Anethole inhibits human U87 Glioma cell proliferation by inducing apoptosis via the PI3K/AKT pathway

Ahmed Abdullah Al Awadh; Elhashimi Eltayb Hassan; Omer Mohamed Shoaib; Osman AE Elnoubi; Saadalnour Abusail Mustafa; Yasir Mohammed Althayrayan; Majed Ahmed Althayrayan; Mohammed Merae Alshahrani

doi:10.1371/journal.pone.0336975

. 2025 Nov 21;20(11):e0336975. doi: 10.1371/journal.pone.0336975

Anethole inhibits human U87 Glioma cell proliferation by inducing apoptosis via the PI3K/AKT pathway

Ahmed Abdullah Al Awadh ¹, Elhashimi Eltayb Hassan ¹, Omer Mohamed Shoaib ¹, Osman AE Elnoubi ¹, Saadalnour Abusail Mustafa ¹, Yasir Mohammed Althayrayan ^2,³, Majed Ahmed Althayrayan ⁴, Mohammed Merae Alshahrani ^1,^*

Editor: Yasmina Abd‐Elhakim⁵

PMCID: PMC12637905 PMID: 41270054

Abstract

Glioma is characterized by rapid progression, resistance to conventional therapies, and poor prognosis. Current treatments are often limited by their inability to selectively target tumor cells. Natural compounds, such as anethole, have shown promising anticancer properties in various cancers, but their efficacy in glioblastoma remains unexplored. This study investigates the anticancer activity of anethole in glioma cells, focusing on its influence on cell proliferation, apoptosis, and the PI3K/Akt pathway. Human glioma cell lines (U87-MG and LN-229) and normal human astrocytes (NHA) were treated with anethole. Cell viability was evaluated using the CCK-8 assay, while colony formation and AO/EB staining assays evaluated proliferation and apoptosis, respectively. Cell viability was evaluated using the CCK-8 assay, while colony formation and AO/EB staining assessed proliferation and apoptosis, respectively. Western blotting was used to analyze apoptosis-related markers and PI3K/AKT pathway proteins. Molecular docking assessed anethole–PI3K binding, and in silico analyses (SwissTargetPrediction, KEGG, RummaGEO) identified putative targets and pathways. Anethole exhibited selective cytotoxicity, with significantly lower IC₅₀ values for U87-MG (10.8 ± 0.42 µM) and LN-229 (12.5 ± 0.51 µM) compared to NHA cells (61.5 ± 1.27 µM), calculated from three independent experiments with triplicate wells. Colony formation was notably inhibited in a dose-dependent manner. AO/EB staining and Western blotting confirmed this with upregulation of Bax, downregulation of Bcl-2, and reduced phosphorylation of PI3K and Akt. Molecular docking revealed strong binding affinity of anethole to PI3K (−9.32 kcal/mol), and Western blot showed inhibition of PI3K and Akt phosphorylation. Anethole selectively inhibits glioma cell proliferation by inducing apoptosis and suppressing the PI3K/Akt cascade. These observations underscore its potential as a novel therapeutic agent for glioblastoma, warranting further preclinical and clinical investigations.

1. Introduction

Gliomas are among the most prevalent primary brain tumors, constituting about 80% of all malignant intracranial neoplasms [1]. Glioblastomas, a subtype of gliomas, are particularly malignant, characterized by excessive proliferation, invasion into adjacent brain parenchyma, and resistance to standard therapies, leading to a grim prognosis and a 5-year survival rate of only 2–10% despite the best possible treatment. The prevalence of gliomas globally is increasing, raising the critical need for new therapeutic approaches to combat this devastating illness [2]. Given the limited efficacy of current treatment modalities, including surgery, radiotherapy, and chemotherapy, there is a critical demand for new agents that can effectively target glioma cells while minimizing toxicity to normal brain tissue [2]. Conventional chemotherapeutics such as temozolomide also have limitations, including drug resistance and side effects [3]. These shortcomings emphasize the need for new therapeutic strategies that overcome these gaps and enhance patient outcomes. In the quest for novel treatments, natural products are recognized as a promising reservoir of bioactive molecules with potent anticancer properties [4]. Plant-derived compounds, in particular, have shown remarkable antitumor activity by inducing multiple mechanisms of action such as apoptosis, cell-cycle arrest, and signaling pathway modulation [5]. The effectiveness of compounds like paclitaxel and vincristine illustrates the value of natural molecules as important resources for cancer drug development [6].

Anethole, a plant-derived phytoconstituent with a broad range of therapeutic applications [7], has recently emerged as a topic of great interest. Several studies have highlighted its ability to target multiple cellular pathways involved in cancer progression, making it a strong candidate for further exploration in oncology. Recent studies have shown the potency of anethole against several types of cancers, such as prostate and oral cancers [8,9]. For example, Contant et al. (2021) showed that anethole activates apoptosis in oral cancer cells, while Nakagawa and Suzuki (2003) reported its ability to impede growth via cell-cycle arrest in prostate cancer cells [8,9]. Furthermore, anethole has also been reported to activate apoptosis in breast cancer cells and inhibit the metastasis of prostate cancer cells [10,11]. In yet another study, anethole has been shown to target STAT3 to impede lung cancer cell growth [12]. These findings underscore the versatility of anethole as an anticancer molecule**.** However, despite its reported efficacy in various cancers, its effects on gliomas remain unexplored. This lack of data highlights a critical gap in understanding the potential of anethole in brain tumors. Among the numerous signaling pathways implicated in glioblastoma pathogenesis, the phosphoinositide 3-kinase (PI3K)/AKT pathway is one of the most frequently activated [13]. This pathway regulates key cellular processes such as proliferation, apoptosis, angiogenesis, metabolism, and therapy resistance [14]. Genetic alterations, such as PTEN loss and PIK3CA amplification, contribute to its hyperactivation in gliomas [15]. Importantly, PI3K/AKT signaling has been associated with tumor aggressiveness, treatment failure, and poor prognosis in glioblastoma patients. Therefore, the PI3K/AKT axis is considered a prime molecular target for therapeutic intervention in glioblastoma. Interfering with this pathway may sensitize glioma cells to apoptosis and reduce their invasive capacity [16].

Accordingly, the aim of this investigation was to evaluate the anticancer efficacy of anethole against glioma cells and to determine its potential mechanism of action. Specifically, this research aimed to unveil the influence of anethole on cell growth and key molecular cascades, such as the PI3K/AKT pathway, which plays a central role in glioma pathogenesis [17]. By targeting this pathway, we sought to determine whether anethole could overcome resistance mechanisms and lay the foundation for its future use as a therapeutic agent in glioma therapy. In doing so, the present study contributes to the expanding literature on natural product-based approaches to cancer therapy and may pave the way for future clinical investigations.

2. Materials and methods

2.1. Drug and reagents

Anethole (99% purity; Sigma-Aldrich, Oakville, ON, Canada) was dissolved in methanol to prepare a 3 mM stock solution and subsequently diluted to the required concentrations for each experiment. U87-MG (ATCC® HTB-14™), LN-229 (ATCC® CRL-2611™), and normal human astrocytes (NHA; ScienCell Cat.#1800) were authenticated and confirmed mycoplasma-free before use. DMEM (Gibco), FBS (Gibco), penicillin-streptomycin (Gibco), CCK-8 (Dojindo Cat.#CK04), crystal violet (Sigma), AO/EB (Sigma), RIPA buffer (Thermo), PVDF membranes (Millipore), and ECL substrate (Thermo) were used for all assays.

2.1. Cell culture

Glioma cell lines U87-MG and LN-229, along with NHA, were cultured in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin at 37 °C in a humidified 5% CO₂ incubator. Prior to anethole treatment, cells were seeded and grown to 70–80% confluence.

2.2. CCK-8 assay

Cell viability was determined using the CCK-8 assay. U87-MG, LN-229, and NHA cells were seeded in 96-well plates (5 × 10³ cells/well) and treated with anethole (0–96 µM) for 24 h [8]. After treatment, 10 µL of CCK-8 solution was added and plates were incubated for 2 h at 37 °C. Absorbance at 450 nm was measured using a microplate reader. Each experiment contained three technical replicate wells per condition; values were averaged to yield one biological replicate.

2.3. Colony assay

U87-MG cells were plated in six-well plates with 500 cells per well for colony development analysis. Cells were exposed to anethole at concentrations of 0, 5.4, 10.8, and 21.6 µM and cultured for 10–14 days, with the medium refreshed every 2–3 days. Colonies were fixed with 4% paraformaldehyde for 20 minutes, stained with 0.5% crystal violet, and observed by a microscope. Those with over 50 cells were manually counted. The assay was repeated n = 3 independent times, each with two technical wells per condition. Colony counts were averaged per experiment before statistical analysis.

2.4. AO/EB staining assay

To determine the induction of apoptosis, AO/EB staining was performed. U87-MG cells were administrated with anethole at 0, 5.4, 10.8, and 21.6 µM for 24 h. The treated cells were washed with PBS and stained with AO/EB solution (1:1 ratio) for 6 min. The stained cells were visualized under a fluorescence microscope, and apoptotic, necrotic, and viable cells were quantified based on fluorescence emission.

Apoptotic, necrotic, and viable cells were differentiated based on nuclear morphology and fluorescence emission: viable cells appeared green with uniform nuclei, early apoptotic cells displayed green nuclei with chromatin condensation, late apoptotic cells showed orange-red fluorescence with nuclear fragmentation, and necrotic cells exhibited uniform orange-red nuclei. At least 300 cells per group were counted in randomly selected fields using ImageJ [18]. The percentage reported represents total apoptosis (early + late) averaged from three independent experiments.

2.5. In Silico analysis

Molecular targets of anethole were identified using the SwissTargetPrediction platform. Identified targets were subjected to KEGG pathway enrichment analysis to elucidate pathways potentially influenced by anethole. Additionally, gene expression profiles were examined using the RummaGEO database to correlate identified targets with cancer related processes. The expression levels of the selected anethole targets were retrieved from the TCGA database. The pharmacokinetic properties of anethole were evaluated using the SwissADME web tool (http://www.swissadme.ch). The SMILES structure of anethole was entered to obtain predictions on lipophilicity (LogP), topological polar surface area (TPSA), gastrointestinal (GI) absorption, and blood–brain barrier (BBB) permeability. A consensus LogP was calculated from five models, and BBB permeability was assessed using the BOILED-Egg model. Additional parameters such as water solubility, CYP enzyme inhibition, and bioavailability score were also recorded.

2.6. Molecular docking

Docking simulations were performed using DockingServer. Anethole was energy-minimized with the MMFF94 force field, and Gasteiger charges were assigned. The protein model was prepared in AutoDock tools with hydrogen atoms and Kollman charges [19,20]. A 20 × 20 Å grid with 0.375 Å spacing was used for affinity maps. Simulations employed the Lamarckian genetic algorithm and Solis & Wets local search method, with random ligand initialization, two runs, 250,000 energy evaluations, and a population size of 150 [20].

AutoDock 4.2 was chosen over more recent tools like AutoDock Vina or Schrödinger due to its well-established reliability, flexibility in algorithm customization, and detailed output of binding energy components. While Vina offers faster performance, AutoDock 4.2 provides more granular control over docking parameters and energy scoring, which was essential for comparative analysis of binding interactions. Moreover, web-based interface of DockingServer ensures reproducibility and accessibility, making it suitable for high-throughput screening in academic settings.

2.7. Western blot analysis

Western blotting was employed to examine expression of specific proteins. Cells were lysed using RIPA buffer and 30 µg of protein was loaded onto an SDS-PAGE gel, transferred to PVDF membranes, and blocked with 5% non-fat milk. Membranes were incubated overnight at 4°C with primary antibodies targeting Bax (Cell Signaling #2772), Bcl-2 (#15071), PI3K p85 (#4257), p-PI3K (Tyr458) (#4228), AKT (#9272), p-AKT (Ser473) (#9271), and β-actin (#4967). After washing, HRP-conjugated secondary antibodies were applied, and protein bands were visualized using ECL. Densitometric analysis was performed using ImageJ. Phospho-proteins were normalized to their respective total proteins (p-PI3K/PI3K and p-AKT/AKT), with β-actin serving only as a loading control. Uncropped blots with visible molecular weight markers were included in (Supplementary S1 Fig).

2.8. Statistical analysis

All statistical analyses were performed using GraphPad Prism software version 9.5.1 (GraphPad Software, LLC, San Diego, CA, USA). Data are presented as mean ± SD of n = 3 independent biological experiments, unless otherwise stated. Comparisons between groups were conducted using either Student’s t-test or one-way ANOVA, followed by Tukey’s post hoc test where appropriate. A p-value less than 0.05 was considered statistically significant.

3. Results

3.1. Anethole inhibits proliferation of glioma cells

The antiproliferative effects of anethole were assessed on U87-MG, LN-229, and normal NHA cells. Anethole treatment for 24 hours significantly decreased glioma cell viability in a concentration-dependently (p < 0.05). The IC₅₀ values, calculated from three independent experiments with triplicate wells (n = 3), were 10.8 ± 0.42 µM for U87-MG cells, 12.5 ± 0.51 µM for LN-229 cells, and 61.5 ± 1.27 µM for NHA cells (Fig 1A). This highlights the selective cytotoxicity of anethole toward glioma cells compared to normal astrocytes. The colony formation assay further confirmed these results, showing a significant reduction in colony numbers in U87-MG cells treated with 5.4, 10.8, and 21.6 µM anethole compared to untreated controls (Fig 1B).

Fig 1 — **(A)** Cell viability assay showing the effects of anethole on glioma (U87-MG, LN-229) and normal (NHA) cells. **(B)** Colony formation assay of U87-MG cells treated with indicated concentrations of anethole. Each experiment was independently repeated three times, with three technical replicates per condition. Data are expressed as the mean ± SD of three independent experiments (*P < 0.05 vs. control).

3.2. Anethole triggers apoptosis in glioma cells

The pro-apoptotic effects of anethole were confirmed through AO/EB staining to visually quantify apoptotic cells. AO/EB staining showed a dose-dependent increase in apoptotic cells, with higher concentrations of anethole resulting in more yellow-orange and red fluorescent cells, indicating apoptosis in U87-MG cells. The percentage of total apoptotic cells (early + late) was ~ 2% at control which increased to 51.3% at 21.8 µM anethole (Fig 2A). Western blot analysis corroborated these findings, revealing a dose-dependent Bax upregulation and Bcl-2 downregulation (Fig 2B-2D).

Fig 2 — **(A)** AO/EB staining assay showing apoptosis in U87-MG cells at indicated concentrations of anethole. **(B)** Western blot analysis of Bax and Bcl-2 protein expression in U87-MG cells treated with indicated concentrations of anethole. **(C)** Densitometric analysis of Bax expression. **(D)** Densitometric analysis of Bcl-2 expression. All experiments were independently repeated three times, and data are presented as mean ± SD (*P < 0.05 vs. control).

3.3. Anethole targets genes involved in cancer pathways

Through SwissTargetPrediction, 98 potential targets of anethole were identified, with functional roles in enzymatic activity, gene regulation, and redox processes (Fig 3A). KEGG pathway enrichment analysis showed that these targets are associated with cancer-related pathways, including chemical carcinogenesis, xenobiotic metabolism, and immune checkpoint regulation, emphasizing their roles in apoptosis and cancer immunotherapy (Fig 3B). RummaGEO analysis further demonstrated that these targets are significantly expressed in glioma tissues and are linked to key processes such as apoptosis, autophagy, cell cycle regulation, EMT, and metastasis, highlighting their relevance in glioma progression and treatment (Fig 3C). KEGG cluster analysis revealed 16 of these genes to be involved in cancer pathways (Fig 4A). Additionally, TCGA data analysis confirmed differential expression patterns of these targets in glioma, with key oncogenic pathways such as PI3K/Akt, JAK/STAT, and hormone biosynthesis being significantly enriched (Fig 4B). These integrated findings corroborate the experimental results of apoptosis induction by anethole, underscoring its potential as an anticancer agent targeting glioma-specific pathways.

Fig 3 — **(A)** Pie chart representing the percentage distribution of targets identified by the SwissTarget Prediction tool across different categories. **(B)** KEGG pathway analysis showing the association of identified targets with various pathways. **(C)** RummaGEO analysis highlighting the relevance of identified targets to molecular processes.

Fig 4 — **(A)** KEGG cluster analysis illustrating 16 cancer-related targets of anethole. **(B)** Expression levels of these 16 targets in 162 glioma tissues retrieved from the TCGA database.

3.4. Anethole targets PI3K and inhibits the PI3K/Akt pathway

Of the targets identified, PI3K and JAK2 were prioritized based on their elevated expression in glioma datasets from TCGA. Anethole demonstrated differential binding affinities with PI3K and JAK2 in molecular docking studies, highlighting its potential as a modulator of key signaling pathways. For PI3K, anethole exhibited a strong binding interaction with an estimated free energy of binding of −9.32 kcal/mol and an inhibition constant (Ki) of 120.40 nM. The binding was stabilized primarily by van der Waals, hydrogen bonding, and desolvation energies (−10.15 kcal/mol), with minor electrostatic contributions (−0.50 kcal/mol), resulting in a total intermolecular energy of −10.65 kcal/mol. The interaction surface was 650.471, and key residues involved were TRP780, ILE800, TYR836, ILE848, VAL850, VAL851, SER854, MET922, and ILE932, emphasizing significant contact with the active site (Fig 5A). In contrast, the interaction with JAK2 was moderate, with a free energy of binding of −4.03 kcal/mol and a Ki of 1.11 mM. The binding energies were mainly derived from van der Waals and hydrogen bonding (−4.36 kcal/mol) with minor electrostatic interactions (−0.26 kcal/mol), leading to a total intermolecular energy of −4.62 kcal/mol. The interaction surface was smaller (539.088), with key residues including ARG975, LEU1001, PRO1002, LYS1005, and PHE1031 (Fig 5B). These findings suggest that anethole has a stronger binding affinity and more favorable interactions with PI3K compared to JAK2, supporting its potential as a selective inhibitor of PI3K-related pathways while exhibiting moderate activity against JAK2. Further optimization could enhance its efficacy and specificity for these targets. Western blot analysis further confirmed the inhibitory influence of anethole on the PI3K/Akt pathway (Fig 5C). Densitometric analysis, performed by normalizing phosphorylated proteins to their respective totals (p-PI3K/PI3K and p-AKT/AKT) with β-actin serving as a loading control, revealed that anethole treatment induced a concentration-dependent decrease in PI3K and AKT phosphorylation in U87-MG cells, indicating effective suppression of this critical survival pathway (Fig 5D and 5E).

Fig 5 — **(A)** Molecular docking analysis showing the interaction of anethole with PI3K. **(B)** Molecular docking analysis showing the interaction of anethole with JAK2. **(C)** Western blot analysis of PI3K/AKT pathway-related proteins in U87-MG cells treated with anethole. **(D)** Densitometric quantification of p-PI3K/PI3K. **(E)** Densitometric quantification of p-AKT/AKT. Data are expressed as mean ± SD (n = 3 independent biological experiments). Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test (*P < 0.05).

3.5. In Silico ADME and BBB Prediction

SwissADME analysis revealed that anethole has a consensus LogP of 2.79, indicating favorable lipophilicity for membrane permeability. The topological polar surface area (TPSA) was calculated as 9.23 Å², well below the 90 Å² threshold for effective blood–brain barrier (BBB) penetration (Supplementary S2 Fig in S1 Fig). The BOILED-Egg model predicted that anethole is BBB permeant and exhibits high gastrointestinal absorption (Supplementary Fig S3 in S1 Fig). Additionally, the compound was found to be soluble across all three solubility models and showed a bioavailability score of 0.55. These findings support the potential of anethole to cross the BBB and exert central nervous system effects.

4. Discussion

Glioma continues to be a major oncology challenge due to its aggressive growth, therapy resistance, and dismal prognosis [21]. Natural compounds such as anethole have gained attention for their potential to modulate critical cancer pathways with minimal toxicity to normal cells [22]. The present study emphasizes the strong anticancer activity of anethole, its selective cytotoxicity, apoptosis induction, and the inhibition of the PI3K/Akt cascade in glioma cells. These results establish a basis for understanding the molecular mechanisms underlying the therapeutic potential of anethole and its broader applications in glioma therapy. In this study, anethole exhibited preferential cytotoxicity toward glioma cells as indicated by significantly lower IC₅₀ values for U87-MG and LN-229 compared with normal human astrocytes (NHA). This apparent selectivity may be attributed to the higher basal activation of the PI3K/AKT pathway and altered membrane permeability in glioma cells. Tumor cells often depend on constitutively active PI3K/AKT signaling due to genetic alterations such as PTEN loss or PIK3CA amplification, rendering them more vulnerable to inhibitors targeting this axis [23,24]. Additionally, changes in lipid composition, increased fluidity, and disrupted oxidative homeostasis in cancer membranes may facilitate greater anethole uptake compared with normal astrocytes [25]. These observations are consistent with earlier studies showing selective cytotoxicity of anethole in oral and breast cancer models [8,26]. The decreased colony formation of U87-MG cells further substantiates its antiproliferative action, in agreement with Elkady (2018), who demonstrated similar effects in prostate cancer cells [27].

Apoptosis induction, a hallmark of anticancer efficacy [28], was validated in the current study through AO/EB staining, revealing a dose-dependent increase in apoptotic cells with typical nuclear condensation and fragmentation. Western blot analysis showed elevated Bax and reduced Bcl-2 expression, indicating activation of intrinsic apoptosis. These findings align with prior reports demonstrating modulation of the Bax/Bcl-2 ratio by anethole [29] and its ability to regulate PTEN and CXCR4 to trigger apoptosis [11].

Unlike earlier suggestions of a pro-oxidant role [27], accumulating evidence indicates that anethole predominantly exhibits antioxidant activity, decreasing reactive oxygen species (ROS) levels and enhancing glutathione (GSH) defenses in cancer cells [8]. These antioxidant effects may indirectly contribute to apoptosis by rebalancing redox signaling and suppressing pro-survival pathways rather than by inducing oxidative stress [30,31]. Hence, the apoptotic mechanism observed in glioma cells in this study is more consistent with direct inhibition of the PI3K/AKT cascade and modulation of apoptotic regulators, rather than ROS elevation.

In the current work, in silico analysis identified 98 potential molecular targets of anethole, with KEGG pathway enrichment revealing 16 cancer-associated targets. This bioinformatic evidence strengthens the experimental findings and suggests that anethole exerts multi-targeted effects beyond a single signaling node. The RummaGEO analysis further linked these targets to apoptosis, cell cycle control, and autophagy, supporting the observed biological outcomes.

The PI3K/Akt cascade, frequently dysregulated in gliomas, is pivotal in regulating survival and proliferation [16]. Molecular docking demonstrated a strong binding affinity of anethole to PI3K with favorable interaction energy, supporting its potential as a direct inhibitor. Western blot analysis confirmed a dose-dependent reduction in PI3K and AKT phosphorylation, consistent with the in silico findings. These results align with previous reports by Ha et al. (2014) and Arumugam et al. (2021), who found that anethole suppressed the PI3K/Akt pathway in prostate and breast cancer cells, respectively [10,32]. Through this pathway, anethole may sensitize glioma cells to apoptosis and enhance the efficacy of combination regimens with standard chemotherapeutics. When compared to established PI3K inhibitors such as Wortmannin and LY294002, anethole displayed moderate but meaningful binding affinity with a lower likelihood of off-target cytotoxicity. These synthetic inhibitors, while potent, are limited by instability, poor solubility, and systemic toxicity. The natural origin, favorable pharmacokinetic properties, and selectivity profile of anethole may provide a therapeutic advantage, supporting its potential as a safer alternative or adjunct agent in glioma management [33–36].

The findings of this study hold important clinical relevance for the development of anethole-based therapies against gliomas. The selective cytotoxicity of anethole toward glioma cells, coupled with its ability to modulate multiple oncogenic signaling pathways, underscores its promise as a novel therapeutic candidate. Moreover, its predicted lipophilicity and potential to traverse the blood–brain barrier (BBB) suggest suitability for treating central nervous system (CNS) tumors [37]. Although previous pharmacokinetic data support possible CNS access for anethole derivatives [37,38], dedicated permeability studies—such as in vitro BBB models and in vivo brain-to-plasma distribution assays—are required to confirm its bioavailability within the brain. Despite favorable computational predictions, clinical translation may be constrained by anethole’s limited aqueous solubility and extensive first-pass metabolism following oral administration. To overcome these barriers, advanced delivery strategies, including lipid-based nanoformulations, prodrug development, or intranasal delivery systems, could enhance its systemic bioavailability and CNS penetration [39,40]. Future pharmacokinetic investigations should focus on validating these approaches to optimize the therapeutic potential of anethole for glioma management.

Collectively, the findings of this study highlight anethole as a promising candidate for glioma therapy. However, certain limitations should be acknowledged. First, the current work did not include apoptosis assays in NHA, which would have provided additional confirmation of cancer-specific selectivity. Second, we did not employ quantitative flow cytometry (Annexin V/PI) to distinguish early and late apoptotic events, and we recognize this as a methodological limitation. Third, while the inhibition of PI3K/AKT phosphorylation supports the proposed mechanism, we did not perform a rescue experiment (e.g., constitutively active AKT transfection) to definitively prove pathway causality. These aspects are important for future validation, along with in vivo efficacy studies, real-time binding assays (ITC, SPR, or BLI), and investigation of immune and angiogenic responses within the tumor microenvironment.

5. Conclusion

In conclusion, this study provides compelling evidence that anethole suppresses glioma cell proliferation and induces apoptosis by inhibiting the PI3K/AKT signaling pathway. Its selective cytotoxicity toward glioma cells, combined with favorable in silico pharmacokinetic predictions, supports its potential as a candidate for glioma therapy. While in vivo and mechanistic rescue experiments remain to be performed, the present findings establish a robust foundation for further translational and preclinical evaluation. Future studies should focus on optimizing drug delivery across the BBB and exploring synergistic effects of anethole with established chemotherapeutic agents to develop safer, more effective strategies against glioblastoma and other CNS malignancies.

Institutional review board approval and informed consent

This study did not involve human participants, patient data, or clinical samples. Only commercially available human glioma cell lines (U87-MG and LN-229) and normal human astrocytes (NHA) were used. Therefore, Institutional Review Board (IRB) approval and informed consent were not required.

Supporting information

S1 Fig. Uncropped blots with visible molecular weight markers.

(DOCX)

pone.0336975.s001.docx^{(104.5KB, docx)}

S2 File. SwissADME analysis of anethole.

Predicted physicochemical, pharmacokinetic, and drug-likeness properties of anethole were evaluated using the SwissADME web tool based on its SMILES structure.

(DOCX)

pone.0336975.s002.docx^{(201.1KB, docx)}

S3 File. BOILED-Egg plot prediction o gastrointestinal absorption and brain penetration of anethole.

The plot shows anethole’s predicted position within the yellow region, indicating blood–brain barrier (BBB) permeability. The white region represents high gastrointestinal absorption (HIA). The red circle (PGP–) marks anethole as a non-substrate of P-glycoprotein.

(DOCX)

pone.0336975.s003.docx^{(125.5KB, docx)}

Acknowledgments

The authors are thankful to the Deanship of Graduate Studies and Scientific Research at Najran University for funding this work.

Data Availability

All relevant data are within the manuscript.

Funding Statement

This work was supported by the Deanship of Graduate Studies and Scientific Research at Najran University under the Easy Funding Program (grant code NU/EFP/MRC/13/220) awarded to A.A.A. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Jayaram MA, Phillips JJ. Role of the Microenvironment in Glioma Pathogenesis. Annu Rev Pathol. 2024;19:181–201. doi: 10.1146/annurev-pathmechdis-051122-110348 [DOI] [PubMed] [Google Scholar]
2.Sadowski K, Jażdżewska A, Kozłowski J, Zacny A, Lorenc T, Olejarz W. Revolutionizing Glioblastoma Treatment: A Comprehensive Overview of Modern Therapeutic Approaches. Int J Mol Sci. 2024;25(11):5774. doi: 10.3390/ijms25115774 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Karve AS, Desai JM, Gadgil SN, Dave N, Wise-Draper TM, Gudelsky GA, et al. A Review of Approaches to Potentiate the Activity of Temozolomide against Glioblastoma to Overcome Resistance. Int J Mol Sci. 2024;25(6):3217. doi: 10.3390/ijms25063217 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Asma ST, Acaroz U, Imre K, Morar A, Shah SRA, Hussain SZ, et al. Natural Products/Bioactive Compounds as a Source of Anticancer Drugs. Cancers (Basel). 2022;14(24):6203. doi: 10.3390/cancers14246203 [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Liu W, Wang Y, Xia L, Li J. Research Progress of Plant-Derived Natural Products against Drug-Resistant Cancer. Nutrients. 2024;16(6):797. doi: 10.3390/nu16060797 [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Chaachouay N, Zidane L. Plant-Derived Natural Products: A Source for Drug Discovery and Development. DDC. 2024;3(1):184–207. doi: 10.3390/ddc3010011 [DOI] [Google Scholar]
7.Aprotosoaie AC, Costache I-I, Miron A. Anethole and Its Role in Chronic Diseases. Adv Exp Med Biol. 2016;929:247–67. doi: 10.1007/978-3-319-41342-6_11 [DOI] [PubMed] [Google Scholar]
8.Contant C, Rouabhia M, Loubaki L, Chandad F, Semlali A. Anethole induces anti-oral cancer activity by triggering apoptosis, autophagy and oxidative stress and by modulation of multiple signaling pathways. Sci Rep. 2021;11(1):13087. doi: 10.1038/s41598-021-92456-w [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Nakagawa Y, Suzuki T. Cytotoxic and xenoestrogenic effects via biotransformation of trans-anethole on isolated rat hepatocytes and cultured MCF-7 human breast cancer cells. Biochem Pharmacol. 2003;66(1):63–73. doi: 10.1016/s0006-2952(03)00208-9 [DOI] [PubMed] [Google Scholar]
10.Ha B, Ko H, Kim B, Sohn EJ, Jung JH, Kim JS, et al. Regulation of crosstalk between epithelial to mesenchymal transition molecules and MMP-9 mediates the antimetastatic activity of anethole in DU145 prostate cancer cells. J Nat Prod. 2014;77(1):63–9. doi: 10.1021/np4006376 [DOI] [PubMed] [Google Scholar]
11.Rhee Y-H, Chung P-S, Kim S-H, Ahn JC. CXCR4 and PTEN are involved in the anti-metastatic regulation of anethole in DU145 prostate cancer cells. Biochem Biophys Res Commun. 2014;447(4):557–62. doi: 10.1016/j.bbrc.2014.01.121 [DOI] [PubMed] [Google Scholar]
12.Kumar V, Venkateswarulu M. Anethole attenuates lung cancer progression by regulating the proliferation and apoptosis through AKT and STAT3 signaling. STEMedicine. 2023;4(1):e153. doi: 10.37175/stemedicine.v4i1.153 [DOI] [Google Scholar]
13.Braccini L, Ciraolo E, Martini M, Pirali T, Germena G, Rolfo K, et al. PI3K keeps the balance between metabolism and cancer. Adv Biol Regul. 2012;52(3):389–405. doi: 10.1016/j.jbior.2012.04.002 [DOI] [PubMed] [Google Scholar]
14.Noorolyai S, Shajari N, Baghbani E, Sadreddini S, Baradaran B. The relation between PI3K/AKT signalling pathway and cancer. Gene. 2019;698:120–8. doi: 10.1016/j.gene.2019.02.076 [DOI] [PubMed] [Google Scholar]
15.Knobbe CB, Reifenberger G. Genetic alterations and aberrant expression of genes related to the phosphatidyl-inositol-3’-kinase/protein kinase B (Akt) signal transduction pathway in glioblastomas. Brain Pathol. 2003;13(4):507–18. doi: 10.1111/j.1750-3639.2003.tb00481.x [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Barzegar Behrooz A, Talaie Z, Jusheghani F, Łos MJ, Klonisch T, Ghavami S. Wnt and PI3K/Akt/mTOR Survival Pathways as Therapeutic Targets in Glioblastoma. Int J Mol Sci. 2022;23(3):1353. doi: 10.3390/ijms23031353 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Mafi A, Khoshnazar SM, Shahpar A, Nabavi N, Hedayati N, Alimohammadi M, et al. Mechanistic insights into circRNA-mediated regulation of PI3K signaling pathway in glioma progression. Pathol Res Pract. 2024;260:155442. doi: 10.1016/j.prp.2024.155442 [DOI] [PubMed] [Google Scholar]
18.Ribble D, Goldstein NB, Norris DA, Shellman YG. A simple technique for quantifying apoptosis in 96-well plates. BMC Biotechnol. 2005;5:12. doi: 10.1186/1472-6750-5-12 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Morris G, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J Comput Chem. 1998;19:1639–62. [Google Scholar]
20.Solis FJ, Wets RJB. Minimization by random search techniques. Math Oper Res. 1981;6:19–30. [Google Scholar]
21.Ijaz M, Hasan I, Jiang Z, Ullah Z, Aslam B, Khurshid M. Combinatory immunotherapy for glioblastoma treatment. Adv Ther. 2024;7:2400217. [Google Scholar]
22.Han M, Li S, Fan H, An J, Peng C, Peng F. Regulated cell death in glioma: promising targets for natural small-molecule compounds. Front Oncol. 2024;14:1273841. doi: 10.3389/fonc.2024.1273841 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Cancer Genome Atlas Research Network. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature. 2008;455(7216):1061–8. doi: 10.1038/nature07385 [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Parsons DW, Jones S, Zhang X, Lin JC-H, Leary RJ, Angenendt P, et al. An integrated genomic analysis of human glioblastoma multiforme. Science. 2008;321(5897):1807–12. doi: 10.1126/science.1164382 [DOI] [PMC free article] [PubMed] [Google Scholar]
25.van Blitterswijk WJ, Verheij M. Anticancer mechanisms and clinical application of alkylphospholipids. Biochim Biophys Acta. 2013;1831(3):663–74. doi: 10.1016/j.bbalip.2012.10.008 [DOI] [PubMed] [Google Scholar]
26.Chen CH, deGraffenried LA. Anethole suppressed cell survival and induced apoptosis in human breast cancer cells independent of estrogen receptor status. Phytomedicine. 2012;19(8–9):763–7. doi: 10.1016/j.phymed.2012.02.017 [DOI] [PubMed] [Google Scholar]
27.Elkady AI. Anethole Inhibits the Proliferation of Human Prostate Cancer Cells via Induction of Cell Cycle Arrest and Apoptosis. Anticancer Agents Med Chem. 2018;18(2):216–36. doi: 10.2174/1871520617666170725165717 [DOI] [PubMed] [Google Scholar]
28.Pfeffer CM, Singh ATK. Apoptosis: A Target for Anticancer Therapy. Int J Mol Sci. 2018;19(2):448. doi: 10.3390/ijms19020448 [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Redza-Dutordoir M, Averill-Bates DA. Activation of apoptosis signalling pathways by reactive oxygen species. Biochim Biophys Acta. 2016;1863(12):2977–92. doi: 10.1016/j.bbamcr.2016.09.012 [DOI] [PubMed] [Google Scholar]
30.Bakkali F, Averbeck S, Averbeck D, Idaomar M. Biological effects of essential oils--a review. Food Chem Toxicol. 2008;46(2):446–75. doi: 10.1016/j.fct.2007.09.106 [DOI] [PubMed] [Google Scholar]
31.Harakeh S, Al-Raddadi R, Alamri T, Al-Jaouni S, Qari M, Qari Y, et al. Apoptosis induction in human hepatoma cell line HepG2 cells by trans- Anethole via activation of mitochondria-mediated apoptotic pathways. Biomed Pharmacother. 2023;165:115236. doi: 10.1016/j.biopha.2023.115236 [DOI] [PubMed] [Google Scholar]
32.Arumugam P, Sampathkumar B, Perumalsamy H, Balusamy SR, Ramesh V, Sundaravadevel S. Synergistic effect of anethole and doxorubicin alleviates cell proliferation, cell cycle arrest, and ER stress and promotes ROS-mediated apoptosis in triple-negative breast cancer cells. J Biochem Mol Toxicol. 2021;35(12):e22928. doi: 10.1002/jbt.22928 [DOI] [PubMed] [Google Scholar]
33.Gharbi SI, Zvelebil MJ, Shuttleworth SJ, Hancox T, Saghir N, Timms JF, et al. Exploring the specificity of the PI3K family inhibitor LY294002. Biochem J. 2007;404(1):15–21. doi: 10.1042/BJ20061489 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Brunn GJ, Williams J, Sabers C, Wiederrecht G, Lawrence JCJ, Abraham RT. Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors wortmannin and LY294002. EMBO J. 1996;15(19):5256–67. [PMC free article] [PubMed] [Google Scholar]
35.Knight ZA, Gonzalez B, Feldman ME, Zunder ER, Goldenberg DD, Williams O, et al. A pharmacological map of the PI3-K family defines a role for p110alpha in insulin signaling. Cell. 2006;125(4):733–47. doi: 10.1016/j.cell.2006.03.035 [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Powis G, Bonjouklian R, Berggren MM, Gallegos A, Abraham R, Ashendel C, et al. Wortmannin, a potent and selective inhibitor of phosphatidylinositol-3-kinase. Cancer Res. 1994;54(9):2419–23. [PubMed] [Google Scholar]
37.Yu H-Z, Han S-F, Li P, Zhu C-L, Zhang X-X, Gan L, et al. An examination of the potential effect of lipids on the first-pass metabolism of the lipophilic drug anethol trithione. J Pharm Sci. 2011;100(11):5048–58. doi: 10.1002/jps.22702 [DOI] [PubMed] [Google Scholar]
38.Patel MM, Patel BM. Crossing the Blood-Brain Barrier: Recent Advances in Drug Delivery to the Brain. CNS Drugs. 2017;31(2):109–33. doi: 10.1007/s40263-016-0405-9 [DOI] [PubMed] [Google Scholar]
39.Chen Y, Zhang C, Huang Y, Ma Y, Song Q, Chen H, et al. Intranasal drug delivery: The interaction between nanoparticles and the nose-to-brain pathway. Adv Drug Deliv Rev. 2024;207:115196. doi: 10.1016/j.addr.2024.115196 [DOI] [PubMed] [Google Scholar]
40.Nance E, Pun SH, Saigal R, Sellers DL. Drug delivery to the central nervous system. Nat Rev Mater. 2022;7(4):314–31. doi: 10.1038/s41578-021-00394-w [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0336975.r001

Decision Letter 0

Zahra Lorigooini

31 Mar 2025

Dear Dr. Alshahrani,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR:

After reviewing the manuscript, I would like to suggest several revisions that will improve its clarity, consistency, and overall alignment with the journal's guidelines.

Adherence to Journal Guidelines: It appears that some sections of the manuscript do not fully comply with the formatting and citation guidelines provided by the journal. Kindly refer to the journal’s style guide and make necessary adjustments, particularly in relation to:
- Correct formatting of the references.
- Consistency in the use of terminology and abbreviations.
- Proper structuring of headings and subheadings.
Language and Clarity: The manuscript would benefit from a thorough review of the language and grammatical structure to enhance clarity. I recommend having the text reviewed for grammatical and syntactical accuracy.
Methodology Section:
- It would be helpful to provide additional details regarding the source and purity of anethole, the solvents used, and any quality control measures taken during its preparation and administration. This will improve reproducibility and transparency.
Figures and Tables: The figures and tables should be clearly labeled, and all axes should be properly defined. I suggest ensuring that figure captions fully describe what is presented, and that they are referenced correctly within the text.
Supplementary Data: If any supplementary data or additional experiments have been conducted, please include them as supplementary files or specify their omission and provide an explanation.

I would greatly appreciate it if you could make these revisions and resubmit the manuscript. Please also ensure that the manuscript is reviewed for consistency in the presentation of the data, particularly concerning statistical analyses and experimental results.

==============================

Please submit your revised manuscript by May 15 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Zahra Lorigooini

Academic Editor

PLOS ONE

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When submitting your revision, we need you to address these additional requirements.

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2. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: No

Reviewer #2: Yes

**********

Reviewer #1: Abstract: "Colony formation was notably inhibited in a dose-dependently." → Grammatical error. Correct it to: "Colony formation was notably inhibited in a dose-dependent manner."

• How many replicates were performed for IC50 determination? Mention explicitly.

• Western blot results should be summarized more concisely.

Introduction:

• Page 2, Paragraph 2: "Gliomas are essentially the most prevalent and therefore malignant primary brain tumors..." → Unclear phrasing. Gliomas are not "therefore malignant"; glioblastomas are. Reword for clarity.

• Page 3, Last Paragraph: "These findings underscore the versatility of anethole as an anticancer molecule..." → Mention gaps in knowledge regarding anethole in gliomas.

• The introduction should provide a clearer rationale for targeting PI3K/Akt in glioblastoma.

Materials and Methods:

• Page 4, "CCK-8 Assay": What was the exact duration of anethole treatment? Specify. Cite the references

• Page 5, "AO/EB Staining": How were apoptotic vs. necrotic cells quantified? State whether manual counting or software analysis was used. Cite the reference

• Page 6, "Molecular Docking": Why was the docking performed with AutoDock instead of newer methods like AutoDock Vina or Schrödinger? Justify.

• Page 7, "Western Blot Analysis": Were the densitometry results normalized to loading controls? Mention which software was used.

Results:

• Page 8, Figure 1: What is the sample size (n) and standard deviation (±SD) for IC50 values? Ensure reproducibility.

• Page 9, "Anethole Triggers Apoptosis": Clarify if apoptosis was confirmed via flow cytometry or just AO/EB staining.

• Page 10, "Molecular Docking Results": Docking energy (-9.32 kcal/mol) is strong, but was in vitro validation performed to confirm target binding?

Discussion:

• Page 12, Paragraph 1: How does anethole compare to existing PI3K inhibitors like Wortmannin or LY294002? A comparative discussion is needed.

• Page 13, "Potential Clinical Application": While anethole has brain permeability potential, is there direct evidence that it crosses the blood-brain barrier (BBB)? Cite relevant studies.

• Page 14, "Limitations": The study lacks animal model validation. Suggest including a statement on future in vivo studies.

Conclusion:

• The conclusion should briefly highlight future directions (e.g., combinational therapies, BBB permeability studies).

Reviewer #2: The authors need to compare the binding constants of anethole with PI3K and JAK2 calculated by MD simulations with experimental data by performing ITC/ BLI/SPR studies if possible which will further supports the mechanism of binding.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: Yes: Jesil Mathew A

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

PLoS One. 2025 Nov 21;20(11):e0336975. doi: 10.1371/journal.pone.0336975.r002

Author response to Decision Letter 1

15 Apr 2025

Dear editor,

We thank the reviewers for their thoughtful and constructive comments, which have significantly improved the quality and clarity of our manuscript. We have addressed each point below and made corresponding changes to the manuscript as indicated.

Reviewer #1

Abstract

1. “Colony formation was notably inhibited in a dose-dependently.” → Grammatical error.

✔ Corrected to “Colony formation was notably inhibited in a dose-dependent manner” in the abstract.

2. How many replicates were performed for IC₅₀ determination? Mention explicitly.

✔ We now state that all IC₅₀ values were calculated based on three independent experiments (n=3), with data expressed as mean ± standard deviation (SD).

3. Western blot results should be summarized more concisely.

✔ The Western blot summary in the abstract has been streamlined to focus on the major findings: induction of Bax and reduction of Bcl-2, and suppression of PI3K/Akt phosphorylation.

Introduction

4. Page 2, Paragraph 2: Unclear phrasing regarding glioma malignancy.

✔ Rephrased the sentence to: “Gliomas are essentially the most prevalent and primary brain tumors, constituting about 80% of all malignant intracranial neoplasms [1].”

5. Page 3: "These findings underscore..." → Mention gaps in knowledge regarding anethole in gliomas.

✔ Revised to note that although anethole shows promise in other cancers, its role in glioma remains unexplored and warrants targeted investigation. This has included in detail

6. Provide a clearer rationale for targeting PI3K/Akt in glioblastoma.

✔ We have added supporting context and references explaining that the PI3K/Akt pathway is frequently dysregulated in glioblastoma and is associated with tumor progression, making it a rational therapeutic target.

Materials and Methods

7. Page 4, “CCK-8 Assay”: Specify exact duration of anethole treatment.

✔ The duration has been specified as 24 hours, in line with Contant et al., 2021 (now cited in the revised manuscript).

8. Page 5, “AO/EB Staining”: Clarify quantification method and cite reference.

✔ We now state that apoptotic, necrotic, and viable cells were quantified using manual cell counting under a fluorescence microscope based on morphological and staining criteria, referencing Ribble et al., 2005.

9. Page 6, “Molecular Docking”: Justify use of AutoDock over newer methods.

✔ We have included a rationale noting that AutoDock remains a validated and widely used tool, particularly suitable for flexible ligand docking and preliminary screening of natural products due to its robust scoring functions and availability.

10. Page 7, “Western Blot”: Were densitometry results normalized?

✔ Yes. We now clarify that ImageJ software (NIH) was used for densitometry, and expression levels were normalized to β-actin as the loading control.

Results

11. Page 8, Figure 1: Include sample size and SD for IC₅₀.

✔ This information has been added: IC₅₀ values were derived from three independent experiments and are expressed as mean ± SD.

12. Page 9, Apoptosis: Was flow cytometry used?

✔ No flow cytometry was used. We clarify that apoptosis was assessed only via AO/EB staining in this study.

13. Page 10, Docking: Was in vitro validation of target binding performed?

✔ We now acknowledge that in vitro validation (e.g., via SPR or ITC) was not performed and have highlighted this as a limitation and future direction in the discussion.

________________________________________

Discussion

14. Compare anethole with existing PI3K inhibitors like Wortmannin and LY294002.

✔ We have added a paragraph discussing that while anethole demonstrates moderate affinity for PI3K, it may offer advantages over conventional inhibitors due to lower toxicity and broader multi-target effects. We also reference the known drawbacks of Wortmannin and LY294002 (e.g., instability, cytotoxicity).

15. BBB permeability: Is there direct evidence for anethole crossing the BBB?

✔ We clarify that while anethole is lipophilic and theoretically capable of BBB penetration, direct experimental evidence is lacking. We reference Yu et al., 2011 and recommend further pharmacokinetic studies.

16. Limitations: No in vivo validation. Suggest adding statement.

✔ We have included a clear statement acknowledging the lack of in vivo data and recommending future studies using animal models to validate the therapeutic potential and pharmacodynamics of anethole.

Conclusion

17. Highlight future directions (e.g., combinational therapy, BBB studies).

✔ The conclusion now includes suggestions for combinational therapy with current chemotherapeutics, investigation of BBB permeability, and in vivo validation as future directions.

Reviewer #2

1. Compare MD-predicted binding constants with experimental data (ITC, BLI, SPR).

✔ We appreciate this important suggestion. In the discussion, we have noted that while molecular docking predicted strong binding to PI3K, experimental confirmation using ITC, BLI, or SPR is necessary to validate these findings. This limitation has been acknowledged, and we propose these methods as next steps in validating the binding mechanism.

Response to Editor’s Comments

We thank the Editor for the valuable suggestions aimed at enhancing the clarity, consistency, and compliance of our manuscript with PLOS ONE guidelines. Below, we provide point-by-point responses to each of the recommendations and describe the revisions made accordingly:

1. Adherence to Journal Guidelines

• Correct formatting of the references

Response: The entire reference list has been reformatted to align with the PLOS ONE style, as per the journal’s formatting requirements.

• Consistency in the use of terminology and abbreviations

Response: All abbreviations and scientific terms have been reviewed for consistent usage throughout the manuscript. A list of abbreviations has also been provided where necessary.

• Proper structuring of headings and subheadings

Response: The manuscript has been revised to ensure uniform formatting of all section headings and subheadings in accordance with the journal’s structure.

2. Language and Clarity

• Grammatical and syntactical accuracy

Response: The manuscript has undergone a detailed language review to improve clarity, correct grammatical issues, and enhance the overall readability of the text.

3. Methodology Section

• Additional details on source, purity, solvents, and quality control of anethole

Response: Anethole (99% purity) was obtained from Sigma-Aldrich (Oakville, ON, Canada) and dissolved in methanol to prepare a 3 mM stock solution. It was subsequently used at different concentrations for the experiments

4. Figures and Tables

• Clear labeling, defined axes, and detailed captions

Response: All figures and tables have been reviewed and revised. Axes are now clearly labeled, units are defined, and figure captions have been expanded to fully describe the contents. Each figure and table is now properly referenced within the manuscript.

5. Supplementary Data

________________________________________

6. Consistency in Presentation of Data

• Statistical analysis and experimental result presentation

Response: The manuscript has been carefully reviewed to ensure consistent reporting of sample size (n), standard deviations (SD), and p-values. All statistical analyses are now clearly defined in both the Methods and Results sections.

7. Original Images for Blot/Gel Data

• Compliance with blot and gel image requirements

Response: As per PLOS ONE’s latest guidelines, the original, uncropped, and unadjusted image data underlying all Western blot figures have been included as Supporting Information files. We have also ensured that the preparation of these images and figure panels adheres to the specific requirements detailed in the journal’s policy.

Attachment

Submitted filename: Response to reviewers.docx

pone.0336975.s005.docx^{(21.4KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0336975.r003

Decision Letter 1

Zahra Lorigooini

4 Jul 2025

Dear Dr. Alshahrani,

Please submit your revised manuscript by Aug 18 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Zahra Lorigooini

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #3: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #3: Yes

**********

Reviewer #3: Although the authors have been responsive, the following high-level suggestions may further strengthen the study:

Add a Note on Flow Cytometry as Future Work

While the authors clarified they did not use flow cytometry, suggesting its inclusion in future validation would demonstrate deeper awareness of methodological robustness in apoptosis detection.

Clarify Lipophilicity Measurement

The statement that anethole is “lipophilic” and may cross the BBB is speculative. Including a brief reference to LogP or in silico BBB permeability prediction (e.g., via SwissADME) would lend more support.

Densitometry Quantification – Loading Control Uniformity

Western blot images (if reviewed) should show uniform β-actin levels across conditions. While the use of ImageJ is stated, it's critical that raw blot bands (provided as supplementary) confirm integrity.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #3: Yes: Dr. Majid Asadi-Samani

**********

PLoS One. 2025 Nov 21;20(11):e0336975. doi: 10.1371/journal.pone.0336975.r004

Author response to Decision Letter 2

10 Jul 2025

Dear Editor,

We thank you for the consideration of our manuscript. We also thank the reviewers for the suggestions for further strengthening of our manuscript and we have incorporated all suggestions of the respected reviewer.

Reviewer #3: Although the authors have been responsive, the following high-level suggestions may further strengthen the study:

Add a Note on Flow Cytometry as Future Work

While the authors clarified they did not use flow cytometry, suggesting its inclusion in future validation would demonstrate deeper awareness of methodological robustness in apoptosis detection.

Response: We appreciate this insightful recommendation. We have revised the Discussion section to acknowledge the value of incorporating flow cytometry (e.g., Annexin V/PI staining) in future studies for quantitative apoptosis detection.

Clarify Lipophilicity Measurement

Response: We agree with the reviewer and have now included in silico SwissADME predictions in the Results and Discussion sections. These predictions show a consensus LogP of 2.79 and classify anethole as BBB permeant, supporting its CNS bioavailability (Supplementary Figure S1 and S2).

Densitometry Quantification – Loading Control Uniformity

Response: We fully agree and have included densitometery quantification in Figure 2C, 2D, 5D-G. The uncropped raw Western blot images as Supplementary file, clearly showing consistent β-actin expression across all experimental conditions. This addition supports the integrity of protein loading and quantification.

Attachment

Submitted filename: Response_to_Reviewers_auresp_2.docx

pone.0336975.s006.docx^{(14.3KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0336975.r005

Decision Letter 2

Yasmina Abd‐Elhakim

22 Sep 2025

Dear Dr. Alshahrani,

Please submit your revised manuscript by Nov 06 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Yasmina Abd‐Elhakim

Academic Editor

PLOS ONE

Journal Requirements:

1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewer's Responses to Questions

Comments to the Author

Reviewer #4: (No Response)

Reviewer #5: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #4: No

Reviewer #5: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #4: Yes

Reviewer #5: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #4: Yes

Reviewer #5: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #4: Yes

Reviewer #5: Yes

**********

Reviewer #4: This manuscript studies the anticancer cell effect of anethole in glioma cell lines and normal human astrocytes. Anethole has been broadly described in several cancer cell types, by different groups and being reviewed, showing antiproliferative and antimetastatic effect on neoplastic cells. As described by others, anethole induces apoptosis, cell cycle arrest, autophagy, antioxidant GHS, reduction of ROS, and metalloproteinases, etc., by interfering in different signaling pathways. The manuscript by Al Alwadh et al., demonstrates that anethole exerts the same effect in glioma cells as in other cancer cells, inducing apoptosis and reduction of PI3K/AKT phosphorylation. The mayor hypothesis and aim of this manuscript is to show the blocking effect of anethole in the PI3K/AKT pathway. for that, the authors analyzed in silico prediction of PI3K and anethole interaction. Although this is an interesting subject, the work should be completed by correcting some errors and adding some data as follows indicated:

Main concerns:

1- The most interesting data is the predictable interaction of anethole and PI3k, some aspects in this regard should be considered. First if the effect of anethole takes place through interaction with PI3K and its inhibition, why does it not affect Normal human astrocytes (NHA)? The authors demonstrate, as previously described in cancer cells, reduction of PI3K and AKT phosphorylation only in one line of cancer cells. Then, the effect of anethole in NHA cells, as new data, would be nice to add in. Second, independent of the result about the effect in NHA, Anethole's stronger effect on cancer cells needs discussion. Could this be due to increased diffusion through cancer cell membranes? It would be interesting to discuss this, as increased anethole affinity to PI3K in cancer cells is unlikely. Finaly, AKT can take place through an independent mechanism as well, and it will be interesting to show either some downstream signal, or upstream signal that demonstrate PI3K direct inhibition.

2- In discussion section, it is indicated “In addition, anethole has also been shown to elevate ROS accretion in cancerous cells, promoting oxidative stress and apoptosis [8]. ROS accumulation has been estimated to impair mitochondrial membrane potential, thereby activating intrinsic apoptotic sequences as described in breast cancer cells [26]”, the reference 8 showed that anethole reduces ROS and increased GSH antioxidant. Therefore, this paragraph and the argument should be changed accordingly.

3- Regarding the treatment with anethole, the most limiting aspect is that, even when anethole can diffuse through the BBB, due to low solubility in water and high elimination when orally administrated, the effect in gliomas looks limited. Some discussion also in this area should be nice to discuss.

4- The figure legends need to indicate clearly whether the graphs show the media plus SD of an independent out of three experiments, or the media plus SD of three independent experiments.

5- The AO/EB analysis is used to indicate early and late apoptosis, the quantification of cells in these two statuses should be indicated in the graph. On the other hand, it would be convenient to show cell cycles of treated cells, also in NHA cells.

6- In general, it is indicated that experiments were performed three times, but whether the graph represents one out three or the media plus SD of three independent experiments need to be indicated.

7- The apoptosis assay needs to be performed with normal NHA cells, to consistently confirm the specific effect of anethole in gliomas. Also, the

8- The relative expression of p-PI3K and p-AKT needs to be calculated according to the total amount of PI3K and AKT protein, rather than to actine, and needs to be indicated in the Figure 5 legend.

Minor concerns:

1- Material, please indicate the precedence of: cell lines, NHA cells, CCK-8 assay, and al used material in the material and methods section.

2- Please, indicate the meaning of acronyms first time used in the text

3- Indication of the antibody clone used, and the company of precedence needs to be included in the material and methods section.

4- Correct the sentence at page 35- “Cells were subsequently lysing was done using RIPA buffer”,

5- Either Figures 5a and 5b have been mixed up, or the text in the legends and in the 'Results' section (3.4) is incorrect.

6- The images in Figures 3, 4, 5a and 5b should be clearer.

Reviewer #5: Based on the authors’ responses to the previous reviewers’ comments, I believe the manuscript is acceptable after some minor revisions, as outlined below.

1- Mechanistic Depth and Specificity: The study compellingly shows that anethole inhibits the PI3K/AKT pathway and induces apoptosis. However, to strengthen the claim that apoptosis is directly caused by PI3K/AKT inhibition (and not a parallel event), a rescue experiment would be highly valuable. Could the pro-apoptotic and anti-proliferative effects of anethole be reversed by introducing a constitutively active form of AKT (e.g., via plasmid transfection) into the U87 cells prior to anethole treatment? If such an experiment is beyond the scope of this revision, explicitly stating this as a key limitation and a requirement for future validation would strengthen the manuscript's conclusions.

Where to place this: In the Discussion (to frame the findings) and/or the Conclusion (as a future direction).

Figure 5A and 5B captions are swapped. Figure 5A describes the JAK2 interaction, while the image is labeled for PI3K, and vice versa. This must be corrected.

3- Statistical Analysis Description: The Methods section (2.9) states that experiments were performed in triplicate and data is presented as mean ± SD. For the colony formation assay, it would be helpful to specify how many technical replicates (wells) and biological replicates (independent experiments) were performed, as this assay typically has fewer replicates.

4- Language and Flow Minor Revisions: The manuscript is well-written but would benefit from a final proofread for minor grammatical redundancies. For example, in the Introduction (page 33): "...impede growth via cell cycle arrest in prostate cancer cells via arresting cell cycle." The phrase "via arresting cell cycle" is redundant and can be removed.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

PLoS One. 2025 Nov 21;20(11):e0336975. doi: 10.1371/journal.pone.0336975.r006

Author response to Decision Letter 3

15 Oct 2025

Dear Editor

We sincerely thank you and the anonymous reviewers for their constructive comments and valuable suggestions, which have significantly improved the quality and clarity of our manuscript. We have carefully addressed all concerns point by point, as detailed below. All textual changes have been incorporated into the revised version of manuscript and have been highlighted in red.

Reviewer #4

This manuscript studies the anticancer cell effect of anethole in glioma cell lines and normal human astrocytes. Anethole has been broadly described in several cancer cell types, by different groups and being reviewed, showing antiproliferative and antimetastatic effect on neoplastic cells. As described by others, anethole induces apoptosis, cell cycle arrest, autophagy, antioxidant GHS, reduction of ROS, and metalloproteinases, etc., by interfering in different signaling pathways. The manuscript by Al Alwadh et al., demonstrates that anethole exerts the same effect in glioma cells as in other cancer cells, inducing apoptosis and reduction of PI3K/AKT phosphorylation. The mayor hypothesis and aim of this manuscript is to show the blocking effect of anethole in the PI3K/AKT pathway. for that, the authors analyzed in silico prediction of PI3K and anethole interaction. Although this is an interesting subject, the work should be completed by correcting some errors and adding some data as follows indicated:

Main concerns

Response: We thank the reviewer for this insightful comment. While we acknowledge that inclusion of NHA apoptosis and phosphorylation data would strengthen the study, new wet-lab experiments are beyond the scope of this revision. To address this point, we have added a detailed explanation in the Discussion section discussing (i) why normal astrocytes show limited PI3K/AKT response, (ii) the potential role of altered membrane permeability and PI3K/AKT hyperactivation in glioma cells as the cause of selective sensitivity, and (iii) possible AKT-independent mechanisms requiring future validation. This clarification provides a mechanistic rationale for cancer-selective action of anethole and outlines future directions for in vivo and molecular validation.

Response: We appreciate this correction. The cited paragraph has been revised in the Discussion section to accurately reflect the findings of reference [8], clarifying that anethole predominantly reduces ROS levels and enhances GSH antioxidant capacity, rather than increasing ROS. The interpretation was rewritten accordingly.

Response: We agree with the reviewer’s important observation. Additional discussion has been incorporated in the Discussion section addressing pharmacokinetic limitations of anethole such as low aqueous solubility and high first-pass metabolism. We have also added potential strategies—such as lipid-based nanoformulations, prodrug development, and intranasal delivery—to improve systemic and brain bioavailability.

4- The figure legends need to indicate clearly whether the graphs show the media plus SD of an independent out of three experiments, or the media plus SD of three independent experiments.

Response: We have revised all figure legends to explicitly state that “data are presented as mean ± SD of three independent biological experiments,” clarifying that each represents the mean of three independent replicates.

Response: We appreciate this valuable suggestion. Quantitative analysis of total apoptosis (early + late) was added in the Methods (AO/EB assay) and described in Figure 2 legend. As additional flow cytometry experiments were not performed, we clarified in the Discussion that detailed early/late apoptosis and NHA apoptosis assays will be considered in future work.

6- In general, it is indicated that experiments were performed three times, but whether the graph represents one out three or the media plus SD of three independent experiments need to be indicated.

Response: This point has been addressed throughout the Results and Figure legends, which now specify that all data represent the mean ± SD of three independent biological experiments.

7- The apoptosis assay needs to be performed with normal NHA cells, to consistently confirm the specific effect of anethole in gliomas. Also, the

Response: We acknowledge the reviewer’s suggestion. While inclusion of NHA apoptosis data would provide valuable confirmation, conducting new experiments exceeds the current revision scope. However, this limitation is now explicitly stated in the Discussion (last paragraph), and future studies will incorporate apoptosis assays on NHA and in vivo models to verify selectivity.

8- The relative expression of p-PI3K and p-AKT needs to be calculated according to the total amount of PI3K and AKT protein, rather than to actine, and needs to be indicated in the Figure 5 legend.

Response: We appreciate this important observation. As suggested, the densitometric analysis was recalculated and normalized to the respective total proteins (p-PI3K/PI3K and p-AKT/AKT). This correction has been clearly indicated in both the Methods (Western blot) and Figure 5 legend.

Minor concerns

1- Material, please indicate the precedence of: cell lines, NHA cells, CCK-8 assay, and al used material in the material and methods section.

Response: The Materials and Methods section was expanded to include the source, catalog numbers, and vendors for all reagents and cell lines (Section 2.1).

2- Please, indicate the meaning of acronyms first time used in the text

Response: All acronyms (e.g., NHA, AO/EB, TPSA, BBB) are now defined at first mention throughout the manuscript.

3- Indication of the antibody clone used, and the company of precedence needs to be included in the material and methods section.

Response: The Western blot subsection (2.7) now lists the antibody source, catalog number, and manufacturer for each primary antibody.

4- Correct the sentence at page 35- “Cells were subsequently lysing was done using RIPA buffer”

Response: The sentence has been corrected to “Cells were lysed using RIPA buffer.”

5- Either Figures 5a and 5b have been mixed up, or the text in the legends and in the 'Results' section (3.4) is incorrect.

Response: Thank you for noticing this. The mix-up was corrected—the figure panels and legends now correctly correspond to PI3K (Figure 5A) and JAK2 (Figure 5B).

6- The images in Figures 3, 4, 5a and 5b should be clearer.

Response: We increased the resolution of Figures 3–5

Reviewer #5

Response: We appreciate this valuable suggestion. As additional transfection experiments could not be performed at this stage, we have explicitly included this as a key limitation in the Discussion (final paragraph) and highlighted it in the Conclusion as a future research direction.

2- The figures (e.g., Figure 2B, 5C) show representative Western blot bands, but the molecular weight markers are not visible. Including a lane with markers in the main figure or the supplementary uncropped blots is essential for verifying the identity of the protein bands. Figure 5A and 5B captions are swapped. Figure 5A describes the JAK2 interaction, while the image is labeled for PI3K, and vice versa. This must be corrected.

Response: We have now included uncropped blots with visible molecular weight markers as supplementary material and corrected the swap between Figure 5A and 5B to align with the text.

Response: We agree and have updated the Colony Formation Assay (Section 2.4) to indicate that each condition included two technical replicates and three independent biological experiments.

Response: We thank the reviewer for this observation. The entire manuscript has been carefully proofread to correct redundancies and improve fluency. The specific phrase mentioned has been revised accordingly in the Introduction.

We are deeply grateful to both reviewers for their thoughtful input, which helped us strengthen the manuscript. We hope the revisions satisfactorily address all concerns.

Sincerely,

Dr. Mohammed Merae Alshahrani (Corresponding Author)

Department of Clinical Laboratory Sciences

Najran University, Saudi Arabia

Email: mmalshahrani@nu.edu.sa

Attachment

Submitted filename: Response_to_Reviewers_auresp_3.docx

pone.0336975.s007.docx^{(19.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0336975.r007

Decision Letter 3

Yasmina Abd‐Elhakim

2 Nov 2025

Anethole Inhibits Human U87 Glioma Cell Proliferation by Inducing Apoptosis via the PI3K/AKT Pathway

PONE-D-25-10072R3

Dear Dr. Alshahrani,

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Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #5: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #5: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #5: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #5: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #5: Yes

**********

Reviewer #5: Thank you for your thorough revisions and detailed point-by-point responses to my comments. I have reviewed the updated manuscript and find that you have addressed all of my concerns satisfactorily.

The manuscript is now significantly improved and I recommend it for acceptance.

**********

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Reviewer #5: Yes: Gad Elsayed Mohamed Salem

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PLoS One. doi: 10.1371/journal.pone.0336975.r008

Acceptance letter

Yasmina Abd‐Elhakim

PONE-D-25-10072R3

PLOS ONE

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Uncropped blots with visible molecular weight markers.

(DOCX)

pone.0336975.s001.docx^{(104.5KB, docx)}

S2 File. SwissADME analysis of anethole.

Predicted physicochemical, pharmacokinetic, and drug-likeness properties of anethole were evaluated using the SwissADME web tool based on its SMILES structure.

(DOCX)

pone.0336975.s002.docx^{(201.1KB, docx)}

S3 File. BOILED-Egg plot prediction o gastrointestinal absorption and brain penetration of anethole.

(DOCX)

pone.0336975.s003.docx^{(125.5KB, docx)}

Attachment

Submitted filename: Response to reviewers.docx

pone.0336975.s005.docx^{(21.4KB, docx)}

Attachment

Submitted filename: Response_to_Reviewers_auresp_2.docx

pone.0336975.s006.docx^{(14.3KB, docx)}

Attachment

Submitted filename: Response_to_Reviewers_auresp_3.docx

pone.0336975.s007.docx^{(19.9KB, docx)}

Data Availability Statement

All relevant data are within the manuscript.

[pone.0336975.ref001] 1.Jayaram MA, Phillips JJ. Role of the Microenvironment in Glioma Pathogenesis. Annu Rev Pathol. 2024;19:181–201. doi: 10.1146/annurev-pathmechdis-051122-110348 [DOI] [PubMed] [Google Scholar]

[pone.0336975.ref002] 2.Sadowski K, Jażdżewska A, Kozłowski J, Zacny A, Lorenc T, Olejarz W. Revolutionizing Glioblastoma Treatment: A Comprehensive Overview of Modern Therapeutic Approaches. Int J Mol Sci. 2024;25(11):5774. doi: 10.3390/ijms25115774 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336975.ref003] 3.Karve AS, Desai JM, Gadgil SN, Dave N, Wise-Draper TM, Gudelsky GA, et al. A Review of Approaches to Potentiate the Activity of Temozolomide against Glioblastoma to Overcome Resistance. Int J Mol Sci. 2024;25(6):3217. doi: 10.3390/ijms25063217 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336975.ref004] 4.Asma ST, Acaroz U, Imre K, Morar A, Shah SRA, Hussain SZ, et al. Natural Products/Bioactive Compounds as a Source of Anticancer Drugs. Cancers (Basel). 2022;14(24):6203. doi: 10.3390/cancers14246203 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336975.ref005] 5.Liu W, Wang Y, Xia L, Li J. Research Progress of Plant-Derived Natural Products against Drug-Resistant Cancer. Nutrients. 2024;16(6):797. doi: 10.3390/nu16060797 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336975.ref006] 6.Chaachouay N, Zidane L. Plant-Derived Natural Products: A Source for Drug Discovery and Development. DDC. 2024;3(1):184–207. doi: 10.3390/ddc3010011 [DOI] [Google Scholar]

[pone.0336975.ref007] 7.Aprotosoaie AC, Costache I-I, Miron A. Anethole and Its Role in Chronic Diseases. Adv Exp Med Biol. 2016;929:247–67. doi: 10.1007/978-3-319-41342-6_11 [DOI] [PubMed] [Google Scholar]

[pone.0336975.ref008] 8.Contant C, Rouabhia M, Loubaki L, Chandad F, Semlali A. Anethole induces anti-oral cancer activity by triggering apoptosis, autophagy and oxidative stress and by modulation of multiple signaling pathways. Sci Rep. 2021;11(1):13087. doi: 10.1038/s41598-021-92456-w [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336975.ref009] 9.Nakagawa Y, Suzuki T. Cytotoxic and xenoestrogenic effects via biotransformation of trans-anethole on isolated rat hepatocytes and cultured MCF-7 human breast cancer cells. Biochem Pharmacol. 2003;66(1):63–73. doi: 10.1016/s0006-2952(03)00208-9 [DOI] [PubMed] [Google Scholar]

[pone.0336975.ref010] 10.Ha B, Ko H, Kim B, Sohn EJ, Jung JH, Kim JS, et al. Regulation of crosstalk between epithelial to mesenchymal transition molecules and MMP-9 mediates the antimetastatic activity of anethole in DU145 prostate cancer cells. J Nat Prod. 2014;77(1):63–9. doi: 10.1021/np4006376 [DOI] [PubMed] [Google Scholar]

[pone.0336975.ref011] 11.Rhee Y-H, Chung P-S, Kim S-H, Ahn JC. CXCR4 and PTEN are involved in the anti-metastatic regulation of anethole in DU145 prostate cancer cells. Biochem Biophys Res Commun. 2014;447(4):557–62. doi: 10.1016/j.bbrc.2014.01.121 [DOI] [PubMed] [Google Scholar]

[pone.0336975.ref012] 12.Kumar V, Venkateswarulu M. Anethole attenuates lung cancer progression by regulating the proliferation and apoptosis through AKT and STAT3 signaling. STEMedicine. 2023;4(1):e153. doi: 10.37175/stemedicine.v4i1.153 [DOI] [Google Scholar]

[pone.0336975.ref013] 13.Braccini L, Ciraolo E, Martini M, Pirali T, Germena G, Rolfo K, et al. PI3K keeps the balance between metabolism and cancer. Adv Biol Regul. 2012;52(3):389–405. doi: 10.1016/j.jbior.2012.04.002 [DOI] [PubMed] [Google Scholar]

[pone.0336975.ref014] 14.Noorolyai S, Shajari N, Baghbani E, Sadreddini S, Baradaran B. The relation between PI3K/AKT signalling pathway and cancer. Gene. 2019;698:120–8. doi: 10.1016/j.gene.2019.02.076 [DOI] [PubMed] [Google Scholar]

[pone.0336975.ref015] 15.Knobbe CB, Reifenberger G. Genetic alterations and aberrant expression of genes related to the phosphatidyl-inositol-3’-kinase/protein kinase B (Akt) signal transduction pathway in glioblastomas. Brain Pathol. 2003;13(4):507–18. doi: 10.1111/j.1750-3639.2003.tb00481.x [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336975.ref016] 16.Barzegar Behrooz A, Talaie Z, Jusheghani F, Łos MJ, Klonisch T, Ghavami S. Wnt and PI3K/Akt/mTOR Survival Pathways as Therapeutic Targets in Glioblastoma. Int J Mol Sci. 2022;23(3):1353. doi: 10.3390/ijms23031353 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336975.ref017] 17.Mafi A, Khoshnazar SM, Shahpar A, Nabavi N, Hedayati N, Alimohammadi M, et al. Mechanistic insights into circRNA-mediated regulation of PI3K signaling pathway in glioma progression. Pathol Res Pract. 2024;260:155442. doi: 10.1016/j.prp.2024.155442 [DOI] [PubMed] [Google Scholar]

[pone.0336975.ref018] 18.Ribble D, Goldstein NB, Norris DA, Shellman YG. A simple technique for quantifying apoptosis in 96-well plates. BMC Biotechnol. 2005;5:12. doi: 10.1186/1472-6750-5-12 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336975.ref019] 19.Morris G, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J Comput Chem. 1998;19:1639–62. [Google Scholar]

[pone.0336975.ref020] 20.Solis FJ, Wets RJB. Minimization by random search techniques. Math Oper Res. 1981;6:19–30. [Google Scholar]

[pone.0336975.ref021] 21.Ijaz M, Hasan I, Jiang Z, Ullah Z, Aslam B, Khurshid M. Combinatory immunotherapy for glioblastoma treatment. Adv Ther. 2024;7:2400217. [Google Scholar]

[pone.0336975.ref022] 22.Han M, Li S, Fan H, An J, Peng C, Peng F. Regulated cell death in glioma: promising targets for natural small-molecule compounds. Front Oncol. 2024;14:1273841. doi: 10.3389/fonc.2024.1273841 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336975.ref023] 23.Cancer Genome Atlas Research Network. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature. 2008;455(7216):1061–8. doi: 10.1038/nature07385 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336975.ref024] 24.Parsons DW, Jones S, Zhang X, Lin JC-H, Leary RJ, Angenendt P, et al. An integrated genomic analysis of human glioblastoma multiforme. Science. 2008;321(5897):1807–12. doi: 10.1126/science.1164382 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336975.ref025] 25.van Blitterswijk WJ, Verheij M. Anticancer mechanisms and clinical application of alkylphospholipids. Biochim Biophys Acta. 2013;1831(3):663–74. doi: 10.1016/j.bbalip.2012.10.008 [DOI] [PubMed] [Google Scholar]

[pone.0336975.ref026] 26.Chen CH, deGraffenried LA. Anethole suppressed cell survival and induced apoptosis in human breast cancer cells independent of estrogen receptor status. Phytomedicine. 2012;19(8–9):763–7. doi: 10.1016/j.phymed.2012.02.017 [DOI] [PubMed] [Google Scholar]

[pone.0336975.ref027] 27.Elkady AI. Anethole Inhibits the Proliferation of Human Prostate Cancer Cells via Induction of Cell Cycle Arrest and Apoptosis. Anticancer Agents Med Chem. 2018;18(2):216–36. doi: 10.2174/1871520617666170725165717 [DOI] [PubMed] [Google Scholar]

[pone.0336975.ref028] 28.Pfeffer CM, Singh ATK. Apoptosis: A Target for Anticancer Therapy. Int J Mol Sci. 2018;19(2):448. doi: 10.3390/ijms19020448 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336975.ref029] 29.Redza-Dutordoir M, Averill-Bates DA. Activation of apoptosis signalling pathways by reactive oxygen species. Biochim Biophys Acta. 2016;1863(12):2977–92. doi: 10.1016/j.bbamcr.2016.09.012 [DOI] [PubMed] [Google Scholar]

[pone.0336975.ref030] 30.Bakkali F, Averbeck S, Averbeck D, Idaomar M. Biological effects of essential oils--a review. Food Chem Toxicol. 2008;46(2):446–75. doi: 10.1016/j.fct.2007.09.106 [DOI] [PubMed] [Google Scholar]

[pone.0336975.ref031] 31.Harakeh S, Al-Raddadi R, Alamri T, Al-Jaouni S, Qari M, Qari Y, et al. Apoptosis induction in human hepatoma cell line HepG2 cells by trans- Anethole via activation of mitochondria-mediated apoptotic pathways. Biomed Pharmacother. 2023;165:115236. doi: 10.1016/j.biopha.2023.115236 [DOI] [PubMed] [Google Scholar]

[pone.0336975.ref032] 32.Arumugam P, Sampathkumar B, Perumalsamy H, Balusamy SR, Ramesh V, Sundaravadevel S. Synergistic effect of anethole and doxorubicin alleviates cell proliferation, cell cycle arrest, and ER stress and promotes ROS-mediated apoptosis in triple-negative breast cancer cells. J Biochem Mol Toxicol. 2021;35(12):e22928. doi: 10.1002/jbt.22928 [DOI] [PubMed] [Google Scholar]

[pone.0336975.ref033] 33.Gharbi SI, Zvelebil MJ, Shuttleworth SJ, Hancox T, Saghir N, Timms JF, et al. Exploring the specificity of the PI3K family inhibitor LY294002. Biochem J. 2007;404(1):15–21. doi: 10.1042/BJ20061489 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336975.ref034] 34.Brunn GJ, Williams J, Sabers C, Wiederrecht G, Lawrence JCJ, Abraham RT. Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors wortmannin and LY294002. EMBO J. 1996;15(19):5256–67. [PMC free article] [PubMed] [Google Scholar]

[pone.0336975.ref035] 35.Knight ZA, Gonzalez B, Feldman ME, Zunder ER, Goldenberg DD, Williams O, et al. A pharmacological map of the PI3-K family defines a role for p110alpha in insulin signaling. Cell. 2006;125(4):733–47. doi: 10.1016/j.cell.2006.03.035 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0336975.ref036] 36.Powis G, Bonjouklian R, Berggren MM, Gallegos A, Abraham R, Ashendel C, et al. Wortmannin, a potent and selective inhibitor of phosphatidylinositol-3-kinase. Cancer Res. 1994;54(9):2419–23. [PubMed] [Google Scholar]

[pone.0336975.ref037] 37.Yu H-Z, Han S-F, Li P, Zhu C-L, Zhang X-X, Gan L, et al. An examination of the potential effect of lipids on the first-pass metabolism of the lipophilic drug anethol trithione. J Pharm Sci. 2011;100(11):5048–58. doi: 10.1002/jps.22702 [DOI] [PubMed] [Google Scholar]

[pone.0336975.ref038] 38.Patel MM, Patel BM. Crossing the Blood-Brain Barrier: Recent Advances in Drug Delivery to the Brain. CNS Drugs. 2017;31(2):109–33. doi: 10.1007/s40263-016-0405-9 [DOI] [PubMed] [Google Scholar]

[pone.0336975.ref039] 39.Chen Y, Zhang C, Huang Y, Ma Y, Song Q, Chen H, et al. Intranasal drug delivery: The interaction between nanoparticles and the nose-to-brain pathway. Adv Drug Deliv Rev. 2024;207:115196. doi: 10.1016/j.addr.2024.115196 [DOI] [PubMed] [Google Scholar]

[pone.0336975.ref040] 40.Nance E, Pun SH, Saigal R, Sellers DL. Drug delivery to the central nervous system. Nat Rev Mater. 2022;7(4):314–31. doi: 10.1038/s41578-021-00394-w [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Anethole inhibits human U87 Glioma cell proliferation by inducing apoptosis via the PI3K/AKT pathway

Ahmed Abdullah Al Awadh

Elhashimi Eltayb Hassan

Omer Mohamed Shoaib

Osman AE Elnoubi

Saadalnour Abusail Mustafa

Yasir Mohammed Althayrayan

Majed Ahmed Althayrayan

Mohammed Merae Alshahrani

Roles

Abstract

1. Introduction

2. Materials and methods

2.1. Drug and reagents

2.1. Cell culture

2.2. CCK-8 assay

2.3. Colony assay

2.4. AO/EB staining assay

2.5. In Silico analysis

2.6. Molecular docking

2.7. Western blot analysis

2.8. Statistical analysis

3. Results

3.1. Anethole inhibits proliferation of glioma cells

Fig 1. Anethole inhibits the proliferation of glioma cells.

3.2. Anethole triggers apoptosis in glioma cells

Fig 2. Anethole induces apoptosis in glioma cells.

3.3. Anethole targets genes involved in cancer pathways

Fig 3. Target identification of anethole.

Fig 4. Identification and expression of cancer-related targets of anethole.

3.4. Anethole targets PI3K and inhibits the PI3K/Akt pathway

Fig 5. Anethole targets the PI3K/AKT pathway in glioma cells.

3.5. In Silico ADME and BBB Prediction

4. Discussion

5. Conclusion

Institutional review board approval and informed consent

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Zahra Lorigooini

Roles

Author response to Decision Letter 1

Decision Letter 1

Zahra Lorigooini

Roles

Author response to Decision Letter 2

Decision Letter 2

Yasmina Abd‐Elhakim

Roles

Author response to Decision Letter 3

Decision Letter 3

Yasmina Abd‐Elhakim

Roles

Acceptance letter

Yasmina Abd‐Elhakim

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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