Hindawi
Evidence-Based Complementary and Alternative Medicine
Volume 2020, Article ID 8567182, 10 pages
https://doi.org/10.1155/2020/8567182
Research Article
Chemopreventive Effects and Antioxidant Capacity of Combined
Leaf Extracts of Sesamum angustifolium (Oliv.) Engl. and
Hibiscus articulatus on Rhabdomyosarcoma
Clayton E. Siamayuwa,1 Loveness K. Nyanga,2 and Cathrine Chidewe
1
2
1
Department of Biochemistry, University of Zimbabwe, P.O. Box MP 167 Mount Pleasant, Harare, Zimbabwe
Institute of Food and Nutritional Sciences, University of Zimbabwe, P.O. Box MP 167 Mount Pleasant, Harare, Zimbabwe
Correspondence should be addressed to Cathrine Chidewe; cchidewe@gmail.com
Received 1 November 2019; Revised 5 February 2020; Accepted 6 March 2020; Published 26 March 2020
Academic Editor: Visweswara Rao Pasupuleti
Copyright © 2020 Clayton E. Siamayuwa et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Sesamum angustifolium (Oliv.) Engl. and Hibiscus articulatus contain compounds that have antimutagenic properties. The rise in
rhabdomyosarcoma in paediatrics and prognosis of the disease in infants compared to adults calls for newer, less toxic alternatives
in treatment of the disease. The aim of this study was to determine the anticancer activity and antioxidant capacity of combined
leaf extracts of Sesamum angustifolium (Oliv.) Engl. and Hibiscus articulatus (SAHA), against rhabdomyosarcoma (RMS) using
rhabdomyosarcoma (RD) cell line and mouse (L20B) cell line. Cytotoxicity, morphology, apoptosis induction, and antioxidant
capacity assays were done. Of the four solvents used for extraction, the dichloromethane SAHA extract was the most cytotoxic
with IC50 of 106 μg/mL after doxorubicin, the reference anticancer drug with IC50 of 0.8 μg/mL. The SAHA extracts had a stronger
cytotoxicity effect on the cancerous RD cells than on normal L20B cells. Morphological assessment showed untreated cells
maintained their normal striated appearance of muscle cells whereas cells treated with doxorubicin or SAHA extracts exhibited
cell shrinkage, loss of surface adherence, reduced cell density along with cell debris, which is a characteristic of apoptosis. Normal
L20B cells when treated with doxorubicin or SAHA extracts, maintained their cell shape, and remained adherent to the surface.
The apoptotic enzyme caspase-3 was induced in a concentration dependent manner upon treatment of the RD cells with SAHA
extracts or doxorubicin. Induction of caspase-3 was ten times less in treated L20B cells compared to the RD cells. Low induction of
caspase-9 enzyme was observed in both treated RD and L20B cells. Treatment of both RD and L20B cells with SAHA extracts or
doxorubicin resulted in increased activity of peroxidase and reduction of oxidative stress. Results of the study show that the SAHA
extracts are potential sources of compounds that may serve as useful agents for treatment of rhabdomyosarcoma.
1. Introduction
Cancer is the third leading cause of death in the world. In 2018,
the cancer morbidity rate was 18.1 million and mortality rate
was 9.6 million [1]. The annual cancer mortality and morbidity
rates are likely to increase in the near future. The World Health
Organization projected an annual increase to 26 million new
cases and 17 million deaths by 2030, with developing countries
bearing the heaviest burden [2].
Rhabdomyosarcoma is listed among the top 20 most
diagnosed cancers in the world. RMS is a soft tissue neoplasm that share a propensity to undergo myogenesis [3].
RMS is the third most common childhood solid tumor after
neuroblastoma and Wilms’ tumor accounting for 4–6% of all
paediatric tumors [4, 5]. About 350 new cases of RMS are
diagnosed yearly in the United States and 60 new cases in the
UK [5]. In Southern Africa, rhabdomyosarcoma and other
soft tissue sarcomas constitute 70% in Kenya, 58% in
Mozambique, and 21% in Zimbabwe of all paediatric cancers
according to 2010–2012 statistics [6].
The survival rate of a patient diagnosed of RMS is
50–70% [7]. The survival rate has been improved by the
available clinical treatment methods which include laser
surgery, radiation therapy, chemotherapy, organ sparing,
2
and transoral robotic surgery [8]. Some of the current
therapies for the treatment of RMS are associated with high
expense, increased toxicity, and numerous side effects [5].
Medicinal plants have been proposed as an alternate
source of medicament, since they have been used for centuries [9]. Medicinal plants are rich in chemical substances
in the form of secondary metabolites or phytochemicals. The
mechanism of action of the phytochemicals in cancer
treatment is drug-like and biologically friendly compared to
synthetic molecules [10]. Phytochemicals have been reported to possess the ability to control cell growth in cancerous cells via the deregulation of cell cycle proteins,
thereby preventing the abnormal cells from proliferating
[11]. Phytochemicals such as triterpenes, flavonoids, and
polyphenols have shown the ability to upregulate apoptotic
proteins, caspase 3/9, involved in an active form of programmed cell death called apoptosis [12–14].
Sesamum angustifolium (Oliv.) Engl. is a perennial wild
vegetable native to tropical and subtropical Africa. The
mucilage of rubbed leaves in water is traditionally used to treat
eye troubles, burns, wounds, and diarrhea in children while
the crushed leaves can be used as a soap substitute due to their
mucilaginous nature [15]. Hibiscus articulatus is an erect
semiwoody herb native to west and tropical Africa [16]. The
leaves of Hibiscus articulatus are of great economic importance as a source of medicinal products, food, and cosmetics.
The aim of the study was to evaluate the combined effect
of SAHA extracts on RD cancer cells and L20B normal cells
in relation to the reduction of high levels of oxidative stress,
cytotoxicity, and induction of apoptosis.
2. Materials and Methods
2.1. Chemicals. Chemicals, media, and drugs used were
purchased from Sigma-Aldrich (Steinheim, Germany) and
were of analytical grade, these include RD cells; L20B cells;
eagle’s minimum essential medium (MEM), Fetal Bovine
Serum (FBS), Gibco Phosphate-Buffered Saline (GPBS)
without Ca2+ and Mg2+ (life technologies), Penicillin/
Streptomycin, trypsin/EDTA, doxorubicin, propidium iodide (PI), Thiazolyl Blue Tetrazolium Bromide (MTT) and
dimethyl sulphoxide (DMSO).
2.2. Preparation of Plant Material. Sesamum angustifolium
(Oliv.) Engl. and Hibiscus articulatus were collected from
two tree farms in Zimbabwe ((17°23′S, 30°24′E), in the
month of December. The plants were identified and authenticated by a botanist from the Botanical gardens of
Zimbabwe. Voucher specimens of the plants were deposited
in the national herbarium, Sesamum angustifolium (Oliv.)
Engl. voucher specimen number (68726) and Hibiscus
articulatus, voucher specimen number (68728). Young
leaves from aerial parts of the plants were air-dried and
pulverized into powder using a laboratory homogeniser
(Christy and Norris Ltd. Chemsford, England).
2.3. Preparation of Extracts. The powdered samples (25 g)
were subjected to exhaustive solvent extraction for 8 hours
Evidence-Based Complementary and Alternative Medicine
using a Soxhlet apparatus and 250 mLs each of the solvents
dichloromethane, acetone, and methanol successively. After
extraction with methanol, the plant residue was extracted
with water at 25°C for 8 hrs with continuous shaking. The
dichloromethane, acetone, and methanol fractions were
concentrated by rotary evaporation in a Buchi R205 rotary
evaporator (Buchi, Switzerland) at 40°C. The aqueous extract
was freeze-dried on a Christ Alpha 1–4 freeze drier (Dorfen,
Germany). To prepare the combined (SAHA) extract solution for testing, equal amounts of the dried extracts of
Sesamum angustifolium (Oliv.) Engl. and Hibiscus articulatus were weighed, mixed, and dissolved in appropriate
amount of solvent to achieve the desired concentration. The
combined extracts were dissolved in dimethyl sulphoxide
(DMSO) and Eagle’s minimum essential medium to a final
concentration of 0.65% DMSO.
2.4. Proliferation of RD and L20B Cells. The human rhabdomyosarcoma (RD) cancer cell line and the mouse (LB20)
normal cell line were cultured in a humid environment at
37°C and 5% CO2 in Eagle’s minimum essential medium
supplemented with 10% fetal bovine serum and 1% streptomycin/penicillin. At 90% confluence, the cells were harvested using 0.25% trypsin/0.53 mM EDTA solution (SigmaAldrich, USA) and subcultured onto 96 well plates.
2.5. Determination of Cytotoxicity. The cytotoxity assay
(MTT assay) was carried out following the procedure previously described by [17] with modifications. The trypsinized
RD and L20B cells were seeded in a 96 well plate at a density
of 5 × 104 cells per well. After 48 hrs incubation, the medium
was removed from each well and 300 μL of fresh medium
added. One hundred microliter of 400 μg/ml crude extract
was serially diluted at concentrations ranging from
200–12.5 μg/mL in rows D to H of the microtiter plate. Row
A served as a blank (containing medium only) while row B
served as a positive control (untreated cells). Row C contained untreated cells in 0.65% DMSO. The cells with and
without extract were incubated in a Biobase QP 160-II CO2
incubator at 37°C for 48 hrs before determining cell viability
(Biobase, China). The test was performed in triplicate.
After incubation, the culture medium with or without
extract was removed from the plates and replaced with
100 μL fresh culture medium. Five microliters of 0.5 mg/ml
of MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide, in PBS were added to each well. After
2 hr incubation at 37°C, the medium with MTT was removed and 100 μL of DMSO added to dissolve the formed
formazan crystals. The cells were incubated for 30 mins and
optical density measured on an ELISA reader (Biobase, China)
at 495 nm. Percentage cell viability was calculated as follows:
% cell viability � (mean absorbance of sample − blank/
mean absorbance of control-blank) × 100
2.6. Morphological Assessment. The confluent RD and
L20B cells in 24 well plates were treated with various
concentrations (IC50) of each SAHA extract and
Evidence-Based Complementary and Alternative Medicine
doxorubicin (0.8 μg/mL) for 48 hrs and morphological alterations observed under an inverted microscope (Leitz,
Germany) at 400x magnification and photographs were
taken using a Japson MD130 digital camera (Japson, China).
The pictures were analyzed using a Future Win Joe software
for windows.
2.7. Determination of Apoptotic Induction Using PI Assay.
The effect of SAHA leaf extracts on RD cells and L20B cells
was analyzed using propidium iodide following the method
described by [18], with some modifications. PI is capable of
passing through damaged cell membranes, and binding to
DNA. RD and L20B cells were harvested at exponential
phase of growth and seeded into 16 well plates at a seeding
density of 1 × 104 viable cells/well. After 48 hr incubation at
37°C, an aliquot of 100 μl of SAHA extracts (50 μg/mL and
200 μg/mL) and doxorubicin (2 μg/mL and 8 μg/mL) were
added to the wells in triplicate. Control wells containing
untreated cells were also included. The plates were incubated
for 48 hours after which the media were aspirated from the
wells. The cells were resuspended in 250 μL of 1X annexin V
binding buffer and 5 μL of propidium iodide were added.
The cells were placed in the dark for 15 minutes. The absorbance was read under an FMAX374 fluorescence
microplate spectrophotometer (Molecular Devices, Sunnyvale, USA) at an excitation wavelength of 544 nm and an
emission wavelength of 620 nm. The plates were frozen to
kill all the cells and then thawed before fluorescence was
measured as before. Growth stimulation/inhibition was
calculated as T/C × 100%, where T and C are the fluorescence readings of the test and control samples, respectively.
T/C greater than 125% indicated stimulation while T/C
<30% indicated cytotoxicity [18].
2.8. Caspase 3 and Caspase 9 Activity Assays. Caspase 3 or
caspase 9 colorimetric assay was conducted as per manufacturer’s instructions (Abcam, UK). RD and L20B cells were
treated with different concentrations of SAHA extracts of 50,
100, and 200 μg/mL and doxorubicin, 2, 4, and 8 μg/mL for
24 hours. The cells were harvested and pelleted at a concentration of 2–5 × 106 cells/mL. The cells were resuspended
in 50 μL lysis buffer and protein concentration determined
using the BCA assay. The protein was diluted to achieve a
concentration of 50–200 μg protein per 50 μL lysis buffer per
each well. To each well was added 50 μL of cytosolic extract
and 50 μL of 2X reaction buffer containing 10 mM
Dithiothreitol (DTT). A control well was included containing cytosolic extract from untreated cells and background well containing 50 μL of reaction buffer (with 10 mM
DTT). An aliquot of 5 μL of DEVD-p-NA (caspase 3 substrate) or LEHD-p-NA (caspase 9 substrate) was added and
the mixture incubated for 60 minutes at 37°C in a CO2
incubator (Biobase, China). After incubation, the absorbance was read at 405 nm in an ELISA reader (Biobase,
China). Background reading was subtracted from the
samples before calculating fold increase. Fold increase in
caspase 3 and caspase 9 was determined by comparing
treated sample results to the level of untreated control.
3
2.9. Antioxidant Capacity of Treated Cells
2.9.1. Peroxidase Assay. The peroxidase assay was done
using a peroxidase assay kit (KPL Inc., Germany) following
the manufacturer’s recommendations with some modifications. Treated and untreated RD and L20B cells were
subjected to lysis buffer and the protein concentration was
determined. An aliquot (10 μL) of sample or standard was
added to 96 well plate followed by 170 μL of 50 mM
phosphate buffer, pH 7.0. An aliquot of 50 μL microwell
peroxidase substrate was added into each well and mixed.
The reaction was initiated by adding 75 μL of hydrogen
peroxide working solution, prepared by mixing 10 mL of
50 mM phosphate buffer and 34 μL of 30% v/v of hydrogen
peroxide. The reaction was done in duplicate. Absorbance
was read at 620 nm at 75 second intervals. Peroxidase activity
was calculated using the equation obtained from linear
regression of the standard curve.
2.9.2. Thiobarbituric Acid Reacting Substance (TBARS)
Assay. This assay was used to monitor lipid peroxidation
which is a major indicator of oxidative stress. TBARS are
expressed in terms of malonaldehyde (MDA) equivalents; as
such MDA standard was used to construct a standard curve.
Treated and untreated RD and L20B cells were subjected to
lysis buffer and protein concentration determined. An aliquot (12.6 μL) of 100% ethanol was added to 100 μL of
supernatant in a tube, followed by 100 μL of orthophosphoric acid. The mixture was vortexed for 10 seconds after
which 12.5 μL of thiobarbituric acid (TBA) reagent (0.11 M
in 0.1 M sodium hydroxide) were added. The mixture was
further mixed for 10 seconds and heated in a 90°C water bath
for 45 minutes. The mixture was transferred to ice for 2
minutes in order to stop the reaction. The mixture was
transferred to room temperature for 5 minutes, after which
1000 μL of n-butanol and 100 μL of 0.14 M NaCI were added.
The mixture was then vortexed for 10 seconds. The samples
were centrifuged at 12,000 ×g for 2 minutes at 4°C. A volume
(250 μL) of the top butanol phase was added into each well of
the 96 well plate. The reaction was done in triplicate. The
absorbance was read at 532 nm on a microplate reader
(Biobase, China). TBARS results were expressed in nmoles/
mg protein.
2.10. Statistical Analysis. Statistical analysis was carried out
with GraphPad Prism (Graphpad Software Inc., San Diago,
California, USA) version 5.0 using ANOVA followed by
Turkey’s test (p values < 0.05 regarded as significant).
3. Results
3.1. Yield of Extracts. The yield of phytochemicals increased
with increase in solvent polarity, as shown in Table 1. The
aqueous fractions had the highest percentage yield of
phytochemicals followed by methanol, acetone, and
dichloromethane fractions in both Sesamum angustifolium
(Oliv.) Engl. and Hibiscus articulatus leaves. The aqueous
fraction of both Sesamum angustifolium (Oliv.) Engl. and
4
Evidence-Based Complementary and Alternative Medicine
Table 1: Yield of Sesamum angustifolium (Oliv.) Engl. and Hibiscus articulatus crude leaf extracts.
Plant species
Sesamum angustifolium (Oliv.) Engl.
Hibiscus articulatus
Dichloromethane
7.1 ± 3.1
7.1 ± 2.0
Yield (%)
Acetone
16.7 ± 1.4
16.9 ± 2.3
Methanol
18.5 ± 1.1
20.5 ± 1.0
Water
23.1 ± 3.0
23.4 ± 3.2
Results are expressed as mean ± SD (n � 3).
Hibiscus articulatus had similar yield of 23%. There was no
significant difference (p > 0.05) in the yield of the methanolic
fractions of Hibiscus articulatus (20.5 ± 1.0%) and Sesamum
angustifolium (Oliv.) Engl. (18.5 ± 1.1%) respectively. The
yield of dichloromethane fractions of both Sesamum
angustifolium (Oliv.) Engl. (7.1 ± 3.1%) and Hibiscus articulatus (7.1 ± 2.0%) was similar. The yield of the acetone
extracts was also similar for both S. angustifolium (Oliv.)
Engl. (16.7 ± 1.4) and H. articulatus (16.9 ± 2.3).
3.2. Determination of Cytotoxicity. As shown in Table 2, the
cell viability in both RD and L20B cells decreased with the
increase in the concentration of extract. L20B cells had
higher cell viability compared to RD cells for all treatments.
Doxorubicin recorded significantly lower cell viability for
RD cells compared to all SAHA extracts at all concentrations
used.
Doxorubicin was used as a reference anticancer drug. It had
the highest cytotoxicity effect on RD cells (IC50 � 0.8 μg/mL),
compared to SAHA fractions (Table 3). Among the SAHA
fractions, dichloromethane extract had the highest cytotoxicity
effect on RD cells with IC50 value of 106 μg/mL followed by
methanol extract (IC50 � 122 μg/mL), aqueous extract
(IC50 � 129 μg/mL), and acetone (IC50 � 158 μg/mL).
3.3. Determination of the Effect of SAHA Extracts on Cell
Morphology. The effect of SAHA extracts and doxorubicin
on RD and L20B cell morphology is shown in Figures 1 and 2
respectively. Both untreated RD and L20B cells appeared in
normal shape with 70–100% confluence. Treated RD cells
showed loss of cell adherence, shrinking of cells, and reduced
cell density along with cell debris (Figure 1). The alteration in
cell morphology was intense in cells treated with doxorubicin, dichloromethane extract, and methanol extract followed by acetone extract and lastly aqueous extract. The
morphological alterations seemed to follow the IC50 trend
observed. Treated L20B cells did not have any loss of cell
adherence, shrinking of cells, or reduced cell density as
shown in Figure 2. The treated cells appeared normal in
shape with 50–70% confluence.
3.4. Determination of the Effect of SAHA Extracts Using
Propidium Iodide. As shown in Table 4, cytotoxicity induction was observed in both SAHA extracts and doxorubicin (T/C% range 93%–48%). Of the extracts, the highest
apoptotic induction effect of T/C 48.4 + 2.7%, was observed
in RD cells treated with 200 μg/mL methanol. Aqueous
extract (50 μg/mL) recorded the lowest cytotoxicity effect on
RD cells of T/C 93.0 + 10.6%. A significant apoptotic
Table 2: Cell viability of RD and L20B cells after 48-hour treatment
with SAHA extracts and doxorubicin.
SAHA extract
Dichloromethane
Dichloromethane
Dichloromethane
Dichloromethane
Dichloromethane
Acetone
Acetone
Acetone
Acetone
Acetone
Methanol
Methanol
Methanol
Methanol
Methanol
Aqueous
Aqueous
Aqueous
Aqueous
Aqueous
Doxorubicin
Doxorubicin
Doxorubicin
Doxorubicin
Doxorubicin
Concentration
(μg/mL)
200
100
50
25
12.5
200
100
50
25
12.5
200
100
50
25
12.5
200
100
50
25
12.5
8
4
2
1
0.5
Cell viability (%)
RD cells
L20B cells
64.5 ± 4.1∗
39.5 ± 5.1a
71.4 ± 6.0∗
45.4 ± 9.7a
72.0 ± 8.6∗
47.1 ± 5.7a
63.1 ± 4.6
78.2 ± 5.8∗
77.0 ± 7.3
85.6 ± 2.1
68.4 ± 7.2∗
43.4 ± 4.9a
61.6 ± 3.5
72.3 ± 9.2∗
73.3 ± 7.3
74.4 ± 12.1
79.2 ± 5.6
74.3 ± 10.2
84.3 ± 5.5
92.9 ± 5.7
40.0 ± 6.2a 79.3 ± 4.4∗∗∗
48.3 ± 5.6a
80.6 ± 8.9∗
64.1 ± 3.5
86.0 ± 5.0∗
63.8 ± 6.3
86.2 ± 3.0∗
76.6 ± 10.1
89.7 ± 4.8
62.5 ± 1.8∗
41.4 ± 3.0a
63.2 ± 3.8
50.7 ± 1.6a
66.9 ± 4.5
58.9 ± 5.8a
74.3 ± 4.0
70.3 ± 2.7
83.6 ± 1.3
79.4 ± 8.6
30.5 ± 2.4a 59.2 ± 7.5b∗
34.2 ± 4.1a 71.0 ± 9.4∗∗∗
40.1 ± 5.4a 73.9 ± 6.1∗∗∗
44.4 ± 6.8a 77.5 ± 10.0∗∗∗
57.2 ± 11.1a 81.9 ± 5.3∗
The number of live negative control cells (0 μg/mL) at 48 h was used as 100%
viability, so all cells were expressed as a percentage of them. Results
are expressed as mean ± SD (n � 3). a and b represents significant difference (p < 0.001), compared to untreated RD and L20B cells, respectively.
∗
indicates p < 0.05 and ∗∗∗ indicates p < 0.0001 when comparing RD cells
to L20B cells.
Table 3: IC50 values for treated RD cells.
Extract
Doxorubicin
Dichloromethane
Methanol
Aqueous
Acetone
RD cells IC50 (μg/mL)
0.8
106
122
129
158
induction effect was observed in RD cells treated with
200 μg/mL of all SAHA extracts compared to treated
L20B cells.
3.5. Induction Effects of SAHA Extracts on Caspase-3 and
Caspase-9 Activity. As shown in Figure 3, caspase 3 activity
in RD cells significantly increased several folds in a
Evidence-Based Complementary and Alternative Medicine
Control
Acetone (0.158mg/ml)
5
Doxorubicin
(0.0008mg/ml)
Dichloromethane
(0.106mg/ml)
Methanol (0.122mg/ml)
Aqueous (0.129 mg/ml)
Figure 1: Morphological alterations on RD cells at IC50 of SAHA extracts and doxorubicin. Photomicrographs taken at 400x magnification
using a Japson MD130 digital camera. The pictures were analyzed using a future win joe software for windows. Untreated RD cells (control)
appeared in normal shape with 95–100% confluence. RD cells treated with SAHA extracts and doxorubicin showed loss of cell adherence,
shrinking of cells, and reduced cell density along with cell debris.
Control
Acetone (0.1 mg/ml)
Dichloromethane
(0.1 mg/ml)
Methanol (0.1mg/ml)
Aqueous (0.1mg/ml)
Doxorubicin
(0.004mg/ml)
Figure 2: Morphological alterations on L20B cells at IC50 of SAHA extracts and doxorubicin. Photomicrographs taken at 400x magnification using a Japson MD130 digital camera. The pictures were analyzed using a future win joe software for windows. Untreated
(control) and treated L20B cells appeared in normal shape with 50–75% confluence. No morphological alterations, characteristic of
apoptosis, were observed in the treated cells.
concentration-dependent manner between 50 μg/mL and
200 μg/mL of SAHA extracts and between 2 μg/mL and 8 μg/
mL of doxorubicin. For the dichloromethane, methanol and
aqueous extracts significant increase in caspase 3 activity
(p < 0.0001) was observed between 50 μg/mL and 200 μg/mL
concentrations of the extract. For the acetone extract significant increase in caspase 3 activity (p < 0.001) was observed between 50 μg/mL and 100 μg/mL concentrations of
extracts but no significant difference was observed beyond
100 μg/mL concentration of the extract. A significant increase in caspase 3 activity (p < 0.0001) was observed between 2 μg/mL and 8 μg/mL concentrations of doxorubicin.
Methanol fraction gave the highest fold increase in caspase 3
activity (3.8-fold increase), followed by dichloromethane
(3.4-fold increase), acetone (0.9-fold increase), and aqueous
(0.8-fold increase) at 200 μg/mL. Doxorubicin gave a fold
increase of 1.6 at 8 μg/mL concentration.
As shown in Figure 4, caspase 3 activity in L20B cells
increased in a concentration-dependent manner between
50 μg/mL and 200 μg/mL of SAHA extracts and between
2 μg/mL and 8 μg/mL of doxorubicin. For the dichloromethane, methanol and aqueous extracts significant increase
in caspase 3 activity (p < 0.0001) was observed between
50 μg/mL and 200 μg/mL concentrations of extracts. A
significant increase in the activity of caspase 3 (p < 0.001)
was observed between 50 μg/mL and 100 μg/mL
Evidence-Based Complementary and Alternative Medicine
Table 4: Propidium iodide assay to determine apoptosis induction
effect of SAHA extracts and doxorubicin in RD and L20B cells after
48 hour treatment.
Viability
RD cells
49.7 ± 5.7
68.2 ± 8.1
55.1 ± 3.2
63.5 ± 12.3
60.9 ± 1.0
66.6 ± 8.6
48.4 ± 2.7
50.9 ± 7.8
72.1 ± 3.2
93.0 ± 10.6
Concentration
(μg/mL)
Extract
Doxorubicin
Dichloromethane
Acetone
Methanol
Aqueous
8
2
200
50
200
50
200
50
200
50
(T/C) (%)
L20B cells
57.7 ± 17.2
68.2 ± 14.7
66.7 ± 14.4∗
60.0 ± 10.1
78.3 ± 33.6∗
60.5 ± 4.5
65.1 ± 10.8∗
55.6 ± 13.0
85.9 ± 13.4∗
83.4 ± 10.0∗
The number of live negative control cells (0 μg/mL) at 48 h was used as 100%
viability, so all cells were expressed as a percentage of them. Results are
mean ± SD of two replicates (n � 2). ∗ indicates p < 0.05 comparing treated
RD cells to treated L20B cells of similar concentration.
∗∗∗
c
4
c
0.4
c
c
c
0.3
c
b
0.2
a
a
∗
b
∗∗
0.1
0.0
500
100
Concentration (µg/mL)
200
Methanol
Aqueous
Doxorubicin
Dichloromethane
Acetone
Figure 4: Effects of SAHA extracts and doxorubicin on the activation of caspase-3 in L20B cells. Results are mean ± SD (n � 2). a
represents significant difference (p < 0.01), b represents p < 0.001,
and c represents p < 0.0001 when compared to 50 μg/ml extract or
2 μg/ml doxorubicin.(∗) represents p < 0.05 and (∗∗) represents
p < 0.01 when comparing to 8 μg/mL doxorubicin.
∗∗
c
3
1.0
2
c
b
b
1
a
c
b
0
50
100
Concentration (µg/mL)
Doxorubicin
Dichloromethane
Acetone
200
Methanol
Aqueous
Figure 3: Effects of SAHA extracts and doxorubicin on the activation of caspase-3 in RD cancer cells. Results are mean ± SD
(n � 2). a represents significant difference (p < 0.01), b represents
p < 0.001, and c represents p < 0.0001 when comparing to 50 μg/ml
extract or 2 μg/ml doxorubicin. (∗∗) represents p < 0.01 and (∗∗∗)
represents p < 0.001 when compared to 8 μg/mL doxorubicin.
concentrations of the acetone extract. No significant increase
in caspase 3 activity was observed beyond 100 μg/mL concentration of the acetone extract. A significant increase in
caspase 3 activity (p < 0.0001) was observed between 2 μg/
mL and 8 μg/mL concentrations of doxorubicin. However,
the fold increase in caspase 3 for L20B cells was significantly
lower than that for RD cells. Doxorubicin (8 μg/mL) and
dichloromethane fraction (200 μg/mL) gave the highest fold
increase of 0.4, followed by methanol (0.36 fold increase),
aqueous (0.2 fold increase), and acetone (0.1 fold increase) at
200 μg/mL.
As shown in Figure 5, caspase 9 activity in RD cells
increased in a concentration-dependent manner between
Relative fold increase of caspase-9
Relative fold increase of caspase-3
5
0.5
Relative fold increase of caspase-3
6
0.8
c
b
0.6
c
a
0.4
c
c
c
a
0.2
0.0
50
100
Concentration (µg/mL)
Doxorubicin
Dichloromethane
Acetone
200
Methanol
Aqueous
Figure 5: Effects of SAHA extracts and doxorubicin on the activation of caspase-9 in RD cells. Results are mean ± SD (n � 2). a
represents significant difference (p < 0.01), b represents p < 0.001,
and c represents p < 0.0001 when compared to 50 μg/ml extract or
2 μg/ml doxorubicin.
50 μg/mL and 200 μg/mL of SAHA extracts and between
2 μg/mL and 8 μg/mL of doxorubicin. For the dichloromethane, acetone, methanol, and aqueous extracts significant increase in caspase 9 activity (p < 0.0001) was observed
between 50 μg/mL and 200 μg/mL concentrations of extracts. A significant increase in the activity of caspase 9
(p < 0.0001) was observed between 2 μg/mL and 4 μg/mL
doxorubicin. No significant increase in caspase 9 activity was
observed beyond 4 μg/mL concentration of doxorubicin. At
Evidence-Based Complementary and Alternative Medicine
0.6
Relative fold increase of caspase-9
200 μg/mL, dichloromethane fraction gave the highest fold
increase of 0.8 followed by aqueous (0.6-fold increase),
acetone (0.5-fold increase), and methanol (0.4-fold increase). Doxorubicin gave a fold increase of 0.5 at 8 μg/mL
concentration.
As shown in Figure 6, caspase 9 activity in L20B cells
increased in a concentration-dependent manner for some of
the SAHA extracts and doxorubicin. For the dichloromethane, methanol and aqueous extracts, significant increase in caspase 9 activity was observed between 50 μg/mL
and 200 μg/mL concentrations of the extract. No significant
difference was observed in caspase 9 activity at increasing
concentrations of the acetone extract. A significant increase
in the activity of caspase 9 (p < 0.0001) was observed between 2 μg/mL and 4 μg/mL doxorubicin. No significant
increase in caspase 9 activity was observed beyond 4 μg/mL
concentration of doxorubicin. The fold increase in caspase 9
activity for L20B cells was lower compared to that for RD
cells. Methanol extract gave the highest fold increase of 0.4,
followed by aqueous (0.3-fold increase), dichloromethane
(0.26-fold increase), and acetone (0.2-fold increase) at
200 μg/ml.
7
c
c
0.4
∗
b
c
∗
c
∗
b
0.2
a
0.0
50
100
Concentration (µg/mL)
Doxorubicin
Dichloromethane
Acetone
200
Methanol
Aqueous
Figure 6: Effects of SAHA extracts and doxorubicin on the activation of caspase-9 in L20B cells. Results are mean ± SD (n � 2). a)
represents significant difference (p < 0.01), b represents p < 0.001,
and c represents p < 0.0001 when compared to 50 μg/ml extract or
2 μg/ml doxorubicin. (∗) represents p < 0.05 when compared to
8 μg/mL doxorubicin.
3.6. Antioxidant Capacity of Treated Cells
3.6.2. Determination of Lipid Peroxidation Levels in Treated
and Untreated RD and L20B Cells. As shown in Figure 8, the
lipid peroxidation levels in treated RD and L20B cells were
significantly lower (p < 0.05) than in untreated RD and
L20B cells. There was no significant difference in the levels of
lipid peroxidation between treated RD cells and treated
L20B cells.
4. Discussion
Rhabdomyosarcoma is among the world’s major public
health burden and accounts for high mortality rates in
paediatrics [19]. Clinical approaches in the treatment of
rhabdomyosarcoma are often associated with high expense,
increased toxicity, and adverse side effects. Anticancer
8
∗
b
6
U/mg protein
∗
∗∗∗
b
b
b
a
a
a
4
a
b
2
Aqueous
Methanol
Acetone
Dichloromethane
Doxorubicin
0
Untreated
3.6.1. Determination of Peroxidase Activity of Treated and
Untreated RD and L20B Cells. As shown in Figure 7, the
activity of peroxidase was significantly higher in treated RD
and L20B cells compared to untreated RD and L20B cells
(p < 0.05). Treated L20B cells had a higher peroxidase activity compared to the treated RD cells, with dichloromethane fraction giving the highest peroxidase activity of
5.4 U/mg in L20B cells which was significantly higher
compared to 3.4 U/mg recorded in RD cells, p < 0.05.
Doxorubicin gave a significantly higher peroxidase activity
of 4.9 U/mg in L20B cells compared to 3.3 U/mg recorded in
RD cells, p < 0.05. Acetone and aqueous extracts gave higher
peroxidase activities of 4.4 U/mg in L20B cells, with the
peroxidase activity of aqueous significantly higher in
L20B cells compared to RD cells (1.1 U/mg, p < 0.001).
Methanol fraction recorded the lowest peroxidase activity of
3.3 U/mg in RD cells and 3.2 U/mg in L20B cells.
RD cells
L20B cells
Figure 7: Peroxidase activity in RD and L20B cells after 24 hour
treatment with SAHA extracts (200 μg/ml) and doxorubicin (8 μg/
ml). Results are mean ± SD (n � 2). a and b represent p < 0.05
compared to untreated RD and L20B cells, respectively. (∗) represents p < 0.05, and (∗∗∗) represents p < 0.001 when comparing
RD cells to L20B cells.
compounds that can arrest the cell cycle and induce apoptosis could pave a way for cancer treatment. Phytochemicals such as terpenes, flavonoids, and polyphenols have
shown the ability to upregulate apoptotic proteins caspase-3/
9 involved in an active form of cell death called apoptosis
[20–22].
8
Evidence-Based Complementary and Alternative Medicine
MDA nmol/mg protein
2.0
1.5
1.0
0.5
Aqueous
Methanol
Acetone
Dichloromethane
Doxorubicin
Untreated
0.0
L20B cells
RD cells
Figure 8: Lipid peroxidation levels in RD and L20B cells after 24hour treatment with SAHA extracts (200 μg/mL) and doxorubicin
(8 μg/mL). Results are mean ± SD (n � 3).
Phytochemicals extracted from the leaves of Hibiscus
articulatus and Sesamum angustifolium (Oliv.) Engl. were
investigated for their combined effects on rhabdomyosarcoma after extraction using different solvents of varying
polarity. Of the four extracts that were studied, the
dichloromethane extract (IC50, 106 μg/mL) recorded the
highest cytotoxicity towards RD cells (Table 3). As shown
in Table 2, the dichloromethane fraction had a stronger
cytotoxic effect on cancerous RD cells compared to the
noncancerous L20B cells. The cancer selective character
displayed by the dichloromethane fraction has been reported by other workers [23, 24]. The cytotoxic selective
character is essential in all potential chemotherapeutic
drugs enabling the killing of cancerous cells without
harming normal cells.
According to [25, 26], cell death is characterized by
morphological changes in a cell. In our study (Figures 6 and
7), untreated RD cells were spindle shaped exhibiting crossstriation, a characteristic of normal muscle cells, and adhered to the surface. Treatment of the cells with IC50 of
SAHA extracts resulted in morphological changes which
included loss of surface adherence, cell shrinkage characterized by round shape, reduced cell density along with cell
debris, as shown in Figure 6. The morphological changes
observed are characteristic of apoptosis in adherent cells
[20]. The results obtained are consistent with those of
[27, 28] showing morphological changes characteristic of
apoptosis in various cancerous cells, breast (MCF-7 cells),
liver (HepG2 cells), lung (NCI-H23), and colon (HT-29).
Morphological changes that characterize cell death were
not observed in L20B cells treated with SAHA extracts
(Figure 7). The treated cells adhered to the surface,
retaining the same morphology as those of the untreated
cells.
Propidium iodide assay can be used to detect cell death.
Propidium iodide stains DNA of cells with damaged
membranes. The number of dead cells was expressed as a
ratio of total cells present and percentage cell viability
expressed as a percentage proportion relative to the untreated cells. The low cell viability obtained at the highest
concentration of extracts and doxorubicin in treated RD
cells (Table 4) shows that apoptosis induction was more
pronounced at higher concentrations of extracts compared
to the low concentrations. This could be due to a higher
concentration of phytochemicals present at a high concentration of extracts. A similar trend was observed by [18]
on Jukart cells after 4 days of treatment with Triumfetta
welwitschii. As doxorubicin was used as the reference cancer
drug, the highest concentration gave a strong apoptosis
induction effect in treated RD cells which was significantly
higher compared to untreated cells. This shows the effectiveness of doxorubicin as a known anticancer drug against
RD cells. The mechanism of cell death induction by
doxorubicin is p-53 mediated apoptosis. The apoptotic induction effect of SAHA extracts and doxorubicin were more
pronounced in treated RD cells than treated L20B cells
(Table 4). In treated L20B cells, the cytotoxicity effect was
above 50%, depicting that there were more viable cells as
compared to cell death by apoptosis. This could be attributed
to the low toxicity of SAHA extracts on L20B cells as
compared to RD cells.
The activation of caspase 3 and caspase 9 was evaluated
to determine the death pathway induced by SAHA extracts.
Caspase 3 is the most important protein involved in the
execution pathway of apoptosis. The activation of caspase 3
regulates cell proliferation and reduces the onset of cancers.
SAHA extracts showed a concentration-dependent increase
in the activation of caspase 3 activity, which may be attributed to the increase in phytochemical content. Phytochemicals have been previously reported for the apoptotic
induction effect [29]. The low caspase 3 induction effect of
acetone and water extract at 200 μg/mL could be due to the
low content of anticancer compounds present. The induction of caspase 3 by SAHA extracts in RD cells was ten times
higher than that observed in L20B cells, Figure 4. The activity
of caspase 3 in normal cells is highly regulated by a group of
proteins collectively known as inhibitors of apoptosis [30].
The level of caspase 3 is usually maintained at low concentrations compared to the level of procaspase 3 [30]. This
result is very low apoptosis induction in normal cells. Apoptosis is a protective mechanism that maintains cell homeostasis by removing only rogue cells from tissue milieu
[31].
In the intrinsic pathway of apoptosis, the activity of
caspase 3 is initiated by initiator caspases such as caspase 9.
From the results obtained in Figures 5 and 6 there was no
direct relationship in the induction levels of caspase 9 and
caspase 3. The activation of caspase 9 was four times lower
than that observed in caspase 3 in RD cells. Badmus and
others reported a low caspase 9 induction effect of Holarrhena floribunda (G. Don) extracts on the breast (MCF-7),
colorectal (HT-29), and cervical (HeLa) cancer cells [32].
Our study also observed a low activity of caspase 9 in
Evidence-Based Complementary and Alternative Medicine
L20B cells (Figure 6). This was expected as normal cells do
not normally undergo apoptosis unless a mutation in DNA
arises. Further work is required to determine the actual
pathway of cell death by apoptosis.
The SAHA extracts enhanced the activity of the antioxidant enzyme peroxidase, as shown in Figure 7. The
peroxidase activity in untreated RD and L20B cells was
significantly lower compared to the treated cells. Normal
cells are protected by antioxidant enzymes from the toxic
effects of high concentrations of reactive oxygen species
generated during cellular metabolism. Even though cancer
cells generate reactive oxygen species, it has been demonstrated biochemically that antioxidant enzyme levels are low
in most animal and human cancers [33].
The activity of antioxidant enzymes in treated L20B cells
was significantly higher than that of treated RD cells in
dichloromethane and aqueous extracts, and doxorubicin.
Lipid peroxidation is one of the major indications of
oxidative stress in cells. One of the byproducts of lipid
peroxidation is MDA which is considered to be a mutagen.
MDA has been previously used as a biomarker for lipid
peroxidation in several in vitro studies [34, 35]. High levels
of lipid peroxidation indicate high oxidative stress and have
been observed in different types of cancers such as breast
cancer and lung cancer [36–38]. As shown in Figure 8,
SAHA extracts demonstrated the ability to significantly
reduce lipid peroxidation in pretreated RD and L20B cells.
Untreated RD and L20B cells had significantly higher levels
of MDA compared to the treated cells. The high levels of
MDA in untreated RD cells are an indication of high oxidative stress in cancerous cells which is a result of depletion
of antioxidant enzymes [39]. The phytochemicals in SAHA
extracts prevent lipid peroxidation by binding to lipid
peroxides. Our findings are in agreement with previously
reported results by other workers [40, 41].
5. Conclusion
The combined leaf extracts of Sesamum angustifolium (Oliv.)
Eng. and Hibiscus articulatus (SAHA) were selectively cytotoxic to RD cells than L20B cells. The leaf extracts demonstrated a powerful induction of caspase 3 in RD cells than
L20B cells. RD and L20B cells pretreated with the combined
leaf extracts showed enhanced antioxidant capacity against
hydrogen peroxide induced damage and orthophosphoric
acid induced lipid peroxidation. Further in vivo studies are
being done to make it clear whether the combined leaf
extracts could be proposed as a natural agent for the prevention and treatment of rhabdomyosarcoma.
Data Availability
The data used to support the findings of this study are
available from the corresponding author upon request.
Conflicts of Interest
The authors declare that there are no conflicts of interest
regarding the publication of this paper.
9
Acknowledgments
The authors would like to acknowledge Mrs. Berejena and
Mr. Ruhanya from the Medical Microbiology Department,
College of Health Sciences, University of Zimbabwe, for
technical assistance and the Department of Preclinical
Veterinary Sciences for laboratory space. This work was
supported by Third World Academy of Sciences (TWAS)
(grant number 17-428 RG/BIO/AF/AC_I-FR3240297762).
References
[1] F. Bray, J. Ferlay, I. Soerjomataram, R. L. Siegel, L. A. Torre,
and A. Jemal, “Global cancer statistics 2018: GLOBOCAN
estimates of incidence and mortality worldwide for 36 cancers
in 185 countries,” CA: A Cancer Journal for Clinicians, vol. 68,
no. 6, pp. 394–424, 2018.
[2] S.-G. Kim, M.-I. Hahm, K.-S. Choi, N.-Y. Seung, H.-R. Shin,
and E.-C. Park, “The economic burden of cancer in Korea in
2002,” European Journal of Cancer Care, vol. 17, no. 2,
pp. 136–144, 2008.
[3] D. Parham and D. Ellison, “Rhabdomyosarcomas in adults
and children: an update,” Archives of Pathology & Laboratory
Medicine, vol. 130, pp. 1454–1465, 2006.
[4] R. Dagher and L. Helman, “Rhabdomyosarcoma: an overview,” The Oncologist, vol. 4, no. 1, pp. 34–44, 1999.
[5] D. Egas-Bejar and W. W. Huh, “Rhabdomyosarcoma in
adolescent and young adult patients: current perspectives,”
Adolescent Health, Medicine and Therapeutics, vol. 5,
pp. 115–125, 2014.
[6] D. C. Stefan, “Patterns of distribution of childhood cancer in
Africa,” Journal of Tropical Pediatrics, vol. 61, no. 3,
pp. 165–173, 2015.
[7] C. Rodrı́guez-Galindo, T. Liu, M. J. Krasin et al., ““Analysis of
prognostic factors in ewing sarcoma family of tumors: review
of St. Jude Children’s Research Hospital studies,” Cancer,
vol. 110, no. 2, pp. 375–384, 2007.
[8] M. L. Blakely, T. E. Lobe, J. R. Anderson et al., “Does
debulking improve survival rate in advanced-stage retroperitoneal embryonal rhabdomyosarcoma?” Journal of Pediatric Surgery, vol. 34, no. 5, pp. 736–742, 1999.
[9] A. G. Atanasov, B. Waltenberger, E.-M. Pferschy-Wenzig
et al., “Discovery and resupply of pharmacologically active
plant-derived natural products: a review,” Biotechnology
Advances, vol. 33, no. 8, pp. 1582–1614, 2015.
[10] A. K. Panda, D. Chakraborty, I. Sarkar, T. Khan, and G. Sa,
“New insights into therapeutic activity and anticancer
properties of curcumin,” Journal of Experimental Pharmacology, vol. 9, pp. 31–45, 2017.
[11] A. Hosseini and A. Ghorbani, “Cancer therapy with phytochemicals: evidence from clinical studies,” Avicenna Journal
of Phytomedicine, vol. 5, no. 2, p. 84, 2015.
[12] M. Abotaleb, S. Samuel, E. Varghese et al., “Flavonoids in
cancer and apoptosis,” Cancers (Basel), vol. 11, no. 1, 2018.
[13] P. Pratheeshkumar, C. Sreekala, Z. Zhang et al., “Cancer
prevention with promising natural products: mechanisms of
action and molecular targets,” Anti-Cancer Agents in Medicinal Chemistry, vol. 12, no. 10, pp. 1159–1184, 2012.
[14] H. J. Park, M.-J. Kim, E. Ha, and J.-H. Chung, “Apoptotic
effect of hesperidin through caspase3 activation in human
colon cancer cells, SNU-C4,” Phytomedicine, vol. 15, no. 1-2,
pp. 147–151, 2008.
10
[15] C. Chidewe, U. F. Castillo, and D. S. Sem, “Structural analysis
and antimicrobial activity of chromatographically separated
fractions of leaves of Sesamum angustifolium(Oliv.) Engl,”
Journal of Biologically Active Products from Nature, vol. 7,
no. 6, pp. 463–474, 2017.
[16] Hibiscus articulatus Hochst. ex A.Rich. (family MALVACEAE) on JSTOR, 2019, https://plants.jstor.org/stable/10.
5555/al.ap.specimen.k000240734.
[17] D. M. Fernando, R. L. Wijesundera, P. Soysa, D. de Silva, and
C. M. Nanayakkara, “Antioxidant potential, in vitro cytotoxicity and apoptotic effect induced by crude organic extract
of Anthracophyllum lateritium against RD sarcoma cells,”
BMC Complementary and Alternative Medicine, vol. 15, no. 1,
2015.
[18] B. Moyo and S. Mukanganyama, “Antiproliferative activity of
T. welwitschii extract on Jurkat T cells in vitro,” BioMed
Research International, vol. 2015, Article ID 817624, 10 pages,
2015.
[19] S. Malempati, D. A. Rodeberg, S. S. Donaldson et al.,
“Rhabdomyosarcoma in infants less than one year of age: a
report from the children’s oncology group,” Cancer, vol. 117,
no. 15, pp. 3493–3501, 2011.
[20] M. Kumar, V. Kaur, S. Kumar, and S. Kaur, “Phytoconstituents as apoptosis inducing agents: strategy to combat
cancer,” Cytotechnology, vol. 68, no. 4, pp. 531–563, 2016.
[21] S. Upadhyay and M. Dixit, “Role of polyphenols and other
phytochemicals on molecular signaling,” Oxidative Medicine
and Cellular Longevity, vol. 2015, Article ID 504253, 15 pages,
2015.
[22] S. Sukumari-Ramesh, J. N. Bentley, M. D. Laird, N. Singh,
J. R. Vender, and K. M. Dhandapani, “Dietary phytochemicals
induce p53- and caspase-independent cell death in human
neuroblastoma cells,” International Journal of Developmental
Neuroscience, vol. 29, no. 7, pp. 701–710, 2011.
[23] M. M. Suhail, W. Wu, A. Cao et al., “Boswellia sacra essential
oil induces tumor cell-specific apoptosis and suppresses tumor aggressiveness in cultured human breast cancer cells,”
BMC Complementary and Alternative Medicine, vol. 11, no. 1,
p. 129, 2011.
[24] S. L. Moses and V. M. Edwards, “Cytotoxicity in MCF-7 and
MDA-MB-231 breast cancer cells, without harming MCF-10a
healthy cells,” Journal of Nanomedicine & Nanotechnology,
vol. 7, no. 2, 2016.
[25] Y. Zhang, X. Chen, C. Gueydan, and J. Han, “Plasma
membrane changes during programmed cell deaths,” Cell
Research, vol. 28, no. 1, pp. 9–21, 2018.
[26] D. Wlodkowic, W. Telford, J. Skommer, and Z. Darzynkiewicz,
“Apoptosis and beyond: cytometry in studies of programmed
cell death,” Methods in Cell Biology, vol. 103, pp. 55–98, 2011.
[27] M. P. Sobantu, The Antioxidative and Cytotoxic Effects of
Hibiscus Sabdariffa on Mcf7 and Mcf12a Breast Cell Lines,
Cape Peninsula University of Technology, Cape Town, South
Africa, 2015.
[28] R. Basha, S. F. Connelly, U. T. Sankpal et al., “Small molecule
tolfenamic acid and dietary spice curcumin treatment enhances antiproliferative effect in pancreatic cancer cells via
suppressing Sp1, disrupting NF-kB translocation to nucleus
and cell cycle phase distribution,” The Journal of Nutritional
Biochemistry, vol. 31, pp. 77–87, 2016.
[29] C. Kandaswami, “The antitumor activities of flavonoids,” In
Vivo, vol. 19, no. 5, pp. 895–909, 2005.
[30] S. J. Riedl, M. Renatus, R. Schwarzenbacher et al., “Structural
basis for the inhibition of caspase-3 by XIAP,” Cell, vol. 104,
no. 5, pp. 791–800, 2001.
Evidence-Based Complementary and Alternative Medicine
[31] C. Dabrowska, M. Li, and Y. Fan, “Apoptotic caspases in
promoting cancer: implications from their roles in development and tissue homeostasis,” Apoptosis in Cancer Pathogenesis and Anti-cancer Therapy, vol. 930, pp. 89–112, 2016.
[32] J. A. Badmus, O. E. Ekpo, A. A. Hussein, M. Meyer, and
D. C. Hiss, “Antiproliferative and apoptosis induction potential of the methanolic leaf extract of Holarrhena floribunda
(G. Don),” Evidence-Based Complementary and Alternative
Medicine, vol. 2015, Article ID 756482, 11 pages, 2015.
[33] T. D. Oberley and L. W. Oberley, “Antioxidant enzyme levels
in cancer,” Histology & Histopathology, vol. 12, no. 2,
pp. 525–535, 1997.
[34] M. Repetto, J. Semprine, and A. Boveris, “Lipid peroxidation:
chemical mechanism, biological implications and analytical
determination,” in Lipid Peroxidation, IntechOpen, London,
UK, 2012.
[35] A. Ayala, M. F. Muñoz, and S. Argüelles, “Lipid peroxidation:
production, metabolism, and signaling mechanisms of
malondialdehyde and 4-hydroxy-2-nonenal,” Oxidative
Medicine and Cellular Longevity, vol. 2014, Article ID 360438,
31 pages, 2014.
[36] M. Kedzierska, B. Olas, B. Wachowicz, A. Jeziorski, and
J. Piekarski, “The lipid peroxidation in breast cancer patients,”
General Physiology and Biophysics, vol. 29, no. 2, pp. 208–210,
2010.
[37] O. O. Erejuwa, S. A. Sulaiman, and M. S. Ab Wahab, “Evidence in support of potential applications of lipid peroxidation products in cancer treatment,” Oxidative Medicine
and Cellular Longevity, vol. 2013, Article ID 931251, 8 pages,
2013.
[38] M. Zieba, D. Nowak, M. Suwalski et al., “Enhanced lipid
peroxidation in cancer tissue homogenates in non-small cell
lung cancer,” Monaldi Archives for Chest Disease, vol. 56,
no. 2, pp. 110–114, 2001.
[39] G.-Y. Liou and P. Storz, “Reactive oxygen species in cancer,”
Free Radical Research, vol. 44, no. 5, pp. 479–496, 2010.
[40] G. A. Engwa, “Free radicals and the role of plant phytochemicals as antioxidants against oxidative stress-related
diseases,” in Phytochemicals—Source of Antioxidants and Role
in Disease Prevention, IntechOpen, London, UK, 2018.
[41] A. Sorice, E. Guerriero, M. Volpe et al., “Differential response
of two human breast cancer cell lines to the phenolic extract
from flaxseed oil,” Molecules, vol. 21, no. 3, p. 319, 2016.