Network pharmacology and experimental verification reveal the mechanism of safranal against glioblastoma (GBM)

Introduction Safranal is an active component of the traditional Tibetan medicine (TTM) saffron, which has potential anticancer activity. Methods and results Here, we studied the therapeutic effect and mechanism of safranal on GBM. CCK-8, GBM-brain organoid coculture experiments and 3D tumour spheroid invasion assays showed that safranal inhibited GBM cell proliferation and invasion in vitro. Network pharmacology, RNA-seq, molecular docking analysis, western blotting, apoptosis, and cell cycle assays predicted and verified that safranal could promote GBM cell apoptosis and G2/M phase arrest and inhibit the PI3K/AKT/mTOR axis. In vivo experiments showed that safranal could inhibit GBM cell growth alone and in combination with TMZ. Conclusion This study revealed that safranal inhibits GBM cell growth in vivo and in vitro, promotes GBM cell apoptosis and G2/M phase arrest, inhibits the PI3K/AKT/mTOR axis and cooperate with TMZ.


Introduction
Gliomas represent 81% of malignant brain tumours; they have a 5year relative survival of 5% and are the most common and aggressive primary brain tumour (1,2).Despite aggressive treatment protocols, including surgery, radiation, and chemotherapy, there are no known measures to prevent the development of gliomas (3).Therefore, new treatment measures and methods are urgently needed.
Disease is not only caused by a genetic abnormality, but reflects the complex network within or between cells (4).Since the success rate of drug discovery is declining.The "one disease-one target-one drug" doctrine has been challenged (5).Network-based method shows potential clinical application value (4).
Traditional Tibetan medicine (TTM) has a history of thousands of years, and many TTMs have been proven to have anticancer properties (6)(7)(8)(9)(10).Saffron is a traditional Tibetan medicine that has been reported to have anticancer effects in previous studies (11 -17).The main ingredients of saffron include crocin, crocetin, picrocrocin and safranal (18).We noticed that the molecular weight of safranal is very low, and it was previously reported in the literature that safranal could relieve various nervous system disorders by systemic administration in vivo, which indicates that safranal could easily enter the nervous system (19)(20)(21).In addition, we found that many articles reported that safranal has anticancer effects (22)(23)(24)(25)(26).However, the effect of safranal on glioma, especially glioblastoma (GBM), has not yet been reported.
In this study, CCK-8 and many other in vitro experiments were used to preliminarily verify the inhibitory effect of safranal on primary GBM cells.Then, we used network pharmacological methods and RNA-seq analysis to predict the possible safranal pathway in glioma.Finally, western blotting, flow cytometry and other experiments were used to verify the mechanism of safranal, and in vivo experiments were used to further verify its effect.In addition, we used in vivo and in vitro experiments to verify the effect of safranal combined with temozolomide (TMZ).The methodology of this research is summarized in the flowchart (Figure 1).

Ethics statement
All animal procedures were approved by the Ethics Committee of Qilu Hospital of Shandong University (permit number: DWLL-2021-095).This study was conducted in full compliance with relevant 100 regulations and guidelines.

FIGURE 1
The flow chart of this study.

Cell invasion in 3D culture
GBM#P3 or BG7 cells were seeded into 3D Culture Qualified 96well spheroid formation plates (Trevigen) with 50 ml of medium, with 3000 cells per well.Then, they were cultured at 37°C and 5% CO2 for 72 hours in a humidified incubator for 72 h to form tumour spheroids.Then, we placed the plate on ice for 15 minutes and added 50 ml of invasion matrix (Trevigen, 3500-096-03) into each well.The plates were centrifuged at 300×g at 4°C for 15 minutes and incubated at 37°C for 1 hour.Then, 100 ml medium with 100 mM safranal or DMSO was added to each well.After 48 h (GBM#P3) or 72 h (BG7), we used a 4× objective to capture the spheroids under a bright field microscope.The images were analysed with ImageJ software.

GBM-brain organoid coculture assay
The GBM-brain organoid coculture invasion ex vivo system was performed as described in a previous study (27).GFP-transfected GBM#P3 cells (3000 cells per well) or BG7 cells (5000 cells per well) were cultured to generate glioma spheroids.Then, mature brain organoids were cocultured for 36 h with GBM#P3 cells or 48 h with BG7 cells.Confocal microscopy (Leica TCS SP8; Wetzlar, Germany) was used to capture GBM cell invasion images.The images were analysed with ImageJ software.
Determination of overlapping glioma-related genes and target genes of safranal and construction of the "Drug-targets-disease" network involved in the antiglioma effect of safranal The "venndiagram" package of R software (version 4.1.3(2022-03-10)) was used to obtain 72 intersecting genes among gliomarelated genes and the safranal target genes, and a Venn diagram was drawn.We used Cytoscape 3.9.1 software to visualize them as a "Drug-Targets-Disease" network.

Construction and analysis of the protein −protein interaction (PPI) network
The STRING database (https://cn.string-db.org/) was used to obtain the relationships between the 72 intersecting genes.PPIs with a combined score greater than 0.4 were selected and used to construct a PPI network.Then, we used the MNC algorithm in the cytohubba plugin in Cytoscape 3.9.1 software to find the key proteins in the network.

GO and KEGG pathway enrichment analysis
The "clusterProfiler" and "org.hs.eg.db" packages in R software were used to analyse the enrichment of drug-disease genes in GO terms and KEGG pathways.Then, we visualized the results with R software.

RNA-Seq and bioinformatics analysis
We treated GBM#P3 cells with safranal (100 mM) or vehicle (DMSO) for 24 h and performed RNA sequencing (LC-Bio; Shanghai, China).Full data accompanying this experiment are available in the Sequence Read Archive (SRA).The accession number is PRJNA991989.Differentially expressed genes were analysed and visualized by R software.The processes for PPI network analysis and GO and KEGG pathway enrichment analysis were the same as above.

Molecular docking
We first searched the UniProt ID of the target protein from the UniProt database (https://www.UniProt.org/),and then searched the structure of target proteins in the PDB database (https:// www.rcsb.org/)using the UniProt ID of the protein.Protein structures were downloaded.PyMOL software were used to remove water and organic.
We used AutoDockTools-1.5.7 to add hydrogens to the protein and simulate potential targets for molecular docking.Then, AutoDockVina were used to perform molecular docking.

Western blotting
Total protein of GBM#P3 or BG7 cells was extracted with cold RIPA Lysis Buffer (Beyotime; Shanghai, China) supplemented with phenylmethanesulfonyl fluoride (PMSF, Beyotime).Western blotting was performed according to previously described protocols (28).We used a chemiluminescence imager (Bio-Rad; Hercules, CA, USA) to detect the chemiluminescent signals according to the manufacturer's protocol.

Cell cycle
Seventy percent ethanol was used to dehydrate the cells overnight at 4°C.Then, the cells were stained with PI/RNase (550825, BD Biosciences; San Jose, CA).After 15 min in the dark, the cells were analysed by flow cytometry (Accuri C6, BD Biosciences).

Apoptosis
We resuspended cells in 300 mL binding buffer and stained them with Annexin V-FITC (BD Biosciences) according to the manufacturer's instructions.Then, the cells were analysed by flow cytometry.

Orthotopic xenograft model
For statistical purposes, three nude mice were used in each group.A total of 1×10 6 luciferase-stable GBM#P3 cells were injected into the brains of 15 4-week-old male nude mice.One week later, tumours were examined using bioluminescence imaging (PerkinElmer IVIS Spectrum; Waltham, MA, USA).We found that in the 15 mice, one had no intracranial tumor formation and two had spinal metastases, so we excluded them.Our inclusion criteria included successful tumor implantation, uniform tumor size, and no spinal metastasis.Exclusion criteria included that there was no intracranial tumor formation, the tumor volume was significantly larger or smaller than that in other mice, and spinal metastasis.Then, we classified the remaining 12 mice as numbers 1-12, used excel software to generate random numbers, arranged the random numbers in reverse order, and then divided the mice into four groups(each group has 3 mice) from top to bottom: control group (n=3), safranal group (n=3), TMZ group (n=3) and TMZ+safranal group (n=3).Tumour volume was monitored using bioluminescence imaging.Mice were euthanized after 2 weeks of treatment.Cages were placed in the same house, keeping the external environment consistent.

Immunohistochemistry
Mice in each group were harvested, and the brains were removed, fixed in 4% formalin, and prepared as paraffinembedded sections.Immunohistochemistry was performed as previously reported (28).

Safranal suppresses the proliferation and invasion of GBM cells
First, we performed in vitro experiments to test whether safranal had any effect on the proliferation and invasion of primary GBM cells.The CCK-8 assay showed that the viability of GBM cells decreased significantly with increasing safranal concentration (Figure 2A).GBM-brain organoid coculture experiments (Figures 2B, C) and 3D tumour spheroid invasion assays (Figures 2D, E) demonstrated that safranal could inhibit the invasion of GBM cells.

Drug-disease intersection targets
We used 308 targets of safranal and 1932 glioma-related targets for Venn analysis (Figure 3B) to obtain 72 intersecting targets and a drug-targets-disease network (Figure 4).The hexagon in violet represents glioma, the "V" in blue represents safranal, and the ellipses in green represent the intersection targets.

PPI network analysis
We used the STRING database to find the protein interaction relationship of 72 intersection targets (Figure 5A) and counted the top 20 nodes connected most in the network (Figure 5B).Then, we imported the results of the STRING database into Cytoscape software and used the MNC algorithm in the CytoHubba plug-in to obtain the top 20 core targets (Figure 5C).We found that the top 20 core targets obtained by the MNC algorithm had 19 of the same targets as the top 20 targets calculated in Figure 5B (Figure 5D), which illustrated that these 19 targets played a key role in the network.

KEGG pathway enrichment analysis
To explore the possible mechanism of safranal in treating glioma, KEGG pathway enrichment analysis was performed on 19 core targets (Figures 5E, F).We found that these 19 targets were enriched in some critical tumour-related pathways, such as apoptosis and the PI3K-Akt signaling pathway.This suggests that safranal may inhibit the growth of glioma cells through these two signaling pathways.

Analysis of mRNA sequencing data
Then, primary GBM cells (GBM#P3) were treated with DMSO or 100 mM safranal, and mRNA sequencing was performed.We obtained 1751 differentially expressed genes between the treatment groups (Figures 6A, B).We used the STRING database to obtain the interaction network between the proteins encoded by these genes.The MNC algorithm in the CytoHubba plug-in of Cytoscape software was used to find the core protein of the network (Figure 6C).Subsequently, KEGG pathway enrichment analysis was performed based on these proteins.We found that these targets were enriched in the cell cycle and PI3K-Akt signaling pathways (Figures 6D, E). continued to perform Gene Ontology (GO) functional analysis.The results showed that the core proteins of the differentially expressed genes were enriched in the cyclin-dependent protein kinase holoenzyme complex, cyclin-dependent protein serine/threonine kinase regulator activity and other cell cyclerelated pathways (Figures 6F-H).

Molecular docking study
Based on the above analysis, we found that the inhibitory effect of safranal on glioma was most likely achieved through apoptosis, the PI3K-Akt signaling pathway and the cell cycle pathway.Therefore, we selected several key proteins in these pathways to perform molecular docking with safranal.As previously reported, safranal can inhibit the PI3K/Akt/mTOR axis, and we docked safranal with the proteins in this axis.The results showed that the binding energies of safranal with AKT1, PI3K and mTOR in the PI3K/AKT/mTOR axis; PARP1, CASP3, and CASP7 in the apoptosis pathway; and CCNB1, CDK1 and CDK6 in the cell cycle pathway were lower than -5 kcal/mol (Figure 7, Table 1).This indicates that safranal's inhibitory effect on glioma is most likely to be achieved by inhibiting the PI3K/Akt/mTOR axis, acting on apoptosis-related proteins to increase tumour cell apoptosis, and acting on cycle-related proteins to regulate the tumour cell cycle.

In vitro experiments verified that safranal promotes the G2/M arrest of GBM cells
According to previous studies, safranal could cause G2/M phase arrest of HepG2 cells (23).In our molecular docking prediction, we found that safranal may have a direct interaction with the key proteins CCNB1 and CDK1 of the G2/M transition.We suspect that safranal may cause G2/M phase arrest in GBM cells, so we used the primary GBM cells (GBM#P3 and BG7 cells) for experimental verification.Figures 8A, B shows that after treatment with safranal for 24 h, GBM#P3 and BG7 cells showed G2/M phase arrest.In addition, the western blotting results showed that safranal could significantly reduce the protein levels of CDK1, CCNB1, and CDK6 in GBM#P3 and BG7 cells (Figures 8E, F).

In vitro experiments verified that safranal promotes the apoptosis of GBM cells
We further assessed the effect of safranal on the apoptosis pathway of GBM cells.In Figures 8C, D we used flow cytometry to detect the effect of safranal on the apoptosis rate of GBM#P3 and BG7 cells.As we previously predicted, the apoptosis rate of GBM cells was significantly increased after safranal treatment.
The Western blotting results also showed that cleaved caspase3, cleaved caspase7, and cleaved PARP1 were significantly upregulated in GBM#P3 and BG7 treated with safranal (Figures 8G, H), which further confirmed that safranal could promote GBM cell apoptosis.
In vitro experiments verified that safranal inhibits the PI3K/AKT/mTOR axis in GBM In our molecular docking studies, we found that the binding energies (BEs) of safranal with AKT1, PI3K and mTOR were less than -5 kcal/mol, indicating that safranal may be involved in regulating the PI3K/AKT/mTOR axis.This prediction was tested experimentally.The Western blotting results showed that the levels of PI3K, AKT, mTOR and p-mTOR in GBM#P3 and BG7 cells decreased significantly after safranal treatment (Figures 8I, J).This confirms that safranal inhibits the PI3K/AKT/mTOR axis in GBM.
In vitro experiments verified that safranal has a synergistic effect with TMZ TMZ is a first-line treatment for newly diagnosed glioblastoma (29,30).However, not all patients respond to TMZ (30,31).It was previously reported that PI3K/AKT axis inhibitors could inhibit temozolomide resistance in glioblastoma cells and activation of the PI3K/AKT axis could promote TMZ resistance in GBM cells (35)(36)(37).Therefore, we suspect that safranal and TMZ may have a synergistic effect against GBM.We used a CCK-8 experiment for verification.The results in Figure 8K and Figure 8L show that the combination of safranal and TMZ had a better effect than either single agent alone, which verified that safranal and TMZ could synergistically inhibit the growth of GBM cells in vitro.
In vivo experiments verified that safranal could inhibit GBM cell growth and that safranal and TMZ could synergistically inhibit the growth of GBM cells To further explore whether systemic administration of safranal is effective for intracranial tumours, we implanted 1×10 6 luciferasestable GBM#P3 cells into the brains of 4-week-old nude mice.One week later, we randomly divided them into four treatment groups and started treatment.In the control group, the vehicle was injected intraperitoneally (twice a day); in safranal group, intraperitoneal injection of safranal (15 mg/kg, twice daily) was used; the TMZ group received intraperitoneal injection of temozolomide(25 mg/ kg) every morning and vehicle every afternoon; TMZ+safranal group received intraperitoneal injection of temozolomide(25 mg/ kg) with safranal (15 mg/kg) every morning and safranal 15 mg/kg every afternoon.Two weeks later, tumour growth was evaluated using luciferase bioluminescence.We found that the tumours in the safranal-treated group were significantly smaller than those in the control group, while the intracranial tumours in the safranal combined with temozolomide group were significantly smaller than those in the temozolomide monotherapy group (Figures 9A,  B).The immunohistochemical results showed that the expression of Ki-67 in tumour cells of safranal-treated mice was significantly lower than that tumour cells of the control mice (Figures 9C, D).This indicated that safranal could inhibit the growth of GBM in vivo.

Discussion
Glioma is the most common malignant tumor in the central nervous system, and there is still no effective cure for it.The bloodbrain barrier separates the microenvironment of the brain from the blood and is the main physical and physiological barrier that hinders the entry of chemotherapy drugs into the brain.It greatly limits the systemic treatment effect of glioma (38,39).At present, traditional herbs are becoming increasingly popular in China and other Asian countries.Researchers are using scientific approaches to elucidate their mechanisms, and efficacy (40).For example, paclitaxel is a potent anticancer drug that originates from the tree Taxus brevifolia (41); b-Elemene is a sesquiterpene compound extracted from the herb Curcuma Rhizoma, which is used to treat several types of cancer with no reported severe adverse effects (42).
Safranal, one of the most extensively studied bioactive compounds present in herbal medicine saffron (42), could cross brain blood barrier.In previous studies, safranal has been reported to have anticancer effects.However, the effect and mechanism of safranal on GBM are not clear.
Our study reflects the effect of safranal on the growth of GBM cells in vitro and in vivo.We first used the CCK-8 assay, GBM-brain  organoid coculture experiment and 3D tumour spheroid invasion assay in vitro to preliminarily find that safranal has inhibitory effect on the proliferation and invasion of primary GBM cells.Subsequently, we used network pharmacology methods and RNAseq bioinformatics analysis to predict that safranal may influence the PI3K-AKT signaling pathway, apoptosis, and cell cycle to inhibit glioblastoma growth.In the process of molecular docking verification, we further found that the binding energy between safranal and many key proteins in the PI3K/AKT/mTOR axis, apoptosis pathway and cell cycle pathway was lower than -5 kcal/ mol.This shows that safranal's ability to inhibit GBM cells is most likely achieved by inhibiting the PI3K/Akt/mTOR axis, increasing cancer cell apoptosis and regulating the cancer cell cycle.Subsequently, we used flow cytometry to discover that safranal can induce G2/M phase arrest in GBM cells, and significantly reduce the expression of G2/M transition related proteins CCNB1 and CDK1.The effect of safranal on promoting apoptosis in GBM cells was also verified through flow cytometry and western blot experiments.Additionally, the western blot experiment also confirmed that safranal can inhibit the PI3K/AKT/mTOR pathway in GBM cells.TMZ is a first-line treatment for GBM and activation of the PI3K/AKT axis can promote temozolomide resistance in glioblastoma (32-34).An increasing in the levels of p-Akt (S473, T308) were found after TMZ treatment in a time-and dose-dependent manner in glioblastoma cell lines, which suggests that the activation of AKT may lead to TMZ resistance (43).Salvianolic acid A treatment could suppress the malignant behaviors of glioma cells and improve TMZ sensitivity through inactivating TAGLN2/PI3K/Akt pathway (32); Bodo Haas at al. also indicated that PI3K pathway seems to play a crucial role in resistance to alkylating agents and might serve as drug target for chemosensitization (33).Therefore, we suspect that safranal may have a synergistic effect with TMZ, which was confirmed by our in vitro experiments.Finally, we verified the effect of safranal on GBM and its synergistic effect with TMZ using in vivo experiments.Above all, our study mainly discussed the effect and mechanism of safranal in inhibiting GBM and proved the synergistic effect of and TMZ.

Conclusion
Safranal inhibited the growth of GBM cells in vitro and in vivo by promoting GBM cell apoptosis, causing G2/M phase arrest, and inhibiting the PI3K/AKT/mTOR axis.Safranal could cooperate with temozolomide to inhibit GBM cell growth in vitro and in vivo.
FIGURE 3 (A) Venn diagram of drug targets in the PharmMapper, SwissTargetPrediction and TCMSP databases.(B) Venn diagram of drug-and disease-related targets.
FIGURE 5 (A) PPI network of targets generated using STRING 11.5.Nodes represent proteins.Edges represent PPIs.(B) Numbers of edges of 20 core genes in the network.(C) Topological analysis of the protein−protein interaction network.(D) The top 20 targets with the highest score obtained by the MNC algorithm have 19 intersections with the 20 targets calculated in Figure 5B.(E, F) KEGG pathway enrichment analysis.

6
FIGURE 6 Analysis of mRNA sequencing data.(A) Heatmap showing differences in gene expression between the control and safranal-treated groups.(B) Volcano plot exhibiting differentially expressed genes between safranal-treated cells and controls.(C) Core proteins of the network of proteins coded by differentially expressed genes.(D, E) KEGG pathway enrichment analysis of core proteins.(F-H) Enriched terms in the biological process (BP), cellular component (CC), and molecular function (MF) categories according to GO enrichment analysis.
FIGURE 8 (A) Flow cytometry showing cell cycle parameters in GBM#P3 and BG7 cells following safranal treatment for 24 h.(B) Graphs representing the proportions of cells in the G2 phase of the cell cycle.(C) Flow cytometry showing Annexin V-FITC staining to detect apoptosis in GBM#P3 and BG7 cells treated with safranal or DMSO for 24 h.(D) Graphs representing quantification of Annexin V-FITC positive staining for treated cells and controls.(E, F) Western blot analysis of cell cycle-related protein markers in GBM#P3 and BG7 cells.(G, H) Western blot analysis of apoptosis-related protein markers in GBM#P3 and BG7 cells.(I, J) Western blot analysis of protein markers in PI3K/AKT/mTOR axis in GBM#P3 and BG7 cells.(K, L) CCK-8 assay for cell viability of GBM#P3 and BG7 after treatment with vehicle (DMSO), safranal, TMZ or safranal combined with TMZ (n = 4) for 24 h (K) or 48 h (L).

TABLE 1
The docking affinity of safranal binding to key targets.