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Cycloartane triterpenoid from Euphorbia macrostegia modulates ER stress signaling pathways to induce apoptosis in MDA-MB231 and MCF-7 breast cancer cell lines

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Abstract

Unfolded protein response (UPR) is involved in breast cancer (BC) progression and drug resistance. Many natural products (NPs) could modulate UPR and used for therapeutic purposes. Herein, we aimed to investigate the molecular mechanism of Cycloart-23E-ene-3β, 25-diol (Cycloart-E25), cytotoxicity, as a NP extracted from Euphorbia macrostegia and focused on endoplasmic-reticulum stress (ERS) and UPR signaling pathways. Reactive oxygen species (ROS) were probed by DCFDA fluorescence dye. Apoptosis was assayed by annexin V/propidium iodide (PI), immunoblotting of anti- and proapoptotic, Bcl-2 and Bax proteins, and mitochondrial transmembrane potential (ΔΨm) changes. Thioflavin T (ThT) staining and immunoblotting of UPR signaling components (CHOP, PERK, ATF6, BiP, and XBP1) were recruited for the assessment of ERS. Our results indicated that Cycloart-E25 noticeably increases ROS levels in both MB-231 MDA-MB-231 and MCF-7 cell lines, p>0.05. Flow cytometry assessments revealed an increase in the cell population undergoing apoptosis. Also, the Bax/Bcl-2 ratio increased in a dose-dependent manner following Cycloart-E25 treatment, significantly, p>0.05. Mitochondrial involvement could be deduced by significant decreases in ΔΨm, p>0.05. Cycloart-E25 potently induces protein aggregation and upregulated CHOP, PERK, ATF6, BiP, and XBP1 factors in both MDA-MB-231 MB-231 and MCF-7 cell lines, indicating the involvement of ERS in Cycloart-E25-mediated apoptosis. In conclusion, Cycloart-E25 increased the accumulation of misfolded proteins and upregulated UPR components. Therefore, induction of ERS may be involved in the trigger of apoptosis in BC cell lines. Cycloart-E25 induced apoptosis in breast cancer cell lines through ERS. More assessments are needed to confirm its in vivo anti-tumoral effects.

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DATA availability

All data and materials were presented in the “Result” section.

Abbreviations

BC:

Breast cancer

UPR:

Unfolded protein response

ERS:

Endoplasmic-reticulum stress

PERK:

Protein kinase RNA-activated (PKR)-like ER kinase

ATF6:

Activating transcription factor 6

BiP:

Binding immunoglobulin protein

XBP1:

x-box-binding protein 1

CHOP:

CCAAT-enhancer-binding protein homologous protein ()

Cycloart-E25:

Cycloart-23E-ene-3β, 25-diol

MTT:

3-(4, 5-dimethylthiazol-2- yl)-2, 5-diphenyl tetrazolium bromide

PI:

Propidium iodide

DCFDA:

2′,7′-dichlorofluorescein diacetate

ΔΨm:

Mitochondrial transmembrane potential

ThT:

ThioflavinT

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Funding

Financial support was made by Isfahan University of Medical Sciences Grants (195001 and 393064).

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Authors and Affiliations

  1. Department of Nutrition & Biochemistry, School of Medical Sciences, Sabzevar University of Medical Sciences, Sabzevar, I.R, Iran

    Mohammad Keyvaloo Shahrestanaki

  2. Department of Pharmacognosy, Isfahan University of Medical Sciences, Isfahan, I.R, Iran

    Mustafa Ghanadian

  3. Department of Clinical Biochemistry, School of Pharmacy & Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, I.R, Iran

    Abdollah Mirjani & Mahmoud Aghaei

Authors
  1. Mohammad Keyvaloo Shahrestanaki
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  2. Abdollah Mirjani
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Contributions

Mohammad Keyvaloo Shahrestanki and Abdollah Mirjani performed the experimental part and prepared the drafts of the manuscript. Mahmoud Aghaei and Mustafa Ghanadian designed the research, finalized the manuscript, and provided funds.

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Correspondence to Mahmoud Aghaei.

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Shahrestanaki, M.K., Mirjani, A., Ghanadian, M. et al. Cycloartane triterpenoid from Euphorbia macrostegia modulates ER stress signaling pathways to induce apoptosis in MDA-MB231 and MCF-7 breast cancer cell lines. Naunyn-Schmiedeberg's Arch Pharmacol 396, 1749–1758 (2023). https://doi.org/10.1007/s00210-023-02426-4

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