CME is orchestrated with a set of scaffolding and adaptor proteins, including high molecular weight dynamins. Although this mechanism is utilized mainly to internalize liganded- membrane receptors, recent evidences demonstrated that some viruses, including SARS-CoV2, entry is established through their attachment with certain membrane receptors, like angiotensin-converting enzyme 2 (ACE2) receptors , followed by CME-mediated internalization. Accordingly, CME and the related proteins were extensively targeted by several small molecules to inhibit CME-mediated viral infection. 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 . 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. Compared to other large dynamins inhibitors (like Dynole 34 − 2 and Mdivi-1), Dynasore (DYN) selectively inhibits both isoforms (Dyn 1 & Dyn 2) in addition to the mitochondrial dynamin-related protein (Drp-1). More importantly, DYN does not affect small GTPases like Ras, Rho, Rab families [25, 26]. The involvement of Dyn2 in insulin secretion and plasma glucose homeostasis  may explain the progressive reduction of cells metabolic activity when Dyn2 was inhibited, as indicated by the MTT assay.
Dynasore was able to induce apoptosis in 18.3% of cells, increase the expression of the apoptotic markers Bax and caspase-3 and downregulated the activation of AKT. Although the inhibition of large GTPase activity represents its main mode of action , some studies revealed that DYN cytotoxic effect is mediated through blocking cytokinesis and/or induction of caspase-mediated apoptosis following cytokinesis failure . Also, the direct involvement of Drp-1 in mitochondrial fission, suggests the mitochondrial dysfunction mediated cell death. In similar manner, Wort exerted a synergistic apoptotic effect with DYN. This is attributed to Bak activation and the subsequent mitochondrial damage . Although breast cancer cells express AVP gene, more apoptosis was observed in cells prestimulated with exogenous AVP. Dyn 2 plays an important role in the completion of the last stages of mitosis , where it is intensely localized at the mitotic spindle via its proline-rich domain (PRD) . Although the Dyn2 GTPase activity is not involved in the microtubule polymerization, the obtained results suggest that DYN may induce cytokinesis failure and inhibit proliferation of TNBC breast cancer, where cells were arrested in G0/G1 phase in absence or presence of AVP. Such effect was previously observed in other cancers including cervical and lung cancers . In agreement with previous reports , Wort alone failed to induced cell cycle arrest, where 28.6% of cells were still dividing (S phase). Moreover, cells cotreated with DYN combined with Wort did not show cell cycle arrest, where 36.4% of cells were still dividing. AVP in contrast was able to accumulate cells in G0/G1 phase, due to the cumulative effects of both the endogenously expressed (Pro-AVP) and the exogenous AVP and their direct inhibitory effect on cyclin D1 expression .
In parallel, the involvement of Dynamin in regulation of autophagy was previously reported, where mutations in Dyn 2, led to the impairment of autophagy in mice . The observed increase of the autophagic marker (LC3II) in Dynasore-treated cells may be explained by the inhibition of Drp-1. Moreover, the increase of LC3II protein was associated 1.8-fold increase in the expression of Beclin1 in Dyn-inhibited cells and more expression (2.5-fold) in presence of AVP, the observation previously reported in senescent Endothelial Cells. In contrast, Wort was associated with the downregulation of both markers, the finding previously reported in brain  and colorectal cancer . In a similar manner, the auophagic effect of AVP was evidenced through 1.6 and 2.5-fold increment in LC3II and Beclin 1. Moreover the enhance AVP-mediated responsiveness of cells could be attributed to V2R activation  and the associated downstream signaling. Although AVP did not induce observable apoptotic changes (about 6%), it led to cell cycle arrest possibly due to the inhibition of cyclin D1 . The obtained data excludes mitogenic effect of AVP, however it enhanced the apoptotic effect of Dynasore in agreement with reports, that suggested the antiproliferative of AVP-mediated activation of V2R in renal collecting duct . AVP binding with V1R, for example, enhanced cellular proliferation, whereas its binding to V2R was associated with antiproliferative effects. Moreover, the downstream signaling effect of AVP varies when it liganded with V1ar, V1br or V2R, that distributed among different cell types . The contradicting effects of AVP may predict its apoptotic or anti-apoptotic effect that varies according to the cell type and the type of VR receptor, it binds with .
To compare the antineoplastic effect of DYN, Wort was utilized, where it inhibits cell proliferation via the inhibition of PI3K/AKT/mTOR signaling . DYN treatment was associated with an observable decrease pAkt. This may be explained by RagA-Raptor binding that reduces the recruitment of mTORC1 to the lysosome and the subsequent reduction of Akt activation . A similar regression in AKT activation was observed in cells prestimulated with AVP, where AVP-mediated activation of the V2R receptor, leads to the recruitment and oligomerzation of Dyn2, around the necks of nascent AVP/V2R containing vesicles . During this process, Dyn2 is anchored to membrane PIP2 (through ithe pleckstrin-homology domain PH domain, shown in Fig. 1) and to the negatively charged lipids through its positive residues. Similar to Wort , the depletion of PIP2 may explain the synergistic effect of AVP with DYN in down-regulating AKT pathway.
The potential of cell migration and invasion gives breast cancer the ability of local, regional and systemic metastasis, that convey to poor prognosis and death. Previous reports predicted the implication of large dynamins in facilitating cell migration and invasion . Also, other studies have indicated that metastasis of breast cancer cells may involve GTP binding and/or hydrolysis by dynamin . The obtained data showed that Dynamin inhibition led to a reduction of cells invasion. As PI3K/Akt signaling is a common pathway regulating cell proliferation, migration, and invasion, the observed down-regulation of the cell’s invasiveness, may be attributed to the associated DYN-mediated PI3K/Akt inhibition.
Although the study did not involve genome or proteome-wide analysis, we thought to mine all observations (13 variables) performed in cells treated with either DYN or Wort using a simple clustering tool. Correlation based clustering using ClustVis demonstrated that DYN and AVP/DYN effects were tightly clustered. Similarly, Wort and combination of Wort/DYN were tightly clustered (Fig. 8) indicating the corresponding anticancer effects of Dynamin inhibition and PI3K/Akt/mTOR inhibition. The overall scenario depicts that the apoptotic and the autophagic effect of Dynamin inhibition by Dynasore. This may represent a beneficial role in limiting the growth rate of invasive breast cancer, especially when cells are prestimulated with AVP. These events are supported with other observations including, limitation of metastasis. Also there is a relative improvement in cell’s sensitivity to chemotherapy, as indicated by the downregulation of the drug resistance gene that encodes the multidrug efflux protein P-glycoprotein (PGY1).