Anti-proliferative effect of leaf phytochemicals of soursop (Annona muricata L.) against human osteosarcoma in vitro

doi:10.21203/rs.3.rs-2951478/v1

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Anti-proliferative effect of leaf phytochemicals of soursop (Annona muricata L.) against human osteosarcoma in vitro

https://doi.org/10.21203/rs.3.rs-2951478/v1

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published 23 Feb, 2024

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Soursop (Annona muricata) is being used in treating various types of cancers and there is no report on effect of soursop leaf phytochemicals against osteosarcoma. Current study identified 28 metabolites from ethyl acetate leaf (EAL) extract through GC-MS chemoprofiling and subjected to in silico analysis against the potential protein target, Platelet Derived Growth Factor Receptor α (PDGFRA) of osteosarcoma, including Absorption, Distribution, Metabolism, and Excretion and Toxicity (ADMET) analysis to identify possible hit compounds. This resulted in three hit leaf bioactives namely, 2’- hydroxy-5’-methyl chalcone, linoleic acid and annonacin showing good binding affinity with a docking score of -7.4, -7.0 and – 6.9 kcal/mol respectively. With ADMET analysis, 2’- hydroxy-5’-methyl chalcone and linoleic acid obeyed Lipkinsi’s rule of five, whereas annonacin showed slight violation. Among the three docked complexes, annonacin exhibited good stability during molecular dynamic simulation performed with PDGFRA. Hence, concentration of the key marker compound, annonacin in EAL concentrate is found to be 5.032± 0.13 mg/g of leaf sample. Further, EAL concentrate exhibited cytotoxicity (IC₅₀ value) on MG-63 osteosarcoma cells in vitro for concentrations ranging from 10 to 25 µg/mL and nuclear imaging of osteoblast cells treated with EAL concentrate at 25 µg/mL concentration exhibited typical symptoms of apoptosis. In vitro cytotoxicity along with nuclear imaging confirmed EAL concentrate from soursop to be a potential drug candidate in developing new anti-cancer agent against osteosarcoma.

Soursop leaves

Annona muricata

GC-MS chemoprofiling

Annonacin

Osteosarcoma

PDGFRA

Molecular docking

ADMET analysis

MG-63 cell lines

Nuclear imaging

In the process of drug discovery from phytomolecules, synergistic effects of phytochemicals are always preferred over pure compounds in Traditional or Complementary or Integrative systems of Medicine across the globe. However, the key challenge is always to identify the compound(s) responsible for specific bioactivity and toxicity. As a multi-disciplinary approach, the present study focused on designating phytochemicals from leaf extracts of soursop (Annona muricata), a plant known for its broad spectrum anti-cancer activity against osteosarcoma or bone cancer through analytical and in silico chemistry approach with confirmation from in vitro cell line studies. Annona muricata L, widely known as graviola or soursop is considered to have potential phytochemicals like phenols, saponins, terpenoids, flavonoids, isoquinoline alkaloids and annonaceous acetogenins (Alali et al. 1999; Bonneau et al. 2017; Shanmugam et al. 2022) that are reported to be effective against various cancer cell lines (Pieme et al. 2014). Several research works have confirmed that the entire soursop tree exhibits selective and significant anticancer activity, with leaves being the most potent (Mishra et al. 2013; Rupprecht et al. 1990). Interestingly, leaf extracts exhibited void/negative impact on normal cells, while showing selective toxicity towards cancerous cells (de Sousa and Vieira, 2010; Oberlies et al. 1995). Of late, nano delivery of plant phytochemicals with antiviral properties are explored in cancer treatment to increase the therapeutic efficiency and to overcome the limitations of chemotherapy drugs (Shanmugam et al. 2022).

Osteosarcoma, a deadly form of musculoskeletal cancer, is the most common type of cancerous tumor in a bone that predominantly affects children and adults (Marques et al. 2014; Mora et al. 2019). After lymphomas and brain tumors, osteosarcoma is considered to be the third most common cancer occurring in adolescence children with an average diagnosis age of 15 years. Osteosarcoma occurs in primitive mesenchymal bone-forming cells with production of malignant osteoid and exhibits an inclination over the metaphysis of long bones commonly found in the distal femur (43%), proximal tibia (23%) and humerus (10%) (Isakoff et al. 2015). Current curative treatment regimens consist of surgery, radiotherapy and intensive chemotherapy. Despite this multi-dimensional approach, survival of osteosarcoma patients is 5 years with 60–65% of survival rate (Blumenthal et al. 2002). Even adjuvant chemotherapies results in more side effects with dose intensification and patients are also at high risk of eventual relapse. Among various chemotherapeutic drugs given to the patients, use of high-dose methotrexate, doxorubicin, cisplatin, ifosfamide /etoposide, rafenib, sorafenib and regorafenib have been reported to be effective (Ferguson and Goorin, 2001; Rathore and Van Tine, 2021a). However, these drugs have different targets proteins that inhibit osteosarcoma cell proliferation. Among the various target proteins, receptor tyrosine kinases (RTK) are considered to be the effective target protein inhibiting osteosarcoma proliferation (Ségaliny et al. 2015).

RTK inhibitors like, Sorafenib and Regorafenib, binds to one of the target receptor, platelet derived growth factor receptors α and β (PDGFR) that has high affinity for various vital polypeptide growth factors, cytokines, hormones and hence, serves as an attractive drug discovery target for osteosarcoma (Zhou et al. 2021). Despite the effective inhibition of osteosarcoma proliferations, the current drugs have very high side effects like nerve damage, cardiotoxicity, damage to lining of bladder, renal and liver failure, damage to the white matter of the brain (Bielack et al. 2015; Rathore and Van Tine, 2021b). Considering the side effects, relapse and the cost associated with these treatments, there is a need to identify phytomolcules having selective inhibitory effect on the affected cancerous cells against osteosarcoma. An approach to identify phytomolecules possessing dual or multiple bioactivities like osteogenic stimulatory activity in bael bioactives (Shanmugam et al. 2019), apart from anti-cancer activity against osteosarcoma will be interesting. However, the current study is to assess the efficiency of leaf phytochemicals of A. muricata against osteosarcoma and to get an understanding of the chemistry behind the bioactivity. Specifically to identify hits from leaf phytochemicals as potential inhibitors against osteosarcoma targeting the receptor binding protein, Platelet Derived Growth Factor Receptor (PDGFR) through in silico analysis. Various types of PDGFR inhibitors like 3,3′Diindolylmethane, Brazilin, Curcumin, Dehydrozingerone, Ellagic acid, Glyceollins, Pterostilbene, and Vitisin B have been reported from various plants and vegetables sources (Ricci and Ferri, 2015). Till date, to the best of our knowledge, there are no reports on the inhibitory effect of A. muricata leaf phytochemicals as potential drug candidate against bone cancer. Hence, assessing the efficiency of leaf phytomolecules of A. muricata against human osteosarcoma and in silico screening of compounds to understand their chemistry could be a potential significant approach in dealing with developing new drug molecules from phytoextracts. To the best of our knowledge, this is the first report on cell proliferation inhibitory effect of leaf phytochemicals of A. muricata against osteosarcoma MG-63 cell lines.

2.1. Preparation of leaf extract

Leaves of A. muricata were collected freshly from the Horticultural Research Station in October 2020, Tamil Nadu Agricultural University, Pechiparai (latitude of 8.44340 N and longitude of 77.30660 E) and transported at room temperature to CANT, TNAU, Coimbatore on the same day. The fresh leaves were graded and shade dried at ambient room temperature of 25 ± 2 ℃ for 5 days, until it reaches constant weight. Dried leaves were extracted with ethyl acetate (AR grade) in the ratio of 1:4 (w/v of leaf powder/solvent) and filtered through filter paper. The filtrates were then concentrated using rotary vacuum evaporator at 45℃ (Heidolph Model-G3, Germany). The obtained leaf concentrates were subjected for further analysis.

2.1.1. GC-MS profiling

Ethyl acetate leaf (EAL) concentrate of A. muricata was dissolved in GC-MS grade ethyl acetate and filtered through microfiltration (0.22micron size) technique. The GC-MS analysis was performed on a Perkin Elmer Clarus SQ8C (Perkin Elmer Clarus SQ8C, CA), interfaced to a mass spectrometer (GC-MS) equipped with fused silica column packed with HP-5MS capillary column (TG-5MS, 30 M in length, 0.5mm Diameter, and 0.25 µm film thicknesses). The helium carrier gas was set to 1mL/min flow rate (constant flow mode). The injector temperature was set at 280⁰C. The oven temperature program was programmed to start at 40°C hold for 5 min then ramp to 40–70°C for 2 min, 70°C for 2 min, 70–120°C at 3°C/min, 120–150°C at 5°C/min, 150–220°C at 10°C/min and then 220°C for 2 min. A 1 µL aliquot of the plant samples were injected onto the instrument with a flow rate of carrier gas 1.0 mL/min. The mass spectrometer operating in electron ionization (EI) mode with transfer line temperature at 280°C; ion-source temperature at 230°C; ionization mode; electron impact at 70eV, scan time of 2.88 sec and scanning range of m/z 29–600. Interpretation on mass-spectrum GC-MS was carried out using the database of National Institute Standard and Technology (NIST) having more than 62,000 patterns. Identification of unknown phytocompounds were carried out by matching the recorded spectra with the data bank mass spectra of NIST library provided with the instrument in- built software.

2.1.2. Quantification of Annonacin

The leaf concentrate (1 mg) dissolved in HPLC grade methanol (1 mL), and filtered through a 0.45µm membrane filter, was subjected to quantitative analysis with UV-Vis spectrophotometer (SPECORD PLUS, Analytik Jena AG, Germany) at λ_max of 214 nm using standard annonacin (98% HPLC purity) purchased from Aobious technologies, United States of America. The working standard solutions of annonacin were prepared with methanol for six different concentrations in the range of 0.5–10.0µg/mL (annonacin) for obtaining Standard calibration curve with a regression coefficient of 0.99. All spectroscopic conditions were performed at room temperature in triplicates. Concentration of annonacin present in the leaf sample of A. muricata is expressed as milligrams of annonacin per gram of leaf concentrate.

2.2. Molecular docking analysis

2.2.1. Selection of receptors

Receptors were selected based on their significant function expressed in the pathway of various types of cancers. The receptor selected for our study was PDGFRA, which is a Type III receptor tyrosine kinase (RTK), a potential target for osteosarcoma. The three-dimensional (3D) coordinates of the selected receptor were retrieved from Protein Data Bank (PDBID: 5K5X) database. The target had a resolution of 2.17 Å, theoretical PI of 8.14, aliphatic index of 86.77, instability index of 46.04, GRAAVY value of -0.256 and a half-life period of 30h.

2.2.2. Structure and active site prediction of proteins

The Computed Atlas of Surface Topography of proteins (CASTp) 3.0 was used to predict the active sites that are present in the protein structure (Tian et al. 2018a). Online server was used for identification and measurement of voids in the 3D protein structures (Tian et al. 2018b). The 3D protein structures were submitted to the server and the binding site residues have been predicted.

2.2.3. Selection of ligands

Compounds from EAL concentrates of A. muricata identified through GC-MS analysis were used for molecular docking study. Structures of 28 phytochemicals including annonacin have been retrieved from PubChem (Kim et al. 2019) (Table S1) and was used for virtual screening against the potential protein target of bone cancer, PDGFRA. Drug-likeliness, rate of ADMET were predicted for all the 28 phytochemicals inclusive of annonacin. In silico drug-likeliness and toxicity prediction for the identified 28 ligands were carried out using Swiss ADME predictor and the compounds screened based on Lipinski rule of five are listed in Table S2.

2.2.4. Virtual screening of phytochemicals

Virtual screening of 28 phytochemicals was performed to study the receptor-ligand interactions, which were considered as the basis for structure-based drug discovery. Docking studies were performed using Python Prescription Virtual Screening tool (PyRx 0.8), which is an open-source software containing AutoDock Vina module (Dallakyan and Olson, 2015). The protein receptors and the ligand molecules were converted into pdbqt file using AutoDock module of PyRx tool. The active site residues of the protein target have been defined for grid generation. The grid was generated in the dimensions of X: 35.6973Å, Y: 29.4151Å, Z: 26.3306Å. All the 28 compounds including annonacin were subjected for virtual screening against the target protein, PDGFRA. Two dimensional (2D) interactions between the protein-ligand complexes of top three hit compounds were analyzed based on their binding score using Biovia Discovery Studio Visualizer.

2.2.5 Molecular dynamics simulation

The docked complexes of the top three hit compounds of A. muricata were subjected to molecular dynamics simulation study using the GROMACS 5.4.1 suite. The topologies of the protein-ligand complex for three hit compounds were generated with GROMACS utilities using Gromcs94 43a1 force field. The protein-ligand complex has been defined within a unit cubic cell box and solvated with water. For adding ions and to neutralize the protein-ligand complex system, genion tool within GROMACS was used. The molecular dynamics simulation was carried out through energy minimization followed by two steps of equilibration. The energy minimization process was performed through the MD engine of GROMACS mdrun. Then, the system was equilibrated in two phases and mdrun was performed for 10 ns.

2.3. Cell culture studies

EAL concentrate containing 28 phytochemicals with estimated amount of annonacin were subjected to in vitro studies against MG-63 human osteosarcoma cell lines (gifted by Dr.Santhini Elango, The South India Textile Research Association, and Coimbatore, India). The cultures were maintained in the medium containing Minimum Essential Media (MEM), Fetal Bovine Serum (FBS) (10% v/v), penicillin (100 units/ml), and streptomycin (100 mg/ml). The cells were incubated at 37°C with 5% CO₂ throughout the study. The cells were then detached after the plates reached 70% confluency using trypsin and seeded as required for the experiments. The experiments were not randomized.

2.3.1 Cytotoxicity assay

Leaf concentrate of A. muricata obtained with ethyl acetate were subjected to MTT assay using (Rusanov et al. 2017) with slight modifications, a colorimetric assay performed to determine cell viability on MG-63 cells of osteosarcoma. The cells were harvested and seeded in 96-well plates at a concentration of 1 × 10⁴ cells/well (Himedia, Mumbai, India) and incubated at 5% CO_2, at 37°C for the cells to adhere to the wells. The culture medium was replaced with the fresh medium containing various IC₅₀ concentrations (5 to 25 µg/mL) of EAL concentrates of A. muricata to determine its dose dependent anticancer property. After 24 h of EAL concentrate treatment, 20 µL of MTT dye (1 mg/mL) (Sigma Aldrich, Bangalore) was loaded to each well. After 4 h of incubation, purple colored formazon crystals were formed under dark conditions. The supernatant was then carefully pipetted out, and 150 µL of dimethyl Sulfoxide (DMSO) was added to each well. The plates were then placed in a microplate shaker for 5 min and the absorbance at 570 nm was recorded using Multiskan™ GO Microplate Spectrophotometer (Thermo Scientific, USA). The culture medium containing cells without EAL concentrate was considered as blank.

2.3.2 Nuclear imaging

To monitor the cell division and morphology of MG-63 osteoblast cells, the nuclei were stained with Hoechst 33258 dye (Himedia, Mumbai, India.). After 24 h of treatment with EAL concentrate, morphology of the osteoblast cells were analyzed using fluorescence microscopy (TiS Nikon, Japan). The cells were gently washed with phosphate-buffered saline and treated with Carnoy’s fixation solution (1: 3 acetic acid: methanol) for 10 min. After fixation, Hoechst stain was added and incubated for 30 min, followed by washing with sterile deionized water. The samples were then air dried and observed under an inverted phase contrast epi-fluorescence microscope fitted with a filter having a wavelength range of 460–490 nm (Purschke et al. 2010).

2.4. Statistical Analysis

All the experiments were carried out in triplicates and data were presented as mean ± SD. The mean difference was tested by One-way analysis of variance (ANOVA), in which level of p< 0.05 was considered as significant.

3.1. Chemo-profiling and quantification of annonacin

According to the in-house standardized protocol, ethyl acetate has been identified as the best solvent for extraction of the major marker annonaceous acetogenins, annonacin (fatty acid derivatives). In the present study, chemoprofiling of EAL concentrate of A. muricata through GC-MS analysis revealed 27 leaf phytochemicals. It shows major peaks representing fatty acid derivatives, sesquiterpenes, phytosterols, carboxylic acids and esters based on the relative abundance of peak area (Fig. 1). Amongst them, Linoleic acid, 2-hydroxy 5-methyl chalcone, Humulene, Tetramethylheptadecan-4-ol, Sitosterol, Phytol, Diethyl Phthalate and Eicosatrienoic acid are found to have reported anti-cancer activity. However, the key and predominant annonaceous acetogenin, annonacin responsible for most anticancer activities are not detected through the GC-MS analysis, as the compounds might have degraded due to the set temperature and duration. Interestingly, annonacin has been detected and quantified through spectroscopic technique with the external standard (98% HPLC purity). Concentration of annonacin present is found to be 5.032 ± 0.13 mg/g of leaf concentrate and the 2D structure of annonacin and is represented in Fig. 1. Hence, altogether 28 phytochemicals detected and identified through studied analytical methods has been taken for in silico screening against the target protein of osteosarcoma, PDGFRA along with ADMET analysis. The other miscellaneous compounds of less abundance are acetic acid derivatives, epoxides and hydrocarbon compounds.

3.2. In silico analysis of leaf phytochemicals against PDGFRA

3.2.1. Active Site Prediction

The promising approach of PDGFR inhibition is directed towards specific targeting of transmembrane RTK present in the bone sarcomas. PDGFRA has been identified as the potential target for osteosarcoma, as selective inhibition of PDGFRA may lead to apoptosis of osteosarcoma cells in vitro (Wilson et al. 2018).The binding site has been predicted using CASTp server by submitting the 3D structure of PDGFRA in its PDB file format. The active site residues have been determined from the predicted binding site. The target protein, PDGFRA consists of 18 amino acids in its binding site viz., TRP, LEU, SER, GLN, VAL, MET, LYS, THR, GLY, PRO, ILE, ASN, GLU, TYR, CYS, PHE, ALA and ASP. The active site amino acid residues and its corresponding atoms are given in Table S3.

3.2.2. Analysis of docked compounds with best ligand hits against PDGFRA

A structure-based screening of docked phytocompounds is a commonly explored tool during early drug discovery phase. In order to understand the efficiency of leaf phytochemicals in A. muricata to have inhibitory activity against osteosarcoma, in silico analysis has been carried out before in vitro study. Among the 28 phytomolecules docked with the target protein, PDGFRA of osteosarcoma, top three hit compounds has been taken up for further investigation based on their binding affinity score with the interacting amino acid residues. The compounds, 2’- hydroxy-5’-methyl chalcone (interacts with active site residue of Lys 833), linoleic acid (interacts with active site residue of ASP 836) and annonacin (interacts with active site residue of Ser 972 and Gln 828) shows good binding affinity against PDGFRA with a docking score of -7.4, -7.0 and − 6.9 kcal/mol respectively. The 2D molecular structure of 2’ hydroxy − 5’- methyl chalcone (flavonoids), linoleic acid (fatty acid derivative) and annonacin (annonaceous acetogenin) are given in (Fig. S1). The 3D view of the complex structure of PDGFRA with the top three hit compounds of A. muricata has been visualized using Pymol and are given in (Table. 1). The docking scores are given in (Table. 2) and the docked images of the interacting residues with the three hit compounds are represented as 2D interaction diagram in (Table. 1). Interestingly, the compound annonacin exhibited a good binding energy of -6.9 Kcal/mol with the target protein, PDGFRA domain by forming 2 hydrogen bonds with the active site residues. Linoleic acid (binding energy of -7.0 Kcal/mol) and 2’- hydroxy-5’-methyl chalcone (binding energy of -7.4kcal/mol) showed lowest binding affinity by forming only single hydrogen bond with the active residue site. The interaction between leaf phytochemicals of A. muricata with the target protein, PDFGRA at specific amino acid residues signifies their role in disruption of oncogenic protein reliability.

Table 2

Details of binding energy, number of hydrogen bonds and their interacting amino acids for the three hit compounds
S. No	Compound	Binding energy (kcal/mol)	Number of hydrogen bonds formed	Interacting amino acids
1.	2’ hydroxy − 5’ methyl chalcone	-7.4	1	(Lys A :833)
2. 3.	Linoleic acid Annonacin	-7.0 -6.9	1 2	(ASP A:836) (Ser A:972, Gln A:828)

3.2.3. In silico ADMET prediction and drug likeness score

Potential drug candidates should have certain desirable pharmacokinetic properties to be an effective therapeutic agent. Analysis of in silico ADMET (absorption, distribution, metabolism, excretion, and transport) properties in the early phases of drug discovery reduces failures associated with drug toxicity. Analysis of ADMET related pharmacokinetic and physico-chemical parameters for the 28 leaf phytochemicals of A. muricata has been computed. As seen from (Table. S2), suitability of the identified compounds has been evaluated based on human oral absorption parameters and Lipinski’s rule of five, an indication of drug-likeness for the 28 compounds. According to the results, 27 phytochemicals complied with the Lipinski’s rule of five except annonacin. Among these 28 compounds, only three compounds like 2’- hydroxy-5’-methyl chalcone (Flavonoid), linoleic acid (Polyunsaturated fatty acids) and annonacin (marker acetogenins) are found to have good binding affinity with the target protein, PDGFRA. ADMET analysis for the three hits compounds revealed that the compound, 2’- hydroxy-5’-methyl chalcone is in full accordance with Lipinski’s rule of five; Linoleic acid with one violation (MLogOP > 5) and is found to be within the acceptable range, except for its lipophilic nature with moderate aqueous solubility. The number of rotatable bonds for the compound, 2’- hydroxy-5’-methyl chalcone < 10, signifies an acceptable molecular flexibility with good permeability as well as good oral bioavailability. However, annonacin with molecular weight (596.9 g/mol) more than 500 g/mol has exhibited low gastrointestinal absorption and poor aqueous solubility, indicating its poor bioavailability. These three compounds might serve as potential hits in developing new anti-cancer agent against osteosarcoma.

3.2.4. Molecular Dynamics Simulation

The best docked complexes for the top 3 hit compounds are chosen for conducting the molecular dynamics simulation study using the GROMACS 5.4.1 suite. The result of the current simulation study includes Root Mean Square Deviation (RMSD) value, Root Mean Square Fluctuation (RMSF) value and radius of gyration (Rg) for analysis. Molecular dynamics simulation study has been performed to evaluate the stability of the docked complexes with three hit compounds. The simulation for 5k5x with three hit compounds, 2’ hydroxy-5’- methyl chalcone, linoleic acid and annonacin were carried out. RMSD is a key parameter to investigate the equilibrium state of molecular dynamics trajectories. From the present results (Fig. 2), RMSD of 5k5x-2’hydroxy-5’- methyl chalcone is found to vary for the first 2.5 ns and then attained an equilibrium state. RMSD of 5k5x-linoleic acid is found to vary for the first 4 ns during the simulation and the attained an equilibrium state. RMSD of 5k5x-annonacin is found to vary only between 6.5 to 7 ns to attain an equilibrium state. Considering the RMSD fluctuations, interaction of annonacin with 5K5X is considered to be stable among the three hit compounds. RMSF of backbone atoms are calculated to check the flexibility of backbone structure in the presence of the ligand and a higher value indicates more flexibility as a result of poor interaction. Mostly similar RMSF has been observed (Fig. 2) with both, 2’ hydroxy-5’- methyl chalcone and linoleic acid complexes indicating lesser flexibility with better interaction of proteins. While comparing the other two complexes of 2’ hydroxy-5’- methyl chalcone and linoleic acid, annonacin showed a lower RMSF value with lesser flexibility and better interaction.

Radius of gyration evaluates how the secondary structure of protein target gets compactly packed into its 3D form. In general, Rg value indicates the compactness of proteins, which in turn reflects its stability. The more it fluctuates, the less stable it is at that point of time. Hence, Rg value plays a significant role during comparative studies and our results indicated that annonacin while interacting with the protein 5k5x did not affect the stability of the target protein. All these results proved stability of the docked complex of 5k5x with annonacin. In silico analysis plays a significant role in identifying promising drug candidates and in the present study, it designates three major compounds that might exhibit inhibitory or anti-proliferative activity, with annonacin being the most desirable against osteosarcoma.

3.3. Cell line studies

3.3.1. Cell viability

The results obtained with little support through in silico analysis, in vitro cytotoxicity assay has been carried out. In the present experiment, the cytotoxicity results of the EAL concentrate of A. muricata with annonacin concentration of 5.032 ± 0.13 mg/g of EAL extract on osteosarcoma cells (MG-63) are summarized in Fig. 3. It shows that EAL concentrate of A. muricata exhibited cytotoxic activity towards MG-63 cells at all studied IC₅₀ concentrations (5 to 25 µg/mL). The cell viability showed a decreasing trend, when the concentration has been increased further indicating its significant anticancer activity in MG-63 cells. The trend of decreasing cell viability supports the principle that the leaf extracts rich in key marker acetogenins, annonacin might be responsible for exhibiting inhibitory anti-proliferative property in vitro in MG-63 cells. In addition, presence of the flavonoid, 2’- hydroxy-5’-methyl chalcone could also have provided additional effect through its synergistic activity with annonacin. As, the synergistic interaction between flavonoids and annonaceous acetogenins obtained from leaves of A. muricata has been proven to exert maximum therapeutic efficiency against breast cancer cell lines with enhanced absorption and bioavailability (Yang et al. 2015), which supports our current finding. Thus, a quantitative increase in EAL concentrate might have increased the concentration of the major anti-cancer bioactives, specifically annonacin, thought to have contributed to apoptosis on osteosarcoma cells in vitro. Ko et al. 2011 reported that annonacin in MCF-7 breast cancer cells induced growth arrest and apoptosis by inhibiting ERα, cyclin D1 and Bcl-2 protein expressions.

3.4. Nuclear morphology of osteoblast cells

Nuclear imaging can be used as a non-destructive molecular visualization tool to know the effect or cytotoxicity caused by the phytomolecules at cellular level. Nuclear morphological abnormalities are considered an essential diagnostic feature to distinguish normal cells from malignant cells. Alterations in nuclear shape of cancer cells can affect transcriptional activity of the cell and thereby gene expressions. In the present study, significant morphological alterations have been observed in the cell nuclei of A. muricata EAL concentrate treated osteoblast cells compared to untreated control cells. Hoechst dye stains the nuclei of the cells regardless of their viability (Baxter et al. 2002) and enables them to distinguish the changes that has happened in the morphology of the nuclei induced by the EAL concentrate of A. muricata. Morphology of normal osteoblast cells are spindle-elongated with long lamellipodia (Bielack et al. 2015). Also, the untreated/control osteoblast cells retained its original shape and parallel orientation. Interestingly, the EAL concentrate of A. muricata treated cells exhibited nuclei condensation as shown in Fig. 4. An increase in number of cleaved nuclei has also been observed in the cells treated with EAL concentrate above 10 µg/mL concentration. Cells treated with 25 µg/mL concentration of EAL concentrate exhibited typical characteristics of apoptosis such as cell shrinkage and fragmentation, segregated bodies and cell decrement. A decrease in cell viability along with nuclear imaging results confirmed the inhibitory or anti-proliferative effect of EAL concentrate of A. muricata on MG-63 osteosarcoma cells

In the present study, 28 leaf phytochemicals identified from A. muricata with known concentration of annonacin content obtained through ethyl acetate has been assessed for its efficacy against osteosarcoma. The in-silico molecular docking analysis of 28 leaf phytochemicals from A. muricata resulted in identification of three hit bioactives namely, 2’- hydroxy-5’-methyl chalcone, linoleic acid and annonacin to be effective against the protein target, PDGFRA of osteosarcoma with a good binding affinity. Amongst the three hit compounds, 2’- hydroxy-5’-methyl chalcone satisfied the Lipinski’s rule of five and ADMET prediction, while linoleic acid and annonacin showed some violations against the rule of five due to their poor aqueous solubility. Though annonacin showed low drug likeness among the three hit compounds due to its lipophilicity, interestingly it showed good binding energy with PDGFRA by forming two hydrogen bonds at the active site of the target protein. On comparing the molecular dynamic behavior of the three hit compounds with target protein (5k5x), annonacin is found to be more stable. Likewise, in-vitro cytotoxicity and nuclear imaging with EAL concentrate of A. muricata treated cells proved its inhibitory action on MG-63 osteosarcoma cells at all studied concentrations. Our in vitro results supports the chemistry of in-silico prediction about the effectiveness of leaf phytochemicals of A. muricata against osteosarcoma. Furthermore, our findings will help researchers to hasten the process of purifying and developing the most efficient therapeutic agent against osteosarcoma from the identified hits or the EAL concentrate without side effects.

Funding sources

This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.

Data Availability Statement

The authors declare that data supporting the findings of this study are available within the article inclusive of supplementary information

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Consent for Publication

All authors have their consent to publish the present work.

Conflicts of Interest

The authors have no conflicts of interest to declare

Author’s contribution

HS conceptualized and planed the study. HS sourced, extracted and characterized leaf extract of soursop used in this study at CANT, TNAU. CR, KA and JM carried out molecular interaction studies at CPMBB, TNAU. RN and RS carried out in vitro anti-proliferative activity in MG-63 cancer cells at PSG IAS, Coimbatore. HS drafted the final version of the manuscript and all the authors approved the same.

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Table 1 is available in the Supplementary Files section.

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Anti-proliferative effect of leaf phytochemicals of soursop (Annona muricata L.) against human osteosarcoma in vitro

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Journal Publication

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Preparation of leaf extract

2.1.1. GC-MS profiling

2.1.2. Quantification of Annonacin

2.2. Molecular docking analysis

2.2.1. Selection of receptors

2.2.2. Structure and active site prediction of proteins

2.2.3. Selection of ligands

2.2.4. Virtual screening of phytochemicals

2.2.5 Molecular dynamics simulation

2.3. Cell culture studies

2.3.1 Cytotoxicity assay

2.3.2 Nuclear imaging

2.4. Statistical Analysis

3. RESULTS AND DISCUSSION

3.1. Chemo-profiling and quantification of annonacin

3.2. In silico analysis of leaf phytochemicals against PDGFRA

3.2.1. Active Site Prediction

3.2.2. Analysis of docked compounds with best ligand hits against PDGFRA

3.2.3. In silico ADMET prediction and drug likeness score

3.2.4. Molecular Dynamics Simulation

3.3. Cell line studies

3.3.1. Cell viability

3.4. Nuclear morphology of osteoblast cells

4. Conclusion

Declarations

References

Table 1

Supplementary Files

Status:

Journal Publication

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