We report new ruthenium complexes bearing the lipophilic bathophenanthroline (BPhen) ligand and dihydroxybipyridine (dhbp) ligands which differ in the placement of the OH groups ([(BPhen)2Ru(n,n′‐dhbp)]Cl2 with n = 6 and 4 in 1A and 2A, respectively). Full characterization data are reported for 1A and 2A and single crystal X‐ray diffraction for 1A. Both 1A and 2A are diprotic acids. We have studied 1A, 1B, 2A, and 2B (B = deprotonated forms) by UV‐vis spectroscopy and 1 photodissociates, but 2 is light stable. Luminescence studies reveal that the basic forms have lower energy 3MLCT states relative to the acidic forms. Complexes 1A and 2A produce singlet oxygen with quantum yields of 0.05 and 0.68, respectively, in acetonitrile. Complexes 1 and 2 are both photocytotoxic toward breast cancer cells, with complex 2 showing EC50 light values as low as 0.50 μM with PI values as high as >200 vs. MCF7. Computational studies were used to predict the energies of the 3MLCT and 3MC states. An inaccessible 3MC state for 2B suggests a rationale for why photodissociation does not occur with the 4,4′‐dhbp ligand. Low dark toxicity combined with an accessible 3MLCT state for 1O2 generation explains the excellent photocytotoxicity of 2.

Circulating tumor cells (CTCs) are cancer cells that detach from the primary site and travel in the blood stream. A higher number of CTCs increases the risk of breast cancer metastasis, and it is inversely associated with the survival rates of patients with breast cancer. Although the numbers of CTCs are generally low and the majority of CTCs die in circulation, the survival of a few CTCs can seed the development of a tumor at a secondary location. An increasing number of studies demonstrate that CTCs undergo modification in response to the dynamic biophysical environment in the blood due in part to fluid shear stress. Fluid shear stress generates reactive oxygen species (ROS), triggers redox-sensitive cell signaling, and alters the function of intracellular organelles. In particular, the mitochondrion is an important target organelle in determining the metastatic phenotype of CTCs. In healthy cells, mitochondria produce adenosine triphosphate (ATP) via oxidative phosphorylation in the electron transport chain, and during oxidative phosphorylation, they produce physiological levels of ROS. Mitochondria also govern death mechanisms such as apoptosis and mitochondrial permeability transition pore opening to, in order eliminate unwanted or damaged cells. However, in cancer cells, mitochondria are dysregulated, causing aberrant energy metabolism, redox homeostasis, and cell death pathways that may favor cancer invasiveness. In this review, we discuss the influence of fluid shear stress on CTCs with an emphasis on breast cancer pathology, then discuss alterations of cellular mechanisms that may increase the metastatic potentials of CTCs.

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Most cancer deaths are caused by secondary metastasized tumors. The cells that spread these tumors are known as circulating tumor cells (CTCs). They exist in a dynamic environment, including exposure to fluid shear stress (FSS) that makes them susceptible to reactive oxygen species (ROS) generation. There are questions about the similarities of CTCs to cancer stem cells (CSCs) and whether the stem cell-like characteristics of CTCs allow them to proliferate and spread despite the biophysical obstacles during the metastatic process. One of those qualities is the ability to undergo the epithelial-to-mesenchymal transition (EMT). Here, MDA-MB-231 and MCF7 were modeled as CTCs by prolonged exposure to FSS using a spinner flask. They were tested for ROS generation, CSC, EMT, and Hippo pathway gene and protein markers using qRT-PCR and flow cytometry. MDA-MB-231 did not show significant changes in CSC markers, but did show significant changes in ROS, EMT, and Hippo markers (<i>p</i> &#x3c; 0.05). Similarly, MCF7 showed significant changes in ROS and EMT markers (<i>p</i> &#x3c; 0.05). Furthermore, both cell lines demonstrated the reverse mesenchymal-to-epithelial transition signature when allowed to recover after FSS. These results suggest that the degree of their stemness or aggressiveness affects their responses to externally applied biophysical forces and demonstrates a potential link between mechanotransduction, the Hippo pathway, and the induction of EMT in breast cancer cells.

Extracellular vesicles (EVs) have shown great potential as cell-free therapeutics and biomimetic nanocarriers for drug delivery. However, the potential of EVs is limited by scalable, reproducible production and in vivo tracking after delivery. Here, we report the preparation of quercetin-iron complex nanoparticle-loaded EVs derived from a breast cancer cell line, MDA-MB-231br, using direct flow filtration. The morphology and size of the nanoparticle-loaded EVs were characterized using transmission electron microscopy and dynamic light scattering. The SDS-PAGE gel electrophoresis of those EVs showed several protein bands in the range of 20–100 kDa. The analysis of EV protein markers by a semi-quantitative antibody array confirmed the presence of several typical EV markers, such as ALIX, TSG101, CD63, and CD81. Our EV yield quantification suggested a significant yield increase in direct flow filtration compared with ultracentrifugation. Subsequently, we compared the cellular uptake behaviors of nanoparticle-loaded EVs with free nanoparticles using MDA-MB-231br cell line. Iron staining studies indicated that free nanoparticles were taken up by cells via endocytosis and localized at a certain area within the cells while uniform iron staining across cells was observed for cells treated with nanoparticle-loaded EVs. Our studies demonstrate the feasibility of using direct flow filtration for the production of nanoparticle-loaded EVs from cancer cells. The cellular uptake studies suggested the possibility of deeper penetration of the nanocarriers because the cancer cells readily took up the quercetin-iron complex nanoparticles, and then released nanoparticle-loaded EVs, which can be further delivered to regional cells.

Objectives The F1Fo ATP synthase is a multienzyme complex that produces mitochondrial ATP. Aberrant expression or assembly of F1Fo ATP synthase subunits leads to alterations in energy metabolism. We recently found that breast cancer cells exposed to fluid shear stress (FSS) have significantly enhanced metastatic behavior including chemoresistance and cell proliferation. Chemoresistance depends upon active transport systems, and cell division and growth require ATP. Therefore, we hypothesized that circulating breast cancer cells undergo altered energy metabolism via FSS-induced changes in F1Fo ATP synthase subunits and subsequent mitochondrial remodeling. Methods Non-metastatic MCF7 and metastatic MDA-MB-231 human breast cancer cells were treated with or without FSS and cultured. Cellular proliferation was assayed by measuring cell number and gap distance. Metabolic profile including intracellular ATP and oxygen consumption rate were analyzed. We also quantified abundance of F1Fo ATP synthase subunits using immunoblotting. Results Treatment with FSS significantly increased proliferation of both MCF7 and MDA-MB-231 human breast cancer cells. FSS significantly increased intracellular ATP in MDA-MB-231 breast cancer cells while ATP levels in MCF7 were not significantly changed. MDA-MB-231 cells retained increased ATP after treatment with the uncoupler FCCP, indicating remodeling and decreased reliance on mitochondrial energy metabolism. Interestingly, oxygen consumption rate was significantly increased in both MCF7 and MDA-MB-231 by FSS. We further quantified the abundance of F1Fo ATP synthase subunits in both cell lines. The β- and c-subunits of the F1Fo ATP synthase were significantly depleted in both lines of FSS-treated breast cancer cells. Conclusions Our data show that FSS alters abundance of the F1Fo ATP synthase subunits leading to metabolic remodeling. We suggest that FSS may influence non-metastatic (MCF7) and metastatic cancer cells (MDA-MB-231) differently. Underlying changes in mitochondrial and cytoplasmic ATP production in these cells is still under investigation. However, it is possible that reactive oxygen species generated during FSS may signal a switch to cytoplasmic intracellular energy metabolism. Funding Sources Alabama Life Research Institute Pilot Project (University of Alabama)

Objectives Fluid sheer stress (FSS) is a physical stimuli of circulating tumor cells responsible for development of and progression to cancer. FSS is reported to enhance chemoresistance and proliferation in breast cancer cells. However, cellular mechanisms explaining how FSS contributes to the metastatic phenotype of breast cancer cell are less known. Chemoresistance is highly dependent upon active transport systems, and cell division and growth require ATP. In this study, we hypothesize that FSS contributes to mitochondrial remodeling and leads to alterations in energy metabolism which favor metastasis. Methods MDA-MB-231 human breast cancer cells were exposed to fluid sheer stress (FSS). MDA-MB-231 cells were then grown in culture media for 24 h, and intracellular energy (ATP) and abundance of ATP synthase were analyzed. Results FSS significantly increases intracellular ATP in MDA-MB-231 breast cancer cells. Interestingly, MDA-MB-231 cells retained increased ATP after treatment with the uncoupler FCCP indicating remodeling and decreased reliance on mitochondrial energy metabolism. We then quantified the abundance of ATP synthase, the key enzyme complex that produces mitochondrial ATP. FSS significantly decreased protein levels of the c-subunit of ATP synthase. Conclusions Our data show that FSS causes metabolic remodeling of mitochondria-dependent ATP production. We suggest that the c-subunit of ATP synthase is an important target of FSS-mediated metastasis. Strategies to enhance the abundance or activity of the c-subunit may prevent metabolic remodeling-associated with metastasis in FSS-exposed circulating cancer cells. Funding Sources Alabama Life Research Institute (ALRI) 14,565.