Extract Regulates the PI3K/AKT Cascade and Inhibits the Proliferation of the Lung Cancer Cell Line.

INTRODUCTION
Lung cancer is one of the leading causes of cancer-related death worldwide. It can be divided into two categories: small-cell lung cancer and non-small-cell lung cancer (NSCLC). Approximately 85% of lung cancers are NSCLCs, which grow more slowly than other types and are often asymptomatic until they get advanced. NSCLC is composed of different histological types including squamous cell cancer, adenocarcinoma, and large cell cancers. Even though a variety of treatment strategies such as chemotherapy and potential compounds have been used, and some treatment options have been successful in controlling the disease, blooming quandaries associated with refractoriness to chemotherapy and drug resistance have made little progress in treating the disease.[12]
The use of naturally occurring substances in the treatment of cancer has a long history, where they usually do not show the problems caused by other treatments and have little or no toxicity to normal cells. The extracts of some plants generally contain many compounds that have been shown to target various cellular and molecular pathways in cancer and many other diseases.[3] Alhagi maurorum is a perennial shrubby legume that has been identified as a potential medicinal herb that contains certain compounds such as polyphenols and flavonoids. Extracts isolated from different parts of this plant have shown various potential effects on several diseases.[45]
The most fundamental aspect of the carcinogenic process is the uncontrolled molecular status of signaling pathways and control of cellular communication. Polyphenolic and flavonoid compounds exert their anticancer effects by affecting various signaling pathways, including proliferative processes, cell differentiation, apoptosis, angiogenesis, and metastasis.[6] PI3K/AKT signaling is an intracellular signal transduction pathway that has been the target of many new inhibitors. Several common cell processes, including proliferation and apoptosis, are regulated by this pathway in a variety of tumors, which plays an important role in tumorigenesis.[7] The PTEN (phosphatase and tensin homolog) tumor suppressor gene, as a key component, is involved in inactivating the PI3K/AKT signaling pathway by dephosphorylation of phosphatidylinositol kinase, and partial or complete loss of PTEN expression has often been observed in lung cancer and 30 to 70% of NSCLC cases.[89] Loss of PTEN expression leads to the downstream activation and phosphorylation of AKT, an inhibitor of apoptosis, and promotes cell proliferation.[10]
By enrichment analysis, KEGG pathways related to NSCLC were investigated to determine the most important signaling pathways of this cancer. Then the hydroalcoholic extract of Alhagi maurorum was extracted, and after measuring the amount of some of its active compounds, its inhibitory effects on the A549 lung cancer cell line were investigated through PTEN on the PI3K/AKT signaling pathway.

MATERIALS AND METHODS

Bioinformatic analysis

Identification of DEGs

GSE50627 dataset was acquired from the GEO website (http://www.ncbi.nlm.nih.gov/geo/) with the keyword lung cancer, by limiting to expression profiling by array, CEL and Homo sapiens at the GEO database to investigate NSCLC gene expression. The cutoff selection criteria for selecting the significant DEGs were P value < 0.05 and | logFC| > +1.

Protein-protein interaction (PPI) network, and pathway enrichment

Gene symbol was obtained from BioDBnet (https://biodbnet-abcc.ncifcrf.gov/) website, and the protein-PPI network was analyzed by STRING software. Finally, DEGs were analyzed using the EnrichR site (https://maayanlab.cloud/Enrichr/), and the signaling pathways were analyzed based on KEGG algorithms (www.genome.jp/kegg/). The cutoff criteria (MCODE score >3) with the default parameters were utilized to identify critical modules inside the PPI network using Molecular Complex Detection (MCODE). The network’s hub genes were chosen based on connection degree using Cytoscape software v. 3.6.0.[11]

Data collection and compound structure

The three-dimensional structures of the substances in the A. maurorum extract (chrysoeriol, isorhamnetin, and kaempferol) were obtained as SDF files from the Pubchem database (https://pubchem.ncbi.nlm.nih.gov/). The structure of proteins involved in most pathways was obtained from Uniprot (https://www.uniprot.org) and Protein Data Bank (PDB) (https://www.rcsb.org/) databases as pdb file and converted to pdbqt using AutoDock tools.

Molecular docking

To prepare the ligands and receptors for docking, the compound structures were converted into the mol2 and then pdbqt formats using the OpenBabel and Raccoon software, respectively. The structures of the proteins involved in the pathways identified through pathway enrichment were obtained and converted to pdbqt using PDB bank and AutoDock software, respectively. The Vina program, hosted in the PyRx software, was used for docking, and PyMol and DiscoveryStudio were used to analyze the docked complexes.[12]

Molecular dynamics simulation

Molecular dynamics (MD) simulation was performed using the GROMACS 5.1.1 software package and the CHARMM36 all-atom force field for analyzing two ligand-protein complexes. The automated official CHARMM general force field server (CGenFF) was used to perceive ligand bonds. The system was solved and the charge was neutralized, and then the energy was carried out. The system was then equilibrated to more stable structures for 100 ps in the canonical (NVT) ensemble and 100 ps in the isothermal–isobaric (NPT) ensemble. Finally, the MD simulation was run for a production of 20 ns. For analyzing the MD production, root mean square fluctuation (RMSF), mean square deviation (RMSD), the radius of gyration (Rg), and the number of hydrogen bonds were calculated.[12]

MM/PBSA binding free energy calculation

Molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) using the g_mmpbsa module was used to predict the binding free energy of simulated protein-ligand complexes by several energies. The total binding energy was calculated by the following formula:
ΔG binding = G complex – G ligand – G receptor

A. maurorum plant and extract preparation
The whole Alhagi maurorum plant was collected and the samples were identified in the Department of Pharmacognosy of Shahrekord University of Medical Sciences. After drying, the extraction process was performed using the maceration method in 70% ethanol. The plant powder was soaked in 70% ethanol and shaken for 72 hours at room temperature. The solution was then filtered, evaporated by rotary evaporation, and the concentrated extract was dried on glass plates in an incubator at 37°C.[13]

Measurements of total flavonoid and phenolic content of the extract
The total flavonoid content was measured using the aluminum chloride colorimetric method. In this method, solutions of 25, 50, 100, 250, and 500 PPm of the routine substance were prepared, and 1 ml of each solution was transferred to a test tube. One milliliter of 2% aluminum chloride solution and 6 ml of 5% potassium acetate solution were added to the test tubes. After 40 min, the light absorption of the samples at 415 nm was read using a spectrophotometer, and a standard curve was prepared. Afterward, 0.01 to 0.02 g of the dried extract was dissolved in 60% methanol, and then 0.01 ml of the extract solution was mixed with 0.5 ml of 2% aluminum chloride and 6 ml of 5% potassium acetate. The absorbance of the samples was measured at 415 nm using a spectrophotometer and compared with the standard curve.
The Folin-Ciocalteu (FC) colorimetric method and gallic acid were used to measure total phenolic compounds. First, gallic acid standard solutions including 12.5, 25, 50, 62.5, 100, and 125 PPm were made, and 0.1 ml of each concentration was transferred to a separate test tube. Second, 0.5 ml of a 10% solution of FC reagent was added to each test tube, and after 3–8 minutes, 0.4 ml of 7.5% sodium carbonate solution was added. Third, the tubes were placed in the dark at room temperature for 30 minutes and the amount of light absorption was measured using a spectrophotometer at 765 nm and a standard curve was prepared.
Then, 0.01 g of the dried extract was dissolved in 60% methanol, and the amount of total phenol was determined by the same method as the FC method, and the absorbance of the samples was compared with the standard curve.[1415]

Determination of the antioxidant activity of the extract
The antioxidant activity of the extracts was evaluated using the radical scavenging method using diphenyl-3-picryl hydrazyl (DPPH) radical scavenging method. In fact, the ability of different compounds and extracts to produce a hydrogen atom or electron is measured in this test by the amount of decolorization of the purple solution of DPPH in methanol. First, different concentrations of the extract were prepared, and a DPPH stock with a concentration of 90 μM was prepared with methanol. 200 μl of the samples with different concentrations were mixed with 1 ml of DPPH stock, made up to 4 ml with 95% methanol, and shaken in the dark for 60 min. The adsorption of all samples and controls (methanol and DPPH) was read at 517 nm using a spectrophotometer, and the percentage of free radical scavenging activity (%IP) was calculated using the amount of sample absorption by the following formula[16]:
%IP=control absorption - sample absorption/control absorption×100

Cell culture and cytotoxicity assay
A549 lung cancer cell line was cultured in DMEM/F12 (Gibco, Life Technologies, USA) supplemented with 5% fetal bovine serum (Grand Island Biological Company (Gibco)), penicillin (100 U/ml), streptomycin (100 μg/ml), and incubated at 37°C in 95% humidified air containing 5% CO2. 5 × 104 cells/well were cultured in 96-well plates. After 24 h, the wells were treated in triplicate with a series of concentrations of A. maurorum extract (50, 100, 200, 400, 600, 800, 1000 μg/μl). MTT assay was used to measure cell viability. This colorimetric test is based on the breakdown of yellow MTT tetrazolium salt by dehydrogenase (mitochondrial enzyme) in metabolically active cells to purple formazan. Briefly, A 10 μl solution of 5 mg/ml MTT solution was added to each well after 48 hours of exposure to the extract. Subsequently, 100 μl of dimethyl sulfoxide (DMSO) (Merck) was replaced with the supernatant of the cells in each well and pipetted slowly to dissolve the formazan crystals. It was then incubated at 37°C in the dark for approximately 15 min. The optical density of each well was read at 630–570 nm using an ELISA reader (Stat fax-2100). The concentration of the tested compounds that reduced cell viability by half was considered the IC50.[16]

AKT, PI3K, and PTEN genes expression
To evaluate the effect of the ethanolic extract of A. maurorum on the mRNA expression of AKT, PI3K, and PTEN genes in A549 cancer cell lines, real-time PCR was performed as follows. Briefly, 6 × 105 cells were cultured on six-well plates and treated with the IC50 concentration of the extract. After 24 and 48 h, 1000 μl of TRIzol Reagent (Invitrogen, USA) was added to the wells, and then the plate was kept at room temperature for 10 minutes. The mixture was transferred to a tube, and 200 μl of chloroform was added and incubated for approximately 2 minutes at room temperature. The mixture was then centrifuged at 13000 rpm for 4 minutes at 4°C. The upper RNA-containing phase was carefully removed and transferred into an RNase-free tube. Next, 500 μl of cold isopropanol was added, and the centrifugation step was repeated. Then, it was washed with 75% ethanol and dissolved in DEPC water. Finally, Nanodrop® 2000 was used to measure the quantity and quality of isolated RNA (Thermo Fisher Scientific, USA). RNA quality and quantity were examined via OD260/OD280 and OD260/OD230 absorption.
cDNA was synthesized using the GeneAll kit (GeneAll Biotechnology, China), according to the manufacturer’s protocol. For this purpose, about 2 μg of RNA was mixed with 1 μl of Oligo dT (50 μM), 1 μl of dNTP (10 μM) in a volume of 14.5 μl and placed in a thermocycler at 65°C for 5 min. Then, RTase reaction buffer, DTT, reverse transcriptase, and RNase inhibitor were added, and the tubes were incubated in a thermocycler at 42°C for 1 h and at 85°C for 5 min.
To perform real-time PCR, primers for the desired genes were designed using literature reviews and Gene Runner software [Supplementary Table S1]. GAPDH was used as the reference gene for normalization. One microliter of cDNA with 6.5 μl of QPCR Master Mix SYBR Green 2X (Takara, Japan), 0.5 μl of Specific forward primer (10 μM), and 0.5 μl of specific reverse primer (10 μM) were mixed with water in a volume of 13 μl, and finally, according to the program, the reactions were performed in the Corbett Rotor Gene 3000 for each gene.

Assessment of apoptosis by flow cytometry
The FITC Annexin V Apoptosis detection kit was prepared by BD Pharmingen Inc., and the manufacturer’s protocol was followed to evaluate apoptosis. In this method, 2 × 105 of the cells were cultured in a six-well plate. After 24 hours, the cells were exposed to IC50 concentrations of the extract at 24- and 48-hour intervals. The cells were trypsinized, transferred to tubes, and centrifuged at 1200 rpm for 5 minutes. The culture medium was removed, and the cells were washed twice with 1 ml of PBS. After the final centrifugation, the supernatant was discarded, 1 ml of 1X binding buffer solution was added, and the cells were resuspended. Next, 100 μl of the cell suspension was transferred to flow cytometry tubes, and 2.5 μl each of FITC Annexin V solution and propidium iodide (PI) solution was added to each tube. After 20 minutes of dark incubation, they were analyzed using flow cytometry (PARTEC Cyflow) and the FlowJo software.[1718]

Data analysis
REST software version 2009 and Linreg PCR version 2017.1 were used to analyze real-time PCR data based on the Pfaffl method. Flomax version 2.7 was used to analyze apoptosis data. GraphPad Prism version 8.0.1 was used for statistical analysis. The t-test was conducted to compare two groups, and the data were expressed as mean ± standard deviation.

RESULTS

Bioinformatic analyses
According to enrichment analysis, the KEGG pathways most abundantly represented in GSE50627, related to NSCLC, were the PI3K-Akt signaling pathway, focal adhesion, mucin-type O-glycan biosynthesis, and the ECM-receptor interaction signaling pathway, respectively [Supplementary Figure S1].
Therefore, the most important genes of the PI3K/AKT pathway, including AKT and PI3K, were selected for further study. The structures of AKT and PI3K were downloaded from the PDB database (PDB IDs 7NH5 and 7RSP). Discovery Studio Visualizer was used for protein essential preparations including removing water, ATP, ligands, and adding hydrogens charges. Critical ligand-binding sites in AKT and PI3K were identified and docked with chrysoeriol, isorhamnetin, and kaempferol using AutoDock software (center x = 19.8, y = −34.2, z = −17.4) and (center x = 40.4, y = 4.8, z = 83.2 for AKT and PI3K, respectively) were calculated as the coordinates of the ligand-binding sites centroid in AKT and PI3K using DiscoveryStudio software. Docked complexes were analyzed using PyMol and DiscoveryStudio software. Supplementary Table S2 displays the findings of docking analysis based on the binding energy (kcal/mol) of compounds with target structures. These findings led to the selection of chrysoeriol for MD simulation, as it formed a more stable complex with a lower energy level than other compounds with AKT and PI3K.
Figure 1 illustrates the binding position and the amino acids involved in the interaction. MD results are shown in Supplementary Table S3 including Van der Waal energy (kJ/mol), electrostatic energy (kJ/mol), polar solvation energy (kJ/mol), SASA energy (kJ/mol), SAV energy (kJ/mol), WCA energy (kJ/mol), and binding energy (kJ/mol). The RMSD values for the AKT and PI3K showed between 0.2 and 0.24 nm. The RMSD score has minor fluctuations, indicating the stability of chrysoeriol within the AKT-chrysoeriol and PI3K-chrysoeriol complex. The RMSF value for both protein structures was computed to accurately determine the impact of chrysoeriol binding on flexibility. Analysis of the chrysoeriol-associated RMSF plot revealed high flexibility in the regions 100–150 and 295–312 in the AKT and 400–500 in the PI3K, which may be due to a lack of interaction between these regions and chrysoeriol. Flexibility was notably higher at residues 100–150 and 295–312 in AKT and 400–500 in PI3K [Figure 2].

Preparation of A. maurorum extract and total content of flavonoids and phenols
After extraction from the plant, the concentrated extract was poured into special glass plates and placed in an incubator at 37°C for drying, then powdered and stored in a refrigerator. The content of phenolic and flavonoid compounds in the extract was determined using gallic acid and routine equivalents. As shown in the Supplementary Table S4, this extract contains high amounts of flavonoid compounds, suggesting that a significant portion of its antioxidant activity is likely due to these flavonoids.

Free radical scavenging activity using DPPH
The inhibitory percentages of different concentrations of A. maurorum ethanolic extract are shown in Figure 3a. The IC50 value was calculated using a linear equation based on the UV-visible spectrophotometer absorption of various concentrations of A. maurorum extract at 517 nm. This value was found to be 40.95 µg/ml, and the antiradical activity increased with higher concentration of the extract.

Determination of cell cytotoxicity of the extract using MTT assay
The cell viability test results revealed that cancer cells treated with the A. maurorum extract showed morphological changes compared to untreated cells. The cytotoxicity curve, determined by MTT assay, was used to calculate IC50 values for A. maurorum extract in the A549 cell line induced after 48 hours, based on an exponential equation [Figure 3b]. A. marourum extract inhibited the cell viability of the A549 cell line in a dose-dependent manner with IC50 values of 277 μg/μl.

Apoptosis assay
Cells were stained with annexin V-FITC/PI and analyzed using a flow cytometer. The percentage of viable, early apoptosis, late apoptosis, and necrosis fractions in treated and untreated (control group) A549 cells with an IC50 concentration of A. maurorum extract was recorded. The results showed that, compared to the control group, the number of apoptotic cells significantly increased with the extended duration of the extract treatment [Figure 4].

Cell signaling pathways in the transcriptome
PTEN was selected as the study target due to its inhibitory role in the PI3K-AKT signaling pathway. The results showed a 0.6-fold decrease in the relative expression of the PTEN gene at 24 hours and a 2-fold increase in its expression at 48 hours after treatment with the IC50 concentration of the extract compared to the control group [Figure 5a]. The PI3K gene was selected as a key gene in the PI3K-AKT signaling pathway. Data analysis for this gene indicated that PI3K expression decreased at 24 hours, but the changes were not significant at 48 hours after treatment [Figure 5b]. The relative expression of the AKT gene decreased by 0.4-fold and 0.75-fold in 24 hours and 48 hours after treatment, respectively, compared to the control group [Figure 5c].

DISCUSSION
The findings showed that the active ingredients of the A. maurorum extract are three compounds: chrysoeriol, isorhamnetin, and kaempferol. Studies indicate that these three substances significantly impact cancer cell migration, apoptosis, and proliferation by regulating the PI3K/AKT pathway and apoptosis genes. Research on the effect of isorhamnetin on cardiac hypertrophy has confirmed that isorhamnetin can reduce angiotensin II-induced cardiomyocyte hypertrophy via the PI3K/AKT signaling pathway. This study was the first to confirm the association between isorhamnetin and the PI3K/AKT signaling pathway.[19] As a natural flavonoid, it has also exhibited anticancer effects in breast cancer, colon cancer, gastric cancer, and gallbladder cancer, through the induction of cell apoptosis and significantly reduced cell proliferation and migration. Investigation of its molecular mechanism revealed that it can suppress Akt phosphorylation and NF-κB translocation, both of which are key factors in apoptosis-related pathways.[2021]
Additionally, isorhamnetin and kaempferol induced apoptosis and significantly inhibited the proliferation of A549 cells by increasing the expression of the apoptotic genes Bax and caspase-3, decreasing the expression of Bcl-2, cyclin D1, and PCNA protein, and regulating PI3K/AKT and MEK-MAPK pathways.[222324]
Chrysoriol significantly induced autophagy in A549 lung cancer cells by changing the expression of LC3II and Beclin-1 protein. Additionally, it prevents A549 cells from migrating and invading.[25] The findings of this investigation were consistent with previous research. This research has shown that A. maurorum extract suppressed PI3K and AKT expression and increased the expression of PTEN.
Measurement of DPPH free radical scavenging is a valid, accurate, and easy method used to evaluate the antioxidant activity of plant extracts in vitro. Phenolic and flavonoid compounds are important plant compounds because of their antioxidant properties, which play important roles in eliminating free radicals and preventing the conversion of hydroperoxides to free radicals. The findings of this investigation demonstrated the significant antioxidant effect of the phenolic extract of A. maurorum. These findings were in line with previous research on the antioxidant effect of A. maurorum extract.[2627]

CONCLUSION
The hydroalcoholic extract of A. maurorum indicated significant inhibitory activity against the A549 lung cancer cell line. According to the experiments, the ability of the extract to inhibit free radicals increased with increasing concentration, and the total phenolic and flavonoid content of the extract was effective for its antioxidant activity. The ethanolic extract of A. maurorum affected the AKT pathway by promoting PTEN expression. To address the possibility that the effects of the A. maurorum extract might influence both cancerous and normal cells, it is essential to recognize the broad-spectrum action of phenolic and flavonoid compounds in the extract. These compounds, well-known for their antioxidant properties, can impact various cellular pathways, including those in normal cells. While the extract effectively inhibited the A549 lung cancer cell line, this inhibition might not be entirely specific to cancer cells, as antioxidant and pathway-modulating compounds like flavonoids and phenolics can act on universal cell mechanisms. In particular, the influence on the AKT pathway via PTEN expression could have implications for cellular function in non-cancerous cells, which warrants careful consideration. This limitation emphasizes the importance of conducting further studies, such as testing on non-cancerous cell lines and validating findings at the protein level, to determine whether the extract’s effects are more specific to cancer cells or if they also affect normal cell processes. This additional analysis would clarify the therapeutic potential of the extract and guide its application in a clinical setting.

Ethics approval and consent to participate
The study was approved by the Ethics Committee of the Shahrekord University of Medical Sciences (Code: IR.SKUMS.REC.1397.313).

Conflicts of interest
There are no conflicts of interest.

Supplementary Figure S1(a). Enrichment analysis of GSE50627. The KEGG pathways that were most particularly abundant in GSE50627 related to non-small-cell lung cancer were the PI3K-Akt signaling pathway, Focal adhesion, mucin-type O-glycan biosynthesis, and the ECM-receptor interaction signaling pathway, respectively. (b). Protein-protein interaction (PPI) network analysis of GSE50627 by STRING software