Anticancer property of β-sitosterol studied in MG 63 Osteosarcoma cell line

 

U.S. Mahadeva Rao1, S. Ragunandhakumar2, G. Shoba3, D. Saravanan4, P. Malliga5 , G. Sriram Prasath6*, Kasim Sakran Abass7

1Professor, School of Basic Medical Sciences, Faculty of Medicine, Kampus Perubatan, Universiti Sultan Zainal Abdin 20400 Kuala Terengganu Malaysia.

2Associate Professor, Cancer and Stem Cell Research Lab, Department of Pharmacology, Saveetha Dental College, Chennai-600 077.

3 Post Graduate Department of Biotechnology, Dwaraka Doss Goverdhan Doss Vaishnav College, Arumbakkam, Chennai - 106, Tamilnadu. India.

4Associate Professor and Head, Department of Biochemistry, Dwaraka Doss Goverdhan Doss Vaishnav College, Arumbakkam, Chennai-106, Tamilnadu. India.

5Post Graduate Student, PG and Research Department of Biochemistry, Dwaraka Doss Goverdhan Doss Vaishnav College, Arumbakkam, Chennai-106 Tamilnadu India.

6Assistant Professor, Department of Biochemistry, Dwaraka Doss Goverdhan Doss Vaishnav College, Arumbakkam, Chennai-106, Tamilnadu. India.

7Department of Physiology, Biochemistry and Pharmacology, College of Veterinary Medicine, University of Kirkuk,  Kirkuk 36001, Iraq

      *Corresponding Author E-mail: sriram@dgvaishnavcollege.edu.in

 

ABSTRACT:

Osteosarcoma is a type of bone cancer that usually develops in the osteoblast cells, which are responsible for making new bone tissue. It is a high-grade primary skeletal cancer caused by spindle cells of mesenchymal origin that deposit immature osteoid matrix. Phytochemicals and their derivatives are promising options to improve treatment efficiency in cancer patients and decrease adverse reactions. β-sitosterol is a prominent dietary phytosterol found in beans, nuts, and seeds known for its significant pharmacological properties. In the present study and attempt has been made to evaluate the   anticancer properties of β sitosterol in MG 63 Osteosarcoma cell line. The MTT assay revealed a significant decrease in cell viability of MG-63 human osteosarcoma cells upon treatment with β-sitosterol in a dose-dependent manner. This reduction in cell viability indicates the potential of β-sitosterol to exert an anticancer effect against osteosarcoma cells. There was dose dependent decrease in percentage of cell viability. The cell viability was around 20% in 320 µg/ml indicating the fact that β-sitosterol  can act as cytotoxic drug against cancer cells. Treatment with the β sitosterol indicated a marked inhibition in cell viability indicating its antiproliferative nature.  DAPI staining revealed changes in nuclear morphology, such as condensation or fragmentation, which are characteristic of apoptotic cells. The result obtained in Docking analysis of β sitosterol and Bcl-2  indicate that the  Binding Energy for beta sitosterol and Bcl-2 shows -7.7 kcal/mol and forms four hydrophobic interactions with ARG107, LEU201  and PHE104.   Therefore, β sitosterol acts as BCL2 inhibitors which holds great promise for the use of the compound in treating cancer. 

 

KEYWORDS: Osteosarcoma, β sitosterol, MTT assay, DAP staining, Anti cancer potential.

 

 

 

 

Received on 24.12.2024      Revised on 30.01.2025

Accepted on 14.03.2025      Published on 27.03.2025

Available online from March 27, 2025

Research J. Pharmacy and Technology. 2025;18(3):1374-1379.

DOI: 10.52711/0974-360X.2025.00198

© RJPT All right reserved

 

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

 

 

INTRODUCTION: 

Osteosarcoma is a type of bone cancer that usually develops in the osteoblast cells, which are responsible for making new bone tissue. It is a high-grade primary skeletal cancer caused by spindle cells of mesenchymal origin that deposit immature osteoid matrix. With a yearly incidence rate of 3.1 cases per million in the US, OS accounts for less than 1% of all newly diagnosed malignancies in adults and 3-5% of those in children1.  It's most commonly found in children and young adults, often occurring in the long bones, such as the arms and legs. Treatment typically involves a combination of surgery, chemotherapy, and sometimes radiation therapy2. Early detection and treatment are crucial for a better prognosis time of their initial assessment, despite the presence of micro metastases3. Phytochemicals and derivatives present in plants are promising options to improve treatment efficiency in cancer patients and decrease adverse reactions. A number of these phytochemicals are naturally occurring biologically active compounds with significant antitumor potential4. The development of effective and side-effects free phytochemical based anticancer therapy begins with the testing of natural extracts (from dry/wet plant material) for potential anticancer biological activity followed by purification of active phytochemicals based on bioassay-guided fractionation and testing for in vitro and in vivo effects5

 

β-sitosterol

The most common plant sterol in the human diet, β-sitosterol is also the main ingredient in many traditional medicines, such as saw palmetto and devil's claw. Despite having anti-inflammatory and anti-cancer properties as well as being effective against enlarged prostates in human clinical trials6. Phytosterols are a type of steroid generated by plants. They are structurally and physiologically related to cholesterol and constitute a significant portion of the human diet. β-sitosterol (24-ethylcholesterol) is a prominent dietary phytosterol found in beans, nuts, and seeds7. Additionally, it is a crucial component of saw palmetto, stinging nettle, devil's claw, and a number of other herbal treatments. It has been claimed that by blocking the intestinal absorption of cholesterol, β-sitosterol ingestion lowers blood cholesterol levels. Additionally, in a number of animal models, it has demonstrated analgesic and anti-inflammatory effects. Additionally, β-sitosterol has shown a significant effect in lowering the symptoms of benign prostatic hyperplasia in both animal models and human clinical trials8. The lower incidence of prostate, colon, and breast cancer among vegetarians and men and women in Asian countries who consume significantly more β-sitosterol than most Westerners may also be largely attributed to β-sitosterol intake. β-sitosterol demonstrates growth inhibitory and lethal effects on many cancer cell lines, so providing evidence in favour of this concept.

 

Cell line for osteosarcoma

Osteosarcoma cell lines, such as MG-63, HOS-MNNG, HOS, OSA, HOS-143b, and G-292, are available for use in experiments9. MG-63 outperformed the other osteosarcoma cell lines in terms of colony-formation ability, fast proliferation, and phenotypic stability. In the present study and attempt has been made to evaluate the anticancer properties of β sitosterol in MG 63 Osteosarcoma cell line. 

 

MATERIALS AND METHODS

Cell line studies

Prior to the in vitro cell culture study, The MG-63 cancer cell line was procured from NCCS, Pune India and ATCC - USA. The high glucose media containing Dulbecco’s Modified Eagle Medium (DMEM), 10% Fetal Bovine Serum (FBS) V/V and 1% antibiotic/antimycotic penicillin and streptomycin (W/V) was used to sub-culture the cancer cells. The cancer cells were grown in CO2 incubator with 5% carbon dioxide and 95% humidity as recommended by NCCS for this particular cell line.

 

Cytotoxicity assay:

The anti-proliferative activity of control and experimental group on MG-63 cells was determined over 24hr by MTT assay were studied by MTT assay. Briefly, after incubation with control and experimental group on MG-63 cells were seeded on 96 well culture plate for 24 hr respectively. To determine the percentage of cell viability, the post incubated cells were replaced with 10 𝜇l of stock MTT dye (10 mg/ml) was added in each well and plate was incubated again at 37 °C for 4 h. The medium was replaced with 100 μl DMSO in each well to dissolve the formazan crystals and absorbance was recorded at 570 nm. with Synergy hybrid Multi-Mode Reader (BioTek, Winooski, VT, US). The percent cell viability was calculated using the following equation:

 

                                 OD(Test sample) – OD (blank)

Cell Viability (%) = ------------------------------------ x 100

                                       OD(PC) – OD (blank)

 

 

Experimental Group:

Group 1

Control

Group 2

β-sitosterol

 

Evaluation of DNA Fragmentation by DAPI Staining:

Cell apoptosis of MG-63 cells induced by control and experimental group were determined by DAPI staining. Cells were seeded on 24-well with a density of 5×104 cells/well and treated with experimental group at low, medium, high concentrations for 24 h. Cells were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature, followed by permeabilization with 0.25% Triton X-100 in PBS for 30 min and stained with DAPI for 4 min in dark. The unstained and stained cells were observed under Phase contrast inverted fluorescence microscopy (Invitrogen, evos).

 

Molecular Docking

The compound structures were drawn and optimised using Chemsketch and saved in MDL-mol format and converted to pdb format using open babel molecular converter program.  AutoDock Vina10 program was used to perform the docking. The receptor BSA with PDB ID:6QS9 were obtained through an online PDB bank and water molecules and co-crystallized ligands were removed. The macromolecule Were open using PyRx software and converted to PDBQT format the ligand (Beta sitosterol) in the PDBQT format were obtained using the OpenBabelIGUI program11. The energy of all of the ligands was minimized through PyRx-Python at 200 steps using the conjugate gradient optimization algorithm and MMFF94 force field12   Through the virtual screening each complex generated 10 conformation least binding affinity were selected and results were analyzed using Biovia Discovery Studio software.

 

Discovery Studio Visualizer

Molecular visualization is a key aspect of the analysis and communication of modelling studies. 

High Performance Publication Quality Graphics

▪     Handle very large macromolecule systems (E.g., Ribosomes)

▪     Support a range of stereo graphics options (E.g., split screen, hardware stereo)

▪     Hardware graphics acceleration support for a range of AMD(ATI) and NVidia cards

▪     Depth cueing, blur and shading capabilities

http://accelrys.com/products/discovery-studio/visualization.html

 

RESULTS AND DISCUSSION:

 

 

Figure 1:The cytotoxic effects of β-sitosterol on MG-63 cells 1

Data are shown as means ± SD (n = 3). *Compared with the control blank group, p < 0.001

The cytotoxic effects of β-sitosterol on MG-63 cells is shown in figure 1. Cells were treated with β-sitosterol (0- 320 µg/ml concentrations) for 24 h, and cell viability was evaluated by MTT assay.

 

Cell viability Assay (MTT)

MTT assay is used to measure cell viability and proliferation. It assesses the metabolic activity of cells by measuring the reduction of a yellow tetrazolium salt (MTT) to formazan crystals by mitochondrial enzymes. A decrease in MTT conversion indicates a reduction in cell viability or proliferation. The cytotoxic effects of β-sitosterol on MG 63 cells is depicted in figure 1. Cells were treated with β-sitosterol (0- 320 µg/ml concentration) for 24 h, and cell viability was evaluated by MTT assay. There was dose dependent decrease in percentage of cell viability. The cell viability was around 20% in 320 µg/ml indicating the fact that β-sitosterol can act as cytotoxic drug against cancer cells.

 

The MTT assay revealed a significant decrease in cell viability of MG-63 human osteosarcoma cells upon treatment with β-sitosterol in a dose-dependent manner. This reduction in cell viability indicates the potential of β-sitosterol to exert an anticancer effect against osteosarcoma cells.

 

Figure 2: Phase contrast microscopic studies of control and treated cells

Cells were treated with β-sitosterol (60 μg/ml) on MG-63 cells for 24 h along with control group.

 

Phase contrast microscopic studies of control and treated cells is depicted in figure 2. The control group cells showed fully confluent growth, as shown. Treatment with the β sitosterol indicated a marked inhibition in cell viability indicating its antiproliferative nature. Apoptosis is characterised by a series of unique morphological and cellular changes in the apoptotic cell, including nuclear condensation and fragmentation, chromatin material suspension, and cell membrane alterations. Treatment with β sitosterol indicated a marked inhibition in cell viability indicating its antiproliferative nature.

 

 

Control

β-sitosterol

 

Figure 3: DAPI staining of control and treated cells

 

Cells were treated with β-sitosterol (60 μg/ml) on MG-63 cells for 24 h along with control group. The cells were stained using DAPI, and the images were obtained using an inverted fluorescent microscope. Scale Bar (100µm).

 

DAPI Staining:

Figure 3 indicates  DAPI staining of control and β sitosterol treated cells. DAPI (4',6-diamidino-2-phenylindole) is a fluorescent dye that binds strongly to DNA. It is commonly used in cell biology to stain cell nuclei. DAPI staining can reveal changes in nuclear morphology, such as condensation or fragmentation, which are characteristic of apoptotic cells.

 

Induction of Apoptosis:

Observations from DAPI staining showed characteristic features of apoptosis in MG-63 cells treated with β-sitosterol. These features include nuclear condensation and fragmentation, which are indicative of programmed cell death. The ability of β-sitosterol to induce apoptosis suggests its potential as a therapeutic agent for osteosarcoma by promoting the elimination of cancerous cells.

 

Dose-Response Relationship:

A dose-response relationship was evident in both the MTT assay and DAPI staining results. Increasing concentrations of β-sitosterol led to a proportional decrease in cell viability and an increase in the apoptotic rate of MG-63 cells. This dose-dependent effect strengthens the evidence supporting the anticancer potential of β-sitosterol against osteosarcoma.

 

Protein structure retrieval- PDB database

The structure of Bcl-2 from Homo sapiens was retrieved from PDB database and its PDB id was 2XA0 -A chain.

 

 

Docking Results

Docking analysis of beta sitosterol and Bcl-2 was carried out using PyRx software – Autodock Vina Module. Binding Energy for beta sitosterol and Bcl-2 shows -7.7 kcal/mol and forms four hydrophobic interactions with ARG107, LEU201 and PHE104. The protein ligand complex was visualized using Biovia Discovery studio visualizer.

 

Beta-sitosterol

-7.7 kcal/mol

 

Beta sitosterol

 

Figure 4: Docking Results between beta sitosterol and Bcl-2 depicting Binding energy and complex

 

 

Figure 5: Beta sitosterol (scaled ball and stick model) and Bcl-2 (Solid ribbon model) complex visualized in Biovia Discovery Studio visualizer

 

 

Figure 6: Beta sitosterol (scaled ball and stick model) and Bcl-2 (stick model) complex visualized in Biovia Discovery Studio visualizer

 

Figure 7: 2D Diagram of Beta sitosterol (line model) and Bcl-2 (active residues) complex visualized in Biovia Discovery Studio visualizer

 

Table 1 Binding interactions of key aminoacid residues of  Bcl2 with β sitosterol

Donor-Acceptor

Distance (A)

Category of bond

Bond type

A:ARG107 - :UNL1

4.38784

Hydrophobic

Alkyl

A:ARG107 - :UNL1

4.19967

Hydrophobic

Alkyl

A:LEU201 - :UNL1

5.17841

Hydrophobic

Alkyl

A:PHE104 - :UNL1

4.39495

Hydrophobic

Pi-Alkyl

 

Osteosarcoma (OS) is the most common primary malignancy of bone. It is responsible for 80-85% of the primary bone tumours affecting dogs and it is characterized by aggressive and invasive behaviour, with a high metastatic potential13 . Several studies on cancer and related tumorigenesis, show an involvement of the mechanisms of programmed cell death and cell survival14. Many signals seem to be involved in the related mechanism of autophagy and in particular, our interest is focused on the expression of a family of Bcl-2 that seems to be involved either in the control of biomolecular mechanisms like autophagy and apoptosis. The expression of Bcl-2 is increased in   Osteosarcoma compared to normal bone tissue15. Bcl-2 activity may play an important role in the formation of OS and as a diagnostic for neoplastic activity. 

 

Computer-aided drug design (CADD) has been widely adopted by biologists and chemists as an essential component of a comprehensive drug discovery approach16.   The B cell lymphoma 2 (BCL2) protein family, which is involved in the regulation of apoptotic cell death, has been linked to the pathogenesis and progression of various cancers17-18 The result obtained in Docking analysis of beta sitosterol and Bcl-2 indicate that the Binding Energy for beta sitosterol and Bcl-2 shows -7.7 kcal/mol and forms four hydrophobic interactions with ARG107, LEU201 and PHE104.   The BCL2 family of proteins plays a crucial role in intrinsic apoptosis.   Elevated expression of BCL2 proteins can thwart resistance to apoptosis and chemotherapeutic agents. Therefore, β sitosterol acts as BCL2 inhibitors which holds great promise for the use of the compound in treating cancer 

 

CONCLUSION:

The exact mechanisms underlying the anticancer effects of β-sitosterol on MG-63 cells require further elucidation. It is plausible that β-sitosterol may interfere with signalling pathways involved in cell proliferation and apoptosis regulation, such as the PI3K/Akt pathway or the Bcl-2 family of proteins. Future studies focusing on the molecular mechanisms of β-sitosterol action will provide valuable insights into its therapeutic potential for osteosarcoma. Comparative studies with standard chemotherapeutic agents commonly used for osteosarcoma treatment, such as doxorubicin or cisplatin, would help assess the efficacy of β-sitosterol relative to conventional treatments. Understanding how β-sitosterol compares to established therapies is essential for evaluating its potential as a novel therapeutic option for osteosarcoma patients. The encouraging results from this in vitro study warrant further exploration of β-sitosterol as a potential therapeutic agent for osteosarcoma in preclinical and clinical settings. If validated in animal models and clinical trials, β-sitosterol could offer a promising alternative or adjunctive treatment option for osteosarcoma patients, potentially improving treatment outcomes and patient survival rates.

 

ACKNOWLEDGEMENTS:

The authors wish to thank the management of Dwaraka Doss Goverdhan Doss Vaishnav College and Saveetha Dental College for having provided the support for carrying out the cell line study. The authors also wish to thank School of Basic Medical Sciences, Kampus Perubatan, Universiti Sultan Zainal Abdin 20400 Kuala Terengganu Malaysia for their support.

 

CONFLICT OF INTEREST:

There is no conflict of interest.

 

REFERENCES:

1.        Campanacci M. Bone and soft tissue tumors: clinical features, imaging, pathology and treatment. Berlin: Springer; 2013.

2.        Lamplot D, Joseph, Sahitya Denduluri, Jiaqiang Qin, Ruidong Li, Xing Liu, Hongyu Zhang, Xiang Chen et al. The current and future therapies for human osteosarcoma. Current cancer therapy reviews. 2013; 9(1): 55-77.

3.        Portlock, C. S. Involved site radiation therapy for the treatment of early-stage Hodgkin lymphoma in adolescents and young adults. Clinical Oncology in Adolescents and Young Adults. 2015; 97. https://doi.org/10.2147/coaya.s70370

4.        Ambulkar, P., Jadhav, P., Tambea, B., and Hande, P. Anticancer Activity of a Poly-Herbal formulation ‘Tulsi and Licorice Herbal Green Tea’ on A549 Human Lung Carcinoma Cell Line. International Journal of Research Publication and Reviews. 2024; 5(1): 1802–1806. https://doi.org/10.55248/gengpi.5.0124.0229

5.        Liang G.P, Li J.X, Zhang J, et al. Retracted: In Vitro Antitumor Activities of Biologically Active Extracts and Isolated Phytochemicals From Rabdosia latifolia C. Y. Wu Et H. W. Li in Addenda. Natural Product Communications. 2020; 15(5). doi:10.1177/1934578X20926277

6.        Bailly, C. Atractylenolides, essential components of Atractylodes-based traditional herbal medicines: Antioxidant, anti-inflammatory and anticancer properties. European Journal of Pharmacology. 2021; 891: 173735. https://doi.org/10.1016/j.ejphar.2020.173735

7.        England, C., Mitchell, A., and Atkinson, C. Diet After Ileostomy Study: an observational study describing dietary intake and stoma‐related symptoms in people with an ileostomy. Journal of Human Nutrition and Dietetics. 2023; 36(4): 1600–1612. https://doi.org/10.1111/jhn.13168

8.        Laine, R. IPX-750, A Dopamine Gluconamine That Binds D1/D5 Receptors and Has Anti-Parkinsonian Effects in Three Animal Models, is Transported across the Blood Brain Barrier. Journal of Biotechnology and Biomaterials. 2012; 2(5). https://doi.org/10.4172/2155-952x.1000142

9.        Lauvrak, S. U., Munthe, E., Kresse, S. H., Stratford, E. W Namlřs, H. M., Meza-Zepeda, L. A., and Myklebost, O. Functional characterisation of osteosarcoma cell lines and identification of mRNAs and miRNAs associated with aggressive cancer phenotypes. British Journal of Cancer. 2013; 109(8): 2228–2236

10.      Eberhardt J., Santos-Martins D., Tillack A.F., Forli S. AutoDock Vina 1.2.0: New Docking Methods, Expanded Force Field, and Python Bindings. J. Chem. Inf. Model. 2021; 61: 3891–3898. doi: 10.1021/acs.jcim.1c00203.

11.      O’Boyle N.M., Banck M., James C.A., Morley C., Vandermeersch T., Hutchison G.R. Open Babel: An open chemical toolbox. J. Cheminform. 2011; 3: 33. doi: 10.1186/1758-2946-3-33

12.      Dallakyan S. PyRx-Python Prescription v. 0.8. Volume 2010 Scripps Research Institute; La Jolla, CA, USA: 2008.

13.      Oh, J. Y., Lee, Y. J., and Kim, E. H. Tumor-Treating Fields Inhibit the Metastatic Potential of Osteosarcoma Cells. Technology in Cancer Research and Treatment. 2020; 19. 153303382094748. https://doi.org/10.1177/1533033820947481

14.      Damron TA, Ward WG, Stewart A. Osteosarcoma, chondrosarcoma, and Ewing’s sarcoma: National Cancer Data Base Report. Clin Orthop Relat Res. 2007; 459: 40–7.

15.      Piro F, Leonardi L. Expression of Bcl-2 in canine osteosarcoma. Open Vet J. 2015; 5(1): 27-9. Epub 2015 Mar 19. PMID: 26623359; PMCID: PMC4629559.

16.      Sabe VT, Ntombela T, Jhamba LA, Maguire GEM, Govender T, et al. Current trends in computer aided drug design and a highlight of drugs discovered via computational techniques: A review. European Journal of Medicinal Chemistry. 2021; 224: 113705.

17.      Belka C, Budach W. Anti-apoptotic Bcl-2 proteins: structure, function and relevance for radiation biology. International Journal of Radiation Biology.  2002; 78(8): 643-658.

18.      Alam M, Ali S, Mohammad T, Hasan GM, Yadav DK, et al. B Cell Lymphoma 2: A Potential Therapeutic Target for Cancer Therapy. International Journal of Molecular Sciences. 2021; 22(19): 10442.

 

 

 

Received on 27.08.2024      Revised on 11.12.2024

Accepted on 28.02.2025      Published on 27.03.2025

Available online from March 27, 2025

Research J. Pharmacy and Technology. 2025;18(3):1368-1373.

DOI: 10.52711/0974-360X.2025.00197

© RJPT All right reserved

 

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.