Debasmita Chatterjee, Banhishikha Singh, Krishnendu Paira, Satadal Das*
Abstract
Background: Acute Myeloid Leukaemia (AML) is considered to be an extremely heterogeneous malignancy of
bone marrow and blood. The first line of therapy for AML is prolonged chemotherapy. Due to the presence of molecular
heterogeneity in AML as confirmed by next-generation sequencing, researchers are planning to develop newer strategies
of therapy. Objective: In the present study we have explored the anti-cancer potentiality of the hydro-ethanolic extract
(50% and 70%) of the whole flower of Nymphaea caerulea against the Acute Myeloid Leukaemia cell line, THP-1
with control of normal human kidney epithelial cell line (HEK 293). The present study is a novel contribution to the
existing scientific knowledge as at present no study as an anti-leukaemic agent is available on N. caerulea (blue lotus)
extract and exploring its action mechanism on in-vitro cell line model. Methods: Some targeted cytokine and apoptotic
genes genes to deduce the anti-cancer mechanism of action of the crude extract (hydro-ethanolic extract (50% and
70%) of the whole flower) were selected as Interferon (IFN) γ, Interleukins – IL-6, IL-8, IL- 10, IL-1β, Transforming
Growth Factor (TGF β1), Tumor Necrosis Factor (TNF α), Caspase 3(CAS 3), Caspase 9 (CAS 9), CD95 (Fas), Tumor
Necrosis Factor Receptor 1 (TNFRSF1A) to observe relative fold changes of the expression using Real-Time PCR
with housekeeping gene β-actin. Cellular cytopathic effect (CPE), cell viability assay by methylene blue assay, and cell
cytotoxicity of the crude extract against the THP-1 cell line were also studied along with it’s bio-active compositional
analysis of the extract was explored using ultra-performance liquid chromatography followed by mass spectra. Results:
The N. caerulea flower extract is capable of inducing apoptosis in AML and it can balance cytokine alterations in such
diseases. Conclusions: Nymphaea caerulea flower extract appears to be a good anti-leukemia agent.
Keywords: Nymphaea caerulea- Acute Myeloid Leukaemia- THP-1 cell line- LC-MS analysis- anti-cancer activity. Asian Pac J Cancer Prev, 25 (1), 123-13
Introduction
The latest data sheet from WHO, 2022 [1] indicated 10 million cancer deaths in 2020, affecting one out of six people. WHO observes that the primary reasons for the rising cases of death due to cancer are worse prognosis and limited access to diagnosis and therapeutic measures at the proper time [1]. The most common cases of cancer are of
breast, colon and rectum, and lung, whereas breast cancer and leukemia are considered to be the most neglected forms of cancer [1]. As of now, there are no curative measures to treat leukemia and therefore, researchers all over the world are trying to develop innovative therapeutic measures and technologies for the targeted or precision therapy
of leukemia [2, 3]. Many studies are being done on immunotherapies to curb the progression of these deadly diseases from the initial stages in some sub-types of
leukemia [4]. Among the varied treatment procedures, alternative or complementary medicines are also considered to be effective measures where conventional therapy fails [4]. Past literature survey has shown that hypertension is a potent risk factor for the disease of erectile dysfunction. The study demonstrated that Nymphaea lotus Linn. can
alleviate the sexual dysfunction in the hypertensive male rat [5]. Nymphaea caerulea is a perennial aquatic plant that grows on the shore of lakes and is commonly
named as blue lotus, Egyptian lotus and blue water lily. According to traditional medicine literatures, the blue lotus have several healing or medicinal properties such
as a tranquilizer, a significant detoxicant, aphrodisiac, act as traditional or complementary medicine for dyspepsia, diarrhea, urinary passage associated problems, palpitations of heart, enteritis, and fever [6]. According to the past historic literatures, when the Napolean expedited Nile River, they found three types of water lilies namely
Nymphaea caerulea, Nymphaea lotos (white in colour), and Nelumbo nucifera (pink in colour). In the early literatures, Nymphaea caerulea was named in varied names such as N. stellata, N. scutifolia [7]. It is evident that the geographical difference leads to the development of heterogeneous cytogenetic nature among the different hematological neoplasms. A study which explored about the cytogenetic profile of the adult
patients suffering from Acute Myeloid Leukaemia (AML) in Egypt reported that the median age of the patients was 36.5 years, and among them 53.3% were males
whereas 46.7% were females and the median WBC count was 42.3 × 109 /L [8]. Leukaemia is the most common hematological neoplasm and nearly 50% of them are
AML cases in Egypt. The major risk factors identified by scientists in a study are agricultural chemicals and electromagnetic fields and therefore in the past 10 years
the cases of hematological neoplasms are constantly rising [9]. Findings on Leukaemia reported by National Institute of Health of the Republic of Armenia within the
period of 2012 - 2018 indicated that there were 259 new cases of ALL and 478 AML [10]. However, the morbidity indicators obtained from the registered incidence of AML
was found to be few in numbers in population based study in Kazakhstan [11]. As mentioned earlier, that the sacred blue lotus N. caerulea is found in several numbers along the banks of River Nile. The flower comprises several important
phytochemicals namely Liensinine, Iso- Liensinine, Neferine, Lotusine, Pronuciferine, Rutin, Hyperin etc [12]. Many medicinal properties of blue lotus which are
reported are antioxidant activity, antisteroids, anti-pyretic, anti-viral, anti-inflammatory, for treatment against erectile dysfunctions, against diabetic complications, as an
anti-ageing, showing hepato-protective activity, etc [12]. Blue lotus exhibited cytotoxic and apoptotic activities on triple-negative breast cancer cells via ROS generation and
p38 MAPK/JNK activation pathway [13]. N. nucifera also showed anti-cancer activity against lung cancer cell line (NSCLC) and also lowered the ratio of B-cell lymphoma
2/Bcl-2-associated X protein (Bcl-2/Bax) pointing towards the pro-apoptosis nature of nuciferin [14]. In the present study, we have explored the anti-cancer activity for the first time of the ethanolic extracts of Nymphaea caerulea flower against the cancer cell line
THP-1 monocytes and normal HEK-293 cell line. The bioactive composition analysis was also carried out with the aid of ultra-performance liquid chromatography
followed by mass spectrometry.
Materials and Methods
Plant Sample collection
50 g of dried petals of the blue lotus was purchased
from authenticated herbal product production company
(approved by ISO 9000-2015; 100% organic herbal; GMP
certified and HACCP certified), Green Earth Products Pvt.
Ltd. India (approved by fssai 23318008001334), collected
and packed in August 2022 (Figure 1).
Cell lines
The cell line, THP -1 was procured from National
Centre for Cell Science (NCCS), Pune, India. The cells
were transported within sterile RPMI 1640 media with
10% fetal bovine serum concentration. The human
embryonic kidney cell line (HEK 293) was also procured from NCCS, Pune, and it was transported in sterile DMEM
media with 10% fetal bovine serum concentration. The
cell lines were authenticated using sixteen short tandem
repeat (STR) loci which was amplified using commercially
available AmpFISTR Identifier Plus PCR Amplification
kit from Applied Bio-systems. The cell line sample was
processed using the Applied Bio-system® 3500 genetic
analyser. The data obtained was analysed using Gene
Mapper® ID –X v1.5 software (Applied Bio-systems).
For confirmation, appropriate positive and negative
controls were used. Mycoplasma testing was done using
Mycoplasma PCR method/ Hoechst staining and it was
confirmed not detected.
Chemicals
Rose well Park Memorial Institute 1640 (RPMI) 1X
(Ref: 11875 -093; Lot: 2436312) and Dulbecco’s Modified
Eagle Medium (DMEM) (1X) along with Glutamax
were purchased from Gibco, UK (Ref: 10567-014; Lot
No: 2522563) for cell culture purposes of THP-1 and
HEK 293 respectively. F12 (1X) nutrient mixture Ham
+ L-glutamine supplement (Product code: AL025A; Lot
No. 0000512083) for the growth of the cell line, was also
purchased from Himedia Pvt. Ltd., India. It was required
for the culture and maintenance of the HEK 293 cell
line. Fetal Bovine Serum (FBS) (Product code: RM1112-
500ML, Himedia Pvt. Ltd., India), antibiotic–antimycotic
solution (Penicillin/Streptomycin/ Amphotericin B)
(A002A – 100ML, Lot No. 0000506599, Himedia Pvt.
Ltd., India) and Phosphate Buffer saline (PBS, 1X) (Ref:
10010-031; Lot no. 2276680; Gibco, UK) were also
procured. The MTT assay EZ count kit was bought from
Hi Media, India (Cat No: CCK003). Molecular biology
reagents and RNA iso plus were purchased from Takara
(TAKARA, USA; Cat. No. 9108; Lot No. ALZ1011N),
and the reverse transcriptase cDNA synthesis kit (Bio-Rad,
USA, Cat. No. 1708841; Batch No. 64449565) and the
iTaq SYBR green supermix RT-PCR reagents (Cat. No.
1725121; Batch No. 64464415) were purchased from
Bio-Rad, USA.
Plant extraction of bioactive constituents
10 g of dried blue lotus petals was weighed and cut
down into small pieces and extracted using 100 mL of 50% and 70% ethanol (molecular biology grade purity) for 72
hours in dark conditions. Following extraction, the crude
extract was filtered through Whatman filter paper no. 1
and then sterilized using a 0.22 micron PES membrane
filter (Merck, Millipore). The extract was then lyophilized
and the crude powder was stored at 4ºC in a refrigerator
for further biological assays [15].
To calculate the yield analysis of the extract, the dried
content of the extract was weighed and then calculated
using the formula:
W2 – W1 / 100mL where, W2 is the weight of the
extract along with the container, W1 is the weight of the
container alone and W0 is the weight of the initial dried
plant sample.
Culturing of Cell Lines
THP-1 cell line, peripheral blood monocyte isolated
from acute monocytic leukaemia patient, a non-adherent
cell line with passage number 28 was seeded in a T25cm2
cell culture flask with filtered sterile RPMI 1640 media
supplemented with 10% FBS and antibiotic–antimycotic
solution. The cells were allowed to become confluent at
37ºC, 5% carbon dioxide, and humidified atmosphere for
the next 48 hours. When the cells reached a confluence
of more than 80%, the cells were re-suspended with
fresh media in a 12-well culture plate for experimental
purposes. The plate was incubated at 37ºC, with 5%
carbon dioxide in the humidified atmosphere for the next
24 hours [16].
The human embryonic kidney cell line (HEK 293) is
an adherent cell line of passage number 27 that was seeded
in a T25cm2
cell culture flask with filtered sterile DMEM
+ F12 along with 10% FBS and antibiotic–antimycotic
solution and incubated at 37ºC, 5% carbon-dioxide in the
humidified atmosphere for the next 48 – 72 hours. HEK
293 cells take time to adhere to the base but grow rapidly
once it gets adhered to the base. When the cells reached
80% confluence, it was trypsinized and re-suspended in
a 12-well culture plate and allowed to grow to maintain
the above-mentioned culture conditions. The cells were
allowed to grow until it reaches a confluence of 106
to
108
[17].
Inoculation of Extracts and Vehicle Control
Following incubation, the experimental sets were
prepared for both the THP-1 and HEK 293 cell lines to
study the anti-cancer activities as well as to observe any
deleterious effect in normal cells (control) - cells were kept
intact and allowed to grow freely for the next 24 hours.
There were also vehicle control sets in which cells were
inoculated with 50µL of 50% and 70% ethanol for the next
24 hours; following that cells were inoculated with 50%
and 70% ethanolic extract of blue lotus. Then the plates
were rotated clockwise and anti-clockwise for proper
mixing, and then kept in a carbon-dioxide incubator at 5%
CO2
level, 37ºC temperature in a humidified environment
[18].
Cytopathic effect Study
After 24 hours of incubation, the cell morphology
was observed under an inverted microscope under 40 X magnification and the photographs were recorded for the
cell size and shape in all the experimental sets with both
cancer and normal cell line [18].
Cell viability Assay
A viable Cell counting assay was done following
the modified methylene blue assay. The modified
stain comprises 1X PBS solution, glutaraldehyde, and
methylene blue stain. After discarding the media, 300 µL
of stain was added to each well and incubated for 1 hour
at 37ºC. Following incubation, the stain was discarded and
the cells were washed with PBS (1X) and observed under
the inverted microscope in 40 X magnification and the
photographs were recorded of the viable and non–viable
cells. The viable cells appeared blue while the non-viable
cells appeared white under the inverted microscope [18].
Gene Expression Assay
The cells after staining of different experimental
sets of both the cell lines were washed with PBS (1X)
and harvested with RNA iso plus following the protocol
of the manufacturer. The RNA was dissolved in 60µL
of nuclease-free water and the yield was quantified
using A260/280 ratio of UV-Vis spectrophotometer.
cDNA was synthesized using the reverse transcriptase
kit following the instructions with thermal cycler T100
(BIO-RAD, USA). The cytokines expression was analyzed
Interferon (IFN) γ, Interleukins – IL-6, IL-8, IL- 10,
IL-1β, Transforming Growth Factor (TGF β1), Tumor
Necrosis Factor (TNF α), Caspase 3(CAS 3), Caspase 9
(CAS 9), CD95 (Fas), Tumor Necrosis Factor Receptor
1 (TNFRSF1A) gene as a relative fold change of the
expression of housekeeping gene β-actin using Real-Time
PCR (BIO-RAD, USA) [18].
Cytotoxicity Assay
The cytotoxicity of the extracts was assayed using an
MTT assay kit in a 96-well microtiter plate. The assay
consisted of the following controls namely medium
control (without THP-1 cell line), cell control (THP-1 cell
line + media), and the vehicle control containing the 50%
and 70% ethanol-based extracts. The inoculum volumes of
the extracts were 2, 4, 6, 8, 10, 12, and 15 µL of the extracts
in triplicates keeping the final volume of the reaction per
well to be 100µL. After completion of dilution, the plate
was kept in the carbon- dioxide incubator (5%) at 37ºC
for the next 24 hrs. 10µL of MTT solution was added
to each well, mixed thoroughly, and kept at incubation
for the next 4 hours. After completion of incubation,
the plate was observed under the inverted microscope
to observe formazan crystal formation. Thereafter, 100
µL of solubilization solution was added to each of the
wells and kept at overnight incubation to maintain the
above-mentioned condition. The absorbance of the 96
well plates was recorded using an ELISA plate reader
(Roboniks, India) and thereafter the data was graphically
plotted [18].
Characterization of bioactive compounds by LCESIQTOF-MS/MS
The bioactive constituents of the extract were analyzed by reverse-phase ultra-performance liquid
chromatography and electrospray ionization mass
spectrometry. The instrument that was used for this
analysis was the Waters Acquity UPLC system and Waters
Acquity SQD mass spectrometer (Single quadrupole
mass spectrometer) system (Waters TM, USA). The
electrospray ionization source of the MS system operated
in the positive ion mode. Based on the experimental m/z
value of the analyte obtained, it was identified with the
m/z value of the standard compounds mentioned in the
previous literature [19].
Statistical Analysis
The T-test and P-value with 95% of Confidence
Interval of each parameter of cytokines were studied and
given in Table 1 for the experimental sets, both row-wise
and column wise interaction were analyzed. The software
used for the analysis was MedCalc, easy to use statistical
software, version 22.
Results
Yield Content analysis
The crude ethanolic extract of different percentages, i.e., 50% and 70% depicted marginal variation in their
yield content of the bioactive compounds (g/dL). It is
found to be high with the 50% ethanolic solvent (2.93
g/dL) in comparison to 70% ethanolic solvent (2.59 g/dL)
(Figure 2).
Cytopathic Study
The control cells of THP-1 (24 h) showed that the
morphology of the cells is round, large, and singlecell. Cells are in the active proliferation stage with
increased numbers. In the 50% and 70% N. caerulea
extract treated sets cells are mostly dead with altered
shape, size, and numbers along with extensive cell
debris. In the vehicle control sets, the cells treated with
50% alcohol are comparatively large and round in size
and shape with lesser cellular debris when compared
with the 70% ethanol-treated cells. The HEK 293 cell
line control (24 h) set depicted proper epithelial celllike morphology with good margins and a negligible
amount of dead cells and debris. With the treatment of
50% N. caerulea extract, the cells are alive with little
round-shaped epithelial morphology but with very
few dead cells or cellular debris. Whereas in the 70%
N. caerulea extract treated sets cells the cells are mostly the 70% ethanol control set the number of dead cells is
comparatively higher (Figures 3 – 6).
Cell viability Study
The control set THP-1 cells have all taken up the modified methylene blue stain and are showing round
large single cellular morphology. Therefore, all the cells
visible in the field are viable. However, in the 50% and
70% N. caerulea extract treated sets, all the cells are
de-shaped with no up taking of stain indicating that all cells are dead. In the vehicle control sets (both 50% and
70% ethanol) the cells are mostly dead with no stain.
In the HEK 293 cell line, the control set cells are all
stained blue (live) with accurate morphology. Whereas
in the 50% N. caerulea extract-treated set, the cellular
morphology has little changes and is all stained but in
the 70% extract-treated set the morphology of cells has
become more rounded and clustered though they are alive.
In the vehicle control sets (50% and 70% ethanol), cells
are marginally round in shape with few clustered together
but are mostly live (stained blue) (Figures 7 – 10).
Moreover before initiating the experiment the total
yield of bioactive compositions was measured and it is
found to be high with the 50% ethanolic solvent (2.93
g/dL) in comparison to 70% ethanolic solvent (2.59 g/dL)
(Figure 2).
The ultra-performance liquid chromatography
followed by mass spectra analysis of the crude extract
revealed the presence of several bioactive constituents
like Paeoniflorin, Hesperidin, Homogentisic acid, 3 –p
coumaroyl – quinic acid, Syringic acid, etc (Table 2) [19, 20].
Cytokine expression analysis
The cytokine gene expression of the targeted genes
namely Interferon (IFN) γ, Interleukins – IL-6, IL8, IL- 10, IL-1β, Transforming Growth Factor (TGF
β1), Tumor Necrosis Factor (TNF α), Caspase 3(CAS
3), Caspase 9 (CAS 9), CD95 (Fas), Tumor Necrosis
Factor Receptor 1 (TNFRSF1A) gene was expressed as
bar diagrams as a relative fold change of the expression
of housekeeping gene β-actin of the five different
experimental sets (Figure 11 – 12). The MTT cytotoxicity
assay showed that the effective dosage (volume) of the
crude extract upon the THP-1 cell line is approximately
40µL for both extracts (50% and 70%) (Table 1).
Thus in brief, after yield content analysis of the extract
we observed 2.93 g/dl and 2.59 g/dl yield of bioactive
chemicals in 50% and 70% ethanol extracts of the flower
respectively. When applied to the THP-1 leukemia cell line
there were significant dead, particularly with 50% extract.
Although some changes were present with 50% and 70% Debasmita Chatterjee et al
130 Asian Pacific Journal of Cancer Prevention, Vol 25
Cytokine gene expressions (fold changes, Mean ± Standard
Deviation) in different experimental sets
P value (between
different sets) with
95% Confidence
Interval (THP-1)
P value (between
different rows) with
95% Confidence
Interval (THP-1)
P value (between different
columns) with 95%
Confidence Interval (THP-1)
Sets HEK 293 (IFN γ) THP -1 (IFN γ) HEK 293 (IFN γ) THP -1 (IFN γ) IFN γ (HEK 293 vs THP-1)
Control 1.00 ± 0.05 1.00 ± 0.05 1 1 1
N. caerulea 50 % 0.44 ± 0.02 0.87 ± 0.04 0.0001 0.0245 0.0001
N. caerulea 70 % 0.02 ± 0.00 0.62 ± 0.03 < 0.0001 0.0004 < 0.0001
ALC 70 % 0.01 ± 0.00 0.27 ± 0.01 < 0.0001 < 0.0001 < 0.0001
ALC 50 % 0.02 ± 0.00 2.40 ± 0.12 < 0.0001 < 0.0001 < 0.0001
Sets HEK 293 (IL 6) THP -1 (IL 6) HEK 293 (IL 6) THP -1 (IL 6) IL-6 (HEK 293 vs THP-1)
Control 1.00 ± 0.05 1.00 ± 0.05 1 1 1
N. caerulea 50 % 0.69 ± 0.03 1.38 ± 0.07 0.0008 0.0016 0.0001
N. caerulea 70 % 0.15 ± 0.01 120.28 ± 6.01 < 0.0001 < 0.0001 < 0.0001
ALC 70 % 0.06 ± 0.00 76.02 ± 3.80 < 0.0001 < 0.0001 < 0.0001
ALC 50 % 0.17 ± 0.01 63.70 ± 3.19 < 0.0001 < 0.0001 < 0.0001
Sets HEK 293 (IL 8) THP -1 (IL 8) HEK 293 (IL 8) THP -1 (IL 8) IL-8 (HEK 293 vs THP-1)
Control 1.00 ± 0.05 1.00 ± 0.05 1 1 1
N. caerulea 50 % 4.66 ± 0.23 0.30 ± 0.01 < 0.0001 < 0.0001 < 0.0001
N. caerulea 70 % 6.07 ± 0.30 18.54 ± 0.93 < 0.0001 < 0.0001 < 0.0001
ALC 70 % 2.57 ± 0.13 0.92 ± 0.05 < 0.0001 0.1216 < 0.0001
ALC 50 % 3.03 ± 0.15 0.26 ± 0.01 < 0.0001 < 0.0001 < 0.0001
HEK 293 Sets HEK 293 (IL 10) THP -1 (IL 10) HEK 293 (IL 10) THP -1 (IL 10) IL-10 (HEK 293 vs THP-1)
Control 1.00 ± 0.05 1.00 ± 0.05 1 1 1
N. caerulea 50 % 0.18 ± 0.01 3.60 ± 0.18 < 0.0001 < 0.0001 < 0.0001
N. caerulea 70 % 1.52 ± 0.08 0.52 ± 0.03 0.0007 0.0001 < 0.0001
ALC 70 % 1.03 ± 0.05 2.63 ± 0.13 < 0.0001 < 0.0001 < 0.0001
ALC 50 % 1.87 ± 0.09 2.91 ± 0.15 < 0.0001 < 0.0001 0.0005
Sets HEK 293 (IL 1B) THP -1 (IL 1B) HEK 293 (IL 1B) THP -1 (IL 1B) IL-1β (HEK 293 vs THP-1)
Control 1.00 ± 0.05 1.00 ± 0.05 1 1 1
N. caerulea 50 % 0.06 ± 0.00 0.43 ± 0.02 < 0.0001 0.0001 < 0.0001
N. caerulea 70 % 0.78 ± 0.04 17.03 ± 0.85 0.004 0.0001 < 0.0001
ALC 70 % 0.30 ± 0.01 7.60 ± 0.38 < 0.0001 < 0.0001 < 0.0001
ALC 50 % 0.63 ± 0.03 0.20 ± 0.01 0.0004 < 0.0001 < 0.0001
Sets HEK 293 (TGF B1) THP -1 (TGF B1) HEK 293 (TGF B1) THP -1 (TGF B1) TGF B1 (HEK 293 vs THP-1)
Control 1.00 ± 0.05 1.00 ± 0.05 1 1 1
N. caerulea 50 % 9.54 ± 0.48 13.45 ± 0.67 < 0.0001 < 0.0001 0.0012
N. caerulea 70 % 2.22 ± 0.11 0.30 ± 0.02 0.0001 < 0.0001 < 0.0001
ALC 70 % 0.21 ± 0.01 7.24 ± 0.36 < 0.0001 < 0.0001 < 0.0001
ALC 50 % 0.68 ± 0.03 2.62 ± 0.13 0.0007 < 0.0001 < 0.0001
Sets HEK 293 (TNF-α) THP -1 (TNF-α) HEK 293 (TNF-α) THP -1 (TNF-α) TNF-α (HEK 293 vs THP-1)
Control 1.00 ± 0.05 1.00 ± 0.05 1 1 1
N. caerulea 50 % 0.01 ± 0.00 0.30 ± 0.01 < 0.0001 < 0.0001 < 0.0001
N. caerulea 70 % 0.02 ± 0.00 0.81 ± 0.04 < 0.0001 0.0068 < 0.0001
ALC 70 % 2.17 ± 0.11 4.20 ± 0.21 0.0001 < 0.0001 0.0001
ALC 50 % 0.01 ± 0.00 3.48 ± 0.17 < 0.0001 < 0.0001 < 0.0001
SETS HEK 293 (CAS 3) THP 1 (CAS 3) HEK 293 (CAS 3) THP 1 (CAS 3) CAS 3 (HEK 293 vs THP-1)
Control 1.00 ± 0.05 1.00 ± 0.05 1 1 1
N. caerulea 50 % 2.53 ± 0.13 173.59 ± 8.68 < 0.0001 < 0.0001 < 0.0001
N. caerulea 70 % 0.47 ± 0.02 5.65 ± 0.28 0.0001 < 0.0001 < 0.0001
ALC 70 % 0.85 ± 0.04 1.33 ± 0.07 0.0154 0.0027 < 0.0001
ALC 50 % 0.82 ± 0.04 0.42 ± 0.02 0.0082 < 0.0001 < 0.0001
Table 1. Changes in Cytokine Gene Expressions Alongwith Statistical Analysis
Asian Pacific Journal of Cancer Prevention, Vol 25 131
DOI:10.31557/APJCP.2024.25.1.123
The Apoptotic Property of Blue Lotus Extract on Leukaemia Cells
Cytokine gene expressions (fold changes, Mean ± Standard
Deviation) in different experimental sets
P value (between
different sets) with 95%
Confidence Interval
(THP-1)
P value (between
different rows) with
95% Confidence
Interval (THP-1)
P value (between different
columns) with 95%
Confidence Interval (THP-1)
Sets HEK 293 (CAS 9) THP 1 (CAS 9) HEK 293 (CAS 9) THP 1 (CAS 9) CAS 9 (HEK 293 vs THP-1)
Control 1.01 ± 0.05 1.00 ± 0.05 1 1 1
N. caerulea 50 % 1.41 ± 0.07 7.67 ± 0.38 0.0012 < 0.0001 < 0.0001
N. caerulea 70 % 0.83 ± 0.04 2.85 ± 0.14 0.01 < 0.0001 < 0.0001
ALC 70 % 0.46 ± 0.02 5.44 ± 0.27 0.0001 < 0.0001 < 0.0001
ALC 50 % 0.97 ± 0.05 8.85 ± 0.44 0.5032 < 0.0001 < 0.0001
Sets HEK 293 - CD 95 (FAS) THP 1 - CD 95 (FAS) HEK 293 - CD 95 (FAS) THP 1 - CD 95 (FAS) CD 95 (HEK 293 vs THP-1)
Control 1.00 ± 0.05 1.00 ± 0.05 1 1 1
N. caerulea 50 % 0.31 ± 0.02 83.20 ± 4.16 < 0.0001 < 0.0001 < 0.0001
N. caerulea 70 % 0.10 ± 0.01 0.08 ± 0.00 < 0.0001 < 0.0001 < 0.0001
ALC 70 % 0.10 ± 0.01 0.10 ± 0.00 < 0.0001 < 0.0001 < 0.0001
ALC 50 % 0.14 ± 0.01 0.03 ± 0.00 < 0.0001 < 0.0001 < 0.0001
Sets HEK 293 (TNFRA1) THP 1 (TNFRA1) HEK 293 (TNFRA1) THP 1 (TNFRA1) TNFRA1 (HEK 293 vs THP-1)
Control 1.00 ± 0.05 1.00 ± 0.05 1 1 1
N. caerulea 50 % 0.94 ± 0.05 21.91 ± 1.10 0.2156 < 0.0001 < 0.0001
N. caerulea 70 % 0.18 ± 0.01 0.59 ± 0.03 0.0003 < 0.0001 < 0.0001
ALC 70 % 0.55 ± 0.03 6.17 ± 0.31 0.0002 < 0.0001 < 0.0001
ALC 50 % 0.32 ± 0.02 1.57 ± 0.08 < 0.0001 < 0.0001 < 0.0001
Table 1. Continued
SL. NO. Experimental m/z value Probable Compounds Identified
1 169.87 Gallic acid
2 168.9 Homogentisic acid
3 166.89 Vanillic acid
4 186.94 Brevifolin
5 198.1 Syringic acid
6 305.05 Gallocatechin
7 339 3 –p coumaroyl – quinic acid
8 340.1 Caffeoyl glucose/ Esculin
9 359.06 Ellagic acid rhamnosyl
10 447.11 Quercetin 3–O-acetyl hexoside
11 448.18 delphinidin 3-O-rhamnosyl-5-Ogalactoside
12 455.09 delphinidin 39-O-(20-O-galloyl-60-Oacetyl-b-galactopyranoside)
13 480.16 Paeoniflorin
14 483 HHDPe
-hexoside
15 610.18 Hesperidin
Table 2. Phytochemical Profiling of the Crude 50% Ethanolic Extract
[21, 22]. It is reported that most AML patients go through
a hard-line clinical course and 28% of them reported
having an overall survival rate of 5 years. There are
varied factors that influence the survival rate of individual
patients such as associated genetic mutations, factors
of individuals that comprises gender, age, exposure to
radiation, and initial stage of chemotherapy [23-25]. It
is also reported previously by researchers that signaling
pathways of cytokines lead to tumor development of
AML and its progression, and incongruence, resulting
in chemo-resistance, and escaping the immune system.
Therefore, we in this study tried to develop some targeted
therapy towards cytokine signaling that might eradicate
the LSC populace along with prevention of relapse of
chemotherapy [23-25].
It has been observed the cytokine IFN γ (a
pro-inflammatory cytokine) causes a reduction in AML
cell production and survival and also increases unplanned
clonogenicity of AML cells [26-28] Binder et al. 2018;.
In the case of AML patients, the gene expression of the
cytokine remains unchanged in peripheral blood (PB)
levels and also reduces levels in the bone marrow (BM)
[29, 27, 28]. In our study, we observed that there is a mild
decrease in IFN γ, both in the N. caerulea extracts (50%
and 70%) treated cells concerning control, however, the
level got mildly enhanced after challenging the THP-1 cell
line with 50% ethanol. When compared with the normal
epithelial HEK 293 cell line, there was a reduction of the alcohol (vehicle), the changes were not so pronounced
and the cells were viable (Figure 3). However, when 50%
and 70% extracts were separately applied on HEK 293
normal cell line there were insignificant changes with
50% and 70% extract. IFN γ gene expression changes
were not significant with the extracts as well as with the
vehicle alcohols. However, IL-6, IL-8, IL-1β, TGF-β1,
and TNF-α gene expressions were much less with 50%
extract although they were markedly increased with 70%
extract. On the contrary, IL-10 gene expression was
significantly increased with 50% extract. Thus 50% extract
appears to be capable of controlling an imbalance between
pro-inflammatory and anti-inflammatory cytokines which
is commonly found in most infectious diseases and many
non-infectious diseases also.
When we studied some markers of the apoptotic
pathway, we found the Cas3, Cas9, CD95, and TNFRSF1A
gene expressions were more with 50% extract, which also
indicates that significant apoptotic action on the leukemia
cells is possible with this extract (Table 1).
Statistical Analysis
All the data of gene expression was found to be
statistically significant with P- value <0.005 (Given in Table 1). Discussion
Acute Myeloid Leukaemia (AML) is considered to
be an extremely aggressive hematological malignancy
that is associated with the gathering of cytogenetic
and molecular mutations within progenitor and or
hematopoietic stem cells (HSPCs) [21]. This condition
leads to the development of leukemic stem cells (LSCs) gene expression level of IFN γ among all five sets.
According to the reports of existing literature, another
pro-inflammatory cytokine, IL-6 partially promotes AML
cell proliferation and the level is also found to be elevated
within the plasma of patients [30, 31]. Our study data
revealed that there are hardly any elevations of the level of
IL-6 in the 50% extract treated THP-1 cells whereas to the
contrary among all the other sets the gene expression level
of IL-6 is found to be profoundly high when compared
with controls, especially in the 70% extract treated THP-1
cell line (120 fold). In the case of the HEK 293 cell line,
there is an overall decrease of IL-6 expression in all the
sets for control.
Chemo-attractant cytokine, IL-8 plays a significant
role in the activation of chemoattracting neutrophils
resulting in acute inflammation, therefore AML patients
with a lower expression level of IL-8 were reported to
have good survival outcomes [31, 27, 30]. Thus, this
factor is also reported to be a prognostic biomarker for the
progress of acute promyelocytic leukemia differentiation
syndrome [27, 30, 32];Çelik et al. 2020. Within our study,
there is a decrease in the gene expression level of IL-8
in the 50% treated THP-1 cell line, whereas the level got
enhanced in the 70% extract-treated cell line (19 fold
approx) concerning the control set. In all the HEK 293
cell sets, the gene expression level of IL-8 was enhanced
for the control set. Therefore, 50% extract of N. caerulea
is considered to have cancer-regulating properties.
Anti-inflammatory cytokine, IL-10 is reported to
inhibit the enhancement of AML cells with the reduction
of associated factors expressions such as Interleukins -
IL-1α, IL-1β, IL-6, TNF-α and Granulocyte-macrophage
colony-stimulating factor (GM-CSF) [33-35]. Moreover,
it is also reported that the level of IL-10 is enhanced in
the PB of AML patients when compared to normal healthy
individuals [36]. Here also in our experimental data, it
can be noticed that the gene expression level in the 50%
extract treated cells is more (3.60 fold) when compared
to the other sets concerning the control set. When the
cell line, HEK 293 is considered, there is a very mild
increase in the gene expression level of IL-10 compared
to the control set.
Another pro-inflammatory cytokine IL-1β is reported
to support the growth survival, and proliferation of AML
cells in vitro and turn also enhances the expression level of
IL-6, GM-CSF, and TNF factors [34, 36]. However, with
AML patients the level is either unchanged or elevated in
PB and remains unchanged in BM [34, 36]. The expression
level of IL-1β is lower in 50% extract treated THP-1
cells (0.43 fold) and higher in 70% extract-treated cell
line (17.03) when compared to normal cell control. In
the HEK 293 cell line, there are no such marked changes
concerning control.
TGF β, an anti-inflammatory cytokine inhibits the
proliferation and survival of AML cells in vitro and
the level is also reported to be lower in the BM and PB
among AML patients [33, 34]. The expression level
of TGF β1 is also lower in 50% extract treated THP-1
cells when compared to the other sets. There is also a
slight increase in the gene expression in the 70% ethanol
(vehicle control) set. In the HEK 293 cell line also there
is an enhancement of gene expression of TGF β1 among
the 50% extract-treated cells. However, these changes
are very mild.
As previously reported TNF α, pro-inflammatory
cytokine favors the development of chemoresistance of
AML cells and proliferation of LSCs in vitro. The level
is also found to be elevated in PB among AML patients
[33, 34, 32]. In our data, there is a reduction of the gene
expression in all the THP -1 cell treated sets except the
one challenged with 70% alcohol. The same observations
have been noticed among the HEK 293 cell line.
The process of apoptosis involves two core pathways
namely extrinsic and intrinsic pathways [37, 38]. The
extrinsic pathway involves the death receptors (DR)
pathway and the intrinsic pathway is via the mitochondrial
mediated pathway [38] (Figure 13). The extrinsic pathway
involves the type 1 TNF receptor (TNFR1), and Fas
(also called CD95/Apo-1) receptors [39, 40]. Moreover,
experimental evidence has also confirmed the extensive
significant role of caspases in apoptosis. Caspases are
considered to be the initiator and the executioners in
the pathway of apoptosis. Death effector domain (DED)
comprises caspase-8 and 10 whereas caspase recruitment
domains (CARD) comprise caspase-1, 2, 4, 5, 9, 11, and 12
[41, 42]. The triggering of caspase 9 leads to the activation
of the caspase 3 signaling network which in turn causes the
destruction of cells and concludes in apoptosis [41, 42]. In
our experimental data there is an elevated gene expression
level of caspase 9 (7.67 fold) in 50% extract treated THP-1
cells and followed by extensive expression of caspase 3
(173.59 fold) that may lead to apoptosis of THP-1 cells.
There is also an elevation of the gene expression level of
CD95 (Fas L) (83.20 fold) and TNFR1 (21.91 fold) in
50% extract treated THP-1 cells indicating the execution
of extrinsic pathway.
Other researchers have reported about the presence
of several phytochemicals namely alkaloids (+++),
anthraquinones (+++), cardiac glycosides (+++),
flavonoids (++), phenolics (++), saponins (+) and tannins
(+) [5]. The extract of blue lotus depicted antibacterial
activity against E .coli, P. aeruginosa, K. pneumonia,
S. aureus and S. pyogenes (Akinjogunla, et al. 2009).
Very few research article are available on the bioactive
composition of N. caerulea and among them one of the
researcher have reported about three novel macrocyclic
flavonoids namely myricetin-3’ -O- (6” - p-coumaroyl)
glucoside, pentagalloyl glucose and myricetin-3-Orhamnoside via NMR study [43]. Similarly in the year,
1999 few authors reported about the confirmation of
two important compounds namely delphinidin 3’- O-
(2”-O- galloyl β galactopyranoside (1) and delphinidin
3’- O- (2”-O- galloyl – 6”- O acetyl β galactopyranoside
(2) [44]. According to the West African cultural beliefs,
the rhizome of the plant N. caerulea was consumed
during the time of famine. Moreover, the seeds and
rhizome was used for the treatment of diabetes. Infusions
prepared from the root and stem of the plant fights with
the disease like gonorrhea and urinary passage diseases
[45]. The tea prepared with rhizome was used to treat
kidney and bladder disorders, also treats insomnia,
problems of the intestine and this tea infusion causes
Asian Pacific Journal of Cancer Prevention, Vol 25 135
DOI:10.31557/APJCP.2024.25.1.123
The Apoptotic Property of Blue Lotus Extract on Leukaemia Cells
drowsiness. Moreover, the decoction prepared from the
flower was used to treat painful urination, reduces libido
and cough problems [45]. Previously some authors have
reported about the phytochemical characterization of
the flower and the reported compounds are - kaempferol
aglycones, quercetin, and myricetin. It also comprises of
compounds like helichrysin B, naringenin, gallic acid,
isosalipurposide, p-coumaric acid, and methoxybenzoic
acid [45, 46]. In our study, we have also reported the
presence of similar bioactive constituents of the whole
flower (petals, thalamus, reproductive parts of Nelumbo
nucifera) by ultra-performance liquid chromatography
followed by mass spectrometry such as homogentisic
acid, gallocatechin, caffeoyl glucose/ esculin, paeoniflorin,
hesperidin, and several others. Thus, the reported
anti-cancer activities of the N. caerulea flower might
be due to the synergistic activities of all such bioactive
compounds (Figures 12 and 14).
In conclusion, N. caerulea flower extract appears
to be a good anti-leukemia drug against AML, and the
extract is capable of inducing apoptosis of these cells
as well as the extract can also control the cytokine
imbalance in leukemia by balancing pro-inflammatory
and anti-inflammatory cytokine gene expressions.
Limitations of the study
The study was done with crude 50% ethanolic extract
of flower N. caerulea. Though phytochemical profiling
has been done of the crude extract, however further study
is needed on animal model with pure marker compounds
reported from this study to see the difference in the
anti-cancer activity of the phyto-chemicals against the
crude extract.
List of abbreviations
THP-1: human monocytic cell line; HEK 293:
Human Embryonic Kidney Cell line; AML: Acute
myeloid leukemia; HSPCs: Hematopoietic Stem Cells;
LSCs: Leukemic Stem Cells; PB: Peripheral Blood;
IFN γ: Interferon Gamma; IL-6: Interleukin – 6;
IL-8: Interleukin – 8; IL-10: Interleukin – 10; IL-1β:
Interleukin – 1β; TGF-β1: Transforming Growth
Factor- β1; TGF-β3: Transforming Growth Factorβ3; TNF-α: Tumor Necrosis Factor Alpha; GM-CSF:
Granulocyte-macrophage colony-stimulating factor ;
DR: Death Receptors ; TNFR1: Type 1 Tumor Necrosis
Factor receptor ; DED: Death Effector Domain ; CARD:
Caspase Recruitment Domains ; RT-PCR: Real Time
Polymerase Chain Reaction; DNA: Deoxy Ribonucleic
Acid; MTT: 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide; HPLC: high-performance liquid
chromatography; LC-MS: Liquid Chromatography and
Mass Spectrometry; Earle’s BSS: Earle’s Balanced Salt
Solution; DMEM: Dulbecco’s Modified Eagle Medium;
RPMI: Rosewell Park Memorial Institute; RNA:
Ribonucleic Acid; cDNA: complementary DNA; FBS:
Fetal Bovine Serum; PBS: Phosphate Buffer Saline.
Author Contribution Statement
Author DC performed the experimental study, initially
analyzed the findings and written the draft manuscript;
author BS assisted her during the entire experiment
and data analysis; author KP provided all the necessary
administrative support for the research investigation and
author SD conceptualized the experimental study, finalized
the data interpretation and checked the final version of the
manuscript. All authors have gone through and accepted
the final version of the manuscript.
Acknowledgements
The authors would like to acknowledge the Heritage
Institute of Technology for providing the laboratory setup
for conducting the experimental study. We would also like
to acknowledge TCG Life Sciences, Pvt. Ltd. for carrying
out the LC-MS analysis of the research samples. No
funding is available for this study. The study was carried
out on in-vitro cell line model, therefore no scientific body
or Institutional Ethical Committee permission was needed.
Ethics Approval
However, the study was ratified by Institutional Ethical
Committee.
Availability of Data
All the data files produced during the study are with
corresponding author and can be produced if required in
future.
Conflict of Interest
The authors declare none.
References
1. World health organization. Key facts on cancer, 3rd february,
(2022).
Https://www.Who.Int/news-room/fact-sheets/detail/cancer
(Accessed on 12.12.2022).
2. Marçais A, Suarez F, Sibon D, Frenzel L, Hermine O,
Bazarbachi A. Therapeutic options for adult t-cell leukemia/
lymphoma. Curr Oncol Rep. 2013;15(5):457-64. https://doi.
org/10.1007/s11912-013-0332-6.
3. Fiore D, Cappelli LV, Broccoli A, Zinzani PL, Chan WC,
Inghirami G. Peripheral t cell lymphomas: From the bench
to the clinic. Nat Rev Cancer. 2020;20(6):323-42. https://
doi.org/10.1038/s41568-020-0247-0.
4. N’Guessan BB, Asiamah AD, Arthur NK, Frimpong-Manso
S, Amoateng P, Amponsah SK, et al. Ethanolic extract
of nymphaea lotus l. (nymphaeaceae) leaves exhibits in
vitro antioxidant, in vivo anti-inflammatory and cytotoxic
activities on jurkat and mcf-7 cancer cell lines. BMC
Complement Med Ther. 2021;21(1):22. https://doi.
org/10.1186/s12906-020-03195-w.
5. Kameni PM, Dzeufiet DPD, Bilanda DC, Mballa MF,
Mengue NYS, Tchoupou TH, et al. Nymphaea lotus
linn. (nymphaeaceae) alleviates sexual disability in
l-name hypertensive male rats. Evid Based Complement
Alternat Med. 2019;2019:8619283. https://doi.
org/10.1155/2019/8619283.
6. Agnihotri VK, Elsohly HN, Khan SI, Smillie TJ, Khan IA,
Walker LA. Antioxidant constituents of nymphaea caerulea
flowers. Phytochemistry. 2008;69(10):2061-6. https://doi.
org/10.1016/j.phytochem.2008.04.009.
7. Harer WB. Pharmacological and biological properties of the
Debasmita Chatterjee et al
136 Asian Pacific Journal of Cancer Prevention, Vol 25
egyptian lotus. JARCE. 1985;22:49-54.
8. Elnaggar MG, Mosad E, Makboul A, Shafik EA. Cytogenetic
profile of adult acute myeloid leukemia in egypt: A singlecenter experience. Mol Cytogenet. 2022;15(1):43. https://
doi.org/10.1186/s13039-022-00621-1.
9. Hussein S, Mohamed D, Hafez R. Risk factors of hematological
malignancies in upper egypt: A case–control study. Egypt J
Intern Med. 2019;31:171-7.
10. Khondkaryan L, Andreasyan D, Hakobyan Y, Bankoglu EE,
Aroutiounian R, Stopper H, et al. Incidence and risk factors
of acute leukemias in armenia: A population-based study.
Asian Pac J Cancer Prev. 2022;23(11):3869-75. https://doi.
org/10.31557/apjcp.2022.23.11.3869.
11. Aitbekov R, Murzakhmetova M, Zhamanbayeva G,
Zhaparkulova N, Seel H. Epidemiological features of
acute myeloid leukemia in five regions of the republic
of kazakhstan: Population study. Asian Pac J Cancer
Prev. 2022;23(12):4163-7. https://doi.org/10.31557/
apjcp.2022.23.12.4163.
12. Paudel KR, Panth N. Phytochemical profile and
biological activity of nelumbo nucifera. Evid Based
Complement Alternat Med. 2015;2015:789124. https://doi.
org/10.1155/2015/789124.
13. Zhang X, Wang X, Wu T, Li B, Liu T, Wang R, et al.
Isoliensinine induces apoptosis in triple-negative human
breast cancer cells through ros generation and p38 mapk/jnk
activation. Sci Rep. 2015;5:12579. https://doi.org/10.1038/
srep12579.
14. Liu W, Yi DD, Guo JL, Xiang ZX, Deng LF, He L.
Nuciferine, extracted from nelumbo nucifera gaertn, inhibits
tumor-promoting effect of nicotine involving wnt/β-catenin
signaling in non-small cell lung cancer. J Ethnopharmacol.
2015;165:83-93. https://doi.org/10.1016/j.jep.2015.02.015.
15. Shukla K, chaturvedi N. In vitro antioxidant properties of
different parts of nelumbo nucifera gaertn. Int J Adv Pharm
Biol Chem. 2016;5(2):196-201.
16. Nascimento CR, Rodrigues Fernandes NA, Gonzalez
Maldonado LA, Rossa Junior C. Comparison of monocytic
cell lines u937 and thp-1 as macrophage models for in vitro
studies. Biochem Biophys Rep. 2022;32:101383. https://doi.
org/10.1016/j.bbrep.2022.101383.
17. Kannan N, Shanmuga Sundar S, Balaji S, Amuthan A,
Anil Kumar NV, Balasubramanian N. Physiochemical
characterization and cytotoxicity evaluation of mercurybased formulation for the development of anticancer
therapeuticals. PLoS One. 2018;13(4):e0195800. https://
doi.org/10.1371/journal.pone.0195800.
18. Chatterjee D, Singh B, Pradhan AK, Paira K, Das S.
Diluted Lycopodium Induced Cell Death and Clinical
Improvement in Hepatocellular Carcinoma. Science
Repository. 2022:5(3):2-8.
19. Zhu Z, Zhong B, Yang Z, Zhao W, Shi L, Aziz A, et al.
Lc-esi-qtof-ms/ms characterization and estimation of the
antioxidant potential of phenolic compounds from different
parts of the lotus (nelumbo nucifera) seed and rhizome.
ACS Omega. 2022;7(17):14630-42. https://doi.org/10.1021/
acsomega.1c07018.
20. Rajauria G, Foley B, Abu-Ghannam N. Identification and
characterization of phenolic antioxidant compounds from
brown irish seaweed himanthalia elongata using lc-dad–esims/ms. Innov Food Sci Emerg Technol. 2016;37:261-8.
21. Döhner H, Weisdorf DJ, Bloomfield CD. Acute myeloid
leukemia. N Engl J Med. 2015;373(12):1136-52. https://
doi.org/10.1056/NEJMra1406184.
22. Papayannidis C, Sartor C, Marconi G, Fontana MC, Nanni
J, Cristiano G, et al. Acute myeloid leukemia mutations:
Therapeutic implications. Int J Mol Sci. 2019;20(11). https://
doi.org/10.3390/ijms20112721.
23. Krause DS, Scadden DT. A hostel for the hostile:
The bone marrow niche in hematologic neoplasms.
Haematologica. 2015;100(11):1376-87. https://doi.
org/10.3324/haematol.2014.113852.
24. Ganesan S, Mathews V, Vyas N. Microenvironment and
drug resistance in acute myeloid leukemia: Do we know
enough? Int J Cancer. 2022;150(9):1401-11. https://doi.
org/10.1002/ijc.33908.
25. Zeisig BB, Fung TK, Zarowiecki M, Tsai CT, Luo
H, Stanojevic B, et al. Functional reconstruction of
human aml reveals stem cell origin and vulnerability of
treatment-resistant mll-rearranged leukemia. Sci Transl
Med. 2021;13(582). https://doi.org/10.1126/scitranslmed.
abc4822.
26. Gu J, Huang X, Zhang Y, Bao C, Zhou Z, Jin J. Cytokine
profiles in patients with newly diagnosed multiple
myeloma: Survival is associated with il-6 and il-17a levels.
Cytokine. 2021;138:155358. https://doi.org/10.1016/j.
cyto.2020.155358.
27. Corradi G, Bassani B, Simonetti G, Sangaletti S,
Vadakekolathu J, Fontana MC, et al. Release of ifnγ by
acute myeloid leukemia cells remodels bone marrow
immune microenvironment by inducing regulatory t
cells. Clin Cancer Res. 2022;28(14):3141-55. https://doi.
org/10.1158/1078-0432.Ccr-21-3594.
28. Ding H, Wang G, Yu Z, Sun H, Wang L. Role of interferongamma (ifn-γ) and ifn-γ receptor 1/2 (ifnγr1/2) in
regulation of immunity, infection, and cancer development:
Ifn-γ-dependent or independent pathway. Biomed
Pharmacother. 2022;155:113683. https://doi.org/10.1016/j.
biopha.2022.113683.
29. Konopleva M, Cardama AQ, Kantarjian H, Cortes J.
Molecular biology and cytogenetics of chronic myeloid
leukemia. Neoplastic Diseases of the Blood. 2018:29-47.
30. Hassanshahi M, Hassanshahi A, Khabbazi S, Su YW, Xian
CJ. Bone marrow sinusoidal endothelium: Damage and
potential regeneration following cancer radiotherapy or
chemotherapy. Angiogenesis. 2017;20(4):427-42. https://
doi.org/10.1007/s10456-017-9577-2.
31. Konopleva M, Cardama A, Kantarjian H, Cortes J. Molecular
biology and cytogenetics of chronic myeloid leukemia.
Neoplastic Diseases of the Blood. 2018:29-47. https://doi.
org/10.1007/978-3-319-64263-5_4.
32. Islam M, Mohamed EH, Esa E, Kamaluddin NR, Zain SM,
Yusoff YM, et al. Circulating cytokines and small molecules
follow distinct expression patterns in acute myeloid
leukaemia. Br J Cancer. 2017;117(10):1551-6. https://doi.
org/10.1038/bjc.2017.316.
33. Savage SA, Walsh MF. Myelodysplastic syndrome, acute
myeloid leukemia, and cancer surveillance in fanconi
anemia. Hematol Oncol Clin North Am. 2018;32(4):657-68.
https://doi.org/10.1016/j.hoc.2018.04.002.
34. Karimdadi Sariani O, Eghbalpour S, Kazemi E, Rafiei Buzhani
K, Zaker F. Pathogenic and therapeutic roles of cytokines
in acute myeloid leukemia. Cytokine. 2021;142:155508.
https://doi.org/10.1016/j.cyto.2021.155508.
35. Wu H, Li P, Shao N, Ma J, Ji M, Sun X, et al. Aberrant
expression of treg-associated cytokine il-35 along with
il-10 and tgf-β in acute myeloid leukemia. Oncol Lett.
2012;3(5):1119-23. https://doi.org/10.3892/ol.2012.614.
36. Grauers Wiktorin H, Aydin E, Christenson K, Issdisai N,
Thorén FB, Hellstrand K, et al. Impact of il-1β and the
il-1r antagonist on relapse risk and survival in aml patients
undergoing immunotherapy for remission maintenance.
Oncoimmunology. 2021;10(1):1944538. https://doi.org/10
.1080/2162402x.2021.1944538.
Asian Pacific Journal of Cancer Prevention, Vol 25 137
DOI:10.31557/APJCP.2024.25.1.123
The Apoptotic Property of Blue Lotus Extract on Leukaemia Cells
This work is licensed under a Creative Commons AttributionNon Commercial 4.0 International License.
37. Kessler CS, Eisenmann C, Oberzaucher F, Forster M,
Steckhan N, Meier L, et al. Ayurvedic versus conventional
dietary and lifestyle counseling for mothers with burnoutsyndrome: A randomized controlled pilot study including a
qualitative evaluation. Complement Ther Med. 2017;34:57-
65. https://doi.org/10.1016/j.ctim.2017.07.005.
38. Elmore S. Apoptosis: A review of programmed cell
death. Toxicol Pathol. 2007;35(4):495-516. https://doi.
org/10.1080/01926230701320337.
39. Jan R, Chaudhry GE. Understanding apoptosis and apoptotic
pathways targeted cancer therapeutics. Adv Pharm Bull.
2019;9(2):205-18. https://doi.org/10.15171/apb.2019.024.
40. Jin Z, El-Deiry WS. Overview of cell death signaling
pathways. Cancer Biol Ther. 2005;4(2):139-63. https://doi.
org/10.4161/cbt.4.2.1508.
41. Guicciardi ME, Gores GJ. Life and death by death receptors.
Faseb J. 2009;23(6):1625-37. https://doi.org/10.1096/fj.08-
111005.
42. Hengartner MO. The biochemistry of apoptosis. Nature.
2000;407(6805):770-6. https://doi.org/10.1038/35037710.
43. Elegami AA, Bates C, Gray AI, Mackay SP, Skellern GG,
Waigh RD. Two very unusual macrocyclic flavonoids from the
water lily nymphaea lotus. Phytochemistry. 2003;63(6):727-
31. https://doi.org/10.1016/s0031-9422(03)00238-3.
44. Fossen T, Andersen ØM. Delphinidin 3′-galloylgalactosides
from blue flowers of nymphaéa caerulea. Phytochemistry.
1999;50(7):1185-8.
45. Oosthuizen CB, Fisher M, Lall N. Nymphaea caerulea. In
Underexplored Medicinal Plants from Sub-Saharan Africa:
Academic Press; 2020. 205-10. https://doi.org/10.1016/
B978-0-12-816814-1.00031-4.
46. Zhu M, Zheng X, Shu Q, Li H, Zhong P, Zhang H, et al.
Relationship between the composition of flavonoids and
flower colors variation in tropical water lily (nymphaea)
cultivars. PLoS One. 2012;7(4):e34335.