Skip to main content

Advertisement

SpringerLink
Log in

Nimbin (N1) and analog N3 from the neem seeds suppress the migration of osteosarcoma MG-63 cells and arrest the cells in a quiescent state mediated via activation of the caspase-modulated apoptotic pathway

  • Original Article
  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Background

Natural products are considered effective sources for new therapeutic research and development. The numerous therapeutic properties of natural substances in traditional medicine compel us to investigate the anti-cancer properties of Nimbin (N1) and its semi-natural analog Nimbic acid (N3) from Azadirachta indica against MG-63 Osteosarcoma cells.

Materials and methods

The therapeutic efficacy of N1 and N3 were screened for their toxicity and cytotoxic activity using L6 myotubes, zebrafish larvae and MG-63 osteosarcoma cells. The mitochondrial membrane potential was evaluated using the Rhodamine 123 stain. Further, the nuclear and cellular damage was distinguished using Hoechst and Acridine orange/EtBr stain. The mechanism of cell cycle progression, cellular proliferation and caspase cascade activation was screened using scratch assay, flow cytometry, and mRNA expression analysis.

Results

The Nimbin and analogue N3 were found to be non-toxic to normal L6 cells (Rat skeletal muscles), exhibited cytotoxicity in MG-63 cells, and were exposed to be an active inhibitor of cell proliferation and migration. Analogs N1 and N3 induced negative mitochondrial membrane potential when stained with Rhodamine 123, leading to nuclear damage and apoptosis stimulation using AO/EtBr and Hoechst. Further, N1 and N3 induced cell cycle arrest in G0/G1 phase in flow cytometry using PI staining and induced apoptosis by activating the caspase cascade and upregulated Caspase 3 and caspase 9.

Conclusion

The study demonstrated cytotoxic activity against MG-63 osteosarcoma cells while being non-toxic to normal L6 cells. These compounds inhibited cell proliferation and migration, induced mitochondrial dysfunction, nuclear damage, and apoptosis stimulation. Furthermore, N1 and N3 caused cell cycle arrest and activated the caspase cascade, ultimately leading to apoptosis. These findings indicate that N1 and N3 hold promise as potential candidates used alone or combined with existing drugs for further investigation and development as anti-cancer agents.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Data Availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

References

  1. Yi C, Li G, Wang W et al (2021) Disruption of yy1-ezh2 interaction using synthetic peptides inhibits breast cancer development. Cancers (Basel) 13:1–23. https://doi.org/10.3390/cancers13102402

    Article  CAS  Google Scholar 

  2. Cooper G (1951) Bone cancer. Va Med Mon (1918) 78:654. https://doi.org/10.6004/jnccn.2013.0088

    Article  PubMed  Google Scholar 

  3. Mantyh P (2013) Bone cancer pain: causes, consequences, and therapeutic opportunities. Pain 154:S54–S62. https://doi.org/10.1016/j.pain.2013.07.044

    Article  PubMed  Google Scholar 

  4. Song X, Zhang Y, feng Wang X et al (2017) Casticin induces apoptosis and G0/G1 cell cycle arrest in gallbladder cancer cells. Cancer Cell Int 17:1–10. https://doi.org/10.1186/s12935-016-0377-3

    Article  CAS  Google Scholar 

  5. Siddiqui MA, Alhadlaq HA, Ahmad J et al (2013) Copper Oxide Nanoparticles Induced Mitochondria mediated apoptosis in human Hepatocarcinoma cells. PLoS ONE 8:e69534. https://doi.org/10.1371/journal.pone.0069534

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Chen Y, McAndrews KM, Kalluri R (2021) Clinical and therapeutic relevance of cancer-associated fibroblasts. Nat Rev Clin Oncol 18:792–804. https://doi.org/10.1038/s41571-021-00546-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Alzohairy MA (2016) Therapeutics role of azadirachta indica (neem) and their active constituents in diseases prevention and treatment. Evidence-based Complement Altern Med. https://doi.org/10.1155/2016/7382506. 2016:

    Article  Google Scholar 

  8. Moga MA, Bălan A, Anastasiu CV et al (2018) An overview on the anticancer activity of azadirachta indica (neem) in gynecological cancers. Int J Mol Sci 19. https://doi.org/10.3390/ijms19123898

  9. Mogana R, Teng-Jin K, Wiart C (2013) The Medicinal timber Canarium patentinervium Miq. (Burseraceae Kunth.) Is an anti-inflammatory bioresource of dual inhibitors of cyclooxygenase (COX) and 5-Lipoxygenase (5-LOX). ISRN Biotechnol 2013:1–8. https://doi.org/10.5402/2013/986361

    Article  CAS  Google Scholar 

  10. Sudhakaran G, Prathap P, Guru A et al (2022) Anti-inflammatory role demonstrated both in vitro and in vivo models using non‐steroidal tetranortriterpenoid, Nimbin (N1) and its analogues (N2 and N3) that alleviate the domestication of alternative medicine. Cell Biol Int 24:327–332. https://doi.org/10.1002/cbin.11769

    Article  CAS  Google Scholar 

  11. Sharma V, Vijay J, Ganesh MR, Sundaramurthy A (2020) Multilayer capsules encapsulating nimbin and doxorubicin for cancer chemo-photothermal therapy. Int J Pharm 582:119350. https://doi.org/10.1016/j.ijpharm.2020.119350

    Article  CAS  PubMed  Google Scholar 

  12. Issac PK, Karan R, Guru A et al (2021) Insulin signaling pathway assessment by enhancing antioxidant activity due to morin using in vitro rat skeletal muscle L6 myotubes cells. Mol Biol Rep 48:5857–5872. https://doi.org/10.1007/s11033-021-06580-x

    Article  CAS  PubMed  Google Scholar 

  13. Zarandi PK, Mirakabadi AZ, Sotoodehnejadnematalahi F (2019) Cytotoxic and anticancer effects of ICD-85 (venom derived peptides) in human breast adenocarcinoma and normal human dermal fibroblasts. Iran J Pharm Res 18:232–240. https://doi.org/10.22037/ijpr.2019.2341

    Article  CAS  Google Scholar 

  14. Yan CH, Li F, Ma YC (2015) Plumbagin shows anticancer activity in human osteosarcoma (MG-63) cells via the inhibition of s-phase checkpoints and down-regulation of c-myc. Int J Clin Exp Med 8:14432–14439

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Liu W, Yang Y, Zhang J et al (2014) Effects of dietary microencapsulated sodium butyrate on growth, intestinal mucosal morphology, immune response and adhesive bacteria in juvenile common carp (Cyprinus carpio) pre-fed with or without oxidised oil. Br J Nutr 112:15–29. https://doi.org/10.1017/S0007114514000610

    Article  CAS  PubMed  Google Scholar 

  16. Velayutham M, Guru A, Gatasheh MK et al (2022) Molecular Docking of SA11, RF13 and DI14 peptides from Vacuolar Protein sorting Associated protein 26B against Cancer Proteins and in vitro investigation of its Anticancer Potency in Hep-2 cells. Int J Pept Res Ther 28. https://doi.org/10.1007/s10989-022-10395-0

  17. Al-Dabbagh B, Elhaty IA, Al Hrout A et al (2018) Antioxidant and anticancer activities of Trigonella foenum-graecum, Cassia acutifolia and rhazya stricta. BMC Complement Altern Med 18:1–12. https://doi.org/10.1186/s12906-018-2285-7

    Article  CAS  Google Scholar 

  18. Song J, Liu K, Yi J et al (2010) Luteolin inhibits Lysophosphatidylcholine-Induced apoptosis in endothelial cells by a Calcium/Mithocondrion/Caspases-Dependent pathway. Planta Med 76:433–438. https://doi.org/10.1055/s-0029-1186197

    Article  CAS  PubMed  Google Scholar 

  19. Umapathy E, Ndebia EJ, Meeme A et al (2010) An experimental evaluation of Albuca setosa aqueous extract on membrane stabilization, protein denaturation and white blood cell migration during acute inflammation. J Med Plants Res 4:789–795. https://doi.org/10.5897/JMPR10.056

    Article  Google Scholar 

  20. Sarkar P, Stefi RV, Pasupuleti M et al (2020) Antioxidant molecular mechanism of adenosyl homocysteinase from cyanobacteria and its wound healing process in fibroblast cells. Mol Biol Rep 47:1821–1834. https://doi.org/10.1007/s11033-020-05276-y

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kazantseva L, Becerra J, Santos-Ruiz L (2022) Traditional Medicinal plants as a source of inspiration for Osteosarcoma Therapy. Molecules 27:5008. https://doi.org/10.3390/molecules27155008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Velayutham M, Haridevamuthu B, Priya PS et al (2022) Serine O-acetyltransferase derived NV14 peptide reduces cytotoxicity in H2O2 induced MDCK cells and inhibits MCF-7 cell proliferation through caspase gene expression. Mol Biol Rep 49:9205–9215. https://doi.org/10.1007/s11033-022-07746-x

    Article  CAS  PubMed  Google Scholar 

  23. Velayutham M, Sarkar P, Sudhakaran G et al (2022) Anti-cancer and anti-inflammatory activities of a short molecule, PS14 derived from the virulent cellulose binding domain of Aphanomyces invadans, on human laryngeal epithelial cells and an in vivo zebrafish embryo model. Molecules 27:7333. https://doi.org/10.3390/molecules27217333

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Koriem KMM (2013) Review on pharmacological and toxicologyical effects of oleum azadirachti oil. Asian Pac J Trop Biomed 3:834–840. https://doi.org/10.1016/S2221-1691(13)60165-3

    Article  PubMed  PubMed Central  Google Scholar 

  25. Sudhakaran G, Prathap P, Guru A et al (2022) Anti-inflammatory role demonstrated both in vitro and in vivo models using nonsteroidal tetranortriterpenoid, Nimbin (N1) and its analogs (N2 and N3) that alleviate the domestication of alternative medicine. Cell Biol Int 46:771–791. https://doi.org/10.1002/cbin.11769

    Article  CAS  PubMed  Google Scholar 

  26. Zorova LD, Popkov VA, Plotnikov EY et al (2018) Mitochondrial membrane potential. Anal Biochem 552:50–59. https://doi.org/10.1016/j.ab.2017.07.009

    Article  CAS  PubMed  Google Scholar 

  27. Velayutham M, Sarkar P, Karuppiah KM et al (2023) PS9, derived from an aquatic Fungus virulent protein, glycosyl hydrolase, arrests MCF-7 proliferation by regulating intracellular reactive oxygen species and apoptotic pathways. ACS Omega 8:18543–18553. https://doi.org/10.1021/acsomega.3c00336

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Guru A, Lite C, Freddy AJ et al (2021) Intracellular ROS scavenging and antioxidant regulation of WL15 from cysteine and glycine-rich protein 2 demonstrated in zebrafish in vivo model. Dev Comp Immunol 114:103863. https://doi.org/10.1016/j.dci.2020.103863

    Article  CAS  PubMed  Google Scholar 

  29. Issac PK, Lite C, Guru A et al (2021) Tryptophan-tagged peptide from serine threonine-protein kinase of Channa striatus improves antioxidant defence in L6 myotubes and attenuates caspase 3–dependent apoptotic response in zebrafish larvae. Fish Physiol Biochem 47:293–311. https://doi.org/10.1007/s10695-020-00912-7

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project Number (PNURSP2023R62), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Author information

Authors and Affiliations

  1. Toxicology and Pharmacology Laboratory, Department of Biotechnology, Faculty of Science and Humanities, SRM Institute of Science and Technology, Chengalpattu District, Kattankulatur, 603203, Tamil Nadu, India

    Gokul Sudhakaran & Jesu Arockiaraj

  2. Department of Medical Biotechnology and Integrative Physiology, Institute of Biotechnology, Saveetha School of Engineering, Thandalam, Chennai, 602 105, Tamil Nadu, India

    Manikandan Velayutham

  3. Department of Biology, College of Science, Princess Nourah bint Abdulrahman University, P.O. Box 84428, Riyadh, 11671, Saudi Arabia

    Nada H. Aljarba

  4. Biology Department, College of Science and Humanities, Prince Sattam bin Abdulaziz University, P.O. Box 83, Al-Kharj, 11940, Saudi Arabia

    Tahani Mohamad AL-Hazani

  5. Department of Food Science & Biotechnology, Sejong University, Seoul, 05006, South Korea

    Selvaraj Arokiyaraj

  6. Department of Cariology, Saveetha Dental College and Hospitals, SIMATS, Chennai, 600 077, Tamil Nadu, India

    Ajay Guru

Authors
  1. Gokul Sudhakaran
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Manikandan Velayutham
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nada H. Aljarba
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tahani Mohamad AL-Hazani
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Selvaraj Arokiyaraj
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ajay Guru
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jesu Arockiaraj
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

G.S and M.V performed most of experiments and wrote the manuscript. N.H.A; T.M.A; and S.A reviewed and edited the manuscript. A.G and J.A supervised and validated the work.

Corresponding authors

Correspondence to Ajay Guru or Jesu Arockiaraj.

Ethics declarations

Conflict of interest

The authors have declared that no competing interests exist.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sudhakaran, G., Velayutham, M., Aljarba, N.H. et al. Nimbin (N1) and analog N3 from the neem seeds suppress the migration of osteosarcoma MG-63 cells and arrest the cells in a quiescent state mediated via activation of the caspase-modulated apoptotic pathway. Mol Biol Rep 50, 7357–7369 (2023). https://doi.org/10.1007/s11033-023-08627-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-023-08627-7

Keywords

Search

Navigation