Antitumor activity of photodynamic therapy with tetracationic derivative of synthetic bacteriochlorin in spheroid culture of liver and colon cancer cells.

Efficient screening of photosensitizers (PS) as well as studying their photodynamic activity, especially PS excited in the near-infrared region, require informative in vitro models to adequately reflect the architecture, thickness, and intercellular interactions in tumors. In our study, we used spheroids formed from human colon cancer HCT-116 cells and liver cancer Huh7 cells to assess the phototoxicity of a new PS based on tetracationic derivative of synthetic bacteriochlorin (BC4). We optimized conditions for the irradiation regime based on the kinetics of BC4 accumulation in spheroids and kinetics of spheroid growth. Although PS accumulated more efficiently in HCT-116 cells, characterized by more aggressive growth and high proliferative potential, they were less susceptible to the photodynamic therapy (PDT) compared to the slower growing Huh7 cells. We also showed that 3D models of spheroids were less sensitive to BC4 than conventional 2D cultures with relatively identical kinetics of drug accumulation. Our findings suggest that BC4 is a perspective agent for photodynamic therapy against cancer cells.

Distinct Effects of Beta-Amyloid, Its Isomerized and Phosphorylated Forms on the Redox Status and Mitochondrial Functioning of the Blood-Brain Barrier Endothelium.

The Alzheimer's disease (AD)-associated breakdown of the blood-brain barrier (BBB) promotes the accumulation of beta-amyloid peptide (Aß) in the brain as the BBB cells provide Aß transport from the brain parenchyma to the blood, and vice versa. The breakdown of the BBB during AD may be caused by the emergence of blood-borne Aß pathogenic forms, such as structurally and chemically modified Aß species; their effect on the BBB cells has not yet been studied. Here, we report that the effects of Aß42, Aß42, containing isomerized Asp7 residue (iso-Aß42) or phosphorylated Ser8 residue (p-Aß42) on the mitochondrial potential and respiration are closely related to the redox status changes in the mouse brain endothelial cells bEnd.3. Aß42 and iso-Aß42 cause a significant increase in nitric oxide, reactive oxygen species, glutathione, cytosolic calcium and the mitochondrial potential after 4 h of incubation. P-Aß42 either does not affect or its effect develops after 24 h of incubation. Aß42 and iso-Aß42 activate mitochondrial respiration compared to p-Aß42. The isomerized form promotes a greater cytotoxicity and mitochondrial dysfunction, causing maximum oxidative stress. Thus, Aß42, p-Aß42 and iso-Aß42 isoforms differently affect the BBBs' cell redox parameters, significantly modulating the functioning of the mitochondria. The changes in the level of modified Aß forms can contribute to the BBBs' breakdown during AD.

Pre-Senescence Induction in Hepatoma Cells Favors Hepatitis C Virus Replication and Can Be Used in Exploring Antiviral Potential of Histone Deacetylase Inhibitors.

Recent evidence suggests that fibrotic liver injury in patients with chronic hepatitis C correlates with cellular senescence in damaged liver tissue. However, it is still unclear how senescence can affect replication of the hepatitis C virus (HCV). In this work, we report that an inhibitor of cyclin-dependent kinases 4/6, palbociclib, not only induced in hepatoma cells a pre-senescent cellular phenotype, including G1 arrest in the cell cycle, but also accelerated viral replicon multiplication. Importantly, suppression of HCV replication by direct acting antivirals (DAAs) was barely affected by pre-senescence induction, and vice versa, the antiviral activities of host-targeting agents (HTAs), such as inhibitors of human histone deacetylases (HDACi), produced a wide range of reactions-from a dramatic reduction to a noticeable increase. It is very likely that under conditions of the G1 arrest in the cell cycle, HDACi exhibit their actual antiviral potency, since their inherent anticancer activity that complicates the interpretation of test results is minimized.

Mitochondrial localization of SESN2.

SESN2 is a member of the evolutionarily conserved sestrin protein family found in most of the Metazoa species. The SESN2 gene is transcriptionally activated by many stress factors, including metabolic derangements, reactive oxygen species (ROS), and DNA-damage. As a result, SESN2 controls ROS accumulation, metabolism, and cell viability. The best-known function of SESN2 is the inhibition of the mechanistic target of rapamycin complex 1 kinase (mTORC1) that plays a central role in support of cell growth and suppression of autophagy. SESN2 inhibits mTORC1 activity through interaction with the GATOR2 protein complex preventing an inhibitory effect of GATOR2 on the GATOR1 protein complex. GATOR1 stimulates GTPase activity of the RagA/B small GTPase, the component of RagA/B:RagC/D complex, preventing mTORC1 translocation to the lysosomes and its activation by the small GTPase Rheb. Despite the well-established role of SESN2 in mTORC1 inhibition, other SESN2 activities are not well-characterized. We recently showed that SESN2 could control mitochondrial function and cell death via mTORC1-independent mechanisms, and these activities might be explained by direct effects of SESN2 on mitochondria. In this work, we examined mitochondrial localization of SESN2 and demonstrated that SESN2 is located on mitochondria and can be directly involved in the regulation of mitochondrial functions.

Isolation and Expansion of Mesenchymal Stem Cells from Murine Adipose Tissue.

Mesenchymal stem cells (MSCs) are currently intensively studied due to significant promise which they represent for successful implementations of future cell therapy clinical protocols. This in turn emphasizes importance of careful preclinical studies of MSC effects in various murine disease models. The appropriate cell preparations with reproducible biological properties are important to minimize variability of results of experimental cell therapies. We describe here a simple protocol for isolation of murine MSCs from adipose tissues and their reproducible multi-log expansion under hypoxia conditions.