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

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-arrstins 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 ow cytometry. Barbadin reduced cell viability to 69.1% and increased the autophagy marker LC3 II 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.Thus, this work was designed to explore the autophagic and apoptosis effects of the inhibition of β-Arr/AP2 interaction in hormonally stimulated breast cancer cells. Materials And MethodsKey Reagents: Barbadin (3-amino-5-(4-benzylphenyl)-3H,4H-thieno[2,3-d]pyrimidin-4-one) (Cat No. B118250), Arginine Vasopressin acetic acid salt (AVP) (Cat No. V991535) and Wortmannin (Cat No. W499400), were purchased from Toronto Research Chemicals, Toronto, Ontario, Canada). Trichostatin A (TSA) and dimethyl sulfoxide (DMSO) were from Sigma Chemicals, USA. Earle's balanced salt solution (EBSS) and other cell culture reagents (DMEM 4.5 g/L glucose with L glutamine, penicillin/streptomycin, fetal bovine serum (FBS) and Trypsin/EDTA) were from Lonza Pharma & Biotech. Cell culture and treatment MDA MB-231 cells [16] was generously provided by Department of Cancer Biology, NCI, Cairo University.Cells were cultured in Dulbecco's modi ed Eagle's Minimal Essential Medium (DMEM) supplemented with 10% heat inactivated FBS, 1% Penicillin/Streptomycin in a humidi ed atmosphere of 95% air and 5% CO 2 at 37 °C. Initially, cells were seeded with a low cell density then subcultured with particular densities in 100 mm, 6 wells, or 96 wells plates according to the experimental settings. Cell treatmentsAutophagy was induced by glucose oxygen deprivation, where cells were starved for 4 hours in EBSS, which does not contain nutrients nor growth factors, at...

Prestimulation of breast cancer cells with arginine vasopressin accentuates the anticancer effect induced by Dynamin 2 inhibitor

“…However, the crosstalk between inhibitors of these proteins and non-endocytic targets is not well explored. Previously, we demonstrated that the inhibition of the interaction between β-arrestin, an endocytic accessory protein, and AP2 adaptor protein, decreased the viability of TNBC cells [22]. In the present study, Dyn2, (integral endocytic proteins with an intrinsic GTPase activity) was inhibited by 3-hydroxynaphthalene-2-carboxylic acid-(3,4-dihydroxybenzylidene)-hydrazide (commonly known as Dynasore) to explore the associated side effects on TNBC cells viability and their metastasis, especially when cells were prestimulated with exogenous AVP hormone.…”

“…Also, other studies have reported that it suppressed cell proliferation, migration and enhanced the antitumor e cacy of cisplatin in osteosarcoma via STAT3 pathway [20], reduced the metastasis of cervical cancer [12] (Lee et al, 2016) and induced apoptosis in NSCLC cells [21]. Recently, we reported that the inhibition of early endocytic events of CME, through inhibiting the interaction between β-arrestin and AP2 adaptor protein induced both apoptosis and autophagy in invasive breast cancer [22].…”

Cold atmospheric pressure plasma treatment combined with starvation increases autophagy and apoptosis in melanoma in vitro and in vivo

“…78 Starvation-induced apoptosis and autophagy also trigger cell cycle arrest in different cancer cell types. 81,82 Recent research has also suggested that plasma exposure inhibits cell cycle progression and promotes apoptosis or autophagy in cancer cells in many types of cancer cells. [83][84][85] Combining CAP with starvation may amplify cell cycle arrest, cell death and therapeutic outcomes in animal models, but more research is needed to pinpoint the exact modes of action.…”

“…The complex of Atg5 and Bcl‐X then contributes to apoptosis induction 78 . Starvation‐induced apoptosis and autophagy also trigger cell cycle arrest in different cancer cell types 81,82 . Recent research has also suggested that plasma exposure inhibits cell cycle progression and promotes apoptosis or autophagy in cancer cells in many types of cancer cells 83–85 .…”

Deciphering the signaling mechanisms of β‐arrestin1 and β‐arrestin2 in regulation of cancer cell cycle and metastasis

“…Proteins have been shown to be scaffolded by β‐arrestin isoforms 1 and 2 (β‐arr1 and β‐arr2) within the cytoplasm and migrate to the nucleus in regulating several cellular processes (Peterson & Luttrell, 2017). β‐Arrs regulate several cellular functionalities such as cell proliferation, cell cycle regulation, inhibition of cell apoptosis, angiogenesis, and others (Donia et al, 2021). These functions are common attributes of a cancer phenotype.…”