Abstract
Introduction: Cervical cancer is one of the leading causes of death
among women globally due to the limitation of current treatment methods
and their associated adverse side effects. Launaea cornuta is used as
traditional medicine for the treatment of a variety of diseases
including cancer. However, there is no scientific validation on the
antiproliferative activity of L. cornuta against cervical cancer.
Objective: This study aimed to evaluate the selective
antiproliferative, cytotoxic and antimigratory effects of L. cornuta
and to explore its therapeutical mechanisms in human cervical cancer
cell lines (HeLa-229) through a network analysis approach.
Materials and methods: The cytotoxic effect of L. cornuta ethyl acetate
fraction on the proliferation of cervical cancer cells was evaluated by
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT)
bioassay and the antimigratory effect was assessed by wound healing
assays. Compounds were analysed using the qualitative colour method and
gas chromatography-mass spectroscopy (GC-MS). Subsequently,
bioinformatic analyses, including the protein-protein interaction (PPI)
network analysis, Gene Ontology (GO), and Kyoto Encyclopaedia of Genes
and Genomes (KEGG) analysis, were performed to screen for potential
anticervical cancer therapeutic target genes of L. cornuta. Molecular
docking (MD) was performed to predict and understand the molecular
interactions of the ligands against cervical cancer. Reverse
transcription-quantitative polymerase chain reaction (RT-qPCR) was
performed to validate the network analysis results.
Results: L. cornuta ethyl acetate fraction exhibited a remarkable
cytotoxic effect on HeLa-229 proliferation (IC[50] of 20.56 ±
2.83 μg/mL) with a selectivity index (SI) of 2.36 with minimal
cytotoxicity on non-cancerous cells (Vero-CCL 81 (IC[50] of 48.83 ±
23.02). The preliminary screening revealed the presence of glycosides,
phenols, saponins, terpenoids, quinones, and tannins. Thirteen
compounds were also identified by GC-MS analysis. 124 potential target
genes associated with the effect of L. cornuta ethyl acetate fraction
on human cervical cancer were obtained, including AKT1, MDM2, CDK2,
MCL1 and MTOR were identified among the top hub genes and PI3K/Akt1,
Ras/MAPK, FoxO and EGFR signalling pathways were identified as the
significantly enriched pathways. Molecular docking results showed that
stigmasteryl methyl ether had a good binding affinity against CDK2,
ATK1, BCL2, MDM2, and Casp9, with binding energy ranging from −7.0 to
−12.6 kcal/mol. Tremulone showed a good binding affinity against TP53
and P21 with −7.0 and −8.0 kcal/mol, respectively. This suggests a
stable molecular interaction of the ethyl acetate fraction of L.
cornuta compounds with the selected target genes for cervical cancer.
Furthermore, RT-qPCR analysis revealed that CDK2, MDM2 and BCL2 were
downregulated, and Casp9 and P21 were upregulated in HeLa-229 cells
treated with L. cornuta compared to the negative control (DMSO 0.2%).
Conclusion: The findings indicate that L. cornuta ethyl acetate
fraction phytochemicals modulates various molecular targets and
pathways to exhibit selective antiproliferative and cytotoxic effects
against HeLa-229 cells. This study lays a foundation for further
research to develop innovative clinical anticervical cancer agents.
Keywords: Launaea cornuta, cervical cancer, phytochemicals,
antiproliferative, network analysis, molecular docking, cytotoxicity,
anticancer activity
Introduction
Cervical cancer is a major global health disease that affects women
([30]Drokow et al., 2022). It is one of the leading causes of cancer
death among women and the fourth most common cancer worldwide
([31]Revathidevi et al., 2021). Human papillomavirus is the primary
aetiological driver in carcinogenesis ([32]Balasubramaniam et al.,
2019). Not all human papillomavirus (HPV) infections in women result in
cervical cancer. High-risk HPV genotypes induce a normal cell to
transform into a precancerous lesion, which subsequently becomes an
invasive lesion ([33]Kasi et al., 2021). HPV infection causes the
overexpression of viral oncogenes, which may inhibit a number of
cellular proteins and affect biological processes such as cell
proliferation, cell cycle, and apoptosis ([34]Martínez-Rodríguez et
al., 2021).
Globally, in 2020, a total of 604127 cases of cervical cancer were
estimated, and 341831 deaths were recorded ([35]Olthof et al., 2024).
The incidence rate was 13.3 cases per 100,000 women, and the mortality
rate was 7.2 deaths per 100,000 women ([36]Singh et al., 2023). Over
58% of cervical cancer cases globally were estimated in Asia, followed
by Africa (20%), Europe (10%) and Latin America (10%) ([37]Singh et
al., 2023). However, the incidence and mortality rates for cervical
cancer are exceptionally high in Sub-Saharan Africa at 19.59% and
24.55% annual global burden, respectively ([38]Black and Richmond,
2018).
Botanicals’ secondary metabolites and their derivatives have been
evaluated in oncology-related clinical research and trials ([39]Omara
et al., 2022). The comprehensive knowledge on the mechanism of action
for some investigated plant compounds provided the basis to develop
novel, efficacious, safer botanical-derived compounds or their
semi-synthetic analogues. Such had additional therapeutic advantages
over the convectional chemotherapeutic drugs, which are associated with
high toxicity not only to cancer cells but also to noncancerous cells
([40]Scatchard et al., 2012; [41]Vordermark, 2016; [42]Johnson et al.,
2018; [43]Paken et al., 2023). Cervical cancer has become resistant to
conventional therapeutic drugs, including cisplatin, paclitaxel, and
carboplatin (Small et al., 2017), thus rendering them less effective.
Hence, it was imperative to speed up research on botanical plants as an
alternative source of less toxic, more tolerable and effective
anticancer drugs for cervical cancer treatment.
The rich biodiversity has contributed significantly to traditional
medicine and medicinal systems since ancient times ([44]Omara, 2020).
Approximately 1,200 medicinal plants are used in Kenya, 41 of which
have anticancer properties, which account for only 10% of the medicinal
plants ([45]Onyancha et al., 2018). Botanical-based drugs are gaining
attention due to their low toxicity, availability, cost-effectiveness,
and tolerability compared to conventional drugs ([46]Tariq et al.,
2017; [47]Ochwang’i et al., 2014; [48]Misonge et al., 2016). In this
study, Launaea cornuta (Hochst. ex Oliv. & Hiern) C. Jeffrey also known
as “mchunga” was investigated. L. cornuta is an erect herbaceous
perennial plant belonging to the Asteraceae family, characterised by a
hollow leafy and stem, slightly succulent, glabrous, with milky juice
and grows to a height of about 1.5 m ([49]J et al., 2015). It is
indigenous to Kenya, Burundi, Cameroon, Uganda, Nigeria, Rwanda, Chad,
Djibouti, Eritrea, Ethiopia, Malawi, Mozambique, Somalia, Sudan,
Tanzania, Zambia, Central African Republic, Zaïre, and Zimbabwe
([50]Machocho et al., 2014; [51]Omara et al., 2022). In Kenyan
communities, this plant is used as a wild vegetable and a source of
vitamin C ([52]Musila et al., 2013). The localised budding root
concoction known as ‘Kipche’ is used to cure throat cancer (“Koroitab
mokto”), typhoid, gonorrhoea, benign prostate hyperplasia, and breast
cancer, among others ([53]J et al., 2015; [54]Fatuma Some, 2014;
[55]Khan et al., 2016; [56]Chemweno et al., 2022). Regardless of the
extensive use of L. cornuta plants in traditional medicine, their
therapeutic efficacy, and toxicity have not been empirically validated,
especially in cervical cancer.
In this study, we systemically evaluated the anticancer activity of the
ethyl acetate fraction of L. cornuta, analysed and identified compounds
using the GC-MS approach and explored the relevant targets and pathways
involved in eliciting the anticervical effects through a network
analysis approach.
Materials and methods
Samples collection and extract preparation
Fresh stems and leaves of L. cornuta (Hochst. ex Oliv. & Hiern) C.
Jeffrey were collected from Embu County, Mbeere South sub-County,
Mavuria ward (Latitude 0°46′27.0″S 37°40′54.9″E and Longitude
−0.774156, 37.681908). The plant was not classified as an endangered
species, and therefore, no prior permission/consent was sought during
the plant material collection phase. Plant identification and
authentication were performed by a botanist at the University of
Nairobi, and a voucher specimen number NSN18 was given and deposited at
the Kenyan National Museums - Eastern Africa Herbarium. The stems and
leaves were washed under running tap water, dried under a shade for
3 weeks and milled into a fine powder using an electric grinder
(Christy 8 MILL, serial No; 51474). The plant powder was extracted
using dichloromethane: methanol solvent following a method described by
Okpako et al. with some modifications to obtain the crude extract
([57]Okpako et al., 2023a) and three fractions: n-hexane, ethyl
acetate, and water were obtained. The crude extract obtained was
macerated in 250 mL of n-hexane solvent, shaken vigorously and left
undisturbed for about 20 min. Thereafter, the hexane fraction was
separated. Another 250 mL was added to the residues from the first
fractionation, and the process was repeated after the two hexane
fractions were pooled together. The resulting residues were further
fractionated with water and ethyl acetate solvents. The resulting
mixtures with intermittent shaking for 30 min were left to stand for
24h and were separated based on the solvent interphase. The organic
solvent fractions were concentrated with a rotor evaporator, as
described before ([58]Okpako et al., 2023a). The crude extract and the
fraction of hexane, water and ethyl acetate were stored at −20°C until
analysis.
Cell lines and culture conditions
A human cervical cancer cell line (HeLa-229) and a kidney epithelial
cell line derived from the African green monkey (Vero-CCL) were
purchased from ATCC and cultured in Essential Modified Eagle Medium
(EMEM) supplemented with 1% L-glutamine (200 mM), 10% fetal bovine
serum (FBS), 1.5% sodium bicarbonate, 1% HEPES (1M), 1%
penicillin-streptomycin and 0.25 μg/mL amphotericin B. The cell culture
was conducted at 37^°C under a humidified atmosphere and 5% CO[2] to
achieve 75% – 80% confluence.
Antiproliferative and cytotoxicity assay
The effects of L. cornuta fractions and crude extract on the
proliferation of HeLa cells were evaluated using the 3-(4,
5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay
(Solarbio, China) following a method described by Nyeru et al. and
Sergazy et al. with some modification ([59]Njeru and Muema, 2021;
[60]Sergazy et al., 2021). The HeLa-229 cells (1.0 × 10^4 cells/mL)
were seeded in a 96-well plate containing fresh EMEM medium and
incubated for 24 h. Subsequently, the medium was aspirated, and 100 µL
of 200 μg/mL of the plant fractions and the total extract were added
and incubated for 48 h. Doxorubicin (200 μg/mL) was used as a positive
control, and 0.2% dimethyl sulfoxide was used as a negative control.
After incubation for 48 h, 10 µL of freshly prepared 5 μg/mL MTT was
added to each well and incubated for 4 h. MTT was then aspirated, and
100 µL of 100% dimethyl sulfoxide (DMSO, Finar Chemicals, India) was
added to dissolve formazan crystals. The absorbance was read at 570 and
720 nm using a plate reader (Infinite M1000 by Tecan). All experiments
were carried out in four replicates, and the percentage cell viability
was calculated using the formula presented below ([61]Juwitaningsih et
al., 2022).
[MATH: Cell viability %=
Absorbance of treated cell – Absorbance of culture medium/Absorbance of untreated cell –<
/mo> Absorbance of culture medium x 100 :MATH]
The L. cornuta ethyl acetate fraction, which gave less than 50% cell
viability after 48 h of incubation, was considered to have
anti-cervical cancer activity and was prioritised for further
downstream investigations. Thereby, ethyl acetate fraction was selected
for dose-dependent tests using a range of concentrations
(2.0–64.0 μg/mL) and (4.0–256 μg/mL) for the cytotoxic effect on
HeLa-229 and Vero-CCL cells, respectively. The half-maximal inhibitory
concentration (IC[50]) and the half-maximal cytotoxicity concentration
(CC[50]) were determined using dose-response and dose-cytotoxicity
graphs and calculated by a non-linear regression method using GraphPad
Prism 8.4 ([62]Panyajai et al., 2024). The selectivity index (SI) was
calculated using the formula below ([63]Mongalo et al., 2017).
[MATH: Selectivity Index SI=(CC50
in µg/mL)
/(IC50 in µg/mL)
:MATH]
Cell morphological study
To investigate the effects of L. cornuta ethyl acetate on HeLa 229
cells, the cells were treated with five different concentrations of the
extract (4, 8, 16, 32, and 64 μg/mL) for a period of 48 h. The
morphological changes in HeLa cells were then determined with a digital
microscope at ×20 magnification.
Wound healing assay
HeLa 229 cells were cultured in a 6-well plate at a density of 1 × 10^6
cells/mL per well and incubated for 24 h to form a monolayer. A wound
was created using a sterile 200 mL pipette tip. The cells were then
treated with L. cornuta ethyl acetate fraction at the concentration
corresponding to the IC[50] (20.56 μg/mL) and incubated for 48 h.
During the treatment period, cells were imaged using a digital
microscope at 0 h, 24 h and 48 h. The distance between the wounds on
the cells was measured and analysed with ImageJ software. The relative
migration distance was calculated and expressed as a percentage, as
shown below.
[MATH: Relative migration distance %=
Distance within the scratch at
0 h – Distance w
ithin the scratch at 24 h)/(
Distance within the scratch at 0 hx 100 :MATH]
Preliminary screening and phytochemical analysis using GC-MS
We performed a preliminary screening for the presence of alkaloids,
glycosides phenols, flavonoids, terpenoids, steroids, quinones,
saponins, and tannins in L. cornuta ethyl acetate fraction, which
exhibited anti-cancer activity using methods described previously
([64]Njeru et al., 2015; [65]Yadav et al., 2017; [66]Shaikh and Patil,
2020) and subsequently, gas chromatography-mass spectrometer system
(Model; Shimadzu, GC-MS QP-2010SE) was used for quantitative analysis.
The system was equipped with a low-polarity BPX5 capillary column (30 m
× 0.25 mm × 0.25 μm film thickness). The oven temperature was set at
55°C for 1 min, then increased by 10°C per minute to reach a constant
temperature of 280°C for 15 min and held for 30 s. The injector
temperature was set at 200°C. Helium was used as the carrier gas with a
1.08 mL/min flow rate. The diluted sample (1% v/v) was injected (1 µL)
into the GC with an AS3000 autosampler in a split ratio of 10:1. The
mass detector was set at 200°C (ion source) and 250°C (interface
temperature). Electron-ionisation mass spectra were collected at 70 eV
in full scan mode at a m/z of 35–550. The identity of phytochemicals
was determined by comparing the mass spectra with the spectra of
compounds in the NIST library database ([67]Wavinya Nyamai et al.,
2015; [68]Njeru and Muema, 2020).
Bioinformatics analysis
Drug likeness screening test
The PubChem CID and canonical SMILES (Simplified Molecular Input Line
Entry System) of the GC-MS identified compounds of L. cornuta ethyl
acetate were retrieved from PubChem
([69]https://pubchem.ncbi.nlm.nih.gov/). The SMILES were then submitted
to SwissADME and pkCSM tools ([70]http://www.swissadme.ch/index.php &
[71]https://biosig.lab.uq.edu.au/pkcsm/prediction) to predict the
druglike (DL), the pharmacokinetics and physicochemical properties of
the compounds. The prediction was based on Lipinski’s rule of five:
blood-brain barrier (BBB), the central nervous system (CNS), human
intestinal absorption, and inhibition of cytochrome P450s
([72]Vishvakarma et al., 2023).
Identification of candidate targets for Launaea cornuta ethyl acetate
fraction against human cervical cancer
The targets of the compounds were predicted from Swiss TargetPrediction
([73]http://www.swisstargetprediction.ch/database), with ‘humans’ (Homo
sapiens) as the study species with p > 0.1 considered potential targets
([74]Daina et al., 2019). Additional targets were predicted using
BindingDB
([75]https://bindingdb.org/rwd/bind/chemsearch/marvin/FMCT.jsp),
potential targets with p > 0.7 were considered and retrieved. The Gene
IDs of the selected targets were retrieved from UniProtKB database
([76]https://www.uniprot.org). Ensemble approach (SEA)
([77]https://sea.bkslab.org/) database was used to predict the
compound’s targets based on the assumption that molecules with similar
structures tend to have similar responses to a target ([78]Wang et al.,
2016). For each database, the term ‘H. sapiens’ was used as the
keyword. The results predicted from the three databases were pooled,
and duplicates were removed ([79]Li and Ren, 2022). The disease targets
of human cervical cancer for development and progression were retrieved
from GeneCards ([80]https://www.genecards.org/), DisGeNET
([81]https://www.disgenet.org/), OMIM ([82]https://www.omim.org/) and
Pharos ([83]https://pharos.nih.gov/diseases/) databases using ‘Cervical
cancer’ the disease type. All retrieved targets were merged, and only
the unique targets were selected. Lastly, the potential targets of the
identified compounds of L. cornuta ethyl acetate and the disease
targets of cervical cancer were intersected using an online
bioinformatics and evolutionary genomics platform
([84]https://bioinformatics.psb.ugent.be/webtools/ven) to obtain the
common target genes.
Protein-protein interaction (PPI) analysis
The STRING database ([85]https://string-db.org/), version 12.0, was
used to build a PPI network for the common target genes between the L.
cornuta ethyl acetate fraction and cervical cancer targets. The species
was set as “H. sapiens”, and the interaction threshold was set to 0.4.
Subsequently, the PPI data was exported as a ‘TSV’ format file and
imported into Cytoscape software (version 3.10) for analysis. The
Cytohubba plug-in in Cytoscape was used to determine and calculate the
score (degree) of each node and the top 30 hub targets were selected
([86]Zhang et al., 2021).
Gene ontology (GO) and Kyoto encyclopaedia of genes and genomes (KEGG)
enrichment analyses
GO and KEGG pathway enrichment analysis was performed following the
method described by Jeddi et al. with some modifications ([87]Jeddi et
al., 2023) for the common targets of between L. cornuta ethyl acetate
fraction and cervical cancer using an online enrichment tool ShinyGO
version 0.77 ([88]https://ge-lab.org/go/), SRplot ([89]Science and
Research online plot (bioinformatics.com.cn) tool was used to identify
the KEGG pathway and mapping associated with the targets, Human was
selected as the species with a false discovery rate FDR cut-off of
0.05, and the number of pathways to be presented was set as 20. The
cellular components (CC), molecular functions (MF), biological process
(BP) and pathway enrichment terms with a p-value of <0.05 were
identified as significantly enriched and considered for subsequent
analysis.
Molecular docking
The 3D structures of AKT1 (1H10), MDM2 (5C5A), CDK2 (4GCJ), P53 (7LIN)
BCL-2 (6O0O), CASP9 (4RHW) AND P21 (5DEW) were retrieved from the
Protein Data Bank ([90]https://www.rcsb.org/). The 3D structures of the
L. cornuta compounds and the native ligands of the targeted proteins
were downloaded from PubChem ([91]https://pubchem.ncbi.nlm.nih.gov/)
and converted to the PDBQT (Protein Data Bank, Partial Charge (Q), and
Atom Type (T)) format using Open Babel plug-in in the PyRx software.
Ligand energy minimisation was performed using the PyRx software. The
grid box for each protein was maximised to fully enclose the protein
structure, and blind docking was performed using the VINA tool in the
PyRx, with an exhaustiveness of 8. The compounds with the binding
affinity less than −7.5 kcal/mol were selected and visualised with
Discovery Studio 2021 Client. Docking validation was conducted by
redocking native ligands to the target proteins.
Relative gene expression using quantitative polymerase chain reaction (qPCR)
assay
In this study, a quantitative RT-PCR assay was conducted to measure the
transcript levels of AKT1, TP53, BCL-2, MDM2, CDK2, Casp9, and P21
proteins. First, total RNA was extracted from HeLa cells using the
SolarBio Total RNA Extraction Kit (Beijing, China) following the
manufacturer’s instructions. Then, the quality of the RNA was assessed
using a nanodrop spectrophotometer (Thermofisher). The RNAs (1 µg) were
then converted to complementary DNA (cDNA) using the SensiFAST cDNA
synthesis kit (Brisbane, Australia) following the manufacturer’s
protocol. Quantitative RT-qPCR was performed using the SensiFAST™
SYBER^@ Lo-ROX Kit (Bioline, Australia). The program for the qPCR
machine (QuantStudio 5; Applied Biosynthesis) was set as follows:
initial denaturation at 95°C for 2 min, 1 cycle; denaturation at 95°C
for 5 s, 40 cycles; annealing at 62°C for 10 s and extension at 72°C
for 20 s, 40 cycles and melt curve at 60 s, 1 cycle. GAPDH was used as
the reference gene and the relative expression was calculated using the
2^^−ΔΔct method ([92]Njeru et al., 2020). The primer sequences used in
this RT-qPCR analysis are presented in [93]Supplementary Table S1.
Statistical analysis
All data described in this study were expressed as mean ± standard
deviation (M±SD). GraphPad Prism 8.4 software (San Diego, CA, USA) was
used to perform all the statistical analyses. Statistical comparisons
were conducted using a one-way analysis of variance (ONE-WAY ANOVA),
followed by the Turkey multiple comparison test. The differences were
then considered statistically significant at p < 0.05.
Results
Screening of Launaea cornuta extract fractions for antiproliferative activity
We performed MTT assay to evaluate the antiproliferative activity of
the crude L. cornuta extract and its solvent fractions (hexane, ethyl
acetate and water fractions) at a fixed concentration (200 μg/mL)
against human cervical cancer cell lines. The ethyl acetate fraction
significantly (p < 0.0001) exhibited a robust antiproliferative effect
on the HeLa cell lines, suppressing cancer cell growth by more than 50%
([94]Figure 1). The crude extract, hexane, had significant
antiproliferative effects (p < 0.002 and p < 0.0001, respectively) but
not within our set tight threshold, while water fractions had no
activity (p > 0.9) compared to the negative control. Based on these
criteria, we, therefore, selected the L. cornuta ethyl acetate fraction
for concentration-dependent tests to obtain the minimum inhibitory
concentration (IC[50]).
FIGURE 1.
[95]FIGURE 1
[96]Open in a new tab
Screening of antiproliferative activity of Launaea cornuta extract and
its fractions at a fixed concentration of 200 μg/mL on HeLa-229 cell
line. Doxorubicin (200 μg/mL) was used as a positive control, and 0.2%
DMSO as the negative control. All treatments were carried out in
triplicate (n = 3). Statistical significance was calculated by
nonlinear regression compared to the negative control (DMSO, 0.2%) and
*, p < 0.05; **, p < 0.01, ***, p < 0.001 and ****, p < 0.0001. DMSO
represents dimethyl sulfoxide; MTN represents Launaea cornuta.
Dose-dependent cytotoxicity
A dose-dependent cytotoxicity test was performed to determine the
concentration of the L. cornuta ethyl acetate fraction that selectively
inhibited the growth of Hela cells by determining both IC[50] and
CC[50]) ([97]Figures 2, [98]3). HeLa cells and Vero cells were treated
with ethyl acetate fraction of L cornuta at concentrations ranging from
1–64 μg/mL (HeLa cells) and 4–256 μg/mL (Vero cells). After 48 h, L.
cornuta ethyl acetate IC[50] and CC[50] for HeLa and Vero cells were
determined as 20.56 ± 2 μg/mL and 48.83 ± 23 μg/mL, respectively.
Doxorubicin exhibited an IC[50] of 2.09 ± 1.35 μg/mL on Hela cells and
a CC[50] of 3.44 ± 1.00 μg/mL on Vero cells, respectively ([99]Table
1). The dose-dependent test results for positive control (doxorubicin)
are presented in the [100]Supplementary Figure S1.
FIGURE 2.
FIGURE 2
[101]Open in a new tab
In vitro antiproliferative assay at different concentrations of the
Launaea cornuta ethyl acetate fraction against HeLa cells after 48 h of
incubation. Mean ± SD values are expressed independently of three
minimum experiments.
FIGURE 3.
[102]FIGURE 3
[103]Open in a new tab
In vitro cytotoxicity assay at different concentrations of the Launaea
cornuta ethyl acetate fraction against Vero cells after 48 h of
incubation. Mean ± SD values are expressed independently of three
minimum experiments.
TABLE 1.
Selectivity Index (SI) of Launaea cornuta ethyl acetate fraction and
doxorubicin.
Extract IC[50] (µg/mL) CC[50] (µg/mL) SI
L. cornuta ethyl acetate fraction 20.56 ± 2.83 48.83 ± 23.02 2.36
Doxorubicin 2.09 ± 1.35 3.44 ± 1.00 1.64
[104]Open in a new tab
Selectivity index (SI) was calculated to assess the selective toxicity
of L. cornuta ethyl acetate fraction toward cervical cancer cells
compared to normal cells. The resulting SI value for the HeLa cell was
2.37 and 1.64 for L. cornuta ethyl acetate fraction and doxorubicin,
respectively ([105]Table 1; [106]Figure 4), demonstrating high
selectivity of L. cornuta ethyl acetate fraction to cancer cells.
FIGURE 4.
[107]FIGURE 4
[108]Open in a new tab
Half-maximal concentration and half-maximal cytotoxic concentration of
Launaea cornuta ethyl acetate and doxorubicin on HeLa and Vero cells
after 48 h of incubation. There is a significant difference between
IC[50] and CC[50] values (p < 0.01). Mean ± SD values are expressed
independently of three minimum experiments.
Morphological study
We confirmed the effect of L. cornuta ethyl acetate fraction on the
morphology of HeLa cells for 48 h under a digital microscope. HeLa
cells exposed to varying concentrations of L. cornuta ethyl acetate
fraction exhibited atypical morphology with cellular shrinkage,
sphere-shaped and surface detachment, indicating that the extract
fraction had cytotoxic effects ([109]Figure 5) as compared to the
negative control. Notably, the effects of the extract fraction was
dose-dependent.
FIGURE 5.
[110]FIGURE 5
[111]Open in a new tab
Morphological changes in HeLa cells after exposure to varying
concentrations of Launaea cornuta ethyl acetate fraction over 48 h.
(magnification ×15). Negative control; 0.2% DMSO and Positive control;
Doxorubicin drug (2.09 μg/mL).
Effect of Launaea cornuta ethyl acetate on cell migration
A wound-healing assay was conducted to investigate the effect of L.
cornuta ethyl acetate fraction on cancer cell migration. The results
indicated a decrease in wound size in the control cells (DMSO, 0.2%),
which eventually closed after 48 h compared with treated HeLa cells. In
contrast, the HeLa cells treated with L. cornuta ethyl acetate fraction
at IC[50] of 20.56 μg/mL inhibited wound closure. The results indicated
that L. cornuta ethyl acetate significantly (p < 0.0001) inhibited cell
migration compared to the negative control after 24 and 48 h. However,
there were no significant differences between L. cornuta ethyl acetate
and the positive control at 24 and 48 h (p > 0.6 and p > 0.9,
respectively). The substantial decrease in migration and minimum
closure in wound size was attributed to the increased cell mortality
and/or inhibition of cell migration mediated by the extract fraction
([112]Figure 6A). The percentage of wound closure for L. cornuta ethyl
acetate fraction (20.56 μg/mL) was 35.9% and 41.9% for 24 and 48 h
([113]Figure 6B).
FIGURE 6.
[114]FIGURE 6
[115]Open in a new tab
Launaea cornuta ethyl acetate inhibited HeLa cells migration ability
after treatment at a concentration of 20.56 μg/mL (IC[50]). Images were
obtained at time points of 0, 24, and 48 h were taken to capture
images. (A) photomicrographs show the anti-migration effects of Launaea
cornuta ethyl acetate fraction on HeLa cells as compared with negative
control. (B) Percentage of wound healing area. Each bar graph shows the
wound closure (%) of HeLa cells. Wound areas were measured at each time
point and expressed as a percentage of reduction area in comparison
with 0 h of incubation. The percentages of wound closure were
statistically compared to the negative control. Each bar represents
mean ± SD of at least three independent experiments performed in
triplicate. ***, p < 0.001 and ****, p < 0.0001.
Preliminary screening and gas chromatography-mass spectrometry (GC-MS)
analysis of phytochemicals in L. cornuta ethyl acetate
Having demonstrated selective antiproliferative activity of L. cornuta
ethyl acetate, we next then identified the phytochemicals that could be
attributed to the established antiproliferative activity of the extract
fraction. The qualitative phytochemical screening showed broad groups
of phytochemicals as follows: glycosides, phenols, saponins,
terpenoids, quinones, and tannins ([116]Supplementary Table S2). GC-MS
analysis was performed to reveal the semi-quantitative presence of
phytochemical profiles. A total of 13 compounds were identified in L.
cornuta ethyl acetate fraction through GC-MS analysis ([117]Table 2),
and the chromatogram showing the 13 peaks is presented in the
[118]Supplementary Figure S2. The compounds identified included
(9Z,12Z) -octadeca-9,12-dienoyl chloride (57.26%),
2-Octylcyclopropene-1-heptanol (14.48%), Tricyclo [20.8.0.0 (7,16)]
triacontane, 1 (22) 7, 7 (16) -diepoxy, Tricyclo [20.8.0.0 (7,16)]
triacontane, 1 (22) 7, (16)-diepoxy (7.39%), ethyl
(9Z,12Z,15Z)-octadeca-9,12,15-trienoate (4.28%), Cycloprop
[e]indene-1a,2(1H)-dimethanol,
3a,4,5,6,6a,6b-hexahydro-5,5,6b-trimethyl-,
(1a.alpha,3a.beta,6a.beta,6b.alpha) (2.94%),
6-Hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4H)-one
(2.73%) and 2-Linoleoylglycerol (2.28%). A previous study reported that
6-Hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4H)-one
exhibited anti-inflammatory effect against lipopolysaccharide
(LPS)-induced raw macrophage ([119]Jayawardena et al., 2019), Glycerol
1,2-dipalmitate possess ant-fungal activity ([120]Sanni and Omotoyinbo,
2016). Ethyl (9Z,12Z,15Z)-octadeca-9,12,15-trienoate exhibited
anticancer activity against oral epidermoid carcinoma, breast, colon,
and lung cancer ([121]Huang et al., 2022), Stigmast-5-ene,
3beta-methoxy- induced a reduction of cell viability by apoptotic cell
death through cell cycle arrest at the sub-G1 stage ([122]Alvarez-sala
et al., 2019; Jaiganesh et al.) and tremulone exhibited anticancer
activity against cervical, colorectal and breast cancers ([123]Lu et
al., 2012; [124]Suliman and El-hddad, 2023). However, most of the
compounds identified in this study have not been reported to have
biological activity, therefore, further investigation is necessary to
evaluate the potential biological activity of each compound. The 2D
structures of these compounds are shown in [125]Figure 7.
TABLE 2.
The phytochemical profile of the Launaea cornuta ethyl acetate fraction
detected by the GC-MS technique.
Peak no. RT (minute) Compound MF MW (g/mol) Relative abundance (%) Type
of compound
1 16.476
6-Hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4H)-one
C[11]H[16]O[3] 196.2 2.73 Benzenoids
2 21.249 Glycerol 1,2-dipalmitate C[35]H[68]O[5] 568.9 1.57 Fatty acid
methyl esters
3 21.955 Cycloprop [e]indene-1a,2(1H)-dimethanol,
3a,4,5,6,6a,6b-hexahydro-5,5,6b-trimethyl-, (1a.
alpha.,3a.beta.,6a.beta.,6b.alpha.)-(−)- C[15]H[24]O[2] 236.4 2.94
Terpenoids
4 22.006 3-Heptafluorobutyriloxy-3,5,10-pregnatrien-20-one
C[25]H[27]F[7]O[3] 505.8 1.21 Aliphatic compound
5 22.361 2-Linoleoylglycerol C[21]H[38]O[4] 354.5 2.28 Glycerolipids
6 22.791 2-Octylcyclopropene-1-heptanol C[18]H[34]O 266.5 14.48 Fatty
alcohols
7 23.798 Tremulone C[29]H[46]O 410.7 1.26 Terpenoids
8 24.799 Spiro [2.6]non-4-ene, 4-ethyl-8-methylene-5-trimethylsilyl
C[15]H[26]Si 234.5 1.98 Alkenes
9 26.127 Cyclohexyldichlorophosphine C[6]H[11]Cl[2]P 185.0 0.96 Fatty
acids
10 26.127 (9Z,12Z)-octadeca-9,12-dienoyl chloride C[18]H[31]ClO 298.9
57.26 Fatty acids
11 27.018 Ethyl (9Z,12Z,15Z)-octadeca-9,12,15-trienoate C[20]H[34]O[2]
306.5 4.28 Fatty acids
12 29.624 Stigmasteryl methyl ether C[30]H[52]O 428.7 1.67 Phytosterols
13 31.846 Tricyclo [20.8.0.0 (7,16)]triacontane, 1(22),7(16)-diepoxy
C[30]H[52]O[2] 444.7 7.39 Fatty acids
[126]Open in a new tab
Key: RT, retention time (minute); MF, molecular formula; MW, molecular
weight (g/mol).
FIGURE 7.
[127]FIGURE 7
[128]Open in a new tab
Numbers 1–13 showed the 2D structures of the compounds identified in
Launaea cornuta ethyl acetate fraction (Details refer to [129]Table 2).
In-silico results
Screening for drug-like compounds in Launaea cornuta ethyl acetate fraction
The chemical compounds of L. cornuta ethyl acetate fraction obtained
from GC-MS analysis were used in the in silico studies. Thirteen
compounds were subjected to screening, and their putative
pharmacokinetics and physicochemical properties (absorption,
distribution, metabolism, excretion and toxicity) were determined using
SwissADME and pkCSM databases. Only 6 met the Lipinski’s rule of 5
(RO5) of MW; <500 g/mol, Log P; <5, HBA<10, HBD<5, and RB < 10. In
addition to Lipinski’s rules, the compounds were also predicted to be
water-soluble and did not interfere with the blood-brain barrier and
central nervous system. Furthermore, the ADMET profile suggest that the
6 compounds do not interfere with metabolism, thus minimising the risk
of drug-drug interactions ([130]Table 3, [131]Supplementary Table S3).
Oral bioavailability was evaluated for the six (6) compounds using a
bioavailability radar ([132]Ahmed Khan et al., 2023) ([133]Figure 8).
The bioavailability radars were based on six ideally adapted
physicochemical properties for oral bioavailability, namely,
lipophilicity, polarity, size, solubility, saturation, and flexibility.
The thresholds were as follows: lipophilicity (LIPO): 0.7 < XLOGP3 <
+5, SIZE: 150 < MV < 500 g/mol, flexibility (FLEX): 0 < Number of
rotatable bonds <9, saturation (INSATU): 0.25 < Fraction Csp3 < 1,
insolubility (INSOLU): 6 < LOG S < 0, and polarity (POLAR): 20 Å2 <
TPSA < 130 Å2. All candidate molecules exhibited oral bioavailability
because they were within the pink zone of bioavailability radars. The
pink-coloured area presents the fit physicochemical space for oral
bioavailability. The graph for each molecule must be adjusted to suit
drug-like properties. The prediction of ADMET pharmacokinetic
properties demonstrated that all six compounds exhibited good
absorption properties as their human intestinal absorptions (HIAs)
exceeded 90%. The inhibitory effect on cytochromes indicated that most
of them did not affect cytochromes except M5, M7, M11 and M12, which
inhibited 3A4, whereas M7, M11, and M12 inhibited 1A2 ([134]Table 3).
The ADMES toxicity confirmed that the six drug candidates were not
toxic inhibitors. M5, M8, and M11 were predicated to be sensitive to
the skin but had no hepatotoxic effect except M13.
TABLE 3.
Prediction of ADMET in silico pharmacokinetic properties of Launaea
cornuta ethyl acetate compounds.
Compounds number Absorption Distribution Metabolism Excretion Toxicity
Intestinal absorption (human) Substrate Inhibitor Total excretion
Permeability Cytochromes
BBB CNS 2D6 3A4 1A2 2C19 2C9 2D6 3A4 Ames toxicity Hepatotoxicity Skin
irritation
Numeric (% absorbed) Numeric (log BB) Numeric (log PS) Categorical
(yes/No) Numeric (log mL/minute/kg Categorical (yes/No)
M1 95.697 −0.198 −3.36 No No No No No No No 1.038 No No Yes
M2 85.807 −0.943 −3.114 No Yes No No No No No 2.171 No No No
M3 92.7 0.091 −2.37 No No No No No No No −0.007 No No No
M4 91.346 0.185 −3.244 No Yes No No No No No −0.103 No No No
M5* 90.602 −0.294 −3.227 No Yes No No No No No 2.165 No No Yes
M6 90.308 0.837 −1.802 No Yes No No No No No 1.715 No No Yes
M7* 97.921 0.827 −1.589 No Yes Yes No No No No 0.571 No No No
M8* 93.522 0.763 −2.141 No No No No No No No 1.11 No No Yes
M9 90.275 0.684 −1.794 No No No Yes No No No 0.418 No No No
M10 91.195 0.81 −1.394 No Yes No No No No No 0.237 No No No
M11* 92.747 0.766 −1.509 No Yes Yes No No No No 2.134 No No Yes
M12* 96.946 0.903 −4.387 No Yes Yes No No No No 0.663 No No No
M13* 91.183 1.466 −3.114 No Yes No No No No No 0.909 No Yes No
[135]Open in a new tab
Key: BBB, Brain-blood barrier; CNS, central nervous system; ADMET,
absorption, distribution, metabolism, excretion, and toxicity; *
Drug-like compounds (DL).
FIGURE 8.
[136]FIGURE 8
[137]Open in a new tab
Bioavailability radars of 6 Launaea cornuta ethyl acetate fraction
compounds, based on the six ideal physicochemical properties for oral
bioavailability, namely, polarity (POLAR), lipophilicity (LIPO),
saturation (INSATU), size (SIZE), flexibility (FLEX), and solubility
(INSOLU).
Prediction of targets for Launaea cornuta ethyl acetate compounds and
cervical cancer
The putative targets of the six (6) compounds of L. cornuta ethyl
acetate fraction were predicted and retrieved from three databases:
SWISS TargetPrediction (STP) with 612 targets, BindingDB (BDB) with 59
targets, and the Similarity Ensemble Approach (SEA) with 503 targets.
The targets obtained were merged to obtain 581 unique potential
targets. We also predicted human cervical cancer-related genes from 4
databases: GeneCards with 714 targets, DisGeNET with 1817 targets,
Pharos with 174 targets, and OMIM with 50 targets to obtain 2253 unique
targets. A Venn diagram was generated to show the intersected targets
between L. cornuta ethyl acetate fraction and cervical cancer as hub
genes for subsequent analyses ([138]Figure 9A).
FIGURE 9.
[139]FIGURE 9
[140]Open in a new tab
Launaea cornuta ethyl acetate-cervical cancer intersection. (A) Venn
diagram and (B) PPI network of the 124 key targets of Launaea cornuta
ethyl acetate and cervical cancer.
Construction of protein-protein interaction (PPI) network for the
compound-disease targets
The intersected targets obtained using the Venn tool were imported into
the STRING database. Cytoscape 3.10 software was used to construct a
PPI network, as shown in [141]Figure 9B. The top 30 key hub targets
were selected according to their degree and score ranking using the
Cytohubba plugin: the greater the degree value, the larger the node
([142]Supplementary Table S4). The PPI exhibited 124 nodes and 1,457
edges. The average node degree and Local Clustering Coefficient were
23.5 and 0.61, respectively. The expected number of edges was 720. The
PPI enrichment p-value was <1.0e-16, implying there were more
interactions of the proteins than expected. This could also indicate
that proteins are biologically linked as cluster proteins. The top 30
hub genes are shown in [143]Figure 10. Among the top 30 targets, AKT1,
MDM2, and CDK2 were selected as therapeutic targets in cervical cancer
due to their established roles in the regulation of apoptosis and BCL2,
Caspase9, TP53 and P21 due to their established role in the regulation
and progression of the cell cycle and apoptosis ([144]Rashmi et al.,
2014; [145]Li et al., 2023; [146]Tatekawa et al., 2024).
FIGURE 10.
[147]FIGURE 10
[148]Open in a new tab
PPI network for the top hub targets. The top 30 targets selected from
Launaea cornuta ethyl acetate-cervical cancer targets. The intensity of
the colour represents the significance (p < 0.05) of the targets, with
darker red indicating a higher degree.
Gene ontology enrichment analysis
GO and KEGG enrichment analyses were conducted for the 124 intersection
targets using ShinyGO 0.77 server (restricted species: H. sapiens; p <
0.05), and a total of 1415 GO enrichment terms were retrieved. The GO
terms were classified into three categories with 1,234 terms in the
biological processes (BP) category, 88 in the cellular components (CC)
category and 93 in the molecular functions (MF) category. The top 20
significant GO terms for each GO category were selected ([149]Figure
11). The top enriched biological processes mainly included MAP Kinase
cascade, regulation of programmed cell death and apoptotic processes,
response to both organic cyclic compounds and hormones, cellular
response to oxygen-containing compounds, and positive regulation of
cell population proliferation. The most enriched cellular compounds
terms included phosphatidylinositol 3-kinase complexes I and IA,
endosome, receptor complexes, transferase complex, and membrane raft,
and the significantly enriched molecular function terms mainly
associated with the core targets included nuclear receptors,
ligand-activated transcription factors, transmembrane receptor protein
tyrosine kinase, and protein tyrosine kinase activities.
FIGURE 11.
[150]FIGURE 11
[151]Open in a new tab
Gene Ontology enrichment terms associated with Launaea cornuta ethyl
acetate compounds. (A) biological process terms, (B) molecular function
terms, and (C) cellular component terms. The bar represents the GO
terms on the vertical axis. The LogP values are shown on the horizontal
axis. The enrichment colour scale defines the FDR, with red colour
indicating a high FDR value and a significant association.
Kyoto encyclopaedia of genes and genomes pathway enrichment analysis
KEGG pathway analysis was performed to explore the pathways associated
with the anticervical cancer effect of the L. cornuta ethyl
acetate-related target genes. The results showed that 124 target genes
were associated with enriched in 123 significantly enriched pathways (p
> 0.05). This implies that the anticervical cancer mechanism of L.
cornuta ethyl acetate involves multiple targets, genes, and pathways.
The top 20 KEGG signalling pathways with lower p-values were selected
as the significant pathways ([152]Figure 12; [153]Supplementary Table
S5). The top enriched cancer-associated pathways included central
carbon metabolism in cancer, Ras signalling pathway, FoxO signalling
pathway and PI3K-Akt signalling pathway. The PI3K-AKT signalling
pathway with a lower p-value was predicted to be highly associated with
cervical cancer and was selected for subsequent analysis. The KEGG
pathway mapped to link molecular interaction, reactions, and relation
network associated with top enriched pathway are presented in
[154]Figure 13. The proposed pathway model-based on the selected
targets is presented in [155]Figure 14.
FIGURE 12.
[156]FIGURE 12
[157]Open in a new tab
Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway enrichment
analysis. A) the enriched pathway associated with Launaea cornuta ethyl
acetate targets. The bar represents the KEGG terms on the vertical
axis. The LogP values are shown on the horizontal axis. The enrichment
colour scale defines the FDR, with red colour indicating a high FDR
value and a significant association.
FIGURE 13.
[158]FIGURE 13
[159]Open in a new tab
Enriched KEGG pathways associated with cervical cancer-related targets
of Launaea cornuta ethyl acetate.
FIGURE 14.
[160]FIGURE 14
[161]Open in a new tab
Proposed pathways and therapeutic modules of Launaea cornuta ethyl
acetate fraction against cervical cancer.
Molecular docking results
Molecular docking was performed to stimulate the possibility of binding
and interactions between L. cornuta ethyl acetate fraction compounds
and the core targets in cervical cancer using PyRx software. Six (6)
compounds of L. cornuta ethyl acetate fraction were docked against 7
target proteins ([162]Supplementary Table S6). We selected 3 target
proteins (AKT1, CDK2, and MDM2 from the top core hub genes and 4 target
proteins (BCL2, TP53, P21, and Casp9) due to their established roles in
regulating cell cycle and apoptosis ([163]Xing et al., 2015; [164]Yuan
et al., 2018; [165]Ghosh et al., 2020; [166]Luo et al., 2023). Two
ligands, stigmasteryl methyl ether (M12) and tremulone (M7), that
exhibited binding affinity less than −7.0 kcal/mol with zero (0) root
mean standard deviation (RMSD), were selected, analysed and presented
in [167]Supplementary Table S7 and [168]Supplementary Figure S3.
Molecular docking results indicated that stigmasteryl methyl ether and
tremulone ligands exhibited good binding affinity with the selected
target proteins (CDK2-M12 with −12.6 kcal/mol, MDM2-M12 with
−9.1 kcal/mol, BCL2-M12 with −10.3 kcal/mol, Casp9-M7 with
−9.9 kcal/mol, P21-M7 with −8 kcal/mol, TP53-M7 with −7 kcal/mol and
AKT-M12 with −9.5 kcal/mol respectively). Their interactions were
mainly through several hydrogen and hydrophobic bonds ([169]Figures 15
and [170]16).
FIGURE 15.
[171]FIGURE 15
[172]Open in a new tab
Molecular docking results showing 2D schematic representation of
docking interactions of stigmasteryl methyl ether (M12) and tremulone
(M7) with selected targets: (A) AKT1-M12; (B) MDM2-M12; (C) CDK2-M12;
(D) BCL2-M12; (E) Casp9-M7; (F) TP53-M7 and (G) P21-M7.
FIGURE 16.
[173]FIGURE 16
[174]Open in a new tab
Molecular docking results showing 3D schematic presentation of M12 and
M7 with target genes (A) AKT1-M12, (B) MDM2-M12, (C) CDK2-M12, (D)
BCL2-M12, (E) Casp9-M7, (F) TP53-M7 and (G) P21-M7).
The redocked results for the native ligands with their target proteins
are presented in [175]Supplementary Figure S4 and [176]Supplementary
Table S8. M12 bonds to AKT1 protein through one hydrogen interaction
with residue Glu40A and two hydrophobic interactions with residues
His13A and Trp11A. Meanwhile, the native ligand NL7 interacted with
AKT1 protein (−5.2 kcal/mol) through several hydrogen bonds with
residues Lys20A, Arg15A, and Glu17A and hydrophobic interactions with
residues Glu85A, Glu17A, Lys20A and Arg15A. The interaction between M12
and MDM2 protein occurred through hydrophobic bonds only with
interacting residues of Val109A, Tyr104A, and Arg105A, whereas the
native ligand NL2 bind to MDM2 (−7.3 kcal/mol) through one hydrogen
bond with residues Hu8201A and hydrophobic interactions with residues
Hu8201A and Lys51A. M12 bind to CDK2 protein through one hydrogen
interaction with residue Leu55A and hydrophobic interactions with
residues Thr15A, Ala151A, Leu148A, Phe152A, and Ile35A. CDK2 interacted
with its co-recrystallised ligand NL1 (−9.3 kcal/mol) through a number
of hydrogen interactions with residues Thr14A, Gln131A, Glu12A,
Asp145A, Asp127A, Phe146A, Lys33A and Leu148A as well as several
hydrophobic interactions with residues Ala151A, Ile35A, Lys33A,
Leu148A, Tyr15A, Asp127A and Asp145A respectively. BCL2 protein
interacted with M12 through several hydrophobic interactions with
residues Trp135C, Phe89C and Ala90C, while the native ligand NL3 binds
to BCL2 (−8.1 kcal/mol) through two hydrogen bonds with residues
Arg142A and His143A and hydrophobic bonds through residues Arg86A,
Ala90A, Phe89A and Glu138A. The interaction between M7 and Casp9
occurred through residue Arg44C to form a hydrogen bond and residue
Ile43 to form a hydrophobic bond. Conversely, the native ligand NL4
interacted with Casp9 protein (−9.3 kcal/mol) through several hydrogen
bonds with residues Arg44C, Arg52C, Asn45C, Glu46C, Ser48E, and Pro47A
only. M7 bind to TP53 protein through two hydrogen bonds with residues
Tys134 and Tys135 and through residue Lys134 to form a hydrophobic
interaction. The co-recrystallised ligand NL6 (−8 kcal/mol) interacted
through residues Lys129A and Gly132A to form hydrogen bonds and through
residues Met117A, Phe102A, Phe133A and Tyr123A to form hydrophobic
bonds and lastly, the interaction between P21 and M7 occurred through
residues Lys308A, Leu311A, A:59N601 forming hydrophobic bonds only
whereas the native ligand NL5 (−8.5 kcal/mol) had interaction through
residues Gly409A, Glu423A and Asp393A to form two hydrogen bonds and
hydrophobic bonds with residues Leu311A, Phe410A and Ile312A. These
results showed that CDK2-M12, BCL2-M12, Casp9-M7 and P21-M7 complexes
shared the same active site residues implicated in the binding of the
native ligand for each protein, whereas ATK1-M12, MDM2-M12 and TP53-M7
complexes did not share any active site residues implicated in the
binding of native ligands.
The effect of L. cornuta ethyl acetate fraction on mRNA expression levels of
selected target genes in HeLa cells
Real-time PCR analysis was performed to validate the putative molecular
targets of L. cornuta ethyl acetate fraction associated with the
cervical cancer cells, as depicted by the network analysis results. We
evaluated the gene expression levels of ATK1, BCL2, CDK2, MDM2, P21,
TP53, and Casp9 in HeLa cells treated with L. cornuta ethyl acetate
fraction and untreated HeLa cells. AKT1, CDK2 and MDM2 were among the
top hub genes. We further selected BCL2, P21, TP53 and Casp9 genes due
to their established role in apoptosis and cell cycle. The
quantification cycle, also known as the threshold cycle (Ct), was
determined, and relative gene expression levels of the target genes
were normalized to GAPDH ([177]Figure 17). There was significant
upregulation of Casp9 (p < 0.0001) and P21 (p < 0.5) expression levels
in HeLa cells treated with L. cornuta ethyl acetate fraction compared
to the negative control (NC). There was a significant downregulation of
BCL-2 (p < 0.01), MDM2 (p < 0.0001), and CDK2 (p < 0.01) expression
levels in the L. cornuta-treated cervical cancer cells compared to the
NC. The gene expression level of TP53 was upregulated, although the
difference was not significant (p > 0.0591) in treated HeLa cells
compared to the NC. Meanwhile, there was no significant expression
level of AKT1 (p > 0.1452) in the L. cornuta-treated cervical cancer
cells compared to the NC. The RT-qPCR results suggest the fact that L.
cornuta ethyl acetate fraction interferes with cervical cancer cell
proliferation by inducing cell cycle arrest and apoptosis.
FIGURE 17.
[178]FIGURE 17
[179]Open in a new tab
Relative gene expression analysis of Launaea cornuta ethyl acetate
treated HeLa cells and the untreated control (0.2% DMSO). Mean ± SD
values represent at least three independent experiments. The relative
expression for each target was compared to their negative control (NC).
ns, p > 0.05; *, p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
Discussion
Cervical cancer, among other cancers, remains one of the leading causes
of mortality in women worldwide ([180]Cohen et al., 2019; [181]Kasi et
al., 2021). Due to the increased global prevalence of cervical cancer
and its poor prognosis, cervical cancer remains undetected in the early
stages, resulting in patients with advanced stages; also, due to
emerging drug resistance in cervical cancer treatment, there is a need
for alternative drug design and development that involves
target-specific therapies ([182]Xi et al., 2023). Regardless of some
chemotherapeutic drugs such as cisplatin, paclitaxel (Taxol), and
carboplatin approved and recognised by the FDA of the United States,
they tend to have adverse effects and resistance in patients with
cervical cancer ([183]Li et al., 2016). Furthermore, there are limited
effective treatments for advanced cervical cancer. However, the high
cost of cancer treatment is a significant constraint on patients’
access to quality healthcare in low-income countries. These populations
tend to prefer traditional herbs that are relatively affordable and
available. Research shows that natural products from medicinal herbs
are increasingly valuable in developing anticancer drugs ([184]Elshamy
et al., 2019; [185]Owolabi et al., 2020). In this regard, L. cornuta
merits our attention because it has been widely used as a traditional
medicine among Kenyan communities to treat various diseases, including
cancers ([186]Ibrahim, 2021; [187]Chemweno et al., 2022).
Using MTT assay, we evaluated the antiproliferative activity of the
crude extract, hexane, ethyl acetate and water fractions from aerial
parts of L. cornuta against cervical cancer cell lines. Our results
demonstrated selective antiproliferative effect of L. cornuta ethyl
acetate fraction by inhibiting cellular proliferation in cervical
cancer cells (HeLa-229) by less than 50% cell viability. The
dose-dependent cytotoxicity test showed antiproliferative effects of L.
cornuta ethyl acetate on HeLa cells in a dose-dependent manner with
minimal cytotoxicity on non-cancerous cells. The ethyl acetate fraction
of L. cornuta showed potent cytotoxic effects on HeLa cells as defined
by the guidelines of the US National Cancer Institute (NCI), where an
extract is generally considered to have in vitro cytotoxic activity if
the IC[50] value is ≤ 20 μg/mL after 48 h of incubation ([188]Ogbole et
al., 2017; [189]Okpako et al., 2023b). We observed that the L. cornuta
ethyl acetate fraction exhibited cytotoxicity on HeLa cells with an
IC[50] value of 20.56 μg/mL, thus presenting L. cornuta ethyl acetate
fraction as a potential chemotherapeutic drug agent for cervical
cancer. Previous studies reported the biological activity of Launaea
species against various types of cancer cell lines, such as breast
cancer, MCF-7, A549, and HCC cell lines ([190]Rawat et al., 2016;
[191]Rezaei Seresht et al., 2016; [192]Abouzied et al., 2021). Notably,
this study is the first to report the anticancer activity of L. cornuta
against HeLa cell line. On the other hand, L. cornuta ethyl acetate
fraction did not display toxicity toward normal cells (CC[50] of
48.83 μg/mL), setting a safety margin (selectivity index) of 2.38,
which agreed with the safety margin threshold for drugs. A selectivity
index greater than 2 is considered highly selective ([193]Canga et al.,
2022). Therefore, the anticancer activity exhibited by L. cornuta ethyl
acetate fraction was not a result of general cellular toxicity compared
with the doxorubicin, having a selective index of 1.64. Furthermore, L.
cornuta ethyl acetate fraction demonstrated a substantial inhibitory
effect on HeLa cell migration. It was noted that the wound size
remained relatively open by almost 50% relative migration distance in
HeLa cells treated with L. cornuta ethyl acetate fraction compared with
the untreated cells. This result is consistent with findings from Khan
et al. (2024) and Lin et al. (2022), where 6-gingerol and puerarin
exhibited antimigration effects by suppressing the migration of PC-3
cancer cells and Ishikawa endometrial cancer cells, respectively
([194]Lin et al., 2022; [195]Khan et al., 2024). Thus, justifying the
extract’s ability to prevent cancer cell metastases. Furthermore, we
also studied the morphological changes in HeLa cells exposed to varying
concentrations of the L. cornuta ethyl acetate fraction for 48 h. The
data showed that L. cornuta ethyl acetate fraction triggered
morphological changes in cells, including diminished HeLa cell
viability with irregular and rounded shapes, detached from each other
in a dose-dependent as compared with untreated HeLa cells, which remain
in an organised monolayer with regular shapes. This finding tallied
with the study reported by Ghosh et al. (2020) noted the atypical
morphological changes of HeLa cells exposed to Hexagonia glabra after
24 h ([196]Ghosh et al., 2020). In summary, these findings indicate the
potential effects of L. cornuta ethyl acetate fraction in inhibiting
proliferation in HeLa cells.
The preliminary screening of L. cornuta showed the presence of phenols,
glycosides, tannins, and terpenoids in the ethyl acetate fraction of L.
cornuta, which were also reported by previous findings in L. cornuta
ethyl acetate aerial extract ([197]Akimat et al., 2021; [198]Maina et
al., 2022). Thirteen (13) photochemical compounds were identified in L.
cornuta ethyl acetate fraction through GC-MS analysis, and fatty acids
and terpenoids were the most abundant compounds. These compounds were
not similar to those reported by Machocho et al. (2014) in the aerial
extract of L. cornuta ethyl acetate ([199]Machocho et al., 2014). Some
of the compounds identified in L. cornuta ethyl acetate fraction have
been previously reported to exhibit anticancer (cytotoxic and
antioxidant) activity against various cancer cell lines; these included
ethyl (9Z,12Z,15Z)-octadeca-9,12,15-trienoate (fatty acids)
([200]Chumkaew et al., 2014; [201]Eid et al., 2021; [202]Huang et al.,
2022), Stigmast-5-ene, 3beta-methoxy- (phytosterols) ([203]Chumkaew et
al., 2014; [204]Alvarez-sala et al., 2019; Jaiganesh et al.) and
tremulone (terpenoids) ([205]Gupta and Chaphalkar, 2016; [206]Lehtonen
and Kaarniranta, 2016). These compounds were reported to induce
cytotoxic effects in cancer cells through at least one of the pathways:
apoptosis, inhibition of cell cycle, invasion and metastasis
([207]Shaikhaldein et al., 2022). However, some of the compounds
identified in our study have not been reported to exhibit anticancer
activity, and their presence in the ethyl acetate fraction of L.
cornuta may suggest that they work synergistically with other compounds
([208]Ibrahim et al., 2022). Given the presence of these compounds in
L. cornuta, ethyl acetate fraction could be attributed to the cytotoxic
effects of the plants on cervical cancer cells. However, further
research should be done to evaluate the anticancer activity of these
individual compounds identified in the ethyl acetate fraction of L.
cornuta.
The Gene Ontology and Kyoto Encyclopaedia of Genes and Genome Analysis
presented several pathways as well as other diseases and disorders for
the top hub genes. GO enrichment analysis revealed the direct
involvement of the identified compounds in the regulation or
progression of cervical cancer through programmed cell death, cell
population, apoptotic processes, positive regulation of molecular
function, and mitogen-activated protein (MAP) signalling. The KEGG
pathway analysis showed that PI3K-AKT1 signalling was a significantly
enriched pathway for L. cornuta ethyl acetate compounds. The EGFR
tyrosine kinase inhibitor pathway, the MAPK signalling pathway, the Ras
signalling pathway, and the FoxO signalling pathway were also enriched,
suggesting the use of L. cornuta ethyl acetate fraction in the
development of multi-target drugs. The MAPK pathway is known to play a
role in inducing apoptosis and cell cycle arrest in tumorigenic cells
([209]Chen, 2020; [210]Huang et al., 2020; [211]Tatekawa et al., 2024).
Adding to the evidence from the pathway enrichment for L. cornuta ethyl
acetate, the plant extracts may be used in targeting prostate cancer,
human cytomegalovirus infection, and endocrine resistance treatment.
PI3K/Akt pathway is known to play a key role in cancer cell
proliferation, metastasis, differentiation, and drug resistance
([212]Xia et al., 2015; [213]Arjumand et al., 2016). Previous studies
have shown that activation of PI3K through Akt phosphorylation results
in the regulation of cell proliferation, thus promoting tumour growth
([214]Arjumand et al., 2016; [215]Jiang et al., 2020; [216]Ma et al.,
2024). Potential drug-like compounds that can inhibit cancer cell
progression by negatively inhibiting Akt phosphorylation and
downregulation of Akt kinase, thus regulating the PI3K-Akt pathway,
could be a potential anticancer drug ([217]Tao et al., 2017; [218]Mart
et al., 2023). Therefore, targeting the PI3K/Akt signalling pathway and
its downstream targets, such as STAT3 and mTOR, may be a therapeutic
option for L. cornuta ethyl acetate.
To validate the results of the network analysis, molecular docking was
performed. As shown by molecular docking results, L. cornuta ethyl
acetate’ stigmasteryl methyl ether (M12) and tremulone (M7) exhibited
good interaction toward AKT1, CDK2, MDM2, BCL2, TP53, P21, and Casp9
proteins with binding affinity ranging from −7.0 to −12.6 kcal/mol. A
docking score of less than −5 kcal/mol has been shown to represent a
good binding affinity ([219]Zhao et al., 2022), which means that the
lower the docking score, the more stable and stronger the affinity
between compounds and targets. As shown by the docking scores, the
binding energies of all docking were less than −7.0 kcal/mol,
indicating that these target genes have good and stable binding
affinity with stigmasteryl methyl ester and tremulone compounds,
respectively. Moreover, the binding energy values of the compounds
docked with the selected target proteins are lower than those of the
native ligands redocked with the selected proteins. Therefore, these
results implies that M7 and M12 compounds exhibited good binding
affinity and can form more stable interactions with the targeted
proteins in cervical cancer cells. M7 and M12 compounds bonds to the
CDK2, BCL2, Casp9 and P21 proteins through the same active binding
pocket implicated in the binding of the native co-recrystallised
ligands, suggesting M7 and M12 compounds maybe orthosteric hits.
Whereas, M7 and M12 interacted with AKT1, MDM2, and TP53 proteins
through different hydrogen and hydrophobic interacting amino acid
residues (active binding pocket), which are not implicated in the
binding of the native co-recrystallised ligand of redocked, thus
suggesting that M7 and M12 compounds could be an allosteric hit toward
AKT1, MDM2 and TP53 proteins. The presence of hydrogen bonds,
hydrophobic interactions, and van der Waals forces in the
ligand-protein interactions are critical for protein-ligand stability.
Hydrogen bonds improve specificity, hydrophobic interactions augment
the drug effects, and van der Waals forces improve structural fit
between molecules, resulting in increased drug efficacy and potency in
therapeutic applications ([220]Dhar and Roy, 2020; [221]Nussinov et
al., 2023).
The gene expression levels of P21 and Casp9 were found to be high in
HeLa cells treated with the ethyl acetate fraction of L. cornuta
(20.56 μg/mL) compared to the untreated control. Studies by Dalghi et
al. (2023), and Zhou et al. (2024) reported that high expression levels
of P21 in cancer cells are implicated in the promotion of cell cycle
arrest and apoptosis ([222]Dalghi et al., 2023; [223]Zhou et al.,
2024). BCL2 is a known inhibitor of apoptosis ([224]Babakanrad et al.,
2023; [225]Branch et al., 2023; [226]Hosseini et al., 2024). Cancer
cells typically evade cell death by upregulating antiapoptotic proteins
like BCL-2 or inhibiting pro-apoptotic proteins such as Casp9. BCL-2
inhibits mitochondrial apoptosis by binding to pro-apoptotic proteins
and blocking pore formation and cytochrome c release. Caspases and
upstream regulatory factors, such as p53, trigger apoptosis ([227]Ghosh
et al., 2020). As observed in our results, there was downregulation of
BCL2 with concomitant upregulation of Casp9 in HeLa cells treated with
L. cornuta ethyl acetate compared to the untreated HeLa cells; this
pattern was also reported by Eltamany et al. (2022). MDM2 is a protein
that directly regulates TP53 function and stability ([228]Hou et al.,
2019; [229]Zhu et al., 2021); as noted in this study, MDM2 was
significantly downregulated (p < 0.0001) and therefore, the expression
level of TP53 was insignificantly expressed (p > 0.05) in treated HeLa
cells compared to the untreated control. Study by Zhu et al. (2021)
reported that downregulation of the expression level of MDM2 results in
upregulation of TP53 expression, resulting in the induction of cell
cycle arrest and apoptosis in cervical cancer cells ([230]Zhu et al.,
2021). CDK is well known to regulate the transition of the cell cycle
from the G1 phase to the S phase, thus accelerating the S phase and the
proliferation efficiency in cells. It is highly expressed in cancer
cells ([231]Liu et al., 2013; [232]Tadesse et al., 2020; [233]Bao et
al., 2023). L. cornuta ethyl acetate fraction elicited a significant
downregulation of CDK2 (p > 0.01), which is consistent with the study
reported by Zhong et al. (2019) ([234]Zhong et al., 2019). Therefore,
the downregulation of CDK2 in HeLa cells could be attributed to the
anticancer effect of the L. cornuta ethyl acetate fraction through
suppression of the cell cycle. Furthermore, the expression level of
AKT1 was observed to be insignificant in treated HeLa cells despite its
crucial role in the regulation of cell survival, angiogenesis, and
tumorigenesis of cancer ([235]Rashmi et al., 2014).
Conclusion
The L. cornuta ethyl acetate fraction demonstrates significant
antiproliferative effects against cervical cancer HeLa cells, with
minimal cytotoxicity on non-cancerous cells and rich composition of
fatty acids and terpenoids identified through GC-MS analysis. Network
analysis has pinpointed drug-like compounds, including
2-linoleolglycerol, tremulone, spiro [2.5] non-4-ene,
4-ethyl-8-methylene-5-trimethylsilyl, ethyl (9Z, 12Z,
15Z)-octadeca-9,12,15-trienoate, stigmasteryl methyl ether and tricylo
[20.8.0.0 (7,16)] triacontane, 1(22),7(16)-diepoxy targeting key
proteins involved in cancer progressions and tumorigenesis, such as
AKT1, MDM STAT3, EGFR, CDK2, MTOR, MAPK3 PTGS2, MCL1 and TNF among
others. Intriguingly, the gene expression levels of BCL2, CDK2, MDM2,
Casp9 and P21 were reversed in HeLa cells treated with L. cornuta ethyl
acetate fraction when compared with the untreated HeLa cells,
indicating its potential to regulate cell proliferation, apoptosis, and
cell cycle in cancer cells. These findings highlight L. cornuta’s
promise as a multi-target anticancer drug, warranting further in-vitro
and in vivo studies to assess its efficacy and safety for clinical use
as well as anticancer activity on other cancer cell lines.
Acknowledgments