-induced apo-, and apoptosis was again completely inhibited byntioxidant NAC. With the addition of BSO, thewas significantly lower in all cell lines (72 hours,ol/L in Ramos; 48 hours, 40 μmol/L in Granta; 48, 5 μmol/L in HF-1; and 48 hours, 20 μmol/L inL4) compared with andrographolide alone. Further,when added to andrographolide in Ramos cells,a striking increase in ROS production comparedndrographolide alone (data not shown).t, to determine if andrographolide-induced apopto-s caspase dependent, the caspase inhibitors Z-VAD-Ac-LEHD-CHO, Ac-IETD-CHO, and Ac-DEVD-CHOpreincubated with Ramos, Granta, HF-1, andL4 cell lines, and then andrographolide was addedindicated doses and time periods (Fig. 3C). In allell lines, the pan-caspase inhibitor Z-VAD-FMK re-in complete inhibition, whereas the caspase-3 in-r Ac-DEVD-CHO significantly (P < 0.05) inhibitedosis in Ramos and Granta. Further, the caspase-8 in-r and the caspase-9 inhibitor (Ac-IETD-CHO and Ac--CHO) also resulted in significant inhibition (P <in Ramos and Granta (Fig. 3C). Together, thesehow that andrographolide-induced apoptosis inoma cell lines is ROS and caspase dependent, andoth the intrinsic and the extrinsic caspase pathwayslevant.

grapholide induces poly(ADP-ribose)erase and caspase cleavagefurther investigate the mechanism of apoptosis, wened caspase activation and poly(ADP-ribose) poly-e (PARP) cleavage by andrographolide using immu-tting (Fig. 4). After 24 hours of andrographolideure in Granta cells, we found that cleavage of PARP,e-3, and caspase-8 was dose dependent and com-y inhibited by NAC (Fig. 4A). PARP cleavage wasime dependent, as shown in Fig. 4B. We begin toRP cleavage after 16 hours, and it is inhibited bynd Z-VAD-FMK. In Fig. 4C and D, after 24 hours ofgrapholide exposure, we observed dose-dependent,inhibitable cleavage of PARP, caspase-8, caspase-9,aspase-3 in Ramos cells. In Fig. 4E and F, we alsotime-dependent cleavage of PARP, caspase-8,e-9, and caspase-3 at 40 and 30 μmol/L, respective-HF-1 and SUDHL4 cells at concentrations that haveshown to induce apoptosis (Fig. 3). As in GrantaNAC and Z-VAD-FMK also inhibited caspase and
cleavage induced by andrographolide in HF-1UDHL4. These data provide further evidence that
loweraffect



grapholide-induced apoptosis is ROS and caspasedent and may proceed by both intrinsic and extrin-thways.

grapholide induces apoptosis in primarynant cells from patients with FL,and DLBCLed on our observations in lymphoma cell lines, wened whether andrographolide also induced apopto-primary lymphoma patient samples and normal hu-ymphocytes and, if so, by which cell death pathways.e treated fresh FL, MCL, and DLBCL malignant cellsndrographolide at the indicated doses and time per-(Fig. 5 shows a representative sample. Similar resultsseen in all patient samples.) Interestingly, we foundrimary malignant cells from patients were moreive to andrographolide than the cell lines. Androgra-e induced significant dose-dependent apoptosis (P <in primary malignant cells at a lower AC50 and earlieroints (5μmol/L, 24hours for FL; 10μmol/L, 18hoursBCL; 10 μmol/L, 14 hours for MCL). Similar to thenes, NAC completely prevented andrographolide-ed apoptosis (P < 0.05) in FL, DLBCL, and MCLA). By contrast, andrographolide did not cause signif-apoptosis in normal human lymphocytes comparedalignant cells at the same andrographolide concen-s (Fig. 5A).t, wepretreatedwith the caspase inhibitors (50μmol/Lto determine if andrographolide-induced apopto-s caspase dependent. The inhibitors of caspase-8,se-9, and pan-caspase inhibitor (Ac-IETD-CHO,HD-CHO, and Z-VAD-FMK) significantly inhibitedgrapholide-induced apoptosis (P < 0.05) in pri-FL cells. Z-VAD-FMK also significantly inhibitedgrapholide-induced apoptosis (P < 0.05) in MCLry cells. The inhibition patterns were similar tosults we observed in the cell lines.ig. 5B, we show that in fresh FL cells, NAC inhib-ARP cleavage starting at concentrations of androgra-e as low as 5 μmol/L, and that BID cleavage wast 10 μmol/L and also inhibited by NAC. Similarly,e-3, caspase-8, and caspase-9 were cleaved at con-tions as low as 10 μmol/L, and this was inhibitedC. In Fig. 5C, after 24-hour treatment of andro-olide in the presence or absence of NAC, weed dose-dependent cleavage of PARP, caspase-8,aspase-9 in malignant cells from a patient withL. In Fig. 5D and E, we show cleavage of PARPaspase-9 starting at 16 hours of incubation withol/L andrographolide in primary MCL cells. BIDge was also time dependent and was first seen athours of exposure. NAC and Z-VAD-FMK inhib-

he activation of PARP and caspase-9 in these cells.together, these data show that as in cell lines,

grapholide-induced apoptosis in primary lymphomas dose, ROS, and caspase dependent. It occurs at
concentrations of andrographolide and does notnormal human lymphocytes.



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grapholide-mediated apoptosis depends onhondrial pathwaysinvestigate the involvement of mitochondrial path-in lymphoma cell death, we determined the role ofAK in andrographolide-induced apoptosis. BAX is aidomain” proapoptotic protein of the Bcl-2 familytriggered by BID to undergo homo-oligomerizationAK, resulting in release of cytochrome c from thehondria (18). To explore the role of Bcl-2 familyns in andrographolide-induced apoptosis in lym-a, we first performed whole-cell protein Westernnoblots. We found that total BAX protein expres-id not change after exposure to andrographolidenot shown). However, we then investigated confor-nal change of BAX following mitochondrial translo-during apoptosis (10, 12, 19). Using the BAXmonoclonal antibody, which specifically binds

AX protein with conformational change (10, 12),grapholide induced BAX conformational changeSUDHL4 (DLBCL cell line) and HF-1 (FL cell line)(Fig. 6A and C, respectively) and, similarly, inry DLBCL and FL samples (Fig. 6B and D, respec-. These data show an increase of BAX fluorescentng at 16 to 18 hours in andrographolide-treated(Fig. 6A-D). Furthermore, BAX conformationale was ROS and caspase dependent, as treatmentAC or pretreatment with the pan-caspase inhibitor-FMK eliminated the andrographolide-inducedonformational change in cell lines (Fig. 6A andd primary patient samples (Fig. 6B and D). Thesendicate that andrographolide-induced apoptosis incell lines and primary lymphoma cells dependst on Bcl-2 family proteins and is ROS and caspasedent.further investigate mitochondrial events duringgrapholide-induced apoptosis, we extracted cyto-and mitochondrial fractions for immunoblotting.e SUDHL4 and HF-1 cell lines, we found Baxulation in the mitochondrial fractions of both cellby 3 hours, whereas BAX in the cytoplasm was re-(Fig. 6E and F). Thus, andrographolide inducesonformational change and mitochondrial trans-on from the cytoplasm in lymphoma, leading tor apoptosis.

grapholide-mediated cell death is regulated by/BAK protein–dependent cell pathwayand BAK and their complexes are known to play al role in facilitating the release of mitochondrialembrane proteins during apoptosis (9). To furtherigate the role of BAX and BAK in andrographolide-ed cell death, we examined apoptosis in wild-type(MEFwt) and in MEFs obtained from Bax/Bak

le-knockout (DKO) mice (MEFBax−/−/Bak−/−) afterent with andrographolide. We found that andro-olide significantly increased cell death in MEFwt

.05), but in MEFBax−/−/Bak−/−, there was minimal cell(Fig. 7). Figure 7A shows that progressive detachment

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unding up of cells after andrographolide exposure isependent in MEFwt and not seen at 40 μmol/L andally at 60 μmol/L in MEFBax−/−/Bak−/− (Fig. 7A). In, apoptosis (Annexin V–FITC and PI) was performed0 hours of andrographolide treatment. The bar graphthat there was a significant difference in apoptosisen MEFwt and MEFBax−/−/Bak−/− at 60 μmol/L andro-olide. Moreover, after 4 hours, 60 μmol/L androgra-de caused significant loss of Δψm in MEFwt (P <, whereas in MEFBax−/−/Bak−/− this was not seen7C). To further confirm that MEFBax−/−/Bak−/− hadX and BAK proteins, we performed Western blotsown in Fig. 7D. These results suggest that BAXr BAK are necessary for andrographolide-mediatedeath, as their absence attenuated cell death in re-e to andrographolide.

ssion

have shown that andrographolide, the active compo-derived from the plant A. paniculata, causes redox-dent apoptosis in several non–Hodgkin's lymphomanes and in primary lymphoma cells. It seems thatechanism is redox dependent, is mediated throughse activation, depends on BAX conformationale, and is accompanied by translocation of BAX fromtoplasm to the mitochondria.fundamental molecular mechanisms of the

ical effects of lactone diterpenoids have been ex-d. Andrographolide targets NF-κB for its anti-matory activity (20, 21). The pharmacokineticsdrographolide in human plasma also have beented by oral administration of 200 mg androgra-e (22), and adducts of andrographolide in humanhave been documented after oral administrationThe extract (>10% or =10% andrographolide) oficulata did not affect the reproductive and fertilityy in male Wistar rats at >1,000 mg/kg per dayRecently, andrographolide has also been shownibit cancer cell proliferation and induce apoptosiscer cell lines, including leukemia (HL-60), pros-

adenocarcinoma (PC-3), breast cancer (MDA-MB-nd MCF 7), liver cancer (HepG2 and Hep3B),al cancer (HeLa), and colorectal cancer (HCT116T-29; refs. 9–11, 25–31), but not previously inoma cell lines.hypothesized that andrographolide might inhibitroliferation and cause apoptosis in lymphoma celland in primary lymphoma cells by mechanismsnvolved cellular redox systems, caspase activation,itochondrial pathways. Indeed, we found that theof andrographolide were dose and time related,ere accompanied by ROS generation (Fig. 2). Thatplayed an important role in apoptosis of lym-a cell lines Ramos, Granta, HF-1, and SUDHL4hown by near-complete abrogation of apoptosis
e antioxidant NAC and by enhancement of apop-y the GSH-depleting agent BSO. These observations



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not restricted to cell lines, as we also found thatgrapholide, at lower concentrations, resulted inand caspase-dependent inhibition in primary

nt samples from patients with FL, MCL, andL.further postulated that andrographolide inducedosis in patient samples and cell lines through in-caspase pathways, and that Bcl-2 family proteinsitochondrial regulation would be required. WeBID cleavage in patient samples (Fig. 5B ande also found that andrographolide induced Δψm

e in Ramos, HF-1, and SUDHL4 (Fig. 2C). Further,owed that andrographolide induced conforma-change of BAX in the SUDHL4 and HF-1 cell

and in malignant cells from patients with DLBCLL. This was inhibited by NAC, indicating that BAXrmational change depended on oxidant mechan-(Fig. 6). We also found that andrographolidees BAX mitochondrial translocation in HF-1 andL4 lymphoma cell lines (Fig. 6), results similarse in HepG2 cells as described by Zhou et al.Additionally, BAX and BAK and their complexesown to play a central role in facilitating the re-of mitochondrial intermembrane proteins duringosis (9). To further characterize the mechanism,ed Bax/Bak MEF DKOs (MEFBax−/−/Bak−/−; Fig. 7)und that MEFwt had reduced Δψm and were killedmilar concentrations of andrographolide, butax−/−/Bak−/− were not, suggesting that apoptosis pro-though the BAX/BAK pathway. We also found

6) that caspase inhibition blocked BAX conforma-change, suggesting that caspases are required for

tep in andrographolide-induced apoptosis.concentrations that achieved 50% growth inhibi-IC50) and 50% apoptosis (AC50; Figs. 1 and 3) arer or even lower than concentrations in other canceres [HL-60 cell line (29), SMMC-7721 human carci-cell line (12), and HepG2 human hepatoma cell

10)] and are clinically relevant. There are data thatst that serum concentrations of between 1.9 andol/L can be achieved with doses of andrographo-mmonly used in China (32) and that 20-fold highercan be given safely (7). This is well within the rangesulted in biological effects and apoptosis in patientes in our studies. Further, based on our data (Fig. 5A),grapholide did not cause significant apoptosis inl lymphocytes compared with patient samples or cellFurther, there was no hematologic toxicity seen in aI trial in HIV and non-HIV patients (7).mechanism of ROS-dependent cell death related tographolide is not clear. Woo et al. (33) have re-that andrographolide upregulates cellular-reduced

n neonatal rat cardiomyocytes. However, androgra-e has been found to react with reduced thiols (33),t initially there is a reduction in GSH, which isollowed by activation of glutamine cysteine ligase
ts modifier subunit as an endogenous antioxidantr defensive response to GSH reduction. However,
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l. (34) have reported that andrographolide initiallysed intracellular GSH levels, followed by a de-, whereas inhibition of cellular GSH synthesis byaugmented andrographolide-induced cytotoxicitypoptosis in Hep3B cells. Our observation thatreverses andrographolide-induced apoptosis andgeneration may be explained by the ability ofto restore GSH levels depleted by andrographolide.ossible that the inherent cellular defense mecha-in neonatal rat cardiomyocytes to increase GSHinsult is not present in lymphoma cells or lym-a cell lines, or that andrographolide may have aanism in lymphoma cell lines and patient samplesr to the liver cancer cell line (Hep3B). We have pre-y found that GSH levels in lymphoma cell lines ared higher than in primary tumors (data not shown).may explain our observations that the AC50 ofgrapholide is much lower in patient samples thanl lines.found that andrographolide induced BAX conforma-change in lymphoma cell lines and in fresh patientles from patients with FL and DLBCL (Fig. 6). BAXrmational change is known to follow caspase-8 activa-nd is accompanied by pore formation in the outer mi-ndrial membrane and precedes the release ofrome c from mitochondria, an important early stepochondrial-mediated apoptosis (18, 19, 35). Zhou et0) have shown that andrographolide induced BIDge and BAX conformational change in HepG2 cellsut upregulation of total BAX protein level. Similarly,und no increase in total BAX (data not shown) butNAC-inhibitable conformational change in lympho-ll lines and fresh patient samples. These data suggesttential clinical relevance to lymphoma biology andt that lactonediterpenoidsmayhave antitumor activityients with lymphoma. Recently, radiation-sensitizingof andrographolide have been published in an in vitrovivo model of Ras-transformed cells (36).interesting that we see both caspase-8 and caspase-9tion with andrographolide, suggesting that bothsic and extrinsic caspase pathways are involved ingrapholide-induced apoptosis in lymphoma. Zhou(37) have found that andrographolide may enhance(tumor necrosis factor–related apoptosis-inducing)–induced apoptosis through death receptor 4 upre-on, and that this is mediated through p53. Our datasting that the pan-caspase inhibitor was required toandrographolide-induced apoptosis are consistentprocess that may involve caspase pathways and/orpathways. Currently, studies of death receptor path-in andrographolide-induced apoptosis of lymphomare ongoing.s also intriguing that our data with andrographo-re extant for cell lines from B-cell lymphomas withifferent biological and molecular signatures and,d, hold true for samples from patients with the
ponding clinicopathologic subtypes of lymphoma.ugh there are marked differences in the clinical



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ior of these three types of lymphoma that wouldagainst categorizing them as a single entity, it isle that the activity of this natural diterpenoide operates through biological pathways that areendent of the heretofore established biology oflymphomas, and thereby will open new pathwaysommon approach to treatment.have shown that andrographolide, the activeonent of the plant A. paniculata, causes cell deathphoma cell lines and in fresh malignant cells fromoma patients. The mechanism is related to thestate of the cells, as it is blocked completely byand it is caspase dependent. Furthermore, apoptosis
iated through mitochondrial pathways in both celland primary patient samples. This novel natural
Recepublish

its-Iacovoni F. Reactive oxygen species and lipoxygenasesulate the oncogenicity of NPM-ALK-positive anaplastic largell lymphomas. Oncogene 2009;28:2690–6.

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e diterpenoid deserves further preclinical and clini-vestigation in lymphoma.

osure of Potential Conflicts of Interest

Gordon: honoraria from speakers bureau, Genentech; consultant/y board, Cure Tech Ltd.

Support

onal Cancer Institute grant K23 CA109613-A1 (A.M. Evens).costs of publication of this article were defrayed in part by thet of page charges. This article must therefore be hereby markedsement in accordance with 18 U.S.C. Section 1734 solely tothis fact.

ived 04/10/2010; revised 07/15/2010; accepted 08/10/2010;ed OnlineFirst 09/28/2010.

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2010;16:4755-4768. Published OnlineFirst August 26, 2010.Clin Cancer Res Shuo Yang, Andrew M. Evens, Sheila Prachand, et al.

Andrographis paniculataof Diterpenoid Lactone Andrographolide, the Active Component Mitochondrial-Mediated Apoptosis in Lymphoma Cells by the

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Research Mitochondrial-Mediated Apoptosis in Lymphoma ...grapholide or andrographolide + BSO where indicated. Fifty micromoles in DMSO or 100 mmol/L in water stock solution were prepared