In this work, Barbadin was used to functionally hinder the interaction between β-Arr1 and β2-adaptin subunit of the clathrin adaptin protein-2 (AP2). This does not interfere with the formation of the membrane receptor/β-Arr complex . The hormonal agonist (AVP) we used, is known to ligand with V2R receptor and stimulates ERK1/2 pathway (independently of heterotrimeric G protein signaling). Also, ERK1/2 stimulation involves β-Arr . Thus, it was anticipated that treatment of cells with Barbadin may block V2R-stimulated ERK1/2 activation and prohibits the intracellular accumulation of cAMP and downstream signaling. MDA MB-231 cells are highly metastatic TNBC cells , expresses V2R receptor  and non-visual arrestins (β-arr 1&2). We have employed this cellular, in vitro, model to reveal the direct impact of β-Arr inhibition on cellular events including autophagy, apoptosis and cell cycle progression and the subsequent anticancer potential of Barbadin.
Growing cells in starvation conditions (in EBSS) is well established protocol to induce autophagy . Limited starvation (4 h) was sufficient to induce autophagy, as indicated by 2-fold increase in the microtubule-associated protein light chain 3 (LC3II) compared to its basal level in cells grown in nutrients rich condition. Starvation-induced autophagy was associated with apoptosis in 28% of cells dually stained with AV/PI. The displacement of Bcl-2 from Beclin-1 and Bax, may be the driving force that triggered both autophagy and apoptosis . In similar work, starved MDA MB-231 cells developed autophagy through the AMBRA1/mTOR pathway leading to an increase of LC3II, increase of autophagosomes but decrease of p62 protein . The PI3K inhibitor (Wort) is commonly used as an autophagy inhibitor, based on its inhibitory effect on class III PI3K activity, which is known to be essential for induction of autophagy. The LC3II in Wort treated cells was significantly reduced. Unexpectedly, both cytotoxic mechanisms were associated with mild increase in the expression of P62 mRNA. Although P62 protein was not assessed in this work, the relative overexpression of its coding gene may help to replenish selective degradation of P62 protein during the initial stages of autophagy . This may occur during the first few hours of starvation and before the turnover of P62 protein. In this process, P62 protein acts as an intracellular receptor for ubiquitinated proteins, where the P62-ubiquitinated complex is merged with LC3II, and the new complex is sequestered in the autophagosomes . Also, the increased P62 mRNA, we observed, may be attributed to the increased response to the proteasome inhibitor (PSI) or prostate-derived Ets factor (PDEF) .
An altered scenario was observed when cells were treated with Wort, where autophagy was inhibited, as indicated by the significant decrease of LC3 II compared to its basal level. Additionally, Wort developed apoptosis in 22% of cells. Wort-mediated autophagy inhibition may occur through inhibiting the conversion of PIP2 into PIP3 and phosphorylation of AKT leading to the inhibition of PI3k/AKT pathway . Cells transiently exposed to harsh conditions followed by Wort have maintained their high LC3II protein and developed more apoptosis (31% of cells) indicating the irreversibility of starvation-induced autophagy. In similar studies, concomitant exposure of mouse embryonic fibroblasts (MEF) cells to Wort or 3-methyladenine during starvation, led to the suppression of starvation-induced autophagy . Cell cycle analysis confirmed the association between autophagy and apoptosis, where about 39% of cells were trapped in Sub-G1 phase, and more cells (54%) were arrested in G0/G1 phase, the observation previously reported in both TNBC (MDA MB-231)  and the less aggressive luminal A cells (MCF-7) .
Nonvisual β-Arrs, on the other hand, play a key role in mediating cellular cytoprotective events through the activation of GPCRs . They participate in cancer invasion and metastasis . Herein, Barbadin mediated β-Arr inhibition, in cells grown in normal condition, was associated with autophagy and developed apoptosis as similar as starved cells (EBSS-incubation). The inhibition of β-Arr/AP2 interaction may provide more chance for β-Arr to act as a caspase substrate, where its cleaved fragments participate in the core mechanism of apoptosis and assist other product of caspase activity in releasing the mitochondrial Cytochrmome . Also, Barbadin autophagic/apoptotic effect was associated in G0/G1 arrest. These observations are supported by some previous reports in which β-Arr1 depletion, markedly induced neuronal apoptosis/necrosis in vivo and in vitro . Also, siRNA-mediated silencing of β-Arr1 &2 reduced ERK1/2 activation and MDA MB-231 cells metastasis . In addition, β-Arr2-associated type III transforming growth factor-β receptor negatively mediated the migration and invasion of MDA-MB-231 breast cancer cells via NF-κB signaling . Our observations indicated that Barbadin did not exert additional autophagic effect after cells were desensitized with EBSS, most probably due to the internalization of their GPCRs. This was evidenced by the insignificant differences in LC3II protein, P62 mRNA and apoptosis in Barbadin treated cells with or without starvation. Moreover, the autophagic role of Barbadin did not involve PI3K signaling, where cells cotreated with Barbadin and Wort did not show significant changes in the magnitude of autophagy. More importantly, the data predicts the dependence of Barbadin-induced apoptosis on autophagy, where cotreatment of cells with Wort and Barbadin resulted in higher proportion of cells in Sub-G1 peak (31.9%) and higher percent of apoptotic cells (32.7%) (Fig 2) The intervening role of P62 seems to be cell type and/or autophagy phase dependent. P62, it is multifunction and involved in other pathways including the UPS . The increased synthesis of P62 could be triggered through the effect of some transcription factors, such as Nrf2, where P62 is phosphorylated to participate in autophagy flux . This hypothesis is supported by the decreased expression of P62 in Wort treated cells. Other expression data revealed low expression of β-Arr1 mRNA is consistent with clinical studies in which β-Arr 1 was inversely correlated with the histological grade of breast cancer and positively associated with TNBC patient survival, suggestive of a tumor-suppressive function of β-Arrs1 in breast cancer patients . Silencing of β-Arr1 increased the migration potential of MDA MB-468 and MDA MB-231 cells . The autophagy/apoptosis effect of Barbadin is exterted through the hindrance of the covalent interaction between β-Arr and AP2 β-adaptin subunit, where Barbadin is superimposited with the β-Arr1 C-terminus peptide where Phe-388, Phe-391 and Arg-395 are the three key residues for β-Arr binding . This may exclude possible direct effect of Barbadin on β-Arr1 expression.
In parallel, histone deacetylases (HDACs) 1, 6 and 8 are deeply involved in invasion of breast cancer . HDAC6, in particular, plays a critical role in the ubiquitinated aggregate formation and autophagosome–lysosome fusion. Although this role is mediated by P62, other studies revealed the involvement of HDAC6 in β-Arr mediated tubulin acetylation/deacetylation that affects cell migration. Thus, we thought to monitor the autophagic/apoptotic potential of HDAC inhibition compared that induced by β-Arr inhibitor. Treatment of cells with HDACI (TSA), resulted in regression of cells viability, induction of autophagy, massive necrosis and cell cycle arrest in G0/G1 phase. These observations are largely consistent with previous reports that nominated TSA as anticancer drug through the induction of both autophagy  and apoptosis . These findings, however, predict that TSA may adopt different anticancer mechanism as indicated by its necrotic effect and the downregulation of P62 mRNA. Correlation based clustering revealed that TSA effects did not tightly cluster with Barb/AVP or Wort effects (Fig 5). This added to its well reported epigenetic mechanism in acetylating genes and transcription factors.