International Journal of
Molecular Sciences
Article
Vernonia calvoana Shows Promise towards the
Treatment of Ovarian Cancer
Ariane T. Mbemi 1 , Jennifer N. Sims 2 , Clement G. Yedjou 1,3, * , Felicite K. Noubissi 4 ,
Christian R. Gomez 5 and Paul B. Tchounwou 4, *
1
2
3
4
5
*
Natural Chemotherapeutics Research Laboratory, NIH/NIMHD RCMI-Center for Environmental Health,
College of Science, Engineering and Technology, Jackson State University, 1400 Lynch Street, Jackson,
MS 39217, USA; ariane.t.mbemi@jsums.edu
School of Public Health, Jackson State University, Jackson Medical Mall-Thad Cochran Center,
350 West Woodrow Wilson Avenue, Jackson, MS 39213, USA; jennifer.n.sims@jsums.edu
Department of Biological Sciences, College of Science and Technology, Florida Agricultural and Mechanical
University, 1610 S. Martin Luther King Blvd, Tallahassee, FL 32307, USA
Department of Biology, College of Science, Engineering and Technology, Jackson State University,
1400 Lynch Street, Jackson, MS 39217, USA; felicite.noubissi_kamdem@jsums.edu
Departments of Pathology and Radiation Oncology, Center for Clinical and Translational Science,
University of Mississippi Medical Center, 2500 N. State St., Jackson, MS 39216, USA; CRGomez@umc.edu
Correspondence: clement.yedjou@famu.edu (C.G.Y.); paul.b.tchounwou@jsums.edu (P.B.T.);
Tel.: +1-850-599-3908 (C.G.Y.); +1-601-979-2095 (P.B.T.); Fax: +1-601-979-5853 (P.B.T.)
Received: 26 May 2020; Accepted: 15 June 2020; Published: 22 June 2020
Abstract: The treatment for ovarian cancers includes chemotherapies which use drugs such as
cisplatin, paclitaxel, carboplatin, platinum, taxanes, or their combination, and other molecular target
therapies. However, these current therapies are often accompanied with side effects. Vernonia calvoana
(VC) is a valuable edible medicinal plant that is widespread in West Africa. In vitro data in our
lab demonstrated that VC crude extract inhibits human ovarian cancer cells in a dose-dependent
manner, suggesting its antitumor activity. From the VC crude extract, we have generated 10 fractions
and VC fraction 7 (F7) appears to show the highest antitumor activity towards ovarian cancer cells.
However, the mechanisms by which VC F7 exerts its antitumor activity in cancer cells remain largely
unknown. We hypothesized that VC F7 inhibits cell proliferation and induces DNA damage and
cell cycle arrest in ovarian cells through oxidative stress. To test our hypothesis, we extracted and
fractionated VC leaves. The effects of VC F7 were tested in OVCAR-3 cells. Viability was assessed by
the means of MTS assay. Cell morphology was analyzed by acridine orange and propidium iodide
(AO/PI) dye using a fluorescent microscope. Oxidative stress biomarkers were evaluated by the
means of lipid peroxidation, catalase, and glutathione peroxidase assays, respectively. The degree of
DNA damage was assessed by comet assay. Cell cycle distribution was assessed by flow cytometry.
Data generated from the MTS assay demonstrated that VC F7 inhibits the growth of OVCAR-3 cells
in a dose-dependent manner, showing a gradual increase in the loss of viability in VC F7-treated
cells. Data obtained from the AO/PI dye assessment revealed morphological alterations and exhibited
characteristics such as loss of cellular membrane integrity, cell shrinkage, cell membrane damage,
organelle breakdown, and detachment from the culture plate. We observed a significant increase
(p < 0.05) in the levels of malondialdhyde (MDA) production in treated cells compared to the control.
A gradual decrease in both catalase and glutathione peroxidase activities were observed in the
treated cells compared to the control. Data obtained from the comet assay showed a significant
increase (p < 0.05) in the percentages of DNA cleavage and comet tail length. The results of the
flow cytometry analysis indicated VC F7 treatment caused cell cycle arrest at the S-phase checkpoint.
Taken together, our results demonstrate that VC F7 exerts its anticancer activity by inhibiting cell
proliferation, inducing DNA damage, and causing cell cycle arrest through oxidative stress in OVAR-3
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cells. This finding suggests that VC F7 may be a potential alternative dietary agent for the prevention
and/or treatment of ovarian cancer.
Keywords: Vernonia calvoana; OVCAR-3 cells; cell viability; oxidative stress; DNA damage; cell cycle
1. Introduction
Ovarian cancer is classified as the leading cause of death in gynecological cancer among
women [1,2]. Family history of breast or ovarian cancer is the strongest risk factor for developing
ovarian cancer [3]. The American College of Obstetricians and Gynecologists and the Society of
Gynecologic Oncology recommend that females with BRCA1 and/or BRCA2 mutations should consider
the use of oral contraceptives which may reduce the risk of developing ovarian cancer by approximately
50% among high-risk females [4–7]. Due to the location of the ovaries in the female reproductive
system, ovarian cancer is considered a “silent killer”, and over 70% of cases are diagnosed at the
advanced stages [8,9]. Last year, there was an approximate of 22,240 new cases of ovarian cancer
diagnosed and about 14,070 ovarian cancer deaths in North America [10]. Ovarian cancer accounts for
about 2.5% of all malignancies among women worldwide. However, 5% of female cancer deaths are
attributed to low survival rates, largely due to late stage diagnoses [11].
The current first line of treatment for ovarian cancers are chemotherapies which use drugs such as
cisplatin, paclitaxel, carboplatin, platinum, taxanes, or their combination, and other molecular target
therapies [10,12]. However, these therapies for ovarian cancer are usually accompanied with side
effects such as hair loss, loss of appetite, and infertility [13]. About 70% of the patients diagnosed
with recurring ovarian cancer will die within five years of their diagnosis [14]. Medicinal plants are
gaining special consideration and value in the discovery for ovarian cancer drugs. Vernonia calvoana
(VC), commonly called sweet bitter leaf in English, is an Asteraceae that has been widely consumed
as vegetable and used to treat diseases such as diabetes, measles, tuberculosis, hyperlipidemia,
and women infertility in many Africa countries [15,16]. Scientific reports indicated that the leaves of VC
contain phytochemicals such as flavonoids, which are good antioxidants [17–19]. In addition, scientific
reports indicated that VC possesses the hepatoprotective effect and hypolipidemic and antidiabetic
activities [20]. Testing the medicinal property of Vernonia amygdalina, a member of the same genus as
Vernonia calvoana, we demonstrated in our lab that this medicinal plant possesses anticancer activity
potential against human breast cancer cells [21,22]. Other Vernonia species including Vernonia divaricate
and Vernonia amygdalina act as potential anticancer agents that inhibit the proliferation of HL-60 cells,
MCF-7 cells and PC-3 cells [23,24]. Although VC have been used traditionally to treat many illnesses,
the mechanisms by which it exerts its antitumor activity in OVCAR-3 cells remain largely unknown.
Therefore, our objective was to test the therapeutic efficacy of the most bioactive compound of VC
against ovarian cancer.
2. Results
2.1. Antiproliferative Effect
The results showed that VC F7 treatment significantly decreased the viability of OVCAR-3 cells
(Figure 1). As seen in Figure 1, the percentages of viability of OVCAR-3 cells treated with VC F7
upon 48 h were 100 ± 5.73%, 63 ± 2.78%, 43 ± 1.27 %, 31 ± 0.92% in 0, 8, 16, 32 µg/mL, respectively.
These results revealed that the OVCAR-3 cells are more sensitive to VC F7 treatment with an estimated
inhibition dose (IC50 ) equal to 18.56 µg/mL. These data showed that VC F7 caused growth arrest of
OVCAR-3 cells, suggesting its potential as an anticancer agent.
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Figure 1. Antiproliferative effect of Vernonia calvoana fraction 7 (VC F7) on OVCAR-3 cells. The OVCAR-3
cells were treated with different doses of VC F7 for 48 h. The cell viability was measured by MTS assay
as described in the Materials and Methods section. Each data point represents the mean value and
standard deviation (n = 3). * Asterisks denote a statistically significant difference (p < 0.05) between the
control and the treated groups according to the ANOVA Dunnett test.
2.2. Morphological Changes
To confirm the antiproliferative effect of VC F7 on OVCAR-3 cells, we examined the cell morphology
by acridine orange/propidium iodide (AO/PI) double staining assay. We observed that VC F7 inhibits
the proliferation of OVCAR-3 cells in a dose-dependent manner (Figure 2). As seen in Figure 2, there
is a strong dose–response relationship in regard to VC F7 treatment, showing a significant increase
in the percentage of dead cells compared to the percentage of live cells in the control. The control
(0 µg/mL) OVCAR-3 cells display a normal round shape and remain firmly attached to the culture
plate. Meanwhile, cells treated with VC F7 resulted in morphological alterations and exhibited
characteristics such as loss of cellular membrane integrity, cell shrinkage, cell membrane damage,
organelle breakdown, and detachment from the culture plate. Acridine orange/propidium iodide is a
rapid, sensitive, and successful method to examine cellular morphology, live and dead cells, apoptosis
and/or necrosis.
Figure 2. Cont.
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Figure 2. Representative fluorescence images of AO/PI-stained OVCAR-3 cells untreated and treated
with VC F7. Live cells are stained in green and dead cells are stained red. (A)—Control, (B)—8 µg/mL,
(C)—16 µg/mL, (D)—32 µg/mL. All fluorescence images were captured under 20× optical resolution of
the microscope.
2.3. Induction of Oxidative Stress
To test whether oxidative stress play a role in VC F7 inducing the antiproliferative effect against
ovarian cancer cells, we measured the levels of lipid peroxidation, catalase, and glutathione peroxidase
in OVCAR-3 cells. Our result obtained from lipid peroxidation assay showed a significant (p < 0.05)
increase in the production of malondialdehyde (a by-product of lipid peroxidation and biomarker of
oxidative stress) in VC F7-treated cells compared to the control (Figure 3). Upon 48 h of treatment,
the MDA values were 0.044 ± 0.058, 0.140 ± 0.034, 0.209 ± 0.010, and 0.324 ± 0.017 nmol in 0, 8, 16,
and 32 µg/mL of VC F7, respectively.
Figure 3. Effect of Vernonia calvoana fraction 7 (VC F7) on MDA production in untreated and treated
OVCAR-3 cells for 48 h. The doses that were found to be statistically significantly different (p < 0.05)
compared to the control are denoted by (*) according to ANOVA Dunnett.
To further understand the ability of VC F7 to induce oxidative stress in OVCAR-3 cells,
we determined the activity of catalase. Data generated from catalase assay demonstrated that
VC F7 slightly decreased the activity of catalase at 8 and 16 µg/mL of treatment. When cells were
treated with VC F7 at 32 µg/mL, catalase activity showed a significant decrease (p < 0.05) compared
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to the control. The catalase activities were found to be 0.14 ± 0.07853, 0.13 ± 0.001, 0.13 ± 0.0001,
and 0.048 ± 0.000416 n/mol in 0, 8, 16, and 32 µg/mL of VC F7, respectively (Figure 4).
Figure 4. Effect of Vernonia calvoana fraction 7 (VC F7) on catalase activity in untreated and treated
OVCAR-3 cells for 48 h. The doses that were found to be statistically significantly different (p < 0.05)
compared to the control are denoted by (*) according to ANOVA Dunnett.
To confirm our observations with the catalase activities, we performed a glutathione peroxidase
assay. Data generated from the glutathione peroxidase assay showed a gradual decrease in glutathione
peroxidase activity in OVCAR-3 cells treated with VC F7 compared to the vehicle control (Figure 5).
Figure 5. Effect of Vernonia calvoana fraction 7 (VC F7) on glutathione peroxidase activity in untreated
and treated OVCAR-3 cells for 48 h. The doses that were found to be statistically significantly different
(p < 0.05) compared to the control are denoted by (*) according to ANOVA Dunnett.
2.4. Induction of DNA Damage
To assess the ability of VC F7 to induce DNA damage in OVCAR-3 cells, we performed a comet
assay. Data generated from this assay showed a gradual increase in the mean values of comet tail
length, tail moment, and percentages of DNA cleavage of OVCAR-3 cells, with increasing doses of
VC F7 (Figure 6). After 48 h of treatment, the percentages of DNA cleavage were computed to be
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3.46 ± 0.043 %, 21 ± 0.953 %, 33.77 ± 0.529 %, and 51.46 ± 0.353% in the respective amounts of 0, 8, 16,
and 32 µg/mL of VC F7 (Figure 7A). In the same order, the mean comet tail lengths computed were
7.47 ± 0.16, 27 ± 0.23, 56.44 ± 0.53, and 69 ± 0.45 µM in the respective amounts of 0, 8, 16, and 32 µg/mL
of VC F7 (Figure 7B).
Figure 6. Representative SYBR green comet assay images of untreated (A-control) and Vernonia calvoana
fraction 7 (VC F7)-treated cells at 8 µg/mL (B), 16 µg/mL (C), and 32 µg/mL (D) for 48 h.
Figure 7. Vernonia calvoana fraction 7-induced DNA damage in OVCAR3 cells was measured by the
comet assay. The OVCAR-3 cells were treated with different doses of VC F7 for 48 h. (A) represents the
percentage of DNA cleavage and (B) represents the comet tail length. * p < 0.05 considered significant.
2.5. Induction of Cell Cycle Arrest
Here, our aim was to analyze the effects of VC F7 on cell cycle phases and find out at which
phase/stage ceases the cycle. Treatment of OVCAR3 cells with VC F7 significantly increases cell
population within the S-phase and significantly reduces it in the G2/M-phase (Figures 8 and 9),
indicating that VC F7 treatment causes cell cycle arrest at the S-phase checkpoint. The cell cycle arrest
at the S-phase implies that the cell is unable to duplicate its DNA. Figure 8 shows the percentage of cells
at the G0/G1, S, and G2/M regions. As seen in Figure 8, a cell cycle arrest at the S-phase is associated
with a reduction in the G2/M-phase. Figure 9 shows representative dots plots and a histogram of cell
cycle distribution of OVCAR-3 cells treated and untreated with VC-F7.
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Figure 8. Bar graph showing percentage (%) of OVCAR-3 cells in different phases of the cell cycle.
Each point represents the mean ± standard deviation of three independent experiments. * denotes
statistically significant difference between the control and the treated group according to ANOVA
(p < 0.05).
Figure 9. Representative dots plots and histogram showing cell cycle distribution of OVCAR-3 cells
treated with VC-F7. The cells were fixed with methanol, stained with PI, and analyzed by flow
cytometry (FACS Calibar; Becton-Dickinson) using CellQuest software as described in the Methods
section. (A) 0 µg/mL, (B) 8 µg/mL, (C) 16 µg/mL, (D) 32 µg/mL. Three experiments were performed,
and one (1) representative experiment is shown.
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3. Discussion
3.1. Antiproliferative Effect
In the present study, we first evaluated the antiproliferative effect of VC F7 on OVCAR-3 cells
by the means of MTS assay. Our results demonstrated that VC F7 significantly (p < 0.05) reduces the
percentage of live cells in a dose-dependent manner, suggesting its antiproliferative effect against
ovarian cancer (Figure 1). We further evaluated the antiproliferative effect of VC F7 and morphological
changes of OVCAR-3 cells by the means of AO/PI assay. We observed that VC F7 gradually inhibits the
proliferation of OVCAR-3 cells and causes morphological changes of these cells (Figure 2). As seen in
Figure 2, the untreated OVCAR-3 cells display a normal round shape, have about 90 % confluency,
and remain firmly attached to the culture plate. However, cells treated with VC F7 resulted in
morphological alterations and exhibited characteristics such as loss of cellular membrane integrity,
cell shrinkage, cell membrane damage, organelle breakdown, apoptotic bodies, and detachment from
the culture plate [25]. We previously observed similar results while testing the antitumor activity of
Vernonia amygdalina, a species of the same family as Vernonia calvoana [26]. In Cameroon, the leaves
of Vernonia amygdalina, Vernonia calvoana, and Vernonia amygdalina Delile plants are used extensively
as leaf vegetables and form a major constituent of a stew called ndole. The medicinal properties of
these plants are well-documented and are commonly recommended by herbalists to patients in African
countries for the treatment of headaches, stomach-aches [27], gastrointestinal tract problems [28], loss of
appetite, breast milk enhancement in nursing mothers [29], bacterial infections, liver diseases, kidney
problems [30], hypertension and diabetes [20,31], and cancer [24,32]. Animals use these medicinal
plants to cure themselves and there is scientific evidence in which chimpanzees inhabiting the Mahale
Mountains National Park in Tanzania have been observed chewing the pith of the leaves of Vernonia
Amygdalina. Possible benefits include Vernonia’s ability to ward off parasites and to treat gastrointestinal
tract infections [33]. Interestingly, the chimpanzees’ health condition gradually improved within
a day and they resumed their normal activity. According to statistics, herbal and plant-derived
medicines are the most frequently used therapies worldwide. A large number of people in developing
countries depend on these natural remedies to maintain healthcare and a 38% increase in usage in
the United States within the last decade of the 20th century alone has been reported [34,35]. It has
been shown that natural medicinal plants work with the body to boost the immune system by killing
unhealthy cells [36,37]. A previous scientific report indicated that VC possesses a hepatoprotective
effect and hypolipidemic and antidiabetic activities [20].
3.2. Induction of Oxidative Stress
Given the effectiveness of VC F7 to inhibit the growth of OVCAR-3 cells, we hypothesized that
the antiproferative effect of VC F7 may be mediated through oxidative stress. To test our hypothesis,
we measured the levels of lipid peroxidation, catalase, and glutathione peroxidase in OVCAR-3 cells
treated with VC F7. Oxidative stress plays an important role in cancer initiation and progression [38].
Our result obtained from lipid peroxidation assay showed a significant (p < 0.05) increase in the
production of malondialdehyde (MDA—a by-product of lipid peroxidation and biomarker of oxidative
stress) in VC F7-treated cells compared to the control. This finding is consistent with previous
studies in our lab showing that Vernonia amygdalina Delile crude extract acts as a pro-oxidant in
prostate cancer (PC-3) cells at a high concentration [39]. We also previously demonstrated that garlic
extract significantly induced MDA production in the HL-60 human leukemia cell in a dose-dependent
manner [39].
Our hypothesis that the antiproferative effect of VC F7 may be mediated through oxidative stress
was also tested by catalase assay. Catalase is an enzyme that neutralizes the burden of H2 O2 in cells by
decomposing this molecule into water and oxygen. Catalase activity intercepts the oxidative damage
that is triggered by high levels of H2 O2 . Studies have shown that the presence of high levels of H2 O2
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increases the speed of DNA mutation [40,41]. Our result revealed a gradual decrease in catalase activity
in VC F7-treated cells when compared to the control.
To further understand the ability of VC F7 to induce oxidative stress in OVCAR-3 cells,
we examined the activity of glutathione peroxidase. Glutathione peroxidase is localized in the cytosol
and mitochondria, and research suggests that it may degrade low levels of hydrogen peroxide—one
of the main ROS involved in arsenic-induced DNA damage [42,43]. Data demonstrated that VC F7
significantly (p < 0.05) decreases the levels of glutathione content. Depletion of glutathione level is
associated with the early stage of the initiation of cell death [44,45].
Taken together, our results of oxidative stress are in agreement with a previous study showing
that extracts of Alhagi maurorum increased the production of MDA levels, decreased the content of
GSH, and decreased the activities of antioxidant enzymes including SOD, GPx, and GST in livers of
STZ-induced diabetic rats [46].
3.3. Induction of DNA Damage
The ability of VC F7 to induce DNA damage in OVCAR-3 cells was determined by the means
of comet assay. Our results showed that VC F7 induces DNA damage in OVCAR-3 cells in a
dose-dependent manner. Untreated cells show low or no DNA migration, indicating that the DNA is
intact and undamaged. However, cells treated with VC F7 revealed a gradual increase in DNA cleavage
as well as an increase in comet tail length when compared to that of the untreated cells (control).
To the best of our knowledge, no data are found in the literature regarding the genotoxic effect of
VC in vitro or in vivo. We revealed for the first time that VC F7 induces DNA damage in OVCAR-3
cells, supporting its ability as a potential DNA-damaging anticancer agent effective against ovarian
cancer. Working with other Vernonia species, previous reports from our laboratory demonstrated that
in vitro Vernonia amygdalina treatment reduces cellular viability, that is, it induces DNA damage leading
to apoptosis accompanied by secondary necrotic cells in human breast cancer (MCF-7) cells [21,22].
In another study, we showed that Vernonia amygdalina Delile induced DNA damage in human leukemia
(HL-60) cells and human prostate cancer (PC-3) cells [24].
3.4. Induction of Cell Cycle Arrest
To assess the effects of VC F7 on cell cycle and population distribution, OVCAR-3 cells were
stained with propidium iodide and analyzed by flow cytometry. We found that 48-h VC F7 treatment
induced significant cell cycle arrest in the S-phase (p < 0.05) in comparison to untreated cells. The cell
cycle arrest in the S-phase is a direct result of VC F7 inhibition of cell growth and induction of DNA
damage in OVCAR-3 cells via oxidative stress (Figure 10). Consistent with our data, many natural
products exhibit inhibitory effects on cancer cells via disruption of cell cycle progression. For example,
studies showed that Ganoderma extract caused cell cycle arrest in cancer cells [47]. The growth inhibitory
effect of celery seed extracts on human gastric cancer BGC-823 cells caused cell cycle arrest at the
S-phase and decreased levels of cyclin A and CDK-2 [48,49]. The crude water extract of Centella asiatica
showed S and G2/M arrest in human colon adenocarcinoma-derived Caco-2 cells, accompanied with
the accumulation of cyclin B1 protein [50,51]. In addition, our results are in agreement with those
of curcumin (diarylheptanoid derivative of turmeric), indicating that it inhibits cell proliferation by
altering the cell cycle in different cancer cells [52,53].
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Figure 10. Underlying mechanisms by which Vernonia calvoana fraction 7 (VC F7) exerts its antitumor
activity in human ovarian cancer (OVAR-3) cells.
4. Materials and Methods
4.1. Chemicals and Media
The growth medium RPMI 1640 containing 1 mmol/L L-glutamine, fetal bovine serum (FBS),
phosphate buffered saline (PBS), and penicillin streptomycin were purchased from the American Type
Culture Collection (ATCC) in Manassas, VA. The MTS assay kit was purchased from Promega Life
Sciences (Madison, WI, USA). The lipid peroxidation, glutathione peroxidase, and catalase assay kits
were purchased from Abcam (Cambridge, MA, USA). The comet assay kit was obtained from Trevigen
(Gaithersburg, MD, USA). The propidium iodide was purchased from Calbiochem (La Jolla, CA, USA).
4.2. Vernonia calvoana Preparation and Fractionation
Leaves of Vernonia calvoana were harvested in Bangou, Cameroon. The leaves were rinsed and
air-dried under the sun for a day. This was followed by shade-drying for 5 days. Five hundred grams
of dried leaves were mixed with 600 mL of methanol and heated at 50 ◦ C. The mixture was filtered
with Whatman No 1 filter paper and evaporate to dryness using a rotary evaporator. Vernonia calvoana
extracts were kept refrigerated at 4 ◦ C until use. Fractionation of plant extract was performed according
to Ogungbe et al. (2014) [54] and Abugri et al. (2016) [55]. The fractions were collected and stored in a
freezer at −4 ◦ C.
4.3. Cell Culture
Human ovarian adenocarcinoma (OVCAR-3) cells were purchased from the American Type
Culture Collection. They were then sub-cultured in RPMI-1640 medium, supplemented with 10% fetal
bovine serum and 1% penicillin/streptomycin (Thermo Scientific, Waltham, MA, USA), and grown in
an incubator at 37 ◦ C in 5% CO2 . Fresh medium was supplemented every 48 h.
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4.4. Cell Treatment and Determination of Cell Viability
The antiproliferative effects of VC F7 on the viability of OVCAR-3 cells was determined by the MTS
colorimetric assay. We used fraction 7 because it showed the highest antitumor activity towards ovarian
cancer cells compared to other fractions present in the crude extract. Cells were seated in 96-well plates
at a density of 5 × 103 cells per well were treated with different doses (0, 8, 16, and 32 µg/mL) of VC F7
for 48 h. After cells treatment, the medium was carefully aspirated from the treated plate and replace
with an aliquot (100 µL) of fresh medium. Then, 20 µL of the MTS solution was added to each well and
incubated for 3 h. The absorbance was read at 490 nm using a Biotex Model micro plate reader.
4.5. Morphological Changes
In this assay, we explore the morphological changes of OVCAR-3 cells treated and untreated
with VC F7. Briefly, cells seated in each polystyrene 6 well-plate were treated with VC F7 for 48 h.
After treatment, cells were washed twice with PBS and stained with a double dye (acridine orange
(AO) and propidium iodide (PI). After staining, cells were examined and photographed under an
Olympus fluorescent microscope.
4.6. Measurement of Lipid Peroxidation/Malondiadehyde
For this experiment, cells seeded in a 6-well plate at a density of 5 × 106 cells/well were treated
with different doses (0, 8, 16, and 32 µg/ mL) of VC F7 for 48 h. Cells were harvested, centrifuged,
and collected in a 15 mL test tube. The cell pellets were lysed in 200 µL malondialdehyde (MDA) lysis
buffer plus 2 µL BHT (100×) The freeze–thaw method was then carried out, and then 200 uL aliquots
of the culture medium was assayed for MDA according to the lipid peroxidation assay protocol as
previously described [56,57]. The absorbance was measured at 586 nm and the concentration of MDA
was estimated from the standard curve. Experiments were performed in triplicates.
4.7. Measurement of Catalase Activity
Catalase activity was estimated by the means of catalase assay activity kit from Abcam company.
Cells treated with different doses (0, 8, 16, and 32 µg/ mL) of VC F7 for 48 h. Treated and untreated
cells were harvested, centrifuged, and collected in a 15-mL test tube. Cells were digested and catalase
activity was determined according to the protocol as previously described with a few modifications [58].
Experiments were performed in triplicates.
4.8. Measurement of Glutathione Peroxidase Activity
To estimate the glutathione activity in this study, OVCAR-3 cells were seeded in a 6-well plate
and treated with different doses of VC F7 for 48 h. Cells were digested and intracellular glutathione
levels were determined using the assay kits purchased from Abcam (Cambridge, MA) according
to the protocol previously described with some modifications [59]. Experiments were performed
in triplicates.
4.9. Assessment of DNA Damage
For this experiment, OVCAR-3 cells were treated with VC F7 at doses of 8, 16, and 32 µg/mL
for 48 h. Briefly, an aliquot 50 µL cell suspension was mixed with 200 µL of agarose. Then, 75 µL of
the mixture was spread on the comet slides and place in the refrigerator for 15 min. The slides were
immersed in lysis solution for 60 min at 4 ◦ C. They were further immersed in a prepared alkaline
solution and kept in the dark for 60 min. Sample slides were electrophoresed at 21 V for 30 min,
dehydrated in 70% ethanol for 5 min, and stained with DNA- bound SYBR green I fluorescence stain
overnight. The samples were visualized for DNA damage under a fluorescent microscope at 494/521 nm
wavelength where several images were taken. The images were analyzed using the Trevigen Comet
Assay software.
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4.10. Assessment of Cell Cycle Distribution
All steps for this experiment were performed at 0 ◦ C. Briefly, cells were seeded into a 6-well plates
at the density of 6 × 106 cells/well and treated with different doses (0, 8, 16, and 32 µg/ mL) of VC F7 for
48 h. After treatment, cells were harvested, washed twice with PBS, and fixed in ice-cold methanol for
30 min at 4 ◦ C. Cells were stained with propidium iodide in the presence of RNase A and incubated for
30 min at room temperature. After staining, cells were analyzed by flow cytometry (BD Biosciences,
San Jose, CA, USA).
5. Conclusions
New therapeutic approaches for the screening of bioactive compounds present in medicinal plants
have received increasing attention due to their chemopreventive properties such as anti-oxidative,
anti-cancer, and anti-inflammatory activities [60–62]. Studies indicated that Vernonia calvoana contained
high concentrations of flavonoids rich in antioxidants which are natural substances that prevent cellular
damage in living organisms. In the present study, we demonstrated that VC F7 is able to inhibit cell
proliferation, induce DNA damage and cell cycle arrest at the S-phase checkpoint of the cell cycle in
human ovarian cancer (OVAR-3) cells through oxidative stress, as demonstrated by an increase in
MDA production and a decrease in catalase and glutathione activities in treated cells compared to the
control. All these unique properties of VC F7 against OVAR-3 cells strongly suggest that VC F7 may be
a novel and potential targeting molecule that can be used as a therapeutic agent for ovarian cancer.
It is noteworthy that VC F7 exerts its anticancer activity by inhibiting cell proliferation, inducing
DNA damage, and causing cell cycle arrest through oxidative stress in OVAR-3 cells. However, future
preclinical and clinical trial studies are needed to test the medicinal properties of VC F7 as an alternative
therapeutic agent for the prevention and/or treatment of ovarian cancer. Future research in our lab will
focus on the identification and characterization of the active constituents present in CV F7 and testing
antitumor activity in an animal model.
Author Contributions: A.T.M. and C.G.Y. conceived, and designed the study. A.T.M. and J.N.S. performed the
experiments and drafted the manuscript. C.G.Y., P.B.T. supervised the experiments and assisted in performing
data interpretation. F.K.N. and C.R.G. provided a critical review and manuscript editing. A.T.M., C.G.Y., and P.B.T.
participated in the implementation of the study and reviewed the manuscript for submission. All authors have
read and agreed to the published version of the manuscript.
Funding: This work was financially supported by a grant from the National Institutes of Health (NIH) under
grant # G12MD007581 through the RCMI Center for Environmental Health, and partly by NIH under grant #
1R03CA223099-01A1 at Jackson State University. The content is solely the responsibility of the authors and does
not necessarily represent the official view of NIH.
Acknowledgments: This research was made possible with the support from the Ph.D. Environmental Science
Program at Jackson State University.
Conflicts of Interest: The authors declare no conflict of interest.
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