Antiproliferative activity of ethyl acetate fraction of Euphorbia ingens against prostate cancer cell line: An in silico and in vitro analysis
Innocent Oluwaseun Okpako a,*, Florence Atieno Ng’ong’a b, Cleophas
Mutinda Kyama c, Sospeter Ngoci Njeru d
a Department of Molecular Biology and Biotechnology, Pan African University Institute for Basic Sciences, Technology and Innovation, Nairobi,
Kenya
b Department of Biochemistry, School of Biomedical Sciences, College of Health Sciences, Jomo Kenyatta University of Agriculture and Technology,
Nairobi, Kenya
c Department of Medical Laboratory Sciences, School of Biomedical Sciences, College of Health Sciences, Nairobi, Kenya
d Centre for Traditional Medicine and Drug Research, Kenya Medical Research Institute, Nairobi, Kenya
A B S T R A C T
Current prostate cancer (PCa) treatments often lead to severe side effects, prompting the exploration
of safer alternatives. Euphorbia ingens is a medicinal plant used for cancer treatment in
African communities; however, there is no scientific validation of its anticancer activity. This
study therefore evaluated the antiproliferative activity of E. ingens on the DU-145 human PCa cell
line. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) bio-assay was
used to determine the antiproliferative activity of E. ingens ethyl acetate-, and water-fractions.
Only the ethyl acetate fraction was considered to be active. It inhibited DU-145 cell growth
selectively without toxicity to non-cancerous Vero E6 cells. The IC50 was 9.71 ± 0.4 μg/ml and
had a selectivity index of 8.26, indicating promising efficacy and cellular safety. Qualitative
phytochemical screening identified the presence of phenols, terpenoids, flavonoids, tannins,
sterols, and saponins in the ethyl acetate fraction. An additional 18 chemical compounds with
potential synergistic roles were identified through gas chromatography-mass spectrometry
analysis. Network pharmacology was then employed to predict molecular targets and mechanisms
of action for drug-like chemical compounds, implicating key targets such as ESR1, IL6,
MMP9, CDK2, MAP2K1, AR, PRKCD, CDK1, CDC25B, and JAK2 with regulation of PI3K/Akt,
MAPK, and p53 signaling pathways suggested to be the potential mechanism of action. Further,
gene expression analysis of selected targets through reverse transcription-quantitative polymerase
chain reaction revealed downregulation of AR and BCL2 levels, along with upregulation of p53
and caspase-3 in fraction-treated DU-145 cells compared to the 0.2 % DMSO-negative control. In
conclusion, the findings of this study validate the traditional use of E. ingens in cancer management.
The identified drug-like compounds, their targets, and associated signaling pathways could
serve as a foundation for developing novel strategies for prostate cancer management. However,
we recommend additional in vitro and in vivo studies to further substantiate these findings.
Introduction
Cancer is one of the major causes of death globally and a significant contributor to decreased life expectancy. Cancer of the prostate
is an adenocarcinoma that often forms in the glandular prostate, it can remain contained in the prostate for a long time or advance by
metastasizing outside the prostate. According to the World Health Organization (WHO), in 2020, there were 1414,259 new cases of
prostate cancer (PCa); it is the second most common cancer in men and the fifth most significant cause of cancer-related mortality
globally [1]. Due to a lack of effective preventive and treatment strategies, data in 2020 show that 77,300 new cases were estimated to
have occurred in sub-Saharan Africa [2].
Prostatectomy and local radiation are used to treat localized PCa, which accounts for 90% of PCa cases. However, PCa is diagnosed
at an advanced stage in around 90% of men with the disease. The common treatment options for advanced PCa are androgen
deprivation therapy (ADT) and chemotherapy. ADT treatment leads to recurrent androgen-independent prostate cancer within 2–3
years, with frequent metastases to regional lymph nodes or the pelvis. Although there are several prostate cancer chemotherapy
treatments, there is still a knowledge gap on the evolution of treatment interventions resistance coupled with limited evidence of the
therapeutic options for advanced prostate cancer. Drugs that target rapidly proliferating cancer cells, such as docetaxel and paclitaxel,
can also damage healthy cells, causing fatigue, hypertension, hot flushes, arthralgia, fractures, peripheral edema, and rash. Therefore,
there is an urgent need for better therapeutic interventions against PCa, and herbal plant sources provide the greatest promise and the
“lowest hanging fruits”.
The use of herbal plants in cancer has gained substantial attention, and recently, ongoing research with the US National Cancer
Institute (NCI) have played a pivotal role in promoting the use of traditional medicine to treat cancer [3]. Herbal plants have various
advantages over chemical products due to their lower chances of inducing adverse effects, cost-effectiveness, tolerability, and reduced
chances of developing resistance. Plants exercise their anticancer properties owing to the existence of phytochemicals that act by
combating lipid peroxidation through their anti-oxidative properties, repairing damaged DNA, boosting the immune system, inducing
apoptosis, and suppressing the cell cycle [4].
Euphorbia ingens E. mey. ex Boiss belongs to the plant family Euphorbiaceae. It is known to contain latex; the leaves, stem, root, and
whole plant are applied in traditional medicine for the treatment of cancer and other pathologies, including swellings, fistula, lesions,
wounds, abscesses, burns and mental disorders [5,6]. In East Africa, E. ingens is also used for the treatment of snakebites, suggesting
that the plant may possess an anti-venom property. Additionally, there are scientific data demonstrating the ichthyoidal, antitubercular,
antimicrobial, and antifungal activities of E. ingens [7–9]. However, despite the aforementioned roles of E. ingens as a viable
therapeutic option for the management and treatment of diseases in ethnomedicine, there is no scientific validation of its ethnobotanical
use in cancer management, and treatment. In this context, we hypothesized that E. ingens could selectively abrogate the
proliferation of prostate cancer cells.
Various plant parts, including stems, leaves, bark, fruits, and seeds, are employed in the formulation of cancer treatments; the roots,
as highlighted by Koduru et al. [6], are mostly used. Also, previous studies have reported E. ingens’ stems and leaves to be inactive
against human hepatoma, human breast adenocarcinoma, and human colon adenocarcinoma cell lines [10]. To test our hypothesis, we
prepared crude (dichloromethane-methanol (1:1, v/v)) extract, hexane-, ethyl acetate-, and water-fractions from the roots of E. ingens,
assessing their inhibitory effects on DU-145 prostate cancer cells. Subsequently, we evaluated the cellular safety of the ethyl acetate
fraction—identified for its robust antiproliferative activity—in non-cancerous Vero E6 cells. Employing network pharmacology, we
probed the likely molecular targets and anticancer mechanisms of action associated with the chemical compounds identified within the
efficacious ethyl acetate fraction. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), we determined the
gene expression levels of selected molecular targets and genes known to play pivotal roles in prostate cancer initiation and progression.
Collectively, our study scientifically validates the traditional use of E. ingens in treating and managing cancer, positioning the plant as a
promising source for discovering bioactive compounds tailored for the treatment and/or management of prostate cancer. This is in line
with Sustainable Development Goal 3, Target 3.4, which aims to reduce the mortality rate attributed to cancer by one-third by 2030. It
is worthy of note that the goal coincides with goal 3 of Africa Agenda 2063, an aspiration to achieve a population that is healthy and
well-nourished across the African continent.
Materials and methods
Plant collection and processing
Fresh root samples of E. ingens were obtained on March 26, 2022, from Embu County, Kenya (0◦ 46′ 27.0″ South, 37◦ 40′ 54.9″ East),
where it grows naturally. Plant identification and authentication were carried out at Egerton University, Kenya by a plant taxonomist,
and a voucher specimen number NSN9 was subsequently deposited in the same place. Approximately 2 kg of plant roots were carefully
taken, washed thoroughly with water to remove sand and any other contaminants. They were cut into little pieces and shade dried at
room temperature for 21 days, with the samples regularly turned upside down to avoid fungal growth. The dried samples were then
milled into fine powder using an electric grinder (Christy 8 MILL, serial number 51,474). The powder of E. ingens (411 g) was soaked in
a volume of 1 L of dichloromethane-methanol (1:1, v/v) solvent. Following percolation and intermittent agitation, the resulting liquid
was filtered with Whatman No. 1 filter paper. This was done repeatedly until the initial deep color of the filtrate faded to indicate an
exhaustive solvent extraction. The filtrate was successively evaporated to dryness at 57 ◦C using a 5-liter rotary vacuum evaporator
(Rotavapor R-300; Buchi, Switzerland). The obtained extract was 149 g and the yield percentage were calculated using the following
equation:
Percentage yield of extracts = [(Weight of the obtained extract material/Weight of original fine plant powder used)* 100]