In vitro evaluation of Annona muricata L. (Soursop) leaf methanol extracts on inhibition of tumorigenicity and metastasis of breast cancer cells

Abstract

Purpose

The present study evaluates the in-vitro anti-tumorigenic potential of leaf methanol extracts of Annona muricata (LMAM).

Materials and methods

The cytotoxic activity was assessed in MCF-7 cells by MTT assay at various concentrations ranging from 25–250µg/mL. MCF-7 cells were treated with 50 and 100 µg/mL LMAM for 24 h. To detect LMAM-induced apoptosis; Hoescht 33342 staining along with Cell cycle analysis, Annexin-PI probe as well as oxidative stress damage by reactive oxygen species (ROS) measurements were determined using flow cytometric analysis. While caspase-3 expression levels were studied employing the qRT-PCR method.

Results

LMAM exhibited significant inhibition of MCF-7 cells with an IC₅₀ value of 85.55 µg/mL. Hoescht staining showed marked morphological features characteristic of apoptosis in LMAM treated cells. Cell cycle analysis confirmed the proven capability of LMAM showing a 30% rise in G₁ phase upon treatment with 100 µg/mL LMAM, thus inducing cell cycle arrest at G₁ phase and a rise in sub G₀–G₁ population paralleled with a decrease in S phase. Flow cytometric analysis with Annexin V-FITC-PI staining indicated an increase in the early and late apoptotic population with a 3.38% and 19.47% rise respectively when treated with 100 µg/mL LMAM. Treatment with 100 µg/mL LMAM caused an increase in intracellular ROS with MFI value 3334.08. Upregulation of caspase-3 was observed with a 2.18 and 32.47 fold increase compared to control in MCF-7 cells cultured at 50 µg/mL and 100 µg/mL LMAM respectively suggesting caspase-dependent apoptosis.

Conclusion

LMAM proved as a potent ethno-chemopreventive agent and a potential lead in cancer treatment attributable to the synergistic interactive properties of phytoconstituents.

Keywords:

• Annona muricata
• Acetogenins
• Apoptosis
• Cancer
• MCF-7

Acknowledgements

The authors are deeply grateful to Dr. Hemant Agrawal, Flowcytometry Solutions for valuable guidance and kind suggestions. The authors also thank Dr. Harikumar K. B, Scientist E-I., Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala for providing facilities and assistance. The authors also wish to thank the International Society for Advancement of Cytometry (ISAC) and Dr. Shyamalina Biswas for kind help and motivation.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Correction Statement

This article has been republished with minor change. This change do not impact the academic content of the article.

Additional information

Funding

The present work was financially supported by the Department of Science and Technology, New-Delhi in the form of the DST INSPIRE Fellowship [IF160005] and by the Council for Scientific and Industrial Research, New-Delhi, India [38(1471)/18/EMR-II].