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RIP1K and RIP3K provoked by shikonin induce cell cycle arrest in the triple negative breast cancer cell line, MDA-MB-468: necroptosis as a desperate programmed suicide pathway

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Tumor Biology

Abstract

Resistance to cell death and reprogramming of metabolism are important in neoplastic cells. Increased resistance to apoptosis and recurrence of tumors are the major roadblocks to effective treatment of triple negative breast cancer. It has been thought that execution of necroptosis involves ROS generation and mitochondrial dysfunction in malignant cells. In this study, the effect of shikonin, an active substance from the dried root of Lithospermum erythrorhizon, on the induction of necroptosis or apoptosis, via RIP1K-RIP3K expressions has been examined in the triple negative breast cancer cell line. The expression levels of RIP1K and RIP3K, caspase-3 and caspase-8 activities, the levels of ROS, and mitochondrial membrane potential have been studied in the shikonin-treated MDA-MB-468 cell line. An increase in the ROS levels and a reduction in mitochondrial membrane potential have been observed in the shikonin-treated cells. Cell death has mainly occurred through necroptosis with a significant increase in the RIP1K and RIP3K expressions, and characteristic morphological changes have been observed. In the presence of Nec-1, caspase-3 mediating apoptosis has occurred in the shikonin-treated cells. The current findings have revealed that shikonin provoked mitochondrial ROS production in the triple negative breast cancer cell line, which works as a double-edged executioner’s ax in the execution of necroptosis or apoptosis. The main route of cell death induced by shikonin is RIP1K-RIP3K-mediated necroptosis, but in the presence of Nec-1, apoptosis has prevailed. The present results shed a new light on the possible treatment of drug-resistant triple negative breast cancer.

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Abbreviations

ER:

Estrogen receptor

PR:

Progesterone receptor

HER-2:

HER-2/neu receptor

Z-VAD-FMK:

Carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone

Nec-1:

Necrostatin-1

ROS:

Reactive oxygen species

Δψm:

Mitochondrial membrane potential

FITC:

Fluorescein isothiocyanate

PI:

Propidium iodide

MTT:

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide

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Correspondence to Fatemeh Karami-Tehrani.

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Part of this work was supported by a Ph.D. grant from Tarbiat Modares University. The authors would like to express their gratitude to Professor Peter Vandenabeele for his valuable comments. The sincere cooperation of Mrs. Batoul Etemadi-kia, lab expert, is much obliged.

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Shahsavari, Z., Karami-Tehrani, F., Salami, S. et al. RIP1K and RIP3K provoked by shikonin induce cell cycle arrest in the triple negative breast cancer cell line, MDA-MB-468: necroptosis as a desperate programmed suicide pathway. Tumor Biol. 37, 4479–4491 (2016). https://doi.org/10.1007/s13277-015-4258-5

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