ORIGINAL PAPER

Mahanine synergistically enhances cytotoxicity of 5-fluorouracilthrough ROS-mediated activation of PTEN and p53/p73 in coloncarcinoma

Ranjita Das • Kaushik Bhattacharya •

Sayantani Sarkar • Suman Kumar Samanta •

Bikas C. Pal • Chitra Mandal

Published online: 20 September 2013

� Springer Science+Business Media New York 2013

Abstract 5-Fluorouracil (5-FU) alone or in combination

with other drugs is the main basis of chemotherapeutic

treatment in colorectal cancer although patients with

microsatellite instability generally show resistance to 5-FU

treatment. The present investigation is focussed on the

mechanistic insight of a pure herbal carbazole alkaloid,

mahanine, as a single or in combination with 5-FU in colon

cancer. We demonstrated that mahanine-induced apoptosis

involved reactive oxygen species (ROS)-mediated nuclear

accumulation of PTEN and its interaction with p53/p73.

Mahanine and 5-FU in combination exerted synergistic

inhibitory effect on cell viability. This combination also

enhanced ROS production, increased tumour suppressor

proteins and suppressed chemo-migration. Taken together,

our results revealed that mahanine can be a potential che-

motherapeutic agent with efficacy to reduce the concen-

tration of toxic 5-FU in colon cancer.

Keywords 5-Fluorouracil � Mahanine � Colon

cancer � PTEN � p53 � p73

Introduction

Colorectal cancer accounts for 10–15 % of all cancers and

is the second leading cause of cancer related death in

Western countries with 35.8 cases per 100,000 individuals.

Incidence rate of this cancer varies around the world [1].

Genetic paradigm for the development of colorectal cancer

embraces chromosomal instability (CIN) and microsatellite

instability (MSI), the two distinct pathways. CIN, resulting

from allelic loss of tumour suppressor genes, is responsible

for *80 % of this cancer. MSI tumours include multiple

errors in short tandem repetitive DNA sequences generated

due to defective mismatch repair system and occur in

*15 % of sporadic and *90 % of hereditary non polyp-

osis colorectal cancers (HNPCC) [2, 3].

p53 is the most common tumour suppressor and most

commonly mutated and functionally inactivated gene in

*50 % of all human cancers. Cellular stresses such as

DNA damage, oncogene activation or hypoxia activates

p53 which acts as transcription factor and induces the

expression of target genes mediating cell cycle arrest or

apoptosis [4]. p63 and p73, two p53 homologues appear to

function similarly to p53. However, unlike p53, the p63

and p73 genes are rarely mutated in human cancer [5].

PTEN is the second most frequent tumour suppressor in

human cancers following p53. PTEN acts both as a lipid

phosphatase and protein phosphatase [6]. Down-regulation

of PTEN is generally associated with invasion, metastasis

and poor patient-survival in colorectal cancers. Therefore,

PTEN could be a possible target in sporadic and HNPCC

with deficient mismatch repair system [7].

The mainstay of chemotherapeutic treatment of patients

with colorectal cancer is 5-FU alone or in combination with

other drugs such as leucovorin, irinotecan, oxaliplatin,

capecitabine etc. [8, 9]. Patients with MSI generally show

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10495-013-0907-6) contains supplementarymaterial, which is available to authorized users.

R. Das � K. Bhattacharya � S. Sarkar � S. K. Samanta �C. Mandal (&)

Cancer Biology and Inflammatory Disorder Division, Council of

Scientific and Industrial Research (CSIR)-Indian Institute of

Chemical Biology, 4, Raja S. C. Mullick Road,

Kolkata 700032, India

e-mail: cmandal@iicb.res.in; chitra_mandal@yahoo.com

B. C. Pal

National Institute of Pharmaceutical Education and Research,

Kolkata, Kolkata, India

123

Apoptosis (2014) 19:149–164

DOI 10.1007/s10495-013-0907-6

resistance to 5-FU treatment. Besides, increase of the

concentration of 5-FU would generate undesirable levels of

toxicity in bone marrow and gastrointestinal tract leading

to severe adverse effects [10, 11].

Mahanine, a carbazole alkaloid, purified from Murraya

koenigii and Micromelum minsutum, well-known medicinal

herbal plants, induced apoptosis in histiocytic lymphoma,

promylocytic leukemia and prostate cancer cells. It

exhibited anti-mutagenic, anti-microbial and cytotoxic

activities [12–16]. It induced apoptosis in leukemic cells

through mitochondrial death cascade [17] and Fas-FasL

mediated extrinsic pathway [18]. It showed inhibition of

Hsp90 through reactive oxygen species (ROS) in pancre-

atic cancer [19]. Recent study reported that C-7-OH and

9-NH functional groups of mahanine were responsible for

its cytotoxicity and responsible for minor groove binding

with DNA [20]. It was an activator of the epigenetically

suppressed tumour suppressor gene RASSF1A [21]. Ma-

hanine could also reduce in vivo xenograft and orthotopic

tumour and showed nontoxicity to total body mass of

normal Balb/c and athymic nude mice [18, 19]. Therefore,

it is worthwhile to explore such a potent carbazole alkaloid

as a strong candidate for the formulation of novel combi-

nation regimens.

Here we demonstrated that mahanine potentially induces

apoptosis in MSI colorectal cancer cells as single agent and

in combination with 5-FU. Mahanine promoted ROS-

mediated nuclear accumulation of PTEN which interacted

with the family of tumour suppressor proteins (p53/p73)

leading to their activation. It also synergistically increased

the cytotoxic effect of 5-FU by *4–5-folds. Furthermore,

mahanine in combination with 5-FU showed the ability to

enhance ROS generation which subsequently increased

PTEN and p53/p73 proteins and suppressed adhesion.

Thus, mahanine can be considered as a potentially active

chemotherapeutic agent in combination with 5-FU for

colon cancers.

Materials and methods

Reagents

The primary antibodies against phospho-PTEN (Ser380),

PTEN, phospho-p53 (Ser15), p53, phospho-p73 (Tyr99),

p73, phospho-MDM2 (Ser166), MDM2, p21/waf1, Bax, b-

actin, poly-ADP ribose polymerase (PARP) and horseradish

peroxidase (HRP)-linked secondary antibodies were all

obtained from Cell Signaling Technology, USA. Primary

antibodies HDAC-3 and Caspase-3 were purchased from BD

Bioscience. IMDM cell culture medium, FCS, antibiotic–

antimycotic, Trypsin–EDTA and H2DCFDA were bought

from Invitrogen, USA. 5-FU, 3-(4,5-dimethylthiazol-2-yl)-

2, 5-diphenyl tetrazolium bromide (MTT), N-acetyl cysteine

(NAC), propidium iodide (PI), esiRNAs, N-TER nanopar-

ticle driven transfection system, molecular grade BSA,

protein A-Sepharose 4B, Tween-20 and dimethyl sulphoxide

(DMSO) were obtained from Sigma–Aldrich, USA. NE-

PER nuclear and cytoplasmic extraction reagents and Su-

perSignal West Pico imaging system were purchased from

Thermo-scientific, USA. Cycle Test Plus kit was obtained

from BD Bioscience, USA. Vascular endothelial growth

factor, epidermal growth factor (EGF) and platelet-derived

growth factor were obtained from PromoCell Heidelberg,

Germany.

Purification and characterization of mahanine

Mahanine was purified from fresh leaves of a native Indian

plant, M. koenigii (Rutaceae family) as described else-

where [16]. The purity was confirmed by HPLC and LC–

MS [18] and the structure of mahanine was identified by1[H] and 13[C] NMR spectral data analysis (Fig 1a).

Cell lines and cell culture

Three colorectal carcinoma cell lines, HCT116 (p53wt),

HCT116 (p53null), SW480 (p53mut) and African green

monkey kidney (Vero) cell line were obtained from

American Type Culture Collection (Manassas, VA). The

cells were cultured in IMDM supplemented with 10 % FCS

and 1 % antibiotic antimycotic mixture (growth medium)

at 37 �C in a humidified incubator containing 5 % CO2.

Cell viability analysis by MTT assay

p53wt (5 9 103 cells/well), p53null (5 9 103 cells/well) and

p53mut (1 9 104 cells/well) cells were seeded into 96-well

microplates. Then cells were treated with mahanine

(0–30 lM) and incubated at 37 �C in a humidified 5 %

CO2/95 % air mixture for 48 h. MTT (0.4 mg/ml) was

added 4 h before the termination of culture and further

incubated in dark at 37 �C. After incubation, the superna-

tant was removed and the formazan complex was dissolved

in DMSO. The optical density was measured by ELISA

reader (Thermo) at 550 nm as described elsewhere [19].

Cell viability was calculated from percentage of MTT

conversion in treated cells relative to untreated control

cells.

Drug combination data analysis

p53wt and p53null (5 9 103 cells/well) cells were treated

either with 5-FU (0–16 lM and 0–32 lM respectively) and

mahanine (3–24 lM) alone or in combination for 48 h. The

combination treatments were done at a fixed concentration

150 Apoptosis (2014) 19:149–164

123

Fig. 1 Mahanine induced apoptotic cell death in colon cancer.

a Chemical structure of mahanine b HCT116 (p53wt) and HCT116

(p53null) cells were seeded into 96-well plates (5 9 103 cells/well)

and treated with mahanine (0–30 lM) for 48 h. The cell growth

inhibition was measured by MTT assay. Mahanine-induced growth

inhibitory effect was expressed as cell viability, inferred from

metabolic activity, relative to untreated controls. Data were repre-

sented as % of cell viability (% of MTT conversion relative to

untreated control cells). Each value is the mean ± SD of three

independent experiments. c–d Cells were seeded in 6-well plates at a

density of 1 9 106 cells/well and treated with mahanine (0–15 lM)

for 24 h. Mahanine-induced apoptosis was confirmed by detection of

a concentration-dependent increase in FITC-Annexin-V and PI

staining assessed by flow cytometry analysis after 24 h. Data

represented the mean ± SD of three independent experiments. ‘‘*’’

indicated a significant difference of p \ 0.05. e Mahanine-treated

both p53wt and p53null cells after 24 h incubation were analysed by

Western blotting to detect the known molecular mediators of

apoptosis

Apoptosis (2014) 19:149–164 151

123

ratio of 1:1.5 (5-FU: mahanine) for p53wt and 1:0.75 (5-

FU: mahanine) for p53null cells. The growth inhibition was

evaluated by MTT assay.

The isobologram and median-effect plot were generated

by using Calcusyn software version 2.1 (Biosoft) and used

to analyze the nature of the drug interactions [22]. Based

on the generated isobologram, combination index (CI) was

calculated using the equation CI = (D)1/(DX)1 ? (D)2/

(DX)2, where (DX)1 and (DX)2 were the concentrations of

each drug alone to exert x% effect and (D)1 and (D)2 were

the concentration of the drugs in combination to elicit the

same effect. CI of \1 indicates synergism, CI = 1 and

CI [ 1 indicate additivity and antagonism respectively.

Data obtained from CI method were used to evaluate the

drug-reduction index (DRI) which represents the fold

decrease of each drug as a result of synergism.

Flow cytometric analysis of exposed membrane

phosphatidylserine

Cells (1 9 106 cells/well) were exposed either to 5-FU

(8 lM for p53wt and 16 lM for p53null cells) and mahanine

(12, 15 and 20 lM) alone or in combination with fixed

ratio for 24 h. Then cells were washed with phosphate

buffer saline (PBS), resuspended in annexin-V binding

buffer according to manufacturer’s instructions and incu-

bated for 45 min at 25 �C. Cells were further incubated

with FITC-Annexin-V and propidium iodide (5 lg/ml) for

20 min at 4 �C in dark. Data acquisition was done on a

FACSCalibur flow cytometer (BD) and analyzed with

CellQuest Pro software. At least 10,000 cells were ana-

lyzed for this experiment.

Measurement of reactive oxygen species (ROS)

Cells (5 9 105 cells/well) were treated either with maha-

nine (0–20 lM) and 5-FU (8 lM for p53wt and 16 lM for

p53null cells) or in combination at fixed ratio for 1 h. They

were washed, resuspended in PBS and incubated with

H2DCFDA (20 lM) at 37 �C for 30 min in dark. ROS

generation was measured flow cytometrically with excita-

tion and emission wavelength at 488 and 530 nm respec-

tively. The percentage of H2DCFDA?ve cells was

calculated. The inhibition of ROS generation was shown by

pretreatment with NAC (2.5 mM) for 30 min.

Cell cycle analysis

Cells (1 9 106 cells/well) were exposed to varying con-

centrations of 5-FU (0–16 lM for p53wt and 0–32 lM for

p53null cells) for 24 h. Next, they were harvested and

processed by Cycle Test Plus kit according to the manu-

facturer’s instructions. Briefly, after treatment, cells were

washed and incubated with trypsin solution (250 ll) for the

membrane permeabilization for 10 min at 25 �C. Then they

were incubated with RNAse solution (200 ll) for 10 min at

25 �C for the degradation of intracellular RNA. Next cells

were incubated with PI solution (200 ll) for 10 min at

4 �C. Then the cells were acquired in FACS Calibur flow

cytometer (BD) and at least 10,000 cells were acquired and

analyzed by CellQuest Pro software.

Subcellular fractionation

Cells (1 9 106 cells/well) were treated with mahanine

(0–20 lM) for 24 h and fractionated into cytosol and

nuclear portion by NE-PER� kit according to manufac-

turer’s protocol. Briefly, washed cells were vortexed in

cytosol extraction reagent, centrifuged and the supernatant

was used as the cytosolic fraction. The pellet incubated in

nuclear extraction reagent, centrifuged and supernatant

served as the nuclear fraction.

Immunoblot and immunoprecipitation analysis

Cells (1 9 106 cells/well) were treated with mahanine

(0–20 lM) for 24 h, washed with cold PBS and lysed in

lysis buffer. The lysates were sonicated, cold centrifuged at

10,000 9 g, and the protein concentrations of the clear

supernatants were estimated [23]. Equal amounts of protein

(50 lg) were resolved on SDS-PAGE (7.5–12 %) and

electrotransferred onto nitrocellulose membrane. Blots

were blocked with TBS-2 % BSA, probed with appropriate

primary antibodies and processed using HRP-secondary

antibodies as described elsewhere [19]. Signals were

detected with the West-pico ECL system (Pierce, Thermo

Scientific, USA).

For the immunoprecipitation analysis, treated cells were

fractionated in cytosol and nucleus and nuclear protein

(300 lg) was incubated with anti-PTEN or anti-MDM2

antibody (1:100) for overnight at 4 �C. This was followed by

further incubation with Protein A-Sepharose 4B. The immune

complex was resolved by SDS-PAGE (7.5 %), transferred

and subsequently identified by appropriate antibodies.

Transient transfection

Both p53wt and p53null cells (5 9 105 cells/well) were

transfected transiently with esiRNA specific for PTEN,

p53, p73 as described in manufacture’s protocol. Briefly,

cells were pre-seeded for overnight. Next, vehicle and

esiRNA transfection reagents were added and incubated for

8 h. Subsequently, they were incubated for another

10–12 h with growth medium followed by mahanine

treatment of 24 h.

152 Apoptosis (2014) 19:149–164

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Transmigration assay

Both the cells (1 9 106 cells/well) were treated either with

5-FU (8 lM for p53wt and 16 lM for p53null) and maha-

nine (12 lM) alone or in combination of both for 24 h and

migration was assessed by Transmigration kit (Promo Cell)

according to manufacture’s protocol. Briefly, 20 ng/ml

EGF was added into the outer well of the 24-transwell

plate. Treated cells (5 9 104 cells/well/750 ll) were added

into the insert and incubated for 6 h. Then, medium was

aspirated from the wells and the wells were washed to

remove the non migrated cells from the upper portion of

the membrane. Next, the wells were air dried using a cotton

swab. Subsequently, migrated cells passed through the

membrane were fixed with chilled methanol and stained

with 0.1 % crystal violet. Random fields were photo-

graphed and quantified under phase contrast microscopy.

Statistical analysis

The data were derived from at least three independent

experiments. Statistical analysis was performed using the

two-tailed Student’s t test. Error-bars represent

mean ± SD from independent experiments. Significant

differences were set at p \ 0.05 and analyzed by Microsoft

Excel and GraphPad Prism.

Results

Mahanine induced apoptosis in colon cancer cells

irrespective of their p53 status

Mahanine exhibited anti-proliferative activity against colon

cancer cells in concentration-dependent manner as evalu-

ated by MTT assay. It potentially inhibited (*75 %)

propagation of HCT116 (p53wt) and HCT116 (p53null) cells

at a concentration of 15 lM after 48 h of treatment

(Fig. 1b). In another colon cancer cell (SW480, p53mut),

the growth inhibition was *70 % at 20 lM of mahanine

(Fig. S1). In Vero cells (served as control), mahanine

inhibited minimal cell proliferation (\20 %) even at

30 lM of concentration (Fig. S1). The IC50 values for

p53wt, p53null and p53mut cells ranged between 12.6 and

16.6 lM (Table 1). This result suggested that mahanine

inhibited proliferation of colon cancer cells independent of

their p53 status.

The appearance of phosphatidylserine on the outer sur-

face of plasma membrane signifies apoptosis of the cell.

Both, p53wt and p53null cells were exposed to mahanine

(0–15 lM) for 24 h. We observed concentration-dependant

increase (*12 to *29 %) in Annexin-V (Fig. 1c) and PI

binding (Fig. 1d) of both p53wt and p53null colon cancer

cells suggesting mahanine-mediated apoptosis.

To investigate the molecular mediators of mahanine-

induced cell death, expression level of PARP and caspase 3

were assessed after 24 h of mahanine treatment. We

observed PARP cleavage and caspase 3 activation at

10 lM (p53wt) and 15 lM (p53null) respectively (Fig. 1e).

This further supported that mahanine-induced cell death

pattern was apoptosis in colon cancer cells irrespective of

their p53 status.

Mahanine synergistically enhanced cytotoxicity

of 5-FU

5-FU is the most important chemotherapeutic drug in colon

cancer with limiting therapeutic success due to its severe

toxic effects and resistance [24, 25]. Accordingly, we

attempted to improve its efficacy of 5-FU in combination

with mahanine. Therefore the growth inhibitory efficacy of

5-FU and mahanine alone or in combination on both p53wt

and p53null cells were assessed by MTT assay.

The concentrations were selected based on the dose

range experiments (supplementary table 1 and 2). 5-FU in

both p53wt (2–16 lM) and p53null cells (4–32 lM),

exhibited *12 to *50 % growth inhibition after 48 h of

treatment (Fig. 2a). Mahanine (3–24 lM) alone inhibited

concentration-dependent growth of p53wt and p53null cells.

This 5-FU-mediated growth inhibition was augmented

when it was used in combination with mahanine at 1:1.5

(5-FU: mahanine) molar ratio in p53wt cells and at 1:0.75

(5-FU: mahanine) molar ratio in p53null cells. 5-FU and

mahanine in combination led to enhanced inhibitory effect

(*40 to *94 %) on both p53wt and p53null cells. These

fixed ratios were used for further experiments. Growth-

inhibition data, was achieved from this combination treat-

ment, were used to evaluate CI values using the Calcusyn

software. The interaction of mahanine and 5-FU was syn-

ergistic as CI values were in the range of 0.6–0.67 and

0.52–0.66 for p53wt and p53null cells respectively (Fig. 2b).

Based on synergism, DRI parameters were also calculated.

Mahanine in combination resulted *4- and *7-folds

reduction in the concentration of 5-FU for 50 and 75 %

Table 1 IC50 values of mahanine in different colon cancer cell lines

Cell lines IC50 (lM)

HCT116 (p53wt) 12.6 ± 0.05

HCT116 (p53null) 13.9 ± 0.1

SW480 (p53mut) 16.6 ± 0.04

The growth inhibitory effect of mahanine on colon cancer cells was

evaluated by MTT assay after treated the cells with varying con-

centrations (0–30 lM) for 48 h. IC50 values shown here are the

mean ± SD from the data of three independent experiments

Apoptosis (2014) 19:149–164 153

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154 Apoptosis (2014) 19:149–164

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inhibition of cell viability in p53wt and p53null respectively

(Table 2).

Furthermore, we demonstrated enhanced FITC-annexin-

V binding of 5-FU and mahanine in combination. Annexin-

V binding of p53wt and p53null cells at 8 lM and 16 lM of

5-FU alone was 17.71 and 16.41 % respectively. These

binding were increased to 43.11 and 35.1 % after combi-

nation treatment of p53wt and p53null cells respectively

(Fig. 2c). Furthermore, the combination led to enhanced

loss of membrane integrity in both p53wt and p53null cells

as demonstrated by the increase of PI?ve cells in the total

cell population (Fig. 2d). Taken together, these results

demonstrated that mahanine has the potential to improve

5-FU-mediated cytotoxicity.

Mahanine in combination with 5-FU enhanced arrest

of p53wt and p53null cells in G1-S phase

5-FU is reported to be an S-phase specific anti-cancer agent

at lower concentrations [26] and in higher concentrations it

induces G1-S phase arrest [27]. Mahanine also induces

arrest in cancer cells at G1-S phase (personal communi-

cation). To verify the specific mode of action of 5FU alone

or in combination with mahanine, cell cycle analyses were

performed with HCT116 (p53wt) and HCT116 (p53null)

cells. We initially observed that p53wt cells exposed to

lower concentration of 5-FU (4 lM) were accumulated in S

phase (68.35 %) compared to cells without 5-FU (35.44 %)

after 24 h. However, at 16 lM of 5-FU arrested more

p53wt (88.18 %) cells in G1-S phase than in absence of

5-FU (47.66 %) (Fig. S3). Similarly, p53null cells also

showed arrest in S phase (53.86 %) after treated with 8 lM

of 5-FU in comparison to untreated (13.96 %) condition. In

contrast, 32 lM of 5-FU resulted in G1-S phase (69.68 %)

arrest compared to untreated cells (46.47 %) (Fig. S3).

Interestingly, even at 4 lM of 5-FU in combination with

mahanine (12 lM) was capable of arresting 90.11 % of

p53wt cells at G1-S phase compared to 21.10 % at 5-FU

alone. Similar G1-S phase arrest (72.45 %) was observed

when p53null cells were treated with 8 lM of 5-FU in

combination with mahanine compared to 27.01 % at 5-FU

alone. Taken together our results suggested that mahanine

increased G1-S phase arrest in combination with 5-FU.

Activation and nuclear accumulation of tumour

suppressor proteins (PTEN and p53/p73) by mahanine

p53 and PTEN are the two most common tumour sup-

pressor proteins, former acting as transcription factor

whereas latter has phosphatase activity [4, 6]. Therefore,

we wanted to investigate the regulation of these two types

of tumour suppressors in mahanine-treated colon cancer

cells. We found that p53 was significantly augmented along

with increased phosphorylation at Ser15 in HCT116

(p53wt) cells. Moreover, in absence of p53, mahanine

eventually activated p73, other p53 family protein in

p53null cells as evident by the enhancement of total and

phospho-p73 level at Tyr99 (Fig. 3a). Mahanine-mediated

higher expression of p73 and p-p73 in p53null cells was

observed compared to p53wt cells. This report was further

supported by an earlier observation in which author

described that p53 inactivation or knockdown leads to

upregulation of p73 at transcriptional level [28]. Addi-

tionally, decrease in Ser166 phosphorylation of murine

double minute 2 (MDM2), the negative regulator of p53

and p73, in mahanine-treated cells further supported the

activation of these tumour suppressor proteins (Fig. 3b).

Similarly, both PTEN and phospho-PTEN level at Ser380

were elevated in mahanine-treated p53wt and p53null cells

(Fig. 3c). Such enhanced accumulation of phospho-PTEN

at this residue possibly indicated increased stabilization of

Table 2 Dose reduction index (DRI) values for 5-FU and mahanine combination

Cell lines Drug:compound Molar ratio

(5-FU:Mahanine)

DRI (50 % fraction

affected level)

DRI (75 % fraction

affected level)

p53wt 5-FU:Mahanine 1:1.5 4.17 8.568

p53null 5-FU:Mahanine 1:0.75 4.536 6.784

The DRI signifies the fold decrease of each drug as a result of synergistic combination in compared with the concentration of a single agent

needed to achieve the same effect. Concentrations of 5-FU was reduced *4- and *7-folds to achieve a 50 and 75 % inhibition of cell

proliferation respectively when the cells were exposed to combination treatment at fixed molar ratios after 48 h

Fig. 2 Mahanine synergistically enhanced efficacy of 5-FU in

HCT116 (p53wt) and HCT116 (p53null) cells. a Both cell lines were

exposed for 48 h to varying concentration of 5-FU (2–16 lM for

p53wt and 4–32 lM for p53null) and mahanine (3–24 lM) alone and

in combinations at a fixed ratio of 1:1.5 (5-FU:mahanine) for p53wt

cells and 1:0.75 for p53null cells. The cell growth inhibition was

measured by MTT assay. Data represented mean ± SD of three

independent experiments. b The combination index (CI) was calcu-

lated by Chou-Talalay method using Calcusyn software. c–d p53wt

and p53null cells were treated with 5-FU (8 lM for p53wt and 16 lM

for p53null) and mahanine (12 lM) alone or in combination for 24 h.

Increased apoptosis by the combination treatment in compared to

single agent alone was assessed by FITC-annexin-V and PI positivity

analysed through flow cytometry. Data represented the mean ± SD of

three independent experiments. ‘‘*’’ indicated a significant difference

of p \ 0.05

b

Apoptosis (2014) 19:149–164 155

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PTEN. This may be due to inactivation of its catalytic

activity [29].

Though cytoplasmic PTEN predominates, it can also be

localized to nucleus and plays role in the tumour sup-

pression [30]. Moreover, activated p53 and p73 also exert

their role in nucleus as transcription factors [5]. We already

observed that the stabilization and activation of PTEN and

p53 family proteins in mahanine-exposed cells (Fig. 3a, b,

c). Next, we determined whether both type of tumour

suppressors could translocated into the nucleus. Increased

nuclear accumulation of PTEN was concomitant with ele-

vated p-PTEN (Ser380) level in both p53wt and p53null

cells. Associated with these, there were increased nuclear

level of p53 and p73 in p53wt and p53null cells respectively

(Fig. 3d). This observation indicated that mahanine not

only activated PTEN and p53/p73 but also promoted their

nuclear localization.

Mahanine-induced ROS modulated nuclear localization

of PTEN and p53/p73

We have reported earlier that mahanine is capable of

generating ROS in leukemia [18] and pancreatic cancer

[19]. To further reveal mahanine-induced oxidative stress

in colon cancer cells, level of ROS was measured and

identified that mahanine exposure for 1 h increased

H2DCFDA?ve HCT116 (p53wt) and HCT116 (p53null) cells

in a concentration-dependent manner (Fig. 4a). This event

was antagonized by pre-treatment with NAC, a scavenger

of ROS (Fig. 4b) confirming that mahanine is responsible

for ROS generation in the colon cancer cells also.

Additionally, we wanted to disclose whether ROS could

regulate PTEN and p53/p73 in these cells. The elevated

levels of PTEN, p53 and p73 were reversed significantly

after NAC pre-treatment indicating the involvement of

ROS in the regulation of these tumour suppressors in colon

cancer (Fig. 4c).

We next also examined the levels of PTEN in the

nucleus after mahanine exposure and found that NAC

prevented the accumulation of this protein in the nuclear

fraction. Decrease in nuclear PTEN level in presence of

NAC also associated with the reduction of p53 and p73

level in nucleus (Fig. 4d). This clearly demonstrated that

mahanine-induced ROS could modulate nuclear accumu-

lation of PTEN and p53/p73. In addition, the abundance of

p-MDM2 (Ser 166) was also regulated by mahanine-

mediated ROS generation in both types of cells (Fig. 4e).

Fig. 3 Mahanine treatment activated tumour suppressor p53/p73 and

PTEN and promoted their nuclear accumulation. HCT116 (p53wt) and

HCT116 (p53null) cells were treated with different concentrations of

mahanine (0–20 lM) for 24 h. The cells were harvested and whole

cell protein lysates were prepared and subjected to Western blot

analysis with a p53, p73, phospho-p53 and phospho-p73 antibodies

b MDM2 and phospho-MDM2 antibodies c PTEN and phospho-

PTEN antibodies d After 24 h of mahanine (0–20 lM) treatment,

both cells extracts were separated into cytosol and nuclear fractions

and subjected to Western blotting with indicated antibodies. Both b-

actin and HDAC3 were used as controls for cytosol and nucleus. The

presented data have been derived from three different experiments,

one of which is shown here

b

156 Apoptosis (2014) 19:149–164

123

Fig. 4 Mahanine induced ROS

generation regulating PTEN and

p53/p73 expression and their

nuclear accumulation in both

p53wt and p53null cells. a Both

cell lines were subjected to

mahanine (0–20 lM) treatment

for 1 h and ROS production was

measured by H2DCF-DA

staining. b Cells were pre-

treated with NAC (2.5 mM) for

30 min and then exposed to

mahanine (20 lM) for 1 h.

Generation of ROS was

analysed using H2DCF-DA by

flow cytometry. Each value

represented the mean ± SD of

three independent experiments.

‘‘*’’ indicated a significant

difference of p \ 0.05.

c–e p53wt and p53null cells were

treated with mahanine (15 lM)

for 24 h after pre-incubation

with or without NAC (2.5 mM,

for 30 min). Cells were

harvested for preparation of

whole cell lysates to perform

Western blot analysis with

PTEN, p53, p73, MDM2 and

phospho-MDM2 antibodies.

d Both cell lines were pre-

treated with NAC (2.5 mM) for

30 min and treated with

mahanine (10 and 15 lM) for

24 h. Cells were harvested,

fractionated into cytosol and

nuclear portions and subjected

to Western blotting with

corresponding antibodies. Both

b-actin and HDAC3 were used

as controls for cytosol and

nucleus. The results were

derived from three different

experiments, of which one is

presented here

Apoptosis (2014) 19:149–164 157

123

Physical interaction of PTEN with p53 and p73

enhanced their accumulation in nucleus for activation

Function of nuclear PTEN as a tumour suppressor is

facilitated through its physical interaction with another

tumour suppressor, p53 family proteins leading to their

activation [29]. Therefore, next we asked whether activa-

tion of p53 and p73 were PTEN-dependent or not. It was

identified that transient knockdown of PTEN led to the

decrease of basal as well as mahanine-induced p53 and p73

levels in HCT116 (p53wt) and HCT116 (p53null) cells

respectively (Fig. 5a). This observation confirmed that

PTEN stabilization was needed for the activation of p53

family.

Subsequently, we wanted to find whether nuclear-

localized PTEN could act as an activator of p53 family

proteins in both p53wt and p53null cells. We found that p53

as well as p73 both co-immunoprecipitated with PTEN and

their association were increased in a concentration-depen-

dent manner in nuclear fraction of mahanine-treated p53wt

and p53null cells (Fig. 5b). These results showed that PTEN

indeed directly interacted with p53 and p73 and thereby

activated them in the nucleus. In cytosol, the association of

PTEN with p53 or p73 was decreased in a concentration-

dependent manner (data not shown).

MDM2 is known to bind with p53 and its family proteins

through its N-terminal region and shuttles it to cytosol from

nucleus acting as ubiquitin ligase [31, 32]. Accordingly, we

further addressed whether PTEN-mediated activation of p53

and p73 were linked with the disruption of MDM2-p53 or

MDM2-p73 complex. Mahanine-treated cells exhibited inhi-

bition of binding of MDM2 with p53 and p73 in a concen-

tration-dependent manner as evident from decreased co-

immunoprecipitation level of p53 and p73 with MDM2 con-

firming their enhanced accumulation in nucleus (Fig. 5c).

Mahanine in combination of 5-FU produced more ROS

Earlier reports stated that 5-FU could generate ROS in

cancer cells [33] and mahanine also reported to exert pro-

oxidant activity [18]. To elucidate whether oxidative stress

was found to be associated with the sensitization of both

HCT116 (p53wt) and HCT116 (p53null) cells to 5-FU by

mahanine, we checked intracellular ROS. Our results

Fig. 5 Mahanine increased physical interaction of PTEN and p53/

p73 in nucleus and dissociated MDM2 from p53/p73. a p53wt and

p53null HCT116 cells were transiently transfected with esiRNA

targeting PTEN and incubated for 8 h. These cells were incubated in

growth medium for 10–12 h followed by mahanine (15 lM) treat-

ment for additional 24 h. They were harvested and whole cell protein

lysates were subjected to Western blot analysis with PTEN, p53 and

p73 antibodies. b–c After 24 h treatment with mahanine (0–20 lM),

cells were separated into cytosol and nuclear fractions. The nuclear

portions were immunoprecipitated with (IP) with anti-PTEN or anti-

MDM2 antibodies. The precipitated proteins were electrophoresed

under non-reducing condition and immunoblotted (IB) with anti-p53,

anti-p73 antibodies. The results were obtained from three different

experiments, of which one is presented here

158 Apoptosis (2014) 19:149–164

123

demonstrated that combination of 5-FU and mahanine

increased ROS production in 1 h by *2-folds in both

p53wt and p53null colon cancer cells in comparison to single

agent (Fig. 6a). We already established that ROS was

critical for mahanine-mediated apoptosis in colon cancer

cells. Here, Additional enhancement of ROS production by

combining 5-FU and mahanine would further support the

potentiating activity of 5-FU by mahanine.

Enhanced expression of PTEN and p53/p73 in HCT116

(p53wt) and HCT116 (p53null) cells after combination

treatment (mahanine and 5-FU)

We have already established that mahanine-mediated

apoptosis include the activation of two tumour suppressors,

PTEN and p53/p73. So, here we explored the capability of

mahanine in combination with 5-FU to determine the

molecular mechanism involved in enhanced anti-cancer

activity.

We observed that mahanine (12 lM) increased the

protein level of PTEN and p53 in HCT116 (p53wt)

whereas, p73 and PTEN were increased in HCT116

(p53null) cells. 5-FU is reported to enhance the expressions

of p53/p73 [34]. Here, we also found that in p53wt cells,

5-FU (8 lM) elevated the level of PTEN and p53 signifi-

cantly however, in p53null cells, even 16 lM of 5-FU was

unable to enhance PTEN though p73 was increased. The

degree of enhancement of PTEN was much less in p53null

cells.

However, after combination treatment of mahanine and

5-FU, we observed additional increase in the expressions of

PTEN and p53 in p53wt and PTEN and p73 in p53null colon

cancer cells (Fig. 6b). These results further supported that

mahanine has the efficacy to potentiate the tumour sup-

pressor activity of 5-FU.

Involvement of PTEN in mahanine alone

and in combination with 5-FU-induced apoptosis

of HCT116 (p53wt) and HCT116 (p53null) cells

By now, we have established that mahanine stabilized

PTEN which further interacted with p53 in p53wt cells

and p73 in p53null cells and activated them. We have

also showed that PTEN knockdown resulted in the

decreased p53/p73 levels (Fig. 5a). Now, we wanted to

address whether PTEN was directly involved in apopto-

sis induced by mahanine alone or in combination.

Accordingly, PTEN knocked down cells were treated

with mahanine (12 lM) for 24 h and percentage of

apoptotic cells were assessed by annexin-V binding.

PTEN knockdown diminished the annexin-V?ve cells

from *16 % and *9 to *8 % and *2 % in p53wt and

p53null cells respectively (Fig. 7a).

Fig. 6 Combination of 5-FU

and mahanine induced more

ROS generation and increased

PTEN and p53 in p53wt cells

and PTEN and p73 in p53null

cells. a Both cell lines were

subjected to mahanine (12 lM)

and 5-FU (8 lM for p53wt and

16 lM for p53null cells) alone

and in combination treatment

for 1 h. The level of ROS

generation was measured using

H2DCF-DA staining by flow

cytometry. b Both the cells were

treated with 5-FU and mahanine

alone and in combination for

24 h as indicated and analysed

by Western blot with indicated

antibodies. Data represented the

mean ± SD of three

independent experiments. ‘‘*’’

indicated a significant

difference of p \ 0.05

Apoptosis (2014) 19:149–164 159

123

Additionally, PTEN knocked down cells were exposed

to mahanine (12 lM) in combination with 5FU (8 lM) for

24 h. These cells showed reduction in annexin-V positivity

from *23 to *10 % compared to PTENwt condition.

PTEN knocked down p53null cells also showed similar

reduction in annexin-V positivity.

Similarly, p53 was knocked down in p53wt cells tran-

siently. The cells after treatment with mahanine reduced

the annexin-V?ve cells from *10 to *6 % compared to

p53wt cells (Fig. 7b). Combination treatment of these cells

showed marginal reduction from *14 to *8 %. Further-

more, p73 knocked down p53null cells also exhibited sim-

ilar effect after mahanine or combination treatment.

Therefore it is clear that in absence of PTEN, the

reductions of apoptotic cells were more compared to p53

and p73 knocked down cells thereby PTEN possibly act as

an upstream of p53/p73 in such apoptosis. Taken together,

these results suggested direct involvement of PTEN both in

mahanine and combination treatment-induced apoptosis.

Mahanine and 5-FU combination suppressed more

migration in HCT116 (p53wt) and HCT116 (p53null)

cells

By now we have established that mahanine enhances

cytotoxic activity of 5-FU by activating tumour suppressors

PTEN and p53/p73. Besides cell cycle arrest and apoptosis,

p53 and its family proteins regulate cell migration and

invasion [35]. Epithelial cell adhesion molecule (EpCAM)

was reported to be over expressed in epithelial carcinomas

and repression of which, p53 was reported to inhibit cell

migration [36]. In view of that, we wanted to study whether

mahanine and 5-FU combination could modulate colon

carcinoma cell-migration. Accordingly, we investigated the

effect of mahanine (12 lM) and 5-FU (8 lM) alone as well

as in combination on the EGF-driven HCT116 (p53wt)

colon cancer cell migration. We found that 5-FU alone has

less inhibitory effect on p53wt cell migration, whereas

mahanine showed its efficacy to reduce the cell migration

in comparison to the untreated one. But in the presence of

mahanine and 5-FU combination, the inhibition of cell

migration was observed more predominantly in both the

cells (Fig. 8). The migration of HCT116 (p53null) cells was

much less when they were exposed to mahanine (12 lM)

and 5-FU (16 lM) in combination compared to either 5-FU

or mahanine.

Discussion

The current study concentrated on the development of

novel combined regimes for colon cancer therapy using an

herbal agent to reduce the concentration-limiting toxicity

of a well known anti-cancer drug and the establishment of

distinct molecular mechanism regulated by the treatment of

these dual agents. 5-FU is the most commonly used che-

motherapeutic drug for the treatment of colon cancer, but

toxic side effects of this agent in association with chemo-

resistance restrict its successful use and leads to major

clinical challenges [24, 25, 33].

Fig. 7 PTEN was involved in

mahanine as well as

combination-induced apoptosis

in p53wt and p53null HCT116

cells. a p53wt and p53null cells

were transiently transfected

with esiRNA targeting PTEN

b p53wt and p53null cells were

transiently transfected with

esiRNA targeting p53 and p73

respectively Transfected cells

were treated either with

mahanine (12 lM) alone or in

combination of 5-FU (8 lM for

p53wt and 16 lM for p53null) for

additional 24 h. The cells were

processed and stained with

FITC-annexin-V followed by

analysis through flow

cytometry. Data represented the

mean ± SD of three

independent experiments. ‘‘*’’

indicated a significant

difference of p \ 0.05

160 Apoptosis (2014) 19:149–164

123

The main achievement of this work is to report that a

nontoxic carbazole alkaloid, mahanine, showed anti-pro-

liferative efficacy against both p53wt and p53null HCT116

colon cancer cells. Furthermore, we demonstrated syner-

gistic increase of cancer cell cytotoxicity to 5-FU after

combining it with mahanine. This study established a novel

role of mahanine as a synergistic modulator of a well

known clinically used toxic chemotherapeutic drug for the

treatment of both p53wt and p53null colon cancer.

Mahanine was reported to be nontoxic towards normal

tissues such as liver, lungs, spleen and total body mass of

normal Balb/c and athymic nude mice [18, 37]. This effi-

cacy of nontoxic mahanine persuaded us to explore it in

adjunct therapy with known toxic chemotherapeutic drugs.

Here, we have found that mahanine was also able to sen-

sitize colon cancer cells to 5-FU-induced cytotoxicity. The

cell growth inhibitory concentrations of 5-FU were

decreased significantly in presence of mahanine to achieve

same percentage of growth inhibition. In combination,

5-FU resulted in significantly greater cell death with CI

values of \1 suggesting synergistic cytotoxic effect. This

clearly established the capability of mahanine to syner-

gistically enhance the cytotoxicity of 5-FU by reducing its

concentration. Thus by decreasing the effective concen-

trations of toxic drug, mahanine would be able to reduce

the concentration-limiting effects of 5-FU.

Natural herbal products are always known to be the

potential source of chemotherapeutics and able to exert

beneficial effects by modulating the actions of pharma-

ceutical drugs either synergistically or antagonistically

[38]. Some natural products such as resveratrol, genistein,

notoginseng, falcarindiol etc. were reported to elicit 5-FU-

mediated apoptosis synergistically in colon cancer [39–42].

However, we reported about a carbazole alkaloid, maha-

nine which in combination with 5-FU produced an overall

synergistic effect on the colon cancer cell apoptosis

Fig. 8 5-FU and mahanine combination treatment mediated more

inhibition of cell-migration in colon cancer. After treated with 5-FU

(8 lM) and mahanine (12 lM) alone or in combination for 24 h,

p53wt cells (5 9 104 cells/well/750 ll) were seeded in the upper

chamber of 24-transwell plate in presence of epidermal growth factor

(EGF, 20 ng/ml) and incubated for 6 h. Then medium was aspirated

and non-migrated cells from the upper portion of the membrane were

also removed. Subsequently, migrated cells passed through the

membrane were fixed with chilled methanol and stained with 0.1 %

crystal violet. Random fields were photographed and quantified under

phase contrast microscopy. The black arrows indicated the migrated

stained cells passed through the membrane pore. The no. of stained

cells was reduced in combination treatment signifying more inhibition

of chemo-migration

Apoptosis (2014) 19:149–164 161

123

irrespective of p53 status. Moreover, this combination

exerted their apoptotic effect by oxidative stress-mediated

activation of two tumour suppressors, PTEN and p53/p73.

This data strongly established the ability of mahanine to

potentiate 5-FU effects through the activation of tumour

suppressors.

In relation to the molecular mechanism, tumour sup-

pressor network is the one of the major determinant of

responsiveness of cancer cells to chemotherapeutic agents.

Mutation or deletion of genes is associated with the

advanced progression of disease as well as tolerance to

anti-cancer drugs [43]. p53 and PTEN are two proteins

which play the primary role in tumour suppression [4, 6].

Mahanine-induced upregulation of Fas and FasL might be

mediated by activation of p53 [18].

Here, we demonstrated mahanine-mediated activation of

p53 as well as PTEN. A significant elevation of p53 level

in p53wt cell and increased phosphorylation at Ser15 of p53

supported the evidence of activation of p53 after mahanine

treatment. In p53null cells mahanine-mediated activation of

p73 suggested that when p53 is absent, p73, a p53 homo-

logue plays an important role in chemo-sensitivity of

cancer cells. Thus p73 might also be a therapeutic target in

chemo-resistant tumours with inactive p53. Phosphoryla-

tion at Tyr 99 of p73 led to the stabilization and activation

of p73 and elicited p73-induced apoptosis in response to

chemotherapy.

The tumour suppressor functions of p53 and p73 is neg-

atively regulated by the MDM2 oncoprotein [31, 32]. From

our study, it was found that mahanine suppressed the phos-

phorylation of Ser 166 on MDM2 suggesting cytoplasmic

retention and destabilization of MDM2 and thereby acti-

vating p53/p73. It was reported that RASSF1A, a tumour

suppressor could enhance self-ubiquitination of MDM2 by

dissociating MDM2–DAXX–HAUSP complex and thus

inhibit degradation of p53 [44]. Activation of RASSF1A

might also support mahanine-mediated destabilization of

MDM2 and activation of p53 here. It was also reported that

mahanine could activate epigenetically silenced RASSF1A

in array of different cancer cells [20]. A similar profile of

elevated PTEN protein levels was observed which was

associated with its nuclear accumulation in response to ma-

hanine established the functional activity of PTEN as a

tumour suppressor. However, association between maha-

nine-induced activation of RASSF1A and PTEN in colon

cancer is yet to be defined.

It is reported that ROS can modulate the activity and

intracellular relocation of several redox-sensitive compo-

nents such as transcription factors, protein kinases, phos-

phatases and nuclear transport factors. Among the

phosphatases, PTEN, accumulated into the nucleus by

oxidative stress [45]. Hyperphosphorylation of PTEN at

Ser 380 by H2O2 treatment resulted in the inhibition of its

nuclear export [29]. Here, it was demonstrated that in

mahanine-treated cells, PTEN phosphorylation was ele-

vated and PTEN was accumulated in nucleus. Scavenging

of ROS resulted in the reversal of nuclear localization

confirming that this nuclear relocation of PTEN was

mediated by ROS. Mahanine-induced ROS also elevated

p53/p73 transcription factor and promoted their nuclear

translocation.

Mahanine treatment activated both PTEN and p53/p73

tumour suppressors. Transient knockdown of PTEN

decreased p53 and p73 levels in mahanine-treated p53wt

and p53null cells respectively demonstrating the depen-

dency of p53/p73 activation on the presence of PTEN.

Furthermore, it is reported that PTEN regulates p53 protein

stability, both in a phosphatase-dependent manner by

impeding Akt-MDM2 pathway and in a phosphatase-

independent manner through protein–protein interaction in

the nucleus [46]. Though MDM2 was deactivated in both

p53wt and p53null cells, we did not get any deactivation of

Akt after mahanine treatment (data not shown). So, there

may be a possibility of phosphatase-independent pathway

of p53 activation. Probably, same phenomenon also occurs

in case of p73 and PTEN in the absence of p53. Further-

more, co-immunoprecipitation study, after mahanine

treatment, revealed enhanced PTEN interaction with p53

and p73 in endogenous nuclear fractions of p53wt and

p53null cells respectively. It is already reported that inter-

action of PTEN with p53 or p73 enhances their DNA

binding activity [46, 47].The increased protein levels of

p21 and Bax (S4), which are direct transcriptional target of

p53 family proteins, strengthen this fact also.

Some anticancer drugs, such as cisplatin, paclitaxel,

5-FU, bleyomycin etc. are known to up regulate ROS gen-

eration to exert their anticancer activities [48]. 5-FU is

reported to generate mitochondrial ROS in the p53-depen-

dent pathway [49] and induction of Romo1 [48]. Here, we

explored the redox behaviour of mahanine to act as a

potential factor for the enhancement of oxidative stress

induced by 5-FU. Our results demonstrated that combination

of 5-FU and mahanine increased ROS production of *2-

folds in both p53wt and p53null colon cancer cells in com-

parison to 5-FU alone. Although, the mechanism by which

mahanine and 5-FU combination generated ROS yet to be

established. Combined treatment also enhanced the expres-

sion of PTEN and p53/p73 proteins, the two key molecules

involved in the mahanine-mediated cytotoxic effect. Fur-

thermore, absence of PTEN led to a reduction in the per-

centage of apoptotic cells in mahanine and combination-

treated p53wt and p53null cells. This established the possible

involvement of PTEN in apoptosis of colon cancer cells

induced by mahanine and combination treatment. From our

findings we can hypothesise that the oxidative stress-medi-

ated activation of tumour suppressor proteins PTEN and p53/

162 Apoptosis (2014) 19:149–164

123

p73 might contribute to the increase cytotoxic activities of

both p53wt and p53null cells after combined therapy. More-

over, functional assay established that mahanine in combi-

nation with 5-FU reduces more in vitro cell migration in

comparison to either single agent alone in colon cancer cells.

Mahanine-mediated activation of p53/p73 might play role in

this regulation of cell migration as p53/p73 is reported to

inhibit migration and invasion through trans-repression of

EpCAM [36]. Furthermore, mahanine-induced inhibition of

invasion and metastasis might involve the down regulation

of MMP9 [50]. Inhibition of the expression of MMP9-

mediated by p53 [19] suggesting further the role of p53 in the

inhibition of cell migration.

In summary, we have demonstrated that herbal com-

pound, mahanine synergistically enhances the cytotoxic

activity of 5-FU in colon cancer by reducing its concen-

trations. The enhancement of the effect of 5-FU was

associated with mahanine-mediated activation of two

tumour suppressors i.e. PTEN and p53/p73. Therefore, our

study highlights that carbazole alkaloid, mahanine can be

used potentially in combination therapy with 5-FU for the

treatment of colon carcinoma.

Acknowledgments This work received financial support from CSIR-

IICB, CSIR under IAP-0001, HCP004, NMITLI, TLP-004 and DBT

under GAP 235, ICMR, Govt. of India. Dr. Chitra Mandal is grateful to

financial support by J.C. Bose Fellowship, DST of Govt. of India and

also mutual grant from ICMR and German Cancer Research Centre.

Conflict of interests None.

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