β-Arrestin inhibition induces autophagy, apoptosis, G0/G1 cell cycle arrest in agonist-activated V2R receptor in breast cancer cells

Mohamed Hessien,Mohamed Kelany,Thoria Donia,Mohamed Abouda

doi:10.1007/s12032-021-01484-z

Mohamed Hessien, Mohamed Kelany + Show 2 more

Open Access

https://doi.org/10.1007/s12032-021-01484-z

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Journal: Medical Oncology	Publication Date: Mar 15, 2021
Citations: 7	License type: cc-by

Affiliation: Tanta University, Ain Shams University

Abstract

Non-visual arrestins (β-arrestins) are endocytic proteins that mediate agonist-activated GPCRs internalization and signaling pathways in an independent manner. The involvement of β-arrestins in cancer invasion and metastasis is increasingly reported. So, it is hypothesized that inhibition of β-arrestins may diminish the survival chances of cancer cells. This study aimed to evaluate the in vitro impact of inhibiting β-arrestins on the autophagic and/or apoptotic responsiveness of breast cancer cells. We used Barbadin to selectively inhibit β-Arr/AP2 interaction in AVP-stimulated V2R receptor of triple-negative breast cancer cells (MDA MB-231). Autophagy was assessed by the microtubule-associated protein 1 light chain 3-II (LC3II), apoptosis was measured by Annexin-V/PI staining and cell cycle distribution was investigated based upon the DNA content using flow cytometry. Barbadin reduced cell viability to 69.1% and increased the autophagy marker LC3II and its autophagic effect disappeared in cells transiently starved in Earle's balanced salt solution (EBSS). Also, Barbadin mildly enhanced the expression of P62 mRNA and arrested 63.7% of cells in G0/G1 phase. In parallel, the drug-induced apoptosis in 29.9% of cells (by AV/PI) and 27.8% of cells were trapped in sub-G1 phase. The apoptotic effect of Barbadin was enhanced when autophagy was inhibited by the PI3K inhibitor (Wortmannin). Conclusively, the data demonstrate the dual autophagic and apoptotic effects of β-βArr/AP2 inhibition in triple-negative breast cancer cells. These observations nominate β-Arrs as selective targets in breast cancer treatment.

Highlights

Autophagy is regarded as protective mechanism when cells experience unfavorable conditions
Cells starved in Earle's balanced salt solution (EBSS) for 4 h demonstrated higher expression of light chain 3-II (LC3II) protein (66.7 ± 2.2) compared to the basal level (30.03 ± 0.95) seen in cells grown in normal nutrients and growth factors (P < 0.001)
In cells transiently grown in EBSS followed by Wort, the LC3II was 54.2 ± 2.61 indicating the irreversible effect of starvation induced autophagy even when phosphoinositide-3 kinase (PI3K) was inhibited (Fig. 1)

Summary

Introduction

Autophagy is regarded as protective mechanism when cells experience unfavorable conditions. These pathways are extremely diverse, derive variable cellular responses through different GPCRs, or independent of G protein activation [8] These events determine life-or-death decisions in the cell. In triple negative breast cancer cells (MDA MB-231), β-Arrs contributed in PAR2 mediated migration via ERK1/2 MAP kinase activation [12] and involved in cellular proliferation and migration via AMPK activation [13]. They contributed in GPR161-mediated proliferation and cell migration via IQGAP1 dependent mechanism [14] and associated with resistance through regulating the expression of multidrug resistance gene (MDR1) [15]. This work was designed to explore the autophagic and apoptosis effects of the inhibition of βArr/AP2 interaction in hormonally stimulated breast cancer cells

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