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. 2024 Jun 28:15:1399885.
doi: 10.3389/fphar.2024.1399885. eCollection 2024.

Phytochemical analysis, in-vitro and in-silico study of antiproliferative activity of ethyl acetate fraction of Launaea cornuta (Hochst. ex Oliv. & Hiern) C. Jeffrey against human cervical cancer cell line

Inyani John Lino Lagu  1 , Dorothy Wavinya Nyamai  2 , Sospeter Ngoci Njeru  3
Affiliations

Phytochemical analysis, in-vitro and in-silico study of antiproliferative activity of ethyl acetate fraction of Launaea cornuta (Hochst. ex Oliv. & Hiern) C. Jeffrey against human cervical cancer cell line

Inyani John Lino Lagu et al. Front Pharmacol. .

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 (IC50 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 (IC50 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; anticancer activity; antiproliferative; cervical cancer; cytotoxicity; molecular docking; network analysis; phytochemicals.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
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.
FIGURE 2
FIGURE 2
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
FIGURE 3
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.
FIGURE 4
FIGURE 4
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 IC50 and CC50 values (p < 0.01). Mean ± SD values are expressed independently of three minimum experiments.
FIGURE 5
FIGURE 5
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).
FIGURE 6
FIGURE 6
Launaea cornuta ethyl acetate inhibited HeLa cells migration ability after treatment at a concentration of 20.56 μg/mL (IC50). 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.
FIGURE 7
FIGURE 7
Numbers 1–13 showed the 2D structures of the compounds identified in Launaea cornuta ethyl acetate fraction (Details refer to Table 2).
FIGURE 8
FIGURE 8
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).
FIGURE 9
FIGURE 9
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.
FIGURE 10
FIGURE 10
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.
FIGURE 11
FIGURE 11
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.
FIGURE 12
FIGURE 12
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
FIGURE 13
Enriched KEGG pathways associated with cervical cancer-related targets of Launaea cornuta ethyl acetate.
FIGURE 14
FIGURE 14
Proposed pathways and therapeutic modules of Launaea cornuta ethyl acetate fraction against cervical cancer.
FIGURE 15
FIGURE 15
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
FIGURE 16
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).
FIGURE 17
FIGURE 17
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.
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Grants and funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This research was supported by the African Union Scholarship Programme through the Pan African University Institute for Basic Sciences, Technology, and Innovation (PAUSTI), funding to IL, REF: PAU/ADM/PAUSTI/9/2022 and the KEMRI Internal Research Grant funding to SN, REF: KEMRI/IRG/EC0017.