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: [email protected]; [email protected]
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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