Ovarian cancer cells with CD133+ phenotype is more resistant against Ngai Bun Boesenbergia pandurata extract than original ovarian cancer cells

Progress in Stem Cell, 7(1):290-295
Open Access Full Text Article Original Research
1Stem Cell Institute, University of Science
Ho Chi Minh City, Viet Nam
2Vietnam National University Ho Chi
Minh City, Viet Nam
3Cancer Research Laboratory, University
of Science Ho Chi Minh City, Viet Nam
4Laboratory of Stem Cell Research and
Application, University of Science Ho
Chi Minh City, Viet Nam
Correspondence
Phuc Van Pham, Stem Cell Institute,
University of Science Ho Chi Minh City,
Viet Nam
Vietnam National University Ho Chi Minh
City, Viet Nam
Cancer Research Laboratory, University
of Science Ho Chi Minh City, Viet Nam
Laboratory of Stem Cell Research and
Application, University of Science Ho Chi
Minh City, Viet Nam
Email: pvphuc@hcmuns.edu.vn;
phucpham@sci.edu.vn
History
 Received: 22 January 2020
 Accepted: 05 March 2020
 Published: 19 March 2020
DOI : 10.15419/psc.v7i1.408
Ovarian cancer cells with CD133+ phenotype is more resistant
against Ngai Bun Boesenbergia pandurata extract than original
ovarian cancer cells
Oanh Thi-Kieu Nguyen1,2, Phuc Van Pham1,2,3,4,* 
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ABSTRACT
Introduction: Ovarian cancer is one of the most common cancers in women. Due to the diffi-
culty in early detection and treatment of ovarian cancer, many research studies and clinical trials
have been developed to discover more efficient therapies. Besides Western medicine, traditional
medicine has gained increased interest as a research fieldwith potential to lead to the production of
marketable therapeutic products. With the diversity of tropical plants in Asia, traditional medicine
has been very popular and has served as a traditional therapy for generations. The Ngai bun (Boe-
senbergia pandurata) root is used not only as a food spice but also in ethnomedicine. This study
aimed to compare the anti-tumor activity of Boesenbergia pandurata root extract against ovarian
cancer cells and CD133+ovarian cancer cells that were enriched from the original ovarian cancer
cells. Methods: Crude extract of Boesenbergia pandurata roots were prepared in two kinds of sol-
vents (methanol and chloroform). The ovarian cancer cells OVP-10 were used in this study. The
population of CD133+ ovarian cancer cells (CD133+OVP-10) were sorted from the OVP-10 cancer
cells. Both OVP-10 cells and CD133+OVP-10 cells were treated with these crude extracts. Adipose-
derived stem cells (ADSCs) were used as control normal cells for all assays. The anti-tumor activity
of extracts were evaluated based on the IC50 values. Results: Based on the IC50 index, the chlo-
roform extract had an anti-tumor activity higher than that of methanol extract, on both OVP-10
and CD133+OPV-10 cells (IC50 of methanol and chloroform extracts were 330.1 16.9 mg/mL and
246.5 21.2 mg/mL, respectively, for OVP-10 cells; IC50 of methanol and chloroform extracts were
411.8  83.7 mg/mL and 307  9.2 mg/mL respectively, for CD133+OVP-10 cells). The results also
showed that CD133+OVP-10 cells weremore resistant to chloroform extract thanwereOVP-10 cells
(307  9.2 mg/mL vs. 246.5  21.2 mg/mL, respectively, for CD133+OVP-10 vs. OVP-10 cells, p <
0.05). Conclusion: The chloroform extract of Boesenbergia pandurata roots displayed strong anti-
tumor activity against ovarian cancer cells OVP-10 and CD133+OVP-10; the latter cells were found
to be more resistant than the original ovarian cancer cells.
Key words: CD133+ cancer stem cells, ovarian cancer stem cells, ovarian cancer, Boesenbergia
pandurata
INTRODUCTION
In 2019, the AmericanCancer Society estimated ovar-
ian cancer as the leading cause of death in gyneco-
logical disease, ranking 5th of the 10 leading cancer
types in women1. Ovarian cancer is a rare disease, in
which early detection is difficult and surgical strate-
gies are the first step in treatment for this cancer2.
However, the extent of surgery depends on how far
the ovarian cancer has spread; chemotherapy must be
used in the next steps to eradicate any residual cancer
cells still present in the body after surgery. The goal
of chemotherapy is to destroy the cancer by inhibit-
ing the proliferation of cancer cells. Chemotherapy
is a potential treatment for prolonging the cancer pa-
tient’s life. There are many kinds of anti-cancer drugs
from natural sources, such as plants (e.g. vincristine,
irinotecan, and camptothecins) and microorganisms
(e.g. doxorubicin, mitomycin, and bleomycin)3.
Doxorubicin (DOX) is the most popular anti-cancer
drug, which is currently widely used for treatment of
many kinds of human cancers, both solid and hema-
tological4. DOX was shown to induce resistance in
3D spheroids, at a rate higher than that exhibited in
standard 2D cell culture5. Additionally, tirapazamine
(TPZ; 3-amino-1,2,4-benzotriazine 1,4 dioxide) is a
new class of cytotoxic drugs with a focus on treating
hypoxic mammalian cells6. When culture ... Z); both were pur-
chased from Sigma-Aldrich. Ngai bun extract was
isolated from fresh root, following a previously pub-
lished protocol, and dissolved in the different sol-
vents7.
Alamar Blue assay
The cell viability of ovarian cancer cells was tested by
Alamar Blue assay. Cells were plated in a 96-well plate
at a density of 2 x 104 cells/well. After plating for
24 h, cells were treated with the drugs at six differ-
ing concentrations (2000, 1000, 500, 250, 125, 62, and
0 mg/mL) for 48 h. The culture medium was then
removed and the wells were replaced with fresh me-
dia. As a negative control, fresh media was also added
to empty wells. All wells were added with 10 m l of
the Alamar Blue solution and then re-incubated at
370C, 5% CO2 for 4 h. Data was collected by using
a DTX880 system (Beckman-Coulter, Brea, CA), and
fluorescence was monitored at 530-560 nm excitation
wavelength and 590 nm emission wavelength.
Statistical analysis
Each experiment was repeated three times. The IC50
and significant differences between mean values were
calculated by using GraphPad Prism 7.0 (GraphPad
Inc., La Jolla, CA), with p-value < 0.05 set as statistical
significance.
RESULTS
Isolation of human ovarian cancer
CD133+OVP-10 cells
Human ovarian cancer OVP-10 cells were cultured
and expanded in 75-cm2 flasks. When cells reached
approximately 70% confluency (Figure 1 B), cells
will be trypsinized and subcultured in DMEM/F12
medium supplemented with 10% FBS. After pass-
ing two to three times, OVP-10 cells were subjected
to bioassays, and/or sorted for CD133+ cells using
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Progress in Stem Cell, 7(1):290-295
MACS (Figure 1A). CD133+OVP-10 cells were la-
belled with CD133 magnetic beads, isolated and cul-
tured to confluency before treatment with the com-
pounds (Figure 1C).Themorphology ofOVP-10 cells
and CD133+OVP-10 cells showed no significant dif-
ferences; however, in culture, the proliferation time of
CD133+OVP-10 cells was slightly longer than that of
OVP-10 cells.
Testing an ovarian cell model for drug
screening with standard drugs
The cell concentration also affected the in vitro bioas-
say for anti-cancer drug screening12. The growth of
OVP-10 cells was tested and the model for screening
the different extracts was optimized. As seen in Fig-
ure 2A, OVP-10 cells continued to proliferate after 7
days. The bioassay was performed in 3 days: on the
first day, cells were plated in wells and incubated for
24 h; next, cells were treated with compounds for 48
h; on the third day, cells were processed for Alamar
blue assay and the IC50 indexes were calculated. Dur-
ing these days, OVP-10 cells were still stable and there
was little increase in cell number (Figure 2A). The
OVP-10 cells were treated with standard drugs (dox-
orubicin or tirapazamine) to confirm that the OVP-
10 cell model could be used for screening. Doxoru-
bicin is popular standard drug which used as a control
in many studies of drug screening. Specifically, dox-
orubicin only affected the monolayer cell model but
not the three-dimensional (3D) cell culture model.
In contrast, tirapazamine was only effective in the
3D cell culture model. As shown in Figure 2B and
Figure 2C, the IC50 of doxorubicin in OVP-10 cells
(168.9  2.3 nM) and that of CD133+OVP-10 cells
(567.7 95.7 nM) were highly different (p < 0.01).
Ngai bun extract dissolved in chloroform
solvent had a greater effect on both OVP-
10 and CD133+OVP-10 cancer cells than
methanol solvent
The IC50 index of Ngai bun extract in chloroform sol-
vent (CHCl3) had a greater effect on killing ovarian
cancer OVP-10 cells when compared with the effect
of this extract on adipose-derived stem cells (ADSCs),
which were used as the control. The IC50 of methanol
(MeOH) and chloroform (CHCl3) solvent were 330.1
 16.9 mg/mL and 246.5  21.2 mg/mL, respec-
tively. As indicated inFigure 3A, the MeOH extract
had a different effect on ADSCs and OVP-10 cells,
as shown by the IC50 index of 497.2  32.4 mg/mL
and 330.1  16.9 mg/mL, respectively. However,
with CD133+OVP-10 cells, no significant differences
were observed between the two cell lines; the IC50
of the corresponding cells (ADSCs vs. CD133+OVP-
10 cells) with MeOH solvent, respectively, were 497.2
 32.4 mg/mL and 411.8  83.7 mg/mL (Figure 3B)
(p > 0.05). In Figure 3C, with the same cells in a
different solvent (CHCl3), the IC50 index of OVP-10
cells was 246.5 21.2 mg/mL, which was significantly
different from the IC50 of 474.6  18.8 mg/mL for
ADSCs (p < 0.05). The results inFigure 3D showed
that CD133+OVP-10 cells treatedwith CHCl3 extract
corresponded to an IC50 index that was significantly
lower than that for ADSC cells (307  9.2 mg/mL vs.
474.6 18.8 mg/mL, respectively) (p < 0.05).
DISCUSSION
Ovarian cancer is the most serious gynecologic can-
cer, typically diagnosed at an advanced stage13,14.
The current standard treatment for ovarian cancer is
surgery and chemotherapy. The chemotherapy strat-
egy is faced with many obstacles including cancer
metastasis and resistance of tumor with drugs. This
has motivated the development of drug discovery to
help find novel potentially therapeutic compounds for
anti-cancer treatment. In drug screening, cells must
be in the proliferation stage and should be stable in
the testing with drugs. In this study, the OVP-10 cells
continued to proliferate for 7 days and were suitable
for our assay. After 24 h of plating, OVP-10 cells
showed a low increase in cell number and after 3 days
(i.e. the day of the Alamar blue assay), cells were still
in log phase of proliferation.
Despite the disadvantage of the 2D model and the
development of the 3D model, the 2D model is still
very popular for drug screening. Evaluation of the 2D
model using in vitro bioassays, such as MTT or Ala-
mar Blue assay, is necessary to assess the efficiency of
the 2D model in anti-cancer drug screening12. The
cell concentration and drug concentration parame-
ters have a great effect on the success of the in vitro
bioassays. As shown in this study, the cell concen-
tration for plating in 96-well plates was 1000-2500
cells/well, and 6 parameters of drugs or extract con-
centrations were at the very least required for calcula-
tion of the IC50 index15.
Use of the traditional extract from plants could kill
cancer cells with fewer effects on normal cells. In
this study, Ngai bun (Boesenbergia pandurata) extract
was demonstrated to be toxic for OVP-10 cells, when
compared with adipose derived stem cells as control.
When comparing different Boesenbergia species (B.
armeniaca, B. rotunda, or B. pulchella var attenuate),
Jing et al. showed that Boesenbergia rotunda extract in
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Progress in Stem Cell, 7(1):290-295
Figure 1: Isolation and expansion of CD133+OVP-10 cells from OVP-10 ovarian cancer cells. (A) Procedure
of CD133+OVP-10 cells isolation by magnetic activated cell sorting. (B) Human ovarian cancer OVP-10 cells. (C)
Human ovarian carcinoma CD133+OVP-10 cells. Pictures were taken at 20 X magnification.
methanol had the strongest inhibitory effects against
CaOV3 ovarian cancer and different types of can-
cers, such as breast cancer MDA-MB231 (IC50 66.5
2.12 mg/mL), MCF7 (IC50 51 mg/mL), cervical can-
cer HeLa (IC50 66.5 2.12 mg/mL), and colon cancer
HT-29 (IC50 52 2.12 mg/mL). Boesenbergia genera
is potentially potent extract for treatment of ovarian
cancer. OVP-10 cells is one of the targets for investi-
gation of drug cytotoxicity against ovarian cancer16.
Moreover, the synergistic anti-tumor effect would be
combined to develop new therapies for ovarian can-
cer treatment17. From different studies, the extract of
Boesenbergia genera exhibit robust potency that can
be utilized as an potential candidates for the devel-
opment of new anti-cancer drugs. Advances in drug
discovery will require identifying and developing new
and innovative marketable pharmaceutical products.
Future studies from this research should focus further
on the discovery of such compounds.
Besides the toxicity towards ovarian cancer OVP-10
cells in a dose-dependent manner, Ngai bun (Boesen-
bergia pandurata) extract in chloroform was demon-
strated to inhibit the cell viability of CD133+OVP-
10 cells, representing ovarian cancer stem cells. At
the IC50 of Ngai bun extract which could kill 50% of
CD133+OVP-10 cells, that concentration could kill
more than 50% of OVP-10 cells but less than 50% of
ADSC cells. This shows that the dose of drug used for
treatment must be chosen carefully.
In this study, two kind of solvents were chosen to eval-
uate which was the best solvent for dissolving Ngai
bun (Boesenbergia pandurata) extract, and still main-
tain the functions of the extract. As observed, the
chloroform-dissolved extract induced better toxicity
than the methanol-dissolved extract. Besides the ap-
propriate concentrations, the suitable solvent is also a
key factor for determining the success of drug discov-
ery.
CONCLUSION
Overall, the data obtained from this study shows
that Ngai bun (Boesenbergia pandurata) chloroform-
dissolved extract is more toxic on OVP-10 cells
than on CD133+OVP-10 cells. The cytotoxicity of
the chloroform extract was also higher that of the
methanol extract.
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Progress in Stem Cell, 7(1):290-295
Figure 2: Model for screening the efficiency of Ngai bun extract. (A) Proliferation of human ovarian cancer
OVP-10 cells in 96-well plates over10 days. (B, C) Effect of standard drugs (doxorubicin or tirapazamine) on human
ovarian cancer OVP-10 cells (B) and CD133+ sorted human ovarian cancer (CD133+OVP-10) cells.  : p < 0.001.
Abbreviations: DOX: dororubicin, TPZ: tirapazamine
Figure 3: The IC50 of human ovarian cancer OVP-10 cells and CD133+OVP-10 cells in different solutions.
(A) methanol (MeOH), (B) chloroform (CCl3). Each experiment was processed three times, and statistical analysis
was performed by GraphPad Prism 7.0 with *p<0.05 (** p<0.0021, ***p<0.0002). Abbreviations: ADSCs: Adipose
derived stem cells.
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Progress in Stem Cell, 7(1):290-295
ABBREVIATIONS
CSC: Cancer stem cell
DOX: Doxorubicin
IC50: the half-maximal inhibitor concentrations
TPZ: Tirapazamine
CONFLICT OF INTEREST
Theauthors report no conflicts of interest in thiswork.
AUTHORS’ CONTRIBUTION
All authors equally contributed in this work and ap-
proved the final version ofmanuscript for submission.
ACKNOWLEDGEMENT
Theauthors thank toVietnamNationalUniversity, Ho
Chi Minh City (VNU-HCM) for funding this project,
under grant number A2015-18-01/HD-KHCN.
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