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Arctigenin-mediated cell death of SK-BR-3 cells is caused by HER2 inhibition and autophagy-linked apoptosis

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Abstract

Background

Human epidermal growth factor receptor 2 (HER2) is well-known as the therapeutic marker in breast cancer. Therefore, we evaluated anti-cancer activity of arctigenin (ATG) on in SK-BR-3 HER2-overexpressing human breast cancer cells.

Methods

Cell viability and cytotoxicity were analyzed with MTT and colony-forming assay and cell cycle analysis was performed by flow cytometry. The expression and/or phosphorylation of proteins in whole cell lysate and mitochondrial fraction were analyzed by Western blotting. Cellular levels of LC3 and sequestosome 1 (SQSTM1/P62) were observed by immunofluorescence analysis.

Results

The result showed that ATG decreased cell viability of SK-BR-3 cells in a concentration-dependent manner. Moreover, ATG increased the sub G1 population linked to the suppression of HER2/EGFR1 signaling pathway. Furthermore, ATG increased the phosphorylation of H2AX and down-regulated RAD51 and survivin expressions, indicating that ATG induced DNA damage and inhibited the DNA repair system. We also found that cleavages of caspase-7 and PARP by releasing mitochondrial cytochrome c into the cytoplasm were induced by ATG treatment for 72 h through the reduction of Bcl-2 and Bcl-xL levels in mitochondria. In contrast, the levels of LC-3 and SQSTM1/P62 were increased by ATG for 24 h through the Akt/mTOR and AMPK signaling pathway.

Conclusions

Taken together, this study indicates that autophagy-linked apoptosis is responsible for the anti-cancer activity of ATG in SK-BR-3 cells, and suggests that ATG is considered a potential therapeutic for the treatment of HER2-overexpressing breast cancer.

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Abbreviations

ATG:

Arctigenin

HER2:

Human epidermal growth factor receptor2

EGFR:

Epidermal growth factor receptor

p-H2A.X:

Phospho-histone 2A.X

PARP:

Poly (ADP-ribose) polymerase

Bcl-xL:

B-cell lymphoma-extra large

Bcl-2:

B-cell lymphoma-2

Cyt c:

Cytochrome c

AMPK:

AMP-activated protein kinase

mTOR:

Mammalian target of rapamycin

S6:

Ribosomal protein s6

S6K1:

S6 kinase1

LC-3:

Microtubule-associated proteins 1A/1B light chain 3 (LC-3)

SQSTM1/P62:

Sequestosome 1

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Acknowledgements

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (Grant no. 2015R1D1A1A01058841).

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Authors and Affiliations

  1. Department of Pharmacology, College of Medicine and Intractable Disease Research Center, Dongguk University, Gyeongju, 38066, Republic of Korea

    Min-Gu Lee, Kyu-Shik Lee & Kyung-Soo Nam

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Contributions

K-SN and K-SL designed the experiments. K-SN, M-GL and K-SL analyzed and interpreted data. M-GL performed the experiments and wrote the manuscript, which was approved by all authors.

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Correspondence to Kyung-Soo Nam.

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Lee, MG., Lee, KS. & Nam, KS. Arctigenin-mediated cell death of SK-BR-3 cells is caused by HER2 inhibition and autophagy-linked apoptosis. Pharmacol. Rep 73, 629–641 (2021). https://doi.org/10.1007/s43440-021-00223-5

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